Avaliação do crescimento de micro-organismos fotossintetizantes em fotobiorreator tubular empregando nutrientes provenientes da vinhaça tratada / Evaluation of the growth of photosynthetic microorganisms in tubular photobioreactor using nutrients from treated vinasse

vinhaça é resultante da produção de álcool, após a fermentação do mosto e destilação do vinho. É um resíduo rico em nutrientes, principalmente matéria orgânica, nitrogênio, fósforo e potássio. Para ser despejado em rios e lagos, esse efluente deve passar por tratamentos para remoção desses nutrientes, pois o excesso desses elementos nos corpos hídricos poderia levar a grandes problemas ambientais, de modo que tem sido utilizado em fertirrigação. Microorganismos fotossintetizantes absorvem nutrientes inorgânicos, podendo absorver nutrientes de águas residuais. Se for removida a parte orgânica da vinhaça, a fração inorgânica ou com baixa carga orgânica pode ser a base ou o próprio meio de cultivo destes micro-organismos, que absorvem gás carbônico e sua biomassa é de interesse industrial. Neste contexto, foi estudado o cultivo de Monoraphidium contortum e Synechocystis salina, oriundos de água de mangue, em frascos de Erlenmeyer e em fotobiorreatores tubulares por processo descontínuo, empregando a vinhaça proveniente do tratamento aeróbio (biológico), acoplada a processos físico-químicos, com diferentes diluições. As características físico-químicas das vinhaças tratadas foram avaliadas. Além disso, foram comparados os crescimentos celulares nas diferentes condições experimentais adotadas para o crescimento da microalga M. contortum e da cianobactéria S. salina na vinhaça tratada com e sem diluição. Em cultivos em frascos de Erlenmeyer, em meio proveniente de tratamento biológico, o crescimento celular não diferiu do cultivo em meio padrão, com diluições de 5 e 2 vezes da vinhaça tratada para M. contortum e S. salina, respectivamente. Em fotobiorreator tubular, independente do tratamento ser apenas biológico ou também com carvão ativado, as concentrações celulares máximas (Xm) de M. contortum e S. salina foram da ordem de 1,86x107 células mL-1 e 7,90x106 células mL-1, respectivamente, valores esses menores que os obtidos em meio padrão, com valores de Xm de 2,69x107 células mL-1 e 1,27x106 células mL-1 para M. contortum e S. salina, respectivamente. Em fotobiorreatores tubulares, os teores de mínimos de lipídios de M. contortum e S. salina foram de 33,4 % e 11,0 %, respectivamente. Adicionalmente, os teores mínimos de proteínas da microalga foram de 15,1 % e da cianobactéria foi de 23,2 %

The vinasse is the result of the production of alcohol after the fermentation of the mash and the distillation of the wine. It is a waste rich in nutrients, mainly organic matter, nitrogen, phosphorus and potassium. To be discharged into rivers and lakes, this effluent must be treated in order to remove nutrients, because the excess of these elements in water bodies can lead to major environmental problems, so that it has been used in fertigation. Photosynthetic microorganisms absorb inorganic nutrients and they can absorb nutrients from wastewater. If the organic fraction of vinasse is removed, the inorganic or low organic fraction may be the basis or a medium of cultivation of these microorganisms, which absorb carbon dioxide and its biomass is of industrial interest. In this context, Monoraphidium contortum and Synechocystis salina from mangrove water were cultivated in Erlenmeyer flasks and in tubular photobioreactors by batch process using vinasse from aerobic biological treatment, coupled to physicochemical treatments with different dilutions. The physicochemical characteristics of the treated vinasse were evaluated. In addition, cell growth was compared under different experimental conditions adopted for growth of microalgae M. contortum and cyanobacteria S. salina in vinasse treated with and without dilution. In Erlenmeyer flask cultivations, in medium from the biological treatment, the cell growth was not different of that one in standard medium cultivation, with dilutions of 5 and 2 times the vinasse treated for M. contortum and S. salina, respectively. In the tubular photobioreactor, irrespective if the treatment is only biological or also is carried out treatment with activated charcoal, they were obtained maximum cell concentrations (Xm) of M. contortum and S. salina of 1.86x107 cells mL-1 and 7.90x106 cells. mL-1 , respectively, lower than the standard, whose Xm values were 2.69x107 cells mL-1 and 1.27x106 cells mL-1 for M. contortum and S. salina, respectively. In tubular photobioreactors, the minimum lipid contents of M. contortum and S. salina were 33.4 % and 11.0 %, respectively. In addition, the minimum protein content of microalgae was 15.1 % and cyanobacterium was 23.2 %

Evaluation of Dunaliella salina growth and corresponding ß-carotene production in tubular photobioreactor / Avaliação do crescimento da Dunaliella salina e correspondente produção de ß-caroteno em fotobiorreator tubula

Microalgae, photosynthetic microorganisms, are rich in lipids, polyunsaturated fatty acids, carbohydrates, proteins, vitamins, as well as carotenoids, which are antioxidants that may protect human body from various diseases including obesity, cardiovascular disease, vision-related diseases such as macular degeneration and certain types of cancer. These natural pigments have applications in the pharmaceutical (nutraceutical), food (coloring, functional food, and supplements), and cosmetics industries (e.g. sunscreen), as well as in aquaculture (animal feed). The Dunaliella salina microalga can synthesize 10% of dry weight in ß-carotene (orange pigment, pro-vitamin A activity) under high light intensity and nitrogen and phosphorus limitation, among other stress conditions. The first chapter of this thesis presents a review focused on microalgae carotenoids: culture systems, mode of operation, and applications. In this bibliographic survey, the advantages of microalgae cultivation in relation to traditional sources (higher plants) were discussed, as well as a discussion of the main cultivation systems and their importance in cell growth. This review presented a critical analysis of the different operational regimes like batch, fed-batch, semi-continuous and continuous. Relevant information on the most important world producers of microalgae carotenoids were presented. Chapter II presents the development of a modified method of dispersive liquid-liquid microextraction (DLLME) for rapid extraction of ß-carotene from Dunaliella salina cultivated in tubular photobioreactor, with subsequent development of a rapid chromatographic screening method using a C4 column for separation of geometric isomer of ß-carotene. The use of benzene as extraction solvent and water with 50% acetone as dispersant provided the best condition for the extraction of this carotenoid. In HPLC (High Performance Liquid Chromatography), employing mobile phase composed of methanol and water (95:5, v/v), it was possible to detect/quantify ß-carotene at 14 min (retention time). Besides the short analysis time (<20 min), by the miniaturized extraction (< 10 mL organic waste) this method abide by green chemistry analytical principles. It is known that nitrogen, phosphorus, as well as carbon and vitamins are vital elements for the growth of microalgae, also determining the biochemical composition of biomass. In this sense, Chapter III presents the study of the influence of different amounts of sodium nitrate (1N = 75 mg L-1; 1.5N = 112.5 mg L-1, and 3N = 225 mg L-1) and phosphate monobasic dehydrate (1P = 5.65 mg L-1, 1.5P = 8.47 mg L-1, and 3P = 16.95 mg L-1) in seawater-based f/2 medium on the growth of Dunaliella salina and ß-carotene biosynthesis, by continuous process with different replenishment proportions (R = 20% and 80%). Best results of cell productivity were obtained by semicontinuous process (mean values of Px up to 6.7 x 104 cells mL-1 d-1 with medium 1N:1P; R =20%) in comparison with batch process cultivation. Maximum cell density (Xm) obtained in this work was not dependent of R, but the best results were obtained when using medium 1.5N:1.5P (mean values up to 5.6 x 105 cells mL-1 with R =80%) instead of 1N:1P. The content of ß-carotene in the cells, in general, was higher in cells grown in medium 1N:1P (mean yield values up to 57.5 mg g-1 with R =80%) in comparison with medium 1.5N:1.5P. The cultivation of D. salina with media 3N:3P led to a long lag phase, followed by decrease in cell density and cell lysis. The use of a tubular photobioreactor contributed to successfully cultivate this microalga without contamination by protozoa. The cultivation of Dunaliella salina in tubular photobioreactor with the use of 12:12 photoperiod was appropriate, as well as to induce carotenogenesis, in the second stage, by increasing the light intensity and absence of pH control

