Treatment of fragile X syndrome.
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Behavior Therapy↗
Biomedical subjects
J Turk
Publications and source records attributed to J Turk.
Behavioural phenotypes in dysmorphic syndromes.
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Animals↗
Cholesterol chlorohydrin synthesis by the myeloperoxidase-hydrogen peroxide-chloride system: potential markers for lipoproteins oxidatively damaged by phagocytes.
Myeloperoxidase, a heme protein secreted by activated phagocytes, uses hydrogen peroxide to produce potent cytotoxins. One important substrate is chloride, which is converted to hypochlorous acid (HOCl). This diffusible oxidant plays a critical role in the destruction of invading pathogens. Under pathological conditions, HOCl may also injure normal tissue. Recent studies have shown that myeloperoxidase is a component of human atherosclerotic lesions. Because oxidized lipoproteins may play a central role in atherogenesis, we have explored the possibility that cholesterol is a target for damage by myeloperoxidase. Three major classes of sterol oxidation products were apparent when cholesterol-phosphatidylcholine multilamellar vesicles which had been exposed to a myeloperoxidase-hydrogen peroxide-chloride system were subsequently analyzed by normal-phase thin layer chromatography. The products were identified by gas chromatography-mass spectrometry as cholesterol alpha- and beta-chlorohydrins (6 beta-chlorocholestane-3 beta,5 alpha-diol and 5 alpha-chlorocholestane-3 beta,6 beta-diol), cholesterol alpha- and beta-epoxides (cholesterol 5 alpha,6 alpha-epoxide and cholesterol 5 beta,6 beta-epoxide), and a novel cholesterol chlorohydrin. Conversion of cholesterol to the oxidation products required active myeloperoxidase, hydrogen peroxide, and halide and could be blocked by catalase or by scavengers of HOCl. Moreover, in the absence of the enzymatic system, reagent HOCl generated the same distribution of products. These results indicate that myeloperoxidase can convert cholesterol to chlorohydrins and epoxides by a reaction involving HOCl. Other oxygenated sterols are cytotoxic and mutagenic and are potent regulators of cholesterol homeostasis in cultured mammalian cells. Cholesterol chlorohydrins might similarly mediate powerful biological effects in the artery wall. Because chlorohydrins are stable under our experimental conditions, chlorinated sterols may prove useful as markers for lipoproteins oxidatively damaged by activated phagocytes.
Catalase↗
Characterization of an ATP-stimulatable Ca(2+)-independent phospholipase A2 from clonal insulin-secreting HIT cells and rat pancreatic islets: a possible molecular component of the beta-cell fuel sensor.
Isolated pancreatic islets from rats and humans express a plasmalogen-preferring ATP-stimulatable, Ca(2+)-independent phospholipase A2 (ASCI-PLA2) enzyme which participates in the glucose-stimulated hydrolysis of arachidonate from membrane phospholipids and in insulin secretion. Here we report that clonal insulin-secreting HIT beta-cells contain substantial amounts of endogenous plasmalogens and express a similar ASCI-PLA2 activity with the following properties: (1) Enzymatic activity as well as glucose-induced eicosanoid release and insulin secretion are inhibited by a mechanism-based suicide substrate directed towards ASCI-PLA2. (2) HIT cell ASCI-PLA2 is selectively activated and protected against thermal denaturation by ATP. (3) The magnitude of ASCI-PLA2 activation by the nonhydrolyzable ATP analog AMP-PCP is similar to that by ATP. (4) The ATP concentrations required to activate ASCI-PLA2 fall within physiologic ranges in the presence of Mg2+. (5) ADP induces a concentration-dependent attenuation of the activation of ASCI-PLA2 by ATP. HIT cell ASCI-PLA2 exhibited an apparent isoelectric point of 7.5 on chromatofocusing analysis and was quantitatively adsorbed to an ATP-agarose matrix and selectively desorbed from this column by ATP. Mono-Q anion-exchange analysis of the active ATP-agarose eluant yielded a peak of ASCI-PLA2 activity associated with a single protein band with an apparent molecular mass of 40 kDa. Similar chromatographic behavior of the rat pancreatic islet ASCI-PLA2 activity was observed during sequential ATP-agarose and Mono-Q anion-exchange steps. These results indicate that HIT cells express an ASCI-PLA2 similar to the analogous islet enzyme and suggest that expression of this enzyme and of its preferred plasmalogen substrates may be a general property of insulin-secreting beta-cells.
