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Biomedical subjects

R Starr

Publications and source records attributed to R Starr.

At least 37 records · Page 2Linked to original sources
PubMed1999

Aberrant hematopoiesis in mice with inactivation of the gene encoding SOCS-1.

Mice with homozygous inactivation of the gene encoding the suppressor of cytokine signaling-1 (SOCS-1) protein die within 21 days of birth with low body weight, fatty degeneration and necrosis of the liver, infiltration of the lung, pancreas, heart and skin by macrophages and granulocytes and a profound depletion of T- and B-lymphocytes. In the present study, SOCS-1 -/- mice were found to have a moderate neutrophilia, and reduced platelet and hematocrit levels. Replacement of the SOCS-1 gene by a lac-Z reporter gene allowed documentation by FACS sorting that at least a proportion of granulocyte-macrophage progenitor cells transcribe SOCS-1. Most hematopoietic progenitor cell frequencies were normal in -/- marrow as were the size and cellular content of colonies formed by -/- progenitor cells in response to various stimulating factors. However, there was an increased frequency of macrophage progenitor cells in -/- mice and, abnormally, one quarter of all progenitor cells were located in the liver. Progenitor cells from -/- mice were hyper-responsive to stimulation by GM-CSF but not by M-CSF or Multi-CSF (IL-3). Progenitor cells from -/- mice were also hypersensitive to inhibition by interferon-gamma (IFN-gamma), the degree of inhibition varying markedly with the stimulating factor used. The suppressive effects of IFN-gamma therefore appear to involve interactions with particular growth factor-initiated signals in -/- cells--interactions that are strongly modulated by the action of the SOCS-1 protein.

Animals↗
PubMed1998

Liver degeneration and lymphoid deficiencies in mice lacking suppressor of cytokine signaling-1.

SOCS-1, a member of the suppressor of cytokine signaling (SOCS) family, was identified in a genetic screen for inhibitors of interleukin 6 signal transduction. SOCS-1 transcription is induced by cytokines, and the protein binds and inhibits Janus kinases and reduces cytokine-stimulated tyrosine phosphorylation of signal transducers and activators of transcription 3 and the gp130 component of the interleukin 6 receptor. Thus, SOCS-1 forms part of a feedback loop that modulates signal transduction from cytokine receptors. To examine the role of SOCS-1 in vivo, we have used gene targeting to generate mice lacking this protein. SOCS-1(-/-) mice exhibited stunted growth and died before weaning with fatty degeneration of the liver and monocytic infiltration of several organs. In addition, the thymus of SOCS-1(-/-) mice was reduced markedly in size, and there was a progressive loss of maturing B lymphocytes in the bone marrow, spleen, and peripheral blood. Thus, SOCS-1 is required for in vivo regulation of multiple cell types and is indispensable for normal postnatal growth and survival.

Abnormalities, Multiple↗
PubMed1998

Growth hormone preferentially induces the rapid, transient expression of SOCS-3, a novel inhibitor of cytokine receptor signaling.

Four members (SOCS-1, SOCS-2, SOCS-3, and CIS) of a family of cytokine-inducible, negative regulators of cytokine receptor signaling have recently been identified. To address whether any of these genes are induced in response to growth hormone (GH), serum-starved 3T3-F442A fibroblasts were incubated with GH for various time points, and the expression of the SOCS gene family was analyzed by Northern blotting. GH stimulated the rapid, transient induction of SOCS-3 mRNA, peaking 30 min after the initiation of GH exposure and declining to basal levels by 2 h. Expression of the other SOCS genes (SOCS-1, SOCS-2, CIS) was also up-regulated by GH, although to a lesser extent than SOCS-3 and with differing kinetics. SOCS-3 expression was also strongly induced in 3T3-F442A cells treated with leukemia-inhibitory factor (LIF), with weaker induction of SOCS-1 and CIS being observed. The preferential induction of SOCS-3 mRNA was also observed in hepatic RNA isolated from the livers of mice that had received a single supraphysiological dose of GH intraperitoneally. Co-transfection studies revealed that constitutive expression of SOCS-1 and SOCS-3, but not SOCS-2 or CIS, blocked GH-induced transactivation of the GH-responsive serine protease inhibitor 2.1 gene promoter.

3T3 Cells↗
PubMed1998

Twenty proteins containing a C-terminal SOCS box form five structural classes.

