Establishment of a Brazilian Line of Human Embryonic Stem Cells in Defined Medium: Implications for Cell Therapy in an Ethnically Diverse Population

Pluripotent human embryonic stem (hES) cells are an important experimental tool for basic and applied research, and a potential source of different tissues for transplantation. However, one important challenge for the clinical use of these cells is the issue of immunocompatibility, which may be dealt with by the establishment of hES cell banks to attend different populations. Here we describe the derivation and characterization of a line of hES cells from the Brazilian population, named BR-I, in commercial defined medium. In contrast to the other hES cell lines established in defined medium, BR-I maintained a stable normal karyotype as determined by genomic array analysis after 6 months in continuous culture (passage 29). To our knowledge, this is the first reported line of hES cells derived in South America. We have determined its genomic ancestry and compared the HLA-profile of BR-I and another 22 hES cell lines established elsewhere with those of the Brazilian population, finding they would match only 0.011% of those individuals. Our results highlight the challenges involved in hES cell banking for populations with a high degree of ethnic admixture.

Male and female odors induce Fos expression in chemically defined neuronal population

Olfactory information modulates innate and social behaviors in rodents and other species. Studies have shown that the medial nucleus of the amygdala (MEA) and the ventral premammillary, nucleus (PMV) are recruited by conspecific odor stimulation. However, the chemical identity of these neurons is not determined. We exposed sexually inexperienced male rats to female or male odors and assessed Fos immunoreactivity (Fos-ir) in neurons expressing NADPH diaphorase activity (NADPHd, a nitric oxide synthase), neuropeptide Urocortin 3, or glutamic acid decarboxylase rnRNA (GAD-67, a GABA-synthesizing enzyme) in the MEA and PMV. Male and female odors elicited Fos-ir in the MEA and PMV neurons, but the number of Fos-immunoreactive neurons was higher following female odor exposure, in both nuclei. We found no difference in odor induced Fos-ir ill the MEA and PMV comparing fed and fasted animals. Ill the MEA, NADPHd neurons colocalized Fos-ir only in response to female odors. In addition, Urocortin 3 neurons comprise a distinct population and they do not express Fos-ir after conspecific odor stimulation. We found that 80% of neurons activated by male odors coexpressed GAD-67 mRNA. Following female odor, 50% of Fos neurons coexpressed GAD-67 rnRNA. The PMV expresses very little GAD-67, and virtually no colocalization with Fos was observed. We found intense NADPHd activity in PMV neurons, some of which coexpressed Fos-ir after exposure to both odors. The majority of the PMV neurons expressing NADPHd colocalized cocaine-and amphetamine-regulated transcript (CART). Our findings suggest that female and male odors engage distinct neuronal populations in the MEA, thereby inducing contextualized behavioral responses according to olfactory cues. In the PMV, NADPHd/CART neurons respond to male and female odors, suggesting a role in neuroendocrine regulation in response to olfactory cues. (C) 2009 Elsevier Inc. All rights reserved.

Preimplantation development and expression of Hsp-70 and Bax genes in bovine blastocysts derived from oocytes matured in alpha-MEM supplemented with growth factors and synthetic macromolecules

In vitro culture conditions affect both the maternal and embryonic expression of genes and is likely to alter both oocyte and embryo developmental competence. The search for better and less variable culture conditions simulating those in vivo has led to the development of defined culture media, with lower impact on the molecular reprogramming of oocytes and embryos. We evaluated embryo development and relative abundance (RA) of Hsp-70 and Bax transcripts in bovine blastocysts produced from oocytes matured in a chemically defined IVM system with synthetic polymers. Immature cumulus oocyte complexes (COCs) were matured for 22-24 h in alpha-MEM supplemented with IGF-1, insulin, 0.1% polyvinyl alcohol (PVA), or 0.1% polyvinylpyrrolidone (PVP), but without FSH or LH. The control group consisted of COCs matured it, TCM plus FSH and 10% estrous cow serum. After fertilization. presumptive zygotes were co-cultured with cumulus cells until 224 h post-insemination. Total RNA was isolated from embryo pools, reverse transcribed into cDNA, and subjected to transcript analysis by real-time PCR. Cleavage rate was higher (P < 0.05) for the control group (68.3%) than for the PVA (54.4%) and PVP-40 (58.3%) groups. Nevertheless. there was no difference among the PVA, PVP-40 and control groups in blastocyst or hatching rates. similarly, no difference in relative abundance of Hsp-70 and Bax transcripts was detected in comparison to the control group. We inferred that bovine oocytes can be matured in serum- and gonadotrophin-free medium supplemented with PVA or PVP, enriched with IGF-I and insulin, without altering post-cleavage development and relative abundance of some genes associated with stress and apoptosis. (C) 2009 Elsevier Inc. All rights reserved.

