Carotenoid production in sugarcane juice and synthetic media supplemented with nutrients by Rhodotorula rubra l02

In order for the use of biological carotenoids to become feasible, it is necessary to have adequate low cost sources and improved methods of cultivation. The aim of this study was to evaluate the effect of supplementation with nitrogen, phosphorus, zinc, and magnesium, on the biomass and carotenoid volumetric production by yeast Rhodotorula rubra L02 using a complex medium (sugarcane juice) and synthetic media (sucrose and maltose) as substrates. The experimental design used for each substrate was randomized in blocks with 16 treatments and 3 repetitions. The treatments were compound for 15 different combinations of nutrients (N; Mg; Zn; P, N + Mg; N + Zn; N + P; Mg + Zn; Mg + P; Zn + P; N + P + Zn; N + P + Mg; N + Zn + Mg; P + Zn + Mg; N + Zn + Mg + P) alone and combined, and a control. The results were submitted to analysis of variance and Tukey test at 5% significance level. Among the treatments evaluated, the highest production of dry biomass, with both maltose and sucrose, was observed for Mg (1.60 g/L and 1.94 g/L, respectively). Additionally, another treatment that stood out in terms of biomass production was the control treatment with maltose (1.54 g/L). After the incubation time, killer activity was not observed since there was no formation of inhibition halo around the L02 yeast.(AU)

Isolation, characterization, and identification of wild killer yeasts from sugarcane juice / Isolamento, caracterização e identificação de leveduras killer de caldo de cana de açúcar

Ethanol produced by the fermentation of sugarcane juice has emerged as an important renewable fuel. The yield of this fermentation is affected by undesirable microbial contaminants, but killer yeasts can be a promising strategy to reduce this problem. The present study aimed to isolate, characterize, and identify wild killer yeasts from sugarcane juice. Samples were inoculated in culture medium containing chloramphenicol, and 140 colonies with different characteristics were selected. These isolates were submitted to the killer phenotype assay, and the positive killers were characterized and identified according to the standard methods. Only two strains showed killer activity, identified as Pichia anomala CE025 and P. membranaefaciens CE088. At 25C, both strains exhibited killer activity at pH 4.0, 4.3, and 4.5, but this activity was not detected at pH 3.0, 3.5, 5.0, and 6.0. The killer phenotype of P. membranaefaciens CE088 was inhibited above 30C, while for P. anomala, CE025 inhibition occurred only at a higher temperature. Both strains were able to grow in 12% ethanol, and P. anomala CE025 was more tolerant than P. membranaefaciens CE 088. Further studies will be conducted to isolate, purify and identify the killer toxins produced by Pichia anomala and Pichia membranaefaciens species.(AU)

O etanol produzido a partir da fermentação do caldo de cana emergiu como um combustível renovável. O rendimento desta fermentação é afetado por micro-organismos indesejáveis e as leveduras killer se constituem uma alternativa promissora para combater essa contaminação. Nesta perspectiva, o presente trabalho teve como objetivo isolar, caracterizar e identificar leveduras killer de caldo de cana. As amostras foram inoculadas em meio de cultura contendo cloranfenicol e 140 colônias com diferentes características foram selecionadas. Esses isolados foram avaliados quanto à presença do fator killer e os isolados positivos caracterizados e identificados por métodos convencionais. Apenas dois isolados apresentaram atividade killer e foram identificados como Pichia anomala CE025 e P. membranaefaciens CE088. A 25C as duas linhagens exibiram atividade killer em pH 4.0, 4.3 e 4.5, mas esta atividade foi inibida a pH 3.0, 3.5, 5.0 e 6.0. Para P. membranaefaciens CE088 o fenótipo killer foi inibido acima de 30C, enquanto que a P. anomala CE025 exibiu essa característica acima deste valor. Ambas as linhagens foram capazes de crescer na presença de 12% de etanol, mas P. anomala CE025 foi mais tolerante do que P. membranaefaciens CE088. Estudos posteriores serão realizados para isolar, purificar e identificar as toxinas killer produzidas pelas espécies Pichia anomala e Pichia membranaefacien.(AU)

Selenium and vitamin E enriched diet increases NK cell cytotoxicity in cattle / Dieta enriquecida com selênio e vitamina E aumenta a citotoxicidade de células NK em bovinos

A number of studies has shown that antioxidants, fatty acids and trace minerals may modulate different immune cell activities, and that their deficiency may be associated with diseases and impaired immune responses. In innate immunity, natural killer (NK) cells have a central role, killing virally infected and cancerous cells, and also secreting cytokines that shape adaptive immune responses. Thus, the aim of this study was to evaluate the effect of enriched diets in selenium plus vitamin E and/or canola oil on complete blood count and on NK cell cytotoxicity from blood lymphocytes of Nellore bulls. Bulls that received selenium plus vitamin E had (P=0.0091) higher NK cell cytotoxicity than control bulls. This result positively correlated with serum selenium levels. To the best of our knowledge, this is the first study that showed immunostimulatory effects of selenium plus vitamin E on NK cell cytotoxicity of Nellore bulls.(AU)

Vários estudos demonstraram que antioxidantes, ácidos graxos e minerais podem modular a atividade de diferentes células do sistema imunológico e que as suas carências podem estar associadas a doenças e a respostas imunes comprometidas. Na imunidade inata, os linfócitos natural killer (NK) têm um papel central matando células infectadas por vírus e células cancerígenas, ao mesmo tempo em que também secretam citocinas que modulam as respostas imunes adaptativas. Assim, o objetivo deste estudo foi avaliar o efeito de dietas enriquecidas em selênio e vitamina E e/ou óleo de canola no hemograma e na citotoxicidade das células NK do sangue de bovinos da raça Nelore. Os animais que receberam selênio e vitamina E tiveram (P = 0,0091) maior citotoxicidade das células NK do que os animais do grupo controle. Este resultado foi positivamente correlacionado com os níveis de selênio no sangue. Para o melhor do nosso conhecimento, este é o primeiro estudo que mostrou efeitos imunoestimulatórios do selênio e vitamina E sobre a citotoxicidade das células NK de bovinos Nelore.(AU)

