The role of major histocompatibility complex Alleles in the susceptibility of Brazilian patients to develop the myogenic type of Graves' orbitopathy

Objective: To assess the frequency of the genetic markers HLA-DRB1 and DQB1 in patients with Graves' orbitopathy ( GO) with and without extraocular muscle involvement. Design: The frequencies of class II HLA-DRB1 and DQB1 allele groups were determined for 81 Brazilian patients with GO and 161 normal subjects. The patients were divided into myogenic and nonmyogenic groups based on the clinical characteristics of the orbitopathy and quantitative computed tomography analysis of the extraocular muscle ( EOM) dimensions. Main outcome: Compared to the frequency obtained for samples of normal subjects of the Brazilian population, HLA-DRB1*16 (p(c)= 0.008) was overrepresented in myogenic and HLA-DRB1*03 (p(c)= 0.02) in nonmyogenic patients. Conclusions: The association between the HLA-DRB1* 16 and the myogenic subtype of GO suggests that EOM involvement in GO may be genetically predisposed.

Proteome analysis of schizophrenia patients Wernicke's area reveals an energy metabolism dysregulation

Background: Schizophrenia is likely to be a consequence of DNA alterations that, together with environmental factors, will lead to protein expression differences and the ultimate establishment of the illness. The superior temporal gyrus is implicated in schizophrenia and executes functions such as the processing of speech, language skills and sound processing. Methods: We performed an individual comparative proteome analysis using two-dimensional gel electrophoresis of 9 schizophrenia and 6 healthy control patients' left posterior superior temporal gyrus (Wernicke's area - BA22p) identifying by mass spectrometry several protein expression alterations that could be related to the disease. Results: Our analysis revealed 11 downregulated and 14 upregulated proteins, most of them related to energy metabolism. Whereas many of the identified proteins have been previously implicated in schizophrenia, such as fructose-bisphosphate aldolase C, creatine kinase and neuron-specific enolase, new putative disease markers were also identified such as dihydrolipoyl dehydrogenase, tropomyosin 3, breast cancer metastasis-suppressor 1, heterogeneous nuclear ribonucleoproteins C1/C2 and phosphate carrier protein, mitochondrial precursor. Besides, the differential expression of peroxiredoxin 6 (PRDX6) and glial fibrillary acidic protein (GFAP) were confirmed by western blot in schizophrenia prefrontal cortex. Conclusion: Our data supports a dysregulation of energy metabolism in schizophrenia as well as suggests new markers that may contribute to a better understanding of this complex disease.

Expression of cell adhesion proteins and proteins related to angiogenesis and fatty acid metabolism in benign, atypical, and anaplastic meningiomas

Most meningiomas are benign tumours of arachnoidal origin, although a small number have high proliferative rates and invasive properties which complicate complete surgical resection and are associated with increased recurrence rates. Few prognostic indicators exist for meningiomas and further research is necessary to identify factors that influence tumour invasion, oedema and recurrence. Paraffin sections from 25 intracranial meningiomas were analysed for expression of the proteins vascular endothelial growth factor (VEGF), VEGF receptors Flt1 and Flk1, E-cadherin, metalloproteinases 2 and 9 (MMP2, MMP9), CD44, receptor for hyaluronic acid-mediated motility (RHAMM), hyaluronic acid (HA), CD45, cyclooxygenase 2 (COX2), brain fatty acid binding protein (BFABP), Ki67, and proliferating cell nuclear antigen (PCNA). Correlations among protein expression were found for several markers of proliferation (Ki67, PCNA, MI) and microvessel density (MVD). COX2 expression increased with increasing with tumour grade and correlated with Ki67, PCNA, MI, MVD, and BFABP. BFABP expression also correlated with Ki67 and PCNA expression. Relationships were also identified among angiogenic factors (VEGF, Flt1, Flk1) and proliferation markers. Oedema was found to correlate with MMP9 expression and MMP9 also correlated with proliferation markers. No correlations were found for MMP2, E-cadherin, or CD44 in meningiomas. In conclusion Ki67, PCNA, MI, MVD, BFABP, and COX2 were significantly correlated with meningioma tumour grade and with each other. These findings, by correlating both intracellular fatty acid transport and eicosanoid metabolism with tumour proliferation, as determined by Ki67 labelling and mitotic index, suggest fatty acids are involved in the progression of meningiomas.