Adenosine Triphosphate↗
omega-Conotoxin inhibits glucose- and arachidonic acid-induced rises in intracellular [Ca2+] in rat pancreatic islet beta-cells.
Earlier studies suggest that the accumulation of non-esterified arachidonic acid (AA) in islets following stimulation with glucose participates in the glucose-induced secretion of insulin. A possible role for AA might include the facilitation of Ca2+ influx into islet beta-cells. Recently, we demonstrated that AA induces Ca2+ influx into purified rat pancreatic islet beta-cells, prepared by fluorescence-activated cell sorting (FACS). This effect was abolished in the presence of the Ca(2+)-chelator EGTA, but was only partially reduced by the dihydropyridine (DHP) L-type Ca(2+)-channel blocker, nifedipine. This raised the possibility that DHP-insensitive Ca2+ entry mechanisms may exist in pancreatic beta-cells, in addition to the known DHP-sensitive L-type Ca2+ channels. Here we report that omega-conotoxin (CTX), which blocks omega-type Ca(2+)-channels, inhibits AA-induced Ca2+ influx by a magnitude similar to that of nifedipine and that the combination of omega-CTX and nifedipine results in a nearly additive decrement in AA-induced increases in beta-cell cytosolic [Ca2+]. We further demonstrate that bovine serum albumin, which complexes free AA and prevents AA-induced increases in cytosolic [Ca2+], also inhibits the glucose-induced increase in beta-cell [Ca2+]. These results suggest that rat pancreatic FACS-purified islet beta-cells express omega-type (DHP-insensitive) Ca(2+)-channels, in addition to DHP-sensitive Ca(2+)-channels. They further suggest that the glucose-induced accumulation of non-esterified AA in the membranes of beta-cells serves to amplify glucose-mediated Ca2+ influx into the beta-cells.
Animals↗
General pharmacology of gemcitabine hydrochloride in animals.
Gemcitabine (2',2'-difluorodeoxycytidine monohydrochloride, LY188011 hydrochloride, CAS 122111-03-9) is a nucleoside analog with a broad spectrum of antitumor activity in murine models and is currently undergoing clinical evaluation. The profile of the pharmacological effects of this agent was assessed in studies evaluating the cardiovascular and respiratory systems, renal function, the gastrointestinal system, the central nervous system, and the autonomic nervous system. In vivo doses ranged from 0.15 to 300 mg/kg given by the intravenous route, while in vitro concentrations up to 1 x 10-3 mol/l were used. Gemcitabine was inactive in the autonomic nervous system, gastrointestinal function, and central nervous system studies. Only minimal changes were seen in the cardiovascular and respiratory study, with a slight decrease in pulmonary arterial pressure at the mid dose and a stroke volume increase at the high dose. In the renal function studies, a slight decrease in the urine pH at the high dose and decreased serum creatinine at the mid dose levels were observed. In summary, gemcitabine had minimal effect in these pharmacodynamic studies. These results indicate that gemcitabine has a low potential to produce adverse pharmacologic effects.
Animals↗
Biochemical evidence for nitric oxide formation from streptozotocin in isolated pancreatic islets.
Streptozotocin (STZ) is selectively toxic to insulin-secreting beta-cells of pancreatic islets and induces impairment of islet glucose oxidation and of glucose-induced insulin secretion. Similar effects are induced by Interleukin-1 (IL-1), and the deleterious effects of IL-1 on islets appear to be mediated by nitric oxide (NO). STZ contains a nitroso moiety and may liberate NO by processes analogous to those for the NO-releasing drug nitroprusside. NO is rapidly transformed to nitrite in aqueous solution, and NO activates heme-containing enzymes such as guanylyl cyclase and inhibits iron-sulfur enzymes such as mitochondrial aconitase. Data presented here indicate that incubation of rat islets with STZ at concentrations that impair insulin secretion results in generation of nitrite, stimulation of islet guanylyl cyclase and accumulation of cGMP, and inhibition of islet mitochondrial aconitase activity to a degree similar to that achieved by IL-1. Effects of STZ on beta-cells may be mediated by local liberation of NO from STZ within islets.
