Activation of members of the mitogen-activated protein kinase family by glucose in endothelial cells.
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To better understand the molecular mechanisms for hyperglycemia-induced proatherogenic changes in endothelial cells, the effect of high glucose on activation of members of the mitogen-activated protein kinase (MAPK) family, including c-Jun NH(2)-terminal kinase (JNK), extracellular signal-regulated kinase (ERK)-1, -2, and -5, and p38 kinase, was examined in bovine pulmonary artery endothelial cells (PAEC). Glucose, fructose, and raffinose induced a concentration-dependent decrease in PAEC growth. Addition of 25 mM glucose, fructose, or raffinose to normal growth medium stimulated an approximately twofold increase in JNK1 activity that was maximal after 24 h, whereas only glucose markedly increased ERK5 activity. Neither ERK1/2 nor p38 kinase activity was increased by glucose, fructose, or raffinose. The antioxidant N-acetylcysteine partially abrogated the glucose-induced increase in ERK5 activity but had no effect on the increase in JNK1 activity. In contrast, azaserine, which prevents increased flux through the hexosamine pathway, decreased glucose-induced JNK1 activity but had no effect on fructose- or raffinose-induced JNK1 activity. Consistent with this finding, glucosamine stimulated a 2.4-fold increase in JNK1 activity and reproduced the inhibitory effect of glucose on PAEC growth. In summary, glucose activates different members of the MAPK family in PAEC via distinct mechanisms. Moreover, the correlation between the ability of different sugars to activate JNK1 and inhibit cell growth suggests that activation of this signaling pathway may contribute to the growth inhibitory effect of glucose in endothelial cells. (+info)
Excessive hexosamines block the neuroprotective effect of insulin and induce apoptosis in retinal neurons.
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In addition to microvascular abnormalities, neuronal apoptosis occurs early in diabetic retinopathy, but the mechanism is unknown. Insulin may act as a neurotrophic factor in the retina via the phosphoinositide 3-kinase/Akt pathway. Excessive glucose flux through the hexosamine biosynthetic pathway (HBP) is implicated in the development of insulin resistance in peripheral tissues and diabetic complications such as nephropathy. We tested whether increased glucose flux through the HBP perturbs insulin action and induces apoptosis in retinal neuronal cells. Exposure of R28 cells, a model of retinal neurons, to 20 mm glucose for 24 h attenuated the ability of 10 nm insulin to rescue them from serum deprivation-induced apoptosis and to phosphorylate Akt compared with 5 mm glucose. Glucosamine not only impaired the neuroprotective effect of insulin but also induced apoptosis in R28 cells in a dose-dependent fashion. UDP-N-acetylhexosamines (UDP-HexNAc), end products of the HBP, were increased approximately 2- and 15-fold after a 24-h incubation in 20 mm glucose and 1.5 mm glucosamine, respectively. Azaserine, a glutamine:fructose-6-phosphate amidotransferase inhibitor, reversed the effect of 20 mm glucose, but not that of 1.5 mm glucosamine, on attenuation of the ability of insulin to promote cell survival and phosphorylate Akt as well as accumulation of UDP-HexNAc. Glucosamine also impaired insulin receptor processing in a dose-dependent manner but did not decrease ATP content. By contrast, in L6 muscle cells, glucosamine impaired insulin receptor processing but did not induce apoptosis. These results suggest that the excessive glucose flux through the HBP may direct retinal neurons to undergo apoptosis in a bimodal fashion; i.e. via perturbation of the neuroprotective effect of insulin mediated by Akt and via induction of apoptosis possibly by altered glycosylation of proteins. The HBP may be involved in retinal neurodegeneration in diabetes. (+info)
Linkage between toxin production and purine biosynthesis in Clostridium difficile.
