CD95 (FAS) and CD178 (FASL) induce the apoptosis of CD4+ and CD8+ cells isolated from the peripheral blood and spleen of dogs naturally infected with Leishmania spp

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

Yeast CA-11 fermentation in musts treated with brown and green propolis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Improvement of the alcohol dehydrogenase from baker's yeast using polymers and salts for alcohol determinations in beverages

An alcohol dehydrogenase (ADH) was purified from dry baker’s yeast. This is a key enzyme of the primary short-chain alcohol metabolism in many organisms. In the present study, the obtained enzymatic preparation of baker’s yeast, containing 2.7 U/mg of ADH, was used in the reactions. The purified extract of the ADH obtained from Fermix commercial dry yeast, presented the highest activity and purification factor when ammonium sulfate was added in the precipitation of protein, in the range 35-60% (w/v). The enzymatic preparation was maintained for 2 months in the lyophilized form at 4ºC (retention of 96.2% of activity) in the presence of 1 mmol/L of sodium azide, and it maintained 47% of activity for 30 days at 30°C in the presence of 15% PEG. The assays of ethanol (detection range 5 mM -150 mM or 2.3 x 10-4 – 6.91 x 10-3g/L) in different samples in alcoholic beverages, presented a maximum deviation of only 2.1%. Assays of recovery of the substrate (99.25%) added in the wine showed that the methodology is viable for this sample type. The standard curve and the analytic curve of this method meet the conditions of precision, sensitivity, simplicity, and low cost, required for a useable analytical method.

Novas alternativas terapêuticas para o tratamento da Criptococose: análogos de Resveratrol e microRNAs

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Correlation between immunoexpression of BCL-2 protein family with apoptosis index, cellular proliferation and survival in colorectal carcinomas

Objectives: To evaluate the Bcl-2, Bax, Bad and Bak immunoexpression in tumor and nontumorous tissue of 130 patients with colorectal carcinoma submitted to surgery at São Paulo Hospital, EPM/ UNIFESP, from 2002 to 2005, and to correlate the immunoexpression data with the apoptotic index (AI, obtained by anti-cleaved caspase 3 and M30 labeling), cell proliferation score (CPS, obtained by Ki-67), immunoexpression of p53 and patient’s clinical prognosis. Results: Positive correlation was verified between Bcl-2 protein family in tumor and nontumor tissue. Only Bcl-2 protein correlated with IA and CPS in the tumor. Positive correlation was observed between pro- -apoptotic proteins and Bcl-2 protein. In the adjacent mucosa, Bcl-2 correlated with Ki-67 and p53, but not with IA. Carcinomas exhibited higher immunoexpression of CPS and IA markers. No correlation occurred between immunoexpression data and patient survival. Conclusion: Positive correlation was observed between the pro-apoptotic proteins of the Bcl-2 family and the anti-apoptotic protein Bcl-2. In the adjacent nontumor mucosa, Bcl-2 correlated with Ki-67 and p53, but not with AI. Carcinomas presented greater immunoexpression for CPS and AI markers; however immunoexpression of these markers was not correlated with patient survival.

Transforming growth factor beta and apoptosis in leprosy skin lesions: possible relationship with the control of the tissue immune response in the Mycobacterium leprae infection

The course of leprosy depends of the host immune response which ranges from the lepromatous pole (LL) to the tuberculoid pole (TT). A comparative study was conducted in 60 patients with the LL and TT The results showed a mean expression of TGF-beta of 339 +/- 99.4 cells/field for TT and of 519.2 +/- 68.2 cells/field for LL. Frequency of apoptosis was 6.3 +/- 1.8 in TT and 14.0 +/- 6.1 in LL. A correlation (p = 0.0251) between TGF-beta and caspase-3 in the LL was found. This finding indicates a role of TGF-beta and apoptosis in the immune response in leprosy. (C) 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Sugars metabolism and ethanol production by different yeast strains from coffee industry wastes hydrolysates

Significant amounts of wastes are generated by the coffee industry, among of which, coffee silverskin (CS) and spent coffee grounds (SCG) are the most abundantly generated during the beans roasting and instant coffee preparation, respectively. This study evaluated the sugars metabolism and production of ethanol by three different yeast strains (Saccharomyces cerevisiae, Pichia stipitis and Kluyveromyces fragilis) when cultivated in sugar rich hydrolysates produced by acid hydrolysis of CS and SCG. S. cerevisiae provided the best ethanol production from SCG hydrolysate (11.7 g/l, 50.2% efficiency). On the other hand, insignificant (<= 1.0 g/l) ethanol production was obtained from CS hydrolysate, for all the evaluated yeast strains, probably due to the low sugars concentration present in this medium (approx. 22 g/l). It was concluded that it is possible to reuse SCG as raw material for ethanol production, which is of great interest for the production of this biofuel, as well as to add value to this agro-industrial waste. CS hydrolysate, in the way that is produced, was not a suitable fermentation medium for ethanol production; however, the hydrolysate concentration for the sugars content increase previous the use as fermentation medium could be an alternative to overcome this problem. (C) 2011 Elsevier Ltd. All rights reserved.

