Kinin B1 Receptor in Adipocytes Regulates Glucose Tolerance and Predisposition to Obesity

Background: Kinins participate in the pathophysiology of obesity and type 2 diabetes by mechanisms which are not fully understood. Kinin B-1 receptor knockout mice (B-1(-/-)) are leaner and exhibit improved insulin sensitivity. Methodology/Principal Findings: Here we show that kinin B-1 receptors in adipocytes play a role in controlling whole body insulin action and glucose homeostasis. Adipocytes isolated from mouse white adipose tissue (WAT) constitutively express kinin B-1 receptors. In these cells, treatment with the B-1 receptor agonist des-Arg(9)-bradykinin improved insulin signaling, GLUT4 translocation, and glucose uptake. Adipocytes from B-1(-/-) mice showed reduced GLUT4 expression and impaired glucose uptake at both basal and insulin-stimulated states. To investigate the consequences of these phenomena to whole body metabolism, we generated mice where the expression of the kinin B-1 receptor was limited to cells of the adipose tissue (aP2-B-1/B-1(-/-)). Similarly to B-1(-/-) mice, aP2-B-1/B-1(-/-) mice were leaner than wild type controls. However, exclusive expression of the kinin B1 receptor in adipose tissue completely rescued the improved systemic insulin sensitivity phenotype of B-1(-/-) mice. Adipose tissue gene expression analysis also revealed that genes involved in insulin signaling were significantly affected by the presence of the kinin B-1 receptor in adipose tissue. In agreement, GLUT4 expression and glucose uptake were increased in fat tissue of aP2-B-1/B-1(-/-) when compared to B-1(-/-) mice. When subjected to high fat diet, aP2-B-1/B-1(-/-) mice gained more weight than B-1(-/-) littermates, becoming as obese as the wild types. Conclusions/Significance: Thus, kinin B-1 receptor participates in the modulation of insulin action in adipocytes, contributing to systemic insulin sensitivity and predisposition to obesity.

Distribution and stability of aflatoxin M-1 during processing and storage of Minas Frescal cheese

Aflatoxin M-1 (AFM(1)) is a hepatocarcinogen found in milk of animals that have consumed feeds with aflatoxin B-1. The carry-over of AFM(1) from milk to Minas Frescal cheese produced with or without starter cultures was determined. 40 L of milk were divided into 10 L each and assigned to the following treatments for cheese manufacture: 0.250 rig AFM(1) mL(-1), 0.500 rig AFM(1) mL(-1), 0.250 ng AFM(1) mL(-1) + starter, 0.500 ng AFM(1) mL(-1) + starter. Quantification of AFM(1) was achieved by high performance liquid chromatography. The carry-over of AFM(1) from milk to cheese ranged from 30.64% to 42.26%. There was no effect of storage time on AFM(1). Milk with AFM(1) in levels studied may concentrate the toxin in Minas Frescal cheese, but at concentrations below the Brazilian tolerance limit. The addition of starter cultures did not influence concentration or stability of the AFM(1) in cheese over 30 days storage. (C) 2011 Elsevier Ltd. All rights reserved.

The adipokine chemerin augments vascular reactivity to contractile stimuli via activation of the MEK-ERK1/2 pathway

