Co-expression of SOX9 and SOX10 during melanocytic differentiation in vitro

Investigations into pigment cell biology have relied on the ability to culture both murine and human melanocytes, numerous melanoma cell lines and more recently, murine and human melanoblasts. Melanoblast culture requires medium supplemented with a range of growth factors including Stem Cell Factor, Endothelin-3 and Fibroblast Growth Factor-2, withdrawal of which causes the cells to differentiate into melanocytes. Using the human melanoblast culture system, we have now examined the expression and/or DNA binding activity of several transcription factors implicated in melanocytic development and differentiation. Of these, the POU domain factor BRN2 and the SOX family member SOX10 are both highly expressed in unpigmented melanocyte precursors but are down-regulated upon differentiation. In contrast, the expression levels of the previously described MITF and PAX3 transcription factors remain relatively constant during the melanoblast-melanocyte transition. Moreover, BRN2 ablated melanoma cells lack expression of SOX10 and MITF but retain PAX3. A novel finding implicates a second SOX protein, SOX9, as a potential melanogenic transcriptional regulator, as its expression level is increased following the down-regulation of BRN2 and SOX10 in differentiated melanoblasts. Our results suggest that a complex network of transcription factor interactions requiring proper temporal coordination is necessary for acquisition and maintenance of the melanocytic phenotype. (c) 2005 Elsevier Inc. All rights reserved.

Biodegradation of naphthalene-2-sulfonic acid present in tannery wastewater by bacterial isolates Arthrobacter sp 2AC and Comamonas sp 4BC

Two bacterial strains, 2AC and 4BC, both capable of utilizing naphthalene-2-sulfonic acid (2-NSA) as a sole source of carbon, were isolated from activated sludges previously exposed to tannery wastewater. Enrichments were carried out in mineral salt medium (MSM) with 2-NSA as the sole carbon source. 16S rDNA sequencing analysis indicated that 2AC is an Arthrobacter sp. and 4BC is a Comamonas sp. Within 33 h, both isolates degraded 100% of 2-NSA in MSM and also 2-NSA in non-sterile tannery wastewater. The yield coefficient was 0.33 g biomass dry weight per gram of 2-NSA. A conceptual model, which describes the aerobic transformation of organic matter, was used for interpreting the biodegradation kinetics of 2-NSA. The half-lives for 2-NSA, at initial concentrations of 100 and 500 mg/l in MSM, ranged from 20 h (2AC) to 26 h (4BC) with lag-phases of 8 h (2AC) and 12 h (4BC). The carbon balance indicates that 75-90% of the initial TOC (total organic carbon) was mineralized, 5-20% remained as DOC (dissolved organic carbon) and 3-10% was biomass carbon. The principal metabolite of 2-NSA biodegradation (in both MSM and tannery wastewater) produced by Comamonas sp. 4BC had a MW of 174 and accounted for the residual DOC (7.0-19.0% of the initial TOC and 66% of the remaining TOC). Three to ten percent of the initial TOC (33% of the remaining TOC) was associated with biomass. The metabolite was not detected when Arthrobacter sp. 2AC was used, and a lower residual DOC and biomass carbon were recorded. This suggests that the two strains may use different catabolic pathways for 2-NSA degradation. The rapid biodegradation of 2-NSA (100 mg/l) added to non-sterile tannery wastewater (total 2-NSA, 105 mg/l) when inoculated with either Arthrobacter 2AC or Comamonas 4BC showed that both strains were able to compete with the indigenous microorganisms and degrade 2-NSA even in the presence of alternate carbon sources (DOC in tannery wastewater = 91 mg/l). The results provide information useful for the rational design of bioreactors for tannery wastewater treatment.

Generation and maturation of dendritic cells for clinical application under serum-free conditions