Characteristics of Saccharomyces cerevisiae yeasts exhibiting rough colonies and pseudohyphal morphology with respect to alcoholic fermentation

Among the native yeasts found in alcoholic fermentation, rough colonies associated with pseudohyphal morphology belonging to the species Saccharomyces cerevisiae are very common and undesirable during the process. The aim of this work was to perform morphological and physiological characterisations of S. cerevisiae strains that exhibited rough and smooth colonies in an attempt to identify alternatives that could contribute to the management of rough colony yeasts in alcoholic fermentation. Characterisation tests for invasiveness in Agar medium, killer activity, flocculation and fermentative capacity were performed on 22 strains (11 rough and 11 smooth colonies). The effects of acid treatment at different pH values on the growth of two strains ("52" -rough and "PE-02" smooth) as well as batch fermentation tests with cell recycling and acid treatment of the cells were also evaluated. Invasiveness in YPD Agar medium occurred at low frequency; ten of eleven rough yeasts exhibited flocculation; none of the strains showed killer activity; and the rough strains presented lower and slower fermentative capacities compared to the smooth strains in a 48-h cycle in a batch system with sugar cane juice. The growth of the rough strain was severely affected by the acid treatment at pH values of 1.0 and 1.5; however, the growth of the smooth strain was not affected. The fermentative efficiency in mixed fermentation (smooth and rough strains in the same cell mass proportion) did not differ from the efficiency obtained with the smooth strain alone, most likely because the acid treatment was conducted at pH 1.5 in a batch cell-recycle test. A fermentative efficiency as low as 60% was observed with the rough colony alone.

Characteristics of Saccharomyces cerevisiae yeasts exhibiting rough colonies and pseudohyphal morphology with respect to alcoholic fermentation

Among the native yeasts found in alcoholic fermentation, rough colonies associated with pseudohyphal morphology belonging to the species Saccharomyces cerevisiae are very common and undesirable during the process. The aim of this work was to perform morphological and physiological characterisations of S. cerevisiae strains that exhibited rough and smooth colonies in an attempt to identify alternatives that could contribute to the management of rough colony yeasts in alcoholic fermentation. Characterisation tests for invasiveness in Agar medium, killer activity, flocculation and fermentative capacity were performed on 22 strains (11 rough and 11 smooth colonies). The effects of acid treatment at different pH values on the growth of two strains ("52" -rough and "PE-02" smooth) as well as batch fermentation tests with cell recycling and acid treatment of the cells were also evaluated. Invasiveness in YPD Agar medium occurred at low frequency; ten of eleven rough yeasts exhibited flocculation; none of the strains showed killer activity; and the rough strains presented lower and slower fermentative capacities compared to the smooth strains in a 48-h cycle in a batch system with sugar cane juice. The growth of the rough strain was severely affected by the acid treatment at pH values of 1.0 and 1.5; however, the growth of the smooth strain was not affected. The fermentative efficiency in mixed fermentation (smooth and rough strains in the same cell mass proportion) did not differ from the efficiency obtained with the smooth strain alone, most likely because the acid treatment was conducted at pH 1.5 in a batch cell-recycle test. A fermentative efficiency as low as 60% was observed with the rough colony alone.

Occurrence of killer Candida glabrata clinical isolates

In this work we characterized the occurrence of killer activity in 64 Candida glabrata clinical isolates under different conditions. We found that only 6.25 % of the clinical isolates tested were positive for killer activity against a Saccharomyces cerevisiae W303 sensitive strain. Sensitivity of killer activity to different values of pH and temperatures was analyzed. We found that the killer activity presented by all isolates was resistant to every pH and temperature tested, although optimal activity was found at a range of pH values from 4 to 7 and at 37ºC. We did not observe extrachromosomal genetic elements associated with killer activity in any of the positive C. glabrata isolates. The killer effect was due to a decrease in viability and DNA fragmentation in sensitive yeast.

ESTUDO DE BIOMARCADORES DE MERCÚRIO EM PEIXES DA AMAZÔNIA POR MEIO DA METALÔMICA E ANÁLISE DO ESTRESSE OXIDATIVO

O mercúrio é um metal tóxico, de distribuição ubíqua, com capacidade para bioacumulação e biomagnificação, que provoca alterações em biomoléculas importantes no metabolismo e contribui para o estabelecimento do estresse oxidativo em organismos aquáticos. Logo, o presente estudo teve por objetivo identificar e avaliar possíveis biomarcadores proteicos e/ou enzimáticos da toxicidade do mercúrio em peixes da região amazônica, por meio do estudo metaloproteômico e avaliação do estresse oxidativo. Foram utilizadas metodologias de fracionamento e identificação de proteínas por eletroforese bidimensional (2D PAGE) associada à espectrometria de massas (MS), mapeamento do mercúrio, em spots proteicos, por espectrometria de absorção atômica em forno de grafite (GFAAS) e avaliação de marcadores de estresse oxidativo. As espécies utilizadas foram o Plagioscion squamosissimus (corvina) e Colossoma macropomum (tambaqui), coletados na área da Usina Hidrelétrica de Jirau (rio Madeira-RO), que foram selecionadas em função da abundância populacional, interesse para a pesca e posição diferente na cadeia trófica (carnívoro e onívoro, respectivamente). Os tecidos amostrados foram o hepático, renal e muscular. Os resultados obtidos demonstraram maior concentração de mercúrio total no P. squamosissimus, espécie carnívora, e padrão de distribuição deste elemento igual para ambas as espécies(fígado>rim>músculo). Há tendência para maior atividade enzimática nos tecidos hepático e renal da espécie com maior concentração de mercúrio. Somente a atividade da glutationa peroxidase no rim e da glutationa-S-transferase no fígado do P.squamosissimus foi inferior, fato que pode ser explicado pela interação do mercúrio com essas enzimas, interferindo em sua conformação e função. Os dados obtidos por MS possibilitaram a identificação dos spots proteicos associados ao mercúrio, revelando proteínas envolvidas no metabolismo energético (gliceraldeído-3-fosfato, malato e Llactato desidrogenase, enolase, creatina quinase e fosfoglicerato mutase), transporte (hemoglobinas e , proteínas de ligação a ácidos graxos e GTPases), síntese e 3 degradação de proteínas (ubiquitina e ribossomal S27a e ribosomal 40S S24)diferenciação celular (nucleosídeo difosfato quinase), regulação gênica (pterina-4--carbinolamina desidratase e histona H4), defesa imunitária (fator de aprimoramento de células natural killer), metabolismo do cálcio (parvalbumina ), adipogênese (UPF0366), metabolismo de xenobióticos e esteroidogênese (citocromo P450scc) e sistema antioxidante (glutationa peroxidase, glutationa-S-transferase, superóxido dismutase, catalase e aldeído desidrogenase). Contribuindo assim para a compreensão dos mecanismos moleculares subjacentes à toxicidade ao mercúrio e fornecendo novas perspectivas sobre possíveis candidatos a biomarcadores de monitoramento ambiental.

