Evidence of caspase-mediated apoptosis induced by L-amino acid oxidase isolated from Bothrops atrox snake venom

The aim of this work was to investigate the involvement of caspases in apoptosis induced by L-amino acid oxidase isolated from Bothrops atrox snake venom. The isolation of LAAO involved three chromatographic steps: molecular exclusion on a G-75 column; ion exchange column by HPLC and affinity chromatography on a Lentil Lectin column. SDS-PAGE was used to confirm the expected high purity level of BatroxLAA0. It is a glycoprotein with 12% sugar and an acidic character, as confirmed by its amino acid composition, rich in ""Asp and Glu"" residues. It displays high specificity toward hydrophobic L-amino acids. The N-terminal amino acid sequence and internal peptide sequences showed close structural homology to other snake venom LAAOs. This enzyme induces in vitro platelet aggregation, which may be due to H(2)O(2) production by LAAOs, since the addition of catalase completely inhibited the aggregation effect. It also showed cytotoxicity towards several cancer cell lines: HL60, Jurkat, B16F10 and PC12. The cytotoxicity activity was abolished by catalase. A fluorescence microscopy evaluation revealed a significant increase in the apoptotic index of these cells after BatroxLAAO treatment. This observation was confirmed by phosphatidyl serine exposure and activation of caspases. BatroxLAAO is a protein with various biological functions that can be involved in envenomation. Further investigations of its function will contribute to toxicology advances. Published by Elsevier Inc.

BjussuSP-I: A new thrombin-like enzyme isolated from Bothrops jararacussu snake venom

A thrombin-like enzyme named BjussuSP-I, isolated from B. jararacussu snake venom, is an acidic single chain glycoprotein with approximately 6% sugar, Mr = 61,000 under reducing conditions and pI similar to 3.8, representing 1.09% of the chromatographic A(280) recovery. BjussuSP-I is a glycosylated scrine protease containing both N-linked carbohydrates and sialic acid in its structure. BjussuSP-I showed a high clotting activity upon human plasma, which was inhibited by PMSF, leupeptin, heparin and 1,10-phenantroline. This enzyme showed high stability regarding coagulant activity when analyzed at different temperatures (-70 to 37 degrees C), pHs (4.5 to 8.0), and presence of two divalent metal ions (Ca2+ and Mg2+). It also displayed TAME esterase and proteolytic activities toward natural (fibrinogen and fibrin) and synthetic (BAPNA) substrates, respectively, being also inhibited by PMSF and leupeptin. BjussuSP-I can induce production of polyclonal antibodies able to inhibit its clotting activity, but unable to inhibit its proteolytic activity on fibrinogen. The enzyme also showed crossed immunoreactivity against I I venom samples of Bothrops, I of Crotalus, and I of Calloselasma snakes, in addition of LAAO isolated from B. moojeni venom. It displayed neither hemorrhagic, myotoxic, edema-inducing profiles nor proteolytic activity on casein. BjussuSP-I showed an N-terminal sequence (VLGGDECDfNEHPFLA FLYS) similar to other thrombin-like enzymes from snake venoms. Based on its biochemical, enzymatic and pharmacological characteristics, BjussuSP-I was identified as a new thrombin-like enzyme isoform from Bothrops jararacussu snake venom. (C) 2007 Elsevier Inc. All rights reserved.

Biochemical and functional properties of a thrombin-like enzyme isolated from Bothrops pauloensis snake venom

In the present study, a thrombin-like enzyme named BpSP-I was isolated from Bothrops pauloensis snake venom and its biochemical, enzymatic and pharmacological characteristics were determined. BpSP-I is a glycoprotein that contains both N-linked carbohydrates and sialic acid in its structure, with M(r) = 34,000 under reducing conditions and pI similar to 6.4. The N-terminal sequence of the enzyme (VIGGDECDINEHPFL) showed high similarity with other thrombin-like enzymes from snake venoms. BpSP-I showed high clotting activity upon bovine and human plasma and was inhibited by PMSF, benzamidine and leupeptin. Moreover, this enzyme showed stability when examined at different temperatures (-70 to 37 degrees C), pH values (3-9) or in the presence of divalent metal ions (Ca(2+), Mg(2+), Zn(2+) and Mn(2+)). BpSP-I showed high catalytic activity upon substrates, such as fibrinogen, TAME, S-2238 and S-2288. It also showed kallikrein-like activity, but was unable to act upon factor Xa and plasmin substrates. Indeed, the enzyme did not induce hemorrhage, myotoxicity or edema. Taken together, our data showed that BpSP-I is in fact a thrombin-like enzyme isoform isolated from Bothrops pauloensis snake venom. (C) 2009 Elsevier Ltd. All rights reserved.

