Loxosceles amazonica Brown Spider venom: Insights into enzymatic activities, immunorecognition, and novel phospholipase D isoforms.

The Loxosceles genus represents one of the main arachnid genera of medical importance in Brazil. Despite the gravity of Loxosceles-related accidents, just a handful of species are deemed medically important and only a few have undergone comprehensive venom characterization. Loxosceles amazonica is a notable example of a potentially dangerous yet understudied Loxosceles species. While there have been limited reports of accidents involving L. amazonica to date, accidents related to Loxosceles are increasing in the North and Northeast regions of Brazil, where L. amazonica has been reported. In this work, we provide a complementary biochemical and immunological characterization of L. amazonica venom, considering its most relevant enzymatic activities and its immunorecognition and neutralization by current therapeutic antivenoms. Additionally, a cDNA library enriched with phospholipase D (PLD) sequences from L. amazonica venom glands was built and subsequently sequenced. The results showed that L. amazonica venom is well immunorecognised by all the tested antibodies. Its venom also displayed proteolytic, hyaluronidase, and sphingomyelinase activities. These activities were at least partially inhibited by available antivenoms. With cDNA sequencing of PLDs, seven new putative isoforms were identified in the venom of L. amazonica. These results contribute to a better knowledge of the venom content and activities of a synanthropic, yet understudied, Loxosceles species. In vivo assays are essential to confirm the medical relevance of L. amazonica, as well as to assess its true toxic potential and elucidate its related pathophysiology.

Microbiological attributes as indicators of soil quality in coffee growing systems in Southwest Bahia, Brazil.

Microorganisms are important indicators of soil quality due to their sensitivity to changes, reflecting the impacts caused by different land uses. The objective of this study was to evaluate the microbiological and physical-chemical attributes of the soil in areas cultivated with coffee under three different management systems (shaded coffee and full sun coffee with two spacings), as well as in adjacent areas under pasture and native forest, in Bahia, Brazil. The microbiological and physicochemical indicators evaluated were basal soil respiration (MBR), soil total organic carbon (TOC), microbial biomass carbon (MBC), metabolic quotient (qCO2), microbial quotient (qMic), enzyme activities (urease, acid phosphatase and fluorescein diacetate hydrolysis (FDA)). Physical and chemical indicators (particle size, texture, pH, P, K+, Ca2+, Mg2+, Al3+, and sum of bases) were also evaluated. Biological and chemical attributes were much more discriminative of study areas in the dry season. Microbial quotient (qMic) and metabolic quotient (qCO2) in the dry season showed that pasture is the most degraded land use. Conversely, nature forest and coffee with Grevillea were similar and were the best ones. In general, soil quality indicators were more sensitive to discriminate pasture and native forest from coffee systems, which, in turn, were not well discriminated among themselves.

Toxic and antigenic characterization of Peruvian Micrurus surinamensis coral snake venom.

Micrurus surinamensis is a semi-aquatic coral snake found in primary forest region and can cause relevant human accidents. In this work we investigated the toxic and antigenic activities of the Peruvian Micrurus surinamensis venom (MsV). We found that MsV show hyaluronidase activity but lack LAAO and PLA2 enzymatic activities. Interestingly, MsV induce edematogenic responses but cannot cause nociceptive effects. Furthermore, MsV can reduce in vitro cell viability in MGSO-3 cell line derived from human breast cancer tissue. To evaluate its antigenic potential, rabbits were immunized with MsV, which proved to be immunogenic. ELISA, immunobloting and in vivo neutralization assays demonstrated that the specific rabbit anti-MsV antivenom is more efficient than the therapeutic Brazilian antivenom in recognizing and neutralizing the lethal activity of MsV. MsV differs in protein profile and biological activities from M. frontalis venom (MfV), used as control, which impairs its recognition and neutralization by Brazilian therapeutic anti-elapidic antivenom. We performed a SPOT immunoassay for the identification of B-cell linear epitopes in the main toxins described for MsV targeted by the elicited neutralizing antibodies previously produced. A membrane containing 15-mer peptides representing the sequences of five 3TFxs and five PLA2s was produced and probed with anti- MsV antibodies. Results revealed important regions in 3FTx toxins for venom neutralization. Identifying the main MsV components and its biological activities can be helpful in guiding the production of antivenoms and in the optimization of treatment for coral snake envenomation in Brazil.

