Modulation of the pharmacological effects of enzymatically-active PLA 2 by BTL-2, an isolectin isolated from the Bryothamnion triquetrum red alga

Background. An interaction between lectins from marine algae and PLA 2 from rattlesnake was suggested some years ago. We, herein, studied the effects elicited by a small isolectin (BTL-2), isolated from Bryothamnion triquetrum, on the pharmacological and biological activities of a PLA 2 isolated from rattlesnake venom (Crotalus durissus cascavella), to better understand the enzymatic and pharmacological mechanisms of the PLA 2 and its complex. Results. This PLA2 consisted of 122 amino acids (approximate molecular mass of 14 kDa), its pI was estimated to be 8.3, and its amino acid sequence shared a high degree of similarity with that of other neurotoxic and enzymatically-active PLA2s. BTL-2 had a molecular mass estimated in approximately 9 kDa and was characterized as a basic protein. In addition, BTL-2 did not exhibit any enzymatic activity. The PLA2 and BTL-2 formed a stable heterodimer with a molecular mass of approximately 24-26 kDa, estimated by molecular exclusion HPLC. In the presence of BTL-2, we observed a significant increase in PLA2 activity, 23% higher than that of PLA2 alone. BTL-2 demonstrated an inhibition of 98% in the growth of the Gram-positive bacterial strain, Clavibacter michiganensis michiganensis (Cmm), but only 9.8% inhibition of the Gram-negative bacterial strain, Xanthomonas axonopodis pv passiflorae (Xap). PLA2 decreased bacterial growth by 27.3% and 98.5% for Xap and Cmm, respectively, while incubating these two proteins with PLA2-BTL-2 inhibited their growths by 36.2% for Xap and 98.5% for Cmm. PLA2 significantly induced platelet aggregation in washed platelets, whereas BTL-2 did not induce significant platelet aggregation in any assay. However, BTL-2 significantly inhibited platelet aggregation induced by PLA2. In addition, PLA 2 exhibited strong oedematogenic activity, which was decreased in the presence of BTL-2. BTL-2 alone did not induce oedema and did not decrease or abolish the oedema induced by the 48/80 compound. Conclusion. The unexpected results observed for the PLA2-BTL-2 complex strongly suggest that the pharmacological activity of this PLA2 is not solely dependent on the presence of enzymatic activity, and that other pharmacological regions may also be involved. In addition, we describe for the first time an interaction between two different molecules, which form a stable complex with significant changes in their original biological action. This opens new possibilities for understanding the function and action of crude venom, an extremely complex mixture of different molecules. © 2008 Oliveira et al; licensee BioMed Central Ltd.

Positive selection results in frequent reversible amino acid replacements in the G protein gene of human respiratory syncytial virus

Human respiratory syncytial virus (HRSV) is the major cause of lower respiratory tract infections in children under 5 years of age and the elderly, causing annual disease outbreaks during the fall and winter. Multiple lineages of the HRSVA and HRSVB serotypes co-circulate within a single outbreak and display a strongly temporal pattern of genetic variation, with a replacement of dominant genotypes occurring during consecutive years. In the present study we utilized phylogenetic methods to detect and map sites subject to adaptive evolution in the G protein of HRSVA and HRSVB. A total of 29 and 23 amino acid sites were found to be putatively positively selected in HRSVA and HRSVB, respectively. Several of these sites defined genotypes and lineages within genotypes in both groups, and correlated well with epitopes previously described in group A. Remarkably, 18 of these positively selected tended to revert in time to a previous codon state, producing a flipflop phylogenetic pattern. Such frequent evolutionary reversals in HRSV are indicative of a combination of frequent positive selection, reflecting the changing immune status of the human population, and a limited repertoire of functionally viable amino acids at specific amino acid sites.

