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S Mahajan

Publications and source records attributed to S Mahajan.

At least 55 records · Page 3Linked to original sources
PubMed1996

Specific association of tyrosine-phosphorylated c-Cbl with Fyn tyrosine kinase in T cells.

Fyn is a Src family protein-tyrosine kinase functionally associated with the T-cell antigen receptor (TcR)/CD3 receptor complex. We have demonstrated earlier that the TcR/CD3-induced activation of Fyn results in tyrosine phosphorylation of several Fyn-associated proteins, including a protein of 116 kDa. In this report, we identify the Fyn-associated 116-kDa phosphoprotein (p116) as c-Cbl. The identity of p116 has been demonstrated by its specific reactivity with anti-Cbl and similarity of phosphopeptides generated by V8 proteolysis of phospho-Cbl and p116. We demonstrate here that the association of Fyn and c-Cbl is direct and does not require the presence of other proteins. We also demonstrate that Fyn is the Src family kinase that preferentially interacts with c-Cbl in T cells. The fraction of c-Cbl capable of coprecipitating with Fyn is increased by TcR/CD3 ligation. This increase is likely due to the involvement of Fyn SH2 in the interactions between Fyn and tyrosine-phosphorylated c-Cbl.

Clone Cells↗
PubMed1996

Nla and Nlb of peanut stripe potyvirus are present in the nucleus of infected cells, but do not form inclusions.

We investigated, by immunological and gene-fusion methods, whether the failure of peanut stripe potyvirus (PStV)-encoded nuclear inclusion proteins a (Nla) and b (Nlb) to form nuclear inclusions is due to the lack of their in vivo accumulation or the inability of one or both proteins to be transported into the nucleus Nla domains (Nla-VPg and Nla-proteinase), full-length Nlb, and full-length cylindrical inclusion (CI) protein of PStV were cloned, expressed in Escherichia coli, and used for antisera production. Immunoblot analysis of accumulation of Nla, Nlb, and CI in time course experiments revealed that they accumulated to similar levels in PStV-infected Nicotiana benthamiana. In immunocytochemical studies with electron microscopy, antiserum against Nla-VPg, Nla-Pro, and Nlb specifically labeled Nla and Nlb proteins throughout the nuclei of PStV-infected cells, in the absence of nuclear inclusions. Translational fusions were made between Nla and Nlb to either the green fluorescence protein or the beta-glucuronidase in vectors for transient gene expression or stable expression in transgenic plants respectively. Fusion proteins containing Nla accumulated in the nucleus, whereas fusion proteins containing Nlb accumulated in a punctate pattern in the cytoplasm. These data indicate that at least Nla possesses a nuclear localization signal.

Animals↗
PubMed1996

BTK as a mediator of radiation-induced apoptosis in DT-40 lymphoma B cells.

Bruton's tyrosine kinase (BTK) is a member of the SRC-related TEC family of protein tyrosine kinases (PTKs). DT-40 lymphoma B cells, rendered BTK-deficient through targeted disruption of the btk gene by homologous recombination knockout, did not undergo radiation-induced apoptosis, but cells with disrupted lyn or syk genes did. Introduction of the wild-type, or a SRC homology 2 domain or a plecstrin homology domain mutant (but not a kinase domain mutant), human btk gene into BTK-deficient cells restored the apoptotic response to radiation. Thus, BTK is the PTK responsible for triggering radiation-induced apoptosis of lymphoma B cells, and its kinase domain is indispensable for the apoptotic response.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1996

Site-directed mutagenesis of monocyte chemoattractant protein-1 identifies two regions of the polypeptide essential for biological activity.