Biocontrol of Phyllosticta citricarpa by Bacillus spp.: biological and chemical aspects of the microbial interaction.

Citrus fruits are the most produced fruits in the world, but they are threatened by several pathogens, including the fungus Phyllosticta citricarpa, the causal agent of citrus black spot (CBS). The fungus affects most citrus species and the infection results in economic losses in citrus-producing areas. This disease causes the aesthetic depreciation of fresh fruit, impairing its commercialization. As an alternative to the use of synthetic fungicides to control the pathogen, the biological control, using bacteria of the genus Bacillus, is highlighted. Such microorganisms enable biocontrol by the production of volatile organic compounds (VOC) or non-volatile. Therefore, this work aimed to evaluate the production of VOC by isolates of Bacillus spp. grown in different culture media; to evaluate the effects of these compounds on the evolution of CBS lesions in orange fruits; to study the effects of VOC on resistance induction in orange fruits; to evaluate the effects of VOC on P. citricarpa morphology in CBS lesions, and to identify the produced VOC. Tryptone soya agar (TSA) and tryptone soya broth (TSB) media used to culture the bacterium resulted in up to 73% pathogen inhibition by VOC. Volatile compounds from Bacillus spp. ACB-65 and Bacillus spp. ACB-73 when cultured in TSB culture medium provided 86% inhibition of freckles that evolved to hard spots. The volatile fractions produced by the bacteria were identified as alcohols, ketones, amines, ethers, aldehydes and carboxylic acids that can serve as arsenal against the phytopathogen. The present work demonstrated the potential of VOC produced by Bacillus spp. in the control of P. citricarpa.

Culture media based on effluent derived from soy protein concentrate production for Lacticaseibacillus paracasei 90 biomass production: statistical optimisation, mineral characterization, and metabolic activities.

The waste and by-products of the soybean industry could be an economic source of nutrients to satisfy the high nutritional demands for the cultivation of lactic acid bacteria. The aims of this work were to maximize the biomass production of Lacticaseibacillus paracasei 90 (L90) in three culture media formulated from an effluent derived from soy protein concentrate production and to assess the effects these media have on the enzymatic activity of L90, together with their influence on its fermentation profile in milk. The presence of essential minerals and fermentable carbohydrates (sucrose, raffinose, and stachyose) in the effluent was verified. L90 reached high levels of microbiological counts (∼ 9 log cfu mL-1) and dry weight (> 1 g L-1) on the three optimized media. Enzymatic activities (lactate dehydrogenase and ß-galactosidase) of L90, and its metabolism of lactose and citric acid, as well as lactic acid and pyruvic acid production in milk, were modified depending on the growth media. The ability of the L90 to produce the key flavour compounds (diacetyl and acetoin) was maintained or improved by growing in the optimized media in comparison with MRS.

Service crops improve a degraded monoculture system by changing common bean rhizospheric soil microbiota and reducing soil-borne fungal diseases.

Intensive agricultural practices have resulted in progressive soil degradation, with consequences on soil ecosystem services. The inclusion of service crops is a promising alternative to support the sustainability of the agricultural system. The aim of this study was to analyze in a six-year field experiment the effect of Brachiaria brizantha (perennial tropical grass) and Zea mays as service crops in a degraded common bean monoculture system in northwest Argentina. After six years, service crop treatments revealed a significant increase in most physical, chemical and biological properties of the soil (enzyme activities, microbial biomass, respiration and glomalin-related soil protein), compared with common bean monoculture. Also, a lower disease incidence was observed under B. brizantha treatments, associated with increased populations of Trichoderma spp. and Gliocladium spp. The phospholipid fatty acid profiles detected higher values of total microbial biomass under service crops. Our results suggest that the inclusion of several cycles of B. brizantha constitutes a promising soil management for recovering degraded agroecosystems.

Evaluation of hydrolytic enzyme activities from digestive fluids of Anthonomus grandis (Coleoptera: Curculionidae).

The cotton boll weevil, Anthonomus grandis, is a major pest of cotton crops in South America. In this work, partial biochemical characterizations of (hemi) cellulases and pectinases activities in the digestive system (head- and gut- extracts) of A. grandis were evaluated. Gut extract section from third instar larvae exhibited endoglucanase, xylanase, ß-glucosidase, and pectinase activities. The endoglucanase and xylanase activities were localized in the foregut, whereas ß-glucosidase activity was mainly detected in the hindgut. In addition, no difference in pectinase activity was observed across the gut sections. Thus, A. grandis digestive system is a potentially interesting reservoir for further lignocellulolytic enzymes research.

