Eighty-four strains of Vibrio cholerae O1, O139 and non-O1/non-O139 from clinical and environmental sources were investigated for the presence of the toxin co-regulated pilus gene, tcpA, the virulence cassette genes ctxA, zot, ace and cep and also for their ability to elaborate haemolysin and protease. The ctxA and zot genes were detected using DNA-DNA hybridization while the ace, cep and tcpA genes were detected using PCR. Production of haemolysin and protease was detected using mammalian erythrocytes and an agar diffusion assay respectively. Analysis of their virulence profiles showed six different groups designated Type I to Type VI and the major distinguishing factor among these profiles was in the in vitro production of haemolysin and/or protease. Clinical O1, O139 and environmental O1 strains were similar with regard to presence of the virulence cassette genes. All environmental O1 strains with the exception of one were found to possess ctxA, zot and ace giving rise to the probability that these strains may actually be of clinical origin. One strain which had only cep but none of the toxin genes may be a true environmental isolate. The virulence cassette and colonization factor genes were absent in all non-O1/non-O139 environmental strains but production of both the haemolysin and protease was present, indicating that these may be putative virulence factors. These findings suggest that with regard to its pathogenic potential, only strains of the O1 and O139 serogroup that possess the tcpA gene which encodes the phage receptor, have the potential to acquire the CTX genetic element and become choleragenic.
The main objective of this study was to use heating method (HM) to prepare liposome without employing any chemical solvent or detergent. Plackett-Burman design (PBD) was applied for the screening of significant process variables including the lecithin proportion, the cholesterol/lecithin ratio, the pH of solution for liposome preparation, the enzyme/lecithin ratio, the stirring time, the process temperature, the speed of stirrer, the ratio of stirrer to the tank diameter, the application of homogenization, the method of adding enzyme and centrifugation conditions on the encapsulation efficiency (EE %) of liposome and the activity of liposomal Flavourzyme (LAPU(-1)) (P
1. The protease, phosphodiesterase, alkaline phosphomonoesterase, L-amino acid oxidase, acetylcholinesterase, phospholipase A, 5'-nucleotidase, hyaluronidase, arginine ester hydrolase, procoagulant, anticoagulant and hemorrhagic activities of ten samples of venoms from seven taxa of sea snakes were examined. 2. The results show that venoms of sea snakes of both subfamilies of Hydrophiinae and Laticaudinae are characterized by a very low level of enzymatic activities, except phospholipase A activity and, for some species, hyaluronidase activity. 3. Because of the low levels of enzymatic activities and the total lack of procoagulant and hemorrhagic activities, venom biological properties are not useful for the differentiation of species of sea snakes. Nevertheless, the unusually low levels of enzymatic activities of sea snake venoms may be used to distinguish sea snake venoms from other elapid or viperid venoms.
1. The biological properties of twelve samples of venoms from all four species of Dendroaspis (mamba) were investigated. 2. Dendroaspis venoms generally exhibited very low levels of protease, phosphodiesterase and alkaline phosphomonoesterase; low to moderately low level of 5'-nucleotidase and very high hyaluronidase activities, but were devoid of L-amino acid oxidase, phospholipase A, acetylcholinesterase and arginine ester hydrolase activities. The unusual feature in venom enzyme content can be used to distinguish Dendroaspis venoms from other snake venoms. 3. All Dendroaspis venoms did not exhibit hemorrhagic or procoagulant activity. Some Dendroaspis venoms, however, exhibited strong anticoagulant activity. The intravenous median lethal dose of the venoms ranged from 0.5 microgram/g mouse to 4.2 micrograms/g mouse. 4. Venom biological activities are not very useful for the differentiation of the Dendroaspis species. The four Dendroaspis venoms, however, can be differentiated by their venom SDS-polyacrylamide gel electrophoretic patterns.
