Excitatory amino acid receptor blockade within the caudal pressor area and rostral ventrolateral medulla alters cardiovascular responses to nucleus raphe obscurus stimulation in rats

Pressor responses elicited by stimulation of the nucleus raphe obscurus (NRO) depend on the integrity of the rostral ventrolateral medulla (RVLM). Therefore, to test the participation of excitatory amino acid (EAA) receptors in the cardiovascular responses evoked by NRO stimulation (1 ms, 100 Hz, 40-70 µA, for 10 s), the EAA antagonist kynurenic acid (Kyn) was microinjected at different sites in the ventrolateral medullar surface (2.7 nmol/200 nl) of male Wistar rats (270-320 g, N = 39) and NRO stimulation was repeated. The effects of NRO stimulation were: hypertension (deltaMAP = +43 ± 1 mmHg, P<0.01), bradycardia (deltaHR = -30 ± 7 bpm, P<0.01) and apnea. Bilateral microinjection of Kyn into the RVLM, which did not change baseline parameters, almost abolished the bradycardia induced by NRO stimulation (deltaHR = -61 ± 3 before vs -2 ± 3 bpm after Kyn, P<0.01, N = 7). Unilateral microinjection of Kyn into the CVLM did not change baseline parameters or reduce the pressor response to NRO stimulation (deltaMAP = +46 ± 5 before vs +48 ± 5 mmHg after Kyn, N = 6). Kyn bilaterally microinjected into the caudal pressor area reduced blood pressure and heart rate and almost abolished the pressor response to NRO stimulation (deltaMAP = +46 ± 4 mmHg before vs +4 ± 2 mmHg after Kyn, P<0.01, N = 7). These results indicate that EAA receptors on the medullary ventrolateral surface play a role in the modulation of the cardiovascular responses induced by NRO stimulation, and also suggest that the RVLM participates in the modulation of heart rate responses and that the caudal pressor area modulates the pressor response following NRO stimulation.

A protein with amino acid sequence homology to bovine insulin is present in the legume Vigna unguiculata (cowpea)

Since the discovery of bovine insulin in plants, much effort has been devoted to the characterization of these proteins and elucidation of their functions. We report here the isolation of a protein with similar molecular mass and same amino acid sequence to bovine insulin from developing fruits of cowpea (Vigna unguiculata) genotype Epace 10. Insulin was measured by ELISA using an anti-human insulin antibody and was detected both in empty pods and seed coats but not in the embryo. The highest concentrations (about 0.5 ng/µg of protein) of the protein were detected in seed coats at 16 and 18 days after pollination, and the values were 1.6 to 4.0 times higher than those found for isolated pods tested on any day. N-terminal amino acid sequencing of insulin was performed on the protein purified by C4-HPLC. The significance of the presence of insulin in these plant tissues is not fully understood but we speculate that it may be involved in the transport of carbohydrate to the fruit.

A leucine-supplemented diet improved protein content of skeletal muscle in young tumor-bearing rats

Cancer cachexia induces host protein wastage but the mechanisms are poorly understood. Branched-chain amino acids play a regulatory role in the modulation of both protein synthesis and degradation in host tissues. Leucine, an important amino acid in skeletal muscle, is higher oxidized in tumor-bearing animals. A leucine-supplemented diet was used to analyze the effects of Walker 256 tumor growth on body composition in young weanling Wistar rats divided into two main dietary groups: normal diet (N, 18% protein) and leucine-rich diet (L, 15% protein plus 3% leucine), which were further subdivided into control (N or L) or tumor-bearing (W or LW) subgroups. After 12 days, the animals were sacrificed and their carcass analyzed. The tumor-bearing groups showed a decrease in body weight and fat content. Lean carcass mass was lower in the W and LW groups (W = 19.9 ± 0.6, LW = 23.1 ± 1.0 g vs N = 29.4 ± 1.3, L = 28.1 ± 1.9 g, P < 0.05). Tumor weight was similar in both tumor-bearing groups fed either diet. Western blot analysis showed that myosin protein content in gastrocnemius muscle was reduced in tumor-bearing animals (W = 0.234 ± 0.033 vs LW = 0.598 ± 0.036, N = 0.623 ± 0.062, L = 0.697 ± 0.065 arbitrary intensity, P < 0.05). Despite accelerated tumor growth, LW animals exhibited a smaller reduction in lean carcass mass and muscle myosin maintenance, suggesting that excess leucine in the diet could counteract, at least in part, the high host protein wasting in weanling tumor-bearing rats.