Inhibition of nuclear factor-kappa B by dehydroxymethylepoxyquinomicin induces schedule-dependent chemosensitivity to anticancer drugs and enhances chemoinduced apoptosis in osteosarcoma cells

Osteosarcoma (OS) is the most common primary malignant bone tumor, usually developing in children and adolescents, and is highly invasive and metastatic, potentially developing chemoresistance. Thus, novel effective treatment regimens are urgently needed. This study was the first to investigate the anticancer effects of dehydroxymethylepoxyquinomicin (DHMEQ), a highly specific nuclear factor-kappa B (NF-kappa B) inhibitor, on the OS cell lines HOS and MG-63. We demonstrate that NF-kappa B blockade by DHMEQ inhibits proliferation, decreases the mitotic index, and triggers apoptosis of OS cells. We examined the effects of combination treatment with DHMEQ and cisplatin, doxorubicin, or methotrexate, drugs commonly used in OS treatment. Using the median effect method of Chou and Talalay, we evaluated the combination indices for simultaneous and sequential treatment schedules. In all cases, combination with a chemotherapeutic drug produced a synergistic effect, even at low single-agent cytotoxic levels. When cells were treated with DHMEQ and cisplatin, a more synergistic effect was obtained using simultaneous treatment. For the doxorubicin and methotrexate combination, a more synergistic effect was achieved with sequential treatment using DHMEQ before chemotherapy. These synergistic effects were accompanied by enhancement of chemoinduced apoptosis. Interestingly, the highest apoptotic effect was reached with sequential exposure in both cell lines, independent of the chemotherapeutic agent used. Likewise, DHMEQ decreased cell invasion and migration, crucial steps for tumor progression. Our data suggest that combining DHMEQ with chemotherapeutic drugs might be useful for planning new therapeutic strategies for OS treatment, mainly in resistant and metastatic cases. Anti-Cancer Drugs 23:638-650 (C) 2012 Wolters Kluwer Health broken vertical bar Lippincott Williams & Wilkins.

Disulfide Biochemistry in 2-Cys Peroxiredoxin: Requirement of Glu50 and Arg146 for the Reduction of Yeast Tsa1 by Thioredoxin

2-Cys peroxiredoxin (Prx) enzymes are ubiquitously distributed peroxidases that make use of a peroxidatic cysteine (Cys(P)) to decompose hydroperoxides. A disulfide bond is generated as a consequence of the partial unfolding of the alpha-helix that contains Cys(P). Therefore, during its catalytic cycle, 2-Cys Prx alternates between two states, locally unfolded and fully folded. Tsa1 (thiol-specific antioxidant protein 1 from yeast) is by far the most abundant Cys-based peroxidase in Saccharomyces cerevisiae. In this work, we present the crystallographic structure at 2.8 angstrom resolution of Tsa1(C47S) in the decameric form [(alpha(2))(5)] with a DTT molecule bound to the active site, representing one of the few available reports of a 2-Cys Prx (AhpC-Prx1 subfamily) (AhpC, alkyl hydroperoxide reductase subunit C) structure that incorporates a ligand. The analysis of the Tsa1(C47S) structure indicated that G1u50 and Arg146 participate in the stabilization of the Cys(P) alpha-helix. As a consequence, we raised the hypothesis that G1u50 and Arg146 might be relevant to the Cys(P) reactivity. Therefore, Tsa1(E50A) and Tsa1(R146Q) mutants were generated and were still able to decompose hydrogen peroxide, presenting a second-order rate constant in the range of 10(6) M-1 S-1. Remarkably, although Tsa1(E50A) and Tsa1(R146Q) were efficiently reduced by the low-molecular-weight reductant DTT, these mutants displayed only marginal thioredoxin (Trx)-dependent peroxidase activity, indicating that G1u50 and Arg146 are important for the Tsa1-Trx interaction. These results may impact the comprehension of downstream events of signaling pathways that are triggered by the oxidation of critical Cys residues, such as Trx. (C) 2012 Elsevier Ltd. All rights reserved.

