Construction of a cell-permeable CDC42 binding fragment of ACK that inhibits v-Ha-Ras transformation.
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3T3 Cells↗
Biomedical subjects
W Montague
Publications and source records attributed to W Montague.
Multiplicity and tissue specific expression of camel cytochrome P450(s).
The presence of multiple forms of the cytochrome P450 was demonstrated enzymatically in camel tissues using a variety of isoenzyme specific substrates and immunochemically using isoenzyme specific antibodies. The maximum catalytic activity using xenobiotics as substrate was observed in the liver followed by the kidney. However, lauric acid hydroxylation was found to be higher in the kidney than in any other tissues. Camel liver microsomal monooxygenase activity using aniline, aminopyrene, ethoxycoumarin, ethoxyresorufin and benzo(a)pyrene as substrates was comparable with those of rat and human livers. The activity of the enzymes in extrahepatic tissues of the camel was comparable with those of the rat extrahepatic tissues. The maximum expression of P450 protein was seen in the camel liver and kidney while the brain and intestine exhibited relatively low levels of expression. P450 expression in camel tissues appeared to be higher than in rat tissues. Immunohistochemical staining of P450 in the camel liver, kidney and brain confirmed the higher expression of P450 enzyme proteins in the liver and kidney as compared to other extrahepatic tissues. The maximum expression of P450 in the liver was observed in hepatocytes around the central vein and in the kidney it was observed in the proximal tubules. These results demonstrate that the multiple forms of P450s are differentially expressed in camel tissues and that the relative levels of expression are comparable with those of rat and human tissues. These observations may be important in understanding the differential susceptibility of camel tissues to the toxic/therapeutic effects of xenobiotics/drugs and environmental pollution.
Animals↗
The GTPase and Rho GAP domains of p190, a tumor suppressor protein that binds the M(r) 120,000 Ras GAP, independently function as anti-Ras tumor suppressors.
p190 is a Tyr-phosphorylatable G protein of M(r) 190,000 that binds NH2-terminal SH2 domains of GAP1, a Ras GAP of M(r) 120,000. p190 contains at least two functional domains: a GTPase domain at the NH2 terminus and a GAP domain at the COOH terminus that can attenuate signal-transducing activity of three distinct G proteins (Rac, Rho, and CDC42). Here, we demonstrate that overexpression of either an antisense p190 RNA or a dominant negative mutant (Asn36) of p190 GTPase domain (residues 1-251) but not the wild-type p190 GTPase domain is able to transform normal NIH/3T3 fibroblasts. Furthermore, overexpression of either the wild-type p190 GTPase domain or the COOH-terminal GAP domain can suppress v-Ha-Ras-induced malignant transformation. These results indicate that p190 contains at least two distinct anti-Ras tumor suppressor domains, the GTPase and GAP domains, and suggest that one of the mechanisms underlying the suppression of Ras-transformation by p190 is the attenuation by p190 GAP domain of Rac/Rho/CDC42 signalings, which are essential for Ras-transformation. In fact, the p190 GAP domain alone suppresses the expression of the c-Fos gene, which is mediated by Rac/Rho/CDC42 and is required for oncogenicity of Ras.
Animals↗
Effect of bitter melon (Momordica charantia) fruit juice on the hepatic cytochrome P450-dependent monooxygenases and glutathione S-transferases in streptozotocin-induced diabetic rats.
Bitter melon (Momordica charantia), commonly known as karela, has been reported to have hypoglycemic, antiviral, antidiabetic, and antitumor activities. In the present study, we have investigated the effects of oral feeding of karela fruit juice on the hepatic cytochrome P450 (CYP) and glutathione S-transferase (GST) drug-metabolizing enzymes in the streptozotocin (STZ)-induced diabetic rat. Hepatic CYP contents, ethoxycoumarin-O-deethylase (ECOD), ethoxyresorufin-O-deethylase (EROD), aniline hydroxylase (AH), and aminopyrene N-demethylase (APD) activities were measured in control, diabetic, and karela juice fed animals. Diabetic rats exhibited a 50-100% increase in AH and EROD activities that was reversed by karela juice feeding. In addition, a decrease (17-20%) in the activities of APD and ECOD was observed in diabetic rat liver. Feeding of karela juice to the diabetic animals brought the level of APD close to that of control animals, while ECOD was further reduced to 60% of the control value. The cytosolic glutathione concentration was decreased in diabetic rats, and karela juice feeding normalized the effect. However, an increase (of 20-30%) in the GST activity was observed in both diabetic and karela juice fed rats. Western immunoblot analysis of CYP and GST isozymes exhibited a differential response during diabetes. The expression of CYP1A1, 2B1, 2E1, 3A4, and 4A2 in diabetes, while a decrease in GST mu was observed. Our results suggest that the changes in hepatic phase I and phase II drug-metabolizing enzyme activities in the STZ-induced diabetic animals may be associated with the altered expression of different CYP and GST isozymes. In addition, we have also observed that karela does not always reverse the effects on drug-metabolizing enzymes in STZ-induced diabetes.
