Isolation and in vitro hydrolysis of lentil protein fractions by trypsin

The albumin and globulin fractions from lentil seeds were isolated and characterised by gel filtration. The latter was shown to be homogeneous and the former heterogeneous on PAGE. The aminoacid analysis revealed high values of amidic amino acids for both fractions with great differences in the sulphur-containing amino acids. Native albumin, globulin and salt-soluble proteins were markedly resistant to trypsin hydrolysis compared to casein. The SDS-PAGE of native salt-soluble proteins indicated that the globulin fragments (20 to 30 kD) were slowly digested in the presence of albumin. The heating increased the hydrolysis of the proteins in the order: salt-soluble, albumin and globulin. The facilitated hydrolysis of the heated salt-soluble fraction seemed to be due to protein-protein interactions induced by heat.

Trypanosoma cruzi: identification and characterization of a novel ribosomal protein L27 (TcrL27) that cross-reacts with an affinity-purified anti-Sm antibody

Small nuclear ribonucleoproteins (snRNPs)are involved in trans-splicing processing of pre-mRNA in Trypanosoma cruzi. To clone T. cruzi snRNPs we screened an epimastigote cDNA library with a purified antibody raised against the Sm-binding site of a yeast sequence. A clone was obtained containing a 507 bp-insert with an ORF of 399 bp and coding for a protein of 133 amino acids. Sequence analysis revealed high identity with the L27 ribosomal proteins from different species including: Canis familiaris, Homo sapiens, Schizosaccharomyces pombe and Saccharomyces cerevisiae. This protein has not been previously described in the literature and seems to be a new ribosomal protein in T. cruzi and was given the code TcrL27. To express this recombinant T. cruzi L27 ribosomal protein in E. coli, the insert was subcloned into the pET32a vector and a 26 kDa recombinant protein was purified. Immunoblotting studies demonstrated that this purified recombinant protein was recognized by the same anti-Sm serum used in the library screening as well as by chagasic and systemic lupus erythemathosus (SLE) sera. Our results suggest that the T. cruzi L27 ribosomal protein may be involved in autoimmunity of Chagas disease.

Genetic structure of Blastocerus dichotomus populations in the Parana River basin (Brazil) based on protein variability

The population structure of 147 marsh deer (Blastocerus dichotomus) from three areas in the Parana River basin, Brazil, was studied by observing protein polymorphism at 17 loci. Six loci were polymorphic and 11 monomorphic. The proportion of polymorphic loci (P) was 35.29% and the average heterozygosity (H) was 6.31%. Wright's F-ST indicated that only 4.9% of the total variation in allelic frequencies was due to genetic differences between the three groups. The high value of F-IS (0.246) indicated inbreeding in the marsh deer. Genetic distance values (D = 0.014-0.051) showed little divergence between the three areas. We suggest that probable mechanisms accounting for the genetic structure are female phylopatry and polygyny and also that inbreeding has resulted from decreasing areas of wetland leading to isolation, overhunting, and diseases transmitted by cattle.

CONCERTED ACTION OF THE HIGH-AFFINITY CALCIUM-BINDING SITES IN SKELETAL-MUSCLE TROPONIN-C

Mutants of each of the four divalent cation binding sites of chicken skeletal muscle troponin C (TnC) were constructed using site directed mutagenesis to convert Asp to Ala at the first coordinating position in each site. With a view to evaluating the importance of site-site interactions both within and between the N- and C-terminal domains, in this study the mutants are examined for their ability to associate with other components of the troponin-tropomyosin regulatory complex and to regulate thin filaments. The functional effects of each mutation in reconstitution assays are largely confined to the domain in which it occurs, where the unmutated site is unable to compensate for the defect, Thus the mutants of sites I and II bind to the regulatory complex but are impaired in ability to regulate tension and actomyosin ATPase activity, whereas the mutants of sites III and IV regulate activity but are unable to remain bound to thin filaments unless Ca2+ is present. When all four sites are intact, free Mg2+ causes a 50-60-fold increase in TnC's affinity for the other components of the regulatory complex, allowing it to attach firmly to thin filaments. Calcium can replace Mg2+ at a concentration ratio of 1:5000, and at this ratio the Ca2 . TnC complex is more tightly bound to the filaments than the Mg2 . TnC form, In the C-terminal mutants, higher concentrations of Ca2+ (above tension threshold) are required to effect this transformation than in the recombinant wild-type protein, suggesting that the mutants reveal an attachment mediated by Ca2+ in the N-domain sites.

