2024-03-28T09:02:51Zhttp://repository.helmholtz-hzi.de/oai/requestoai:repository.helmholtz-hzi.de:10033/84032019-08-30T11:32:38Zcom_10033_6823com_10033_6820col_10033_6891
Ben-Asouli, Yitzhak
Banai, Yona
Hauser, Hansjoerg
Kaempfer, Raymond
2007-02-14T15:44:56Z
2000-02-15
2007-02-14T15:44:56Z
2000-02-15
Nucleic Acids Research 2000 28(4):1011-1018
0305-1048
1362-4962
10648795
http://hdl.handle.net/10033/8403
102579
en_US
Oxford University Press
http://www.oup.co.uk/nar/Volume_28/Issue_4/html/gkd196.htm
Copyright © 2000 Oxford University Press
Recognition of 5′-terminal TAR structure in human immunodeficiency virus-1 mRNA by eukaryotic translation initiation factor 2
YES2018-06-13T01:07:07Zoai:repository.helmholtz-hzi.de:10033/85032019-08-30T11:32:38Zcom_10033_6824com_10033_6823com_10033_6820col_10033_6892
Liebich, I.
Bode, J.
Reuter, I.
Wingender, E.
2007-02-19T08:57:29Z
2002-08-01
2007-02-19T08:57:29Z
2002-08-01
Nucleic Acids Research 2002 30(15):3433-3442
0305-1048
1362-4962
12140328
http://hdl.handle.net/10033/8503
137072
en_US
Oxford University Press
http://www.nar.oupjournals.org/content/vol30/issue15/
Copyright © 2002 Oxford University Press
Evaluation of sequence motifs found in scaffold/matrix-attached regions (S/MARs)
YES2018-06-13T00:13:57Zoai:repository.helmholtz-hzi.de:10033/86072019-08-30T11:32:38Zcom_10033_6823com_10033_6820col_10033_6891
Kollmus, H
Flohé, L
McCarthy, J E
2007-02-20T13:00:28Z
1996-04-01
2007-02-20T13:00:28Z
1996-04-01
Nucleic Acids Research 1996 24(7):1195-1201
0305-1048
1362-4962
8614619
http://hdl.handle.net/10033/8607
145795
en_US
Analysis of eukaryotic mRNA structures directing cotranslational incorporation of selenocysteine.
YES2018-06-12T20:05:19Zoai:repository.helmholtz-hzi.de:10033/85212019-08-30T11:32:38Zcom_10033_6823com_10033_6820col_10033_6891
Karreman, S
Hauser, Hansjoerg
Karreman, C
2007-02-19T09:49:19Z
1996-05-01
2007-02-19T09:49:19Z
1996-05-01
Nucleic Acids Research 1996 24(9):1616-1624
0305-1048
1362-4962
8649977
http://hdl.handle.net/10033/8521
145857
en_US
On the use of double FLP recognition targets (FRTs) in the LTR of retroviruses for the construction of high producer cell lines.
YES2018-06-12T17:32:13Zoai:repository.helmholtz-hzi.de:10033/85912019-08-30T11:24:24Zcom_10033_6823com_10033_6820col_10033_6891
Feng, Xuesheng
Guo, Zhong
Nourbakhsh, Mahtab
Hauser, Hansjorg
Ganster, Ray
Shao, Lifang
Geller, David A.
2007-02-20T12:43:10Z
2002-10-29
2007-02-20T12:43:10Z
2002-10-29
Proceedings of the National Academy of Sciences of the United States of America 2002 99(22):14212-14217
0027-8424
1091-6490
12381793
10.1073/pnas.212306199
http://hdl.handle.net/10033/8591
137863
en_US
National Academy of Sciences
Copyright © 2002, The National Academy of Sciences
Identification of a negative response element in the human inducible nitric-oxide synthase (hiNOS) promoter: The role of NF-κB-repressing factor (NRF) in basal repression of the hiNOS gene
YES2018-06-13T17:04:27Zoai:repository.helmholtz-hzi.de:10033/85942019-08-30T11:24:31Zcom_10033_6823com_10033_6820col_10033_6891
Wirth, M
Grannemann, R
Klehr, D
Hauser, Hansjoerg
2007-02-20T12:45:49Z
1994-01
2007-02-20T12:45:49Z
1994-01
Journal of Virology 1994 68(1):566-569
0022-538X
1098-5514
8254773
http://hdl.handle.net/10033/8594
236323
Images
en_US
Screening retroviral packaging cells for highly efficient virus production by using a combined selection procedure.
YES2018-06-13T19:35:23ZImagesoai:repository.helmholtz-hzi.de:10033/85952019-08-30T11:37:44Zcom_10033_6823com_10033_6820col_10033_6891
Kollmus, H
Honigman, A
Panet, A
Hauser, H
2007-02-20T12:46:19Z
1994-09
2007-02-20T12:46:19Z
1994-09
Journal of Virology 1994 68(9):6087-6091
0022-538X
1098-5514
8057488
http://hdl.handle.net/10033/8595
237019
Images
en_US
The sequences of and distance between two cis-acting signals determine the efficiency of ribosomal frameshifting in human immunodeficiency virus type 1 and human T-cell leukemia virus type II in vivo.
YES2018-06-12T23:19:16ZImagesoai:repository.helmholtz-hzi.de:10033/85962019-08-30T11:25:43Zcom_10033_6823com_10033_6820col_10033_6891
Reil, H
Kollmus, H
Weidle, U H
Hauser, Hansjoerg
2007-02-20T12:46:45Z
1993-09
2007-02-20T12:46:45Z
1993-09
Journal of Virology 1993 67(9):5579-5584
0022-538X
1098-5514
8350413
http://hdl.handle.net/10033/8596
237961
Images
en_US
A heptanucleotide sequence mediates ribosomal frameshifting in mammalian cells.
YES2018-06-12T17:18:31ZImagesoai:repository.helmholtz-hzi.de:10033/86512019-08-30T11:31:23Zcom_10033_6823com_10033_6820col_10033_6891
Kirchhoff, S
Schaper, F
Hauser, Hansjoerg
2007-02-20T13:40:42Z
1993-06-25
2007-02-20T13:40:42Z
1993-06-25
Nucleic Acids Research 1993 21(12):2881-2889
0305-1048
1362-4962
8332497
http://hdl.handle.net/10033/8651
309674
Images
en_US
Interferon regulatory factor 1 (IRF-1) mediates cell growth inhibition by transactivation of downstream target genes.
YES2018-06-12T23:39:49ZImagesoai:repository.helmholtz-hzi.de:10033/86532019-08-30T11:26:13Zcom_10033_6823com_10033_6820col_10033_6891
Oliveira, C C
Goossen, B
Zanchin, N I
McCarthy, J E
Hentze, M W
Stripecke, R
2007-02-20T13:42:21Z
1993-11-25
2007-02-20T13:42:21Z
1993-11-25
Nucleic Acids Research 1993 21(23):5316-5322
0305-1048
1362-4962
8265343
http://hdl.handle.net/10033/8653
310564
Images
en_US
Translational repression by the human iron-regulatory factor (IRF) in Saccharomyces cerevisiae.
YES2018-06-12T18:08:48ZImagesoai:repository.helmholtz-hzi.de:10033/86542019-08-30T11:26:13Zcom_10033_6823com_10033_6820col_10033_6891
Hobom, G.
Schwarz, E.
Melzer, M.
Mayer, H.
2007-02-20T13:43:15Z
1981-10-10
2007-02-20T13:43:15Z
1981-10-10
Nucleic Acids Research 1981 9(19):4823-4832
0305-1048
1362-4962
6273787
http://hdl.handle.net/10033/8654
327482
Images
en_US
© IRL Press Limited, 1 Falconberg Court, London W1V 5FG, U.K.
Restriction endonuclease EcaI from Enterobacter cloacae
YES2018-06-13T21:42:00ZImagesoai:repository.helmholtz-hzi.de:10033/86552019-08-30T11:26:13Zcom_10033_6823com_10033_6820col_10033_6891
Rupp, E
Mayer, H
Wingender, E
2007-02-20T13:43:40Z
1990-10-11
2007-02-20T13:43:40Z
1990-10-11
Nucleic Acids Research 1990 18(19):5677-5683
0305-1048
1362-4962
1977134
http://hdl.handle.net/10033/8655
332300
Images
en_US
The promoter of the human parathyroid hormone gene contains a functional cyclic AMP-response element.
YES2018-06-13T00:30:54ZImagesoai:repository.helmholtz-hzi.de:10033/86812019-08-30T11:25:40Zcom_10033_6824com_10033_6823com_10033_6820col_10033_6892
Jenke, Andreas C. W.
Stehle, Isa M.
Herrmann, Frank
Eisenberger, Tobias
Baiker, Armin
Bode, Jürgen
Fackelmayer, Frank O.
Lipps, Hans J.
2007-02-21T08:17:26Z
2004-08-03
2007-02-21T08:17:26Z
2004-08-03
Proceedings of the National Academy of Sciences of the United States of America 2004 101(31):11322-11327
0027-8424
1091-6490
15272077
10.1073/pnas.0401355101
http://hdl.handle.net/10033/8681
509201
en_US
National Academy of Sciences
Copyright © 2004, The National Academy of Sciences
Nuclear scaffold/matrix attached region modules linked to a transcription unit are sufficient for replication and maintenance of a mammalian episome
YES2018-06-13T03:56:42Zoai:repository.helmholtz-hzi.de:10033/86932019-08-30T11:31:49Zcom_10033_6823com_10033_6820col_10033_6891
Nourbakhsh, M
Hoffmann, K
Hauser, Hansjoerg
2007-02-21T08:30:21Z
1993-02
2007-02-21T08:30:21Z
1993-02
The EMBO Journal 1993 12(2):451-459
0261-4189
1460-2075
8440236
http://hdl.handle.net/10033/8693
413228
Images
en_US
Interferon-beta promoters contain a DNA element that acts as a position-independent silencer on the NF-kappa B site.
YES2018-06-12T23:14:39ZImagesoai:repository.helmholtz-hzi.de:10033/86952019-08-30T11:24:27Zcom_10033_6824com_10033_6823com_10033_6820col_10033_6892
Goetze, Sandra
Baer, Alexandra
Winkelmann, Silke
Nehlsen, Kristina
Seibler, Jost
Maass, Karin
Bode, Jürgen
2007-02-21T08:35:35Z
2005-03
2007-02-21T08:35:35Z
2005-03
Molecular and Cellular Biology 2005 25(6):2260-2272
0270-7306
1098-5549
15743822
10.1128/MCB.25.6.2260-2272.2005
http://hdl.handle.net/10033/8695
1061597
en_US
American Society for Microbiology
Copyright © 2005, American Society for Microbiology
Performance of Genomic Bordering Elements at Predefined Genomic Loci
YES2018-06-12T17:57:39Zoai:repository.helmholtz-hzi.de:10033/87012019-08-30T11:24:27Zcom_10033_6823com_10033_6820col_10033_6891
Nourbakhsh, M
Hauser, Hansjoerg
2007-02-21T08:40:15Z
1999-11-15
2007-02-21T08:40:15Z
1999-11-15
The EMBO Journal 1999 18(22):6415-6425
0261-4189
1460-2075
10562553
10.1093/emboj/18.22.6415
http://hdl.handle.net/10033/8701
1171704
en_US
Constitutive silencing of IFN-beta promoter is mediated by NRF (NF-kappaB-repressing factor), a nuclear inhibitor of NF-kappaB.
YES2018-06-13T07:45:43Zoai:repository.helmholtz-hzi.de:10033/87432019-08-30T11:30:29Zcom_10033_6824com_10033_6823com_10033_6820col_10033_6892
Klar, Martin
Bode, Juergen
2007-02-22T13:38:14Z
2005-11
2007-02-22T13:38:14Z
2005-11
Molecular and Cellular Biology 2005 25(22):10159-10170
0270-7306
1098-5549
16260628
10.1128/MCB.25.22.10159-10170.2005
http://hdl.handle.net/10033/8743
1280260
en_US
American Society for Microbiology
Copyright © 2005, American Society for Microbiology
Enhanceosome Formation over the Beta Interferon Promoter Underlies a Remote-Control Mechanism Mediated by YY1 and YY2
YES2018-06-12T16:46:08Zoai:repository.helmholtz-hzi.de:10033/87482019-08-30T11:25:09Zcom_10033_6823com_10033_6820col_10033_6891
Kollmus, H
Hentze, M W
Hauser, Hansjoerg
2007-02-22T14:45:19Z
1996-04
2007-02-22T14:45:19Z
1996-04
RNA 1996 2(4):316-323
1355-8382
1469-9001
8634912
http://hdl.handle.net/10033/8748
1369374
en_US
Regulated ribosomal frameshifting by an RNA-protein interaction.
YES2018-06-13T07:38:51Zoai:repository.helmholtz-hzi.de:10033/87542019-08-30T11:25:09Zcom_10033_6823com_10033_6820col_10033_6891
Hoffmann, Andrea
Pelled, Gadi
Turgeman, Gadi
Eberle, Peter
Zilberman, Yoram
Shinar, Hadassah
Keinan-Adamsky, Keren
Winkel, Andreas
Shahab, Sandra
Navon, Gil
Gross, Gerhard
Gazit, Dan
2007-02-22T14:52:43Z
2006-04-03
2007-02-22T14:52:43Z
2006-04-03
Journal of Clinical Investigation 2006 116(4):940-952
0021-9738
16585960
10.1172/JCI22689
http://hdl.handle.net/10033/8754
1421340
en_US
American Society for Clinical Investigation
Copyright © 2006, American Society for Clinical Investigation
Neotendon formation induced by manipulation of the Smad8 signalling pathway in mesenchymal stem cells
YES2018-06-12T17:42:57Zoai:repository.helmholtz-hzi.de:10033/123102019-08-30T11:37:00Zcom_10033_6822com_10033_6821com_10033_6820col_10033_6895
He, Feng
Zeng, An-Ping
2007-06-08T14:30:36Z
2007-06-08T14:30:36Z
2006
BMC Bioinformatics 2006, 7:69
1471-2105
16478547
10.1186/1471-2105-7-69
http://hdl.handle.net/10033/12310
BACKGROUND: The increasing availability of time-series expression data opens up new possibilities to study functional linkages of genes. Present methods used to infer functional linkages between genes from expression data are mainly based on a point-to-point comparison. Change trends between consecutive time points in time-series data have been so far not well explored. RESULTS: In this work we present a new method based on extracting main features of the change trend and level of gene expression between consecutive time points. The method, termed as trend correlation (TC), includes two major steps: 1, calculating a maximal local alignment of change trend score by dynamic programming and a change trend correlation coefficient between the maximal matched change levels of each gene pair; 2, inferring relationships of gene pairs based on two statistical extraction procedures. The new method considers time shifts and inverted relationships in a similar way as the local clustering (LC) method but the latter is merely based on a point-to-point comparison. The TC method is demonstrated with data from yeast cell cycle and compared with the LC method and the widely used Pearson correlation coefficient (PCC) based clustering method. The biological significance of the gene pairs is examined with several large-scale yeast databases. Although the TC method predicts an overall lower number of gene pairs than the other two methods at a same p-value threshold, the additional number of gene pairs inferred by the TC method is considerable: e.g. 20.5% compared with the LC method and 49.6% with the PCC method for a p-value threshold of 2.7E-3. Moreover, the percentage of the inferred gene pairs consistent with databases by our method is generally higher than the LC method and similar to the PCC method. A significant number of the gene pairs only inferred by the TC method are process-identity or function-similarity pairs or have well-documented biological interactions, including 443 known protein interactions and some known cell cycle related regulatory interactions. It should be emphasized that the overlapping of gene pairs detected by the three methods is normally not very high, indicating a necessity of combining the different methods in search of functional association of genes from time-series data. For a p-value threshold of 1E-5 the percentage of process-identity and function-similarity gene pairs among the shared part of the three methods reaches 60.2% and 55.6% respectively, building a good basis for further experimental and functional study. Furthermore, the combined use of methods is important to infer more complete regulatory circuits and network as exemplified in this study. CONCLUSION: The TC method can significantly augment the current major methods to infer functional linkages and biological network and is well suitable for exploring temporal relationships of gene expression in time-series data.
984387 bytes
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In search of functional association from time-series microarray data based on the change trend and level of gene expression.
Article2018-06-13T05:37:18ZBACKGROUND: The increasing availability of time-series expression data opens up new possibilities to study functional linkages of genes. Present methods used to infer functional linkages between genes from expression data are mainly based on a point-to-point comparison. Change trends between consecutive time points in time-series data have been so far not well explored. RESULTS: In this work we present a new method based on extracting main features of the change trend and level of gene expression between consecutive time points. The method, termed as trend correlation (TC), includes two major steps: 1, calculating a maximal local alignment of change trend score by dynamic programming and a change trend correlation coefficient between the maximal matched change levels of each gene pair; 2, inferring relationships of gene pairs based on two statistical extraction procedures. The new method considers time shifts and inverted relationships in a similar way as the local clustering (LC) method but the latter is merely based on a point-to-point comparison. The TC method is demonstrated with data from yeast cell cycle and compared with the LC method and the widely used Pearson correlation coefficient (PCC) based clustering method. The biological significance of the gene pairs is examined with several large-scale yeast databases. Although the TC method predicts an overall lower number of gene pairs than the other two methods at a same p-value threshold, the additional number of gene pairs inferred by the TC method is considerable: e.g. 20.5% compared with the LC method and 49.6% with the PCC method for a p-value threshold of 2.7E-3. Moreover, the percentage of the inferred gene pairs consistent with databases by our method is generally higher than the LC method and similar to the PCC method. A significant number of the gene pairs only inferred by the TC method are process-identity or function-similarity pairs or have well-documented biological interactions, including 443 known protein interactions and some known cell cycle related regulatory interactions. It should be emphasized that the overlapping of gene pairs detected by the three methods is normally not very high, indicating a necessity of combining the different methods in search of functional association of genes from time-series data. For a p-value threshold of 1E-5 the percentage of process-identity and function-similarity gene pairs among the shared part of the three methods reaches 60.2% and 55.6% respectively, building a good basis for further experimental and functional study. Furthermore, the combined use of methods is important to infer more complete regulatory circuits and network as exemplified in this study. CONCLUSION: The TC method can significantly augment the current major methods to infer functional linkages and biological network and is well suitable for exploring temporal relationships of gene expression in time-series data.oai:repository.helmholtz-hzi.de:10033/123672019-08-30T11:25:43Zcom_10033_6823com_10033_6820col_10033_6891
Mueller, Peter P
May, Tobias
Perz, Angela
Hauser, Hansjörg
Peuster, Matthias
2007-06-14T09:39:14Z
2007-06-14T09:39:14Z
2006-04-01
Biomaterials 2006, 27(10):2193-200
0142-9612
16310850
10.1016/j.biomaterials.2005.10.042
http://hdl.handle.net/10033/12367
This study was conducted to determine the interaction of individual corrosion products from biodegradable iron stents with cells from the adjacent tissue. The response of human umbilical venous smooth muscle cells (SMCs) to an excess of ferrous ions was investigated in a cell culture model at the phenotypic and at the molecular level. When soluble ferrous ions were added to the cell culture medium the cell growth rate was reduced. Gene expression profiling indicated a reduction in the amounts of mRNA from genes that are required for cell proliferation. In addition, mRNA was regulated from multiple genes involved in iron homeostasis, DNA replication and lipid metabolism. In conclusion, ions released from iron stents could reduce the vascular SMC proliferation rate by influencing growth-related gene expression and may therefore play a beneficial role in antagonizing restenosis in vivo.
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Control of smooth muscle cell proliferation by ferrous iron.
Article2018-06-13T15:20:10ZThis study was conducted to determine the interaction of individual corrosion products from biodegradable iron stents with cells from the adjacent tissue. The response of human umbilical venous smooth muscle cells (SMCs) to an excess of ferrous ions was investigated in a cell culture model at the phenotypic and at the molecular level. When soluble ferrous ions were added to the cell culture medium the cell growth rate was reduced. Gene expression profiling indicated a reduction in the amounts of mRNA from genes that are required for cell proliferation. In addition, mRNA was regulated from multiple genes involved in iron homeostasis, DNA replication and lipid metabolism. In conclusion, ions released from iron stents could reduce the vascular SMC proliferation rate by influencing growth-related gene expression and may therefore play a beneficial role in antagonizing restenosis in vivo.oai:repository.helmholtz-hzi.de:10033/123992019-08-30T11:25:43Zcom_10033_6823com_10033_6820col_10033_6891
Ma, Bin
Winkelbach, Simon
Lindenmaier, Werner
Dittmar, Kurt E J
2007-06-20T07:44:01Z
2007-06-20T07:44:01Z
2006
Acta Histochem. 2006, 108(4):243-57
0065-1281
16730369
10.1016/j.acthis.2006.02.002
http://hdl.handle.net/10033/12399
Multi-colour imaging of immunofluorescently labelled tissue using confocal microscopy was accomplished by using colour addition theory. This new technique includes several improvements for immunolabelling: (1) the co-localization of two or more markers on one cell for the identification of specific cell populations; (2) the co-localization of two fluorescent dyes from secondary reagents for the identification of the cells; (3) a multi-step staining protocol with two primary antibodies originating from the same host species or with two or three biotin-conjugated primary antibodies. After image acquisition, colour segmentation/unmixing are applied to the single multi-colour image to generate multi-pseudo-channels for individual or co-localized fluorescent dyes. With this new technique, we have been able to visualize six cell populations simultaneously in the mouse lymph node and intestine. The efficiency of this method has also been demonstrated in the three-dimensional reconstruction of thick sections from mouse ileum. Our method is simple, efficient, and may be indispensable in experimental cell and tissue studies requiring multiple immunolabelling.
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Six-colour fluorescent imaging of lymphoid tissue based on colour addition theory.
Article2018-05-23T12:10:51ZMulti-colour imaging of immunofluorescently labelled tissue using confocal microscopy was accomplished by using colour addition theory. This new technique includes several improvements for immunolabelling: (1) the co-localization of two or more markers on one cell for the identification of specific cell populations; (2) the co-localization of two fluorescent dyes from secondary reagents for the identification of the cells; (3) a multi-step staining protocol with two primary antibodies originating from the same host species or with two or three biotin-conjugated primary antibodies. After image acquisition, colour segmentation/unmixing are applied to the single multi-colour image to generate multi-pseudo-channels for individual or co-localized fluorescent dyes. With this new technique, we have been able to visualize six cell populations simultaneously in the mouse lymph node and intestine. The efficiency of this method has also been demonstrated in the three-dimensional reconstruction of thick sections from mouse ileum. Our method is simple, efficient, and may be indispensable in experimental cell and tissue studies requiring multiple immunolabelling.oai:repository.helmholtz-hzi.de:10033/134582019-08-30T11:34:48Zcom_10033_6823com_10033_6820col_10033_6891
Frahm, Thomas
Hauser, Hansjörg
Köster, Mario
2007-09-05T08:56:35Z
2007-09-05T08:56:35Z
2006-03-15
J. Cell. Sci. 2006, 119(Pt 6):1092-104
0021-9533
16507591
10.1242/jcs.02822
http://hdl.handle.net/10033/13458
Signaling through the IFN type I receptor is mediated by assembly of the ISGF3 complex consisting of STAT1, STAT2 and IRF9. Whereas STAT1 is instrumentalized by many cytokines, STAT2 is specifically used by type I IFNs. Here, we report that the main regulatory mechanism of nuclear accumulation of STAT2 is nuclear export. We determined the kinetics of nucleocytoplasmic shuttling of STAT2 in living cells. In the absence of IFN, a virtually exclusive cytoplasmic localisation of STAT2 can be detected. Nevertheless, STAT2 is permanently and rapidly shuttling between the cytoplasm and the nucleus. The steady-state localization is explained by a very efficient nuclear export. Our studies indicate that at least two pathways (one of which is CRM1-dependent, the other not yet identified) are responsible for clearing the nucleus from STAT2. The constitutive nucleocytoplasmic shuttling of STAT2 does neither depend on the presence of IRF9 or STAT1, nor does it require tyrosine phosphorylation. Upon treatment with IFN type I, nuclear export of STAT2 is completely abolished in cells used within this study, whereas nuclear import is functioning. This explains the observed nuclear accumulation of STAT2. We have identified a region in the C-terminus of STAT2 that is essential for its almost exclusively cytoplasmic localization in the absence of IFN and responsible for CRM1-specific export. In comparative studies we show that nucleocytoplasmic shuttling of STAT2 is significantly different from that of STAT1. STAT1 is also shuttling in the absence of IFN, but the exchange rate in unstimulated cells is more than ten times lower. We further show that the latent STAT2 protein has stronger intrinsic nuclear-export activity than STAT1. Together, these observations lead to a model for IFN-type-I-induction in which the receptor-mediated heterodimerization overcomes the slow nuclear import of STAT1 and blocks the strong STAT2 export activity that leads to the accumulation of both signal transducers in the nucleus.
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en
IFN-type-I-mediated signaling is regulated by modulation of STAT2 nuclear export.
