Purification and binding analysis of the nitrogen fixation regulatory NifA protein from Azospirillum brasilense

NifA protein activates transcription of nitrogen fixation operons by the alternative sigma54 holoenzyme form of RNA polymerase. This protein binds to a well-defined upstream activator sequence (UAS) located at the -200/-100 position of nif promoters with the consensus motif TGT-N10-ACA. NifA of Azospirillum brasilense was purified in the form of a glutathione-S-transferase (GST)-NifA fusion protein and proteolytic release of GST yielded inactive and partially soluble NifA. However, the purified NifA was able to induce the production of specific anti-A. brasilense NifA-antiserum that recognized NifA from A. brasilense but not from K. pneumoniae. Both GST-NifA and NifA expressed from the E. coli tac promoter are able to activate transcription from the nifHDK promoter but only in an A. brasilense background. In order to investigate the mechanism that regulates NifA binding capacity we have used E. coli total protein extracts expressing A. brasilense nifA in mobility shift assays. DNA fragments carrying the two overlapping, wild-type or mutated UAS motifs present in the nifH promoter region revealed a retarded band of related size. These data show that the binding activity present in the C-terminal domain of A. brasilense NifA protein is still functional even in the presence of oxygen.

Structural, functional and immunological studies on a polymeric bacterial protein

The characterization of proteins from Brucella spp, the causative agent of brucellosis, has been the subject of intensive research. We have described an 18-kDa cytoplasmic protein of Brucella abortus and shown the potential usefulness of this protein as an antigen for the serologic diagnosis of brucellosis. The amino acid sequence of the protein showed a low but significant homology with that of lumazine synthases. Lumazine is an intermediate product in bacterial riboflavin biosynthesis. The recombinant form of the 18-kDa protein (expressed in E. coli) folds like the native Brucella protein and has lumazine-synthase enzymatic activity. Three-dimensional analysis by X-ray crystallography of the homolog Bacillus subtilis lumazine synthase has revealed that the enzyme forms an icosahedral capsid. Recombinant lumazine synthase from B. abortus was crystallized, diffracted X rays to 2.7-Å resolution at room temperature, and the structure successfully solved by molecular replacement procedures. The macromolecular assembly of the enzyme differs from that of the enzyme from B. subtilis. The Brucella enzyme remains pentameric (90 kDa) in its crystallographic form. Nonetheless, the active sites of the two enzymes are virtually identical at the structural level, indicating that inhibitors of these enzymes could be viable pharmaceuticals across a broad species range. We describe the structural reasons for the differences in their quaternary arrangement and also discuss the potential use of this protein as a target for the development of acellular vaccines.

Extracellular calcium-sensing receptor: structural and functional features and association with diseases

The recently cloned extracellular calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays an essential role in the regulation of extracellular calcium homeostasis. This receptor is expressed in all tissues related to this control (parathyroid glands, thyroid C-cells, kidneys, intestine and bones) and also in tissues with apparently no role in the maintenance of extracellular calcium levels, such as brain, skin and pancreas. The CaR amino acid sequence is compatible with three major domains: a long and hydrophilic aminoterminal extracellular domain, where most of the activating and inactivating mutations described to date are located and where the dimerization process occurs, and the agonist-binding site is located, a hydrophobic transmembrane domain involved in the signal transduction mechanism from the extracellular domain to its respective G protein, and a carboxyterminal intracellular tail, with a well-established role for cell surface CaR expression and for signal transduction. CaR cloning was immediately followed by the association of genetic human diseases with inactivating and activating CaR mutations: familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism are caused by CaR-inactivating mutations, whereas autosomal dominant hypoparathyroidism is secondary to CaR-activating mutations. Finally, we will comment on the development of drugs that modulate CaR function by either activating (calcimimetic drugs) or antagonizing it (calcilytic drugs), and on their potential therapeutic implications, such as medical control of specific cases of primary and uremic hyperparathyroidism with calcimimetic drugs and a potential treatment for osteoporosis with a calcilytic drug.