Amino acid composition of parturient plasma, the intervillous space of the placenta and the umbilical vein of term newborn infants

The objective of the present study was to determine the levels of amino acids in maternal plasma, placental intervillous space and fetal umbilical vein in order to identify the similarities and differences in amino acid levels in these compartments of 15 term newborns from normal pregnancies and deliveries. All amino acids, except tryptophan, were present in at least 186% higher concentrations in the intervillous space than in maternal venous blood, with the difference being statistically significant. This result contradicted the initial hypothesis of the study that the plasma amino acid levels in the placental intervillous space should be similar to those of maternal plasma. When the maternal venous compartment was compared with the umbilical vein, we observed values 103% higher on the fetal side which is compatible with currently accepted mechanisms of active amino acid transport. Amino acid levels of the placental intervillous space were similar to the values of the umbilical vein except for proline, glycine and aspartic acid, whose levels were significantly higher than fetal umbilical vein levels (average 107% higher). The elevated levels of the intervillous space are compatible with syncytiotrophoblast activity, which maintain high concentrations of free amino acids inside syncytiotrophoblast cells, permitting asymmetric efflux or active transport from the trophoblast cells to the blood in the intervillous space. The plasma amino acid levels in the umbilical vein of term newborns probably may be used as a standard of local normality for clinical studies of amino acid profiles.

Elevated amniotic fluid amino acid levels in fetuses with gastroschisis

Our objective was to measure maternal plasma and amniotic fluid amino acid concentrations in pregnant women diagnosed as having fetuses with gastroschisis in the second trimester of pregnancy. Twenty-one pregnant women who had fetuses with gastroschisis detected by ultrasonography (gastroschisis group) in the second trimester and 32 women who had abnormal triple screenings indicating an increased risk for Down syndrome but had healthy fetuses (control group) were enrolled in the study. Amniotic fluid was obtained by amniocentesis, and maternal plasma samples were taken simultaneously. The chromosomal analysis of the study and control groups was normal. Levels of free amino acids and non-essential amino acids were measured in plasma and amniotic fluid samples using EZ:fast kits (EZ:fast GC/FID free (physiological) amino acid kit) by gas chromatography (Focus GC AI 3000 Thermo Finnigan analyzer). The mean levels of essential amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine) and non-essential amino acids (alanine, glycine, proline, and tyrosine) in amniotic fluid were found to be significantly higher in fetuses with gastroschisis than in the control group (P < 0.05). A significant positive correlation between maternal plasma and amniotic fluid concentrations of essential and nonessential amino acids was found only in the gastroschisis group (P < 0.05). The detection of significantly higher amino acid concentrations in the amniotic fluid of fetuses with a gastroschisis defect than in healthy fetuses suggests the occurrence of amino acid malabsorption or of amino acid leakage from the fetus into amniotic fluid.

Amniotic fluid amino acid levels in non-immune hydrops fetalis: a case-control study

