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Biomedical subjects

D Kaplan

Publications and source records attributed to D Kaplan.

At least 73 records · Page 4Linked to original sources
PubMed1996

Identification of residues that control specific binding of the Shc phosphotyrosine-binding domain to phosphotyrosine sites.

The Shc adaptor protein contains two phosphotyrosine [Tyr(P)]binding modules--an N-terminal Tyr(P) binding (PTB) domain and a C-terminal Src homology 2 (SH2) domain. We have compared the ability of the Shc PTB domain to bind the receptors for nerve growth factor and insulin, both of which contain juxtamembrane Asn-Pro-Xaa-Tyr(P) motifs implicated in PTB binding. The Shc PTB domain binds with high affinity to a phosphopeptide corresponding to the nerve growth factor receptor Tyr-490 autophosphorylation site. Analysis of individual residues within this motif indicates that the Asn at position -3 [with respect to Tyr(P)], in addition to Tyr(P), is critical for PTB binding, while the Pro at position -2 plays a less significant role. A hydrophobic amino acid 5 residues N-terminal to the Tyr(P) is also essential for high-affinity binding. In contrast, the Shc PTB domain does not bind stably to the Asn-Pro-Xaa-Tyr(P) site at Tyr-960 in the activated insulin receptor, which has a polar residue (Ser) at position -5. Substitution of this Ser at position -5 with Ile markedly increased binding of the insulin receptor Tyr-960 phosphopeptide to the PTB domain. These results suggest that while the Shc PTB domain recognizes a core sequence of Asn-Pro-Xaa-Tyr(P), its binding affinity is modulated by more N-terminal residues in the ligand, which therefore contribute to the specificity of PTB-receptor interactions. An analysis of residues in the Shc PTB domain required for binding to Tyr(P) sites identified a specific and evolutionarily conserved Arg (Arg-175) that is uniquely important for ligand binding and is potentially involved in Tyr(P) recognition.

3T3 Cells↗
PubMed1996

Nerve growth factor signal transduction in human B lymphocytes is mediated by gp140trk.

Nerve growth factor (NGF) plays an important role in the regulation of the immune system. Recent studies from this laboratory demonstrated the presence of functional NGF receptors on human B lymphocytes; in addition, NGF has been shown to enhance B lymphocyte proliferation. NGF caused both concentration- and time-dependent increases in tyrosine phosphorylation of five proteins of 140, 110, 85, 60 and 42 kDa, which were identified as phospholipase C-gamma 1, phosphatidylinositol-3 kinase and mitogen-activated protein kinase. To elucidate the contribution of the Trk family of tyrosine kinases to the phosphorylation events induced by NGF, we identified gp140trk in human B cells and in human B cell lines. Analysis of specific gp140trk immunoprecipitates indicated that addition of NGF to B cells induced a rapid increase in the tyrosine phosphorylation of gp140trk and inhibition of this phosphorylation prevented the tyrosine phosphorylation of other proteins. These data identify the central role of gp40trk in NGF signaling of human B lymphocytes.

B-Lymphocytes↗
PubMed1996

Effects of delayed administration of octreotide in acute experimental pancreatitis.

Multiple therapeutic modalities studied for acute pancreatitis often show a poor correlation between results obtained in experimental studies and results of clinical trials. One of the main reasons for this discrepancy is that in most experimental studies the drugs were administered immediately after induction of pancreatitis, whereas in the clinical setting there is almost always a delay between the onset of the disease and initiation of the treatment. We studied the effects of a delayed treatment with octreotide, the synthetic analogue of the hormone somatostatin, on acute experimental pancreatitis in rats. The disease was induced by intraparenchymal injections of 0.5 ml 5% sodium taurocholate, and octreotide (10 mg/kg/day s.c.) was started either 4 or 12 hr later. Subcutaneous saline injections were used in controls. One-half of the animals of each study group was sacrificed after 36 hr, and the following parameters were examined: pancreatic weight, plasma pH, serum calcium and amylase, and histopathological damage. The same parameters, as well as survival, were assessed after 20 days in the remaining rats. Neither intrapancreatic saline injections, nor octreotide administration without the induction of pancreatitis, caused any biochemical or histological alterations. Hypocalcemia and acidosis in pancreatitis-induced rats were improved by octreotide, but, as expected, it had no effect on amylase levels. Octreotide ameliorated pancreatic edema, intestinal dilatation, and the histopathological injury score 36 hr after induction of pancreatitis. Mortality was 40% in control animals, and only 20% in rats treated with octreotide. Overall, octreotide had beneficial effects in acute experimental pancreatitis, and was more effective when started earlier. These results indicate that octreotide may have a role in the management of acute pancreatitis.

