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Biomedical subjects

M Welsh

Publications and source records attributed to M Welsh.

At least 55 records · Page 3Linked to original sources
PubMed1995

Molecular interactions of the Src homology 2 domain protein Shb with phosphotyrosine residues, tyrosine kinase receptors and Src homology 3 domain proteins.

The molecular interactions of the Src homology 2 (SH2) domain and the N-terminal proline-rich sequence motifs (pro-1 to pro-5) of the SH2 protein Shb with other components were presently characterised. Using a degenerate phosphopeptide library the preferred binding site for the Shb SH2 domain was determined to pTyr-Thr/Val/Ile-X-Leu at positions +1 to +3 relative the phosphotyrosine residue. Experiments with competing peptides and platelet-derived growth factor (PDGF) beta-receptor mutants with Y to F substitutions in autophosphorylation sites revealed multiple binding sites for the Shb SH2 domain in the receptor. The Shb SH2 domain also binds to in vitro phosphorylated fibroblast growth factor receptor-1 (FGFR-1) mainly through position Y776. The receptor experiments suggest that other residues besides the +1 to +3 positions may also be of significance for Shb binding. The pro-4/pro-5 motif of Shb binds in vitro particularly well to the Src, p85 alpha PI3-kinase and Eps8 SH3 domains expressed as GST fusion proteins. However, the GST-SH3 domain fusion proteins tested bind in vitro to peptides corresponding to the pro-1 to pro-5 motifs of Shb with low affinity and selectivity, suggesting that sequences outside the core proline motif may also be important for Shb-SH3 domain interactions. In vivo association between Shb-SH3 domain proteins v-Src and Eps8 was detected by coimmunoprecipitation. PDGF treatment did not affect the association between Eps8 and Shb. The data suggest that Shb is an adaptor protein linking SH3 domain proteins to tyrosine kinases or other tyrosine phosphorylated proteins.

Amino Acid Sequence↗
PubMed1995

Cloning of BSK, a murine FRK homologue with a specific pattern of tissue distribution.

A novel protein tyrosine kinase (PTK) was previously identified by us from the rat insulin-producing cell line, RINm5F, by polymerase chain reaction (PCR). By using this PCR fragment to screen a cDNA library from the mouse insulin-producing cell line beta TC-1, a cDNA clone of about 2.0 kb was obtained which encodes the entire amino acid (aa) sequence of the corresponding PTK. The deduced aa sequence reveals strong homology with the members of the SRC family of intracellular PTKs. We have designated the gene as BSK (beta-cell Src-homology tyrosine kinase). Southern blot analysis after PCR with primers specific for BSK confirmed its expression in fetal and adult islets of Langerhans, in RINm5F cells and in mouse kidney. Northern blots using poly(A)+RNA from non-beta-cell tissues showed that the BSK cDNA hybridized to three mRNA transcripts (2.9, 3.1 and 5.0 kb) present in kidney, liver and lung. Extensive homology of BSK with the recently identified human gene FRK was observed. It is concluded that Bsk is a murine Frk homologue with a specific pattern of tissue expression.

Amino Acid Sequence↗
PubMed1995

Comparison of mRNA contents of interleukin-1 beta and nitric oxide synthase in pancreatic islets isolated from female and male nonobese diabetic mice.

Interleukin-1 beta (IL-1 beta) has been suggested to mediate beta-cell destruction in insulin-dependent diabetes mellitus (IDDM) by inducing nitric oxide production. In this study, we assessed the levels of IL-1 beta and the inducible form of nitric oxide synthase (iNOS), using a semi-quantitative polymerase chain reaction assay, and performed determinations of nitrite accumulation and IL-1 beta bioactivity, on pancreatic islets isolated from 5- and 16-week-old female and male nonobese diabetic (NOD) mice and from nondiabetes prone NMRI mice. NOD mouse islets contained notable amounts of IL-1 beta mRNA. At 5 weeks of age, but not at 16 weeks, the values were higher in islets isolated from NOD females compared to males. The IL-1 beta bioactivity showed differences roughly reflecting the mRNA levels in the NOD mouse islets. In the NMRI mouse islets the IL-1 beta bioactivity was very low. The expression of iNOS mRNA increased in both male and female islets between 5 and 16 weeks of age. Immunocytochemistry of pancreatic sections indicated the presence of macrophages especially in the peri-insular area of the NOD mice which suggests that IL-1 beta was produced by macrophages. The levels of IL-1 beta activity and mRNA in freshly isolated islets from NOD 5-weeks-old females did not correlate to the iNOS mRNA content or to the nitrite production. However, after incubation with IL-1 beta in vitro, both NOD and NMRI islets responded with a marked increase in nitric oxide production.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗
PubMed1995