Na+-glucose transporter-2 messenger ribonucleic acid expression in kidney of diabetic rats correlates with glycemic levels: Involvement of hepatocyte nuclear factor-1 alpha expression and activity

Mutations in Na+-glucose transporters (SGLT)-2 and hepatocyte nuclear factor (HNF)-1 alpha genes have been related to renal glycosuria and maturity-onset diabetes of the young 3, respectively. However, the expression of these genes have not been investigated in type 1 and type 2 diabetes. Here in kidney of diabetic rats, we tested the hypotheses that SGLT2 mRNA expression is altered; HNF-1 alpha is involved in this regulation; and glycemic homeostasis is a related mechanism. The in vivo binding of HNF-1 alpha into the SGLT2 promoter region in renal cortex was confirmed by chromatin immunoprecipitation assay. SGLT2 and HNF-1 alpha mRNA expression (by Northern and RT-PCR analysis) and HNF-1 binding activity of nuclear proteins (by EMSA) were investigated in diabetic rats and treated or not with insulin or phlorizin (an inhibitor of SGLT2). Results showed that diabetes increases SGLT2 and HNF-1 alpha mRNA expression (similar to 50%) and binding of nuclear proteins to a HNF-1 consensus motif (similar to 100%). Six days of insulin or phlorizin treatment restores these parameters to nondiabetic-rat levels. Moreover, both treatments similarly reduced glycemia, despite the differences in plasma insulin and urinary glucose concentrations, highlighting the plasma glucose levels as involved in the observed modulations. This study shows that SGLT2 mRNA expression and HNF-1 alpha expression and activity correlate positively in kidney of diabetic rats. It also shows that diabetes-induced changes are reversed by lowering glycemia, independently of insulinemia. Our demonstration that HNF-1 alpha binds DNA that encodes SGLT2 supports the hypothesis that HNF-1 alpha, as a modulator of SGLT2 expression, may be involved in diabetic kidney disease.

Overexpression of Nrp/b (nuclear restrict protein in brain) suppresses the malignant phenotype in the C6/ST1 glioma cell line

Upon searching for glucocorticoid-regulated cDNA sequences associated with the transformed to normal phenotypic reversion of C6/ST1 rat glioma cells, we identified Nrp/b (nuclear restrict protein in brain) as a novel rat gene. Here we report on the identification and functional characterization of the complete sequence encoding the rat NRP/B protein. The cloned cDNA presented a 1767 nucleotides open-reading frame encoding a 589 aminoacids residues sequence containing a BTB/POZ (broad complex Tramtrack bric-a-brac/Pox virus and zinc finger) domain in its N-terminal region and kelch motifs in its C-terminal region. Sequence analysis indicates that the rat Nrp/b displays a high level of identity with the equivalent gene orthologs from other organisms. Among rat tissues, Nrp/b expression is more pronounced in brain tissue. We show that overexpression of the Nrp/b cDNA in C6/ST1 cells suppresses anchorage independence in vitro and tumorigenicity in vivo, altering their malignant nature towards a more benign phenotype. Therefore, Nrp/b may be postulated as a novel tumor suppressorgene, with possible relevance for glioblastoma therapy. (C) 2009 Elsevier Ltd. All rights reserved.

Bleaching of melanin in the epidermis of South American fur seal and its application on enzyme immunohistochemistry

The South American fur seal (Arctocephalus australis) is an amphibious marine mammal distributed along the Atlantic and Pacific coasts of South America. The species is well adjusted to different habitats due to the morphology of its fin-like members and due to some adaptations in their integumentary system. Immunohistochemical studies are very important to evaluate the mechanisms of skin adaptation due the differential expression of the antigens present in the tissue depending of the region of the body surface. However, its strongly pigmented (melanin) epidermis prevents the visualization of the immuno-histochemical chromogens markers. In this study a melanin bleaching method was developed aimed to allow the visualization of the chromogens without interfering in the antigen-antibody affinity for immunohistochemistry. The analysis of PCNA (proliferating cell nuclear antigen) index in the epidermis of A. australis by immunohistochemistry with diaminobenzidine (DAB) as chromogen was used to test the method. The bleaching of the melanin allowed to obtain the cell proliferation index in epidermis and to avoid false positive results without affecting the immunohistochemical results.

