Purification, characterization, and enzyme kinetics of a glutathione S transferase from larvae of the camel tick Hyalomma dromedarii.

BACKGROUND: Glutathione s-transferases (GSTs) perform an essential role in detoxification of xenobiotics and endogenous compounds via their conjugation to reduce glutathione. RESULTS: A GST enzyme, designated tick larvae glutathione S transferase (TLGST), was purified from larvae of the camel tick Hyalomma dromedarii via ammonium sulfate precipitation, glutathione-Sepharose affinity column and Sephacryl S-300 chromatography. TLGST-specific activity was found to be 1.56 Umg-1 which represents 39 folds and 32.2% recovery. The molecular weight of TLGST purified from camel tick larvae was found as 42 kDa by gel filtration. TLGST has a pI value of 6.9 and was found a heterodimeric protein of 28 and 14 kDa subunits as detected on SDS-PAGE. The Lineweaver-Burk plot calculated the km for CDNB to be 0.43 mM with Vmax value of 9.2 Umg-1. TLGST exhibited its optimal activity at pH 7.9. Co2+, Ni2+ and Mn2+ increased the activity of TLGST while Ca2+, Cu2+, Fe2+ and Zn2+ inhibited it. TLGST was inhibited by cumene hydroperoxide, p-hydroxymercuribenzoate, lithocholic acid, hematin, triphenyltin chloride, p-chloromercuribenzoic acid (pCMB), N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK), iodoacetamide, EDTA and quercetin. pCMB inhibited TLGST competitively with Ki value of 0.3 mM. CONCLUSIONS: These findings will help to understand the various physiologic conditions of ticks and targeting TLGST could be significant tool for development of prospective vaccines against ticks as a bio-control strategy to overcome the rapid grows in pesticide-resistant tick populations.

Influence of probiotics on water quality in intensified Litopenaeus vannamei ponds under minimum-water exchange.

The effects of two probiotics on NH3 degradation, as well as the magnetic field (21.56 m tesla) on the germination and proliferation of Bacillus spores, were studied in-vitro. Additionally, the effect of these probiotics on water quality maintenance in Litopenaeus vannamei holding ponds was investigated. For 180 min, NH3 degradation was assessed as follows: Set 1: ammonia-free tap water with NH3; Probiotic A (5 × 1010 viable Bacillus spores/g) with NH3; Probiotic B (multi spp. 2 × 109 CFU/g) with NH3; and Set 2: same as set 1 with 30 mg L-1 OM. The magnetic field was tested on Probiotic A (3.5 × 107 CFU) for 36 h in triplicate. In the presence of organic matter, both probiotics degrade NH3. The viable Bacillus count increased within 6 h of being exposed to the magnetic field, reaching its peak after 36 h. Firstly, fifteen ponds (250,000 PL/acre) were investigated, then 360 water samples were collected from the same corresponding pond for 8 weeks, and subjected to T1: control; T2: Probiotic A (0.007 g/m3/2 weeks); T3: Probiotic B (0.03 g/m3/2 weeks). Both probiotics with TVC and NH3 demonstrated a negative correlation, on the other hand, they showed a significant (P ≤ 0.01) improvement in DO and pH. Overall, both probiotics were able to degrade NH3 and the magnetic field (21.56 m tesla) was efficient to improve the germination and proliferation of Bacillus spores in-vitro. Probiotics were also effective for reducing TVC and NH3 levels by increasing dissolved oxygen and pH in pond water.

Phospholipase A2 enzyme from the venom of Egyptian honey bee Apis mellifera lamarckii with anti-platelet aggregation and anti-coagulation activities.

BACKGROUND: Honey bee venom contains various enzymes with wide medical and pharmaceutical applications. RESULTS: The phospholipase A2 (PLA2) has been apparently purified from the venom of Egyptian honey bee (Apis mellifera lamarckii) 8.9-fold to a very high specific activity of 6033 U/mg protein using DEAE-cellulose and Sephacryl S-300 columns. The purified bee venom PLA2 is monomeric 16 kDa protein and has isoelectric point (pI) of 5.9. The optimal activity of bee venom PLA2 was attained at pH 8 and 45 °C. Cu2+, Ni2+, Fe2+, Ca2+, and Co2+ exhibited a complete activating effect on it, while Zn2+, Mn2+, NaN3, PMSF, N-Methylmaleimide, and EDTA have inhibitory effect. CONCLUSIONS: The purified bee venom PLA2 exhibited anti-platelet aggregation and anti-coagulation activities which makes it promising agent for developing novel anti-clot formation drugs in future.

A simple and efficient purification method of native immunoreactive antigen for diagnosis of camel hydatidosis.

BACKGROUND: Cystic echinococcosis (CE), a zoonotic disease that affects animal and human health, is of increasing economic importance due to high morbidity rates and high economic losses in the livestock industry. AIM: The present study was conducted to purify the antigen from hydatid cyst fluid (HCF) with high diagnostic efficacy of camel hydatidosis using indirect enzyme-linked immunosorbent assay (ELISA). MATERIALS AND METHODS: The HCF antigen was purified using Sephacryl S-300 column chromatography. Characterization of fractions was performed using reducing and non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Further, antibodies against Echinococcus granulosus cysts in camel serum were detected using indirect ELISA. RESULTS: The purification process resulted in three fractions of antigens: FI, FII, and FIII. Indirect ELISA showed that higher diagnostic efficacy was observed in FI than in FII and FIII. Indirect ELISA, in which FI was utilized, showed 88% sensitivity and 91.7% specificity. Non-reducing SDS-PAGE showed that FI had two bands of molecular weights 120 and 60 kDa. Western blot analysis of FI demonstrated that 60, 38, and 22 kDa were antigenic bands when reacted with naturally infected camel sera with E. granulosus cysts. Using indirect ELISA, F1 recorded an infection percentage of 81.7% in randomly collected camel serum samples. CONCLUSION: FI is a promising antigen for accurate diagnosis of camel CE using indirect ELISA.

Apyrase with anti-platelet aggregation activity from the nymph of the camel tick Hyalomma dromedarii.

Apyrase is one of the essential platelet aggregation inhibitors in hematophagous arthropods due to its ability to hydrolyze ATP and ADP molecules. Here, an apyrase (TNapyrase) with antiplatelet aggregation activity was purified and characterized from the nymphs of the camel tick Hyalomma dromedarii through anion exchange and gel filtration columns. The homogeneity of TNapyrase was confirmed by native-PAGE, SDS-PAGE as well as with isoelectric focusing. Purified TNapyrase had a molecular mass of 25 kDa and a monomer structure. TNapyrase hydrolyzed various nucleotides in the order of ATP > PPi > ADP > UDP > 6GP. The Km value was 1.25 mM ATP and its optimum activity reached at pH 8.4. The influence of various ions on TNapyrase activity showed that FeCl2, FeCl3 and ZnCl2 are activators of TNapyrase. EDTA inhibited TNapyrase activity competitively with a single binding site on the molecule and Ki value of 2 mM. Finally, TNapyrase caused 70% inhibition of ADP-stimulated platelets aggregation and is a possible target for antibodies in future tick vaccine studies.