Isolation and characterization of a serine proteinase with thrombin-like activity from the venom of the snake Bothrops asper

A serine proteinase with thrombin-like activity was isolated from the venom of the Central American pit viper Bothrops asper. Isolation was performed by a combination of affinity chromatography on aminobenzamidine-Sepharose and ion-exchange chromatography on DEAE-Sepharose. The enzyme accounts for approximately 0.13% of the venom dry weight and has a molecular mass of 32 kDa as determined by SDS-PAGE, and of 27 kDa as determined by MALDI-TOF mass spectrometry. Its partial amino acid sequence shows high identity with snake venom serine proteinases and a complete identity with a cDNA clone previously sequenced from this species. The N-terminal sequence of the enzyme is VIGGDECNINEHRSLVVLFXSSGFL CAGTLVQDEWVLTAANCDSKNFQ. The enzyme induces clotting of plasma (minimum coagulant dose = 4.1 µg) and fibrinogen (minimum coagulant dose = 4.2 µg) in vitro, and promotes defibrin(ogen)ation in vivo (minimum defibrin(ogen)ating dose = 1.0 µg). In addition, when injected intravenously in mice at doses of 5 and 10 µg, it induces a series of behavioral changes, i.e., loss of the righting reflex, opisthotonus, and intermittent rotations over the long axis of the body, which closely resemble the `gyroxin-like' effect induced by other thrombin-like enzymes from snake venoms.

Reduced cortical renal GLUT1 expression induced by angiotensin-converting enzyme inhibition in diabetic spontaneously hypertensive rats

Diabetes in spontaneously hypertensive rats is associated with cortical renal GLUT1 and GLUT2 overexpression. Our objective was to evaluate the effect of the angiotensin-converting enzyme blockade on cortical renal GLUT1 and GLUT2 expression, urinary albumin and urinary TGF-β1. Streptozotocin, 50 mg/kg, or citrate buffer (N = 16) was administered as a single injection into the tail vein in adult spontaneously hypertensive rats (~260 g). Thirty days later, these diabetic spontaneously hypertensive rats received ramipril by gavage: 0.01 mg·kg-1·day-1 (D0.01, N = 14), 1 mg·kg-1·day-1 (D1, N = 9) or water (D, N = 11) for 15 days. Albumin and TGF-β1 (24-h urine), direct arterial pressure, renal tissue angiotensin-converting enzyme activity (fluorometric assay), and GLUT1 and GLUT2 protein levels (Western blot, renal cortex) were determined. Glycemia and glycosuria were higher (P < 0.05) in the diabetic rats compared with controls, but similar between the diabetic groups. Diabetes in spontaneously hypertensive rats lowered renal tissue angiotensin-converting enzyme activity (40%), which was reduced further when higher ramipril doses were used. Diabetes associated with hypertension raised GLUT1 by 28% (P < 0.0001) and GLUT2 by 76% (P = 0.01), and both doses of ramipril equally reduced cortical GLUT1 (D vs D1 and vs D0.01, P ≤ 0.001). GLUT2 levels were reduced in D0.01 (P < 0.05 vs D). Diabetes increased urinary albumin and TGF-β1 urinary excretion, but the 15-day ramipril treatment (with either dose) did not reduce them. In conclusion, ramipril is effective in lowering renal tissue angiotensin-converting enzyme activity, as well as blocking cortical GLUT1 overexpression, which may be beneficial in arresting the development of diabetic nephropathy.

Inhibition of cyclooxygenase activity by standardized hydroalcoholic extracts of four Asteraceae species from the Argentine Puna

