Analysis of Paracoccidioides lutzii mitochondria: a proteomic approach.

The genus Paracoccidioides is composed of thermal dimorphic fungi, causative agents of paracoccidioidomycosis, one of the most frequent systemic mycoses in Latin America. Mitochondria have sophisticated machinery for ATP production, which involves metabolic pathways such as citric acid and glyoxylate cycles, electron transport chain and oxidative phosphorylation. In addition, this organelle performs a variety of functions in the cell, working as an exceptional metabolic signalling centre that contributes to cellular stress responses, as autophagy and apoptosis in eukaryotic organisms. The aim of this work was to perform a descriptive proteomic analysis of mitochondria in Paracoccidioides lutzii yeast cells. After mitochondria fractionation, samples enriched in mitochondrial proteins were digested with trypsin and analysed using a NanoUPLC-MSE system (Waters Corporation, Manchester, UK). Ours results revealed that the established protocol for purification of mitochondria was very effective for P. lutzii, and 298 proteins were identified as primarily mitochondrial, in our analysis. To our knowledge, this is the first compilation of mitochondrial proteins from P. lutzii, to date. Copyright © 2016 John Wiley & Sons, Ltd.

In silico characterization of hypothetical proteins from Paracoccidioides lutzii.

Nearly 60% of Paracoccidioides lutzii genes encode products annotated as hypothetical or predicted proteins (HPs). In this study, we describe the global detection and functional inference of HPs, using computational methods based on sequence similarity, identification of targeting signals, presence of known protein domains, and use of the Gene Ontology functional classification scheme. Our analysis enabled a high-throughput characterization of predicted cellular localization and presence of protein domains, clustering HPs into different functional categories including metabolism, localization, cell cycle, response to stimulus, and signaling. To investigate P. lutzii HP expression profiles, we used data obtained from the expressed sequence tag database (dbEST). These analyses revealed 2364 HPs expressed in different situations, namely in mycelial and yeast forms, during the transition from mycelium to yeast, and under conditions mimicking infection. Based on this transcriptomic data, we performed a functional enrichment analysis according to the domains present in the HPs expressed in each condition. The most overrepresented functional domains were those involved in the regulation of gene expression, suggesting important and as yet undescribed roles for these HPs in the adaptation of P. lutzii to environmental conditions. In addition, the expression profiles of six randomly selected HPs were analyzed by quantitative real-time polymerase chain reaction in order to verify their expression in the complementary DNA libraries analyzed in this investigation. The approach used in this study should improve functional characterization of P. lutzii HPs.

Occurrence of group A rotavirus mixed P genotypes infections in children living in Goiânia-Goiás, Brazil.

Group A rotaviruses (RVA) are the main causing agents of acute gastroenteritis worldwide, having a great impact on childhood mortality in developing countries. The objective of this study was to identify RVA-positive fecal samples with mixed P genotypes by hemi-nested reverse transcriptase-polymerase chain reaction (RT-PCR), followed by sequencing confirmation. Our results showed that, from the 81 RVA-positive samples, 25 were positive for more than one P genotype by hemi-nested RT-PCR. Of these 25 samples, 12 (48%) had their mixed P genotypes confirmed by sequencing and, from these, 10 were identified as P[6]P[8], one as P[4]P[6], and one as P[4]P[6]P[8]. Our results confirm the occurrence of RVA mixed infections among children in Brazil and reinforce the importance of the constant monitoring of RVA circulating strains for the efficacy of control/prevention against these agents.

Ab initio determination of the crystal structure of cytochrome c6 and comparison with plastocyanin.

