Use of constant denaturant capillary electrophoresis of pooled blood samples to identify single-nucleotide polymorphisms in the genes (Scnn1a and Scnn1b) encoding the alpha and beta subunits of the epithelial sodium channel.

BACKGROUND: The epithelial sodium channel (ENaC) is composed of three homologous subunits: alpha, beta, and gamma. Mutations in the Scnn1b and Scnn1g genes, which encode the beta and the gamma subunits of ENaC, cause a severe form of hypertension (Liddle syndrome). The contribution of genetic variants within the Scnn1a gene, which codes for the alpha subunit, has not been investigated. METHODS: We screened for mutations in the COOH termini of the alpha and beta subunits of ENaC. Blood from 184 individuals from 31 families participating in a study on the genetics of hypertension were analyzed. Exons 13 of Scnn1a and Scnn1b, which encode the second transmembrane segment and the COOH termini of alpha- and beta-ENaC, respectively, were amplified from pooled DNA samples of members of each family by PCR. Constant denaturant capillary electrophoresis (CDCE) was used to detect mutations in PCR products of the pooled DNA samples. RESULTS: The detection limit of CDCE for ENaC variants was 1%, indicating that all members of any family or up to 100 individuals can be analyzed in one CDCE run. CDCE profiles of the COOH terminus of alpha-ENaC in pooled family members showed that the 31 families belonged to four groups and identified families with genetic variants. Using this approach, we analyzed 31 rather than 184 samples. Individual CDCE analysis of members from families with different pooled CDCE profiles revealed five genotypes containing 1853G-->T and 1987A-->G polymorphisms. The presence of the mutations was confirmed by DNA sequencing. For the COOH terminus of beta-ENaC, only one family showed a different CDCE profile. Two members of this family (n = 5) were heterozygous at 1781C-->T (T594M). CONCLUSION: CDCE rapidly detects point mutations in these candidate disease genes.

Complementary DNA cloning of complement C8 beta and its sequence homology to C9.

The complete amino acid sequence of mature C8 beta has been derived from the DNA sequence of a cDNA clone identified by expression screening of a human liver cDNA library. Comparison with the amino acid sequence of C9 shows an overall homology with few deletions and insertions. In particular, the cysteine-rich domains and membrane-inserting regions of C9 are well conserved. These findings are discussed in relation to a possible mechanism of membrane attack complex formation.

Paired activation of two components within muscarinic M3 receptor dimers is required for recruitment of beta-arrestin-1 to the plasma membrane.

beta-Arrestins regulate the functioning of G protein-coupled receptors in a variety of cellular processes including receptor-mediated endocytosis and activation of signaling molecules such as ERK. A key event in these processes is the G protein-coupled receptor-mediated recruitment of beta-arrestins to the plasma membrane. However, despite extensive knowledge in this field, it is still disputable whether activation of signaling pathways via beta-arrestin recruitment entails paired activation of receptor dimers. To address this question, we investigated the ability of different muscarinic receptor dimers to recruit beta-arrestin-1 using both co-immunoprecipitation and fluorescence microscopy in COS-7 cells. Experimentally, we first made use of a mutated muscarinic M(3) receptor, which is deleted in most of the third intracellular loop (M(3)-short). Although still capable of activating phospholipase C, this receptor loses almost completely the ability to recruit beta-arrestin-1 following carbachol stimulation in COS-7 cells. Subsequently, M(3)-short was co-expressed with the M(3) receptor. Under these conditions, the M(3)/M(3)-short heterodimer could not recruit beta-arrestin-1 to the plasma membrane, even though the control M(3)/M(3) homodimer could. We next tested the ability of chimeric adrenergic muscarinic alpha(2)/M(3) and M(3)/alpha(2) heterodimeric receptors to co-immunoprecipitate with beta-arrestin-1 following stimulation with adrenergic and muscarinic agonists. beta-Arrestin-1 co-immunoprecipitation could be induced only when carbachol or clonidine were given together and not when the two agonists were supplied separately. Finally, we tested the reciprocal influence that each receptor may exert on the M(2)/M(3) heterodimer to recruit beta-arrestin-1. Remarkably, we observed that M(2)/M(3) heterodimers recruit significantly greater amounts of beta-arrestin-1 than their respective M(3)/M(3) or M(2)/M(2) homodimers. Altogether, these findings provide strong evidence in favor of the view that binding of beta-arrestin-1 to muscarinic M(3) receptors requires paired stimulation of two receptor components within the same receptor dimer.

PVHL is a regulator of glucose metabolism and insulin secretion in pancreatic beta cells.