Adjustment of Mineral Elements in the Culture Medium for the Micropropagation of Three Vriesea Bromeliads from the Brazilian Atlantic Forest: The Importance of Calcium

Many different species of Bromeliaceae are endangered and their conservation requires specific knowledge of their growth habits and propagation. In vitro culture of bromeliads is an important method for efficient clonal propagation and ill vitro seed g,germination can be used to maintain genetic variability. The present work aims to evaluate the in vitro growth and nutrient concentration in leaves of the epiphyte bromeliads Vriesea friburguensis Mez, Vriesea hieroglyphica (Carriere) E. Morren, and Vriesea unilateralis Mez, which exhibit slow rates of growth in vivo and in vitro. Initially, we compared the endogenous mineral composition of bromeliad plantlets grown in half-strength Murashige and Skoog (MS) medium and the mineral composition considered adequate in the literature. This approach suggested that calcium (Ca) is a critical nutrient and this was considered for new media formulation. Three new culture media were defined in which the main changes to half-strength MS medium were an increase in Ca, magnesium, sulfur, copper, and chloride and a decrease in iron, maintaining the nitrate: ammonium rate at approximate to 2:1. The main difference among the three new media formulated was Ca concentration, which varied from 1.5 mm in half-strength MS to 3.0, 6.0, and 12 mm in M2, M3, and M4 media, respectively. Consistently, all three species exhibited significantly higher fresh and dry weight on M4, the newly defined medium with the highest level of Ca (12 mm). Leaf nitrogen, potassium, zinc, magnesium and boron concentrations increased as Ca concentration in the medium increased from 1.5 to 12 mm.

Comparison of the efficacy of two commercially available media for culturing one-cell embryos in the in vitro fertilization mouse model

Objective: To examine the effects of two commercial media on the development of mouse ova fertilized in vitro to the blastocyst stage. Design: Animal model. Setting: Academic institution. Animal(s): Eight-week old, superovulated mice. Intervention(s): One-cell embryos cultured in vitro up to the blastocyst stage in potassium-enriched simplex optimized medium (KSOM) or G1/G2 medium. Main Outcome Measure(s): Blastocyst and hatching rates, total cell number count, and proportion of allocation of cells to the inner cell mass (ICM) and trophectoderm (TE). Result(s): The percentage of zygotes that developed to the blastocyst stage 96 and 120 hours after insemination was statistically significantly higher in the KSOM group. The percentage of blastocysts that partially or completely hatched by day 5 of culture was 84% and 71% for the KSOM and G1/G2 groups, respectively, showing a statistically significant difference between the groups. The mean number of ICM cells was 11.7 +/- 4.0 and 9.2 +/- 5.2 for the zygotes cultured in KSOM and G1/G2 media, respectively, revealing a statistically significantly higher cell number in the ICM of blastocysts derived from culture in KSOM medium. The ICM/TE ratio in the blastocysts cultured in KSOM or G1/G2 media was similar in both groups. Conclusion(s): Commercially available KSOM medium is superior to sequential G1/G2 media for culturing one-cell embryos up to the blastocyst stage in the mouse IVF model.