Monocyte chemoattractant protein-1 (MCP-1) mediates monocyte migration into tissues in inflammatory diseases and atherosclerosis. We have investigated structure-activity relationships for human MCP-1. Mutations were introduced based upon differences between MCP-1 and the structurally related but functionally distinct molecule interleukin-8 (IL-8). Mutant proteins produced using the baculovirus/insect cell expression system were purified and their ability to stimulate monocyte chemotaxis and elevation of intracellular calcium in THP-1 monocytic leukaemia cells was measured. Two regions in MCP-1 were identified as important for its biological activity. One region consists of the sequence Thr-Cys-Cys-Tyr (amino acids 10-13). Point mutations of Thr-10 to Arg and Tyr-13 to Ile greatly lowered MCP-1 activity. The second functionally important region is formed by Ser-34 and Lys-35. Insertion of a Pro between these two residues, or their substitution by the sequence Gly-Pro-His, caused nearly complete loss of MCP-1 activity. Competition binding experiments showed that the mutations that affected activity also lowered the ability to compete with wild-type MCP-1 for receptors on THP-1 cells. Point mutations at positions 8, 15, 30, 37, 38 and 68 had little effect on MCP-1 activity. The important regions that we have identified in MCP-1 correspond with previously identified functionally important regions of IL-8, suggesting that the two molecules bind to their respective receptors by similar contacts.

Amino Acid Sequence↗
PubMed1996

The shocking lung mass: a case report.

Two years after implantation of an ICD with epicardial patch leads, a patient presented with hemoptysis. The posterior left ventricular patch was found to have eroded into lingular segmental bronchus. During thoracotomy, the hardware was removed and the bronchus successfully repaired.

Aged↗
PubMed1996

Roles of the sequence encoding tobacco etch virus capsid protein in genome amplification: requirements for the translation process and a cis-active element.

The roles of the capsid protein (CP) and the CP coding sequence of tobacco etch potyvirus (TEV) in genome amplification were analyzed. A series of frameshift-stop codon mutations that interrupted translation of the CP coding sequence at various positions were introduced into the TEV genome. A series of 3' deletion mutants that lacked the CP coding sequence beyond each of the frameshift-stop codon mutations were also produced. In addition, a series of 5' CP deletion mutants were generated. Amplification of genomes containing either frameshift-stop codon insertions after codons 1, 59, 103, and 138 or genomes containing the corresponding 3' deletions of the CP coding sequence was reduced by 100- to 1,000-fold relative to that of the parental genome in inoculated protoplasts. In contrast, a mutant containing a frameshift-stop codon after CP position 189 was amplified to 27% of the level of the parental virus, but the corresponding 3' deletion mutant lacking codons 190 to 261 was nonviable. Deletion mutants lacking CP codons 2 to 100, 2 to 150, 2 to 189, and 2 to 210 were amplified relatively efficiently in protoplasts, but a deletion mutant lacking codons 2 to 230 was nonviable. None of the amplification-defective frameshift-stop codon or deletion mutants was rescued in transgenic cells expressing TEV CP, although the transgenic CP was able to rescue intercellular movement defects of replication-competent CP mutants. Coupled with previous results, these data led to the conclusions that (i) TEV genome amplification requires translation to a position between CP codons 138 and 189 but does not require the CP product and (ii) the TEV CP coding sequence contains a cis-active RNA element between codons 211 and 246. The implications of these findings on mechanisms of RNA replication and genome evolution are discussed.

Capsid↗
PubMed1995

Reconstitution of the B cell antigen receptor signaling components in COS cells.

To elucidate interactions occurring between B cell protein tyrosine kinases and the signaling components of the B cell antigen receptor, we have co-transfected into COS cells individual tyrosine kinases together with chimeric cell surface receptors containing the cytoplasmic domains of Ig alpha or Ig beta. Of the tyrosine kinases transfected (Lyn, Blk, Hck, Syk, Fyn), only Blk was able to phosphorylate and subsequently associate with cotransfected Ig alpha and Ig beta chimeras in vivo. Association between Blk and the Ig alpha and Ig beta cytoplasmic domains was shown by mutational analyses to be the result of an SH2-phosphotyrosine interaction. We identified the tyrosine residues of the Ig alpha and Ig beta cytoplasmic domains was shown by mutational analyses to be the result of an SH2-phosphotyrosine interaction. We identified the tyrosine residues of the Ig alpha and Ig beta cytoplasmic domains phosphorylated by Blk. The enzymatic activity and membrane association of Blk were required for the observed phosphorylation of the Ig alpha and Ig beta chimeras. Sequences within the amino-terminal unique domain of Blk are responsible for recognition and subsequent phosphorylation of the Ig alpha chimera since transfer of the unique region of Blk to Fyn results in the chimeric kinase's ability to phosphorylate the cytoplasmic domain of Ig alpha. These findings indicate that the unique domain of Src family kinases may direct recognition of certain substrates leading to their phosphorylation.

