Superprodução, purificação e caracterização parcial de complexos proteína-polissacáridos inovadores a partir de estirpes de basidiomicetos

O presente trabalho teve como objectivo o estudo de meios de cultura, tendo em vista a superprodução de complexos proteína-polissacáridos, intracelulares e extracelulares, a partir dos basidiomicetos Pleurotus ostreatus e Lentinula edodes. O meio de cultura suplementado com soro de leite apresentou diversas vantagens para a obtenção dessas macromoléculas biológicas, face aos restantes meios analisados, pelo que foi seleccionado para a produção em fermentador de laboratório em regime descontínuo. O crescimento da cultura foi efectuado a 28°C, 200 rpm e durante 10 dias, e as produtividades de complexos proteína-polissacáridos obtidas para o Pleurotus ostreatus foram de 0,008 ± 8,12 x 10-5 g/(L.dia) e de 0,324 ± 0,005 g/(L.dia), para os complexos precipitados a partir da biomassa e do caldo de fermentação, respectivamente. Posteriormente, os complexos intracelulares e extracelulares foram purificados por cromatografia de filtração em gel e parcialmente caracterizados, revelando concentrações de polissacáridos superiores à concentração proteica, pseudo-actividade de superóxido dismutase, e valores de Mr entre 64 e 9 000kDa. As amostras de complexos proteína-polissacáridos foram caracterizadas por HPLC e apresentaram picos de UV e IR com tempos de retenção de aproximadamente 6 e 12 minutos, respectivamente. Após hidrólise ácida, os complexos heteropolissacáridos obtidos apresentaram na sua constituição glucose, ramnose e arabinose. Adicionalmente, a análise dos complexos proteína-polissacáridos por FT-IR revelou bandas de absorção características destas macromoleculas biológicas, designadamente, a 846,5; 1032,3; 1186,4; 1471,0; 1648,8; 2739,6 e 3419,3 cm-1 para o Pleurotus ostreatus e 862,3; 1044,1; 1474,9; 1644,8; 2372,0; 2992,5; e 3415,4 cm-1 para Lentinula edodes.

Cobalt and Zinc Compounds Bearing 1,10-Phenanthroline-5,6-dione or 1,3,5-Triaza-7-phosphaadamantane Derivatives - Synthesis, Characterization, Cytotoxicity, and Cell Selectivity Studies

The compounds [mPTA][CoCl4] (1, mPTA = N-methyl-1,3,5-triaza-7-phosphaadamantane cation), [CoCl(H2O)(DION)(2)][BF4] (2, DION = 1,10-phenanthroline-5,6-dione), [Zn(DION)(2)]Cl-2 (3) and [ZnCl(O-PTA=O)(DION)][BF4] (4) were synthesized by reaction of CoCl2 with [mPTA]I or DION and ZnCl2 with DION or 1,3,5-triaza-7-phosphaadamantane-7-oxide (PTA=O) and DION, respectively. All complexes are water soluble and have been characterized by IR, far-IR, H-1, C-13 and P-31{H-1} NMR spectroscopy, ESI-MS, elemental analyses and single-crystal X-ray diffraction structural analysis (for 1). They were screened against the human tumour cell lines HCT116, HepG2 and MCF7. Complexes 2 and 3 exhibit the highest in vitro cytotoxicity and show lower cytotoxic activities in normal human fibroblast cell line than in HCT116 tumour cell line, which demonstrates their slight specificity for this type of tumour cell.

Regulation of antioxidant enzymes gene expression in the yeast Saccharomyces cerevisiae during stationary phase

Gene expression of three antioxidant enzymes, Mn superoxide dismutase (MnSOD), Cu,Zn superoxide dismutase (Cu,ZnSOD), and glutathione reductase (GR) was investigated in stationary phase Saccharomyces cerevisiae during menadione-induced oxidative stress. Both GR and Cu,ZnSOD mRNA steady state levels increased, reaching a plateau at about 90 min exposure to menadione. GR mRNA induction was higher than that of Cu,ZnSOD (about 14-fold and 9-fold after 90 min, respectively). A different pattern of response was obtained for MnSOD mRNA, with a peak at about 15 min (about 8-fold higher) followed by a decrease to a plateau approximately 4-fold higher than the control value. However, these increased mRNA levels did not result in increased protein levels and activities of these enzymes. Furthermore, exposure to menadione decreased MnSOD activity to half its value, indicating that the enzyme is partially inactivated due to oxidative damage. Cu,ZnSOD protein levels were increased 2-fold, but MnSOD protein levels were unchanged after exposure to menadione in the presence of the proteolysis inhibitor phenylmethylsulfonyl fluoride. These results indicate that the rates of Cu,ZnSOD synthesis and proteolysis are increased, while the rates of MnSOD synthesis and proteolysis are unchanged by exposure to menadione. Also, the translational efficiency for both enzymes is probably decreased, since increases in protein levels when proteolysis is inhibited do not reflect the increases in mRNA levels. Our results indicate that oxidative stress modifies MnSOD, Cu,ZnSOD, and GR gene expression in a complex way, not only at the transcription level but also at the post-transcriptional, translational, and post-translational levels.

Production, purification and characterisation of polysaccharides from pleurotus ostreatus with antitumor activity

Background: Mushroom polysaccharides play an important role in functional foods because they exhibit biological modulator properties such as antitumour, antiviral and antibacterial activities. The present study involved the production, purification and characterisation of intracellular and extracellular free and protein-bound polysaccharides from Pleurotus ostreatus and the investigation of their growth-inhibitory effect on human carcinoma cell lines. Results: Several fermentation parameters were obtained: batch polysaccharide productivities of 0.013 +/- 8.12 x 10-5 and 0.037 +/- 0.0005 g L-1 day-1 for intracellular and extracellular polysaccharides respectively, a maximum biomass concentration of 9.35 +/- 0.18 g L-1, Pmax = 0.935 +/- 0.018 g L-1 day-1, µmax = 0.218 +/- 0.02 day-1, YEP/X = 0.040 +/- 0.0015 g g-1 and YIP/X = 0.014 +/- 0.0003 g g-1. Some polysaccharides exhibited superoxide dismutase (SOD)-like activity of 50-200 units. Fourier transform infrared analysis of the polysaccharides revealed absorption bands characteristic of such biological macromolecules. Cytotoxicity assays showed that both intracellular and extracellular polysaccharides exhibited antitumour activity towards several tested human carcinoma cell lines in a dose-dependent manner. Conclusion - The polysaccharides of P. ostreatus exhibited high SOD-like activity, which strongly supports their biological effect on tumour cell lines. The extracellular polysaccharides presented the highest antitumour activity towards the RL95 carcinoma cell line and should be further investigated as an antitumour agent.