Casitas b-lineage lymphoma-deficient mice are pretected against high-fat diet-induuced obesity and insulin resistance

Casitas b-lineage lymphoma (c-Cbl) is a multiadaptor protein with E3-ubiquitin ligase activity involved in regulating the degradation of receptor tyrosine kinases. We have recently reported that c-Cbl^–/– mice exhibit a lean phenotype and enhanced peripheral insulin action likely due to elevated energy expenditure. In the study reported here, we examined the effect of a high-fat diet on energy homeostasis and glucose metabolism in these animals. When c-Cbl^–/– mice were fed a high-fat diet for 4 weeks, they maintained hyperphagia, higher whole-body oxygen consumption (27%), and greater activity (threefold) compared with wild-type animals fed the same diet. In addition, the activity of several enzymes involved in mitochondrial fat oxidation and the phosphorylation of acetyl CoA carboxylase was significantly increased in muscle of high-fat–fed c-Cbl–deficient mice, indicating a greater capacity for fat oxidation in these animals. As a result of these differences, fat-fed c-Cbl^–/– mice were 30% leaner than wild-type animals and were protected against high-fat diet–induced insulin resistance. These studies are consistent with a role for c-Cbl in regulating nutrient partitioning in skeletal muscle and emphasize the potential of c-Cbl as a therapeutic target in the treatment of obesity and type 2 diabetes.

c-Cbl-deficient mice have reduced adiposity, higher energy expenditure, and improved peripheral insulin action

Casitas b-lineage lymphoma (c-Cbl) is an E3 ubiquitin ligase that has an important role in regulating the degradation of cell surface receptors. In the present study we have examined the role of c-Cbl in whole-body energy homeostasis. c-Cb^-/- mice exhibited a profound increase in whole-body energy expenditure as determined by increased core temperature and whole-body oxygen consumption. As a consequence, these mice displayed a decrease in adiposity, primarily due to a reduction in cell size despite an increase in food intake. These changes were accompanied by a significant
increase in activity (2- to 3-fold). In addition, cc-Cb-/- mice displayed a marked improvement in whole-body insulin action, primarily due to changes in muscle metabolism. We observed increased protein levels of the insulin receptor (4-fold) and uncoupling protein-3 (2-fold) in skeletal muscle and a significant increase in the phosphorylation of AMP-activated protein kinase and acetyl-CoA carboxylase. These fmdings suggest that c-Cbl plays an integral role in whole-body fuel homeostasis by regulating whole-body energy expenditure and insulin action.

Human skeletal muscle atrophy in amyotrophic lateral sclerosis reveals a reduction in Akt and an increase in atrogin-1

The molecular mechanisms influencing muscle atrophy in humans are poorly understood. Atrogin-1 and MuRF1, two ubiquitin E3-ligases, mediate rodent and cell muscle atrophy and are suggested to be regulated by an Akt/Forkhead (FKHR) signaling pathway. Here we investigated the expression of atrogin-1, MuRF1, and the activity of Akt and its catabolic (FKHR and FKHRL1) and anabolic (p70s6k and GSK-3β) targets in human skeletal muscle atrophy. The muscle atrophy model used was amyotrophic lateral sclerosis (ALS). All measurements were performed in biopsies from 22 ALS patients and 16 healthy controls as well as in G93A ALS mice. ALS patients had a significant increase in atrogin-1 mRNA and protein content, which was associated with a decrease in Akt activity. There was no difference in the mRNA and protein content of FKHR, FKHRL1, p70s6k, and GSK-3β. Similar observations were made in the G93A ALS mice. Human skeletal muscle atrophy, as seen in the ALS model, is associated with an increase in atrogin-1 and a decrease in Akt. The transcriptional regulation of human atrogin-1 may be controlled by an Akt-mediated transcription factor other than FKHR or via another signaling pathway.

Muscle atrophy and hypertrophy signaling in patients with chronic obstructive pulmonary disease