Acute Disease↗
PubMed1996

A rapid-flow perfusion chamber for high-resolution microscopy.

Perfusion chambers employing laminar flow have dead volumes and unstirred layers which limit the minimum time required to effect a change in the local chemical environment of the sample. We have fabricated and tested a chamber capable of developing turbulent flow at reasonable flow rates of aqueous solutions. Transition to turbulence occurred at approximately equal to 1 mLs-1. To minimize dead space, a dual-exit cross-flow pattern was employed. The chamber was designed to mount on optical microscope stages for visual sample observation supplemented by a variety of techniques, such as fluorescence, light scattering and electrochemical monitoring. As indicated by fluorescence from a fluorescein-labelled protein film adherent to the chamber wall, local pH changes were produced within 200 ms. Use of the chamber is illustrated by measurements of stopped-flow kinetics in both calcium-triggered cortical granule exocytosis and influenza virus haemagglutinin-mediated cell-cell fusion.

Animals↗
PubMed1996

Parathyroid hormone resistance and B cell lymphopenia in propionic acidemia.

The mechanisms of hypocalcemia, recurrent infections and hypogammaglobulinemia associated with metabolic decompensation of propionic acidemia due to propionyl-CoA carboxylase deficiency have not been defined. A 7-week-old infant with this disorder presented with severe hypocalcemia and B cell lymphopenia during an episode of metabolic acidosis and hyperammonemia. Hypocalcemia (1.1 mmol l-1) was associated with elevated serum intact parathyroid hormone (122 ng l-1), hyperphosphatemia, hypophosphaturia and hypercalcuria, indicating parathyroid hormone resistance. B cell lymphopenia (20 cells microliters-1) was associated with transient neutropenia, anemia and subsequent hypogamma-globulinemia (IgG < 294 mg dl-1, IgM < 8 mg dl-1, IgA < 8 mg dl-1), while T cells were normal. Parathyroid hormone resistance and B cell lymphopenia resolved following treatment with hemodialysis, diet and carnitine. These complications may be due to interference with parathyroid hormone renal tubular action and B cell maturation/proliferation by accumulated organic acids.

Amino Acid Metabolism, Inborn Errors↗
PubMed1996

Herpes simplex virus type 2 inhibition of Fas ligand expression.

Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) are common human pathogens. In this report we demonstrate the capacity of HSV-2, but not HSV-1, to inhibit the activity and cell surface expression of Fas ligand, an important molecule involved in T-cell apoptosis and cell-mediated cytotoxicity. Cells infected with HSV-2 retained Fas ligand intracellularly instead of expressing it on the cell surface. Addition of anti-Fas antibodies markedly inhibited HSV-2 viral production, suggesting that the capacity of the virus to regulate Fas ligand expression, and thereby programmed cell death, may represent a powerful mechanism for the virus to enhance viral replication.

Animals↗
PubMed1996

The role of interleukin-10 in the inhibition of T-cell proliferation and apoptosis mediated by parainfluenza virus type 3.

We have previously demonstrated that parainfluenza virus type 3 (PIV3), a significant respiratory pathogen, can markedly inhibit T-cell function in vitro. We now report that the virus potently induces interleukin-10 (IL-10) production by peripheral blood mononuclear cells. The IL-10 produced contributes to viral inhibition of T-cell proliferation and protects T cells from PIV3-mediated apoptosis. These findings suggest that IL-10 is likely to play an important immunoregulatory role in PIV3 infections.

Adult↗
PubMed1996

Cytochrome P4502E1- and cytochrome P4502B1/2B2-catalyzed carbon tetrachloride metabolism: effects on signal transduction as demonstrated by altered immediate-early (c-Fos and c-Jun) gene expression and nuclear AP-1 and NF-kappa B transcription factor levels.

