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Biomedical subjects

K A Siminovitch

Publications and source records attributed to K A Siminovitch.

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

Structure of Cdc42 in complex with the GTPase-binding domain of the 'Wiskott-Aldrich syndrome' protein.

The Rho-family GTP-hydrolysing proteins (GTPases), Cdc42, Rac and Rho, act as molecular switches in signalling pathways that regulate cytoskeletal architecture, gene expression and progression of the cell cycle. Cdc42 and Rac transmit many signals through GTP-dependent binding to effector proteins containing a Cdc42/Rac-interactive-binding (CRIB) motif. One such effector, the Wiskott-Aldrich syndrome protein (WASP), is postulated to link activation of Cdc42 directly to the rearrangement of actin. Human mutations in WASP cause severe defects in haematopoletic cell function, leading to clinical symptoms of thrombocytopenia, immunodeficiency and eczema. Here we report the solution structure of a complex between activated Cdc42 and a minimal GTPase-binding domain (GBD) from WASP. An extended amino-terminal GBD peptide that includes the CRIB motif contacts the switch I, beta2 and alpha5 regions of Cdc42. A carboxy-terminal beta-hairpin and alpha-helix pack against switch II. The Phe-X-His-X2-His portion of the CRIB motif and the alpha-helix appear to mediate sensitivity to the nucleotide switch through contacts to residues 36-40 of Cdc42. Discrimination between the Rho-family members is likely to be governed by GBD contacts to the switch I and alpha5 regions of the GTPases. Structural and biochemical data suggest that GBD-sequence divergence outside the CRIB motif may reflect additional regulatory interactions with functional domains that are specific to individual effectors.

Amino Acid Sequence↗
PubMed1999

Negative regulation of myeloid cell proliferation and function by the SH2 domain-containing tyrosine phosphatase-1.

The SH2 domain containing tyrosine phosphatase SHP-1 has been implicated in the regulation of a multiplicity of signaling pathways involved in hemopoietic cell growth, differentiation, and activation. A pivotal contribution of SHP-1 in the modulation of myeloid cell signaling cascades has been revealed by the demonstration that SHP-1 gene mutation is responsible for the overexpansion and inappropriate activation of myelomonocytic populations in motheaten mice. To investigate the role of SHP-1 in regulation of myeloid leukocytes, an HA epitope-tagged dominant negative (interfering) SHP-1 (SHP-1C453S) was expressed in the myelo-monocytic cell line U937 using the pcDNA3 vector. Overexpression of this protein in SHP-1C453S transfectants was demonstrated by Western blot analysis and by detection of decreased specific activity. Growth, proliferation, and IL-3-induced proliferative responses were substantially increased in the SHP-1C453S-overexpressing cells relative to those in control cells. The results of cell cycle analysis also revealed that the proportion of cells overexpressing SHP-1C453S in S phase was greater than that of control cells. The SHP-1C453S-expressing cells also displayed diminished rates of apoptosis as detected by flow cytometric analysis of propidium iodide-stained cells and terminal deoxynucleotidyltransferase-mediated fluorescein-dUTP nick end-labeling assay. While motility and phagocytosis were not affected by SHP-1C453S overexpression, adhesion and the oxidative burst in response to PMA were enhanced in the SHP-1C453S compared with those in the vector alone transfectants. Taken together, these results suggest that SHP-1 exerts an important negative regulatory influence on cell proliferation and activation while promoting spontaneous cell death in myeloid cells.

Animals↗
PubMed1999

Leishmania-induced increases in activation of macrophage SHP-1 tyrosine phosphatase are associated with impaired IFN-gamma-triggered JAK2 activation.

Leishmania-induced macrophage (Mphi) dysfunctions have been correlated with altered signaling events. Recent findings from our laboratory suggest that modulation of host protein tyrosine phosphatase (PTP) following Leishmania infection could lead to these Mphi defects. To address this issue, Mphi PTP activity and IFN-gamma-inducible signaling events were evaluated in Leishmania donovani (Ld)-infected cells. We observed that Ld promastigotes can rapidly trigger host PTP activity simultaneously with dephosphorylation of Mphi protein tyrosyl residues and inhibition of protein tyrosine kinase (PTK). Our results further revealed that Mphi SHP-1 PTP was rapidly activated by the infection. This Ld-evoked signaling alteration was reflected by absence of IFN-gamma-induced intracellular phosphorylation. IFN-gamma-inducible JAK2 PTK phosphorylation was also markedly diminished in Ld-infected cells. We also observed that co-immunoprecipitation of JAK2 with SHP-1 was considerably higher in infected as compared to uninfected cells. Altogether, these results suggest that SHP-1-mediated JAK2 dephosphorylation triggered by Leishmania is partly responsible for abnormal Mphi IFN-gamma signaling and represent an important mechanism supporting persistent parasitic infection.