Insulin secretion from pancreatic beta cells is stimulated by glucose metabolism. However, the relative importance of metabolizing glucose via mitochondrial oxidative phosphorylation versus glycolysis for insulin secretion remains unclear. von Hippel-Lindau (VHL) tumor suppressor protein, pVHL, negatively regulates hypoxia-inducible factor HIF1alpha, a transcription factor implicated in promoting a glycolytic form of metabolism. Here we report a central role for the pVHL-HIF1alpha pathway in the control of beta-cell glucose utilization, insulin secretion, and glucose homeostasis. Conditional inactivation of Vhlh in beta cells promoted a diversion of glucose away from mitochondria into lactate production, causing cells to produce high levels of glycolytically derived ATP and to secrete elevated levels of insulin at low glucose concentrations. Vhlh-deficient mice exhibited diminished glucose-stimulated changes in cytoplasmic Ca(2+) concentration, electrical activity, and insulin secretion, which culminate in impaired systemic glucose tolerance. Importantly, combined deletion of Vhlh and Hif1alpha rescued these phenotypes, implying that they are the result of HIF1alpha activation. Together, these results identify pVHL and HIF1alpha as key regulators of insulin secretion from pancreatic beta cells. They further suggest that changes in the metabolic strategy of glucose metabolism in beta cells have profound effects on whole-body glucose homeostasis.

Radiação gama na conservação pós-colheita da nectarina (Prunus persica var. Nucipersica) frigoconservada

O trabalho foi desenvolvido no laboratório de Frutas e Hortaliças do Departamento de Gestão e Tecnologia Agroindustrial da Faculdade de Ciências Agronômicas - UNESP - Câmpus de Botucatu, tendo como objetivo principal a caracterização do comportamento da radiação gama, na conservação pós-colheita da nectarina cv. "Sunred". Os frutos foram colhidos no início do estádio de maturação, selecionados, limpos, pré-resfriados (4ºC por 12 horas) e submetidos a diferentes doses de radiação gama, constituindo assim os tratamentos: 1 0,0 kGy, 2 - 0,2 kGy, 3 - 0,4 kGy, 4 - 0,6 kGy, 5 - 0,8 kGy, sendo após armazenados em câmara fria com temperaturas de 0ºC e 90-95% de UR, por 28 dias. As análises foram realizadas a cada sete dias, determinando-se o aspecto visual dos frutos, a perda de massa fresca, a firmeza de polpa, a acidez total titulável (ATT), os sólidos solúveis totais (STT) e a razão STT/ATT. Após 28 dias de armazenamento, verificou-se que os frutos submetidos à dose de 0,4 kGy apresentaram o melhor aspecto visual, as menores perdas de massa fresca, e a maior firmeza de polpa, não ocorrendo, entretanto, variações significativas nos teores de acidez total titulável, sólidos solúveis totais e nos valores da razão SST/ATT.

Quinupristin-dalfopristin combined with beta-lactams for treatment of experimental endocarditis due to Staphylococcus aureus constitutively resistant to macrolide-lincosamide-streptogramin B antibiotics.

Quinupristin-dalfopristin (Q-D) is an injectable streptogramin active against most gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). In experimental endocarditis, however, Q-D was less efficacious against MRSA isolates constitutively resistant to macrolide-lincosamide-streptogram B (C-MLS(B)) than against MLS(B)-susceptible isolates. To circumvent this problem, we used the checkerboard method to screen drug combinations that would increase the efficacy of Q-D against such bacteria. beta-Lactams consistently exhibited additive or synergistic activity with Q-D. Glycopeptides, quinolones, and aminoglycosides were indifferent. No drugs were antagonistic. The positive Q-D-beta-lactam interaction was independent of MLS(B) or beta-lactam resistance. Moreover, addition of Q-D at one-fourth the MIC to flucloxacillin-containing plates decreased the flucloxacillin MIC for MRSA from 500 to 1,000 mg/liter to 30 to 60 mg/liter. Yet, Q-D-beta-lactam combinations were not synergistic in bactericidal tests. Rats with aortic vegetations were infected with two C-MLS(B)-resistant MRSA isolates (isolates AW7 and P8) and were treated for 3 or 5 days with drug dosages simulating the following treatments in humans: (i) Q-D at 7 mg/kg two times a day (b.i.d.) (a relatively low dosage purposely used to help detect positive drug interactions), (ii) cefamandole at constant levels in serum of 30 mg/liter, (iii) cefepime at 2 g b.i.d., (iv) Q-D combined with either cefamandole or cefepime. Any of the drugs used alone resulted in treatment failure. In contrast, Q-D plus either cefamandole or cefepime significantly decreased valve infection compared to the levels of infection for both untreated controls and those that received monotherapy (P < 0.05). Importantly, Q-D prevented the growth of highly beta-lactam-resistant MRSA in vivo. The mechanism of this beneficial drug interaction is unknown. However, Q-D-beta-lactam combinations might be useful for the treatment of complicated infections caused by multiple organisms, including MRSA.

