Thiol and Sulfenic Acid Oxidation of AhpE, the One-Cysteine Peroxiredoxin from Mycobacterium tuberculosis: Kinetics, Acidity Constants, and Conformational Dynamics

Drug resistance and virulence of Mycobacterium tuberculosis are partially related to the pathogen`s antioxidant systems. Peroxide detoxification in this bacterium is achieved by the heme-containing catalase peroxidase and different two-cysteine peroxiredoxins. M. tuberculosis genome also codifies for a putative one-cysteine peroxiredoxin, alkyl hydroperoxide reductase E (MtAhpE). Its expression was previously demonstrated at a transcriptional level, and the crystallographic structure of the recombinant protein was resolved under reduced and oxidized states. Herein, we report that the conformation of MtAhpE changed depending on its single cysteine redox state, as reflected by different tryptophan fluorescence properties and changes in quaternary structure. Dynamics of fluorescence changes, complemented by competition kinetic assays, were used to perform protein functional studies. MtAhE reduced peroxynitrite 2 orders of magnitude faster than hydrogen peroxide (1.9 x 10(7) M(-1) s(-1) vs 8.2 x 10(4) M(-1) s(-1) at pH 7.4 and 25 degrees C, respectively). The latter also caused cysteine overoxidation to sulfinic acid, but at much slower rate constant (40 M(-1) s(-1)). The pK(a) of the thiol in the reduced enzyme was 5.2, more than one unit lower than that of the sulfenic acid in the oxidized enzyme. The pH profile of hydrogen peroxide-mediated thiol and sulfenic acid oxidations indicated thiolate and sulfenate as the reacting species. The formation of sulfenic acid as well as the catalytic peroxidase activity of MtAhpE was demonstrated using the artificial reducing substrate thionitrobenzoate. Taken together, our results indicate that MtAhpE is a relevant component in the antioxidant repertoire of M. tuberculosis probably involved in peroxide and specially peroxynitrite detoxification.

Diet and food selection by small mammals in an old-growth Atlantic forest of south-eastern Brazil

Here we describe the stomach contents of nine small mammal species (seven rodents and two didelphid marsupials) co-occurring in an old-growth Atlantic forest area. For four terrestrial rodents, we also compared the importance of arthropods in the diet and the selection of arthropod groups by comparing consumption with availability. Small mammals and arthropods were sampled in a 36-ha grid containing 25 sampling stations spaced every 150 m, and 47 stomach contents were analysed. While plant matter was the predominant item in the stomach contents of two rodents (Oligoryzomys nigripes and Rhipidomys mastacalis), four species presented arthropods as the main food item (the rodents Brucepattersonius soricinus and Oxymycterus dasytrichus, and the marsupials Monodelphis n. sp. and Marmosops incanus) and three consumed more plant matter than arthropods, but had significant amounts of both items (the rodents Delomys sublineatus, Euryoryzomys russatus and Thaptomys nigrita). Our results suggest that differences in diet, coupled with differences in habit and microhabitat preferences, are important factors allowing resource partition among species of the diverse group of co-occurring terrestrial small mammals in Atlantic forest areas. Moreover, arthropods were not preyed opportunistically by any of the four terrestrial rodents, since consumption was not proportional to availability. Rather, selection or rejection of arthropod groups seems to be determined by aspects other than availability, such as nutritional value, easiness of capture and handling or palatability.