Structural and functional properties of Bp-LAAO, a new L-amino acid oxidase isolated from Bothrops pauloensis snake venom

An L-amino acid oxidase (Bp-LAAO) from Bothrops pauloensis snake venom was highly purified using sequential chromatography steps on CM-Sepharose, Phenyl-Sepharose CL4B, Benzamidine Sepharose and C18 reverse-phase HPLC. Purified Bp-LAAO showed to be a homodimeric acidic glycoprotein with molecular weight around 65 kDa under reducing conditions in SDS-PAGE. The best substrates for Bp-LAAO were L-Met, L-Leu, L-Phe and L-Ile and the enzyme showed a strong reduction of its catalytic activity upon L-Met and L-Phe substrates at extreme temperatures. Bp-LAAO showed leishmanicidal, antitumoral and bactericidal activities dose dependently. Bp-LAAO induced platelet aggregation in platelet-rich plasma and this activity was inhibited by catalase. Bp-LAAC-cDNA of 1548 bp codified a mature protein with 516 amino acid residues corresponding to a theoretical isoelectric point and molecular weight of 6.3 and 58 kDa, respectively. Additionally, structural and phylogenetic studies identified residues under positive selection and their probable location in Elp-LAAO and other snake venom LAAOs (svLAAOs). Structural and functional investigations of these enzymes can contribute to the advancement of toxinology and to the elaboration of novel therapeutic agents. (C) 2009 Elsevier Masson SAS. All rights reserved.

Molecular cloning and biochemical characterization of a myotoxin inhibitor from Bothrops alternatus snake plasma

Phospholipases A(2) (PLA(2)s) are important components of Bothrops snake venoms, that can induce several effects on envenomations such as myotoxicity, inhibition or induction of platelet aggregation and edema. It is known that venomous and non-venomous snakes present PLA(2) inhibitory proteins (PLIs) in their blood plasma. An inhibitory protein that neutralizes the enzymatic and toxic activities of several PLA2s from Bothrops venoms was isolated from Bothrops alternatus snake plasma by affinity chromatography using the immobilized myotoxin BthTX-I on CNBr-activated Sepharose. Biochemical characterization of this inhibitory protein, denominated alpha BaltMIP, showed it to be a glycoprotein with Mr of similar to 24,000 for the monomeric subunit. CD spectra of the PLA(2)/inhibitor complexes are considerably different from those corresponding to the individual proteins and data deconvolution suggests that the complexes had a relative gain of helical structure elements in comparison to the individual protomers, which may indicate a more compact structure upon complexation. Theoretical and experimental structural studies performed in order to obtain insights into the structural features of aBaltMIP indicated that this molecule may potentially trimerize in solution, thus strengthening the hypothesis previously raised by other authors about snake PLIs oligomerization. (C) 2010 Elsevier Masson SAS. All rights reserved.

Influence of Plasma Protein Binding on Pharmacodynamics: Estimation of In Vivo Receptor Affinities of beta Blockers Using a New Mechanism-Based PK-PD Modelling Approach

The objective of this investigation was to examine in a systematic manner the influence of plasma protein binding on in vivo pharmacodynamics. Comparative pharmacokinetic-pharmacodynamic studies with four beta blockers were performed in conscious rats, using heart rate under isoprenaline-induced tachycardia as a pharmacodynamic endpoint. A recently proposed mechanism-based agonist-antagonist interaction model was used to obtain in vivo estimates of receptor affinities (K(B),(vivo)). These values were compared with in vitro affinities (K(B),(vitro)) on the basis of both total and free drug concentrations. For the total drug concentrations, the K(B),(vivo) estimates were 26, 13, 6.5 and 0.89 nM for S(-)-atenolol, S(-)-propranolol, S(-)-metoprolol and timolol. The K(B),(vivo) estimates on the basis of the free concentrations were 25, 2.0, 5.2 and 0.56 nM, respectively. The K(B),(vivo)-K(B),(vitro) correlation for total drug concentrations clearly deviated from the line of identity, especially for the most highly bound drug S(-)-propranolol (ratio K(B),(vivo)/K(B),(vitro) similar to 6.8). For the free drug, the correlation approximated the line of identity. Using this model, for beta-blockers the free plasma concentration appears to be the best predictor of in vivo pharmacodynamics. (C) 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3816-3828, 2009