Animals↗
beta-Naphthoflavone-inducible cytochrome P4501A1 activity in liver microsomes of the marine safi fish (Siganus canaliculatus).
The cytochrome P450-dependent metabolism of benzo(a)pyrene and other xenobiotics has been investigated in liver microsomes prepared from a local marine safi fish, Siganus canaliculatus. The safi fish was found to have a well-developed microsomal monooxygenase system consisting of cytochrome P450, cytochrome b5 and NADPH-cytochrome c reductase. The fish microsomal enzyme system was able to metabolize benzo(a)pyrene, 7-ethoxycoumarin and 7-ethoxyresorufin. Male fish were found to exhibit a higher monooxygenase activity than female fish. Treatment of fish with beta-naphthoflavone was found to induce (2- to 4-fold) the activities of aryl hydrocarbon hydroxylase, ethoxycoumarin O-deethylase and ethoxyresorufin O-deethylase. HPLC analysis of the metabolites produced by incubation of benzo(a)pyrene with the liver microsomal preparation showed a predominant formation of 3-OH and 9-OH benzo(a)pyrene. There was an increased formation of benzo(a)pyrene 7,8-diol and benzo(a)pyrene 7,8,9,10-tetrol in liver microsomes prepared from beta-naphthoflavone-treated fish. Western immunoblot analysis of liver microsomes from beta-naphthoflavone-treated fish using an antibody to rat liver cytochrome P4501A1 (CYP1A1) suggested the presence of an inducible cytochrome P450 enzyme that was comparable with that of rat liver enzyme. Our results suggest that liver microsomes from the safi fish have multiple forms of cytochrome P450 with a specific beta-naphthoflavone-inducible CYP1A1 homologous protein that can metabolize a variety of substrates.
Animals↗
Alteration of glutathione, glutathione S-transferase and lipid peroxidation in mouse skin and extracutaneous tissues after topical application of gasoline.
The skin is the major environmental interface of the human body and is repeatedly exposed to a broad array of exogenous chemicals potentially capable of causing toxicity. In the present study we have applied 3, 6 or 12 ml leaded gasoline/kg body weight to the skin of adult male Swiss mice for 7 consecutive days and then sacrificed the animals on 8th day after an overnight fast. Glutathione (GSH) concentration, lipid peroxidation and other GSH-dependent enzyme activities were measured in skin, liver, brain and blood tissues of the mice. Topical application of 12 ml/kg gasoline caused a significant increase in water consumption by the animals, although, their body weight and food consumption was not significantly affected. A 40-60% decrease in blood concentration of glucose, triglyceride, and cholesterol was also observed after the treatment. The hemoglobin concentration, GSH content, lipid peroxidation and glucose 6-phosphate dehydrogenase activity of erythrocytes were not significantly affected by the gasoline treatment. However, a decrease in GSH concentration (16-21%), lipid peroxidation (30-60%) and glutathione S-transferase (GST) activity (30-40%) was observed in skin, liver and brain after gasoline application. Western blot analysis of tissues using antibodies against GST isoenzymes demonstrated an alteration in the expression of various GST isoenzymes after gasoline treatment. Our results suggest that topical exposure of gasoline causes some deleterious effects on skin and extracutaneous tissues.
Animals↗
"How much biochemistry should a good doctor know?". Experiences from two new medical schools.
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Biochemistry↗
Metabolism of benzo(a)pyrene, dimethylbenzanthracene and aflatoxin B1 by camel liver microsomes.