We have previously reported that carbon tetrachloride (CCI4) stimulates c-fos, c-jun, and Ca(2+)-activated neutral protease gene expression in rat hepatic tissue (Zawaski et al., Biochem. Biophys. Res. Comm. 197, 585-590, 1993). The proteins c-Fos and c-Jun constitute inducible transcription factors in signal transduction and regulate the transcriptional activation of a battery of genes involved in cell growth and division. The present study was initiated to characterize the role of cytochrome P450 expression and metabolic activation on the magnitude of immediate-early (i.e. c-fos and c-jun) gene expression. Animals were treated either with diallyl sulfide, N-acetylcysteine, pyridine, or phenobarbital before treatment with CCI4. Total and poly(A)+ RNA were isolated, and c-fos and c-jun mRNA levels were analyzed by Northern blot and reverse transcriptase-polymerase chain reaction analyses. Treatment of animals with CCI4 increased c-fos and c-jun mRNA levels from below the limit of detection in control tissue to intense bands within 30 min of treatment, with maximal expression monitored at 1 and 2 hr posttreatment. Treatment of animals with diallyl sulfide alone also elevated c-fos and c-jun mRNA expression to detectable levels. However, pretreatment of animals with diallyl sulfide before treatment with CCI4 produced a 76-92% decrease in c-fos and c-jun mRNA levels, relative to that monitored for CCI4-treated animals. Pretreatment with N-acetylcysteine did not affect c-fos or c-jun mRNA levels and diminished CCI4-stimulated c-fos and c-jun gene expression by 44 and 55%, respectively, relative to the immediate-early gene mRNA levels monitored in the hepatic tissue of CCI4-treated animals. Pretreatment of animals with the CYP2E1 inducer pyridine for 24 hr had only a marginal effect on c-fos mRNA levels, but increased CCI4-stimulated c-fos and c-jun mRNA levels by an additional approximately 2- to approximately 4-fold over those monitored in the uninduced hepatic tissue of CCI4-treated animals. Whereas phenobarbital treatment alone enhanced c-fos expression only marginally, CCI4 treatment of phenobarbital-pretreated animals increased c-fos expression by up to an additional approximately 8-fold and c-jun mRNA levels by up to an additional approximately 5-fold over the respective levels monitored in the hepatic tissue of CCI4-treated animals. Enhanced CYP2E1 or CYP2B1/2B2 levels after treatment with pyridine or phenobarbital elevated c-fos mRNA over untreated controls. This increase was marginal, however, and detectable only with reverse transcriptase-polymerase chain reaction. Examination of nuclear levels of the heterodimeric c-Fos and c-Jun AP-1 transcription factor complex revealed a time-dependent increase in AP-1 levels. AP-1 transcription factor binding was confirmed using competitor consensus sequences and antibody supershifts. Nuclear levels of NF-kappa B, a transcription factor complex implicated in hepatocyte proliferation and apoptotic or programmed cell death, were also examined. NF-kappa B, which consists of the p50 and p65/Rel A polypeptides, was increased in hepatic nuclear extracts at 2 and 24 hr after CCI4 administration, with a concomitant decrease in the p50 polypeptide. Thus, the magnitude of CCI4 stimulation of the immediate-early genes c-fos and c-jun is dependent on metabolic activation by the P450s, and the magnitude of the effect is dependent on the levels and isozyme composition of P450s in the tissue. Furthermore, nuclear transcription factor levels of AP-1 and NF-kappa B are elevated in response to this toxicant.

Animals↗
PubMed1995

N-acylated alpha-amino acids as novel oral delivery agents for proteins.

A series of N-acylated alpha-amino acids were synthesized and shown to improve the oral delivery of two protein drugs, salmon calcitonin (sCT) and interferon-alpha. Forty-five compounds in this series were tested in vivo in rats and primates. A significant positive correlation was found between the log P of the acylated amino acids and the decrease in serum calcium following oral dosage of sCT in rats. Such a correlation was not found for interferon-alpha. These derivatized amino acids only weakly inhibited the activity of trypsin or leucine aminopeptidase. Histological examinations of rat intestinal tissue after oral dosing of acylated amino acid/protein combinations revealed no detectable pathology.

Acylation↗
PubMed1995

Tyrosine phosphorylation and activation of STAT5, STAT3, and Janus kinases by interleukins 2 and 15.

The cytokines interleukin 2 (IL-2) and IL-15 have similar biological effects on T cells and bind common hematopoietin receptor subunits. Pathways that involve Janus kinases (JAKs) and signal transducers and activators of transcription (STATs) have been shown to be important for hematopoietin receptor signaling. In this study we identify the STAT proteins activated by IL-2 and IL-15 in human T cells. IL-2 and IL-15 rapidly induced the tyrosine phosphorylation of STAT3 and STAT5, and DNA-binding complexes containing STAT3 and STAT5 were rapidly activated by these cytokines in T cells. IL-4 induced tyrosine phosphorylation and activation of STAT3 but not STAT5. JAK1 and JAK3 were tyrosine-phosphorylated in response to IL-2 and IL-15. Hence, the JAK and STAT molecules that are activated in response to IL-2 and IL-15 are similar but differ from those induced by IL-4. These observations identify the STAT proteins activated by IL-2 and IL-15 and therefore define signaling pathways by which these T-cell growth factors may regulate gene transcription.