Article2018-06-13T00:00:18ZSignaling through the IFN type I receptor is mediated by assembly of the ISGF3 complex consisting of STAT1, STAT2 and IRF9. Whereas STAT1 is instrumentalized by many cytokines, STAT2 is specifically used by type I IFNs. Here, we report that the main regulatory mechanism of nuclear accumulation of STAT2 is nuclear export. We determined the kinetics of nucleocytoplasmic shuttling of STAT2 in living cells. In the absence of IFN, a virtually exclusive cytoplasmic localisation of STAT2 can be detected. Nevertheless, STAT2 is permanently and rapidly shuttling between the cytoplasm and the nucleus. The steady-state localization is explained by a very efficient nuclear export. Our studies indicate that at least two pathways (one of which is CRM1-dependent, the other not yet identified) are responsible for clearing the nucleus from STAT2. The constitutive nucleocytoplasmic shuttling of STAT2 does neither depend on the presence of IRF9 or STAT1, nor does it require tyrosine phosphorylation. Upon treatment with IFN type I, nuclear export of STAT2 is completely abolished in cells used within this study, whereas nuclear import is functioning. This explains the observed nuclear accumulation of STAT2. We have identified a region in the C-terminus of STAT2 that is essential for its almost exclusively cytoplasmic localization in the absence of IFN and responsible for CRM1-specific export. In comparative studies we show that nucleocytoplasmic shuttling of STAT2 is significantly different from that of STAT1. STAT1 is also shuttling in the absence of IFN, but the exchange rate in unstimulated cells is more than ten times lower. We further show that the latent STAT2 protein has stronger intrinsic nuclear-export activity than STAT1. Together, these observations lead to a model for IFN-type-I-induction in which the receptor-mediated heterodimerization overcomes the slow nuclear import of STAT1 and blocks the strong STAT2 export activity that leads to the accumulation of both signal transducers in the nucleus.oai:repository.helmholtz-hzi.de:10033/142832019-08-30T11:34:48Zcom_10033_6823com_10033_6820col_10033_6891
Yu, Zheng
Beer, Christiane
Koester, Mario
Wirth, Manfred
2007-10-25T13:26:43Z
2007-10-25T13:26:43Z
2006
Virol. J. 2006, 3:73
1743-422X
16956408
10.1186/1743-422X-3-73
http://hdl.handle.net/10033/14283
BACKGROUND: Retroviral Gag determines virus assembly at the plasma membrane and the formation of virus-like particles in intracellular multivesicular bodies. Thereby, retroviruses exploit by interaction with cellular partners the cellular machineries for vesicular transport in various ways. RESULTS: The retroviral Gag precursor protein drives assembly of murine leukaemia viruses (MLV) at the plasma membrane (PM) and the formation of virus like particles in multivesicular bodies (MVBs). In our study we show that caveolin-1 (Cav-1), a multifunctional membrane-associated protein, co-localizes with Gag in a punctate pattern at the PM of infected NIH 3T3 cells. We provide evidence that Cav-1 interacts with the matrix protein (MA) of the Gag precursor. This interaction is mediated by a Cav-1 binding domain (CBD) within the N-terminus of MA. Interestingly, the CBD motif identified within MA is highly conserved among most other gamma-retroviruses. Furthermore, Cav-1 is incorporated into MLV released from NIH 3T3 cells. Overexpression of a GFP fusion protein containing the putative CBD of the retroviral MA resulted in a considerable decrease in production of infectious retrovirus. Moreover, expression of a dominant-negative Cav-1 mutant affected retroviral titres significantly. CONCLUSION: This study demonstrates that Cav-1 interacts with MLV Gag, co-localizes with Gag at the PM and affects the production of infectious virus. The results strongly suggest a role for Cav-1 in the process of virus assembly.
587001 bytes
application/pdf
YES
en
Caveolin-1 interacts with the Gag precursor of murine leukaemia virus and modulates virus production.
Article2018-06-13T09:27:56ZBACKGROUND: Retroviral Gag determines virus assembly at the plasma membrane and the formation of virus-like particles in intracellular multivesicular bodies. Thereby, retroviruses exploit by interaction with cellular partners the cellular machineries for vesicular transport in various ways. RESULTS: The retroviral Gag precursor protein drives assembly of murine leukaemia viruses (MLV) at the plasma membrane (PM) and the formation of virus like particles in multivesicular bodies (MVBs). In our study we show that caveolin-1 (Cav-1), a multifunctional membrane-associated protein, co-localizes with Gag in a punctate pattern at the PM of infected NIH 3T3 cells. We provide evidence that Cav-1 interacts with the matrix protein (MA) of the Gag precursor. This interaction is mediated by a Cav-1 binding domain (CBD) within the N-terminus of MA. Interestingly, the CBD motif identified within MA is highly conserved among most other gamma-retroviruses. Furthermore, Cav-1 is incorporated into MLV released from NIH 3T3 cells. Overexpression of a GFP fusion protein containing the putative CBD of the retroviral MA resulted in a considerable decrease in production of infectious retrovirus. Moreover, expression of a dominant-negative Cav-1 mutant affected retroviral titres significantly. CONCLUSION: This study demonstrates that Cav-1 interacts with MLV Gag, co-localizes with Gag at the PM and affects the production of infectious virus. The results strongly suggest a role for Cav-1 in the process of virus assembly.oai:repository.helmholtz-hzi.de:10033/145942019-08-30T11:26:12Zcom_10033_6823com_10033_6820col_10033_6891
Schucht, R
Coroadinha, A S
Zanta-Boussif, M A
Verhoeyen, E
Carrondo, M J T
Hauser, Hansjoerg
Wirth, Dagmar
2007-11-16T14:34:49Z
2007-11-16T14:34:49Z
2006-08-01
Mol. Ther. 2006, 14(2):285-92
1525-0016
16697259
10.1016/j.ymthe.2005.12.003
http://hdl.handle.net/10033/14594
We developed a new strategy that provides well-defined high-titer producer cells for recombinant retroviruses in a minimum amount of time. The strategy involves the targeted integration of the retroviral vector into a chromosomal locus with favorable properties. For proof of concept we established a novel HEK293-based retroviral producer cell line, called Flp293A, with a single-copy retroviral vector integrated at a selected chromosomal locus. The vector was flanked by noninteracting Flp-recombinase recognition sites and was exchanged for different retroviral vectors via Flp-mediated cassette exchange. All analyzed cell clones showed correct integration and identical titers for each of the vectors, confirming that the expression characteristics from the parental cell were preserved. Titers up to 2.5 x 10(7) infectious particles/10(6) cells were obtained. Also, high-titer producer cells for a therapeutic vector that encodes the 8.9-kb collagen VII cDNA in a marker-free cassette were obtained within 3 weeks without screening. Thus, we provide evidence that the precise integration of viral vectors into a favorable chromosomal locus leads to high and predictable virus production. This method is compatible with other retroviral vectors, including self-inactivating vectors and marker-free vectors. Further, it provides a tool for evaluation of different retroviral vector designs.
387713 bytes
application/pdf
YES
en
A new generation of retroviral producer cells: predictable and stable virus production by Flp-mediated site-specific integration of retroviral vectors.
Article2018-06-12T21:40:14ZWe developed a new strategy that provides well-defined high-titer producer cells for recombinant retroviruses in a minimum amount of time. The strategy involves the targeted integration of the retroviral vector into a chromosomal locus with favorable properties. For proof of concept we established a novel HEK293-based retroviral producer cell line, called Flp293A, with a single-copy retroviral vector integrated at a selected chromosomal locus. The vector was flanked by noninteracting Flp-recombinase recognition sites and was exchanged for different retroviral vectors via Flp-mediated cassette exchange. All analyzed cell clones showed correct integration and identical titers for each of the vectors, confirming that the expression characteristics from the parental cell were preserved. Titers up to 2.5 x 10(7) infectious particles/10(6) cells were obtained. Also, high-titer producer cells for a therapeutic vector that encodes the 8.9-kb collagen VII cDNA in a marker-free cassette were obtained within 3 weeks without screening. Thus, we provide evidence that the precise integration of viral vectors into a favorable chromosomal locus leads to high and predictable virus production. This method is compatible with other retroviral vectors, including self-inactivating vectors and marker-free vectors. Further, it provides a tool for evaluation of different retroviral vector designs.oai:repository.helmholtz-hzi.de:10033/150152019-08-30T11:31:20Zcom_10033_6823com_10033_6820col_10033_6891
Peuster, Matthias
Beerbaum, Phillip
Bach, Friedrich-Wilhelm
Hauser, Hansjoerg
Clinic for Congenital Heart Defects and Cardiovascular Implant Research Unit, Heart and Diabetes Center, Ruhr-University Bochum, Bad Oeynhausen, Germany. mpeuster@hdz-nrw.de
2007-12-06T14:45:17Z
2007-12-06T14:45:17Z
2006-04
Are resorbable implants about to become a reality? 2006, 16 (2):107-16notCardiol Young
1047-9511
16553970
10.1017/S1047951106000011
http://hdl.handle.net/10033/15015
Cardiology in the young
en
Absorbable Implants
Alloys
Angioplasty, Transluminal, Percutaneous Coronary
Animals
Aorta, Thoracic
Blood Vessel Prosthesis
Cardiovascular Diseases
Coronary Vessels
Corrosion
Humans
Iron
Magnesium
Stents
Are resorbable implants about to become a reality?
Article2018-06-12T16:49:33Zoai:repository.helmholtz-hzi.de:10033/162742019-08-30T11:36:05Zcom_10033_6823com_10033_6820col_10033_6891
Norgall, Susanne
Papoutsi, Maria
Rössler, Jochen
Schweigerer, Lothar
Wilting, Jörg
Weich, Herbert A
Department Gene Regulation and Differentiation, Helmholtz Centre for Infection Research, Braunschweig, Germany. susannenorgall@gmx.de <susannenorgall@gmx.de>
2008-01-17T13:50:03Z
2008-01-17T13:50:03Z
2007
Elevated expression of VEGFR-3 in lymphatic endothelial cells from lymphangiomas. 2007, 7:105 BMC Cancer
1471-2407
17584927
10.1186/1471-2407-7-105
http://hdl.handle.net/10033/16274
BMC cancer
BACKGROUND: Lymphangiomas are neoplasias of childhood. Their etiology is unknown and a causal therapy does not exist. The recent discovery of highly specific markers for lymphatic endothelial cells (LECs) has permitted their isolation and characterization, but expression levels and stability of molecular markers on LECs from healthy and lymphangioma tissues have not been studied yet. We addressed this problem by profiling LECs from normal dermis and two children suffering from lymphangioma, and also compared them with blood endothelial cells (BECs) from umbilical vein, aorta and myometrial microvessels. METHODS: Lymphangioma tissue samples were obtained from two young patients suffering from lymphangioma in the axillary and upper arm region. Initially isolated with anti-CD31 (PECAM-1) antibodies, the cells were separated by FACS sorting and magnetic beads using anti-podoplanin and/or LYVE-1 antibodies. Characterization was performed by FACS analysis, immunofluorescence staining, ELISA and micro-array gene analysis. RESULTS: LECs from foreskin and lymphangioma had an almost identical pattern of lymphendothelial markers such as podoplanin, Prox1, reelin, cMaf and integrin-alpha1 and -alpha9. However, LYVE-1 was down-regulated and VEGFR-2 and R-3 were up-regulated in lymphangiomas. Prox1 was constantly expressed in LECs but not in any of the BECs. CONCLUSION: LECs from different sources express slightly variable molecular markers, but can always be distinguished from BECs by their Prox1 expression. High levels of VEGFR-3 and -2 seem to contribute to the etiology of lymphangiomas.
en
Adolescent
Biopsy, Needle
Case-Control Studies
Child
Endothelial Cells
Endothelium, Lymphatic
Enzyme-Linked Immunosorbent Assay
Female
Fluorescent Antibody Technique
Humans
Lymphangioma
Male
Prognosis
Reference Values
Sampling Studies
Sensitivity and Specificity
Skin Neoplasms
Tumor Cells, Cultured
Tumor Markers, Biological
Vascular Endothelial Growth Factor Receptor-2
Vascular Endothelial Growth Factor Receptor-3
Elevated expression of VEGFR-3 in lymphatic endothelial cells from lymphangiomas.
Article2018-06-12T17:58:24ZBACKGROUND: Lymphangiomas are neoplasias of childhood. Their etiology is unknown and a causal therapy does not exist. The recent discovery of highly specific markers for lymphatic endothelial cells (LECs) has permitted their isolation and characterization, but expression levels and stability of molecular markers on LECs from healthy and lymphangioma tissues have not been studied yet. We addressed this problem by profiling LECs from normal dermis and two children suffering from lymphangioma, and also compared them with blood endothelial cells (BECs) from umbilical vein, aorta and myometrial microvessels. METHODS: Lymphangioma tissue samples were obtained from two young patients suffering from lymphangioma in the axillary and upper arm region. Initially isolated with anti-CD31 (PECAM-1) antibodies, the cells were separated by FACS sorting and magnetic beads using anti-podoplanin and/or LYVE-1 antibodies. Characterization was performed by FACS analysis, immunofluorescence staining, ELISA and micro-array gene analysis. RESULTS: LECs from foreskin and lymphangioma had an almost identical pattern of lymphendothelial markers such as podoplanin, Prox1, reelin, cMaf and integrin-alpha1 and -alpha9. However, LYVE-1 was down-regulated and VEGFR-2 and R-3 were up-regulated in lymphangiomas. Prox1 was constantly expressed in LECs but not in any of the BECs. CONCLUSION: LECs from different sources express slightly variable molecular markers, but can always be distinguished from BECs by their Prox1 expression. High levels of VEGFR-3 and -2 seem to contribute to the etiology of lymphangiomas.oai:repository.helmholtz-hzi.de:10033/172322019-08-30T11:36:05Zcom_10033_6823com_10033_6820col_10033_6891
Adden, Nina
Hoffmann, Andrea
Gross, Gerhard
Windhagen, Henning
Thorey, Fritz
Menzel, Henning
Institut für Technische Chemie, Abt. TC Makromolekularer Stoffe, Technische Universität Braunschweig, Hans-Sommer-Strasse 10, D-38106 Braunschweig, Germany.
2008-01-31T14:30:10Z
2008-01-31T14:30:10Z
2007
Screening of photochemically grafted polymer films for compatibility with osteogenic precursor cells. 2007, 18 (3):303-16notJ Biomater Sci Polym Ed
0920-5063
17471767
http://hdl.handle.net/10033/17232
Journal of biomaterials science. Polymer edition
Surfaces of biomaterials often do not have the ideal properties for direct application in vivo. Although titanium and its alloys show a good biocompatibility, in some applications there is still need to improve the osteoblast adhesion to titanium implants. A polymeric surface coating is an ideal solution because the polymer can be adjusted to the needs of the application and can be bound to the surface by the photochemical grafting method. Therefore, 22 different polymers were tested for their compatibility using a murine mesenchymal progenitor cell line and three polymers were identified for which more elaborate investigations are reasonable. It was investigated whether or not the results of the cell culture test can be correlated with, e.g., the wetting properties. Indeed it was found that a contact angle above approx. 45 degrees was necessary for good cell adhesion and proliferation. However, otherwise no clear correlation between the contact angle hysteresis or the functionalities of the polymers and the cell growth was observed.
en
Acrylamides
Alloys
Animals
Biocompatible Materials
Biopolymers
Bone and Bones
Cell Adhesion
Humans
Molecular Conformation
Osteogenesis
Photochemistry
Polymethacrylic Acids
Styrenes
Surface Properties
Titanium
Screening of photochemically grafted polymer films for compatibility with osteogenic precursor cells.
Article2018-06-13T00:36:13ZSurfaces of biomaterials often do not have the ideal properties for direct application in vivo. Although titanium and its alloys show a good biocompatibility, in some applications there is still need to improve the osteoblast adhesion to titanium implants. A polymeric surface coating is an ideal solution because the polymer can be adjusted to the needs of the application and can be bound to the surface by the photochemical grafting method. Therefore, 22 different polymers were tested for their compatibility using a murine mesenchymal progenitor cell line and three polymers were identified for which more elaborate investigations are reasonable. It was investigated whether or not the results of the cell culture test can be correlated with, e.g., the wetting properties. Indeed it was found that a contact angle above approx. 45 degrees was necessary for good cell adhesion and proliferation. However, otherwise no clear correlation between the contact angle hysteresis or the functionalities of the polymers and the cell growth was observed.oai:repository.helmholtz-hzi.de:10033/192382019-08-30T11:37:00Zcom_10033_6823com_10033_6820col_10033_6891
Reboll, Marc René
Oumard, André
Gazdag, Aniko Carla
Renger, Isabelle
Ritter, Birgit
Schwarzer, Michael
Hauser, Hansjoerg
Wood, Monika
Yamada, Michiyuki
Resch, Klaus
Nourbakhsh, Mahtab
Institute of Pharmacology, Hannover Medical School, Hannover, Germany.
2008-02-27T14:16:06Z
2008-02-27T14:16:06Z
2007-08
NRF IRES activity is mediated by RNA binding protein JKTBP1 and a 14-nt RNA element. 2007, 13 (8):1328-40 RNA
1355-8382
17592041
10.1261/rna.545407
http://hdl.handle.net/10033/19238
RNA (New York, N.Y.)
The mRNA of human NF-kappaB repressing factor (NRF) contains a long 5'-untranslated region (UTR) that directs ribosomes to the downstream start codon by a cap-independent mechanism. Comparison of the nucleotide (nt) sequences of human and mouse NRF mRNAs reveals a high degree of identity throughout a fragment of 150 nt proximal to the start codon. Here, we show that this region constitutes a minimal internal ribosome entry segment (IRES) module. Enzymatic RNA structure analysis reveals a secondary structure model of the NRF IRES module. Point mutation analysis of the module determines a short, 14-nt RNA element (nt 640-653) as a mediator of IRES function. Purification of IRES binding cellular proteins and subsequent ESI/MS/MS sequence analysis led to identification of the RNA-binding protein, JKTBP1. EMSA experiments show that JKTBP1 binds upstream to the 14-nt RNA element in the NRF IRES module (nt 579-639). Over-expression of JKTBP1 significantly enhances activity of the NRF IRES module in dicistronic constructs. Moreover, siRNA experiments demonstrate that down-regulation of endogenous JKTBP1 decreases NRF IRES activity and the level of endogenous NRF protein. The data of this study show that JKTBP1 and the 14-nt element act independently to mediate NRF IRES activity.
en
http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=17592041
5' Untranslated Regions
Animals
Base Sequence
DNA-Binding Proteins
Genes, Reporter
Humans
Mice
Molecular Sequence Data
Nucleic Acid Conformation
Protein Isoforms
Repressor Proteins
Ribonucleoproteins
Ribosomes
NRF IRES activity is mediated by RNA binding protein JKTBP1 and a 14-nt RNA element.
Article2018-06-13T00:16:48ZThe mRNA of human NF-kappaB repressing factor (NRF) contains a long 5'-untranslated region (UTR) that directs ribosomes to the downstream start codon by a cap-independent mechanism. Comparison of the nucleotide (nt) sequences of human and mouse NRF mRNAs reveals a high degree of identity throughout a fragment of 150 nt proximal to the start codon. Here, we show that this region constitutes a minimal internal ribosome entry segment (IRES) module. Enzymatic RNA structure analysis reveals a secondary structure model of the NRF IRES module. Point mutation analysis of the module determines a short, 14-nt RNA element (nt 640-653) as a mediator of IRES function. Purification of IRES binding cellular proteins and subsequent ESI/MS/MS sequence analysis led to identification of the RNA-binding protein, JKTBP1. EMSA experiments show that JKTBP1 binds upstream to the 14-nt RNA element in the NRF IRES module (nt 579-639). Over-expression of JKTBP1 significantly enhances activity of the NRF IRES module in dicistronic constructs. Moreover, siRNA experiments demonstrate that down-regulation of endogenous JKTBP1 decreases NRF IRES activity and the level of endogenous NRF protein. The data of this study show that JKTBP1 and the 14-nt element act independently to mediate NRF IRES activity.oai:repository.helmholtz-hzi.de:10033/194572019-08-30T11:37:00Zcom_10033_6823com_10033_6820col_10033_6891
Jablonska, Jadwiga
Dittmar, Kurt E
Kleinke, Tanja
Buer, Jan
Weiss, Siegfried
Molecular Immunology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany. jja@gbf.de
2008-03-03T14:08:14Z
2008-03-03T14:08:14Z
2007-01
Essential role of CCL2 in clustering of splenic ERTR-9+ macrophages during infection of BALB/c mice by Listeria monocytogenes. 2007, 75 (1):462-70 Infect. Immun.
0019-9567
17074847
10.1128/IAI.00443-06
http://hdl.handle.net/10033/19457
Infection and immunity
Early interactions between pathogens and host cells are often decisive for the subsequent course of infection. Here we investigated early events during infection by Listeria monocytogenes, a ubiquitously occurring facultative intracellular microorganism that exhibits severe pathogenicity, mainly in immunocompromised individuals. We show that the inflammatory chemokine CCL2 is highly up-regulated early after Listeria infection in spleens of BALB/c mice. ERTR-9+ macrophages of the marginal zone were identified as the only infected cells and exclusive producers of CCL2 at the early time point. Consequently, clusters of different cell types were formed around infected ERTR-9+ cells. Metallophilic MOMA-1+ marginal zone macrophages were, however, excluded from the clusters and migrated into the B-cell follicles. Depletion of CCL2 during infection resulted in a different composition of cell clusters in the spleen and increased the mortality rate of treated mice. Interestingly, ERTR-9+ macrophages no longer were part of clusters in such mice but remained at their original location in the marginal zone.
en
Animals
Chemokine CCL2
Female
Flow Cytometry
Host-Parasite Interactions
Immunohistochemistry
Listeria Infections
Listeria monocytogenes
Macrophages
Mice
Mice, Inbred BALB C
Microscopy, Confocal
Reverse Transcriptase Polymerase Chain Reaction
Spleen
Essential role of CCL2 in clustering of splenic ERTR-9+ macrophages during infection of BALB/c mice by Listeria monocytogenes.
Article2018-06-12T17:19:46ZEarly interactions between pathogens and host cells are often decisive for the subsequent course of infection. Here we investigated early events during infection by Listeria monocytogenes, a ubiquitously occurring facultative intracellular microorganism that exhibits severe pathogenicity, mainly in immunocompromised individuals. We show that the inflammatory chemokine CCL2 is highly up-regulated early after Listeria infection in spleens of BALB/c mice. ERTR-9+ macrophages of the marginal zone were identified as the only infected cells and exclusive producers of CCL2 at the early time point. Consequently, clusters of different cell types were formed around infected ERTR-9+ cells. Metallophilic MOMA-1+ marginal zone macrophages were, however, excluded from the clusters and migrated into the B-cell follicles. Depletion of CCL2 during infection resulted in a different composition of cell clusters in the spleen and increased the mortality rate of treated mice. Interestingly, ERTR-9+ macrophages no longer were part of clusters in such mice but remained at their original location in the marginal zone.oai:repository.helmholtz-hzi.de:10033/197732019-08-30T11:27:16Zcom_10033_6823com_10033_6820col_10033_6891
Dror, Natalie
Alter-Koltunoff, Michal
Azriel, Aviva
Amariglio, Ninette
Jacob-Hirsch, Jasmine
Zeligson, Sharon
Morgenstern, Avigail
Tamura, Tomohiko
Hauser, Hansjörg
Rechavi, Gideon
Ozato, Keiko
Levi, Ben-Zion
Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa, Israel.
2008-03-05T10:06:02Z
2008-03-05T10:06:02Z
2007-01
Identification of IRF-8 and IRF-1 target genes in activated macrophages. 2007, 44 (4):338-46 Mol. Immunol.
0161-5890
16597464
10.1016/j.molimm.2006.02.026
http://hdl.handle.net/10033/19773
Molecular immunology
Interferon regulatory factor 1 (IRF-1) and IRF-8, also known as interferon consensus sequence binding protein (ICSBP), are important regulators of macrophage differentiation and function. These factors exert their activities through the formation of heterocomplexes. As such, they are coactivators of various interferon-inducible genes in macrophages. To gain better insights into the involvement of these two transcription factors in the onset of the innate immune response and to identify their regulatory network in activated macrophages, DNA microarray was employed. Changes in the expression profile were analyzed in peritoneal macrophages from wild type mice and compared to IRF-1 and IRF-8 null mice, before and following 4 h exposure to IFN-gamma and LPS. The expression pattern of 265 genes was significantly changed (up/down) in peritoneal macrophages extracted from wild type mice following treatment with IFN-gamma and LPS, while no changes in the expression levels of these genes were observed in samples of the same cell-type from both IRF-1 and IRF-8 null mice. Among these putative target genes, numerous genes are involved in macrophage activity during inflammation. The expression profile of 10 of them was further examined by quantitative RT-PCR. In addition, the promoter regions of three of the identified genes were analyzed by reporter gene assay for the ability to respond to IRF-1 and IRF-8. Together, our results suggest that both IRF-1 and IRF-8 are involved in the transcriptional regulation of these genes. We therefore suggest a broader role for IRF-1 and IRF-8 in macrophages differentiation and maturation, being important inflammatory mediators.
en
Animals
Cell Line
Gene Expression Profiling
Interferon Regulatory Factor-1
Interferon Regulatory Factors
Interferon Type II
Lipopolysaccharides
Macrophage Activation
Macrophages, Peritoneal
Mice
Mice, Inbred C57BL
Promoter Regions (Genetics)
Trans-Activation (Genetics)
Identification of IRF-8 and IRF-1 target genes in activated macrophages.
Article2018-06-12T16:50:12ZInterferon regulatory factor 1 (IRF-1) and IRF-8, also known as interferon consensus sequence binding protein (ICSBP), are important regulators of macrophage differentiation and function. These factors exert their activities through the formation of heterocomplexes. As such, they are coactivators of various interferon-inducible genes in macrophages. To gain better insights into the involvement of these two transcription factors in the onset of the innate immune response and to identify their regulatory network in activated macrophages, DNA microarray was employed. Changes in the expression profile were analyzed in peritoneal macrophages from wild type mice and compared to IRF-1 and IRF-8 null mice, before and following 4 h exposure to IFN-gamma and LPS. The expression pattern of 265 genes was significantly changed (up/down) in peritoneal macrophages extracted from wild type mice following treatment with IFN-gamma and LPS, while no changes in the expression levels of these genes were observed in samples of the same cell-type from both IRF-1 and IRF-8 null mice. Among these putative target genes, numerous genes are involved in macrophage activity during inflammation. The expression profile of 10 of them was further examined by quantitative RT-PCR. In addition, the promoter regions of three of the identified genes were analyzed by reporter gene assay for the ability to respond to IRF-1 and IRF-8. Together, our results suggest that both IRF-1 and IRF-8 are involved in the transcriptional regulation of these genes. We therefore suggest a broader role for IRF-1 and IRF-8 in macrophages differentiation and maturation, being important inflammatory mediators.oai:repository.helmholtz-hzi.de:10033/230922019-08-30T11:31:23Zcom_10033_6823com_10033_6820col_10033_6891
Schliephake, Henning
Weich, Herbert A
Dullin, Christian
Gruber, Rudolf
Frahse, Sarah
Department of Oral and Maxillofacial Surgery, George-Augusta-University, Robert-Koch-Strasse 40, 37075 Göttingen, Germany. schliephake.henning@med.uni-goettingen.de
2008-04-11T13:59:56Z
2008-04-11T13:59:56Z
2008-01
Mandibular bone repair by implantation of rhBMP-2 in a slow release carrier of polylactic acid--an experimental study in rats. 2008, 29 (1):103-10 Biomaterials
0142-9612
17936352
10.1016/j.biomaterials.2007.09.019
http://hdl.handle.net/10033/23092
Biomaterials
The aim of the present study was to test the hypothesis that human recombinant bone morphogenic protein 2 (rhBMP-2) implanted in a slow release carrier of polylactic acid (PLA) can repair a non-healing defect in the rat mandible and maintain the thickness of an augmented volume. p-DL-lactic acid discs were produced and loaded with 48 and 96 microg rhBMP-2 and inserted into non-healing defects of the mandible of 45 Wistar rats. Fifteen rats received implants with 96 microg rhBMP-2 (Group 2), 48 microg rhBMP-2 (Group 1) and blank implants without BMP (Group 0) each on one side of the mandible. Unfilled defects of the same size on the contralateral sides of the mandibles served as empty controls. After 6, 13 and 26 weeks, implants of each group were retrieved from five animals each and submitted to flat panel detector computed tomography. Bone formation and thickness of augmentation was assessed by computer-assisted histomorphometry. In Group 2 significantly more bone was produced than in Group 1. Implants of Group 1 induced significantly more bone than the blank controls only after 6 weeks, whereas the difference was not significant after 13 and 26 weeks. Differences between Group 2 and Group 1 were clearly significant after 26 weeks. The thickness of bone tissue was maintained in Group 2 whereas it decreased in Group 1 and was negligible in Group 0. It is concluded that the PLA implants with 96 microg rhBMP-2 were able to bridge a non-healing defect in the rat mandible and maintained the thickness of an augmented volume. However, continuous supply of osteogenic signals appears to be required to compensate for adverse effects during polymer degradation.
en
Animals
Bone Morphogenetic Proteins
Bone and Bones
Drug Carriers
Humans
Implants, Experimental
Lactic Acid
Male
Mandible
Polymers
Rats
Rats, Wistar
Recombinant Proteins
Tomography Scanners, X-Ray Computed
Transforming Growth Factor beta
Mandibular bone repair by implantation of rhBMP-2 in a slow release carrier of polylactic acid--an experimental study in rats.
