Myotoxic phospholipases A(2) 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 A(2) (PLA(2)S) 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 M-r similar to 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 PLA(2) class, enzymatically active, and MTX-II to Lys49 PLA(2)S, catalytically inactive. Treatment of MTX-I with BPB and EDTA reduced drastically its PLA(2) and anticoagulant activities, corroborating the importance of residue His48 and Ca2+ ions for the enzymatic catalysis. Both PLA(2)S 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 PLA(2) 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. (C) 2008 Elsevier Inc. All rights reserved.

Antitumor effects of snake venom chemically modified Lys49 phospholipase A(2)-like BthTX-I and a synthetic peptide derived from its C-terminal region

The present work evaluates both in vitro and in vivo antitumor activity of BPB-modified BthTX-I and its cationic synthetic peptide derived from the 115-129 C-terminal region. BPB-BthTX-1 presented cytotoxicity of 10-40% on different tumor cell lines, which were also susceptible to the lytic action of the synthetic peptide. Injection of the modified protein or the peptide in mice, 5 days after transplantation of S 180 tumor cells, reduced 30 and 36% of the tumor size on day 14th and 76 and 79% on day 60th, respectively, when compared to the untreated control group. Thus, these antitumor properties might be of interest in the development of therapeutic strategies against cancer. (C) 2009 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.

Computer-aided Drug Design of Novel PLA(2) Inhibitor Candidates for Treatment of Snakebite

Phospholipases A(2) (PLA(2)) are enzymes commonly found in snake venoms from Viperidae and Elaphidae families, which are major components thereof. Many plants are used in traditional medicine its active agents against various effects induced by snakebite. This article presents the PLA(2) BthTX-I structure prediction based on homology modeling. In addition, we have performed virtual screening in a large database yielding a set of potential bioactive inhibitors. A flexible docking program was used to investigate the interactions between the receptor and the new ligands. We have performed molecular interaction fields (MIFs) calculations with the phospholipase model. Results confirm the important role of Lys49 for binding ligands and suggest three additional residues as well. We have proposed a theoretically nontoxic, drug-like, and potential novel BthTX-I inhibitor. These calculations have been used to guide the design of novel phospholipase inhibitors as potential lead compounds that may be optimized for future treatment of snakebite victims as well as other human diseases in which PLA(2) enzymes are involved.

The interaction of bothropstoxin-I (Lys49-PLA(2)) with liposome membranes

Bothropstoxin-I (BthTx-I) is a Lys49-PLA(2) from the venom of the snake Bothrops jararacussu, which permeabilizes biological and artificial membranes by a mechanism independent of lipid hydrolysis. This mechanism has been investigated by studying the interaction of nine single tryptophan BthTx-I mutants with negatively charged phospholipid membranes. Changes in the solvent exposure of the tryptophan in each mutant were evaluated comparing the rate of chemical modification (k(mod)) by bromosuccinamide with the maximum intrinsic tryptophan fluorescence emission wavelength (lambda(max)) in buffer and in the presence of 10% DMPA/90% DPPC liposomes. No changes in lambda(max). were observed, whereas k(mod) values for tryptophans at positions 7, 10, 31 and 125 were significantly reduced in the presence of lipids, suggesting that bound phospholipid decreases solvent accessibility at these positions. Since the half-lives of the fluorescence and chemical modification effects differ by at least six orders of magnitude, these results suggest that the bound phospholipid may interact with multiple locations on the protein surface over micro- to millisecond timescales. (C) 2009 Elsevier Ltd. All rights reserved.

Insights on calcium-independent phospholipid membrane damage by Lys49-PLA(2) using tryptophan scanning mutagenesis of bothropstoxin-I from Bothrops jararacussu

Bothropstoxin-I (BthTx-I) is a homodimerie Lys49-PLA(2) from the venom of the snake Bothrops jararacussu, which lacks hydrolytic activity against phospholipid substrates, yet permeabilizes membranes by a Ca2+- independent mechanism. The interaction of the BthTx-I with model membranes has been studied by intrinsic tryptophan fluorescence emission (ITFE) spectroscopy. Nine separate mutants have been created each with a unique tryptophan residue located at a different position in the interfacial recognition site (IRS) of the protein. The rapid and efficient Ca2+-independent membrane damage against unilamellar liposomes composed of DPPC/DMPA in a 9:1 molar ratio was unaffected by these substitutions. Binding studies revealed low protein affinity for these liposomes and no changes were observed in the ITFE properties. In contrast, the binding of all mutants to DPPC/DMPA liposomes in a 1:1 molar ratio was stronger, and was correlated with altered ITFE properties. The blue-shifted emission spectra and increased emission intensity of mutants at positions 31, 67 and 115-117 in the interface recognition surface of the protein suggest these regions are partially inserted into the membrane. These results are consistent with a model for the Ca2+-independent membrane damaging mechanism that involves a transient interaction of the protein with the outer phospholipid leaflet of the target membrane. (C) 2007 Elsevier Masson SAS. All rights reserved.