Aconitate Hydratase↗
IL-1 beta induces the coexpression of both nitric oxide synthase and cyclooxygenase by islets of Langerhans: activation of cyclooxygenase by nitric oxide.
Autoimmune diabetes is characterized by an early infiltration of lymphocytes into and around islets, which is followed by selective destruction of the insulin-secreting beta-cell. Cytokines released during this inflammatory reaction have been implicated as effector molecules which mediate beta-cell destruction. In vitro treatment of rat islets with the cytokine IL-1 beta results in an inhibition of glucose-stimulated insulin secretion that is mediated by the overproduction of nitric oxide. IL-1 beta also stimulates the production of the cyclooxygenase (COX) product prostaglandin E2 (PGE2). In this study we have examined the effects of IL-1 beta on both inducible nitric oxide synthase (iNOS) and inducible cyclooxygenase (iCOX) expression, and the direct effects of nitric oxide on the activity of COX. Treatment of rat islets with 5 units/mL IL-1 beta induces a similar time-dependent production of both nitrite and PGE2. IL-1 beta-induced nitrite and PGE2 production is attenuated by the NOS inhibitor NG-monomethyl-L-arginine (NMMA), but NMMA has no inhibitory effect on the expression of either iCOX or iNOS as determined by immunoprecipitation. Actinomycin D prevents IL-1 beta-induced iCOX and iNOS expression and the production of both nitrite and PGE2 by islets, suggesting that mRNA transcription is required for IL-1 beta-induced expression of both iNOS and iCOX. The effects of exogenous arachidonic acid on both constitutive COX (cCOX) and iCOX activity were also investigated.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Oxidoreductases↗
Mass spectrometric characterization of arachidonate-containing plasmalogens in human pancreatic islets and in rat islet beta-cells and subcellular membranes.
Pancreatic islets, when stimulated with D-glucose, secrete insulin by processes requiring glycolytic metabolism and generation of ATP. Hydrolysis of membrane phospholipids also occurs in glucose-stimulated islets, resulting in accumulation of nonesterified arachidonate, which facilitates Ca2+ entry and the rise in beta-cell [Ca2+] that triggers insulin secretion. Glucose-induced hydrolysis of arachidonate from islet phospholipids is mediated in part by an ATP-stimulated, Ca(2+)-independent (ASCI) phospholipase A2 (PLA2) which prefers plasmenyl over diacyl phospholipid substrates. Here we characterize the endogenous plasmalogen content of islet cells and subcellular membranes. Fast atom bombardment mass spectrometric analyses demonstrated that three of the most abundant molecular species of ethanolamine phospholipids in rat pancreatic islets were plasmalogens with sn-2 arachidonate residues and palmitic, oleic, or stearic aldehyde residues, respectively, in the sn-1 position. Purified populations of beta-cells prepared by fluorescence-activated cell sorting were also found to contain these plasmenylethanolamine molecular species in abundance similar to that in intact islets and greater than that in islet alpha-cells. Both islet plasma membranes (PM) and endoplasmic reticulum (ER) also contained these plasmenylethanolamine species, which accounted for 42% (PM) to 64% (ER) of the ethanolamine phospholipid arachidonate content of these membranes, as measured by stable isotope dilution mass spectrometry. Plasmenylethanolamine species were also abundant constituents of human pancreatic islets (accounting for 58% of their ethanolamine phospholipid arachidonate content) and were hydrolyzed more rapidly than diacyl ethanolamine phospholipid by human islet cytosolic ASCI-PLA2. Both secretagogue-induced eicosanoid release and insulin secretion from human islets were attenuated by an ASCI-PLA2 suicide substrate which sterically resembles plasmalogens. These observations are consistent with the hypotheses that islet beta-cell ASCI-PLA2-catalyzed hydrolysis of arachidonate from endogenous plasmenylethanolamine substrates may occur in membrane compartments which participate in regulation of the beta-cell cytosolic [Ca2+] and that this may be an intermediary biochemical event in the induction of insulin secretion.