Animals↗
PubMed1995

Src family protein tyrosine kinases induce autoactivation of Bruton's tyrosine kinase.

Bruton's tyrosine kinase (Btk) is tyrosine phosphorylated and enzymatically activated following ligation of the B-cell antigen receptor. These events are temporally regulated, and Btk activation follows that of various members of the Src family of protein tyrosine kinases, thus raising the possibility that Src kinases participate in the Btk activation process. We have evaluated the mechanism underlying Btk enzyme activation and have explored the potential regulatory relationship between Btk and Src protein kinases. We demonstrate in COS transient-expression assays that Btk can be activated through intramolecular autophosphorylation at tyrosine 551 and that Btk autophosphorylation is required for Btk catalytic functions. Coexpression of Btk with members of the Src family of protein tyrosine kinases, but not Syk, led to Btk tyrosine phosphorylation and activation. Using a series of point mutations in Blk (a representative Src protein kinase) and Btk, we show that Src kinases activate Btk through an indirect mechanism that requires membrane association of the Src enzymes as well as functional Btk SH3 and SH2 domains. Our results are compatible with the idea that Src protein tyrosine kinases contribute to Btk activation by indirectly stimulating Btk intramolecular autophosphorylation.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1994

Temporal differences in the activation of three classes of non-transmembrane protein tyrosine kinases following B-cell antigen receptor surface engagement.

We evaluated in WEHI 231 B cells the time-dependent responses of Lyn, Blk, Btk, Syk, and three members of the Jak family of protein tyrosine kinases following antibody-mediated surface engagement of the B-cell antigen receptor. Our results show that the enzyme activities of Lyn and Blk were stimulated within seconds of antigen receptor engagement and correlated with the initial tyrosine phosphorylation of the Ig alpha and Ig beta subunits of the B-cell antigen receptor. Btk enzyme activity was also transiently stimulated and was maximal at approximately 5 min after B-cell receptor surface binding. Syk activity gradually increased to a maximum at 10-30 min following receptor ligation and was found to parallel the association of Syk with the tyrosine phosphorylated Ig alpha and Ig beta subunits of the receptor. While the specific activities of the Jak1, Jak2, and Tyk2 protein tyrosine kinases were unaltered following B-cell receptor ligation, the abundance of Jak1 and Jak2 were increased 3- to 4-fold within 10 min of receptor engagement. These results demonstrate that multiple families of non-transmembrane protein tyrosine kinases are temporally regulated during the process of B-cell antigen receptor-initiated intracellular signal transduction.

Amino Acid Sequence↗
PubMed1994

Temporal regulation of non-transmembrane protein tyrosine kinase enzyme activity following T cell antigen receptor engagement.

We evaluated in Jurkat T cells the time-dependent responses of Fyn, Lck, Syk, and Zap following antibody-mediated cross-linking of the T cell antigen receptor. Our results show that the protein kinase activities of Fyn and Lck were activated within seconds of receptor cross-linking. Fyn activity, as measured by autophosphorylation and tyrosine phosphorylation of an exogenous substrate, was maximal 5 s to 1 min following receptor cross-linking. Lck was also found to be activated within 5 s of antigen receptor cross-linking but differed from Fyn in that Lck activity was elevated for at least 30 min. Syk and Zap protein kinase activities were found to peak between 5 and 10 min following receptor cross-linking, returning to approximately basal activity levels by 60 min. The protein kinase activities of both Syk and Zap were found to parallel their reactivity in immunoblotting experiments with anti-phosphotyrosine antibodies. Both Syk and Zap were found to associate with the tyrosine-phosphorylated zeta subunit of the T cell antigen receptor. These observations imply that T cell antigen receptor signal transduction involves the activation of multiple members of at least two different families of non-transmembrane protein tyrosine kinases.

Amino Acid Sequence↗
PubMed1993

Signal transduction through a biomolecular receptor tyrosine protein kinase composed of a platelet-derived growth factor receptor-CD4 chimera and the nonreceptor tyrosine protein kinase Lck.

