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Artikel ; Online: Utilizing antagomiR (antisense microRNA) to knock down microRNA in murine bone marrow cells.

Velu, Chinavenmeni S / Grimes, H Leighton

Methods in molecular biology (Clifton, N.J.)

2012 Band 928, Seite(n) 185–195

Abstract: MicroRNAs (miRNAs) are highly conserved small RNAs which regulate gene expression primarily through base pairing to the 3' untranslated region of target messenger RNA (mRNA), leading to mRNA degradation or translation inhibition depending on the ... ...

Abstract	MicroRNAs (miRNAs) are highly conserved small RNAs which regulate gene expression primarily through base pairing to the 3' untranslated region of target messenger RNA (mRNA), leading to mRNA degradation or translation inhibition depending on the complementarity between the miRNA and target mRNA. Single miRNA regulates multiple target mRNA. miRNAs have been shown to regulate gene expression in the hematopoietic stem cells, as well as at key decision points for various lineages. However, aberrant expression of miRNAs has been documented in cancer and disease models. Rigorous dissection of miRNA pathways and biology requires facile loss of function modeling. This chapter describes detailed protocol for knockdown miRNA-21 which is involved in myelopoiesis using antagomiRs in primary murine bone marrow stem/progenitor cells.
Mesh-Begriff(e)	Animals ; Bone Marrow Cells/drug effects ; Bone Marrow Cells/metabolism ; Mice ; MicroRNAs/antagonists & inhibitors ; MicroRNAs/genetics ; Oligoribonucleotides/genetics ; Oligoribonucleotides/pharmacology
Chemische Substanzen	MicroRNAs ; Oligoribonucleotides
Sprache	Englisch
Erscheinungsdatum	2012-09-06
Erscheinungsland	United States
Dokumenttyp	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-62703-008-3_15
Datenquelle	MEDical Literature Analysis and Retrieval System OnLINE

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Artikel ; Online: Gfi1 regulates miR-21 and miR-196b to control myelopoiesis.

Velu, Chinavenmeni S / Baktula, Avinash M / Grimes, H Leighton

Blood

2009 Band 113, Heft 19, Seite(n) 4720–4728

Abstract: The zinc finger protein growth factor independent-1 (Gfi1) is a transcriptional repressor that is critically required for normal granulocytic differentiation. GFI1 loss-of-function mutations are found in some patients with severe congenital neutropenia ( ... ...

Abstract	The zinc finger protein growth factor independent-1 (Gfi1) is a transcriptional repressor that is critically required for normal granulocytic differentiation. GFI1 loss-of-function mutations are found in some patients with severe congenital neutropenia (SCN). The SCN-associated GFI1-mutant proteins act as dominant negatives to block granulopoiesis through selective deregulation of a subset of GFI1 target genes. Here we show that Gfi1 is a master regulator of microRNAs, and that deregulated expression of these microRNAs recapitulates a Gfi1 loss-of-function block to granulocyte colony-stimulating factor (G-CSF)-stimulated granulopoiesis. Specifically, bone marrow cells from a GFI1-mutant SCN patient and Gfi1(-/-) mice display deregulated expression of miR-21 and miR-196B expression. Flow cytometric analysis and colony assays reveal that the overexpression or depletion of either miR induces changes in myeloid development. However, coexpression of miR-21 and miR-196b (as seen in Gfi1(-/-) mice and a GFI1N382S SCN patient) completely blocks G-CSF-induced granulopoiesis. Thus, our results not only identify microRNAs whose regulation is required during myelopoiesis, but also provide an example of synergy in microRNA biologic activity and illustrate potential mechanisms underlying SCN disease pathogenesis.
Mesh-Begriff(e)	Animals ; Bone Marrow/physiology ; Bone Marrow Transplantation ; Chromatin Immunoprecipitation ; Colony-Forming Units Assay ; DNA-Binding Proteins/physiology ; Electrophoretic Mobility Shift Assay ; Female ; Flow Cytometry ; Gene Expression Profiling ; Granulocyte Colony-Stimulating Factor/pharmacology ; Granulocytes/cytology ; Granulocytes/physiology ; Hematopoietic Stem Cells ; Immunoblotting ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Mice, Knockout ; MicroRNAs/genetics ; MicroRNAs/physiology ; Myelopoiesis/physiology ; Oligonucleotide Array Sequence Analysis ; RNA, Small Interfering/pharmacology ; Transcription Factors/physiology
Chemische Substanzen	DNA-Binding Proteins ; Gfi1 protein, mouse ; MIRN21 microRNA, mouse ; MicroRNAs ; RNA, Small Interfering ; Transcription Factors ; Granulocyte Colony-Stimulating Factor (143011-72-7)
Sprache	Englisch
Erscheinungsdatum	2009-03-10
Erscheinungsland	United States
Dokumenttyp	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	80069-7
ISSN	1528-0020 ; 0006-4971
ISSN (online)	1528-0020
ISSN	0006-4971
DOI	10.1182/blood-2008-11-190215
Datenquelle	MEDical Literature Analysis and Retrieval System OnLINE

