eIF5A binds to translational machinery components and affects translation in yeast

The putative translation factor eIF5A is essential for cell viability and is highly conserved from archebacteria to mammals. Although this protein was originally identified as a translation initiation factor, subsequent experiments did not support a role for eIF5A in general translation. In this work, we demonstrate that eIF-5A interacts with structural components of the 80S ribosome, as well as with the translation elongation factor 2 (eEF2). Moreover, eIF5A is further shown to cofractionate with monosomes in a translation-dependent manner. Finally, eIF5A mutants show altered polysome profiles and are sensitive to translation inhibitors. Our results re-establish a function for eIF5A in translation and suggest a role for this factor in translation elongation instead of translation initiation. (c) 2006 Elsevier B.V. All rights reserved.

Valor prognóstico de células TCD8+ E natural killer em carcinoma epidermóide oral e orofaringeano tratado com radioterapia e quimioterapia

The most common malignant neoplasm of the oral cavity and oropharynx are squamous cell carcinoma. Injuries to the same stage and subjected to the same treatment protocol have sometimes different evolutionary courses. The scope of this study was to investigate, through a retrospective cohort, associations between the number of CD8 + T cells and natural killer, identified immunohistochemically in the inflammatory infiltrate in a series of cases of oral squamous cell carcinoma and orofaringeano, and the level of tumor response to radiotherapy and chemotherapy, overall survival and relapse-free survival of patients. We identified 54 patients with unresectable disease were treated exclusively with radiotherapy and chemotherapy. The median follow-up was 22 months. The sample was characterized by the predominance of male subjects, median age 60 years, all were smokers. The most frequent site was the tongue and 81.5% were in stage IV. Patients with disease in the oral cavity had a worse response to treatment (p = 0.006), worse relapse-free survival (p = 0.007), worse overall survival (p = 0.007). The advanced T stage was shown a negative prognostic factor (p= 0.006) for the clinical treatment response made. Immunohistochemistry was performed to select CD8 + cells (anti-CD8) and NK cells (anti-CD57). Lymphocytes positive and negative markings were counted using the program ImageJ ®. Two groups were created for each marking evaluated: Group I patients with more than 50% cells positive, Group II: less than 50% of labeled cells. For CD8 + cells detected in 38 (70.3%) of Group I were CD8 + and 16 (29.7%) Group II CD8 +. For NK cells, 26 (48.15%) Group I NK and 28 (51.85%) Group II NK. Regarding the clinical response to treatment, we observed that 39% of patients achieved a complete response and 25.9% remained without recurrence at the end of follow-up. These results were better in Group I CD8 + (p = 0.2). Identified that 72.2% of patients progressed to death, this finding had no association with the immunohistochemical data. There was no statistically significant differences between the number of CD8 + and NK cells and the ability of tumor response to radiotherapy and chemotherapy, or with overall survival and relapse-free survival of patients. However, especially in relation to a learned response, we found that this group of patients with advanced disease have a low count of CD8 + T cells active. Believing in the role that the immune response plays in the local fight against neoplastic cells, however, our results do not support the use of quantitative analysis of CD8 + T cells and NK cells as a prognostic factors for oral squamous cell carcinoma and oropharynx

Granulocyte macrophage colony-stimulating factor enhances the modulatory effect of cytokines on monocyte-derived multinucleated giant cell formation and fungicidal activity against Paracoccidioides brasiliensis

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

Infiltrating CD8(+) T lymphocytes, natural killer cells, and expression of IL-10 and TGF-beta 1 in chemically induced neoplasms in male Wistar rats