Mercury is a toxic metal, of ubiquitous distribution, with capacity for bioaccumulation and biomagnification, that causes changes in important biomolecules in the metabolismo and contributes to the establishment of oxidative stress in aquatic organisms. Therefore, the present study aimed to identify and evaluate possible proteic and/or enzymatic biomarkers of the mercury toxicity in fish from the Amazon region, through a metalloproteomic study and evaluation of oxidative stress. Were used protein fractionation and identification me-thodologies by two-dimensional electrophoresis (2D PAGE) associated with mass spectrome-try (MS), mapping of mercury in protein spots by atomic absorption spectrometry in graphite furnace (GFAAS) and evaluation of oxidative stress markers. The species used were Plagios-cion squamosissimus (corvina) and Colossoma macropomum (tambaqui), collected in the area of the Jirau Hydroelectric Power Plant (Madeira-RO), which were selected due to the population abundance, interest for fishing and different position in the food chain (carnivo-rous and omnivorous, respectively). The tissues sampled were hepatic, renal and muscular. The results showed higher concentration of total mercury in P. squamosissimus, carnivorous species, and distribution pattern of this element equal to both species (liver> kid-ney>muscle). There is a tendency for greater enzymatic activity in the hepatic and renal tis-sues of the specie with the highest concentration of mercury. Only the activity of glutathione peroxidase in the kidney and glutathione-S-transferase in the liver of P.squamosissimus was lower, a fact that can be explained by the interaction of Mercury with these enzymes, inter-fering in their conformation and function. The data obtained by MS allowed the identification of the protein spots associated with mercury, revealing proteins involved in energy metabo-lism (glyceraldehyde-3-phosphate, malate and Llactate dehydrogenase, enolase, creatine kinase and phosphoglycerate mutase), transport ( and hemoglobins, fatty acid binding proteins and GTPases), synthesis and degradation of proteins (ubiquitin and ribosomal S27a and ribosomal 40S S24), cell differentiation (nucleoside diphosphate kinase), gene regulation (pterin-4--5 carbinolamine dehydratase and histone H4), immune defense (natural killer cell enhancement factor), calcium metabolism (-parvalbumin), adipogenesis (UPF0366), xeno-biotic metabolism and steroidogenesis (cytochrome P450scc) and antioxidante system (Glu-tathione peroxidase, glutathione-S-transferase, superoxide dismutase, catalase and al-dehyde dehydrogenase). Thus contributing to the understanding of the molecular mecha-nisms underlying mercury toxicity and providing new perspectives on potential candidates for environmental monitoring biomarkers.

Evaluation of potential antagonistism in yeasts, seeking biocontrol of spoilage by Penicillium expansum / Avaliação do potencial antagônico de leveduras, visando biocontrole de deterioração por Penicillium expansum

Considerable losses during apple fruit storage occur due to microbiological diseases, mainly caused by Penicillium expansum, which in addition to fruit pulp deterioration produces patulin, a mycotoxin with carcinogenic and teratogenic activity. Biological control of post-harvest disease by antagonist yeasts focused on killer toxins is an appreciable alternative to the chemical fungicides, due to the low possibility of toxic residues demonstrated during fermentative processes. Twenty out of 44 yeasts (16 isolated from fruits, 10 from corn silage and 18 from laboratory anthill), showed antagonism against spores of P. expansum. The assay in solid medium pointed the strongest nutrient competition antagonism by D. hansenii strain C1 (31 mm inhibition diameter), while D. hansenii strain C7 (15 mm) showed higher antibiosis and parasitism pattern. In the following step the extracellular activity was tested performing the assay with culture supernatant in Yeast Medium agar, where C. guilliermondii P3 was more effective against conidia germination (inhibition rate of 58.15%) while P. ohmeri showed better inhibition on micelial growth (66.17%). The antibiosis showed by both yeasts could suggest probable mechanism associated with killer phenomenon, once both strains were killer positive against sensitive reference strains (S. cerevisiae NCYC 1006 and P. kluyveri CAY-15). In order to enhanc

As perdas consideráveis no armazenamento de maçãs decorrem principalmente de desordens microbiológicas, causadas por Penicillium expansum, que além de colonizar o fruto e causar dano à polpa, produz a patulina, micotoxina teratogênica e cancerígena. Entre as alternativas ao tradicional tratamento químico de doenças pós-colheita de frutos, enfoca-se o biocontrole por leveduras antagonistas, com ênfase em linhagens killer, em função da baixa possibilidade de resíduos tóxicos e com ampla inocuidade demonstrada nos processos fermentativos. O objetivo deste trabalho foi analisar o potencial antagônico de leveduras no controle de P. expansum, mediante antifungigrama em meio sólido e líquido. Do total de 44 leveduras isoladas (16 de frutas, 10 de silagem de milho e 18 de formigueiro de laboratório), 20 apresentaram antagonismo perante esporos de P. expansum em ágar Meio Para Levedura, sendo Debaryomyces hansenii C1 responsável por maior atividade associada à competição por nutrientes (zona de inibição de 31 mm) e D. hansenii C7 por antibiose/hiperparasitismo (15 mm). Entretanto, o ensaio realizado com o sobrenadante de cultivo reduziu o número de cepas ativas em cinco, sendo Pichia ohmeri 158 e Candida guilliermondii P3 as de maior atividade antagônica. No antifungigrama em meio líquido (caldo MPL) o sobrenadante do cultivo de C. guilliermondii (25ºC/72 horas) inibiu 58,15% da germina