The ability of camel liver microsomes to metabolise a range of common environmental carcinogens including benzo(a)pyrene, dimethylbenzanthracene and aflatoxin B1 has been investigated. The camel liver has shown the ability to metabolise benzo(a)pyrene, dimethylbenzanthracene and aflatoxin B1 to a number of metabolites. The major metabolites of benzo(a)pyrene produced by camel liver enzymes were identified as its mono-hydroxy derivatives and suggest that the metabolic detoxification pathways of carcinogen metabolism are predominant in this species. Benzo(a)pyrene metabolising activity in camel liver required NADPH and was inhibited by CO and alpha-naphthoflavone suggesting the involvement of cytochrome P450 in the metabolism of this carcinogen by camel liver. The cytochrome P450-dependent metabolism of carcinogen and other specific substrates such as ethoxyresorufin and ethoxycoumarin, by camel liver enzymes, was about 50% higher than that of rat liver enzymes. The cytochrome P450-dependent metabolism of a variety of carcinogenic and other substrates by camel liver demonstrated that there are multiple forms of cytochrome P450 enzymes involved in the metabolism of a wide array of xenobiotics and pollutants.
9,10-Dimethyl-1,2-benzanthracene↗
Drug and xenobiotic metabolising enzymes in camel liver: multiple forms and species specific expression.
1. Previous studies have demonstrated the presence of phase I mixed-function oxidases (cytochrome P450-dependent) and phase II conjugation (glutathione S-transferase) enzymes in camel liver. This study represents further characterisation of these drug metabolising enzyme systems in camel liver by comparing their catalytic and immunochemical properties with enzymes of rat and mouse liver. 2. Using the specific P450 substrate aniline, the microsomal aniline hydroxylase activity of camel liver was found to be significantly lower than that of rat and mouse. The Km values of the enzyme for aniline was similar in rat and camel liver; however, the Vmax for camel liver enzyme was 50% of the rat liver enzyme. Aminopyrene N-demethylase activity in camel liver, was lower than that of rat but higher than in mouse. Microsomal NADPH cytochrome C-reductase and NADPH-supported lipid peroxidation activities were similar in all three species. 3. The cytosolic phase II conjugation enzyme glutathione S-transferase and glutathione peroxidase activities in camel liver were markedly lower than those of rat and mouse enzymes. However, GSH concentration was similar in all three species. 4. Immunodot blot and Western blot analysis of liver cytosols, using antibodies to specific GST isoenzymes, have shown that camel liver like mouse and rat, expresses predominantly the Alpha and Mu classes of GST. GST Pi on the other hand, was abundant in mouse liver and was underexpressed in camel and rat liver. 5. Our results demonstrate that there are multiple forms of phase I (P450) and phase II (GST) enzymes in camel liver and that they are comparable with the drug metabolising enzymes of rat and mouse.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Academic selection criteria and subsequent performance.
In this paper we have attempted to determine if there are any aspects of the academic history of a student, prior to admission to the University of Leicester medical course, that correlate with his or her subsequent performance in that course. The analyses presented suggest that a student's overall performance in GCE O- and A-level examinations correlates with subsequent performance at various stages of the medical course. In addition, there is a correlation between performance in the medical course and performance in A-level chemistry and biology but not mathematics, physics or general studies. Students who resat two or more A-level subjects performed at a lower level throughout the course than those with only one or no resits. These results emphasize the importance of academic criteria in the medical student selection process.
Achievement↗
Biochemical studies on the release of catecholamines from the rat carotid body in vitro.
The effects of hypoxia and carbachol on the release of newly synthesized catecholamines from superfused rat carotid bodies have been examined. Hypoxic superfusion medium was found to evoke catecholamine release which was dependent on the extracellular calcium concentration and was reduced by nitrendipine and atropine. Superfusion with the muscarinic agonist, carbachol, stimulated catecholamine release independently of the oxygen tension of the medium. The effect of carbachol on catecholamine release was abolished by atropine, suggesting that it was mediated by activation of cholinergic receptors of the muscarinic type. Both hypoxia and carbachol stimulated the release of 45Ca from carotid bodies prelabelled with 45Ca. The release of 45Ca with either stimulus was reduced by atropine and nitrendipine. These results suggest that although extracellular calcium plays an important role in the exocytotic secretory process of the carotid body, the mobilization of intracellular calcium pools may also contribute to the secretory response.
Animals↗
Calcium handling by stimulated islets of Langerhans.
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Animals↗
Mechanisms involved in intracellular calcium mobilization in isolated rat islets of Langerhans.
