Interaction of the Nav1.2a subunit of the voltage-dependent sodium channel with nodal ankyrinG. In vitro mapping of the interacting domains and association in synaptosomes

Voltage-dependant sodium channels at the axon initial segment and nodes of Ranvier colocalize with the nodal isoforms of ankyrin(G) (Ank(G) node). Using fusion proteins derived from the intracellular regions of the Nav1.2a subunit and the Ank repeat domain of Ank(G) node, we mapped a major interaction site in the intracellular loop separating alpha subunit domains I-II. This 57-amino acid region binds the Ank repeat region with a K(D) value of 69 nm. We identified another site in intracellular loop III-IV, and we mapped both Nav1.2a binding sites on the ankyrin repeat domain to the region encompassing repeats 12-22. The ankyrin repeat domain did not bind the beta(1) and beta(2) subunit cytoplasmic regions. We showed that in cultured embryonic motoneurons, expression of the beta(2) subunit is not necessary for the colocalization of Ank(G) node with functional sodium channels at the axon initial segment. Antibodies directed against the beta(1) subunit intracellular region, alpha subunit loop III-IV, and Ank(G) node could not co-immunoprecipitate Ank(G) node and sodium channels from Triton X-100 solubilisates of rat brain synaptosomes. Co-immunoprecipitation of sodium channel alpha subunit and of the 270- and 480-kDa AnkG node isoforms was obtained when solubilization conditions that maximize membrane protein extraction were used. However, we could not find conditions that allowed for co-immunoprecipitation of ankyrin with the sodium channel beta(1) subunit.     

Effects of mitogens and co-mitogens on the formation of small-cell colonies in primary cultures of rat hepatocytes

Degradation of collagen by fibroblast phagocytosis is an important pathway for physiological remodelling of soft connective tissues. Perturbations of this pathway may provide a mechanism for the development of fibrotic lesions. As collagen phagocytosis may be regulated by either a change of the proportions or the activity of phagocytic cells, we quantified phagocytosis with an in vitro model system. Collagen-coated fluorescent latex beads were incubated with human gingival fibroblasts and the fluorescence associated with internalized beads was measured by flow cytometry. Cells from normal tissues that had been incubated with beads for 3 hours contained a mean of 64% phagocytic cells; however, a small subpopulation (10% of phagocytic cells) contained more than threefold higher numbers of beads per cell than the mean. In contrast, cells from fibrotic lesions exhibited a large reduction of the proportions of phagocytic cells (mean = 13.8%) and there were no cells with high numbers of beads. On the basis of ³H-Tdr labeling, cells from fibrotic lesions that had internalized beads failed to proliferate, in contrast to phagocytic cells from normal tissues, which underwent repeated cell divisions. This result was not due to variations of cell cycle phase as there was no preferential internalization of beads during different phases of the cell cycle. The low phagocytic rate of cells from fibrotic lesions was also not due to asymmetric partitioning of phagosomes at mitosis as videocinemicrography of bead-labeled phagosomes in single, pre-mitotic cells demonstrated that > 90% of phagocytic cells equally partitioned beads to daughter cells. To investigate if inhibition of phagocytosis could be replicated in vitro, cells were incubated with the fibrosis-inducing drugs nifedipine or dilantin. These cultures exhibited marked (15–75%), dose-dependent reductions in the proportions of phagocytic cells, but there was no reduction in bead number per cell. Fibrotic lesions appear to contain fibroblasts with marked deficiencies in phagocytosis and the reduced phagocytic activity of these cells may contribute to unbalanced degradation and fibrosis. © 1993 Wiley-Liss, Inc.     