Polymorphonuclear leukocytes CH138+apoptosis evaluation in milk with high and low somatic cell count - preliminary data

Polymorphonuclear leukocyte (PMNL) apoptosis is central to the successful resolution of inflammation. Since Somatic Cell Count (SCC) is an indicator of the mammary gland's immune status, this study sought to clarify the influence that these factors have on each other and on the evolution of the inflammatory process. Milk samples were stained with annexin-V, propidium iodide (PI), primary antibody anti-CH138A. Negative correlation between SCC and PMNL apoptosis was found, and a statistical difference between high SCC group and low SCC group was observed concerning the rate of viable PMNL, apoptotic PMNL, necrotic PMNL and necrotic and/or apoptotic PMNL. Overall, the high cellularity group presented lower proportions of CH138+ cells undergoing apoptosis and higher proportions of viable and necrotic CH138+ cells. Thus, it can be concluded that PMNL apoptosis and SCC are related factors, and that in high SCC, milk apoptosis is delayed. Although there is a greater amount of active phagocytes in this situation, apoptosis' anti-inflammatory effects are decreased, while necrosis' pro-inflammatory effects are increased, which can contribute to chronic inflammation.

Immunohistochemistry Analysis of Proteins Related with Apoptosis as Prognostic Factor in Epidermoid Carcinoma of Penis

The aim was to analyze the protein expression of apoptotic genes caspase-3, caspase-8 and bcl-2 with the immunohistochemistry technique, correlating with tumor grade (I, II and III) and with the patient survival in order to understand the basic mechanism of tumoral transformation. The immunohistochemistry reactions on 50 samples of squamous cell carcinoma were carried out with the avidin-biotin immunoperoxidase method and antigen recovery. The analyses were made using the graduation method "in crosses" (0 to 4 crosses - no stain to more than 75% of positives cells) and in categories (low, intermediate, high) of the cytoplasm immunoreactivity of the epidermoid penile carcinoma cells. It was observed a statistically significant difference when the expression of caspase-3 were compared with the grades land II of the tumor (p=0.0010) and when comparing the patient survival with the grades I and II of the tumor (p=0.0212). The protein bcl-2 was more expressed than caspase-3 and caspase-8 proteins, suggesting that the apoptotic rate in this carcinoma is low. The higher expression of the anti-apoptotic protein bcl-2 suggests a higher preservation of the tumoral cells.

Identification of Regions Involved in Substrate Binding and Dimer Stabilization within the Central Domains of Yeast Hsp40 Sis1

Protein folding, refolding and degradation are essential for cellular life and are regulated by protein homeostatic processes such those that involve the molecular chaperone DnaK/Hsp70 and its co-chaperone DnaJ. Hsp70 action is initiated when proteins from the DnaJ family bind an unfolded protein for delivery purposes. In eukaryotes, the DnaJ family can be divided into two main groups, Type I and Type II, represented by yeast cytosolic Ydj1 and Sis1, respectively. Although sharing some unique features both members of the DnaJ family, Ydj1 and Sis1 are structurally and functionally distinct as deemed by previous studies, including the observation that their central domains carry the structural and functional information even in switched chimeras. In this study, we combined several biophysical tools for evaluating the stability of Sis1 and mutants that had the central domains (named Gly/Met rich domain and C-terminal Domain I) deleted or switched to those of Ydj1 to gain insight into the role of these regions in the structure and function of Sis1. The mutants retained some functions similar to full length wild-type Sis1, however they were defective in others. We found that: 1) Sis1 unfolds in at least two steps as follows: folded dimer to partially folded monomer and then to an unfolded monomer. 2) The Gly/Met rich domain had intrinsically disordered characteristics and its deletion had no effect on the conformational stability of the protein. 3) The deletion of the C-terminal Domain I perturbed the stability of the dimer. 4) Exchanging the central domains perturbed the conformational stability of the protein. Altogether, our results suggest the existence of two similar subdomains in the C-terminal domain of DnaJ that could be important for stabilizing each other in order to maintain a folded substrate-binding site as well as the dimeric state of the protein.