Obese women on a low energy rice and bean diet: effects of leucine, arginine or glycine supplementation on protein turnover

This study examined if leucine, arginine or glycine supplementation in adult obese patients (body mass index of 33 ± 4 kg/m²) consuming a Brazilian low energy and protein diet (4.2 MJ/day and 0.6 g protein/kg) affects protein and amino acid metabolism. After four weeks adaptation to this diet, each subject received supplements of these amino acids (equivalent to 0.2 g protein kg-1 day-1) in random order. On the seventh day of each amino acid supplementation, a single-dose 15N-glycine study was carried out. There were no significant differences in protein flux, synthesis or breakdown. The protein flux (grams of nitrogen, gN/9 h) was 55 ± 24 during the nonsupplemented diet intake and 39 ± 10, 44 ± 22 and 58 ± 35 during the leucine-, glycine- and arginine-supplemented diet intake, respectively; protein synthesis (gN/9 h) was 57 ± 24, 36 ± 10, 41 ± 22 and 56 ± 36, respectively; protein breakdown (gN/9 h) was 51 ± 24, 34 ± 10, 32 ± 28 and 53 ± 35, respectively; kinetic balance (gN/9 h) was 3.2 ± 1.8, 4.1 ± 1.7, 3.4 ± 2.9 and 3.9 ± 1.6. There was no difference in amino acid profiles due to leucine, arginine or glycine supplementation. The present results suggest that 0.6 g/kg of dietary protein is enough to maintain protein turnover in obese women consuming a reduced energy diet and that leucine, arginine or glycine supplementation does not change kinetic balance or protein synthesis.

Can organic and transgenic soy be used as a substitute for animal protein by rats?

We evaluated the protein quality of organic and transgenic soy fed to rats throughout life. Thirty female Wistar rats were divided into three groups (N = 10): organic soy group (OSG) receiving organic soy-based diet, genetically modified soy group (GMSG) receiving transgenic soy-based diet, and a control group (CG) receiving casein-based diet. All animals received water and isocaloric diet (10% protein), ad libitum for 291 days. After this, the weight of GMSG animals (290.9 ± 9.1 g) was significantly lower (P <= 0.04) than CG (323.2 ± 7.9 g). The weight of OSG (302.2 ± 8.7 g) was between that of the GMSG and the CG. Protein intake was similar for OSG (308.4 ± 6.8 g) and GMSG (301.5 ± 2.5 g), and significantly lower (P <= 0.0005) than the CG (358.4 ± 8.1 g). Growth rate was similar for all groups: OSG (0.80 ± 0.02 g), GMSG (0.81 ± 0.03 g) and CG (0.75 ± 0.02 g). In addition to providing a good protein intake and inducing less weight gain, both types of soy were utilized in a manner similar to that of casein, suggesting that the protein quality of soy is similar to that of the standard protein casein. The groups fed soy-based diet gained less weight, which may be considered to be beneficial for health. We conclude that organic and transgenic soy can be fed throughout life to rats in place of animal protein, because contain high quality protein and do not cause a marked increase in body weight.

Volume and energy folding landscape of prion protein revealed by pressure

The main hypothesis for prion diseases proposes that the cellular protein (PrP C) can be altered into a misfolded, ß-sheet-rich isoform, the PrP Sc (from scrapie). The formation of this abnormal isoform then triggers the transmissible spongiform encephalopathies. Here, we discuss the use of high pressure as a tool to investigate this structural transition and to populate possible intermediates in the folding/unfolding pathway of the prion protein. The latest findings on the application of high pressure to the cellular prion protein and to the scrapie PrP forms will be summarized in this review, which focuses on the energetic and volumetric properties of prion folding and conversion.

