Isolation, characterization and antigenic cross-reactivities of the major hemorrhagin from Cryptelytrops purpureomaculatus venom.

The major hemorrhagin from C. purpureomaculatus (mangrove pit viper) venom was purified to homogeneity and termed Maculatoxin. Maculatoxin has a molecular weight of 38 kDa as determined by SDS-PAGE. It is an acidic protein (pI= 4.2) and exhibited proteolytic and hemorrhagic activities (MHD10 = 0.84 μg in mice) but was not lethal to mice at a dose of 1 μg/g. The hemorrhagic activity of Maculatoxin was completely inactivated by EDTA and partially inhibited by ATP and citrate. The N-terminal sequence of Maculatoxin (TPEQQRFPPTYIDLGIFVDHGMYAT) shares a significant degree of homology with the metalloprotease domain of other venom hemorrhagins. Indirect ELISA showed anti-Maculatoxin cross reacted with protein components of many snake venoms. In the double-sandwich ELISA, however, anti-Maculatoxin cross-reacted only with venoms of certain species of the Trimeresurus (Asia lance-head viper) complex, and the results support the recent proposed taxonomy changes concerning the Trimeresurus complex

Optimization and characterization of alkaline protease and carboxymethyl-cellulase produced by Bacillus pumillus grown on Ficus nitida wastes

The potentiality of 23 bacterial isolates to produce alkaline protease and carboxymethyl-cellulase (CMCase) on Ficus nitida wastes was investigated. Bacillus pumillus ATCC7061 was selected as the most potent bacterial strain for the production of both enzymes. It was found that the optimum production of protease and CMCase were recorded at 30 °C, 5% Ficus nitida leaves and incubation period of 72 h. The best nitrogen sources for protease and CMCase production were yeast extract and casein, respectively. Also maximum protease and CMCase production were reported at pH 9 and pH 10, respectively. The enzymes possessed a good stability over a pH range of 8-10, expressed their maximum activities at pH10 and temperature range of 30-50 °C, expressed their maximum activities at 50 °C. Ions of Hg2+, Fe2+ and Ag+ showed a stimulatory effect on protease activity and ions of Fe2+, Mg2+, Ca2+, Cu2+ and Ag+ caused enhancement of CMCase activity. The enzymes were stable not only towards the nonionic surfactants like Triton X-100 and Tween 80 but also the strong anionic surfactant, SDS. Moreover, the enzymes were not significantly inhibited by EDTA or cystein. Concerning biotechnological applications, the enzymes retained (51-97%) of their initial activities upon incubation in the presence of commercials detergents for 1 h. The potential use of the produced enzymes in the degradation of human hair and cotton fabric samples were also assessed.

Caracterización de factores asociados con comportamiento suicida en adolescentes estudiantes de octavo grado, en tres colegios bogotanos / Factor Characterization Associated with Suicidal Behavior in 8"' Grade Adolescent Students in Three Schools from Bogotá (Colombia)

Objetivos: Determinar la probabilidad de riesgo suicida y/o enfermedad mental y factores asociados en estudiantes de secundaria de tres colegios bogotanos. Métodos: Estudio de corte transversal con 309 adolescentes. Resultados: El promedio de edad fue de 13,83 ± 0,9 años, predominó el género femenino (58,6%) y el estrato socioeconómico 3 (68,3%). La probabilidad de riesgo para comportamiento suicida y/o síntomas mentales fue de 47,6%; 26,5% tuvo alguna manifestación suicida; 14,23% tuvo ideación suicida en los últimos tres meses; 3,55% tuvo intentos suicidas alguna vez en la vida, y 8,73% tuvo ideación suicida e intentos suicidas en los últimos tres meses. El riesgo de comportamiento suicida y/o enfermedad mental fue explicado conjuntamente por la depresión (OR = 27,9, IC95% = 3,5-223,1), la baja autoestima (OR = 11,8, IC95% = 2,5-56,5), la disfunción familiar severa (OR = 3,4, IC95% = 1,2-9,7), el sexo femenino (OR = 2,1, IC95% = 1,2-3,8) y la edad mayor o igual a 15 años (OR = 1,9, IC95% = 0,9-3,9). El maltrato psicológico seguido del abuso físico se asociaron con manifestación suicida y/o enfermedad mental, y la buena relación familiar, con menor probabilidad. Conclusión: La depresión, la baja autoestima, la disfuncionalidad familiar, el género femenino, la edad > 15 y la violencia intrafamiliar son factores asociados al riesgo suicida y/o enfermedad mental en adolescentes, y las buenas relaciones familiares se asocian con menor riesgo.