SEASONAL MICROHABITAT SELECTION BY TERRESTRIAL RODENTS IN AN OLD-GROWTH ATLANTIC FOREST

We tested the hypothesis that microhabitat variables, abundance of terrestrial rodents, and microhabitat selection patterns of terrestrial rodents vary between the cool-dry and warm-wet season in the Atlantic forest of Brazil. We selected variables associated with ecological factors potentially important to terrestrial rodents (physical structure of litter and woody debris, and arthropod availability) and established 25 small, independent sampling units covering 36 ha of a homogenous, mature Atlantic forest patch. Litter humidity and height, amount of small woody debris, arthropod availability, and terrestrial rodent abundance increased, whereas the quantity of large woody debris decreased in the warm-wet season. Greater spatial segregation among terrestrial rodents also was observed in this season, especially between morphologically similar species. The distribution of 3 of the 4 most common terrestrial rodents was influenced by microhabitat variables in at least I of the seasons, and these species also differed in their pattern of microhabitat selection between seasons. In general, the amount of small woody debris and litter humidity were more important for the microscale distribution of terrestrial rodents in the cool-dry season, whereas in the mild warm-wet season species distributions were associated with food availability or were not clearly influenced by the measured variables. The patterns of microhabitat selection by 3 common terrestrial rodents, which were associated with features that characterize old-growth forest, may be responsible for their vulnerability to forest fragmentation.

Muscle Protein Alterations in LGMD21 Patients With Different Mutations in the Fukutin-related Protein Gene

Fukutin-related protein (FKRP) is a protein involved in the glycosylation of cell surface molecules. Pathogenic mutations in the FKRP gene cause both the more severe congenital muscular dystrophy Type 1C and the milder Limb-Girdle Type 21 form (LGMD21). Here we report muscle histological alterations and the analysis of 11 muscle proteins: dystrophin, four sarcoglycans, calpain 3, dysferlin, telethonin, collagen VI, alpha-DG, and alpha 2-laminin, in muscle biopsies from 13 unrelated LGMD21 patients with 10 different FKRP mutations. In all, a typical dystrophic pattern was observed. In eight patients, a high frequency of rimmed vacuoles was also found. A variable degree of alpha 2-laminin deficiency was detected in 12 patients through immunofluorescence analysis, and 10 patients presented a-DG deficiency on sarcolemmal membranes. Additionally, through Western blot analysis, deficiency of calpain 3 and dystrophin bands was found in four and two patients, respectively. All the remaining proteins showed a similar pattern to normal controls. These results suggest that, in our population of LGMD21 patients, different mutations in the FKRP gene are associated with several secondary muscle protein reductions, and the deficiencies of alpha 2-laminin and alpha-DG on sections are prevalent, independently of mutation type or clinical severity.

Structural and Biochemical Characterization of Peroxiredoxin Q beta from Xylella fastidiosa CATALYTIC MECHANISM AND HIGH REACTIVITY

The phytopathogenic bacterium Xylella fastidiosa is the etiological agent of various plant diseases. To survive under oxidative stress imposed by the host, microorganisms express antioxidant proteins, including cysteine-based peroxidases named peroxiredoxins. This work is a comprehensive analysis of the catalysis performed by PrxQ from X. fastidiosa (XfPrxQ) that belongs to a peroxiredoxin class still poorly characterized and previously considered as moderately reactive toward hydroperoxides. Contrary to these assumptions, our competitive kinetics studies have shown that the second-order rate constants of the peroxidase reactions of XfPrxQ with hydrogen peroxide and peroxynitrite are in the order of 107 and 106 M(-1) s(-1), respectively, which are as fast as the most efficient peroxidases. The XfPrxQ disulfides were only slightly reducible by dithiothreitol; therefore, the identification of a thioredoxin system as the probable biological reductant of XfPrxQ was a relevant finding. We also showed by site-specific mutagenesis and mass spectrometry that an intramolecular disulfide bond between Cys-47 and Cys-83 is generated during the catalytic cycle. Furthermore, we elucidated the crystal structure of XfPrxQ C47S in which Ser-47 and Cys-83 lie similar to 12.3 angstrom apart. Therefore, significant conformational changes are required for disulfide bond formation. In fact, circular dichroism data indicated that there was a significant redox-dependent unfolding of alpha-helices, which is probably triggered by the peroxidatic cysteine oxidation. Finally, we proposed a model that takes data from this work as well data as from the literature into account.