Animals↗
PubMed1999

Molecular intensive care medicine.

The development of methods for the analysis of gene structure and function, referred to as recombinant DNA (deoxyribonucleic acid) technology, has created unprecedented opportunities for significantly improving the prevention or treatment of human diseases. Both practitioners working in this field and interested observers cannot fail to recognize that the remarkable progress in understanding disease pathogenesis has placed us on the threshold of a new, revolutionary era of clinical practice. In this context, molecular medicine--that is, the application of molecular biology to elucidating the causes and potential cures of disease, has become a major thrust of research at virtually all medical schools. Incorporating the techniques of molecular biology into the research arsenal of the physician should provide new opportunities to dissect out and define the reversible and irreversible intracellular processes giving rise to acute respiratory distress syndrome, sepsis, septic shock, or multiple system organ failure, the major causes of mortality in most intensive care units.

Critical Care↗
PubMed1999

Gene therapy and dermatology: more than just skin deep.

BACKGROUND: Recent advances in the molecular characterization of dermatologic disease have substantively augmented the understanding of the pathogenetic processes underlying disorders of the skin. This new knowledge coupled with progress in gene delivery technologies has paved the way for introducing cutaneous gene therapy into the dermatologic therapeutic armamentorium. OBJECTIVE: This review article includes an overview of the current strategies for delivery of gene therapy with an emphasis on the potential role of cutaneous gene delivery in the treatment of skin and systemic diseases. CONCLUSIONS: Accessibility for gene delivery, clinical evaluation, and topical modulation of gene expression render the skin a very attractive tissue for therapeutic gene delivery. However, there are several key hurdles to be overcome before cutaneous gene therapy becomes a viable clinical option. These include difficulties in inducing sustained expression of the desired gene in vivo, the challenge of targeting genes to long-lived stem cells, and the difficulty in achieving specific and uniform transfer to different compartments of the skin. However, these problems are not insurmountable and will likely be resolved in conjunction with ongoing advances in delineating gene expression profiles and other molecular properties of the skin, strategies for stem cell isolation, and improved approaches to regulating gene delivery and expression. These advances should create the framework for translating the enormous potential of cutaneous gene therapy into the clinical arena and, thereby, substantively improving the management of both cutaneous and systemic disease.

Animals↗
PubMed1998

The major SHP-1-binding, tyrosine-phosphorylated protein in macrophages is a member of the KIR/LIR family and an SHP-1 substrate.

The SH2 domain-containing cytoplasmic protein tyrosine phosphatase, SHP-1, negatively regulates hematopoietic cell signaling. SHP-1 is associated with a tyrosine phosphorylated, plasma membrane-spanning glycoprotein, pp130, in colony stimulating factor-1 stimulated or unstimulated macrophages. This association is phosphotyrosine dependent and is mediated by the amino-terminal SH2 domain of SHP-1. pp130 behaves as a substrate of SHP-1 in vitro and is hyperphosphorylated on tyrosine in SHP-1 deficient macrophages from viable-motheaten mice. Co-immunoprecipitation data indicate that pp130 is the product of the mouse p91/PIR-B gene that encodes a member of the killer cell inhibitory receptor (KIR)/leukocyte immunoglobulin-like receptor (LIR) family. By analogy to the KIRs, p91/PIR-B may represent a novel class of macrophage receptors which act to suppress macrophage activation. These observations identify SHP-1 interactions with and regulation of p91/PIR-B as a potential mechanism for inhibiting the signaling cascades linking extracellular stimuli to macrophage activation and/or development.

Amino Acid Sequence↗
PubMed1998

Regulation of B cell signal transduction by SH2-containing protein-tyrosine phosphatases.

Tyrosyl phosphorylation plays a key role in B lymphocyte signaling. The mechanisms by which protein tyrosine kinases (PTKs) regulate signaling pathways in B cells have been investigated extensively. More recently, attention has turned to the protein--tryosine phosphatases (PTPs), particularly those containing SH2 domains. SHP-1 has been shown to be a critical regulator of antigen receptor signaling, acting, at least in part, via inhibitory co-receptors containing SHP-1 binding sites. These studies have been aided considerably by the analysis of mice carrying naturally-arising mutations in the SHP-1 gene as well as mice bearing targeted mutations in other components of Be cells signaling pathways. The function of SHP-2 in B cells in less clear, although studies in other cell systems suggests that it may play a signal-enhancing role.