Base Sequence↗
PubMed1995

A conserved amino-terminal Shc domain binds to phosphotyrosine motifs in activated receptors and phosphopeptides.

BACKGROUND: Signal transduction by growth factor receptor protein-tyrosine kinases is generally initiated by autophosphorylation on tyrosine residues following ligand binding. Phosphotyrosines within activated receptors form binding sites for the Src homology 2 (SH2) domains of cytoplasmic signalling proteins. One such protein, Shc, is tyrosine phosphorylated in response to a large number of growth factors and cytokines. Phosphorylation of Shc on tyrosine residue Y317 allows binding to the SH2 domain of Grb2, and hence stimulation of the Ras pathway. Shc is therefore implicated as an adaptor protein able to couple normal and oncogenic protein-tyrosine kinases to Ras activation. Shc itself contains an SH2 domain at its carboxyl terminus, but the function of the amino-terminal half of the protein is unknown. RESULTS: We have found that the Shc amino-terminal region binds to a number of tyrosine-phosphorylated proteins in v-src-transformed cells. This domain also bound directly to the activated epidermal growth factor (EGF) receptor. A phosphotyrosine (pY)-containing peptide modeled after the Shc-binding site in polyoma middle T antigen (LLSNPTpYSVMRSK) was able to compete efficiently with the activated EGF receptor for binding to the Shc amino terminus. This competition was dependent on phosphorylation of the tyrosine residue within the peptide, and was abrogated by deletion of the leucine residue at position -5. The Shc amino-terminal domain also bound to the autophosphorylated nerve growth factor receptor (Trk), but bound significantly less well to a mutant receptor in which tyrosine Y490 in the receptor's Shc-binding site had been substituted by phenylalanine. CONCLUSION: These data implicate the amino-terminal region of Shc in binding to activated receptors and other tyrosine-phosphorylated proteins. Binding appears to be specific for phosphorylated tyrosine residues within the sequence NPXpY, which is conserved in many Shc-binding sites. The Shc amino-terminal region bears only very limited sequence identify to known SH2 domains, suggesting that it represents a new class of phosphotyrosine-binding modules. Consistent with this view, the amino-terminal Shc domain is highly conserved in a Drosophila Shc homologue. Binding of Shc to activated receptors through its amino terminus could leave the carboxy-terminal SH2 domain free for other interactions. In this way, Shc may function as an adaptor protein to bring two tyrosine-phosphorylated proteins together.

3T3 Cells↗
PubMed1995

Management of recurrent malignant pleural effusions. The complementary role talc pleurodesis and pleuroperitoneal shunting.

BACKGROUND: Recurrent pleural effusions in patients with advanced cancer is a common problem that causes significant morbidity and can negatively affect patients' quality of life for their remaining months. Several palliative treatment options are available. METHODS: The results of a 10-year experience with 180 patients referred for the surgical palliation of their condition were retrospectively reviewed. Their mean age was 60 years (range, 20-90 years). One hundred and thirty-four patients (74%) had been treated before referral with one or more of the following modalities: repeated needle thoracocentesis (87 patients), tube thoracostomy (24 patients), chemical or biologic pleurodesis (22 patients), and pleurectomy (1 patient). One hundred and seventeen patients demonstrated full lung expansion at thoracoscopy/mini-thoracotomy and underwent talc pleurodesis, whereas the other 63 patients had the "trapped lung syndrome" and required the insertion of a pleuroperitoneal shunt (Denver, Biomedical, Inc). RESULTS: There were no intraoperative deaths and the early death rate was 5.9% for the talc pleurodesis group and 3.2% for the group that received shunts. The mean hospital stay for the patients receiving talc and shunts was 7.3 days (range, 3-15 days) and 5.9 days (range, 2-12 days), respectively. Follow-up data were available in 60% of the patients and showed that effective palliation was achieved in more than 95% of patients in each group. There were eight patients (12%) with blocked shunts (five requiring replacement or renovation and three requiring removal and open drainage) at 1 week to 4 months after insertion. Two patients (one from each group) required one further episode of treatment by thoracocentesis. The median survival for the talc and shunt groups was 4.9 months (range, 1-36 months) and 5.4 months (range, 1-53 months). Patients with effusions because of secondary breast carcinoma or lymphomas survived the longest. CONCLUSION: In patients with malignant pleural effusions in whom pleurodesis is precluded by limited lung expansion, effective palliation can be achieved by pleuro-peritoneal shunt insertion.

