We have inactivated the nuclear gene coding for a putative NAD(P)H dehydrogenase from the inner membrane of Neurospora crassa mitochondria by repeat-induced point mutations. The respiratory rates of mitochondria from the resulting mutant (nde-1) were measured, using NADH or NADPH as substrates under different assay conditions. The results showed that the mutant lacks an external calcium-dependent NADPH dehydrogenase. The observation of NADH and NADPH oxidation by intact mitochondria from the nde-1 mutant suggests the existence of a second external NAD(P)H dehydrogenase. The topology of the NDE1 protein was further studied by protease accessibility, in vitro import experiments, and in silico analysis of the amino acid sequence. Taken together, it appears that most of the NDE1 protein extends into the intermembrane space in a tightly folded conformation and that it remains anchored to the inner mitochondrial membrane by an Nterminal transmembrane domain.In nonphotosynthetic eukaryotes, the mitochondrion is the cellular organelle responsible for producing most of the energy required for cellular metabolism. The process of oxidative phosphorylation takes place in the inner mitochondrial membrane, whereby the electrons produced by the oxidation of substrates like NAD(P)H are transported through the electron transport chain to oxygen, coupled to the generation of a transmembrane proton gradient that eventually leads to ATP synthesis (1). In contrast to mammals, the electron transport chains of plants and fungi possess several nonproton-pumping NAD(P)H dehydrogenases for transferring electrons to ubiquinone (2). In the case of potato tubers, four rotenone-insensitive NAD(P)H dehydrogenases have been identified in the inner mitochondrial membrane, two with the catalytic site facing the matrix (3, 4) and two facing the intermembrane space (5). In mitochondria from Saccharomyces cerevisiae, where the proton-pumping complex I is not present, the oxidation of NADH and NADPH is performed exclusively by three nonproton-pumping enzymes, one facing the matrix and two facing the intermembrane space (6 -8). In addition, the genome analysis of Synechocystis revealed three open reading frames that may code for such type II NAD(P)H dehydrogenases (9). On the other hand, only one external type II NADH dehydrogenase was reported for the fungus Yarrowia lipolytica (10). Although NAD(P)H dehydrogenases have been studied for a long time, our understanding of protein function at the molecular level is still very incomplete. The cloning of genes encoding several of these rotenoneinsensitive NAD(P)H dehydrogenases from mitochondria of different organisms (7, 10 -13) provides important tools for further research in this field. These enzymes might constitute a wasteful system acting to prevent the overreduction of the electron transport components and the production of reactive oxygen species, but their exact roles remain unclear.Both proton-pumping and nonproton-pumping NAD(P)H dehydrogenases have been described in Neurospora crassa mitochon...

Craft beers are known for their distinct flavor, brew, and regional distribution. They are made using top-fermenting (ale) yeast, bottom-fermenting (lager) yeast, or through spontaneous fermentation. Craft beers are consumed and produced in Brazil in large quantities. However, they present a high level of polyphenols, which affects consumer preference as they may yield a taste of bitterness to beers. In this study, we analyzed the relationship between polyphenols and bitterness as well as the composition of the main styles of craft beers and consumer preference for them. Six different styles were analyzed according to their polyphenol content, bitterness, chemical composition, sensory profile, and preference. For preference, a panel of 62 untrained assessors was used. For sensory profile, quantitative descriptive analysis was performed using expert assessors (n = 8). The most preferred style was classic American pilsner, and the least preferred was standard American lager. The most preferred style showed less bitterness (9.52) and lower polyphenol content (0.61 mg EAG/mL), total solids (6.75 °Brix), and turbidity (7.27 NTU). This beer also exhibited reduced sensory notes of malty, fruity, smoked, hoppy, and phenolic but a higher perception of floral, sweet, and yeast notes; the bitterness attribute had a reduced perception. This study advances the understanding and complexity of the sensory profile of different styles of craft beers from Southern Brazil.

No abstract

The control of Acromyrmex leaf-cutting ants is necessary due to the severe damage they cause to diverse crops. A possibility was to control them using the bacterium Bacillus thuringiensis (Bt) that characteristically produces insecticidal crystal proteins (ICPs). The ICPs have been effective in controlling lepidopterans, dipterans, and coleopterans, but their action against hymenopterans is unknown. This paper describes an attempt to isolate Bt from ants of two Acromyrmex species, to evaluate its pathogenicity towards these ants, and to test isolates by PCR. Bacterial isolates of Bt obtained from A. crassispinus and A. lundi have been assayed against A. lundi in the laboratory. The bioassays were carried out in BOD at 25°C, with a 12-hour photoperiod, until the seventh day after treatment. The Bt isolates obtained were submitted to total DNA extraction and tested by PCR with primers specific to cry genes. The results showed Bt presence in 40% of the assessed samples. The data from the in vivo assays showed a mortality rate higher than 50% in the target population, with the Bt HA48 isolate causing 100% of corrected mortality. The PCR results of Bt isolates showed a magnification of DNA fragments relative to cry1 genes in 22% of the isolates, and cry9 in 67%. Cry2, cry3, cry7, and cry8 genes were not detected in the tested samples, and 22% had no magnified DNA fragments corresponding to the assessed cry genes. The results are promising not only regarding allele identification in new isolates, but also fort the assays aimed at determining the Bt HA48 LC 50 's, which can eventually be applied in controlling of Acromyrmex leaf-cutting ants.Key words: bioassay, leaf-cutting ants, Bacillus thuringiensis. RESUMO Patogenicidade de Bacillus thuringiensis isolados de duas espécies deAcromyrmex (Hymenoptera, Formicidae)O controle das formigas-cortadeiras do gênero Acromyrmex é necessário em razão dos severos danos que causam a diversas culturas. Uma alternativa é a utilização da bactéria Bacillus thuringiensis (Bt), caracterizada pela produção de proteínas inseticidas eficazes para lepidópteros, dípteros e coleópteros, mas cuja ação é ainda desconhecida para himenópteros. O presente trabalho teve por objetivo isolar Bt a partir de formigas de duas espécies de Acromyrmex, avaliar sua patogenicidade a formigas-cortadeiras e testar por PCR.