The four members of the recently identified suppressor of cytokines signaling family (SOCS-1, SOCS-2, SOCS-3, and CIS, where CIS is cytokine-inducible SH2-containing protein) appear, by various means, to negatively regulate cytokine signal transduction. Structurally, the SOCS proteins are composed of an N-terminal region of variable length and amino acid composition, a central SH2 domain, and a previously unrecognized C-terminal motif that we have called the SOCS box. By using the SOCS box amino acid sequence consensus, we have searched DNA databases and have identified a further 16 proteins that contain this motif. These proteins fall into five classes based on the protein motifs found N-terminal of the SOCS box. In addition to four new SOCS proteins (SOCS-4 to SOCS-7) containing an SH2 domain and a SOCS box, we describe three new families of proteins that contain either WD-40 repeats (WSB-1 and -2), SPRY domains (SSB-1 to -3) or ankyrin repeats (ASB-1 to -3) N-terminal of the SOCS box. In addition, we show that a class of small GTPases also contains a SOCS box. The expression of representative members of each class of proteins differs markedly, as does the regulation of expression by cytokines. The function of the WSB, SSB, and ASB protein families remains to be determined.

Amino Acid Sequence↗
PubMed1998

SOCS: suppressors of cytokine signalling.

Regulation of many aspects of cell behaviour occurs through the interaction of cytokines with specific cell surface receptors, resulting in the activation of cytoplasmic signal transduction pathways. Although cellular responses to cytokines are tightly controlled, few molecules have been identified which are able to switch these signals off. The suppressors of cytokine signalling (SOCS) proteins are a new family of negative regulators of cytokine signal transduction. SOCS proteins contain a variable amino-terminal region, a central Src-homology 2 (SH2) domain and a novel conserved carboxy-terminal motif termed the SOCS box. The expression of SOCS proteins is induced by cytokine. Once expressed, SOCS downregulate JAK/STAT pathways and hence the biological response. Recent studies, primarily reliant on overexpression of proteins, indicate that SOCS may be involved in modulating additional pathways, suggesting that they may play a more general role in regulating cellular responses to cytokine. The analysis of knockout mice will clarify the physiological role of SOCS in regulating cytokine responsiveness. Mutations leading to the loss of SOCS activity may give rise to cytokine hyperresponsiveness and may contribute to the development of diseases such as diabetes and cancer. Small molecule effectors which modify SOCS function may potentially be useful therapeutics for the treatment of certain diseases.

Animals↗
PubMed1997

Distinct roles for leukemia inhibitory factor receptor alpha-chain and gp130 in cell type-specific signal transduction.

Leukemia inhibitory factor (LIF) induces a variety of disparate biological responses in different cell types. These responses are thought to be mediated through the functional LIF receptor (LIFR), consisting of a heterodimeric complex of LIFR alpha-chain (LIFRalpha) and gp130. The present study investigated the relative capacity of the cytoplasmic domains of each receptor subunit to signal particular responses in several cell types. To monitor the signaling potential of LIFRalpha and gp130 individually, we constructed chimeric receptors by linking the extracellular domain of granulocyte colony-stimulating factor receptor (GCSFR) to the transmembrane and cytoplasmic regions of either LIFRalpha or gp130. Both chimeric receptors and the full-length GCSFR in expressed in M1 myeloid leukemic cells to measure differentiation induction, in embryonic stem cells to measure differentiation inhibition, and in Ba/F3 cells to measure cell proliferation. Our results demonstrated that whereas GCSFR-gp130 receptor homodimer mediated a GCSF-induced signal in all three cell types, the GCSFR-LIFRalpha receptor homodimer was only functional in embryonic stem cells. These findings suggest that the signaling potential of gp130 and LIFRalpha cytoplasmic domains may differ depending upon the tissue and cellular response initiated.

Antigens, CD↗
PubMed1997

A family of cytokine-inducible inhibitors of signalling.

Cytokines are secreted proteins that regulate important cellular responses such as proliferation and differentiation. Key events in cytokine signal transduction are well defined: cytokines induce receptor aggregation, leading to activation of members of the JAK family of cytoplasmic tyrosine kinases. In turn, members of the STAT family of transcription factors are phosphorylated, dimerize and increase the transcription of genes with STAT recognition sites in their promoters. Less is known of how cytokine signal transduction is switched off. We have cloned a complementary DNA encoding a protein SOCS-1, containing an SH2-domain, by its ability to inhibit the macrophage differentiation of M1 cells in response to interleukin-6. Expression of SOCS-1 inhibited both interleukin-6-induced receptor phosphorylation and STAT activation. We have also cloned two relatives of SOCS-1, named SOCS-2 and SOCS-3, which together with the previously described CIS form a new family of proteins. Transcription of all four SOCS genes is increased rapidly in response to interleukin-6, in vitro and in vivo, suggesting they may act in a classic negative feedback loop to regulate cytokine signal transduction.

