Role of CFTR and ClC-5 in Modulating Vacuolar H(+)-ATPase Activity in Kidney Proximal Tubule

Background/Aims: It has been widely accepted that chloride ions moving along chloride channels act to dissipate the electrical gradient established by the electrogenic transport of H(+) ions performed by H(+)-ATPase into subcellular vesicles. Largely known in intracellular compartments, this mechanism is also important at the plasma membrane of cells from various tissues, including kidney. The present work was performed to study the modulation of plasma membrane H(+)-ATPase by chloride channels, in particular, CFTR and ClC-5 in kidney proximal tubule. Methods and Results: Using in vivo stationary microperfusion, it was observed that ATPase-mediated HCO(3)(-) reabsorption was significantly reduced in the presence of the Cl(-) channels inhibitor NPPB. This effect was confirmed in vitro by measuring the cell pH recovery rates after a NH(4)Cl pulse in immortalized rat renal proximal tubule cells, IRPTC. In these cells, even after abolishing the membrane potential with valinomycin, ATPase activity was seen to be still dependent on Cl(-). siRNA-mediated CFTR channels and ClC-5 chloride-proton exchanger knockdown significantly reduced H(+)-ATPase activity and V-ATPase B2 subunit expression. Conclusion: These results indicate a role of chloride in modulating plasma membrane H(+)-ATPase activity in proximal tubule and suggest that both CFTR and ClC-5 modulate ATPase activity. Copyright (C) 2010 S. Karger AG, Basel

Interaction with calmodulin is important for the secretion of thimet oligopeptidase following stimulation

Thimet oligopeptidase (EC 3.4.24.15; EP24.15) was originally described as a neuropeptide-metabolizing enzyme, highly expressed in the brain, kidneys and neuroendocrine tissue. EP24.15 lacks a typical signal peptide sequence for entry into the secretory pathway and is secreted by cells via an unconventional and unknown mechanism. In this study, we identified a novel calcium-dependent interaction between EP24.15 and calmodulin, which is important for the stimulated, but not constitutive, secretion of EP24.15. We demonstrated that, in vitro, EP24.15 and calmodulin physically interact only in the presence of Ca(2+), with an estimated K(d) value of 0.52 mu m. Confocal microscopy confirmed that EP24.15 colocalizes with calmodulin in the cytosol of resting HEK293 cells. This colocalization markedly increases when cells are treated with either the calcium ionophore A23187 or the protein kinase A activator forskolin. Overexpression of calmodulin in HEK293 cells is sufficient to greatly increase the A23187-stimulated secretion of EP24.15, which can be inhibited by the calmodulin inhibitor calmidazolium. The specific inhibition of protein kinase A with KT5720 reduces the A23187-stimulated secretion of EP24.15 and inhibits the synergistic effects of forskolin with A23187. Treatment with calmidazolium and KT5720 nearly abolishes the stimulatory effects of A23187 on EP24.15 secretion. Together, these data suggest that the interaction between EP24.15 and calmodulin is regulated within cells and is important for the stimulated secretion of EP24.15 from HEK293 cells.

Evaluation of Experimental Autoimmune Uveitis in Mice Treated with FTY720

PURPOSE. FTY720 (fingolimod) is an immunomodulatory drug capable of preventing T-cell migration to inflammatory sites by binding to and subsequently downregulating the expression of sphingosine-1 phosphate receptor 1 (S1P(1)) leading in turn to T-cell retention in lymphoid organs. Additional effects of FTY720 by increasing functional activity of regulatory T cells have recently been demonstrated, raising the conversion of conventional T cells into regulatory T cells and affecting the sequestration of regulatory T cells in normal mice. In this study, the action of FTY720 in the ocular autoimmune model in mice was investigated. METHODS. Mice were immunized with 161-180 peptide and pertussis toxin and were treated with 1 mg/kg/d FTY720 by gavage (7-21 days postimmunization [dpi]) or left untreated. Spleen cells, harvested 21 dpi, were cultured and assayed for cytokine production. Draining lymph node, spleen, and eye cells 21 dpi were assayed for quantification of T-cell populations. Disease severity was evaluated by histologic examination of the enucleated eyes at 21 and 49 dpi. In addition, anti-IRBP antibodies were analyzed by ELISA. RESULTS. FTY720 was effective in suppressing the experimental autoimmune uveitis score. Although there was a reduction in the number of eye-infiltrating cells, FTY did not prevent Treg accumulation at this site. FTY720 leads to a significant increase of CD4(+)IFN-gamma(+) and CD4(+)Foxp3(+) cell percentages in lymph nodes, suggesting that this site could be the source of Treg cells found in the eye. CONCLUSIONS. The data showed that treatment in vivo with FTY720 was able to suppress EAU in mice. These results are indicative of the possible therapeutic use of FTY720 in ocular autoimmune processes. (Invest Ophthalmol Vis Sci. 2010;51:2568-2574) DOI:10.1167/iovs.09-4769

