Protease-mediated prophenoloxidase activation in the haemolymph of American cockroach Periplaneta americana

Prophenoloxidase has been successfully obtained from the haemolymph of the cockroach Periplaneta americana using cane sugar saline solution. The proenzyme was activated by various exogenously added proteases such as chymotrypsin, trypsin, subtilisin and thermolysin. Thermolysin was found to be the greatest activator, followed by chymotrypsin and subtilisin. Chymotrypsin activation showed a lag period when compared with the other proteases tested, indicating that activation by chymotrypsin followed an indirect path, whereas, subtilisin and thermolysin activated the proenzyme directly.Exogenously added protease inhibitor showed inhibition towards protease-mediated prophenoloxidase activation. Benzamidine inhibited chymotrypsin and trypsin activation, whereas soybean trypsin inhibitor inhibited trypsin. In situ inhibitor isolated from the haemocytes of Periplaneta americana inhibited the prophenoloxidase activation and showed evidence for the presence of a built-in inhibition system for the release of the components of the prophenoloxidase activating system of P. americana. Electrophoretic localization of activated phenoloxidase showed two bands, suggesting the dimeric condition of high mol. wt prophenoloxidase.     

Cumulative exposure and dietary risk assessment of phthalates in bottled water and bovine milk samples: A preliminary case study in Tamil Nadu,India

Exposure to phthalates may cause adverse health effects in wildlife and humans. Study on phthalates exposure and risk is limited in the Indian context. Therefore, this preliminary investigation was performed to ascertain the phthalates exposure through bottled water and milk among the Indian sub-population. Phthalates were extracted from water and milk by solid-phase and ultrasonication methods, respectively, and analysis was performed using gas chromatography–mass spectrometry. Total phthalates in bottled water and milk were in the range of 39–7820 ng/L and 56–686 ng/g, respectively, with the highest contribution from diethylhexyl phthalate (DEHP). A substantial increase in phthalates concentration in bottled water was observed with increased shelf life. Total mean phthalates in packed milk (245 ng/g) and raw milk (134 ng/g) shows potential enrichment during “farm to table” process. Among phthalates, the lowest risk was expected from diethyl phthalate, whereas the highest risk was observed for DEHP with cumulative dietary exposure of 0.23 μg/kg bw/day (median). The human health risk based on tolerable daily intake and reference dose was found safe. This is the first study reporting phthalates migration in packed commodities from a developing country, India, which further warrants extensive phthalates exposure assessment to understand its effect on public health.     

Protective Effects of 7‐Hydroxycoumarin on Dyslipidemia and Cardiac Hypertrophy in Isoproterenol‐Induced Myocardial Infarction in Rats

This study evaluates the protective effects of 7‐hydroxycoumarin (7‐HC) on dyslipidemia and cardiac hypertrophy in isoproterenol (ISO) induced myocardial infarction (MI) in rats. Rats were pre‐ and co treated with 7‐HC (16 mg/kg) daily for 8 days. ISO (100 mg/kg) was subcutaneously injected into rats on seventh and eighth days to induce MI. Increased activity/levels of serum creatine kinase‐MB (CK‐MB), troponin‐T, plasma lipid peroxidation products, and altered levels of lipids in the serum and heart and serum lipoproteins were noted in ISO‐induced rats. ISO‐induced myocardial infarcted rats revealed increased hypertrophy (cardiac and left ventricular) and hepatic 3‐hydroxyl 3‐methylglutaryl‐coenzyme‐A reductase (HMG‐CoA reductase) activity. Pre and cotreatment with 7‐HC revealed significant protective effects on all the biochemical parameters evaluated. The in vitro study demonstrated its free radical scavenging property. Thus, 7‐HC protects ISO‐induced MI in rats by its free radical scavenging and antihyperlipidaemic and antihypertrophic properties.     