Primary Metabolism Is Distinctly Modulated by Plant Resistance Inducers in Coffea arabica Leaves Infected by Hemileia vastatrix.

Epidemics of coffee leaf rust (CLR) leads to great yield losses and huge depreciation of coffee marketing values, if no control measures are applied. Societal expectations of a more sustainable coffee production are increasingly imposing the replacement of fungicide treatments by alternative solutions. A protection strategy is to take advantage of the plant immune system by eliciting constitutive defenses. Based on such concept, plant resistance inducers (PRIs) have been developed. The Greenforce CuCa formulation, similarly to acibenzolar-S-methyl (ASM), shows promising results in the control of CLR (Hemileia vastatrix) in Coffea arabica cv. Mundo Novo. The molecular mechanisms of PRIs action are poorly understood. In order to contribute to its elucidation a proteomic, physiological (leaf gas-exchange) and biochemical (enzymatic) analyses were performed. Coffee leaves treated with Greenforce CuCa and ASM and inoculation with H. vastatrix were considered. Proteomics revealed that both PRIs lead to metabolic adjustments but, inducing distinct proteins. These proteins were related with photosynthesis, protein metabolism and stress responses. Greenforce CuCa increased photosynthesis and stomatal conductance, while ASM caused a decrease in these parameters. It was further observed that Greenforce CuCa reinforces the redox homeostasis of the leaf, while ASM seems to affect preferentially the secondary metabolism and the stress-related proteins. So, the PRIs prepare the plant to resist CLR but, inducing different defense mechanisms upon pathogen infection. The existence of a link between the primary metabolism and defense responses was evidenced. The identification of components of the plant primary metabolism, essential for plant growth and development that, simultaneously, participate in the plant defense responses can open new perspectives for plant breeding programs.

Molecular composition of the paralyzing venom of three solitary wasps (Hymenoptera: Pompilidae) collected in southeast Mexico.

The chemical and biological characterization of peptide and protein components of the paralyzing venom from three Pompilidae solitary spider wasps (Pepsis mexicana, Pepsis terminata, and Anoplius nigritus) is described for the first time. The molecular masses of the most abundant peptides were determined. The N-terminal sequences of two cysteine-rich peptides were obtained from Pepsis. Metalloproteinase and hyaluronidase activities were identified in the venom of P. mexicana. A novel non-lethal method to collect venom is described.

Perfil lipídico da carne de frangos de corte de diferentes cruzamentos criados em sistema alternativo / Lipid profile of broiler meat from different crosses raised in alternative system

Objetivou-se avaliar o perfil lipídico da carne de frangos de diferentes genótipos. O delineamento foi inteiramente ao acaso (DIC), disposto em esquema fatorial (5x2), sendo cinco genótipos (New Hampshire ˗ NHS; Gigante Negra de Jersey ˗ GNJ; Índio Gigante ˗ IG; cruzamento entre as raças IG e NHS ˗ IG x NHS; e entre as raças IG e GNJ ˗ IG x GNJ) e dois sexos, com cinco repetições, sendo cada uma representada por três aves, totalizando 150 aves, abatidas aos 105 dias. As análises de perfil lipídico foram realizadas no peito e na coxa. Foram calculadas as estimativas das atividades enzimáticas, os índices de aterogenicidade e de trombogenicidade. Os genótipos IG e IG x NHS apresentaram maiores teores de ácido araquidônico e DHA. Foram observados maiores teores de ácidos graxos saturados e monoinsaturados no peito para os genótipos IG x NHS e NHS, respectivamente. Maiores médias de ácidos graxos poli-insaturados e ômega 3 foram observadas para os genótipos IG e IG x NHS. O genótipo IG x NHS e as fêmeas apresentaram melhores características de qualidade de carne, por oferecerem uma maior fonte de ômega 3.(AU)