Aims: Cytokines interfere with signaling pathways and mediators of vascular contraction. Endothelin-1 (ET-1) plays a major role on vascular dysfunction in conditions characterized by increased circulating levels of adipokines. In the present study we tested the hypothesis that the adipokine chemerin increases vascular contractile responses via activation of ET-1/ET-1 receptors-mediated pathways. Main methods: Male, 10-12 week-old Wistar rats were used. Endothelium-intact and endothelium-denuded aortic rings were incubated with chemerin (0.5 ng/mL or 5 ng/mL, for 1 or 24 h), and isometric contraction was recorded. Protein expression was determined by Western blotting. Key findings: Constrictor responses to phenylephrine (PE) and ET-1 were increased in vessels treated for 1 h with chemerin. Chemerin incubation for 24 h decreased PE contractile response whereas it increased the sensitivity to ET-1. Endothelium removal significantly potentiated chemerin effects on vascular contractile responses to PE and ET-1. Incubation with either an ERK1/2 inhibitor (PD98059) or ETA antagonist (BQ123) abolished chemerin effects on PE- and ET-1-induced vasoconstriction. Phosphorylation of MEK1/2 and ERK1/2 was significantly increased in vessels treated with chemerin for 1 and 24 h. Phosphorylation of these proteins was further increased in vessels incubated with ET-1 plus chemerin. ET-1 increased MEK1/2, ERK1/2 and MKP1 protein expression to values observed in vessels treated with chemerin. Significance: Chemerin increases contractile responses to PE and ET-1 via ERK1/2 activation. Our study contributes to a better understanding of the mechanisms by which the adipose tissue affects vascular function and, consequently, the vascular alterations present in obesity and related diseases. (c) 2012 Elsevier Inc. All rights reserved.

Voriconazole, Combined with Amphotericin B, in the Treatment for Pulmonary Cryptococcosis Caused by C. neoformans (Serotype A) in Mice with Severe Combined Immunodeficiency (SCID)

Cryptococcosis is a subacute or chronic systemic mycosis with a cosmopolitan nature, caused by yeast of the genus Cryptococcus neoformans. The model of systemic cryptococcosis in mice with severe combined immunodeficiency (SCID) is useful for immunological and therapeutic study of the disease in immunodeficient hosts. Amphotericin B, fluconazole and flucytosine are the drugs most commonly used to treat cryptococcosis. Voriconazole is a triazole with high bioavailability, large distribution volume, and excellent penetration of the central nervous system. The objective of this study was to evaluate treatment with amphotericin B (AMB), voriconazole (VRC), and AMB, used in combination with VRC, of experimental pulmonary cryptococcosis in a murine model (SCID). The animals were inoculated intravenously (iv) with a solution containing 3.0 x 10(5) viable cells of C. neoformans ATCC 90112, (serotype A). Treatments were performed with amphotericin B (1.5 mg/kg/day), voriconazole (40.0 mg/kg/day) and AMB (1.5 mg/kg/day) combined with VRC (40.0 mg/kg/day); began 1 day after the initial infection; were daily; and lasted 15 days. Evaluations were performed using analysis of the survival curve and isolation of yeast in the lung tissue. There was a significant increase in survival in groups treated with AMB combined with VRC, compared with the untreated group and groups receiving other treatments (P < 0.05). In the group treated only with VRC and AMB combined with VRC, there was a significant reduction (P < 0.05) in the isolation of C. neoformans in lung tissue. Amphotericin B combined with voriconazole may be an effective alternative to increasing survival and may reduce yeast in the lung tissue of mice with pulmonary cryptococcosis and SCID.

Endothelium dependent expression and underlying mechanisms of des-Arg(9)-bradykinin-induced B1R-mediated vasoconstriction in rat portal vein

Endothelial dysfunction has been implicated in portal vein obstruction, a condition responsible for major complications in chronic portal hypertension. Increased vascular tone due to disruption of endothelial function has been associated with an imbalance in the equilibrium between endothelium-derived relaxing and contracting factors. Herein, we assessed underlying mechanisms by which expression of bradykinin B-1 receptor (B1R) is induced in the endothelium and how its stimulation triggers vasoconstriction in the rat portal vein. Prolonged in vitro incubation of portal vein resulted in time- and endothelium-dependent expression of B1R and cyclooxygenase-2 (COX-2). Inhibition of protein kinase C (PKC) or phosphatidylinositol 3-kinase (PI3K) significantly reduced expression of B1R through the regulation of transcription factors, activator protein-1 (AP-1) and cAMP response element-binding protein (CREB). Moreover, pharmacological studies showed that B1R-mediated portal vein contraction was reduced by COX-2, but not COX-1, inhibitors. Notably, activation of endothelial B1R increased phospholipase A(2)/COX-2-derived thromboxane A(2) (TXA(2)) levels, which in turn mediated portal vein contraction through binding to TXA(2) receptors expressed in vascular smooth muscle cells. These results provide novel molecular mechanisms involved in the regulation of B1R expression and identify a critical role for the endothelial B1R in the modulation of portal vein vascular tone. Our study suggests a potential role for B1R antagonists as therapeutic tools for diseases where portal hypertension may be involved. (C) 2012 Elsevier Inc. All rights reserved.