Structural and phylogenetic relationship of ORF 31 from the Anticarsia gemmatalis MNPV to poly (ADP-ribose) polymerases (PARP)

ORF 31 is a unique baculovirus gene in the genome of Anticarsia gemmatalis multiple nucleopolyhedrovirus isolate 2D (AgMNPV-2D). It encodes a putative polypeptide of 369 aa homologous to poly (ADP-ribose) polymerase (PARP) found in the genomes of several organisms. Moreover, we found a phylogenetic association with Group I PARP proteins and a 3D homology model of its conserved PARP C-terminal catalytic domain indicating that had almost an exact spatial superimposition of < 1 angstrom with other PARP available structures. The 5` end of ORF 31 mRNA was located at the first nucleotide of a CATT motif at position -27. Using real-time PCR we detected transcripts at 3 h post-infection (p.i.) increasing until 24 h p.i., which coincides with the onset of DNA replication, suggestive of a possible role in DNA metabolism.

Molecular characterization of human erythrovirus B19 strains obtained from patients with several clinical presentations in the Amazon region of Brazil

Background: Human erythrovirus B 19, endemic in the Amazon region since 1990, is associated with a wide spectrum of clinical presentations. Objectives: To assess the prevalence of erythrovirus B 19 infection and the relative frequency of erythrovirus B 19 genotypes in patients in the Amazon region with various clinical presentations. Study design: A total of 487 clinical samples obtained from patients with symptoms suggestive of erythrovirus infection were tested using specific IgM and IgG antibody assays (ELISA) and PCR for viral DNA detection. Partial VP1 and VP2 regions were sequenced and genotyped by phylogenetic reconstruction. Results: B 19 DNA was detected in 117 (24%) of 487 samples. Of these, 106 (91%) isolates were genotype I and II (9%) were genotype 3. No genotype 2 was found. Genotype I had three clusters (A1, A2 and 13) and all genotype 3 sequences were subtype 3b. All patients with hernatological disorders within cluster B of genotype I were infected by the sarne B 19 lineage, suggesting that this lineage of B 19 may have been transmitted via transfusion of blood products. Conclusion: We reported two genotypes, I and 3b, with three genotype I clusters co-circulating in the Amazon region during the past 10 years. (C) 2008 Elsevier B.V. All rights reserved.

Bayesian coalescent analysis reveals a high rate of molecular evolution in GB virus C

GB virus C/hepatitis G (GBV-C) is an RNA virus of the family Flaviviridae. Despite replicating with an RNA-dependent RNA polymerase, some previous estimates of rates of evolutionary change in GBV-C suggest that it fixes mutations at the anomalously low rate of similar to 100(-7) nucleotide substitution per site, per year. However, these estimates were largely based on the assumption that GBV-C and its close relative GBV-A (New World monkey GB viruses) codiverged with their primate hosts over millions of years. Herein, we estimated the substitution rate of GBV-C using the largest set of dated GBV-C isolates compiled to date and a Bayesian coalescent approach that utilizes the year of sampling and so is independent of the assumption of codivergence. This revealed a rate of evolutionary change approximately four orders of magnitude higher than that estimated previously, in the range of 10(-2) to 10(-3) sub/site/year, and hence in line with those previously determined for RNA viruses in general and the Flaviviridae in particular. In addition, we tested the assumption of host-virus codivergence in GBV-A by performing a reconciliation analysis of host and virus phylogenies. Strikingly, we found no statistical evidence for host-virus codivergence in GBV-A, indicating that substitution rates in the GB viruses should not be estimated from host divergence times.