Nuclear and mitochondrial DNA markers in traceability of retail beef samples

Several characteristics are important in a traceability system of animal products, such as age at slaughter, breed composition, besides information of the productive chain. In general, the certification agent records information about the animals and the system which it came from, although cannot guarantee that the slaughtering, meat processing and distribution are error proof. Besides, there is a differential price, at least at the international market, based on sex and breed composition of the animals. Genetic markers allow identification of characteristics controlled in the beef cattle traceability program, as sex and breed composition, in order to correctly identify and appraise the final product for the consumer. The hypothesis of this study was that the majority beef samples retailed in the local market originate from female with a great participation of zebu breeds. Therefore, the objective of this work was to characterize retail beef samples with DNA markers that identify cattle sex and breed composition. Within 10 beef shops localized in Pirassununga, SP, Brazil, 61 samples were collected, all were genotyped as harboring Bos taurus mitochondrial DNA and 18 were positive for the Y chromosome amplification (male). For the marker sat1711b-Msp I the frequency of the allele A was 0.278 and for the marker Lhr-Hha I the frequency of the allele T was 0.417. The results of sat1711b-Msp I and Lhr-Hha I allelic frequencies are suggestive that the proportion of indicus genome compared with the taurine genome in the market meat is smaller than the observed in the Nellore breed. The procedure described in this study identified sex and subspecies characteristics of beef meat samples, with potential application in meat products certification in special as an auxiliary tool in beef cattle traceability programs.

Efeito do número da passagem e do gênero das células doadoras de núcleo no desenvolvimento de bovinos produzidos por transferência nuclear

Objetivou-se com este trabalho avaliar o efeito do número da passagem e do sexo das células doadoras de núcleo no desenvolvimento embrionário e fetal após transferência nuclear. Para isso, oócitos bovinos foram maturados, enucleados e reconstruídos com células somáticas de animal adulto. Após a fusão e ativação química, os zigotos reconstituídos foram cultivados em Charles Rosenkranz 2 (CR2) com monocamada de células da granulosa a 38,8ºC em atmosfera umidificada a 5% de CO2 em ar, durante sete dias, e transferidos para receptoras sincronizadas. As taxas de clivagem e desenvolvimento a blastocisto de embriões reconstruídos com células cultivadas por tempo maior foram inferiores às obtidas com os demais tempos de cultivo. Além disso, os blastocistos produzidos não resultaram no desenvolvimento de uma gestação a termo. Embora a taxa de clivagem em embriões fêmeas tenha sido maior, o número de embriões que atingiram o estádio de blastocisto foi maior nos embriões machos. No período gestacional, fêmeas apresentaram maior taxa de aborto entre 90 e 120 dias de gestação. Esses resultados indicam que células doadoras de núcleos cultivados por longos períodos dificultam a produção de blastocistos e aumentam as chances de perdas durante a gestação. Embriões clonados machos têm maior competência para se desenvolver a blastocisto e resultam em menor taxa de perda gestacional.

Importância da gordura alimentar na prevenção e no controle de distúrbios metabólicos e da doença cardiovascular

A Organização Mundial da Saúde (OMS) reiterou recentemente que o consumo de dietas inadequadas e a inatividade física estão entre os dez principais fatores de mortalidade. Diversos ensaios aleatorizados demonstram que intervenções alimentares adequadas podem diminuir ou prevenir significativamente o aparecimento de várias doenças crônicas não transmissíveis. Neste contexto, o papel da dieta vem sendo exaustivamente avaliado em estudos clínicos e epidemiológicos. Assim, já foi bem estabelecido na literatura que a quantidade e o tipo de gordura alimentar exercem influência direta sobre fatores de risco cardiovascular, tais como a concentração de lípides e de lipoproteínas plasmáticas, bem como sua associação a processos inflamatórios. Os ácidos graxos participam de complexos sistemas de sinalização intracelular, função que vem sendo bastante explorada. Os ácidos graxos poli-insaturados não somente influenciam a composição das membranas, metabolismo celular e sinais de tradução, mas também modulam a expressão de genes, regulando a atividade e a produção de diversos fatores de transcrição. A proposta deste artigo é rever tópicos relevantes referentes ao metabolismo de lípides e os relacionar a terapias nutricionais que possam contribuir para a prevenção e o tratamento de doenças associadas.

