Thr(118)Met amino acid substitution in the peripheral myelin protein 22 does not influence the clinical phenotype of Charcot-Marie-Tooth disease type 1A due to the 17p11.2-p12 duplication

The Thr(118)Met substitution in the peripheral myelin protein 22 (PMP22) gene has been detected in a number of families with demyelinating Charcot-Marie-Tooth (CMT1) neuropathy or with the hereditary neuropathy with liability to pressure palsy, but in none of them has it consistently segregated with the peripheral neuropathy. We describe here a CMT1 family (a 63-year-old man, his brother and his niece) in which two mutations on different chromosomes were found in the PMP22 gene, the 17p duplication, detected by fluorescent semiquantitative polymerase chain reaction (PCR) of microsatellite markers localized within the duplicated region on chromosome 17p11.2-p12, and the Thr(118)Met substitution, detected by direct sequencing the four coding exons of the PMP22 gene. A genotype/phenotype correlation study showed that the neuropathy segregates with the duplication and that the amino acid substitution does not seem to modify the clinical characteristics or the severity of the peripheral neuropathy. We did not find any evidence to characterize this substitution as a polymorphism in the population studied and we propose that the high frequency reported for this point mutation in the literature suggests that the Thr(118)Met substitution may be a hotspot for mutations in the PMP22 gene.

A first case of protease codon 35 amino acid insertion in a HIV-1 subtype B sequence detected in the Bauru region, state of São Paulo, Brazil: case report

Amino acid insertions in the protease have rarely been described in HIVinfected patients. One of these insertions has recently been described in codon 35, although its impact on resistance remains unknown. This study presents a case of an HIV variant with an insertion in codon 35 of the protease, described for the first time in Bauru, State of Sao Paulo, Brazil, circulating in a 38-year-old caucasian male with asymptomatic HIV infection since 1997. The variant isolated showed a codon 35 insertion of two amino acids in the protease: a threonine and an aspartic acid, resulting in the amino acid sequence E35E_TD.

Immunogenicity and antigenicity of the N-term repeat amino acid sequence of the Plasmodium falciparum P126 antigen

The P126 protein, a parasitosphorus vacuole antigen of Plasmodium falciparum has beenshoen to induce protective immunity in Saimiri and Aotus monkeys. In the present work we investigated its immunogenicity. Our results suggest that the N-term of P126 is poorly immunogenic and antibody response against the P126 could be under a MHC restricted control in C57BL/6(H-2b) mice, which could be problematic in ternms of a use of the P126 in a vaccine program. However, we observed that a synthetic peptide, copying the 6 octapeptide repeat corresponding to the N-term of the P126, induces an antibody response to the native molecule in C57BL/6 non-responder mice. Moreover, the vaccine-P126 recombinant induced anmtibodies against the N-term of the molecule in rabbits while the unprocessed P126 did not.

N-terminal amino acid sequences of the major outer membrane proteins from a Neisseria meningitidis group B strain isolated in Brazil

The four dominant outer membrane proteins (46, 38, 33 and 28 kDa) were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in a semi-purified preparation of vesicle membranes of a Neisseria meningitidis (N44/89, B:4:P1.15:P5.5,7) strain isolated in Brazil. The N-terminal amino acid sequence for the 46 kDa and 28 kDa proteins matched that reported by others for class 1 and 5 proteins respectively, whereas the sequence (25 amino acids) for the 38 kDa (class 3) protein was similar to class 1 meningococcal proteins. The sequence for the 33 kDa (class 4) was unique and not homologous to any known protein.

Amino acid sequences of proteins from Leptospira serovar pomona

This report describes a partial amino acid sequences from three putative outer envelope proteins from Leptospira serovar pomona. In order to obtain internal fragments for protein sequencing, enzymatic and chemical digestion was performed. The enzyme clostripain was used to digest the proteins 32 and 45 kDa. In situ digestion of 40 kDa molecular weight protein was accomplished using cyanogen bromide. The 32 kDa protein generated two fragments, one of 21 kDa and another of 10 kDa that yielded five residues. A fragment of 24 kDa that yielded nineteen residues of amino acids was obtained from 45 kDa protein. A fragment with a molecular weight of 20 kDa, yielding a twenty amino acids sequence from the 40 kDa protein.