We determined the anti-inflammatory activity of standardized extracts of four medicinal plant species (Baccharis incarum, B. boliviensis, Chuquiraga atacamensis, Parastrephia lucida) that grow in the Argentine Puna (3800 m above sea level) and that are used to reduce oxidative stress and alleviate gout and arthritic pain. The extracts of plant aerial parts were standardized in terms of total phenolic compounds and flavone/flavanone content and free radical scavenging activity. All extracts showed high phenolic compound concentration (0.5-1.6 mg/mL), mainly flavones and flavonols (0.1-0.8 mg/mL). The extracts showed hydrogen donating ability (DPPH and ABTS) and reactive oxygen species scavenging activity (O2●-, OH-, H2O2). The ability of the extracts to inhibit cyclooxygenase enzymes (COX-1 and COX-2) was determined by calculating percent inhibition of PGE2 production measured by enzyme immunoassay. All extracts inhibited both enzymes with IC50 values of 2.0 to 16.7 µg/mL. The anti-inflammatory activity of B. incarum and C. atacamensis extracts was higher than that of B. boliviensis and P. lucida. The IC50 values obtained for indomethacin were 0.11 and 0.78 µM for COX-1 and COX-2, respectively. The present results are consistent with the anecdotal use of these species in phytotherapic preparations.

Construction of a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen and mouse 4-1BBL genes and its effect on dendritic cells

Our aim was to construct a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen (tPSMA) and mouse 4-1BBL genes and to determine its effect on dendritic cells (DCs) generated from bone marrow suspensions harvested from C57BL/6 mice for which the effect of 4-1BBL on DCs is not clear, especially during DCs processing tumor-associated antigen. Replication deficient adenovirus AdMaxTM Expression System was used to construct recombinant adenovirus Ad-tPSMA-internal ribosome entry site-mouse 4-1BBL (Ad-tPSMA-IRES-m4-1BBL) and Ad-enhanced green fluorescent protein. Day 7 proliferating DC aggregates generated from C57BL/6 mice were collected as immature DCs and further mature DCs were obtained by lipopolysaccharide activated immature DCs. After DCs were exposed to the recombinant adenovirus with 250 multiplicity of infection, the expression of tPSMA and m4-1BBL proteins were detected by Western blot, and the apoptosis and phenotype of DCs were analyzed by flow cytometry. Cytokines (IL-6 and IL-12) in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Proliferation of T cells was detected by allogeneic mixed lymphocyte reactions. The tPSMA and m4-1BBL proteins were expressed correctly. The apoptosis rate of DCs transfected with Ad-tPSMA-IRES-m4-1BBL was 14.6%, lower than that of control DCs. The expression of co-stimulatory molecules [CD80 (81.6 ± 5.4%) and CD86 (80.13 ± 2.81%)] up-regulated in Ad-tPSMA-IRES-m4-1BBL-pulsed DCs, and the level of IL-6 (3960.2 ± 50.54 pg/mL) and IL-12 (249.57 ± 12.51 pg/mL) production in Ad-tPSMA-IRES-m4-1BBL-transduced DCs were significantly higher (P < 0.05) than those in control DCs. Ad-tPSMA-IRES-m4-1BBL induced higher T-cell proliferation (OD450 = 0.614 ± 0.018), indicating that this recombinant adenovirus can effectively enhance the activity of DCs.

The chronic blockade of angiotensin I-converting enzyme eliminates the sex differences of serum cytokine levels of spontaneously hypertensive rats

Sex hormones modulate the action of both cytokines and the renin-angiotensin system. However, the effects of angiotensin I-converting enzyme (ACE) on the proinflammatory and anti-inflammatory cytokine levels in male and female spontaneously hypertensive rats (SHR) are unclear. We determined the relationship between ACE activity, cytokine levels and sex differences in SHR. Female (F) and male (M) SHR were divided into 4 experimental groups each (n = 7): sham + vehicle (SV), sham + enalapril (10 mg/kg body weight by gavage), castrated + vehicle, and castrated + enalapril. Treatment began 21 days after castration and continued for 30 days. Serum cytokine levels (ELISA) and ACE activity (fluorimetry) were measured. Male rats exhibited a higher serum ACE activity than female rats. Castration reduced serum ACE in males but did not affect it in females. Enalapril reduced serum ACE in all groups. IL-10 (FSV = 16.4 ± 1.1 pg/mL; MSV = 12.8 ± 1.2 pg/mL), TNF-α (FSV = 16.6 ± 1.2 pg/mL; MSV = 12.8 ± 1 pg/mL) and IL-6 (FSV = 10.3 ± 0.2 pg/mL; MSV = 7.2 ± 0.2 pg/mL) levels were higher in females than in males. Ovariectomy reduced all cytokine levels and orchiectomy reduced IL-6 but increased IL-10 concentrations in males. Castration eliminated the differences in all inflammatory cytokine levels (IL-6 and TNF-α) between males and females. Enalapril increased IL-10 in all groups and reduced IL-6 in SV rats. In conclusion, serum ACE inhibition by enalapril eliminated the sexual dimorphisms of cytokine levels in SV animals, which suggests that enalapril exerts systemic anti-inflammatory and anti-hypertensive effects.

