CztR, a LysR-Type Transcriptional Regulator Involved in Zinc Homeostasis and Oxidative Stress Defense in Caulobacter crescentus

Caulobacter crescentus is a free-living alphaproteobacterium that has 11 predicted LysR-type transcriptional regulators (LTTRs). Previously, a C. crescentus mutant strain with a mini-Tn5lacZ transposon inserted into a gene encoding an LTTR was isolated; this mutant was sensitive to cadmium. In this work, a mutant strain with a deletion was obtained, and the role of this LTTR (called CztR here) was evaluated. The transcriptional start site of this gene was determined by primer extension analysis, and its promoter was cloned in front of a lacZ reporter gene. beta-Galactosidase activity assays, performed with the wild-type and mutant strains, indicated that this gene is 2-fold induced when cells enter stationary phase and that it is negatively autoregulated. Moreover, this regulator is essential for the expression of the divergent cztA gene at stationary phase, in minimal medium, and in response to zinc depletion. This gene encodes a hypothetical protein containing 10 predicted transmembrane segments, and its expression pattern suggests that it encodes a putative zinc transporter. The cztR strain was also shown to be sensitive to superoxide (generated by paraquat) and to hydrogen peroxide but not to tert-butyl hydroperoxide. The expression of katG and ahpC, but not that of the superoxide dismutase genes, was increased in the cztR mutant. A model is proposed to explain how CztR binding to the divergent regulatory regions could activate cztA expression and repress its own transcription.

Slower rescue of ER homeostasis by the unfolded protein response pathway associated with common variable immunodeficiency

Common variable immunodeficiency (CVID) is a primary immunodeficiency characterized by hypogammaglobulinemia and recurrent infections. Herein we addressed the role of unfolded protein response (UPR) in the pathogenesis of the disease. Augmented unspliced X-box binding protein 1 (XBP-1) mRNA concurrent with co-localization of IgM and BiP/GRP78 were found in one CVID patient. At confocal microscopy analysis this patient`s cells were enlarged and failed to present the typical surface distribution of IgM, which accumulated within an abnormally expanded endoplasmic reticulum. Sequencing did not reveal any mutation on XBP-1, neither on IRE-1 alpha that could potentially prevent the splicing to occur. Analysis of spliced XBP-1, IRE-1 alpha and BiP messages after LPS or Brefeldin A treatment showed that, unlike healthy controls that respond to these endoplasmic reticulum (ER) stressors by presenting waves of transcription of these three genes, this patient`s cells presented lower rates of transcription, not reaching the same level of response of healthy subjects even after 48 h of ER stress. Treatment with DMSO rescued IgM and IgG secretion as well as the expression of spliced XBP-1. Our findings associate diminished splicing of XBP-1 mRNA with accumulation of IgM within the ER and lower rates of chaperone transcription, therefore providing a mechanism to explain the observed hypogammaglobulinemia. (C) 2008 Elsevier Ltd. All rights reserved.

Fur controls iron homeostasis and oxidative stress defense in the oligotrophic alpha-proteobacterium Caulobacter crescentus

In most bacteria, the ferric uptake regulator (Fur) is a global regulator that controls iron homeostasis and other cellular processes, such as oxidative stress defense. In this work, we apply a combination of bioinformatics, in vitro and in vivo assays to identify the Caulobacter crescentus Fur regulon. A C. crescentus fur deletion mutant showed a slow growth phenotype, and was hypersensitive to H(2)O(2) and organic peroxide. Using a position weight matrix approach, several predicted Fur-binding sites were detected in the genome of C. crescentus, located in regulatory regions of genes not only involved in iron uptake and usage but also in other functions. Selected Fur-binding sites were validated using electrophoretic mobility shift assay and DNAse I footprinting analysis. Gene expression assays revealed that genes involved in iron uptake were repressed by iron-Fur and induced under conditions of iron limitation, whereas genes encoding iron-using proteins were activated by Fur under conditions of iron sufficiency. Furthermore, several genes that are regulated via small RNAs in other bacteria were found to be directly regulated by Fur in C. crescentus. In conclusion, Fur functions as an activator and as a repressor, integrating iron metabolism and oxidative stress response in C. crescentus.