Rationale: The molecular mechanisms of muscle atrophy in chronic obstructive pulmonary disease (COPD) are poorly understood. In wasted animals, muscle mass is regulated by several AKT-related signaling pathways.
Objectives: To measure the protein expression of AKT, forkhead box class O (FoxO)-1 and -3, atrogin-1, the phosphophrylated form of AKT, p70^S6K glycogen synthase kinase-3ß (GSK-3ß), eukaryotic translation initiation factor 4E binding protein-1 (4E-BP1), and the mRNA expression of atrogin-1, muscle ring finger (MuRF) protein 1, and FoxO-1 and -3 in the quadriceps of 12 patients with COPD with muscle atrophy and 10 healthy control subjects. Five patients with COPD with preserved muscle mass were subsequently recruited and were compared with six patients with low muscle mass.
Methods: Protein contents and mRNA expression were measured by Western blot and quantitative polymerase chain reaction, respectively.
Measurements and Main Results: The levels of atrogin-1 and MuRF1 mRNA, and of phosphorylated AKT and 4E-BP1 and FoxO-1 proteins, were increased in patients with COPD with muscle atrophy compared with healthy control subjects, whereas atrogin-1, p70^S6K, GSK-3ß, and FoxO-3 protein levels were similar. Patients with COPD with muscle atrophy showed an increased expression of p70^S6K, GSK-3ß, and 4E-BP1 compared with patients with COPD with preserved muscle mass.
Conclusions: An increase in atrogin-1 and MuRF1 mRNA and FoxO-1 protein content was observed in the quadriceps of patients with COPD. The transcriptional regulation of atrogin-1 and MuRF1 may occur via FoxO-1, but independently of AKT. The overexpression of the muscle hypertrophic signaling pathways found in patients with COPD with muscle atrophy could represent an attempt to restore muscle mass.

Characterization of the Wheat Grain PKABA1-Interacting Protein TaWD40

Abscisic acid (ABA)-mediated gene expression is a critical component of plant responses to this important hormone, which affects plant growth, development, and responses to environmental stresses. Plant responses to ABA are mediated by a number of factors including PKABA1, an ABA induced protein kinase involved in ABA-suppressed gene expression in cereal grains, and TaWD40, which has previously been shown to physically interact with PKABA1. A full-length 1.9 kb TaWD40 cDNA, CK210682, was sequenced as part of this project. Based on the deduced protein sequence, it is thought that TaWD40 may belong to the family of E3 ubiquitin ligases, possibly targeting PKABA1 for destruction. Construction of expression plasmids for overproduction of the TaWD40 polypeptide in E. coli is currently underway. The TaWD40 cDNA has been successfully amplified from the source plasmid and inserted into an intermediate plasmid, pCR2.1. The TaWD40 cDNA is currently being cloned from the pCR2.1 intermediate plasmid into two different expression vectors, pRSET-A and pMAL-c2x, for future protein production and purification.

Antihypertensive treatments obscure familial contributions to blood pressure variation

The linkage and association between inherent blood pressure and underlying genotype is potentially confounded by antihypertensive treatment. We estimated blood pressure variance components (genetic, shared environmental, individual-specific) in 767 adult volunteer families by using a variety of approaches to adjusting blood pressure of the 244 subjects (8.2%) receiving antihypertensive medications. The additive genetic component of variance for systolic pressure was 73.9 mm Hg(2) (SE, 8.8) when measured pressures (adjusted for age by gender within each generation) were used but fell to 61.4 mm Hg(2) (SE, 8.0) when treated subjects were excluded. When the relevant 95th percentile values were substituted for treated systolic pressures, the additive genetic component was 81.9 mm Hg(2) (SE, 9.5), but individual adjustments in systolic pressure ranged from -53.5 mm Hg to +64.5 mm Hg (mean, +17.2 mm Hg). Instead, when 10 mm Hg was added to treated systolic pressure, the additive genetic component rose to 86.6 mm Hg(2) (SE, 10.1). Similar changes were seen in the shared environment component of variance for systolic pressure and for the combined genetic and shared environmental (ie, familial) components of diastolic pressure. There was little change in the individual-specific variance component across any of the methods. Therefore, treated subjects contribute important information to the familial components of blood pressure variance. This information is lost if treated subjects are excluded and obscured by treatment effects if unadjusted measured pressures are used. Adding back an appropriate increment of pressure restores familial components, more closely reflects the pretreatment values, and should increase the power of genomic linkage and linkage disequilibrium analyses.