Global gene expression under nitrogen starvation in Xylella fastidiosa: contribution of the sigma(54) regulon

Background: Xylella fastidiosa, a Gram-negative fastidious bacterium, grows in the xylem of several plants causing diseases such as citrus variegated chlorosis. As the xylem sap contains low concentrations of amino acids and other compounds, X. fastidiosa needs to cope with nitrogen limitation in its natural habitat. Results: In this work, we performed a whole-genome microarray analysis of the X. fastidiosa nitrogen starvation response. A time course experiment (2, 8 and 12 hours) of cultures grown in defined medium under nitrogen starvation revealed many differentially expressed genes, such as those related to transport, nitrogen assimilation, amino acid biosynthesis, transcriptional regulation, and many genes encoding hypothetical proteins. In addition, a decrease in the expression levels of many genes involved in carbon metabolism and energy generation pathways was also observed. Comparison of gene expression profiles between the wild type strain and the rpoN null mutant allowed the identification of genes directly or indirectly induced by nitrogen starvation in a sigma(54)-dependent manner. A more complete picture of the sigma(54) regulon was achieved by combining the transcriptome data with an in silico search for potential sigma(54)-dependent promoters, using a position weight matrix approach. One of these sigma(54)-predicted binding sites, located upstream of the glnA gene (encoding glutamine synthetase), was validated by primer extension assays, confirming that this gene has a sigma(54)-dependent promoter. Conclusions: Together, these results show that nitrogen starvation causes intense changes in the X. fastidiosa transcriptome and some of these differentially expressed genes belong to the sigma(54) regulon.

ETHANOL PRODUCTION FROM XYLOSE BY Pichia stipitis NRRL Y-7124 IN A STIRRED TANK BIOREACTOR

The ethanol production by Pichia stipitis was evaluated in a stirred tank bioreactor using semi-defined medium containing xylose (90.0 g/l) as the main carbon source. Experimental assays were performed according to a 2(2) full factorial design to evaluate the influence of aeration (0.25 to 0.75 vvm) and agitation (150 to 250 rpm) conditions on ethanol production. In the studied range of values, the agitation increase and aeration decrease favored ethanol production, which was maximum (26.7 g/l) using 250 rpm and 0.25 vvm, conditions that gave a volumetric oxygen transfer coefficient (k(L)a value) of 4.9 h(-1). Under these conditions, the ethanol yield factor, ethanol productivity, and the process efficiency were 0.32 g/g, 0.32 g/l.h, and 63%, respectively. These results are promising and contribute to the development of a suitable process for ethanol production from xylose by Pichia stipitis.

Insufficient uracil supply in fully aerobic chemostat cultures of Saccharomyces cerevisiae leads to respiro-fermentative metabolism and double nutrient-limitation

A combination of chemostat cultivation and a defined medium was used to demonstrate that uracil limitation leads to a drastic alteration in the physiology of auxotrophic cells of Saccharomyces cerevisiae. Under this condition, the carbon source is dissimilated mainly to ethanol and acetate, even in fully aerobic cultures grown at 0.1 h(-1), which is far below the critical dilution rate. Differently from nitrogen-, sulphur-, or phosphate-limited cultures, uracil limitation leads to residual sugar (either glucose or sucrose) concentrations below 2 mM, which characterizes a situation of double-limitation: by the carbon source and by uracil. Furthermore, the specific rates of CO(2) production and O(2) consumption are increased when compared to the corresponding prototrophic strain. We conclude that when auxotrophic strains are to be used for quantitative physiological studies, special attention must be paid to the cultivation conditions, mainly regarding medium formulation, in order to avoid limitation of growth by the auxotrophic nutrient.

Physiological diversity within the kluyveromyces marxianus species

The Kluyveromyces marxianus strains CBS 6556, CBS 397 and CBS 712(T) were cultivated on a defined medium with either glucose, lactose or sucrose as the sole carbon source, at 30 and 37A degrees C. The aim of this work was to evaluate the diversity within this species, in terms of the macroscopic physiology. The main properties evaluated were: intensity of the Crabtree effect, specific growth rate, biomass yield on substrate, metabolite excretion and protein secretion capacity, inferred by measuring extracellular inulinase activity. The strain Kluyveromyces lactis CBS 2359 was evaluated in parallel, since it is the best described Kluyveromyces yeast and thus can be used as a control for the experimental setup. K. marxianus CBS 6556 presented the highest specific growth rate (0.70 h(-1)) and the highest specific inulinase activity (1.65 U mg(-1) dry cell weight) among all strains investigated, when grown at 37A degrees C with sucrose as the sole carbon source. The lowest metabolite formation and highest biomass yield on substrate (0.59 g dry cell weight g sucrose(-1)) was achieved by K. marxianus CBS 712(T) at 37A degrees C. Taken together, the results show a systematic comparison of carbon and energy metabolism among three of the best known K. marxianus strains, in parallel to K. lactis CBS 2359.