1. The rate of 45Ca2+ efflux from prelabelled rat islets of Langerhans was stimulated by carbachol in a dose-dependent manner. 2. Significant stimulation occurred in the presence of 0.2 microM-carbachol; the response was half-maximal at 3-5 microM and was maximal at 20 microM. 3. Stimulation of 45Ca2+ efflux by carbachol was not dependent on the presence of extracellular Ca2+ and was enhanced in Ca2+-depleted medium. 4. Stimulation of 45Ca2+ efflux by 5 microM-carbachol occurred independently of any change in [3H]arachidonic acid release in prelabelled islets, and probably reflected generation of inositol trisphosphate in the cells. 5. The amphipathic peptide melittin failed to increase islet-cell 45Ca2+ efflux at a concentration of 1 microgram/ml, and caused only a modest increase at 10 micrograms/ml. 6. Despite its failure to increase 45Ca2+ efflux, melittin at 1 microgram/ml caused a marked enhancement of 3H release from islets that had been prelabelled with [3H]arachidonic acid. 7. The stimulation of 3H efflux caused by melittin correlated with a dose-dependent increase in the unesterified [3H]arachidonic acid content of prelabelled islets and with a corresponding decrease in the extent of labelling of islet phospholipids. 8. Combined addition of melittin (1 microgram/ml) and 5 microM-carbachol to perifused islets failed to augment 45Ca2+ efflux relative to that elicited by carbachol alone. 9. The data indicate that melittin promotes an increase in arachidonic acid availability in intact rat islets. They do not, however, support the proposal that this can either directly reproduce or subsequently modify the extent of intracellular Ca2+ mobilization induced by agents that cause an increase in inositol trisphosphate.
Animals↗
Effects of the calcium-channel agonist CGP 28392 on insulin secretion from isolated rat islets of Langerhans.
The rate of insulin secretion from isolated rat islets of Langerhans was affected by a number of dihydropyridine derivatives known to interact with voltage-sensitive Ca2+ channels in excitable cells. The channel antagonists nifedipine and nitrendipine were potent inhibitors of glucose-induced insulin secretion in response to both 8 mM- and 20 mM-glucose, although they did not lower the basal secretion rate observed in the presence of 4 mM-glucose. The Ca2+-channel agonist, CGP 28392, also failed to alter the basal rate of insulin secretion. In the presence of 8 mM-glucose, however, 1 microM-CGP 28392 enhanced the insulin-secretion rate to a value approximately double that with 8 mM-glucose alone. This effect was dose-dependent, with half the maximal response elicited by 0.1 microM-CGP 28392, and full enhancement at 10 microM. The response was rapid in onset, with an increase in insulin secretion evident within 2 min of CGP 28392 infusion in perifused islets. Stimulation of insulin secretion by CGP 28392 was correlated with a rapid enhancement of glucose-stimulated 45Ca2+ uptake into islets cells, and with a transiently increased rate of 45Ca2+ efflux from pre-loaded islets. Stimulation of insulin secretion by CGP 28392 was abolished in the presence of noradrenaline, although under these conditions the rapid stimulation of 45Ca2+ influx induced by CGP 28392 was only partially inhibited. In contrast with these results, when islets were incubated in the presence of 20 mM-glucose, CGP 28392 caused a dose-dependent inhibition of insulin secretion. Half-maximal inhibition required approx. 0.2 microM-CGP 28392, with maximal effects observed at 10 microM. Under these conditions, however, the extent of insulin secretion was still only decreased by about 50%, to a value which was similar to that seen in the presence of 8 mM-glucose and CGP 28392. These results suggest that dihydropyridine derivatives can alter the activity of voltage-dependent Ca2+ channels in islet cells, and are consistent with the possibility that gating of these channels plays an important role in regulating the rate of insulin secretion after glucose stimulation.
Animals↗
Studies on the mechanism by which melittin stimulates insulin secretion from isolated rat islets of Langerhans.
Incubation of isolated rat islets of Langerhans with melittin resulted in a dose-dependent stimulation of insulin secretion with half the maximal response occurring at 4 micrograms/ml melittin. The effect of melittin on insulin secretion was dependent on extracellular calcium, was inhibited by the phospholipase A2 inhibitor quinacrine and by the lipoxygenase inhibitor nordihydroguaiaretic acid. Stimulation of insulin secretion by melittin was associated with a calcium-dependent loss of [3H]arachidonic acid from phospholipids in islet cells prelabelled with [3H]arachidonic acid. Analysis of the islet phospholipids involved in this response revealed that the [3H]arachidonic acid was released predominantly from phosphatidylcholine. These results suggest that melittin may stimulate insulin secretion by activating phospholipase A2 in islet cells, causing the release of arachidonic acid from membrane phospholipid. The results are consistent with suggestions that the subsequent metabolism of arachidonic acid via the lipoxygenase pathway may be involved in regulating the insulin secretory response.
