Calcium Hydroxide Promotes Cementogenesis and Induces Cementoblastic Differentiation of Mesenchymal Periodontal Ligament Cells in a CEMP1-and ERK-Dependent Manner

Periodontal tissue engineering is a complex process requiring the regeneration of bone, cementum, and periodontal ligament (PDL). Since cementum regeneration is poorly understood, we used a dog model of dental pulpal necrosis and in vitro cellular wounding and mineralization assays to determine the mechanism of action of calcium hydroxide, Ca(OH)(2), in cementogenesis. Laser capture microdissection (LCM) followed by qRT-PCR were used to assay responses of periapical tissues to Ca(OH)(2) treatment. Additionally, viability, proliferation, migration, and mineralization responses of human mesenchymal PDL cells to Ca(OH)(2) were assayed. Finally, biochemical inhibitors and siRNA were used to investigate Ca(OH)(2)-mediated signaling in PDL cell differentiation. In vivo, Ca(OH)(2)-treated teeth formed a neocementum in a STRO-1- and cementum protein-1 (CEMP1)-positive cellular environment. LCM-harvested tissues adjacent to the neocementum exhibited higher mRNA levels for CEMP1, integrin-binding sialoprotein, and Runx2 than central PDL cells. In vitro, Ca(OH)(2) and CEMP1 promoted STRO-1-positive cell proliferation, migration, and wound closure. Ca(OH)(2) stimulated expression of the cementum-specific proteins CEMP1 and PTPLA/CAP in an ERK-dependent manner. Lastly, Ca(OH)(2) stimulated mineralization by CEMP1-positive cells. Blocking CEMP1 and ERK function abolished Ca(OH)(2)-induced mineralization, confirming a role for CEMP1 and ERK in the process. Ca(OH)(2) promotes cementogenesis and recruits STRO-1-positive mesenchymal PDL cells to undergo cementoblastic differentiation and mineralization via a CEMP1- and ERK-dependent pathway.

Distinct subsets of hypothalamic genes are modulated by two different thermogenesis-inducing stimuli

Obesity results from an imbalance between food intake and energy expenditure, two vital functions that are tightly controlled by specialized neurons of the hypothalamus. The complex mechanisms that integrate these two functions are only beginning to be deciphered. The objective of this study was to determine the effect of two thermogenesis-inducing conditions, i.e., ingestion of a high-fat (HF) diet and exposure to cold environment, on the expression of 1,176 genes in the hypothalamus of Wistar rats. Hypothalamic gene expression was evaluated using a cDNA macroarray approach. mRNA and protein expressions were determined by reverse-transcription PCR (RT-PCR) and immunoblot. Cold exposure led to an increased expression of 43 genes and to a reduced expression of four genes. HF diet promoted an increased expression of 90 genes and a reduced expression of 78 genes. Only two genes (N-methyl-D-aspartate (NMDA) receptor 2B and guanosine triphosphate (GTP)-binding protein G-alpha-i1) were similarly affected by both thermogenesis-inducing conditions, undergoing an increment of expression. RT-PCR and immunoblot evaluations confirmed the modulation of NMDA receptor 2B and GTP-binding protein G-alpha-i1, only. This corresponds to 0.93% of all the responsive genes and 0.17% of the analyzed genes. These results indicate that distinct environmental thermogenic stimuli can modulate predominantly distinct profiles of genes reinforcing the complexity and multiplicity of the hypothalamic mechanisms that regulate energy conservation and expenditure.

Efeito dos períodos de envelhecimento na lixiviação de íons e de proteínas solúveis em sementes de milho

Este trabalho foi conduzido no Laboratório de Análise de Sementes, do Departamento de Produção Vegetal, da Universidade Estadual Paulista (Botucatu/SP), com o objetivo de avaliar os efeitos causados por diferentes períodos de envelhecimento acelerado na lixiviação de íons e de proteínas solúveis em sementes de milho. Sementes de milho do híbrido BR 3123 foram colocadas sobre tela em gerbox, contendo 40mL de água destilada, e mantidas a temperatura de 42°C por períodos de 0, 24, 48, 72, 96, 120, 144 e 166 horas. Após o envelhecimento, parte das sementes foi colocada para germinar, empregando quatro repetições para cada período, obtendo-se a porcentagem de plântulas normais no quarto dia. em outra parte realizou-se, após manutenção em copos plásticos com 75mL de H2O destilada por 24 horas à 25°C, leituras de condutividade elétrica (µS.cm-1.g-1), determinação de íons (mg.L-1.g de semente-1) e de proteínas solúveis totais (µproteina.g-1.mL-1) lixiviadas na solução. Os resultados indicaram que a elevação dos valores de condutividade elétrica e de lixiviado de proteínas totais ocorreu a partir de 72 horas de envelhecimento acelerado. A lixiviação de potássio foi mais acentuada que a dos íons cálcio, zinco, manganês, cobre, ferro e magnésio.