Challenges in cyclosporine therapy: the role of therapeutic monitoring by area under the curve monitoring.

Cyclosporine has revolutionized the practice of transplantation, but its clinical application has been beclouded by a narrow therapeutic window between immunosuppressive and nephrotoxic concentrations. Marked intra- and interindividual pharmacokinetic differences preclude the use of routine dosing regimens. For example, at the intraindividual level, cyclosporine absorption improves during the first 90 days after institution of therapy. A wide range of demographic factors, namely, age, race, and concomitant drug therapy, as well as individual-specific factors produce unique pharmacokinetic behaviors in any given patient. We introduced a pharmacokinetic strategy for cyclosporine administration almost 10 years ago based on the observation that the best estimate of drug exposure was the area under the concentration-time kinetic curve (AUC) not the trough level. Early studies documented the relation between AUC and the incidence of acute rejection. Subsequent studies revealed that not only is the AUC an important predictor, but so is the consistency of drug absorption over time; namely, patients with variations > 25% among AUC determinations display an increased risk of chronic rejection episodes. Therefore therapeutic drug monitoring plays an important role in the optimal care of patients under cyclosporine therapy.

Cyclosporine↗
PubMed1995

Expression of an insulin/interleukin-1 receptor antagonist hybrid gene in insulin-producing cell lines (HIT-T15 and NIT-1) confers resistance against interleukin-1-induced nitric oxide production.

A hybrid gene consisting of the insulin gene enhancer/promoter region, the signal sequence, the insulin B- and C-chains, and the human interleukin-1 receptor antagonist (IL-1ra) gene was constructed. This hybrid gene was transfected together with the pSV2-neo construct into the insulin-producing cell lines HIT-T15 and NIT-1. One of the geneticin-selected clones, HITra2, expressed a 1.4-kb mRNA, which hybridized both to insulin and IL-1ra-cDNA in Northern blot analysis. Three proteins, with the mol wt 23, 17, and 14 kD, were immunoprecipitated with anti-IL-1ra antibodies from [35S]methionine-labeled HITra2 cells. Both at a low and at a high glucose concentration, 4-5 ng of IL-1ra/10(6) cells (ELISA) was released from these cells. On the other hand, a high glucose concentration evoked a three-fold increase in the release of insulin, suggesting that IL-1ra was released constitutively. Measured by nitrite production, transfected HIT, and NIT-1 cells exhibited a more than 10-fold decrease in IL-1 beta sensitivity. Since the conditioned culture media from the HITra2 cells exhibited an anti-IL-1 beta activity of only 0.5 U/ml, and mixed culture of HITra2 cells and isolated rat islets prevented IL-1 beta induced inhibition of insulin release, it is likely that IL-1ra acts locally at the cell surface. It is concluded that expression of a hybrid insulin/IL-1ra gene confers resistance to IL-1 and that this technique may be used to elucidate the role of IL-1 in autoimmune disorders such as insulin-dependent diabetes mellitus.

Animals↗
PubMed1995

Mitochondrial deoxyribonucleic acid content is specifically decreased in adult, but not fetal, pancreatic islets of the Goto-Kakizaki rat, a genetic model of noninsulin-dependent diabetes.