The objective was to evaluate the lipid profile of chickens from different genotypes. The design was completely randomized arranged in factorial scheme (5x2), being 5 genotypes (New Hampshire - NHS, Gigante Negra de Jersey - GNJ, Índio Gigante - IG; poultry from the cross between IG and NHS breeds - IG x NHS and between IG and GNJ breeds - IG x GNJ) and two genders, with five replicates and three poultry per replicate, totaling 150 birds, slaughtered at 105 days. Lipid profile analyzes were performed on the breast and thigh. Estimates of the enzymatic activities related to lipid metabolism were calculated, in addition to the atherogenicity and thrombogenicity indexes. The IG and IG x NHS genotypes showed higher levels of arachidonic acid and DHA. Higher levels of saturated and monounsaturated fatty acids were observed in the breast for IG x NHS and NHS genotypes, respectively. Higher averages of polyunsaturated fatty acids and omega 3 were observed for the IG and IG x NHS genotypes. The genotype IG x NHS and the females presented better characteristics of meat quality, for offering a greater source of omega 3.(AU)

Perfil lipídico da carne de frangos de corte de diferentes cruzamentos criados em sistema alternativo / Lipid profile of broiler meat from different crosses raised in alternative system

Objetivou-se avaliar o perfil lipídico da carne de frangos de diferentes genótipos. O delineamento foi inteiramente ao acaso (DIC), disposto em esquema fatorial (5x2), sendo cinco genótipos (New Hampshire ˗ NHS; Gigante Negra de Jersey ˗ GNJ; Índio Gigante ˗ IG; cruzamento entre as raças IG e NHS ˗ IG x NHS; e entre as raças IG e GNJ ˗ IG x GNJ) e dois sexos, com cinco repetições, sendo cada uma representada por três aves, totalizando 150 aves, abatidas aos 105 dias. As análises de perfil lipídico foram realizadas no peito e na coxa. Foram calculadas as estimativas das atividades enzimáticas, os índices de aterogenicidade e de trombogenicidade. Os genótipos IG e IG x NHS apresentaram maiores teores de ácido araquidônico e DHA. Foram observados maiores teores de ácidos graxos saturados e monoinsaturados no peito para os genótipos IG x NHS e NHS, respectivamente. Maiores médias de ácidos graxos poli-insaturados e ômega 3 foram observadas para os genótipos IG e IG x NHS. O genótipo IG x NHS e as fêmeas apresentaram melhores características de qualidade de carne, por oferecerem uma maior fonte de ômega 3.(AU)

The objective was to evaluate the lipid profile of chickens from different genotypes. The design was completely randomized arranged in factorial scheme (5x2), being 5 genotypes (New Hampshire - NHS, Gigante Negra de Jersey - GNJ, Índio Gigante - IG; poultry from the cross between IG and NHS breeds - IG x NHS and between IG and GNJ breeds - IG x GNJ) and two genders, with five replicates and three poultry per replicate, totaling 150 birds, slaughtered at 105 days. Lipid profile analyzes were performed on the breast and thigh. Estimates of the enzymatic activities related to lipid metabolism were calculated, in addition to the atherogenicity and thrombogenicity indexes. The IG and IG x NHS genotypes showed higher levels of arachidonic acid and DHA. Higher levels of saturated and monounsaturated fatty acids were observed in the breast for IG x NHS and NHS genotypes, respectively. Higher averages of polyunsaturated fatty acids and omega 3 were observed for the IG and IG x NHS genotypes. The genotype IG x NHS and the females presented better characteristics of meat quality, for offering a greater source of omega 3.(AU)

Bothrops fonsecai snake venom activities and cross-reactivity with commercial bothropic venom.

In this work, we examined some biochemical and biological activities of Bothrops fonsecai venom, a pitviper endemic to southeastern Brazil, and assessed their neutralization by commercial bothropic antivenom (CAv). Cross-reactivity of venom with CAv was also assessed by immunoblotting and size-exclusion high performance chromatography (SE-HPLC). Bothrops fonsecai venom had PLA2, proteolytic and esterase activities that were neutralized to varying extents by venom:antivenom ratios of 5:1 and 5:2 (PLA2 and esterase activities) or not significantly by either venom:antivenom ratio (proteolytic activity). The minimum hemorrhagic dose (69.2µg) was totally neutralized by both ratios. Clotting time in rat citrated plasma was 33±10.5s (mean±SD; n=5) and was completely neutralized by a 5:2 ratio. Edema formation was dose-dependent (1-30µg/site) and significantly inhibited by both ratios. Venom (10-300µg/mL) caused neuromuscular blockade in extensor digitorum longus preparations; this blockade was inhibited best by a 5:2 ratio. Venom caused myonecrosis and creatine kinase release in vivo (gastrocnemius muscle) and in vitro (extensor digitorum longus) that was effectively neutralized by both venom:antivenom ratios. Immunoblotting showed that venom components of ~25-100kDa interacted with CAv. SE-HPLC profiles for venom incubated with CAv or specific anti-B. fonsecai antivenom raised in rabbits (SAv) indicated that CAv had a higher binding capacity than SAv, whereas SAv had higher affinity than CAv. These findings indicate that B. fonsecai venom contains various activities that are neutralized to different extents by CAv and suggest that CAv could be used to treat envenoming by B. fonsecai.