UMP kinase from Mycobacterium tuberculosis: Mode of action and allosteric interactions, and their likely role in pyrimidine metabolism regulation

The pyrH-encoded uridine 5′-monophosphate kinase (UMPK) is involved in both de novo and salvage synthesis of DNA and RNA precursors. Here we describe Mycobacterium tuberculosis UMPK (MtUMPK) cloning and expression in Escherichia coli. N-terminal amino acid sequencing and electrospray ionization mass spectrometry analyses confirmed the identity of homogeneous MtUMPK. MtUMPK catalyzed the phosphorylation of UMP to UDP, using ATP-Mg 2+ as phosphate donor. Size exclusion chromatography showed that the protein is a homotetramer. Kinetic studies revealed that MtUMPK exhibits cooperative kinetics towards ATP and undergoes allosteric regulation. GTP and UTP are, respectively, positive and negative effectors, maintaining the balance of purine versus pyrimidine synthesis. Initial velocity studies and substrate(s) binding measured by isothermal titration calorimetry suggested that catalysis proceeds by a sequential ordered mechanism, in which ATP binds first followed by UMP binding, and release of products is random. As MtUMPK does not resemble its eukaryotic counterparts, specific inhibitors could be designed to be tested as antitubercular agents. © 2010 Elsevier Inc. All rights reserved.

Salmonella enterica serovar typhimurium colonizing the lumen of the chicken intestine grows slowly and upregulates a unique set of virulence and metabolism genes

The pattern of global gene expression in Salmonella enterica serovar Typhimurium bacteria harvested from the chicken intestinal lumen (cecum) was compared with that of a late-log-phase LB broth culture using a whole-genome microarray. Levels of transcription, translation, and cell division in vivo were lower than those in vitro. S. Typhimurium appeared to be using carbon sources, such as propionate, 1,2-propanediol, and ethanolamine, in addition to melibiose and ascorbate, the latter possibly transformed to D-xylulose. Amino acid starvation appeared to be a factor during colonization. Bacteria in the lumen were non- or weakly motile and nonchemotactic but showed upregulation of a number of fimbrial and Salmonella pathogenicity island 3 (SPI-3) and 5 genes, suggesting a close physical association with the host during colonization. S. Typhimurium bacteria harvested from the cecal mucosa showed an expression profile similar to that of bacteria from the intestinal lumen, except that levels of transcription, translation, and cell division were higher and glucose may also have been used as a carbon source. © 2011, American Society for Microbiology.

Structural insights into selectivity and cofactor binding in snake venom l-amino acid oxidases

l-Amino acid oxidases (LAAOs) are flavoenzymes that catalytically deaminate l-amino acids to corresponding α-keto acids with the concomitant production of ammonia (NH 3) and hydrogen peroxide (H 2O 2). Particularly, snake venom LAAOs have been attracted much attention due to their diverse clinical and biological effects, interfering on human coagulation factors and being cytotoxic against some pathogenic bacteria and Leishmania ssp. In this work, a new LAAO from Bothrops jararacussu venom (BjsuLAAO) was purified, functionally characterized and its structure determined by X-ray crystallography at 3.1å resolution. BjsuLAAO showed high catalytic specificity for aromatic and aliphatic large side-chain amino acids. Comparative structural analysis with prokaryotic LAAOs, which exhibit low specificity, indicates the importance of the active-site volume in modulating enzyme selectivity. Surprisingly, the flavin adenine dinucleotide (FAD) cofactor was found in a different orientation canonically described for both prokaryotic and eukaryotic LAAOs. In this new conformational state, the adenosyl group is flipped towards the 62-71 loop, being stabilized by several hydrogen-bond interactions, which is equally stable to the classical binding mode. © 2012 Elsevier Inc.

Development of sustainable precision farming systems for swine: Estimating realtime individual amino acid requirements in growing-finishing pigs