Short-Term effect of COX-2 selective inhibitor as an adjunct for the treatment of periodontal disease: a clinical double-blind study in humans

Adjunctive therapeutic strategies that modulate the inflammatory mediators can play a significant role in periodontal therapy. In this double-blind, placebo-controlled study, 60 subjects diagnosed as periodontitis patients were evaluated for 28 days after periodontal treatment combined with selective cyclooxygenase-2 (COX-2) inhibitor. The experimental group received scaling and root planning (SRP) combined with the Loxoprofen antiinflammatory drug (SRP+Loxoprofen). The control group received SRP combined with placebo (SRP+placebo). Plaque index (PI), probing pocket depth (PD) and bleeding on probing (BOP) were monitored with an electronic probe at baseline and after 14 and 28 days. Both groups displayed clinical improvement in PD, PI and BOP. They also showed statistically similar values (p>0.05) of PD reduction on day 14 (0.4 mm) and on day 28 (0.6 mm). At the baseline, few deeper sites (>7 mm) from SRP+Loxoprofen group were responsible and most PD reduction was observed after 14 days (p<0.05). The percentage of remaining deep pockets (>7 mm) after 14 days in the SRP+Loxoprofen group was significantly lower (p<0.05) than in the SRP+placebo group. Loxoprofen presents potential effect as an adjunct of periodontal disease treatment, but long-term clinical trials are necessary to confirm its efficacy.

Shelf-life of a 2.5% sodium hypochlorite solution as determined by arrhenius equation

Accelerated stability tests are indicated to assess, within a short time, the degree of chemical degradation that may affect an active substance, either alone or in a formula, under normal storage conditions. This method is based on increased stress conditions to accelerate the rate of chemical degradation. Based on the equation of the straight line obtained as a function of the reaction order (at 50 and 70 ºC) and using Arrhenius equation, the speed of the reaction was calculated for the temperature of 20 ºC (normal storage conditions). This model of accelerated stability test makes it possible to predict the chemical stability of any active substance at any given moment, as long as the method to quantify the chemical substance is available. As an example of the applicability of Arrhenius equation in accelerated stability tests, a 2.5% sodium hypochlorite solution was analyzed due to its chemical instability. Iodometric titration was used to quantify free residual chlorine in the solutions. Based on data obtained keeping this solution at 50 and 70 ºC, using Arrhenius equation and considering 2.0% of free residual chlorine as the minimum acceptable threshold, the shelf-life was equal to 166 days at 20 ºC. This model, however, makes it possible to calculate shelf-life at any other given temperature.

Expressão da proteína erbB-2 e dos receptores de hormônios na transição das regiões in situ para invasora de tumores da mama

OBJETIVOS: avaliar a expressão de erbB-2 e dos receptores hormonais para estrógeno e progesterona (RE/RP) nas regiões de transição entre as frações in situ e invasoras de neoplasias ductais da mama (CDIS e CDI, respectivamente). MÉTODOS: oitenta e cinco casos de neoplasias mamárias, contendo regiões contíguas de CDIS e CDI, foram selecionados. Espécimes histológicos das áreas de CDIS e de CDI foram obtidos através da técnica de tissue microarray (TMA). As expressões da erbB-2 e dos RE/RP foram avaliadas por meio de imunoistoquímica convencional. A comparação da expressão da erbB-2 e dos RE/RP nas frações in situ e invasoras da mama foi realizada com emprego do teste de McNemar. Os intervalos de confiança foram determinados em 5% (p=0,05). Foram calculados coeficientes de correlação intraclasse (ICC) para avaliar a concordância na tabulação cruzada da expressão de erbB-2 e RE/RP nas frações de CDIS e CDI. RESULTADOS: a expressão da erbB-2 não diferiu entre as áreas de CDIS e CDI (p=0,38). Comparando caso a caso suas áreas de CDIS e CDI, houve boa concordância na expressão da erbB-2 (coeficiente de correlação intraclasse, ICC=0,64), dos RP (ICC = 0,71) e dos RE (ICC = 0,64). Considerando apenas tumores cujo componente in situ apresentasse áreas de necrose (comedo), o ICC para erbB-2 foi de 0,4, comparado a 0,6 no conjunto completo de casos. Os ICC não diferiram substancialmente daqueles obtidos com o conjunto completo de espécimes em relação aos RE/RP: para RE, ICC=0,7 (versus 0,7 no conjunto completo), e para RP, ICC=0,7 (versus 0,6 no conjunto completo). CONCLUSÕES: nossos achados sugerem que as expressões de erbB-2 e RE/RP não diferem nos componentes contíguos in situ e invasivo em tumores ductais da mama.