As microalgas, micro-organismos fotossintetizantes, são ricas em lipídios, ácidos graxos poli-insaturados, carboidratos, proteínas, vitaminas, além de carotenoides que são antioxidantes com potencial de proteger o organismo humano de várias doenças incluindo a obesidade, doenças cardiovasculares, doenças relacionadas à visão como a degeneração macular e certos tipos de câncer, entre outras. Esses pigmentos naturais têm aplicações em indústrias farmacêuticas (nutracêuticos), alimentícias (colorantes, alimentos funcionais e suplementos) e de cosméticos (exemplo: filtro solar) e na aquacultura (ração animal). A microalga Dunaliella salina é capaz de sintetizar, sob alta intensidade luminosa e limitação de nutrientes como fontes de fósforo e nitrogênio, dentre outras condições de estresse, 10 % do peso seco em ß-caroteno (pigmento laranja com atividade pró-vitamina A). Assim, neste trabalho, numa primeira etapa, foi feita uma revisão da literatura abordando a produção de carotenoides por microalgas, bem como sua aplicação. Nesse levantamento bibliográfico abordou-se, dentre outros assuntos, as vantagens do cultivo de microalgas em relação as fontes tradicionais (plantas superiores), assim como uma discussão dos diferentes sistemas de cultivos e sua importância no crescimento celular. Esse review apresentou uma análise crítica dos principais regimes operacionais como batch, fed-batch, semicontínuo e contínuo. Apresentou-se também informações relevantes sobre os mais importantes produtores mundiais de carotenoides de microalgas. Numa segunda etapa, foi desenvolvido um método modificado de microextração líquido-líquido dispersivo modificado (DLLME) para a rápida extração de ß-caroteno de Dunaliella salina cultivada em fotobiorreatores tubulares, com subsequente desenvolvimento de método cromatográfico em uma coluna C4 para a separação do isômero geométrico de ß-caroteno. A extração ótima de ß-caroteno foi obtida com benzeno como solvente extrator e água com 50% de acetona como dispersante. Empregando uma fase móvel composta por metanol e água (95:5, v/v) em HPLC, foi possível a detecção/quantificação de ß-caroteno com 14 minutos de tempo de retenção. Além dos tempos curtos de análises (<20 min), pela extração em volume reduzido (< 10 mL resíduos orgânicos) este método obedece aos princípios da química verde. Sabe-se que nitrogênio, fósforo, assim como carbono e vitaminas são elementos vitais para o crescimento das microalgas e também exercem influência na composição bioquímica da biomassa. Assim, na terceira etapa deste trabalho, estudou-se a influência das quantidades de nitrato de sódio (75 mg L-1, denominado 1N; 112,5 mg L-1, denominado 1,5N; 225 mg L-1, denominado 3N) e de fosfato monobásico dihidratado (5,65 mg L-1, denominado 1P; 8,47 mg L-1, denominado 1,5P; 16,95 mg L-1, denominado 3P) em meio f/2, que tem como base a água do mar, no crescimento e na síntese de ß-caroteno da Dunaliella salina por processo semicontínuo, com uso de frações de corte (R) de 20% e 80%. Foram obtidas produtividades celulares mais elevadas em processos semicontínuos do que em processo descontínuo, com produtividades médias de até 6,7 x 104 células mL-1 d-1 (meio 1N:1P; R =20%). A máxima concentração celular (Xm) obtida neste trabalho não foi dependente de R. Os melhores resultados de Xm foram obtidos quando se usou meio 1,5N:1,5P em vez de meio, com 1N:1P, com valores médios de até 5,6 x 105 células m L-1 (R =80%). O conteúdo de ß-caroteno nas células, de maneira geral, foi maior nas células cultivadas em meio 1N:1P do que no meio 1,5N:1,5P, com valores até 57,5 mg g-1 (R =80%). O cultivo de D. salina com o meio 3N:3P levou a uma longa fase lag, seguida por uma diminuição na concentração celular e sua lise. O cultivo de células em um fotobiorreator tubular contribuiu para um crescimento celular sem contaminação por protozoários. O cultivo de Dunaliella salina em fotobiorreator tubular com o uso de fotoperíodo 12:12 foi apropriado, assim como induzir a carotenogênese, no segundo estágio, por meio do aumento da intensidade luminosa e ausência de controle de pH

Reconstrucción compleja con matriz de regeneración dérmica acelular en el paciente quemado / Complex reconstruction with acellular dermal regeneration matrix in the burned patient

INTRODUCCIÓN. Las quemaduras de gran extensión que han afectado a las extremidades produciendo efectos funcionales y de calidad de vida permanente, han sido el reto que enfrenta la cirugía reparadora. OBJETIVO. Demostrar la efectividad del uso de la matriz de regeneración dérmica acelular para lograr una cobertura cutánea definitiva y funcional en el tratamiento de las heridas profundas producidas por quemaduras. CASO CLINICO. Paciente masculino de 29 años de edad, que sufrió quemadura térmica con cemento de contacto y fuego, presentó una quemadura del 45% de superficie corporal total, se realizó el tratamiento con matriz dérmica acelular con enfoque en lesión de tercer grado en toda una extremidad superior. RESULTADOS. El paciente tras 6 meses de reconstrucción no presentó limitación funcional, cicatrices estéticamente aceptables. DISCUSIÓN. Los sustitutos dérmicos ayudaron en el tratamiento de defectos de cobertura cutánea. La matriz dérmica acelular y su uso en el paciente quemado, permitió brindar una cobertura cutánea precoz y permanente, con resultados óptimos, evitando generar heridas profundas en sitios donantes, lo que disminuyó la morbilidad en el paciente con quemaduras graves. CONCLUSIÓN. El uso de matriz dérmica acelular permitió la regeneración de nueva dermis y tejido en corto tiempo concediendo cobertura definitiva de las lesiones sin dejar secuelas funcionales y con cicatrices aceptables.