Structure and Ionic Conductivity in the Mixed-Network Former Chalcogenide Glass System [Na2S](2/3)[(B2S3)(x)(P2S5)(1-x)](1/3)

Glasses in the system [Na2S](2/3)[(B2S3)(x)(P2S5)(1-x)](1/3) (0.0 <= x <= 1.0) were prepared by the melt quenching technique, and their properties were characterized by thermal analysis and impedance spectroscopy. Their atomic-level structures were comprehensively characterized by Raman spectroscopy and B-11, P-31, and Na-23 high resolution solid state magic-angle spinning (MAS) NMR techniques. P-31 MAS NMR peak assignments were made by the presence or absence of homonuclear indirect P-31-P-31 spin-spin interactions as detected using homonuclear J-resolved and refocused INADEQUATE techniques. The extent of B-S-P connectivity in the glassy network was quantified by P-31{B-11} and B-11{P-31} rotational echo double resonance spectroscopy. The results clearly illustrate that the network modifier alkali sulfide, Na2S, is not proportionally shared between the two network former components, B and P. Rather, the thiophosphate (P) component tends to attract a larger concentration of network modifier species than predicted by the bulk composition, and this results in the conversion of P2S74-, pyrothiophosphate, Na/P = 2:1, units into PS43-, orthothiophosphate, Na/P = 3:1, groups. Charge balance is maintained by increasing the net degree of polymerization of the thioborate (B) units through the formation of covalent bridging sulfur (BS) units, B S B. Detailed inspection of the B-11 MAS NMR spectra reveals that multiple thioborate units are formed, ranging from neutral BS3/2 groups all the way to the fully depolymerized orthothioborate (BS33-) species. On the basis of these results, a comprehensive and quantitative structural model is developed for these glasses, on the basis of which the compositional trends in the glass transition temperatures (T-g) and ionic conductivities can be rationalized. Up to x = 0.4, the dominant process can be described in a simplified way by the net reaction equation P-1 + B-1 reversible arrow P-0 + B-4, where the superscripts denote the number of BS atoms for the respective network former species. Above x = 0.4, all of the thiophosphate units are of the P-0 type and both pyro-(B-1) and orthothioborate (B-0) species make increasing contributions to the network structure with increasing x. In sharp contrast to the situation in sodium borophosphate glasses, four-coordinated thioborate species are generally less abundant and heteroatomic B-S-P linkages appear to not exist. On the basis of this structural information, compositional trends in the ionic conductivities are discussed in relation to the nature of the charge-compensating anionic species and the spatial distribution of the charge carriers.

Effect of neoadjuvant chemotherapy on low-density lipoprotein (LDL) receptor and LDL receptor-related protein 1 (LRP-1) receptor in locally advanced breast cancer

Low-density lipoprotein (LDL) receptors are overexpressed in most neoplastic cell lines and provide a mechanism for the internalization and concentration of drug-laden nanoemulsions that bind to these receptors. The aim of the present study was to determine whether the administration of standard chemotherapeutic schemes can alter the expression of LDL and LDL receptor-related protein 1 (LRP-1) receptors in breast carcinoma. Fragments of tumoral and normal breast tissue from 16 consecutive volunteer women with breast cancer in stage II or III were obtained from biopsies before the beginning of neoadjuvant chemotherapy and after chemotherapy, from fragments excised during mastectomy. Tissues were analyzed by immunohistochemistry for both receptors. Because complete response to treatment was achieved in 4 patients, only the tumors from 12 were analyzed. Before chemotherapy, there was overexpression of LDL receptor in the tumoral tissue compared to normal breast tissue in 8 of these patients. LRP-1 receptor overexpression was observed in tumors of 4 patients. After chemotherapy, expression of both receptors decreased in the tumors of 6 patients, increased in 4 and was unchanged in 2. Nonetheless, even when chemotherapy reduced receptors expression, the expression was still above normal. The fact that chemotherapy does not impair LDL receptors expression supports the use of drug carrier systems that target neoplastic cells by the LDL receptor endocytic pathway in patients on conventional chemotherapy.