Bottle feeding, increased overjet and Class 2 primary canine relationship: is there any association?

The aim of this study was to investigate the association between bottle feeding and prevalence rates of increased overjet and Class 2 primary canine relationship. The sample consisted of 911 children (461 boys, 450 girls) aged 3 (13.9%), 4 (40.8%), 5 (34%) and 6 (11.3%) years, with complete primary dentition. Information about nutritive and nonnutritive (pacifier and/or digit) sucking habits was collected through questionnaires. Three calibrated dentists (κ: 0.9-1.0 and Rs > 0.90) performed the clinical assessments. The children were divided into four groups: G1 - not bottle-fed; G2 - exclusively bottle-fed; G3 - breast- and bottle-fed, bottle feeding ceased before 3 years of age; and G4 - breast- and bottle-fed, bottle feeding ceased between 3 and 4 years of age. Associations between nutritive and nonnutritive sucking behaviors and the malocclusions studied were analyzed by multiple binary logistic regression (α= 0.05). The frequencies of increased overjet were: 25.3% (G1), 38.8% (G2), 39.2% (G3) and 47.8% (G4). The percentages of Class 2 canine relationship were: 27.9% (G1), 48.8% (G2), 43.4% (G3) and 43% (G4). No significant effect of bottle feeding was found. The chances of diagnosing increased overjet (O.R. = 4.42, p < 0.001) and Class 2 canine relationship (O.R. = 4.02, p < 0.001) were greater for children with pacifier and/or digit-sucking habits, compared to those without a history of nonnutritive sucking behavior. It may be suggested that bottle feeding alone is not directly associated with higher prevalence rates of increased overjet and Class 2 canine relationship in the primary dentition.

Association of polymorphisms at the ADIPOR1 regulatory region with type 2 diabetes and body mass index in a Brazilian population with European or African ancestry

Association studies between ADIPOR1 genetic variants and predisposition to type 2 diabetes (DM2) have provided contradictory results. We determined if two single nucleotide polymorphisms (SNP c.-8503G>A and SNP c.10225C>G) in regulatory regions of ADIPOR1 in 567 Brazilian individuals of European (EA; N = 443) or African (AfA; N = 124) ancestry from rural (quilombo remnants; N = 439) and urban (N = 567) areas. We detected a significant effect of ethnicity on the distribution of the allelic frequencies of both SNPs in these populations (EA: -8503A = 0.27; AfA: -8503A = 0.16; P = 0.001 and EA: 10225G = 0.35; AfA: 10225G = 0.51; P < 0.001). Neither of the polymorphisms were associated with DM2 in the case-control study in EA (SNP c.-8503G>A: DM2 group -8503A = 0.26; control group -8503A = 0.30; P = 0.14/SNP 10225C>G: DM2 group 10225G = 0.37; control group 10225G = 0.32; P = 0.40) and AfA populations (SNP c.-8503G>A: DM2 group -8503A = 0.16; control group -8503A = 0.15; P = 0.34/SNP 10225C>G: DM2 group 10225G = 0.51; control group 10225G = 0.52; P = 0.50). Similarly, none of the polymorphisms were associated with metabolic/anthropometric risk factors for DM2 in any of the three populations, except for HDL cholesterol, which was significantly higher in AfA heterozygotes (GC = 53.75 ± 17.26 mg/dL) than in homozygotes. We conclude that ADIPOR1 polymorphisms are unlikely to be major risk factors for DM2 or for metabolic/anthropometric measurements that represent risk factors for DM2 in populations of European and African ancestries.

COL18A1 is highly expressed during human adipocyte differentiation and the SNP c.1136C > T in its "frizzled" motif is associated with obesity in diabetes type 2 patients

Collagen XVIII can generate two fragments, NC11-728 containing a frizzled motif which possibly acts in Wnt signaling and Endostatin, which is cleaved from the NC1 and is a potent inhibitor of angiogenesis. Collagen XVIII and Wnt signaling have recently been associated with adipogenic differentiation and obesity in some animal models, but not in humans. In the present report, we have shown that COL18A1 expression increases during human adipogenic differentiation. We also tested if polymorphisms in the Frizzled (c.1136C>T; Thr379Met) and Endostatin (c.4349G>A; Asp1437Asn) regions contribute towards susceptibility to obesity in patients with type 2 diabetes (113 obese, BMI =30; 232 non-obese, BMI < 30) of European ancestry. No evidence of association was observed between the allele c.4349G>A and obesity, but we observed a significantly higher frequency of homozygotes c.1136TT in obese (19.5%) than in non-obese individuals (10.9%) [P = 0.02; OR = 2.0 (95%CI: 1.07-3.73)], suggesting that the allele c.1136T is associated to obesity in a recessive model. This genotype, after controlling for cholesterol, LDL cholesterol, and triglycerides, was independently associated with obesity (P = 0.048), and increases the chance of obesity in 2.8 times. Therefore, our data suggest the involvement of collagen XVIII in human adipogenesis and susceptibility to obesity.