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Artikel: Improving the fatty acid profile of fairy shrimp, Streptocephalus dichotomus, using a lipid emulsion rich in highly unsaturated fatty acids.

Velu, Chinavenmeni S / Munuswamy, Natesan

Journal of agricultural and food chemistry

2004 Band 52, Heft 23, Seite(n) 7033–7038

Abstract: Fatty acids are the largest component of lipids and have become a useful tool in the determination of live feeds to a variety of cultured species. Bioencapsulation is a technique which allows high-level incorporation of desired components (i.e., fatty ... ...

Abstract	Fatty acids are the largest component of lipids and have become a useful tool in the determination of live feeds to a variety of cultured species. Bioencapsulation is a technique which allows high-level incorporation of desired components (i.e., fatty acids, vitamins, antibiotics, etc.) in live feeds, which in turn can be supplemented to the consumer organisms. The procedure described in the present study serves as a platform of technology for enriching the Streptocephalus dichotomus. Uptake of two enrichment diets (ALGAMAC2000 and DHA-SELCO) by adult S. dichotomus was investigated. The fatty acid profile supports the hypothesis that the enrichment diet increases the level of essential fatty acids, such as linolic, linolenic, eicosapentenoic, and docosahexaenoic acids. The average content (percent of total fatty acids detected) of the enriched organism by different highly unsaturated fatty acid (HUFA) products were as follows: ALGAMAC2000 showed 14-22% saturated fatty acid (SFA), 17-18% monounsaturated fatty acid (MUFA), 28-41% polyunsaturated fatty acid (PUFA), 23-34% n-3, and 4.9-7.5% n-6, whereas DHA-SELCO showed about 20-23% SFA, 20-26% MUFA, 38% PUFA, 28-31% n-3, and 7.5-10% n-6. Our present investigation proves that both HUFA-rich diets appear to be an appropriate enrichment diet, and further provides an additional rationale for using fairy shrimp as a maturation diet for any cultivable freshwater organism.
Mesh-Begriff(e)	Animals ; Anostraca/chemistry ; Anostraca/growth & development ; Diet ; Emulsions/administration & dosage ; Emulsions/chemistry ; Fatty Acids/analysis ; Fatty Acids, Unsaturated/administration & dosage ; Fatty Acids, Unsaturated/analysis
Chemische Substanzen	Emulsions ; Fatty Acids ; Fatty Acids, Unsaturated
Sprache	Englisch
Erscheinungsdatum	2004-11-17
Erscheinungsland	United States
Dokumenttyp	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	241619-0
ISSN	1520-5118 ; 0021-8561
ISSN (online)	1520-5118
ISSN	0021-8561
DOI	10.1021/jf0490605
Datenquelle	MEDical Literature Analysis and Retrieval System OnLINE

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Artikel ; Online: SKI

Muench, David E / Ferchen, Kyle / Velu, Chinavenmeni S / Pradhan, Kith / Chetal, Kashish / Chen, Xiaoting / Weirauch, Matthew T / Colmenares, Clemencia / Verma, Amit / Salomonis, Nathan / Grimes, H Leighton

Blood

2018 Band 132, Heft 21, Seite(n) e24–e34

Abstract: The transforming growth factor beta (TGF-β) signaling pathway controls hematopoietic stem cell (HSC) behavior in the marrow niche; however, TGF-β signaling becomes chronic in early-stage myelodysplastic syndrome (MDS). Although TGF-β signaling normally ... ...