PREDICTION OF METAL CATION TOXICITY TO THE BIOLUMINESCENT FUNGUS GERRONEMA VIRIDILUCENS

A correlation between the physicochemical properties of mono- [Li(I), K(I), Na(I)] and divalent [Cd(II), Cu(II), Mn(II), Ni(II), Co(II), Zn(II), Mg(II), Ca(II)] metal cations and their toxicity (evaluated by the free ion median effective concentration. EC50(F)) to the naturally bioluminescent fungus Gerronema viridilucens has been studied using the quantitative ion character activity relationship (QICAR) approach. Among the 11 ionic parameters used in the current study, a univariate model based on the covalent index (X(m)(2)r) proved to be the most adequate for prediction of fungal metal toxicity evaluated by the logarithm of free ion median effective concentration (log EC50(F)): log EC50(F) = 4.243 (+/-0.243) -1.268 (+/-0.125).X(m)(2)r (adj-R(2) = 0.9113, Alkaike information criterion [AIC] = 60.42). Additional two- and three-variable models were also tested and proved less suitable to fit the experimental data. These results indicate that covalent bonding is a good indicator of metal inherent toxicity to bioluminescent fungi. Furthermore, the toxicity of additional metal ions [Ag(I), Cs(I), Sr(II), Ba(II), Fe(II), Hg(II), and Pb(II)] to G. viridilucens was predicted, and Pb was found to be the most toxic metal to this bioluminescent fungus (EC50(F)): Pb(II) > Ag(I) > Hg(I) > Cd(II) > Cu(II) > Co(II) Ni(II) > Mn(II) > Fe(II) approximate to Zn(II) > Mg(II) approximate to Ba(II) approximate to Cs(I) > Li(I) > K(I) approximate to Na(I) approximate to Sr(II)> Ca(II). Environ. Toxicol. Chem. 2010;29:2177-2181. (C) 2010 SETAC

The isatin-Schiff base copper(II) complex Cu(isaepy)(2) acts as delocalized lipophilic cation, yields widespread mitochondrial oxidative damage and induces AMP-activated protein kinase-dependent apoptosis

We previously demonstrated that Bis[(2-oxindol-3-ylimino)-2-(2-aminoethyl) pyridine-N, N`] copper(II) [Cu(isaepy)(2)] was an efficient inducer of the apoptotic mitochondrial pathway. Here, we deeply dissect the mechanisms underlying the ability of Cu(isaepy)(2) to cause mitochondriotoxicity. In particular, we demonstrate that Cu(isaepy)(2) increases NADH-dependent oxygen consumption of isolated mitochondria and that this phenomenon is associated with oxy-radical production and insensitive to adenosine diphosphate. These data indicate that Cu(isaepy)(2) behaves as an uncoupler and this property is also confirmed in cell systems. Particularly, SH-SY5Y cells show: (i) an early loss of mitochondrial transmembrane potential; (ii) a decrease in the expression levels of respiratory complex components and (iii) a significant adenosine triphosphate (ATP) decrement. The causative energetic impairment mediated by Cu(isaepy)(2) in apoptosis is confirmed by experiments carried out with rho(0) cells, or by glucose supplementation, where cell death is significantly inhibited. Moreover, gastric and cervix carcinoma AGS and HeLa cells, which rely most of their ATP production on oxidative phosphorylation, show a marked sensitivity toward Cu(isaepy)(2). Adenosine monophosphate-activated protein kinase (AMPK), which is activated by events increasing the adenosine monophosphate: ATP ratio, is deeply involved in the apoptotic process because the overexpression of its dominant/negative form completely abolishes cell death. Upon glucose supplementation, AMPK is not activated, confirming its role as fuel-sensing enzyme that positively responds to Cu(isaepy)(2)-mediated energetic impairment by committing cells to apoptosis. Overall, data obtained indicate that Cu(isaepy)(2) behaves as delocalized lipophilic cation and induces mitochondrial-sited reactive oxygen species production. This event results in mitochondrial dysfunction and ATP decrease, which in turn triggers AMPK-dependent apoptosis.