Efeitos de beta-bloqueadores sobre parâmetros comportamentais e imunes no modelo de coabitação com parceiros doentes

A existência de associações bidirecionais entre o Sistema Nervoso e o Sistema Imune reforça a hipótese de que mudanças no Sistema Nervoso Central (SNC) influenciam a resposta imune e a susceptibilidade orgânica às doenças. De fato, em contextos de estresse físico ou psicológico, glicocorticóides liberados pelo córtex das adrenais e catecolaminas produzidas pelo Sistema Nervoso Autônomo Simpático (SNAS) e pela medula das adrenais, modulam a atividade do Sistema Imune. O estresse psicológico associado no ser humano ao ato de cuidar e conviver com companheiros acometidos por diversos tipos de enfermidades crônicas ou transtornos psíquicos têm sido correlacionado ao aparecimento de alterações comportamentais, tais como depressão e ansiedade, assim como alterações imunes e queda da resistência orgânica às infecções. Em nosso laboratório, camundongos que conviveram por 14 dias com coespecíficos portadores de um tumor ascítico de Ehrlich (TAE) apresentaram importantes alterações comportamentais e de imunidade inata, atribuídas tanto à estimulação do sistema catecolaminérgico central e do SNAS. Desta forma, pareceu-nos relevante estudar mais profundamente e dentro de uma perspectiva neuroimune o envolvimento do SNAS com os efeitos desencadeados pela convivência com dois portadores do TAE usando como ferramentas farmacológicas o propranolol (antagonista -adrenérgico) e o ICI-118,551 (antagonista 2-adrenérgico). Os resultados obtidos mostraram que o bloqueio dos receptores -adrenérgicos nos animais companheiros do doente: 1) reverteu o comportamento do tipo-ansioso, uma vez que os camundongos voltaram a frequentar a zona central do campo aberto e reduziu a velocidade média dos animais no campo aberto, porém, não alterou de forma significante a distância total percorrida; 2) reverteu a diminuição nos níveis de noradrenalina e aumentou os níveis do metabólito VMA no hipotálamo, mas não alterou o turnover hipotalâmico de noradrenalina ou os níveis de monoaminas e de seus metabólitos no córtex frontal e no bulbo olfatório; 3) não alterou os níveis séricos de corticosterona; 4) não alterou o peso relativo das adrenais; 5) não alterou a contagem de células na medula óssea; 6) modulou a porcentagem de células natural killer (NK) sanguíneas e esplênicas e diminuiu a expressão da molécula CD69 em células NK esplênicas, mas não alterou a expressão da molécula CD69 em células NK sanguíneas; 7) não alterou o burst basal ou induzido por PMA ou por SAPI e a fagocitose de neutrófilos. O bloqueio dos receptores 2-adrenérgicos nos animais companheiros do doente alterou apenas a expressão da molécula CD69 em células NK sanguíneas, mas não modificou de forma significante quaisquer dos parâmetros comportamentais e neuroquímicos analisados. Conclui-se, então, que algumas das alterações imunes e comportamentais desencadeadas pela convivência com coespecíficos portadores de TAE sejam mediadas pelo SNAS via receptores beta-adrenérgicos.

The existence of bi-directional interactions between the Nervous System and the Immune System reinforces the hypothesis that changes in Central Nervous System (CNS) activity modify not only the immune response but also the susceptibility to diseases. Indeed, in presence of physical or psychological stress, glucocorticoids released by the cortex of the adrenal gland and catecholamines released by the Autonomic Sympathetic Nervous System (ASNS) activation and the adrenal glands were reported to modulate the activity of the Immune System. Psychological stress was described in human caregivers of sick partners, especially when they present chronic diseases and/or psychological disorders, such as depression and anxiety; immune disorders and decreased host resistance to infections were also reported in caregivers. In our laboratory, Swiss mice that lived for 14 days with Ehrlich ascetic tumor bearing conspecifics showed interesting behavioral and innate immune changes that were attributed to catecholaminergic activation within the Central Nervous System or to ASNS activation. Thus, it seemed relevant to study more deeply and under a neuroimmune perspective the involvement of the ASNS in the effects triggered by cohabitation with two sick partners using as pharmacological tools: propranolol (-adrenergic antagonist) and ICI-118.551 (2-adrenergic antagonist). Our results showed that -adrenergic receptors blockade: 1) reversed the anxiety-like behavior induced by cohabitation, once the animals returned to visit the central area of the open field arena and abrogated the effects of the housing condition on the average speed of locomotion measured in those animals; 2) reversed the decrease in norepinephrine levels and increased VMA levels in the hypothalamus; 3) modulated the percentage of natural killer (NK) cells in both, spleen and blood; also, decreased the expression of CD69 molecule on splenic NK cells. Further analysis showed no changes in: 4) the total distance traveled in the open field; 5) the hypothalamic norepinephrine turnover and the levels of monoamines and their metabolites in the frontal cortex and olfactory bulb; 6) the serum corticosterone levels; 7) the relative adrenal glands; 8) cell number in the bone marrow; 9) the basal oxidative burst or those induced by PMA or SAPI in neutrophils and in neutrophils phagocytosis. The blockade of 2-adrenergic receptors by ICI-118.551 changed only the CD69 expression in NK blood cells, being unable to modify any of the behavioral and neurochemical parameters analyzed. Therefore, it seems that only some of the immune and behavioral changes triggered by cohabitation with two sick partners present a relevant participation of SNAS through beta-adrenergic receptors

