Chronic kidney disease induced by an adenine rich diet upregulates integrin linked kinase (ILK) and its depletion prevents the disease progression

Kidney fibrosis is one of the main pathological findings of progressive chronic kidney disease (CKD) although the pathogenesis of renal scar formation remains incompletely explained. Integrin-linked kinase (ILK), a major scaffold protein between the extracellular matrix (ECM) and intracellular signaling pathways, is involved in several pathophysiological processes during renal damage. However, ILK contribution in the CKD progress remains to be fully elucidated. In the present work, we studied 1) the renal functional and structural consequences of CKD genesis and progression when ILK is depleted and 2) the potential of ILK depletion as a therapeutic approach to delay CKD progression. We induced an experimental CKD model, based on an adenine-supplemented diet on adult wild-type (WT) and ILK-depleted mice, with a tubulointerstitial damage profile resembling that is observed in human CKD. The adenine diet induced in WT mice a progressive increase in plasma creatinine and urea concentrations. In the renal cortex it was also observed tubular damage, interstitial fibrosis and progressive increased ECM components, pro-inflammatory and chemo-attractant cytokines, EMT markers and TGF-β1 expressions. These observations were highly correlated to a simultaneous increase of ILK expression and activity. In adenine-fed transgenic ILK-depleted mice, all these changes were prevented. Additionally, we evaluated the potential role of ILK depletion to be applied after the disease induction, as an effective approach to interventions in human CKD subjects. In this scenario, two weeks after the establishment of adenine-induced CKD, ILK was abrogated in WT mice and stabilized renal damage, avoiding CKD progression. We propose ILK to be a potential target to delay renal disease progression.     

Photoperiod modulates growth,morphoanatomy, and linalool content in Lippia alba L. (Verbenaceae) cultured in vitro

Biotechnology provides valuable tools to support conservation of plant species, especially in case of threatened taxa or when dealing with seed unavailability, low viability or sterility. However, plant cell culture methods have often to face problems associated with tissue recalcitrance to in vitro systems. Recalcitrance can be related to a variety of triggering factors, involving many efforts and manipulations within one or more of the micropropagation stages before obtaining successful results. An in vitro propagation protocol was developed for Zelkova sicula, a very rare and endangered relict tree, endemic to Sicily (Southern Italy). The species revealed extremely recalcitrant to in vitro culture approaches, but after many trials throughout a number of years an effective micropropagation protocol was completed. The rooting rate was about 84% of the treated explants, 8% of which were successfully acclimatized outdoor and reintroduced in the wild within a comprehensive conservation project. The technique allowed to overcome the problems of sexual sterility of this species, hence contributing concretely to contrast the problems connected with its conservation. However, additional efforts need to be carried out in order to refine the acclimatization step and further improve the whole process effectiveness.

     

Factor analysis models for structuring covariance matrices of additive genetic effects: a Bayesian implementation

Multivariate linear models are increasingly important in quantitative genetics. In high dimensional specifications, factor analysis (FA) may provide an avenue for structuring (co)variance matrices, thus reducing the number of parameters needed for describing (co)dispersion. We describe how FA can be used to model genetic effects in the context of a multivariate linear mixed model. An orthogonal common factor structure is used to model genetic effects under Gaussian assumption, so that the marginal likelihood is multivariate normal with a structured genetic (co)variance matrix. Under standard prior assumptions, all fully conditional distributions have closed form, and samples from the joint posterior distribution can be obtained via Gibbs sampling. The model and the algorithm developed for its Bayesian implementation were used to describe five repeated records of milk yield in dairy cattle, and a one common FA model was compared with a standard multiple trait model. The Bayesian Information Criterion favored the FA model.     

Testis-mediated gene transfer in mice: comparison of transfection reagents regarding transgene transmission and testicular damage

Testis-mediated gene transfer (TMGT) has been used as in vivo gene transfer technology to introduce foreign DNA directly into testes, allowing mass gene transfer to offspring via mating. In this study, we used plasmid DNA (pEGFP-N1) mixed with dimethylsulfoxide (DMSO), N,N-dimethylacetamide (DMA) or liposome (Lipofectin) in an attempt to improve TMGT. Males receiving consecutive DNA complex injections were mated to normal females to obtain F0 progeny. In vivo evaluation of EGFP expression, RT-PCR and PCR were used to detect the expression and the presence of exogenous DNA in the progeny. We also evaluated possible testicular damage by histological procedures. PC R and RT-PCR analyses revealed that liposome and DMSO increased the rate of TMGT. Histological analyses demonstrated that repeated (4 times) injections of DNA complexes can affect spermatogenesis. DMSO was the most deleterious among the reagents tested. In this study, we detected the presence of transgene in the progeny, and its expression in blood cells. Consecutive injections of DNA complexes were associated with impaired spermatogenesis, suggesting requirement of optimal conditions for DNA delivery through TMGT.     