Functional Diversity of Heat-labile Toxins (LT) Produced by Enterotoxigenic Escherichia coli

Heat-labile toxins (LTs) have ADP-ribosylation activity and induce the secretory diarrhea caused by enterotoxigenic Escherichia coli (ETEC) strains in different mammalian hosts. LTs also act as adjuvants following delivery via mucosal, parenteral, or transcutaneous routes. Previously we have shown that LT produced by human-derived ETEC strains encompass a group of 16 polymorphic variants, including the reference toxin (LT1 or hLT) produced by the H10407 strain and one variant that is found mainly among bacterial strains isolated from pigs (LT4 or pLT). Herein, we show that LT4 ( with six polymorphic sites in the A (K4R, K213E, and N238D) and B (S4T, A46E, and E102K) subunits) displays differential in vitro toxicity and in vivo adjuvant activities compared with LT1. One in vitro generated LT mutant (LTK4R), in which the lysine at position 4 of the A subunit was replaced by arginine, showed most of the LT4 features with an similar to 10-fold reduction of the cytotonic effects, ADP-ribosylation activity, and accumulation of intracellular cAMP in Y1 cells. Molecular dynamic studies of the A subunit showed that the K4R replacement reduces the N-terminal region flexibility and decreases the catalytic site crevice. Noticeably, LT4 showed a stronger Th1-biased adjuvant activity with regard to LT1, particularly concerning activation of cytotoxic CD8(+) T lymphocytes when delivered via the intranasal route. Our results further emphasize the relevance of LT polymorphism among human-derived ETEC strains that may impact both the pathogenicity of the bacterial strain and the use of these toxins as potential vaccine adjuvants.

Distinctive Immunomodulatory and Inflammatory Properties of the Escherichia coli Type II Heat-Labile Enterotoxin LT-IIa and Its B Pentamer following Intradermal Administration

The type I and type II heat-labile enterotoxins (LT-I and LT-II) are strong mucosal adjuvants when they are coadministered with soluble antigens. Nonetheless, data on the parenteral adjuvant activities of LT-II are still limited. Particularly, no previous study has evaluated the adjuvant effects and induced inflammatory reactions of LT-II holotoxins or their B pentameric subunits after delivery via the intradermal (i.d.) route to mice. In the present report, the adjuvant and local skin inflammatory effects of LT-IIa and its B subunit pentamer (LT-IIaB(5)) were determined. When coadministered with ovalbumin (OVA), LT-IIa and, to a lesser extent, LT-IIaB(5) exhibited serum IgG adjuvant effects. In addition, LT-IIa but not LT-IIaB(5) induced T cell-specific anti-OVA responses, particularly in respect to induction of antigen-specific cytotoxic CD8(+) T cell responses. LT-IIa and LT-IIaB(5) induced differential tissue permeability and local inflammatory reactions after i.d. injection. Of particular interest was the reduced or complete lack of local reactions, such as edema and tissue induration, in mice i.d. inoculated with LT-IIa and LT-IIaB(5), respectively, compared with mice immunized with LT-I. In conclusion, the present results show that LT-IIa and, to a lesser extent, LT-IIaB(5) exert adjuvant effects when they are delivered via the i.d. route. In addition, the low inflammatory effects of LT-IIa and LT-IIaB(5) in comparison to those of LT-I support the usefulness of LT-IIa and LT-IIaB(5) as parenterally delivered vaccine adjuvants.