Construction of killer industrial yeast Saccharomyces cerevisiae HAU-1 and its fermentation performance

Saccharomyces cerevisiae HAU-1, a time tested industrial yeast possesses most of the desirable fermentation characteristics like fast growth and fermentation rate, osmotolerance, high ethanol tolerance, ability to ferment molasses, and to ferment at elevated temperatures etc. However, this yeast was found to be sensitive against the killer strains of Saccharomyces cerevisiae. In the present study, killer trait was introduced into Saccharomyces cerevisiae HAU-1 by protoplast fusion with Saccharomyces cerevisiae MTCC 475, a killer strain. The resultant fusants were characterized for desirable fermentation characteristics. All the technologically important characteristics of distillery yeast Saccharomyces cerevisiae HAU-1 were retained in the fusants, and in addition the killer trait was also introduced into them. Further, the killer activity was found to be stably maintained during hostile conditions of ethanol fermentations in dextrose or molasses, and even during biomass recycling.

Correlation between serum levels of interleukins 10 and 12 and thrombocytopenia in hepatitis C cirrhotic (class A) patients

Hepatitis C virus (HCV) patients commonly have low platelet counts; however, the exact role of HCV infection in thrombocytopenia is unknown. This work aimed to study the serum levels of interleukins (IL) 10 and 12 in patients with mild and moderate thrombocytopenia associated with chronic hepatitis C infection. Our study included 15 patients with chronic HCV infection and newly diagnosed isolated autoimmune thrombocytopenia (Group I) and 15 patients with chronic HCV infection and normal platelet count as controls (Group II). All patients were examined for personal history and clinical aspects, complete blood count, bone marrow aspiration, liver function tests, HCV antibody assay by ELISA and polymerase chain reaction (PCR), abdominal ultrasound, Helicobacter pylori stool antigen test, evaluation of serum levels of IL-10, IL-12 and platelet specific antibodies. Our results revealed that eight patients from Group l had mild thrombocytopenia and seven patients had moderate thrombocytopenia. Serum IL-10 level was significantly elevated (t = 9.301, p < 0.001) while serum IL-12 showed a significant decrease (t = 6.502, p < 0.001) in Group I compared to the control group. No correlation was detected between platelet counts and the serum levels of either IL-10 [r = 0.454, p = 0.089 (Group I), r = 0.038, p = 0.89 (Group II)] or IL-12 [r = 0.497, p = 0.06 (Group I), r = 0.499, p = 0.058 (Group II)]. However, in Group I, a significant correlation was present only between moderate thrombocytopenia and serum levels of either IL-10 (r = 0.794, p = 0.033) or IL-12 (r = 0.967, p = 0.001), while no correlation was detected between these interleukin parameters and mild thrombocytopenia (r = 0.311 and p = 0.453 for IL-10 and r = -0.08 and p = 0.851 for IL-12). Based on our data, we may conclude that interleukins 10 and 12 are involved in low platelet levels.(AU)

Correlation between serum levels of interleukins 10 and 12 and thrombocytopenia in hepatitis C cirrhotic (class A) patients

Hepatitis C virus (HCV) patients commonly have low platelet counts; however, the exact role of HCV infection in thrombocytopenia is unknown. This work aimed to study the serum levels of interleukins (IL) 10 and 12 in patients with mild and moderate thrombocytopenia associated with chronic hepatitis C infection. Our study included 15 patients with chronic HCV infection and newly diagnosed isolated autoimmune thrombocytopenia (Group I) and 15 patients with chronic HCV infection and normal platelet count as controls (Group II). All patients were examined for personal history and clinical aspects, complete blood count, bone marrow aspiration, liver function tests, HCV antibody assay by ELISA and polymerase chain reaction (PCR), abdominal ultrasound, Helicobacter pylori stool antigen test, evaluation of serum levels of IL-10, IL-12 and platelet specific antibodies. Our results revealed that eight patients from Group l had mild thrombocytopenia and seven patients had moderate thrombocytopenia. Serum IL-10 level was significantly elevated (t = 9.301, p < 0.001) while serum IL-12 showed a significant decrease (t = 6.502, p < 0.001) in Group I compared to the control group. No correlation was detected between platelet counts and the serum levels of either IL-10 [r = 0.454, p = 0.089 (Group I), r = 0.038, p = 0.89 (Group II)] or IL-12 [r = 0.497, p = 0.06 (Group I), r = 0.499, p = 0.058 (Group II)]. However, in Group I, a significant correlation was present only between moderate thrombocytopenia and serum levels of either IL-10 (r = 0.794, p = 0.033) or IL-12 (r = 0.967, p = 0.001), while no correlation was detected between these interleukin parameters and mild thrombocytopenia (r = 0.311 and p = 0.453 for IL-10 and r = –0.08 and p = 0.851 for IL-12). Based on our data, we may conclude that interleukins 10 and 12 are involved in low platelet levels.(AU)

Killer yeasts inhibit the growth of the phytopathogen Moniliophthora perniciosa, the causal agent of Witches' Broom disease

Fruit and soil yeasts isolated from the Amazon, Atlantic Rainforests and an organic farm were screened for killer activity against yeasts. Killer yeasts were then tested against the phytopathogen Moniliophthora perniciosa (syn. Crinipellis perniciosa) and a Dipodascus capitatus strain and a Candida sp strain inhibited its growth.

Leveduras de frutas e de solo isoladas da Floresta Amazônica, Mata Atlântica e de uma fazenda orgânica foram selecionadas em uma triagem para atividade micocinogênica. As estirpes micocinogênicas foram posteriormente testadas frente a Moniliophthora perniciosa (syn. Crinipellis perniciosa). Uma estirpe de Dipodascus capitatus e outra de Candida sp.inibiram o crescimento deste fitopatógeno.