The objective of this study was to develop and evaluate a mathematical model used to estimate the daily amino acid requirements of individual growing-finishing pigs. The model includes empirical and mechanistic model components. The empirical component estimates daily feed intake (DFI), BW, and daily gain (DG) based on individual pig information collected in real time. Based on DFI, BW, and DG estimates, the mechanistic component uses classic factorial equations to estimate the optimal concentration of amino acids that must be offered to each pig to meet its requirements. The model was evaluated with data from a study that investigated the effect of feeding pigs with a 3-phase or daily multiphase system. The DFI and BW values measured in this study were compared with those estimated by the empirical component of the model. The coherence of the values estimated by the mechanistic component was evaluated by analyzing if it followed a normal pattern of requirements. Lastly, the proposed model was evaluated by comparing its estimates with those generated by the existing growth model (InraPorc). The precision of the proposed model and InraPorc in estimating DFI and BW was evaluated through the mean absolute error. The empirical component results indicated that the DFI and BW trajectories of individual pigs fed ad libitum could be predicted 1 d (DFI) or 7 d (BW) ahead with the average mean absolute error of 12.45 and 1.85%, respectively. The average mean absolute error obtained with the InraPorc for the average individual of the population was 14.72% for DFI and 5.38% for BW. Major differences were observed when estimates from InraPorc were compared with individual observations. The proposed model, however, was effective in tracking the change in DFI and BW for each individual pig. The mechanistic model component estimated the optimal standardized ileal digestible Lys to NE ratio with reasonable between animal (average CV = 7%) and overtime (average CV = 14%) variation. Thus, the amino acid requirements estimated by model are animal- and time-dependent and follow, in real time, the individual DFI and BW growth patterns. The proposed model can follow the average feed intake and feed weight trajectory of each individual pig in real time with good accuracy. Based on these trajectories and using classical factorial equations, the model makes it possible to estimate dynamically the AA requirements of each animal, taking into account the intake and growth changes of the animal. © 2012 American Society of Animal Science. All rights reserved.

Advances in drug design based on the amino acid approach: Taurine analogues for the treatment of CNS diseases

Amino acids are well known to be an important class of compounds for the maintenance of body homeostasis and their deficit, even for the polar neuroactive aminoacids, can be controlled by supplementation. However, for the amino acid taurine (2-aminoethanesulfonic acid) this is not true. Due its special physicochemical properties, taurine is unable to cross the blood-brain barrier. In addition of injured taurine transport systems under pathological conditions, CNS supplementation of taurine is almost null. Taurine is a potent antioxidant and anti-inflammatory semi-essential amino acid extensively involved in neurological activities, acting as neurotrophic factor, binding to GABA A/glycine receptors and blocking the excitotoxicity glutamate-induced pathway leading to be a neuroprotective effect and neuromodulation. Taurine deficits have been implicated in several CNS diseases, such as Alzheimer's, Parkinson's, epilepsy and in the damage of retinal neurons. This review describes the CNS physiological functions of taurine and the development of new derivatives based on its structure useful in CNS disease treatment.&; 2012 by the authors; licensee MDPI, Basel, Switzerland.

The role of fibres and the hypodermis in Compositae melanin secretion

Melanins are dark, insoluble pigments that are resistant to concentrated acids and bleaching by oxidising agents. Phytomelanin (or phytomelan) is present in the seed coat of some Asparagales and in the fruits of some Compositae. In Compositae fruits, melanin is deposited in the schizogenous spaces between the hypodermis and underlying fibrous layer. Phytomelanin in Compositae is poorly understood, and there are only speculations regarding the cells that produce the pigment and the cellular processes involved in the secretion and polymerisation of phytomelanin. This report describes the cellular processes involved in the secretion of phytomelanin in the pericarp of Praxelis diffusa, a species with a structure typical of the family. The ovaries and fruits at different stages were fixed and processed according to the standard methods of studies of light microscopy and transmission electron microscopy. Hypodermal cells have abundant rough endoplasmic reticulum and mitochondria, and the nuclei have chromatin that is less dense than other cells. These characteristics are typical of cells that synthesise protein/amino acids and suggest no carbohydrate secretion. The fibres, however, have a dense cytoplasm rich in the Golgi bodies that are associated with vesicles and smooth endoplasmic reticulum, common characteristics of carbohydrate secretory cells. Our results indicate that the hypodermal cells are not responsible for the secretion of phytomelanin, as previously described in the literature; in contrast, this function is assigned to the adjacent fibres, which have an organisation typical of cells that secrete carbohydrates. © 2012 Elsevier Ltd.

