Schistosoma mansoni Tegument Protein Sm29 Is Able to Induce a Th1-Type of Immune Response and Protection against Parasite Infection

Background: Schistosomiasis continues to be a significant public health problem. This disease affects 200 million people worldwide and almost 800 million people are at risk of acquiring the infection. Although vaccine development against this disease has experienced more failures than successes, encouraging results have recently been obtained using membrane-spanning protein antigens from the tegument of Schistosoma mansoni. Our group recently identified Sm29, another antigen that is present at the adult worm tegument surface. In this study, we investigated murine cellular immune responses to recombinant (r) Sm29 and tested this protein as a vaccine candidate. Methods and Findings: We first show that Sm29 is located on the surface of adult worms and lung-stage schistosomula through confocal microscopy. Next, immunization of mice with rSm29 engendered 51%, 60% and 50% reduction in adult worm burdens, in intestinal eggs and in liver granuloma counts, respectively (p<0.05). Protective immunity in mice was associated with high titers of specific anti-Sm29 IgG1 and IgG2a and elevated production of IFN-gamma, TNF-alpha and IL-12, a typical Th1 response. Gene expression analysis of worms recovered from rSm29 vaccinated mice relative to worms from control mice revealed a significant (q<0.01) down-regulation of 495 genes and up-regulation of only 22 genes. Among down-regulated genes, many of them encode surface antigens and proteins associated with immune signals, suggesting that under immune attack schistosomes reduce the expression of critical surface proteins. Conclusion: This study demonstrates that Sm29 surface protein is a new vaccine candidate against schistosomiasis and suggests that Sm29 vaccination associated with other protective critical surface antigens is the next logical strategy for improving protection.

Solution structure of the human signaling protein RACK1

Background: The adaptor protein RACK1 (receptor of activated kinase 1) was originally identified as an anchoring protein for protein kinase C. RACK1 is a 36 kDa protein, and is composed of seven WD repeats which mediate its protein-protein interactions. RACK1 is ubiquitously expressed and has been implicated in diverse cellular processes involving: protein translation regulation, neuropathological processes, cellular stress, and tissue development. Results: In this study we performed a biophysical analysis of human RACK1 with the aim of obtaining low resolution structural information. Small angle X-ray scattering (SAXS) experiments demonstrated that human RACK1 is globular and monomeric in solution and its low resolution structure is strikingly similar to that of an homology model previously calculated by us and to the crystallographic structure of RACK1 isoform A from Arabidopsis thaliana. Both sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation techniques showed that RACK1 is predominantly a monomer of around 37 kDa in solution, but also presents small amounts of oligomeric species. Moreover, hydrodynamic data suggested that RACK1 has a slightly asymmetric shape. The interaction of RACK1 and Ki1/57 was tested by sedimentation equilibrium. The results suggested that the association between RACK1 and Ki-1/57(122-413) follows a stoichiometry of 1:1. The binding constant (KB) observed for RACK1-Ki-1/57(122-413) interaction was of around (1.5 +/- 0.2) x 10(6) M(-1) and resulted in a dissociation constant (KD) of (0.7 +/- 0.1) x 10(-6) M. Moreover, the fluorescence data also suggests that the interaction may occur in a cooperative fashion. Conclusion: Our SAXS and analytical ultracentrifugation experiments indicated that RACK1 is predominantly a monomer in solution. RACK1 and Ki-1/57(122-413) interact strongly under the tested conditions.

Conformational stability of peanut agglutinin using small angle X-ray scattering

In this work, quaternary conformational studies of peanut agglutinin (PNA) have been carried out using small-angle X-ray scattering (SAXS). PNA was submitted to three different conditions: pH variation (2.5, 4.0, 7.4 and 9.0), guanidine hydrochloride presence (0.5-2 M) at each pH value, and temperature ranging from 25 to 60 degrees C. All experiments were performed in the absence and presence of T-antigen to evaluate its influence on the lectin stability. At room temperature and pH 4.0,7.4 and 9.0, the SAXS curves are consistent with the PNA scattering in its crystallographic native homotetrameric structure, with monomers in a jelly roll fold, associated by non-covalent bonds resulting in an open structure. At pH 2.5, the results indicate that PNA tends to dissociate into smaller sub-units, as dimers and monomers, followed by a self-assembling into larger aggregates. Furthermore, the conformational stability under thermal denaturation follows the pH sequence 7.4 > 9.0 > 4.0 > 2.5. Such results are consistent with the conformational behavior found upon GndHCl influence. The presence of T-antigen does not affect the protein quaternary structure in all studied systems within the SAXS resolution. (C) 2010 Elsevier B.V. All rights reserved.