Digestible lysine levels in diets for pigs from 24 to 50 kg under sanitary segregation

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Modeling energy utilization and growth parameter description for broiler chickens

Two experiments were conducted to develop and evaluate a model to estimate ME requirements and determine Gompertz growth parameters for broilers. The first experiment was conducted to determine maintenance energy requirements and the efficiencies of energy utilization for fat and protein deposition. Maintenance ME (ME m) requirements were estimated to be 157.8, 112.1, and 127.2 kcal of ME/kg 0.75 per day for broilers at 13, 23, and 32°C, respectively. Environmental temperature (T) had a quadratic effect on maintenance requirements (ME m = 307.87 - 15.63T + 0.3105T 2; r 2= 0.93). Energy requirements for fat and protein deposition were estimated to be 13.52 and 12.59 kcal of ME/g, respectively. Based on these coefficients, a model was developed to calculate daily ME requirements: ME = BW 0.75 (307.87 - 15.63T + 0.3105 T 2) + 13.52 G f + 12.59 G p. This model considers live BW, the effects of environmental temperature, and fractional fat (G f) and protein (G p) deposition. The second experiment was carried out to estimate the growth parameters of Ross broilers and to collect data to evaluate the ME requirement model proposed. Live BW, empty feather-free carcass, weight of the feathers, and carcass chemical compositions were analyzed until 16 wk of age. Parameters of Gompertz curves for each component were estimated. Males had higher growth potential and higher capacity to deposit nutrients than females, except for fat deposition. Data of BW and body composition collected in this experiment were fitted into the energy model proposed herein and the equations described by Emmans (1989) and Chwalibog (1991). The daily ME requirements estimated by the model determined in this study were closer to the ME intake observed in this trial compared with other models. ©2005 Poultry Science Association, Inc.

Proteínas, enzimas e minerais na secreção láctea de cabras e vacas, nos primeiros 30 dias pós-parto, congelada ou não

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

Estudo de potenciais inibidores de inibidores de infecção causada por Vírus Sincicial Respiratório em cultura de célula

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Caracterização molecular de cepas do vírus respiratório sincicial isoladas de casos de infecção respiratória aguda na cidade de Belém, Pará, Brasil nos anos de 2000 a 2006

As doenças do trato respiratório são as principais queixas nos serviços de atendimento médico, sendo as infecções respiratórias agudas (IRA) as manifestações mais comuns, principalmente em crianças menores de cinco anos de idade. Em países em desenvolvimento as IRA constituem um sério problema de saúde pública. Em todo mundo estima-se que ocorram cerca de dois milhões de mortes devido as IRA a cada ano. Dentre os agentes causais das mesmas, destaca-se o Vírus Respiratório Sincicial (VRS), especialmente por causar doença grave em crianças menores de dois anos. Com o objetivo de gerar dados sobre a epidemiologia molecular deste vírus, foram analisadas amostras colhidas de pacientes com IRA no período de 2000 a 2006 na cidade de Belém, Pará. Foram utilizados testes de imunofluorescência indireta (IFI) para caracterização antigênica dos vírus isolados e RT-PCR para os genes codificadores das proteínas G e F, que foram em seguida parcialmente seqüenciados. Dentro do período estudado, 153 amostras positivas para VRS foram detectadas. A faixa etária de 0-4 anos foi a que concentrou maior número de casos (n=138; 90,19%). Em relação ao perfil sazonal, o pico de atividade do VRS ocorreu nos primeiros seis meses do ano, estando associado principalmente ao período de troca da estação chuvosa para um período de menor pluviosidade. Houve co-circulação dos subgrupos A e B nos anos de 2001 e 2003. Em 2000, 2005 e 2006 somente o subgrupo A circulou. Entretanto no ano de 2004 foi registrada a ocorrência somente do subgrupo B. Dentro do período estudado, genótipos distintos da proteína G do subgrupo A (GA2 e GA5) e do subgrupo B (SAB1 e SAB3) foram detectados, indicando o primeiro relato da circulação do genótipo SAB1 na América do Sul. Em 2004, um cluster diferenciado dos demais genótipos circulantes foi encontrado, sendo este denominado BRB1. A análise do gene codificador da proteína F permitiu a identificação de mutações na sequência nucleotídica resultando em trocas na cadeia aminoacídica da mesma. Este estudo representa o primeiro relato sobre dados da epidemiologia molecular do Vírus Respiratório Sincicial na região Norte do Brasil.