Functional and immunological characterization of a natural polymorphic variant of a heat-labile toxin (LT-I) produced by enterotoxigenic Escherichia coli (ETEC)

Heat-labile toxins (LT) encompass at least 16 natural polymorphic toxin variants expressed by wild-type enterotoxigenic Escherichia coli (ETEC) strains isolated from human beings, but only one specific form, produced by the reference ETEC H10407 strain (LT1), has been intensively studied either as a virulence-associated factor or as a mucosal/transcutaneous adjuvant. In the present study, we carried out a biological/immunological characterization of a natural LT variant (LT2) with four polymorphic sites at the A subunit (S190L, G196D, K213E, and S224T) and one at the B subunit (T75A). The results indicated that purified LT2, in comparison with LT1, displayed similar in vitro toxic activities (adenosine 3`,5`-cyclic monophosphate accumulation) on mammalian cells and in vivo immunogenicity following delivery via the oral route. Nonetheless, the LT2 variant showed increased adjuvant action to ovalbumin when delivered to mice via the transcutaneous route while antibodies raised in mice immunized with LT2 displayed enhanced affinity and neutralization activity to LT1 and LT2. Taken together, the results indicate that the two most frequent LT polymorphic forms expressed by wild ETEC strains share similar biological features, but differ with regard to their immunological properties.

Trypanosoma evansi: Adenosine deaminase activity in the brain of infected rats

The study was undertaken to evaluate changes in the activity of adenosine deaminase (ADA) in brains of rats infected by Trypanosoma evansi. Each rat was intraperitoneally infected with 10(6) trypomastigotes either suspended in fresh (group A; n = 13) and cryopreserved blood (group B; n = 13). Thirteen animals were used as control (group C). ADA activity was estimated in the cerebellum, cerebral cortex, striatum and hippocampus. No differences (P > 0.05) in ADA activity were observed in the cerebellum between infected and non-infected animals. Significant (P < 0.05) reductions in ADA activity occurred in cerebral cortex in acutely (day 4 post-infection; PI) and chronically (day 20 PI) infected rats. ADA activity was significantly (P < 0.05) decreased in the hippocampus in acutely infected rats, but significantly (P < 0.05) increased in the chronically infected rats. Significant (P < 0.05) reductions in ADA activity occurred in the striatum of chronically infected rats. Parasites could be found in peripheral blood and brain tissue through microscopic examination and PCR assay, respectively, in acutely and chronically infected rats. The reduction of ADA activity in the brain was associated with high levels of parasitemia and anemia in acute infections. Alterations in ADA activity of the brain in T. evansi-infected rats may have implications for pathogenesis of the disease. (C) 2010 Elsevier Inc. All rights reserved.

Leishmania amazonensis META2 protein confers protection against heat shock and oxidative stress

The META cluster of Leishmania amazonensis contains both META1 and META2 genes, which are upregulated in metacyclic promastigotes and encode proteins containing the META domain. Previous studies defined META2 as a 48.0-kDa protein, which is conserved in other Leishmania species and in Trypanosoma brucei. In this work, we demonstrate that META2 protein expression is regulated during the Leishmania life cycle but constitutive in T. brucei. META2 protein is present in the cytoplasm and flagellum of L amazonensis promastigotes. Leishmania META2-null replacement mutants are more sensitive to oxidative stress and, upon heat shock, assume rounded morphology with shortened flagella. The increased susceptibility of null parasites to heat shock is reversed by extra-chromosomal expression of the META2 gene. Defective Leishmania promastigotes exhibit decreased ability to survive in macrophages. By contrast, META2 expression is decreased by 80% in RNAi-induced T. brucei bloodstream forms with no measurable effect on survival or resistance to heat shock. (C) 2010 Elsevier Inc. All rights reserved.

Malaria on the Amazonian frontier: Transmission dynamics, risk factors, spatial distribution, and prospects for control

Little follow-up data on malaria transmission in communities originating from frontier settlements in Amazonia are available. Here we describe a cohort study in a frontier settlement in Acre, Brazil, where 509 subjects contributed 489.7 person-years of follow-up. The association between malaria morbidity during the follow-up and individual, household, and spatial covariates was explored with mixed-effects logistic regression models and spatial analysis. Incidence rates for Plasmodium vivax and Plasmodium falciparum malaria were 30.0/100 and 16.3/100 person-years at risk, respectively. Malaria morbidity was strongly associated with land clearing and farming, and decreased after five years of residence in the area, suggesting that clinical immunity develops among subjects exposed to low malaria endemicity. Significant spatial clustering of malaria was observed in the areas of most recent occupation, indicating that the continuous influx of nonimmune settlers to forest-fringe areas perpetuates the cycle of environmental change and colonization that favors malaria transmission in rural Amazonia.