Article2018-06-12T22:02:01ZThe aim of the present study was to test the hypothesis that human recombinant bone morphogenic protein 2 (rhBMP-2) implanted in a slow release carrier of polylactic acid (PLA) can repair a non-healing defect in the rat mandible and maintain the thickness of an augmented volume. p-DL-lactic acid discs were produced and loaded with 48 and 96 microg rhBMP-2 and inserted into non-healing defects of the mandible of 45 Wistar rats. Fifteen rats received implants with 96 microg rhBMP-2 (Group 2), 48 microg rhBMP-2 (Group 1) and blank implants without BMP (Group 0) each on one side of the mandible. Unfilled defects of the same size on the contralateral sides of the mandibles served as empty controls. After 6, 13 and 26 weeks, implants of each group were retrieved from five animals each and submitted to flat panel detector computed tomography. Bone formation and thickness of augmentation was assessed by computer-assisted histomorphometry. In Group 2 significantly more bone was produced than in Group 1. Implants of Group 1 induced significantly more bone than the blank controls only after 6 weeks, whereas the difference was not significant after 13 and 26 weeks. Differences between Group 2 and Group 1 were clearly significant after 26 weeks. The thickness of bone tissue was maintained in Group 2 whereas it decreased in Group 1 and was negligible in Group 0. It is concluded that the PLA implants with 96 microg rhBMP-2 were able to bridge a non-healing defect in the rat mandible and maintained the thickness of an augmented volume. However, continuous supply of osteogenic signals appears to be required to compensate for adverse effects during polymer degradation.oai:repository.helmholtz-hzi.de:10033/231552019-08-30T11:30:58Zcom_10033_6822com_10033_6821com_10033_6820col_10033_6895
Liu, YH
Bi, JX
Zeng, An-Ping
Yuan, JQ
Department of Automation, Shanghai Jiao Tong University, 800 Dongchuan Rd., 200240, Shanghai, People’s Republic of China.
2008-04-14T09:17:54Z
2008-04-14T09:17:54Z
2008-02-06
A simple kinetic model for myeloma cell culture with consideration of lysine limitation. 2008:notBioprocess Biosyst Eng
1615-7591
18253755
10.1007/s00449-008-0204-x
http://hdl.handle.net/10033/23155
Bioprocess and biosystems engineering
A simple kinetic model is developed to describe the dynamic behavior of myeloma cell growth and cell metabolism. Glucose, glutamine as well as lysine are considered as growth limiting substrates. The cell growth was restricted as soon as the extracellular lysine is exhausted and then intracellular lysine becomes a growth limiting substrate. In addition, a metabolic regulator model together with the Monod model is used to deal with the growth lag phase after inoculation or feeding. By using these models, concentrations of substrates and metabolites, as well as densities of viable and dead cells are quantitatively described. One batch cultivation and two fed-batch cultivations with pulse feeding of nutrients are used to validate the model.
ENG
null
A simple kinetic model for myeloma cell culture with consideration of lysine limitation.
Article2018-06-13T03:48:20ZA simple kinetic model is developed to describe the dynamic behavior of myeloma cell growth and cell metabolism. Glucose, glutamine as well as lysine are considered as growth limiting substrates. The cell growth was restricted as soon as the extracellular lysine is exhausted and then intracellular lysine becomes a growth limiting substrate. In addition, a metabolic regulator model together with the Monod model is used to deal with the growth lag phase after inoculation or feeding. By using these models, concentrations of substrates and metabolites, as well as densities of viable and dead cells are quantitatively described. One batch cultivation and two fed-batch cultivations with pulse feeding of nutrients are used to validate the model.oai:repository.helmholtz-hzi.de:10033/257142019-08-30T11:28:51Zcom_10033_6823com_10033_6820col_10033_6891
Klöpper, Jonas
Lindenmaier, Werner
Fiedler, Ulrike
Mehlhorn, Alexander
Stark, G Björn
Finkenzeller, Günter
Department of Plastic and Hand Surgery, University of Freiburg Medical Center, Freiburg, Germany.
2008-05-13T10:42:15Z
2008-05-13T10:42:15Z
2008-01
High efficient adenoviral-mediated VEGF and Ang-1 gene delivery into osteogenically differentiated human mesenchymal stem cells. 2008, 75 (1):83-90 Microvasc. Res.
0026-2862
17603084
10.1016/j.mvr.2007.04.010
http://hdl.handle.net/10033/25714
Microvascular research
Survival of ex vivo constructed tissues after transplantation is limited by insufficient oxygen and nutrient supply. Therefore, strategies aiming at improvement of neovascularization of engineered tissues are a key issue in tissue engineering applications. This in vitro study aimed at exploring the usability of osteogenically differentiated human mesenchymal stem cells (MSCs) as carriers of the angiogenic growth factor genes vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) for therapeutic angiogenesis in bone tissue engineering. The ex vivo adenoviral vector mediated transduction into osteogenically differentiated MSCs revealed a highly efficient and long lasting expression of the transgenes. Biological activity of VEGF and Ang-1 secreted from transduced cells was confirmed by analyzing the sprouting, proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs) in response to conditioned medium obtained from transduced cells. The transduced osteogenically differentiated MSCs described in this report may be suitable for inducing neovascularization in bone tissue engineering applications.
en
Adenoviridae
Adult Stem Cells
Angiopoietin-1
Apoptosis
Cell Differentiation
Cell Proliferation
Cells, Cultured
Culture Media, Conditioned
Endothelial Cells
Genetic Vectors
Humans
Mesenchymal Stem Cells
Neovascularization, Physiologic
Osteoblasts
Osteogenesis
Spheroids, Cellular
Time Factors
Tissue Engineering
Transduction, Genetic
Vascular Endothelial Growth Factor A
High efficient adenoviral-mediated VEGF and Ang-1 gene delivery into osteogenically differentiated human mesenchymal stem cells.
Article2018-06-13T00:30:21ZSurvival of ex vivo constructed tissues after transplantation is limited by insufficient oxygen and nutrient supply. Therefore, strategies aiming at improvement of neovascularization of engineered tissues are a key issue in tissue engineering applications. This in vitro study aimed at exploring the usability of osteogenically differentiated human mesenchymal stem cells (MSCs) as carriers of the angiogenic growth factor genes vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) for therapeutic angiogenesis in bone tissue engineering. The ex vivo adenoviral vector mediated transduction into osteogenically differentiated MSCs revealed a highly efficient and long lasting expression of the transgenes. Biological activity of VEGF and Ang-1 secreted from transduced cells was confirmed by analyzing the sprouting, proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs) in response to conditioned medium obtained from transduced cells. The transduced osteogenically differentiated MSCs described in this report may be suitable for inducing neovascularization in bone tissue engineering applications.oai:repository.helmholtz-hzi.de:10033/360922019-08-30T11:26:05Zcom_10033_6824com_10033_6823com_10033_6820col_10033_6892
Schneider, B
Nagel, S
Kaufmann, M
Winkelmann, S
Bode, J
Drexler, H G
MacLeod, R A F
2008-08-21T10:35:07Z
2008-08-21T10:35:07Z
2008-06
T(3;7)(q27;q32) fuses BCL6 to a non-coding region at FRA7H near miR-29. 2008, 22 (6):1262-6 Leukemia
1476-5551
17989715
10.1038/sj.leu.2405025
http://hdl.handle.net/10033/36092
Leukemia : official journal of the Leukemia Society of America, Leukemia Research Fund, U.K
en
Base Sequence
Chromosome Fragile Sites
Chromosomes, Human, Pair 3
Chromosomes, Human, Pair 7
DNA-Binding Proteins
Humans
In Situ Hybridization, Fluorescence
Karyotyping
Lymphoma, B-Cell
Lymphoma, Large B-Cell, Diffuse
Male
MicroRNAs
Middle Aged
Molecular Sequence Data
Open Reading Frames
RNA, Messenger
Reverse Transcriptase Polymerase Chain Reaction
Sequence Homology, Nucleic Acid
Translocation, Genetic
Tumor Cells, Cultured
Untranslated Regions
T(3;7)(q27;q32) fuses BCL6 to a non-coding region at FRA7H near miR-29.
Article2008-12-05T00:00:00Zoai:repository.helmholtz-hzi.de:10033/766532019-08-30T11:34:22Zcom_10033_6824com_10033_6823com_10033_6820col_10033_6892
Qiao, Junhua
Oumard, André
Wegloehner, Wolfgang
Bode, Juergen
Department of Molecular Biotechnology/Epigenetic Regulation, Helmholtz Centre for Infection Research, Braunschweig, Germany.
2009-08-07T09:05:59Z
2009-08-07T09:05:59Z
2009-07-24
Novel tag-and-exchange (RMCE) strategies generate master cell clones with predictable and stable transgene expression properties. 2009, 390 (4):579-94 J. Mol. Biol.
1089-8638
19447116
10.1016/j.jmb.2009.05.012
http://hdl.handle.net/10033/76653
Journal of molecular biology
Site-specific recombinases have revolutionized the systematic generation of transgenic cell lines and embryonic stem cells/animals and will ultimately also reveal their potential in the genetic modification of induced pluripotent stem cells. Introduced in 1994, our Flp recombinase-mediated cassette exchange strategy permits the exchange of a target cassette for a cassette with the gene of interest, introduced as a part of an exchange vector. The process is "clean" in the sense that it does not co-introduce prokaryotic vector parts; neither does it leave behind a selection marker. Stringent selection principles provide master cell lines permitting subsequent recombinase-mediated cassette exchange cycles in the absence of a drug selection and with a considerable efficiency (approximately 10%). Exemplified by Chinese hamster ovary cells, the strategy proves to be successful even for cell lines with an unstable genotype.
en
Novel tag-and-exchange (RMCE) strategies generate master cell clones with predictable and stable transgene expression properties.
Article2018-06-13T00:22:50ZSite-specific recombinases have revolutionized the systematic generation of transgenic cell lines and embryonic stem cells/animals and will ultimately also reveal their potential in the genetic modification of induced pluripotent stem cells. Introduced in 1994, our Flp recombinase-mediated cassette exchange strategy permits the exchange of a target cassette for a cassette with the gene of interest, introduced as a part of an exchange vector. The process is "clean" in the sense that it does not co-introduce prokaryotic vector parts; neither does it leave behind a selection marker. Stringent selection principles provide master cell lines permitting subsequent recombinase-mediated cassette exchange cycles in the absence of a drug selection and with a considerable efficiency (approximately 10%). Exemplified by Chinese hamster ovary cells, the strategy proves to be successful even for cell lines with an unstable genotype.oai:repository.helmholtz-hzi.de:10033/847562019-08-30T11:31:46Zcom_10033_6823com_10033_6820col_10033_6891
Garritsen, Henk S.P.
Fan, Alex Xiu-Cheng
Lenz, Daniela
Hanning, Horst
Zhong, Xiao Yan
Geffers, Robert
Lindenmaier, Werner
Dittmar, Kurt E.J.
Institute for Clinical Transfusion Medicine, Department of Hematology/Oncology Städtisches Klinikum Braunschweig gGmbH, Braunschweig, Germany
2009-10-23T11:20:22Z
2009-10-23T11:20:22Z
2009-05-18
2009-03-03
Molecular diagnostics in transfusion medicine: In capillary, on a chip, in silico, or in flight? 2009 36 (3): 181-187 Transfusion Medicine and Hemotherapy.
16603796
16603818
10.1159/000217719
http://hdl.handle.net/10033/84756
Transfusion Medicine and Hemotherapy
Karger
Molecular Diagnostics in Transfusion Medicine: In Capilary, on a Chip, in Silico, or in Flight?
Article2018-05-23T10:10:32Zoai:repository.helmholtz-hzi.de:10033/901542019-08-30T11:35:13Zcom_10033_6823com_10033_6820col_10033_6891
Hoffmann, Andrea
Gross, Gerhard
Department of Molecular Biotechnology, Helmholtz Centre for Infection Research, Braunschweig, Germany. Andrea.Hoffmann@helmholtz-hzi.de
2010-01-20T15:25:54Z
2010-01-20T15:25:54Z
2009
Innovative strategies for treatment of soft tissue injuries in human and animal athletes. 2009, 54:150-65 Med Sport Sci
0254-5020
19696513
10.1159/000235702
http://hdl.handle.net/10033/90154
Medicine and sport science
Our aim is to review the recent progress in the management of musculoskeletal disorders. We will cover novel therapeutic approaches based on growth factors, gene therapy and cells, including stem cells, which may be combined with each other as appropriate. We focus mainly on the treatment of soft tissue injuries - muscle, cartilage, and tendon/ligament for both human and animal athletes. The need for innovative strategies results from the fact that despite all efforts, the current strategies for cartilage and tendon/ligament still result in the formation of functionally and biomechanically inferior tissues after injury (a phenomenon called 'repair' as opposed to proper 'regeneration'), whereas the outcome for muscle is more favorable. Innovative approaches are urgently needed not only to enhance the outcome of conservative or surgical procedures but also to speed up the healing process from the very long disabling periods, which is of special relevance for athletes.
en
Animals
Athletic Injuries
Bone Morphogenetic Proteins
Cartilage
Exercise Therapy
Gene Therapy
Gene Transfer Techniques
Humans
Ligaments
Mesenchymal Stem Cell Transplantation
Muscle, Skeletal
Physical Conditioning, Animal
Regeneration
Soft Tissue Injuries
Tendon Injuries
Tendons
Wound Healing
Innovative strategies for treatment of soft tissue injuries in human and animal athletes.
Article2010-08-15T00:00:00ZOur aim is to review the recent progress in the management of musculoskeletal disorders. We will cover novel therapeutic approaches based on growth factors, gene therapy and cells, including stem cells, which may be combined with each other as appropriate. We focus mainly on the treatment of soft tissue injuries - muscle, cartilage, and tendon/ligament for both human and animal athletes. The need for innovative strategies results from the fact that despite all efforts, the current strategies for cartilage and tendon/ligament still result in the formation of functionally and biomechanically inferior tissues after injury (a phenomenon called 'repair' as opposed to proper 'regeneration'), whereas the outcome for muscle is more favorable. Innovative approaches are urgently needed not only to enhance the outcome of conservative or surgical procedures but also to speed up the healing process from the very long disabling periods, which is of special relevance for athletes.oai:repository.helmholtz-hzi.de:10033/1189092019-08-30T11:33:27Zcom_10033_6823com_10033_6820col_10033_6891
Dittmar, Kurt E.J.
Simann, Meike
Zghoul, Nadia
Schön, Oliver
Meyring, Wilhelm
Hanning, Horst
Dirks, Wilhelm G.
Miller, Konstantin
Garritsen, Henk S.P.
Lindenmaier, Werner
Helmholtz Center for Infection Research, Inhoffenstr. 7, D-38124 Braunschweig, Germany
2011-01-07T12:12:08Z
2011-01-07T12:12:08Z
2010-04
2009-07
Quality of cell products: Authenticity, identity, genomic stability and status of differentiation 2010 (37/2):57-64 Transfusion Medicine and Hemotherapy.
http://hdl.handle.net/10033/118909
Transfusion Medicine and Hemotherapy
null
Karger
http://content.karger.com/ProdukteDB/produkte.asp?Aktion=JournalHome&ProduktNr=224170&ContentOnly=false
Quality of cell products: Authenticity, Identity, Genomic stability and status of differentiation
Article2011-04-15T00:00:00Zoai:repository.helmholtz-hzi.de:10033/1207882019-08-30T11:34:22Zcom_10033_6823com_10033_6820col_10033_6891
Thorey, Fritz
Menzel, Henning
Lorenz, Corinna
Gross, Gerhard
Hoffmann, Andrea
Windhagen, Henning
Department of Orthopaedic Surgery, Hannover Medical School, Hannover, Germany.
2011-02-01T12:37:40Z
2011-02-01T12:37:40Z
2011-01
Osseointegration by bone morphogenetic protein-2 and transforming growth factor beta2 coated titanium implants in femora of New Zealand white rabbits. 2011, 45 (1):57-62 Indian J Orthop
1998-3727
21221225
10.4103/0019-5413.73659
http://hdl.handle.net/10033/120788
Indian journal of orthopaedics
Intramembranous bone formation is essential in uncemented joint replacement to provide a mechanical anchorage of the implant. Since the discovery of bone morphogenic proteins (BMPs) by Urist in 1965, many studies have been conducted to show the influence of growth factors on implant ingrowth. In this study, the influence of bone morphogenetic protein-2 (rhBMP-2) and transforming growth factor β2 (TGF-β2) on implant osseointegration was investigated.
en
Osseointegration by bone morphogenetic protein-2 and transforming growth factor beta2 coated titanium implants in femora of New Zealand white rabbits.
Article2018-06-12T21:30:47ZIntramembranous bone formation is essential in uncemented joint replacement to provide a mechanical anchorage of the implant. Since the discovery of bone morphogenic proteins (BMPs) by Urist in 1965, many studies have been conducted to show the influence of growth factors on implant ingrowth. In this study, the influence of bone morphogenetic protein-2 (rhBMP-2) and transforming growth factor β2 (TGF-β2) on implant osseointegration was investigated.oai:repository.helmholtz-hzi.de:10033/1225252019-08-30T11:27:42Zcom_10033_6823com_10033_6820col_10033_6891
Koutinas, Michalis
Kiparissides, Alexandros
Lam, Ming-Chi
Silva-Rocha, Rafael
Martins dos Santos, Vítor A P
Pistikopoulos, Estradios N.
Mantalaris, Athanasios
Helmholtz Center for Infection Research, Chemical Microbiology, Inhoffenstrasse 7, 38124 Braunschweig, Germany.
2011-02-21T12:20:12Z
2011-02-21T12:20:12Z
2010
Combining genetic circuit and microbial growth kinetic models: A challenge for biological modelling. (2010), 28 (C):301-306 Computer Aided Chemical Engineering
1570-7946
http://hdl.handle.net/10033/122525
European Syposium on Computer Aided Process Engineering
Elsevier B.V.
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B8G5G-505XT0T-1X&_user=104184&_coverDate=12%2F31%2F2010&_rdoc=54&_fmt=high&_orig=browse&_origin=browse&_zone=rslt_list_item&_srch=doc-info%28%23toc%2341850%232010%23999719999%232073736%23FLP%23display%23Volume%29&_cdi=41850&_sort=d&_docanchor=&_ct=226&_acct=C000007378&_version=1&_urlVersion=0&_userid=104184&md5=2a412e73bdca06763d2d7c6e5f0d6315&searchtype=a
Combining genetic circuit and microbial growth Kinetic models: Achallenge for biological modelling
Article2018-06-13T07:24:22Zoai:repository.helmholtz-hzi.de:10033/1427562019-08-30T11:36:32Zcom_10033_6823com_10033_6820col_10033_6891
van Duuren, J B J H
Brehmer, B
Mars, A E
Eggink, G
Dos Santos, V A P Martins
Sanders, J P M
Wageningen UR Food & Biobased Research, Wageningen, The Netherlands. joost.vanduuren@helmholtz-hzi.de
2011-09-20T14:37:01Z
2011-09-20T14:37:01Z
2011-06
A limited LCA of bio-adipic acid: manufacturing the nylon-6,6 precursor adipic acid using the benzoic acid degradation pathway from different feedstocks. 2011, 108 (6):1298-306 Biotechnol. Bioeng.
1097-0290
21328320
10.1002/bit.23074
http://hdl.handle.net/10033/142756
Biotechnology and bioengineering
A limited life cycle assessment (LCA) was performed on a combined biological and chemical process for the production of adipic acid, which was compared to the traditional petrochemical process. The LCA comprises the biological conversion of the aromatic feedstocks benzoic acid, impure aromatics, toluene, or phenol from lignin to cis, cis-muconic acid, which is subsequently converted to adipic acid through hydrogenation. Apart from the impact of usage of petrochemical and biomass-based feedstocks, the environmental impact of the final concentration of cis, cis-muconic acid in the fermentation broth was studied using 1.85% and 4.26% cis, cis-muconic acid. The LCA focused on the cumulative energy demand (CED), cumulative exergy demand (CExD), and the CO(2) equivalent (CO(2) eq) emission, with CO(2) and N(2) O measured separately. The highest calculated reduction potential of CED and CExD were achieved using phenol, which reduced the CED by 29% and 57% with 1.85% and 4.26% cis, cis-muconic acid, respectively. A decrease in the CO(2) eq emission was especially achieved when the N(2) O emission in the combined biological and chemical process was restricted. At 4.26% cis, cis-muconic acid, the different carbon backbone feedstocks contributed to an optimized reduction of CO(2) eq emissions ranging from 14.0 to 17.4 ton CO(2) eq/ton adipic acid. The bulk of the bioprocessing energy intensity is attributed to the hydrogenation reactor, which has a high environmental impact and a direct relationship with the product concentration in the broth.
en
Adipic Acids
Benzoic Acid
Biomass
Biotechnology
Caprolactam
Environment
Fossil Fuels
Polymers
Pseudomonas putida
A limited LCA of bio-adipic acid: manufacturing the nylon-6,6 precursor adipic acid using the benzoic acid degradation pathway from different feedstocks.
Article2018-06-13T19:50:38ZA limited life cycle assessment (LCA) was performed on a combined biological and chemical process for the production of adipic acid, which was compared to the traditional petrochemical process. The LCA comprises the biological conversion of the aromatic feedstocks benzoic acid, impure aromatics, toluene, or phenol from lignin to cis, cis-muconic acid, which is subsequently converted to adipic acid through hydrogenation. Apart from the impact of usage of petrochemical and biomass-based feedstocks, the environmental impact of the final concentration of cis, cis-muconic acid in the fermentation broth was studied using 1.85% and 4.26% cis, cis-muconic acid. The LCA focused on the cumulative energy demand (CED), cumulative exergy demand (CExD), and the CO(2) equivalent (CO(2) eq) emission, with CO(2) and N(2) O measured separately. The highest calculated reduction potential of CED and CExD were achieved using phenol, which reduced the CED by 29% and 57% with 1.85% and 4.26% cis, cis-muconic acid, respectively. A decrease in the CO(2) eq emission was especially achieved when the N(2) O emission in the combined biological and chemical process was restricted. At 4.26% cis, cis-muconic acid, the different carbon backbone feedstocks contributed to an optimized reduction of CO(2) eq emissions ranging from 14.0 to 17.4 ton CO(2) eq/ton adipic acid. The bulk of the bioprocessing energy intensity is attributed to the hydrogenation reactor, which has a high environmental impact and a direct relationship with the product concentration in the broth.oai:repository.helmholtz-hzi.de:10033/1242052019-08-30T11:36:32Zcom_10033_6823com_10033_6820col_10033_6891
May, Tobias
Eccleston, Lee
Herrmann, Sabrina
Hauser, Hansjörg
Goncalves, Jorge
Wirth, Dagmar
Department of Gene Regulation and Differentiation, Helmholtz Centre for Infection Research, Braunschweig, Germany.
2011-03-11T09:19:18Z
2011-03-11T09:19:18Z
2008
Bimodal and hysteretic expression in mammalian cells from a synthetic gene circuit. 2008, 3 (6):e2372 PLoS ONE
1932-6203
18523635
10.1371/journal.pone.0002372
http://hdl.handle.net/10033/124205
PloS one
In order to establish cells and organisms with predictable properties, synthetic biology makes use of controllable, synthetic genetic devices. These devices are used to replace or to interfere with natural pathways. Alternatively, they may be interlinked with endogenous pathways to create artificial networks of higher complexity. While these approaches have been already successful in prokaryotes and lower eukaryotes, the implementation of such synthetic cassettes in mammalian systems and even animals is still a major obstacle. This is mainly due to the lack of methods that reliably and efficiently transduce synthetic modules without compromising their regulation properties. To pave the way for implementation of synthetic regulation modules in mammalian systems we utilized lentiviral transduction of synthetic modules. A synthetic positive feedback loop, based on the Tetracycline regulation system was implemented in a lentiviral vector system and stably integrated in mammalian cells. This gene regulation circuit yields a bimodal expression response. Based on experimental data a mathematical model based on stochasticity was developed which matched and described the experimental findings. Modelling predicted a hysteretic expression response which was verified experimentally. Thereby supporting the idea that the system is driven by stochasticity. The results presented here highlight that the combination of three independent tools/methodologies facilitate the reliable installation of synthetic gene circuits with predictable expression characteristics in mammalian cells and organisms.
en
Animals
Genes, Synthetic
Humans
Lentivirus
Mice
NIH 3T3 Cells
Transduction, Genetic
Bimodal and hysteretic expression in mammalian cells from a synthetic gene circuit.
Article2018-06-12T23:35:54ZIn order to establish cells and organisms with predictable properties, synthetic biology makes use of controllable, synthetic genetic devices. These devices are used to replace or to interfere with natural pathways. Alternatively, they may be interlinked with endogenous pathways to create artificial networks of higher complexity. While these approaches have been already successful in prokaryotes and lower eukaryotes, the implementation of such synthetic cassettes in mammalian systems and even animals is still a major obstacle. This is mainly due to the lack of methods that reliably and efficiently transduce synthetic modules without compromising their regulation properties. To pave the way for implementation of synthetic regulation modules in mammalian systems we utilized lentiviral transduction of synthetic modules. A synthetic positive feedback loop, based on the Tetracycline regulation system was implemented in a lentiviral vector system and stably integrated in mammalian cells. This gene regulation circuit yields a bimodal expression response. Based on experimental data a mathematical model based on stochasticity was developed which matched and described the experimental findings. Modelling predicted a hysteretic expression response which was verified experimentally. Thereby supporting the idea that the system is driven by stochasticity. The results presented here highlight that the combination of three independent tools/methodologies facilitate the reliable installation of synthetic gene circuits with predictable expression characteristics in mammalian cells and organisms.oai:repository.helmholtz-hzi.de:10033/1298362019-08-30T11:26:13Zcom_10033_6823com_10033_6820col_10033_6891
Gama-Norton, L
Herrmann, S
Schucht, R
Coroadinha, A S
Löw, R
Alves, P M
Bartholomae, C C
Schmidt, M
Baum, C
Schambach, A
Hauser, Hansjoerg
Wirth, D
Helmholtz Center for Infection Research (HZI), 38124 Braunschweig, Germany.