The role and regulation of MAFbx/atrogin-1 and MuRF1 in skeletal muscle atrophy

Skeletal muscle atrophy occurs in many chronic diseases and disuse conditions. Its severity reduces patient recovery, independence and quality of life. The discovery of two muscle-specific E3 ubiquitin ligases, MAFbx/ atrogin-1 and Muscle RING Finger-1 (MuRF1), promoted an expectation of these molecules as targets for therapeutic development. While numerous studies have determined the conditions in which MAFbx/atrogin-1 and MuRF1 mRNA levels are regulated, few studies have investigated their functional role in skeletal muscle. Recently, studies identifying new target substrates for MAFbx/atrogin-1 and
MuRF1, outside of their response to the initiation of muscle atrophy, suggest that there is more to these proteins than
previously appreciated. This review will highlight our present knowledge of MAFbx/atrogin-1 and MuRF1 in skeletal muscle atrophy, the impact of potential therapeutics and their known regulators and substrates. Finally, we will comment on new approaches that may expand our knowledge of these two molecules in their control of skeletal muscle function.

c-Cbl facilitates endocytosis and lysosomal degradation of cystic fibrosis transmembrane conductance regulator in human airway epithelial cells

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl− channel expressed in the apical membrane of fluid-transporting epithelia. The apical membrane density of CFTR channels is determined, in part, by endocytosis and the postendocytic sorting of CFTR for lysosomal degradation or recycling to the plasma membrane. Although previous studies suggested that ubiquitination plays a role in the postendocytic sorting of CFTR, the specific ubiquitin ligases are unknown. c-Cbl is a multifunctional molecule with ubiquitin ligase activity and a protein adaptor function. c-Cbl co-immunoprecipitated with CFTR in primary differentiated human bronchial epithelial cells and in cultured human airway cells. Small interfering RNA-mediated silencing of c-Cbl increased CFTR expression in the plasma membrane by inhibiting CFTR endocytosis and increased CFTR-mediated Cl− currents. Silencing c-Cbl did not change the expression of the ubiquitinated fraction of plasma membrane CFTR. Moreover, the c-Cbl mutant with impaired ubiquitin ligase activity (FLAG-70Z-Cbl) did not affect the plasma membrane expression or the endocytosis of CFTR. In contrast, the c-Cbl mutant with the truncated C-terminal region (FLAG-Cbl-480), responsible for protein adaptor function, had a dominant interfering effect on the endocytosis and plasma membrane expression of CFTR. Moreover, CFTR and c-Cbl co-localized and co-immunoprecipitated in early endosomes, and silencing c-Cbl reduced the amount of ubiquitinated CFTR in early endosomes. In summary, our data demonstrate that in human airway epithelial cells, c-Cbl regulates CFTR by two mechanisms: first by acting as an adaptor protein and facilitating CFTR endocytosis by a ubiquitin-independent mechanism, and second by ubiquitinating CFTR in early endosomes and thereby facilitating the lysosomal degradation of CFTR.

The growth performance of Jade Tiger cultured abalone fed diets supplemented with fish oil and vegetable oils

Background
The effects of fish oil (FO) supplementation and the dietary replacement of FO with flaxseed oil (FlaxO) and canola oil (CO) on the growth of cultured abalone was investigated. The study involved three growth experiments: (E1) diets containing 0.5, 1.0, 1.5, 2.0 and 2.5% of FO, respectively; (E2) diets in which FO was serially replaced by 25, 50, 75 and 100% FlaxO, respectively; and (E3) diets in which FO was serially replaced by 25, 50, 75 and 100% CO, respectively.

Results
In Experiment 1, abalone fed a diet supplemented with 1.5% FO showed a significantly higher (121.2 ± 1.1 mg day−1) daily growth rate of weight (DGRw) compared to control (70.1 ± 1.71 mg day−1). In Experiment 2, abalone fed 1.5% FO diet and diets containing 25–75% FlaxO showed no significant differences in DGRw. The diet containing 100% FlaxO showed significantly lower (63.3 ± 6.7 mg day−1) DGRw. In Experiment 3, abalone fed diets containing 25% and 50% CO showed similar DGRw as those fed a 1.5% FO diet. The diet containing 75% and 100% CO showed significantly lower (63.7 ± 5.0 to 95.4 ± 5.1 mg day−1) DGRw.

Conclusion
Supplementation with 1.5% of dietary FO can improve growth performance in cultured abalone. It is feasible to replace 75% of dietary FO with FlaxO and 50% of dietary FO with CO, without negative effect on growth performance.

