Genomic DNA microarray comparison of gene expression patterns in Paracoccidioides brasiliensis mycelia and yeasts in vitro

Paracoccidioides brasiliensis is a thermally dimorphic fungus, and causes the most prevalent systemic mycosis in Latin America. Infection is initiated by inhalation of conidia or mycelial fragments by the host, followed by further differentiation into the yeast form. Information regarding gene expression by either form has rarely been addressed with respect to multiple time points of growth in culture. Here, we report on the construction of a genomic DNA microarray, covering approximately 25% of the genome of the organism, and its utilization in identifying genes and gene expression patterns during growth in vitro. Cloned, amplified inserts from randomly sheared genomic DNA (gDNA) and known control genes were printed onto glass slides to generate a microarray of over 12 000 elements. To examine gene expression, mRNA was extracted and amplified from mycelial or yeast cultures grown in semi-defined medium for 5, 8 and 14 days. Principal components analysis and hierarchical clustering indicated that yeast gene expression profiles differed greatly from those of mycelia, especially at earlier time points, and that mycelial gene expression changed less than gene expression in yeasts over time. Genes upregulated in yeasts were found to encode proteins shown to be involved in methionine/cysteine metabolism, respiratory and metabolic processes (of sugars, amino acids, proteins and lipids), transporters (small peptides, sugars, ions and toxins), regulatory proteins and transcription factors. Mycelial genes involved in processes such as cell division, protein catabolism, nucleotide biosynthesis and toxin and sugar transport showed differential expression. Sequenced clones were compared with Histoplasma capsulatum and Coccidioides posadasii genome sequences to assess potentially common pathways across species, such as sulfur and lipid metabolism, amino acid transporters, transcription factors and genes possibly related to virulence. We also analysed gene expression with time in culture and found that while transposable elements and components of respiratory pathways tended to increase in expression with time, genes encoding ribosomal structural proteins and protein catabolism tended to sharply decrease in expression over time, particularly in yeast. These findings expand our knowledge of the different morphological forms of P. brasiliensis during growth in culture.

In vitro effects of propolis on Giardia duodenalis trophozoites

In order to improve the current chemotherapy of Giardia infection, potential antigiardial agents have been screened, including natural products. Propolis, a resinous hive product collected by bees, has attracted attention as a useful and popular substance with several therapeutic activities. The present study was carried out aiming to evaluate the in vitro effects of an ethanolic extract of propolis on the growth and adherence of Giardia duodenalis trophozoites. Propolis inhibited the growth of trophozoites and the level of inhibition varied according to the extract concentration and incubation times. The highest reduction of parasite growth was observed in cultures exposed to 125, 250 and 500 mu g/ml of propolis, in all incubation periods (24, 48, 72 and 96 h). Growth reduction by 50% was observed in 125 mu g/ml propolis-treated cultures, while the concentrations of 250 and 500 mu g/ml were able to inhibit growth by more than 60%. Propolis also inhibited parasite adherence and all assayed propolis concentrations promoted the detachment of trophozoites. Light microscope observations revealed changes of the pear-shaped aspect of the cell and reduction of flagellar beating frequency in the great part of the trophozoites. Our results hold the perspective for the utilization of propolis as an antigiardial agent. (c) 2005 Elsevier GmbH. All rights reserved.

In vitro effects of Crotalus durissus terrificus and Bothrops jararaca venoms on Giardia duodenalis trophozoites

Considering the snake venoms' pharmacological properties and chemotherapeutic potential as well as the need for new alternatives for Giardia infection treatment, the present study was carried out aiming to evaluate the in vitro effects of crude Crotalus durissus terrificus and Bothrops jararaca venoms on the growth and adherence of Giardia duodenalis trophozoites. Trophozoites (10(6)) were exposed to serial twofold dilutions of C. durissus terrificus and B. jararaca venoms that ranged from 3.125 to 200 mu g/ml and from 5 to 320 mu g/ml, respectively. The two venoms inhibited the growth of trophozoites, and the level of inhibition varied according to the assayed concentrations and incubation times. The highest reduction of parasite growth was observed with increasing concentrations of the venoms. No effect on parasite adherence was observed. Light microscope observations revealed changes of the pear-shape aspect of the cell and reduction of flagellar beating frequency in the great part of the trophozoites. This is the first attempt to evaluate the in vitro effects of snake venoms on Giardia trophozoites. The findings stress the need for more investigations and prompt us to perform further studies to identify active principles with antigiardial effect.