An alpha-type phospholipase A(2) inhibitor from Bothrops jararacussu snake plasma: Structural and functional characterization

An inhibitory protein that neutralizes the enzymatic, toxic and pharmacological activities of several phospholipases A(2) from Bothrops venoms was isolated from B. jararacussu snake plasma by affinity chromatography using the immobilized myotoxin BthTX-I on Sepharose gel. Biochemical characterization of this inhibitory protein, denominated alpha BjussuMIP, showed it to be an oligomeric glycoprotein with M-r of 24,000 for the monomeric subunit. Secondary structural analysis by circular dichroism revealed 44% alpha-helix, 18% beta-sheet, 10% beta-turn and 28% random coil structures. Circular dichroism spectroscopy indicated that no significant alterations in the secondary structure of either alpha BjussuMIP or the target protein occur following their interaction. The product from the reaction with reverse transcriptase produced a cDNA fragment of 432 bp that codifies for a mature protein of 144 amino acid residues. The first 21 amino acid residues from the N-terminal and five tryptic peptides were characterized by mass spectrometry of the mature protein and confirmed by the nucleotide sequence. Alignment of alpha BjussuMIP with other snake inhibitors showed a sequence similarity of 73-92% with these alpha PLIs. alpha BjussuMIP was relatively stable within the pH range of 6-12 and temperatures from 0 degrees C to 80 degrees C, even after deglycosylation. The results showed effects against Bothrops phospholipase A(2) activities (enzymatic, edema inducing, myotoxic, cytotoxic and bactericidal), suggesting that alpha BjussuMIP may prove useful in the treatment of snakebite envenomations. (C) 2008 Elsevier Masson SAS. All rights reserved.

Purification and characterization of a thermostable alpha-amylase produced by the fungus Paecilomyces variotii

An alpha-amylase produced by Paecilomyces variotii was purified by DEAE-cellulose ion exchange chromatography, followed by Sephadex G-100 gel filtration and electroelution. The alpha-amylase showed a molecular mass of 75 kDa (SDS-PAGE) and pl value of 4.5. Temperature and pH optima were 60 degrees C and 4.0, respectively. The enzyme was stable for 1 h at 55 degrees C, showing a t(50) of 53 min at 60 degrees C. Starch protected the enzyme against thermal inactivation. The a-amylase was more stable in alkaline pH. It was activated mainly by calcium and cobalt, and it presented as a glycoprotein with 23% carbohydrate content. The enzyme preferentially hydrolyzed starch and, to a lower extent, amylose and amylopectin. The K(m) of alpha-amylase on Reagen (R) and Sigma (R) starches were 4.3 and 6.2 mg/mL, respectively. The products of starch hydrolysis analyzed by TLC were oligosaccharides such as maltose and maltotriose. The partial amino acid sequence of the enzyme presented similarity to alpha-amylases from Bacillus sp. These results confirmed that the studied enzyme was an a-amylase ((1 -> 4)-alpha-glucan glucanohydrolase). (C) 2010 Elsevier Ltd. All rights reserved.