Immunoglobulin production is impaired in protein-deprived mice and can be restored by dietary protein supplementation

Most contacts with food protein and microbiota antigens occur at the level of the gut mucosa. In animal models where this natural stimulation is absent, such as germ-free and antigen-free mice, the gut-associated lymphoid tissue (GALT) and systemic immunological activities are underdeveloped. We have shown that food proteins play a critical role in the full development of the immune system. C57BL/6 mice weaned to a diet in which intact proteins are replaced by equivalent amounts of amino acids (Aa diet) have a poorly developed GALT as well as low levels of serum immunoglobulins (total Ig, IgG, and IgA, but not IgM). In the present study, we evaluated whether the introduction of a protein-containing diet in 10 adult Aa-fed C57BL/6 mice could restore their immunoglobulin levels and whether this recovery was dependent on the amount of dietary protein. After the introduction of a casein-containing diet, Aa-fed mice presented a fast recovery (after 7 days) of secretory IgA (from 0.33 to 0.75 mg/mL, while in casein-fed mice this value was 0.81 mg/mL) and serum immunoglobulin levels (from 5.39 to 10.25 mg/mL of total Ig). Five percent dietary casein was enough to promote the restoration of secretory IgA and serum immunoglobulin levels to a normal range after 30 days feeding casein diet (as in casein-fed mice - 15% by weight of diet). These data suggest that the defect detected in the immunoglobulin levels was a reversible result of the absence of food proteins as an antigenic stimulus. They also indicate that the deleterious consequences of malnutrition at an early age for some immune functions may be restored by therapeutic intervention later in life.

Correlation of serum leptin and insulin levels of pregnant protein-restricted rats with predictive obesity variables

During pregnancy and protein restriction, changes in serum insulin and leptin levels, food intake and several metabolic parameters normally result in enhanced adiposity. We evaluated serum leptin and insulin levels and their correlations with some predictive obesity variables in Wistar rats (90 days), up to the 14th day of pregnancy: control non-pregnant (N = 5) and pregnant (N = 7) groups (control diet: 17% protein), and low-protein non-pregnant (N = 5) and pregnant (N = 6) groups (low-protein diet: 6%). Independent of the protein content of the diet, pregnancy increased total (F1,19 = 22.28, P < 0.001) and relative (F1,19 = 5.57, P < 0.03) food intake, the variation of weight (F1,19 = 49.79, P < 0.000) and final body weight (F1,19 = 19.52, P < 0.001), but glycemia (F1,19 = 9.02, P = 0.01) and the relative weight of gonadal adipose tissue (F1,19 = 17.11, P < 0.001) were decreased. Pregnancy (F1,19 = 18.13, P < 0.001) and low-protein diet (F1,19 = 20.35, P < 0.001) increased the absolute weight of brown adipose tissue. However, the relative weight of this tissue was increased only by protein restriction (F1,19 = 15.20, P < 0.001) and the relative lipid in carcass was decreased in low-protein groups (F1,19 = 4.34, P = 0.05). Serum insulin and leptin levels were similar among groups and did not correlate with food intake. However, there was a positive relationship between serum insulin levels and carcass fat depots in low-protein groups (r = 0.37, P < 0.05), while in pregnancy serum leptin correlated with weight of gonadal (r = 0.39, P < 0.02) and retroperitoneal (r = 0.41, P < 0.01) adipose tissues. Unexpectedly, protein restriction during 14 days of pregnancy did not alter the serum profile of adiposity signals and their effects on food intake and adiposity, probably due to the short term of exposure to low-protein diet.

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.