Leptospira spp., rotavirus,norovirus, and hepatitis E virus surveillance in a wild invasive golden‐headed lion tamarin (Leontopithecus chrysomelas; Kuhl, 1820) population from an urban park in Niterói,Rio de Janeiro,Brazil

The world currently faces severe biodiversity losses caused by anthropogenic activities such as deforestation, pollution, the introduction of exotic species, habitat fragmentation, and climate changes. Disease ecology in altered environments is still poorly understood. The golden‐headed lion tamarin (GHLT, Leontopithecus chrysomelas) is an endangered species that became invasive in an urban park in Niterói, Rio de Janeiro, Brazil. The initially few invasive GHLT individuals became hundreds, adapted to living in proximity to humans and domestic animals. These GHLTs were captured as part of a conservation project; some animals were translocated to Bahia and some were kept in captivity. This study tested 593 GHLT for Leptospira serology; 100 and 95 GHLT for polymerase chain reaction (PCR) toLeptospira and hepatitis E virus genotype 3 (HEV‐3), respectively, and 101 familiar groups for PCR to viruses (rotavirus A, norovirus GI and GII, and HEV‐3). One animal had antibodies for Leptospira serovar Shermani and another for serovar Hebdomadis. One saprophyticLeptospira was found by the 16S PCR and sequencing. Viruses were not detected in samples tested. Findings suggest that the epidemiological importance of such pathogens in this GHLT population is either low or nonexistent. These data are important to understand the local disease ecology, as well as monitoring a translocation project, and to contribute data for species conservation.     

Liquid vs Solid Culture Medium to Evaluate Proportion and Time to Change in Management of Suspects of Tuberculosis—A Pragmatic Randomized Trial in Secondary and Tertiary Health Care Units in Brazil

Background

The use of liquid medium (MGIT960) for tuberculosis (TB) diagnosis was recommended by WHO in 2007. However, there has been no evaluation of its effectiveness on clinically important outcomes.

Methods and Findings

A pragmatic trial was carried out in a tertiary hospital and a secondary health care unit in Rio de Janeiro City, Brazil. Participants were 16 years or older, suspected of having TB. They were excluded if only cerebral spinal fluid or blood specimens were available for analysis. MGIT960 technique was compared with the Lowenstein-Jensen (LJ) method for laboratory diagnosis of active TB. Primary outcome was the proportion of patients who had their initial medical management changed within 2 months after randomisation. Secondary outcomes were: mean time for changing the procedure, patient satisfaction with the overall treatment and adverse events. Data were analysed by intention-to-treat. Between April 2008 and September 2011, 693 patients were enrolled (348 to MGIT, 345 to LJ). Smear and culture results were positive for 10% and 15.7% of participants, respectively. Patients in the MGIT arm had their initial medical management changed more frequently than those in the LJ group (10.1% MGIT vs 3.8% LJ, RR 2.67 95% CI 1.44–.96, p = 0.002, NNT 16, 95% CI 10–39). Mean time for changing the initial procedure was greater in LJ group at both sites: 20.0 and 29.6 days in MGIT group and 52.2 and 64.3 in LJ group (MD 33.5, 95% CI 30.6–36.4, p = 0.0001). No other important differences were observed.

Conclusions

This study suggests that opting for the MGIT960 system for TB diagnosis provides a promising case management model for improving the quality of care and control of TB.

Trial Registration

Controlled-Trials.com ISRCTN79888843     

Protection against T1DM-Induced Bone Loss by Zinc Supplementation: Biomechanical,Histomorphometric, and Molecular Analyses in STZ-Induced Diabetic Rats