Effect of glycosylation on the biochemical properties of beta-xylosidases from Aspergillus versicolor

Aspergillus versicolor grown on xylan or xylose produces two beta-xylosidases with differences in biochemical properties and degree of glycosylation. We investigated the alterations in the biochemical properties of these beta-xylosidases after deglycosylation with Endo-H or PNGase F. After deglycosylation, both enzymes migrated faster in PAGE or SDS-PAGE exhibiting the same R(f). Temperature optimum of xylan-induced and xylose-induced beta-xylosidases was 45A degrees C and 40A degrees C, respectively, and 35A degrees C after deglycosylation. The xylan-induced enzyme was more active at acidic pH. After deglycosylation, both enzymes had the same pH optimum of 6.0. Thermal resistance at 55A degrees C showed half-life of 15 min and 9 min for xylose- and xylan-induced enzymes, respectively. After deglycosylation, both enzymes exhibited half-lives of 7.5 min. Native enzymes exhibited different responses to ions, while deglycosylated enzymes exhibited identical responses. Limited proteolysis yielded similar polypeptide profiles for the deglycosylated enzymes, suggesting a common polypeptide core with differential glycosylation apparently responsible for their biochemical and biophysical differences.

Biochemical properties of an extracellular beta-D-fructofuranosidase II produced by Aspergillus phoenicis under Solid-Sate Fermentation using soy bran as substrate

The filamentous fungus A. phoenicis produced high levels of beta-D-fructofuranosidase (FFase) when grown for 72 hrs under Solid-State Fermentation (SSF), using soy bran moistened with tap water (1:0.5 w/v) as substrate/carbon source. Two isoforms (I and II) were obtained, and FFase II was purified 18-fold to apparent homogeneity with 14% recovery. The native molecular mass of the glycoprotein (12% of carbohydrate content) was 158.5 kDa with two subunits of 85 kDa estimated by SDS-PAGE. Optima of temperature and pH were 55 degrees C and 4.5. The enzyme was stable for more than 1 hr at 50 degrees C and was also stable in a pH range from 7.0 to 8.0. FFase II retained 80% of activity after storage at 4 degrees C by 200 hrs. Dichroism analysis showed the presence of random and beta-sheet structure. A. phoenicis FFase II was activated by Mn(2+), Mg(2+) and Co(2+), and inhibited by Cu(2+), Hg(2+) and EDTA. The enzyme hydrolyzed sucrose, inulin and raffinose. K(d) and V(max) values were 18 mM and 189 U/mg protein using sucrose as substrate.

Proteoliposomes Harboring Alkaline Phosphatase and Nucleotide Pyrophosphatase as Matrix Vesicle Biomimetics

We have established a proteoliposome system as an osteoblast-derived matrix vesicle (MV) biomimetic to facilitate the study of the interplay of tissue-nonspecific alkaline phosphatase (TNAP) and NPP1 (nucleotide pyrophosphatase/phosphodiesterase-1) during catalysis of biomineralization substrates. First, we studied the incorporation of TNAP into liposomes of various lipid compositions (i.e. in pure dipalmitoyl phosphatidylcholine (DPPC), DPPC/dipalmitoyl phosphatidylserine (9:1 and 8:2), and DPPC/dioctadecyl-dimethylammonium bromide (9:1 and 8:2) mixtures. TNAP reconstitution proved virtually complete in DPPC liposomes. Next, proteoliposomes containing either recombinant TNAP, recombinant NPP1, or both together were reconstituted in DPPC, and the hydrolysis of ATP, ADP, AMP, pyridoxal-5`-phosphate (PLP), p-nitrophenyl phosphate, p-nitrophenylthymidine 5`-monophosphate, and PP(i) by these proteoliposomes was studied at physiological pH. p-Nitrophenylthymidine 5`-monophosphate and PLP were exclusively hydrolyzed by NPP1-containing and TNAP-containing proteoliposomes, respectively. In contrast, ATP, ADP, AMP, PLP, p-nitrophenyl phosphate, and PPi were hydrolyzed by TNAP-, NPP1-, and TNAP plus NPP1- containing proteoliposomes. NPP1 plus TNAP additively hydrolyzed ATP, but TNAP appeared more active in AMP formation than NPP1. Hydrolysis of PPi by TNAP-, and TNAP plus NPP1- containing proteoliposomes occurred with catalytic efficiencies and mild cooperativity, effects comparable with those manifested by murine osteoblast-derived MVs. The reconstitution of TNAP and NPP1 into proteoliposome membranes generates a phospholipid microenvironment that allows the kinetic study of phosphosubstrate catabolism in a manner that recapitulates the native MV microenvironment.