Treinamento aeróbio de natação aumenta a atividade de enzimas antioxidantes e o conteúdo de glicogênio no musculoesquelético de ratos

INTRODUCTION: Studies which verified the effects of physical exercise on oxidative stress biomarkers and its relation to muscle glycogen are lack. OBJECTIVE: The present study verified the effects of aerobic swimming training (AST) on biomarkers of oxidative stress, glycogen content and cell growth in the skeletal muscle of rats. METHODS: Eighteen male Wistar rats (60 days) were divided into two groups: Sedentary Group (SG; n = 10): sedentary rats; and Trained Group (TG; n = 8): rats subjected to AST (5.0% of body weight), 1h/day, 5x/week, during 8 weeks. The activity of antioxidant enzymes (AOE) superoxide dismutase (SOD; U/ml), catalase (CAT; µmol/min/100mg), glutathione peroxidase (GPx; nmol/min/100mg), and substances that react with thiobarbituric acid levels (TBARs; nmolMDA/mg protein) were determined in the right gastrocnemius muscle. Glycogen (mg/100mg), protein (g/100g), and DNA (g/100g) contents were evaluated in the left gastrocnemius muscle. Data were analyzed by Student t-test (p < 0.05). RESULTS: AOE activity was higher in the TG group (CAT: 0.87 ± 0.04; SOD: 6.49 ± 0.45; GPX: 6.49 ± 0.52) when compared to SG group (CAT: 0.52 ± 0.03; SOD: 4.10 ± 0.37; GPx: 2.87 ± 0.35). TBARs levels was lower in TG (TG: 2.35 ± 0.45; SG: 8.90 ± 0.47). Gastrocnemius glycogen content (SG: 0.108 ± 0.013; TG: 0.320 ± 0.012) and protein/DNA ratio (SG: 24.94 ± 3.25; TG: 41.68 ± 4.02) were higher in TG group. CONCLUSION: Altogether, these data provide evidence that AST improved antioxidant defense, which may be associated to higher glycogen content of skeletal muscle of the animals.

Produção de ligno-hemi-celulases por fermentação em estado sólido e avaliação dos efeitos da aplicação das enzimas na composição química e estrutura do bagaço e da palha de cana

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

Desenvolvimento de dispositivos eletrônico-orgânicos e sua aplicação como sensores em meio aquoso

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

Multiscale sensing of antibody - antigen interactions by organic transistors and single-molecule force spectroscopy

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

THE GLYCOPROTEINS OF PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS AND THEIR ROLE IN INFECTION AND IMMUNITY

The porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important pathogen of swine and is known to cause abortion and infertility in pregnant sows and respiratory distress in piglets. PRRSV contains a major glycoprotein (GP5) and three minor glycoproteins (GP2a, GP3, and GP4) on the virion envelope, all of which are required for infectious virus production. To study their interactions amongst each other and with a cellular receptor for PRRSV, CD163, I cloned each of the viral glycoproteins and CD163 in various expression vectors. My studies have shown that while the GP2a, GP3, and GP4 are co-translationally glycosylated, the GP5 is post-translationally glycosylated. By using co-immunoprecipitation (co-IP) assays, strong interaction was demonstrated between GP4 and GP5 proteins, although weak interactions among the other envelope glycoproteins were also detected. Further, GP4 was found to mediate interactions leading to formation of multiprotein glycoprotein complex. My results also show that GP2a and GP4 proteins are the only two GPs that specifically interact with the CD163 molecule and that glycosylation of these GPs is required for efficient interaction. Based on these studies, I have developed an interactome map of the viral GPs and CD163 and have proposed a model of the viral glycoprotein complex and its interaction with CD163. Studies reported here also show that glycan addition at residue 184 (N184) of GP2a, and residues N42, N50, and N131 of GP3 is essential for recovery of infectious virus. Although single site glycosylation mutants of GP4 had no effect on infectious virus production, introduction of double mutations was lethal. The loss of glycan moieties of GP2a, GP3, and GP4 proteins had no effect on host neutralizing antibody production. Overall, I conclude that the PRRSV glycoproteins are co-translationally and post-translationally glycosylated, the GP4 protein is central to mediating interglycoprotein interactions, and along with GP2a, serves as the viral attachment protein that is responsible for interactions with the viral receptor, CD163. Further, glycosylation of GP2a, GP3, and GP4 proteins is required for infectious virus production, efficient interaction with CD163, but does not play any role in neutralizing antibody response in infected animals.