2011-05-20T10:42:53Z
2011-05-20T10:42:53Z
2010-08
Retroviral vector performance in defined chromosomal Loci of modular packaging cell lines. 2010, 21 (8):979-91 Hum. Gene Ther.
1557-7422
20222806
10.1089/hum.2009.089
http://hdl.handle.net/10033/129836
Human gene therapy
The improvement of safety and titer of retroviral vectors produced in standard retroviral packaging cell lines is hampered because production relies on uncontrollable vector integration events. The influences of chromosomal surroundings make it difficult to dissect the performance of a specific vector from the chromosomal surroundings of the respective integration site. Taking advantage of a technology that relies on the use of packaging cell lines with predefined integration sites, we have systematically evaluated the performance of several retroviral vectors. In two previously established modular packaging cell lines (Flp293A and 293 FLEX) with single, defined chromosomal integration sites, retroviral vectors were integrated by means of Flp-mediated site-specific recombination. Vectors that are distinguished by different long terminal repeat promoters were introduced in either the sense or reverse orientation. The results show that the promoter, viral vector orientation, and integration site are the main determinants of the titer. Furthermore, we exploited the viral production systems to evaluate read-through activity. Read-through is thought to be caused by inefficient termination of vector transcription and is inherent to the nature of retroviral vectors. We assessed the frequency of transduction of sequences flanking the retroviral vectors from both integration sites. The approach presented here provides a platform for systematic design and evaluation of the efficiency and safety of retroviral vectors optimized for a given producer cell line.
en
Cell Line
Chromosomes
Gene Targeting
Gene Therapy
Genetic Loci
Genetic Vectors
Promoter Regions, Genetic
Retroviridae
Transduction, Genetic
Virus Assembly
Virus Integration
Retroviral vector performance in defined chromosomal Loci of modular packaging cell lines.
Article2018-06-13T01:36:56ZThe improvement of safety and titer of retroviral vectors produced in standard retroviral packaging cell lines is hampered because production relies on uncontrollable vector integration events. The influences of chromosomal surroundings make it difficult to dissect the performance of a specific vector from the chromosomal surroundings of the respective integration site. Taking advantage of a technology that relies on the use of packaging cell lines with predefined integration sites, we have systematically evaluated the performance of several retroviral vectors. In two previously established modular packaging cell lines (Flp293A and 293 FLEX) with single, defined chromosomal integration sites, retroviral vectors were integrated by means of Flp-mediated site-specific recombination. Vectors that are distinguished by different long terminal repeat promoters were introduced in either the sense or reverse orientation. The results show that the promoter, viral vector orientation, and integration site are the main determinants of the titer. Furthermore, we exploited the viral production systems to evaluate read-through activity. Read-through is thought to be caused by inefficient termination of vector transcription and is inherent to the nature of retroviral vectors. We assessed the frequency of transduction of sequences flanking the retroviral vectors from both integration sites. The approach presented here provides a platform for systematic design and evaluation of the efficiency and safety of retroviral vectors optimized for a given producer cell line.oai:repository.helmholtz-hzi.de:10033/1411142019-08-30T11:37:44Zcom_10033_6823com_10033_6820col_10033_6891
Carrondo, Manuel
Panet, Amos
Wirth, Dagmar
Coroadinha, Ana Sofia
Cruz, Pedro
Falk, Haya
Schucht, Roland
Dupont, Francis
Geny-Fiamma, Cécile
Merten, Otto-Wilhelm
Hauser, Hansjörg
Instituto de Biologia Experimental e Tecnológica/Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, P-2781-901 Oeiras, Portugal.
2011-08-29T12:31:56Z
2011-08-29T12:31:56Z
2011-03
Integrated strategy for the production of therapeutic retroviral vectors. 2011, 22 (3):370-9 Hum. Gene Ther.
1557-7422
21043806
10.1089/hum.2009.165
http://hdl.handle.net/10033/141114
Human gene therapy
The broad application of retroviral vectors for gene delivery is still hampered by the difficulty to reproducibly establish high vector producer cell lines generating sufficient amounts of highly concentrated virus vector preparations of high quality. To enhance the process for producing clinically relevant retroviral vector preparations for therapeutic applications, we have integrated novel and state-of-the-art technologies in a process that allows rapid access to high-efficiency vector-producing cells and consistent production, purification, and storage of retroviral vectors. The process has been designed for various types of retroviral vectors for clinical application and to support a high-throughput process. New modular helper cell lines that permit rapid insertion of DNA encoding the therapeutic vector of interest at predetermined, optimal chromosomal loci were developed to facilitate stable and high vector production levels. Packaging cell lines, cultivation methods, and improved medium composition were coupled with vector purification and storage process strategies that yield maximal vector infectivity and stability. To facilitate GMP-grade vector production, standard of operation protocols were established. These processes were validated by production of retroviral vector lots that drive the expression of type VII collagen (Col7) for the treatment of a skin genetic disease, dystrophic epidermolysis bullosa. The potential efficacy of the Col7-expressing vectors was finally proven with newly developed systems, in particular in target primary keratinocyte cultures and three-dimensional skin tissues in organ culture.
en
Animals
Cell Culture Techniques
Cell Line
Collagen Type VII
DNA Nucleotidyltransferases
Epidermolysis Bullosa Dystrophica
Gene Therapy
Genetic Vectors
HEK293 Cells
Humans
Industrial Microbiology
Mice
Mice, Inbred BALB C
Recombination, Genetic
Reproducibility of Results
Retroviridae
Integrated strategy for the production of therapeutic retroviral vectors.
Article2018-06-12T17:21:16ZThe broad application of retroviral vectors for gene delivery is still hampered by the difficulty to reproducibly establish high vector producer cell lines generating sufficient amounts of highly concentrated virus vector preparations of high quality. To enhance the process for producing clinically relevant retroviral vector preparations for therapeutic applications, we have integrated novel and state-of-the-art technologies in a process that allows rapid access to high-efficiency vector-producing cells and consistent production, purification, and storage of retroviral vectors. The process has been designed for various types of retroviral vectors for clinical application and to support a high-throughput process. New modular helper cell lines that permit rapid insertion of DNA encoding the therapeutic vector of interest at predetermined, optimal chromosomal loci were developed to facilitate stable and high vector production levels. Packaging cell lines, cultivation methods, and improved medium composition were coupled with vector purification and storage process strategies that yield maximal vector infectivity and stability. To facilitate GMP-grade vector production, standard of operation protocols were established. These processes were validated by production of retroviral vector lots that drive the expression of type VII collagen (Col7) for the treatment of a skin genetic disease, dystrophic epidermolysis bullosa. The potential efficacy of the Col7-expressing vectors was finally proven with newly developed systems, in particular in target primary keratinocyte cultures and three-dimensional skin tissues in organ culture.oai:repository.helmholtz-hzi.de:10033/1411152019-08-30T11:37:44Zcom_10033_6823com_10033_6820col_10033_6891
Sester, Urban
Fousse, Mathias
Dirks, Jan
Mack, Ulrich
Prasse, Antje
Singh, Mahavir
Lalvani, Ajit
Sester, Martina
Department of Internal Medicine IV, Saarland University, Homburg, Germany.
2011-08-29T12:55:10Z
2011-08-29T12:55:10Z
2011
Whole-blood flow-cytometric analysis of antigen-specific CD4 T-cell cytokine profiles distinguishes active tuberculosis from non-active states. 2011, 6 (3):e17813 PLoS ONE
1932-6203
21423578
10.1371/journal.pone.0017813
http://hdl.handle.net/10033/141115
PloS one
T-cell based IFN-γ release assays do not permit distinction of active tuberculosis (TB) from successfully treated disease or latent M. tuberculosis infection. We postulated that IFN-γ and IL-2 cytokine profiles of antigen-specific T cells measured by flow-cytometry ex vivo might correlate with TB disease activity in vivo. Tuberculin (PPD), ESAT-6 and CFP-10 were used as stimuli to determine antigen-specific cytokine profiles in CD4 T cells from 24 patients with active TB and 28 patients with successfully treated TB using flow-cytometry. Moreover, 25 individuals with immunity consistent with latent M. tuberculosis infection and BCG-vaccination, respectively, were recruited. Although the frequency of cytokine secreting PPD reactive CD4 T cells was higher in patients with active TB compared to patients with treated TB (median 0.81% vs. 0.39% of CD4 T cells, p = 0.02), the overlap in frequencies precluded distinction between the groups on an individual basis. When assessing cytokine profiles, PPD specific CD4 T cells secreting both IFN-γ and IL-2 predominated in treated TB, latent infection and BCG-vaccination, whilst in active TB the cytokine profile was shifted towards cells secreting IFN-γ only (p<0.0001). Cytokine profiles of ESAT-6 or CFP-10 reactive CD4 T cells did not differ between the groups. Receiver operator characteristics (ROC) analysis revealed that frequencies of PPD specific IFN-γ/IL-2 dual-positive T cells below 56% were an accurate marker for active TB (specificity 100%, sensitivity 70%) enabling effective discrimination from non-active states. In conclusion, a frequency lower than 56% IFN-γ/IL-2 dual positive PPD-specific circulating CD4 T-cells is strongly indicative of active TB.
en
Antigens, Bacterial
Bacterial Proteins
CD4-Positive T-Lymphocytes
Demography
Diagnosis, Differential
Female
Flow Cytometry
Humans
Interferon-gamma
Interleukin-2
Male
Middle Aged
Tuberculosis
Whole-blood flow-cytometric analysis of antigen-specific CD4 T-cell cytokine profiles distinguishes active tuberculosis from non-active states.
Article2018-06-12T17:53:34ZT-cell based IFN-γ release assays do not permit distinction of active tuberculosis (TB) from successfully treated disease or latent M. tuberculosis infection. We postulated that IFN-γ and IL-2 cytokine profiles of antigen-specific T cells measured by flow-cytometry ex vivo might correlate with TB disease activity in vivo. Tuberculin (PPD), ESAT-6 and CFP-10 were used as stimuli to determine antigen-specific cytokine profiles in CD4 T cells from 24 patients with active TB and 28 patients with successfully treated TB using flow-cytometry. Moreover, 25 individuals with immunity consistent with latent M. tuberculosis infection and BCG-vaccination, respectively, were recruited. Although the frequency of cytokine secreting PPD reactive CD4 T cells was higher in patients with active TB compared to patients with treated TB (median 0.81% vs. 0.39% of CD4 T cells, p = 0.02), the overlap in frequencies precluded distinction between the groups on an individual basis. When assessing cytokine profiles, PPD specific CD4 T cells secreting both IFN-γ and IL-2 predominated in treated TB, latent infection and BCG-vaccination, whilst in active TB the cytokine profile was shifted towards cells secreting IFN-γ only (p<0.0001). Cytokine profiles of ESAT-6 or CFP-10 reactive CD4 T cells did not differ between the groups. Receiver operator characteristics (ROC) analysis revealed that frequencies of PPD specific IFN-γ/IL-2 dual-positive T cells below 56% were an accurate marker for active TB (specificity 100%, sensitivity 70%) enabling effective discrimination from non-active states. In conclusion, a frequency lower than 56% IFN-γ/IL-2 dual positive PPD-specific circulating CD4 T-cells is strongly indicative of active TB.oai:repository.helmholtz-hzi.de:10033/1427402019-08-30T11:37:44Zcom_10033_6823com_10033_6820col_10033_6891
Oberhardt, Matthew A
Puchałka, Jacek
Martins dos Santos, Vítor A P
Papin, Jason A
Department of Biomedical Engineering, University of Virginia, Charlottesville, Virginia, United States of America.
2011-09-20T11:51:50Z
2011-09-20T11:51:50Z
2011-03
Reconciliation of genome-scale metabolic reconstructions for comparative systems analysis. 2011, 7 (3):e1001116 PLoS Comput. Biol.
1553-7358
21483480
10.1371/journal.pcbi.1001116
http://hdl.handle.net/10033/142740
PLoS computational biology
In the past decade, over 50 genome-scale metabolic reconstructions have been built for a variety of single- and multi- cellular organisms. These reconstructions have enabled a host of computational methods to be leveraged for systems-analysis of metabolism, leading to greater understanding of observed phenotypes. These methods have been sparsely applied to comparisons between multiple organisms, however, due mainly to the existence of differences between reconstructions that are inherited from the respective reconstruction processes of the organisms to be compared. To circumvent this obstacle, we developed a novel process, termed metabolic network reconciliation, whereby non-biological differences are removed from genome-scale reconstructions while keeping the reconstructions as true as possible to the underlying biological data on which they are based. This process was applied to two organisms of great importance to disease and biotechnological applications, Pseudomonas aeruginosa and Pseudomonas putida, respectively. The result is a pair of revised genome-scale reconstructions for these organisms that can be analyzed at a systems level with confidence that differences are indicative of true biological differences (to the degree that is currently known), rather than artifacts of the reconstruction process. The reconstructions were re-validated with various experimental data after reconciliation. With the reconciled and validated reconstructions, we performed a genome-wide comparison of metabolic flexibility between P. aeruginosa and P. putida that generated significant new insight into the underlying biology of these important organisms. Through this work, we provide a novel methodology for reconciling models, present new genome-scale reconstructions of P. aeruginosa and P. putida that can be directly compared at a network level, and perform a network-wide comparison of the two species. These reconstructions provide fresh insights into the metabolic similarities and differences between these important Pseudomonads, and pave the way towards full comparative analysis of genome-scale metabolic reconstructions of multiple species.
en
Algorithms
Biotechnology
Computational Biology
Computer Simulation
Databases, Factual
Gene Expression Regulation, Bacterial
Genome
Genome, Bacterial
Metabolic Networks and Pathways
Phenotype
Pseudomonas
Reproducibility of Results
Software
Species Specificity
Reconciliation of genome-scale metabolic reconstructions for comparative systems analysis.
Article2018-06-12T23:17:27ZIn the past decade, over 50 genome-scale metabolic reconstructions have been built for a variety of single- and multi- cellular organisms. These reconstructions have enabled a host of computational methods to be leveraged for systems-analysis of metabolism, leading to greater understanding of observed phenotypes. These methods have been sparsely applied to comparisons between multiple organisms, however, due mainly to the existence of differences between reconstructions that are inherited from the respective reconstruction processes of the organisms to be compared. To circumvent this obstacle, we developed a novel process, termed metabolic network reconciliation, whereby non-biological differences are removed from genome-scale reconstructions while keeping the reconstructions as true as possible to the underlying biological data on which they are based. This process was applied to two organisms of great importance to disease and biotechnological applications, Pseudomonas aeruginosa and Pseudomonas putida, respectively. The result is a pair of revised genome-scale reconstructions for these organisms that can be analyzed at a systems level with confidence that differences are indicative of true biological differences (to the degree that is currently known), rather than artifacts of the reconstruction process. The reconstructions were re-validated with various experimental data after reconciliation. With the reconciled and validated reconstructions, we performed a genome-wide comparison of metabolic flexibility between P. aeruginosa and P. putida that generated significant new insight into the underlying biology of these important organisms. Through this work, we provide a novel methodology for reconciling models, present new genome-scale reconstructions of P. aeruginosa and P. putida that can be directly compared at a network level, and perform a network-wide comparison of the two species. These reconstructions provide fresh insights into the metabolic similarities and differences between these important Pseudomonads, and pave the way towards full comparative analysis of genome-scale metabolic reconstructions of multiple species.oai:repository.helmholtz-hzi.de:10033/1429692019-08-30T11:27:16Zcom_10033_6823com_10033_6820col_10033_6891
Kugel, Daniela
Pulverer, Julia Elisabeth
Köster, Mario
Hauser, Hansjörg
Staeheli, Peter
Department of Virology, University of Freiburg, Freiburg, Germany.
2011-09-23T12:09:53Z
2011-09-23T12:09:53Z
2011-04
Novel nonviral bioassays for mouse type I and type III interferon. 2011, 31 (4):345-9 J. Interferon Cytokine Res.
1557-7465
21138377
10.1089/jir.2010.0079
http://hdl.handle.net/10033/142969
Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research
We used embryo fibroblasts from Mx2-Luc transgenic mice that express Firefly luciferase under control of the interferon (IFN)-regulated mouse Mx2 promoter to develop simple nonviral bioassays for type I and type III IFN. Since type III IFN is acid-labile, Mx2-Luc fibroblasts detected the presence of type I IFN in acid-treated biological samples with high sensitivity and selectivity. For selective detection of type III IFN, we employed embryo fibroblasts from Mx2-Luc mutant mice that lack functional receptors for type I IFN. The sensitivity of this latter assay remained comparatively low, presumably because type III IFN receptors are not abundantly present on fibroblasts. The main advantages of our novel IFN assays are that they are easy to perform, yield fast results, and can be used in laboratories that are not licensed for work with infectious agents. Further, the type I IFN assay has superior sensitivity than commercially available enzyme-linked immunosorbent assay systems.
en
Novel nonviral bioassays for mouse type I and type III interferon.
Article2018-06-12T22:19:11ZWe used embryo fibroblasts from Mx2-Luc transgenic mice that express Firefly luciferase under control of the interferon (IFN)-regulated mouse Mx2 promoter to develop simple nonviral bioassays for type I and type III IFN. Since type III IFN is acid-labile, Mx2-Luc fibroblasts detected the presence of type I IFN in acid-treated biological samples with high sensitivity and selectivity. For selective detection of type III IFN, we employed embryo fibroblasts from Mx2-Luc mutant mice that lack functional receptors for type I IFN. The sensitivity of this latter assay remained comparatively low, presumably because type III IFN receptors are not abundantly present on fibroblasts. The main advantages of our novel IFN assays are that they are easy to perform, yield fast results, and can be used in laboratories that are not licensed for work with infectious agents. Further, the type I IFN assay has superior sensitivity than commercially available enzyme-linked immunosorbent assay systems.oai:repository.helmholtz-hzi.de:10033/1539592019-08-30T11:26:13Zcom_10033_6823com_10033_6820col_10033_6891
Schucht, Roland
Lydford, Simon
Andzinski, Lisa
Zauers, Jeannette
Cooper, James
Hauser, Hansjörg
Wirth, Dagmar
May, Tobias
Department of Gene Regulation and Differentiation, HZI-Helmholtz Centre for Infection Research, Braunschweig, Germany. roland.schucht@inscreenex.com
2011-10-26T12:38:25Z
2011-10-26T12:38:25Z
2011-03
Rapid establishment of G-protein-coupled receptor-expressing cell lines by site-specific integration. 2011, 16 (3):323-31 J Biomol Screen
1552-454X
21335600
10.1177/1087057110396371
http://hdl.handle.net/10033/153959
Journal of biomolecular screening
The establishment of mammalian cell lines reliably expressing G-protein-coupled receptors (GPCRs) can be a tedious and often time-consuming process. A strategy has been developed to allow the rapid production of such cell lines. The first step of this approach was the generation of a specialized master cell line, characterized by optimized stable expression of a membrane-bound reporter protein. In the second step, this reporter gene was exchanged for that of the GPCR of interest by a DNA recombinase "cut-and-paste" engineering step. It has been demonstrated that the resulting GPCR cell lines inherit the advantages of the master cell line, expressing the GPCR in a homogeneous and stable manner. The case studies presented demonstrate the functionality of the established GPCR cell lines, and most important, because of the highly efficient integration event, these recombinant GPCR-expressing cell lines were generated within a timeframe of 2 to 4 weeks. The advantages of this cut-and-paste approach versus other strategies such as Flp-In or Jump-In are compared.
en
Animals
CHO Cells
Cell Line
Cricetinae
Cricetulus
Gene Expression Regulation
Gene Order
Gene Targeting
Genetic Vectors
High-Throughput Screening Assays
Receptors, G-Protein-Coupled
Recombinases
Rapid establishment of G-protein-coupled receptor-expressing cell lines by site-specific integration.
Article2012-03-15T00:00:00ZThe establishment of mammalian cell lines reliably expressing G-protein-coupled receptors (GPCRs) can be a tedious and often time-consuming process. A strategy has been developed to allow the rapid production of such cell lines. The first step of this approach was the generation of a specialized master cell line, characterized by optimized stable expression of a membrane-bound reporter protein. In the second step, this reporter gene was exchanged for that of the GPCR of interest by a DNA recombinase "cut-and-paste" engineering step. It has been demonstrated that the resulting GPCR cell lines inherit the advantages of the master cell line, expressing the GPCR in a homogeneous and stable manner. The case studies presented demonstrate the functionality of the established GPCR cell lines, and most important, because of the highly efficient integration event, these recombinant GPCR-expressing cell lines were generated within a timeframe of 2 to 4 weeks. The advantages of this cut-and-paste approach versus other strategies such as Flp-In or Jump-In are compared.oai:repository.helmholtz-hzi.de:10033/1963102019-08-30T11:28:24Zcom_10033_6823com_10033_6820col_10033_6891
Koutinas, Michalis
Kiparissides, Alexandros
Silva-Rocha, Rafael
Lam, Ming-Chi
Martins Dos Santos, Vitor A P
de Lorenzo, Victor
Pistikopoulos, Efstratios N
Mantalaris, Athanasios
Centre for Process Systems Engineering, Department of Chemical Engineering, South Kensington Campus, Imperial College London, UK.
2011-12-07T14:02:51Z
2011-12-07T14:02:51Z
2011-07
Linking genes to microbial growth kinetics: an integrated biochemical systems engineering approach. 2011, 13 (4):401-13 Metab. Eng.
1096-7184
21315172
10.1016/j.ymben.2011.02.001
http://hdl.handle.net/10033/196310
Metabolic engineering
The majority of models describing the kinetic properties of a microorganism for a given substrate are unstructured and empirical. They are formulated in this manner so that the complex mechanism of cell growth is simplified. Herein, a novel approach for modelling microbial growth kinetics is proposed, linking biomass growth and substrate consumption rates to the gene regulatory programmes that control these processes. A dynamic model of the TOL (pWW0) plasmid of Pseudomonas putida mt-2 has been developed, describing the molecular interactions that lead to the transcription of the upper and meta operons, known to produce the enzymes for the oxidative catabolism of m-xylene. The genetic circuit model was combined with a growth kinetic model decoupling biomass growth and substrate consumption rates, which are expressed as independent functions of the rate-limiting enzymes produced by the operons. Estimation of model parameters and validation of the model's predictive capability were successfully performed in batch cultures of mt-2 fed with different concentrations of m-xylene, as confirmed by relative mRNA concentration measurements of the promoters encoded in TOL. The growth formation and substrate utilisation patterns could not be accurately described by traditional Monod-type models for a wide range of conditions, demonstrating the critical importance of gene regulation for the development of advanced models closely predicting complex bioprocesses. In contrast, the proposed strategy, which utilises quantitative information pertaining to upstream molecular events that control the production of rate-limiting enzymes, predicts the catabolism of a substrate and biomass formation and could be of central importance for the design of optimal bioprocesses.
en
Gene Expression Regulation, Bacterial
Gene Expression Regulation, Enzymologic
Kinetics
Models, Biological
Oxidation-Reduction
Plasmids
Pseudomonas putida
Transcription, Genetic
Xylenes
Linking genes to microbial growth kinetics: an integrated biochemical systems engineering approach.
Article2018-06-13T05:42:13ZThe majority of models describing the kinetic properties of a microorganism for a given substrate are unstructured and empirical. They are formulated in this manner so that the complex mechanism of cell growth is simplified. Herein, a novel approach for modelling microbial growth kinetics is proposed, linking biomass growth and substrate consumption rates to the gene regulatory programmes that control these processes. A dynamic model of the TOL (pWW0) plasmid of Pseudomonas putida mt-2 has been developed, describing the molecular interactions that lead to the transcription of the upper and meta operons, known to produce the enzymes for the oxidative catabolism of m-xylene. The genetic circuit model was combined with a growth kinetic model decoupling biomass growth and substrate consumption rates, which are expressed as independent functions of the rate-limiting enzymes produced by the operons. Estimation of model parameters and validation of the model's predictive capability were successfully performed in batch cultures of mt-2 fed with different concentrations of m-xylene, as confirmed by relative mRNA concentration measurements of the promoters encoded in TOL. The growth formation and substrate utilisation patterns could not be accurately described by traditional Monod-type models for a wide range of conditions, demonstrating the critical importance of gene regulation for the development of advanced models closely predicting complex bioprocesses. In contrast, the proposed strategy, which utilises quantitative information pertaining to upstream molecular events that control the production of rate-limiting enzymes, predicts the catabolism of a substrate and biomass formation and could be of central importance for the design of optimal bioprocesses.oai:repository.helmholtz-hzi.de:10033/2100292019-08-30T11:31:49Zcom_10033_6823com_10033_6820col_10033_6891
Hemmen, Katherina
Reinl, Tobias
Buttler, Kerstin
Behler, Friederike
Dieken, Hauke
Jänsch, Lothar
Wilting, Jörg
Weich, Herbert A
Department of Gene Regulation, HZI, Build. D, Inhoffenstr. 7, 38124, Braunschweig, Germany. katharina.hemmen@ewetel.net
2012-02-08T15:14:21Z
2012-02-08T15:14:21Z
2011-05
High-resolution mass spectrometric analysis of the secretome from mouse lung endothelial progenitor cells. 2011, 14 (2):163-72 Angiogenesis
1573-7209
21234671
10.1007/s10456-011-9200-x
http://hdl.handle.net/10033/210029
Angiogenesis
Recently, we isolated and characterized resident endothelial progenitor cells from the lungs of adult mice. These cells have a high proliferation potential, are not transformed and can differentiate into blood- and lymph-vascular endothelial cells under in vitro and in vivo conditions. Here we studied the secretome of these cells by nanoflow liquid chromatographic mass spectrometry (LC-MS). For analysis, 3-day conditioned serum-free media were used. We found 133 proteins belonging to the categories of membrane-bound or secreted proteins. Thereby, several of the membrane-bound proteins also existed as released variants. Thirty-five proteins from this group are well known as endothelial cell- or angiogenesis-related proteins. The MS analysis of the secretome was supplemented and confirmed by fluorescence activated cell sorting analyses, ELISA measurements and immunocytological studies of selected proteins. The secretome data presented in this study provides a platform for the in-depth analysis of endothelial progenitor cells and characterizes potential cellular markers and signaling components in hem- and lymphangiogenesis.
en
Animals
Culture Media, Conditioned
Endothelial Cells
Flow Cytometry
Fluorescent Antibody Technique
Lung
Mass Spectrometry
Mice
Mice, Inbred BALB C
Neovascularization, Physiologic
Proteome
Reproducibility of Results
Solubility
Stem Cells
Subcellular Fractions
Vascular Endothelial Growth Factor Receptor-1
Vascular Endothelial Growth Factor Receptor-2
High-resolution mass spectrometric analysis of the secretome from mouse lung endothelial progenitor cells.
