Nedd4-2 ubiquitin ligase: a contributor to experimental neuropathic pain?

Background: Neuropathic pain is associated with altered expression of voltage-gated sodium channels (VGSCs) leading to peripheral nerve hyperexcitability. Interestingly, in cell expression systems, the ubiquitin ligase Nedd4-2 regulates the cell membrane density of the most abundant peripheral and pain-related VGSC, namely Nav1.7, and decreases its sodium current. Yet nothing is known about the involvement of Nedd4-2 in nociception and chronic pain. Therefore, the goal of this study is (i) to characterize Nedd4-2 and Nav1.7 expression in an experimental model of neuropathic pain (ii) to design by viral vector-mediated gene therapy an approach to depict the implication of Nedd4-2 in chronic pain. Methods: Western Blot and immunohistochemistry experiments detecting Nav1.7 and Nedd4-2 were performed in rodent DRGs 7 days after spared nerve injury (SNI). For the viral vector-mediated gene therapy, a recombinant Adeno-Associated Virus (rAAV2/6) was generated expressing the Nedd4-2 gene. Intrathecal injection of rAAV2/6 was followed 2 weeks after by the SNI surgery. Data are expressed in mean ± SEM, n = 4 in each condition. Results: Immunofluorescence on DRGs neurons reveals a decreased number of positive Nedd4-2 cells in the SNI model (27.0 ± 1.2%) versus sham group (43.4 ± 3.5%; p <0.005), as well as an increase in positive Nav1.7 cells in SNI (50.1 ± 2.9%) versus Sham (41.6 ± 1.8%; p <0.05). The change of Nedd4-2 expression was confirmed by western-blot analysis. In addition, we show that Nedd4-2 and Nav1.7 are largely expressed in overlapping cell populations, chiefly colocalizing with markers of small nociceptive neurons. Furthermore, we report that intrathecal injection of rAAV is able to counteract the reduction of Nedd4-2 expression in SNI animals. Conclusion: Our results indicate that Nedd4-2 is mainly expressed in nociceptors and downregulated after nerve injury. Moreover, our data suggest that the reduction of Nedd4-2, after nerve injury, may modulate Nav1.7 activity and contribute to hyperexcitability in neuropathic pain. A normal level of Nedd4-2 can be restored using a viral vector and we will further assess its functional effect on pain sensitivity.

Polymorphisms cyclooxygenase-2 -765G>C and interleukin-6 -174G>C are associated with serum inflammation markers in a high cardiovascular risk population and do not modify the response to a Mediterranean diet supplemented with virgin olive oil or nuts

Inflammation is involved in cardiovascular diseases. Some studies have found that the Mediterranean diet (MD) can reduce serum concentrations of inflammation markers. However, none of these studies have analyzed the influence of genetic variability in such a response. Our objective was to study the effect of the -765G.C polymorphism in the cyclooxygenase-2 (COX-2) gene and the -174G.C polymorphism in the interleukin-6 (IL-6) gene on serum concentrations of IL-6, C-reactive protein, intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule-1 as well as their influence on the response toa nutritional interventionwithMD.An intervention study ina high cardiovascular riskMediterranean population (314 men and 407 women) was undertaken. Participants were randomly assigned to consume a low-fat control diet or a MD supplementedwith virgin olive oil ornuts.Measureswereobtained at baseline and after a 3-mointervention period.At baseline, the COX-2 -765G.C polymorphismwas associated with lower serum IL-6 (5.85 6 4.82 in GG vs. 4.74 6 4.14 ng/L in C-allele carriers; P ¼ 0.002) and ICAM-1 (265.8 6 114.8 in GG vs. 243.0 6 107.1 mg/L in C-carriers; P ¼ 0.018) concentrations. These differences remained significant aftermultivariate adjustment. The IL-6 -174G.C polymorphism was associatedwith higher (CC vs. G-carriers) serumICAM-1concentrations in bothmenandwomenandwithhigherserumIL-6 concentrations inmen.Following the dietary intervention, no significant gene x diet interactions were found. In conclusion, although COX-2 -765G.C and IL-6 -174G.C polymorphismswere associatedwith inflammation, consuming aMD(either supplemented with virgin olive oil or nuts) reduced the concentration of inflammation markers regardless of these polymorphisms.