Animals↗
Studies on the role of inositol trisphosphate in the regulation of insulin secretion from isolated rat islets of Langerhans.
Glucose (20 mM) and carbachol (1 mM) produced a rapid increase in [3H]inositol trisphosphate (InsP3) formation in isolated rat islets of Langerhans prelabelled with myo-[3H]inositol. The magnitude of the increase in InsP3 formation was similar when either agent was used alone and was additive when they were used together. In islets prelabelled with 45Ca2+ and treated with carbachol (1 mM), the rise in InsP3 correlated with a rapid, transient, release of 45Ca2+ from the cells, consistent with mobilization of 45Ca2+ from an intracellular pool. Under these conditions, however, insulin secretion was not increased. In contrast, islets prelabelled with 45Ca2+ and exposed to 20mM-glucose exhibited a delayed and decreased 45Ca2+ efflux, but released 7-8-fold more insulin than did those exposed to carbachol. Depletion of extracellular Ca2+ failed to modify the increase in InsP3 elicited by either glucose or carbachol, whereas it selectively inhibited the efflux of 45Ca2+ induced by glucose in preloaded islets. Under these conditions, however, glucose was still able to induce a small stimulation of the first phase of insulin secretion. These results demonstrate that polyphosphoinositide metabolism, Ca2+ mobilization and insulin release can all be dissociated in islet cells, and suggest that glucose and carbachol regulate these parameters by different mechanisms.
Animals↗
Effect of glucose on polyphosphoinositide metabolism in isolated rat islets of Langerhans.
The metabolism of inositol-containing phospholipids during insulin secretion was studied in rat islets of Langerhans preincubated with [3H]inositol to label their phospholipids. Glucose (20 mM) caused a rapid breakdown of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate and an accumulation of inositol trisphosphate and inositol bisphosphate. This effect was maximal at 60s, did not require the presence of extracellular Ca2+, and was abolished by mannoheptulose (15 mM), but not by noradrenaline (1 microM). Mannose (20 mM) and DL-glyceraldehyde (10 mM) produced similar effects to those of glucose, but galactose (20 mM) and KCl (30 mM) were without effect. These results are compatible with the hypothesis that an early event in the stimulus-secretion coupling mechanism in the pancreatic B-cell is the rapid breakdown of polyphosphoinositides catalysed by phospholipase C. Moreover, they suggest that the breakdown of polyphosphoinositides is linked to sugar metabolism in the B-cell. This observation is important, since it demonstrates that events in a cell other than plasma-membrane receptor occupancy can promote polyphosphoinositide hydrolysis.
Animals↗
Studies on the mechanism of inhibition of glucose-stimulated insulin secretion by noradrenaline in rat islets of Langerhans.
Noradrenaline (norepinephrine) was shown to be a potent inhibitor of glucose-induced insulin release from rat pancreatic islets, with half-maximal inhibition of the secretory response to 20 mM-glucose occurring at approx. 0.3 microM, and complete suppression of the response occurring at 4 microM-noradrenaline. Inhibition of insulin secretion by noradrenaline was antagonized by the alpha 2-adrenergic antagonist yohimbine (half maximally effective dose approximately 1 microM), but was largely unaffected by the alpha 1-adrenergic antagonist prazosin at concentrations up to 50 microM, suggesting that the response was mediated by alpha 2-adrenergic receptors. Noradrenaline significantly reduced the extent of 45Ca2+ accumulation in glucose-stimulated islets, although as much as 5 microM-noradrenaline was required for 50% inhibition of this response. The ability of noradrenaline to inhibit islet-cell 45Ca2+ uptake was totally abolished in media containing 1 mM-dibutyryl cyclic AMP, suggesting that the response may have been secondary to lowering of islet cyclic AMP. Under these conditions, however, noradrenaline was still able to inhibit insulin secretion maximally. The data suggest that the site(s) at which noradrenaline acts to mediate inhibition of insulin secretion in rat islets lies distal to both islet-cell cyclic AMP accumulation and Ca2+ uptake.
Animals↗