Sistema purinérgico em plaquetas de ratos adultos e interações com o sistema renina-angiotensina

As plaquetas sangüíneas são fragmentos citoplasmáticos, oriundos da ruptura dos megacariócitos, cuja principal função está relacionada à manutenção da integridade vascular. Os nucleotídeos extracelulares, ATP e ADP, bem como a adenosina, têm sido implicados em um grande número de funções fisiológicas: o ADP é o principal fator recrutador de plaquetas, enquanto que o ATP é um inibidor competitivo da agregação induzida por ADP. A adenosina é uma molécula capaz de induzir vasodilatação e inibir a agregação plaquetária. Desta maneira, a manutenção da sinalização purinérgica normal tem se mostrado importante para o tratamento de doenças cardiovasculares. Os nucleosídeos di e trifosfatos circulantes podem ser hidrolisados por membros de várias famílias de ectonucleotidases de membrana e solúveis, incluindo as ecto-nucleosídeo trifosfato difosfoidrolases (E-NTPDases) e ecto-nucleotídeo pirofosfatase/fosfodiesterases (E-NPPs), que em conjunto com a ecto-5’-nucleotidase, levam à formação de adenosina. Na superfície das plaquetas, ambas enzimas, E-NTPDase e ecto-5’-nucleotidase, estão descritas. O sistema renina-angiotensina é o principal regulador da função renal e cardiovascular, desenvolvendo um papel fundamental na homeostasia da pressão arterial e do balanço eletrolítico. A angiotensina II (ANGII) induz fisiologicamente a ativação das plaquetas, possivelmente devido às suas propriedades vasoconstritoras. Os objetivos deste trabalho foram, portanto: 1) caracterizar cineticamente a enzima E-NPP em plaquetas de ratos, utilizando o substrato marcador p-Nph-5’TMP e 2) esclarecer, mesmo que em parte, os possíveis efeitos da ANGII sobre a hidrólise extracelular de nucleotídeos por plaquetas de ratos. No primeiro capítulo deste trabalho, descrevemos uma atividade enzimática em plaquetas de ratos que compartilha as principais características bioquímicas já descritas para as E-NPPs: pH ótimo alcalino; valores de KM e Vmax calculados de aproximadamente 106.22 ± 17.83 μM e 3.44 ± 0.18 nmol p-nitrophenol/min/mg, respectivamente; e dependência de cátions divalentes. Além disso, o AMP inibiu somente a hidrólise do p-Nph-5’TMP. Por outro lado, a azida de sódio, em altas concentrações, a angiotensina II e o cloreto de gadolínio alteraram apenas as hidrólises de ATP ou ADP ou de ambos. No segundo capítulo, mostramos que a ANGII foi capaz de aumentar as hidrólises de ATP, ADP e AMP em plaquetas em todas as doses testadas (5, 50, 500 e 5000 picomóis). Entretanto, nenhuma alteração foi observada com relação à hidrólise do p-Nph-5'TMP. Em adição, observamos um aumento na hidrólise de AMP e uma diminuição na hidrólise de p-Nph-5'TMP em plaquetas de ratos espontaneamente hipertensos (SHR) quando comparados a ratos Wistar normotensos. De maneira geral, esta dissertação traz a caracterização bioquímica da enzima E-NPP na superfície de plaquetas intactas de ratos como sendo parte de um complexo sistema para a hidrólise de nucleotídeos nestes fragmentos citoplasmáticos, podendo, assim, contribuir para o desenvolvimento de terapias antiplaquetárias e para o tratamento de doenças vasculares. Adicionalmente, apresentamos alguns resultados demonstrando interações entre os sistemas angiotensinérgico e adenosinérgico de plaquetas de ratos, o que poderá contribuir para o entendimento e o tratamento de doenças cardiovasculares como hipertensão e arteriosclerose.