BACKGROUND: Electron transfer between cytochrome f and photosystem I (PSI) can be accomplished by the heme-containing protein cytochrome c6 or by the copper-containing protein plastocyanin. Higher plants use plastocyanin as the only electron donor to PSI, whereas most green algae and cyanobacteria can use either, with similar kinetics, depending on the copper concentration in the culture medium. RESULTS: We report here the determination of the structure of cytochrome c6 from the green alga Monoraphidium braunii. Synchrotron X-ray data with an effective resolution of 1.2 A and the presence of one iron and three sulfur atoms enabled, possibly for the first time, the determination of an unknown protein structure by ab initio methods. Anisotropic refinement was accompanied by a decrease in the 'free' R value of over 7% the anisotropic motion is concentrated at the termini and between residues 38 and 53. The heme geometry is in very good agreement with a new set of heme distances derived from the structures of small molecules. This is probably the most precise structure of a heme protein to date. CONCLUSIONS: On the basis of this cytochrome c6 structure, we have calculated potential electron transfer pathways and made comparisons with similar analyses for plastocyanin. Electron transfer between the copper redox center of plastocyanin to PSI and from cytochrome f is believed to involve two sites on the protein. In contrast, cytochrome c6 may well use just one electron transfer site, close to the heme unit, in its corresponding reactions with the same two redox partners.

Comparative analyses of the structure of the 1,3-beta-glucan synthase gene in Paracoccidioides brasiliensis isolates.

The evolutionary origin and significance of spliceosomal introns have been the subject of many investigations. Two theories, "introns-early" theory and "introns-late" theory, have been proposed to explain the evolution of introns in eukaryotic genes. Intron position is generally conserved in paralogue and orthologue genes. Some introns occur at similar but not necessarily identical positions in homologous genes, which were separated by great evolutionary distances. This event can be explained by insertion, loss or movement of the intron over short distances. Intron loss and gain events are unique in evolution and can be useful as markers for phylogenetic analyses. The insertion of introns at an identical position suggests a common ancestor gene. Here we analyzed, using PCR and RT-PCR, the structure of the 1,3-beta-glucan synthase gene (FKS) in several clinical isolates of Paracoccidioides brasiliensis (Pb): isolates Pb 01, Pb 4940, Pb 8515, Pb 8311, Pb 8334, Pb 4268, Pb 1668, and Pb E. Our results showed that seven of the isolates examined showed identical structures concerning the position of introns in PbFKS1. PbFKS4940 showed the intron described at the 3' end and had lost that one at the 5' end. The presence of the PbFKS4940 transcript suggests that it could be a functional gene. These data suggest a divergent evolution for introns with regard to the 1,3-beta-glucan synthase gene in P. brasiliensis isolates.

The caa3 terminal oxidase of the thermohalophilic bacterium Rhodothermus marinus: a HiPIP:oxygen oxidoreductase lacking the key glutamate of the D-channel.

The respiratory chain of the thermohalophilic bacterium Rhodothermus marinus contains a novel complex III and a high potential iron-sulfur protein (HiPIP) as the main electron shuttle (Pereira et al., Biochemistry 38 (1999) 1268-1275 and 1276-1283). In this paper, one of the terminal oxidases expressed in this bacterium is extensively characterised. It is a caa3-type oxidase, isolated with four subunits (apparent molecular masses of 42, 19 and 15 kDa and a C-haem containing subunit of 35 kDa), which has haems of the A(s) type. This oxidase is capable of using TMPD and horse heart cytochrome c as substrates, but has a higher turnover with HiPIP, being the first example of a HiPIP:oxygen oxidoreductase. The oxidase has unusually low reduction potentials of 260 (haem C), 255 (haem A) and 180 mV (haem A3). Subunit I of R. marinus caa3 oxidase has an overall significant homology with the subunits I of the COX type oxidases, namely the metal binding sites and most residues considered to be functionally important for proton uptake and pumping (K- and D-channels). However, a major difference is present: the putative essential glutamate (E278 in Paraccocus denitrificans) of the D-channel is missing in the R. marinus oxidase. Homology modelling of the R. marinus oxidase shows that the phenol group of a tyrosine residue may occupy a similar spatial position as the glutamate carboxyl, in relation to the binuclear centre. Moreover, sequence comparisons reveal that several enzymes lacking that glutamate have a conserved substitution pattern in helix VI: -YSHPXV- instead of -XGHPEV-. These observations are discussed in terms of the mechanisms for proton uptake and it is suggested that, in these enzymes, tyrosine may play the role of the glutamate in the proton channel.

Local water bridges and protein conformational stability.

