Identification and molecular characterization of a novel stage-specific surface protein of Trypanosoma congolense epimastigotes

The cattle pathogen Trypanosoma congolense expresses life cycle stage-specific surface molecules involved in adaptation to different host and vector environments. Here we report the discovery and molecular characterization of a novel stage-specific GPI-anchored surface glycoprotein that is selectively expressed in the epimastigote (EMF) life cycle stage of T. congolense. Culture supernatants of EMF but not of procyclic culture forms (PCFs) promoted adhesion of PCF parasites in an in vitro assay. Biosynthetic labeling experiments showed that these EMF culture supernatants contained a 100kDa trypanosome-derived protein that was not present in supernatants from PCF. We named this molecule "congolense epimastigote-specific protein" (CESP). The gene encoding CESP was isolated from an EMF cDNA library after immunoscreening. The multicopy gene had a 2070-bp open reading frame that encodes a polypeptide of 689 amino acids with a predicted mass of 72.9kDa. The discrepancy between the predicted (72.9kDa) and observed (100kDa) masses may be explained partially by glycosylation of the molecule which has six potential N-glycosylation sites and a predicted GPI anchor. Indeed, metabolic labeling of CESP with [(3)H] ethanolamine revealed that CESP was a GPI-anchored protein. Confocal laser scanning microscopy showed that CESP was expressed only on the surface of the EMF stage of the parasite. The identification of CESP as a unique component of culture supernatants from EMF and that such supernatants can confer plastic-adhesive ability on PCF suggest that CESP is worth further investigation as an adhesion molecule that perhaps allows T. congolense EMF to adhere to the tsetse proboscis.     

Behavioral and gene expression analyses of Wfs1 knockout mice as a possible animal model of mood disorder

Wolfram disease is a rare genetic disorder frequently accompanying depression and psychosis. Non-symptomatic mutation carriers also have higher rates of depression and suicide. Because WfS1, the causative gene of Wolfram disease, is located at 4p16, a linkage locus for bipolar disorder, mutations of WfS1 were suggested to be involved in the pathophysiology of bipolar disorder. In this study, we performed behavioral and gene expression analyses of Wfs1 knockout mice to assess the validity as an animal model of mood disorder. In addition, the distribution of Wfs1 protein was examined in mouse brain. Wfs1 knockout mice did not show abnormalities in circadian rhythm and periodic fluctuation of wheel-running activity. Behavioral analysis showed that Wfs1 knockout mice had retardation in emotionally triggered behavior, decreased social interaction, and altered behavioral despair depending on experimental conditions. Wfs1-like immunoreactivity in mouse brain showed a similar distribution pattern to that in rats, including several nuclei potentially relevant to the symptoms of mood disorders. Gene expression analysis showed down-regulation of Cdc42ep5 and Rnd1, both of which are related to Rho GTPase, which plays a role in dendrite development. These findings may be relevant to the mood disorder observed in patients with Wolfram disease.     

NOX enzymes and diabetic complications

Several molecular mechanisms have been identified that mediate the tissue-damaging effects of hyperglycemia. These are increased flux through the polyol and hexosamine pathways, increased formation of advanced glycation end products, activation of protein kinase C, and augmented generation of reactive oxygen species (ROS). Increased production of ROS not only causes cellular damage but also activates the signal transduction cascade that activates specific target genes. Based on recent experimental data, it is now accepted that increased NADPH oxidase activity in tissues vulnerable to hyperglycemia takes place downstream of the advanced glycation end products and protein kinase C pathways, two of the primary mechanisms involved in the pathogenesis of diabetic complications. Thus, compounds that suppress NADPH oxidase activity may offer therapeutic benefits to ameliorate diabetic complications, highlighting the significance of NADPH oxidase as a new therapeutic target.     