1. The hemorrhagic, procoagulant, anticoagulant, protease, arginine ester hydrolase, phosphodiesterase, alkaline phosphomonoesterase, 5'-nucleotidase, hyaluronidase, phospholipase A and L-amino acid oxidase activities of 50 venom samples from 20 taxa of rattlesnake (genera Crotalus and Sistrurus) were examined. 2. The results show that notwithstanding individual variations in the biological activities of Crotalus venoms and the wide ranges of certain biological activities observed, there are some common characteristics at the genus and species levels. 3. The differences in biological activities of the venoms compared can be used for differentiation of the species. Particularly useful for this purpose are the thrombin-like enzyme, protease, arginine ester hydrolase, hemorrhagic and phospholipase A activities and kaolin-cephalin clotting time measurements.
1. The hemorrhagic, procoagulant, anticoagulant, phosphodiesterase, hyaluronidase, alkaline phosphomonoesterase, 5'-nucleotidase, arginine ester hydrolase, phospholipase A, L-amino acid oxidase and protease activities of 26 samples of venoms of 13 taxa of Vipera were determined and the Sephadex G-75 gel filtration patterns for some of the venoms were also examined. 2. The results indicate the presence of certain common characteristics among the venoms, particularly if V. russelli is excluded from the comparison. The results also support the recently proposed reassignment of V. russelli to a separate genus. 3. The data show that information on venom biological properties can be used for differentiation of venoms of many species of Vipera. Particularly useful for this purpose are the protease, phosphodiesterase, phospholipase A and the procoagulant activities and the Sephadex G-75 gel filtration patterns of the venoms.
Indirect ELISA shows that the antibodies to Calloselasma rhodostoma venom hemorrhagin (CR-HMG), thrombin-like enzyme (CR-TLE) and L-amino acid oxidase (CR-LAAO) exhibited strong to moderate cross-reactions with most crotalid and viperid venoms, but only anti-CR-LAAO cross-reacted with the elapid venoms. However, the indirect ELISA failed to detect some antigenic similarities demonstrable by cross-neutralization study. The double-sandwich ELISA for the three anti-C. rhodostoma venom components exhibited a much lower level of cross-reactions than the indirect ELISA.
The aims of the present study were to determine whether Allium sativum (garlic) extract has any effect on the morphology transformation of Candida albicans, and to investigate whether it could alter the gene expression level of SIR2, a morphogenetic control gene and SAP4, a gene encoding secreted aspartyl proteinase.
In eukaryotes, serine proteases are cellular localized hydrolases reported to regulate essential biological reactions. Improved industrial applications of proteins are aided by prediction and analysis of their 3-dimensional structures (3D). A serine protease was identified from CTG-clade yeast Meyerozyma guilliermondii strain SO and its 3D structure as well as its catalytic attributes have not been fully understood yet, thus we seek to report on the catalytic mechanism of M. guilliermondii strain SO MgPRB1 using substrate PMSF via in silico docking as well as its stability by way of disulfide bonds formation. Herein, bioinformatics tools and techniques were used to predict, validate and analyze the possible changes of CUG ambiguity (if any) in strain SO using template PDB ID: 3F7O. Structural assessments confirmed the classic catalytic triad Asp305, His337, and Ser499. Superimposition of MgPRB1 and template 3F7O structures revealed the unlinked cysteine residues between Cys341, Cys440, Cys471 and Cys506 of MgPRB1 compared to template 3F7O with two disulfide bonds formation, which confers structural stability. In conclusion, serine protease structure from strain SO was successfully predicted and studies towards understanding at the molecular level may be undertaken for its potential applications in the degradation of peptide bonds.
Unripe and ripe bilimbi (Averrhoa bilimbi. L) were ground and the extracted juices were partially purified by ammonium sulfate precipitation at the concentrations of 40 and 60% (w/v). The collected proteases were analysed for pH, temperature stability, storage stability, molecular weight distribution, protein concentration and protein content. Protein content of bilimbi fruit was 0.89 g. Protease activity of both the unripe and ripe fruit were optimum at pH 4 and 40ºC when the juice were purified at 40 and 60% ammonium sulfate precipitation. A decreased in protease activity was observed during the seven days of storage at 4°C. Molecular weight distribution indicated that the proteases protein bands fall between 10 to 220 kDa. Protein bands were observed at 25, 50 and 160 kDa in both the unripe and ripe bilimbi proteases purified with 40% ammonium sulfate, however, the bands were more intense in those from unripe bilimbi. No protein bands were seen in proteases purified with 60% ammonium sulfate. Protein concentration was higher for proteases extracted with 40% ammonium sulfate at both ripening stages. Thus, purification using 40% ammonium sulfate precipitation could be a successful method to partially purify proteases from bilimbi especially from the unripe stage.