Recent studies have pointed out the important role of local water structures in protein conformational stability. Here, we present an accurate and computationally effective way to estimate the free energy contribution of the simplest water structure motif--the water bridge. Based on the combination of empirical parameters for accessible protein surface area and the explicit consideration of all possible water bridges with the protein, we introduce an improved protein solvation model. We find that accounting for water bridge formation in our model is essential to understand the conformational behavior of polypeptides in water. The model formulation, in fact, does not depend on the polypeptide nature of the solute and is therefore applicable to other flexible biomolecules (i.e., DNAs, RNAs, polysaccharides, etc.).

Control of tubulin gene expression during metacyclogenesis of Trypanosoma cruzi.

During differentiation of the dividing epimastigote to the non-dividing metacyclic trypomastigote form of the parasitic protozoan Trypanosoma cruzi there is a marked reduction in the rate of synthesis of the major proteins alpha- and beta-tubulin. Our results indicate that the control of synthesis of these proteins during the differentiation event is exerted at the level of alpha- and beta-tubulin mRNA accumulation.

Alpha- and beta-tubulin mRNAs of Trypanosoma cruzi originate from a single multicistronic transcript.

The cluster of alternated alpha- and beta-tubulin genes in the genome of Trypanosoma cruzi was shown to be transcribed into a single RNA molecule which upon processing gives rise to the mature alpha- and beta-tubulin mRNAs. This conclusion was based on: (i) nuclear RNA species with the same molecular mass hybridize to both alpha- and beta-tubulin cDNA probes; (ii) S1 nuclease assay of the clustered tubulin genes has shown protected DNA fragments of the same size and of greater molecular mass than that corresponding to the mRNAs, hybridizable to both alpha- and beta-tubulin cDNA probes; (iii) beta-tubulin hybrid selected RNA is still able to hybridize to alpha-tubulin probe.

Two-dimensional electrophoresis and characterization of antigens from Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is a fungal pathogen of humans. To identify antigens from P. brasiliensis we fractionated a crude preparation of proteins from the fungus and detected the IgG reactive proteins by immunoblot assays of yeast cellular extracts with sera of patients with paracoccidioidomycosis (PCM). We identified and characterized six new antigens by amino acid sequencing and homology search analyses with other proteins deposited in a database. The newly characterized antigens were highly homologous to catalase, fructose-1,6-biphosphate aldolase (aldolase), glyceraldehyde-3-phosphate dehydrogenase, malate dehydrogenase and triosephosphate isomerase from several sources. The characterized antigens presented preferential synthesis in yeast cells, the host fungus phase.

Genotyping of group A rotavirus samples from Brazilian children by probe hybridization.

The G genotyping of 74 group A rotavirus samples was done by RNA-DNA hybridization (dot-blot) using oligonucleotide probes for the VP7 gene region of the human rotavirus serotypes/genotypes 1, 2, 3 and 4. Thirty-one samples could be genotyped by dot-blot showing the following results: G1 = 16, G4 = 6, G3 = 5, and G2 = 4. The data show circulation of genotypes G1-G4 and the predominance of G1. The knowledge of genotypes provides important information concerning rotavirus circulation in Central Brazil.

Serotypes and subgroups of rotavirus isolated from children in central Brazil.

Group A rotavirus, obtained from children of Goiânia, Brazil, during 1987-1994, were analyzed for subgroup and G serotype by enzyme-linked immunosorbent assay with monoclonal antibodies. The index of serotyping obtained was 61.4% with the following proportions: G1--19.7%, G2--28.0%, G3--9.8%, G4--1.5%, and G5--2.3%. It was observed that G1 occurred from 1987 to 1989 and from 1993 to 1994, and G2 from 1990 to 1993. About 94% of the samples (85/90) could be subgrouped with the following results: 55.5% for SG II, 7.8% SG I, and 31.1% for SG non-I-non-II. Unusual relationship patterns were also detected among serotypes, subgroups, and profiles of electropherotypes in 57.0% of the samples: 20 of them were G2/SG II/"long" profile. The results suggest that variation in temporal and regional characteristics should be considered in the development of rotavirus vaccine.

Characterization and utilization of Candida rugosa lipase immobilized on controlled pore silica.