Aldehydes↗
Arachidonic acid release from aortic smooth muscle cells induced by [Arg8]vasopressin is largely mediated by calcium-independent phospholipase A2.
To identify the phospholipase mediating the majority of [Arg8]vasopressin (AVP)-induced release of arachidonic acid in A-10 smooth muscle cells, we exploited the specificity inherent in the mechanism-based inhibitor, (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (HELSS), which possesses a 1,000-fold selectivity for inhibition of calcium-independent versus calcium-dependent phospholipases A2. Utilizing [3H]arachidonic acid-labeled A-10 smooth muscle cells, one-half of AVP-inducible [3H]arachidonic acid release was inhibited by pretreatment with only 1 microM HELSS and two-thirds of AVP-stimulated [3H]arachidonic acid release was inhibited by 5 microM HELSS. The inhibition of [3H]arachidonic acid release by HELSS was saturable (i.e. no additional inhibition of [3H]arachidonic acid release was present at 10 microM HELSS), specific (i.e. the activities of six intracellular enzymes, as well as the rate of glucose oxidation, were not altered by HELSS treatment), and nontoxic (i.e. HELSS-treated cells excluded trypan blue dye and did not leak intracellular enzymes into the medium). Collectively, these results demonstrate that HELSS blocks AVP-induced arachidonic acid release by specific and irreversible inhibition of calcium-independent phospholipase A2 and underscore the importance of calcium-independent phospholipase A2 in agonist-induced arachidonic acid release in at least some cell types.
Animals↗
Mass spectrometric identification and quantitation of arachidonate-containing phospholipids in pancreatic islets: prominence of plasmenylethanolamine molecular species.
D-Glucose induces insulin secretion from beta-cells of pancreatic islets by processes involving glycolytic metabolism and generation of ATP. Glucose also induces hydrolysis of beta-cell membrane phospholipids and accumulation of nonesterified arachidonate, which facilitates Ca2+ entry and the rise in beta-cell Ca2+ concentration that is a critical signal in the induction of insulin secretion. Glucose-induced hydrolysis of arachidonate from beta-cell phospholipids is mediated in part by an ATP-stimulated, Ca(2+)-independent (ASCI)-phospholipase A2 (PLA2), which, in vitro, prefers plasmalogen over diacylphospholipid substrates, but it is not known whether islets contain plasmalogens. We have identified and quantitated the major species of arachidonate-containing phospholipids in pancreatic islets by high-performance liquid chromatographic and mass spectrometric analyses. Arachidonate has been found to constitute 30% of the total islet glycerolipid fatty acyl mass. Ethanolamine phospholipids contain 30% of total islet arachidonate, and 44% of that amount resides in three plasmenylethanolamine molecular species with residues of palmitic, oleic, or stearic aldehydes in the sn-1 position. These endogenous islet plasmenylethanolamine species are hydrolyzed more rapidly than phosphatidylethanolamine species by islet ASCI-PLA2 in vitro and are also hydrolyzed in intact islets stimulated with secretagogues. ASCI-PLA2-catalyzed hydrolysis of islet plasmenylethanolamine species in vitro is inhibited by a selective haloenol lactone suicide substrate (HELSS) which is sterically similar to plasmalogens, and HELSS also inhibits all temporal phases of both eicosanoid release and insulin secretion from secretagogue-stimulated pancreatic islets. Islet beta-cell ASCI-PLA2-catalyzed hydrolysis of arachidonate from endogenous plasmenylethanolamine substrates may be an intermediary biochemical event in the induction of insulin secretion.
Animals↗
Rat and human pancreatic islet cells contain a calcium ion independent phospholipase A2 activity selective for hydrolysis of arachidonate which is stimulated by adenosine triphosphate and is specifically localized to islet beta-cells.