Efeitos imunotóxicos da Pteridium aquilinum em células natural killer de camundongos e a reversão destes efeitos com selênio / Immunotoxic effects of Pteridium aquilinum in natural killer cells from mice and the reversion of these effects by selenium

Os resultados obtidos no mestrado mostraram que a samambaia do campo (Pteridium aquilinum) reduz a citotoxicidade das células natural killer (NK) esplênicas e a resposta imune celular do tipo tardia (DTH) de camundongos. Entretanto, até aquele momento não era sabido qual a célula afetada pela planta causava a diminuição da DTH. Assim, o objetivo inicial deste estudo foi verificar qual a célula envolvida na diminuição da DTH. Além disto, buscou-se descobrir o mecanismo de ação imunotóxico da P. aquilinum, o principio tóxico envolvido e se este efeito poderia ser revertido pelo selênio (Se). Para tal, camundongos C57BL/6 foram administrados com extrato de P. aquilinum, por gavage, durante 30 dias e suplementados com Se por mais 30 dias e a análise histológica revelou redução significativa na área de polpa branca esplênica que foi completamente revertida pelo tratamento com Se. Ainda, foi possível verificar que a diminuição da DTH foi causada pela redução da produção de IFNγ pelas células NK durante a indução da resposta imune celular. Além disto, camundongos administrados com ptaquilosídeo, por gavage, durante 14 dias mostraram a mesma redução na atividade das células NK causada pelo extrato de P. aquilinum, assim como a prevenção deste efeito pela co-administração de Se. Por fim, na análise da expressão gênica das células NK esplênicas dos camundongos tratados com ptaquilosídeo e/ou selênio pôde-se observar o aumento da expressão dos genes Mt1 e Mt2, possíveis responsáveis pelo mecanismo imunotóxico da planta, sendo posteriormente confirmado pelo aumento de metalotioneína e consequente redução de Zn2+ livre no espaço intracelular das células NK. Os resultados deste estudo claramente mostram que os efeitos imunossupressores da P. aquilinum são induzidos pelo ptaquilosídeo e são decorrentes do aumento da expressão dos genes da Mt1 e Mt2 e que a suplementação com Se pode prevenir e reverter estes efeitos tóxicos

The results obtained in the master showed that the bracken fern (Pteridium aquilinum) reduces both the cytotoxicity of splenic natural killer cells (NK) and the delayed-type hypersensitivity (DTH) from mice. However, it was not known until that time, which cell affected by the plant that caused a decrease in DTH. Thus, the initial goal of this study was to determine which cell was involved in the reduction of DTH. Moreover, we sought to discover the mechanism of action of the P. aquilinum, the toxic principle involved and whether this effect could be reversed by selenium (Se). For that, C57BL/6 mice were treated with extract of P. aquilinum, by gavage, for 30 days and supplemented with Se for following 30 days. The histological analysis revealed a significant reduction in the splenic white pulp area that was completely reversed by treatment with Se. Still, it was verified that the decrease in DTH was caused by reduced production of IFNγ by NK cells during the induction of cellular immune response. In addition, the mice administered with ptaquiloside, by gavage, for 14 days showed the same reduction in the NK cell activity caused by the extract of P. aquilinum, as well as the prevention of this effect by co-administration of Se. Finally, we could observe an increase in the expression of Mt1 and Mt2 genes in the gene expression analysis of splenic NK cells from mice treated with ptaquiloside and/or selenium. These genes were probably responsible for immunotoxic mechanism of the plant, which was confirmed later by the augment of metallothionein and consequent reduction of free Zn2+ into the intracellular space of NK cells. The results of this study clearly show that the immunosuppressive effects of P. aquilinum are induced by ptaquiloside and they are a consequence of the augment in the gene expression of Mt1 and Mt2 and that the supplementation with Se can prevent and reverse these toxic effects

'Killer' character of yeasts isolated from ethanolic fermentations

The number of killer, neutral and sensitive yeasts was determined from strains isolated from substrates related to alcoholic fermentations. From 113 isolates, 24 showed killer activity against NCYC 1006 (standard sensitive strain), while 30 were sensitive to NCYC 738 (standard killer strain), and 59 had no reaction in assays at 25-27°C. Two wild yeast strains of Saccharomyces cerevisiae and one of Candida colliculosa were tested against 10 standard killer strains and one standard sensitive strain in a cell x cell and well-test assays at four different pHs. None of the isolates displayed strong killer activity or were sensitive to the standard strains. All belonged to the neutral type. It was concluded that although the number of killer strains was high, this character cannot be used to protect ethanol fermentation processes against yeast contaminants like those which form cell clusters.

Avaliou-se o número de linhagens 'killer', sensíveis e neutras em leveduras isoladas de substratos relacionados à fermentação etanólica. Das 113 linhagens, 24 mostraram atividade 'Killer' contra o isolado NCYC 1006 (padrão de sensibilidade), 30 foram sensíveis ao isolado NCYC 738 (padrão 'Killer') e 59 apresentaram reação neutra para ambos os isolados. Três cepas de leveduras selvagens do processo (duas de Sacch. cerevisiae e uma de Candida colliculosa, que formam cachos de células não separáveis por tratamentos químicos ou físicos), foram testadas contra 10 isolados padrões do tipo 'Killer' e um isolado padrão de sensibilidade. Os ensaios células x células e células X toxinas foram realizados em diferentes pH e a 30ºC. As três cepas contaminantes mostraram reação neutra a todos os isolados padrões testados. Apesar do alto número de linhagens 'Killer' entre aquelas testadas, concluiu-se que este caráter não pode ser utilizado na proteção do processo de fermentação etanólica contra essas leveduras selvagens contaminantes.

'Killer' character of yeasts isolated from ethanolic fermentations

The number of killer, neutral and sensitive yeasts was determined from strains isolated from substrates related to alcoholic fermentations. From 113 isolates, 24 showed killer activity against NCYC 1006 (standard sensitive strain), while 30 were sensitive to NCYC 738 (standard killer strain), and 59 had no reaction in assays at 25-27°C. Two wild yeast strains of Saccharomyces cerevisiae and one of Candida colliculosa were tested against 10 standard killer strains and one standard sensitive strain in a cell x cell and well-test assays at four different pHs. None of the isolates displayed strong killer activity or were sensitive to the standard strains. All belonged to the neutral type. It was concluded that although the number of killer strains was high, this character cannot be used to protect ethanol fermentation processes against yeast contaminants like those which form cell clusters.