Abstract	The transforming growth factor beta (TGF-β) signaling pathway controls hematopoietic stem cell (HSC) behavior in the marrow niche; however, TGF-β signaling becomes chronic in early-stage myelodysplastic syndrome (MDS). Although TGF-β signaling normally induces negative feedback, in early-stage MDS, high levels of microRNA-21 (miR-21) contribute to chronic TGF-β signaling. We found that a TGF-β signal-correlated gene signature is sufficient to identify an MDS patient population with abnormal RNA splicing (eg,
Mesh-Begriff(e)	DNA-Binding Proteins/genetics ; DNA-Binding Proteins/metabolism ; Gene Deletion ; Gene Expression Regulation ; Hematopoietic Stem Cells/metabolism ; Hematopoietic Stem Cells/pathology ; Humans ; MicroRNAs/genetics ; MicroRNAs/metabolism ; Myelodysplastic Syndromes/genetics ; Myelodysplastic Syndromes/metabolism ; Myelodysplastic Syndromes/pathology ; Proto-Oncogene Proteins/genetics ; Proto-Oncogene Proteins/metabolism ; RNA Splicing ; RNA, Messenger/genetics ; Signal Transduction ; Transforming Growth Factor beta/metabolism
Chemische Substanzen	DNA-Binding Proteins ; MIRN21 microRNA, human ; MicroRNAs ; Proto-Oncogene Proteins ; RNA, Messenger ; Transforming Growth Factor beta ; SKI protein, human (126648-96-2)
Sprache	Englisch
Erscheinungsdatum	2018-09-24
Erscheinungsland	United States
Dokumenttyp	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	80069-7
ISSN	1528-0020 ; 0006-4971
ISSN (online)	1528-0020
ISSN	0006-4971
DOI	10.1182/blood-2018-06-860890
Datenquelle	MEDical Literature Analysis and Retrieval System OnLINE

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Artikel ; Online: Rho GTPase Cdc42 is essential for B-lymphocyte development and activation.

Guo, Fukun / Velu, Chinavenmeni S / Grimes, H Leighton / Zheng, Yi

Blood

2009 Band 114, Heft 14, Seite(n) 2909–2916

Abstract: Cdc42 is a member of the Rho GTPase family that has been implicated in several cell functions including proliferation and migration, but its physiologic role needs to be dissected in each cell type. We achieved B-cell and hematopoietic stem cell deletion ...

Abstract	Cdc42 is a member of the Rho GTPase family that has been implicated in several cell functions including proliferation and migration, but its physiologic role needs to be dissected in each cell type. We achieved B-cell and hematopoietic stem cell deletion of Cdc42 by conditional gene targeting in mice. Deletion of Cdc42 from proB/preB-cell stage significantly blocked B-cell development at T1 and later stages, resulting in reduced mature B-cell populations and reduced antigen-specific immunoglobulin M (IgM), IgG1, and IgG3 production. The Cdc42(-/-) B cells, themselves, were abnormal with impaired proliferation and survival. The mutant B cells were further characterized by a B-cell receptor (BCR) signaling defect with increased Erk and decreased Akt activation, as well as a defect in BCR-mediated B-cell-activating factor (BAFF) receptor up-regulation and subsequent BAFF receptor signaling in mature resting B cells. Surprisingly, Cdc42 was dispensable for stromal cell-derived factor-1alpha (SDF-1alpha)- or B-lymphocyte chemoattractant (BLC)-induced B-cell migration. Finally, loss of Cdc42 from hematopoietic stem cells did not alter common lymphoid progenitor production but severely reduced proB/preB- and immature B-cell populations, indicating that Cdc42 is also involved in B-cell precursor differentiation. These results reveal multifaceted roles of Cdc42 in B-cell development and activation.
Mesh-Begriff(e)	Animals ; B-Cell Activation Factor Receptor/metabolism ; B-Lymphocytes/cytology ; B-Lymphocytes/metabolism ; Blotting, Western ; Cell Differentiation ; Cell Movement ; Cell Proliferation ; Chemokine CXCL12/metabolism ; Enzyme-Linked Immunosorbent Assay ; Gene Expression Profiling ; Hematopoietic Stem Cells/metabolism ; Immunoglobulin G/blood ; Immunoglobulin M/blood ; Immunoglobulin M/deficiency ; Integrases/metabolism ; Lymphocyte Activation ; Mice ; Mice, Knockout ; Mice, Transgenic ; Oligonucleotide Array Sequence Analysis ; Precursor Cells, B-Lymphoid/metabolism ; Receptors, Antigen, B-Cell/metabolism ; Signal Transduction ; cdc42 GTP-Binding Protein/physiology
Chemische Substanzen	B-Cell Activation Factor Receptor ; Chemokine CXCL12 ; Immunoglobulin G ; Immunoglobulin M ; Receptors, Antigen, B-Cell ; Cre recombinase (EC 2.7.7.-) ; Integrases (EC 2.7.7.-) ; cdc42 GTP-Binding Protein (EC 3.6.5.2)
Sprache	Englisch
Erscheinungsdatum	2009-08-11
Erscheinungsland	United States
Dokumenttyp	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	80069-7
ISSN	1528-0020 ; 0006-4971
ISSN (online)	1528-0020
ISSN	0006-4971
DOI	10.1182/blood-2009-04-214676
Datenquelle	MEDical Literature Analysis and Retrieval System OnLINE