Amino Acid Sequence↗
PubMed1996

Tyrosine residues in the granulocyte colony-stimulating factor (G-CSF) receptor mediate G-CSF-induced differentiation of murine myeloid leukemic (M1) cells.

The cytoplasmic tyrosine residues of many growth factor receptors have been shown to be important for receptor signal transduction via the recruitment of proteins containing phosphotyrosine-binding domains. This study demonstrates the importance of specific tyrosine residues in the granulocyte colony-stimulating factor (G-CSF) receptor cytoplasmic domain in G-CSF-induced macrophage cell differentiation. Site-directed mutagenesis was used to generate a series of G-CSF receptor (G-CSF-R) mutants in which the tyrosine residues were replaced with phenylalanine either singly or in combination. The mouse myeloid leukemic cell line (M1) transfected with G-CSF-R cDNA can be induced to differentiate into macrophages in response to G-CSF. The effect of the tyrosine mutations on this differentiation response was assessed by examining cell morphology and differentiation in soft agar colony assays. Although three of the four cytoplasmic tyrosine residues appeared to contribute to the differentiation response, mutation of a single residue (Tyr744) significantly reduced the ability of the M1 cells to differentiate. The STAT family of signaling molecules (Stat1, Stat3, and Stat5) were activated by G-CSF in M1 cells expressing those G-CSF-R tyrosine mutants unable to mediate G-CSF-induced differentiation. Furthermore, activation of STAT proteins was shown to occur in the absence of all four cytoplasmic tyrosine residues, suggesting an alternative mechanism for STAT activation other than direct interaction with receptor phosphotyrosines.

Animals↗
PubMed1996

Prolonged in vivo gene expression driven by a tyrosine hydroxylase promoter in a defective herpes simplex virus amplicon vector.

A 9.0-kb fragment of the tyrosine hydroxylase (TH) promoter, previously shown to direct tissue-specific expression in transgenic mice, was fused to an Escherichia coli LacZ reporter gene in a defective herpes simplex virus type-1 (HSV-1) amplicon vector (THlac). The HSV immediate early (IE) 4/5 promoter (HSVlac) was used as a control. LacZ gene expression was visualized by X-Gal histochemical and TH immunocytochemical analysis. Two days and 10 weeks after THlac injection into rat caudate nucleus (CN), X-Gal-stained cells were observed in the substantia nigra (SN) and locus ceruleus (LC) ipsilateral to the injection site. These blue cells were TH-positive neurons as evidenced by double labeling with immunocytochemistry. Moreover, the number of X-Gal+, TH+ (double-positive) neurons in the SN increased at 10 weeks as compared to that seen 2 days after THlac injection. In marked contrast, few double-positive nigral neurons were observed either 2 days or 10 weeks after direct injection of THlac into SN. However, neither nigral nor striatal injection of HSVlac resulted in prolonged gene expression. These results suggest that a neuronal, but not a viral, promoter in an HSV vector can produce cell-type-specific, prolonged, and stable gene expression following retrograde transport. In addition, THlac produced infrequent gene expression in TH-negative cells (CN and dorsal to SN) after THlac injection into CN and SN, respectively. Overall, these results suggest that in some in vivo contexts cell-type-preferred expression can be achieved by a cellular promoter in an amplicon vector. Moreover, they underscore the need for the careful and systematic study of neuronal promoters in HSV vectors.

Defective Viruses↗
PubMed1996

Neurofilament phosphorylation is modulated by myelination.