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The production of toxins A and B by Clostridium difficile was greatly enhanced under biotin-limited conditions, in which a 140-kDa protein was expressed strongly. Gene cloning revealed that this protein was a homologue of formylglycinamidine ribonucleotide synthetase (FGAM synthetase, EC 6.3.5.3), which is known as PurL in Escherichia coli and catalyses the fourth step of the de novo purine biosynthesis pathway. This enzyme consisted of a single polypeptide, although FGAM synthetases of gram-positive bacteria usually consist of two subunits. Inhibition of the enzymic activity of C. difficile PurL by O-diazoacetyl-L-serine (azaserine) resulted in enhanced toxin B production even in biotin-sufficient conditions. In contrast, blockade of the preceding step of the PurL catalysing step by sulfamethoxazole inhibited toxin B production almost completely. These results suggest that accumulation of formylglycinamide ribonucleotide (FGAR), a substrate of FGAM synthetase, enhances toxin production by C difficile and depletion of FGAR reduces toxin production. (+info)
EFFECT OF HEAT AND PLATING MEDIUM ON SURVIVAL OF ESCHERICHIA COLI AFTER TREATMENT WITH RADIOMIMETIC CHEMICALS.
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Zampieri, Antonio (Palo Alto Medical Research Foundation, Palo Alto, Calif.), and Joseph Greenberg. Effect of heat and plating medium on survival of Escherichia coli after treatment with radiomimetic chemicals. J. Bacteriol. 89:931-936. 1965.-Survival of Escherichia coli strain S and its radioresistant mutant R(4) after treatment with mitomycin C, azaserine, nitrogen mustard, 1-methyl-3-nitro-1-nitrosoguanidine, nitrofurazone, and proflavine was studied. With all agents except proflavine, R(4) was more resistant than was S. Survival of strain S was greater on minimal glucose-salts medium than on Penassay (Difco), and greater on the latter than on Tryptone (Difco) agar; survival of S was greater when posttreatment incubation temperature was 45 C than when it was 37 C. Post-treatment plating medium or temperature had no effect on survival of R(4). Visible light did not affect survival of S or R(4). The survival curves of R(4) were exponential; those of S exhibited decreasing sensitivity with time of exposure. With proflavine, photoactivation by visible light was demonstrable, but there was no difference in survival between S and R(4). Survival of either strain was not affected by post-treatment plating medium or incubation temperature. (+info)
Novel expression of gastrin (cholecystokinin-B) receptors in azaserine-induced rat pancreatic carcinoma: receptor determination and characterization.
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Many reports have emphasized the role of gastrin as a growth factor for normal gastrointestinal mucosa and gastrointestinal cancers. Recent studies have pointed out that this peptide acts also as a growth factor for the pancreatic cancer cell line AR42J. This effect is mediated by gastrin [cholecystokinin (CCK)-B] receptors. In the present study, we investigated gastrin (CCK-B) receptor expression in the azaserine-induced rat pancreatic carcinoma DSL-6, comparing it to normal rat pancreas, and we also characterized CCK receptor subtypes in this tumor. The results showed that there is extensive gastrin binding to the DSL-6 pancreatic carcinoma. No evidence of specific gastrin binding to normal pancreas was found. Analysis of the ability of gastrin-17-I to inhibit 125I-gastrin-I binding demonstrated that gastrin bound to a single class of receptors with a Kd of 0.21 +/- 0.04 nM and a binding capacity of 184 +/- 29 fmol/mg protein. 125I-Gastrin-I binding was inhibited by the specific CCK-B receptor antagonist L365,260 approximately 40 times more effectively than by the specific CCK-A receptor antagonist L364,718. Analysis of the ability of cholecystokinin octapeptide (CCK-8) to inhibit 125I-Bolton-Hunter-CCK-8 binding revealed two CCK binding sites, i.e., a high affinity site and a low affinity site. The observed binding affinities of CCK-8 were then introduced into the computer analysis of the dose-inhibition curve of the ability of gastrin-17-I to inhibit binding of 125I-Bolton-Hunter-CCK-8, which was significantly better fit by a three-site model than by a two-site model. The three sites meet the criteria for CCK-B, high affinity CCK-A, and low affinity CCK-A receptors. The binding capacity of CCK-B receptors constitutes 34% of the total high affinity CCK binding sites. This study demonstrated that DSL-6 pancreatic carcinoma expresses three subtypes of CCK receptors. Gastrin (CCK-B) receptors, which were not detected in normal rat pancreas, constitute about one third of the total high affinity CCK receptors. We suggest that novel expression of gastrin (CCK-B) receptors may be generated by gene mutation or amplification during carcinogenesis and may play an important role in promoting tumor growth. (+info)
Affinity labeling of gamma-glutamyl transpeptidase and location of the gamma-glutamyl binding site on the light subunit.