Thermogenic effect of the new beta-adrenoreceptor agonist Ro 16-8714 in healthy male volunteers.

Previous investigations in experimental animals have shown that a new type of beta-adrenoceptor agonist (Ro 16-8714) possesses both thermogenic and antihyperglycemic properties. The aim of the study was to assess the thermogenic capacity of the compound in man after acute administration. Following an overnight fast three different doses (5, 10 and 20 mg) and a placebo were given per os to six normal-weight young men. The rate of energy expenditure (EE) and substrate utilization were determined by indirect calorimetry (hood system) before and for 6 h following the drug administration. Heart rate and blood pressure as well as plasma glucose, insulin and free fatty acid (FFA) concentrations were also measured at regular intervals throughout the study. The increment relative to base-line (mean +/- s.e.m.) in EE with placebo, 5, 10 and 20 mg was 4 +/- 3, 10 +/- 2, 11 +/- 2 and 21 +/- 2 percent respectively whereas heart rate was enhanced by 2 +/- 2, 8 +/- 3, 22 +/- 2, and 49 +/- 8 percent. Systolic blood pressure increased less (1 +/- 2, 8 +/- 1, 11 +/- 1 and 13 +/- 2 percent), and diastolic blood pressure did not change significantly. Simultaneously we observed a slight and transient increase in blood glucose, insulin and FFA concentrations. It is concluded that in lean individuals Ro 16-8714 is a potent thermogenic agent; however, new beta-adrenoceptor agonists should be developed in order to avoid the tachycardia associated with the thermogenic effect.

Qualidade do mamão cv. Solo submetido ao choque térmico e tratamento quarentenário por radiação gama

Para se exportar, é necessário não só atingir padrões de qualidade exigidos pelo país importador, mas também padrões fitossanitários. A irradiação tem como principal função o controle quarentenário de moscas-das-frutas, portanto é necessário que se associe a tratamentos que visem à manutenção da qualidade do fruto. O objetivo deste trabalho foi avaliar a qualidade dos mamões submetidos à imersão em água quente seguidos de irradiação em doses de tratamento quarentenário. Os mamões verdes selecionados foram submetidos aos seguintes tratamentos: a) controle; b) 250 Gy; c) 500 Gy; d) choque térmico (60º C por 30 segundos); e) 250 Gy + choque térmico, e f) 500 Gy + choque térmico. Os mamões foram armazenados a 21±1ºC e umidade relativa de 85-90%, e, após 8 dias, foram avaliados quanto ao teor de sólidos solúveis, pH, acidez titulável, firmeza, índice de doenças e coloração interna. As análises de perda de massa e coloração externa foram realizadas a 0; 2; 4 e 7 dias após a irradiação. Foi observado nos frutos submetidos a choque térmico (d, e, f) quantidade menor de sintomas de antracnose e podridão peduncular e, nos tratamentos combinados (e, f), foi verificada manutenção da firmeza do mamão. Somente no segundo dia os frutos irradiados (b, c, e, f) estavam mais amarelados que os demais. Para os demais parâmetros avaliados, não houve diferença entre os tratamentos.

Disponibilidade hídrica, radiação solar e fotossíntese em videiras 'Cabernet Sauvignon' sob cultivo protegido

O objetivo deste trabalho foi avaliar os efeitos da cobertura de videira 'Cabernet Sauvignon' com lona plástica translúcida sobre a disponibilidade de luz e água, a concentração foliar de clorofila e a fotossíntese. As plantas com cinco anos de idade foram conduzidas em sistema 'Y' sobre porta-enxerto Paulsen 1103. O experimento seguiu o delineamento em blocos ao acaso, com dois tratamentos (plantas sem e com cobertura plástica) e quatro repetições de 15 plantas (unidade experimental). A cobertura plástica reduziu o suprimento às plantas de radiações ultravioleta (UV), azul, verde, vermelho, vermelho distante e total (300-750 nm), bem como a disponibilidade hídrica nas camadas superficiais do solo (0-30 cm). As plantas cobertas apresentaram maior taxa fotossintética máxima e condutância estomática em relação às plantas descobertas. Os pontos de compensação e de saturação de luz, a eficiência quântica aparente, a respiração no escuro, a concentração foliar de clorofilas e o potencial hídrico foliar de base não foram influenciados pelo uso da cobertura plástica. A cobertura plástica reduziu a radiação e a disponibilidade hídrica nas camadas superficiais do solo, porém favoreceu a assimilação foliar de CO2.