The present study aimed to estimate the number of CD8(+) T and natural killer (NK) infiltrating cells and the expression of interleukin-10 (IL-10) and transforming growth factor beta 1 (TGF-beta1) in chemically induced neoplasms in an initiation-promotion bioassay for carcinogenesis. Male Wistar rats were treated with N-nitrosodiethylamine, N-methyl-N-nitrosourea, N-butyl-N-(4-hydroxybutyl) nitrosamine, dihydroxy-di-N-propylnitrosamine, and 1,2-dimethylhydrazine for 4 weeks. Two groups were subsequently exposed through diet to phenobarbital (0.05%) or 2-acetylaminofluorene (0.01%) for 25 weeks. An untreated group was used as a control. Immune cells and cytokines were immunohistochemically evaluated in neoplasms and in surrounding normal tissues at the liver, kidneys, lung, and small and large intestines. When compared to the respective normal tissues, an increased number of NK cells was verified infiltrating the colon, lung, and kidney neoplasms, while the number of CD8+ T cells decreased in the intestine and lung neoplasms. Expression of IL-10 was found mainly in kidney tumors. TGF-beta1 was expressed mainly in the liver and kidneys tumors. The results indicate that the differential occurrence of immune cells between neoplastic and normal tissues could be dependent upon tumor microenvironment.

Enhanced natural killer activity and production of pro-inflammatory cytokines in mice selected for high acute inflammatory response (AIRmax)

Strains of mice with maximal and minimal acute inflammatory responsiveness (AIRmax and AIRmin, respectively) were developed through selective breeding based on their high- or low-acute inflammatory responsiveness. Previous reports have shown that AIRmax mice are more resistant to the development of a variety of tumours than AIRmin mice, including spontaneous metastasis of murine melanoma. Natural killer activity is involved in immunosurveillance against tumour development, so we analysed the number and activity of natural killer cells (CD49b(+)), T-lymphocyte subsets and in vitro cytokine production by spleen cells of normal AIRmax and AIRmin mice. Analysis of lymphocyte subsets by flow cytometry showed that AIRmax mice had a higher relative number of CD49b(+) cells than AIRmin mice, as well as cytolytic activity against Yac.1 target cells. The number of CD3(+) CD8(+) cells was also higher in AIRmax mice. These findings were associated with the ability of spleen cells from AIRmax mice in vitro to produce higher levels of the pro-inflammatory cytokines tumour necrosis factor-alpha, interleukin-12p40 and interferon-gamma but not the anti-inflammatory interleukin-10. Taken together, our data suggest that the selective breeding to achieve the AIRmax and AIRmin strains was able to polarize the genes associated with cytotoxic activity, which can be responsible for the antitumour resistance observed in AIRmax mice.

Evaluation of the biological control by the yeast Torulaspora globosa against Colletotrichum sublineolum in sorghum

The yeasts are microorganisms with great potential for biotechnological applications in diverse areas. The biological control of phytopathogens by yeasts has showed satisfactory results under laboratory conditions, and it has already produced commercial formulations. With this as focus, this work aims to perform in vitro and in vivo evaluations of the action of a Torulaspora globosa yeast strain (1S112), isolated from sugarcane rhizosphere, against the phytopathogenic mold Colletotrichum sublineolum, the causative agent of anthracnose in sorghum. In vitro experiments included the antagonism test in Petri dishes with morphological hyphal evaluation; yeast killer activity; siderophore, volatile compound and hydrolytic enzyme production. In vivo experiments were conducted in greenhouse conditions with a sorghum variety susceptible to C. sublineolum by evaluating the anthracnose disease for 6 weeks. The results indicated that the yeast strain significantly controlled the fungal growth, either in vitro or in vivo. The strain of T. globosa exhibited killer activity against two sensitive strains, which is a novel capacity for this species. The yeast did not produce siderophores, volatile compounds or hydrolytic enzymes, although it has reduced the mycelial growth, resulting in hyphal deformities but not cell death. The yeast controlled the anthracnose disease in sorghum, either inoculated before or after the fungal spores, suggesting that the competition for space and nutrients to dominate the mold and killer toxin production, altering the hyphal morphology, are mechanisms utilized by the yeast in the biocontrol.