A set of keys for biochemical identification of environmental Vibrio species

A set of biochemical keys which provide fast and presumptive identification for Vibrio spp. is presented. They have been specially designed for environmental isolates, and can be used for strains that are Gram-negative, give a positive oxidase test, grow on TCBS medium and are facultative anaerobes. The keys are constituted by 28 tests and a maximum of 10 tests are needed for the most complicated identification. They have been designed for routine purposes, especially for studies with a high number of isolates. Some tests are included in enzyme-activity based kits that could be used with these keys through certain results, principally for environmental isolates, should be confirmed by standard methods.     

Cytokine-driven regulation of NK cell functions in tumor immunity: role of the MICA-NKG2D system

Natural killer (NK) cells are critical players during tumor growth control in immunocompetent hosts. These cells also establish a cross-talk with dendritic cells (DCs) and promote a Th1-mediated immunity. NKG2D is a pivotal receptor that directs the tumoricidal activity of NK cells through the recognition of a group of ligands such as MICA widely expressed on different tumors. Here we will review the most important tumor immune escape mechanisms that compromise the functionality of NKG2D and its cognate ligands, including TGF-beta secretion, tumor shedding of soluble MICA, and additional mechanisms that compromise the tumoricidal activity of NKG2D-expressing cells. Such mechanisms may also dampen the cross-talk between NK cells and DCs during the anti-tumor immune responses. Recent knowledge may lead to innovative approaches to promote efficient NK cell-mediated anti-tumor immune responses.     

Phosphorus cycling in a small watershed in the Brazilian Cerrado: impacts of frequent burning

Plant productivity in many tropical savannas is phosphorus limited. The biogeochemical cycling of P in these ecosystems, however, has not been well quantified. In the present study, we characterized P stocks and fluxes in a well-preserved small watershed in the Brazilian Cerrado. As the Cerrado is also a fire-dominated ecosystem, we measured the P stocks and fluxes in a cerrado stricto sensu plot with complete exclusion of fire for 26 years (unburned plot) and then tested some predictions about the impacts of fire impacts on P cycling in an experimental plot that was burned three times since 1992 (burned plot). The unburned area is an ecosystem with large soil stocks of total P (1,151 kg ha^?1 up to 50 cm depth), but the largest fraction is in an occluded form. Readily extractable P was found up to 3 m soil depth suggesting that deep soil is more important to the P cycle than has been recognized. The P stock in belowground biomass (0?C800 cm) was 9.9 kg ha^?1. Decomposition of fine litter released 0.97 kg P ha^?1 year^?1. Fluxes of P through bulk atmospheric deposition, throughfall and litter leachate were very low (0.008, 0.006 and 0.028 kg ha^?1 year^?1, respectively) as was stream export (0.001 kg ha^?1 year^?1). Immobilization of P by microbes during the rainy season seems to be an important mechanism of P conservation in this ecosystem. Fire significantly increased P flux in litter leachate to 0.11 kg ha^?1 year^?1, and added 1.2 kg ha^?1 of P in ash deposition after fire. We found an increase of P concentration in soil solution at 100 cm depth (from 0.03 ??g l^?1 in unburned plot to 0.3 ??g l^?1 in the burned plot). In surface soils (0?C10 cm) of the burned plot, fire decreased the concentrations of extractable organic-P fractions, but did not significantly increase inorganic-P fractions. The reduction of extractable soil organic P in the burned plot in topsoil and the increase of P in the soil solution at greater depths indicated a reduction of P availability and may increase P fixation in deep soils. Repeated fire events over the long term may result in significant net loss of available forms of phosphorus from this ecosystem.     