Axons undergo substantial changes in radial growth during the course of development. Recent evidence suggests that axonal diameter may be controlled by the state of neurofilament (NF) phosphorylation. Using dorsal root ganglion (DRG)-Schwann cell co-cultures, we provide direct evidence that phosphorylation of NF is regulated by myelination. NF phosphorylation increased upon myelination of DRG neurons by Schwann cells. The increase in NF phosphorylation was reflected both as an increase in immunoreactivity with the antibody SMI31, specific for phosphorylation-dependent NF epitopes, and a concomitant decrease in immunoreactivity with SMI32, specific for nonphosphorylated NF epitopes. The increase in NF phosphorylation induced by myelination in the neuron-glia co-cultures was similar to NF phosphorylation seen in sciatic nerve extracts of mice with normal myelination compared to Trembler J mouse littermates in which myelination of peripheral nerves is compromised. Using an in situ gel kinase assay, we have detected changes in individual NF kinase activities during myelination. In particular, a 35-kDa kinase activity was induced by myelination, whereas a 42-kDa kinase decreased in activity. We discuss the possibility that these and other kinases may be involved in signaling processes between neurons and glia during myelination.

Animals↗
PubMed1996

A cdc2-like kinase distinct from cdk5 is associated with neurofilaments.

An immunoprecipitation assay was used to identify protein kinases which are physically associated with neurofilaments (NF) in mouse brain extracts. Using this approach, we show that a cdc2-related kinase is associated with NF. The cdc2-related kinase was found to be distinct from cdk5 and the authentic cdc2 by a number of criteria. Firstly, it has a molecular mass on SDS-PAGE gels of 34 kDa, similar to that of cdc2, but differing from cdk5 (31 kDa). Secondly, it is not recognized by an antibody specific for cdk5. Thirdly, it is recognized by an antibody raised against the C-terminal region of authentic cdc2, but not by an antibody specific for the PSTAIRE motif. Using immunoblotting, we further show that the cdc2-related kinase copurifies with NF isolated from rat tissues. In vitro kinase assays further demonstrated that immunoprecipitated cdc2-related kinase phosphorylates recombinant NF-H protein. Phosphorylation of NF-H by the cdc2-like activity was not affected by 3 microM olomoucine but was inhibited by 10 microM of this kinase inhibitor. Phosphoamino acid analysis of in vitro phosphorylated NF-H indicates that the immunoprecipitated cdc2-related kinase phosphorylates serine residues.

Animals↗
PubMed1995

Production of monoclonal antibodies against neurofilament-associated proteins: demonstration of association with neurofilaments by a coimmunoprecipitation method.

A panel of monoclonal antibodies (MAbs) was produced against mouse brain proteins that bind to the tail domain of the neurofilament (NF) heavy (200-kDa) subunit (NF-H) in vitro. An in vivo association of the MAb ligands with NFs was confirmed by examining reactivity of the MAbs with immunoprecipitated NF-H complexes. Using this method we demonstrated association of the ligands of three of the MAbs with NFs. In contrast, glial fibrillary acidic protein and an unknown 97-kDa brain protein were not associated with NFs by this criterion. An 80-kDa doublet that coimmunoprecipitated with NF-H complexes, recognized by MAb 223, was shown by immunocytochemistry and immunoblotting to be synapsin Ia and Ib. Using a complementary approach, we confirmed an association of synapsin with NFs by demonstrating that immunoprecipitated synapsin I complexes contained NF-H and NF medium (160-kDa) subunits. MAbs 63 and 105 recognized a more complex set of proteins that had predominantly synaptic localizations. These data suggest that NFs may provide important support for attachment and/or transport of synaptic proteins in brain.

Animals↗
PubMed1994

Characterization of illudin S sensitivity in DNA repair-deficient Chinese hamster cells. Unusually high sensitivity of ERCC2 and ERCC3 DNA helicase-deficient mutants in comparison to other chemotherapeutic agents.

Illudins, novel natural products with a structure unrelated to any other known chemical, display potent in vitro and in vivo anti-cancer activity against even multi-drug resistant tumors, and are metabolically activated to an unstable intermediate that binds to DNA. The DNA damage produced by illudins, however, appears to differ from that of other known DNA damaging toxins. The sensitivity pattern of the various UV-sensitive cell lines differs from previously studied DNA cross-linking agents. Normally, the ERCC1- (excision repair cross complementing) and ERCC4-deficient cell lines are most sensitive to DNA cross-linking agents, with ERCC2-, ERCC3- and ERCC5-deficient cell lines having minimal sensitivity. With illudins the pattern is reversed, with ERCC2 and ERCC3 being the most sensitive. The sensitivity to illudins in complementation groups 1 through 3 is due to a deficiency of the ERCC1-3 gene products, as cellular drug accumulation studies revealed no differences in transport capacity or total drug accumulation. Also, a transgenic cell line in which ERCC2 activity was expressed through an expression vector regained its relative resistance to the illudins. The EM9 cell line, which displays sensitivity to monoadduct producing chemicals, was not sensitive. Thus, excision repair is involved in repair of illudin-induced damage and, unlike other anti-cancer agents, the involvement of ERCC2 and ERCC3 helicases is critical for repair to occur. The requirement for ERCC2 and ERCC3, combined with the finding that ERCC1 but not ERCC2 is upregulated in drug-resistant tumors, may explain the efficacy of illudins against drug-resistant tumors. The inhibition of DNA synthesis in cells within minutes after exposure to illudins at nanomolar concentrations may be related to the finding that the ERCC3 gene product is actually the p89 helicase component of the BTF2 (TFII) basic transcription factor and the high sensitivity of ERCC3-deficient cells to illudins.

