A SequenceSpace analysis of Lys49 phospholipases A2: Clues towards identification of residues involved in a novel mechanism of membrane damage and in myotoxicity

'SequenceSpace' analysis is a novel approach which has been used to identify unique amino acids within a subfamily of phospholipases A2 (PLA2) in which the highly conserved active site residue Asp49 is substituted by Lys (Lys49-PLA2s). Although Lys49-PLA2s do not bind the catalytic co-factor Ca2+ and possess extremely low catalytic activity, they demonstrate a Ca2+-independent membrane damaging activity through a poorly understood mechanism, which does not involve lipid hydrolysis. Additionally, Lys49-PLA2s possess combined myotoxic, oedema forming and cardiotoxic pharmacological activities, however the structural basis of these varied functions is largely unknown. Using the 'SequenceSpace' analysis we have identified nine residues highly unique to the Lys49-PLA2 sub-family, which are grouped in three amino acid clusters in the active site, hydrophobic substrate binding channel and homodimer interface regions. These three highly specific residue clusters may have relevance for the Ca2+-independent membrane damaging activity. Of a further 15 less stringently conserved residues, nine are located in two additional clusters which are well isolated from the active site region. The less strictly conserved clusters have been used in predictive sequence searches to correlate amino acid patterns in other venom PLA2s with their pharmacological activities, and motifs for presynaptic and combined toxicities are proposed.

Effects of glucose infusion on the endocrine, metabolic and cardiorespiratory responses to halothane anaesthesia of ponies

Glucose was infused intravenously into six ponies during halothane anaesthesia, to evaluate its effect on their endocrine response to anaesthesia. The ponies were premedicated with acepromazine, and anaesthesia was induced with thiopentone and maintained with halothane in oxygen for two hours. Glucose was infused to maintain the plasma glucose concentration above 20 mmol/litre. Anaesthesia was associated with hypothermia, a decrease in haematocrit, hypotension, hyperoxaemia, respiratory acidosis and an increase in the plasma concentrations of lactate and arginine vasopressin. The concentration of β-endorphin in plasma increased transiently after 20 minutes but there were no changes in concentrations of adrenocorticotrophic hormone, dynorphin, cortisol or catecholamines. These data suggest that the glucose infusion attenuated the normal adrenal response of ponies to halothane anaesthesia.

Structure of tick anticoagulant peptide at 1.6 Å resolution complexed with bovine pancreatic trypsin inhibitor

The structure of tick anticoagulant peptide (TAP) has been determined by X-ray crystallography at t.6 Å resolution complexed with bovine pancreatic trypsin inhibitor (BPTI). The TAP-BPTI crystals are tetragonal, a = b = 46.87, c = 50.35 Å, space group P41, four complexes per unit cell. The TAP molecules are highly dipolar and form an intermolecular helical array along the c-axis with a diameter of about 45 Å. Individual TAP units interact in a head-to-tail fashion, the positive end of one molecule associating with the distal negative end of another, and vice versa. The BPTI molecules have a uniformly distributed positively charged surface that interacts extensively through 14 hydrogen bonds and two hydrogen bonded salt bridges with the helical groove around the helical TAP chains. Comparing the structure of TAP in TAP-BPTI with TAP bound to factor Xa(Xa) suggests a massive reorganization in the N-terminal tetrapeptide and the first disulfide loop of TAP (CyS5(T)- Cys 15(T)) upon binding to Xa. The Tyr1(T)OH atom of TAP moves 14.2 Å to interact with Asp189 of the S1 specificity site, Arg3(T)CZ moves 5.0 Å with the guanidinium group forming a cation-π-electron complex in the S4 subsite of Xa, while Lys7(T)NZ differs in position by 10.6 Å in TAP-BPTI and TAP-Xa, all of which indicates a different pre-Xa-bound conformation for the N- terminal of TAP in its native state. In contrast to TAP, the BPTI structure of TAP-BPTI is practically the same as all those of previously determined structures of BPTI, only arginine and lysine side-chain conformations showing significant differences.

