Biomedical subjects
Publications and source records attributed to J Ostrowski.
A case of rare facial dysplasia due to renal osteodystrophy of end stage renal failure is described in a 19-year old boy treated with chronic haemodialyses. The favourable effects of surgical correction are shown.
Nitric oxide is produced by the NO synthase, which catalyses the conversion of arginine to citrulline and NO using tetrahydrobiopterin as an essential cofactor. N-Acetylserotonin, an inhibitor of the tetrahydrobiopterin biosynthesis, given 30 min before bacterial lipopolysaccharide to anesthetized rats, inhibited both the decrease in blood pressure and the increase in nitrite plasma levels induced by lipopolysaccharide. Thus, during endotoxemia the availability of tetrahydrobiopterin appears to be essential for the activity of NO synthase.
To identify inducible protein kinases localized exclusively in the nucleus, nuclear and cytosolic extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted to an Immobilon-P membranes were incubated in phosphorylation buffer containing [gamma-32P]ATP. Autoradiographs of the membranes revealed an 85-kDa 32P-labeled band; the intensity of this band was transiently increased in nuclear but not in cytosolic extracts from interleukin-1 alpha-treated cells. Incorporation of 32P label into a blotted protein band suggested the presence of an interleukin-1 alpha-responsive 85-kDa nuclear protein kinase. Fractionation of nuclear extracts by Mono Q failed to separate the kinase activity from the substrate, indicating that the 85-kDa band identified on the Immobilon-P membrane represents a protein kinase that undergoes autophosphorylation. Phosphoamino acid analysis of the 85-kDa band showed that this enzyme is a serine/threonine kinase. Purified pp90RSK could not be identified by the denaturation-renaturation method, indicating that the 85-kDa kinase identified here is not pp90RSK. This observation, nuclear but not cytoplasmic localization, and the fact that antibodies to known protein kinases kinase failed to recognize it suggest that the enzyme identified here is a novel protein kinase.
The actions of many hormones and neurotransmitters are mediated by the members of a superfamily of receptors coupled to heterotrimeric guanine nucleotide-binding proteins (G proteins). These receptors are characterized by a highly conserved topographical arrangement in which seven transmembrane domains are connected by intracellular and extracellular loops. The interaction between these receptors and G proteins is mediated in large part by the third intracellular loop of the receptor. Coexpression of the third intracellular loop of the alpha 1B-adrenergic receptor with its parent receptor inhibited receptor-mediated activation of phospholipase C. The inhibition extended to the closely related alpha 1C-adrenergic receptor subtype, but not the phospholipase C-coupled M1 muscarinic acetylcholine receptor nor the adenylate cyclase-coupled D1A dopamine receptor. These results suggest that the receptor-G protein interface may represent a target for receptor antagonist drugs.
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We present 3.1 kb of the nucleotide sequence from the 5'-flanking region of the human beta 1-adrenergic receptor gene. The first 1.0 kb upstream from the translational start site is composed of 72% G + C residues. The sequence was analyzed for the presence of transcriptional regulatory elements and contains putative thyroid hormone, glucocorticoid hormone and cAMP response elements. These putative hormone response elements support physiological evidence that thyroid and glucocorticoid hormones regulate beta 1AR function by affecting receptor expression in tissues such as heart and adipose, where beta 1-adrenergic receptors are important regulators of heart rate and lipolysis, respectively.
Patients suffering from congestive heart failure combined with gastrointestinal congestion often present irregular resorption and metabolism of orally applicated cardiovascular drugs. Intravenous therapy therefore is used very often for those patients. In this trial we compared the efficiency of molsidomine given either orally or intravenously in patients suffering from congestive heart failure. We investigated ten patients (54.6 +/- 13 years) with congestive heart failure (NYHA III and IV) and a central venous pressure > 10 mmHg. Five patients of group A were given 4 mg of molsidomine intravenously on the first day and an oral dose of 4 mg of molsidomine on the second day. The five patients of group B received the oral dose on the first day and intravenous treatment on the second day. Central venous pressure and plasma levels of molsidomine and of the effective metabolite SIN-1 were measured before as well as ten, 20, 40, 60 minutes, two, four and eight hours after application of molsidomine. Central venous pressure decreased significantly up to two hours after molsidomine in both oral and intravenous groups (p < or = 0.01). There was no relevant difference between the oral and the intravenous group. The hemodynamic parameters correlated with molsidomine plasma levels. According to our results the efficiency of oral molsidomine does not differ from intravenous molsidomine in patients with congestive heart failure.