Aerobic and anaerobic metabolism for the zebrafish, Danio rerio, reared under normoxic and hypoxic conditions and exposed to acute hypoxia during development

In order to verify the influence of chronic and acute ambient oxygen levels from egg to adult stage of the zebrafish, in vivo oxygen consumption (MO2), critical tensions of oxygen (Pcrit), heart rate (fH) and total body lactate concentration (Lc) were determined for Danio rerio (Hamilton, 1822) raised at 28 °C under normoxic (7.5 mgO2.L-1 or 80 mm.Hg-1) and hypoxic conditions (4.3 mgO2.L-1) and exposed to acute hypoxia during different developmental stages. Our findings confirmed that very early stages do not respond effectively to ambient acute hypoxia. However, after the stage corresponding to the age of 30 days, D. rerio was able to respond to acute hypoxia through effective physiological mechanisms involving aerobic and anaerobic metabolism. Such responses were more efficient for the fishes reared under hypoxia which showed that D. rerio survival capability increased during acclimation to mild hypoxia. Measurements of body mass and length showed that moderate hypoxia did not affect growth significantly until the fish reached the stage of 60 days. Moreover, a growth delay was verified for the hypoxic-reared animals. Also, the D. rerio eggs-to-larvae survival varied from 87.7 to 62.4% in animals reared under normoxia and mild hypoxia, respectively. However, the surviving animals raised under moderated hypoxia showed a better aptitude to regulate aerobic and anaerobic capacities when exposed to acute hypoxia.

Influence of food components on lipid metabolism: scenarios and perspective on the control and prevention of dyslipidemias

Cardiovascular diseases (CVD) are the main causes of death in the Western world. Among the risk factors that are modifiable by diet, for reducing cardiovascular disease risks, the total plasma concentrations of cholesterol, triglycerides, LDL-C, and HDL-C are the most important. Dietary measures can balance these components of the lipid profile thus reducing the risk of cardiovascular diseases. The main food components that affect the lipid profile and can be modified by diet are the saturated and trans fats, unsaturated fats, cholesterol, phytosterols, plant protein, and soluble fiber. A wealth of evidence suggests that saturated and trans fats and cholesterol in the diet raise the total plasma cholesterol and LDL-C. Trans fats also reduce HDL-C, an important lipoprotein for mediating the reverse cholesterol transport. On the other hand, phytosterols, plant proteins, isoflavones, and soluble fiber are protective diet factors against cardiovascular diseases by modulating plasma lipoprotein levels. These food components at certain concentrations are able to reduce the total cholesterol, TG, and LDL-C and raise the plasma levels of HDL-C. Therefore, diet is an important tool for the prevention and control of cardiovascular diseases, and should be taken into account as a whole, i.e., not only the food components that modulate plasma concentrations of lipoproteins, but also the diet content of macro nutrients and micronutrients should be considered.

Memantine prevents cardiomyocytes nuclear size reduction in the left ventricle of rats exposed to cold stress

OBJECTIVES: Memantine is an N-methyl-d-aspartate (NMDA) glutamate receptor antagonist used to treat Alzheimer's disease. Previous studies have suggested that receptor blockers act as neuroprotective agents; however, no study has specifically investigated the impact that these drugs have on the heart. We sought to evaluate the effects of memantine on nuclear size reduction in cardiac cells exposed to cold stress. METHOD: We used male EPM-Wistar rats (n=40) divided into 4 groups: 1) Matched control (CON); 2) Memantine-treated rats (MEM); 3) Rats undergoing induced hypothermia (IH) and 4) Rats undergoing induced hypothermia that were also treated with memantine (IHM). Animals in the MEM and IHM groups were treated by oral gavage administration of 20 mg/kg/day memantine over an eight-day period. Animals in the IH and IHM groups were submitted to 4 hours of hypothermia in a controlled environment with a temperature of - 8ºC on the last day of the study. RESULTS: The MEM group had the largest cardiomyocyte nuclear size (151 ± 3.5 μm³ vs. CON: 142 ± 2.3 μm³; p<0.05), while the IH group had the smallest mean value of nuclear size. The nuclear size of the IHM group was preserved (125 ± 2.9 μm³) compared to the IH group (108 ± 1.7 μm³; p<0.05). CONCLUSION: Memantine prevented the nuclear size reduction of cardiomyocytes in rats exposed to cold stress.

PDIA3, HSPA5 and viementin, proteins identified by 2-DE in the valvular tissue, are the target antigens of peripheral and heart infiltrating T cells from chronic rheumatic heart disease patients