Digestible lysine levels in diets for pigs from 24 to 50 kg under sanitary segregation

This study evaluated the relationship between digestible lysine and metabolizable energy for barrow and gilts from 24 to 50 kg. Performance, digestibility and blood profile were studied. The experimental design was of randomized blocks, with five treatments, eight replicates and two animals per experimental unit in the performance assay and four replicates and one animal per experimental unit in the digestibility assay. The blood profile was chosen at 20 random animals of each sex, with four replicates per treatment and the animal as experimental unit. The treatments were 0.80, 0.90, 1.00, 1.10 and 1.20% digestible lysine. There was a linear effect of lysine levels on weight gain and feed conversion in females and crude protein in both sexes, gross energy excreted in the urine (kcal) and digestible energy (kcal). A quadratic effect of the amino acid studied in the daily consumption of crude protein (g) in both sexes, weight gain and feed conversion of barrows, as well as the nitrogen excreted in the urine (g) and nitrogen retained, and absorbed and retained. The relationship between neutrophils and lymphocytes had quadratic responses with increasing levels of lysine. The maximum performance for the studied genotype was obtained with 0.88 and 0.91% of digestible lysine or 2.60 and 2.67 g of digestible lysine/Mcal of metabolizable energy for females and barrows, respectively, corrected for digestibility trial.

Funktionelle Rolle von SNARE-Proteinen bei der Myelinisierung

Die Hauptfunktion der Oligodendrozyten im zentralen Nervensystem ist die Myelinisierung. Dabei umwickelt die Zelle mit Ausläufern ihrer Plasmamembran mehrmals die Axone. In den Phasen der aktiven Myelinisierung produziert ein Oligodendrozyt eine Fläche von 5000-50000 µm2 Myelin pro Tag, wobei große Mengen der Myelinkomponenten über vesikulären Transport zur Zelloberfläche transportiert werden müssen. Die Fusion der Vesikel mit ihrer Zielmembran wird duch SNARE-Proteine (soluble N-ethylmaleimide-sensitive-factor attachment protein receptor proteins) kontrolliert, die durch spezifische Interaktionen zwischen R- (Vesikel) und Q- (Zielmembran) SNAREs auch zur Spezifität der Fusion beitragen. Um die SNAREs den oligodendroglialen Transportrouten zuzuordnen, wurde deren Expression, Regulation und subzelluläre Lokalisation in primären Oligodendrozyten, in Oli-neu Zellen und im Myelin untersucht. Die Plasmamembran-Q-SNAREs Syntaxin 3, Syntaxin 4 und SNAP23, sowie das endosomale R-SNAREs VAMP3 zeigten eine zunehmende Expression im Verlauf der Oligodendrozytenreifung, die Expression von SNAP29 hingegen verminderte sich. Zudem akkumulierten die SNARE-Proteine Syntaxin 2, Syntaxin 3, Syntaxin 4 und VAMP7 im adulten Myelin, was für ihre Beteiligung an der Myelinisierung spricht. Co-Immunpräzipitationen ergaben u.a. Interaktionen zwischen den SNARE-Proteinen VAMP3 (R), Syntaxin 4 (Qa) und SNAP23 (Qbc). Anhand der beschriebenen Analyse konnten die SNARE-Proteine den oligodendroglialen Transportwegen zugeordnet werden. Immunzytochemische Analysen zeigten, dass das Hauptmyelinprotein PLP mit dem R-SNARE des Recycling Endosoms VAMP3, und mit dem spät endosomal, lysosomalen SNARE VAMP7 kolokalisiert. Um deren Rolle im PLP-Transport zu untersuchen, wurden verschiedene VAMP3- bzw. VAMP7-Silencing-Experimente durchgeführt. In beiden Fällen führte dies zu einer reduzierten Menge an PLP an der Zelloberfläche. Die Ergebnisse lassen somit auf zwei unabhängige Transportwege für PLP zur Plasmamembran von Oligodendrozyten schließen. Der VAMP7-abhängige PLP-Transport wurde zusätzlich in vivo in AP3-defizienten Mäusen, welche VAMP7 fehlsortieren, überprüft. Die Gehirne der mutanten Mäuse enthielten weniger Myelin, das isolierte Myelin enthielt weniger PLP und CNP. VAMP7 scheint also bei der Myelinisierung die Fusion von PLP- und CNP-enthaltenden Vesikeln mit der Myelinmembran zu vermitteln.