INTRODUCTION. Extensive burns that have affected the extremities producing functional defects and permanent quality of life, have been the challenge facing reconstructive surgery. OBJECTIVE. Demonstrate the effectiveness of the use of the dermal acellular regeneration matrix to achieve a definitive and functional skin coverage in the treatment of deep injuries caused by burns. CLINICAL CASE. A 29-year-old male patient, who suffered thermal burn with contact cement and fire, presented a burn of 45% of total body surface area, was treated with acellular dermal matrix with focus on third degree lesion in an entire upper extremity. RESULTS. The patient after 6 months of reconstruction didn´t present functional limitation, aesthetically acceptable scars. DISCUSSION. Dermal substitutes helped in the treatment of skin coverage defects. The acellular dermal matrix and its use in the burned patient, allowed to provide a precocious permanent skin coverage, with optimal results, avoiding generating deep wounds in donor sites, which decreased the morbidity in the patient with severe burns.

Changes in adipose cells of Longissimus dorsi muscle in Iberian pigs raised under extensive conditions

ABSTRACT Twenty-four Iberian castrated male pigs were used to characterize and evaluate the effect of the duration of "Montanera" in the adipocytes size and its relation with the backfat thickness and intramuscular fat. The animals were fed under extensive conditions during 30, 60 and 90 days in the "Dehesa" before slaughtered. Carcass weight, percentage of intramuscular fat, thickness of backfat and its three layers and adipocytes size of the intramuscular fat were obtained. The group which expended 90 days on fattening obtained the highest adipocytes, with an area higher by a 50% than those that only expended 30 days. The differences in diameter and perimeter adipocyte were not as marked as area. A significant positive correlation between the diameter, area and perimeter of adipocyte with the backfat thickness were found. The fat cells in Iberian pig hypertrophy during the "montanera stage", being this increase significant from month to month in this period of fattening. Also, this adipocyte increase is correlated with the animal weight. The correlation between adipocyte size and inner layer of backfat shows that the inner layer obtained in live pig by ultrasound techniques could be a good marker of fat infiltration in pigs fattening in "montanera" system.

Evaluación de las condiciones de crecimiento celular para la producción de astaxantina a patir de la microalga Haematococcus pluvialis / Evaluation of Cell Growth Conditions for the Astaxanthin Production as of Haematococcus pluvialis Microalgae

Resumen En la actualidad la astaxantina de origen natural es uno de los pigmentos carotenoides con importantes aplicaciones en la industria alimenticia, farmacéutica y cosmética, debido a sus grandes propiedades dentro de las que se destaca su gran poder antioxidante, efecto preventivo del cáncer, incremento de la respuesta inmune, inhibición de los radicales libres entre muchas otras. Haematococcus pluvialis es una microalga verde de agua dulce y es una de las fuentes naturales con mayor producción de astaxantina ya que es capaz de acumular hasta un 3% de astaxantina en peso seco. El objetivo del presente trabajo fue determinar el medio de cultivo y las condiciones óptimas para el crecimiento y la producción de astaxantina a partir de Haematococcus pluvialis. La influencia de diferentes factores como el pH, temperatura, agitación, aireación CO2 e iluminación favorecen el crecimiento celular, al darle un ambiente óptimo a la microalga. Para determinar las condiciones nutricionales óptimas, se evaluó el efecto de diferentes medios de cultivo (BBM, OHM, RM) en Birreactores de 500mL con 350mL de medio y 1x104cel/ml de inóculo en fase exponencial, las condiciones de cultivo empleadas fueron: pH. 6.7 a 7, CO2 al 5%, fotoperiodo de 16 horas luz 8 oscuridad, irradianza 70μE/m2s; Los resultados mostraron que el mayor crecimiento o producción celular se obtuvo en el medio RM con 7,5 x 105 cel/ml en el día 36, y la mayor producción de astaxantina se obtuvo en el medio RM con una concentración de 8.3 μg/ml en el tratamiento 4.

Abstract Currently astaxanthin of natural origin is one of the carotenoids with important applications in the food, pharmaceutical and cosmetics, due to their large properties within its powerful antioxidant, cancer preventive effect, increase the immune response, inhibiting free radicals among many others. Haematococcus pluvialis is a freshwater green microalgae and is one of the largest natural sources of astaxanthin production as it is able to accumulate up to 3% astaxanthin by dry weight. The aim of this study was to determine the culture medium and the optimal conditions for growth and production of astaxanthin from Haematococcus pluvialis. The influence of different factors such as pH, temperature, agitation, aeration and lighting, CO2, promote cell growth, by giving an optimal environment to microalgae. To determine the optimal nutritional conditions for cell growth the effect of different culture media (BBM, OHM, RM) in bioreactors of 500 ml with 350mL of medium and 1x104 cells/ml inoculum was evaluated in exponential phase, the culture conditions employed they were: pH. 6.7 to 7, 5% CO2, 16 hours photoperiod Light 8 dark, irradiance 70 μE / m2s; The results showed that the highest growth and cell reproduction was obtained in the middle RM with 7.5 x 105 cells /ml on day 36, and increased production of astaxanthin was obtained in the middle RM with a concentration of 8.3 ug / ml in the treatment 4.

Efeito da composição de bactérias láticas e da suplementação do soro de leite na cinética de acidificação, crescimento celular e produção de nisina / Effect of lactic acid bacteria composition and the supplementation of milk whey on acidification kinetics, cell growth and nisin production