Rheumatic fever (RF) is a post-infectious autoimmune disease due to sequel of group A streptococcus (GAS) pharyngitis. Rheumatic heart disease (RHD), the major manifestation of RF, is characterized by inflammation of heart valves and myocardium. Molecular mimicry between GAS antigens and host proteins has been shown at B and T cell level. However the identification of the autoantigens recognized by B and T cells within the inflammatory microenvironment of heart tissue in patients with RHD is still incompletely elucidated. In the present study, we used two-dimensional gel electrophoresis (2-DE) and mass spectrometry to identify valvular tissue proteins target of T cells from chronic RHD patients. We could identify three proteins recognized by heart infiltrating and peripheral T cells as protein disulfide isomerase ER-60 precursor (PDIA3), 78 kD glucose-regulated protein precursor (HSPA5) and vimentin, with coverage of 45%, 43 and 34%, respectively. These proteins were recognized in a proliferation assay by peripheral and heart infiltrating T cells from RHD patients suggesting that they may be involved in the autoimmune reactions that leads to valve damage. We also observed that several other proteins isolated by 2-DE but not identified by mass spectrometry were also recognized by T cells. The identified cardiac proteins are likely relevant antigens involved in T cell-mediated autoimmune responses in RF/RHD that may contribute to the development of RHD

Effects of trans-10, cis-12 conjugated linoleic acid on gene expression and lipid metabolism of adipose tissue of growing pigs

The objective of the present study was to determine the effects of trans-10, cis-12 conjugated linoleic acid (CLA) in adipose tissue explant cultures of growing pigs on the following responses: lipogenesis (measured as rate of C-14-labeled glucose incorporation over a subsequent 2-h incubation in the presence or absence of insulin), lipolysis (release of non-esterified fatty acid over a 2-h incubation in the presence or absence of isoproterenol), activities of lipogenic enzymes, and mRNA abundance of fatty acid synthase (FAS). Adipose tissue explants from nine growing pigs (78 +/- 3 kg) were cultured in 199 medium with insulin, dexamethasone and antibiotics for 4, 12, 24, and 48 h. The treatments were 1) control: 100 mu M polyvinyl alcohol (PVA); 2) pGH: 100 ng/mL porcine growth hormone (pGH) plus 100 mu M PVA; 3) CLA200: 200 mu M trans-10, cis-12 CLA; 4) CLA50: 50 mu M trans-10, cis-12 CLA, and 5) LA: 200 mu M linoleic acid. Fatty acids were added along with PVA (2: 1), respectively, for 24 h. Explants were collected after each culture period and assayed for lipogenesis. Transcripts of FAS mRNA were quantified by real-time RT-PCR after 24 and 48 h. Lipolysis and activities of FAS, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and NADP-malate dehydrogenase were determined after 48 h. As expected, glucose incorporation was decreased (P < 0.05) in response to pGH treatment (positive control). LA had no effect on any parameter evaluated. Treatment with trans-10, cis-12 CLA decreased FAS activity (P < 0.05), but NADPH-generating enzymes were unaffected by treatments. Consistent with reduction in FAS activity, both lipid synthesis and FAS mRNA abundance were reduced with chronic CLA treatment, pGH increased baseline and stimulated lipolysis (P < 0.05) after 48 h of culture, while CLA treatment had no effect on non-esterified fatty acid release. Results of this study showed that trans-10, cis-12 CLA alters lipogenesis but has no effect on lipolysis in cultures of pig adipose tissue.

Cell-Free Antigens from Paracoccidioides brasiliensis Drive IL-4 Production and Increase the Severity of Paracoccidioidomycosis

The thermally dimorphic fungus Paracoccidioides brasiliensis (Pb) is the causative agent of paracoccidioidomycosis (PCM), one of the most frequent systemic mycosis that affects the rural population in Latin America. PCM is characterized by a chronic inflammatory granulomatous reaction, which is consequence of a Th1-mediated adaptive immune response. In the present study we investigated the mechanisms involved in the immunoregulation triggered after a prior contact with cell-free antigens (CFA) during a murine model of PCM. The results showed that the inoculation of CFA prior to the infection resulted in disorganized granulomatous lesions and increased fungal replication in the lungs, liver and spleen, that paralleled with the higher levels of IL-4 when compared with the control group. The role of IL-4 in facilitating the fungal growth was demonstrated in IL-4-deficient- and neutralizing anti-IL-4 mAb-treated mice. The injection of CFA did not affect the fungal growth in these mice, which, in fact, exhibited a significant diminished amount of fungus in the tissues and smaller granulomas. Considering that in vivo anti-IL-4-application started one week after the CFA-inoculum, it implicates that IL-4-CFA-induced is responsible by the mediation of the observed unresponsiveness. Further, the characterization of CFA indicated that a proteic fraction is required for triggering the immunosuppressive mechanisms, while glycosylation or glycosphingolipids moieties are not. Taken together, our data suggest that the prior contact with soluble Pb antigens leads to severe PCM in an IL-4 dependent manner.