Candida rugosa lipase was immobilized by covalent binding on controlled pore silica (CPS) using glutaraldehyde as cross-linking agent under aqueous and nonaqueous conditions. The immobilized C. rugosa was more active when the coupling procedure was performed in the presence of a nonpolar solvent, hexane. Similar optima pH (7.5-8.0) was found for both free and immobilized lipase. The optimum temperature for the immobilized lipase was about 10 degrees C higher than that for the free lipase. The thermal stability of the CPS lipase was also greater than the original lipase preparation. Studies on the operational stability of CPS lipase revealed good potential for recycling under aqueous (olive-oil hydrolysis) and nonaqueous (butyl butyrate synthesis) conditions.

Fusel oil as precursor for aroma generation by biotransformation using lipase: scientific note.

The feasibility of using mixtures of C4 and C5 chain-length aliphatic alcohols from fusel oil as the bulk organic media for lipase-mediated synthesis of laurate esters was assessed. Reaction mixtures consisted of lauric acid, lipase, solvent (if added), and appropriate amount of fusel oil (previously dehydrated with inorganic salts and molecular sieves). The influence of the reaction conditions such as substrate concentrations and temperature were investigated. Increased molar ratio of acyl donor to acyl acceptor allowed the esterification to proceed with no need for solvent addition.

Selection of stabilizing additive for lipase immobilization on controlled pore silica by factorial design.

Candida rugosa lipase was covalently immobilized on silanized controlled pore silica (CPS) previously activated with glutaraldehyde in the presence of several additives to improve the performance of the immobilized form in long-term operation. Proteins (albumin and lecithin) and organic molecules (beta-cyclodextrin and polyethylene glycol [PEG]-1500) were added during the immobilization procedure, and their effects are reported and compared to the behavior of the immobilized biocatalyst in the absence (lacking) of additive. The selection of the most efficient additive at different lipase loadings (150-450 U/g of dry support) was performed by experimental design. Two 22 full factorial designs with two repetitions at the center point were employed to evaluate the immobilization yield. A better stabilizing effect was found when small amounts of albumin or PEG-1500 were added simultaneously to the lipase onto the support. The catalytic activity had a maximum (193 U/mg) for lipase loading of 150 U/g of dry support using PEG-1500 as the stabilizing additive. This immobilized system was used to perform esterification reactions under repeated batch cycles (for the synthesis of butyl butyrate as a model). The half-life of the lipase immobilized on CPS in the presence of PEG-1500 was found to increase fivefold compared with the control (immobilized lipase on CPS without additive).

Ureia e glicerina bruta como aditivos na ensilagem de cana-de-açúcar / Urea and crude glycerin as additive in sugar cane silage

Objetivou-se avaliar a inclusão de ureia e glicerina bruta como aditivos na ensilagem da cana-de-açúcar, na composição químico-bromatológica, pH, N-amoniacal (N-NH3) e digestibilidade in vitro (DIV). Os tratamentos foram quatro doses de ureia, 0, 10, 20 e 30 g de ureia por kg de cana-de-açúcar na ensilagem, e cinco doses de glicerina bruta, 0, 10, 20, 30 e 40g de glicerina bruta por kg de cana-de-açúcar na ensilagem. As silagens foram armazenadas por 180 dias. O tratamento com ureia afetou a maioria das variáveis relacionadas ao valor nutritivo, aumentando os teores de matéria seca (MS) e proteína (PB) (2,58; 7,76; 18,70 e 19,31%), reduzindo os teores de fibra em detergente neutro (FDN) e melhorando a DIV da MS (42,61; 48,53; 50,69 e 51,18%) e FDN (38,81; 39,23; 41,06 e 43,46%), e as características fermentativas da silagem, apresentando valores de pH de 3,49; 3,86; 4,18 e 3,93 e de N-NH3 de 1,72; 3,80; 7,88 e 9,00, para as dose de 0, 10, 20 e 30 g, respectivamente. A glicerina bruta aumentou os teores de MS e extrato etéreo (1,45; 3,03; 3,62; 3,41 e 4,38%), melhorou a DIV da MS com valores de 49,61; 52,24; 53,28; 55,60 e 56,09% e reduziu perdas por gases durante o processo de fermentação, apresentando médias de 6,69; 5,97; 5,89; 5,51 e 5,48% da MS para as doses 0, 10, 20, 30 e 40g, respectivamente. Assim, a ureia e a glicerina bruta podem ser utilizadas como aditivos na ensilagem da cana-de-açúcar...