Article2018-05-23T10:10:09ZRecently, we isolated and characterized resident endothelial progenitor cells from the lungs of adult mice. These cells have a high proliferation potential, are not transformed and can differentiate into blood- and lymph-vascular endothelial cells under in vitro and in vivo conditions. Here we studied the secretome of these cells by nanoflow liquid chromatographic mass spectrometry (LC-MS). For analysis, 3-day conditioned serum-free media were used. We found 133 proteins belonging to the categories of membrane-bound or secreted proteins. Thereby, several of the membrane-bound proteins also existed as released variants. Thirty-five proteins from this group are well known as endothelial cell- or angiogenesis-related proteins. The MS analysis of the secretome was supplemented and confirmed by fluorescence activated cell sorting analyses, ELISA measurements and immunocytological studies of selected proteins. The secretome data presented in this study provides a platform for the in-depth analysis of endothelial progenitor cells and characterizes potential cellular markers and signaling components in hem- and lymphangiogenesis.oai:repository.helmholtz-hzi.de:10033/2227372019-08-30T11:30:54Zcom_10033_6823com_10033_6820col_10033_6891
Nehlsen, Kristina
da Gama-Norton, Leonor
Schucht, Roland
Hauser, Hansjörg
Wirth, Dagmar
Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany. dagmar.wirth@helmholtz-hzi.de.
2012-05-09T13:54:23Z
2012-05-09T13:54:23Z
2011-11-22
Towards rational engineering of cells: Recombinant gene expression in defined chromosomal loci. 2011, 5 Suppl 8:O6notBMC Proc
1753-6561
22373348
10.1186/1753-6561-5-S8-O6
http://hdl.handle.net/10033/222737
BMC proceedings
ENG
Archived with thanks to BMC proceedings
Towards rational engineering of cells: Recombinant gene expression in defined chromosomal loci.
Meetings and Proceedings2018-06-12T23:52:09Zoai:repository.helmholtz-hzi.de:10033/2276742019-08-30T11:37:23Zcom_10033_6823com_10033_6820col_10033_6891
Rand, Ulfert
Rinas, Melanie
Schwerk, Johannes
Nöhren, Gesa
Linnes, Melanie
Kröger, Andrea
Flossdorf, Michael
Kály-Kullai, Kristóf
Hauser, Hansjörg
Höfer, Thomas
Köster, Mario
1] Department of Gene Regulation and Differentiation, Helmholtz Centre for Infection Research, Braunschweig, Germany [2] Division of Theoretical Systems Biology, German Cancer Research Center (DKFZ) and BioQuant Center, Heidelberg, Germany.
2012-06-06T14:11:36Z
2012-06-06T14:11:36Z
2012
Multi-layered stochasticity and paracrine signal propagation shape the type-I interferon response. 2012, 8:584 Mol. Syst. Biol.
1744-4292
22617958
10.1038/msb.2012.17
http://hdl.handle.net/10033/227674
Molecular systems biology
The cellular recognition of viruses evokes the secretion of type-I interferons (IFNs) that induce an antiviral protective state. By live-cell imaging, we show that key steps of virus-induced signal transduction, IFN-β expression, and induction of IFN-stimulated genes (ISGs) are stochastic events in individual cells. The heterogeneity in IFN production is of cellular-and not viral-origin, and temporal unpredictability of IFN-β expression is largely due to cell-intrinsic noise generated both upstream and downstream of the activation of nuclear factor-κB and IFN regulatory factor transcription factors. Subsequent ISG induction occurs as a stochastic all-or-nothing switch, where the responding cells are protected against virus replication. Mathematical modelling and experimental validation show that reliable antiviral protection in the face of multi-layered cellular stochasticity is achieved by paracrine response amplification. Achieving coherent responses through intercellular communication is likely to be a more widely used strategy by mammalian cells to cope with pervasive stochasticity in signalling and gene expression.
en
Archived with thanks to Molecular systems biology
Multi-layered stochasticity and paracrine signal propagation shape the type-I interferon response.
Article2018-06-12T22:58:51ZThe cellular recognition of viruses evokes the secretion of type-I interferons (IFNs) that induce an antiviral protective state. By live-cell imaging, we show that key steps of virus-induced signal transduction, IFN-β expression, and induction of IFN-stimulated genes (ISGs) are stochastic events in individual cells. The heterogeneity in IFN production is of cellular-and not viral-origin, and temporal unpredictability of IFN-β expression is largely due to cell-intrinsic noise generated both upstream and downstream of the activation of nuclear factor-κB and IFN regulatory factor transcription factors. Subsequent ISG induction occurs as a stochastic all-or-nothing switch, where the responding cells are protected against virus replication. Mathematical modelling and experimental validation show that reliable antiviral protection in the face of multi-layered cellular stochasticity is achieved by paracrine response amplification. Achieving coherent responses through intercellular communication is likely to be a more widely used strategy by mammalian cells to cope with pervasive stochasticity in signalling and gene expression.oai:repository.helmholtz-hzi.de:10033/2304562019-08-30T11:37:00Zcom_10033_6823com_10033_6820col_10033_6891
Reich, Uta
Fadeeva, Elena
Warnecke, Athanasia
Paasche, Gerrit
Müller, Peter
Chichkov, Boris
Stöver, Timo
Lenarz, Thomas
Reuter, Günter
Department of Otorhinolaryngology, Head and Neck Surgery, Hannover Medical School, Hannover, Germany.
2012-06-25T09:51:13Z
2012-06-25T09:51:13Z
2012-05
Directing neuronal cell growth on implant material surfaces by microstructuring. 2012, 100 (4):940-7 J. Biomed. Mater. Res. Part B Appl. Biomater.
1552-4981
22287482
10.1002/jbm.b.32656
http://hdl.handle.net/10033/230456
Journal of biomedical materials research. Part B, Applied biomaterials
For best hearing sensation, electrodes of auditory prosthesis must have an optimal electrical contact to the respective neuronal cells. To improve the electrode-nerve interface, microstructuring of implant surfaces could guide neuronal cells toward the electrode contact. To this end, femtosecond laser ablation was used to generate linear microgrooves on the two currently relevant cochlear implant materials, silicone elastomer and platinum. Silicone surfaces were structured by two different methods, either directly, by laser ablation or indirectly, by imprinting using laser-microstructured molds. The influence of surface structuring on neurite outgrowth was investigated utilizing a neuronal-like cell line and primary auditory neurons. The pheochromocytoma cell line PC-12 and primary spiral ganglion cells were cultured on microstructured auditory implant materials. The orientation of neurite outgrowth relative to the microgrooves was determined. Both cell types showed a preferred orientation in parallel to the microstructures on both, platinum and on molded silicone elastomer. Interestingly, microstructures generated by direct laser ablation of silicone did not influence the orientation of either cell type. This shows that differences in the manufacturing procedures can affect the ability of microstructured implant surfaces to guide the growth of neurites. This is of particular importance for clinical applications, since the molding technique represents a reproducible, economic, and commercially feasible manufacturing procedure for the microstructured silicone surfaces of medical implants.
en
Archived with thanks to Journal of biomedical materials research. Part B, Applied biomaterials
Directing neuronal cell growth on implant material surfaces by microstructuring.
Article2013-05-15T00:00:00ZFor best hearing sensation, electrodes of auditory prosthesis must have an optimal electrical contact to the respective neuronal cells. To improve the electrode-nerve interface, microstructuring of implant surfaces could guide neuronal cells toward the electrode contact. To this end, femtosecond laser ablation was used to generate linear microgrooves on the two currently relevant cochlear implant materials, silicone elastomer and platinum. Silicone surfaces were structured by two different methods, either directly, by laser ablation or indirectly, by imprinting using laser-microstructured molds. The influence of surface structuring on neurite outgrowth was investigated utilizing a neuronal-like cell line and primary auditory neurons. The pheochromocytoma cell line PC-12 and primary spiral ganglion cells were cultured on microstructured auditory implant materials. The orientation of neurite outgrowth relative to the microgrooves was determined. Both cell types showed a preferred orientation in parallel to the microstructures on both, platinum and on molded silicone elastomer. Interestingly, microstructures generated by direct laser ablation of silicone did not influence the orientation of either cell type. This shows that differences in the manufacturing procedures can affect the ability of microstructured implant surfaces to guide the growth of neurites. This is of particular importance for clinical applications, since the molding technique represents a reproducible, economic, and commercially feasible manufacturing procedure for the microstructured silicone surfaces of medical implants.oai:repository.helmholtz-hzi.de:10033/2322312019-08-30T11:30:56Zcom_10033_6823com_10033_6820col_10033_6891
Behrens, Peter
Müller, Peter P.
Stieve, Martin
Besdo, Silke
Ehlert, Nina
Lenarz, Thomas
Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124, Braunschweig, Germany.
2012-07-05T10:54:05Z
2012-07-05T10:54:05Z
2010
Funktionalisierte Mittelohrprothesen 2010, :13 Sonderforschungsbereich 599
978-3-00-032924-1
http://hdl.handle.net/10033/232231
Druckerei der Medizinischen Hochschule
Funktionalisierte Mittelohrprothesen
Book chapter2018-06-13T03:43:38Zoai:repository.helmholtz-hzi.de:10033/2322542019-08-30T11:30:52Zcom_10033_6823com_10033_6820col_10033_6891
Windhagen, Henning
Dempwolf, Wiebke
Gross, Gerhard
Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124, Braunschweig, Germany.
2012-07-05T14:44:34Z
2012-07-05T14:44:34Z
2010
2010
Implantatoberflächen 2010, :85 Sonderforschungsbereich 599
978-3-00-032924-1
http://hdl.handle.net/10033/232254
Druckerei der Medizinischen Hochschule Hannover
Implantatoberflächen
Book chapter2018-06-13T21:37:05Zoai:repository.helmholtz-hzi.de:10033/2449992019-08-30T11:27:16Zcom_10033_6823com_10033_6820col_10033_6891
Lam, Carolyn M C
Suárez Diez, María
Godinho, Miguel
Martins Dos Santos, Vítor A P
Systems and Synthetic Biology Group, Helmholtz Centre for Infection Research, Inhoffenstraße 7, D-38124 Braunschweig, Germany; Systems and Synthetic Biology, Wageningen University, Dreijenplein 10, Building number 316, 6703 HB Wageningen, The Netherlands.
2012-09-19T13:37:40Z
2012-09-19T13:37:40Z
2012-07-16
Programmable bacterial catalysis - designing cells for biosynthesis of value-added compounds. 2012, 586 (15):2184-90 FEBS Lett.
1873-3468
22710181
10.1016/j.febslet.2012.02.030
http://hdl.handle.net/10033/244999
FEBS letters
Bacteria have long been used for the synthesis of a wide range of useful proteins and compounds. The developments of new bioprocesses and improvements of existing strategies for syntheses of valuable products in various bacterial cell hosts have their own challenges and limitations. The field of synthetic biology has combined knowledge from different science and engineering disciplines and facilitated the advancement of novel biological components which has inspired the design of targeted biosynthesis. Here we discuss recent advances in synthetic biology with relevance to biosynthesis in bacteria and the applications of computational algorithms and tools for manipulation of cellular components. Continuous improvements are necessary to keep up with increasing demands in terms of complexity, scale, and predictability of biosynthesis products.
en
Archived with thanks to FEBS letters
Programmable bacterial catalysis - designing cells for biosynthesis of value-added compounds.
Article2018-06-13T05:39:56ZBacteria have long been used for the synthesis of a wide range of useful proteins and compounds. The developments of new bioprocesses and improvements of existing strategies for syntheses of valuable products in various bacterial cell hosts have their own challenges and limitations. The field of synthetic biology has combined knowledge from different science and engineering disciplines and facilitated the advancement of novel biological components which has inspired the design of targeted biosynthesis. Here we discuss recent advances in synthetic biology with relevance to biosynthesis in bacteria and the applications of computational algorithms and tools for manipulation of cellular components. Continuous improvements are necessary to keep up with increasing demands in terms of complexity, scale, and predictability of biosynthesis products.oai:repository.helmholtz-hzi.de:10033/2400512019-08-30T11:27:16Zcom_10033_6823com_10033_6820col_10033_6891
Leprince, Audrey
de Lorenzo, Víctor
Völler, Petra
van Passel, Mark W J
Martins dos Santos, Vitor A P
Systems and Synthetic Biology Group, Helmholtz-Centre for Infection Research, Braunschweig, Germany.
2012-08-27T12:34:58Z
2012-08-27T12:34:58Z
2012-06
Random and cyclical deletion of large DNA segments in the genome of Pseudomonas putida. 2012, 14 (6):1444-53 Environ. Microbiol.
1462-2920
22429517
10.1111/j.1462-2920.2012.02730.x
http://hdl.handle.net/10033/240051
Environmental microbiology
Cumulative site-directed mutagenesis is of limited suitability for the global analysis of the gene functions in the microbe's cellular network. In order to simplify and stabilize the genome of the soil bacterium Pseudomonas putida, we developed a recyclable three-step excision method based on the combination of customized mini-transposons and the FLP-FRT site-specific recombination system. To demonstrate the powerful potential of these tools, we first established insertion mutant libraries that allow users to study gene functions with respect either to phenotypic characteristics (single insertions) or to their involvement in predicted networks (double insertions). Based on these libraries, we generated as a proof-of-principle, single-deletion mutants lacking ~4.1% of the genome (~3.7% of the gene repertoire). A cyclical application of the method generated four double-deletion mutants of which a maximum of ~7.4% of the chromosome (~6.9% of the gene count) was excised. This procedure demonstrates a new strategy for rapid genome streamlining and gain of new insights into the molecular interactions and regulations.
en
Archived with thanks to Environmental microbiology
Random and cyclical deletion of large DNA segments in the genome of Pseudomonas putida.
Article2013-06-15T00:00:00ZCumulative site-directed mutagenesis is of limited suitability for the global analysis of the gene functions in the microbe's cellular network. In order to simplify and stabilize the genome of the soil bacterium Pseudomonas putida, we developed a recyclable three-step excision method based on the combination of customized mini-transposons and the FLP-FRT site-specific recombination system. To demonstrate the powerful potential of these tools, we first established insertion mutant libraries that allow users to study gene functions with respect either to phenotypic characteristics (single insertions) or to their involvement in predicted networks (double insertions). Based on these libraries, we generated as a proof-of-principle, single-deletion mutants lacking ~4.1% of the genome (~3.7% of the gene repertoire). A cyclical application of the method generated four double-deletion mutants of which a maximum of ~7.4% of the chromosome (~6.9% of the gene count) was excised. This procedure demonstrates a new strategy for rapid genome streamlining and gain of new insights into the molecular interactions and regulations.oai:repository.helmholtz-hzi.de:10033/2405912019-08-30T11:24:31Zcom_10033_6823com_10033_6820col_10033_6891
Poblete-Castro, Ignacio
Escapa, Isabel F
Jäger, Christian
Puchalka, Jacek
Lam, Carolyn Ming Chi
Schomburg, Dietmar
Prieto, María Auxiliadora
Martins dos Santos, Vítor A P
Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany. ignacio.pobletecastro@helmholtz-hzi.de
2012-08-30T09:24:05Z
2012-08-30T09:24:05Z
2012
The metabolic response of P. putida KT2442 producing high levels of polyhydroxyalkanoate under single- and multiple-nutrient-limited growth: highlights from a multi-level omics approach. 2012, 11:34 Microb. Cell Fact.
1475-2859
22433058
10.1186/1475-2859-11-34
http://hdl.handle.net/10033/240591
Microbial cell factories
Pseudomonas putida KT2442 is a natural producer of polyhydroxyalkanoates (PHAs), which can substitute petroleum-based non-renewable plastics and form the basis for the production of tailor-made biopolymers. However, despite the substantial body of work on PHA production by P. putida strains, it is not yet clear how the bacterium re-arranges its whole metabolism when it senses the limitation of nitrogen and the excess of fatty acids as carbon source, to result in a large accumulation of PHAs within the cell. In the present study we investigated the metabolic response of KT2442 using a systems biology approach to highlight the differences between single- and multiple-nutrient-limited growth in chemostat cultures.
en
Archived with thanks to Microbial cell factories
Biomass
Carbon
Energy Metabolism
Fatty Acids
Gene Expression Regulation, Bacterial
Metabolomics
Nitrogen
Polyhydroxyalkanoates
Proteomics
Pseudomonas putida
Transcriptome
The metabolic response of P. putida KT2442 producing high levels of polyhydroxyalkanoate under single- and multiple-nutrient-limited growth: highlights from a multi-level omics approach.
Article2018-06-12T23:51:27ZPseudomonas putida KT2442 is a natural producer of polyhydroxyalkanoates (PHAs), which can substitute petroleum-based non-renewable plastics and form the basis for the production of tailor-made biopolymers. However, despite the substantial body of work on PHA production by P. putida strains, it is not yet clear how the bacterium re-arranges its whole metabolism when it senses the limitation of nitrogen and the excess of fatty acids as carbon source, to result in a large accumulation of PHAs within the cell. In the present study we investigated the metabolic response of KT2442 using a systems biology approach to highlight the differences between single- and multiple-nutrient-limited growth in chemostat cultures.oai:repository.helmholtz-hzi.de:10033/2459522019-08-30T11:27:46Zcom_10033_6823com_10033_6820col_10033_6891
Thiele, Wilko
Krishnan, Jaya
Rothley, Melanie
Weih, Debra
Plaumann, Diana
Kuch, Vanessa
Quagliata, Luca
Weich, Herbert A
Sleeman, Jonathan P
Universität Heidelberg, Medizinische Fakultät Mannheim, Mannheim, Germany;
2012-09-26T14:24:10Z
2012-09-26T14:24:10Z
2012-08-30
VEGFR-3 is expressed on megakaryocyte precursors in the murine bone marrow and plays a regulatory role in megakaryopoiesis. 2012, 120 (9):1899-907 Blood
1528-0020
22797697
10.1182/blood-2011-09-376657
http://hdl.handle.net/10033/245952
Blood
VEGFR-3 is a transmembrane receptor tyrosine kinase that is activated by its ligands VEGF-C and VEGF-D. Although VEGFR-3 has been linked primarily to the regulation of lymphangiogenesis, in the present study, we demonstrate a role for VEGFR-3 in megakaryopoiesis. Using a human erythroleukemia cell line and primary murine BM cells, we show that VEGFR-3 is expressed on megakaryocytic progenitor cells through to the promegakaryoblast stage. Functionally, specific activation of VEGFR-3 impaired the transition to polyploidy of CD41(+) cells in primary BM cultures. Blockade of VEGFR-3 promoted endoreplication consistently. In vivo, long-term activation or blockade of VEGFR-3 did not affect steady-state murine megakaryopoiesis or platelet counts significantly. However, activation of VEGFR-3 in sublethally irradiated mice resulted in significantly elevated numbers of CD41(+) cells in the BM and a significant increase in diploid CD41(+) cells, whereas the number of polyploid CD41(+) cells was reduced significantly. Moreover, activation of VEGFR-3 increased platelet counts in thrombopoietin-treated mice significantly and modulated 5-fluorouracil-induced thrombocytosis strongly, suggesting a regulatory role for VEGFR-3 in megakaryopoiesis.
en
Archived with thanks to Blood
VEGFR-3 is expressed on megakaryocyte precursors in the murine bone marrow and plays a regulatory role in megakaryopoiesis.
Article2018-06-12T17:24:21ZVEGFR-3 is a transmembrane receptor tyrosine kinase that is activated by its ligands VEGF-C and VEGF-D. Although VEGFR-3 has been linked primarily to the regulation of lymphangiogenesis, in the present study, we demonstrate a role for VEGFR-3 in megakaryopoiesis. Using a human erythroleukemia cell line and primary murine BM cells, we show that VEGFR-3 is expressed on megakaryocytic progenitor cells through to the promegakaryoblast stage. Functionally, specific activation of VEGFR-3 impaired the transition to polyploidy of CD41(+) cells in primary BM cultures. Blockade of VEGFR-3 promoted endoreplication consistently. In vivo, long-term activation or blockade of VEGFR-3 did not affect steady-state murine megakaryopoiesis or platelet counts significantly. However, activation of VEGFR-3 in sublethally irradiated mice resulted in significantly elevated numbers of CD41(+) cells in the BM and a significant increase in diploid CD41(+) cells, whereas the number of polyploid CD41(+) cells was reduced significantly. Moreover, activation of VEGFR-3 increased platelet counts in thrombopoietin-treated mice significantly and modulated 5-fluorouracil-induced thrombocytosis strongly, suggesting a regulatory role for VEGFR-3 in megakaryopoiesis.oai:repository.helmholtz-hzi.de:10033/2461522019-08-30T11:28:24Zcom_10033_6824com_10033_6823com_10033_6820col_10033_6892
Doetsch, Martina
Gluch, Angela
Poznanović, Goran
Bode, Juergen
Vidaković, Melita
Helmholtz Centre for Infection Research/Epigenetic Regulation, Braunschweig, Germany.
2012-09-27T13:01:41Z
2012-09-27T13:01:41Z
2012
YY1-Binding Sites Provide Central Switch Functions in the PARP-1 Gene Expression Network. 2012, 7 (8):e44125 PLoS ONE
1932-6203
22937159
10.1371/journal.pone.0044125
http://hdl.handle.net/10033/246152
PloS one
Evidence is presented for the involvement of the interplay between transcription factor Yin Yang 1 (YY1) and poly(ADP-ribose) polymerase-1 (PARP-1) in the regulation of mouse PARP-1 gene (muPARP-1) promoter activity. We identified potential YY1 binding motifs (BM) at seven positions in the muPARP-1 core-promoter (-574/+200). Binding of YY1 was observed by the electrophoretic supershift assay using anti-YY1 antibody and linearized or supercoiled forms of plasmids bearing the core promoter, as well as with 30 bp oligonucleotide probes containing the individual YY1 binding motifs and four muPARP-1 promoter fragments. We detected YY1 binding to BM1 (-587/-558), BM4 (-348/-319) and a very prominent association with BM7 (+86/+115). Inspection of BM7 reveals overlap of the muPARP-1 translation start site with the Kozak sequence and YY1 and PARP-1 recognition sites. Site-directed mutagenesis of the YY1 and PARP-1 core motifs eliminated protein binding and showed that YY1 mediates PARP-1 binding next to the Kozak sequence. Transfection experiments with a reporter gene under the control of the muPARP-1 promoter revealed that YY1 binding to BM1 and BM4 independently repressed the promoter. Mutations at these sites prevented YY1 binding, allowing for increased reporter gene activity. In PARP-1 knockout cells subjected to PARP-1 overexpression, effects similar to YY1 became apparent; over expression of YY1 and PARP-1 revealed their synergistic action. Together with our previous findings these results expand the PARP-1 autoregulatory loop principle by YY1 actions, implying rigid limitation of muPARP-1 expression. The joint actions of PARP-1 and YY1 emerge as important contributions to cell homeostasis.
en
Archived with thanks to PloS one
YY1-Binding Sites Provide Central Switch Functions in the PARP-1 Gene Expression Network.
Article2018-06-13T17:08:04ZEvidence is presented for the involvement of the interplay between transcription factor Yin Yang 1 (YY1) and poly(ADP-ribose) polymerase-1 (PARP-1) in the regulation of mouse PARP-1 gene (muPARP-1) promoter activity. We identified potential YY1 binding motifs (BM) at seven positions in the muPARP-1 core-promoter (-574/+200). Binding of YY1 was observed by the electrophoretic supershift assay using anti-YY1 antibody and linearized or supercoiled forms of plasmids bearing the core promoter, as well as with 30 bp oligonucleotide probes containing the individual YY1 binding motifs and four muPARP-1 promoter fragments. We detected YY1 binding to BM1 (-587/-558), BM4 (-348/-319) and a very prominent association with BM7 (+86/+115). Inspection of BM7 reveals overlap of the muPARP-1 translation start site with the Kozak sequence and YY1 and PARP-1 recognition sites. Site-directed mutagenesis of the YY1 and PARP-1 core motifs eliminated protein binding and showed that YY1 mediates PARP-1 binding next to the Kozak sequence. Transfection experiments with a reporter gene under the control of the muPARP-1 promoter revealed that YY1 binding to BM1 and BM4 independently repressed the promoter. Mutations at these sites prevented YY1 binding, allowing for increased reporter gene activity. In PARP-1 knockout cells subjected to PARP-1 overexpression, effects similar to YY1 became apparent; over expression of YY1 and PARP-1 revealed their synergistic action. Together with our previous findings these results expand the PARP-1 autoregulatory loop principle by YY1 actions, implying rigid limitation of muPARP-1 expression. The joint actions of PARP-1 and YY1 emerge as important contributions to cell homeostasis.oai:repository.helmholtz-hzi.de:10033/2465362019-08-30T11:27:46Zcom_10033_6823com_10033_6820col_10033_6891
Poblete-Castro, Ignacio
Becker, Judith
Dohnt, Katrin
dos Santos, Vitor Martins
Wittmann, Christoph
HZI-Helmholtz Centre for Infection Research, Systems and Synthetic Biology, Braunschweig, Germany.
2012-10-02T12:56:23Z
2012-10-02T12:56:23Z
2012-03
Industrial biotechnology of Pseudomonas putida and related species. 2012, 93 (6):2279-90 Appl. Microbiol. Biotechnol.
1432-0614
22350258
10.1007/s00253-012-3928-0
http://hdl.handle.net/10033/246536
Applied microbiology and biotechnology
Since their discovery many decades ago, Pseudomonas putida and related subspecies have been intensively studied with regard to their potential application in industrial biotechnology. Today, these Gram-negative soil bacteria, traditionally known as well-performing xenobiotic degraders, are becoming efficient cell factories for various products of industrial relevance including a full range of unnatural chemicals. This development is strongly driven by systems biotechnology, integrating systems metabolic engineering approaches with novel concepts from bioprocess engineering, including novel reactor designs and renewable feedstocks.
en
Archived with thanks to Applied microbiology and biotechnology
Genetic Engineering
Industrial Microbiology
Metabolic Engineering
Pseudomonas putida
Industrial biotechnology of Pseudomonas putida and related species.