Monocyte-derived dendritic cells (MoDCs) in clinical use for cancer immunotherapy are ideally generated in serum-free medium (SFM) with inclusion of a suitable maturation factor toward the end of the incubation period. Three good manfacturing practice (GMP) grade SFMs (AIM-V, X-VIVO 15, and X-VIVO 20) were compared with RPMI-1640, supplemented with 10% fetal bovine serum or 10% human serum. DCs generated for 7 days in SFM were less mature and secreted less interleukin (IL) 12p70 and IL-10 than DCs generated in 10% serum. DC yield was comparable in SFMs, and a greater proportion of cells was viable after maturation. Toll-like receptor (TLR) ligands were compared for their ability to induce cytokine secretion under serum-free conditions in the presence of interferon (IFN) gamma. With the exception of Poly I:C, TLR ligands stimulated high levels of IL-10 secretion. High levels of IL-12p70 were induced by two TLR4-mediated stimuli, lipopolysaccharide and Ribomunyl, a clinical-grade bacterial extract. When T-cell responses were compared in allogeneic mixed leukocyte reaction, DCs stimulated with Ribomunyl induced higher levels of IFN gamma than DCs stimulated with the cytokine cocktail: tumor necrosis factor-alpha, IL-1 beta, IL-6, and prostaglandin E-2. In the presence of IL-10 neutralizing antibodies, DC IL-12p70 production and T-cell IFN gamma were increased in vitro. Similarly, DCs stimulated with Ribomunyl, IFN gamma, and anti-IL-10 induced high levels of tetanus toxoid-specific T-cell proliferation and IFN gamma secretion. Thus, MoDCs generated ill SFM efficiently stimulate T-cell IFN gamma production after maturation in the presence of a clinical-grade TLR4 agonist and IL-10 neutralization.

Thermal strain in the mushy zone related to hot tearing

A volume-averaged two-phase model addressing the main transport phenomena associated with hot tearing in an isotropic mushy zone during solidification of metallic alloys has recently been presented elsewhere along with a new hot tearing criterion addressing both inadequate melt feeding and excessive deformation at relatively high solid fractions. The viscoplastic deformation in the mushy zone is addressed by a model in which the coherent mush is considered as a porous medium saturated with liquid. The thermal straining of the mush is accounted for by a recently developed model taking into account that there is no thermal strain in the mushy zone at low solid fractions because the dendrites then are free to move in the liquid, and that the thermal strain in the mushy zone tends toward the thermal strain in the fully solidified material when the solid fraction tends toward one. In the present work, the authors determined how variations in the parameters of the constitutive equation for thermal strain influence the hot tearing susceptibility calculated by the criterion. It turns out that varying the parameters in this equation has a significant effect on both liquid pressure drop and viscoplastic strain, which are key parameters in the hot tearing criterion. However, changing the parameters in this constitutive equation will result in changes in the viscoplastic strain and the liquid pressure drop that have opposite effects on the hot tearing susceptibility. The net effect on the hot tearing susceptibility is thus small.

Anomalous scattering analysis of Agrobacterium radiobacter phosphotriesterase: the prominent role of iron in the heterobinuclear active site

Bacterial phosphotriesterases are binuclear metalloproteins for which the catalytic mechanism has been studied with a variety of techniques, principally using active sites reconstituted in vitro from apoenzymes. Here, atomic absorption spectroscopy and anomalous X-ray scattering have been used to determine the identity of the metals incorporated into the active site in vivo. We have recombinantly expressed the phosphotriesterase from Agrobacterium radiobacter (OpdA) in Escherichia coli grown in medium supplemented with 1 mM CoCl2 and in unsupplemented medium. Anomalous scattering data, collected from a single crystal at the Fe-K, Co-K and Zn-K edges, indicate that iron and cobalt are the primary constituents of the two metal-binding sites in the catalytic centre (alpha and P) in the protein expressed in E. coli grown in supplemented medium. Comparison with OpdA expressed in unsupplemented medium demonstrates that the cobalt present in the supplemented medium replaced zinc at the beta-position of the active site, which results in an increase in the catalytic efficiency of the enzyme. These results suggest an essential role for iron in the catalytic mechanism of bacterial phosphotriesterases, and that these phosphotriesterases are natively heterobinuclear iron-zinc enzymes.