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Artikel: Coordination of IL-7 receptor and T-cell receptor signaling by cell-division cycle 42 in T-cell homeostasis

Guo, Fukun / Hildeman, David / Tripathi, Pulak / Velu, Chinavenmeni S / Grimes, H. Leighton / Zheng, Yi

Proceedings of the National Academy of Sciences of the United States of America. 2010 Oct. 26, v. 107, no. 43

2010

Abstract: T-cell homeostasis is essential for normal functioning of the immune system. IL-7 receptor (IL-7R) and T-cell receptor (TCR) signaling are pivotal for T-cell homeostatic regulation. The detailed mechanisms regulating T-cell homeostasis and how IL-7R and ... ...

Abstract	T-cell homeostasis is essential for normal functioning of the immune system. IL-7 receptor (IL-7R) and T-cell receptor (TCR) signaling are pivotal for T-cell homeostatic regulation. The detailed mechanisms regulating T-cell homeostasis and how IL-7R and TCR signaling are coordinated are largely unknown. Here we demonstrate that T cell-specific deletion of cell-division cycle 42 (Cdc42) GTPase causes a profound loss of mature T cells. Deletion of Cdc42 leads to a markedly increased expression of growth factor independence-1 (Gfi-1) and represses expression of IL-7Rα. In the absence of Cdc42, aberrant ERK1/2 MAP kinase activity results in enhanced, TCR-mediated T-cell proliferation. In vivo reconstitution of effector-binding-defective Cdc42 mutants and the effector p21 protein-activated kinase 1 (PAK1) into Cdc42-deficient T cells showed that PAK1 is both necessary and sufficient for Cdc42-regulated T-cell homeostasis. Thus, T-cell homeostasis is maintained through a concerted regulation of Gfi-1-IL-7R-controlled cytokine responsiveness and ERK-mediated TCR signaling strength by the Cdc42-PAK1 signaling axis.
Schlagwörter	T-lymphocytes ; cell division ; guanosinetriphosphatase ; homeostasis ; interleukin-7 ; mitogen-activated protein kinase ; mutants
Sprache	Englisch
Erscheinungsverlauf	2010-1026
Umfang	p. 18505-18510.
Erscheinungsort	National Academy of Sciences
Dokumenttyp	Artikel
ZDB-ID	209104-5
ISSN	1091-6490 ; 0027-8424
ISSN (online)	1091-6490
ISSN	0027-8424
DOI	10.1073/pnas.1010249107
Datenquelle	NAL Katalog (AGRICOLA)

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Artikel ; Online: Coordination of IL-7 receptor and T-cell receptor signaling by cell-division cycle 42 in T-cell homeostasis.

Guo, Fukun / Hildeman, David / Tripathi, Pulak / Velu, Chinavenmeni S / Grimes, H Leighton / Zheng, Yi