Screening for antifungal activities of extracts of the brazilian seaweed genus Laurencia (Ceramiales, Rhodophyta)

The resistance of pathogens to commonly used antibiotics has enhanced morbidity and mortality and has triggered the search for new drugs. Several species of the red alga genus Laurencia are very interesting candidates as potential sources of natural products with pharmaceutical activity because they are known to produce a wide range of chemically interesting halogenated secondary metabolites. This is an initial report of the antifungal activities of the secondary metabolites of five species of Laurencia, collected in the state of Espírito Santo, against three strains of pathogenic fungi: Candida albicans (CA), Candida parapsilosis (CP), and Cryptococcus neoformans (CN). Minimum inhibitory concentrations (MIC) of the algal extracts were determined by serial dilution method in RPMI 1640 Medium in 96-well plates according to the NCCLS and microbial growth was determined by absorbance at 492nm. A result showing maintenance or reduction of the inoculum was defined as fungistatic, while fungicidal action was no observed growth in the 10 µL fungistatic samples subcultured in Sabouraud Agar. Our results indicate that apolar extracts of Laurencia species possess antifungal properties and encourage continued research to find new drugs for therapy of infectious diseases in these algae.

Effect of recombinant human bone morphogenetic protein-7 (rhBMP-7) on the viability, proliferation and differentiation of osteoblast-like cells cultured on a chemically modified titanium surface

Aim: The aim of the present study was to assess the influence of the chemical characteristics and roughness of titanium surfaces on the viability, proliferation and differentiation of osteoblast-like cells cultured in a medium supplemented with recombinant human bone morphogenetic protein-7 (rhBMP-7). Material and methods: Osteo-1 cells were grown on titanium disks presenting with the following surfaces: (1) machined, (2) coarse grit-blasted and acid-attacked (SLA) and (3) chemically modified SLA (SLAmod) in the absence or presence of 20 ng/ml rhBMP-7 in culture medium. The viability and number of osteo-1 cells were evaluated after 24 h. Analyses of total protein content (TP) and alkaline phosphatase (AP) activity at 7, 14 and 21 days, collagen content at 7 and 21 days and mineralized matrix formation at 21 days were performed. Results: Cell viability (P=0.5516), cell number (P=0.3485), collagen content (P=0.1165) and mineralized matrix formation (P=0.5319) were not affected by the different surface configurations or by the addition of rhBMP-7 to the medium. Osteo-1 cells cultured on SLA surfaces showed a significant increase in TP at 21 days. The ALPase/TP ratio (P=0.00001) was affected by treatment and time. Conclusion: The results suggest that the addition of rhBMP-7 to the culture medium did not exert any effect on the viability, proliferation or differentiation of osteoblast-like cells grown on the different surfaces tested. All titanium surfaces analyzed allowed the complete expression of the osteoblast phenotype such as matrix mineralization by osteo-1 cells.

In vitro effect of low intensity laser on the cytotoxicity produced by substances released by bleaching gel

This in vitro study aimed to analyze the effect of different parameters of phototherapy with low intensity laser on the viability of human dental pulp fibroblasts under the effect of substances released by bleaching gel. Cells were seeded into 96 wells plates (1 x 10³ cells/well) and placed in contact with culture medium conditioned by a 35 % hydrogen peroxide bleaching gel for 40 minutes, simulating the clinical condition of the in-office bleaching treatment. Cells cultured in ideal growth conditions served as positive control group (PC), and the cells grown in conditioned medium and non-irradiated served as negative control group (NC). Cells grown in conditioned medium were submitted to a single irradiation with a diode laser (40 mW, 0.04 cm²) emitting at visible red (660 nm; RL) or near infrared (780 nm; NIR) using punctual technique, in contact mode and energy densities of 4, 6 or 10 J/cm². The cell viability was analyzed through the MTT reduction assay immediately and 24 hours after the irradiation. The data was compared by ANOVA followed by the Tukey's test (p < 0.05). The cell viability increased significantly in 24 hours within each group. The PC presented cell viability significantly higher than NC in both experimental times. Only the NIR/10 J/cm² group presented cell viability similar to that of PC in 24 hours. The phototherapy with low intensity laser in defined parameters is able to compensate the cytotoxic effects of substances released by 35 % hydrogen peroxide bleaching gel.