Adult↗
PubMed1995

Self-association of interleukin 2 bound to its receptor.

Although radioiodinated interleukin 2 (IL-2) has been used to define the binding characteristics of the cytokine to the alpha chain of the receptor complex, we have found that unsubstituted IL-2 behaves differently. Whereas previous investigations with radioiodinated IL-2 have shown binding to the alpha chain with a Kd of 10 nM, we show that unsubstituted IL-2 binds to the alpha chain but does not reach saturation between 100 and 1000 nM. The explanation for the discrepancy between the analysis of radioiodinated and unsubstituted cytokine involves the propensity of unsubstituted IL-2 for self-association, a property that is abrogated by radioiodination. The functional relevance of our findings is indicated by the different capacities of unsubstituted and iodinated cytokine to induce prolonged proliferation of human T lymphocytes.

Artifacts↗
PubMed1995

Prevalence of anti-R-13 antibodies in human Trypanosoma cruzi infection.

Infection with Trypanosoma cruzi develops in three phases: acute, indeterminate or asymptomatic, and chronic phase (with cardiac or digestive manifestations). Moreover, transmission may occur from infected mothers to newborn, the so-called congenital form. In the present study, humoral responses against T. cruzi total extract and against the 13 amino acid peptide named R-13 derived from the parasite ribosomal P protein, previously described as a possible marker of chronic Chagas heart disease, were determined in chagasic patients and in blood bank donors from endemic areas. While in sera from acute phase, only IgM anti-T.cruzi response was observed, both IgM and IgG anti-T. cruzi antibodies were detected in sera from congenitally infected newborns. The percentage of positive response in sera from blood bank donors was relatively high in endemic regions. Antibodies against the R-13 peptide were present in a large proportion of cardiac chagasic patients but were totally lacking in patients with digestive form of Chagas' disease. Furthermore, anti-R-13 positive responses were detected in congenitally infected newborns.

Acute Disease↗
PubMed1995

Specific inhibition of granzyme B by parainfluenza virus type 3.

T-cell-mediated cytotoxicity is an important means of defense against viral pathogens; however, several viruses possess mechanisms to disrupt cytotoxicity, thereby allowing them to avoid immune clearance. These viruses have been shown to inhibit cytotoxicity by interfering with the capacity of T lymphocytes to specifically recognize infected cells. An alternative mechanism for virally induced cytotoxic dysfunction is identified in this report. We show that parainfluenza virus type 3, a negative-stranded RNA virus, can inhibit cytotoxicity by causing a defect in the cytotoxic effector apparatus. This defect is identified as a selective inhibition of granzyme B mRNA.

Adult↗
PubMed1995

The role of the NPXY motif in the insulin receptor in tyrosine phosphorylation of insulin receptor substrate-1 and Shc.

The insulin receptor phosphorylates insulin receptor substrate-1 (IRS-1) and Shc on tyrosine residues, both of which associate with the protein-abundant Src homology/growth factor receptor-bound protein 2(ASH/GRB2) leading to p21ras activation. Juxtamembrane Tyr960 of the insulin receptor required for tyrosine phosphorylation of both IRS-1 and Shc is contained in the NPXY motif, which is also present in other tyrosine kinase receptors and oncogene products. In this study, the role of this motif in insulin's signaling was examined in Chinese hamster ovary cells expressing insulin receptors with mutations in this motif. All alterations in Tyr960 examined decreased tyrosine phosphorylation of both IRS-1 and Shc to a similar extent. The replacements of Asn957 and the deletion of NPE impaired tyrosine phosphorylation of Shc and IRS-1, although tyrosine phosphorylation of Shc was more severely affected than that of IRS-1. The amount of ASH/GRB2 bound to IRS-1 and Shc in vitro and in vivo was also decreased in these cells. These data suggest that the NPXY motif in the insulin receptor is important for tyrosine phosphorylation of both IRS-1 and Shc as well as subsequent signaling.

Amino Acid Sequence↗