Identification of the nuclear factor kappa-beta (NF-kB) in cortical of mice Wistar using Technovit 7200 VCR (R)

Objective: this study aimed to develop a nondecalcified bone sample processing technique enabling immunohistochemical labeling of proteins by kappa-beta nuclear factor (NF-kB) utilizing the Technovit 7200 VCR (R) in adult male Wistar rats. Study Method: A 1.8 mm diameter defect was performed 0.5mm from the femur proximal joint by means of a round bur. Experimental groups were divided according to fixing solution prior to histologic processing: Group 1- ethanol 70%; Group 2-10% buffered formalin; and Group 3- Glycerol diluted in 70% ethanol at a 70/30 ratio + 10% buffered formalin. The post-surgical periods ranged from 01 to 24 hours. Control groups included a nonsurgical procedure group (NSPG) and surgical procedures where bone exposure was performed (SPBE) without drilling. Prostate carcinoma was the positive control (PC) and samples subjected to incomplete immunohistochemistry protocol were the negative control (NC). Following euthanization, all samples were kept at 4 degrees C for 7 days, and were dehydrated in a series of alcohols at -20 degrees C. The polymer embedding procedure was performed at ethanol/polymer ratios of 70%-30%, 50%-50%, 30%-70%, 100%, and 100% for 72 hours at -20 degrees C. Polymerization followed the manufacturer`s recommendation. The samples were grounded and polished to 10-15 mu m thickness, and were deacrylated. The sections were rehydrated and were submitted to the primary polyclonal antibody anti-NF-kB on a 1:75 dilution for 12 hours at room temperature. Results: Microscopy showed that the Group 2 presented positive reaction to NF-kB, diffuse reactions for NSPG and SPBE, and no reaction for the NC group. Conclusion: The results obtained support the feasibility of the developed immunohistochemistry technique.

Thyroid hormones alter the adenine nucleotide hydrolysis in adult rat blood serum

The effects of ATP, ADP, and adenosine in the processes of platelet aggregation, vasodilatation, and coronary flow have been known for many years. The sequential hydrolysis of ATP to adenosine by soluble nucleotidases constitutes the main system for rapid inactivation of circulating adenine nucleotides. Thyroid disorders affect a number of biological factors including adenosine levels in different fractions. Then, we intend to investigate if the soluble nucleotidases responsible for the ATP, ADP, and AMP hydrolysis are affected by variations in the thyroid hormone levels in blood serum from adult rats. Hyperthyroidism was induced by daily intraperitoneal injections of L-thyroxine (T4) (2.5 and 10.0 mu g/100 g body weight, respectively) for 7 or 14 days. Hypothyroidism was induced by thyroidectomy and methimazole (0.05%) added to their drinking water during 7 or 14 days. The treatments efficacy was confirmed by determination of hemodynamic parameters and cardiac hypertrophy evaluation. T4 treatment predominantly inhibited, and hypothyroidism (14 days after thyroidectomy) predominantly increased the ATP, ADP, and AMP hydrolysis in rat blood serum. These results suggest that both excess and deficiency of thyroid hormones can modulate the ATP diphosphohydrolase and 5`-nucleotidase activities in rat blood serum and consequently modulate the effects mediated by these enzymes and their products in vascular system. (C) 2010 International Union of Biochemistry and Molecular Biology, Inc.