Bem-estar subjetivo e bem-estar no trabalho em profissionais de educação física

Atualmente, bem-estar cada vez mais ganha espaço como parte importante e integrante da vida das pessoas. Pesquisas vêm buscando destacar fatores positivos que exerçam influencia em sua vida pessoal, e conseqüentemente em seu trabalho. O objetivo deste estudo foi verificar as relações entre bem-estar subjetivo (satisfação geral com a vida, afetos positivos e afetos negativos) e bem-estar no trabalho (satisfação no trabalho, envolvimento com o trabalho e comprometimento organizacional afetivo) em professores de educação física que atuavam em escolas e academias, descrevendo os níveis de bem-estar subjetivo e bem-estar no trabalho, análises de variância e correlação entre os construtos. A amostra foi composta por 124 professores de educação física, 34 atuavam em escolas particulares e 90 atuavam em academias, sendo 81 do sexo masculino e 43 do sexo feminino, solteiros e casados e faixa etária entre 21 e 59 anos, com escolaridade distribuída desde o ensino superior completo até o mestrado concluído. O instrumento de coleta foi um questionário auto-aplicavel composto por cinco escalas que mediram as variáveis do estudo e um questionário de dados complementares. Os resultados deste estudo revelaram que BES e BET guardam relações entre si. Os professores são considerados pessoas relativamente felizes com sua vida pessoal e revelaram uma relação mediana no que se refere a sua vida profissional. Os resultados das correlações relacionadas ao BES sinalizaram que o acúmulo de experiências negativas ao longo da vida poderia reduzir a vivencia de experiências positivas e de avaliações positivas sobre a vida em geral e vice-versa e ao BET, satisfação no trabalho, envolvimento com o trabalho e comprometimento organizacional afetivo são interdependentes. No que se refere às comparações, para BES os professores que atuavam em academias obtiveram médias significativamente maiores de afetos negativos do que os de escolas e por outro lado, a média de satisfação geral com a vida dos professores de escolas foi significativamente superior à dos que atuavam em academias demonstrando que estes vivenciaram mais experiências negativas do que os que trabalhavam em escolas e que os professores de escola eram mais satisfeitos com sua vida do que os de academias. Para BET, o quadro geral se mantém semelhante para professores de educação física que atuavam em escolas e academias. Futuros estudos deveriam aumentar o número de professores bem como, investigar outras áreas de atuação da educação física e compara-las com o presente estudo ampliando os estudos envolvendo bemestar e professores de educação física.(AU)

Investigation of the role of protein kinase C (PKC) in cytostasis induced by tumour promoting phorbol esters and related compounds

Protein kinase C (PKC) is considered to be the major receptor for tumour promoting phorbol esters such as 12-0- tetradecanoylphorbol-13-acetate (TPA). These agents evoke a plethora of biological effects on cells in culture. The growth of A549 human lung carcinoma cells maintained in medium fortified with 10% foetal calf serum (FCS) is arrested for 6 days by TPA and other biologically active phorbol esters. In the work described in this thesis, the hypothesis was tested that modulation of PKC activity is closely related to events pivotal for cytostasis to occur. The effect of several phorbol esters, of newly synthesized analogues of diacylglycerols (DAG) and of bryostatins (bryos) on cell growth and ability to modulate activity of PKC has been investigated.Determination of the subcellular distribution of PKC following treatment of cells with TPA and partial enzyme purification by non-denaturing poly-acrylamide gel electrophoresis revealed translocation of enzyme activity from cytosoUc to paniculate fraction. Chronic exposure of cells to TPA resulted in a time and concentration dependent degradation of enzyme activity. Synthetic DAG and DAG analogues, unable to arrest the growth of cells at non-toxic concentrations, were neither able to affect subcellular PKC distribution nor compete effectively for phorbol ester binding sites at physiologically relevant concentrations. Bryos 1,2,4 and 5, natural products, possessing antineoplastic activity in mice, elicited transient arrest of A549 cell growth in vitro. They successfully competed for phorbol ester receptors in A549 cells with exquisite affinity and induced a shift in sub-cellular PKC distribution, though not to the same extent as PTA. Enzyme down-regulation resulted from prolonged exposure of cells to nanomolar concentrations of bryos. In vivo studies demonstrated that neither PDBu nor bryo 1 was able to inhibit A549 xenograft growth in athymic mice. The growth of A549 cell populations cultured under conditions of serum-deprivation was inhibited only transiently by biologically active phorbol esters. Fortification of serum-free medium with EGF or fetuin was able to partially restore sensitivity to maintained growth arrest by PTA. PKC translocation to the paniculate cellular fraction and subsequent enzyme down-regulation, induced by TPA, occurred in a manner similar to that observed in serum-supplemented cells. However, total PKC activity and cytosolic phorbol ester binding potential were greatly reduced in the serum-deprived cell population. Western blot analysis using monospecific monoclonal antibodies revealed the presence of PKC-a in both A549 cell populations, with significantly reduced protein levels in serum- deprived cells. PKC-/9 was not detected in either cell population.