Article2018-06-13T02:43:15ZSince their discovery many decades ago, Pseudomonas putida and related subspecies have been intensively studied with regard to their potential application in industrial biotechnology. Today, these Gram-negative soil bacteria, traditionally known as well-performing xenobiotic degraders, are becoming efficient cell factories for various products of industrial relevance including a full range of unnatural chemicals. This development is strongly driven by systems biotechnology, integrating systems metabolic engineering approaches with novel concepts from bioprocess engineering, including novel reactor designs and renewable feedstocks.oai:repository.helmholtz-hzi.de:10033/2469932019-08-30T11:27:46Zcom_10033_6823com_10033_6820col_10033_6891
Hogan, Louise
Bhuju, Sabin
Jones, Des C
Laing, Ken
Trowsdale, John
Butcher, Philip
Singh, Mahavir
Vordermeier, Martin
Allen, Rachel L
Centre for Infection, Division of Clinical Sciences, St George's, University of London, Cranmer Terrace, London, United Kingdom. p0904768@sgul.ac.uk
2012-10-04T11:26:46Z
2012-10-04T11:26:46Z
2012
Characterisation of bovine leukocyte Ig-like receptors. 2012, 7 (4):e34291 PLoS ONE
1932-6203
22485161
10.1371/journal.pone.0034291
http://hdl.handle.net/10033/246993
PloS one
Leukocyte Immunoglobulin-like receptors (LILR) are innate immune receptors involved in regulating both innate and adaptive immune functions. LILR show more interspecies conservation than the closely related Killer Ig-like receptors, and homologues have been identified in rodents, primates, seals and chickens. The murine equivalents, paired Ig-like receptors (PIR), contain two additional immunoglobulin domains, but show strong sequence and functional similarities to human LILR. The bovine genome was recently sequenced, with preliminary annotations indicating that LILR were present in this species. We therefore sought to identify and characterize novel LILR within the Bos taurus genome, compare these phylogenetically with LILR from other species and determine whether they were expressed in vivo. Twenty six potential bovine LILR were initially identified using BLAST and BLAT software. Phylogenetic analysis constructed using the neighbour-joining method, incorporating pairwise deletion and confidence limits estimated from 1000 replicates using bootstrapping, indicated that 16 of these represent novel bovine LILR. Protein structures defined using protein BLAST predict that the bovine LILR family comprises seven putative inhibitory, four activating and five soluble receptors. Preliminary expression analysis was performed by mapping the predicted sequences with raw data from total transcript sequence generated using Genome Analyzer IIx (Illumina) to provide evidence that all 16 of these receptors are expressed in vivo. The bovine receptor family appears to contain receptors which resemble the six domain rodent PIR as well as the four domain LILR found in other species.
en
Archived with thanks to PloS one
Animals
Cattle
Cells, Cultured
Chromosome Mapping
Gene Expression
Humans
Leukocytes, Mononuclear
Mice
Models, Molecular
Phylogeny
Protein Structure, Tertiary
Receptors, Immunologic
Sequence Analysis, Protein
Sequence Homology, Amino Acid
Characterisation of bovine leukocyte Ig-like receptors.
Article2018-06-12T21:49:14ZLeukocyte Immunoglobulin-like receptors (LILR) are innate immune receptors involved in regulating both innate and adaptive immune functions. LILR show more interspecies conservation than the closely related Killer Ig-like receptors, and homologues have been identified in rodents, primates, seals and chickens. The murine equivalents, paired Ig-like receptors (PIR), contain two additional immunoglobulin domains, but show strong sequence and functional similarities to human LILR. The bovine genome was recently sequenced, with preliminary annotations indicating that LILR were present in this species. We therefore sought to identify and characterize novel LILR within the Bos taurus genome, compare these phylogenetically with LILR from other species and determine whether they were expressed in vivo. Twenty six potential bovine LILR were initially identified using BLAST and BLAT software. Phylogenetic analysis constructed using the neighbour-joining method, incorporating pairwise deletion and confidence limits estimated from 1000 replicates using bootstrapping, indicated that 16 of these represent novel bovine LILR. Protein structures defined using protein BLAST predict that the bovine LILR family comprises seven putative inhibitory, four activating and five soluble receptors. Preliminary expression analysis was performed by mapping the predicted sequences with raw data from total transcript sequence generated using Genome Analyzer IIx (Illumina) to provide evidence that all 16 of these receptors are expressed in vivo. The bovine receptor family appears to contain receptors which resemble the six domain rodent PIR as well as the four domain LILR found in other species.oai:repository.helmholtz-hzi.de:10033/2518122019-08-30T11:28:51Zcom_10033_6823com_10033_6820col_10033_6891
Cudmore, Melissa J
Hewett, Peter W
Ahmad, Shakil
Wang, Ke-Qing
Cai, Meng
Al-Ani, Bahjat
Fujisawa, Takeshi
Ma, Bin
Sissaoui, Samir
Ramma, Wenda
Miller, Mark R
Newby, David E
Gu, Yuchun
Barleon, Bernhard
Weich, Herbert
Ahmed, Asif
University/BHF Centre for Cardiovascular Science, Queen's Medical Research Institute, College of Medicine and Veterinary Medicine, University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4TJ, UK.
2012-11-12T12:02:48Z
2012-11-12T12:02:48Z
2012
The role of heterodimerization between VEGFR-1 and VEGFR-2 in the regulation of endothelial cell homeostasis. 2012, 3:972 Nat Commun
2041-1723
22828632
10.1038/ncomms1977
http://hdl.handle.net/10033/251812
Nature communications
VEGF-A activity is tightly regulated by ligand and receptor availability. Here we investigate the physiological function of heterodimers between VEGF receptor-1 (VEGFR-1; Flt-1) and VEGFR-2 (KDR; Flk-1) (VEGFR(1-2)) in endothelial cells with a synthetic ligand that binds specifically to VEGFR(1-2). The dimeric ligand comprises one VEGFR-2-specific monomer (VEGF-E) and a VEGFR-1-specific monomer (PlGF-1). Here we show that VEGFR(1-2) activation mediates VEGFR phosphorylation, endothelial cell migration, sustained in vitro tube formation and vasorelaxation via the nitric oxide pathway. VEGFR(1-2) activation does not mediate proliferation or elicit endothelial tissue factor production, confirming that these functions are controlled by VEGFR-2 homodimers. We further demonstrate that activation of VEGFR(1-2) inhibits VEGF-A-induced prostacyclin release, phosphorylation of ERK1/2 MAP kinase and mobilization of intracellular calcium from primary endothelial cells. These findings indicate that VEGFR-1 subunits modulate VEGF activity predominantly by forming heterodimer receptors with VEGFR-2 subunits and such heterodimers regulate endothelial cell homeostasis.
en
Archived with thanks to Nature communications
Blotting, Western
Cells, Cultured
Endothelial Cells
Enzyme-Linked Immunosorbent Assay
Flow Cytometry
Humans
Immunoprecipitation
Protein Multimerization
RNA, Small Interfering
Vascular Endothelial Growth Factor Receptor-1
Vascular Endothelial Growth Factor Receptor-2
The role of heterodimerization between VEGFR-1 and VEGFR-2 in the regulation of endothelial cell homeostasis.
Article2018-06-13T19:57:20ZVEGF-A activity is tightly regulated by ligand and receptor availability. Here we investigate the physiological function of heterodimers between VEGF receptor-1 (VEGFR-1; Flt-1) and VEGFR-2 (KDR; Flk-1) (VEGFR(1-2)) in endothelial cells with a synthetic ligand that binds specifically to VEGFR(1-2). The dimeric ligand comprises one VEGFR-2-specific monomer (VEGF-E) and a VEGFR-1-specific monomer (PlGF-1). Here we show that VEGFR(1-2) activation mediates VEGFR phosphorylation, endothelial cell migration, sustained in vitro tube formation and vasorelaxation via the nitric oxide pathway. VEGFR(1-2) activation does not mediate proliferation or elicit endothelial tissue factor production, confirming that these functions are controlled by VEGFR-2 homodimers. We further demonstrate that activation of VEGFR(1-2) inhibits VEGF-A-induced prostacyclin release, phosphorylation of ERK1/2 MAP kinase and mobilization of intracellular calcium from primary endothelial cells. These findings indicate that VEGFR-1 subunits modulate VEGF activity predominantly by forming heterodimer receptors with VEGFR-2 subunits and such heterodimers regulate endothelial cell homeostasis.oai:repository.helmholtz-hzi.de:10033/2673322019-08-30T11:28:51Zcom_10033_6823com_10033_6820col_10033_6891
Mueller, Peter P
Arnold, Sylvia
Badar, Muhammad
Bormann, Dirk
Bach, Friedrich-Wilhelm
Drynda, Andreas
Meyer-Lindenberg, Andrea
Hauser, Hansjörg
Peuster, Matthias
Helmholtz Centre for Infection Research, Braunschweig, Germany. pmu@gbf.de
2013-01-28T15:17:07Z
2013-01-28T15:17:07Z
2012-11
Histological and molecular evaluation of iron as degradable medical implant material in a murine animal model. 2012, 100 (11):2881-9 J Biomed Mater Res A
1552-4965
22623368
10.1002/jbm.a.34223
http://hdl.handle.net/10033/267332
Journal of biomedical materials research. Part A
A small animal model was established to evaluate the potential of iron as a degradable implant material. After insertion into the tail of mice, the implants gradually degraded over a clinically relevant time period of several months. Histological analysis and gene expression data from whole-genome microarray analyses indicated a limited inflammatory reaction. No evidence of cellular responses to excess iron ions was detected, suggesting that the iron degradation products were metabolically inactive. Iron-rich compounds could be detected in the vicinity of the implant and in individual cells distant from the implantation site. These results demonstrate that the mouse model could be useful for the primary in vivo evaluation of novel implant materials and that iron degradation products can accumulate in diverse organs of the body.
en
Archived with thanks to Journal of biomedical materials research. Part A
Histological and molecular evaluation of iron as degradable medical implant material in a murine animal model.
Article2013-11-15T00:00:00ZA small animal model was established to evaluate the potential of iron as a degradable implant material. After insertion into the tail of mice, the implants gradually degraded over a clinically relevant time period of several months. Histological analysis and gene expression data from whole-genome microarray analyses indicated a limited inflammatory reaction. No evidence of cellular responses to excess iron ions was detected, suggesting that the iron degradation products were metabolically inactive. Iron-rich compounds could be detected in the vicinity of the implant and in individual cells distant from the implantation site. These results demonstrate that the mouse model could be useful for the primary in vivo evaluation of novel implant materials and that iron degradation products can accumulate in diverse organs of the body.oai:repository.helmholtz-hzi.de:10033/2690122019-08-30T11:33:05Zcom_10033_6823com_10033_6820col_10033_6891
Wöhl-Bruhn, Stefanie
Badar, Muhammad
Bertz, Andreas
Tiersch, Brigitte
Koetz, Joachim
Menzel, Henning
Mueller, Peter P
Bunjes, Heike
Technische Universität Braunschweig, Institute of Pharmaceutical Technology, Mendelssohnstraße 1, 38106 Braunschweig, Germany.
2013-02-11T14:43:02Z
2013-02-11T14:43:02Z
2012-08-20
Comparison of in vitro and in vivo protein release from hydrogel systems. 2012, 162 (1):127-33 J Control Release
1873-4995
22687287
10.1016/j.jconrel.2012.05.049
http://hdl.handle.net/10033/269012
Journal of controlled release : official journal of the Controlled Release Society
Hydrogel systems based on hydroxyethyl starch-polyethylene glycol methacrylate (HES-P(EG)(6)MA) or hydroxyethyl starch methacrylate (HES-MA) were used to assess the protein release behavior. Here, we analyzed the in vitro release of FITC-anti-human antibodies incorporated in either HES-P(EG)(6)MA or HES-MA hydrogel delivery systems in PBS or human serum. In addition, hydrogel disks and microparticles prepared from the two polymers were subcutaneously implanted in BALB/c mice. The in vivo release of FITC-IgG was non-invasively monitored by an in vivo imaging system (IVIS 200) over a time period of up to 3 months. The imaging system allowed to asses individual animals over time, therefore only a small number of animals was required to obtain high quality data. The reduction in fluorescence intensity at the site of administration was compared to in vitro release profiles. These investigations demonstrated a sustained release from HES-MA hydrogel disks compared to rapidly degrading HES-P(EG)(6)MA disks and microparticles. The sustained release from HES-MA disks could be further optimized by using increased polymer concentrations. Human serum as in vitro release medium reflected better the in vivo release from HES-P(EG)(6)MA systems than PBS, suggesting that the presence of organic substances like proteins or lipids may play a significant role for the release kinetics.
en
Archived with thanks to Journal of controlled release : official journal of the Controlled Release Society
Absorbable Implants
Animals
Drug Delivery Systems
Fluorescein-5-isothiocyanate
Goats
Hetastarch
Humans
Hydrogel
Immunoglobulin G
Male
Methacrylates
Mice
Mice, Inbred BALB C
Polyethylene Glycols
Comparison of in vitro and in vivo protein release from hydrogel systems.
Article2018-06-12T23:38:19ZHydrogel systems based on hydroxyethyl starch-polyethylene glycol methacrylate (HES-P(EG)(6)MA) or hydroxyethyl starch methacrylate (HES-MA) were used to assess the protein release behavior. Here, we analyzed the in vitro release of FITC-anti-human antibodies incorporated in either HES-P(EG)(6)MA or HES-MA hydrogel delivery systems in PBS or human serum. In addition, hydrogel disks and microparticles prepared from the two polymers were subcutaneously implanted in BALB/c mice. The in vivo release of FITC-IgG was non-invasively monitored by an in vivo imaging system (IVIS 200) over a time period of up to 3 months. The imaging system allowed to asses individual animals over time, therefore only a small number of animals was required to obtain high quality data. The reduction in fluorescence intensity at the site of administration was compared to in vitro release profiles. These investigations demonstrated a sustained release from HES-MA hydrogel disks compared to rapidly degrading HES-P(EG)(6)MA disks and microparticles. The sustained release from HES-MA disks could be further optimized by using increased polymer concentrations. Human serum as in vitro release medium reflected better the in vivo release from HES-P(EG)(6)MA systems than PBS, suggesting that the presence of organic substances like proteins or lipids may play a significant role for the release kinetics.oai:repository.helmholtz-hzi.de:10033/2693132019-08-30T11:33:05Zcom_10033_6823com_10033_6820col_10033_6891
Lensing, Rebecca
Bleich, André
Smoczek, Anna
Glage, Silke
Ehlert, Nina
Luessenhop, Tammo
Behrens, Peter
Müller, Peter Paul
Kietzmann, Manfred
Stieve, Martin
Department of Otolaryngology, Hannover Medical School, Hannover, Germany. Lensing.Rebecca@mh-hannover.de
2013-02-13T10:17:45Z
2013-02-13T10:17:45Z
2013-01
Efficacy of nanoporous silica coatings on middle ear prostheses as a delivery system for antibiotics: an animal study in rabbits. 2013, 9 (1):4815-25 Acta Biomater
1878-7568
22906623
10.1016/j.actbio.2012.08.016
http://hdl.handle.net/10033/269313
Acta biomaterialia
Nanoporous silica layers are able to host molecules and release them over a certain period of time. These local drug delivery systems for antibiotics could be a new approach in the treatment of chronic otitis media. The aim of this study was to examine the efficacy of nanoporous silica coatings on middle ear prostheses as a delivery system for antibiotics in vivo. Pseudomonas aeruginosa was inoculated into the middle ear of rabbits to induce an otitis media. The control group received coated Bioverit®II implants without antibiotics. Coated prostheses with loaded ciprofloxacin were implanted into the middle ears of the study group. After 1 week, the rabbits were sacrificed. The clinical examination as well as the microbiological and histological examinations of organs and middle ear irrigation revealed clear differences between the two groups. P. aeruginosa was detected in every middle ear of the control group and was almost completely eliminated in the study group. Organ examinations revealed the presence of P. aeruginosa in the control group and a prevention of a bacterial spread in the study group. The nanoporous silica layer as antibiotic delivery system showed convincing efficacy in induced pseudomonal otitis media in the rabbit.
en
Archived with thanks to Acta biomaterialia
Efficacy of nanoporous silica coatings on middle ear prostheses as a delivery system for antibiotics: an animal study in rabbits.
Article2018-06-12T23:27:02ZNanoporous silica layers are able to host molecules and release them over a certain period of time. These local drug delivery systems for antibiotics could be a new approach in the treatment of chronic otitis media. The aim of this study was to examine the efficacy of nanoporous silica coatings on middle ear prostheses as a delivery system for antibiotics in vivo. Pseudomonas aeruginosa was inoculated into the middle ear of rabbits to induce an otitis media. The control group received coated Bioverit®II implants without antibiotics. Coated prostheses with loaded ciprofloxacin were implanted into the middle ears of the study group. After 1 week, the rabbits were sacrificed. The clinical examination as well as the microbiological and histological examinations of organs and middle ear irrigation revealed clear differences between the two groups. P. aeruginosa was detected in every middle ear of the control group and was almost completely eliminated in the study group. Organ examinations revealed the presence of P. aeruginosa in the control group and a prevention of a bacterial spread in the study group. The nanoporous silica layer as antibiotic delivery system showed convincing efficacy in induced pseudomonal otitis media in the rabbit.oai:repository.helmholtz-hzi.de:10033/2698522019-08-30T11:27:16Zcom_10033_6823com_10033_6820col_10033_6891
Schniedermann, Judith
Rennecke, Moritz
Buttler, Kerstin
Richter, Georg
Städtler, Anna-Maria
Norgall, Susanne
Badar, Muhammad
Barleon, Bernhard
May, Tobias
Wilting, Jörg
Weich, Herbert A
Division Molecular Biotechnology, Department of Gene Regulation, Helmholtz Centre for Infection Research, Braunschweig, Germany.
2013-02-20T11:47:50Z
2013-02-20T11:47:50Z
2010
Mouse lung contains endothelial progenitors with high capacity to form blood and lymphatic vessels. 2010, 11:50 BMC Cell Biol.
1471-2121
20594323
10.1186/1471-2121-11-50
http://hdl.handle.net/10033/269852
BMC cell biology
Postnatal endothelial progenitor cells (EPCs) have been successfully isolated from whole bone marrow, blood and the walls of conduit vessels. They can, therefore, be classified into circulating and resident progenitor cells. The differentiation capacity of resident lung endothelial progenitor cells from mouse has not been evaluated.
en
Archived with thanks to BMC cell biology
Adult Stem Cells
Animals
Antigens, CD31
Antigens, Differentiation
Blood Cells
Cell Differentiation
Cell Line, Tumor
Cell Proliferation
Endothelial Cells
Lung
Lymphatic Vessels
Mice
Mice, Inbred BALB C
Mice, Inbred C57BL
Neovascularization, Physiologic
Stem Cell Niche
Stem Cell Transplantation
Mouse lung contains endothelial progenitors with high capacity to form blood and lymphatic vessels.
Article2018-06-13T00:30:11ZPostnatal endothelial progenitor cells (EPCs) have been successfully isolated from whole bone marrow, blood and the walls of conduit vessels. They can, therefore, be classified into circulating and resident progenitor cells. The differentiation capacity of resident lung endothelial progenitor cells from mouse has not been evaluated.oai:repository.helmholtz-hzi.de:10033/2954872019-08-30T11:34:48Zcom_10033_6823com_10033_6820col_10033_6891
Badar, Muhammad
Lünsdorf, Heinrich
Evertz, Florian
Rahim, Muhammad Imran
Glasmacher, Birgit
Hauser, Hansjörg
Mueller, Peter P
Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany.
2013-07-08T14:21:38Z
2013-07-08T14:21:38Z
2013-07
The formation of an organic coat and the release of corrosion microparticles from metallic magnesium implants. 2013, 9 (7):7580-9 Acta Biomater
1878-7568
23518475
10.1016/j.actbio.2013.03.012
http://hdl.handle.net/10033/295487
Acta biomaterialia
Magnesium alloys have been proposed as prospective degradable implant materials. To elucidate the complex interactions between the corroding implants and the tissue, magnesium implants were analyzed in a mouse model and the response was compared to that induced by Ti and by the resorbable polymer polyglactin, respectively. One month after implantation, distinct traces of corrosion were apparent but the magnesium implants were still intact, whereas resorbable polymeric wound suture implants were already fragmented. Analysis of magnesium implants 2weeks after implantation by energy-dispersive X-ray spectroscopy indicated that magnesium, oxygen, calcium and phosphate were present at the implant surface. One month after implantation, the element composition of the outermost layer of the implant was indicative of tissue without detectable levels of magnesium, indicating a protective barrier function of this organic layer. In agreement with this notion, gene expression patterns in the surrounding tissue were highly similar for all implant materials investigated. However, high-resolution imaging using energy-filtered transmission electron microscopy revealed magnesium-containing microparticles in the tissue in the proximity of the implant. The release of such corrosion particles may contribute to the accumulation of calcium phosphate in the nearby tissue and to bone conductive activities of magnesium implants.
en
Archived with thanks to Acta biomaterialia
The formation of an organic coat and the release of corrosion microparticles from metallic magnesium implants.
Article2018-06-13T00:05:52ZMagnesium alloys have been proposed as prospective degradable implant materials. To elucidate the complex interactions between the corroding implants and the tissue, magnesium implants were analyzed in a mouse model and the response was compared to that induced by Ti and by the resorbable polymer polyglactin, respectively. One month after implantation, distinct traces of corrosion were apparent but the magnesium implants were still intact, whereas resorbable polymeric wound suture implants were already fragmented. Analysis of magnesium implants 2weeks after implantation by energy-dispersive X-ray spectroscopy indicated that magnesium, oxygen, calcium and phosphate were present at the implant surface. One month after implantation, the element composition of the outermost layer of the implant was indicative of tissue without detectable levels of magnesium, indicating a protective barrier function of this organic layer. In agreement with this notion, gene expression patterns in the surrounding tissue were highly similar for all implant materials investigated. However, high-resolution imaging using energy-filtered transmission electron microscopy revealed magnesium-containing microparticles in the tissue in the proximity of the implant. The release of such corrosion particles may contribute to the accumulation of calcium phosphate in the nearby tissue and to bone conductive activities of magnesium implants.oai:repository.helmholtz-hzi.de:10033/3019982019-08-30T11:36:32Zcom_10033_6823com_10033_6820col_10033_6891
Schwerk, Johannes
Köster, Mario
Hauser, Hansjörg
Rohde, Manfred
Fulde, Marcus
Hornef, Mathias W
May, Tobias
Department of Gene Regulation and Differentiation, Helmholtz Centre for Infection Research, Braunschweig, Germany.
2013-09-20T12:05:26Z
2013-09-20T12:05:26Z
2013
Generation of mouse small intestinal epithelial cell lines that allow the analysis of specific innate immune functions. 2013, 8 (8):e72700 PLoS ONE
1932-6203
23940817
10.1371/journal.pone.0072700
http://hdl.handle.net/10033/301998
PloS one
Cell lines derived from the small intestine that reflect authentic properties of the originating intestinal epithelium are of high value for studies on mucosal immunology and host microbial homeostasis. A novel immortalization procedure was applied to generate continuously proliferating cell lines from murine E19 embryonic small intestinal tissue. The obtained cell lines form a tight and polarized epithelial cell layer, display characteristic tight junction, microvilli and surface protein expression and generate increasing transepithelial electrical resistance during in vitro culture. Significant up-regulation of Cxcl2 and Cxcl5 chemokine expression upon exposure to defined microbial innate immune stimuli and endogenous cytokines is observed. Cell lines were also generated from a transgenic interferon reporter (Mx2-Luciferase) mouse, allowing reporter technology-based quantification of the cellular response to type I and III interferon. Thus, the newly created cell lines mimic properties of the natural epithelium and can be used for diverse studies including testing of the absorption of drug candidates. The reproducibility of the method to create such cell lines from wild type and transgenic mice provides a new tool to study molecular and cellular processes of the epithelial barrier.
en
Archived with thanks to PloS one
Generation of mouse small intestinal epithelial cell lines that allow the analysis of specific innate immune functions.
Article2018-06-13T14:08:46ZCell lines derived from the small intestine that reflect authentic properties of the originating intestinal epithelium are of high value for studies on mucosal immunology and host microbial homeostasis. A novel immortalization procedure was applied to generate continuously proliferating cell lines from murine E19 embryonic small intestinal tissue. The obtained cell lines form a tight and polarized epithelial cell layer, display characteristic tight junction, microvilli and surface protein expression and generate increasing transepithelial electrical resistance during in vitro culture. Significant up-regulation of Cxcl2 and Cxcl5 chemokine expression upon exposure to defined microbial innate immune stimuli and endogenous cytokines is observed. Cell lines were also generated from a transgenic interferon reporter (Mx2-Luciferase) mouse, allowing reporter technology-based quantification of the cellular response to type I and III interferon. Thus, the newly created cell lines mimic properties of the natural epithelium and can be used for diverse studies including testing of the absorption of drug candidates. The reproducibility of the method to create such cell lines from wild type and transgenic mice provides a new tool to study molecular and cellular processes of the epithelial barrier.oai:repository.helmholtz-hzi.de:10033/3048382019-08-30T11:37:24Zcom_10033_6823com_10033_6820col_10033_6891
Gross, Gerhard
Hoffmann, Andrea
Department of Molecular Biotechnology, Signalling and Gene Regulation, Helmholtz Centre for Infection Research, Braunschweig, Germany.
2013-10-31T10:36:45Z
2013-10-31T10:36:45Z
2013
Therapeutic strategies for tendon healing based on novel biomaterials, factors and cells. 2013, 80 (4):203-10 Pathobiology
1423-0291
23652284
10.1159/000347059
http://hdl.handle.net/10033/304838
Pathobiology : journal of immunopathology, molecular and cellular biology
The repair of tendon injuries still presents a major clinical challenge to orthopedic medicine. Tendons, like some other tissues, are poorly vascularized and heal slowly. In addition, healing often leads to the formation of fibrous tissue and scar tissue which lack flexibility and biomechanical properties. So the treatment of tendon injuries is challenging. We give an overview of the structure and composition of tendons, pathological states of tendon and natural healing, as well as therapeutic options. We focus in particular on biomaterials that have been specifically developed or suggested for the successful repair of tendon injuries. In addition, we also review factor- and cell-dependent strategies to heal tendon and ligament disorders. Although brief, we hope that this review will be helpful, particularly for those readers who are new to the field of tendon tissue engineering.
en
Archived with thanks to Pathobiology : journal of immunopathology, molecular and cellular biology
Therapeutic strategies for tendon healing based on novel biomaterials, factors and cells.