Modulatory Effect of Taurine on 7,12‐Dimethylbenz(a)Anthracene‐Induced Alterations in Detoxification Enzyme System,Membrane Bound Enzymes,Glycoprotein Profile and Proliferative Cell Nuclear Antigen in Rat Breast Tissue

The modulatory effect of taurine on 7,12‐dimethylbenz(a)anthracene (DMBA)‐induced breast cancer in rats was studied. DMBA (25 mg/kg body weight) was administered to induce breast cancer in rats. Protein carbonyl levels, activities of membrane bound enzymes (Na⁺/K⁺ATPase, Ca²⁺ATPase, and Mg²⁺ATPase), phase I drug metabolizing enzymes (cytochrome P450, cytochrome b5, NADPH cytochrome c reductase), phase II drug metabolizing enzymes (glutathione‐S‐transferase and UDP‐glucuronyl transferase), glycoprotein levels, and proliferative cell nuclear antigen (PCNA) were studied. DMBA‐induced breast tumor bearing rats showed abnormal alterations in the levels of protein carbonyls, activities of membrane bound enzymes, drug metabolizing enzymes, glycoprotein levels, and PCNA protein expression levels. Taurine treatment (100 mg/kg body weight) appreciably counteracted all the above changes induced by DMBA. Histological examination of breast tissue further supported our biochemical findings. The results of the present study clearly demonstrated the chemotherapeutic effect of taurine in DMBA‐induced breast cancer.     

Importance of long-range interactions in protein folding

Long-range interactions play an active role in the stability of protein molecules. In this work, we have analyzed the importance of long-range interactions in different structural classes of globular proteins in terms of residue distances. We found that 85% of residues are involved in long-range contacts. The residues occurring in the range of 4-10 residues apart contribute more towards long-range contacts in all-alpha proteins while the range is 11-20 in all-beta proteins. The hydrophobic residues Cys, Ile and Val prefer the 11-20 range and all other residues prefer the 4-10 range. The residues in all-beta proteins have an average of 3-8 long-range contacts whereas the residues in other classes have 1-4 long-range contracts. Furthermore, the preference of residue pairs to the folding and stability will be discussed.     

Pathogenicity, antibiotic susceptibility and genetic similarity of environmental and clinical isolates of Vibrio cholerae

Isolates of Vibrio cholerae were obtained from clinical and environmental samples and the pathogenicity of these isolates was confirmed by hemolytic assay. The clinical isolates were more pathogenic than environmental isolates. Antibiotic susceptibility of V. cholerae to a set of antibiotics showed a marked variation. The environmental isolates exhibited more resistance to the antibiotics than clinical isolates. The plasmid curing technique was used to check the encoding of antibiotic resistance gene in genome. In both isolates, the resistance to vancomycin and co-trimaxazole was not mediated by plasmid and it may probably be encoded in genome. RAPD method was adopted to find out the variation in the genome of the clinical isolates and environmental isolates of V. cholerae. The genomic similarity pattern revealed that the environmental Ogawa isolates were closely related to clinical Ogawa isolates. This study confirmed the existence of the complex nature of V. cholerae in its pathogenicity, response to a set of antibiotics and genetic similarity.     

Pairwise contact energy statistical potentials can help to find probability of point mutations

To adopt a particular fold, a protein requires several interactions between its amino acid residues. The energetic contribution of these residue–residue interactions can be approximated by extracting statistical potentials from known high resolution structures. Several methods based on statistical potentials extracted from unrelated proteins are found to make a better prediction of probability of point mutations. We postulate that the statistical potentials extracted from known structures of similar folds with varying sequence identity can be a powerful tool to examine probability of point mutation. By keeping this in mind, we have derived pairwise residue and atomic contact energy potentials for the different functional families that adopt the (α/β)₈ TIM‐Barrel fold. We carried out computational point mutations at various conserved residue positions in yeast Triose phosphate isomerase enzyme for which experimental results are already reported. We have also performed molecular dynamics simulations on a subset of point mutants to make a comparative study. The difference in pairwise residue and atomic contact energy of wildtype and various point mutations reveals probability of mutations at a particular position. Interestingly, we found that our computational prediction agrees with the experimental studies of Silverman et al. (Proc Natl Acad Sci 2001;98:3092–3097) and perform better prediction than i_Mutant and Cologne University Protein Stability Analysis Tool. The present work thus suggests deriving pairwise contact energy potentials and molecular dynamics simulations of functionally important folds could help us to predict probability of point mutations which may ultimately reduce the time and cost of mutation experiments. Proteins 2016; 85:54–64. © 2016 Wiley Periodicals, Inc.