Association Between Increased Platelet P-Selectin Expression and Obesity in Patients With Type 2 Diabetes A BARI 2D (Bypass Angioplasty Revascularization Investigation 2 Diabetes) substudy

OBJECTIVE- To determine whether obesity increases platelet reactivity and thrombin activity in patients with type 2 diabetes plus stable coronary artery disease. RESEARCH DESIGN AND METHODS- We assessed platelet reactivity and markers of thrombin generation and activity in 193 patients from nine clinical sites of the Bypass Angioplasty Revascularization Investigation 2 Diabetes (BARI 2D). Blood taken at the time of enrollment was used for assay of the concentration of prothrombin fragment 1.2 (PT1.2, released when prothrombin is activated) and fibrinopeptide A (FPA, released when fibrinogen is cleaved). Platelet activation was identified with the use of flow cytometry in response to 0, 0.2, and 1 mu mol/l adenosine diphosphate (ADP). RESULTS- Concentrations of FPA, PT1.2, and platelet activation in the absence of agonist were low. Greater BMI was associated with higher platelet reactivity in response to 1 mu m ADP as assessed by surface expression of P-selectin (r = 0.29, P < 0.0001) but not reflected by the binding of fibrinogen to activated glycoprotein IIb-IIIa. BMI was not associated with concentrations of FPA or PT1.2. Platelet reactivity correlated negatively with A1C (P < 0.04), was not related to the concentration Of triglycerides in blood, and did not correlate with the concentration of C-reactive peptide. CONCLUSIONS- Among patients enrolled in this substudy of BARI 2D, a greater BMI was associated with higher platelet reactivity at the time of enrollment. Our results suggest that obesity and insulin resistance that accompanies obesity may influence platelet reactivity in patients with type 2 diabetes.

Investigation on Brazilian Clinical Practices in Rheumatoid Arthritis The Brazilian Rheumatoid Arthritis Clinical Practices Investigation-BRACTICE

Rheumatoid arthritis (RA) is a systemic inflammatory autoimmune disease causing significant social, medical, and economic impact. Several therapeutic regimens are available within the medical arsenal. The rational and reasoned use of various medications approved for their treatment is imperative. This study aimed to evaluate how Brazilian rheumatologists use the drugs available to combat the disease. For this, 128 Brazilian rheumatologists from public and private health services responded to an 18-item questionnaire, sent over the Internet, about different situations of drug treatment of RA. The answers helped to confirm the trends among Brazilian rheumatologists in the drug treatment of RA. The study results have shown that most Brazilian rheumatologists follow the guidelines and consensus established by the Brazilian Society of Rheumatology for the treatment of RA. A small proportion, however, start the biologic therapy in early stages of the disease, including the very early stage, as the first treatment option. Most experts use corticosteroids in low doses early in the treatment. Conclusions: This study confirms that the majority but not all Brazilian rheumatologists follow, in their daily practice, established guidelines and consensus for the treatment of RA. However, it also shows that some few rheumatologists start with anti-tumor necrosis factor therapy in very early arthritis independently of disease severity or prognostic factors.

The p.A2215D Thyroglobulin Gene Mutation Leads to Deficient Synthesis and Secretion of the Mutated Protein and Congenital Hypothyroidism with Wide Phenotype Variation

Context: Thyroglobulin (TG) is a large glycoprotein and functions as a matrix for thyroid hormone synthesis. TG gene mutations give rise to goitrous congenital hypothyroidism (CH) with considerable phenotype variation. Objectives: The aim of the study was to report the genetic screening of 15 patients with CH due to TG gene mutations and to perform functional analysis of the p. A2215D mutation. Design: Clinical evaluation and DNA sequencing of the TG gene were performed in all patients. TG expression was analyzed in the goitrous tissue of one patient. Human cells were transfected with expression vectors containing mutated and wild-type human TG cDNA. Results: All patients had an absent rise of serum TG after stimulation with recombinant human TSH. Sequence analysis revealed three previously described mutations (p. A2215D, p. R277X, and g. IVS30 + 1G > T), and two novel mutations (p. Q2142X and g. IVS46-1G > A). Two known (g. IVS30 + 1G/p. A2215D and p. A2215D/p. R277X) and one novel (p. R277X/g. IVS46-1G > A) compound heterozygous constellations were also identified. Functional analysis indicated deficiency in TG synthesis, reduction of TG secretion, and retention of the mutant TG within the cell, leading to an endoplasmic reticulum storage disease, whereas small amounts of mutant TG were still secreted within the cell system. Conclusion: All studied patients were either homozygous or heterozygous for TG gene mutations. Two novel mutations have been detected, and we show that TG mutation p. A2215D promotes the retention of TG within the endoplasmic reticulum and reduces TG synthesis and secretion, causing mild hypothyroidism. In the presence of sufficient iodine supply, some patients with TG mutations are able to compensate the impaired hormonogenesis and generate thyroid hormone. (J Clin Endocrinol Metab 94: 2938-2944, 2009)