Protease was extracted from two maturity stages of noni fruits (Morinda citrifolia L.), unripe (stage 1) and ripe (stage 5). The crude extract was partially purified by acetone precipitation method followed by dialysis, gel filtration chromatography and freeze drying. Protein concentrations, proteolytic activity, molecular weight distribution, pH stability, temperature stability and storage efficiency of the resulting protease were evaluated. The unripe and ripe noni fruit contains 0.65 and 0.35% protein, respectively. Molecular weight of the proteases from both stages ranged approximately between 3 to 28 kDa based on the SDS-PAGE results. The optimum activity were at pH 7s and 6, temperatures of 40 and 50°C, respectively for proteases obtained from the unripe and ripe fruit. Analysis from the freeze dried protease indicated that protease from ripe noni fruits had higher protein concentration and specific activity compared to those from unripe fruit. However, it is more sensitive to pH and temperature and less stable during storage as it shows lower proteolytic activity compared to protease from unripe fruit. Based on its high proteolytic activity reaching up to 70.31 U/mg and storage stability (30% lost of activity), noni fruit could be an alternative source of plant protease.
Threadfin bream (Nemipterus japonicas) muscle was hydrolysed using protease extracted from
bilimbi (Averrhoa bilimbi L.) fruit. This study was performed in order to compare the efficiency of bilimbi protease in producing threadfin bream protein hydrolysate with the commercial protease; alcalase 2.4 L. Initially, protease was extracted and then purified using 40% ammonium sulfate precipitation method. The proteolytic activity of the crude extract and purified protease was determined. Precipitation using 40% ammonium sulfate resulted in bilimbi protease specific activity of 2.36 U/mg and 23.13% recovery. Threadfin bream hydrolysate was prepared based on the pH-stat method by hydrolysis for 2 hrs. Hydrolysis using bilimbi protease produced 34.76% degree of hydrolysis (DH) and 3.75% yield while hydrolysis using alcalase resulted in 86.6% DH with 22.78% yield. Alcalase hydrolysate showed higher solubility than bilimbi protease hydrolysate at pH 7 with 70.87 and 32.16% solubility, respectively. Results also showed that protein content of threadfin bream hydrolysate produced using alcalase was higher (86.86%) than those produced using bilimbi protease (22.12%). However, both hydrolysates showed low moisture content between 3.93 to 7.00%. The molecular weight distribution analysis using SDS–PAGE indicated the distribution of smaller peptides especially in alcalase hydrolysate. Overall, the results showed that alcalase is more efficient enzyme choice than bilimbi protease for preparing threadfin bream hydrolysates. However, both hydrolysates could play an important role thus contribute to the food industry.
There are few studies on sick building syndrome (SBS) including clinical measurements for atopy and fractional exhaled nitric oxide (FeNO). Our aim was to study associations between SBS symptoms, selected personal factors, office characteristics and indoor office exposures among office workers from a university in Malaysia. Health data were collected by a questionnaire (n=695), skin prick test (SPT) (n=463) and FeNO test (n=460). Office settled dust was vacuumed and analyzed for endotoxin, (1,3)-β-glucan and house dust mites (HDM) allergens group 1 namely Dermatophagoides pteronyssinus (Der p 1) and Dermatophagoides farinae (Der f 1). Office indoor temperature, relative air humidity (RH), carbon monoxide (CO) and carbon dioxide (CO2) were measured by a direct reading instrument. Associations were studied by two-levels multiple logistic regression with mutual adjustment and stratified analysis. The prevalence of weekly dermal, mucosal and general symptoms was 11.9%, 16.0% and 23.0% respectively. A combination of SPT positivity (allergy to HDM or cat) and high FeNO level (≥25 ppb) was associated with dermal (p=0.002), mucosal (p<0.001) and general symptoms (p=0.05). Der f1 level in dust was associated with dermal (p<0.001), mucosal (p<0.001) and general (p=0.02) symptoms. Among those with allergy to D. farinae, associations were found between Der f 1 levels in dust and dermal (p=0.003), mucosal (p=0.001) and general symptoms (p=0.007). Office-related symptoms were associated with Der f 1 levels in dust (p=0.02), low relative air humidity (p=0.04) and high office temperature (p=0.05). In conclusion, a combination of allergy to cat or HDM and high FeNO is a risk factor for SBS symptoms. Der f 1 allergen in dust can be a risk factor for SBS in the office environment, particularly among those sensitized to Der f 1 allergen.