Determination of pectin methylesterase activity in commercial pectinases and study of the inactivation kinetics through two potentiometric procedures

Pectinases are enzymes that degrade pectic substances and are widely used in juice and fruit beverages to improve the quality of the process. The objective of this study was to determine the optimum pH and temperature of two samples of commercial pectinases and propose an alternative procedure to determine the residual activity comparing the data with those of the traditional procedure. The pectin methylesterase (PME) activity in Pectinex 100 L Plus and Panzyn Clears was determined by potentiometry. The reaction consisted of 5.00 mg.mL-1 apple pectin, 0.100 mol.L-1 NaCl, and 50 µL enzyme to a total volume of 30 mL. The pectin reaction in the presence of PME in all experiments revealed a first order kinetics. The PME in the two enzyme preparations showed higher activity at pH 4.0 to 4.5 and temperature of 45 ºC. From the results of both procedures ΔV NaOH/Δt and ΔpH/Δt, it was concluded that the inactivation of PME occurred at 75 ºC. The results obtained from the ratio ΔpH/Δt showed good correlation with those obtained from the ratio ΔV NaOH/Δt. In the reaction accompanied by the ratio ΔpH/Δt, the release of H3O+ occurred in the real time reaction.

Influence of carbon source, pH, and temperature on the polygalacturonase activity of Kluyveromyces marxianus CCMB 322

Microbial pectinolytic enzymes are known to play a commercially important role in a number of industrial processes. Two kinds of yeast can be discerned regarding the production of enzymes. One group includes those which can produce enzymes in the absence of an inducer, and the other group comprises the yeasts that produce enzymes in the presence of an inducer. The objective of this study was to investigate the influence of pectic substances, glucose, pH, and temperature on the polygalacturonase activity by Kluyveromyces marxianus CCMB 322. The yeast was grown in a fermentation broth containing different concentrations of glucose and pectic substances. The polygalacturonase activity was determined by the DNS method, and the pH and temperature were optimized using a central composite experimental design. The polygalacturonase secreted by K. marxianus CCMB 322 was partially constitutive showing optimum pH and temperature of 7.36 and 70 °C, respectively, and maintained approximately 93% of its original activity for 50 minutes at 50 °C. Thermal stability of the polygalacturonase enzyme was studied at different temperatures (50, 60, 70, and 80 °C) and different incubation times (0, 10, 20, 30, 40, and 50 minutes). This study showed that glucose can influence the regulation of the synthesis of polygalacturonase.

Investigation of glutathione peroxidase activity in chicken meat under different experimental conditions

Due to the fact that previous studies on the enzymatic activity of Glutathione peroxidase (GSH-Px) diverge widely in their methodology and results, this study aimed to investigate the influence of different analytical conditions on GSH-Px activity in chicken thighs from broilers that were fed different diets with different sources and concentrations of selenium. GSH-Px activity was evaluated six hours after slaughter and 120 days after frozen storage at -18 ºC. The different analytical conditions included time of pre-incubation (0, 10 and 30 minutes), reaction medium, types of substrate (H2O2 (0.72 mM, 7.2 mM, and 72 mM) and Terc-butil hydroperoxide 15 mM), and different buffer concentrations (buffer 1, potassium phosphate 50 mM pH 7.0 + EDTA 1 mM + mercaptoethanol 1 mM, and buffer 2, tris-HCl 50 mM pH 7.6 + EDTA 1 mM + mercapthanol 5 mM). The results show that the highest GSH-Px activity was observed when enzyme and substrate were in contact at 22 ºC without any pre-incubation, and that, when used at concentrations above 0.72 mM, hydrogen peroxide saturated the GSH-Px enzyme and inhibited its activity. The enzyme presented higher affinity to hydrogen peroxide when compared to terc-butil peroxide, and the addition of a buffer containing mercaptoethanol did not increase GSH-Px enzymatic activity. The activity of GSH-Px was not influenced by the source and concentration of selenium in the diet either. The obtained results allowed the determination of the best temperature of contact between the enzyme and substrate (22 ºC), the optimum concentration, and the type of substrate and buffer to be used. This information is extremely useful for future studies on GSH-Px activity in meat due to the divergence and little information found in the literature.