The recent demonstration that myocardial Ca(2+)-independent phospholipase A2 exists as a complex of catalytic and regulatory polypeptides that is modulated by ATP has suggested a novel mechanisms through which alterations in glycolytic flux can be coupled to the generation of eicosanoids which facilitate insulin secretion. To determine the potential relevance of this mechanism, we examined the kinetic characteristics, substrate specificities, and cellular locus of phospholipase A2 activity in pancreatic islets. Rat pancreatic islets contain a Ca(2+)-independent phospholipase A2 activity which is optimal at physiologic pH, preferentially hydrolyzes phospholipid substrates containing a vinyl ether linkage at the sn-1 position, and prefers arachidonic acid compared to oleic acid in the sn-2 position. Rat islet Ca(2+)-independent phospholipase A2 activity is inhibited by the mechanism-based inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one and is stimulated by ATP. Purification of beta-cells from dispersed pancreatic islet cells by fluorescence-activated cell sorting demonstrated that beta-cells (but not non-beta-cells) contain Ca(2+)-independent, ATP-stimulated phospholipase A2 activity. Remarkably, clonal RIN-m5f insulinoma cells, which possess a defect in glucose-induced insulin secretion, contain a Ca(2+)-independent phospholipase A2 which is not modulated by alterations in ATP concentration. Collectively, these results and those of an accompanying paper [Ramanadham et al. (1993) Biochemistry (following paper in this issue)] implicate Ca(2+)-independent phospholipase A2 as a putative glucose sensor which can couple alterations in glycolytic metabolism to the generation of biologically active eicosanoids and thereby facilitate glucose-induced insulin secretion.
Adenosine Triphosphate↗
Inhibition of arachidonate release by secretagogue-stimulated pancreatic islets suppresses both insulin secretion and the rise in beta-cell cytosolic calcium ion concentration.
Fuel secretagogues induce hydrolysis of esterified arachidonic acid from pancreatic islet cell phospholipids and accumulation of nonesterified arachidonate at concentrations up to 35 microM. Exogenous arachidonate (5-30 microM) amplifies depolarization-induced insulin secretion from islets. Fuel secretagogue-induced hydrolysis of arachidonate from islet phospholipids occurs in Ca(2+)-free medium, suggesting the possible involvement of a Ca(2+)-independent phospholipase. In the companion paper [Gross et al. (1993) Biochemistry (preceding paper in this issue)], we demonstrated that the major islet phospholipase A2 is Ca(2+)-independent, ATP-stimulated, and inhibited by the haloenol lactone suicide substrate (HELSS) (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one. Here we demonstrate that HELSS suppressed both release of the arachidonate metabolite prostaglandin E2 and insulin secretion from islets stimulated with D-glucose and the muscarinic agonist carbachol. Both prostaglandin E2 release and insulin secretion were suppressed with similar concentration profiles and time courses. Islet oxidation of [14C]-glucose to [14C]CO2, activities of islet lactate dehydrogenase and alanine and aspartate aminotransferases, and carbachol-induced inositol phosphate accumulation in islets were all unaffected by HELSS. Depolarization of isolated beta-cells with 40 mM KCl induced a rise in cytosolic [Ca2+] that was also unaffected by HELSS. In contrast, the 17 mM D-glucose-induced rise in beta-cell [Ca2+] was inhibited by HELSS in a concentration-dependent manner, but that induced by exogenous arachidonate (15 microM) was not. These results suggest that fuel secretagogues activate the islet Ca(2+)-independent phospholipase A2, resulting in release of nonesterified arachidonate, which facilitates Ca2+ entry into beta-cells and promotes insulin secretion.
Animals↗
The psychiatric, psychological and behavioural functioning of a boy with terminal deletion of the long arm of chromosome 10.
The developmental and behavioural functioning of a six-year-old boy with deletion of the long arm of chromosome 10 was evaluated using reliable, standardised, psychological inventories. The information obtained clarified his complex pattern of strengths and needs; it also contributes scientifically derived data to the literature on behavioural correlates of this condition.
Child Behavior Disorders↗
Regulation of paracellular permeability in Caco-2 cell monolayers by protein kinase C.