Several studies have established an association between diabetes and alterations in bone metabolism; however, the underlying mechanism is not well established. Although zinc is recognized as a potential preventive agent against diabetes-induced bone loss, there is no evidence demonstrating its effect in chronic diabetic conditions. This study evaluated the effects of zinc supplementation in a chronic (90 days) type 1 diabetes-induced bone-loss model. Male Wistar rats were distributed in three groups: control, type 1 diabetes mellitus (T1DM), and T1DM plus zinc supplementation (T1DMS). Serum biochemical analysis; tibia histomorphometric, biomechanical, and collagen-content analyses; and femur mRNA expression were evaluated. Relative to T1DM, the zinc-supplemented group showed increased histomorphometric parameters such as TbWi and BAr and decreased TbSp, increased biomechanical parameters (maximum load, stiffness, ultimate strain, and Young’s modulus), and increased type I collagen content. Interestingly, similar values for these parameters were observed between the T1DMS and control groups. These results demonstrate the protective effect of zinc on the maintenance of bone strength and flexibility. In addition, downregulation of OPG, COL1A, and MMP-9 genes was observed in T1DMS, and the anabolic effects of zinc were evidenced by increased OC expression and serum ALP activity, both related to osteoblastogenesis, demonstrating a positive effect on bone formation. In contrast, T1DM showed excessive bone loss, observed through reduced histomorphometric and biomechanical parameters, characterizing diabetes-associated bone loss. The bone loss was also observed through upregulation of OPG, COL1A, and MMP-9 genes. In conclusion, zinc showed a positive effect on the maintenance of bone architecture and biomechanical parameters. Indeed, OC upregulation and control of expression of OPG, COL1A, and MMP-9 mRNAs, even in chronic hyperglycemia, support an anabolic and protective effect of zinc under chronic diabetic conditions. Furthermore, these results indicate that zinc supplementation could act as a complementary therapy in chronic T1DM.     

Aerobic Exercise Training Prevents the Onset of Endothelial Dysfunction via Increased Nitric Oxide Bioavailability and Reduced Reactive Oxygen Species in an Experimental Model of Menopause

Objective

Previous studies have shown that estrogen deficiency, arising in postmenopause, promotes endothelial dysfunction. This study evaluated the effects of aerobic exercise training on endothelial dependent vasodilation of aorta in ovariectomized rats, specifically investigating the role of nitric oxide (NO) and reactive oxygen species (ROS).

Methods

Female Wistar rats ovariectomized (OVX – n=20) or with intact ovary (SHAM – n=20) remained sedentary (OVX and SHAM) or performed aerobic exercise training on a treadmill 5 times a week for a period of 8 weeks (OVX-TRA and SHAM-TRA). In the thoracic aorta the endothelium-dependent and –independent vasodilation was assessed by acetylcholine (ACh) and sodium nitroprusside (SNP), respectively. Certain aortic rings were incubated with L-NAME to assess the NO modulation on the ACh-induced vasodilation. The fluorescence to dihydroethidium in aortic slices and plasma nitrite/nitrate concentrations were measured to evaluate ROS and NO bioavailability, respectively.

Results

ACh-induced vasodilation was reduced in OVX rats as compared SHAM (Rmax: SHAM: 86±3.3 vs. OVX: 57±3.0%, p<0.01). Training prevented this response in OVX-TRA (Rmax: OVX-TRA: 88±2.0%, p<0.01), while did not change it in SHAM-TRA (Rmax: SHAM-TRA: 80±2.2%, p<0.01). The L-NAME incubation abolished the differences in ACh-induced relaxation among groups. SNP-induced vasodilation was not different among groups. OVX reduced nitrite/nitrate plasma concentrations and increased ROS in aortic slices, training as effective to restore these parameters to the SHAM levels.

Conclusions

Exercise training, even in estrogen deficiency conditions, is able to improve endothelial dependent vasodilation in rat aorta via enhanced NO bioavailability and reduced ROS levels.     

Down Regulation of NO Signaling in Trypanosoma cruzi upon Parasite-Extracellular Matrix Interaction: Changes in Protein Modification by Nitrosylation and Nitration

Background

Adhesion of the Trypanosoma cruzi trypomastigotes, the causative agent of Chagas'' disease in humans, to components of the extracellular matrix (ECM) is an important step in host cell invasion. The signaling events triggered in the parasite upon binding to ECM are less explored and, to our knowledge, there is no data available regarding •NO signaling.

Methodology/Principal Findings

Trypomastigotes were incubated with ECM for different periods of time. Nitrated and S-nitrosylated proteins were analyzed by Western blotting using anti-nitrotyrosine and S-nitrosyl cysteine antibodies. At 2 h incubation time, a decrease in NO synthase activity, •NO, citrulline, arginine and cGMP concentrations, as well as the protein modifications levels have been observed in the parasite. The modified proteins were enriched by immunoprecipitation with anti-nitrotyrosine antibodies (nitrated proteins) or by the biotin switch method (S-nitrosylated proteins) and identified by MS/MS. The presence of both modifications was confirmed in proteins of interest by immunoblotting or immunoprecipitation.