In a prospective case-control study, we compared the amniotic fluid amino acid levels in non-immune hydrops fetalis (NIHF) and normal fetuses. Eighty fetuses underwent amniocentesis for different reasons at the prenatal diagnosis unit of the Department of Obstetrics and Gynecology, Faculty of Medicine, Dicle University. Forty of these fetuses were diagnosed with NIHF. The study included 40 women each in the NIHF (mean age: 27.69 ± 4.56 years) and control (27.52 ± 5.49 years) groups, who had abnormal double- or triple-screening test values with normal fetuses with gestational ages of 23.26 ± 1.98 and 23.68 ± 1.49 weeks at the time of sample collection, respectively. Amniotic fluid amino acid concentrations (intra-assay variation: 2.26-7.85%; interassay variation: 3.45-8.22%) were measured using EZ:faast kits (EZ:faast GC/FID free (physiological) amino acid kit; Phenomenex, USA) by gas chromatography. The standard for quantitation was a mixture of free amino acids from Phenomenex. The levels of 21 amino acids were measured. The mean phosphoserine and serine levels were significantly lower in the NIHF group, while the taurine, α-aminoadipic acid (aaa), glycine, cysteine, NH4, and arginine (Arg) levels were significantly higher compared to control. Significant risk variables for the NIHF group and odds coefficients were obtained using a binary logistic regression method. The respective odds ratios and 95% confidence intervals for the risk variables phosphoserine, taurine, aaa, Arg, and NH4 were 3.31 (1.84-5.97), 2.45 (1.56-3.86), 1.78 (1.18-2.68), 2.18 (1.56-3.04), and 2.41 (1.66-3.49), respectively. The significant difference between NIHF and control fetuses suggests that the amniotic fluid levels of some amino acids may be useful for the diagnosis of NIHF.

Protein turnover, amino acid requirements and recommendations for athletes and active populations

Skeletal muscle is the major deposit of protein molecules. As for any cell or tissue, total muscle protein reflects a dynamic turnover between net protein synthesis and degradation. Noninvasive and invasive techniques have been applied to determine amino acid catabolism and muscle protein building at rest, during exercise and during the recovery period after a single experiment or training sessions. Stable isotopic tracers (13C-lysine, 15N-glycine, ²H5-phenylalanine) and arteriovenous differences have been used in studies of skeletal muscle and collagen tissues under resting and exercise conditions. There are different fractional synthesis rates in skeletal muscle and tendon tissues, but there is no major difference between collagen and myofibrillar protein synthesis. Strenuous exercise provokes increased proteolysis and decreased protein synthesis, the opposite occurring during the recovery period. Individuals who exercise respond differently when resistance and endurance types of contractions are compared. Endurance exercise induces a greater oxidative capacity (enzymes) compared to resistance exercise, which induces fiber hypertrophy (myofibrils). Nitrogen balance (difference between protein intake and protein degradation) for athletes is usually balanced when the intake of protein reaches 1.2 g·kg-1·day-1 compared to 0.8 g·kg-1·day-1 in resting individuals. Muscular activities promote a cascade of signals leading to the stimulation of eukaryotic initiation of myofibrillar protein synthesis. As suggested in several publications, a bolus of 15-20 g protein (from skimmed milk or whey proteins) and carbohydrate (± 30 g maltodextrine) drinks is needed immediately after stopping exercise to stimulate muscle protein and tendon collagen turnover within 1 h.

The N-terminal 33 amino acid domain of Siva-1 is sufficient for nuclear localization

Siva-1 induces apoptosis in multiple pathological processes and plays an important role in the suppression of tumor metastasis, protein degradation, and other functions. Although many studies have demonstrated that Siva-1 functions in the cytoplasm, a few have found that Siva-1 can relocate to the nucleus. In this study, we found that the first 33 amino acid residues of Siva-1 are required for its nuclear localization. Further study demonstrated that the green fluorescent protein can be imported into the nucleus after fusion with these 33 amino acid residues. Other Siva-1 regions and domains showed less effect on Siva-1 nuclear localization. By site-mutagenesis of all of these 33 amino acid residues, we found that mutants of the first 1-18 amino acids affected Siva-1 nuclear compartmentalization but could not complete this localization independently. In summary, we demonstrated that the N-terminal 33 amino acid residues were sufficient for Siva-1 nuclear localization, but the mechanism of this translocation needs additional investigation.