A prevalence study was conducted among office workers in Malaysia (N= 695). The aim of this study was to examine associations between asthma, airway symptoms, rhinitis and house dust mites (HDM) and cat allergy and HDM levels in office dust. Medical data was collected by a questionnaire. Skin prick tests were performed for HDM allergens (Dermatophagoides pteronyssinus, Dermatophagoides farinae) and cat allergen Felis domesticus. Indoor temperature and relative air humidity (RH) were measured in the offices and vacuumed dust samples were analyzed for HDM allergens. The prevalence of D. pteronyssinus, D. farinae and cat allergy were 50.3%, 49.0% and 25.5% respectively. Totally 9.6% had doctor-diagnosed asthma, 15.5% had current wheeze and 53.0% had current rhinitis. The Der p 1 (from D. pteronyssinus) and Der f 1 (from D. farinae) allergens levels in dust were 556 ng/g and 658 ng/g respectively. Statistical analysis was conducted by multilevel logistic regression, adjusting for age, gender, current smoking, HDM or cat allergy, home dampness and recent indoor painting at home. Office workers with HDM allergy had more wheeze (p= 0.035), any airway symptoms (p= 0.032), doctor-diagnosed asthma (p= 0.005), current asthma (p= 0.007), current rhinitis (p= 0.021) and rhinoconjuctivitis (p< 0.001). Cat allergy was associated with wheeze (p= 0.021), wheeze when not having a cold (p= 0.033), any airway symptoms (p= 0.034), doctor-diagnosed asthma (p= 0.010), current asthma (p= 0.020) and nasal allergy medication (p= 0.042). Der f 1 level in dust was associated with daytime breathlessness (p= 0.033) especially among those with HDM allergy. Der f 1 levels were correlated with indoor temperature (p< 0.001) and inversely correlated with RH (p< 0.001). In conclusion, HDM and cat allergies were common and independently associated with asthma, airway symptoms and rhinitis. Der f 1 allergen can be a risk factor for daytime breathlessness.
The epigenetic changes of RELN that are involved in the development of dopaminergic neurons may fit the developmental theory of schizophrenia. However, evidence regarding the association of RELN DNA methylation with schizophrenia is far from sufficient, as studies have only been conducted on a few limited brain samples. As DNA methylation in the peripheral blood may mirror the changes taking place in the brain, the use of peripheral blood for a DNA methylation study in schizophrenia is feasible due to the scarcity of brain samples. Therefore, the aim of our study was to examine the relationship of DNA methylation levels of RELN promoters with schizophrenia using genomic DNA derived from the peripheral blood of patients with the disorder. The case control studies consisted of 110 schizophrenia participants and 122 healthy controls who had been recruited from the same district. After bisufhite conversion, the methylation levels of the DNA samples were calculated based on their differences of the Cq values assayed using the highly sensitive real-time MethyLight TaqMan® procedure. A significantly higher level of methylation of the RELN promoter was found in patients with schizophrenia compared to controls (p = 0.005) and also in males compared with females (p = 0.004). Subsequently, the RELN expression of the methylated group was 25 fold less than that of the non-methylated group. Based upon the assumption of parallel methylation changes in the brain and peripheral blood, we concluded that RELN DNA methylation might contribute to the pathogenesis of schizophrenia. However, the definite effects of methylation on RELN function during development and also in adult life still require further elaboration.