Intracellular mechanisms of specific beta-adrenoceptor antagonists involved in improved cardiac function and survival in a genetic model of heart failure

beta-blockers, as class, improve cardiac function and survival in heart failure (HF). However, the molecular mechanisms underlying these beneficial effects remain elusive. In the present study, metoprolol and carvedilol were used in doses that display comparable heart rate reduction to assess their beneficial effects in a genetic model of sympathetic hyperactivity-induced HF (alpha(2A)/alpha(2C)-ARKO mice). Five month-old HF mice were randomly assigned to receive either saline, metoprolol or carvedilol for 8 weeks and age-matched wild-type mice (WT) were used as controls. HF mice displayed baseline tachycardia, systolic dysfunction evaluated by echocardiography, 50% mortality rate, increased cardiac myocyte width (50%) and ventricular fibrosis (3-fold) compared with WT. All these responses were significantly improved by both treatments. Cardiomyocytes from HF mice showed reduced peak [Ca(2+)](i) transient (13%) using confocal microscopy imaging. Interestingly, while metoprolol improved [Ca(2+)](i) transient, carvedilol had no effect on peak [Ca(2+)](i) transient but also increased [Ca(2+)] transient decay dynamics. We then examined the influence of carvedilol in cardiac oxidative stress as an alternative target to explain its beneficial effects. Indeed, HF mice showed 10-fold decrease in cardiac reduced/oxidized glutathione ratio compared with WT, which was significantly improved only by carvedilol treatment. Taken together, we provide direct evidence that the beneficial effects of metoprolol were mainly associated with improved cardiac Ca(2+) transients and the net balance of cardiac Ca(2+) handling proteins while carvedilol preferentially improved cardiac redox state. (C) 2008 Elsevier Inc. All rights reserved.

The impact of a 17-day training period for an international championship on mucosal immune parameters in top-level basketball players and staff members

This investigation examined the impact of a 17-d training period (that included basketball-specific training, sprints, intermittent running exercises, and weight training, prior to an international championship competition) on salivary immunoglobulin A (SIgA) levels in 10 subjects (athletes and staff members) from a national basketball team, as a biomarker for mucosal immune defence. Unstimulated saliva samples were collected at rest at the beginning of the preparation for the Pan American Games and 1 d before the first game. The recovery interval from the last bout of exercise was 4 h. The SIgA level was measured using enzyme-linked immunosorbent assay and expressed as absolute concentrations, secretion rate, and SIgA level relative to total protein. The decrease in SIgA levels following training was greater in athletes than in support staff; however, no significant differences between the two groups were detected. A decrease in SIgA level, regardless of the method used to express IgA results, was verified for athletes. Only one episode of upper respiratory tract illness symptoms was reported, and it was not associated with changes in SIgA levels. In summary, a situation of combined stress for an important championship was found to decrease the level of SIgA-mediated immune protection at the mucosal surface in team members, with greater changes observed in the athletes.

Recombinant rabies virus glycoprotein synthesis in bioreactor by transfected Drosophila melanogaster S2 cells carrying a constitutive or an inducible promoter