Considerable interest has recently been focused on the putative role of mutations in the mitochondrial genome for the development of noninsulin-dependent diabetes. The Goto-Kakizaki (GK) rat, a genetic model of defective insulin secretion and hyperglycemia, is characterized by partial maternal inheritance. Because the mitochondrial genome is known to be maternally transmitted, the aim of this study was to investigate whether the GK syndrome can be explained in terms of alterations of the mitochondrial DNA (mtDNA). For this purpose, pancreatic islets were isolated from adult and fetal control Wistar and diabetic GK rats. Using electron microscopy, the ultrastructural morphology of beta-cell mitochondria was analyzed in control and GK islets. It was found that the beta-cells of adult GK rats had a significantly smaller mitochondrial volume and an increased number of mitochondria per unit tissue volume as compared with the beta-cells of corresponding control islets. Moreover, mtDNA and mtRNA were isolated from the islets and, as a control tissue, from liver, and subsequently analyzed using Southern and Northern blot techniques. No major deletions or restriction fragment polymorphism could be detected in mtDNA from both GK liver and GK islets. The mtDNA sequence of the transfer RNAleu(UUS) gene was identical in both strains of rats. mtDNA contents of fetal GK islets and fetal GK liver were not different from those of fetal Wistar rats. However, adult GK islets contained markedly less mtDNA than the corresponding control islets, contrary to the mtDNA contents of adult liver, which were similar in the two strains. The lower islet mtDNA contents were paralleled by a decreased content of islet mtRNA (12S ribosomal RNA and cytochrome b messenger RNA). Islet insulin messenger RNA contents were similar in GK and Wistar rats. In conclusion, our results do not support a role of a genetic defect in mtDNA as a cause of the GK syndrome. Instead, mtDNA damage may occur specifically in islet cells as a consequence of the disturbed metabolic environment of the adult GK rat. It is speculated that a long-lasting metabolic dysfunction may induce mtDNA damage and/or inhibition of mtDNA replication leading to a gradual and late decrease in the mitochondrial volume fraction and subsequently an impaired capacity for oxidative metabolism.

Animals↗
PubMed1995

Ventroposterior medial pallidotomy in patients with advanced Parkinson's disease.

In a preliminary study, the effects of ventroposterior medial pallidotomy were evaluated in five patients with advanced Parkinson's disease in whom medical therapy had failed. The mean age was 67.0 +/- 5.6 years, and the mean Hoehn and Yahr stage when "off" was 3.9 +/- 1.3. Three patients received unilateral pallidotomies; two of these received another pallidotomy after 8 weeks. Two other patients received staged bilateral pallidotomies. No significant differences in overall function could be seen before and after the first surgical procedure. All three patients with peak-dose dyskinesias or dystonia had marked contralateral reduction in these symptoms. Ventroposterior medial pallidotomy can ameliorate peak-dose dyskinesias in patients with advanced Parkinson's disease. Overall function improvement is not remarkable.

Aged↗
PubMed1994

Protein kinase C modulates the insulin secretory process by maintaining a proper function of the beta-cell voltage-activated Ca2+ channels.

In the present study an attempt was made to further elucidate the molecular mechanisms whereby protein kinase C (PKC) modulates the beta-cell stimulus-secretion coupling. Regulation of Ca2+ channel activity, [Ca2+]i, and insulin release were investigated in both normal pancreatic mouse beta-cells and in similar beta-cells deprived of PKC activity. [Ca2+]i was measured with the intracellular fluorescent Ca2+ indicator fura-2 and the Ca2+ channel activity was estimated by the whole cell configuration of the patch-clamp technique. To reveal the various isoenzymes of PKC present in the mouse beta-cell, proteins were separated by one-dimensional gel electrophoresis and Western blotting was performed. The production of inositol phosphates was measured by ion-exchange chromatography and insulin release was measured radioimmunologically. Acute stimulation with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate resulted in suppression of both the carbamylcholine-induced increase in [Ca2+]i and production of inositol 1,4,5-trisphosphate. Under these conditions the increase in [Ca2+]i in response to glucose was similar to that found in control cells. When beta-cells were deprived of PKC, by exposure to 200 nM 12-O-tetradecanoylphorbol-13-acetate for 24-48 h, there was an enhanced response to carbamylcholine. This response constituted increases in both the [Ca2+]i signal and production of inositol 1,4,5-trisphosphate. Interestingly, cells with down-regulated PKC activity responded more slowly to glucose stimulation, when comparing the initial increase in [Ca2+]i, than control cells. On the other hand, the maximal increase in [Ca2+]i was similar whether or not PKC was present. Moreover, PKC down-regulated cells exhibited a significant reduction of maximal whole cell Ca2+ currents, a finding that may explain the altered kinetics with regard to the [Ca2+]i increase in response to the sugar. Both the alpha and beta 1 forms of the PKC isoenzymes were present in the mouse beta-cell and were also subjected to PKC down-regulation. Hence, either of these isoenzymes or both may be involved in the modulation of phospholipase C and Ca2+ channel activity. Since insulin release under physiological conditions is critically dependent on Ca(2+)-influx through the voltage-gated L-type Ca2+ channels, the kinetics of hormone release was expected to demonstrate a similar delay as that of the [Ca2+]i increase. Although not as pronounced, such a delay was indeed also observed in the onset of insulin release. There was, however, no effect on the total amounts of hormone released.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Expression of protein tyrosine kinases in islet cells: possible role of the Flk-1 receptor for beta-cell maturation from duct cells.