Animals↗
PubMed1998

Absence of linkage between inflammatory bowel disease and selected loci on chromosomes 3, 7, 12, and 16.

BACKGROUND & AIMS: Linkage data derived from genome-wide scans of inflammatory bowel disease (IBD) sibling-pair families have identified 4 loci on chromosomes 3, 7, 12, and 16 as potential sites for IBD susceptibility genes. The aim of this study was to investigate whether linkage analysis of another independently collected set of sibling pairs with IBD would provide further evidence of linkage between these previously reported loci and IBD. METHODS: Using the MAPMAKER/SIBS program, the segregation of 21 microsatellite marker loci spanning the 4 putative IBD gene loci was analyzed in a study population comprising 161 families with 114 Crohn's disease, 36 ulcerative colitis, and 50 mixed IBD sibling pairs from the Greater Toronto area. RESULTS: The results of multipoint linkage analysis showed no evidence for linkage between IBD and each of the 21 marker loci studied; the logarithm of odds scores in all instances were less than 0.8. These linkage data were found, by exclusion mapping analysis, to exclude values of lambdas ranging from 1.5 to 3.0, depending on the locus evaluated. CONCLUSIONS: The loci previously suggested as representing IBD susceptibility loci are not linked to IBD in the Toronto population examined in this analysis.

Chromosome Mapping↗
PubMed1998

SHP-1 binds and negatively modulates the c-Kit receptor by interaction with tyrosine 569 in the c-Kit juxtamembrane domain.

The SH2 domain-containing SHP-1 tyrosine phosphatase has been shown to negatively regulate a broad spectrum of growth factor- and cytokine-driven mitogenic signaling pathways. Included among these is the cascade of intracellular events evoked by stem cell factor binding to c-Kit, a tyrosine kinase receptor which associates with and is dephosphorylated by SHP-1. Using a series of glutathione S-transferase (GST) fusion proteins containing either tyrosine-phosphorylated segments of the c-Kit cytosolic region or the SH2 domains of SHP-1, we have shown that SHP-1 interacts with c-Kit by binding selectively to the phosphorylated c-Kit juxtamembrane region and that the association of c-Kit with the larger of the two SHP-1 isoforms may be mediated through either the N-terminal or C-terminal SHP-1 SH2 domain. The results of binding assays with mutagenized GST-Kit juxtamembrane fusion proteins and competitive inhibition assays with phosphopeptides encompassing each c-Kit juxtamembrane region identified the tyrosine residue at position 569 as the major site for binding of SHP-1 to c-Kit and suggested that tyrosine 567 contributes to, but is not required for, this interaction. By analysis of Ba/F3 cells retrovirally transduced to express c-Kit receptors, phenylalanine substitution of c-Kit tyrosine residue 569 was shown to be associated with disruption of c-Kit-SHP-1 binding and induction of hyperproliferative responses to stem cell factor. Although phenylalanine substitution of c-Kit tyrosine residue 567 in the Ba/F3-c-Kit cells did not alter SHP-1 binding to c-Kit, the capacity of a second c-Kit-binding tyrosine phosphatase, SHP-2, to associate with c-Kit was markedly reduced, and the cells again showed hyperproliferative responses to stem cell factor. These data therefore identify SHP-1 binding to tyrosine 569 on c-Kit as an interaction pivotal to SHP-1 inhibitory effects on c-Kit signaling, but they indicate as well that cytosolic protein tyrosine phosphatases other than SHP-1 may also negatively regulate the coupling of c-Kit engagement to proliferation.

Cell Membrane↗
PubMed1997

Estimating the strength of genetic effects: a comparison of maximum likelihood and transmission disequilibrium methods in the study of ankylosing spondylitis.

Here we compare several methods for evaluating associations in the analysis of family data. We applied the transmission disequilibrium test (TDT), and logistic regression, familial aggregation, and segregation analyses for the study of Ankylosing Spondylitis. All methods detected effects from B27 upon susceptibility for Ankylosing Spondylitis. However, the TDT was unable to detect effects from B40 and also could not include sex as a covariate during the analysis. Conditioning or not conditioning the data for the ascertainment event led to little difference in the estimated covariate effects, although the genetic model for residual effects not due to the covariates did change. Results from these studies provide further characterization of the relationship between HLA B27, B40, and sex upon risk for developing Ankylosing Spondylitis. We also identified potential effects from further genetic susceptibility.