Objective: To establish the probability for suicide risk and/or mental disorders, together with related factors among high school students in 3 schools in Bogota. Methods: Cross sectional study of 309 adolescents. Results: The average age was 13.83 ± 0.9, female dominance (58.6%) and a 3rd socioeconomic stratum (68.3%). The suicidal risk behavioral probability and/or mental symptoms was 47.6%, 26.5% exhibited some suicide manifestations, 14.23% had experienced suicidal ideas in the last 3 months, 3.55% had had suicide attempts at least once in life, and 8.73% had suicidal ideas in the last 3 months with suicide attempts. The risk of suicidal behavior and /or mental disorders was explained jointly by depression (OR=27.9, 95% CI: 3.5-223. 1), low self-esteem (OR=11.8, 95% CI: 2.5-56.5), severe family dysfunction (OR=3.4, 95%CI 1.2-9.7), being female (OR=2.1, 95% CI: 1.2-3.8) and being 15 or older (OR=1.9, 95% CI: 0.967-3.9). Psychological abuse followed by physical mistreatment was associated with suicidal behavior and /or mental illness while good family relationships were associated to lower probability. Conclusion: Depression, low self-esteem, severe family dysfunction, female gender, older age (> 15) and domestic violence are risk factors associated with suicide and/or mental disorders in adolescents; good family relationships are associated with lower risk.

Isolation and characterization of a metal ion-dependent alkaline protease from a halotolerant Bacillus aquimaris VITP4.

A halotolerant bacterium Bacillus acquimaris VITP4 was used for the production of extracellular protease. Fractional precipitation using ammonium chloride was used to obtain the enzyme. The protease exhibited optimum activity at pH 8.0 and 40°C and retained 50% of its optimal proteolytic activity even in the presence of 4 M NaCl, suggesting that it is halotolerant. The molecular mass of protease, as revealed by SDS-PAGE was found to be 34 kDa and the homogeneity of the enzyme was confirmed by gelatin zymography and reverse-phase HPLC. Upon purification, the specific activity of th enzyme increased from 533 U/mg to 1719 U/mg. Protease inhibitors like phenyl methane sulphonyl fluoride and 2-mercaptoethanol did not affect the activity of the enzyme, but EDTA inhibited the activity, indicating the requirement of metal ions for activity. Cu­­­2+, Ni2+ and Mn2+ enhanced the enzyme activity, but Zn2+, Hg2+ and Fe2+ decreased the activity, while Mg2+, Ca2+ and K+ had no effect on the enzyme activity. The protease was quite stable in the presence of cationic (CTAB), anionic (SDS) and neutral detergents (Triton X-100 and Tween-20) and exhibited antimicrobial activity against selected bacterial and fungal strains. The stability characteristics and broad spectrum antimicrobial activity indicated the potential use of this protease in industrial applications.

Properties of alkaline protease genetically engineered on cell surface of the yeast Yarrowia lipolytica.

ALP2 gene encoding alkaline protease cloned from Aureobasidium pullulans HN2-3 was ligated into the surface display plasmid and expressed in the cells of the yeast Yarrowia lipolytica. The expressed alkaline protease was immobilized on the yeast cells. The activity of the immobilized enzyme with 6 His tag was found to be significantly higher than that of without 6 His tag. The immobilized enzyme showed lower optimal temperature and a lower affinity for azocasein than the free enzyme purified from A. pullulans HN2-3. The thermal stability of the immobilized enzyme enhanced and the pH stability decreased, compared to that of the free enzyme.