Mouse embryo culture induces changes in postnatal phenotype including raised systolic blood pressure

A key factor in the use of assisted reproductive technologies (ART) for diverse species is the safety of procedures for long-term health. By using a mouse model, we have investigated the effect of in vitro culture and embryo transfer (ET) of superovulated embryos on postnatal growth and physiological activity compared with that of embryos developing in vivo. Embryo culture from two-cell to blastocyst stages in T6 medium either with or without a protein source reduced blastocyst trophectoderm and inner cell mass cell number compared with that of embryos developing in vivo. Embryo culture and ET had minimal effects on postnatal growth when compared with in vivo development with an equivalent litter size. However, embryo culture, and to a lesser extent ET, led to an enhanced systolic blood pressure at 21 weeks compared with in vivo development independent of litter size, maternal origin, or body weight. Moreover, activity of enzymatic regulators of cardiovascular and metabolic physiology, namely, serum angiotensin-converting enzyme and the gluconeogenesis controller, hepatic phosphoeno/pyruvate carboxykinase, were significantly elevated in response to embryo culture and/or ET in female offspring at 27 weeks, independent of maternal factors and postnatal growth. These animal data indicate that postnatal physiological criteria important in cardiovascular and metabolic health may be more sensitive to routine ART procedures than growth. © 2007 by The National Academy of Sciences of the USA.

High permeability of the anionic form restricts accumulation of indomethacin by cultured gastric surface epithelial cells exposed to low apical pH

The 'ion-trapping' hypothesis suggests that the intracellular concentration of acidic non-steroidal anti-inflammatory drugs in gastric epithelial cells could be much higher than in the gastric lumen, and that such accumulation could contribute to their gastrotoxicity. Our aim was to examine the effect of the pH of the apical medium on the apical to basal transfer of the acidic drug indomethacin (pK a 4.5) across a gastric mucous epithelial cell monolayer, and to determine whether indomethacin accumulated in cells exposed to a low apical pH. Guinea-pig gastric mucous epithelial cells were grown on porous membrane culture inserts (Transwells®) for 72 h. Transfer and accumulation of [ 14C] indomethacin were assessed by scintillation counting. Transfer of [ 3H]mannitol and measurement of trans-epithelial electrical resistance were used to assess integrity of the monolayer. Distribution of [ 14C] urea was used to estimate the intracellular volume of the monolayer. The monolayer was not disrupted by exposure of the apical face to media of pH ≥ 3, or by indomethacin. Transfer of indomethacin (12 μM) to the basal medium increased with decreasing apical medium pH. The apparent permeability of the undissociated acid was estimated to be five times that of the anion. The intracellular concentration of indomethacin was respectively 5.3, 4.1 and 4.3 times that in the apical medium at pH 5.5, 4.5 and 3.0. In conclusion, this study represents the first direct demonstration that indomethacin accumulates in gastric epithelial cells exposed to low apical pH. However, accumulation of indomethacin was moderate and the predictions of the ion-trapping hypothesis were not met, probably due to the substantial permeability of anionic indomethacin across membranes. © 2006 Elsevier B.V. All rights reserved.

Serum-free process development:improving the yield and consistency of human mesenchymal stromal cell production

Background aims: The cost-effective production of human mesenchymal stromal cells (hMSCs) for off-the-shelf and patient specific therapies will require an increasing focus on improving product yield and driving manufacturing consistency. Methods: Bone marrow-derived hMSCs (BM-hMSCs) from two donors were expanded for 36 days in monolayer with medium supplemented with either fetal bovine serum (FBS) or PRIME-XV serum-free medium (SFM). Cells were assessed throughout culture for proliferation, mean cell diameter, colony-forming potential, osteogenic potential, gene expression and metabolites. Results: Expansion of BM-hMSCs in PRIME-XV SFM resulted in a significantly higher growth rate (P < 0.001) and increased consistency between donors compared with FBS-based culture. FBS-based culture showed an inter-batch production range of 0.9 and 5 days per dose compared with 0.5 and 0.6 days in SFM for each BM-hMSC donor line. The consistency between donors was also improved by the use of PRIME-XV SFM, with a production range of 0.9 days compared with 19.4 days in FBS-based culture. Mean cell diameter has also been demonstrated as a process metric for BM-hMSC growth rate and senescence through a correlation (R² = 0.8705) across all conditions. PRIME-XV SFM has also shown increased consistency in BM-hMSC characteristics such as per cell metabolite utilization, in vitro colony-forming potential and osteogenic potential despite the higher number of population doublings. Conclusions: We have increased the yield and consistency of BM-hMSC expansion between donors, demonstrating a level of control over the product, which has the potential to increase the cost-effectiveness and reduce the risk in these manufacturing processes.