Natural killer cell activity in experimental paracoccidioidomycosis of the Syrian hamster

Com o objetivo de avaliar a atividade de células natural killer na paracoccidioidomicose experimental do hamster, 80 hamsters foram infectados por via intratesticular com Paracoccidioides brasiliensis e sacrificados após 24h, 48h, 96h, 1, 2, 4, 8 e 11 semanas de infecção. Como controle foram avaliados 40 hamsters normais, não infectados. Os animais foram submetidos ao estudo da atividade citotóxica de células NK pela técnica de single-cell assay e da resposta imune humoral pelas técnicas de imunodifusão dupla e Elisa. A produção do fator inibidor da migração de macrófagos em presença de Phytohemaglutinina e antígeno de P. brasiliensis e a histopatologia das lesões foram estudadas após 1,4, 8e 11 semanas de infecção. Os animais infectados, quando comparados aos controles, apresentaram níveis de atividade NK significativamente elevados durante as 4 primeiras semanas de infecção, havendo diminuição dessa atividade a partir da 8ª semana. Foi observada correlação inversa entre atividade NK e níveis de anticorpos específicos e, associação entre diminuição da atividade NK, depressão de resposta imune celular e aumento da extensão das lesões histopatológicas. Os resultados sugerem que após ativação inicial, as células NK são incapazes de controlar a disseminação do fungo pelos tecidos. A depressão da atividade NK na fase tardia da infecção parece estar relacionada com os distúrbios imunorregulatórios associados à paracoccidioidomicose.

EARLY ACTIVATION OF SPLENIC MACROPHAGES BY TUMOR-NECROSIS-FACTOR-ALPHA IS IMPORTANT IN DETERMINING THE OUTCOME OF EXPERIMENTAL HISTOPLASMOSIS IN MICE

Experimental infection of animals with Histoplasma capsulatum caused a massive macrophage infiltration into the spleen and induced the production of tumor necrosis factor alpha (TNF-alpha) locally. The cytokine was also produced in vitro by peritoneal exudate macrophages exposed to a large inoculum of yeast cells. Depletion of the cytokine by injection of polyclonal sheep anti-TNF-alpha antibody was detrimental to sublethally infected mice. Fungous burdens in the spleens of TNF-alpha-depleted mice were higher than they were in the infected control mice at days 2, 7, and 9 after infection, and the antibody-treated animals succumbed to the infection. Histopathological study of spleen sections revealed that splenic macrophages were not able to control proliferation of intracellular yeasts as a result of TNF-alpha depletion. It seems that TNF-alpha plays a role in early activation of splenic macrophages which is important in controlling the outcome of an infection.

AN IMPROVED CULTURE-MEDIUM FOR DETECTING LIVE YEAST PHASE CELLS OF PARACOCCIDIOIDES-BRASILIENSIS

The plating efficiency of standard mycological media such as brain heart infusion (BHI) agar is poor for Paracoccidioides brasiliensis. We prepared a water-extract of yeast phase cells of P. brasiliensis and examined it for growth-enhancing activity for the fungus. The water-extract, when added to BHI agar to a concentration of 5%, improved the plating efficiency of the medium for the fungus to some extent, but the degree of improvement was considerably varied among P. brasiliensis isolates. By contrast, when the water-extract was added in combination with horse serum (4%), the plating efficiency was highly improved (to 94-99%) for all the P. brasiliensis isolates employed. The growth-enhancing factor(s) in the water-extract was heat-stable and heating at 120-degrees-C for 15 min had little, if any, effect on growth-enhancing activity.

Conserved sequences in the β subunit of archaeal and eukaryal translation initiation factor 2 (elF2), absent from elF5, mediate interaction with elF2γ