Efeito da extrusão termoplástica no teor de usina disponível da farinha desengordurada de grão-de-bico (Cicer arietinum, L.)

The aim of this research was to evaluate lysine availability of chickpea (Cicer arietinum, L.) flour submitted to thermoplastic extrusion at three feed moisture levels (13%, 18% and 27%). It was verified that extrusion treatments reduced available lysine by 58% and 55% at 13% and 18% feed moisture levels. The major lysine loss, 71%, was verified at 27% feed moisture level.

Efeito do extrato aquoso de sete-sangrias [Cuphea mesostemon (Koehne) Lourt] sobre o fluxo de água, medido em bexiga urinária de sapo in vitro

The Cuphea mesostemon specie, known as sete-sangrias, is widely used as a diuretic substance in popular medicine. As the toad urinary bladder is an epithelium analogous to the distal nephron of mammals, it is used in order to study the transport water and electrolytes in many laboratories. This preparation permits excellent observation in water flow, from the urinary bladder lumen to the external side or the serosal one (water re-absorption), by means of gravimetrical measures. In the present work the hydrosmotic effect of aqueous extract (AE) of sete-sangrias leaf was studied. A 20% solution was added to the serosal side (S) of the bladder preparation, and the water flow was measured every 15 minutes after that. The results showed that 4mL of AE in the S side, increased the JH20 in a significant manner (p<0,05). This effect had a dose - response shape, with the volumes of 0,2mL, 0,4mL and 0,8mL of AE in the S bath. The hydro-osmotic effect of the anti-diuretic hormone (ADH) was studied as well and a significant stimulation (p<0,05) in the JH2O was observed with the magnitude of 150%. The AE effect was similar to the ADH one, and was not antagonized by this hormone. We concluded that Cuphea possesses an anti-diuretic activity similar to that presented by ADH, in toad urinary bladder, in vitro.

ZmPUMP encodes a fully functional monocot plant uncoupling mitochondrial protein whose affinity to fatty acid is increased with the introduction of a His pair at the second matrix loop

Uncoupling proteins (UCPs) are specialized mitochondrial transporter proteins that uncouple respiration from ATP synthesis. In this study, cDNA encoding maize uncoupling protein (ZmPUMP) was expressed in Escherichia coli and recombinant ZmPUMP reconstituted in liposomes. ZmPUMP activity was associated with a linoleic acid (LA)-mediated H+ efflux with Km of 56.36 ± 0.27 μM and Vmax of 66.9 μmol H+ min-1 (mg prot)-1. LA-mediated H+ fluxes were sensitive to ATP inhibition with Ki of 2.61 ± 0.36 mM (at pH 7.2), a value similar to those for dicot UCPs. ZmPUMP was also used to investigate the importance of a histidine pair present in the second matrix loop of mammalian UCP1 and absent in plant UCPs. ZmPUMP with introduced His pair (Lys155His and Ala157His) displayed a 1.55-fold increase in LA-affinity while its activity remained unchanged. Our data indicate conserved properties of plant UCPs and suggest an enhancing but not essential role of the histidine pair in proton transport mechanism. © 2006 Elsevier Inc. All rights reserved.

Different criteria for feed formulation based on digestible amino acids for broilers

An experiment was carried out to evaluate different criteria in feed formulation based on digestible amino acids for broilers during the grower phase. Diets were formulated according to the recommendations for digestible methionine, methionine + cysteine, lysine, and threonine. A total number of six hundred Cobb 500 male day-old chicks were distributed in a completely randomized experimental design, with three formulation criteria supplying the recommendations established by Baker & Chung (1992), Degussa (1997), and Rostagno et al. (2000), with 4 replicates of 50 birds each. No significant differences were observed for weight gain and feed intake. However, feed conversion ratio improved when birds were fed the diets containing the profiles recommended by Baker & Chung (1992) and Degussa (1997). No difference was observed in terms of leg, wings, back, and head yields. Broilers fed with the profile of Baker & Chung (1992) presented worst breast yield, whereas those fed the Degussa (1997) standard had better carcass yield.