Mechanisms underlying the long-term regulation of NHE3 by parathyroid hormone

The activity of the Na(+)/H(+) exchanger NHE3 is regulated by a number of factors including parathyroid hormone (PTH). In the current study, we used a renal epithelial cell line, the opossum kidney (OKP) cell, to elucidate the mechanisms underlying the long-term effects of PTH on NHE3 transport activity and expression. We observed that NHE3 activity was reduced 6 h after addition of PTH, and this reduction persisted almost unaltered after 24 h. The decrease in activity was associated with diminished NHE3 cell surface expression at 6, 16, and 24 h after PTH addition, total cellular NHE3 protein at 16 and 24 h, and NHE3 mRNA abundance at 24 h. The lower levels of NHE3 mRNA were associated to a small, but significant, decrease in mRNA stability. Additionally, by analyzing the rat NHE3 gene promoter activity in OKP cells, we verified that the regulatory region spanning the segment -152 to +55 was mildly reduced under the influence of PTH. This effect was completely abolished by the presence of the PKA inhibitor KT 5720. In conclusion, long-term exposure to PTH results in reduction of NHE3 mRNA levels due to a PKA-dependent inhibitory effect on the NHE3 promoter and a small reduction of mRNA half-life, and decrease in the total amount of protein which is preceded by endocytosis of the apical surface NHE3. The decreased NHE3 expression is likely to be responsible for the reduction of sodium, bicarbonate, and fluid reabsorption in the proximal tubule consistently perceived in experimental models of PTH disorders.

Role of MMP9 on Invadopodia Formation in Cells From Adenoid Cystic Carcinoma. Study by Laser Scanning Confocal Microscopy

Migration, invasion and protease activity are essential for tumor progression and metastasis. Metastatic cells rely on invadopodia to degrade and invade extracellular matrix (ECM). Invadopodia are membrane protrusions with enzymes required for ECM degradation. These protrusions contain cortactin and membrane type I matrix metalloproteinase (MT1-MMP) superimposed to areas of digested matrix. Here we characterized invadopodia in a cell line (CAC2) derived from human adenoid cystic carcinoma. We carried out fluorescent-substrate degradation assay to assess in situ protease activity of CAC2 cells. Digestion spots in fluorescent substrate appear as black areas in green background. Cells were cultured on Matrigel-gelatin-FITC and fixed after 1 h and 3 h. CAC2 cells were double labeled to actin and cortactin. Cells were also double stained to actin and MT1-MMR Samples were studied by laser scanning confocal microscopy. In all time points CAC2 cells showed actin, cortactin, and MT1-MMP colocalized with digestion spots in fluorescent substrate. We searched for other proteases involved in invadopodia activity. We have previously demonstrated that MMP9 influences adenoid cystic carcinoma behavior. This prompted us to investigate role played by MMP9 on invadopodia formation. CAC2 cells had MMP9 silenced by siRNA. After I h in fluorescent substrate, cells with silenced MMP9 showed clear decrease in matrix digestion compared with controls. No differences were found in cells with silenced MMP9 grown for 3 h on fluorescent substrate. Our results showed that CAC2 cells exhibit functional invadopodia containing cortactin and MT1-MMR Furthermore, MMP9 would be required in the initial steps of invadopodia formation. Microsc. Res. Tech. 73:99-108, 2010. (C) 2009 Wiley-Liss, Inc.

Hypotheses testing for structural calibration model

The main objective of this paper is to discuss maximum likelihood inference for the comparative structural calibration model (Barnett, in Biometrics 25:129-142, 1969), which is frequently used in the problem of assessing the relative calibrations and relative accuracies of a set of p instruments, each designed to measure the same characteristic on a common group of n experimental units. We consider asymptotic tests to answer the outlined questions. The methodology is applied to a real data set and a small simulation study is presented.