Auto-antibodies do not influence development of atherosclerotic plaques in rheumatoid arthritis

Background: Many questions remain unanswered about premature atherosclerosis in rheumatoid arthritis (RA). Besides inflammation, some studies have suggested the role of autoantibodies on its pathogenesis. Objective: The aim of this study was to investigate the presence of antibodies against phospholipids, beta2-glycoproteinl (beta2-gpl), lipoprotein lipase, and heat shock proteins (Hsp) in RA patients and to evaluate their possible association with subclinical carotid atherosclerosis. Methods: Seventy-one RA patients and 53 age- and sex-matched controls were selected to perform anticardiolipin antibodies (aCL) (IgG and IgM), anti-beta2-gpl (IgG, IgM, and IgA), anti-lipoprotein lipase (anti-LPL), anti-Hsp 60, and anti-Hsp 65 by ELISA tests. Intima-medial thickness (IMT) of common carotid and presence of plaques were assessed by high-resolution B-mode ultrasonography. Exclusion criteria were smoking, diabetes, and arterial hypertension. Lipoproteins, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and fibrinogen levels, as well as health assessment questionnaire (HAQ) and disease activity score (DAS) 28 were also evaluated. Results: Age (48.93 +/- 12.31 vs. 45.37 +/- 9.37 years; p = 0.20) and body mass index (BMI) (p = 0.69) were similar in RA and controls, as well as female gender (p = 0.56). The mean IMT was similar between RA and controls (0. 721 +/- 0.16 vs. 0.667 +/- 0.14 turn, p = 0.07) but the frequency of plaques was higher in RA (14.1% vs. 1.9%; p = 0.02). In RA patients, IMT measurements did not differ according to the presence or absence of these antibodies: IgG aCL (0.62 +/- 0.64 vs. 0.72 +/- 0.17 mm, p = 0.24), IgM aCL (0.65 +/- 0.79 vs. 0.73 +/- 0.17 mm, p = 0.33), anti-Hsp 60 (0.78 +/- 0.20 vs. 0.71 +/- 0.16 mm, p = 0.27), anti-Hsp 65 (0.73 +/- 0.16 vs. 0.72 +/- 0.17 mm, p = 0.77), IgG anti-beta2-gpl (0.73 +/- 0.16 vs. 0.71 +/- 0.17 mm, p = 0.72), and anti-CCP (0.71 +/- 0.16 vs. 0.76 +/- 0.20 mm, p = 0.36). In addition, IMT did not correlate with antibodies titers: IgG aCL (r = -0.09, p = 0.47), IgM aCL (r = - 0.15, p = 0.21), anti-Hsp 60 (r = 0.10, p = 0.42), anti-Hsp 65 (r = 0.05, p = 0.69), IgG anti-beta2-gpl (r = - 0.07, p = 0.57), IgM anti-beta2-gpl (r = - 0.05, p = 0.69), IgA anti-beta2-gpl (r = 0.03, p = 0.79), and anti-CCP (r = - 0.07, p = 0.57). RA patients with plaques had a significantly higher age compared to those without plaques (p = 0.001), as well as higher mean IMT (p < 0.001), total cholesterol (p = 0.001), and LDL (p = 0.003). Conclusions: In RA a clear association between all autoantibodies studied herein and increased IMT or presence of plaques was not observed. The great prevalence of carotid atherosclerosis in RA was related to age, total and LDL cholesterol, as identified in normal population. (c) 2008 Elsevier Masson SAS. All rights reserved.