Animals↗
PubMed1994

A 17S multiprotein form of murine cell DNA polymerase mediates polyomavirus DNA replication in vitro.

We have identified and purified a multiprotein form of DNA polymerase from the murine mammary carcinoma cell line (FM3A) using a series of centrifugation, polyethylene glycol precipitation, and ion-exchange chromatography steps. Proteins and enzymatic activities associated with this mouse cell multiprotein form of DNA polymerase include the DNA polymerases alpha and delta, DNA primase, proliferating cell nuclear antigen (PCNA), DNA ligase I, DNA helicase, and DNA topoisomerases I and II. The sedimentation coefficient of the multiprotein form of DNA polymerase is 17S, as determined by sucrose density gradient analysis. The integrity of the murine cell multiprotein form of DNA polymerase is maintained after treatment with detergents, salt, RNase, DNase, and after chromatography on DE52-cellulose, suggesting that the association of the proteins with one another is independent of nonspecific interaction with other cellular macromolecular components. Most importantly, we have demonstrated that this complex of proteins is fully competent to replicate polyomavirus DNA in vitro. This result implies that all of the cellular activities required for large T-antigen dependent in vitro polyomavirus DNA synthesis are present within the isolated 17S multiprotein form of the mouse cell DNA replication activities. A model is proposed to represent the mammalian Multiprotein DNA Replication Complex (MRC) based on the fractionation and chromatographic profiles of the individual proteins found to co-purify with the complex.

Animals↗
PubMed1993

DNA ligase III is the major high molecular weight DNA joining activity in SV40-transformed human fibroblasts: normal levels of DNA ligase III activity in Bloom syndrome cells.

The phenotypes of cultured cell lines established from individuals with Bloom syndrome (BLM), including an elevated spontaneous frequency of sister chromatid exchanges (SCEs), are consistent with a defect in DNA joining. We have investigated the levels of DNA ligase I and DNA ligase III in an SV40-transformed control and BLM fibroblast cell line, as well as clonal derivatives of the BLM cell line complemented or not for the elevated SCE phenotype. No differences in either DNA ligase I or DNA ligase III were detected in extracts from these cell lines. Furthermore, the data indicate that in dividing cultures of SV40-transformed fibroblasts, DNA ligase III contributes > 85% of high molecular weight DNA joining activity. This observation contrasts with previous studies in which DNA ligase I was reported to be the major DNA joining activity in extracts from proliferating mammalian cells.

Bloom Syndrome↗
PubMed1992

Nucleotide sequences of the HLA-DRw12 and DRw8 B1 chains from an Australian aborigine.

To gain a more detailed understanding of the molecular structure of the HLA genes in Australian aborigines, the polymorphic first-domain sequences of the DR B alleles were determined in an aborigine who was tissue typed as HLA-DRw8 and a probable DRw12; DRw52; DQw1,7. Both peripheral blood leukocytes and a lymphoblastoid cell line were reactive with the majority of DRw12-specific sera, but also with half of the DRw11-specific sera. With the use of primers specific for the conserved regions flanking the first domain, the polymerase chain reaction technique was used to amplify first-strand synthesis products prepared from the cell line. Two distinct DRB1 sequences were obtained. One was virtually identical to the reported DRw8,Dw8.3 sequence present in an Asian haplotype, differing only by a single silent nucleotide substitution at the third position of codon 36 (A to G). A second DRB allele was closely related to two recently published and nearly identical sequences for DRw12, with amino acid differences at positions 67 and 85 of the first domain. DRB RFLP studies on this cell line using the Taq I restriction enzyme indicated bands previously described for the DRw8 and DRw12 haplotypes.

Amino Acid Sequence↗