To elucidate the expression of genes of importance for beta-cell replication and the production of insulin, single-stranded cDNAs from different preparations of insulin producing cells were used as template for the polymerase chain reaction (PCR) using primers specific for protein tyrosine kinases (PTKs). In RINm5F cells, as well as in fetal rat islets, the receptor PTK fetal liver kinase-1 (Flk-1) was expressed among other receptor and cytoplasmic tyrosine kinases. To elucidate the putative effects of stimulation of the Flk-1 receptor, fetal rat islet-like structures were cultured in the presence of the ligand for this receptor, vascular endothelial growth factor (VEGF). VEGF was found to stimulate both the insulin content/islet DNA ratio and the accumulation of insulin in the culture medium without affecting the rates of beta-cell replication. To investigate the localization of expression of the Flk-1 receptor in the pancreas, serial sections of fetal pancreata were immunostained for Flk-1 and insulin. Expression of Flk-1 was detected in endothelial-like cells and cells lining pancreatic ducts. The latter are considered to contain precursor cells for the endocrine pancreas. In conclusion, specific protein tyrosine kinases are expressed in islet cells, and are presumably participating in the regulation of islet function. Specifically, the receptor PTK Flk-1 may play a role of beta-cell maturation from pancreatic duct cells.

Amino Acid Sequence↗
PubMed1994

Shb is a ubiquitously expressed Src homology 2 protein.

To identify serum-inducible genes in the insulin-producing cell line beta TC-1, a library subtraction screening procedure was performed on serum-deprived (G0) and serum-restimulated (G1) insulin-producing beta TC-1 cells. A cDNA containing a motif with strong homology to Src homology 2 (SH2) domains was found using this procedure and called Shb. The Shb cDNA contains two methionine codons in its N-terminus and thus may code for two proteins of 67 and 56 kDa, each with one SH2 domain in its C-terminus. No other structural similarity to proteins with catalytic activity could be detected, suggesting that Shb is a so called adaptor. Shb contains the proline-rich sequence PPPGPGR between the two proposed initiator methionines which resembles a sequence for binding to Src homology 3 (SH3) domains. A second proline-rich sequence was detected after the second methionine codon. The Shb cDNA hybridized to a similar or identical mRNA of 3.1 kb expressed in mouse brain, liver, kidney, heart, NIH3T3 fibroblasts and beta TC-1 cells. Western blot analysis of the same tissues using an antiserum directed against a synthetic peptide corresponding to a part of the SH2 domain of Shb, revealed reactivity with two proteins of 56 and 67 kDa. In addition, a third reactive component of 40 kDa was detected in most tissues. Transfection and transient expression of the Shb cDNA in COS-1 cells yielded increased expression of the 67, 56 and 40 kDa proteins. Transfection and stable expression of the Shb cDNA in pig aortic endothelial cells showed increased expression primarily of the 67 kDa protein. A fusion protein consisting of the SH2 domain of Shb linked to glutathione S-transferase showed increased binding to glycoproteins of cells stimulated with platelet-derived growth factor (PDGF-BB). Furthermore, the autophosphorylated PDGF beta-receptor but not the autophosphorylated epidermal growth factor (EGF) receptor bound specifically to immobilized fusion protein. It is concluded that Shb is a novel SH2-containing protein with proline-rich domains and therefore probably involved in the signal-transduction of some ligand-activated tyrosine kinase receptors.