Adolescent↗
PubMed1997

Src kinase activity is regulated by the SHP-1 protein-tyrosine phosphatase.

Activation of the cellular Src tyrosine kinase depends upon dephosphorylation of the carboxyl-terminal inhibitory tyrosine phosphorylation site. Herein we show that Src isolated from human platelets and Jurkat T cells is preferentially dephosphorylated at its inhibitory phosphotyrosine site by the SHP-1 tyrosine phosphatase. The data also revealed association of Src with SHP-1 in both platelets and lymphocytes and the capacity of Src to phosphorylate SHP-1 and interact with the SHP-1 NH2-terminal SH2 domain in vitro. Analysis of Src activity in thymocytes from SHP-1-deficient motheaten and viable motheaten mice revealed this kinase activity to be substantially lower than that detected in wild-type thymocytes, but to be enhanced by in vitro exposure to SHP-1. Similarly, immunoblotting analysis of thymocyte Src expression before and after selective depletion of active Src protein indicated that the proportion of active relative to inactive Src protein is markedly reduced in motheaten compared with wild-type cells. These observations, together with the finding of reduced Src activity in HEY cells expressing a dominant negative form of SHP-1, provide compelling evidence that SHP-1 functions include the positive regulation of Src activation.

Animals↗
PubMed1997

The motheaten mutation rescues B cell signaling and development in CD45-deficient mice.

The cytosolic SHP-1 and transmembrane CD45 protein tyrosine phosphatases (PTP) play critical roles in regulating signal transduction via the B cell antigen receptor (BCR). These PTPs differ, however, in their effects on BCR function. For example, BCR-mediated mitogenesis is essentially ablated in mice lacking CD45 (CD45(-)), but is enhanced in SHP-1-deficient motheaten (me) and viable motheaten (mev) mice. To determine whether these PTPs act independently or coordinately in modulating the physiologic outcome of BCR engagement, we assessed B cell development and signaling in CD45-deficient mev (CD45-/SHP-1-) mice. Here we report that the CD45-/SHP-1-) cells undergo appropriate induction of protein kinase activity, mitogen-activated protein kinase activation, and proliferative responses after BCR aggregation. However, BCR-elicited increases in the tyrosine phosphorylation of several SHP-1-associated phosphoproteins, including CD19, were substantially enhanced in CD45-/SHP-1-, compared to wild-type and CD45- cells. In addition, we observed that the patterns of cell surface expression of mu, delta, and CD5, which distinguish the PTP-deficient from normal mice, are largely restored to normal levels in the double mutant animals. These findings indicate a critical role for the balance of SHP-1 and CD45 activities in determining the outcome of BCR stimulation and suggest that these PTPs act in a coordinate fashion to couple antigen receptor engagement to B cell activation and maturation.

Animals↗
PubMed1997

Regulation of Src homology 2-containing tyrosine phosphatase 1 during activation of human neutrophils. Role of protein kinase C.

The tyrosine phosphorylation of several proteins induced in neutrophils by soluble and particulate stimuli is thought to be crucial for initiating antimicrobial responses. Although activation of tyrosine kinases is thought to mediate this event, the role of tyrosine phosphatases in the initiation and modulation of neutrophil responses remains largely undefined. We investigated the role of Src homology 2-containing tyrosine phosphatase 1 (SHP-1; also known as protein tyrosine phosphatase 1C (PTP1C), hematopoetic cell phosphatase, PTP-N6, and SHPTP-1), a phosphatase expressed primarily in hemopoietic cells, in the activation of human neutrophils. SHP-1 mRNA and protein were detected in these cells, and the enzyme was found to be predominantly localized to the cytosol in unstimulated cells. Following stimulation with neutrophil agonists such as phorbol ester, chemotactic peptide, or opsonized zymosan, a fraction of the phosphatase redistributed to the cytoskeleton. Agonist treatment also induced significant decreases (30-60%) in SHP-1 activity, which correlated temporally with increases in the cellular phosphotyrosine content. Phosphorylation of SHP-1 on serine residues was associated with the inhibition of its enzymatic activity, suggesting a causal relationship. Accordingly, both the agonist-evoked phosphorylation of SHP-1 and the inhibition of its catalytic activity were blocked by treatment with bisindolylmaleimide I, a potent and specific inhibitor of protein kinase C (PKC) activity. Immunoprecipitated SHP-1 was found to be phosphorylated efficiently by purified PKC in vitro. Such phosphorylation also caused a decrease in the phosphatase activity of SHP-1. Together, these data suggest that inhibition of SHP-1 by PKC-mediated serine phosphorylation plays a role in facilitating the accumulation of tyrosine-phosphorylated proteins following neutrophil stimulation. These findings provide a new link between the PKC and tyrosine phosphorylation branches of the signaling cascade that triggers antimicrobial responses in human neutrophils.