Paraoxonase 1 gene polymorphisms and enzyme activities in coronary artery disease and its relationship to serum lipids and glycemia / Polimorfismos en el gen de la paraoxonasa 1 y sus actividades enzimáticas en la enfermedad coronaria. Su relación con el perfil lipídico y la glucemia

Abstract Objectives Oxidative stress and inflammation are important processes in development of atherosclerosis. Paraoxonase 1 (PON1) is a bioscavenger enzyme associated with inflammation and oxidative stress. We evaluate the association of two single nucleotide polymorphisms in PON1 gene, and enzyme activities with lipid profile and glycemia. Methods This case-control study consisted of 126 patients with coronary artery disease (CAD) and 203 healthy controls. PON Q192R and L55M polymorphisms were detected by real-time PCR. Paraoxonase and arylesterase activities were determined spectrophotometrically. Blood glucose, cholesterol, triglycerides, HDL, and LDL were measured. Results PON1 QR192 polymorphism had a major effect on paraoxonase but no effect on arylesterase serum activities. Paraoxonase activity was higher in RR genotype and lowest in QQ genotype. Paraoxonase and arylesterase activities were higher in LL and lower in MM genotypes of PON1 LM55 polymorphism. RQ and LM variants showed intermediate activities between respective homozygous. Elevated concentrations of triglycerides in cases correlate with QQ variant or the presence of M allele. Glucose levels were elevated in cases with QQ variant or with the presence of M allele. Cholesterol and LDL did not show variations in control and cases with any variant of both polymorphisms. HDL is lower in cases with respect to controls independently of genotypes. All differences were significant with p < 0.05. Conclusions Our results confirm the relationship between variations in PON1 activities and lipid metabolism, and showed that genetically programmed low PON1 activities would have certain responsibility in the increase in glycemia and concomitantly the aggravation of atherosclerotic disease.

Resumen Objetivos La enzima paraoxonasa 1 (PON1), está asociada con el estrés oxidativo y la inflamación, procesos importantes en el desarrollo de la aterosclerosis. Evaluamos la asociación de 2 polimorfismos de un solo nucleótido en el gen PON1 y sus actividades enzimáticas con el perfil lipídico y la glucemia. Métodos Estudio caso-control en 126 pacientes con enfermedad coronaria y 203 controles sanos. Los polimorfismos PON Q192R y L55M fueron detectados por PCR en tiempo real y las actividades de paraoxonasa y arilesterasa por espectrofotometría. Se midieron glucemia, colesterol, triglicéridos, HDL y LDL. Resultados El polimorfismo PON1 QR192 afectó la actividad de paraoxonasa pero no la de arilesterasa. La actividad de paraoxonasa fue mayor en el genotipo RR y menor en QQ. Ambas actividades fueron mayores en el genotipo LL y menores en MM del polimorfismo PON1 LM55. Las variantes RQ y LM mostraron actividades intermedias entre los respectivos homocigotos. Concentraciones elevadas de triglicéridos en los casos correlacionaron con la variante QQ o la presencia del alelo M. Los niveles de glucosa fueron elevados en los casos QQ o con la presencia del alelo M. El colesterol y el LDL no variaron ni en los casos ni en los controles con ambos polimorfismos. El HDL fue menor en los casos respecto de los controles, independientemente del genotipo. Conclusiones Los resultados confirman la relación entre las variaciones en las actividades de PON1 y el metabolismo lipídico y mostraron que las bajas actividades de PON1 genéticamente programadas tendrían cierta responsabilidad en el aumento de la glucemia y, concomitantemente, en la agravación de la enfermedad aterosclerótica.

Paraoxonase 1 gene polymorphisms and enzyme activities in coronary artery disease and its relationship to serum lipids and glycemia.

