Purification and characterization of extracellular alkaline protease from Streptomyces sp. LCJ12A isolated from Pichavaram mangroves

Extracellular protease from culture supernatants of Streptomyces sp. LCJ12A was purified and characterized in thisstudy. The collected supernatant of Streptomyces sp. LCJ12A was purified by ammonium sulfate precipitation anddiethylaminoethyl cellulose chromatography. The molecular weight of the enzyme was 20.1 kDa. Sodium dodecylsulfate-polyacrylamide gel electrophoresis was used to determine the molecular weight of the enzyme. The culture filtrate,partially purified enzyme, and ammonium sulfate precipitate displayed catalysis and stability over pH 3–11.5 and showedoptimal activity at pH 10. The culture filtrate, ammonium sulfate precipitate, and partially purified enzyme were goodbetween 30 and 40°C and the optimum temperature was 35°C. The purified protease exhibited high catalytic activityand stability under different pH and temperature. The partially purified protease from Streptomyces sp. LCJ12A showeda substantial relative activity of 78% with bovine serum albumin (BSA), 43% with gelatin, and 96% relative activitywith casein. Lineweaver–Burk plot was used to calculate the Km and Vmax values for the partially purified protease.The Km values of Streptomyces sp. LCJ12A were 73.5 mM for casein, 44.54 mM for BSA, and 43.45 mM for gelatin.The Vmax values were 500 mM min−1 for casein, 303.03 mM min−1 for BSA, and 270.27 mM min−1 for gelatin fromStreptomyces sp. LCJ12A. The statistical analysis confirmed that compared to the other substrates, casein was significant.

Specification of Bacteriophage Isolated Against Clinical Methicillin-Resistant Staphylococcus Aureus

OBJECTIVES: The emergence of resistant bacteria is being increasingly reported around the world, potentially threatening millions of lives. Amongst resistant bacteria, methicillin-resistant Staphylococcus aureus (MRSA) is the most challenging to treat. This is due to emergent MRSA strains and less effective traditional antibiotic therapies to Staphylococcal infections. The use of bacteriophages (phages) against MRSA is a new, potential alternate therapy. In this study, morphology, genetic and protein structure of lytic phages against MRSA have been analysed. METHODS: Isolation of livestock and sewage bacteriophages were performed using 0.4 μm membrane filters. Plaque assays were used to determine phage quantification by double layer agar method. Pure plaques were then amplified for further characterization. Sulfate-polyacrylamide gel electrophoresis and random amplification of polymorphic DNA were run for protein evaluation, and genotyping respectively. Transmission electron microscope was also used to detect the structure and taxonomic classification of phage visually. RESULTS: Head and tail morphology of bacteriophages against MRSA were identified by transmission electron microscopy and assigned to the Siphoviridae family and the Caudovirales order. CONCLUSION: Bacteriophages are the most abundant microorganism on Earth and coexist with the bacterial population. They can destroy bacterial cells successfully and effectively. They cannot enter mammalian cells which saves the eukaryotic cells from lytic phage activity. In conclusion, phage therapy may have many potential applications in microbiology and human medicine with no side effect on eukaryotic cells.

Alterations in Calcium-Binding Properties of Sarcoplasmic Reticulum Membrane Proteins Following Cardiac Injury.