Effect of medroxy-progesterone acetate on follicular growth and endometrial cycloxygenase-2 (COX-2) expression during the bovine estrous cycle

The objective of this study was to evaluate the effect of medroxy-progesterone acetate (MAP) with or without estradiol benzoate (EB) on follicular growth during the estrous cycle in cattle. In the first experiment, Hereford cows were synchronized with a synthetic analogue of PGF2 alpha and were treated with two different doses of MAP (250 or 500 mg) with or without EB for 7 days starting on day 8 of the estrous cycle. Follicular growth was inhibited (P<0.05) in all cows except controls and those receiving 250mg MAP without EB. Seventy-five percent of the animals (15/20) showed estrus on days 21 and 22 of the cycle rather than at MAP withdrawal, demonstrating that these treatments did not induce estrus. To determine whether the EB treatment altered endometrial sensitivity to oxytocin and thus the luteolytic cascade, multiparous pre-synchronized cows received 5 mg of EB followed 6 hours later with 50 IU of oxytocin (OT; n=9). Eight hours after EB injection, endometrial fragments were collected from the cows on days 4, 13 and 17 of the estrous cycle and COX-2 gene expression was measured by PCR. EB increased COX-2 mRNA levels only on day 17 of the estrous cycle (P<0.05). In conclusion, MAP alone or associated with EB is able to suppress bovine follicular growth. However, EB in the presence of MAP is not efficient to induce luteolysis in cows when injected on day 8 of the estrous cycle.

Avaliação da expressão da proteína bcl-2 no carcinoma de mama: estudo em punção aspirativa por agulha fina; correlação com grau histológico em espécimes cirúrgicos correspondentes

INTRODUÇÃO: O gene bcl-2 codifica uma proteína envolvida no processo de controle da apoptose. Inicialmente descrito em linfomas e posteriormente em tecidos epiteliais, sua expressão é freqüentemente encontrada em carcinomas de mama, associada a fatores de prognóstico favorável. Como a punção aspirativa por agulha fina (PAAF) tem sido utilizada como um método confiável na investigação de carcinomas de mama, acessamos a expressão de bcl-2 em material assim obtido e correlacionamos sua positividade com o grau histológico, avaliado em material cirúrgico correspondente, das respectivas pacientes, seguindo a classificação de SBR (Scarff, Bloom e Richardson). OBJETIVOS: Avaliar a expressão de bcl-2 em PAAF e correlacionar com grau histológico. METODOLOGIA: A positividade do bcl-2 foi analisada, por imunocitoquímica, em 118 casos consecutivos de PAAF e correlacionada com grau histológico em material cirúrgico correspondente, segundo classificação de SBR. RESULTADOS: A positividade para bcl-2 foi encontrada em 77 de 118 casos de PAAF (65,25%) e foi inversamente proporcional ao grau histológico (84,37%, p = 0,0022). CONCLUSÃO: A expressão de bcl-2 em PAAF correlaciona-se com fator de bom prognóstico. O índice de positividade encontrado, assim como a correlação inversa com grau histológico, está de acordo com dados publicados previamente. O fácil e rápido manejo do material obtido por PAAF permite a aplicação de técnicas complementares, de maneira confiável, como demonstra este estudo. A positividade do bcl-2 correlacionada com baixo grau histológico, assim como com outros fatores de bom prognóstico, pode, no futuro, proporcionar informação preditiva e prognóstica para pacientes candidatas a tratamento quimioterápico neo-adjuvante.

Quantitative real-time RT-PCR and chromogenic in situ hybridization: precise methods to detect HER-2 status in breast carcinoma

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Origins of sequence diversity in the malaria vaccine candidate merozoite surface protein-2 (MSP-2) in Amazonian isolates of Plasmodium falciparum

The recent evolution of Plasmodium falciparum is at odds with the extensive polymorphism found in most genes coding for antigens. Here, we examined the patterns and putative mechanisms of sequence diversification in the merozoite surface protein-2 (MSP-2), a major malarial repetitive surface antigen. We compared the msp-2 gene sequences from closely related clones derived from sympatric parasite isolates from Brazilian Amazonia and used microsatellite typing to examine, in these same clones, the haplotype background of chromosome 2, where msp-2 is located. We found examples of msp-2 sequence rearrangements putatively created by nonreciprocal recombinational events, such as replication slippage and gene conversion, while maintaining the chromosome haplotype. We conclude that these nonreciprocal recombination events may represent a major source of antigenic diversity in MSP-2 in P falciparum populations with low rates of classical meiotic recombination. (c) 2006 Elsevier B.V. All rights reserved.

