Impaired proteostasis contributes to renal tubular dysgenesis

Protein conformational disorders are associated with the appearance, persistence, accumulation, and misprocessing of aberrant proteins in the cell. The etiology of renal tubular dysgenesis (RTD) is linked to mutations in the angiotensin-converting enzyme (ACE). Here, we report the identification of a novel ACE mutation (Q1069R) in an RTD patient. ACE Q1069R is found sequestered in the endoplasmic reticulum and is also subject to increased proteasomal degradation, preventing its transport to the cell surface and extracellular fluids. Modulation of cellular proteostasis by temperature shift causes an extension in the processing time and trafficking of ACE Q1069R resulting in partial rescue of the protein processing defect and an increase in plasma membrane levels. In addition, we found that temperature shifting causes the ACE Q1069R protein to be secreted in an active state, suggesting that the mutation does not affect the enzyme's catalytic properties.     

Sir2 is induced by oxidative stress in a yeast model of Huntington disease and its activation reduces protein aggregation

Huntington disease (HD) is a neurodegenerative disorder caused by expansion of CAG trinucleotide repeats, leading to an elongated polyglutamine sequence (polyQ) in the huntingtin protein. Misfolding of mutant polyQ proteins with expanded tracts results in aggregation, causing cytotoxicity. Oxidative stress in HD has been documented in humans as important to disease progression. Using yeast cells as a model of HD, we report that when grown at high glucose concentration, cells expressing mutant polyQ do not show apparent oxidative stress. At higher cell densities, when glucose becomes limiting and cells are metabolically shifting from fermentation to respiration, protein oxidation and catalase activity increases in relation to the length of the polyQ tract. Oxidative stress, either endogenous as a result of mutant polyQ expression or exogenously generated, increases Sir2 levels. Δ sir2 cells expressing expanded polyQ lengths show signs of oxidative stress even at the early exponential phase. In a wild-type background, isonicotinamide, a Sir2 activator, decreases mutant polyQ aggregation and the stress generated by expanded polyQ. Taken together, these results describe mutant polyQ proteins as being more toxic in respiring cells, causing oxidative stress and an increase in Sir2 levels. Activation of Sir2 would play a protective role against this toxicity.     

Sequence analysis of 0.4 megabases of the rabbit germline immunoglobulin kappa1 light chain locus

Two rabbit germline bacterial artificial chromosome (BAC) libraries from animals with the b5 and b4 allotype were screened with probes specific for the immunoglobulin kappa1 light chain locus. Two partially overlapping BAC clones containing Vkappa elements of b5 allotype were isolated from the b5 library and one BAC clone containing Jkappa1, Ckappa and Vkappa was isolated from the b4 library. These three BAC clones were sequenced. They span about 0.4 MB of the rabbit Ig kappa1 light chain locus including 36 Vkappa elements, five J elements and the coding region of Ckappa1. The organization of the locus and the potential function of newly identified functional and structural elements are discussed.     

Xylem parenchyma cells deliver the H<Subscript>2</Subscript>O<Subscript>2</Subscript> necessary for lignification in differentiating xylem vessels

Lignification in Zinnia elegans L. stems is characterized by a burst in the production of H₂O₂, the apparent fate of which is to be used by xylem peroxidases for the polymerization of p-hydroxycinnamyl alcohols into lignins. A search for the sites of H₂O₂ production in the differentiating xylem of Z. elegans stems by the simultaneous use of optical (bright field, polarized light and epi-polarization) and electron-microscope tools revealed that H₂O₂ is produced on the outer-face of the plasma membrane of both differentiating (living) thin-walled xylem cells and particular (non-lignifying) xylem parenchyma cells. From the production sites it diffuses to the differentiating (secondary cell wall-forming) and differentiated lignifying xylem vessels. H₂O₂ diffusion occurs mainly through the continuous cell wall space. Both the experimental data and the theoretical calculations suggest that H₂O₂ diffusion from the sites of production might not limit the rate of xylem cell wall lignification. It can be concluded that H₂O₂ is produced at the plasma membrane in differentiating (living) thin-walled xylem cells and xylem parenchyma cells associated to xylem vessels, and that it diffuses to adjacent secondary lignifying xylem vessels. The results strongly indicate that non-lignifying xylem parenchyma cells are the source of the H₂O₂ necessary for the polymerization of cinnamyl alcohols in the secondary cell wall of lignifying xylem vessels.     

Antifungal activity from Ocimum gratissimum L. towards Cryptococcus neoformans

Cryptococcal infection had an increased incidence in last years due to the explosion of acquired immune deficiency syndrome epidemic and by using new and effective immunosuppressive agents. The currently antifungal therapies used such as amphotericin B, fluconazole, and itraconazole have certain limitations due to side effects and emergence of resistant strains. So, a permanent search to find new drugs for cryptococcosis treatment is essential. Ocimum gratissimum, plant known as alfavaca (Labiatae family), has been reported earlier with in vitro activity against some bacteria and dermatophytes. In our work, we study the in vitro activity of the ethanolic crude extract, ethyl acetate, hexane, and chloroformic fractions, essential oil, and eugenol of O. gratissimum using an agar dilution susceptibility method towards 25 isolates of Cryptococcus neoformans. All the extracts of O. gratissimum studied showed activity in vitro towards C. neoformans. Based on the minimal inhibitory concentration values the most significant results were obtained with chloroformic fraction and eugenol. It was observed that chloroformic fraction inhibited 23 isolates (92%) of C. neoformans at a concentration of 62.5 microg/ml and eugenol inhibited 4 isolates (16%) at a concentration of 0.9 microg/ml. This screening may be the basis for the study of O. gratissimum as a possible antifungal agent.     