Proceedings of the National Academy of Sciences of the United States of America

2010 Band 107, Heft 43, Seite(n) 18505–18510

Abstract	T-cell homeostasis is essential for normal functioning of the immune system. IL-7 receptor (IL-7R) and T-cell receptor (TCR) signaling are pivotal for T-cell homeostatic regulation. The detailed mechanisms regulating T-cell homeostasis and how IL-7R and TCR signaling are coordinated are largely unknown. Here we demonstrate that T cell-specific deletion of cell-division cycle 42 (Cdc42) GTPase causes a profound loss of mature T cells. Deletion of Cdc42 leads to a markedly increased expression of growth factor independence-1 (Gfi-1) and represses expression of IL-7Rα. In the absence of Cdc42, aberrant ERK1/2 MAP kinase activity results in enhanced, TCR-mediated T-cell proliferation. In vivo reconstitution of effector-binding-defective Cdc42 mutants and the effector p21 protein-activated kinase 1 (PAK1) into Cdc42-deficient T cells showed that PAK1 is both necessary and sufficient for Cdc42-regulated T-cell homeostasis. Thus, T-cell homeostasis is maintained through a concerted regulation of Gfi-1-IL-7R-controlled cytokine responsiveness and ERK-mediated TCR signaling strength by the Cdc42-PAK1 signaling axis.
Mesh-Begriff(e)	Animals ; Base Sequence ; Cell Differentiation ; Cell Proliferation ; DNA Primers/genetics ; DNA-Binding Proteins/genetics ; DNA-Binding Proteins/metabolism ; Gene Expression ; Homeostasis ; Lymphocyte Activation ; MAP Kinase Signaling System ; Mice ; Mice, Knockout ; Mice, Transgenic ; Receptors, Antigen, T-Cell/metabolism ; Receptors, Interleukin-7/metabolism ; Recombinant Proteins/genetics ; Recombinant Proteins/metabolism ; Signal Transduction ; T-Lymphocytes/cytology ; T-Lymphocytes/immunology ; T-Lymphocytes/metabolism ; Transcription Factors/genetics ; Transcription Factors/metabolism ; cdc42 GTP-Binding Protein/deficiency ; cdc42 GTP-Binding Protein/genetics ; cdc42 GTP-Binding Protein/metabolism ; p21-Activated Kinases/genetics ; p21-Activated Kinases/metabolism
Chemische Substanzen	DNA Primers ; DNA-Binding Proteins ; Gfi1 protein, mouse ; Receptors, Antigen, T-Cell ; Receptors, Interleukin-7 ; Recombinant Proteins ; Transcription Factors ; Pak1 protein, mouse (EC 2.7.11.1) ; p21-Activated Kinases (EC 2.7.11.1) ; cdc42 GTP-Binding Protein (EC 3.6.5.2)
Sprache	Englisch
Erscheinungsdatum	2010-10-11
Erscheinungsland	United States
Dokumenttyp	Journal Article
ZDB-ID	209104-5
ISSN	1091-6490 ; 0027-8424
ISSN (online)	1091-6490
ISSN	0027-8424
DOI	10.1073/pnas.1010249107
Datenquelle	MEDical Literature Analysis and Retrieval System OnLINE

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Artikel ; Online: The 3' region of the chicken hypersensitive site-4 insulator has properties similar to its core and is required for full insulator activity.

Arumugam, Paritha I / Urbinati, Fabrizia / Velu, Chinavenmeni S / Higashimoto, Tomoyasu / Grimes, H Leighton / Malik, Punam

PloS one

2009 Band 4, Heft 9, Seite(n) e6995

Abstract: Chromatin insulators separate active transcriptional domains and block the spread of heterochromatin in the genome. Studies on the chicken hypersensitive site-4 (cHS4) element, a prototypic insulator, have identified CTCF and USF-1/2 motifs in the ... ...

Abstract	Chromatin insulators separate active transcriptional domains and block the spread of heterochromatin in the genome. Studies on the chicken hypersensitive site-4 (cHS4) element, a prototypic insulator, have identified CTCF and USF-1/2 motifs in the proximal 250 bp of cHS4, termed the "core", which provide enhancer blocking activity and reduce position effects. However, the core alone does not insulate viral vectors effectively. The full-length cHS4 has excellent insulating properties, but its large size severely compromises vector titers. We performed a structure-function analysis of cHS4 flanking lentivirus-vectors and analyzed transgene expression in the clonal progeny of hematopoietic stem cells and epigenetic changes in cHS4 and the transgene promoter. We found that the core only reduced the clonal variegation in expression. Unique insulator activity resided in the distal 400 bp cHS4 sequences, which when combined with the core, restored full insulator activity and open chromatin marks over the transgene promoter and the insulator. These data consolidate the known insulating activity of the canonical 5' core with a novel 3' 400 bp element with properties similar to the core. Together, they have excellent insulating properties and viral titers. Our data have important implications in understanding the molecular basis of insulator function and design of gene therapy vectors.
Mesh-Begriff(e)	3' Untranslated Regions ; Amino Acid Motifs ; Animals ; Cell Line ; Chickens ; Epigenesis, Genetic ; Genetic Vectors ; Hematopoietic Stem Cells/cytology ; Insulator Elements ; Lentivirus/genetics ; Mice ; Promoter Regions, Genetic ; Structure-Activity Relationship ; Transgenes
Chemische Substanzen	3' Untranslated Regions
Sprache	Englisch
Erscheinungsdatum	2009-09-10
Erscheinungsland	United States
Dokumenttyp	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ISSN	1932-6203
ISSN (online)	1932-6203
DOI	10.1371/journal.pone.0006995
Datenquelle	MEDical Literature Analysis and Retrieval System OnLINE