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The present studies were designed to evaluate the parietal cell acid production in response to short-time stimulation by epidermal growth factor (EGF). Studies were performed in vitro using isolated cells from rat stomachs, and acid production was indirectly determined by [14C]aminopyrine accumulation. EGF inhibited histamine-stimulated aminopyrine accumulation from standard incubation medium (K+ = 5 mM) but not from that with increased K+ concentration (K+ = 70 mM). EGF significantly stimulated ornithine decarboxylase (ODC) activity, an effect that was blocked by the specific ODC inhibitor, difluoromethylornithine (DFMO). In the presence of DFMO, EGF failed to inhibit histamine-stimulated aminopyrine uptake. Like EGF, the polyamine spermine, which is a direct product of enhanced ODC activity, also inhibited histamine-stimulated aminopyrine uptake. Unlike EGF, the spermine-induced inhibition of aminopyrine accumulation was not altered by DFMO. Thus the DFMO effect was specific to EGF. Taken together, these results are consistent with the postulate that EGF inhibits parietal cell secretory response through the induction of ODC activity and increased synthesis of polyamines.
Mucosal gastric injury was induced in vivo by oral administration of 75% ethanol to rats that were intraperitoneally pretreated with saline (controls), 4 mg/kg of verapamil, 0.4 mg/kg of nifedipine or 160 mg/kg of MgCl2. The glandular stomachs were used for macroscopic and histologic evaluation of mucosal lesions. The gastroprotective effects of substances tested against ethanol-induced mucosal damage in vitro were examined: 1). in rats intraperitoneally pretreated with calcium antagonists and 2). after adding of 10(-4) M verapamil, 10(-6) M nifedipine or 10(-2) M MgCl2 directly to minced gastric mucosa of untreated rats. This effect was measured by DNA synthesis. Gross and histologic evaluation showed that rats pretreated with nifedipine or MgCl2 had significantly decreased ethanol-induced gastric injury compared to controls, whereas those pretreated with verapamil had significantly increased injury. On the contrary, all verapamil, nifedipine and MgCl2 treatments, administrated intraperitoneally or by exposure to an incubation mixture were equally effective in reducing gastric mucosal damage following to ethanol treatment in vitro. We conclude that the differential effects of verapamil and nifedipine on ethanol-damaged gastric mucosa in vivo, but not in vitro, suggests the existence of different actions of these calcium antagonists more on systemic rather than local protective mechanisms, such as gastric mucosal blood flow, mucosal barrier, or cell renewal.
The aim of this study was to determine pepsinogen secretion by isolated rat chief cells in relation to changes of membrane potential obtained by the use of potassium ionophore valinomycin and different extracellular K+ concentrations, or protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) at different pH. While valinomycin or CCCP decreased basal and cAMP-stimulated secretory response of the chief cells, they increased secretion of pepsinogen stimulated by calcium ionophore A23187. When Ca2+ was absent from the incubation media, A23187-stimulated secretion of pepsinogen significantly decreased. Moreover, the omission extracellular calcium reversed the potentiating effects of valinomycin or CCCP on A23187-stimulated secretory response. It is known that pepsinogen secretion is a biphasic process. In the first phase, Ca2+ is released from its intracellular stores, while the second one depends on extracellular calcium entering the cell. Our results suggest that this phase is dependent also on membrane potential.
Pharmacokinetic measurements were performed in two groups of patients with coronary heart disease (CHD) after single and multiple dosing of 2 mg linsidomine (SIN 1). The drug was administered by intravenous short time infusion in 12 CHD-patients with renal insufficiency (RI group, Clcr: 11 +/- 6 ml/min) and in 12 CHD-patients with normal kidney function (control group, Clcr: 88 +/- 22 ml/min). The measurement of plasma concentration time courses of total SIN 1C (SIN 1 + SIN 1C) was found to be suitable for an estimation of the SIN 1C related half-life of the terminal phase (t50% = 1.5 +/- 0.5 h), as SIN 1 was eliminated from plasma rapidly (t50% = 12 to 20 min). Furthermore, the mean total SIN 1C plasma profiles were equal after single and multiple administration of the drug giving evidence that SIN 1C is not accumulating during repetitive dosing of SIN 1 in patients with renal disease. The mean maximum renal fraction of total SIN 1C excretion of RI-subjects (fe = 0.8 +/- 0.8% of dose) was significantly different from the corresponding mean value of the control group (fe(N) = 5.8 +/- 5.1% of dose). No differences were found for fe and fe(N) between day 1 and day 4. As SIN 1 is degraded in plasma very rapidly and as SIN 1C is cleared mainly extrarenally, any restrictions concerning repetitive SIN 1 dosage regimen should not be considered for CHD-patients with renal failure.