The eukaryotic translation initiation factor 2 (eIF2) binds the methionyl-initiator tRNA in a GTP-dependent mode. This complex associates with the 40 S ribosomal particle, which then, with the aid of other factors, binds to the 5' end of the mRNA and migrates to the first AUG codon, where eIF5 promotes GTP hydrolysis, followed by the formation of the 80 S ribosome. Here we provide a comparative sequence analysis of the β subunit of eIF2 and its archaeal counterpart (aIF2β). aIF2β differs from eIF2β in not possessing an N-terminal extension implicated in binding RNA, eIF5 and eIF2B. The remaining sequences are highly conserved, and are shared with eIF5. Previously isolated mutations in the yeast eIF2β, which allow initiation of translation at UUG codons due to the uncovering of an intrinsic GTPase activity in eIF2, involve residues that are conserved in aIF2β, but not in eIF5. We show that the sequence of eIF2B homologous to aIF2β is sufficient for binding eIF2γ, the only subunit with which it interacts, and comprises, at the most, 78 residues, eIF5 does not interact with eIF2γ, despite its similarity with eIF2β, probably because of a gap in homology in this region. These observations have implications for the evolution of the mechanism of translation initiation.

Role of natural killer cells in antitumor resistance

Natural killer cells constitute a population of lymphocytes able to non-specifically destroy virus-infected and some kinds of tumor cells. Since this lytic activity was shown by non-immunized animals the phenomenon is denominated natural killer (NK) activity and contrasts with specific cytotoxicity performed by cytolytic T lymphocytes (CTLs) because it does not depends on MHC-restricted peptides recognition. In fact, the main feature of most functional receptors of NK cells (NKRs) is their ability to be inhibited by different kinds of class I MHC antigens. In the middle of the 1950's, Burnet & Thomas forged the concept of tumor immunosurveillance and NK cells can be considered one of the main figures in this phenomenon both for effector and regulatory functions. In the present review the early studies on the biology of NK cells were revisited and both their antitumor activity and dependence on the activation by cytokines are discussed.

The eukaryotic translation initiation factor 3 subunit L protein interacts with Flavivirus NS5 and may modulate yellow fever virus replication

Background: Yellow fever virus (YFV) belongs to the Flavivirus genus and causes an important disease. An alarming resurgence of viral circulation and the expansion of YFV-endemic zones have been detected in Africa and South America in recent years. NS5 is a viral protein that contains methyltransferase and RNA-dependent RNA polymerase (RdRp) domains, which are essential for viral replication, and the interactions between NS5 and cellular proteins have been studied to better understand viral replication. The aim of this study was to characterize the interaction of the NS5 protein with eukaryotic translation initiation factor 3 subunit L (eIF3L) and to evaluate the role of eIF3L in yellow fever replication. Methods. To identify interactions of YFV NS5 with cellular proteins, we performed a two-hybrid screen using the YFV NS5 RdRp domain as bait with a human cDNA library, and RNApol deletion mutants were generated and analyzed using the two-hybrid system for mapping the interactions. The RNApol region involved was segmented into three fragments and analyzed using an eIF3L-expressing yeast strain. To map the NS5 residues that are critical for the interactions, we performed site-direct mutagenesis in segment 3 of the interaction domain (ID) and confirmed the interaction using in vitro assays and in vivo coimmunoprecipitation. The significance of eIF3L for YFV replication was investigated using eIF3L overexpression and RNA interference. Results: In this work, we describe and characterize the interaction of NS5 with the translation factor eIF3L. The interaction between NS5 and eIF3L was confirmed using in vitro binding and in vivo coimmunoprecipitation assays. This interaction occurs at a region (the interaction domain of the RNApol domain) that is conserved in several flaviviruses and that is, therefore, likely to be relevant to the genus. eIF3L overexpression and plaque reduction assays showed a slight effect on YFV replication, indicating that the interaction of eIF3L with YFV NS5 may play a role in YFV replication. Conclusions: Although the precise function of eIF3L on interactions with viral proteins is not entirely understood, these results indicate an interaction of eIF3L with YF NS5 and that eIF3L overexpression facilitates translation, which has potential implications for virus replication. © 2013 Morais et al.; licensee BioMed Central Ltd.

Hanseniaspora nectarophila sp nov., a yeast species isolated from ephemeral flowers

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

In situ selective determination of methylmercury in river water by diffusive gradient in thin films technique (DGT) using baker's yeast (Saccharomyces cerevisiae) immobilized in agarose gel as binding phase

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

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.