The objective of present work is to investigate metabolic alterations associated with heart failure, particularly one of its manifestations, a sustained hypocalcemia that causes hemodynamic changes contributed to subsequent myocardial injury. Comparative study was carried out using experimental models of pancreatic necrosis (PN) and crush syndrome (CS) accompanied by cardiac damage down to myocardial infarction. Study design: Wistar adult male rats randomly divided into groups (n=12/group). The controls are healthy intact animals. The pancreatic necrosis (PN) and crush syndrome (CS) groups were then randomly subdivided: PN group- into 3, 24 and 72 h groups concerning hemorrhage, early and late pancreatic necrosis respectively; CS group – into 2, 4, 24, and 48 h decompression stages. The rats were sacrificed to analyze spectra and calcium-binding properties of the membrane proteins isolated from the cardiomyocyte sarcoplasmic reticulum (SR). Development of pathological changes in the heart and pancreas were also monitored. Place and Duration of Study: Department of Pathological Biochemistry and Radioisotope Methods, H. Buniatyan Institute of Biochemistry of Natl. Acad. Sci (NAS), Republic of Armenia (RA). Experiments conducted between May 2011 and October 2013. Methodology: To study pathogenesis of hypocalcemia underlying myocardial damage a translocation of radioactive 45CaCI2 into cardiomyocytes and its intracellular distribution was examined. Binding of 45Ca2+ to the SR membrane proteins was measured after proteins separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) and radioactivity from the gel plates was counted by a gas-flow meter Berthold–II. Isoelectric focusing of the protein isolated from the SR of cardiomyocyte was performed. Results: Statistically significant changes in mean radio labeled calcium incorporation into a total protein fraction of the cardiomyocyte SR from control (13682±271) were determined by3h of PN (23055±168, P<.001), 24 h of PN (22876±240, P<.01), and by 72 h (3851±271,P<.01), P vs. control. Similarly, these parameters were detected following CS by 2h decompression (24179±225, P<.01), 4-24 hours decompression (21666±124, P<.001) and 48 h decompression (2941±189, P<.001), P vs. control. We demonstrate that drop in the binding calcium level observed was partially due to impaired affinity to calcium of the cardiomyocyte SR calcium-binding proteins during development of both PN and CS despite a simultaneous manifestation of affinity to calcium of the SR 32-kDa protein. Conclusion: In the present study we have clearly shown that both experimental acute pancreatitis and long-term compression injury may cause similar changes,а loss the calcium-binding properties of the cardiomyocyte proteins, particularly those of SR serving as a main calcium depot under physiological circumstances and appear to be involved in common cellular and molecular mechanisms of myocardial injury contributing to hypocalcemia. Simultaneously, both PN and/or CS cause similar manifestations of the new calcium-binding properties of the cardiomyocyte SR 32-kDa membrane protein, and mirrored dynamic changes in its calcium affinity suggested by Scatchard plot analysis indicating a common mechanism that would be a transient attempt of certain heart cells to compensate hypocalcemia, and thus emerge from an otherwise pathological outcome. Thus, the above mentioned changes could be used to identify patients at high risk of cardiovascular disease in different pathologies.

Characterization of salivary protein during ovulatory phase of menstrual cycle through MALDI-TOF/MS.

Context: Predicting ovulation is the basis on which the fertile period is determined. Nowadays there are many methods available to detect the ovulatory period. Unfortunately, these methods are not always effective for accurate detection of ovulation. Hence, an attempt was made to detect ovulation through single dimension sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of protein with the help of saliva ferning. Aims: The aim of this study was to determine the association of protein level with endogenous reproductive hormone level across the menstrual cycle. Settings and Design: Salivary protein and its confirmation were evaluated during menstrual cycle followed by SDS-PAGE and Mass spectrometry. Statistical Method Used: The protein content present in saliva throughout menstrual cycle is trail by SPSS statistical software version. Materials and Methods: Salivary proteins were investigated serially during pre-ovulatory, ovulatory and post-ovulatory periods of normal menstrual cycle in eighteen healthy volunteers. The samples were collected in three consecutive menstrual cycles. Salivary protein was estimated and analyzed by single dimension SDS-PAGE. Results: The results revealed significant variations in protein concentrations during the menstrual cycle. Protein levels were maximum during ovulation and minimum during postovulatory phase. Further, single dimension SDS-PAGE analysis showed seven different fractions of proteins is from 14-90 kilo Dalton (kDa) in the three phases of the menstrual cycle. Conclusions: Among the proteins, 48 kDa protein was more predominantly exhibited during ovulatory phase than pre and post-ovulatory phase. The present study indicates that the protein level and the specific protein band (48 kDa) through MALDI-TOF MS analysis might serve as an indicator for ovulation.