The bovine brain A1 adenosine receptor (A1AR) is distinct from other A1ARs in that it displays the unique agonist potency series of N6-R-phenylisopropyladenosine (R-PIA) greater than N6-S-phenylisopropyladenosine (S-PIA) greater than 5'-N-ethylcarboxamidoadenosine and has a 5-10-fold higher affinity for both agonists and antagonists. The cDNA for this receptor has been cloned from a size-selected (2-4-kb) bovine brain library and sequenced. The 2.0-kb cDNA encodes a protein of 326 amino acid residues with a molecular mass of 36,570 daltons. The amino acid sequence fits well into the seven-transmembrane domain motif typical of G protein-coupled receptors. Northern analysis in bovine tissue using the full length cDNA demonstrates mRNAs of 3.4 and 5.7 kb with a tissue distribution consistent with A1AR binding. Subcloning of the cDNA in a pCMV5 expression vector with subsequent transfection into both COS7 and Chinese hamster ovary cells revealed a fully functional A1AR which could inhibit adenylylcyclase and retained the unique pharmacologic properties of the bovine brain A1AR. The A1AR was found to have a single histidine residue in each of transmembrane domains 6 and 7. Histidine residues have been postulated by biochemical studies to be important for ligand binding. Mutation of His-278 to Leu-278 (seventh transmembrane domain) dramatically decreased both agonist and antagonist binding by greater than 90%. In contrast, mutation of His-251 to Leu-251 decreased antagonist affinity and the number of receptors recognized by an antagonist radioligand. In contrast, agonist affinity was not perturbed but the number of receptors detected by an agonist radioligand was also reduced. These data suggest that both histidines are important for both agonist and antagonist binding, but His-278 appears critical for ligand binding to occur.
To investigate the mechanisms of agonist-promoted desensitization of the alpha 2-adrenergic receptor (alpha 2AR), the human alpha 2AAR and a mutated form of the receptor were expressed in CHW cells. After cells were exposed to epinephrine for 30 min, the ability of the wild type alpha 2AAR to mediate inhibition of forskolin-stimulated adenylyl cyclase was depressed by approximately 78%. To assess the role of receptor phosphorylation during desensitization, cells were incubated with 32Pi, exposed to agonist, and alpha 2AAR purified by immunoprecipitation with a fusion protein antibody. Agonist-promoted desensitization was found to be accompanied by phosphorylation of the alpha 2AAR in vivo. The beta-adrenergic receptor kinase (beta ARK) is known to phosphorylate purified alpha 2AAR in vitro. We found that heparin, a beta ARK inhibitor, ablated short term agonist-induced desensitization of alpha 2AAR, while such desensitization was unaffected by inhibition of protein kinase A. To further assess the role of beta ARK, we constructed a mutated alpha 2AAR which has a portion of the third intracellular loop containing 9 serines and threonines (potential phosphorylation sites) deleted. This mutated alpha 2AAR failed to undergo short term agonist-induced desensitization. Agonist promoted in vivo phosphorylation of this mutated receptor was reduced by 90%, consistent with the notion that receptor phosphorylation at sites in the third intracellular loop plays a critical role in alpha 2AAR desensitization. After 24 h of agonist exposure, an even more profound desensitization of alpha 2AAR occurred, which was not accompanied by a decrease in receptor expression. Rather, long term agonist-induced desensitization was found to be due in part to a decrease in the amount of cellular Gi, which was not dependent on receptor third loop phosphorylation sites.