Pressure Dependence of N-15 Chemical Shifts in Model Peptides Ac-Gly-Gly-X-Ala-NH2

High pressure NMR spectroscopy has developed into an important tool for studying conformational equilibria of proteins in solution. We have studied the amide proton and nitrogen chemical shifts of the 20 canonical amino acids X in the random-coil model peptide Ac-Gly-Gly-X-Ala-NH2, in a pressure range from 0.1 to 200 MPa, at a proton resonance frequency of 800 MHz. The obtained data allowed the determination of first and second order pressure coefficients with high accuracy at 283 K and pH 6.7. The mean first and second order pressure coefficients <B-1(15N)> and <B-2(15N)> for nitrogen are 2.91 ppm/GPa and -2.32 ppm/GPa(2), respectively. The corresponding values <B-1(1H)> and <B-2(1H)> for the amide protons are 0.52 ppm/GPa and -0.41 ppm/GPa(2). Residual dependent (1)J(1H15N)-coupling constants are shown.

Molecular characterization by amplified fragment length polymorphism and aflatoxin production of Aspergillus flavus isolated from freshly harvested peanut in Brazil

Brazil contributes substantially to the global peanut production, and the state of Sao Paulo is the largest producer in the country. Peanut crops can be contaminated by Aspergillus flavus strains producing aflatoxins, which are highly toxic and carcinogenic. Thus, the production of high-quality peanuts is crucial both for the commercial peanut industry and as a matter of public health. In this study, we used amplified fragment length polymorphism analysis (AFLP) to investigate the genetic variability among A. flavus strains isolated from fresh peanuts harvested in four different regions in the state of Sao Paulo, and to determine whether the molecular genetic profiles correlated with aflatoxin production or sclerotia formation. AFLP analysis generated 78 fragments ranging from 27 to 365 base pairs in length. Thirteen percent were not polymorphic. Genotyping identified twelve groups of A. flavus. On the basis of the polymorphisms identified, similarity between the isolates ranged from 37% to 100%. Of all isolates collected, 91.7% produced aflatoxins and 83.9% produced small sclerotia. Statistical analysis failed to suggest any relationship between the presence of sclerotia and mean levels of aflatoxins B-1 and B-2. Furthermore, a dendrogram based on AFLP data revealed substantial genetic variability among the A. flavus strains, but showed no correlation between dendrogram groups separated by molecular genetic features and production of aflatoxins B-1 or B-2 or the formation of sclerotia.

Interaction between toxigenic fungi and weevils in corn grain samples

This study investigated the ability of weevils to transmit Aspergillus flavus and Fusarium verticillioides fungal spores and the consequent production of mycotoxins. For this purpose, corn grain samples were stored in flasks connected to a hose to form a closed system (flasks A and B). Flasks A were inoculated with the following groups: group 1 (corn + weevil); group 2 (corn + A. flavus); group 3 (corn + A. flavus + weevil); group 4 (corn + F. verticillioides); group 5 (corn + E verticillioides + weevil), and group 6 (corn + A. flavus + E verticillioides + weevil). Flasks B contained sterile grains. The samples were incubated for 10, 20 and 30 days, posteriorly, weight, water activity, mycoflora, aflatoxins and fumonisins. The corn grain samples were also submitted to scanning electron microscopy. Our results showed that weevils could enhance corn grains contamination by these fungi, hence, could increase mycotoxins production. These findings demonstrate the importance of weevils as fungal vectors and the need for good manipulation and storage practices of grains. (C) 2012 Elsevier Ltd. All rights reserved.