Ação do BAP na regeneração in vitro de Blc Owen Holmes Ponkan x Brassavola digbiana nº 2

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

In Vitro Antibacterial Activity of Adhesive Systems on Streptococcus Mutans

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

In vitro evaluation of the effectiveness of irrigants and intracanal medicaments on microorganisms within root canals

Aim To evaluate in vitro the effectiveness of sodium hypochlorite (NaOCl). chlorhexidine (CHX) and live intracanal medicaments on microorganisms within root canals.Methodology Ninety-six human single-rooted extracted teeth were used. After removing the crowns, canal preparation was completed and the external root Surfaces were coated with epoxy resin. Following sterilization. The teeth were contaminated with Candida albicans and enterococcus faecalis. and were incubated at 37 +/- 1 degreesC for 7 days. The teeth were divided according to the irrigant solution or intracanal medicament: group 1. sterile physiologic solution (SPS) and calcium hydroxide (Ca(OH)(2)) paste: group 2. SPS and camphorated paramonochlorophenol (CPMC): group 3.SPS and tricresol formalin: group 4, SPS and CaOH2 + CPMC paste: group 5, SPS and PMC furacin; group 6.2.5%, NaOCl without intracanal medication: group 7, 2.0% CHX without intracanal medication and group 8, SPS Without intracanal medication (control group). Microbiological samples were collected with sterile paper points, and bacterial growth was determined. The data were submitted to the analysis of variance (ANOVA. P = 0.05).Results For C. albicans, groups 3 and S were statistically less effective than groups 1, 2. 4 and 5 (Kruskal-Wallis (K-W) = 65.241; gl = 7; P = 0.001). For E. faecalis, groups 6 and 8 were statistically less effective than groups 1-4 and 7 (K-W = 61.048; gl = 7; P = 0.001).Conclusions Ca(OH)(2) + CPMC paste was the most effective intracanal medicament for the elimination of the two microorganisms; 2.0% CHX solution was more effective than 2.5% NaOCl against E. faecalis.

Alchornea glandulosa Ethyl Acetate Fraction Exhibits Antiangiogenic Activity: Preliminary Findings from In Vitro Assays Using Human Umbilical Vein Endothelial Cells

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Penicillium expansum versus antagonist yeasts and patulin degradation in vitro

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Eficácia do diflubenzuron 25% no controle da Haematobia irritans (Diptera: Muscidae): desafio in vitro e a campo

Avaliou-se, neste experimento, a eficácia in vitro e in vivo do diflubenzuron a 25% para uso em bovinos, no controle da infestação por Haematobia irritans. Para o teste in vitro, ovos de moscas-dos-chifres foram mantidos em recipientes contendo fezes de animais não-tratados ou tratados com diflubenzuron a 25%, e acompanhados até emergência dos adultos. No teste in vivo, foram utilizadas 40 fêmeas aneloradas, divididas em dois grupos: controle (C) e tratado (T) com intensidade parasitária equivalente. Durante o experimento, o grupo C recebeu apenas suplementação mineral, enquanto o grupo T recebeu suplementação mineral e diflubenzuron a 25%. A contagem de moscas nos animais foi realizada na região dorsal, desde a nuca até as pontas da anca de cada animal, no início e ao final de um período de cinco meses. Na avaliação in vitro, o grupo controle apresentou média de emergência de 86% (± 8,4%), enquanto o grupo cultivado em fezes de bovinos tratados com diflubenzuron a 25% apresentou taxa de emergência média de 1% (± 0,2%), sendo a eficácia calculada de 98,83%. No teste in vivo, não foi observada redução significativa na contagem de moscas no grupo C, porém, no grupo T houve significativa redução da infestação por H. irritans (t = 16,46, p < 0,0001). A eficácia do produto, em condições de campo, foi de 99,20%. O diflubenzuron a 25% adicionado ao sal mineral mostrou-se eficaz contra H. irritans, sendo indicado para esse fim.

In vitro evaluation of three different biomaterials as scaffolds for canine mesenchymal stem cells

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Reguladores vegetais e teores endógenos de poliaminas durante o desenvolvimento de taro cultivado in vitro

Neste trabalho foi investigada a assepsia para obtenção de explantes oriundos de tubérculos e a ação das poliaminas espermidina e espermina exógenas associadas aos reguladores vegetais AIA e BA no desenvolvimento, na tuberização in vitro e nos níveis endógenos de putrescina (Put), espermidina (Spd) e espermina (Spm) de taro (Colocasia esculenta). Plantas crescidas em meio contendo espermidina e espermina mostraram tuberização e a associação dessas poliaminas com AIA e BA induziu aumento do número de brotações. Para o estímulo da rizogênese, não foi necessário o uso de reguladores vegetais. Altos teores de putrescina foram encontrados durante a emissão de brotações, enquanto que altos teores de espermidina foram observados durante a formação de rizomas in vitro.