Free Fatty Acids and Energy Metabolites in Ischemic Cerebral Cortex with Noradrenaline Depletion

Abstract: We tested whether cerebral noradrenaline (NA) may play a central role in mediating the increased production of free fatty acids (FFAs) during cerebral ischemia. Levels of FFAs, cyclic AMP, and NA, as well as ATP, ADP, and AMP, were measured in cerebral cortex during decapitation ischemia in rats 2 weeks after unilateral locus ceruleus lesion. Comparisons were made between the results obtained from the contralateral cortex with normal NA content and the NA-depleted ipsilateral cortex. Although NA depletion was associated with a diminished transient rise of cyclic AMP in response to ischemia, it failed to influence the magnitude of FFA increase or the decline of energy state within the 15-min period of ischemia. A more than twofold increase of total FFAs (sum of palmitic, stearic, oleic, arachidonic, and docosahexaenoic acids) was observed in both hemispheres at 1 min after decapitation, when energy failure became manifest. The increased production of FFAs continued throughout the 15 min of ischemia, with a preferential rise in the levels of stearic and arachidonic acids. There was an inverse correlation between FFA levels and total adenylate pool. The results do not support a major role for NA and cyclic AMP in increasing cortical FFAs during complete ischemia. Instead, they are consistent with the view that impaired oxidative phosphorylation activates deacylating enzymes. Disturbance of reacylation due to energy depletion is probably another factor contributing to the continuous increase of FFAs during prolonged ischemia.     

Functional effects of the hypertrophic cardiomyopathy R403Q mutation are different in an alpha- or beta-myosin heavy chain backbone

The R403Q mutation in the beta-myosin heavy chain (MHC) was the first mutation to be linked to familial hypertrophic cardiomyopathy (FHC), a primary disease of heart muscle. The initial studies with R403Q myosin, isolated from biopsies of patients, showed a large decrease in myosin motor function, leading to the hypothesis that hypertrophy was a compensatory response. The introduction of the mouse model for FHC (the mouse expresses predominantly alpha-MHC as opposed to the beta-isoform in larger mammals) created a new paradigm for FHC based on finding enhanced motor function for R403Q alpha-MHC. To help resolve these conflicting mechanisms, we used a transgenic mouse model in which the endogenous alpha-MHC was largely replaced with transgenically encoded beta-MHC. A His(6) tag was cloned at the N terminus of the alpha-and beta-MHC to facilitate protein isolation by Ni(2+)-chelating chromatography. Characterization of the R403Q alpha-MHC by the in vitro motility assay showed a 30-40% increase in actin filament velocity compared with wild type, consistent with published studies. In contrast, the R403Q mutation in a beta-MHC backbone showed no enhancement in velocity. Cleavage of the His-tagged myosin by chymotrypsin made it possible to isolate homogeneous myosin subfragment 1 (S1), uncontaminated by endogenous myosin. We find that the actin-activated MgATPase activity for R403Q alpha-S1 is approximately 30% higher than for wild type, whereas the enzymatic activity for R403Q beta-S1 is reduced by approximately 10%. Thus, the functional consequences of the mutation are fundamentally changed depending upon the context of the cardiac MHC isoform.     

Quantitative analysis of AgNOR proteins in exfoliative cytology specimens of oral mucosa from smokers and nonsmokers

OBJECTIVE: To determine the cell proliferation rate and possible effects of cigarette smoking on the oral mucosa lining through analysis of silver-stained nucleolar organizer regions (AgNORs) in exfoliative cytology specimens. STUDY DESIGN: Exfoliative cytology was performed on the left side of the border of the tongue and of the floor of the mouth in 25 smoking patients and 25 nonsmoking patients. The inclusion criterion for smokers was the consumption of more than 20 cigarettes per day for a minimum of 30 years. RESULTS: The slides were stained by histochemical AgNOR method. In the nonsmoking group the mean number of AgNORs per nucleus was 2.732 +/- 0.236 in the tongue border and 2.918 +/- 0.195 in the floor of the mouth. In smoking patients the mean number of AgNORs per nucleus was 3.372 +/- 0.375 in the tongue border and 3.245 +/- 0.237 in the floor of the mouth. CONCLUSION: The results suggest higher cell proliferation quantified by the histochemical AgNOR technique in exfoliative cytology specimens obtained from the oral mucosa lining of smokers presenting no clinical alterations.