BCL2A1a Over-Expression in Murine Hematopoietic Stem and Progenitor Cells Decreases Apoptosis and Results in Hematopoietic Transformation

We previously reported the development of a lethal myeloid sarcoma in a non-human primate model utilizing retroviral vectors to genetically modify hematopoietic stem and progenitor cells. This leukemia was characterized by insertion of the vector provirus into the BCL2A1 gene, with resultant BCL2A1 over-expression. There is little information on the role of this anti-apoptotic member of the BCL2 family in hematopoiesis or leukemia induction. Therefore we studied the impact of Bcl2a1a lentiviral over-expression on murine hematopoietic stem and progenitor cells. We demonstrated the anti-apoptotic function of this protein in hematopoietic cells, but did not detect any impact of Bcl2a1a on in vitro cell growth or cell cycle kinetics. In vivo, we showed a higher propensity of HSCs over-expressing Bcl2a1a to engraft and contribute to hematopoiesis. Mice over-expressing Bcl2a1a in the hematologic compartment eventually developed an aggressive malignant disease characterized as a leukemia/lymphoma of B-cell origin. Secondary transplants carried out to investigate the primitive origin of the disease revealed the leukemia was transplantable. Thus, Bcl2a1 should be considered as a protooncogene with a potential role in both lymphoid and myeloid leukemogenesis, and a concerning site for insertional activation by integrating retroviral vectors utilized in hematopoietic stem cell gene therapy.

Cannabidiol protects oligodendrocyte progenitor cells from inflammation-induced apoptosis by attenuating endoplasmic reticulum stress

Cannabidiol (CBD) is the most abundant cannabinoid in Cannabis sativa that has no psychoactive properties. CBD has been approved to treat inflammation, pain and spasticity associated with multiple sclerosis (MS), of which demyelination and oligodendrocyte loss are hallmarks. Thus, we investigated the protective effects of CBD against the damage to oligodendrocyte progenitor cells (OPCs) mediated by the immune system. Doses of 1 mu M CBD protect OPCs from oxidative stress by decreasing the production of reactive oxygen species. CBD also protects OPCs from apoptosis induced by LPS/IFN gamma through the decrease of caspase 3 induction via mechanisms that do not involve CB1, CB2, TRPV1 or PPAR gamma receptors. Tunicamycin-induced OPC death was attenuated by CBD, suggesting a role of endoplasmic reticulum (ER) stress in the mode of action of CBD. This protection against ER stress-induced apoptosis was associated with reduced phosphorylation of eiF2 alpha, one of the initiators of the ER stress pathway. Indeed, CBD diminished the phosphorylation of PKR and eiF2 alpha induced by LPS/IFN gamma. The pro-survival effects of CBD in OPCs were accompanied by decreases in the expression of ER apoptotic effectors (CHOP, Bax and caspase 12), and increased expression of the anti-apoptotic Bcl-2. These findings suggest that attenuation of the ER stress pathway is involved in the 'oligoprotective' effects of CBD during inflammation. Cell Death and Disease (2012) 3, e331; doi:10.1038/cddis.2012.71; published online 28 June 2012

Synthetic phosphoethanolamine a precursor of membrane phospholipids reduce tumor growth in mice bearing melanoma B16-F10 and in vitro induce apoptosis and arrest in G2/M phase

Phosphoethanolamine (Pho-s) is a compound involved in phospholipid turnover, acting as a substrate for many phospholipids of the cell membranes, especially phosphatidylcholine. We recently reported that synthetic Pho-s has potent effects on a wide variety of tumor cells. To determine if Pho-s has a potential antitumor activity, in this study we evaluated the activity of Pho-s against the B16-F10 melanoma both in vitro and in mice bearing a dorsal tumor. The treatment of B16F10 cells with Pho-s resulted in a dose-dependent inhibition of cell proliferation. At low concentrations, this activity appears to be involved in the arrest of the cell cycle at G2/M, while at high concentrations Pho-s induces apoptosis. In accordance with these results, the loss of mitochondrial potential and increased caspase-3 activity suggest that Phos has dual antitumor effects; i.e. it induces apoptosis at high concentrations and modulates the cell cycle at lower concentrations. In vivo, we evaluated the effect of Pho-s in mice bearing B16-F10 melanoma. The results show that Pho-s reduces the tumoral volume increasing survival rate. Furthermore, the tumor doubling time and tumor delays were substantially reduced when compared with untreated mice. Histological analyses reveal that Pho-s induces changes in cell morphology, typical characteristics of apoptosis, in addition the large areas of necrosis correlating with a reduction of tumor size. The results presented here support the hypothesis that Pho-s has antitumor effects by the induction of apoptosis as well as the inhibition of cell proliferation by arrest at G2/M. Thus, Pho-s can be regarded as a promising agent for the treatment of melanoma. Published by Elsevier Masson SAS.