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We studied an involvement of various cellular ceramide pools in signaling of immunoreceptor FcγII (FcγRII). The cell surface ceramide level was assessed by a technique based on binding of ceramide probes to intact cells. Total cellular ceramide was estimated by radioactive measurements. The activity of sphingomyelinases was measured by NBDceramide release while immunoprecipitation and immunoblotting were applied to analyze protein tyrosine phosphorylation. A complex pattern of protein phosphorylation was found to accompany FcγRII activation and the phosphorylation was either diminished by imipramine or increased by B13, modulators of acid sphingomyelinase and acid ceramidase activity. The effects of the drugs on the phosphorylation of FcγRII and NTAL were prominent and correlated with a reduction of the cell surface ceramide production by imipramine and an augmentation of the ceramide generation by B13. The ceramide generation followed activation of acid sphingomyelinase and preceded that of neutral sphingomyelinase. The level of cell surface ceramide was additionally elevated by exogenous bacterial sphingomyelinase, but only at later stages of the receptor activation. The total mass of ceramide was diminished in the course of receptor activation pointing to an engagement of enzymes metabolizing ceramide. The data indicate that FcγRII activates enzymes of the sphingomyelin cycle which affect various sphingomyelin/ceramide pools in a cell.

Lysenin is 297 amino acid long toxin derived from the earthworm Eisenia foetida which specifically recognizes sphingomyelin and induces cell lysis. We synthesized lysenin gene supplemented with a polyhistidine tag, subcloned it into the pT7RS plasmid and the recombinant protein was produced in Escherichia coli . In order to obtain lysenin devoid of its lytic activity, the protein was mutated by substitution of tryptophan 20 by alanine. The recombinant mutant lysenin-His did not evoke cell lysis, although it retained the ability to specifically interact with sphingomyelin, as demonstrated by immunofluorescence microscopy and by dot blot lipid overlay and liposome binding assays. We found that the lytic activity of wild-type lysenin-His was correlated with the protein oligomerization during interaction with sphingomyelin-containing membranes and the amount of oligomers was increased with an elevation of sphingomyelin/lysenin ratio. Blue native gel electrophoresis indicated that trimers can be functional units of the protein, however, lysenin hexamers and nanomers were stabilized by chemical cross-linking of the protein and by sodium dodecyl sulfate. When incorporated into planar lipid bilayers, wild type lysenin-His formed cation-selective channels in a sphingomyelin-dependent manner. We characterized the channel activity by establishing its various open/closed states. In contrast, the mutant lysenin-His did not form channels and its correct oligomerization was strongly impaired. Based on these results we suggest that lysenin oligomerizes upon interaction with sphingomyelin in the plasma membrane, forming cation-selective channels. Their activity disturbs the ion balance of the cell, leading eventually to cell lysis.

The relationship between diabetes and cancer development is not clear. But the risk to develop cancer is increased if the person is diabetic. In the present study we induced type 1 and type 2 diabetes in rats by streptozotocin and high fat diet, respectively. Many cell growth and apoptosis related antigens were studied including tumour suppressor antigen; P53, anti-apoptotic protein bcl-2, pro-apoptotic protein bax, cyp1a2 and cyp2e1 and caspase 3. In type 1 and type 2 diabetes cyp2e1, bax, p53 and active caspase 3 were up regulated but active caspase 3 was prominent active in type 1 compared to type 2 diabetes. Both cyp1a2 and bcl-2 were down regulated in both types of diabetes. Bcl-2 was dramatically decreased in type 1 than type 2 diabetes. Normal or high insulin dose treatment could restore the basal line of most parameters but partially restored the level of cyp1a2 and could not restore the level of p53 in type 1 diabetes. High P53 level in both types of diabetes indicates cellular stress and damage. The high level of P53 was accompanied with active caspas3 and reduced bcl-2 in type 1 but not type 2 diabetes.

The aim of the present study is to investigate the role of hyperglycemia and hyperinsulinemia in autophagy induction in the liver of male mice. Autophagy is a catabolic cellular process that recycles the aged or damaged cellular organelles and inclusions under certain circumstances. Hyperglycemia is induced by a single dose of alloxan IP injection (180 mg/kg) and hyperinsulinemia is induced by high fat diet together with glucose feeding for short period (2 weeks) and long period (3 months). Hyperglycemia and hyperinsulinemia were estimated by measuring blood glucose level by glucometer and insulin level by specific ELISA kit, respectively. Autophagy induction was investigated morphologically by electron microscopy examination and biochemically by immunodetection of microtubular associated light chain protein 3 (LC3) conversion from LC3I to LC3II form and by immunodetection of the phosphorylated and nonphosphorylated forms of mammalian target of Rapamycin (mTOR). Our results revealed that hyperglycemia and hyperinsulinemia independently induced hepatocellular autophagy as indicated by the accumulation of autophagosomes and autolysosomes in EM examination and by the increase of the level of LC3II and decrease of the phosphorylated form of mTOR in western blot analysis. This study throw the light on the autophagy of hepatocytes as a cellular mechanism induced under diabetic conditions which may contribute in better understanding of our knowledge concerning nutrients metabolic disorders.

The aim of the present study is to investigate the role of hyperglycemia and hyperinsulinemia in autophagy induction in the liver of male mice. Autophagy is a catabolic cellular process that recycles the aged or damaged cellular organelles and inclusions under certain circumstances. Hyperglycemia is induced by a single dose of alloxan IP injection (180 mg/kg) and hyperinsulinemia is induced by high fat diet together with glucose feeding for short period (2 weeks) and long period (3 months). Hyperglycemia and hyperinsulinemia were estimated by measuring blood glucose level by glucometer and insulin level by specific ELISA kit, respectively. Autophagy induction was investigated morphologically by electron microscopy examination and biochemically by immunodetection of microtubular associated light chain protein 3 (LC3) conversion from LC3I to LC3II form and by immunodetection of the phosphorylated and nonphosphorylated forms of mammalian target of Rapamycin (mTOR). Our results revealed that hyperglycemia and hyperinsulinemia independently induced hepatocellular autophagy as indicated by the accumulation of autophagosomes and autolysosomes in EM examination and by the increase of the level of LC3II and decrease of the phosphorylated form of mTOR in western blot analysis. This study throw the light on the autophagy of hepatocytes as a cellular mechanism induced under diabetic conditions which may contribute in better understanding of our knowledge concerning nutrients metabolic disorders.