S2 cell populations (S2AcRVGP2K and S2MtRVGP-Hy) were selected after transfection of gene expression vectors carrying the cDNA encoding the rabies virus glycoprotein (RVGP) gene under the control of the constitutive (actin) or inductive (metallothionein) promoters. These cell populations were cultivated in a 1 L bioreactor mimicking a large scale bioprocess. Cell cultures were carried out at 90 rpm and monitored/controlled for temperature (28 degrees C) and dissolved oxygen (10 or 50% air saturation). Cell growth attained similar to 1.5-3 x 10(7) cells/mL after 3-4 clays of cultivation. The constitutive synthesis of RVGP in S2AcRVGP2K cells led to values of 0.76 mu g/10(7) cells at day 4 of culture. The RVGP synthesis in S2MtRVGP-Hy cell fraction increased upon CuSO(4) induction attaining specific productivities of 1.5-2 mu g/10(7) cells at clays 4-5. RVGP values in supernatant as a result of cell lysis were always very low (<0.2 mu g/mL) indicating good integrity of cells in culture. Overall the RVGP productivity was of 1.5-3 mg/L. Our data showed an important influence of dissolved oxygen on RVGP synthesis allowing a higher and sustained productivity by S2MtRVGP-Hy cells when cultivated with a DO of 10% air saturation. The RVGP productivity in bioreactors shown here mirrors those previously observed for T-flasks and shaker bottles and allow the preparation of the large RVGP quantities required for studies of structure and function. (C) 2010 Elsevier B.V. All rights reserved.

MULTIVARIATE STATISTICAL METHOD IN OPTIMIZATION OF PROTEIN EXTRACTION FROM SOYBEAN FLOUR WITH DAIRY WHEY

The aim objective of this project was to evaluate the protein extraction of soybean flour in dairy whey, by the multivariate statistical method with 2(3) experiments. Influence of three variables were considered: temperature, pH and percentage of sodium chloride against the process specific variable ( percentage of protein extraction). It was observed that, during the protein extraction against time and temperature, the treatments at 80 degrees C for 2h presented great values of total protein (5.99%). The increasing for the percentage of protein extraction was major according to the heating time. Therefore, the maximum point from the function that represents the protein extraction was analysed by factorial experiment 2(3). By the results, it was noted that all the variables were important to extraction. After the statistical analyses, was observed that the parameters as pH, temperature, and percentage of sodium chloride, did not sufficient for the extraction process, since did not possible to obtain the inflection point from mathematical function, however, by the other hand, the mathematical model was significant, as well as, predictive.

Identification of protein coding regions using the modified Gabor-wavelet transform

An important topic in genomic sequence analysis is the identification of protein coding regions. In this context, several coding DNA model-independent methods based on the occurrence of specific patterns of nucleotides at coding regions have been proposed. Nonetheless, these methods have not been completely suitable due to their dependence on an empirically predefined window length required for a local analysis of a DNA region. We introduce a method based on a modified Gabor-wavelet transform (MGWT) for the identification of protein coding regions. This novel transform is tuned to analyze periodic signal components and presents the advantage of being independent of the window length. We compared the performance of the MGWT with other methods by using eukaryote data sets. The results show that MGWT outperforms all assessed model-independent methods with respect to identification accuracy. These results indicate that the source of at least part of the identification errors produced by the previous methods is the fixed working scale. The new method not only avoids this source of errors but also makes a tool available for detailed exploration of the nucleotide occurrence.

FtsH2 and FtsH5: two homologous subunits use different integration mechanisms leading to the same thylakoid multimeric complex

P>The Arabidopsis thylakoid FtsH protease complex is composed of FtsH1/FtsH5 (type A) and FtsH2/FtsH8 (type B) subunits. Type A and type B subunits display a high degree of sequence identity throughout their mature domains, but no similarity in their amino-terminal targeting peptide regions. In chloroplast import assays, FtsH2 and FtsH5 were imported and subsequently integrated into thylakoids by a two-step processing mechanism that resulted in an amino-proximal lumenal domain, a single transmembrane anchor, and a carboxyl proximal stromal domain. FtsH2 integration into washed thylakoids was entirely dependent on the proton gradient, whereas FtsH5 integration was dependent on NTPs, suggesting their integration by Tat and Sec pathways, respectively. This finding was corroborated by in organello competition and by antibody inhibition experiments. A series of constructs were made in order to understand the molecular basis for different integration pathways. The amino proximal domains through the transmembrane anchors were sufficient for proper integration as demonstrated with carboxyl-truncated versions of FtsH2 and FtsH5. The mature FtsH2 protein was found to be incompatible with the Sec machinery as determined with targeting peptide-swapping experiments. Incompatibility does not appear to be determined by any specific element in the FtsH2 domain as no single domain was incompatible with Sec transport. This suggests an incompatible structure that requires the intact FtsH2. That the highly homologous type A and type B subunits of the same multimeric complex use different integration pathways is a striking example of the notion that membrane insertion pathways have evolved to accommodate structural features of their respective substrates.