Cleavage of the plasma membrane Na+/Ca2+ exchanger in excitotoxicity

In brain ischemia, gating of postsynaptic glutamate receptors and other membrane channels triggers intracellular Ca2+ overload and cell death. In excitotoxic settings, the initial Ca2+ influx through glutamate receptors is followed by a second uncontrolled Ca2+ increase that leads to neuronal demise. Here we report that the major plasma membrane Ca2+ extruding system, the Na+/Ca2+ exchanger (NCX), is cleaved during brain ischemia and in neurons undergoing excitotoxicity. Inhibition of Ca2+-activated proteases (calpains) by overexpressing their endogenous inhibitor protein, calpastatin or the expression of an NCX isoform not cleaved by calpains, prevented Ca2+ overload and rescued neurons from excitotoxic death. Conversely, down-regulation of NCX by siRNA compromised neuronal Ca2+ handling, transforming the Ca2+ transient elicited by non-excitotoxic glutamate concentrations into a lethal Ca2+overload. Thus, proteolytic inactivation of NCX-driven neuronal Ca2+ extrusion is responsible for the delayed excitotoxic Ca2+ deregulation and neuronal death.     

Evaluation of enzyme-linked immunosorbent assay using crude Leishmania and recombinant antigens as a diagnostic marker for canine visceral leishmaniasis

The performances of ELISA assays with different antigen preparations, such as Leishmania amazonensis or L. chagasi lysates and the recombinant antigens rK-39 and rK-26, were compared using sera or eluates from dried blood collected on filter paper to detect anti-Leishmania antibodies in dogs from a visceral leishmaniasis-endemic area in Brazil. Of 115 IFAT-reactive dogs at 1:40 titre, 106 (92.2%) were positive in parasitological exams (skin and/or spleen). These animals were compared to healthy animals (n = 25), negative for IFAT at a titre of 1:40 and parasitological exams. The sensitivities of crude and recombinant antigens were similar and remarkably high for both sera and eluates (97-100%). Specificity was higher than 96% for sera and eluates for different antigens, except for L. chagasi antigen using eluates (88%). Concordance values among the tests were higher either for sera or eluates (J = 0.95-1.00). High concordances were observed between sera and eluates tested with different antigens (kappa = 0.93-0.97). Crude and recombinant antigens identified different clinical phases of canine leishmaniasis. These results show that eluates could be used in canine surveys to identify L. chagasi infection. Recombinant antigens added little when compared to crude antigen in identifying positive dogs. Cross-reactivity with other diseases whose distribution often overlaps VL-endemic areas is a limitation of crude antigen use however.     

Effects of short-term fasting on energy reserves of vampire bats (Desmodus rotundus)

Studies on metabolic responses to fasting in common vampire bats (Desmodus rotundus) have demonstrated the susceptibility of this species when subjected to long-term fasting. We investigated the effects of short-term fasting (12 h), a period similar to what they face in the field, on their energy reserves. Blood glucose (BG) levels in fed bats were similar to other mammals, but after 12 h without food, these levels were reduced. Plasma lactate and free fatty acids levels in fed bats were higher than in other mammals, although no changes in these levels were detected in response to fasting. Liver glycogen content decreased significantly following fasting. Muscle glycogen, as well as liver and muscle lipid and protein levels, remained unaltered for up to 12 h of fasting. Although BG levels decreased after short-term fasting, body energy reserves do not seem to play an important role for maintenance of glycemic homeostasis during fasting. Despite the decrease in liver glycogen, this small reserve seems insufficient to maintain adequate levels of BG, even during short periods of fasting. Because other reserves were not decreased after fasting, it is possible that the main source of glucose for common vampire bats might be the glucose content of their blood diet.     

Biopesticide effect of green compost against fusarium wilt on melon plants

AIMS: The biopesticide effect of four green composts against fusarium wilt in melon plants and the effect of soil quality in soils amended with composts were assayed. METHODS AND RESULTS: The composts consisted of pruning wastes, with or without addition of coffee wastes (3/1 and 4/1, dry wt/dry wt) or urea (1000/1, dry wt/dry wt). In vitro experiments suggested the biopesticide effect of the composts against Fusarium oxysporum, while only the compost of pine bark and urea (1000/1dry wt/dry wt) had an abiotic effect. Melon plant growth with composts and F. oxysporum was one to four times greater than in the non-amended soil, although there was no significant decrease in the level of the F. oxysporum in the soil. The addition of composts to the soil also improved its biological quality, as assessed by microbiological and biochemical parameters: ATP and hydrolases involved in the P (phosphatase), C (beta-glucosidase) and N (urease) cycles. CONCLUSIONS: Green composts had greater beneficial characteristics, improved plant growth and controlled fusarium wilt in melon plants. These composts improve the soil quality of semi-arid agricultural soils. SIGNIFICANCE AND IMPACT OF THE STUDY: Biotic and abiotic factors from composts have been tested as responsible of their biopesticide activity against fusarium wilt.