We have generated a novel "receptor tyrosine kinase" by fusing the extracellular and transmembrane domain of the mouse platelet-derived growth factor receptor (PDGFR) to the cytoplasmic domain of CD4 and coexpressing the construct with the murine cytoplasmic tyrosine protein kinase p56lck. NMuMG cells, which are mouse mammary gland epithelial cells that lack endogenous platelet-derived growth factor (PDGF) receptor expression, were stably transfected with both PDGFR-CD4 and p56lck. The PDGFR-CD4 chimeric protein was expressed at the cell surface and formed a complex with p56lck. Addition of PDGF to these cells led to increased tyrosine phosphorylation of a 56-kDa protein likely to be p56lck and several unidentified cellular proteins. The enzymatic activity of p56lck was increased after treatment with PDGF, indicating that dimerization (or oligomerization) mediated by ligand binding at the cell surface is capable of inducing the activation not only of receptor tyrosine kinases but nonreceptor tyrosine kinases as well. However, the PDGFR-CD4.p56lck complex was, in contrast to the wild type PDGF receptor, not able to induce a PDGF-dependent mitogenic response or DNA synthesis in NMuMG cells. Analysis of several known substrates of the PDGFR-signaling pathway indicates an early block in the transduction of the signal generated by p56lck.

Amino Acid Sequence↗
PubMed1993

Signal transduction by immunoglobulin is mediated through Ig alpha and Ig beta.

Immunoglobulin (Ig) antigen receptors are composed of a noncovalently-associated complex of Ig and two other proteins, Ig alpha and Ig beta. The cytoplasmic domain of both of these Ig associated proteins contains a consensus sequence that is shared with the signaling proteins of the T cell and Fc receptor. To test the idea that Ig alpha-Ig beta heterodimers are the signaling components of the Ig receptor, we have studied Ig mutations that interfere with signal transduction. We find that specific mutations in the transmembrane domain of Ig that inactivate Ca2+ and phosphorylation responses also uncouple IgM from Ig alpha-Ig beta. These results define amino acid residues that are essential for the assembly of the Ig receptor. Further, receptor activity can be fully reconstituted in Ca2+ flux and phosphorylation assays by fusing the cytoplasmic domain of Ig alpha with the mutant Igs. In contrast, fusion of the cytoplasmic domain of Ig beta to the inactive Ig reconstitutes only Ca2+ responses. Thus, Ig alpha and Ig beta are both necessary and sufficient to mediate signal transduction by the Ig receptor in B cells. In addition, our results suggest that Ig alpha and Ig beta can activate different signaling pathways.

Amino Acid Sequence↗
PubMed1993

Photosystem II reaction center particle from Spirodela stroma lamellae.

A Photosystem II (PSII) reaction center particle from the stroma lamellae of Spirodela oligorrhiza has been isolated. The stroma lamellar PSII reaction center contained the same proteins found in granal PSII reaction centers, namely D1, D2, and cytochrome b559; however, the cytochrome b559 content was half of that in the granal centers. The pigment composition, 77 K fluorescence emission, and excitation spectra of the stroma lamellar reaction centers were determined. Our results indicate a fully functional PSII particle in the stromal lamellae.

Peptides↗
PubMed1993

Gastric intramucosal acidosis in patients with chronic kidney failure.

BACKGROUND: Patients with chronic kidney failure have an increased incidence of gastrointestinal complications, particularly bleeding from the stomach. Diminished mucosal blood flow is thought to be an important etiologic factor for such bleeding. METHODS: Eleven patients with kidney failure on maintenance dialysis underwent placement of a gastric tonometer for the determination of gastric intramucosal pH (pHi) before and during dialysis. The arterial pH (pHa), calculated pHi, and pHa-pHi differences were compared with the results in a control group of seven normal volunteers. RESULTS: The patients with chronic kidney failure had a mean (+/- SD) pHa (7.36 +/- 0.04) similar to that found in seven control subjects (7.37 +/- 0.04). However, the gastric mucosal pH in the patients on dialysis (7.20 +/- 0.17) was lower than in the seven control subjects (7.38 +/- 0.06) (p < 0.01). A gastric mucosal pH thought to be predictive of either bleeding from stress ulceration (pHi < 7.32) or mucosal ischemia (pHi-pHa difference > 0.13) was found in nine (82%) of the patients with kidney failure and in only one (14%) of the control subjects (p < 0.001). CONCLUSIONS: This new preliminary finding suggests that the high incidence of gastric bleeding in chronic kidney failure may be related to mucosal ischemia.

Acidosis↗