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Artikel: S-thiolation mimicry: quantitative and kinetic analysis of redox status of protein cysteines by glutathione-affinity chromatography.

Niture, Suryakant K / Velu, Chinavenmeni S / Bailey, Nathan I / Srivenugopal, Kalkunte S

Archives of biochemistry and biophysics

2005 Band 444, Heft 2, Seite(n) 174–184

Abstract: S-Glutathionylation is emerging as a novel regulatory and adoptive mechanism by which glutathione (GSH or GSSG) conjugation can modify functionally important reactive cysteines in redox-sensitive proteins. The dynamics of generation and reversal of this ... ...

Abstract	S-Glutathionylation is emerging as a novel regulatory and adoptive mechanism by which glutathione (GSH or GSSG) conjugation can modify functionally important reactive cysteines in redox-sensitive proteins. The dynamics of generation and reversal of this modification in cells is poorly understood. This study describes the ability and applicability of GSH- and GSSG-affinity matrices to quantitatively bind proteins which harbor reactive cysteines and undergo glutathionylation. We showed that purified proteins, known to be modified by S-thiolation, bind to these matrices, are selectively eluted by dithiothreitol and rapidly incorporate biotin-labeled GSH or GSSG in vitro. Chromatography of extracts from tumor cells that had been treated with oxidants (diamide, H(2)O(2), tert-butyl hydroperoxide) on GSH-Sepharose showed the specific binding of many proteins, whose levels increased transiently (2- to 6-fold) soon after treatments. However, when these cells were post-incubated in drug/oxidant-free media, protein binding decreased gradually to control levels over 3-12h, thereby demonstrating the central role of cysteine redox status in the binding. Immunoblotting of eluates from GSH-Sepharose showed the presence of known (actin, ubiquitin-activating enzyme E1, NF-kappaB, and proteasome) and putative (p53, glutathione-S-transferase P1) targets for glutathionation. After oxidant withdrawal, many of these proteins displayed unique kinetics in their loss of binding to GSH-matrix, reflecting their differential abilities to recover from cysteine redox changes in cellular milieu. Further, we correlated the kinetics of S-thiolation susceptibility of the proteasome and ubiquitin-E1 proteins with altered levels of protein ubiquitination in H(2)O(2)-treated cells. Our study reveals the hitherto underutilized ability of glutathione matrices for analyzing the kinetics of cysteine redox in cellular proteins and allows easy identification of S-thiolatable proteins.
Mesh-Begriff(e)	Binding Sites ; Biomimetics/methods ; Cell Line, Tumor ; Cysteine/chemistry ; Cysteine/metabolism ; Glutathione/chemistry ; Glutathione/metabolism ; Humans ; Kinetics ; Medulloblastoma/chemistry ; Medulloblastoma/metabolism ; Neoplasm Proteins/chemistry ; Neoplasm Proteins/metabolism ; Oxidation-Reduction ; Protein Binding ; Protein Interaction Mapping/methods ; Sulfhydryl Compounds/chemistry ; Sulfhydryl Compounds/metabolism
Chemische Substanzen	Neoplasm Proteins ; Sulfhydryl Compounds ; Glutathione (GAN16C9B8O) ; Cysteine (K848JZ4886)
Sprache	Englisch
Erscheinungsdatum	2005-12-15
Erscheinungsland	United States
Dokumenttyp	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	523-x
ISSN	1096-0384 ; 0003-9861
ISSN (online)	1096-0384
ISSN	0003-9861
DOI	10.1016/j.abb.2005.10.013
Datenquelle	MEDical Literature Analysis and Retrieval System OnLINE

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Artikel: Human p53 is inhibited by glutathionylation of cysteines present in the proximal DNA-binding domain during oxidative stress.