A revision of the Neotropical Solenopsidini ant genus Oxyepoecus Santschi, 1926 (Hymenoptera: Formicidae: Myrmicinae): 2. Final. Key for species and revision of the Rastratus species-group

In the first paper of this series (Albuquerque & Brandão, 2004) we revised the Vezenyii species group of the exclusively Neotropical solenopsidine (Myrmicinae) ant genus Oxyepoecus. In this closing paper we update distribution information on the Vezenyii group species and revise the other Oxyepoecus species-group (Rastratus). We describe two species (Oxyepoecus myops n. sp. and O. rosai n. sp.) and redescribe previously known species of the group [O. daguerrei (Santschi, 1933), O. mandibularis (Emery, 1913), O. plaumanni Kempf, 1974, O. rastratus Mayr, 1887, and O. reticulatus Kempf, 1974], adding locality records and comments on the meagre biological data of these species. We also present an identification key to Oxyepoecus species based on workers.

EPR and magnetic studies of a carboxylate-bridged dinuclear copper(II) compound: [cu2(flu)4(dmf)2]

We report magnetic and EPR (electron paramagnetic resonance) spectroscopy studies of [Cu2(flu)4(dmf)2] (flu = flufenamate and dmf = dimethylformamide), which has CuII ions in tetracarboxylate "paddle wheel" dinuclear units. Susceptibility measurements at 10 < T < 275 K allowed the evaluation of an antiferromagnetic intradinuclear exchange coupling J0 = -294 ± 5 cm-1 between CuII ions (Hex = "J0 S1·S2). EPR experiments at 300 K in powder and single-crystals at 9.5 and 34.4 GHz indicated g// = 2.373, g⊥ = 2.073 and zero field splitting parameters D = (-0.334 ± 0.001) cm"1 and E ca. 0. EPR signal intensity measurements at X-band in the range 4 < T < 295 K indicated that J0 = "283 ± 5 cm"1. A higher limit |J´| < 5×10-3 cm-1 for the interdinuclear exchange coupling between neighbor units at ca.14.24 Å was estimated from the angular variation of the single crystal spectra around the magic angles. The results are discussed in terms of the structure of the dinuclear unit and the bridges connecting CuII ions and compared with values reported for similar compounds.

Different coordination modes for disulfoxides towards diorganotin(IV) dichlorides. X-ray crystal structures of 1,2-cis-bis-(phenylsulfinyl)ethene (rac-,cis-cbpse) and adducts [{Ph2SnCl2(meso-bpse)}n] and [{n-Bu2SnCl2(pdtd)}2]

The reactions of meso-1,2-bis(phenylsulfinyl)ethane (meso-bpse) with Ph2SnCl2, 2-phenyl-1,3-dithiane trans-1-trans-3-dioxide (pdtd) with n-Bu2SnCl2 and 1,2-cis-bis-(phenylsulfinyl)ethene (rac-,cis-cbpse) with Ph2SnCl2, in 1:1 molar ratio, yielded [{Ph2SnCl2(meso-bpse)}n], [{n-Bu2SnCl2(pdtd)}2] and [{Ph2SnCl2(rac,cis-cbpse)}x] (x = 2 or n), respectively. All adducts were studied by IR, Mössbauer and 119Sn NMR spectroscopic methods, elemental analysis and single crystal X-ray diffractometry. The X-ray crystal structure of [{Ph2SnCl2(meso-bpse)}n] revealed the occurrence of infinite chains in which the tin(IV) atoms appear in a distorted octahedral geometry with Cl atoms in cis and Ph groups in trans positions. The X-ray crystal structure of [{n-Bu2SnCl2(pdtd)}2] revealed discrete centrosymmetric dimeric species in which the tin(IV) atoms possess a distorted octahedral geometry with bridging disulfoxides in cis and n-butyl moieties in trans positions. The spectroscopic data indicated that the adduct containing the rac,cis-cbpse ligand can be dimeric or polymeric. The X-ray structural analysis of the free rac-,cis-cbpse sulfoxide revealed that the crystals belong to the C2/c space group.