Genetic parameters of milk, fat, and protein yields in the first three lactations, using an animal model and restricted maximum likelihood

Milk, fat, and protein yields of Holstein cows from the States of New York and California in the United States were used to estimate (co)variances among yields in the first three lactations, using an animal model and a derivative-free restricted maximum likelihood (REML) algorithm, and to verify if yields in different lactations are the same trait. The data were split in 20 samples, 10 from each state, with means of 5463 and 5543 cows per sample from California and New York. Mean heritability estimates for milk, fat, and protein yields for California data were, respectively, 0.34, 0.35, and 0.40 for first; 0.31, 0.33, and 0.39 for second; and 0.28, 0.31, and 0.37 for third lactations. For New York data, estimates were 0.35, 0.40, and 0.34 for first; 0.34, 0.44, and 0.38 for second; and 0.32, 0.43, and 0.38 for third lactations. Means of estimates of genetic correlations between first and second, first and third, and second and third lactations for California data were 0.86, 0.77, and 0.96 for milk; 0.89, 0.84, and 0.97 for fat; and 0.90, 0.84, and 0.97 for protein yields. Mean estimates for New York data were 0.87, 0.81, and 0.97 for milk; 0.91, 0.86, and 0.98 for fat; and 0.88, 0.82, and 0.98 for protein yields. Environmental correlations varied from 0.30 to 0.50 and were larger between second and third lactations. Phenotypic correlations were similar for both states and varied from 0.52 to 0.66 for milk, fat and protein yields. These estimates are consistent with previous estimates obtained with animal models. Yields in different lactations are not statistically the same trait but for selection programs such yields can be modelled as the same trait because of the high genetic correlations.

Expression of heat shock protein in broiler embryo tissues after acute cold or heat stress

This study evaluated the expression of heat shock protein 70 kD (hsp70) in broiler chicken embryos subjected to cold (Experiment 1) or high incubation temperature (Experiment 11). In each experiment, fertile eggs were distributed in three incubators kept at 37.8degreesC. At day 13 (D13), D16, and D19 of incubation, the embryos were subjected to acute cold (32degreesC) or heat (40degreesC) for 4-6 hr. Immediately after cold or heat exposure, samples from the liver, heart, breast muscle, brain, and lungs of 40 embryos were taken per age and treatment (control or stressed embryos), A tissue pool from 10 embryos was used as 1 replication. The levels of hsp70 in each tissue sample was quantified by Western blot analysis. The data were analyzed in a 3 x 2 factorial arrangement of treatments with four replications. hsp70 was detected in all embryo tissues, and the brain contained 2- to 5-times more hsp70 protein compared to the other tissues in either cold or heat stressed embryos. hsp70 increases were observed in the heart and breast muscle of cold stressed embryos at D16 and D19, respectively. Heat stressed embryos showed an increase of hsp70 in the heart at D13 and D19, and in the lung at D19 of incubation. Younger embryos had higher hsp70 synthesis than older embryos, irrespective of the type of thermal stressor. The results indicate that the expression of hsp70 in broiler chicken embryos is affected by cold and heat distress, and is tissue- and age-dependent. (C) 2004 Wiley-Liss, Inc.

Probing the kinetics of single molecule protein folding

We propose an approach to integrate the theory, simulations, and experiments in protein-folding kinetics. This is realized by measuring the mean and high-order moments of the first-passage time and its associated distribution. The full kinetics is revealed in the current theoretical framework through these measurements. In the experiments, information about the statistical properties of first-passage times can be obtained from the kinetic folding trajectories of single molecule experiments ( for example, fluorescence). Theoretical/simulation and experimental approaches can be directly related. We study in particular the temperature-varying kinetics to probe the underlying structure of the folding energy landscape. At high temperatures, exponential kinetics is observed; there are multiple parallel kinetic paths leading to the native state. At intermediate temperatures, nonexponential kinetics appears, revealing the nature of the distribution of local traps on the landscape and, as a result, discrete kinetic paths emerge. At very low temperatures, exponential kinetics is again observed; the dynamics on the underlying landscape is dominated by a single barrier. The ratio between first-passage-time moments is proposed to be a good variable to quantitatively probe these kinetic changes. The temperature-dependent kinetics is consistent with the strange kinetics found in folding dynamics experiments. The potential applications of the current results to single-molecule protein folding are discussed.