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Gamma-Glutamyl transpeptidase, which consists of two nonidentical subunits, is rapidly inactivated with respect to its transpeptidase and hydrolase activities by the gamma-glutamyl analogs 6-diazo-5-oxo-L-norleucine and L-azaserine. Inactivation, which is prevented by gamma-glutamyl substrates (but not by acceptor substrates), is accelerated by maleate, which was previously shown to enhance utilization of glutamine by transpeptidase. 6-Diazo-5-oxo--norleucine reacts specifically, covalently, and stoichiometrically at the gamma-glutamyl site of the enzyme, which was localized through studies with 6-diazo-5-OXO-[14C]norleucine to the light subunits of both the transpeptidase of rat kidney (which has subunits of molecular weights 22,000 and 46,000) and the transpeptidase of human kidney (which has subunits of molecular weights 22,000 and 62,000). The findings, which indicate that these enzymes have similar gamma-glutamyl binding subunits, are relevant to the structure-function relationships of this membrane-bound enzyme and its physiological role. (+info)
Expression of cholecystokinin-2/gastrin receptor in the murine pancreas modulates cell adhesion and cell differentiation in vivo.
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The presence of gastrin and cholecystokinin-2 (CCK2) receptors in human preneoplastic and neoplastic gastrointestinal lesions suggests a role in cancer development. In addition to the growth-promoting action of gastrin, recently a role of the cholecystokinin-2/gastrin receptor (CCK2-R) modulating cellular morphology in cultured epithelial cells has been shown. Here, we have investigated in transgenic (ElasCCK2) mice whether ectopic expression of human CCK2-R in the exocrine pancreas affected epithelial differentiation. Cellular localization of cell adhesion molecules, differentiation markers, and transcription factors was determined using immunofluorescence techniques. Before tumor formation, expression and subcellular localization of proteins of the adherens junction complex, differentiation markers, and transcription factors were altered in ElasCCK2 exocrine pancreas, indicating an evolution from an acinar to a ductal phenotype. Loss of cell polarity, defective secretion, and loss of intercellular adhesion in acini of ElasCCK2 mice was confirmed by ultrastructural analysis. Finally, expression of the transgene in mice treated with the carcinogen azaserine resulted in enhanced size of preneoplastic lesions as well as an increased degree of acinar-ductal transdifferentiation. Thus, these data represent the first evidence for the CCK2-R modulating intercellular adhesion and cell fate in vivo and show that these alterations may contribute to enhanced sensitivity of ElasCCK2 pancreas to chemical carcinogens. (+info)
Enhancing effect of partial gastrectomy on pancreatic carcinogenesis.
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The controversial issue of enhanced pancreatic carcinogenesis following partial gastrectomy has been explored in male Wistar rats (n = 40) weighing 250-300 g. Animals were randomised to receive either 60% distal gastrectomy with Roux-en-Y reconstruction or gastrotomy and resuture (control). Immediately after operation each group was further divided into two subgroups, receiving i.p. injections of either saline or azaserine (30 mg kg-1 wk-1 for 3 weeks). At 15 months blood was obtained at 0, 5, 15 and 30 min after a fatty meal for cholecystokinin (CCK) assay; rats were then killed. Pancreatic wet weight was measured, and histological sections were examined for atypical acinar cell foci (AACF), the putative precursor lesion of carcinoma. There were no significant differences in body weight or pancreatic weight between controls and rats with gastrectomy. Only azaserine-treated rats had acidophilic AACF. Partial gastrectomy substantially increased the number of acidophilic AACF per pancreas (median 26.05 vs 2.09; P less than 0.005), with a 9-fold increase in their volume (P less than 0.005). Basal and postprandial plasma CCK concentrations were higher after gastrectomy than in controls (P less than 0.05). Partial gastrectomy has an enhancing effect on azaserine-induced pancreatic carcinogenesis, probably by means of increased CCK release. (+info)