Padrões de interceptação de radiação solar em vinhedos com e sem cobertura plástica

O trabalho teve por objetivo avaliar padrões de interceptação da radiação fotossinteticamente ativa (RFA) em vinhedos de 'Moscato Giallo', com e sem cobertura plástica sobre o dossel vegetativo. O experimento foi conduzido no município de Flores da Cunha, na "Serra Gaúcha" (29°06'S, 51°20'W, 541 m), na safra de 2005/2006. Consistiu de fileiras de plantas descobertas e cobertas com filme de polietileno de baixa densidade transparente com 160µm de espessura. Foram feitas medições contínuas de RFA em diferentes alturas do dossel, nos dois sistemas. O índice de área foliar (IAF) foi determinado por método não destrutivo, tendo-se ajustado modelos relacionando IAF com graus-dia acumulados. Também foram ajustados modelos relacionando a eficiência de interceptação de RFA com IAF, nos vinhedos cobertos e descobertos. A cobertura plástica promoveu aumento de 12% na eficiência de interceptação da radiação pelo vinhedo. O vinhedo coberto apresentou menor coeficiente de extinção de RFA que em céu aberto.

The role of fibroblast growth factor (FGF) and type beta transforming growth factot beta (TGF beta1, beta2 and beta3) during rat craniofacial development

Growth factors seem to be part of a complex cellular signalling language, in which individual growth factors are the equivalents of the letters that compose words. According to this analogy, informational content lies, not in an individual growth factor, but in the entire set of growth factors and others signals to which a cell is exposed. The ways in which growth factors exert their combinatorial effects are becoming clearer as the molecular mechanisms of growth factors actions are being investigated. A number of related extracellular signalling molecules that play widespread roles in regulating development in both invertebrates and vertebrates constitute the Fibroblast Growth Factor (FGF) and type beta Transforming Growth Factor ((TGF beta). The latest research literature about the role and fate of these Growth factors and their influence in the craniofacial bone growth ad development is reviewed

The role of fibroblast growth factor (FGF) and type beta transforming growth factot beta (TGF beta1, beta2 and beta3) during rat craniofacial development

Growth factors seem to be part of a complex cellular signalling language, in which individual growth factors are the equivalents of the letters that compose words. According to this analogy, informational content lies, not in an individual growth factor, but in the entire set of growth factors and others signals to which a cell is exposed. The ways in which growth factors exert their combinatorial effects are becoming clearer as the molecular mechanisms of growth factors actions are being investigated. A number of related extracellular signalling molecules that play widespread roles in regulating development in both invertebrates and vertebrates constitute the Fibroblast Growth Factor (FGF) and type beta Transforming Growth Factor ((TGF beta). The latest research literature about the role and fate of these Growth factors and their influence in the craniofacial bone growth ad development is reviewed

Poly(beta,L-malic acid)Doxorubicin ionic complex: A pH-dependent delivery system.

Poly(ß,L-malic acid) (PMLA) was made to interact with the cationic anticancer drug Doxorubicin (DOX) in aqueous solution to form ionic complexes with different compositions and an efficiency near to 100%. The PMLA/DOX complexes were characterized by spectroscopy, thermal analysis, and scanning electron microscopy. According to their composition, the PMLA/DOX complexes spontaneously self-assembled into spherical micro or nanoparticles with negative surface charge. Hydrolytic degradation of PMLA/DOX complexes took place by cleavage of the main chain ester bond and simultaneous release of the drug. In vitro drug release studies revealed that DOX delivery from the complexes was favored by acidic pH and high ionic strength

N=1 SQCD-like theories with N_f massive flavors from AdS/CFT and beta functions

We study new supergravity solutions related to large-N c N=1 supersymmetric gauge field theories with a large number N f of massive flavors. We use a recently proposed framework based on configurations with N c color D5 branes and a distribution of N f flavor D5 branes, governed by a function N f S(r). Although the system admits many solutions, under plausible physical assumptions the relevant solution is uniquely determined for each value of x ≡ N f /N c . In the IR region, the solution smoothly approaches the deformed Maldacena-Núñez solution. In the UV region it approaches a linear dilaton solution. For x < 2 the gauge coupling β g function computed holographically is negative definite, in the UV approaching the NSVZ β function with anomalous dimension γ 0 = −1/2 (approaching − 3/(32π 2)(2N c  − N f )g 3)), and with β g  → −∞ in the IR. For x = 2, β g has a UV fixed point at strong coupling, suggesting the existence of an IR fixed point at a lower value of the coupling. We argue that the solutions with x > 2 describe a"Seiberg dual" picture where N f  − 2N c flips sign.