Involvement of the AT(1) receptor in the venoconstriction induced by angiotensin II in both the inferior vena cava and femoral vein

Although angiotensin II-induced venoconstriction has been demonstrated in the rat vena cava and femoral vein, the angiotensin II receptor subtypes (AT(1) or AT(2)) that mediate this phenomenon have not been precisely characterized. Therefore, the present study aimed to characterize the pharmacological receptors involved in the angiotensin II-induced constriction of rat venae cavae and femoral veins, as well as the opposing effects exerted by locally produced prostanoids and NO upon induction of these vasomotor responses. The obtained results suggest that both AT(1) and AT(2) angiotensin II receptors are expressed in both veins. Angiotensin II concentration-response curves were shifted toward the right by losartan but not by PD 123319 in both the vena cava and femoral vein. Moreover, it was observed that both 10(-5) M indomethacin and 10(-4) M L-NAME improve the angiotensin II responses in the vena cava and femoral vein. In conclusion, in the rat vena cava and femoral vein, angiotensin II stimulates AT(1) but not AT(2) to induce venoconstriction, which is blunted by vasodilator prostanoids and NO. (C) 2010 Elsevier Inc. All rights reserved.

Thyroid hormone stimulates NO production via activation of the PI3K/Akt pathway in vascular myocytes

Aims Thyroid hormone (TH) rapidly relaxes vascular smooth muscle cells (VSMCs). However, the mechanisms involved in this effect remain unclear. We hypothesize that TH-induced rapid vascular relaxation is mediated by VSMC-derived nitric oxide (NO) production and is associated with the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signalling pathway. Methods and results NO levels were determined using a NO-specific fluorescent dye (DAF-2) and nitrite (NO(2)) levels. Expression of NO synthase (NOS) isoforms and proteins of the PI3K/Akt pathway was determined by both western blotting and immunocytochemistry. Myosin light chain (MLC) phosphorylation levels were also investigated by western blotting. Exposure of cultured VSMCs from rat thoracic aortas to triiodothyronine (T3) resulted in a significant decrease of MLC phosphorylation levels. T3 also induced a rapid increase in Akt phosphorylation and increased NO production in a dose-dependent manner (0.001-1 mu M). VSMCs stimulated with T3 for 30 min showed an increase in the expression of all three NOS isoforms and augmented NO production, effects that were prevented by inhibitors of PI3K. Vascular reactivity studies showed that vessels treated with T3 displayed a decreased response to phenylephrine, which was reversed by NOS inhibition. These data suggest that T3 treatment induces greater generation of NO both in aorta and VSMCs and that this phenomenon is endothelium independent. In addition, these findings show for the first time that the PI3K/Akt signalling pathway is involved in T3-induced NO production by VSMCs, which occurs with expressive participation of inducible and neuronal NOS. Conclusion Our data strongly indicate that T3 causes NO-dependent rapid relaxation of VSMC and that this effect is mediated by the PI3K/Akt signalling pathway.

Chemical identity and connections of medial preoptic area neurons expressing melanin-concentrating hormone during lactation

Lactation is an energy-demanding process characterized by massive food and water consumption, cessation of the reproductive cycle and induction of maternal behavior. During lactation, melanin-concentrating hormone (MCH) mRNA and peptide expression are increased in the medial preoptic area (MPO) and in the anterior paraventricular nucleus of the hypothalamus. Here we show that MCH neurons in the MPO coexpress the GABA synthesizing enzyme GAD-67 mRNA. We also show that MCH neurons in the MPO of female rats are innervated by neuropeptides that control energy homeostasis including agouti-related protein (AgRP), alpha-melanocyte stimulating hormone (alpha MSH) and cocaine- and amphetamine-regulated transcript (CART). Most of these inputs originate from the arcuate nucleus neurons. Additionally, using injections of retrograde tracers we found that CART neurons in the ventral premammillary nucleus also innervate the MPO. We then assessed the projections of the female MPO using injections of anterograde tracers. The MPO densely innervates hypothalamic nuclei related to reproductive control including the anteroventral periventricular nucleus, the ventrolateral subdivision of the ventromedial nucleus (VMHvl) and the ventral premammillary nucleus (PMV). We found that the density of MCH-ir fibers is increased in the VMHvl and PMV during lactation. Our findings suggest that the expression of MCH in the MPO may be induced by changing levels of neuropeptides involved in metabolic control. These MCH/GABA neurons may, in turn, participate in the suppression of cyclic reproductive function and/or sexual behavior during lactation through projections to reproductive control sites. (C) 2009 Elsevier B.V. All rights reserved.