Detection of P. aeruginosa harboring bla(CTX-M-2), bla(GES-1) and bla(GES-5), bla(IMP-1) and bla(SPM-1) causing infections in Brazilian tertiary-care hospital

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

Association between IGF2 and CYP21 gene polymorphisms and characteristics of economic interest in Nellore cattle

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

Partial purification and characterization of lysine-ketoglutarate reductase in normal and opaque-2 maize endosperms

Lysine-ketoglutaratc reductase catalyzes the first step of lysine catabolism in maize (Zea mays L.) endosperm. The enzyme condenses L-lysine and α-ketoglutarate into saccharopine using NADPH as cofactor. It is endosperm-specific and has a temporal pattern of activity, increasing with the onset of kernel development, reaching a peak 20 to 25 days after pollination, and thereafter decreasing as the kernel approaches maturity. The enzyme was extracted from the developing maize endosperm and partially purified by ammonium-sulfate precipitation, anion-exchange chromatography on DEAE-cellulose, and affinity chromatography on Blue-Sepharose CL-6B. The preparation obtained from affinity chromatography was enriched 275-fold and had a specific activity of 411 nanomoles per minute per milligram protein. The native and denaturated enzyme is a 140 kilodalton protein as determined by polyacrylamide gel electrophoresis. The enzyme showed specificity for its substrates and was not inhibited by either aminoethyl-cysteine or glutamate. Steady-state product-inhibition studies revealed that saccharopine was a noncompetitive inhibitor with respect to α-ketoglutarate and a competitive inhibitor with respect to lysine. This is suggestive of a rapid equilibriumordered binding mechanism with a binding order of lysine, α-ketoglutarate, NADPH. The enzyme activity was investigated in two maize inbred lines with homozygous normal and opaque-2 endosperms. The pattern of lysine-ketoglutarate reductase activity is coordinated with the rate of zein accumulation during endosperm development. A coordinated regulation of enzyme activity and zein accumulation was observed in the opaque-2 endosperm as the activity and zein levels were two to three times lower than in the normal endosperm. Enzyme extracted from L1038 normal and opaque-2 20 days after pollination was partially purified by DEAE-cellulose chromatography. Both genotypes showed a similar elution pattern with a single activity peak eluted at approximately 0.2 molar KCL. The molecular weight and physical properties of the normal and opaque-2 enzymes were essentially the same. We suggest that the Opaque-2 gene, which is a transactivator of the 22 kilodalton zein genes, may be involved in the regulation of the lysine-ketoglutarate reductase gene in maize endosperm. In addition, the decreased reductase activity caused by the opaque-2 mutation may explain, at least in part, the elevated concentration of lysine found in the opaque-2 endosperm.

Circulation of Different Lineages of Dengue Virus 2, Genotype American/Asian in Brazil: Dynamics and Molecular and Phylogenetic Characterization

The American/Asian genotype of Dengue virus type 2 (DENV-2) was introduced into the Americas in the 80′s. Although there is no data showing when this genotype was first introduced into Brazil, it was first detected in Brazil in 1990. After which the virus spread throughout the country and major epidemics occurred in 1998, 2007/08 and 2010. In this study we sequenced 12 DENV-2 genomes obtained from serum samples of patients with dengue fever residing in São José do Rio Preto, São Paulo (SJRP/SP), Brazil, in 2008. The whole open reading frame or envelope sequences were used to perform phylogenetic, phylogeographic and evolutionary analyses. Isolates from SJRP/SP were grouped within one lineage (BR3) close to isolates from Rio de Janeiro, Brazil. Isolates from SJRP were probably introduced there at least in 2007, prior to its detection in the 2008 outbreak. DENV-2 circulation in Brazil is characterized by the introduction, displacement and circulation of three well-defined lineages in different times, most probably from the Caribbean. Thirty-seven unique amino acid substitutions were observed among the lineages, including seven amino acid differences in domains I to III of the envelope protein. Moreover, we dated here, for the first time, the introduction of American/Asian genotype into Brazil (lineage BR1) to 1988/89, followed by the introduction of lineages BR2 (1998-2000) and BR3 (2003-05). Our results show a delay between the introduction and detection of DENV-2 lineages in Brazil, reinforcing the importance and need for surveillance programs to detect and trace the evolution of these viruses. Additionally, Brazilian DENV-2 differed in genetic diversity, date of introduction and geographic origin and distribution in Brazil, and these are important factors for the evolution, dynamics and control of dengue. © 2013 Drumond et al.

Detecção do genoma do vírus de Epstein Barr (EBV) em tecidos de pacientes com doença de Hodgkin da região Norte do Brasil