Scanning electron microscopic analysis of the effect of Carisolv TM gel on periodontally compromised human root surfaces

The aim of this study was to analyze, under scanning electron microscopy (SEM), the morphologic characteristics of root surfaces after application of CarisolvTM gel in association with scaling and root planing (SRP). Sixty periodontally compromised extracted human teeth were randomly assigned to 6 groups: 1) SRP alone; 2) passive topical application of CarisolvTM + SRP; 3) active topical application of CarisolvTM + SRP; 4) multiple applications of CarisolvTM + SRP; 5) SRP + 24% EDTA; 6) topical application of CarisolvTM + SRP + 24% EDTA. CarisolvTM gel was applied to root surfaces for 30 s, followed by scaling and root planing, consisting of 50 strokes with Gracey curettes in an apical-coronal direction, parallel to the long axis of the tooth. The only exception was group 4, in which the roots were instrumented until a smooth, hard and glass-like surface was achieved. All specimens were further analyzed by SEM. The results showed that the treatment with CarisolvTM caused significant changes in root surface morphology of periodontally compromised teeth only when the chemical agent was actively applied (burnishing technique). CarisolvTM failed to remove the smear layer completely, especially with a single application, independently of the method of application. Multiple applications of CarisolvTM were necessary to achieve a smear layer reduction comparable to that obtained with 24% EDTA conditioning.

Efficacy of Carisolv™ as an adjunctive therapy to scaling and root planing on subgingival calculus removal

The purpose of this study was to evaluate the effectiveness of subgingival application of Carisolv™ gel as an adjunctive therapy to scaling and root planing (SRP) on calculus removal compared to conventional instrumentation. Forty-five teeth requiring extraction due to severe periodontal disease were randomized to the following treatments: 1) SRP alone; 2) placebo gel + SRP; 3) Carisolv™ gel + SRP. Either test or placebo gel was applied subgingivally for 1 min and then the root were instrumented until a smooth and calculus-free surface was achieved. Instrumentation time and the number of strokes required were recorded. After extraction, the efficacy of root surface instrumentation was measured by percentage of remaining calculus. There was no statistically significant difference (p>0.05) between the treatment groups regarding either time required for instrumentation or the percentage of residual calculus. The subgingival application of Carisolv™ gel prior to SRP did not provide any additional benefit to root instrumentation compared to scaling and root planing alone.

In vitro evaluation of marginal leakage in bonded restorations, with mechanical or chemical-mechanical (Carisolv) removal of carious tissue

This study carried out an in vitro evaluation and comparison of the occurrence of marginal leakage in bonded restorations using mechanical or chemical-mechanical (Carisolv) removal of carious tissue. For that purpose, 40 extracted decayed human molars were divided into 4 groups: GI (burs + Prime & Bond NT + TPH), GII (Carisolv + Prime & Bond NT + TPH), GIII (burs + SBMP + Z100) and GIV (Carisolv + SBMP + Z100). After accomplishment of the restorations and thermal cycling, the teeth were exposed to dye, sectioned and qualitatively evaluated. The results demonstrated that the system of removal of carious tissue did not influence the results of microleakage at any of the cavity margins. At dentinal margins, use of the Prime & Bond NT + TPH restorative system allowed the occurrence of less microleakage than the SBMP + Z100 system.

Biomolecular information, brain activity and cognitive functions

Molecular neurobiology has provided an explanation of mechanisms supporting mental functions as learning, memory, emotion and consciousness. However, an explanatory gap remains between two levels of description: molecular mechanisms determining cellular and tissue functions, and cognitive functions. In this paper we review molecular and cellular mechanisms that determine brain activity, and then hypothetize about their relation with cognition and consciousness. The brain is conceived of as a dynamic system that exchanges information with the whole body and the environment. Three explanatory hypotheses are presented, stating that: a) brain tissue function is coordinated by macromolecules controlling ion movements, b) structured (amplitude, frequency and phase-modulated) local field potentials generated by organized ionic movement embody cognitive information patterns, and c) conscious episodes are constructed by a large-scale mechanism that uses oscillatory synchrony to integrate local field patterns. © by São Paulo State University.

