apoptotic effect of extracellular zinc sequestration on HT29/219 and SW742 cell lines

Zn [II] is an important regulator of caspase-3, as well as an antioxidant, microtubule stabilizer, growth cofactor, and anti-inflammatory agent. Over the past 30 years, many researchers have demonstrated the important role of Zn [II] in a variety of physiological processes, including growth and development, maintenance and priming of the immune system, and in tissue repair and regeneration. In this study, we present evidence that chelation of extracellular zinc by diethylenetriaminepentacetic acid [DTPA] in different concentrations causes cell death in carcinoma cell lines, HT29/219 and SW742. Hoechst 33258 staining revealed that cell death was mainly by apoptosis. Additionally, significant increases in the activity of caspase-3 and -9 were observed in both cell lines. Caspase-8 activation was negligible in both cell lines. The cytotoxicity/apoptotic effect of DTPA was inhibited significantly by Zn [II], Cu [II] and N-Acetyl-L-Cysteine [NAC] [P<0.05]. Therefore, DTPA, the membrane-impermeable metal ion chelator, induces apoptosis through the depletion of extracellular zinc ion

Effects of glucosamine on pancreatic glucokinase and hexokinase activities and their relationship to insulin secretion from isolated islets of normal and type 2 diabetic rat

Glucokinase serves as a glucose sensor in pancreatic beta-cells and plays a key role in glucose homeostasis and glucose-stimulated insulin secretion [GSIS]. In the present study we examined the effect of glucosamine, a glucokinase inhibitor, on the pancreatic glucokinase and hexokinase activities and on insulin secretion from freshly rat pancreatic islets of Langerhans. Insulin concentration was measured by rat insulin ELISA kit. The pancreatic islets from normal and type 2 diabetic [nSTZ] rats were isolated by collagenase digestion method. Glucose phosphorylation was quantitated by measuring the rate of glucose-6-phosphate formation in the fluorometric assay. Insulin secretion from hand-picked islets was evaluated in static incubation system. Insulin concentration was measured by rat insulin ELISA kit. Our findings demonstrate that glucosamine in a dose dependent manner, reduced glucokinase activity in islet extract, but had no effect on hexokinase activity. The glucose-stimulated insulin secretion was inhibited by glucosamine but it had no effect on the basal insulin secretion. In diabetic rats glucokinase was decreased while the basal insulin secretion and the activity of hexokinase were higher than normals. Based on results obtained from the present study, the assumption could be made that the decrease in the activity of glucokinase of pancreatic islets could be related to the impaired glucose stimulated insulin secretion. The increase in basal insulin secretion of diabetic rats may be due to an increase in pancreatic hexokinase activity

[Study of the effect of Zn2+,W6+ and V5+ on insulin secretion and glukokinase activation of pancreatic islets obtained from normal and diabetic rats]

Insulin dependant Diabetes Mellitus [IDDM] is associated with a reduction in production or secretion of insulin from pancreatic Islets. On the other hand increase in insulin secretion will result in destruction of islets that will lead to Diabetes .Therefore the factors that could regulate secretion will prevent the onset of diabetes. Glucose metabolism that commences with the glucokinase action is closely related to insulin secretion. The aim of this study is to survey the effect of eat ions Zn2+, V5+ and W5+ on insulin secretion and on the key enzyme, glucokinase, which is known to play an important role in insulin secretion

Methods: 20 single islets were seprated from pancreatic tissues of each normal and diabetic rats and placed in tubes containing perfusion medium . Method ions with different concentrations/ and controls were provided with each set of experiment. Insulin secretion were determined using IDMA method. Glucokinaes activity in homogenate supernatant of normal and diabetic rats was assayed by spectropho to metric methods

Results: the level of insulin concernlyation was shown to increase with vandate and tungsten treatment in normal and diabetic rats / and decreased by zinc .The effect of zinc on glucokinase activity was similarly reducing. Tungsten caused an increase in glucokinase activity [p<.001] while vandate showed no effect on the activity of enzyme

Conclusion: the inhibitory effect of zinc on insulin secretion and the enhancing effect of tungsten correlate closely with their effect on glucokinase activity. There for the effect in insulin secretion could be assumed to be via glucokinase activation or inhibition. The effect of vandate on insulin secretion may be through other mechanisms which is not yet clarified

Purification of alpha-amylase from bacillus subtilis line No 1024 with ATCC 465

Alpha-amylase E.C.3.2.1.1. [1,4 glucan, 4 glucanohydrolase] can be obtained from salivary glands, pancreas and microorganisms such as Pseudomonas and Asperigllus, as well as muscles and ovarian tubes. Alpha-amylase from Bacillus subtilis #1024[ATCC 465] was purified with a highest degree of purity in our laboratory [31.59 U/mg]. The extracellular alpha-amylase was subjected to different purification techniques such as anionic and cationic exchange chromatography and preparative electrophoresis. The final fold of purification was equivalent to 38, which was higher than the previous reported values, polyacrylamide gel electrophoresis with sodium dodecyl sulphate [PAGESDS], Laemmeli method, gave a molecular weight of 82000 Daltons