Velu, Chinavenmeni S / Niture, Suryakant K / Doneanu, Catalin E / Pattabiraman, Nagarajan / Srivenugopal, Kalkunte S

Biochemistry

2007 Band 46, Heft 26, Seite(n) 7765–7780

Abstract: The cellular mechanisms that modulate the redox state of p53 tumor suppressor remain unclear, although its DNA binding function is known to be strongly inhibited by oxidative and nitrosative stresses. We show that human p53 is subjected to a new and ... ...

Abstract	The cellular mechanisms that modulate the redox state of p53 tumor suppressor remain unclear, although its DNA binding function is known to be strongly inhibited by oxidative and nitrosative stresses. We show that human p53 is subjected to a new and reversible posttranslational modification, namely, S-glutathionylation in stressed states, including DNA damage. First, a rapid and direct incorporation of biotinylated GSH or GSSG into the purified recombinant p53 protein was observed. The modified p53 had a significantly weakened ability to bind its consensus DNA sequence. Reciprocal immunoprecipitations and a GST overlay assay showed that p53 in tumor cells was marginally glutathionylated; however, the level of modification increased greatly after oxidant and DNA-damaging treatments. GSH modification coexisted with the serine phophorylations in activated p53, and the thiol-conjugated protein was present in nuclei. When tumor cells treated with camptothecin or cisplatin were subsequently exposed to glutathione-enhancing agents, p53 underwent dethiolation accompanied by detectable increases in the level of p21waf1 expression, relative to the DNA-damaging drugs alone. Mass spectrometry of GSH-modified p53 protein identified cysteines 124, 141, and 182, all present in the proximal DNA-binding domain, as the sites of glutathionylation. Biotinylated maleimide also reacted rapidly with Cys141, implying that this is the most reactive cysteine on the p53 surface. The glutathionylatable cysteines were found to exist in a negatively charged microenvironment in cellular p53. Molecular modeling studies located Cys124 and -141 at the dimer interface of p53 and showed glutathionylation of either residue would inhibit p53-DNA association and also interfere with protein dimerization. These results show for the first time that shielding of reactive cysteines contributes to a negative regulation for human p53 and imply that such an inactivation of the transcription factor may represent an acute defensive response with significant consequences for oncogenesis.
Mesh-Begriff(e)	Acetylcysteine/pharmacology ; Amino Acid Sequence ; Binding Sites ; Buthionine Sulfoximine/pharmacology ; Camptothecin/pharmacology ; Cell Line, Tumor ; Cross-Linking Reagents/chemistry ; Cysteine/chemistry ; DNA Damage/drug effects ; DNA-Binding Proteins/metabolism ; Diamide/pharmacology ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli/metabolism ; Glutaral/chemistry ; Glutathione/analogs & derivatives ; Glutathione/chemistry ; Glutathione/pharmacology ; Glutathione Disulfide/chemistry ; Humans ; Hydrogen Peroxide/pharmacology ; Models, Molecular ; Oxidative Stress/drug effects ; Oxidative Stress/physiology ; Phosphorylation ; Recombinant Proteins/metabolism ; Tandem Mass Spectrometry ; Tumor Suppressor Protein p53/antagonists & inhibitors ; tert-Butylhydroperoxide/pharmacology
Chemische Substanzen	Cross-Linking Reagents ; DNA-Binding Proteins ; Recombinant Proteins ; Tumor Suppressor Protein p53 ; Diamide (10465-78-8) ; Buthionine Sulfoximine (5072-26-4) ; S-ethyl glutathione (7W95D60F4J) ; tert-Butylhydroperoxide (955VYL842B) ; Hydrogen Peroxide (BBX060AN9V) ; Glutathione (GAN16C9B8O) ; Cysteine (K848JZ4886) ; Glutaral (T3C89M417N) ; Glutathione Disulfide (ULW86O013H) ; Acetylcysteine (WYQ7N0BPYC) ; Camptothecin (XT3Z54Z28A)
Sprache	Englisch
Erscheinungsdatum	2007-06-08
Erscheinungsland	United States
Dokumenttyp	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	1108-3
ISSN	1520-4995 ; 0006-2960
ISSN (online)	1520-4995
ISSN	0006-2960
DOI	10.1021/bi700425y
Datenquelle	MEDical Literature Analysis and Retrieval System OnLINE

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