Avaliou-se o número de linhagens 'killer', sensíveis e neutras em leveduras isoladas de substratos relacionados à fermentação etanólica. Das 113 linhagens, 24 mostraram atividade 'Killer' contra o isolado NCYC 1006 (padrão de sensibilidade), 30 foram sensíveis ao isolado NCYC 738 (padrão 'Killer') e 59 apresentaram reação neutra para ambos os isolados. Três cepas de leveduras selvagens do processo (duas de Sacch. cerevisiae e uma de Candida colliculosa, que formam cachos de células não separáveis por tratamentos químicos ou físicos), foram testadas contra 10 isolados padrões do tipo 'Killer' e um isolado padrão de sensibilidade. Os ensaios células x células e células X toxinas foram realizados em diferentes pH e a 30ºC. As três cepas contaminantes mostraram reação neutra a todos os isolados padrões testados. Apesar do alto número de linhagens 'Killer' entre aquelas testadas, concluiu-se que este caráter não pode ser utilizado na proteção do processo de fermentação etanólica contra essas leveduras selvagens contaminantes.

Characterization of the Saccharomyces cerevisae Y500-4L killer toxin

The strain Saccharomyces cerevisiae Y500-4L, selected from the must of alcohol producing plants, liberates a toxin which is lethal to the commercial yeast produced by Fleischmann Royal Nabisco and other strains of yeast. This toxin was characterized, and the maximum production was obtained after 24 hours of incubation at 25ºC in YEPD medium. The maximum activity was achieved between pH 4.1 and 4.5 and between 22 and 25ºC and maximum stability in the pH range 3.8 to 4.5 at -10ºC. The killer toxin was inactivated by heating at 40ºC for 1 hour at pH 4.1. After concentration by ultrafiltration of culture supernatants and purification by gel filtration chromatography, the molecular weight of the purified toxin was estimated by SDS-PAGE to be about 18-20 kDa.

A linhagem de Saccharomyces cerevisiae Y500-4L, selecionada de mosto de fermentação de usina de álcool, produz toxina "killer", letal à levedura comercial Fleischmann Royal Nabisco e outras linhagens de leveduras. Esta proteína foi caracterizada, verificando-se que a produção máxima foi obtida após 24 horas de incubação a 25ºC em meio YEPD. A toxina "killer" apresentou maior atividade na faixa de pH 4,1-4,5 e temperatura de 22-25ºC; e maior estabilidade na faixa de pH 3,8-4,5 a -10ºC, sendo totalmente inativada após 1 hora de incubação a 40ºC em pH 4,1. Após concentração a partir do sobrenadante do meio de cultura através de ultrafiltração e purificação por cromatografia de filtração em gel, estimou-se, através de SDS-PAGE, que o peso molecular desta toxina é cerca de 18 a 20 kDa.

Isolation, characterization, and identification of wild killer yeasts from sugarcane juice / Isolamento, caracterização e identificação de leveduras killer de caldo de cana de açúcar

Ethanol produced by the fermentation of sugarcane juice has emerged as an important renewable fuel. The yield of this fermentation is affected by undesirable microbial contaminants, but killer yeasts can be a promising strategy to reduce this problem. The present study aimed to isolate, characterize, and identify wild killer yeasts from sugarcane juice. Samples were inoculated in culture medium containing chloramphenicol, and 140 colonies with different characteristics were selected. These isolates were submitted to the killer phenotype assay, and the positive killers were characterized and identified according to the standard methods. Only two strains showed killer activity, identified as Pichia anomala CE025 and P. membranaefaciens CE088. At 25C, both strains exhibited killer activity at pH 4.0, 4.3, and 4.5, but this activity was not detected at pH 3.0, 3.5, 5.0, and 6.0. The killer phenotype of P. membranaefaciens CE088 was inhibited above 30C, while for P. anomala, CE025 inhibition occurred only at a higher temperature. Both strains were able to grow in 12% ethanol, and P. anomala CE025 was more tolerant than P. membranaefaciens CE 088. Further studies will be conducted to isolate, purify and identify the killer toxins produced by Pichia anomala and Pichia membranaefaciens species.

O etanol produzido a partir da fermentação do caldo de cana emergiu como um combustível renovável. O rendimento desta fermentação é afetado por micro-organismos indesejáveis e as leveduras killer se constituem uma alternativa promissora para combater essa contaminação. Nesta perspectiva, o presente trabalho teve como objetivo isolar, caracterizar e identificar leveduras killer de caldo de cana. As amostras foram inoculadas em meio de cultura contendo cloranfenicol e 140 colônias com diferentes características foram selecionadas. Esses isolados foram avaliados quanto à presença do fator killer e os isolados positivos caracterizados e identificados por métodos convencionais. Apenas dois isolados apresentaram atividade killer e foram identificados como Pichia anomala CE025 e P. membranaefaciens CE088. A 25C as duas linhagens exibiram atividade killer em pH 4.0, 4.3 e 4.5, mas esta atividade foi inibida a pH 3.0, 3.5, 5.0 e 6.0. Para P. membranaefaciens CE088 o fenótipo killer foi inibido acima de 30C, enquanto que a P. anomala CE025 exibiu essa característica acima deste valor. Ambas as linhagens foram capazes de crescer na presença de 12% de etanol, mas P. anomala CE025 foi mais tolerante do que P. membranaefaciens CE088. Estudos posteriores serão realizados para isolar, purificar e identificar as toxinas killer produzidas pelas espécies Pichia anomala e Pichia membranaefacien.