Corn germ with pericarp in relation to whole corn: nutrient contents, food and protein efficiency, and protein digestibility-corrected amino acid score

The germ fraction with pericarp (bran) is generated in the industrial processing of corn kernel, and it is used for oil extraction and animal feed. This study evaluated the nutritional and protein quality of this fraction in relation to whole corn. The proximate composition, mineral contents, and amino acid profile of the germ fraction with pericarp and of whole corn were determined. A 4-week experiment was conducted using 36 weanling male Wistar rats, and three 10%-protein diets (reference, germ with 15% lipids and casein with 15% lipids), two 6%-protein diets (whole corn and casein), and a protein-free diet were prepared. The germ showed higher contents of proteins, lipids, dietary fiber (27.8 g.100 g-1), ash, minerals (Fe and Zn- approximately 5 mg.100 g-1), and lysine (57.2 mg.g-1 protein) than those of corn. The germ presented good quality protein (Relative Protein Efficiency Ratio-RPER = 80%; Protein Digestibility-Corrected Amino Acid Score-PDCAAS = 86%), higher than that of corn (RPER = 49%; PDCAAS = 60%). The corn germ fraction with pericarp is rich in dietary fiber, and it is a source of good quality protein as well as of iron and zinc, and its use as nutritive raw material is indicated in food products for human consumption.

Successful domino liver transplantation in maple syrup urine disease using a related living donor

Maple syrup urine disease (MSUD) is an autosomal recessive disease associated with high levels of branched-chain amino acids. Children with MSUD can present severe neurological damage, but liver transplantation (LT) allows the patient to resume a normal diet and avoid further neurological damage. The use of living related donors has been controversial because parents are obligatory heterozygotes. We report a case of a 2-year-old child with MSUD who underwent a living donor LT. The donor was the patient's mother, and his liver was then used as a domino graft. The postoperative course was uneventful in all three subjects. DNA analysis performed after the transplantation (sequencing of the coding regions of BCKDHA, BCKDHB, andDBT genes) showed that the MSUD patient was heterozygous for a pathogenic mutation in the BCKDHB gene. This mutation was not found in his mother, who is an obligatory carrier for MSUD according to the family history and, as expected, presented both normal clinical phenotype and levels of branched-chain amino acids. In conclusion, our data suggest that the use of a related donor in LT for MSUD was effective, and the liver of the MSUD patient was successfully used in domino transplantation. Routine donor genotyping may not be feasible, because the test is not widely available, and, most importantly, the disease is associated with both the presence of allelic and locus heterogeneity. Further studies with this population of patients are required to expand the use of related donors in MSUD.

α-N-acetylglucosamine-linked O-glycans of sialoglycoproteins (Tc-mucins) from Trypanosoma cruzi Colombiana strain

Trypanosoma cruzi sialoglycoproteins (Tc-mucins) are mucin-like molecules linked to a parasite membrane via a glycosylphosphatidylinositol anchor. We previously determined the structures of Tc-mucin O-glycan domains from several T. cruzi strains and observed significant differences among them. We now report the amino acid content and structure of Tc-mucin O-glycan chains from T. cruzi Colombiana, a strain resistant to common trypanocidal drugs. Amino acid analysis demonstrated the predominance of threonine residues (42%) and helped to identify the O-glycans as belonging to a Tc-mucin family that contain a ²-galactofuranose (²-Galf) residue attached to an α-N-acetylglucosamine (α-GlcNAc) O-4, with the most complex glycan, a pentasaccharide-GlcNAc-ol with a branched trigalactopyranose chain, on the GlcNAc O-6. The presence of ²-Galf on O-glycans from T. cruzi Colombiana mucins supports the use of glycosylation as a phylogenetic marker for the classification of Colombiana in the T. cruzi I group.

Modulating the electronic structure of amino acids: interaction of model lewis acids with anthranilic acid

On the basis of theoretical B3LYP calculations, Yáñez and co-workers (J. Chem. Theory Comput. 2012, 8, 2293) illustrated that beryllium ions are capable of significantly modulating (changing) the electronic structures of imidazole. In this computational organic chemistry study, the interaction of this β-amino acid and five model Lewis acids (BeF1+, Be2+, AlF2(1+), AlF2+, and Al3+) were investigated. Several aspects were addressed: natural bond orbitals, including second order perturbation analysis of intra-molecular charge delocalization and the natural population analysis atomic charges; molecular geometries; selected infrared stretching frequencies (C-N, C-O, and N-H), and selected ¹H-NMR chemical shifts. The data illustrate that this interaction can weaken the H-O bond and goes beyond strengthening the intra-molecular hydrogen bond (N...H-O) to cause a spontaneous transfer of the proton to the nitrogen atom in five cases generating zwitterion structures. Many new features are observed. Most importantly, the zwitterion structures include a stabilizing hydrogen bond (N-H...O) that varies in relative strength according to the Lewis acid. These findings explain the experimental observations of α-amino acids (for example: J. Am. Chem. Soc. 2001, 123, 3577) and are the first reported fundamental electronic structure characterization of β-amino acids in zwitterion form.