Adaptor Proteins, Signal Transducing↗
PubMed1993

The adverse impact of high cyclosporine. Clearance rates on the incidences of acute rejection and graft loss.

The influence of cyclosporine pharmacokinetic parameters on clinical events and outcome after transplantation was studied in 100 renal transplant recipients who underwent a pre- as well as posttransplant CsA pharmacokinetic evaluation. Among the patients, 30 were black and 50 were white. Black recipients had significantly lower bioavailability (F) pre- as well as posttransplantation than white recipients, the posttransplant mean F values being 25.8 +/- 9.0% and 38.1 +/- 16.7%, respectively (P < 0.002). The posttransplant CsA clearance rate (CL) and oral clearance (clearance/bioavailability; CLoral) were significantly higher in patients who had acute rejection than in those who did not, with CL mean values of 425 +/- 141 ml/min and 359 +/- 131 ml/min, respectively (P < 0.02). The initial posttransplant F was significantly lower, and the CLoral higher in patients who eventually lost their grafts than in those who did not, the mean F values being 26.5 +/- 12.8% and 38.7 +/- 17.5%, respectively (P < 0.002). Thus, several important relationships between CsA pharmacokinetic parameters and clinical events following renal transplantation were documented. The CLoral decreased during the first 3 months after transplantation (P < 0.0001), but it was stable thereafter. Neither the bioavailability nor the clearance of CsA showed a correlation with administered dose. These results indicate that certain recipient groups, such as black patients, and individuals with rapid CL, may benefit from larger CsA doses and/or shorter dosage intervals, in order to compensate for these interpatient variabilities.

Administration, Oral↗
PubMed1993

Enhanced stimulus-secretion coupling in polyamine-depleted rat insulinoma cells. An effect involving increased cytoplasmic Ca2+, inositol phosphate generation, and phorbol ester sensitivity.

To extend previous observations on the role of polyamines in insulin production, metabolism, and replication of insulin-secreting pancreatic beta cells, we have studied the role of polyamines in the regulation of the stimulus-secretion coupling of clonal rat insulinoma cells (RINm5F). For this purpose, RINm5F cells were partially depleted in their polyamine contents by use of the specific ornithine decarboxylase inhibitor difluoromethylornithine (DFMO), which led to an increase in cellular insulin and ATP contents. Analysis of different parts of the signal transduction pathway revealed that insulin secretion and the increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) after K(+)-induced depolarization were markedly enhanced in DFMO-treated cells. These effects were paralleled by increased voltage-activated Ca2+ currents, as judged by whole-cell patch-clamp analysis, probably reflecting increased channel activity rather than elevated number of channels per cell. DFMO treatment also rendered phospholipase C in these cells more sensitive to the muscarinic receptor agonist carbamylcholine, as evidenced by enhanced generation of inositol phosphates, increase in [Ca2+]i and insulin secretion, despite an unaltered ligand binding to muscarinic receptors and lack of effect on protein kinase C activity. In addition, the tumor promoter 12-O-tetradecanoylphorbol 13-acetate, at concentrations suggested to be specific for protein kinase C activation, evoked an increased insulin output in polyamine-deprived cells compared to control cells. The stimulatory effects of glucose or the cyclic AMP raising agent theophylline on insulin release were not increased by DFMO treatment. In spite of increased binding of sulfonylurea in DFMO-treated cells, there was no secretory response or altered increase in [Ca2+]i in response to the drug in these cells. It is concluded that partial polyamine depletion sensitizes the stimulus-secretion coupling at multiple levels in the insulinoma cells, including increased voltage-dependent Ca2+ influx and enhanced responsiveness to activators of phospholipase C and protein kinase C. In their entirety, our present results indicate that the behavior of the stimulus-secretion coupling of polyamine-depleted RINm5F insulinoma cells changes towards that of native beta cells, thus improving the usefulness of this cell line for studies of beta cell insulin secretion.

Adenosine Triphosphate↗