The screening of Bacillus thuringiensis (Bt) Cry proteins with high potential to control insect pests has been the goal of numerous research groups. In this study, we evaluated six monogenic Bt strains (Bt dendrolimus HD-37, Bt kurstaki HD-1, Bt kurstaki HD-73, Bt thuringiensis 4412, Bt kurstaki NRD-12 and Bt entomocidus 60.5, which codify the cry1Aa, cry1Ab, cry1Ac, cry1Ba, cry1C, cry2A genes respectively) as potential insecticides for the most important insect pests of irrigated rice: Spodoptera frugiperda, Diatraea saccharalis, Oryzophagus oryzae, Oebalus poecilus and Tibraca limbativentris. We also analyzed their compatibility with chemical insecticides (thiamethoxam, labdacyhalothrin, malathion and fipronil), which are extensively used in rice crops. The bioassay results showed that Bt thuringiensis 4412 and Bt entomocidus 60.5 were the most toxic for the lepidopterans, with a 93% and 82% mortality rate for S. frugiperda and D. saccharalis, respectively. For O. oryzae, the Bt kurstaki NRD-12 (64%) and Bt dendrolimus HD-37 (62%) strains were the most toxic. The Bt dendrolimus HD-37 strain also caused high mortality (82%) to O. poecilus, however the strains assessed to T. limbativentris caused a maximum rate of 5%. The assays for the Bt strains interaction with insecticides revealed the compatibility of the six strains with the four insecticides tested. The results from this study showed the high potential of cry1Aa and cry1Ba genes for genetic engineering of rice plants or the strains to biopesticide formulations.

We have isolated and characterised the nuclear gene that codes for the 30.4-kDa subunit of the peripheral arm of complex I from Neurospora crassa. The single-copy gene was localised on chromosome VI of the fungal genome by restriction fragment length polymorphism mapping. An extra copy of the gene was introduced into a strain of N. crassa by transformation. This strain was crossed with another strain in order to inactivate, by repeat-induced point mutations, both copies of the duplication carried by the parental transformant. Ascospore progeny from the cross were analysed and a mutant strain lacking the 30.4-kDa protein, nuo30.4, was isolated and further characterised. The mutant appears to assemble the membrane arm of complex I, while formation of the peripheral arm is prevented. Nevertheless, the mutant grows reasonably well--indicating that this well conserved protein is not essential for vegetative growth--and is able to mate with other strains both as male or female. Strains with multiple mutations are readily obtained from heterozygous crosses between different complex I mutants of N. crassa. On the other hand, homozygous crosses between several mutants, including nuo30.4, fail to produce ascospores. These results suggest that complex I plays an essential role during the sexual phase of the life cycle of the fungus.

No abstract

Benzoic acids preservatives may be converted to benzene in soft drinks. The use of alternative antimicrobial compounds, specifically nisin and natamycin, would reduce benzene formation. Onitially, doses of nisin and natamycin were tested against Lactobacillus plantarum lactic bacteria and Zygosaccharomyces bailii yeast. Using 167 UO/mL of nisin and 0.017% w/v of natamycin no microbial growth was reached on lemon-flavored soft drinks. The benzene formation was analyzed in a standard formulation (380 mg/L of sodium benzoate and 180 mg/L of potassium sorbate) and nisin and natamycn formulation (167 UO/mL of nisin and 0.017% w/v of natamycin). The soft drinks were stored without UV exposure at 20 °C and with UV sunlight exposure at 30 °C, for 120 days at PET bottles. The initial benzene content was 1.29 μg/kg for standard and 0.09 μg/kg for nisin and natamycin formulation; these values increased to 11.00 μg/kg and 0.18 μg/kg, respectively. The sensory attributes did not differ from each formulation, either initially or after 120 days of storage. The lower benzene concentrations obtained using nisin and natamycin suggest that these compounds are attractive alternatives to benzoic acids, keeping the sensory quality and microbial stability during shelf life.