Conclusions/Significance

For the first time it was shown that T. cruzi proteins are amenable to modifications by S-nitrosylation and nitration. When T. cruzi trypomastigotes are incubated with the extracellular matrix there is a general down regulation of these reactions, including a decrease in both NOS activity and cGMP concentration. Notwithstanding, some specific proteins, such as enolase or histones had, at least, their nitration levels increased. This suggests that post-translational modifications of T. cruzi proteins are not only a reflex of NOS activity, implying other mechanisms that circumvent a relatively low synthesis of •NO. In conclusion, the extracellular matrix, a cell surrounding layer of macromolecules that have to be trespassed by the parasite in order to be internalized into host cells, contributes to the modification of •NO signaling in the parasite, probably an essential move for the ensuing invasion step.     

1H, 15N and 13C resonance assignments of PpdD,a type IV pilin from enterohemorrhagic Escherichia coli

Bacterial type 4 pili (T4P) are long flexible fibers involved in adhesion, DNA uptake, phage transduction, aggregation and a flagella-independent movement called “twitching motility”. T4P comprise thousands of copies of the major pilin subunit, which is initially inserted in the plasma membrane, processed and assembled into dynamic helical filaments. T4P are crucial for host colonization and virulence of many Gram-negative bacteria. In enterohemorrhagic Escherichia coli the T4P, called hemorrhagic coli pili (HCP) promote cell adhesion, motility, biofilm formation and signaling. To understand the mechanism of HCP assembly and function, we analyzed the structure of the major subunit prepilin peptidase-dependent protein D (PpdD) (also called HcpA), a 15 kDa pilin with two potential disulfide bonds. Here we present the ¹H, ¹⁵N and ¹³C backbone and side chain resonance assignments of the C-terminal globular domain of PpdD as a first step to its structural determination.     

Sterilization of culture media for orchids using a microwave oven

Production of orchid seedlings often requires complex laboratory infrastructure; therefore, a simple and low-cost method would be of general benefit to many small-scale producers and orchid enthusiasts. This article describes a protocol for preparing culture media using a domestic microwave oven. Two alternative culture media were evaluated for a range of factors, including the duration of boiling, the efficacy of antiseptics applied to jars and media, the concentration of the antiseptic hydrogen peroxide required for sterilization, and the growth of Oncidium cebolleta and Phalaenopsis amabilis plantlets in media prepared using a microwave oven compared with conventionally autoclaved media. It was found that addition of 2 mL of 30% hydrogen peroxide (Peridrol® solution) per liter of culture medium, an 8-min boiling time in the microwave oven, and 8 g/L agar were sufficient to produce solidified culture media, which facilitated orchard seed germination and growth without contamination. Furthermore, the microwaved media exhibited superior plantlet growth to autoclaved media.     

Extracellular Inosine is Modulated by H2O2 and Protects Sertoli Cells against Lipoperoxidation and Cellular Injury

Extracellular purines are involved in the regulation of a wide range of physiological processes, including cytoprotection, ischemic preconditioning, and cell death. These actions are usually mediated via triggering of membrane purinergic receptors, which may activate antioxidant enzymes, conferring cytoprotection. Recently, it was demonstrated that the oxidative stress induced by cisplatin up-regulated A¹ receptor expression in rat testes, suggesting an involvement of purinergic signaling in the response of testicular cells to oxidant injury. In this article, we report the effect of hydrogen peroxide on purinergic agonist release by cultured Sertoli cells. Extracellular inosine levels are strongly increased in the presence of H²O², suggesting an involvement of this nucleoside on Sertoli cells response to oxidant treatment. Inosine was observed to decrease H²O²-induced lipoperoxidaton and cellular injury, and it also preserved cellular ATP content during H²O² exposure. These effects were abolished in the presence of nucleoside uptake inhibitors, indicating that nucleoside internalisation is essential for its action in preventing cell damage.