As bactérias ácido-láticas (BAL) são micro-organismos que auxiliam nas características organolépticas, funcionais e de bioconservação de produtos fermentados. A utilização do soro de leite como meio de cultivo natural enaltece o conceito da produção de biomoléculas de alto valor agregado, como bacteriocinas, já que é um subproduto gerado por indústrias de laticínios e considerado um agente poluidor. A inulina é um ingrediente prebiótico que promove seletivamente o crescimento de culturas probióticas. Nesse âmbito, o objetivo deste estudo foi avaliar o efeito da composição da cultura de Lactococcus lactis (LL) em cocultura com Streptococcus thermophilus (ST) e da suplementação da base de soro de leite com inulina: (i) nos parâmetros cinéticos de acidificacão, (ii) no crescimento celular, (iii) na viscosidade do produto e (iv) na atividade antimicrobiana da nisina. A fermentação do soro de leite com Lactococcus lactis em cocultura com Streptococcus thermophilus proporcionou a maior taxa de acidificação (Vmax=7,93x10-3 upH/min), assim como apresentou o menor tempo para atingir a velocidade máxima de acidificação (Tvmax=1,13 h). A adição de 2% de inulina ao soro de leite fermentado pela cocultura binária fez com que o tempo para completar o cultivo fosse o mais curto (TpH4,5=4,43 h) quando comparado aos demais ensaios. Quanto ao crescimento celular, pode-se observar que a inulina não afetou significativamente a contagem microbiológica, quando as cepas ST e LL foram utilizadas separadamente no processo fermentativo. Em particular, a adição de 4% de inulina reduziu em 1,2 LogUFC/mL e 0,92 LogUFC/mL a contagem de ST e LL (em monocultura), respectivamente. Por outro lado, em coculturas binárias (ST-LL), percebeu-se ganho na contagem microbiológica nos ensaios que receberam suplementação do ingrediente prebiótico, ou seja, quando adicionados 2% e 4% de inulina, houve aumento de 1 LogUFC/mL e de 1,34 LogUFC/mL na contagem de ST, respectivamente. No caso da cepa LL em cocultura com ST, a suplementação de 2% e 4% do prebiótico aumentou em 0,31 LogUFC/mL e 0,75 LogUFC/mL, respectivamente. A concentração de ácido lático também foi mais elevada nos cultivos realizados com a cocultura binária, sendo 4,56 g/L (na ausência de inulina), 5,28 g/L (com adição de 2% de inulina) e 5,71 g/L (com suplementação de 4% de inulina). A viscosidade foi influenciada tanto pela adição de inulina como pelo efeito sinérgico da cocultura, sendo que o maior valor (7,38 mPas) foi obtido pela cocultura ST-LL e pela adição de 4% do ingrediente prebiótico. Quanto à produção de nisina, observou-se que, no cultivo em cocultura (ST-LL), a concentração de 2% de inulina aumentou em 102% a atividade antimicrobiana quando comparada com a cultura pura LL. Vale ressaltar que ambas as cepas satisfizeram os requisitos tecnológicos relativos à produção de laticínios funcionais

Lactic acid bacteria (LAB) are microorganisms that help in the organoleptic and functional characteristics and in the biopreservation of fermented products. The use of milk whey as a culture medium extols the concept of the production of high value-added biomolecules, such as bacteriocins, since it is a by-product generated by the dairy industry and considered a pollutant. Inulin is a prebiotic ingredient that promotes selectively the growth of probiotic cultures. In this context, the aim of this study was to evaluate the effect of culture composition Lactococcus lactis (LL) in co-culture with Streptococcus thermophilus (ST) and the supplementation of milk whey with inulin on: (i) the acidification kinetic parameters, (ii) the cell growth, (iii) the product viscosity, and (iv) the antimicrobial activity of nisin. The fermentation of milk whey by Lactococcus lactis in coculture with Streptococcus thermophilus provided the highest acidification rate (Vmax = 7.93x10-3 upH/min) and the shortest time to reach the maximum acidification rate ( TVmax = 1.13 h). The addition of 2% inulin in the binary coculture binary led to the shorter time to complete the fermentation (TpH4,5 = 4.43) compared to the other tests. With regard to cell growth, it can be observed that the addition of inulin did not affect the microbiological count of pure cultures of ST and LL strains in the fermentation process. In particular, the addition of 4% inulin reduced by 1.2 Log CFU/mL and 0.92 Log CFU/mL the counts of ST and LL (monoculture), respectively. In the other hand, the binary co-cultures cultivations (ST-LL) with the addition of 2% and 4% inulin increased by 1 LogCFU/mL and 1.34 Log CFU/mL in the case of the ST counts and 0.31 log CFU/mL and 0.75 log CFU/mL the counts of LL, respectively. Lactic acid concentration was higher in cultivations carried out by binary cocultures, thus being 4.56 g/L (in the absence of inulin), 5.28 g/L (with addition of 2% inulin) and 5.71g/L (supplemented with 4% inulin). The viscosity was influenced by the addition of prebiotic ingredient and by the synergistic effect of binary coculture, being the highest value (7.38 mPas) obtained by the addition of 4% inulin. Finally, as regards the production of nisin noted that in the binary coculture cultivations (ST-LL), the concentration of 2% inulin increased at 102% the antimicrobial activity when compared to the pure culture LL. It is worth mentioning that both strains met the technological requirements as regards the production of functional dairy products

Isolation, expansion and differentiation of cellular progenitors obtained from dental pulp of agouti (Dasyprocta prymnolopha Wagler, 1831) / Isolamento, expansão e diferenciação de progenitors celulares obtidos da polpa dentária de cutia (Dasyprocta prymnolopha Wagler, 1831)

The study aimed to isolate, expand, differentiate and characterize progenitor cells existent in the dental pulp of agouti. The material was washed with PBS solution and dissociated mechanically with the aid of a scalpel blade on plates containing culture medium D-MEM/F-12, and incubated at 5% CO2-37⁰C. The growth curve, CFU assay, osteogenic/adipogenic differentiation and characterization were obtained from the isolation. The cells began to be released from the explant tissue around the 7th day of culture. By day 22 of culture, cells reached 80% confluence. At the UFC test, 81 colonies were counted with 12 days of cultivation. The growth curves before and after freezing showed a regular growth with intense proliferation and clonogenic potential. The cell differentiation showed formation of osteoblasts and fat in culture, starting at 15 days of culture in a specific medium. Flow cytometry (FACs) was as follows: CD34 (positive), CD14 (negative), CD45 (negative), CD73 (positive), CD79 (negative), CD90 (positive), CD105 (positive), demonstrating high specificity and commitment of isolated cells with mesenchymal stem cells strains. These results suggest the existence of a cell population of stem cells with mesenchymal features from the isolated tissue in the explants of agouti dental pulp, a potential model for study of stem cell strains obtained from the pulp tissue.

Isolation, expansion and differentiation of cellular progenitors obtained from dental pulp of agouti (Dasyprocta prymnolopha Wagler, 1831). Este estudo teve como objetivo isolar, expandir, diferenciar e caracterizar células progenitoras existentes na polpa dentária de cutia. O material foi lavado em solução de PBS e dissociado mecanicamente, com o auxílio de uma lâmina de bisturi, em placas contendo meio de cultura D-MEM/F-12, e incubadas em 5% de CO2-37⁰C. A curva de crescimento, o ensaio de CFU, a diferenciação osteogênica/adipogênica e a caracterização foram obtidas a partir do isolamento. As células começaram a ser liberadas, a partir do explante, em torno do sétimo dia de cultura. A partir do 22o dia, as células atingiram 80% de confluência. No teste para UFC, 81 colônias foram contadas aos 12 dias de cultivo. As curvas de crescimento pré- e pós-congelamento apresentaram crescimento regular, com intensa proliferação e potencial clonogênico. A diferenciação das células mostrou a formação de osteoblastos e de células de gordura, a partir de 15 dias de cultura em meio específico. A citometria de fluxo (FACS) apresentou-se como segue: CD34 (positivo), CD14 (negativo), CD45 (negativo), CD73 (positivo), CD79 (negativo), CD90 (positivo), CD105 (positivo), demonstrando a grande especificidade e comprometimento das células isoladas com linhagens de células-tronco mesenquimais. Estes resultados sugerem a existência de uma população de células-tronco mesenquimais isolada a partir de explantes da polpa dentária cutia, um modelo potencial para o estudo de linhagens de células-tronco obtidas a partir do tecido pulpar.