Production and partial characterization of keratinase produced by a microorganism isolated from poultry processing plant wastewater

A Streptomyces was isolated from poultry plant wastewater, showed high keratinolytic activity when cultured on feather meal medium. Optimum keratinolytic activity was observed at 40°C and pH 8.0. The enzyme also showed to be stable between 40 and 60°C. The keratinolytic activity was not inhibited by EDTA, DMSO and Tween 80. On the other hand, CaCl2, ZnCl2, and BaCl2 slightly inhibited the keratinolytic activity. The Streptomyces isolated might be useful in leather, keratin waste treatment, animal feeding industry, and also cosmetic industry. © 2008 Academic Journals.

Efeito dos níveis de triptofano digestível em dietas para codornas japonesas em postura

The adequate level (0.12, 0.16, 0.20, 0.24, and 0.28%) of the dietary digestible tryptophan was evaluated in 400 laying Japanese quail from 21 to 30 weeks old. The animals were housed in laying cages, with initial weight of 158.50 g and egg production average of 84.50%. A completely randomized blocks design, with eight blocks, five diets, eight replicates of ten birds per replicate and three experimental periods of 21 days each was used. Feed intake (g/bird/day), digestible tryptophan intake (mg/bird/day), egg production (%/bird/day), egg weight (g), egg mass (kg/bird/day) and feed conversion (kg feed intake per kg eggs and dozen eggs) were the characteristics evaluated. Only the characteristics digestible tryptophan intake and egg production rate show significant effects of digestible tryptophan levels in the diets. Performance response of the Japanese quails in posture, regarding the adjustment obtained through models of linear regression and broken-line regression model, and the biological interpretation, allow to conclude that to obtain the best productive performance, the Japanese quails diets should contain the level of 0.21% digestible tryptophan, that results in a daily intake of 45.0 mg/ bird of digestible tryptophan, corresponding to the digestible tryptophan: digestible lysine ratio of 21%. © 2008 Sociedade Brasileira de Zootecnia.

Myotoxic phospholipases A2 isolated from Bothrops brazili snake venom and synthetic peptides derived from their C-terminal region: Cytotoxic effect on microorganism and tumor cells

This paper reports the purification and biochemical/pharmacological characterization of two myotoxic phospholipases A2 (PLA2s) from Bothrops brazili venom, a native snake from Brazil. Both myotoxins (MTX-I and II) were purified by a single chromatographic step on a CM-Sepharose ion-exchange column up to a high purity level, showing Mr ∼ 14,000 for the monomer and 28,000 Da for the dimer. The N-terminal and internal peptide amino acid sequences showed similarity with other myotoxic PLA2s from snake venoms, MTX-I belonging to Asp49 PLA2 class, enzymatically active, and MTX-II to Lys49 PLA2s, catalytically inactive. Treatment of MTX-I with BPB and EDTA reduced drastically its PLA2 and anticoagulant activities, corroborating the importance of residue His48 and Ca2+ ions for the enzymatic catalysis. Both PLA2s induced myotoxic activity and dose-time dependent edema similar to other isolated snake venom toxins from Bothrops and Crotalus genus. The results also demonstrated that MTXs and cationic synthetic peptides derived from their 115-129 C-terminal region displayed cytotoxic activity on human T-cell leukemia (JURKAT) lines and microbicidal effects against Escherichia coli, Candida albicans and Leishmania sp. Thus, these PLA2 proteins and C-terminal synthetic peptides present multifunctional properties that might be of interest in the development of therapeutic strategies against parasites, bacteria and cancer. © 2008 Elsevier Inc. All rights reserved.