Caco-2 cells are an enterocyte-like cell line derived from a human colonic adenocarcinoma. Paracellular permeability was assessed in monolayers of these cells by transmonolayer resistance and by the permeation of [3H]mannitol across the monolayer. Paracellular permeability was increased by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (50 nM), carbachol (500 microM), and the combination of carbachol (50 microM) and monolein (100 microM), an inhibitor of diacylglycerol kinase, as manifested by a decrease in transmonolayer resistance and an increase in mannitol permeation. The effects of all of these stimuli on transmonolayer resistance were inhibited by staurosporine (3 nM), an inhibitor of PKC. The effects of carbachol plus monolein were also inhibited by atropine (0.1 microM), a muscarinic antagonist. Treatment of the monolayers with each of the stimuli was associated with translocation of PKC activity from cytosol to a membrane-associated state. Stimulation of Caco-2 cell monolayers with phorbol myristate acetate or with the combination of carbachol and monolein was also associated with phosphorylation of the MARCKS protein, an endogenous substrate of PKC. These data support the hypothesis that intestinal paracellular permeability is regulated by the activity of enterocyte PKC and demonstrate that the increase in paracellular permeability induced by binding of carbachol to the muscarinic receptor is mediated by activation of PKC.
Adenocarcinoma↗
Amplification of insulin secretion by lipid messengers.
D-glucose induces a rise in pancreatic islet beta-cell cytosolic [Ca2+] by processes requiring both glucose metabolism and Ca2+ entry from the extracellular space, and this Ca2+ signal is thought to be critical to the induction of insulin secretion. Insulin secretagogues also induce phospholipid hydrolysis and accumulation of phospholipid-derived mediators in islets, including the lipid messengers DAG, nonesterified arachidonic acid, and arachidonate 12-LO products. This study offers the following viewpoints on potential roles of these lipid messengers in insulin secretion as working hypotheses: 1) the Ca2+ signal provided to the beta-cell by D-glucose induces insulin secretion only in the context of amplifying background signals provided by the beta-cell content of messengers including DAG; 2) muscarinic receptor agonists amplify glucose-induced insulin secretion in part by altering the beta-cell content of DAG; 3) the Ca2+ signal provided by metabolism of D-glucose is amplified by the level of nonesterified arachidonic acid in beta-cell membranes, which acts to facilitate Ca2+ entry; 4) metabolism of glucose induces accumulation of nonesterified arachidonate in beta-cells via activation of a recently identified ASCI-PLA2 enzyme, which may be a component of the beta-cell fuel sensor apparatus; and 5) arachidonate 12-LO metabolites are potential candidates as adjunctive modulators of beta-cell K(+)-channel activity.
Animals↗
Successful therapy of iron intoxication in pregnancy with intravenous deferoxamine and whole bowel irrigation.
We describe the successful treatment of a severely iron-poisoned adult patient in week 26 of gestation with 10.2 g deferoxamine administered iv over 14 h and whole bowel irrigation (2 L/h of polyethylene glycol-electrolyte solution/nasogastric tube for 12 h) with a good maternal outcome and no adverse effects on the fetus.
Adult↗
Constraints on the enhancement of elimination of drugs with activated charcoal.
Both extracorporeal hemoperfusion through charcoal-containing columns and repeated oral administration of charcoal can accelerate clearance of some drugs or toxins from the systemic circulation. The efficacy of these 2 interventions is limited by a variety of factors, and the complex kinetic equations describing charcoal-induced clearance provide little practical clinical guidance about the potential efficacy of charcoal in accelerating clearance of a specific drug or toxin without previous empiric data. We derive here simple rules that place an upper limit on the maximal fraction of an absorbed dose of drug that can be removed (FRmax) by charcoal in terms of the volume of distribution (Vd), a parameter which is known for most drugs. For 4 h of hemoperfusion, a theoretical upper limit of FRmax is (1/Vd), where Vd is expressed in L/kg of body weight, and actual fractional removal (FR) will not exceed [1/(2 x Vd)]. Drug removal by 24 h of repeated po administration of charcoal exhibits similar relationships between FRmax and Vd, when charcoal-induced clearance derives primarily from removal of drug from blood perfusing in gastrointestinal mucosa. These relationships offer a simple means to evaluate the potential efficacy of acceleration of drug clearance by activated charcoal for drugs with a known value for Vd, and the relationships indicate that such interventions are impractical for drugs with very large values for Vd, such as tricyclic antidepressants.
Absorption↗