The interplay between folding-facilitating mechanisms in Trypanosoma cruzi endoplasmic reticulum

Lectin (calreticulin [CRT])-N-glycan-mediated quality control of glycoprotein folding is operative in trypanosomatid protozoa but protein-linked monoglucosylated N-glycans are exclusively formed in these microorganisms by UDP-Glc:glycoprotein glucosyltransferase (GT)-dependent glucosylation. The gene coding for this enzyme in the human pathogen Trypanosoma cruzi was identified and sequenced. Even though several of this parasite glycoproteins have been identified as essential components of differentiation and mammalian cell invasion processes, disruption of both GT-encoding alleles did not affect cell growth rate of epimastigote form parasites and only partially affected differentiation and mammalian cell invasion. The cellular content of one of the already identified T. cruzi glycoprotein virulence factors (cruzipain, a lysosomal proteinase) only showed a partial (5-20%) decrease in GT null mutants in spite of the fact that >90% of all cruzipain molecules interacted with CRT during their folding process in wild-type cells. Although extremely mild cell lysis and immunoprecipitation procedures were used, no CRT-cruzipain interaction was detected in GT null mutants but secretion of the proteinase was nevertheless delayed because of a lengthened interaction with Grp78/BiP probably caused by the detected induction of this chaperone in GT null mutants. This result provides a rationale for the absence of a more drastic consequence of GT absence. It was concluded that T. cruzi endoplasmic reticulum folding machinery presents an exquisite plasticity that allows the parasite to surmount the absence of the glycoprotein-specific folding facilitation mechanism.     

Diabetes and mitochondrial bioenergetics: alterations with age

Several studies have been carried out to evaluate the alterations in mitochondrial functions of diabetic rats. However, some of the results reported are controversial, since experimental conditions, such as aging, and/or strain of animals used were different. The purpose of this study was to evaluate the metabolic changes in liver mitochondria, both in the presence of severe hyperglycaemia (STZ-treated rats) and mild hyperglycaemia (Goto-Kakizaki (GK) rats). Moreover, metabolic alterations were evaluated both at initial and at advanced states of the disease. We observed that both models of type 1 and type 2 diabetes presented alterations on respiratory chain activity. Because of continual severe hyperglycaemia, 9 weeks after the induction of diabetes, the respiratory function declined in STZ-treated rats, as observed by membrane potential and respiratory ratios (RCR, P/O, and FCCP-stimulated respiration) assessment. In contrast, GK rats of 6 months age presented increased respiratory ratios. To localize which respiratory complexes are affected by diabetes, enzymatic respiratory chain activities were evaluated. We observed that succinate dehydrogenase and cytochrome c oxidase activities were significantly augmented both in STZ-treated rats and GK rats of 6 months age. Moreover, H(+)-ATPase activity was also significantly increased in STZ-treated rats with 3 weeks of diabetes and in GK rats of 6 months age as compared to controls. Therefore, these results clearly suggest that both animal models of diabetes present some metabolic adjustments in order to circumvent the deleterious effects promoted by the high glucose levels typical of the disease.     

Origin,precursors and differentiation of mouse dendritic cells

Functional specialization allows defined dendritic-cell (DC) subsets to induce efficient defence mechanisms against pathogens and tumour cells, and maintain T-cell tolerance by inducing the inactivation of autoreactive T cells. A crucial question, which has important implications for both our understanding of the induction and control of immunity by DCs, as well as the use of DCs for immunotherapy, is whether the functional diversity of DCs results from the existence of developmentally independent DC subpopulations, or whether DC subsets that share a common differentiation origin acquire specific functions in response to environmental signals. This review discusses recent findings on mouse DC development.     

Mutant mouse models and their contribution to our knowledge of corpus luteum development,function and regression

The corpus luteum is a unique organ, which is transitory in nature. The development, maintenance and regression of the corpus luteum are regulated by endocrine, paracrine and autocrine signaling events. Defining the specific mediators of luteal development, maintenance and regression has been difficult and often perplexing due to the complexity that stems from the variety of cell types that make up the luteal tissue. Moreover, some regulators may serve dual functions as a luteotropic and luteolytic agent depending on the temporal and spatial environment in which they are expressed. As a result, some confusion is present in the interpretation of in vitro and in vivo studies. More recently investigators have utilized mutant mouse models to define the functional significance of specific gene products. The goal of this mini-review is to identify and discuss mutant mouse models that have luteal anomalies, which may provide some clues as to the significance of specific regulators of corpus luteum function.     