Enzyme Activation↗
PubMed1997

Protein tyrosine phosphatase roles in the regulation of lymphocyte signaling.

Tyrosine phosphorylation-based signaling cascades represent an integral component of the signaling circuitry connecting extracellular stimuli to cell response. As the molecular elements which drive such cascades have become increasingly well-characterized, appreciation has grown for the critical roles played by protein tyrosine phosphatases (PTPs) in intracellular signal relay and for the capacity of PTPs to act not only as a counterbalance for protein kinase activities, but also as pivotal enzymes in directing and modulating signal relay and the translation of given stimuli to cell behaviour. PTP function has been particularly well studied in relation to lymphocyte antigen receptor signaling and the results of these studies have provided many novel and significant insights into the biochemical mechanisms whereby PTPs participate in the integration and interpretation of the complex transmembrane stimulatory signals driving cell function and development.

Animals↗
PubMed1997

Preliminary report on the Mount Sinai Hospital Inflammatory Bowel Disease Genetics Project.

BACKGROUND: Although the etiology of inflammatory bowel disease (IBD) is unknown, there is increasing evidence that genetic predisposition plays a major etiologic role. To provide the framework for gene identification using a positional cloning approach, ascertainment of families with multiple affected members and careful documentation of pedigrees are essential. OBJECTIVE: To report the initial findings of the IBD Genetics Project of the Mount Sinai Hospital IBD Research Unit. METHODS: All records of patients with ulcerative colitis and Crohn's disease followed at the Mount Sinai Hospital IBD Unit were reviewed. A questionnaire was sent to all patients to ascertain those with a family history of IBD. Patients with a presumed family history were contacted by a research assistant, and after confirmation of diagnosis, relevant clinical information, pedigrees, and consent to contact family members were obtained. Blood for DNA and cell line preparation were collected from affected and nonaffected family members. RESULTS: Of 2,504 patients registered in the IBD database, 231 (9.2 percent) were found to have an affected family member: 96 of 964 (10 percent) with Crohn's disease (CD) and 135 of 1,540 (8.8 percent) with ulcerative colitis (UC). A mean of 2.4 family members were affected. In families in which the proband had CD, 82.3 percent had only two affected family members, 78.1 percent had only family members affected with CD, and 82.3 percent had only first-degree family members affected. In families in which the proband had UC, 70.4 percent had only two affected family members, 71.1 percent had only family members affected with UC, and 65.2 percent had only first-degree family members affected. In the 231 families, there were 103 sibling pairs: 46 percent with CD, 28 percent with UC, and 26 percent with CD/UC. CONCLUSION: These data suggest that approximately 10 percent of IBD patients have affected family members, with the rate being similar in UC and CD. Future research is directed to genome scanning and linkage analysis in this cohort of patients.

Colitis, Ulcerative↗
PubMed1997

Flow cytometric analysis of platelets from children with the Wiskott-Aldrich syndrome reveals defects in platelet development, activation and structure.

The pathophysiology of platelet dysfunction in the Wiskott-Aldrich immune deficiency syndrome (WAS) remains unclear. Using flow cytometry, we have characterized the functional properties of platelets from 10 children with WAS. Patients with WAS had thrombocytopenia, small platelets, increased platelet-associated IgG and reduced platelet-dense granule content. Levels of reticulated 'young' platelets were normal in the WAS patients. Although the mean numbers of platelet glycoprotein (GP) Ib, GPIIbIIIa and GPIV molecules per platelet appeared lower in WAS patients than in healthy controls, analysis of similar-sized platelets revealed the mean number of GPIb molecules per platelet to be comparable in patients and normal controls. Surface GPIIbIIIa and GPIV expression was, however, significantly lower on the WAS platelets than on normal platelets. Compared with normal platelets, WAS platelets showed a reduced ability to modulate GPIIbIIIa expression following thrombin stimulation. In addition, thrombin- and ADP-induced expression of CD62P and CD63 was defective in WAS platelets. Phallacidin staining of the WAS platelets revealed less F-actin content than in normal platelets. Together, these data suggest that the reduced platelet number and function in WAS reflects, at least in part, a defect in bone marrow production as well as an intrinsic platelet abnormality.

Actins↗