Mapping the molecular characteristics of Brazilian human T-cell lymphotropic virus type 1 Env (gp46) and Pol amino acid sequences for vaccine design

This study was carried out to evaluate the molecular pattern of all available Brazilian human T-cell lymphotropic virus type 1 Env (n = 15) and Pol (n = 43) nucleotide sequences via epitope prediction, physico-chemical analysis, and protein potential sites identification, giving support to the Brazilian AIDS vaccine program. In 12 previously described peptides of the Env sequences we found 12 epitopes, while in 4 peptides of the Pol sequences we found 4 epitopes. The total variation on the amino acid composition was 9 and 17% for human leukocyte antigen (HLA) class I and class II Env epitopes, respectively. After analyzing the Pol sequences, results revealed a total amino acid variation of 0.75% for HLA-I and HLA-II epitopes. In 5 of the 12 Env epitopes the physico-chemical analysis demonstrated that the mutations magnified the antigenicity profile. The potential protein domain analysis of Env sequences showed the loss of a CK-2 phosphorylation site caused by D197N mutation in one epitope, and a N-glycosylation site caused by S246Y and V247I mutations in another epitope. Besides, the analysis of selection pressure have found 8 positive selected sites (w = 9.59) using the codon-based substitution models and maximum-likelihood methods. These studies underscore the importance of this Env region for the virus fitness, for the host immune response and, therefore, for the development of vaccine candidates.

Differences in ureide and amino acid content of water stressed soybean inoculated with Bradyrhizobium japonicum and B. elkanii

The objective of this work was to study the response to water stress of a drought sensitive soybean cultivar inoculated with Bradyrhizobium japonicum (strain CB1809, Semia 586) and B. elkanii (strain 29W, Semia 5019). CB1809 nodulated plants produced a significantly higher root fraction (19%) than 29W (14.6%). Plants inoculated with CB1809 produced less nodules and accumulated more nitrogen than those inoculated with 29W. In general, low amounts of ureides in nodules were found in watered plants inoculated with either CB1809 or 29W strains, but those levels were five-fold increased in stressed plants inoculated with CB1809. Nodules formed by strain CB1809 had aspartate and glutamate as major amino acids, while those formed by 29W had glutamate, asparagine and alanine. In nodules of plants inoculated with CB1809 aspartate showed the highest accumulation (5 µmol g-1); in stressed plants this amino acid reached a value of 26 µmol g-1, and asparagine was not detected. Nodules formed by the strain 29W accumulated 1 µmol g-1 of aspartate, whether plants were stressed or not. Asparagine was the major amino acid found in nodules from watered plants (6 µmol g-1) and the amount of this amino acid was six-fold increased when plants were water stressed.

Application of 1-methylcyclopropene, calcium chloride and calcium amino acid chelate on fresh-cut cantaloupe muskmelon

The objective of this work was to determine the effects of postharvest application of 1-methylcyclopropene (1-MCP) and two calcium salts, applied individually or combined, on firmness and visual quality of fresh-cut muskmelon stored in air, for 18 days. Two sets of fruits, one of them exposed to 1-MCP at 300 nL L-1, were cut into cubes, dipped in deionized water, or in 1% Ca solutions as CaCl2, or in calcium amino acid chelate (Ca-chelate), placed in clamshell containers, and stored in air at 5±1ºC and 90±5% RH, for 18 days. The assay was conducted using an entirely randomized design, with three replications, in a split plot array. Evaluation of visual appearance, color, flesh firmness, total soluble solids, titratable acidity, and pH was performed right after treatments, and every period of three days, up to eighteen days. Application of 1-MCP at 300 nL L-1, calcium chloride or Ca-chelate, or the combination 1-MCP and calcium, preserved initial freshness and reduced softening of the samples. Ca-chelate synergistically enhanced the effect of 1-MCP on firmness after nine days of storage, while calcium chloride improved firmness of the samples throughout storage. Ca-chelate may serve as an alternative for shelf life extension of cantaloupe fresh-cut muskmelon.

Amino acid composition of soybean seeds as affected by climatic variables

The objective of this work was to perform a quantitative analysis of the amino acid composition of soybean seeds as affected by climatic variables during seed filling. Amino acids were determined from seed samples taken at harvest in 31 multi-environment field trials carried out in Argentina. Total amino acids ranged from 31.69 to 49.14%, and total essential and nonessential amino acids varied from 12.83 to 19.02% and from 18.86 to 31.15%, respectively. Variance components expressed as the percentage of total variation showed that the environment was the most important source of variation for all traits, followed by the genotype x environment interaction. Significant explanatory linear regressions were detected for amino acid content regarding: average daily mean air temperature and cumulative solar radiation, during seed filling; precipitation minus potential evapotranspiration, during the whole reproductive period; and the combinations of these climatic variables. Each amino acid behaves differently according to environmental conditions, indicating compensatory effects among them.