We have constructed a variety of chimeric beta 2/alpha 1 adrenergic receptors (AR) in which selected portions of the third intracellular loop of the alpha (1B)AR were substituted into the corresponding regions of the beta 2AR. The mutant receptors were both transiently and permanently expressed in COS-7 or L-cells, respectively, and tested for their ability to mediate epinephrine-induced activation of polyphosphoinositide (PI) hydrolysis and adenylylcyclase. We have determined that 27 amino acids of the alpha (1B)AR (residues 233-259) derived from the N-terminal portion of the third intracellular loop represent the structural determinant conferring to the beta 2AR the ability to activate PI hydrolysis. This finding suggests that in the alpha (1B)AR the N-terminal portion of the third intracellular loop plays a major role in determining the selectivity of receptor-G protein coupling. However, replacement of alpha 1B sequences in the third intracellular loop of the beta 2AR did not abolish the latter receptor's coupling to activation of adenylylcyclase, thus resulting in chimeric adrenergic receptors which activated both PI hydrolysis and adenylylcyclase. These results indicate that, even if the N-terminal portion of the third intracellular loop is a major determinant of the selectivity of receptor-G protein coupling, other structural domains of the receptors also modulate this property. The comparison of the amino acid sequences which determine the selectivity of G protein coupling in functionally similar receptors may help to elucidate the structural basis for activation of specific G protein-effector systems.
Mutations in an intracellular region of the alpha 1B-adrenergic receptor constitutively activate the receptor, resulting in G protein coupling in the absence of agonist, as evidenced by elevated levels of polyphosphoinositide hydrolysis. Remarkably, all 19 possible amino acid substitutions at a single site in this region (alanine 293) confer constitutive activity. This set of mutated receptors exhibits a graded range of elevated biological activities, apparently representing a spectrum of receptor conformations which mimic the "active" state of the wild type receptor. In addition to their constitutive activities, these mutated receptors all demonstrate a higher affinity for agonists, another primary characteristic of the "active" conformation of G protein-coupled receptors. The fact that all possible mutations at this particular site result in increased activity suggests that this region may function to constrain the G protein coupling of the receptor, a constraint which is normally relieved by agonist occupancy.
Inactivation of tumour suppressor genes may be an important aetiological factor in many human cancers including breast. In a study of 197 breast cancer patients, tumour tissue was snap-frozen at the time of surgery and immunohistochemical labelling for p53 protein and retinoblastoma (Rb) gene product carried out using an indirect immunohistochemical technique. Tumours were scored by two independent observers for the intensity of nuclear staining for each antibody. Expression of p53 protein showed a significant association with a shorter time to relapse (P = 0.03) and death (P = 0.02) (log rank test). p53 expression did not correlate with nodal status but showed a significant association with high tumour grade (P = 0.001). Rb gene expression showed no relationship to relapse or survival but loss of expression showed a significant correlation with positive lymph node status. The manner by which these proteins might act to determine tumour behaviour remains to be established.
We have examined signal transduction via membrane IgM (mIgM) in resting and cycling human B cells. Crosslinking mIgM on all of the cell types studied transduced a signal through the phosphatidylinositol pathway, producing inositol 1,4,5-trisphosphate and release of intracellular free calcium. These second messengers were formed regardless of quantitative or qualitative differences in the surface expression of mIgM: cells that had low levels of surface IgM (T-51) or had no light chain associated with surface heavy chain (DB) signaled phosphatidylinositol pathway activation after mIgM crosslinking. Production of specific lipid products in nonquiescent B cells differed from that in normal resting cells. Ligation of surface immunoglobulin on resting B cells resulted in sustained increases of both diacylglycerol and phosphatidic acid, two lipids that can influence PKC activation. Whereas PKC was strongly activated in normal tonsillar B cells, several cell lines had reduced PKC activation following crosslinking of mIgM. The reduction in protein kinase C activation correlated with the absence or reduced levels of phosphatidic acid or diacylglycerol following stimulation: protein kinase C translocated and was activated only in cells that had elevated levels of both diacylglycerides and phosphatidic acid. Anti-IgM-induced phosphorylation of a protein kinase C substrate protein CD20, also increased in those cells having PKC activation and not in cells in which kinase activity was reduced. CD20 phosphorylation also increased following the direct addition of exogenous phosphatidic acid to resting B cells. Together, these observations show that the generation of lipid products following mIgM crosslinking in resting cells can vary from that in cycling cells and may relate to the different levels of PKC activation. In a companion study we report that ligation of surface IgM activates both an acyltransferase and phospholipase D to form phosphatidic acid.