Involvement of Src tyrosine kinase and protein kinase C in the expression of macrophage migration inhibitory factor induced by H2O2 in HL-1 mouse cardiac muscle cells

Macrophage migration inhibitory factor (MIF), a pleiotropic cytokine, plays an important role in the pathogenesis of atrial fibrillation; however, the upstream regulation of MIF in atrial myocytes remains unclear. In the present study, we investigated whether and how MIF is regulated in response to the renin-angiotensin system and oxidative stress in atrium myocytes (HL-1 cells). MIF protein and mRNA levels in HL-1 cells were assayed using immunofluorescence, real-time PCR, and Western blot. The result indicated that MIF was expressed in the cytoplasm of HL-1 cells. Hydrogen peroxide (H2O2), but not angiotensin II, stimulated MIF expression in HL-1 cells. H2O2-induced MIF protein and gene levels increased in a dose-dependent manner and were completely abolished in the presence of catalase. H2O2-induced MIF production was completely inhibited by tyrosine kinase inhibitors genistein and PP1, as well as by protein kinase C (PKC) inhibitor GF109203X, suggesting that redox-sensitive MIF production is mediated through tyrosine kinase and PKC-dependent mechanisms in HL-1 cells. These results suggest that MIF is upregulated by HL-1 cells in response to redox stress, probably by the activation of Src and PKC.

Abundant immunohistochemical expression of dopamine D2 receptor and p53 protein in meningiomas: follow-up, relation to gender, age, tumor grade, and recurrence

Meningiomas are common, usually benign tumors, with a high postoperative recurrence rate. However, the genesis and development of these tumors remain controversial. We aimed to investigate the presence and implications of a mutated p53 protein and dopamine D2 receptor in a representative series of meningiomas and to correlate these findings with age, gender, tumor grade, and recurrence. Tumor tissue samples of 157 patients diagnosed with meningioma (37 males and 120 females, mean age 53.6±14.3 years) who underwent surgical resection between 2003 and 2012 at our institution were immunohistochemically evaluated for the presence of p53 protein and dopamine D2 receptor and were followed-up to analyze tumor recurrence or regrowth. Tumors were classified as grades I (n=141, 89.8%), II (n=13, 8.3%), or grade III (n=3, 1.9%). Dopamine D2 receptor and p53 protein expression were positive in 93.6% and 49.7% of the cases, respectively. Neither of the markers showed significant expression differences among different tumor grades or recurrence or regrowth statuses. Our findings highlight the potential role of p53 protein in meningioma development and/or progression. The high positivity of dopamine D2 receptor observed in this study warrants further investigation of the therapeutic potential of dopamine agonists in the evolution of meningiomas.

In vitro protein digestibility of enzymatically pre-treated bean (Phaseolus vulgaris L.) flour using commercial protease and Bacillus sp. protease

The common bean (Phaseolus vulgaris L.) is a staple food in the Brazilian diet and represents the major source of dietary protein and other micronutrients and minerals. Despite the considerable protein concentration in beans, the food is considered of low biological value when compared to animal proteins and other plant protein sources. To improve the availability of protein in beans, enzymatic treatments were performed in four cultivars (ON, OPNS, TAL and VC3). The approach was a completely randomized design with four replicates. We used a 4 × 3 factorial arrangement (four cultivars and three treatments: treatment 1-addition of commercial protease (Trypsin 250, Difco), treatment 2-addition of protease from Bacillus sp., and treatment 3:-control without enzyme addition). The enzyme: substrate ratio was 5% w/w (amount of enzyme per total protein in bean flour). The approach was a completely randomized design with four replicates. A 4 × 3 factorial arrangement (four cultivars and three treatments, the same as those mentioned above) was used. The concentration of total protein (g.100 g-1 of dry matter) in the samples ranged from 16.94 to 18.06%, while the concentration of total phenolics was between 0.78 and 1.12% (g Eq. tannic acid.100 g-1 dry matter). The in vitro protein digestibility of enzymatically untreated bean flour (control) ranged from 47.30 to 56.17% based on the digestibility of casein. Concentrations of P, K, Ca, Mg, and Zn observed in the four cultivars tested were within the average values available in the literature. Treatment 2 with protease from Bacillus sp. induced decreases in the levels of Cu and Mn. The average Fe content increased in all bean flour samples when treated with proteases, reaching a maximum increase of 102% in the TAL flour treated with protease from Bacillus sp. The digestibility of all beans tested was significantly increased (p < 0.05) after the enzyme treatment. The greatest change was observed in the OPNS cultivar treated with protease from Bacillus sp., which increased its digestibility from 54.4% (control treatment) to 81.6%.