Caracterização das proteínas caveolinas -1 e -2 na placenta de conceptos bovinos clonados transgênicos / Caracterization of caveolin -1 and -2 proteins in cloned and transgenic placenta of cattle

A utilização da transgenia com a proteína fluorescente verde (GFP) como marcador de células de origem fetal nas placentas de clones bovinos servirá de modelo inédito para estudo morfofisiológico e imunológico da interação materno-fetal, visto que possibilitará o seu mapeamento, diferenciando as células fetais das maternas. Tal modelo terá aplicação direta, principalmente porque estes são animais que apresentam problemas em relação ao seu desenvolvimento. Com o auxílio deste modelo, pretende-se verificar o transporte de substâncias entre a mãe e o feto via endocitose, pela imunolocalização das proteínas chamadas de caveolinas. Para tanto foram utilizados 06 bovinos clonados e 30 bovinos de inseminação artificial (IA) com idade até 90 dias de gestação, os quais tiveram seu desenvolvimento interrompido mediante abate humanitário das receptoras e ovariosalpingohisterectomia, com posterior recuperação do útero gestante. Foram coletados os placentônios e o cório. Uma parte das amostras foi recortada e fixada, por imersão, em solução de parafolmaldeído a 4% ou formoldeído a 10% em tampão fosfato de sódio (PBS) a 0,1M pH 7.4, solução de Zamboni (4% de paraformoldeído, 15% de ácido pícrico, em tampão fosfato de sódio a 0,1M pH 7.4), metacarn (60% de metanol, 30% de clorofórmio, e 10% de ácido acético glacial), para verificação da morfologia e realização de imuno-histoquímica para as proteínas caveolinas -1 e -2 (CAV -1 e CAV-2)...

The transgenic application of green fluorescent protein (GFP) as fetal cell marker on cattle cloned placenta could provide an exclusive model for studying the morphologic and immunologic maternal-fetal interactions, providing information about its mapping, distinguishing the fetal from maternal cells. This model will have direct application, mainly because these animals present problems during its development. With this model's support, we intend to verify the substances transport between mother and fetus during endocytosis, through the immunolocalization of protein named caveolae. For these, we used 06 cloned bovine and 30 cattle samples of artificial insemination (AI) with 90 days of pregnancy, which had been their development interrupted by humanitarian slaughter of the recipient and recovery of the pregnant uterus. We collected the placentome and the chorion. A part of the samples was cut and fixed, by immersion, on a solution containing 4% of parafomaldehyde or 10% of formaldehyde on a sodium phosphate buffer (PBS), at 0,1M pH 7.4, Zamboni solution (4% of paraformaldehyde, 15% of picric acid, on sodium phosphate buffer 0,1M pH 7.4), metacarn (60% of metanol, 30% of chloroform, and 10% glacial acetic acid), for morphologic and immunohistochemistry verification for caveolinas proteins -1 and -2 (CAV -1 and CAV- 2). The caveolins -1 were found in fetal and maternal villi, but its strongest staining was observed in the endometrial stroma. The caveolins -2 had positive staining in trophoblast and chorioallantoic membrane, and specifically in giant trophoblastic binucleated cell. Therefore the results were compared between cloned cattle and from AI or natural mating, for assisting on detection of the reason of many placental alterations, embryonic losses, spontaneous abortion, post-natal mortality and large offspring syndrome on laboratory-manipulated animals. The result suggests that the proteins caveolins -1 and -2 (CAV-1 and CAV-2)...

Papel dos corpúsculos lipídicos na proliferação e transformação celular / [Role of lipid bodies in cell proliferation and transformation]

Corpúsculos lipídicos são organelas dinâmicas envolvidas no metabolismo de lipídeos, no tráfego de membrana e na sinalização intracelular. A lipogênese está associada com um mau prognóstico em diversas doenças neoplásicas, sugerindo um papel dessas organelas no desenvolvimento do câncer. Foi mostrado anteriormente que corpúsculos lipídicos são elementos centrais na síntese de prostaglandina E2 e na proliferação celular em linhagens celulares de câncer de cólon, e podem estar implicados na patologia desta neoplasia. Baseado nesses dados, avaliamos a regulação dos corpúsculos lipídicos na progressão do ciclo celular e na transformação celular. Células NIH3T3 foram sincronizadas por confluência e privação de soro, e progressão pelo ciclo celular foi avaliada após suplementação com soro por incorporação de BrdU e por análise de qRT-PCR ou western blot de genes e proteínas do ciclo celular. Foi observado que, após suplementação, células NIH3T3 progridem para fase S em 24 horas, seguindo para as fases G2 e M após 36-48 horas. Além disso, análise de expressão de ciclinas confirmou que células sincronizadas progridem uniformemente pelo ciclo celular após suplementação com soro. Usando este modelo, avaliamos a regulação de corpúsculos lipídicos durante o ciclo celular. Quantificação e análise de localização subcelular mostraram que células em arresto na fase G1 apresentam menor quantidade e localização perinuclear de corpúsculos lipídicos, enquanto que um maior número e localização dispersa dessas organelas foram observados durante a fase S. Os mesmos resultados foram observados em células IEC-6. Além disso, estes dados foram confirmados em NIH3T3 através de microscopia de fluorescência com marcação simultânea de corpúsculos lipídicos e BrdU incorporado, ou através de sincronização específica na fase S por timidina. A linhagem transformada NIH3T3-H-rasV12 foi também submetida à confluência e privação de soro para análise de corpúsculos lipídicos, onde foi possível observar um aumento no número e localização dispersa de corpúsculos lipídicos, além do acúmulo da proteína estrutural ADRP, quando comparadas com a linhagem selvagem NIH3T3. Por outro lado, células NIH3T3 com superexpressão de ADRP humana não apresentaram características de transformação in vitro, como observado em ensaios de proliferação e morte celular, formação de focos, ou ensaio de crescimento independente de ancoragem. Juntos, esses resultados mostram que corpúsculos lipídicos são regulados durante a progressão do ciclo celular e que em células transformadas esta regulação está alterada, embora tenha sido visto que a superexpressão de ADRP não é capaz de induzir transformação celular em nosso modelo. Não obstante, esses dados dão evidências de que um mecanismo coordenado regula a progressão de ciclo celular e biogênese de corpúsculos lipídicos, que pode estar desregulado durante o desenvolvimento neoplásico. (AU)