HPLC-DAD methodology for the quantification of organic acids, furans and polyphenols by direct injection of wine samples

This article proposes a simple and sensitive HPLC method with photo-diode array detection for the analysis of organic acids, monomeric polyphenols and furanic compounds in wine samples by direct injection. The chromatographic separation of 8 organic acids, 2 furans and 22 phenolic compounds was carried out with a buffered solution (pH 2.70) and acetonitrile as mobile phases and a difunctionally bonded C18 stationary phase, Atlantis dC18 (250 4.6 mm, 5mm) column. The elution was performed in 12 min for the organic acids and in 60 min for the phenolic compounds, including phenolic acids, stilbenes and flavonoids. Target compounds were detected at 210 nm (organic acids, flavan-3-ols and benzoic acids), 254 nm (ellagic acid), 280 nm (furans and cinnamic acid), 315 nm (hydroxycinnamic acids and trans-resveratrol) and 360 nm (flavonoids). The RSD for the repeatability test (n55) of peak area and retention times were below 3.1 and 0.3%, respectively, for phenolics and below 1.0 and 0.2% for organic acids. The RSDs expressing the reproducibility of the method were higher than for the repeatability results but all below 9.0%. Method accuracy was evaluated by the recovery results, with averaged values between 80 and 104% for polyphenols and 97–105% for organic acids. The calibration curves, obtained by triplicate injection of standard solutions, showed good linearity with regression coefficients higher than 0.9982 for polyphenols and 0.9997 for organic acids. The LOD was in the range of 0.07–0.49 mg/L for polyphenols (cinnamic and gallic acids, respectively) and 0.001–0.046 g/L for organic acids (oxalic and lactic acids, respectively). The method was successfully used to measure and assess the polyphenolic fingerprint and organic acids profile of red, white, rose ´ and fortified wines.

Kinetic Analysis of Gill (Na+,K+)-ATPase Activity in Selected Ontogenetic Stages of the Amazon River Shrimp, (Decapoda, Palaemonidae): Interactions at ATP- and Cation-Binding Sites

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Electrochemical investigation of the dimeric oxo-bridged ruthenium complex in aqueous solution and its incorporation within a cation-exchange polymeric film on the electrode surface for electrocatalytic activity of hydrogen peroxide oxidation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Biochemical, histopathological and clinical evaluation of delayed effects caused by methamidophos isoforms and TOCP in hens: Ameliorative effects using control of calcium homeostasis

This work evaluated the potential of the isoforms of methamidophos to cause organophosphorus-induced delayed neuropathy (OPIDN) in hens. In addition to inhibition of neuropathy target esterase (NTE) and acetylcholinesterase (AChE), calpain activation, spinal cord lesions and clinical signs were assessed. The isoforms (+)-, (+/-)- and (-)-methamidophos were administered at 50 mg/kg orally; tri-ortho-cresyl phosphate (TOCP) was administered (500 mg/kg, po) as positive control for delayed neuropathy. The TOCP hens showed greater than 80% and approximately 20% inhibition of NTE and AChE in hen brain, respectively. Among the isoforms of methamidophos, only the (+)-methamidophos was capable of inhibiting NTE activity (approximately 60%) with statistically significant difference compared to the control group. Calpain activity in brain increased by 40% in TOCP hens compared to the control group when measured 24h after dosing and remained high (18% over control) 21 days after dosing. Hens that received (+)-methamidophos had calpain activity 12% greater than controls. The histopathological findings and clinical signs corroborated the biochemical results that indicated the potential of the (+)-methamidophos to be the isoform responsible for OPIDN induction. Protection against OPIDN was examined using a treatment of 2 doses of nimodipine (1 mg/kg, i.m.) and one dose of calcium gluconate (5 mg/kg, iv.). The treatment decreased the effect of OPIDN-inducing TOCP and (+)-methamidophos on calpain activity, spinal cord lesions and clinical signs. (C) 2012 Elsevier B.V. All rights reserved.

Effect of cyclosporin A on alveolar bone homeostasis in a rat periodontitis model