Comunidade de moluscos bentônicos na área de abrangência da Usina Hidrelétrica de Dona Francisca, Rio Jacuí, Rio Grande do Sul, Brasil : fase de pré e pós-enchimento do reservatório

A malacofauna límnica exerce importante papel como constituinte da comunidade bentônica e alterações em sua estrutura podem tornar-se prejudiciais para a vida nesses ecossistemas. Com o objetivo de avaliar quali-quantitativamente a malacofauna límnica na área de abrangência da Usina Hidrelétrica de Dona Francisca (UHEDF) (29°26’50’’S e 53°16’50’’W), Agudo, Rio Grande do Sul, Brasil, tomadas as amostras nas fases de pré-enchimento do reservatório em oito estações de coleta em áreas localizadas à montante (E3B, E4B, E5B), à jusante (E1) e na calha central do futuro lago da barragem (E2, E3, E4, E5) e pós-enchimento nas estações E1, E3B e E4B. No período de junho a outubro de 2000 (fase de pré-enchimento) e de junho a outubro de 2001 (fase de pós-enchimento) foram realizadas amostragens mensais, consistindo de três réplicas por estação de coleta (margens-centro), através do uso do amostrador de Surber modificado com área de 60 cm² e 15 cm de altura, delimitando o tamanho amostral. A fauna foi retirada manualmente dos clastos maiores sendo o sedimento de granulometria mais fina, passada através de peneira com malha de 1mm. Em laboratório foi realizada a identificação específica dos moluscos e da fauna acompanhante de macroinvertebrados , quando possível até família. Os organismos foram fixados em álcool 70%. Foram identificadas 10 famílias de Mollusca com quinze espécies. Para Gastropoda foram registradas seis famílias com nove espécies: Ampullariidae – Pomacea canaliculata (Lamarck, 1801); Hydrobiidae – Potamolithus aff. catharinae Pilsbry, 1911, Potamolithus ribeirensis Pilsbry, 1911, Potamolithus sp.1 e Heleobia sp.; Chilinidae – Chilina parva Martens, 1868; Lymnaeidae – Lymnaea columella Say, 1817; Ancylidae – Gundlachia concentrica (Orbigny, 1835); e Physidae – Stenophysa marmorata (Guilding, 1938). Para Bivalvia: Corbiculidae – Corbicula fluminea (Müller, 1774); Mycetopodidae – Anodontites iheringi (Clessin, 1882), Anodontites lucidus (Orbigny, 1835); Hyriidae – Diplodon charruanus (Orbigny, 1835); Sphaeriidae – Pisidium punctiferum (Guppy, 1817) e Pisidium sp. Na fase de pré-enchimento do reservatório, a correlação de Pearson apontou correlação positiva entre a granulometria e a densidade de moluscos (r=0,15) e correlação negativa entre os fatores físico-químicos e a densidade de moluscos bentônicos (r=-0,28). A análise de agrupamento das espécies de moluscos na fase de pré-enchimento evidenciou a formação de dois grupos distintos: o primeiro formado por espécies acidentais, segundo o cálculo da Constância, e o segundo formado por espécies constantes, xviii acessórias e uma acidental (Potamolithus sp.1) que no entanto foi abundante em E1 e E2. O resultado da freqüência relativa em relação à fase de pré-enchimento do reservatório mostrou a família Hydrobiidae, com quatro espécies, com maior abundância e riqueza de espécies, seguida por Chilinidae, representada por C. parva. Ambas as famílias representaram um total de 93,9% em relação à comunidade de moluscos amostrados. Tais resultados refletem o tipo de ambiente da área: leito formado por matacões e calhaus, favoráveis à instalação e manutenção daqueles moluscos. Na fase de pós-enchimento do reservatório foram registrados o aumento na riqueza e diversidade de espécies nas três estações de coleta amostradas (exceto em E3B – com menor diversidade no pós-enchimento) e diminuição significativa nas densidades mensais e totais de moluscos bentônicos. Os testes de aleatorização, entretanto, não revelaram diferenças significativas entre as duas fases e em relação ao funcionamento da UHEDF. Os fatores físico-químicos apontaram correlação positiva com a densidade de moluscos (r=0,19), ressaltando as diferenças significativas nos valores de pH, oxigênio dissolvido, oxigênio saturado nesta fase. Em E1 mais atingida pelo funcionamento da UHEDF, registrou-se uma acentuada diminuição nas densidades mensais e totais de moluscos bentônicos, não obstante, a riqueza e diversidade apresentaram maiores valores nesta fase. As espécies mantiveram-se as mesmas em ambas as fases.

Caracterização biológica e molecular da interação de Drechslera avenae (Eidam) Sharif com cultivares e linhagens de aveia (Avena sativa L.)