The aim of this study was to evaluate the inclusion of urea and crude glycerin as an additive in ensiling of sugar cane, in chemical composition, pH, ammonia-N (N-NH3) and in vitro digestibility (IVD). The treatments were four doses of urea 0, 10, 20 and 30 g per kg of sugar cane ensiling and five doses of crude glycerin 0, 10, 20, 30 and 40g per kg of crude glycerin sugar cane ensiling. The silages were stored for 180 days. Treatment with urea affected most variables related to nutritional value, increasing the concentrations of dry matter (DM) and protein (CP) (2.58, 7.76, 18.70 and 19.31%) and reduced levels of neutral detergent fiber (NDF) and improved IVDDM (42.61, 48.53, 50.69 and 51.18%) and NDF (38.81, 39.23, 41.06 and 43.46%) and fermentation characteristics of silage, with pH values of 3.49, 3.86, 4.18 and 3.93 and NH3 1.72, 3.80, 7.88 and 9.00 for the dose of 0, 10, 20 and 30 g, respectively. The crude glycerin increased in DM and ether extract (1.45, 3.03, 3.62, 3.41 and 4.38%), improved IVDDM with values of 49.61, 52.24, 53 28; 55.60 and 56.09% and reduced gas losses during the fermentation process with mean of 6.69, 5.97, 5.89, 5.51 and 5.48% of DM for the doses 0, 10 , 20, 30 and 40g, respectively. Urea and crude glycerin can be used as an additive in ensiling of sugar cane...

Imunidade passiva, ingestão de colostro e mortalidade em cabritos Moxotó criados em sistemas extensivo e intensivo / Passive immunity, colostrum ingestion, and mortality of Moxotó kids raised under extensive and intensive breeding systems

Dosou-se a proteína sérica total para avaliar a aquisição de imunidade passiva em cabritos Moxotó. Para tal, formaram-se quatro grupos experimentais, sendo dois sistemas de criação, extensivo e intensivo, e dois manejos de colostro, ingestão natural e artificial. Tanto no sistema intensivo quanto no extensivo, os teores de proteína no soro foram significativamente mais altos nos animais com ingestão natural de colostro, 7,11±0,2g/dL, do que nos com ingestão artificial, 6,35±0,17g/dL. Independentemente da forma de ingestão de colostro, os cabritos do sistema intensivo tiveram teores de proteína sérica total, 7,21±0,19g/dL, mais elevados que os do sistema extensivo, 6,25±0,18g/dL, no entanto a imunidade passiva foi satisfatória nos dois grupos de animais. Ocorreu alta mortalidade de crias no sistema extensivo, 37 por cento, devido ao complexo hipotermia/inanição em decorrência dos baixos níveis de colostro ingeridos. No sistema intensivo de criação não ocorreu mortalidade de cabritos. A produção de colostro das cabras criadas intensivamente, 163,5±14,71mL, foi mais alta que das cabras criadas extensivamente, 53,75±19,12mL. O peso total dos cabritos foi semelhante nos dois sistemas de criação, 2881±252,78g no sistema extensivo, e 2297±194,59g no sistema intensivo. Conclui-se que a ingestão de colostro nos dois sistemas de produção permitiu adequada aquisição de imunidade em cabritos, porém o sistema extensivo determinou severa deficiência nutricional nas mães, com baixa produção de colostro e graves perdas de neonatos.

The acquisition of passive immunity in Moxotó kids was determined by dosages of total serum proteins. Four experimental groups were formed in two breeding systems - extensive and intensive - and two managements of colostrum intake - suckling from the mother or supplying in bottles. In both breeding systems, the serum protein levels were significantly higher in kids with natural ingestion of colostrum, 7.11±0.2g/dL, than in kids with artificial ingestion, 6.35±0.17g/dL. The kids of the intensive system had levels of total serum protein of 7.21±0.19 g/dL which was higher than the one of the extensive breeding system, 6.25±0.18g/dL. However, the passive immunity was satisfactory in all groups. There was high mortality of kids, 37 percent, due to starvation/hypothermia, in the extensive breeding system. This mortality was apparently due to the low levels of colostrum ingestion, 55.83±8.7mL. The production of colostrum by does from intensive breeding sistem, 163.5±14.71mL, was significantly higher than those from extensive breeding system, 53.75±19.12mL. The total weight of the kids born in the extensive breeding system, 2,881±252.78g, was similar to those born in the intensive breeding system, 2,297±194.59g. The colostrum ingestion allowed appropriate immunity acquisition by kid raised under both systems. However, the extensive breeding system determined a severe nutritional deficiency in the does with low colostrum production and high neonatal losses.