Role of endothelial cell apoptosis in regulation of skeletal muscle angiogenesis during high and low salt intake

Angiogenesis, under normal conditions, is a tightly regulated balance between pro- and antiangiogenic factors. The goal of this study was to investigate the mechanisms involved in the control of the skeletal muscle angiogenic response induced by electrical stimulation during the suppression of plasma renin activity (PRA) with a high-salt diet. Rats fed 0.4% or 4% salt diets were exposed to electrical stimulation for 7 days. The tibialis anterior ( TA) muscles from stimulated and unstimulated hindlimbs were removed and prepared for gene expression analysis, CD31-terminal deoxynucleotide transferase-mediated dUTP nick-end labeling ( TUNEL) double-staining assay, and Bcl-2 and Bax protein expression by Western blot. Rats fed a low-salt diet showed a dramatic angiogenesis response in the stimulated limb compared with the unstimulated limb. This angiogenesis response was significantly attenuated when rats were placed on a high-salt diet. Microarray analysis showed that in the stimulated limb of rats fed a low-salt diet many genes related to angiogenesis were upregulated. In contrast, in rats fed a high-salt diet most of the genes upregulated in the stimulated limb function in apoptosis and cell cycle arrest. Endothelial cell apoptosis, as analyzed by CD31-TUNEL staining, increased by fourfold in the stimulated limb compared with the unstimulated limb. There was also a 48% decrease in the Bcl-2-to-Bax ratio in stimulated compared with unstimulated limbs of rats fed a high-salt diet, confirming severe apoptosis. This study suggests that the increase in endothelial cell apoptosis in TA muscle might contribute to the attenuation of angiogenesis response observed in rats fed a high-salt diet.

EFFECTS OF INTRAUTERINE AND POSTNATAL PROTEIN-CALORIE MALNUTRITION ON METABOLIC ADAPTATIONS TO EXERCISE IN YOUNG-RATS

The effect of intrauterine and postnatal protein-calorie malnutrition on the biochemical ability to perform exercise was investigated in young male rats. Malnourished rats were obtained by feeding dams a low-protein (6%) casein-based diet prepared in the laboratory during pregnancy and lactation. Control rats received an isocaloric diet containing 25% protein. The low-protein diet contained additional starch and glucose. At 45 days of age, malnourished rats showed lower body weight, serum protein, albumin and glucose levels, hematocrit values and heart glycogen content but higher circulating free fatty acids and gastrocnemius muscle glycogen than control rats. In response to exercise (50 min of swimming), control rats displayed lower heart, gastrocnemius and liver glycogen levels whereas malnourished rats showed low glycogen levels only in the gastrocnemius muscle. Both control and malnourished rats showed high serum glucose and free fatty acid levels after exercise. In conclusion, protein-calorie malnutrition improved muscle glycogen storage but this substrate was broken down to a greater extent in response to exercise. Malnourished rats were able to perform exercise maintaining high blood glucose levels, as observed in control rats, perhaps as a consequence of the elevated availability of circulating free fatty acids.

Single dose of a vaccine based on DNA encoding mycobacterial hsp65 protein plus TDM-loaded PLGA microspheres protects mice against a virulent strain of Mycobacterium tuberculosis

The high incidence of tuberculosis around the world and the inability of BCG to protect certain populations clearly indicate that an improved vaccine against tuberculosis is needed. A single antigen, the mycobacterial heat shock protein hsp65, is sufficient to protect BALB/c mice against challenge infection when administered as DNA vaccine in a three-dose-based schedule. In order to simplify the vaccination schedule, we coencapsulated hsp65-DNA and trehalose dimicolate (TDM) into biodegradable poly(DL-lactide-co-glycolide) (PLGA) microspheres. BALB/c mice immunized with a single dose of DNA-hsp65/TDM-1oaded microspheres produced high levels of IgG2a subtype antibody and high amounts of IFN-gamma in the supernatant of spleen cell cultures. DNA-hsp65/TDM-loaded microspheres were also able to induce high IFN-gamma production in bulk lung cells from challenged mice and confer protection as effective as that attained after three doses of naked DNA administration. This new formulation also allowed a ten-fold reduction in the DNA dose when compared to naked DNA. Thus, this combination of DNA vaccine and adjuvants with immunomodulatory and carrier properties holds the potential for an improved vaccine against tuberculosis.