Clinical and molecular study of a new form of hereditary myotonia in Murrah water buffalo

Hereditary myotonia caused by mutations in CLCN1 has been previously described in humans, goats, dogs, mice and horses. The goal of this study was to characterize the clinical, morphological and genetic features of hereditary myotonia in Murrah buffalo. Clinical and laboratory evaluations were performed on affected and normal animals. CLCN1 cDNA and the relevant genomic region from normal and affected animals were sequenced. The affected animals exhibited muscle hypertrophy and stiffness. Myotonic discharges were observed during EMG, and dystrophic changes were not present in skeletal muscle biopsies; the last 43 nucleotides of exon-3 of the CLCN1 mRNA were deleted. Cloning of the genomic fragment revealed that the exclusion of this exonic sequence was caused by aberrant splicing, which was associated with the presence of a synonymous SNP in exon-3 (c.396C>T). The mutant allele triggered the efficient use of an ectopic 5' splice donor site located at nucleotides 90-91 of exon-3. The predicted impact of this aberrant splicing event is the alteration of the CLCN1 translational reading frame, which results in the incorporation of 24 unrelated amino acids followed by a premature stop codon. Copyright © 2012 Elsevier B.V. All rights reserved.

Predictive formulas for food base excess and urine pH estimations of cats

Food base excess (BE, mEq/kg) can be calculated from the diet macroelements, together with either the sulfur amino acids methionine and cysteine (BEaa) or total sulfur (BEs) concentrations. The present study compared the use of sulfur or methionine and cysteine for calculating the food BE (experiment 1) and investigated the influence of food BE on blood gas analysis and the urine pH of cats, and proposes a prediction equation to estimate the urine pH of cats fed kibble diets based on the calculated food BE (experiments 2 and 3). In experiment 1, nine healthy, adult cats were used in a change-over design and fed with nine commercial dry cat foods. The cats were housed in metabolism cages over seven days for adaptation and three days for total urine collection. All of the urine produced over 24h was pooled by cat and diet. The cats' acid-base status was assessed through blood gas analysis after 10 days of diet consumption. A mean difference of -115mEq/kg between BEs and BEaa was observed, which could be explained by a greater concentration of sulfur in the whole diet than in methionine and cysteine. Urine pH presented a stronger correlation with food BEs (R2=0.95; P<0.001) than with food BEaa (R2=0.86; P<0.001). Experiment 2 included 30 kibble diets, and each diet was tested in six cats. The food BEs varied between -180 and +307mEq/kg, and the urine pH of the cats varied between 5.60 and 7.74. A significant correlation was found between the measured urine pH and the food BEs (urinary pH=6.269+[0.0036×BEs]+[0.000003×BEs2]; R2=0.91; P<0.001). In experiment 3, eight kibble diets were tested (food BEs between -187mEq/kg and +381mEq/kg) to validate the equation proposed in experiment 2 and to compare the obtained results with previously published formulae. The results of the proposed formula presented a high concordance correlation coefficient (0.942) and high accuracy (0.979) with the measured values, and the estimates of urine pH did not differ from the values obtained in cats (P>0.05). The cats' venous blood pH, bicarbonate, and blood BE were correlated with food BEs (P<0.001); the consumption of diets with low food BEs induced a reduction in these parameters. In conclusion, food macroelement composition has a strong influence on cats' acid-base equilibrium and food BEs calculation is a useful tool to formulate and balance kibble diets for felines. © 2013 Elsevier B.V.

Estado nutricional e níveis plasmáticos de trauma e seus precursores em pacientes portadores de neoplasias malignas de esôfago

Pós-graduação em Bases Gerais da Cirurgia - FMB

Metabolismo energético do camarão da amazonia, Macrobrachium amazonicum

Pós-graduação em Biologia Geral e Aplicada - IBB

Efeito da lisina na produção de compostos beta-lactâmicos produzidos por Sterptomuces clavuligerus

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Sequenciamento do RNAm codificante da hepcidina e análise de sua expressão gênica em diferentes tecidos de ovinos saudáveis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Influência da deficiência de taurina no coração

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)