A helmintosporiose, incitada por Drechslera avenae (teleomorfo, Pyrenophora avenae), constitui fator limitante à produção e à qualidade do grão de aveia (Avena sativa L). A caracterização biológica e molecular da interação aveia - D. avenae pode contribuir para o manejo e a determinação de estratégias de controle da doença. Para a caracterização biológica, foram avaliadas trinta cultivares de aveia quanto à percentagem de área foliar com sintomas da doença e o tipo de lesão, inicialmente sob condições de inoculação artificial com três isolados do fungo e, após, no campo, sob condições naturais de infecção. Os resultados demonstraram haver interações significativas entre os genótipos da planta e isolados do patógeno. Classificaram-se como mais resistentes ao fungo OR 4, UFRGS 7, UFRGS 14, UFRGS 18 e UPF 19. Para a caracterização molecular da interação, cinco genótipos (CTC 5, ORLA 975, preta-comum, UFRGS 18 e UPF 17), não inoculados e inoculados com dois isolados do fungo, foram investigados para a análise de expressão diferencial de genes após 12, 24 e 72 horas após a inoculação. A partir do RNA total extraído das plantas submetidas aos tratamentos, realizaram-se a síntese de cDNA (RT-PCR), o isolamento de cDNAs expressados diferencialmente e a produção de sondas para a verificação de mRNAs induzidos, os quais foram hibridizados por meio de “Southern blot” e “Reverse Northern”. Foram observadas diferenças na expressão de genes, em nível de mRNA, entre plantas sadias e infectadas, tendo-se obtido sete fragmentos de cDNAs relacionados à interação com D. avenae, os quais foram comparados quanto a sua similaridade com seqüências depositadas no banco de dados do Genbank. Pela análise de “Reverse Northern”, pôde-se identificar um cDNA denominado E3-IFCO, relacionado com a resposta de defesa da planta.

Os estilos de liderança nas organizações não governamentais da região metropolitana de Belém

This study aimed to analyze the leadership style adopted by managers of nongovernmental organizations in the metropolitan region of Belem on the theory of Hersey and Blanchard. This theory is called situational leadership ranks E1, E2, E3, E4 and the styles of leadership and maturity in parallel classes M1, M2, M3 and M4. This study examined the relationship of leadership styles with the maturity of work, identified the relationship of leadership styles as related to psychological maturity and job maturity and psychological maturity. The main objectives were to analyze and relate leadership styles with the maturity of the leaders and understand the phenomenon of leadership from the self-perception of those who lead the organizations studied. To achieve the objectives we used a questionnaire already validated the theory of situational leadership and applied in 320 non-governmental organizations in the metropolitan region of Belem The methodology was quantitative, descriptive and exploratory. The analysis was by descriptive statistics and inferential statistics for univariate and bivariate form, applying the chi-square, the V Crammer and Spearman correlation. The data analysis shows safety, attested to the frequencies, and average margin of error and after application of the tests it was found that a relationship between the leadership style of work with the maturity and psychological maturity. The managers of nongovernmental organizations practicing various styles of leadership and focus on the quadrant of high maturity. It was diagnosed when the manager uses only one style of leadership was the predominance of E3 "share or support", which represents 24% of the sample. As uses two styles of leadership is the predominance of E3 and E2, which represents 76%. So the managers of nongovernmental organizations in the metropolitan region of Belem, practicing a style of leadership support, sharing ideas for decision making using a democratic style

CRYOPRESERVATION of RED BROCKET DEER SEMEN (MAZAMA AMERICANA): COMPARISON BETWEEN THREE EXTENDERS

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Comportamento de adesão da madeira de um híbrido clonal de Eucalyptus urophylla x Eucalyptus grandis proveniente de três condições de manejo

This study aimed to evaluate the adhesion ability of eucalyptus lumber from three tillage systems, using adhesives: resorcinol formaldehyde and two adhesives in water emulsion based on vinyl poly-acetate. The management systems were characterized by three strata, the stratum one (E1) characterized by wood from coppice and 70 months of age, the stratum two (E2) characterized by wood and retirement age of 166 months and stratum three (E3), also characterized by retirement at 70 months of age. The wood was derived from a random mixture of the first two sawn logs, each three feet from the base, which comprised three treatments on the adhesive used. We evaluated the shear strength by compression tests and the percentage of wood failure in the glue line. Based on the results obtained, it can be said that the adhesion had satisfactory performance with all the resins used, and the average values of shear strength of the glue line were shown to be equivalent to the shear strength of solid wood only for the samples which adhered with 'Wonderbond' adhesive and also provide higher values for wood failure (97.64%). The highest density present in the wood of the second stratum (E2) influenced only sticking with the resorcinol formaldehyde resin. For polyvinyl acetate (Cascorez 2590), shear values decreased in the third management condition (E3).