Digestibility of plant protein-based diets by largemouth bass Micropterus salmoides

Commercial farming of carnivorous fish demands the reduction of environmental impact of feeds; that requires minimal use of dietary animal protein. This study investigated the digestibility of diets formulated exclusively out of plant protein, added feed attractants, by the carnivore largemouth bass, Micropterus salmoides. Juvenile largemouth bass (14.0 +/- 1.0 cm) conditioned to accept artificial, dry feed were confined in polypropylene cages and fed ad libitum in three daily meals, seven experimental diets containing varying levels of vegetable and animal protein sources, added of different feed stimulants. After last daily meal, cages were transferred to cylindrical-conical-bottomed, 200-L aquaria, where faeces were collected by sedimentation into refrigerated containers, preserved and later analysed for chemical composition. Soybean meal can be used as partial substitute of animal protein in diets for largemouth bass; the poultry by-product meal shows as a good option as animal protein source in these rations. Control treatment - 50PP : 50AP - yielded best performances; the need for the use of fish meal in the formulation for carnivorous diets is, at least, questionable. Results of the digestibility trials demonstrated the importance of determining the diet digestibility, if precision in the formulation of least-cost feeds for carnivorous fish is the ultimate goal.

Apparent digestibility coefficient of protein and amino acids of acid, biological and enzymatic silage for Nile tilapia (Oreochromis niloticus)

Using the fish silage to partially replace proteic feedstuff in aquafeeds is an alternative to mitigate sanitary and environmental problems caused by the lack of adequate destination for fisheries residues. It would also lower feed costs, consequently improving fish culture profitability. However, using fish silages in aquafeeds depends on determination of its apparent digestibility coefficients (ADC). This work aimed to determining the ADC of crude protein and amino acids of acid silage (AS), biological silage (BS) and enzymatic silage (ES) for juvenile Nile tilapia (94.5 +/- 12.7 g). The ADC(CP) was: 92.0%, 89.1% and 93.7% for AS, BS and SE respectively. The average ADC of amino acids was: 91.8%, 90.8% and 94.6% for AS, BS and ES respectively. Results encourage the use of AS, BS and ES to partially replace protein sources in balanced diets for neotropical fish.

ADAPTABILITY AND STABILITY OF SOYBEAN GENOTYPES UNDER DIFFERENT TIMES OF SOWING

This study aimed to evaluate the average behavior, the genotype x environment (GxE), adaptability and stability of seven soybean cultivars at three sowing dates in Uberlandia-MG. The tests were conducted at Capim Branco Farm, belonging to the Federal University of Uberlandia. Sowing was held on october 29 (1st season), november 24 (2nd season) and december 17 (3rd season) 2007. The experimental design was a randomized, seven genotypes (UFUS Xavante, UFUS Riqueza, UFUS Guarani, UFUS Milionaria, Msoy 8001, Msoy 8411 and Msoy 8914) with three replications in each of three sowing dates. Means were compared by Tukey test at 5% probability. Analysis of adaptability and phenotypic stability of genotypes was performed using the Eberhart and Russell (1966), Lin and Binns (1988) modified by Carneiro (1998) and centroid (NASCIMENTO et al., 2009). For grain yield, the cultivar UFUS Xavante was classified as specific adaptability to environment and high stability. The other cultivars were classified as being of general adaptability. For oil content, the cultivars Msoy 8914 and UFUS Xavante behaved as high stability and was classified as having high adaptability. For the character content of protein, all cultivars behaved as wide adaptability and low stability.