Effect of culture medium on biocalcification by Pseudomonas Putida, Lysinibacillus Sphaericus and Bacillus Subtilis

The objective of this study is to investigate the efficiency of calcium carbonate bioprecipitation by Lysinibacillus sphaericus, Bacillus subtilis and Pseudomonas putida, obtained from the Coleção de Culturas do Instituto Nacional de Controle de Qualidade em Saúde (INCQS), as a first step in determining their potential to protect building materials against water uptake. Two culture media were studied: modified B4 containing calcium acetate and 295 with calcium chloride. Calcium consumption in the two media after incubation with and without the bacterial inoculum was determined by atomic absorption analysis. Modified B4 gave the best results and in this medium Pseudomonas putida INQCS 113 produced the highest calcium carbonate precipitation, followed by Lysinibacillus sphaericus INQCS 414; the lowest precipitation was produced by Bacillus subtilis INQCS 328. In this culture medium XRD analysis showed that Pseudomonas putida and Bacillus subtilis precipitated calcite and vaterite polymorphs while Lysinibacillus sphaericus produced only vaterite. The shape and size of the crystals were affected by culture medium, bacterial strain and culture conditions, static or shaken. In conclusion, of the three strains Pseudomonas putida INQCS 113 in modified B4 medium gave the best results precipitating 96% of the calcium, this strain thus has good potential for use on building materials.

Dipeptide synthesis in biphasic medium: evaluating the use of commercial porcine pancreatic lipase preparations and the involvement of contaminant proteases

Dipeptide syntheses starting from Ac-L-Tyr-OEt or Z-L-X-OMe (X: Asp, Tyr, Phe, Arg, Lys or Thr) and glycine amide in biphasic reaction media were achieved using two commercially available porcine pancreatic lipase (PPL) preparations (crude (cPPL) and purified PPL (pPPL)). Under the mild conditions employed, α-chymotrypsin, a pancreatic protease that also presents esterase activity, catalyzed Ac-L-Tyr-Gly-NH2 synthesis with high productivity. Product hydrolysis also occurred in most of the syntheses studied. Polyacrylamide gel electrophoresis, enzymatic assays employing specific chromogenic substrates and size-exclusion chromatography revealed that cPPL and pPPL contain contaminant proteases and, therefore, exhibit esterase and amidase activities. Overall, these data indicate that those contaminants may be the main catalysts of peptide bond synthesis when Nα-blocked-L-amino acid esters and the commercial PPL preparations are used. On the other hand, such data do not contest the possibility of using such enzyme preparations as an inexpensive source of catalysts for dipeptide synthesis under soft conditions.

Time trends and predictors of mortality from ill-defined causes in old age: 9 year folllow-up of the Bambuí cohort study (Brazil)

The study objective was to examine differentials in time trends and predictors of deaths assigned to symptoms, signs and ill-defined conditions in comparison with other ill-defined conditions (ill-defined cardiovascular diseases, cancer and injury) in a population-based cohort study. Of 1,606 baseline participants aged 60 years and over, 524 died during 9-year follow-up and were included in this study. Deaths coded to "symptoms" declined by 77% in the period from 1997-1999 to 2003-2005. Deaths coded to other ill-defined conditions remained unchanged. The calendar period 2003-2005 (RR = 0.25; 95%CI: 0.09-0.70) and in-hospital deaths (RR = 0.16; 95%CI: 0.08-0.34) were independently associated with "symptoms", but not with other ill-defined conditions. Baseline socio-demographic characteristics and chronic diseases were not predictors of these outcomes. International and national agencies have focused on the reduction of deaths assigned to "symptoms" to improve the registration of vital statistics, while other ill-defined conditions have received little attention. Our data provide evidence supporting the need to redress this situation.