On Disruption of Fear Memory by Reconsolidation Blockade: Evidence from Cannabidiol Treatment

The search for reconsolidation blockers may uncover clinically relevant drugs for disrupting memories of significant stressful life experiences, such as those underlying the posttraumatic stress disorder. Considering the safety of systemically administered cannabidiol (CBD), the major non-psychotomimetic component of Cannabis sativa, to animals and humans, the present study sought to investigate whether and how this phytocannabinoid (3-30 mg/kg intraperitoneally; i.p.) could mitigate an established memory, by blockade of its reconsolidation, evaluated in a contextual fear-conditioning paradigm in rats. We report that CBD is able to disrupt 1- and 7-days-old memories when administered immediately, but not 6 h, after their retrieval for 3 min, with the dose of 10 mg/kg being the most effective. This effect persists in either case for at least 1 week, but is prevented when memory reactivation was omitted, or when the cannabinoid type-1 receptors were antagonized selectively with AM251 (1.0 mg/kg). Pretreatment with the serotonin type-1A receptor antagonist WAY100635, however, failed to block CBD effects. These results highlight that recent and older fear memories are equally vulnerable to disruption induced by CBD through reconsolidation blockade, with a consequent long-lasting relief in contextual fear-induced freezing. Importantly, this CBD effect is dependent on memory reactivation, restricted to time window of <6h, and is possibly dependent on cannabinoid type-1 receptor-mediated signaling mechanisms. We also observed that the fear memories disrupted by CBD treatment do not show reinstatement or spontaneous recovery over 22 days. These findings support the view that reconsolidation blockade, rather than facilitated extinction, accounts for the aforementioned CBD results in our experimental conditions. Neuropsychopharmacology (2012) 37, 2132-2142; doi:10.1038/npp.2012.63; published online 2 May 2012

Characterization of aflatoxigenic and non-aflatoxigenic strains of Aspergillus section Flavi isolated from corn grains of different geographic origins in Brazil

Aflatoxins can cause great economic losses and serious risks to humans and animals health. The largest aflatoxin producers belong to Aspergillus section Flavi and can occur naturally in food commodities. Studies showed that molecular tools as well as the type of sclerotia produced by the strains could be helpful for identification of Aspergillus species and could be correlated with levels of toxin production. The purpose of this work was to characterize the genetic diversity using AFLP technique, the type of sclerotia and the ability of aflatoxin production by isolated strains from corn of different origins in Brazil, and to verify whether qPCR based on aflR and aflP genes is appropriate for estimating the level of aflatoxin production. All the 75 strains were classified as A. flavus and the AFLP technique showed a wide intraspecific variability within them. Regarding sclerotia production, 34% were classified as S and 66% as L type. Among the aflatoxin-producers, 52.8% produced aflatoxin B-1, while 47.2% aflatoxins B-1 and B-2. Statistical analysis showed no correlation between sclerotia production and aflatoxigenicty, and no correlation between the phylogenetic clusters and aflatoxin production. Concerning the relative expression of aflR and aflP, Pearson's correlation test demonstrated low positive correlation between the expression of the aflR and aflP genes and the production of AFB(1) and AFB(2), but showed high positive correlation between aflR and aflP expression. In contrast to the other reference strains, A. oryzae ATCC 7282 showed no amplification of aflR and aflP. The results highlight the need for detection of reliable and reproducible markers with a high positive correlation with aflatoxin production.