Antioxidant activity of black bean (Phaseolus vulgaris L.) protein hydrolysates

Abstract The objective of this work was to study the effect of enzymatic hydrolysis of black bean protein concentrate using different enzymes. Bean proteins were extracted and hydrolyzed over a period of 120 min using the enzymes pepsin or alcalase. The protein hydrolysates’ molecular weight was assayed by electrophoresis and the antioxidant activity was evaluated by the capturing methods of free radicals ABTS●+ and DPPH. Electrophoretic results showed that the bands above 50 kDa disappeared, when the beans protein was subjected to hydrolysis with pepsin. The bean protein hydrolysate obtained by hydrolysis with alcalase enzyme, showed higher antioxidant activity for inhibition of the radical ABTS●+. However, the hydrolysates obtained by hydrolysis with pepsin had higher antioxidant activity for inhibition of the radical DPPH. The use of pepsin and alcalase enzymes, under the same reaction time, produced black bean protein hydrolysates with different molecular weight profiles and superior antioxidant activity than the native bean protein.

Action of gibberellic acid (GA3) on dormancy and activity of alpha-amylase in rice seeds

To evaluate the effectiveness of gibberellic acid (GA3) in breaking rice seed dormancy and the use of alpha-amylase enzyme activity as an indicator of the dormancy level, seed from the intensively dormant irrigated cultivar Urucuia were used. The seeds were submitted to a pre-drying process in a forced air circulation chamber under 40ºC during 7 days and submersed in 30 mL of GA3 solution under 0, 10, 30 and 60 mg/L H2O concentrations, during 2, 24 and 36 hours. After the treatments, the alpha-amylase activity was determined by using the polyacrilamide electrophoresis and spectrophotometry. At the same time, the germination test was made. The results indicated a gain in germination and in alpha-amylase activity in higher concentrations and soaking time of seeds in GA3. These observations support the conclusion that soaking seed in 60 mg GA3/L during 36 hours can be used as a quick and efficient treatment in breaking rice seed dormancy and is equivalent to the forced air circulation chamber at 40ºC during 7 days. The alpha-amylase enzyme activity proved to be as an efficient marker of the seeds dormancy level.

Intra-population plasticity of Anopheles darlingi's (Diptera, Culicidae) biting activity patterns in the state of Amapá, Brazil

OBJECTIVE: To assess the variation in Anopheles darlingi's biting activity compared to An. marajoara in the same locality and to biting activity data from other regions. METHODS: Using human bait, eight observations of the biting activity of An. darlingi and An. marajoara were carried out during 1999 and 2000 in the municipality of São Raimundo do Pirativa, state of Amapá, Brazil. Each observation consisted of three consecutive 13-hour collections, close to full moon. There were shifts of collectors in the observation points and nocturnal periods. RESULTS: An. darlingi revealed considerable plasticity of biting activity in contrast to An. marajoara, which showed well-defined crepuscular biting peaks. No significant correlation between density and biting activity was found, but a significant correlation existed between time and proportional crepuscular activity, indicating underlying ecological processes not yet understood. Two of the four available data sets having multiple observations at one locality showed considerable plasticity of this species' biting patterns as well. CONCLUSION: Intra-population variation of biting activity can be as significant as inter-population variation. Some implications in malaria vector control and specific studies are also discussed.