A light and scanning electron microscopy study of bone healing following inferior alveolar nerve lateralization: An experimental study in rabbits

Purpose: The purpose of this study was to evaluate the bone healing kinetics around commercially pure titanium implants following inferior alveolar nerve (IAN) lateralization in a rabbit model. Materials and Methods: Inferior alveolar nerve lateralization was performed in 16 adult female rabbits (Oryctolagus cuniculus). During the nerve lateralization procedure, 1 implant was placed through the mandibular canal, and the IAN was replaced in direct contact with the implant. During the 8-week healing period, various bone labels were administered for fluorescent microscopy analysis. The animals were euthanized by anesthesia overdose, and the mandibular blocks were exposed by sharp dissection. Nondecalcified samples were prepared for optical light and scanning electron microscopy (SEM) evaluation. Results: SEM evaluation showed bone modeling/remodeling between the IAN and implant surface. Fluorochrome area fraction labeling at different times during the healing period showed that bone apposition mainly occurred during the first 2 weeks after implantation. Conclusions: The results obtained showed that bone healing/deposition occurred between the alveolar nerves in contact with a commercially pure titanium implant. No interaction between the nerve and the implant was detected after the 8-week healing period. Appositional bone healing occurred around the nerve bundle structure, restoring the mandibular canal integrity and morphology.

Effect of meloxicam and diclofenac sodium on peri-implant bone healing in rats

Background: This study evaluated the effects of diclofenac sodium and meloxicam on peri-implant bone healing. Methods: Thirty male rats were divided into three groups: the control group (CG) received no drug; the diclofenac sodium group (DSG) received 1.07 mg/kg twice a day for 5 days; and the meloxicam group (MG) received 0.2 mg/kg daily for 5 days. A screw-shaped titanium implant was placed in the tibia. Fluorochromes, oxytetracycline (OxT), calcein (CA), and alizarin (AL), were injected at 7, 14, and 21 days, respectively, after implantation, and the animals were sacrificed 28 days after implant placement. The percentages of OxT-, CA-, and AL-labeled bone as well as the percentages of bone-to-implant contact (BIC), cortical bone area (CBA), and trabecular bone area (TBA) within the implant threads were evaluated. Results: Bone healing was delayed in the DSG during the first 14 days after implant placement (OxT-labeled bone: DSG: 5.3% +/- 7.3% versus CG: 13.2% +/- 9.8%, P= 0.002, and versus MG: 14.4% +/- 13.1%, P = 0.05). The percentages of BIC (DSG: 49.6% +/- 21.9%; MG: 67.1% +/- 22.8%; and CG: 68.1% +/- 22.8%) and CBA (DSG: 63.7% +/- 21.2%; MG: 82.7% +/- 12.4%; CG: 84.9% +/- 10.6%) were lower in the DSG compared to the MG and CG (P<0.001). The percentage of TBA was significantly greater in the DSG compared to the MG and CG (DSG: 36.3% +/- 21.2% versus MG: 17.3% +/- 12.7% and versus CG: 15.1% +/- 10.6%; P<0.001). Conclusion: Diclofenac sodium seemed to delay peri-implant bone healing and to decrease BIC, whereas meloxicam had no negative effect on peri-implant bone healing.