L3 loop-mediated mechanisms of pore closing in porin: a molecular dynamics perturbation approach.

L3 loop-mediated mechanisms for pore closing in porin are investigated with molecular dynamics simulation, using an approach that can be related to the phenomenon of voltage gating. Voltage gating is seen as a perturbation of the electrostatic screening inside the porin pore where, by the influence of the potential gradient, water and counter-ion distribution can be slightly displaced from their equilibrium distribution. This is simulated by perturbing the screening electrostatics of ionizable groups inside the pore. Under these conditions, a localized conformational change takes place, involving 12 (Ile102-Ala113) out of the 44 residues of the loop. The pore is reduced to a sixth of its open state size. The conformational change can be achieved with a small perturbation and it is reversible once the perturbation is switched off (relaxation process). Other types of behaviour predominating at higher simulation temperatures are found for the loop, involving an extra conformational change in the Thr92-Asp96 loop segment. This conformational change completely closes the pore, but is not reversible under the simulation conditions. Both zones involved in the conformational changes contain or overlap the zones which were described previously, using other techniques, to be the most flexible zones of the loop.

On the stability and plastic properties of the interior L3 loop in R. capsulatus porin. A molecular dynamics study.

Structural properties of Rhodobacter capsulatus porin are studied by molecular dynamics simulation using the GROMOS force field. Unconstrained simulations of the trimer and monomer show the trimer to be more stable than the isolated monomer. Simulations of the L3 loop inside the pore are used to assess its stability and plastic properties. Simulated annealing shows that the conformational space available to the L3 loop inside the pore is very large. Simulations at different temperatures show that the energy hypersurface around the open state is complex and flat. These studies also indicate four zones that are more flexible than the rest of the loop. Two of these are stabilized by the addition of the detergent molecule present in the X-ray structure. It is possible that the two remaining flexible zones, situated in the half of the loop facing the extracellular end of the porin molecule, residues Asp93-Gly98 and Arg110-Leu111, are involved in a mechanism for opening and closing of the pore.

Simulation of electron-proton coupling with a Monte Carlo method: application to cytochrome c3 using continuum electrostatics.

A new method is presented for simulating the simultaneous binding equilibrium of electrons and protons on protein molecules, which makes it possible to study the full equilibrium thermodynamics of redox and protonation processes, including electron-proton coupling. The simulations using this method reflect directly the pH and electrostatic potential of the environment, thus providing a much closer and realistic connection with experimental parameters than do usual methods. By ignoring the full binding equilibrium, calculations usually overlook the twofold effect that binding fluctuations have on the behavior of redox proteins: first, they affect the energy of the system by creating partially occupied sites; second, they affect its entropy by introducing an additional empty/occupied site disorder (here named occupational entropy). The proposed method is applied to cytochrome c3 of Desulfovibrio vulgaris Hildenborough to study its redox properties and electron-proton coupling (redox-Bohr effect), using a continuum electrostatic method based on the linear Poisson-Boltzmann equation. Unlike previous studies using other methods, the full reduction order of the four hemes at physiological pH is successfully predicted. The sites more strongly involved in the redox-Bohr effect are identified by analysis of their titration curves/surfaces and the shifts of their midpoint redox potentials and pKa values. Site-site couplings are analyzed using statistical correlations, a method much more realistic than the usual analysis based on direct interactions. The site found to be more strongly involved in the redox-Bohr effect is propionate D of heme I, in agreement with previous studies; other likely candidates are His67, the N-terminus, and propionate D of heme IV. Even though the present study is limited to equilibrium conditions, the possible role of binding fluctuations in the concerted transfer of protons and electrons under nonequilibrium conditions is also discussed. The occupational entropy contributions to midpoint redox potentials and pKa values are computed and shown to be significant.