OBJECTIVES: Oxidative stress and inflammation are important processes in development of atherosclerosis. Paraoxonase 1 (PON1) is a bioscavenger enzyme associated with inflammation and oxidative stress. We evaluate the association of two single nucleotide polymorphisms in PON1 gene, and enzyme activities with lipid profile and glycemia. METHODS: This case-control study consisted of 126 patients with coronary artery disease (CAD) and 203 healthy controls. PON Q192R and L55M polymorphisms were detected by real-time PCR. Paraoxonase and arylesterase activities were determined spectrophotometrically. Blood glucose, cholesterol, triglycerides, HDL, and LDL were measured. RESULTS: PON1 QR192 polymorphism had a major effect on paraoxonase but no effect on arylesterase serum activities. Paraoxonase activity was higher in RR genotype and lowest in QQ genotype. Paraoxonase and arylesterase activities were higher in LL and lower in MM genotypes of PON1 LM55 polymorphism. RQ and LM variants showed intermediate activities between respective homozygous. Elevated concentrations of triglycerides in cases correlate with QQ variant or the presence of M allele. Glucose levels were elevated in cases with QQ variant or with the presence of M allele. Cholesterol and LDL did not show variations in control and cases with any variant of both polymorphisms. HDL is lower in cases with respect to controls independently of genotypes. All differences were significant with p<0.05. CONCLUSIONS: Our results confirm the relationship between variations in PON1 activities and lipid metabolism, and showed that genetically programmed low PON1 activities would have certain responsibility in the increase in glycemia and concomitantly the aggravation of atherosclerotic disease.

Biological attributes of rehabilitated soils contaminated with heavy metals.

This study aimed to evaluate the effects of two rehabilitation systems in sites contaminated by Zn, Cu, Pb, and Cd on biological soil attributes [microbial biomass carbon (Cmic), basal and induced respiration, enzymatic activities, microorganism plate count, and bacterial and fungal community diversity and structure by denaturing gradient gel electrophoresis (DGGE)]. These systems (S1 and S2) consisted of excavation (trenching) and replacement of contaminated soil by uncontaminated soil in rows with Eucalyptus camaldulensis planting (S1-R and S2-R), free of understory vegetation (S1-BR), or completely covered by Brachiaria decumbens (S2-BR) in between rows. A contaminated, non-rehabilitated (NR) site and two contamination-free sites [Cerrado (C) and pasture (P)] were used as controls. Cmic, densities of bacteria and actinobacteria, and enzymatic activities (ß-glucosidase, acid phosphatase, and urease) were significantly higher in the rehabilitated sites of system 2 (S2-R and S2-BR). However, even under high heavy metal contents (S1-R), the rehabilitation with eucalyptus was also effective. DGGE analysis revealed similarity in the diversity and structure of bacteria and fungi communities between rehabilitated sites and C site (uncontaminated). Principal component analysis showed clustering of rehabilitated sites (S2-R and S2-BR) with contamination-free sites, and S1-R was intermediate between the most and least contaminated sites, demonstrating that the soil replacement and revegetation improved the biological condition of the soil. The attributes that most explained these clustering were bacterial density, acid phosphatase, ß-glucosidase, fungal and actinobacterial densities, Cmic, and induced respiration.

Enzymatic activities produced by mixed Saccharomyces and non-Saccharomyces cultures: relationship with wine volatile composition.

During certain wine fermentation processes, yeasts, and mainly non-Saccharomyces strains, produce and secrete enzymes such as ß-glucosidases, proteases, pectinases, xylanases and amylases. The effects of enzyme activity on the aromatic quality of wines during grape juice fermentation, using different co-inoculation strategies of non-Saccharomyces and Saccharomyces cerevisiae yeasts, were assessed in the current study. Three strains with appropriate enological performance and high enzymatic activities, BSc562 (S. cerevisiae), BDv566 (Debaryomyces vanrijiae) and BCs403 (Candida sake), were assayed in pure and mixed Saccharomyces/non-Saccharomyces cultures. ß-Glucosidase, pectinase, protease, xylanase and amylase activities were quantified during fermentations. The aromatic profile of pure and mixed cultures was determined at the end of each fermentation. In mixed cultures, non-Saccharomyces species were detected until day 4-5 of the fermentation process, and highest populations were observed in MSD2 (10% S. cerevisiae/90% D. vanrijiae) and MSC1 (1% S. cerevisiae/99% C. sake). According to correlation and multivariate analysis, MSD2 presented the highest concentrations of terpenes and higher alcohols which were associated with pectinase, amylase and xylanase activities. On the other hand, MSC1 high levels of ß-glucosidase, proteolytic and xylanolytic activities were correlated to esters and fatty acids. Our study contributes to a better understanding of the effect of enzymatic activities by yeasts on compound transformations that occur during wine fermentation.