Are hummingbirds facultatively ammonotelic? Nitrogen excretion and requirements as a function of body size

Most birds are uricotelic. An exception to this rule may be nectar-feeding birds, which excrete significant amounts of ammonia under certain conditions. Although ammonia is toxic, because it is highly water soluble its excretion may be facilitated in animals that ingest and excrete large amounts of water. Bird-pollinated plants secrete carbohydrate- and water-rich floral nectars that contain exceedingly little protein. Thus, nectar-feeding birds are faced with the dual challenge of meeting nitrogen requirements while disposing of large amounts of water. The peculiar diet of nectar-feeding birds suggests two hypotheses: (1) these birds must have low protein requirements, and (2) when they ingest large quantities of water their primary nitrogen excretion product may be ammonia. To test these hypotheses, we measured maintenance nitrogen requirements (MNR) and total endogenous nitrogen losses (TENL) in three hummingbird species (Archilochus alexandri, Eugenes fulgens, and Lampornis clemenciae) fed on diets with varying sugar, protein, and water content. We also quantified the form in which the by-products of nitrogen metabolism were excreted. The MNR and TENL of the hummingbirds examined were exceptionally low. However, no birds excreted more than 50% of nitrogen as ammonia or more nitrogen as ammonia than urates. Furthermore, ammonia excretion was not influenced by either water or protein intake. The smallest species (A. alexandri) excreted a significantly greater proportion (>25%) of their nitrogenous wastes as ammonia than the larger hummingbirds ( approximately 4%). Our results support the hypothesis that nectar-feeding birds have low protein requirements but cast doubt on the notion that they are facultatively ammonotelic. Our data also hint at a possible size-dependent dichotomy in hummingbirds, with higher ammonia excretion in smaller species. Differences in proportionate water loads and/or postrenal modification of urine may explain this dichotomy.     

Increased activity and expression of matrix metalloproteinase-9 in a rat model of distal colitis

Matrix metalloproteinases may play a role in tissue remodelling and destruction associated with inflammation. We investigated activity and expression of matrix metalloproteinases in a rat model of colitis and tested the therapeutic potential of a synthetic inhibitor (CGS-27023-A). Colitis was induced by dextran sulphate sodium (at 5% in drinking water for 5 days) in a group of eight rats, whereas a matched control group received plain water. Activity and expression of matrix metalloproteinases were measured in colonic tissue homogenates using zymography and Western blot on days 3 and 5 after induction of colitis. In another set of experiments, two groups of colitic rats (20 per group) were treated with CGS-27023-A (20 mg/kg) or vehicle, respectively. On days 5 and 14, colonic mucosal lesions were blindly scored by microscopic examination. Induction of colitis led to a significant upregulation of matrix metalloproteinase-9 protein and its activity, but no change in matrix metalloproteinase-2 activity was observed. Treatment with CGS-27023-A significantly decreased the extent and severity of epithelial injury but did not influence mucosal repair. We conclude that increased activity of matrix metalloproteinases may contribute to epithelial damage in this model of colitis.     

Genistein inhibits Ca2+ influx and glutamate release from hippocampal synaptosomes: putative non-specific effects

The role of protein tyrosine kinases on glutamate release was investigated by determining the effect of broad range inhibitors of tyrosine kinases on the release of glutamate from rat hippocampal synaptosomes. We found that lavendustin A and herbimycin A did not inhibit glutamate release stimulated by 15 mM KCl, but genistein, also a broad range inhibitor of tyrosine kinases did inhibit the intracellular Ca(2+) concentration response to KCl and, concomitantly, decreased glutamate release evoked by the same stimulus, in a dose-dependent manner. These effects were not observed with the inactive analogue genistin. Therefore, we investigated the mechanism whereby genistein modulates Ca(2+) influx and glutamate release. Studies with voltage-gated Ca(2+) channel inhibitors showed that omega-conotoxin GVIA did not further inhibit glutamate release or the Ca(2+) influx stimulated by KCl in the presence of genistein. This tyrosine kinase inhibitor and omega-agatoxin IVA had a partially additive effect on those events. Nitrendipine did not reduce significantly the KCl-induced responses. Genistein further reduced Ca(2+) influx in response to KCl in the presence of nitrendipine, omega-conotoxin GVIA and omega-agatoxin IVA, simultaneously. The effect of tyrosine phosphatase inhibitors was also tested on the influx of Ca(2+) and on glutamate release stimulated by KCl-depolarization. We found that the broad range inhibitors sodium orthovanadate and dephostatin did not significantly affect these KCl-evoked events.Our results suggest that genistein inhibits glutamate release and Ca(2+) influx in response to KCl independently of tyrosine kinase inhibition, and that tyrosine kinases and phosphatases are not key regulators of glutamate release in hippocampal nerve terminals.     

Characterization of angiotensin-(1-7) receptor subtype in mesenteric arteries

Mesenteric arteries from male Sprague-Dawley rats were mounted in a pressurized myograph system. Ang-(1-7) concentration-dependent responses were determined in arteries preconstricted with endothelin-1 (10(-7)M). The receptor(s) mediating the Ang-(1-7) evoked dilation were investigated by pretreating the mesenteric arteries with specific antagonists of Ang-(1-7), AT(1) or AT(2) receptors. The effects of Ang-(3-8) and Ang-(3-7) were also determined. Ang-(1-7) caused a concentration-dependent dilation (EC(50): 0.95 nM) that was blocked by the selective Ang-(1-7) receptor antagonist D-[Ala(7)]-Ang-(1-7). Administration of a specific antagonist to the AT(2) receptor (PD123319) had no effect. On the other hand, losartan and CV-11974 attenuated the Ang-(1-7) effect. These results demonstrate that Ang-(1-7) elicits potent dilation of mesenteric resistance vessels mediated by a D-[Ala(7)]-Ang-(1-7) sensitive site that is also sensitive to losartan and CV-11974.