Characterization of ß-trypsin at acid pH by differential scanning calorimetry

Trypsin is a serino-protease with a polypeptide chain of 223 amino acid residues and contains six disulfide bridges. It is a globular protein with a predominance of antiparallel ß-sheet and helix in its secondary structure and has two domains with similar structures. We assessed the stability of ß-trypsin in the acid pH range using microcalorimetric (differential scanning calorimetry) techniques. Protein concentrations varied in the range of 0.05 to 2.30 mg/ml. Buffer solutions of 50.0 mM ß-alanine and 20.0 mM CaCl2 at different pH values (from 2.0 to 4.2) and concentrations of sorbitol (1.0 and 2.0 M), urea (0.5 M) or guanidinium hydrochloride (0.5 and 1.0 M) were used. The data suggest that we are studying the same conformational transition of the protein in all experimental situations using pH, sorbitol, urea and guanidinium hydrochloride as perturbing agents. The observed van't Hoff ratios (deltaHcal/deltaHvH) of 1.0 to 0.5 in the pH range of 3.2 to 4.2 suggest protein aggregation. In contrast, deltaHcal/deltaHvH ratios equal to one in the pH range of 2.0 to 3.2 suggest that the protein unfolds as a monomer. At pH 3.00, ß-trypsin unfolded with Tm = 54ºC and deltaH = 101.8 kcal/mol, and the change in heat capacity between the native and unfolded forms of the protein (deltaCp) was estimated to be 2.50 ± 0.07 kcal mol-1 K-1. The stability of ß-trypsin calculated at 298 K was deltaG D = 5.7 kcal/mol at pH 3.00 and deltaG D = 15.2 kcal/mol at pH 7.00, values in the range expected for a small globular protein.

Radiometric studies on the oxidation of (U-14C) L-amino acids by drug-susceptible and drug-resistant mycobacteria

A radiometric assay system has been used to study oxidation patterns of (U-14C) L-amino acids by drug-susceptible and drug-resistant mycobacteria. Drug-susceptible M. tuberculosis (H37Rv TMC 102 and Erdman) along with the drug-resistant organism M. tuberculosis (H37 Rv TMC 303), M. bovis, M. avium, M. intracellulare, M. kansasii and M. chelonei were used. The organisms were inoculated into a sterile reaction system with liquid 7H9 medium and one of the (U-14C) L-amino acids. Each organism displayed a different pattern of amino acid oxidation, but these patterns were not distinctive enough for identification of the organism. Complex amino acids such as proline, phenylalanine and tyrosine were of no use in identification of mycobacteria, since virtually all organisms failed to oxidize them. There was no combination of substrates able to separate susceptible from resistant organisms.

A recombinant hybrid protein as antigen for an anti-blood stage malaria vaccine: a study on the conservation of a protective component

Recently we have shown that two hybrid proteins expressed in Escherichia coli confer protective immunity to Aotus monkeys against an experimental Plasmodium falciparum infection (Knapp et al., 1992). Both hybrid proteins carry a sequence containing amino acids 631 to 764 of the serine stretch protein SERP (Knapp et al., 1989b). We have studied the diversity of this SERP region in field isolates of P. falciparum. Genomic DNA was extracted from the blood of six donors from different endemic areas of Brazil and West Africa. The SERP region encoding amino acids 630 to 781 was amplified by polymerase chain reaction (PCR) and sequenced. Only conserved amino acid substitutions in maximally two positions of the analyzed SERP fragment could be detected which supports the suitability of this SERP region as a component of anti-blood stage malaria vaccine.