Avaliação do efeito das concentrações de sacarose e dos estádios de desenvolvimento do fruto no cultivo in vitro de embriões de frutos de cafeeiro

A cultura in vitro é uma técnica controlada que proporciona estudar os processos nutricionais, fisiológicos e bioquímicos de embriões em vários estádios de desenvolvimento. O objetivo foi avaliar a relação entre estádio de desenvolvimento do fruto e concentração de sacarose, no meio, durante o desenvolvimento, in vitro, de embriões de cafeeiro. Frutos de Coffea arabica cv. Acaiá foram colhidos, lavados, desinfestados, seus embriões excisados e inoculados em meio de cultivo MS, com pH ajustado para 5,8. Os tratamentos consistiram em combinação de concentrações de sacarose (0, 15, 30, 60, 90 e 120 g L-1) e estádios de desenvolvimento do fruto (chumbinho, chumbo, verde, verde-cana, cereja e passa). Após a inoculação, os embriões foram incubados em sala de crescimento, a 27 ± 1 ºC, fotoperíodo de 16 horas e 35 µ mol m-2 s-1 de intensidade luminosa. O delineamento experimental utilizado foi inteiramente casualizado, em esquema fatorial 6 x 6, com seis repetições constituídas por quatro tubos cada. Após 60 dias de desenvolvimento, as plântulas foram avaliadas com base no comprimento da parte aérea, massa de matéria fresca total da parte aérea e das raízes. Observaram-se influências das concentrações de sacarose e dos estádios de desenvolvimento do fruto no crescimento e desenvolvimento das plântulas. Resultados satisfatórios para todas as variáveis estudadas foram obtidos com embriões excisados no estádio verde, inoculados em meio de cultivo suplementado com 51 a 70 g L-1 de sacarose.

Alterações na atividade da peroxidase e do conteúdo de carboidratos em mandioca cultivada in vitro sob estresse salino

A influência de estresse induzido por cloreto de sódio (75 e 150 mM) sobre o conteúdo de carboidratos solúveis e atividade da peroxidase, foi estudada em plântulas de mandioca cultivadas in vitro. Os resultados mostraram que a atividade da peroxidase diminuiu gradualmente durante o crescimento de plântulas em todos os tratamentos. O conteúdo de açúcares redutores foi menor em plântulas submetidas a 75 mM de NaCl, nas fases mais adiantadas do desenvolvimento, em comparação com a dose mais elevada do sal (150 mM de NaCl) ou sua omissão. Os resultados obtidos indicaram que o NaCl alterou o metabolismo de carboidratos, atividade da peroxidase e o crescimento de plântulas cultivadas in vitro.

Multiplicação in vitro DE Ficus carica L.: efeito da cinetina e do ácido giberélico

A cultura da figueira é afetada pelo vírus-do-mosaico e a cultura de tecidos é uma alternativa para se proceder à limpeza clonal. Neste trabalho, objetivou-se estudar o efeito da cinetina e GA3 na multiplicação in vitro da figueira. Segmentos nodais foram inoculados em meio de cultura WPM contendo as seguintes combinações de cinetina (0; 0,5; 1; 2 e 4 mg.L-1) e GA3 (0, 2, 4, 6 e 8 mg.L-1). Avaliaram-se número e comprimento dos brotos, peso da matéria fresca e seca da parte aérea, número de raízes, peso da matéria fresca e seca do sistema radicular e de calos. A utilização de 0,5 mg.L-1 de cinetina promoveu melhor taxa de multiplicação in vitro de Ficus carica. O GA3 reduziu a formação e multiplicação dos brotos e induziu ao estiolamento, à hiperidricidade, clorose e necrose apical das plântulas.

Nutritionally induced changes in endosperm of shrunken-1 and brittle-2 maize kernels grown in vitro

Although mineral nutrition affects maize (Zea mays L.) yield by controlling starch deposition in kernels, the mechanisms involved are largely unknown. Our objectives were to examine this relationship by nutritionally and genetically altering starch production in the endosperm. Kernels of W64A and two starch-deficient mutants, shrunken-1 and brittle-2, were grown in vitro with varying supplies of N (0-50 mM) or P (0-6 mM) to produce different degrees of endosperm starch production, and the levels of enzyme activities and metabolites associated with carbohydrate and N metabolism were examined. In vitro grown kernels exhibited the expected starch phenotypes, and a minimum level of media N (25 mM) and P (2 mM) was required for optimal growth. However, increasing the availability of N or P could not overcome the genetically induced decrease in starch deposition of the mutants. Nitrogen deficiency enhanced sugar accumulation, but decreased amino acid levels, soluble protein, enzyme activity, starch synthesis, and endosperm dry weight. Phosphorous deficiency also decreased starch production and endosperm dry weight, but with only a minimal effect on the activities of ADP-glucose pyrophosphorylase and alanine transaminase. Genotypic differences in endosperm starch, and the increases induced by N and P supply, Here closely associated with the level of endosperm N, but not endosperm P. Thus, while both N and P are crucial for optimal yield of maize grain, they appear to act by different means, and with different importance in governing starch deposition in the endosperm.