Effects of Plasticizers and Plastic Bags on Granulocyte Function during Storage

The influence of the plasticizers, di-(2-ethylhexyl)phthalate (DEHP) and tri-(2-ethylhexyl)trimellitate (TOTM), on granulocyte function was examined. Polyvinyl chloride (PVC) bags with DEHP (DEHP-PVC) leaked DEHP into plasma, but TOTM did not dissolve in plasma under the same conditions. Glow discharge treatment inhibited the leakage of DEHP from DEHP-PVC bags. Depending on the amount of DEHP added into granulocyte suspension, chemotaxis and bactericidal activity decreased, but cell counts and phagocytosis were not affected. During storage for 24 h at 22 degrees C, granulocyte function decreased greatly in DEHP-PVC, but was well maintained in the bags which did not leak plasticizers, TOTM-PVC and glow-discharged DEHP-PVC.     

Differential effects of diacylglycerols and 12-O-tetradecanoylphorbol-13-acetate on protein phosphorylation and cell proliferation of tumor promoter-dependent leukemia cell line A65T

The Growth of A65T cells, a mouse thymic leukemia cell line,was stricktly dependent on the presence of tumor promoters,such as 12-O-tetradecanoylphorbol-13-acetate (TPA), teleocidinand mezerein. In the presence of the tumor promoters, A65T cellsproliferated rapidly in a concentration dependent manner. Whenthe cells were cultured with a synthetic diacylglycerol, 1-oleoyl-2-acetylglycerol(OAG) or 1, 2-dicaprylin, cell proliferation was not stimulated.In addition, bihourly cumulative addition of OAG or 1, 2-dicaprylinagain failed to induce A65T cell proliferation. PhospholipaseC(PLC) induced a concentration dependent stimulation of A65Tcell proliferation, though the effect was slight compared withthat of the tumor promoters. Protein kinase C activity was detectedboth in cytosol and particulate fractions of A65T cells. Whenthe cells were incubated in the TPA-free medium for 5 h, theprotein kinase C activity in the cytosol fraction increasedwhereas a the activity in the particulate fraction decreased.Treatment of the cells with the tumor promoters mentioned aboveresulted in the increased phosphorylation of proteins with apparentmol;. wts of 27 000 and 68 000. The effects were concentrationdependent and the half maximal phosphorylation was observedeither with 3.6 nM TPA, 4.5 ng/ml teleocidin or 0.33 µMmezerein, respectively. On the other hand, a half maximal effecton cell proliferation was observed either with 0.14 nM TPA,47 pg/ml teleocidin or 6.3 nM mezerein, respectively. At theseconcentrations, these tumor promoters never induced the detectablestimulations of the protein phosphorylation. A synthetic diacylglycerol1, 2-dicaprylin failed to stimulate the phosphorylation of 27-and 68-kd proteins, but stimulated the phosphorylation of proteinswith apparent mol. wts of 100 000 and 54 000. The effect wasconcentration-dependent and a half maximal stimulation was observedwith 35 µg/ml 1, 2-dicaprylin. Neither TPA, teleocidinnor mezerein stimulated the phosphorylation of these 100- and54-kd proteins. OAG and PLC failed to induce any detectablealterations in the protein phosphorylation in A65T cells. BothOAG and 1, 2-dicaprylin, which we used, actually activated partiallypurified mouse brain protein kinase C in a concentration dependentmanner. These results clearly demonstrate that in A65T cellsTPA and diacylglycerol mutually stimulate the phosphorylationof the completely different set of endogenous proteins, andalso suggest that the cell-proliferating effects of the tumorpromoters do not appear to be mediated through the phosphorylationof 27-and 68-kd proteins.     

Physical and Chemical Changes of ACD-Preserved Blood: A Comparison of Blood in Glass Bottles and Plastic Bags

ACD blood was preserved in glass bottles with or without aeration and in plastic bags in air or nitrogen gas at 4.5 degrees C. The blood was examined for physical and chemical changes of erythrocyte membrane resistance, hemoglobin in the plasma, the viscosity of the blood, pH of plasma, and ATP and 2,3-DPG content of erythrocytes. The blood preserved in plastic bags showed less changes than blood in glass bottles. The presence of air or nitrogen gas in blood seems to increase the pH perhaps by elimination of carbon dioxide (CO2) which in turn causes the different rates of glycolysis in the erythrocytes.