Article2018-06-12T23:31:26ZThe repair of tendon injuries still presents a major clinical challenge to orthopedic medicine. Tendons, like some other tissues, are poorly vascularized and heal slowly. In addition, healing often leads to the formation of fibrous tissue and scar tissue which lack flexibility and biomechanical properties. So the treatment of tendon injuries is challenging. We give an overview of the structure and composition of tendons, pathological states of tendon and natural healing, as well as therapeutic options. We focus in particular on biomaterials that have been specifically developed or suggested for the successful repair of tendon injuries. In addition, we also review factor- and cell-dependent strategies to heal tendon and ligament disorders. Although brief, we hope that this review will be helpful, particularly for those readers who are new to the field of tendon tissue engineering.oai:repository.helmholtz-hzi.de:10033/3056592019-08-30T11:36:59Zcom_10033_6823com_10033_6820col_10033_6891
Roesner, Lennart M
Mielke, Christian
Fähnrich, Silke
Merkhoffer, Yvonne
Dittmar, Kurt E J
Drexler, Hans G
Dirks, Wilhelm G
Department of Human and Animal Cell Lines, Leibniz-Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany. roesner.lennart@mh-hannover.de
2013-11-21T11:45:20Z
2013-11-21T11:45:20Z
2013-10
Stable expression of MutLγ in human cells reveals no specific response to mismatched DNA, but distinct recruitment to damage sites. 2013, 114 (10):2405-14 J. Cell. Biochem.
1097-4644
23696135
10.1002/jcb.24591
http://hdl.handle.net/10033/305659
Journal of cellular biochemistry
The human DNA mismatch repair (MMR) gene family comprises four MutL paralogues capable of forming heterodimeric MutLα (MLH1-PMS2), MutLβ (MLH1-PMS1), and MutLγ (MLH1-MLH3) protein complexes. Human MutL subunits PMS2 and MLH3 contain an evolutionarily conserved amino acid motif DQHA(X)2E(X)4E identified as an endonucleolytic domain capable of incising a defective DNA strand. PMS2 of MutLα is generally accepted to be the sole executor of endonucleolytic activity, but since MLH3 was shown to be able to perform DNA repair at low levels in vitro, our aim was to investigate whether or not MLH3 is activated as a backup under MutLα-deficient conditions. Here, we report stable expression of GFP-tagged MLH3 in the isogenic cell lines 293 and 293T which are functional or defective for MLH1 expression, respectively. As expected, MLH3 formed dimeric complexes with endogenous and recombinant MLH1. MutLγ dimers were recruited to sites of DNA damage induced by UVA micro-irradiation as shown for MutLα. Surprisingly, splicing variant MLH3Δ7 lacking the endonucleolytic motif displayed congruent foci formation, implying that recruitment is not necessarily representing active DNA repair. As an alternative test for repair enzyme activity, we combined alkylation-directed DNA damage with comet formation assays. While recombinant MutLα led to full recovery of DNA damage response in MMR deficient cells, expression of MutLγ or single MLH3 failed to do so. These experiments show recruitment and persistence of MutLγ-heterodimers at UVA-induced DNA lesions. However, we demonstrate that in a MutLα-deficient background no DNA repair-specific function carried out by MutLγ can be detected in living cells.
en
Archived with thanks to Journal of cellular biochemistry
Stable expression of MutLγ in human cells reveals no specific response to mismatched DNA, but distinct recruitment to damage sites.
Article2014-10-15T00:00:00ZThe human DNA mismatch repair (MMR) gene family comprises four MutL paralogues capable of forming heterodimeric MutLα (MLH1-PMS2), MutLβ (MLH1-PMS1), and MutLγ (MLH1-MLH3) protein complexes. Human MutL subunits PMS2 and MLH3 contain an evolutionarily conserved amino acid motif DQHA(X)2E(X)4E identified as an endonucleolytic domain capable of incising a defective DNA strand. PMS2 of MutLα is generally accepted to be the sole executor of endonucleolytic activity, but since MLH3 was shown to be able to perform DNA repair at low levels in vitro, our aim was to investigate whether or not MLH3 is activated as a backup under MutLα-deficient conditions. Here, we report stable expression of GFP-tagged MLH3 in the isogenic cell lines 293 and 293T which are functional or defective for MLH1 expression, respectively. As expected, MLH3 formed dimeric complexes with endogenous and recombinant MLH1. MutLγ dimers were recruited to sites of DNA damage induced by UVA micro-irradiation as shown for MutLα. Surprisingly, splicing variant MLH3Δ7 lacking the endonucleolytic motif displayed congruent foci formation, implying that recruitment is not necessarily representing active DNA repair. As an alternative test for repair enzyme activity, we combined alkylation-directed DNA damage with comet formation assays. While recombinant MutLα led to full recovery of DNA damage response in MMR deficient cells, expression of MutLγ or single MLH3 failed to do so. These experiments show recruitment and persistence of MutLγ-heterodimers at UVA-induced DNA lesions. However, we demonstrate that in a MutLα-deficient background no DNA repair-specific function carried out by MutLγ can be detected in living cells.oai:repository.helmholtz-hzi.de:10033/3063812019-08-30T11:25:11Zcom_10033_6823com_10033_6820col_10033_6891
Reifenrath, Janin
Badar, Muhammad
Dziuba, Dina
Müller, Peter P
Heidenblut, Torsten
Bondarenko, Alexander
Meyer-Lindenberg, Andrea
Small Animal Clinic, University of Veterinary Medicine Hannover, Hannover - Germany.
2013-12-05T12:07:43Z
2013-12-05T12:07:43Z
2013
Assessment of cellular reactions to magnesium as implant
material in comparison to titanium and to glyconate using
the mouse tail model. 2013, 11 (2):e89-94 J Appl Biomater Funct Mater
2280-8000
23728545
10.5301/JABFM.5000150
http://hdl.handle.net/10033/306381
Journal of applied biomaterials & functional materials
Nowadays, research in magnesium alloys as a biodegradable implant material has increased. The aim of this study was to examine osteoinductive properties and tissue responses to pure magnesium in comparison to conventional permanent (titanium) and to degradable (glyconate) implant materials.
en
Archived with thanks to Journal of applied biomaterials & functional materials
Assessment of cellular reactions to magnesium as implant
material in comparison to titanium and to glyconate using
the mouse tail model.
Article2014-05-15T00:00:00ZNowadays, research in magnesium alloys as a biodegradable implant material has increased. The aim of this study was to examine osteoinductive properties and tissue responses to pure magnesium in comparison to conventional permanent (titanium) and to degradable (glyconate) implant materials.oai:repository.helmholtz-hzi.de:10033/3117172019-08-30T11:25:43Zcom_10033_6823com_10033_6820col_10033_6891
Harari, Daniel
Abramovich, Renne
Zozulya, Alla
Smith, Paul
Pouly, Sandrine
Köster, Mario
Hauser, Hansjörg
Schreiber, Gideon
2014-01-23T08:38:49Z
2014-01-23T08:38:49Z
2014
Bridging the species divide: transgenic mice humanized for type-I interferon response. 2014, 9 (1):e84259 PLoS ONE
1932-6203
24416207
10.1371/journal.pone.0084259
http://hdl.handle.net/10033/311717
PloS one
We have generated transgenic mice that harbor humanized type I interferon receptors (IFNARs) enabling the study of type I human interferons (Hu-IFN-Is) in mice. These "HyBNAR" (Hybrid IFNAR) mice encode transgenic variants of IFNAR1 and IFNAR2 with the human extracellular domains being fused to transmembrane and cytoplasmic segments of mouse sequence. B16F1 mouse melanoma cells harboring the HyBNAR construct specifically bound Hu-IFN-Is and were rendered sensitive to Hu-IFN-I stimulated anti-proliferation, STAT1 activation and activation of a prototypical IFN-I response gene (MX2). HyBNAR mice were crossed with a transgenic strain expressing the luciferase reporter gene under the control of the IFN-responsive MX2 promoter (MX2-Luciferase). Both the HyBNAR and HyBNAR/MX2-Luciferase mice were responsive to all Hu-IFN-Is tested, inclusive of IFNα2A, IFNβ, and a human superagonist termed YNSα8. The mice displayed dose-dependent pharmacodynamic responses to Hu-IFN-I injection, as assessed by measuring the expression of IFN-responsive genes. Our studies also demonstrated a weak activation of endogenous mouse interferon response, especially after high dose administration of Hu-IFNs. In sharp contrast to data published for humans, our pharmacodynamic readouts demonstrate a very short-lived IFN-I response in mice, which is not enhanced by sub-cutaneous (SC) injections in comparison to other administration routes. With algometric differences between humans and mice taken into account, the HyBNAR mice provides a convenient non-primate pre-clinical model to advance the study of human IFN-Is.
en
Archived with thanks to PloS one
Bridging the species divide: transgenic mice humanized for type-I interferon response.
Article2018-06-12T23:47:42ZWe have generated transgenic mice that harbor humanized type I interferon receptors (IFNARs) enabling the study of type I human interferons (Hu-IFN-Is) in mice. These "HyBNAR" (Hybrid IFNAR) mice encode transgenic variants of IFNAR1 and IFNAR2 with the human extracellular domains being fused to transmembrane and cytoplasmic segments of mouse sequence. B16F1 mouse melanoma cells harboring the HyBNAR construct specifically bound Hu-IFN-Is and were rendered sensitive to Hu-IFN-I stimulated anti-proliferation, STAT1 activation and activation of a prototypical IFN-I response gene (MX2). HyBNAR mice were crossed with a transgenic strain expressing the luciferase reporter gene under the control of the IFN-responsive MX2 promoter (MX2-Luciferase). Both the HyBNAR and HyBNAR/MX2-Luciferase mice were responsive to all Hu-IFN-Is tested, inclusive of IFNα2A, IFNβ, and a human superagonist termed YNSα8. The mice displayed dose-dependent pharmacodynamic responses to Hu-IFN-I injection, as assessed by measuring the expression of IFN-responsive genes. Our studies also demonstrated a weak activation of endogenous mouse interferon response, especially after high dose administration of Hu-IFNs. In sharp contrast to data published for humans, our pharmacodynamic readouts demonstrate a very short-lived IFN-I response in mice, which is not enhanced by sub-cutaneous (SC) injections in comparison to other administration routes. With algometric differences between humans and mice taken into account, the HyBNAR mice provides a convenient non-primate pre-clinical model to advance the study of human IFN-Is.oai:repository.helmholtz-hzi.de:10033/3167332019-08-30T11:26:42Zcom_10033_6823com_10033_6820col_10033_6891
Mullen, Lisa
Rigby, Anne
Sclanders, Michelle
Adams, Gill
Mittal, Gayatri
Colston, Julia
Fatah, Rewas
Subang, Cristina
Foster, Julie
Francis-West, Philippa
Köster, Mario
Hauser, Hansjörg
Layward, Lorna
Vessillier, Sandrine
Annenkov, Alex
Al-Izki, Sarah
Pryce, Gareth
Bolton, Chris
Baker, David
Gould, David J
Chernajovsky, Yuti
Experimental Rheumatology, Charité University Medicine, Berlin, Germany
2014-05-13T08:18:00Z
2014-05-13T08:18:00Z
2014-01
Latency can be conferred to a variety of cytokines by fusion with latency-associated peptide from TGF-β. 2014, 11 (1):5-16 Expert Opin Drug Deliv
1744-7593
24073618
10.1517/17425247.2013.839655
http://hdl.handle.net/10033/316733
Expert opinion on drug delivery
Targeting cytokines to sites of disease has clear advantages because it increases their therapeutic index. We designed fusion proteins of the latent-associated peptide (LAP) derived from TGF-β with various cytokines via a matrix metalloproteinase (MMP) cleavage site. This design confers latency, increased half-life and targeting to sites of inflammation. The aim of this study is to determine whether this approach can be applied to cytokines of different molecular structures and sizes.
en
Archived with thanks to Expert opinion on drug delivery
Latency can be conferred to a variety of cytokines by fusion with latency-associated peptide from TGF-β.
Article2015-01-15T00:00:00ZTargeting cytokines to sites of disease has clear advantages because it increases their therapeutic index. We designed fusion proteins of the latent-associated peptide (LAP) derived from TGF-β with various cytokines via a matrix metalloproteinase (MMP) cleavage site. This design confers latency, increased half-life and targeting to sites of inflammation. The aim of this study is to determine whether this approach can be applied to cytokines of different molecular structures and sizes.oai:repository.helmholtz-hzi.de:10033/3241432019-08-30T11:36:05Zcom_10033_6823com_10033_6820col_10033_6891
Kayser, Hartmut
Wray, Victor
Nimtz, Manfred
2014-08-04T13:04:45Z
2014-08-04T13:04:45Z
2014-05
Structure of a novel farnesylated bilin from an insect--formation by α-cleavage of heme A of mitochondrial cytochrome c oxidases? 2014, 281 (10):2366-76 FEBS J.
1742-4658
24655573
10.1111/febs.12789
http://hdl.handle.net/10033/324143
The FEBS journal
Biliproteins are present in almost all forms of life, and many of them play vital roles in photobiology. The bilin ligand of a recently characterized 500-kDa biliprotein from an insect has been isolated and its structure elucidated with chemical and spectroscopic techniques (UV-visible, IR, MS, NMR, and CD). This blue pigment, named CV-bilin, represents a unique high molecular mass derivative of biliverdin IXα, with an unusual 10E-configuration and a molecular mass of 852 Da, corresponding to C48H60N4O10. The high mass of this open-chain tetrapyrrole results from the presence of an epoxi-dihydroxyethylfarnesyl substituent at C-18 and a hydroxymethyl substituent at C-13. This substitution pattern exactly reflects that of heme A of mitochondrial cytochrome c oxidases with a hydroxyethylfarnesyl chain and a formyl group at corresponding positions of the cyclic tetrapyrrole. As no other natural product is known to show these structural features (heme O, the precursor of heme A, has a methyl group at C-13), this bilin is presumed to be derived from heme A by cleavage of the α-methine bridge and oxidative modifications at C-13 and the hydroxyethylfarnesyl chain. Possibly, a bilin structurally related to this insect bilin is also produced in other organisms as a result of mitochondrial turnover or degradation. As CV-bilin in complex with a specific protein is accumulated at the end of larval life, stored in the pupa, and finally transferred to the oocytes, a possible role of the free or protein-bound pigment in egg or embryonic development is discussed.
en
Archived with thanks to The FEBS journal
Structure of a novel farnesylated bilin from an insect--formation by α-cleavage of heme A of mitochondrial cytochrome c oxidases?
Article2015-05-15T00:00:00ZBiliproteins are present in almost all forms of life, and many of them play vital roles in photobiology. The bilin ligand of a recently characterized 500-kDa biliprotein from an insect has been isolated and its structure elucidated with chemical and spectroscopic techniques (UV-visible, IR, MS, NMR, and CD). This blue pigment, named CV-bilin, represents a unique high molecular mass derivative of biliverdin IXα, with an unusual 10E-configuration and a molecular mass of 852 Da, corresponding to C48H60N4O10. The high mass of this open-chain tetrapyrrole results from the presence of an epoxi-dihydroxyethylfarnesyl substituent at C-18 and a hydroxymethyl substituent at C-13. This substitution pattern exactly reflects that of heme A of mitochondrial cytochrome c oxidases with a hydroxyethylfarnesyl chain and a formyl group at corresponding positions of the cyclic tetrapyrrole. As no other natural product is known to show these structural features (heme O, the precursor of heme A, has a methyl group at C-13), this bilin is presumed to be derived from heme A by cleavage of the α-methine bridge and oxidative modifications at C-13 and the hydroxyethylfarnesyl chain. Possibly, a bilin structurally related to this insect bilin is also produced in other organisms as a result of mitochondrial turnover or degradation. As CV-bilin in complex with a specific protein is accumulated at the end of larval life, stored in the pupa, and finally transferred to the oocytes, a possible role of the free or protein-bound pigment in egg or embryonic development is discussed.oai:repository.helmholtz-hzi.de:10033/3243302019-08-30T11:30:58Zcom_10033_6822com_10033_6821com_10033_6820col_10033_6895
He, Feng
Buer, Jan
Zeng, An-Ping
Balling, Rudi
Helmholtz Centre of infection research, Inhoffenstr. 7, D38124 Braunschweig, Germany
2014-08-06T14:02:12Z
2014-08-06T14:02:12Z
2007
Dynamic cumulative activity of transcription factors as a mechanism of quantitative gene regulation. 2007, 8 (9):R181 Genome Biol.
1465-6914
17784952
10.1186/gb-2007-8-9-r181
http://hdl.handle.net/10033/324330
Genome biology
The regulation of genes in multicellular organisms is generally achieved through the combinatorial activity of different transcription factors. However, the quantitative mechanisms of how a combination of transcription factors controls the expression of their target genes remain unknown.
en
Archived with thanks to Genome biology
Algorithms
Amino Acid Motifs
Binding Sites
Biotechnology
Cell Cycle
False Positive Reactions
Fungal Proteins
Gene Expression Regulation, Fungal
Genes, Fungal
Models, Genetic
Models, Theoretical
Saccharomyces cerevisiae
Transcription Factors
Transcription, Genetic
Dynamic cumulative activity of transcription factors as a mechanism of quantitative gene regulation.
Article2018-06-12T22:09:32ZThe regulation of genes in multicellular organisms is generally achieved through the combinatorial activity of different transcription factors. However, the quantitative mechanisms of how a combination of transcription factors controls the expression of their target genes remain unknown.oai:repository.helmholtz-hzi.de:10033/3248022019-08-30T11:30:32Zcom_10033_6822com_10033_6821com_10033_6820col_10033_6895
Stelzer, Michael
Sun, Jibin
Kamphans, Tom
Fekete, Sándor P
Zeng, An-Ping
2014-08-14T12:52:18Z
2014-08-14T12:52:18Z
2011-11
An extended bioreaction database that significantly improves reconstruction and analysis of genome-scale metabolic networks. 2011, 3 (11):1071-86 Integr Biol (Camb)
1757-9708
21952610
10.1039/c1ib00008j
http://hdl.handle.net/10033/324802
Integrative biology : quantitative biosciences from nano to macro
The bioreaction database established by Ma and Zeng (Bioinformatics, 2003, 19, 270-277) for in silico reconstruction of genome-scale metabolic networks has been widely used. Based on more recent information in the reference databases KEGG LIGAND and Brenda, we upgrade the bioreaction database in this work by almost doubling the number of reactions from 3565 to 6851. Over 70% of the reactions have been manually updated/revised in terms of reversibility, reactant pairs, currency metabolites and error correction. For the first time, 41 spontaneous sugar mutarotation reactions are introduced into the biochemical database. The upgrade significantly improves the reconstruction of genome scale metabolic networks. Many gaps or missing biochemical links can be recovered, as exemplified with three model organisms Homo sapiens, Aspergillus niger, and Escherichia coli. The topological parameters of the constructed networks were also largely affected, however, the overall network structure remains scale-free. Furthermore, we consider the problem of computing biologically feasible shortest paths in reconstructed metabolic networks. We show that these paths are hard to compute and present solutions to find such paths in networks of small and medium size.
en
Archived with thanks to Integrative biology : quantitative biosciences from nano to macro
Algorithms
Aspergillus niger
Computational Biology
Databases, Factual
Databases, Genetic
Escherichia coli
Genome
Glucose
Humans
Metabolic Networks and Pathways
Models, Biological
Software
An extended bioreaction database that significantly improves reconstruction and analysis of genome-scale metabolic networks.
Article2018-06-13T04:22:27ZThe bioreaction database established by Ma and Zeng (Bioinformatics, 2003, 19, 270-277) for in silico reconstruction of genome-scale metabolic networks has been widely used. Based on more recent information in the reference databases KEGG LIGAND and Brenda, we upgrade the bioreaction database in this work by almost doubling the number of reactions from 3565 to 6851. Over 70% of the reactions have been manually updated/revised in terms of reversibility, reactant pairs, currency metabolites and error correction. For the first time, 41 spontaneous sugar mutarotation reactions are introduced into the biochemical database. The upgrade significantly improves the reconstruction of genome scale metabolic networks. Many gaps or missing biochemical links can be recovered, as exemplified with three model organisms Homo sapiens, Aspergillus niger, and Escherichia coli. The topological parameters of the constructed networks were also largely affected, however, the overall network structure remains scale-free. Furthermore, we consider the problem of computing biologically feasible shortest paths in reconstructed metabolic networks. We show that these paths are hard to compute and present solutions to find such paths in networks of small and medium size.oai:repository.helmholtz-hzi.de:10033/3390342019-08-30T11:36:05Zcom_10033_6823com_10033_6820col_10033_6891
Charbord, Pierre
Livne, Erella
Gross, Gerhard
Häupl, Thomas
Neves, Nuno M
Marie, Pierre
Bianco, Paolo
Jorgensen, Christian
2015-01-29T12:08:13Z
2015-01-29T12:08:13Z
2011-03
Human bone marrow mesenchymal stem cells: a systematic reappraisal via the genostem experience. 2011, 7 (1):32-42 Stem Cell Rev
1558-6804
20198518
10.1007/s12015-010-9125-6
http://hdl.handle.net/10033/339034
Stem cell reviews
Genostem (acronym for "Adult mesenchymal stem cells engineering for connective tissue disorders. From the bench to the bed side") has been an European consortium of 30 teams working together on human bone marrow Mesenchymal Stem Cell (MSC) biological properties and repair capacity. Part of Genostem activity has been dedicated to the study of basic issues on undifferentiated MSCs properties and on signalling pathways leading to the differentiation into 3 of the connective tissue lineages, osteoblastic, chondrocytic and tenocytic. We have evidenced that native bone marrow MSCs and stromal cells, forming the niche of hematopoietic stem cells, were the same cellular entity located abluminally from marrow sinus endothelial cells. We have also shown that culture-amplified, clonogenic and highly-proliferative MSCs were bona fide stem cells, sharing with other stem cell types the major attributes of self-renewal and of multipotential priming to the lineages to which they can differentiate (osteoblasts, chondrocytes, adipocytes and vascular smooth muscle cells/pericytes). Extensive transcription profiling and in vitro and in vivo assays were applied to identify genes involved in differentiation. Thus we have described novel factors implicated in osteogenesis (FHL2, ITGA5, Fgf18), chondrogenesis (FOXO1A) and tenogenesis (Smad8). Another part of Genostem activity has been devoted to studies of the repair capacity of MSCs in animal models, a prerequisite for future clinical trials. We have developed novel scaffolds (chitosan, pharmacologically active microcarriers) useful for the repair of both bone and cartilage. Finally and most importantly, we have shown that locally implanted MSCs effectively repair bone, cartilage and tendon.
en
Animals
Bone Marrow Cells
Cell Culture Techniques
Cell Differentiation
Cell Proliferation
Humans
Mesenchymal Stromal Cells
Tissue Engineering
Human bone marrow mesenchymal stem cells: a systematic reappraisal via the genostem experience.
Article2018-06-12T23:42:48ZGenostem (acronym for "Adult mesenchymal stem cells engineering for connective tissue disorders. From the bench to the bed side") has been an European consortium of 30 teams working together on human bone marrow Mesenchymal Stem Cell (MSC) biological properties and repair capacity. Part of Genostem activity has been dedicated to the study of basic issues on undifferentiated MSCs properties and on signalling pathways leading to the differentiation into 3 of the connective tissue lineages, osteoblastic, chondrocytic and tenocytic. We have evidenced that native bone marrow MSCs and stromal cells, forming the niche of hematopoietic stem cells, were the same cellular entity located abluminally from marrow sinus endothelial cells. We have also shown that culture-amplified, clonogenic and highly-proliferative MSCs were bona fide stem cells, sharing with other stem cell types the major attributes of self-renewal and of multipotential priming to the lineages to which they can differentiate (osteoblasts, chondrocytes, adipocytes and vascular smooth muscle cells/pericytes). Extensive transcription profiling and in vitro and in vivo assays were applied to identify genes involved in differentiation. Thus we have described novel factors implicated in osteogenesis (FHL2, ITGA5, Fgf18), chondrogenesis (FOXO1A) and tenogenesis (Smad8). Another part of Genostem activity has been devoted to studies of the repair capacity of MSCs in animal models, a prerequisite for future clinical trials. We have developed novel scaffolds (chitosan, pharmacologically active microcarriers) useful for the repair of both bone and cartilage. Finally and most importantly, we have shown that locally implanted MSCs effectively repair bone, cartilage and tendon.oai:repository.helmholtz-hzi.de:10033/3473152019-08-30T11:25:11Zcom_10033_6823com_10033_6820col_10033_6891
Jenke, Bok Hee C
Fetzer, Christian P
Stehle, Isa M
Jönsson, Franziska
Fackelmayer, Frank O
Conradt, Harald
Bode, Jürgen
Lipps, Hans J
Institute of Cell Biology, Stockumer Strasse 10, University of Witten/Herdecke, D-58448 Witten.
2015-03-30T13:25:41Z
2015-03-30T13:25:41Z
2002-04
An episomally replicating vector binds to the nuclear matrix protein SAF-A in vivo. 2002, 3 (4):349-54 EMBO Rep.
1469-221X
11897664
10.1093/embo-reports/kvf070
http://hdl.handle.net/10033/347315
EMBO reports
pEPI-1, a vector in which a chromosomal scaffold/matrix-attached region (S/MAR) is linked to the simian virus 40 origin of replication, is propagated episomally in CHO cells in the absence of the virally encoded large T-antigen and is stably maintained in the absence of selection pressure. It has been suggested that mitotic stability is provided by a specific interaction of this vector with components of the nuclear matrix. We studied the interactions of pEPI-1 by crosslinking with cis-diamminedichloroplatinum II, after which it is found to copurify with the nuclear matrix. In a south-western analysis, the vector shows exclusive binding to hnRNP-U/SAF-A, a multifunctional scaffold/matrix specific factor. Immunoprecipitation of the crosslinked DNA-protein complex demonstrates that pEPI-1 is bound to this protein in vivo. These data provide the first experimental evidence for the binding of an artificial episome to a nuclear matrix protein in vivo and the basis for understanding the mitotic stability of this novel vector class.
en
Animals
Blotting, Southwestern
Blotting, Western
CHO Cells
Cisplatin
Cricetinae
Genetic Vectors
Heterogeneous-Nuclear Ribonucleoprotein U
Heterogeneous-Nuclear Ribonucleoproteins
Ribonucleoproteins
An episomally replicating vector binds to the nuclear matrix protein SAF-A in vivo.