Simultaneous α-amylase and protease production by the soil bacterium Bacillus sp. SMIA-2 under submerged culture using whey protein concentrate and corn steep liquor: compatibility of enzymes with commercial detergents

Protease and α-amylase production by a thermophilic Bacillus sp. SMIA-2 cultivated in liquid cultures containing 0.25% (w/v) starch as a carbon source reached a maximum at 18 hours (47 U.mg-1 Protein) and 36 hours (325 U.mg-1 Protein), respectively. Culture medium supplementation with whey protein concentrate (0.1%, w/v) and corn steep liquor (0.3%, w/v) not only improved the production of both enzymes but also enabled them to be produced simultaneously. Under these conditions, α-amylase and protease production reached a maximum in 18 hours with levels of 401 U.mg-1 protein and 78 U.mg-1 protein, respectively. The compatibility of the enzymes produced with commercial laundry detergent was investigated. In the presence of Campeiro® detergent, α-amylase activity increased while protease activity decreased by about 27%. These enzymes improved the cleaning power of Campeiro® detergent since they were able to remove egg yolk and tomato sauce stains when used in this detergent.

Study of the proteins in the defatted flour and protein concentrate of baru nuts (Dipteryx alata Vog)

Baru (Dipteryx alata Vog.) is an abundant legume in the Brazilian Savanna. Its nuts can be exploited sustainably using its protein and lipid fractions. This study aimed to analyze the proteins of the nuts present in the defatted flour and protein concentrate in terms of their functional properties, the profile of their fractions, and the in vitro digestibility. The flour was defatted with hexane and extracted at the pH of higher protein solubility to obtain the protein concentrate. The electrophoretic profile of the protein fractions was evaluated in SDS-PAGE gel. The functional properties of the proteins indicate the possibility of their use in various foods, like soybeans providing water absorption capacity, oil absorption capacity, emulsifying properties, and foamability. Globulins, followed by the albumins, are the major fractions of the flour and protein concentrate, respectively. Digestibility was greater for the concentrate than for the defatted flour.

Physicochemical properties and mineral and protein content of honey samples from Ceará state, Northeastern Brazil

This study evaluated the physicochemical properties and protein and mineral content of honey samples from Ceará State, Northeastern Brazil, one of the major honey exporters in the country. Nutritional importance of the minerals detected was also analyzed. Physicochemical properties were examined according to the AOAC and CAC official methods; the protein content was determined using the Bradford method, and the minerals were analyzed by atomic absorption spectrometry. All analyses were performed in triplicate. The levels of macrominerals sodium (Na), potassium (K), calcium (Ca), and magnesium (Mg) varied from 1.80-47.20, 21.30-1513.30, 14.58-304.82, and 2.48-28.33 mg/kg, respectively, and the trace elements iron (Fe), copper (Cu), manganese (Mn), zinc (Zn), selenium (Se), and chromium (Cr) varied from 0.12-8.76, 0.07-1.29, 0.06-1.96, 0.07-1.85 mg/kg, 0.36 × 10-3-62.00 × 10-3 and 22.50 × 10-3-170.33 × 10-3 µg/kg, respectively. Myracrodruon urundeuva honey sample had high contents of macrominerals (Na, K, Ca, and Mg). Protein content of the Anacardium occidentale honey sample was the highest (1121.00 µg/g) among the samples analyzed. Among the minerals detected in the honey samples, K showed the highest concentration, followed by Ca, Na, and Mg. The presence of trace elements can show environmental contamination. The honey samples studied were free of trace elements contamination, except for Mn; the Piptadenia moniliformis was the only honey sample that was in compliance with the law requirements. The variations of the chemical constituents in the honey samples are probably related to differences in the floral origin and mineral and protein contents and confirm the nutritional importance of Ceará State honey.