Storage protein profile and amino acid content in wild rice Oryza glumaepatula

- The objective of this work was to determine the total protein profile and the contents of the four major protein fractions (albumin, globulin, prolamin and glutelin) and of the amino acids in the endosperm of the rice wild species Oryza glumaepatula. The experiment was performed with 29 accessions of this species, collected from 13 Brazilian locations, and two commercial cultivars. Protein samples were prepared using dried, polished, and ground grains to obtain homogeneous, dry flour used in the preparation of extracts. Oryza glumaepatula accessions were identified with the highest levels of total protein, albumin and glutelin protein fractions, and amino acids (with the exception of tryptophan) in comparison to the two analized rice cultivars. The albumin and glutelin profiles in SDS-Page were distinct between rice cultivars and O. glumaepatula. This wild species has the potential to increase the nutritional quality of rice storage protein through interspecific crosses.

Apparent digestibility of nutrients, energy, and amino acid of nontoxic and detoxified physic nut cakes for Nile tilapia

Abstract:The objective of this work was to evaluate the apparent digestibility coefficients of nutrients, energy, and amino acids of nontoxic and detoxified physic nut cakes treated with solvent plus posterior extrusion, for Nile tilapia. The apparent digestibility coefficients of crude protein and gross energy were higher for detoxified than for nontoxic physic nut cake. However, the apparent digestibility coefficient of ether extract of the nontoxic physic nut cake was higher than that of the detoxified one. The apparent digestibility coefficient of amino acids of both feed ingredients was superior to 80%, except for glycine, for the nontoxic psychic nut cake, and for threonine, for the detoxified one. Nontoxic and detoxified physic nut cakes show apparent digestibility coefficient values equivalent to those of the other evaluated oilseeds and potential for inclusion in Nile tilapia diets.

Flavianate, an amino acid precipitant, is a competitive inhibitor of trypsin at pH 3.0

Textile dyes bind to proteins leading to selective co-precipitation of a complex involving one protein molecule and more than one dye molecule of opposite charge in acid solutions, in a process of reversible denaturation that can be utilized for protein fractionation. In order to understand what occurs before the co-precipitation, a kinetic study using bovine ß-trypsin and sodium flavianate was carried out based on reaction progress curve techniques. The experiments were carried out using a-CBZ-L-Lys-p-nitrophenyl ester as substrate which was added to 50 mM sodium citrate buffer, pH 3.0, containing varying concentrations of ß-trypsin and dye. The reaction was recorded spectrophotometrically at 340 nm for 30 min, and the families of curves obtained were analyzed simultaneously by fitting integrated Michaelis-Menten equations. The dye used behaved as a competitive inhibitor of trypsin at pH 3.0, with Ki = 99 µM; kinetic parameters for the substrate hydrolysis were: Km = 32 µM, and kcat = 0.38/min. The competitive character of the inhibition suggests a specific binding of the first dye molecule to His-57, the only positively charged residue at the active site of the enzyme.

Important amino acid residues of potato plant uncoupling protein (StUCP)

Chemical modifications were used to identify some of the functionally important amino acid residues of the potato plant uncoupling protein (StUCP). The proton-dependent swelling of potato mitochondria in K+-acetate in the presence of linoleic acid and valinomycin was inhibited by mersalyl (Ki = 5 µM) and other hydrophilic SH reagents such as Thiolyte MB, iodoacetate and 5,5'-dithio-bis-(2-nitrobenzoate), but not by hydrophobic N-ethylmaleimide. This pattern of inhibition by SH reagents was similar to that of brown adipose tissue uncoupling protein (UCP1). As with UCP1, the arginine reagent 2,3-butadione, but not N-ethylmaleimide or other hydrophobic SH reagents, prevented the inhibition of StUCP-mediated transport by ATP in isolated potato mitochondria or with reconstituted StUCP. The results indicate that the most reactive amino acid residues in UCP1 and StUCP are similar, with the exception of N-ethylmaleimide-reactive cysteines in the purine nucleotide-binding site.