Protease is an enzyme that catalysed the hydrolysis of protein into peptide. Application of protease in industry has been linked with cost effective substrates and complex of enzyme-substrate stability. Molecular docking approach has identified casein as a preference substrates. However, lack of data on casein mode of binding to protease and enzyme stability represents a limitation for its production and structural optimization. In this study, we have used a molecular dynamic (MD) to examine the stability of complex enzyme-substrate of protease from Bacillus lehensis G1. The 3D structure of protease (BleG1_1979) was docked with substrate casein using AutoDock Vina. Structural analysis of the substrate-binding cleft revealed a binding site of casein was predominantly at the hydrophobic region of BleG1_1979. The MD of complex BleG1_1979-casein was tested with two temperatures; 298 K and 310 K using GROMACS v5.1.4. MD simulation showed a stable behaviour of BleG1_1979 over the 20 ns simulation period. The molecular docking and MD simulation suggested that the production of protease from B. lehensis G1 by utilization of casein and the stability of complex protease-casein could be a potential application to generate a cost effective enzyme to be develop for industrial use.
Pencirian enzim ekstraselular protease daripada bakteria Alkalophilic Bacillus lehensis G1 dari Malaysia telah dikaji. Enzim protease yang dirembeskan diuji pada agar susu skim 2%. Keputusan menunjukkan protease ekstraselular mampu mengekalkan aktiviti sehingga suhu 60°C di dalam julat pH yang luas iaitu 3 hingga 11 dengan suhu optimum pada 40°C dan pH optimum pada 7.0. Aktiviti enzim juga diperhatikan akan meningkat dengan penambahan beberapa ion iaitu Mn2+, Fe2+, Cu2+, Mg2+ dan Co2+. Manakala aktiviti protease didapati sedikit direncat dengan kehadiran ion Ca2+, K+ dan Ni2+ dengan baki aktiviti sebanyak 85%, 81% dan 75%. Protease ekstraselular juga didapati serasi dengan beberapa cecair detergen komersial dari Malaysia, yang menunjukkan protease ini boleh dimanfaatkan sebagai pembersih kotoran pada pakaian. Selain itu, potensi kegunaan protease yang dihasilkan oleh B. lehensis G1 ke atas penguraian gelatin dari filem X-ray yang telah digunakan juga telah dilakukan di dalam kajian ini.
Physicochemical properties of mud clam (Polymesoda erosa) hydrolysate produced using two microbial enzymes; alcalase and flavourzyme were determined. Hydrolysis using alcalase at 20.28% degree of hydrolysis (DH) resulted in 25.06 % yield and 45.37% protein while flavourzyme hydrolysis showed 22.93 % DH, 46.67 % protein and 30.68 % yield. Both hydrolysates were yellowish. Better emulsifying properties, foaming properties and water and oil holding capacity were exhibited by flavourzyme hydrolysate compared to the alcalase hydrolysate. However, in terms of amino acid composition, alcalase hydrolysate contained higher amino acid composition (75.06%) than flavourzyme hydrolysate (62.37%). The study suggested that mud clam hydrolysate had the potential to be used in food formulations for human consumption.
Pyrenochaeta, classified under the order Pleosporales, is known to cause diseases in plants and humans. Here, we report a draft genome sequence of a Pyrenochaeta sp. isolated from a skin scraping, with an estimated genome size of 39.4 Mb. Genes associated with the synthesis of proteases, toxins, plant cell wall degradation, and multidrug resistance were found.
Filamentous phage random peptide libraries were used to identify the epitopes of Burkholderia pseudomallei protease by panning against IgG polyclonal sera that exhibited protease neutralizing properties. The isolated fusion peptides presented a consensus peptide sequence, TKSMALSG, which closely resembles part of the active site sequence, 435GTSMATPHVAG445, of B. pseudomallei serine metalloprotease. By comparing the consensus sequence, TKSMALSG, with the predicted three-dimensional molecular model of B. pseudomallei serine metalloprotease, it appears that the potential antibody binding epitope was buried within the molecule. This active site was conformational whereby one continuous sub-region (SMA) was located between two discontinuous sub-regions, supplied by the flanking residues in the same polypeptide. All phages selected from the biopanning with IgG polyclonal sera showed good binding towards the polyclonal antibodies when compared to the negative control. In addition, these peptide-bearing phages showed competitive inhibition of B. pseudomallei serine metalloprotease binding to the polyclonal IgG.