Lipid bodies (or lipid droplets) are organelles involved in lipid turnover, membrane traffic and signaling. Lipogenesis has been associated with poor prognosis in several neoplasic diseases, suggesting a role for these organelles in cancer development. We have previously reported that lipid bodies are involved in prostaglandin E2 synthesis and cell proliferation in colon cancer cells, and may have implications to colon adenocarcinoma pathogenesis. Based on this, we evaluated the role of lipid bodies in cell cycle regulation and during cellular transformation. NIH3T3 cells were synchronized through confluence and serum starvation, and progression through cell cycle was assayed before or after 12, 24, 36 or 48 hours of serum supplementation by BrdU incorporation, and by qRT-PCR and western blot analysis of cell cycle genes and proteins. We observed that, upon serum supplementation, NIH3T3 cells reached S phase after 24 hours and G2/M phase after 36-48 hours, confirming that synchronized NIH3T3 progressed uniformly through cell cycle after stimulus. Using the same model, we evaluated the regulation of lipid bodies during cell cycle progression by fluorescence microscopy and flux citometry. We observed that cells arrested on G1 phase showed a lower number and perinuclear localization of lipid bodies, whereas an increased number of lipid bodies with a dispersed distribution through the cytoplasm were observed during S phase. The same results were seen in IEC-6 cells. These data were also confirmed in NIH3T3 cells by microscopy analysis after simultaneous staining of lipid bodies and incorporated BrdU, and with thymidine synchronization to assess regulation of lipid bodies in S cell cycle phase. The transformed cell lineage NIH3T3-H-rasV12 was also submitted to confluence and serum starvation for lipid bodies' analysis, showing increased number and dispersed localization of lipid bodies, along with increased ADRP protein accumulation, when compared with NIH3T3 cells. In the other hand, NIH3T3 cells overexpressing human ADRP did not display transformed phenotype in vitro, as observed in cell death and proliferation, foci formation, and anchorage-independent growth assays. Taken together, these results suggest that lipid bodies are highly regulated during cell cycle, and that this regulation is altered in transformed cells, although ADRP overexpression was not able to induce cell transformation in our model. Nevertheless, these data provide evidence for a coordinate mechanism that regulates cell cycle progression and lipid body biogenesis, which might be deregulated during cancer development. (AU)

MicroRNA-133a attenuates isoproterenol-induced neonatal rat cardiomyocyte hypertrophy by downregulating L-type calcium channel α1C subunit gene expression / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate the effects of microRNA-133a on isoproterenol (ISO)-induced neonatal rat cardiomyocyte hypertrophy and related molecular mechanism focusing on the changes of L-type calcium channel α1C subunit.</p><p><b>METHODS</b>Neonatal rat cardiomyocytes were cultured, cardiomyocyte hypertrophy was induced by isoproterenol (ISO, 10 µmol/L). The cell surface area was measured by phase contrast microscope and Leica image analysis system. The mRNA expressions of atrial natriuretic peptide (ANP), β-myosin heavy chain (β-MHC), miR-133a and the α1C were detected by qRT-PCR. The protein expression of α1C was evaluated by Western blot. MiR-133a mimic was transfected into cardiomyocytes to investigate the effects of miR-133a on ISO-induced cardiomyocyte hypertrophy. The targets of miR-133a were predicted by online database Targetscan. The 3' untranslated region sequence of α1C was cloned into luciferase reporter vector and then transiently transfected into HEK293 cells. The luciferase activities of samples were measured to verify the expression of luciferase reporter vector. The expression level of α1C was inhibited by RNAi to determine the effects of α1C on cardiomyocyte hypertrophy. Intracellular Ca(2+) content was measured by confocal laser microscope.</p><p><b>RESULTS</b>(1) The expression of miR-133a was significantly reduced in ISO-induced cardiomyocyte hypertrophy (P < 0.01) . Upregulating miR-133a level could suppress the increase of cell surface area, the mRNA expression of ANP and β-MHC (P < 0.01) . (2) α1C was the one of potential target of miR-133a by prediction using online database Targetscan. The luciferase activities of HEK293 cells with the plasmid containing wide type α1C 3'UTR sequence were significantly decreased compared with control group (P < 0.01) . Upregulation of the miR-133a level by miR-133a mimic transfection could suppress the protein expression of α1C (P < 0.05) . (3) The expression of α1C was significantly increased in ISO treated cardiomyocytes (P < 0.05) . Downregulation of α1C by RNAi could markedly inhibit the increase of cell surface area, the mRNA expression of ANP and β-MHC (P < 0.01, P < 0.05, P < 0.05). (4) Downregulation of α1C expression by RNAi or upregulation of miR-133a level by miR-133a mimic transfection significantly inhibited intracellular Ca(2+) content (P < 0.01) .</p><p><b>CONCLUSIONS</b>Our data confirms that α1C is the target of miR-133a. MiR-133a can negatively regulate the expression of L-type calcium α1C subunit, resulting in the decrease of intracellular Ca(2+) content and the attenuation of ISO-induced cardiomyocyte hypertrophy.</p>

Bradykinin attenuates mechanical stress-induced myocardial hypertrophy through inhibiting the Ca²⁺/ calcineurin pathway / 中华心血管病杂志

<p><b>OBJECTIVE</b>To evaluate the inhibitory effect and related mechanism of bradykinin on mechanical stress induced myocardial hypertrophy.</p><p><b>METHODS</b>Neonatal rat cardiomyocytes were isolated and cultured in silicon plates. All cardiomyocytes were randomly divided into three groups: control group, mechanical stretch group (mechanical stretch of silicon plates to 120% for 30 min) and mechanical stretch plus bradykinin group (1×10(-8) mol/L for 24 h before stretch). The protein synthesis and surface area of cardiomyocytes were detected by [(3)H] leucine incorporation and immunofluorescence of α-MHC, respectively. mRNA expression of atrial natriuretic peptide (ANP) and sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2) was detected by real time-PCR, the phosphorylation of calcineurin (CaN), the expression of Angiotensin II receptor 1 (AT1R) and angiotensin converting enzyme (ACE)by Western blot.</p><p><b>RESULTS</b>The surface area of cardiomyocytes of mechanical stretch group [(973 ± 103) µm(2)] was significantly enlarged than in control group [(312 ± 29) µm(2)] and this effect could be partly attenuated by bradykinin [(603 ± 74) µm(2), all P < 0.05]. Mechanical stretch also significantly increased the protein synthesis, up-regulated the expression of ANP and decreased the expression of SERCA2, and these effects could be partly reversed by pretreatment with bradykinin. Moreover, bradykinin partly abolished the mechanical stretch-induced increases in CaN phosphorylation, up-regulation of AT1R but preserved the expression of ACE.</p><p><b>CONCLUSIONS</b>Bradykinin significantly attenuates mechanical stretch-induced myocardial hypertrophy through inhibition of Ca(2+)/CaN pathway.</p>

Statistical optimisation of cell growth and carotenoid production by Rhodotorula mucilaginosa

Sequential statistical methods were used to maximise carotenoid production by a strain of Rhodotorula mucilaginosa, isolated from the Brazilian ecosystem. Initially, a factorial 2(5-1) experimental design was used, and the variables were pH and the levels of glucose, yeast extract, MgSO4.7H2O and KH2PO4. The nitrogen source (yeast extract) was the most important variable in enhancing carotenoid production; MgSO4.7H2O and KH2PO4 had a negative influence. The initial pH had no significant effect on carotenoid and cell productions. We further investigated the effects of glucose and yeast extract effects, using a second-order central composite design (CCD) to optimise carotenoid production, which was adequately approximated with a full quadratic equation obtained from a two-factor-2-level design. The analysis of quadratic surfaces showed that after 5 days of cultivation at 25ºC, the maximum carotenoid concentration (745 µg l-1) was obtained with 15 g l-1 of yeast extract and 20 g l-1 of glucose. The maximum carotenoid production (152 µg g-1) was obtained with 5 g l-1 yeast extract and 10 g l-1 glucose. Carotenoid formation was more sensitive to changes in yeast extract concentration than to changes in glucose concentration. Maximum cell production was achieved with 15-17 g l-1 of yeast extract and 15-20 g l-1 of glucose.