Relation between coumarate decarboxylase and vinylphenol reductase activity with regard to the production of volatile phenols by native Dekkera bruxellensis strains under 'wine-like' conditions.

Dekkera/Brettanomyces bruxellensis is considered a major cause of wine spoilage, and 4-ethylphenol and 4-ethylguaiacol are the most abundant off-aromas produced by this species. They are produced by decarboxylation of the corresponding hydroxycinnamic acids (HCAs), followed by a reduction of the intermediate 4-vinylphenols. The aim of the present study was to examine coumarate decarboxylase (CD) and vinylphenol reductase (VR) enzyme activities in 5 native D. bruxellensis strains and determine their relation with the production of ethylphenols under 'wine-like' conditions. In addition, biomass, cell culturability, carbon source utilization and organic acids were monitored during 60 days. All strains assayed turned out to have both enzyme activities. No significant differences were found in CD activity, whilst VR activity was variable among the strains. Growth of D. bruxellensis under 'wine-like' conditions showed two growth phases. Sugars were completely consumed during the first growth phase. Transformation of HCAs into ethylphenols also occurred during active growth of the yeast. No statistical differences were observed in volatile phenol levels produced by the strains growing under 'wine-like' conditions, independently of the enzyme activity previously recorded. Furthermore, our results demonstrate a relationship between the physiological state of D. bruxellensis and its ability to produce ethylphenols. Inhibition of growth of D. bruxellensis in wine seems to be the most efficient way to avoid ethylphenol production and the consequent loss of wine quality.

Scorpion envenomation in pygmies from Democratic Republic of Congo, the example of Pelenge Center, Lomela, DRC

In a survey among the pygmies of central Democratic Republic of Congo, the incidence of scorpion stings seemed very high with a severity greater than expected. Species responsible were not identified. Specific studies are needed to clarify the risk emerging in the equatorial African forest.

Scorpion envenomation in pygmies from Democratic Republic of Congo, the example of Pelenge Center, Lomela, DRC

In a survey among the pygmies of central Democratic Republic of Congo, the incidence of scorpion stings seemed very high with a severity greater than expected. Species responsible were not identified. Specific studies are needed to clarify the risk emerging in the equatorial African forest.

Production and some properties of crude alkaline proteases of indigenous Central Amazonian rhizobia strains

Two rhizobia strains isolated from soils of the Central Amazonian floodplain produced appreciable quantities of crude alkaline protease extracts with inexpensive carbon and nitrogen sources. These protease crude extracts were optimally active at pH 9.0-11.0. The optimum temperatures were 35 ºC for Rhizobium sp. strain R-986 and 55 ºC for Bradyrhizobium sp. strain R-993. Protease activities in the crude extracts were enhanced in the presence of 5 mM metal ions, such as Na+, Ca2+, Mg2+ and Mn2+. Rhizobia proteases were strongly inhibited by PMSF, a serine-protease inhibitor. The enzymes were active in the presence of surfactants (SDS and Triton X-100) and stable in oxidizing (H2O2) and reducing agents (β-mercaptoethanol), and organic solvents (acetone, hexane, methanol, 1-propanol and toluene).

Duas estirpes de rizóbia isoladas de solos de várzea da Amazônia Central produziram grandes quantidades de proteases alcalinas extracelulares, usando fontes baratas de carbono e nitrogênio. Os extratos brutos de proteases foram ativos em pH 9,0-11,0. As temperaturas ótimas foram de 35 ºC para a enzima do Rhizobium R-986 e de 55 ºC para a do Bradyrhizobium R-993. As atividades proteolíticas aumentaram na presença de 5 mM dos íons Na+, Ca2+ , Mg2+ e Mn2+ . As proteases secretadas pelos rizóbios foram fortemente inibidas por PMSF, um inibidor de serina protease. As enzimas foram ativas na presença de surfactantes (SDS e Triton X-100), e estáveis na presença de agentes oxidantes (H2O2) e redutores (β-mercaptoetanol) e solventes orgânicos (acetona, hexano, metanol, 1-propanol e tolueno).