O vírus de Epstein Barr (EBV) é o agente causador da mononucleose infecciosa e está associado com várias desordens proliferativas malignas tais como: linfoma de Burkitt, linfoma de Hodgkin e linfomas não Hodgkin. Um total de 118 casos de linfomas diagnosticados no Hospital Ofir Loyola no período de 1996 e 2005 foram analisados no Instituto Evandro Chagas, Ananindeua, Brasil; com o objetivo de detectar o genoma do EBV mediante a identificação dos genes EBER 1 e EBNA1 em casos de doença de Hodgkin. Os espécimes parafinizados foram analisados por hibridização in situ (gene EBER 1) e PCR em tempo real (EBNA 1). Do total, 61% (72/118) dos pacientes eram do sexo masculino e 39% (46/118) do sexo feminino com faixa etária variando entre 3- 98 anos. Sessenta e cinco (55%) foram diagnosticados como doença de Hodgkin e cinqüenta e três (45%) como linfomas não-Hodgkin. O EBV foi identificado nas células Reed Sternberg e variantes em 76,9% (50/65) dos casos de linfoma de Hodgkin com idade média de 28,3 anos (variação, 2-84 anos). Os subtipos histológicos de casos EBV-positivos foram o seguinte: esclerose nodular em 50% (25/50), celularidade mista em 28% (14/50), depleção linfocitária em 14% (7/50) e predominância linfocitária em 8% (4/50). O DNA do EBV foi detectado em 53% (26/49) com um coeficiente de regressão para a curva padrão de 0,99. Este estudo foi a primeira descrição do vírus de Epstein Barr em casos de doença de Hodgkin na região Norte do Brasil; reforçando a hipótese de que o EBV seja um co-fator no processo de transformação neoplásica em conjunto com a predisposição genética e imunidade do paciente, justificando a condução de estudos posteriores a nível molecular.

Avaliação da expressão gênica e de lesões no DNA de índividuos portadores de diabetes mellitus tipo 2, dislipidemia e periodontite crônica

Pós-graduação em Odontologia - FOAR

Caracterização funcional e estrutural das proteínas RUV-1 e RUV-2 do fungo Neurospora crassa

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Cocaine effects on mouse incentive-learning and human addiction are linked to alpha 2 subunit-containing GABA(A) receptors

Because GABA(A) receptors containing alpha 2 subunits are highly represented in areas of the brain, such as nucleus accumbens (NAcc), frontal cortex, and amygdala, regions intimately involved in signaling motivation and reward, we hypothesized that manipulations of this receptor subtype would influence processing of rewards. Voltage-clamp recordings from NAcc medium spiny neurons of mice with alpha 2 gene deletion showed reduced synaptic GABA(A) receptor-mediated responses. Behaviorally, the deletion abolished cocaine`s ability to potentiate behaviors conditioned to rewards (conditioned reinforcement), and to support behavioral sensitization. In mice with a point mutation in the benzodiazepine binding pocket of alpha 2-GABA(A) receptors (alpha 2H101R), GABAergic neurotransmission in medium spiny neurons was identical to that of WT (i.e., the mutation was silent), but importantly, receptor function was now facilitated by the atypical benzodiazepine Ro 15-4513 (ethyl 8-amido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5-a] [1,4] benzodiazepine-3-carboxylate). In alpha 2H101R, but not WT mice, Ro 15-4513 administered directly into the NAcc-stimulated locomotor activity, and when given systemically and repeatedly, induced behavioral sensitization. These data indicate that activation of alpha 2-GABA(A) receptors (most likely in NAcc) is both necessary and sufficient for behavioral sensitization. Consistent with a role of these receptors in addiction, we found specific markers and haplotypes of the GABRA2 gene to be associated with human cocaine addiction.

Effects of Matrix Metalloproteinase (MMP)-2 Polymorphisms on Responsiveness to Antihypertensive Therapy of Women with Hypertensive Disorders of Pregnancy

Imbalanced matrix metalloproteinase (MMP) expression, including MMP-2, has been demonstrated in pre-eclampsia. However, little is known about the effect of polymorphisms in MMP-2 gene on hypertensive disorders of pregnancy. We examined whether two functional MMP-2 polymorphisms (g.-1306C>T and g.-735C>T) are associated with pre-eclampsia and/or gestational hypertension and whether these polymorphisms affect therapeutic responses in women with these conditions. We studied 216 healthy pregnant women (HP), 185 patients with gestational hypertension (GH) and 216 patients with pre-eclampsia (PE). They were stratified as responsive or non-responsive to antihypertensive therapy according to clinical and laboratorial parameters of therapeutic responsiveness. Genomic DNA was extracted from whole blood and genotypes for g-1306C>T and g.-735C>T polymorphisms were determined by real-time PCR using Taqman allele discrimination assays. Haplotype frequencies were inferred using the PHASE 2.1 program. The distributions of MMP-2 genotypes and haplotypes were similar in HP, GH and PE patients (p > 0.05). In addition, we found no significant differences in MMP-2 genotype or haplotype frequencies when GH or PE patients were classified as responsive or non-responsive to antihypertensive therapy (p > 0.05). Our results suggest that MMP-2 polymorphisms do not affect the susceptibility to hypertensive disorders of pregnancy. In parallel, MMP-2 polymorphisms apparently do not affect the responsiveness to antihypertensive therapy of women with these hypertensive disorders of pregnancy.