Avaliação do potencial da fibra e casca de coco maduro, casca de coco verde e cacto pré-tratados visando à produção de etanol

The present work investigated the potential of different residual lignocellulosic materials generated in rural and urban areas (coconut fibre mature, green coconut shell and mature coconut shell), and vegetable cultivated in inhospitable environments (cactus) aimed at the production of ethanol, being all materials abundant in the Northeast region of Brazil. These materials were submitted to pretreatments with alkaline hydrogen peroxide followed by sodium hydroxide (AHP-SHP), autohydrolysis (AP), hydrothermal catalyzed with sodium hydroxide (HCSHP) and alkali ethanol organosolv (AEOP). These materials pretreated were submitted to enzymatic hydrolysis and strategies of simultaneous saccharification and fermentation (SSF) and saccharification and fermentation semi-simultaneous (SSSF) by Saccharomyces cerevisiae, Zymomonas mobilis and Pichia stipitis. It was also evaluated the presence of inhibitory compounds (hydroxymethylfurfural, furfural, acetic acid, formic acid and levulinic acid) and seawater during the fermentative process. Materials pretreated with AHP-SHP have resulted in delignification of the materials in a range between 54 and 71%, containing between 51.80 and 54.91% of cellulose, between 17.65 and 28.36% of hemicellulose, between 7.99 and 10.12% of lignin. Enzymatic hydrolysis resulted in the conversions in glucose between 68 and 76%. Conversion yields in ethanol using SSF and SSSF for coconut fibre mature pretreated ranged from 0.40 and 0.43 g/g, 0.43 and 0.45 g/g, respectively. Materials pretreated by AP showed yields of solids between 42.92 and 92.74%, containing between 30.65 and 51.61% of cellulose, 21.34 and 41.28% of lignin. Enzymatic hydrolysis resulted in glucose conversions between 84.10 and 92.52%. Proceeds from conversion into ethanol using green coconut shell pretreated, in strategy SSF and SSSF, were between 0.43 and 0.45 g/g. Coconut fibre mature pretreated by HCSHP presented solids yields between 21.64 and 60.52%, with increased in cellulose between 28.40 and 131.20%, reduction of hemicellulose between 43.22 and 69.04% and reduction in lignin between 8.27 and 89.13%. Enzymatic hydrolysis resulted in the conversion in glucose of 90.72%. Ethanol yields using the SSF and SSSF were 0.43 and 0.46 g/g, respectively. Materials pretreated by AEOP showed solid reductions between 10.75 and 43.18%, cellulose increase up to 121.67%, hemicellulose reduction up to 77.09% and lignin reduced up to 78.22%. Enzymatic hydrolysis resulted in the conversion of glucose between 77.54 and 84.27%. Yields conversion into ethanol using the SSF and SSSF with cactus pretreated ranged from 0.41 and 0.44 g/g, 0.43 and 0.46 g/g, respectively. Fermentations carried out in bioreactors resulted in yields and ethanol production form 0.42 and 0.46 g/g and 7.62 and 12.42 g/L, respectively. The inhibitory compounds showed negative synergistic effects in fermentations performed by P. stipitis, Z. mobilis and S. cerevisiae. Formic acid and acetic acid showed most significant effects among the inhibitory compounds, followed by hydroxymethylfurfural, furfural and levulinic acid. Fermentations carried out in culture medium diluted with seawater showed promising results, especially for S. cerevisiae (0.50 g/g) and Z. mobilis (0.49 g/g). The different results obtained in this study indicate that lignocellulosic materials, pretreatments, fermentative processes strategies and the microorganisms studied deserve attention because they are promising and capable of being used in the context of biorefinery, aiming the ethanol production.

Jery und Bätely: Oper in einem Act

von Göthe ; Musik von Ingeborg von Bronsart ; Clavierauszug.

Lo schiavo: dramma lirico in quattro atti

di Rodolfo Paravicini ; musica di A. Carlos Gomes ; opera completa per canto e pianoforte, riduzione di G. Loscar.

Die Jahrszeiten

von Joseph Haydn.

Lutèce: symphonie dramatique

Le départ -- Le champ de bataille -- Après la défaite.