Evaluation of potential antagonistism in yeasts, seeking biocontrol of spoilage by Penicillium expansum / Avaliação do potencial antagônico de leveduras, visando biocontrole de deterioração por Penicillium expansum

Considerable losses during apple fruit storage occur due to microbiological diseases, mainly caused by Penicillium expansum, which in addition to fruit pulp deterioration produces patulin, a mycotoxin with carcinogenic and teratogenic activity. Biological control of post-harvest disease by antagonist yeasts focused on killer toxins is an appreciable alternative to the chemical fungicides, due to the low possibility of toxic residues demonstrated during fermentative processes. Twenty out of 44 yeasts (16 isolated from fruits, 10 from corn silage and 18 from laboratory anthill), showed antagonism against spores of P. expansum. The assay in solid medium pointed the strongest nutrient competition antagonism by D. hansenii strain C1 (31 mm inhibition diameter), while D. hansenii strain C7 (15 mm) showed higher antibiosis and parasitism pattern. In the following step the extracellular activity was tested performing the assay with culture supernatant in Yeast Medium agar, where C. guilliermondii P3 was more effective against conidia germination (inhibition rate of 58.15%) while P. ohmeri showed better inhibition on micelial growth (66.17%). The antibiosis showed by both yeasts could suggest probable mechanism associated with killer phenomenon, once both strains were killer positive against sensitive reference strains (S. cerevisiae NCYC 1006 and P. kluyveri CAY-15). In order to enhanc

As perdas consideráveis no armazenamento de maçãs decorrem principalmente de desordens microbiológicas, causadas por Penicillium expansum, que além de colonizar o fruto e causar dano à polpa, produz a patulina, micotoxina teratogênica e cancerígena. Entre as alternativas ao tradicional tratamento químico de doenças pós-colheita de frutos, enfoca-se o biocontrole por leveduras antagonistas, com ênfase em linhagens killer, em função da baixa possibilidade de resíduos tóxicos e com ampla inocuidade demonstrada nos processos fermentativos. O objetivo deste trabalho foi analisar o potencial antagônico de leveduras no controle de P. expansum, mediante antifungigrama em meio sólido e líquido. Do total de 44 leveduras isoladas (16 de frutas, 10 de silagem de milho e 18 de formigueiro de laboratório), 20 apresentaram antagonismo perante esporos de P. expansum em ágar Meio Para Levedura, sendo Debaryomyces hansenii C1 responsável por maior atividade associada à competição por nutrientes (zona de inibição de 31 mm) e D. hansenii C7 por antibiose/hiperparasitismo (15 mm). Entretanto, o ensaio realizado com o sobrenadante de cultivo reduziu o número de cepas ativas em cinco, sendo Pichia ohmeri 158 e Candida guilliermondii P3 as de maior atividade antagônica. No antifungigrama em meio líquido (caldo MPL) o sobrenadante do cultivo de C. guilliermondii (25ºC/72 horas) inibiu 58,15% da germina

Avaliação dos efeitos imunotóxicos da Pteridium aquilinum. Estudo em camundongos / Evaluation of the immunotoxic effects of Pteridium aquilinum. Study in mice

Pteridium aquilinum, conhecida popularmente como "samambaia-do-campo" ou simplesmente "samambaia", é considerada uma das plantas tóxicas mais importantes no mundo, não só pela sua distribuição cosmopolita e intoxicação de rebanhos em diversas partes do mundo, mas também pelo seu alto potencial carcinogênico observado em animais e seres humanos que se alimentam com esta planta. Por outro lado, não havia dados na literatura a respeito dos possíveis efeitos tóxicos desta planta sobre o sistema imune, o qual se sabe, tem papel fundamental não só para o controle de doenças infecciosas, como também, para impedir a proliferação de células mutantes e, conseqüentemente o desenvolvimento de câncer. Assim, o presente estudo avaliou os efeitos da P. aquilinum sobre as respostas imune inata e adaptativa em camundongos, através dos seguintes protocolos: produção e titulação de anticorpos T - dependente, proliferação de linfócitos T e B, resposta de hipersensibilidade tardia, fenotipagem linfocítica e citotoxicidade de células NK. Além disso, foram feitas a avaliação histológica e a contagem da celularidade dos órgãos linfóides. Resultados mostraram diminuição da resposta de hipersensibilidade tardia (resposta celular) nos grupos tratados com 10 e 30 g/kg de samambaia, redução da citotoxicidade das células NK, redução da polpa branca do baço, diminuição da camada celular do timo e desorganização dos folículos linfóides nos linfonodos mesentéricos e placas de Peyer dos camundongos tratados com a dose de 30 g/kg de samambaia e diminuição na celularidade da medula óssea em todos os grupos tratados com a samambaia, por 14 dias. Os dados obtidos na presente pesquisa permitem sugerir que a diminuição da resposta imune celular foi decorrente do efeito tóxico da P. aquilinum sobre as células NK e não um efeito tóxico direto sobre os linfócitos Th1

Pteridium aquilinum, known popularly as \"bracken fern\" is considered one of the more important toxic plants in the world, not only for its cosmopolite distribution and poisoning of flocks in diverse parts of the world, but also for its high potential carcinogenicity observed in animals and human that feed with this plant. On the other hand, there are not data available in the literature regarding the possible toxic effects of this plant on the immune system that is fairly known to be important not also for the control of infectious illnesses but also to hinder the proliferation of mutant cells and, consequently cancer development. Thus, the present study evaluated the effect of the P. aquilinum on the innate and acquired immune responses in mice, through the following protocols: production and titer of T - dependent antibody, proliferation of T and B lymphocytes, delayed-type hypersensitivity, lymphocyte subset analysis and natural killer-cell activity. Moreover, histophatological evaluation and cellularity of the lymphoid organs were performed. Results showed reduction of the delayed-type hypersensitivity (cellular immune response) of the mice treated with 10 and 30 g/kg of bracken fern. Besides, it was observed reduction of the natural killer-cell cytotoxicity, decrease of white pulp of spleen and of the cellular layer of the thymus, disorganization of the lymphoid follicle in the mesenteric lymph nodes and Peyer?s patches of the mice treated with 30 g/kg. It was also observed decrease of bone marrow cellularity of all animals treated with bracken fern up to 14 days. Thus, these data found here permit to suggest that P. aquilinum produced reduction of the cellular immune response by a direct toxic effect on the natural-killer cells and not on the Th1 lymphocytes