Análise de mutações nos genes TAC3 e TACR3 em pacientes com distúrbios puberais centrais idiopáticos

OBJETIVO: Investigar a presença de variantes nos genes TAC3 e TACR3, os quais codificam a NKB e seu receptor (NK3R), respectivamente, em uma coorte de pacientes com distúrbios puberais centrais idiopáticos. SUJEITOS E MÉTODOS: Duzentos e trinta e sete pacientes foram estudados: 114 com puberdade precoce central (PPC), 73 com hipogonadismo hipogonadotrófico isolado normósmico (HHI) e 50 com retardo constitucional do crescimento e desenvolvimento (RCCD). O grupo controle consistiu de 150 indivíduos brasileiros que apresentaram desenvolvimento puberal normal. O DNA genômico foi extraído de sangue periférico, e as regiões codificadoras dos genes TAC3 e TACR3 foram amplificadas e sequenciadas automaticamente. RESULTADOS: Uma variante (p.A63P) foi identificada na NKB, e quatro variantes, p.G18D, p.L58L (c.172C>T), p.W275X e p.A449S, foram identificadas no NK3R, as quais foram ausentes no grupo controle. A variante p.A63P foi identificada em uma menina com PPC, e a variante p.A449S, em uma menina com RCCD. As variantes previamente descritas, p.G18D, p.L58L e p.W275X, foram identificadas em três indivíduos com HHI normósmico do sexo masculino não relacionados. CONCLUSÃO: Variantes raras nos genes TAC3 e TACR3 foram identificadas em pacientes com distúrbios puberais centrais idiopáticos. Mutações de perda de função no gene TACR3 foram associadas com o fenótipo de HHI normósmico. Arq Bras Endocrinol Metab. 2012;56(9):646-52

Disseminated histoplasmosis in a juvenile lupus erythematosus patient

Introduction: Histoplasmosis is an infection caused by dimorphic fungus, Histoplasma capsulatum, and it has not been reported in juvenile systemic lupus erythematosus (JSLE) patients, particularly progressive disseminated histoplasmosis (PDH) subtype. Case report: We reported herein a 14-year old girl who was diagnosed with JSLE. Six months later, she had abdominal distension and received prednisone, hydroxychloroquine and azathioprine. Computer tomography evidenced hepatosplenomegaly and multiple mesenteric, mediastinal and retroperitoneal enlarged lymph nodes, forming large conglomerates at the mesentery, suggestive of lymphoproliferative disorder. After 10 days, she had acute surgical abdominal, and underwent a laparotomy and intestinal perforation and conglomerates of lymph nodes were observed. The jejunum biopsy showed perforated acute enteritis with hemorrhage and necrosis, and Grocott staining identified Histoplasma sp. and the culture showed a heavy growth of Histoplasma capsulatum. At that moment liposomal amphotericin B (1.0 mg/Kg/day) was introduced. Despite this treatment she died due to septic shock eight days later. Diffuse Histoplasma capsulatum was evidenced at autopsy. Conclusion: We reported a severe opportunistic infection in JSLE patient with adenopathy and multiple intestinal perforations. This study reinforces the importance of early diagnosis and antifungal therapy, especially in patients with these uncommon clinical manifestations.

Biomonitoring of Microcystin and Aflatoxin Co-Occurrence in Aquaculture Using Immunohistochemistry and Genotoxicity Assays

This work investigated the effects of co-occurring aflatoxin B-1 (AFB(1)) and microcystin (MC) in aquaculture, using immunohistochemistry and genotoxicity methods. Tilapia (Oreochromis niloticus) were exposed to AFB(1) by intraperitoneal and MC (cell extract of Microcystis aeruginosa) by intraperitoneal and immersion routes. The interaction of MC-AFB(1) was evaluated co-exposing the intraperitoneal doses. Blood samples were collected after 8, 24, and 48h to analyze the micronucleus frequency and comet score. The interaction of MC-AFB(1) showed a synergic mutagenic response by higher micronucleus frequency of co-exposed group. A slight genotoxic synergism was also observed in the comet score. Immunohistochemistry detected MC in al lthe fish liver tissues exposed to MC by intraperitoneal route, and only the immersed group with the highest dose of MC showed a positive response. Although MC was non-detectable in the edible muscle, the combination of immunohistochemistry with genotoxicity assay was an attractive biomonitoring tool in aquaculture, where the animals were frequently exposed to co-occurring synergic hazards.