Endopeptidases of Bacillus subtilis IBTC-3 and B. alcalophilus PB92 in synthesis of precursors of biologically active peptides.

Two endopeptidases (from Bacillus subtilis IBTC-3 and from B. alcalophilus PB92-commercial preparation) efficiently synthesized amino acid esters (NAc-Tyr-OEt and NAc-Phe-OEt) and dipeptides (NAc-Tyr-Gly-NH2 and NAc-Tyr-Arg-NH2) in organic solvent/water systems. The rate of NAc-Tyr-OEt synthesis mediated by the native subtilisin IBTC-3 was maximum (0.23 Umg-1) in ethanol/5-7% w/v water system, while the highest activity of the freeze-dried enzyme (0.18 Umg-1) was achieved, when water content was 9-10% w/v. The preferred system for dipeptide synthesis (using NAc-Tyr-OEt as acyl donor) by both the enzymes was acetonitrile/4% w/v water. In this system, the maximum yield of NAc-Tyr-GlyNH2 was 71 and 80% and that of NAc-Tyr-Arg-NH2 was 53 and 40% for subtilisin IBTC-3 and peptidase PB92, respectively. In contrast to the peptidase PB92, the subtilisin efficiently catalyzed esterification of NAc-Tyr with 1-butanol and isopropanol.

Purification and characterization of a small size protease from Bacillus sp. APR-4.

A thermostable extracellular protease of Bacillus sp. APR-4 was purified by size-exclusion and ion-exchange chromatographic methods and its properties were studied. The purified enzyme had a specific activity of 21,000 U/mg of protein and gave single band on SDS/PAGE with a molecular mass of 16.9 KDa. This protease had an optimal pH of 9 and exhibited its highest activity at 60 degrees C. The enzyme activity was inhibited by EDTA, suggesting the presence of metal residue at the active site. Ca2+ (5 mM) had stabilising effect on the activity of protease, but Cu2+ (5 mM) had inhibitory effect. The enzyme exhibited highest specificity towards casein (1%) and had a Km of 26.3 mg/ml and a Vmax of 47.6 U/mg with casein as a substrate. The stability of this enzyme was evaluated in the presence of some organic solvents and the enzyme was stable in methanol, petroleum ether and ethanol. Detergents (Wheel, Farishta) had stimulatory effect on the activity of this enzyme.

Vaccine potential of 56-66 kDa protease secreted by Entamoeba histolytica.

Excretory/secretory (ES) antigens and sub-cellular fractions of E. histolytica (HM1:IMSS strain) were tested for the presence of common proteases using substrate gel electrophoresis. We obtained two E. histolytica proteases (56-66 kDa and 29 kDa) from ES material, soluble components and plasma membrane. Protease 56-66 kDa from ES antigen was selected for immunizing hamsters because it gave a consistent broad band. We observed 62.5 per cent protection in immunized animals, compared to 0 per cent in unimmunized controls. Although all vaccinated golden hamsters showed high antibody response, there was no correlation between antibody titres and protection. 56-66 kDa ES protease could thus prevent disease and could be a candidate molecular vaccine against amoebiasis.

Changes in protein profile during coagulation of latex from Carica papaya

We describe the changes in peptide composition by SDS-PAGE analysis of latex from Carioca papaya collected at various times after incision of the unripe fruit. The data show that during latex coagulation several peptides are processed in an orderly fashion.

Comparison of thermostable phosphatase and proteases from thermophilically disposed transition species and thermophilic actinomycetes.

Thermoactinomycetes vulgaris is a thermophilic actinomycetes growing optimally at 50 degrees C and Streptomyces albus, S. coelicolor, S. fasciculus and S. olivochromogenes are thermophilically disposed transition species of actinomycetes, which have optimum biomass at 40 degrees C. The acid/alkaline phosphatase and acid/alkaline/neutral protease enzyme from Streptomycetes species showed enzyme activity up to 90 degrees C. In comparison to phosphatases and proteases from T. vulgaris it was concluded that these thermophilically disposed transition species showed macromolecular thermostability i.e. thermostable enzymes and protein.