Article2018-06-12T17:16:11ZpEPI-1, a vector in which a chromosomal scaffold/matrix-attached region (S/MAR) is linked to the simian virus 40 origin of replication, is propagated episomally in CHO cells in the absence of the virally encoded large T-antigen and is stably maintained in the absence of selection pressure. It has been suggested that mitotic stability is provided by a specific interaction of this vector with components of the nuclear matrix. We studied the interactions of pEPI-1 by crosslinking with cis-diamminedichloroplatinum II, after which it is found to copurify with the nuclear matrix. In a south-western analysis, the vector shows exclusive binding to hnRNP-U/SAF-A, a multifunctional scaffold/matrix specific factor. Immunoprecipitation of the crosslinked DNA-protein complex demonstrates that pEPI-1 is bound to this protein in vivo. These data provide the first experimental evidence for the binding of an artificial episome to a nuclear matrix protein in vivo and the basis for understanding the mitotic stability of this novel vector class.oai:repository.helmholtz-hzi.de:10033/6210112019-08-30T11:31:49Zcom_10033_6823com_10033_6820col_10033_6891
Martins, José P
Santos, Jorge M
Almeida, Joana M d
Filipe, Mariana A
de Almeida, Mariana V T
Almeida, Sílvia C C
Água-Doce, Ana
Varela, Alexandre
Gilljam, Mari
Stellan, Birgitta
Pohl, Susanne
Dittmar, Kurt
Lindenmaier, Werner
Alici, Evren
Graça, Luís
Cruz, Pedro E
Cruz, Helder J
Bárcia, Rita N
2017-07-13T11:59:23Z
2017-07-13T11:59:23Z
2014-01-17
2015-09-04T08:21:57Z
Stem Cell Research & Therapy. 2014 Jan 17;5(1):9
http://dx.doi.org/10.1186/scrt398
http://hdl.handle.net/10033/621011
Abstract Introduction Standardization of mesenchymal stromal cells (MSCs) manufacturing is urgently needed to enable translational activities and ultimately facilitate comparison of clinical trial results. In this work we describe the adaptation of a proprietary method for isolation of a specific umbilical cord tissue-derived population of MSCs, herein designated by its registered trademark as UCX®, towards the production of an advanced therapy medicinal product (ATMP). Methods The adaptation focused on different stages of production, from cell isolation steps to cell culturing and cryopreservation. The origin and quality of materials and reagents were considered and steps for avoiding microbiological and endotoxin contamination of the final cell product were implemented. Cell isolation efficiency, MSCs surface markers and genetic profiles, originating from the use of different medium supplements, were compared. The ATMP-compliant UCX® product was also cryopreserved avoiding the use of dimethyl sulfoxide, an added benefit for the use of these cells as an ATMP. Cells were analyzed for expansion capacity and longevity. The final cell product was further characterized by flow cytometry, differentiation potential, and tested for contaminants at various passages. Finally, genetic stability and immune properties were also analyzed. Results The isolation efficiency of UCX® was not affected by the introduction of clinical grade enzymes. Furthermore, isolation efficiencies and phenotype analyses revealed advantages in the use of human serum in cell culture as opposed to human platelet lysate. Initial decontamination of the tissue followed by the use of mycoplasma- and endotoxin-free materials and reagents in cell isolation and subsequent culture, enabled the removal of antibiotics during cell expansion. UCX®-ATMP maintained a significant expansion potential of 2.5 population doublings per week up to passage 15 (P15). They were also efficiently cryopreserved in a DMSO-free cryoprotectant medium with approximately 100% recovery and 98% viability post-thaw. Additionally, UCX®-ATMP were genetically stable upon expansion (up to P15) and maintained their immunomodulatory properties. Conclusions We have successfully adapted a method to consistently isolate, expand and cryopreserve a well-characterized population of human umbilical cord tissue-derived MSCs (UCX®), in order to obtain a cell product that is compliant with cell therapy. Here, we present quality and safety data that support the use of the UCX® as an ATMP, according to existing international guidelines.
Towards an advanced therapy medicinal product based on mesenchymal stromal cells isolated from the umbilical cord tissue: quality and safety data
Journal Article
en
Martins et al.; licensee BioMed Central Ltd.2018-06-12T21:49:05ZAbstract
Introduction
Standardization of mesenchymal stromal cells (MSCs) manufacturing is urgently needed to enable translational activities and ultimately facilitate comparison of clinical trial results. In this work we describe the adaptation of a proprietary method for isolation of a specific umbilical cord tissue-derived population of MSCs, herein designated by its registered trademark as UCX®, towards the production of an advanced therapy medicinal product (ATMP).
Methods
The adaptation focused on different stages of production, from cell isolation steps to cell culturing and cryopreservation. The origin and quality of materials and reagents were considered and steps for avoiding microbiological and endotoxin contamination of the final cell product were implemented. Cell isolation efficiency, MSCs surface markers and genetic profiles, originating from the use of different medium supplements, were compared. The ATMP-compliant UCX® product was also cryopreserved avoiding the use of dimethyl sulfoxide, an added benefit for the use of these cells as an ATMP. Cells were analyzed for expansion capacity and longevity. The final cell product was further characterized by flow cytometry, differentiation potential, and tested for contaminants at various passages. Finally, genetic stability and immune properties were also analyzed.
Results
The isolation efficiency of UCX® was not affected by the introduction of clinical grade enzymes. Furthermore, isolation efficiencies and phenotype analyses revealed advantages in the use of human serum in cell culture as opposed to human platelet lysate. Initial decontamination of the tissue followed by the use of mycoplasma- and endotoxin-free materials and reagents in cell isolation and subsequent culture, enabled the removal of antibiotics during cell expansion. UCX®-ATMP maintained a significant expansion potential of 2.5 population doublings per week up to passage 15 (P15). They were also efficiently cryopreserved in a DMSO-free cryoprotectant medium with approximately 100% recovery and 98% viability post-thaw. Additionally, UCX®-ATMP were genetically stable upon expansion (up to P15) and maintained their immunomodulatory properties.
Conclusions
We have successfully adapted a method to consistently isolate, expand and cryopreserve a well-characterized population of human umbilical cord tissue-derived MSCs (UCX®), in order to obtain a cell product that is compliant with cell therapy. Here, we present quality and safety data that support the use of the UCX® as an ATMP, according to existing international guidelines.oai:repository.helmholtz-hzi.de:10033/6210432019-08-30T11:33:30Zcom_10033_6823com_10033_6820col_10033_6891col_10033_6891col_10033_6891col_10033_118931
Nehlsen, Kristina
Schucht, Roland
da Gama-Norton, Leonor
Krömer, Wolfgang
Baer, Alexandra
Cayli, Aziz
Hauser, Hansjörg
Wirth, Dagmar
2017-08-04T08:33:49Z
2017-08-04T08:33:49Z
2009-12-14
2015-09-04T08:23:39Z
BMC Biotechnology. 2009 Dec 14;9(1):100
http://dx.doi.org/10.1186/1472-6750-9-100
http://hdl.handle.net/10033/621043
Abstract Background Recombinant protein expression in mammalian cells is mostly achieved by stable integration of transgenes into the chromosomal DNA of established cell lines. The chromosomal surroundings have strong influences on the expression of transgenes. The exploitation of defined loci by targeting expression constructs with different regulatory elements is an approach to design high level expression systems. Further, this allows to evaluate the impact of chromosomal surroundings on distinct vector constructs. Results We explored antibody expression upon targeting diverse expression constructs into previously tagged loci in CHO-K1 and HEK293 cells that exhibit high reporter gene expression. These loci were selected by random transfer of reporter cassettes and subsequent screening. Both, retroviral infection and plasmid transfection with eGFP or antibody expression cassettes were employed for tagging. The tagged cell clones were screened for expression and single copy integration. Cell clones producing > 20 pg/cell in 24 hours could be identified. Selected integration sites that had been flanked with heterologous recombinase target sites (FRTs) were targeted by Flp recombinase mediated cassette exchange (RMCE). The results give proof of principle for consistent protein expression upon RMCE. Upon targeting antibody expression cassettes 90-100% of all resulting cell clones showed correct integration. Antibody production was found to be highly consistent within the individual cell clones as expected from their isogenic nature. However, the nature and orientation of expression control elements revealed to be critical. The impact of different promoters was examined with the tag-and-targeting approach. For each of the chosen promoters high expression sites were identified. However, each site supported the chosen promoters to a different extent, indicating that the strength of a particular promoter is dominantly defined by its chromosomal context. Conclusion RMCE provides a powerful method to specifically design vectors for optimized gene expression with high accuracy. Upon considering the specific requirements of chromosomal sites this method provides a unique tool to exploit such sites for predictable expression of biotechnologically relevant proteins such as antibodies.
Recombinant protein expression by targeting pre-selected chromosomal loci
Journal Article
en
Nehlsen et al.2018-06-13T00:54:46ZAbstract
Background
Recombinant protein expression in mammalian cells is mostly achieved by stable integration of transgenes into the chromosomal DNA of established cell lines. The chromosomal surroundings have strong influences on the expression of transgenes. The exploitation of defined loci by targeting expression constructs with different regulatory elements is an approach to design high level expression systems. Further, this allows to evaluate the impact of chromosomal surroundings on distinct vector constructs.
Results
We explored antibody expression upon targeting diverse expression constructs into previously tagged loci in CHO-K1 and HEK293 cells that exhibit high reporter gene expression. These loci were selected by random transfer of reporter cassettes and subsequent screening. Both, retroviral infection and plasmid transfection with eGFP or antibody expression cassettes were employed for tagging. The tagged cell clones were screened for expression and single copy integration. Cell clones producing > 20 pg/cell in 24 hours could be identified. Selected integration sites that had been flanked with heterologous recombinase target sites (FRTs) were targeted by Flp recombinase mediated cassette exchange (RMCE). The results give proof of principle for consistent protein expression upon RMCE. Upon targeting antibody expression cassettes 90-100% of all resulting cell clones showed correct integration. Antibody production was found to be highly consistent within the individual cell clones as expected from their isogenic nature. However, the nature and orientation of expression control elements revealed to be critical. The impact of different promoters was examined with the tag-and-targeting approach. For each of the chosen promoters high expression sites were identified. However, each site supported the chosen promoters to a different extent, indicating that the strength of a particular promoter is dominantly defined by its chromosomal context.
Conclusion
RMCE provides a powerful method to specifically design vectors for optimized gene expression with high accuracy. Upon considering the specific requirements of chromosomal sites this method provides a unique tool to exploit such sites for predictable expression of biotechnologically relevant proteins such as antibodies.oai:repository.helmholtz-hzi.de:10033/6210332019-08-30T11:32:16Zcom_10033_6823com_10033_6820com_10033_620652col_10033_6891col_10033_620672
Nehlsen, Kristina
da Gama-Norton, Leonor
Schucht, Roland
Hauser, Hansjörg
Wirth, Dagmar
2017-08-02T09:37:20Z
2017-08-02T09:37:20Z
2011-11-22
2015-09-04T08:22:40Z
BMC Proceedings. 2011 Nov 22;5(Suppl 8):O6
http://dx.doi.org/10.1186/1753-6561-5-S8-O6
http://hdl.handle.net/10033/621033
Towards rational engineering of cells: Recombinant gene expression in defined chromosomal loci
en
Nehlsen et al; licensee BioMed Central Ltd.2018-06-13T03:44:51Zoai:repository.helmholtz-hzi.de:10033/6208412019-08-30T11:32:59Zcom_10033_6823com_10033_6820col_10033_6891
Hauser, Hansjörg
Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany.
2017-02-24T11:32:05Z
2017-02-24T11:32:05Z
2011-11-22
22nd European Society for Animal Cell Technology (ESACT) Meeting on Cell Based Technologies Vienna, Austria. 15-18 May 2011. Abstracts. 2011, 5 Suppl 8:I1-P134 BMC Proc
1753-6561
22166093
http://hdl.handle.net/10033/620841
BMC proceedings
en
22nd European Society for Animal Cell Technology (ESACT) Meeting on Cell Based Technologies Vienna, Austria. 15-18 May 2011. Abstracts.
Article2018-06-13T03:57:36Zoai:repository.helmholtz-hzi.de:10033/6207912019-08-30T11:26:12Zcom_10033_6823com_10033_6820col_10033_6891
Quentmeier, Hilmar
Eberth, Sonja
Romani, Julia
Weich, Herbert A
Zaborski, Margarete
Drexler, Hans G
2017-01-27T12:16:17Z
2017-01-27T12:16:17Z
2012-01-17
2015-09-04T08:27:17Z
BMC Cancer. 2012 Jan 17;12(1):19
http://dx.doi.org/10.1186/1471-2407-12-19
http://hdl.handle.net/10033/620791
Abstract Background Vascular Endothelial Growth Factors (VEGFs) and their receptors (VEGF-Rs) are important regulators for angiogenesis and lymphangiogenesis. VEGFs and VEGF-Rs are not only expressed on endothelial cells but also on various subtypes of solid tumors and leukemias contributing to the growth of the malignant cells. This study was performed to examine whether VEGF-R2 (KDR) and VEGF-R3 (FLT4) are regulated by DNA methylation. Methods Real-time (RT) PCR analysis was performed to quantify KDR and FLT4 expression in some ninety leukemia/lymphoma cell lines, human umbilical vein endothelial cells (HUVECs) and dermal microvascular endothelial cells (HDMECs). Western blot analyses and flow cytometric analyses confirmed results at the protein level. After bisulfite conversion of DNA we determined the methylation status of KDR and FLT4 by DNA sequencing and by methylation specific PCR (MSP). Western blot analyses were performed to examine the effect of VEGF-C on p42/44 MAPK activation. Results Expression of KDR and FLT4 was observed in cell lines from various leukemic entities, but not in lymphoma cell lines: 16% (10/62) of the leukemia cell lines expressed KDR, 42% (27/65) were FLT4 positive. None of thirty cell lines representing six lymphoma subtypes showed more than marginal expression of KDR or FLT4. Western blot analyses confirmed KDR and FLT4 protein expression in HDMECs, HUVECs and in cell lines with high VEGF-R mRNA levels. Mature VEGF-C induced p42/44 MAPK activation in the KDR- /FLT4+ cell line OCI-AML1 verifying the model character of this cell line for VEGF-C signal transduction studies. Bisulfite sequencing and MSP revealed that GpG islands in the promoter regions of KDR and FLT4 were unmethylated in HUVECs, HDMECs and KDR + and FLT4 + cell lines, whereas methylated cell lines did not express these genes. In hypermethylated cell lines, KDR and FLT4 were re-inducible by treatment with the DNA demethylating agent 5-Aza-2'deoxycytidine, confirming epigenetic regulation of both genes. Conclusions Our data show that VEGF-Rs KDR and FLT4 are silenced by DNA methylation. However, if the promoters are unmethylated, other factors (e.g. transactivation factors) determine the extent of KDR and FLT4 expression.
DNA methylation regulates expression of VEGF-R2 (KDR) and VEGF-R3 (FLT4)
Journal Article
en
Quentmeier et al; licensee BioMed Central Ltd.2018-06-12T16:48:21ZAbstract
Background
Vascular Endothelial Growth Factors (VEGFs) and their receptors (VEGF-Rs) are important regulators for angiogenesis and lymphangiogenesis. VEGFs and VEGF-Rs are not only expressed on endothelial cells but also on various subtypes of solid tumors and leukemias contributing to the growth of the malignant cells. This study was performed to examine whether VEGF-R2 (KDR) and VEGF-R3 (FLT4) are regulated by DNA methylation.
Methods
Real-time (RT) PCR analysis was performed to quantify KDR and FLT4 expression in some ninety leukemia/lymphoma cell lines, human umbilical vein endothelial cells (HUVECs) and dermal microvascular endothelial cells (HDMECs). Western blot analyses and flow cytometric analyses confirmed results at the protein level. After bisulfite conversion of DNA we determined the methylation status of KDR and FLT4 by DNA sequencing and by methylation specific PCR (MSP). Western blot analyses were performed to examine the effect of VEGF-C on p42/44 MAPK activation.
Results
Expression of KDR and FLT4 was observed in cell lines from various leukemic entities, but not in lymphoma cell lines: 16% (10/62) of the leukemia cell lines expressed KDR, 42% (27/65) were FLT4 positive. None of thirty cell lines representing six lymphoma subtypes showed more than marginal expression of KDR or FLT4. Western blot analyses confirmed KDR and FLT4 protein expression in HDMECs, HUVECs and in cell lines with high VEGF-R mRNA levels. Mature VEGF-C induced p42/44 MAPK activation in the KDR- /FLT4+ cell line OCI-AML1 verifying the model character of this cell line for VEGF-C signal transduction studies. Bisulfite sequencing and MSP revealed that GpG islands in the promoter regions of KDR and FLT4 were unmethylated in HUVECs, HDMECs and KDR
+ and FLT4
+ cell lines, whereas methylated cell lines did not express these genes. In hypermethylated cell lines, KDR and FLT4 were re-inducible by treatment with the DNA demethylating agent 5-Aza-2'deoxycytidine, confirming epigenetic regulation of both genes.
Conclusions
Our data show that VEGF-Rs KDR and FLT4 are silenced by DNA methylation. However, if the promoters are unmethylated, other factors (e.g. transactivation factors) determine the extent of KDR and FLT4 expression.oai:repository.helmholtz-hzi.de:10033/6207942019-08-30T11:33:05Zcom_10033_6823com_10033_6820col_10033_6891
Poblete-Castro, Ignacio
Escapa, Isabel F
Jäger, Christian
Puchalka, Jacek
Chi Lam, Carolyn M
Schomburg, Dietmar
Prieto, María A
Martins dos Santos, Vítor A
2017-01-30T15:27:39Z
2017-01-30T15:27:39Z
2012-03-20
2015-09-04T08:26:49Z
Microbial Cell Factories. 2012 Mar 20;11(1):34
http://dx.doi.org/10.1186/1475-2859-11-34
http://hdl.handle.net/10033/620794
Abstract Background Pseudomonas putida KT2442 is a natural producer of polyhydroxyalkanoates (PHAs), which can substitute petroleum-based non-renewable plastics and form the basis for the production of tailor-made biopolymers. However, despite the substantial body of work on PHA production by P. putida strains, it is not yet clear how the bacterium re-arranges its whole metabolism when it senses the limitation of nitrogen and the excess of fatty acids as carbon source, to result in a large accumulation of PHAs within the cell. In the present study we investigated the metabolic response of KT2442 using a systems biology approach to highlight the differences between single- and multiple-nutrient-limited growth in chemostat cultures. Results We found that 26, 62, and 81% of the cell dry weight consist of PHA under conditions of carbon, dual, and nitrogen limitation, respectively. Under nitrogen limitation a specific PHA production rate of 0.43 (g·(g·h)-1) was obtained. The residual biomass was not constant for dual- and strict nitrogen-limiting growth, showing a different feature in comparison to other P. putida strains. Dual limitation resulted in patterns of gene expression, protein level, and metabolite concentrations that substantially differ from those observed under exclusive carbon or nitrogen limitation. The most pronounced differences were found in the energy metabolism, fatty acid metabolism, as well as stress proteins and enzymes belonging to the transport system. Conclusion This is the first study where the interrelationship between nutrient limitations and PHA synthesis has been investigated under well-controlled conditions using a system level approach. The knowledge generated will be of great assistance for the development of bioprocesses and further metabolic engineering work in this versatile organism to both enhance and diversify the industrial production of PHAs.
The metabolic response of P. putida KT2442 producing high levels of polyhydroxyalkanoate under single- and multiple-nutrient-limited growth: Highlights from a multi-level omics approach
Journal Article
en
Poblete-Castro et al; licensee BioMed Central Ltd.2018-06-12T22:47:37ZAbstract
Background
Pseudomonas putida KT2442 is a natural producer of polyhydroxyalkanoates (PHAs), which can substitute petroleum-based non-renewable plastics and form the basis for the production of tailor-made biopolymers. However, despite the substantial body of work on PHA production by P. putida strains, it is not yet clear how the bacterium re-arranges its whole metabolism when it senses the limitation of nitrogen and the excess of fatty acids as carbon source, to result in a large accumulation of PHAs within the cell. In the present study we investigated the metabolic response of KT2442 using a systems biology approach to highlight the differences between single- and multiple-nutrient-limited growth in chemostat cultures.
Results
We found that 26, 62, and 81% of the cell dry weight consist of PHA under conditions of carbon, dual, and nitrogen limitation, respectively. Under nitrogen limitation a specific PHA production rate of 0.43 (g·(g·h)-1) was obtained. The residual biomass was not constant for dual- and strict nitrogen-limiting growth, showing a different feature in comparison to other P. putida strains. Dual limitation resulted in patterns of gene expression, protein level, and metabolite concentrations that substantially differ from those observed under exclusive carbon or nitrogen limitation. The most pronounced differences were found in the energy metabolism, fatty acid metabolism, as well as stress proteins and enzymes belonging to the transport system.
Conclusion
This is the first study where the interrelationship between nutrient limitations and PHA synthesis has been investigated under well-controlled conditions using a system level approach. The knowledge generated will be of great assistance for the development of bioprocesses and further metabolic engineering work in this versatile organism to both enhance and diversify the industrial production of PHAs.oai:repository.helmholtz-hzi.de:10033/5797352019-08-30T11:31:23Zcom_10033_6823com_10033_6820col_10033_6891
Martins, José Paulo
Santos, Jorge Miguel
de Almeida, Joana Marto
Filipe, Mariana Alves
de Almeida, Mariana Vargas Teixeira
Almeida, Sílvia Cristina Paiva
Água-Doce, Ana
Varela, Alexandre
Gilljam, Mari
Stellan, Birgitta
Pohl, Susanne
Dittmar, Kurt
Lindenmaier, Werner
Alici, Evren
Graça, Luís
Cruz, Pedro Estilita
Cruz, Helder Joaquim
Bárcia, Rita Nogueira
2015-10-15T08:26:57Z
2015-10-15T08:26:57Z
2014
Towards an advanced therapy medicinal product based on mesenchymal stromal cells isolated from the umbilical cord tissue: quality and safety data. 2014, 5 (1):9 Stem Cell Res Ther
1757-6512
24438697
10.1186/scrt398
http://hdl.handle.net/10033/579735
Stem cell research & therapy
Standardization of mesenchymal stromal cells (MSCs) manufacturing is urgently needed to enable translational activities and ultimately facilitate comparison of clinical trial results. In this work we describe the adaptation of a proprietary method for isolation of a specific umbilical cord tissue-derived population of MSCs, herein designated by its registered trademark as UCX®, towards the production of an advanced therapy medicinal product (ATMP).
en
Cells, Cultured
Cryopreservation
Humans
Mesenchymal Stem Cell Transplantation
Mesenchymal Stromal Cells
Quality Control
Stem Cell Research
Tissue and Organ Harvesting
Umbilical Cord
Towards an advanced therapy medicinal product based on mesenchymal stromal cells isolated from the umbilical cord tissue: quality and safety data.
Article2018-06-13T14:17:14ZStandardization of mesenchymal stromal cells (MSCs) manufacturing is urgently needed to enable translational activities and ultimately facilitate comparison of clinical trial results. In this work we describe the adaptation of a proprietary method for isolation of a specific umbilical cord tissue-derived population of MSCs, herein designated by its registered trademark as UCX®, towards the production of an advanced therapy medicinal product (ATMP).oai:repository.helmholtz-hzi.de:10033/5933912019-08-30T11:37:24Zcom_10033_6823com_10033_6820col_10033_6891
Sun, Lijing
Hemgård, Gun-Viol
Susanto, Sony A
Wirth, Manfred
Helmholtz Centre for infection research, Inhoffenstr. 7, D-38124 Braunschweig, Germany.
2016-01-14T12:21:25Z
2016-01-14T12:21:25Z
2010
Caveolin-1 influences human influenza A virus (H1N1) multiplication in cell culture. 2010, 7:108 Virol. J.
1743-422X
20504340
10.1186/1743-422X-7-108
http://hdl.handle.net/10033/593391
Virology journal
The threat of recurring influenza pandemics caused by new viral strains and the occurrence of escape mutants necessitate the search for potent therapeutic targets. The dependence of viruses on cellular factors provides a weak-spot in the viral multiplication strategy and a means to interfere with viral multiplication.
en
Amino Acid Sequence
Animals
Caveolin 1
Cell Line
Dogs
Humans
Influenza A Virus, H1N1 Subtype
Influenza, Human
Mice
Molecular Sequence Data
NIH 3T3 Cells
Protein Binding
Sequence Alignment
Viral Matrix Proteins
Virus Replication
Caveolin-1 influences human influenza A virus (H1N1) multiplication in cell culture.
Article2018-06-12T23:37:13ZThe threat of recurring influenza pandemics caused by new viral strains and the occurrence of escape mutants necessitate the search for potent therapeutic targets. The dependence of viruses on cellular factors provides a weak-spot in the viral multiplication strategy and a means to interfere with viral multiplication.oai:repository.helmholtz-hzi.de:10033/6208422019-08-30T11:29:17Zcom_10033_6823com_10033_6820col_10033_6891
Quentmeier, Hilmar
Eberth, Sonja
Romani, Julia
Weich, Herbert A
Zaborski, Margarete
Drexler, Hans G
Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany.
2017-02-27T11:01:09Z
2017-02-27T11:01:09Z
2012-01-17
DNA methylation regulates expression of VEGF-R2 (KDR) and VEGF-R3 (FLT4). 2012, 12:19 BMC Cancer
1471-2407
22251800
10.1186/1471-2407-12-19
http://hdl.handle.net/10033/620842
BMC cancer
Vascular Endothelial Growth Factors (VEGFs) and their receptors (VEGF-Rs) are important regulators for angiogenesis and lymphangiogenesis. VEGFs and VEGF-Rs are not only expressed on endothelial cells but also on various subtypes of solid tumors and leukemias contributing to the growth of the malignant cells. This study was performed to examine whether VEGF-R2 (KDR) and VEGF-R3 (FLT4) are regulated by DNA methylation.
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Blotting, Western
Cell Line, Tumor
DNA Methylation
Endothelial Cells
Gene Expression Regulation
Human Umbilical Vein Endothelial Cells
Humans
Leukemia
Lymphoma
Real-Time Polymerase Chain Reaction
Sequence Analysis, DNA
Vascular Endothelial Growth Factor Receptor-2
Vascular Endothelial Growth Factor Receptor-3
DNA methylation regulates expression of VEGF-R2 (KDR) and VEGF-R3 (FLT4).
Article2018-06-13T09:26:06ZVascular Endothelial Growth Factors (VEGFs) and their receptors (VEGF-Rs) are important regulators for angiogenesis and lymphangiogenesis. VEGFs and VEGF-Rs are not only expressed on endothelial cells but also on various subtypes of solid tumors and leukemias contributing to the growth of the malignant cells. This study was performed to examine whether VEGF-R2 (KDR) and VEGF-R3 (FLT4) are regulated by DNA methylation.