High hydrostatic pressure processing on microstructure of probiotic low-fat yogurt

The effect of milk processing on the microstructure of probiotic low-fat yogurt was studied. Skim milk fortified with skim milk powder was subjected to three treatments prior to innoculation: thermal treatment at 85 degrees C for 30 min, high hydrostatic pressure at 676 MPa for 5 min, and combined treatments of high hydrostatic pressure (HHP) and heat. The processed milk was then fermented by using two different starter cultures containing Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus acidophilus, and Bifidobacterium longum. The microstructure of heat-treated milk yogurt had fewer interconnected chains of irregularly shaped casein micelles, forming a network that enclosed the void spaces. on the other hand, microstructure of HHP yogurt had more interconnected clusters of densely aggregated protein of reduced particle size, with an appearance more spherical in shape, exhibiting a smoother more regular surface and presenting more uniform size distribution. The combined HHP and heat milk treatments led to compact yogurt gels with increasingly larger casein micelle clusters interspaced by void spaces, and exhibited a high degree of cross-linking. The rounded micelles tended to fuse and form small irregular aggregates in association with clumps of dense amorphous material, which resulted in improved gel texture and viscosity. (C) 2007 Elsevier Ltd. All rights reserved.

Effects of a lectin-like protein isolated from Acacia farnesiana seeds on phytopathogenic bacterial strains and root-knot nematode

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Apparent digestibility of protein, energy, and amino acids in some selected feed ingredients for pacu Piaractus mesopotamicus

The apparent digestibility coefficients (ADCs) of amino acids (AA), protein, and energy in six feed ingredients (Brazilian fish meal, soybean meal, corn gluten meal, alcoholic yeast, corn, and wheat bran) we evaluated for pacu juveniles. In general, all ingredients showed high digestibility values for all AA, and differences among ADCs of individual amino acid were detected (P < 0.01). Corn gluten, soybean, and fish meals had the highest ADCs of AA. The ADCs of protein in fish meal, yeast, and corn gluten meal should not be used as AA digestibility indicators, because those showed differences up to 6.7% between the ADCs of protein and AA. All ingredients had lower ADCs of energy than corn gluten meal (P < 0.01). Lysine was the first limiting amino acid in fish meal, corn gluten meal, wheat bran, and corn, and the second limiting amino acid in soybean meal, as methionine was the first limiting amino acid in soybean meal and yeast. However, the soybean meal was the best quality protein source, as it had the highest digestible essential amino acid index. This demonstrated that digestible amino acid values can be used to formulate practical diets for pacu, preventing potential deficiencies or excess that might cause environmental and economic losses.

Magneto Immunoassays for Plasmodium falciparum Histidine-Rich Protein 2 Related to Malaria based on Magnetic Nanoparticles

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

NO EVIDENCE of PATHOLOGICAL AUTOIMMUNITY FOLLOWING MYCOBACTERIUM LEPRAE HEAT-SHOCK PROTEIN 65-DNA VACCINATION IN MICE

Heat-shock proteins (HSPs) are currently one of the most promising targets for the development of immunotherapy against tumours and autoimmune disorders. This protein family has the capacity to activate or modulate the function of different immune system cells. They induce the activation of monocytes, macrophages and dendritic cells, and contribute to cross-priming, an important mechanism of presentation of exogenous antigen in the context of MHC class I molecules, These various immunological properties of HSP have encouraged their use in several clinical trials. Nevertheless, an important issue regarding these proteins is whether the high homology among HSPs across different species may trigger the breakdown of immune tolerance and induce autoimmune diseases. We have developed a DNA vaccine codifying the Mycobacterium leprae Hsp65 (DNAhsp65), which showed to be highly immunogenic and protective against experimental tuberculosis. Here, we address the question of whether DNAhsp65 immunization could induce pathological autoimmunity in mice. Our results show that DNAhsp65 vaccination induced antibodies that can recognize the human Hsp60 but did not induce harmful effects in 16 different organs analysed by histopathology up to 210 days after vaccination. We also showed that anti-DNA antibodies were not elicited after DNA vaccination. The results are important for the development of both HSP and DNA-based immunomodulatory agents.