Objectives: the administration of cyclosporin A has been associated with significant bone loss and increased bone remodeling. The present investigation was designed to evaluate the effects of cyclosporin A on alveolar bone of rats subjected to experimental periodontitis, using serum, stereometric and radiographic analysis.Methods: Twenty-four rats were divided into one of the following groups with six animals each: group I, control rats; group II, in which the animals received a cotton ligature around the lower first molars; group III, in which the rats received a cotton ligature around the lower first molars and were treated with 10 mg/(kg body weight day) of cyclosporin A; group IV, in which the rats were treated with 10 mg/(kg body weight day) of cyclosporin A. At the end of experimental period, at 30 days, animals were killed and the serum calcium and alkaline phosphatase levels were measured in all groups. The distance from the alveolar bone crest to the cemento-enamel junction was measured radiographically for each mesial surface of the lower first molars of each rat. After histological processing, the stereological parameters: volume densities of multinucleated osteoclasts (V-o), alveolar bone (V-b), marrow (V-m), and relation of eroded surface/bone surface (Es/Bs) were assessed at the mesial region of the alveolar bone.Results: Significant decreases in serum calcium were observed in those groups that received cyclosporin A therapy. No significant changes in serum alkaline phosphatase were observed. The therapy with cyclosporin A combined with the ligature placement decreased the V-b and increased the V-o, V-m and Es/Bs at the mesial surface of lower first molars. on the other hand, the radiographic data showed that cyclosporin A therapy diminished the alveolar bone loss at the mesial surface of the lower first molars.Conclusions: Therefore, within the limits of this study, we suggest that cyclosporin A at immunosuppressive levels can bring about an imbalance in the alveolar bone homeostasis in rats. However, in the presence of inflammatory stimulation, the inhibition of the immune system by cyclosporin A may decrease the initial periodontal breakdown.

Functional relationship between subfrnical organ cholinergic stimulation and nitrergic activation influencing cardiovascular and body fluid homeostasis

We have studied the effects of L-NG-nitro arginine methyl esther (L-NAME), L-arginine (LAR), inhibitor and a donating nitric oxide agent on the alterations of salivary flow, water intake, arterial blood pressure (MAP) and heart rate (HR) induced by the injection pilocarpine into the subfornical organ (SFO). Rats (Holtzman 250-300 g) were anesthetized with 2, 2, 2-tribromoethanol (20 mg/100 kg b. wt.) and a stainless steel carmula were implanted into their SFO. The volume of injection was 0.2 mu l. The amount of saliva secretion was studied over a 5-min period. Pilocarpine (40 mu g), L-NAME (40 mu g) and LAR (30 mu g) were used in all experiments for the injection into the SFO. Pilocarpine (10, 20, 40, 80 and 160 mu g) injected into SFO elicited a concentration-dependent increase in salivary secretion. L-NAME injected prior to pilocarpine into the SFO increased salivary secretion and water intake due to the effect of pilocarpine. LAR injected prior to pilocarpine into the SFO attenuated the salivary secretion and water intake. Pilocarpine, injected into the SFO increased the MAP and decreased heart rate (HR). L-NAME injected prior to pilocarpine into the SFO potentiated the pressor effect of pilocarpine with a decrease in HR. LAR injected into the SFO prior to pilocarpine attenuated the increase in MAP with no changes in HR. The present study suggests that the SFO nitrergic cells interfere in the cholinergic pathways implicated in the control of salivary secretion, fluid and cardiovascular homeostasis. (c) 2007 Elsevier B.V All rights reserved.

SIRT1 Deacetylase Activity and the Maintenance of Protein Homeostasis in Response to Stress: An Overview

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Growth factors and glucose homeostasis in diabetic rats: effects of exercise training

To investigate the alterations of glucose homeostasis and variables of the insulin-like growth factor-I (IGF- 1) growth system in sedentary and trained diabetic (TD) rats, Wistar rats were divided into sedentary control (SC), trained control (TC), sedentary diabetic (SD), and TD groups. Diabetes was induced by Alloxan (35 mg kg(-1) b.w.). Training program consisted of swimming 5 days week(-1), 1 h day(-1), during 8 weeks. Rats were sacrificed and blood was collected for determinations of serum glucose, insulin, growth hormone (GH), IGF-1, and IGF binding protein-3(IGFBP-3). Muscle and liver were removed to evaluate glycogen content. Cerebellum was extracted to determinate IGF-1 content. Diabetes decreased serum GH, IGF-1, IGFBP-3, liver glycogen, and cerebellum IGF-1 peptide content in baseline condition. Physical training recovered liver glycogen and increased serum and cerebellum IGF-1 peptide in diabetic rats. Physical training induces important metabolic and hormonal alterations that are associated with an improvement in glucose homeostasis and serum and cerebellum IGF-1 concentrations. Copyright (C) 2009 John Wiley & Sons, Ltd.