Impacto de diferentes fontes proteicas sobre o desenvolvimento celular hepático de ratos recém-desmamados submetidos à dieta restritiva seguida de recuperação alimentar / Impact of different protein sources on the development of hepatic cells in newly-weaned rats submitted to a restriction diet followed by food repletion

The aim of this study was to evaluate the effect of different protein sources with different amino-acid profiles on liver cell development in "Wistar" rats submitted to a food restriction and recovery model. The food restriction model was based on a 50% ingestion restriction for the rats fed with the control diet (21 days) and "ad libitum" recovery (a period of 21 days). The protein sources used in this study were: Yeast autolysate (YA), whey protein concentrate from bovine milk (WPC), a mixture containing the YA and WPC in the proportion of 64:36 (protein base), commercial casein (CC) which was used as the experimental treatment (EC) and control treatment (CP). The following parameters were evaluated in this study: the amino-acid profile of the protein sources, the development of liver cells (liver weight and growth rates - RNA, DNA, protein), weight and number of hepatocytes in the whole organ. The results showed that the treatment with (YA) was the most affected by the food restriction, showing an incomplete (leucine-deficient) amino-acid profile, a lower development of liver cells, lower growth of the liver due to a lower growth by cellular hyperplasia (number of cells), lower capacity of cell division and DNA synthesis. However, it showed a higher ability for RNA synthesis, thus indicating that growth in the restricted phase was mainly due to increase in the size of hepatocytes (cell hypertrophy). During the repletion period, all food treatments showed normal liver development, i.e. cell growth and organ hyperplasia and hypertrophy.

El objetivo de este estudio fue evaluar el efecto que diversas fuentes de proteínas con diferentes perfiles de aminoácidos ejercían en el desarrollo de las células hepáticas en ratas "Wistar" sometidas a restricción y reposición de la ingestión de alimentos. El modelo de restricción alimentar consistía en disminuir 50% del consumo de los animales control (período de 21 días) y la recuperación con ingestión "ad libitum" (período de 21 días). Las fuentes de proteínas utilizadas en este estudio fueron: autolisado de levadura (ATL); concentrado proteico de suero de leche bovino (CPL); mezcla de CPL y ATL, en la proporción de 64:36 (base proteica), caseína comercial (CC), que fue utilizada como tratamiento experimental (CE) y como estándar (CP). Fueron evaluados el perfil de aminoácidos de las proteínas, el desarrollo de la célula hepática (peso del hígado y las tasas de crecimiento: ARN, ADN y proteína total), el peso y número de hepatocitos en el órgano. Los resultados mostraron que el grupo tratado con (ATL) fue el más afectado por el proceso de restricción de alimentos, la proteína presenta un perfil incompleto de aminoácidos(deficiente en leucina). Había menor desarrollo de las células del hígado, menor crecimiento del hígado debido a un menor crecimiento por hiperplasia celular (número de células), menor capacidad de división celular y de síntesis de ADN, sin embargo, mostraron una mayor capacidad para sintetizar ARN indicando que el crecimiento en la fase de restricción se debió principalmente al aumento en el tamaño de los hepatocitos (hipertrofia celular). Durante la fase de recuperación alimentar de todos los tratamientos hubo un desarrollo hepático normal, o sea crecimiento de las células y del órgano por hiperplasia e hipertrofia.

O objetivo deste estudo foi avaliar o efeito de diferentes fontes proteicas com diferentes perfis de aminoácidos sobre o desenvolvimento celular hepático de ratos "Wistar" submetidos à restrição e recuperação alimentar. O modelo de restrição alimentar foi baseado na restrição de 50% da ingestão dos animais controle (período de 21 dias), e recuperação ad libitum (período de 21 dias). As fontes proteicas utilizadas neste estudo foram: autolisado de levedura (ATL), concentrado proteico de soro de leite bovino (CSL), mistura contendo CSL e ATL na porcentagem de 64:36 (base proteica), caseína comercial (CC), a qual foi utilizada como tratamento experimental (CE) e como tratamento padrão (CP). Avaliouse, neste estudo, o perfil de aminoácidos das fontes proteicas, o desenvolvimento celular hepático (peso do fígado e dos índices de crescimento - RNA, DNA, proteína total), peso dos hepatócitos e número de hepatócitos em todo órgão. Os resultados mostram que o tratamento com (ATL) foi o mais afetado pelo processo de restrição alimentar, apresentando um perfil de aminoácido incompleto (deficiência em leucina); apresentou menor desenvolvimento celular hepático; menor crescimento do fígado em função do menor crescimento por hiperplasia celular (número de células), menor capacidade de divisão celular e síntese de DNA. Entretanto, apresentou maior capacidade de síntese de RNA, indicando que o crescimento na fase de restrição ocorreu principalmente por aumento no tamanho dos hepatócitos (hipertrofia celular). Durante o período de restauração alimentar todos os tratamentos apresentaram desenvolvimento hepático normal, ou seja, crescimento de células e do órgão por hiperplasia e hipertrofia.

Adipocyte morphometric evaluation and angiogenesis in the omentum transposed to the breast: a preliminary study

OBJECTIVE: The purpose of this study was to describe the probable mechanism of the volume increase of laparoscopically harvested omentum flaps used to treat breast deformities. METHODS: A histological analysis of omentum samples was performed to study the volume increase of laparoscopically harvested omentum flaps. Samples were harvested immediately after the transposition of the omentum from the abdominal cavity to the breast region and during the second surgical procedure for breast symmetrization of eight patients submitted to the transposition of the omentum flap. Changes in the morphometric measurements of the adipocytes (perimeter, diameter, and area), microvascular density (as measured by the CD31 endothelial marker), and immunohistochemical expression of VEGF were documented. RESULTS: The increases in adipocyte size and microvascular density were statistically significant (P < 0.012). The expression levels of VEGF were lower in the second set of samples when compared to the first set, but the differences were not statistically significant (P < 0.093). CONCLUSION: These results demonstrate an increase in cellular volume as measured by adipocyte perimeter, diameter, and area. Moreover, the increase in the number of vessels in the second set of samples suggests that neoangiogenesis was stimulated by the initial increase in VEGF expression levels observed in the first set of samples. The increase in VEGF expression in the flap may have been caused by adipocyte hypertrophy resulting from neoangiogenesis.