Effects of Protein-Energy Malnutrition on NF-KappaB Signalling in Murine Peritoneal Macrophages

Protein-energy malnutrition (PEM) is an important public health problem affecting millions of people worldwide. PEM decreases resistance to infection, impairing a number of physiological processes. In unstimulated cells, NF-kappa B is kept from binding to its consensus sequence by the inhibitor I kappa B alpha, which retains NF-kappa B in the cytoplasm. Upon various signals, such as lipopolysaccharide (LPS), I kappa B alpha is rapidly degraded and NF-kappa B is induced to translocate into the nucleus, where it activates expression of various genes that participate in the inflammatory response, including those involved in the synthesis of TNF-alpha. TRAF-6 is a cytoplasmic adapter protein that links the stimulatory signal from Toll like receptor-4 to NF-kappa B. The aim of this study was to evaluate the effect of malnutrition on induction of TNF-a by LPS in murine peritoneal macrophages. We evaluated peritoneal cellularity, the expression of MyD88, TRAF-6, IKK, I kappa B alpha and NF-kappa B, NF-kappa B activation and TNF-alpha mRNA and protein synthesis inmacrophages. Two-month-old male BALB/Cmice were submitted to PEM with a low-protein diet that contained 2% protein, compared to 12% protein in the control diet. When the experimental group had lost about 20% of the original body weight, it was used in the subsequent experiments. Malnourished animals presented anemia, leucopenia and severe reduction in peritoneal cavity cellularity. TNF-a mRNA and protein levels of macrophages stimulated with LPS were significantly lower in malnourished animals. PEM also decreased TRAF-6 expression and NF-kappa B activation after LPS stimulation. These results led us to conclude that PEM changes NF-kappa B signalling pathway in macrophages to LPS stimulus.

Taenia crassiceps cysticerci: Characterization of the 14-kDa glycoprotein with homologies to antigens from Taenia solium cysticerci

Glycoproteins from the total vesicular fluid of Taenia crassiceps (VF-Tc) were prepared using three different purification methods, consisting of ConA-lectin affinity chromatography (ConA-Tc), preparative electrophoresis (SDS-PAGE) (14gp-Tc), and monoclonal antibody immunoaffinity chromatography (18/14-Tc). The complex composition represented by the VF-Tc and ConA-Tc antigens revealed peptides ranging from 101 - to 14-kDa and from 92- to 12-kDa, respectively. Immunoblotting using lectins confirmed glucose/mannose (glc/man) residues in the 18- and 14-kDa peptides, which are considered specific and immunodominant for the diagnosis of cysticercosis, and indicated that these fractions are glycoproteins. Serum antibodies from a patient with neurocysticercosis that reacted to the 14gp band from T. crassiceps (Tc) were eluted from immunoblotting membranes and showed reactivity to 14gp from Taenia solium. In order to determine the similar peptide sequence, the N-terminal amino acid was determined and analyzed with sequences available in public databases. This sequence revealed partial homology between T. crassiceps and T solium peptides. In addition, mass spectrometry along with theoretical M(r) and pI of the 14gp-Tc point suggested a close relationship to some peptides of a 150-kDa protein complex of the T solium previously described. The identification of these common immunogenic sites will contribute to future efforts to develop recombinant antigens and synthetic peptides for immunological assays. (C) 2009 Elsevier Inc. All rights reserved.

Citrate and Phosphate Influence on Green Fluorescent Protein Thermal Stability

Protein structure and function can be regulated by no specific interactions, such as ionic interactions in the presence of salts. Green fluorescent protein (GFP) shows remarkable structural stability and high fluorescence; its stability can be directly related to its fluorescence output, among other characteristics. GFP is stable under increasing temperatures, and its thermal denaturation is highly reproducible. The aim of this study was to evaluate the thermal stability of GFP in the presence of different salts at several concentrations and exposed to constant temperatures, in a range of 70-95 degrees C. Thermal stability was expressed in decimal reduction time. It was observed that the D-values obtained were higher in the presence of citrate and phosphate, when compared with that obtained in their absence, indicating that these salts stabilized the protein against thermal denaturation. (C) 2010 American Institute of Chemical Engineers Biotechnol. Prog., 27: 269-272, 2011