Hyperglycaemia protects the heart after myocardial infarction: aspects of programmed cell survival and cell death

Exposure to a high glucose medium or diabetes has been found to protect the heart against ischaemia. The activation of antiapoptotic and proliferative factors seems to be involved in this cardioprotection. This study was designed to evaluate the role of hyperglycaemia in cardiac function, programmed cell survival, and cell death in diabetic rats after myocardial infarction (MI). Male Wistar rats were divided into four groups (n = 8): control (C), diabetic (D), myocardial infarcted (MI), and diabetic myocardial infarcted (DI). The following measures were assessed in the left ventricle: size of MI, systolic and diastolic function by echocardiography, cytokines by ELISA (TNF-alpha, IL-1 beta, IL-6, and IL-10), gene expression by real-time PCR (Bax, Fas, p53, Bcl-2, HIF1-alpha, VEGF, and IL8r), caspase-3 activity by spectrofluorometric assay, glucose transporter type 1 and 4 (GLUT-1 and GLUT-4) protein expression by western blotting, and capillary density and fibrosis by histological analysis. Systolic function was improved by hyperglycaemia in the DI group, and this was accompanied by no improvement in diastolic dysfunction, a reduction of 36% in MI size, reduced proinflammatory cytokines, apoptosis activation, and an increase in cell survival factors (HIF1-alpha, VEGFa and IL8r) assessed 15 days post-MI. Moreover, hyperglycaemia resulted in angiogenesis (increased capillary density) before and after MI, accompanied by a reduction in fibrosis. Together, these results suggest that greater plasticity and cellular resistance to ischaemic injury result from chronic diabetic hyperglycaemia in rat hearts.

The role of hemocytes in the immunity of the spider Acanthoscurria gomesiana

Invertebrates protect themselves against microbial infection through cellular and humoral immune defenses. Since the available information on the immune system of spiders is scarce, the main goat of the present study was to investigate the role of hemocytes and antimicrobial peptides (AMPs) in defense against microbes of spider Acanthoscurria gomesiana. We previously described the purification and characterization of two AMPs from the hemocytes of naive spider A. gomesiana, gomesin and acanthoscurrin. Here we show that 57% of the hemocytes store both gomesin and acanthoscurrin, either in the same or in different granules. Progomesin labeling in hemocyte granules indicates that gomesin is addressed to those organelles as a propeptide. In vivo and in vitro experiments showed that lipopolysaccharide (LPS) and yeast caused the hemocytes to migrate. Once they have reached the infection site, hemocytes may secrete coagulation cascade components and AMPs to cell-free hemolymph. Furthermore, our results suggest that phagocytosis is not the major defense mechanism activated after microbial challenge. Therefore, the main reactions involved in the spider immune defense might be coagulation and AMP secretion. (C) 2007 Elsevier Ltd. All rights reserved.

Metformin reduces the stimulatory effect of obesity on in vivo Walker-256 tumor development and increases the area of tumor necrosis

Aims: The objective of this study was to analyze the influence of obesity and insulin resistance on tumor development and, in turn, the effect of insulin sensitizing agents. Main methods: Male offspring of Wistar rats received monosodium glutamate (400 mg/kg) (obese) or saline (control) from the second to sixth day after birth. Sixteen-week-old control and obese rats received 5 x 10(5) Walker-256 tumor cells, subcutaneously injected into the right flank. Some of the obese and control rats received concomitant treatment with metformin (300 mg/kg) by gavage. At the 18th week, obesity was characterized. The percentage of rats that developed tumors, the tumor relative weight and the percentage of cachexia incidence were analyzed. The tumor tissue was evaluated histologically by means of hematoxylin and eosin staining. Key findings: Metformin did not correct the insulin resistance in obese rats. The tumor development was significantly higher in the obese group, whereas metformin treatment reduced it. After pathological analysis, we observed that the tumor tissues were similar in all groups except for adipocytes, which were found in greater quantity in the obese and metformin-treated obese groups. The area of tumor necrosis was higher in the group treated with metformin when compared with the untreated one. Significance: Metformin reduced Walker-256 tumor development but not cachexia in obese rats. The reduction occurred independently of the correction of insulin resistance. Metformin increased the area of necrosis in tumor tissues, which may have contributed to the reduced tumor development. (C) 2011 Elsevier Inc. All rights reserved.