Comparing effects of U50488H, prazosin and/or propranolol on cardiac hypertrophy induced by NE in rat / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To demonstrate the inhibitory effect of kappa-opioid receptor activation by U50488H on hypertrophy induced by NE in cultured neonatal rat cardiac myocytes and compare its effect with that of prazosin and propranolol.</p><p><b>METHODS</b>The cellular proliferation was determined with crystal violet staining. The protein content was assayed with Lowry's method. The cardiomyocytes volumes were measured by computer photograph analysis system. The protein synthesis was assayed with [3H]-lencine incorporation method.</p><p><b>RESULTS</b>(1) NE significantly induced the increase of protein content, [3H]-leucine incorporation and cell size without a concomitant increase in cell number in low serum medium. OThese responses were partially suppressed by prazosin or propranolol alone and completely abolished by both in combination. U50488H significantly inhibited the NE-induced increase of protein content, [3H]-leucine incorporation and cell size. The inhibitory effects of U50488H on NE-induced cardiac hypertrophy were greater than either prazosin or propranolol, but comparable to combination of both.</p><p><b>CONCLUSION</b>NE, acting via both alpha1- and beta-adrenergic pathway, stimulates myocyte hypertrophy. Stimulating kappa-opioid receptor significantly inhibits NE-induced cardiac hypertrophy, which may be related with alpha1- and beta1-adrenergic pathway.</p>

Cardiac hypertrophy and changes in contractile function of cardiomyocyte / 生理学报

To investigate the cellular mechanisms of pressure-overload cardiac hypertrophy transition to heart failure, we observed time course of changes in morphology and contractile function of cardiomyocytes in transverse abdominal aortic constriction (TAC) rats. Since TAC rats suffered higher stress, body weight had a slower growth rate compared with that of synchronous control rats. Therefore, the left ventricular to body weight ratio produced experimental bias to evaluate the degree of cardiac hypertrophy. Length and width of collagenase-isolated cardiomyocyte were directly measured. Length, width and calculated surface area of cardiomyocyte showed a progressive increase in 8-, 16-, and 20-week TAC rats. The increasing rate of surface area in cardiomyocytes was higher at the middle stage of TAC (from the eighth to sixteenth week). Due to the constraint of fibrosis formation, the increasing rate of surface area in cardiomyocytes was slower at the late stage of TAC (from the sixteenth to twentieth week). The sarcomere length of cardiomyocytes was unchanged, whereas sarcomere numbers were significantly increased in 8-, 16-, and 20-week TAC rats. Shortening amplitude of unloaded contraction in single cardiomyocyte was significantly enhanced in 1-week TAC rats, but not altered in 8-week TAC rats compared with that in the synchronous control rats. On the contrary, unloaded shortening amplitude of single cardiomyocyte was significantly reduced in 16- and 20-week TAC rats. The above results suggest that the reduced shortening amplitude may be associated with intrinsic molecular alterations in hypertrophied cardiomyocytes.

Role of Na(+)-K(+)-ATPase in lipopolysaccharide-induced cardiomyocyte hypertrophy in rats / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the possible mechanism of lipopolysaccharide (LPS)-induced cardiomyocyte hypertrophy in rats.</p><p><b>METHODS</b>Neonatal rat cardiomyocytes cultured in vitro were stimulated with 100 µg/L LPS for 1, 4 or 8 h and scanned by atomic force microscopy (AFM) for measurement of the two-dimensional area, three-dimensional surface area and volume of each cell. The total proteins and Na(+)-K(+)-ATPase activity in the cardiomyocytes were determined. The same measurements were also carried out in neonatal rat cardiomyocyte cultures stimulated by 0.5 µmol/L ouabain for 8 h and the total protein levels were measured.</p><p><b>RESULTS</b>Following a 8-hour stimulation with LPS, the two-dimensional area, three-dimensional surface area and volume of the single cardiomyocyte became enlarged and the total cellular proteins increased significantly as compared with those in the normal control cells (P < 0.05). LPS treatment for 4 and 8 h resulted in significantly decreased activity of Na(+)-K(+)-ATPase in the cardiomyocytes (P < 0.05). In the cells treated with ouabain for 8 h, the two-dimensional area, three-dimensional surface area, volume of the single cardiomyocyte and the total cellular proteins increased significantly in comparison with the normal control group (P < 0.05).</p><p><b>CONCLUSION</b>LPS can result in cardiomyocyte hypertrophy in rats possibly in relation to lowered Na(+)-K(+)-ATPase activity in the cardiomyocytes after LPS exposure.</p>

Production of alkaline protease from Cellulosimicrobium cellulans / Produção de protease alcalina porCellulosimicrobium cellulans

Cellulosimicrobium cellulans is one of the microorganisms that produces a wide variety of yeast cell walldegradingenzymes, β-1,3-glucanase, protease and chitinase. Dried cells of Saccharomyces cerevisiae were used as carbon and nitrogen source for cell growth and protease production. The medium components KH2PO4, KOH and dried yeast cells showed a significant effect (p<0.05) on the factorial fractional design. A second design was prepared using two factors: pH and percentage of dried yeast cells. The results showedthat the culture medium for the maximum production of protease was 0.2 g/l of MgSO4.7H2O, 2.0 g/l of(NH4)2SO4 and 8% of dried yeast cells in 0.15M phosphate buffer at pH 8.0. The maximum alkaline protease production was 7.0 ± 0.27 U/ml over the center point. Crude protease showed best activity at 50ºC and pH 7.0-8.0, and was stable at 50ºC.

Cellulosimicrobium cellulans é um microrganismo que produz uma variedade de enzimas que hidrolisam a parede celular de leveduras: β-1,3-glucanase, protease e quitinase. Célulasdesidratadas de Saccharomyces cerevisiae foram usadas como fonte de carbono e nitrogênio para o crescimento celular e produção de protease. Os componentes do meio de cultura: KH2PO4, KOH e células de levedura desidratadas mostraram efeitos significativos (p<0,05) no planejamento experimental fracionário. Um segundo planejamento foi preparado usandodois fatores: pH e porcentagem de células de levedura desidratadas. Os resultados mostraram que o meio de cultura para a produção máxima de protease foi 0,2 g/L de MgSO4.7H2O;2,0 g/L de (NH4)2SO4 e 8% de células de levedura desidratadas em tampão fosfato 0,15M e pH 8,0. A produção máxima de protease alcalina foi 7,0 ± 0,27 U/mL no ponto central. A proteasebruta apresentou atividade ótima a 50ºC e pH 7,0-8,0; e foi estável a 50ºC.