Protein-rich by-products from the canning industry, especially dark flesh of skipjack, have limited uses due to several factors such as darken color, susceptibility to oxidation and off flavour. Protein hydrolysates from skipjack dark flesh was produced with different type of industrial proteases (Alcalase®2.4L FG, Protamex®, Neutrase®1.5MG and Flavourzyme®500MG) for 60, 120, 180 and 240 min with level of proteases used of 0.5, 1, 1.5 and 2% per weight of raw material. The degree of hydrolysis and free tryptophan content of hydrolysate were investigated. The results shows longer time with higher concentration of enzyme has increased the degree of hydrolysis. Alcalase®2.4L FG had the highest degree of hydrolysis among all proteases followed by Protamex®, Flavourzyme®500MG and Neutrase® 1.5MG. All enzymes increase free tryptophan content linearly with the increament of protease enzyme level. The longer the hydrolysis time, the higher the content of free tryptophan produced.
The proteolytic specificity of rhodostoxin, the major hemorrhagin from Calloselasma rhodostoma (Malayan pit viper) venom was investigated using oxidized B-chain of bovine insulin as substrate. Six peptide bonds were cleaved: Ser9-Hist10, His10-Leu11, Ala14-Leu15, Tyr16-Leu17, Gly20-Glu21 and Phe24-Phe25. Deglycosylated rhodostoxin, however, cleaved primarily at Arg22-Gly23.
Burkholderia pseudomallei is resistant to a diverse group of antimicrobials including third generation cephalosporins whilst quinolones and aminoglycosides have no reliable effect. As therapeutic options are limited, development of more effective forms of immunotherapy is vital to avoid a fatal outcome. In an earlier study, we reported on the B. pseudomallei serine MprA protease, which is relatively stable over a wide pH and temperature range and digests physiological proteins. The present study was carried out to evaluate the immunogenicity and protective efficacy of the MprA as a potential vaccine candidate. In BALB/c mice immunized with recombinant MprA protease (smBpF4), a significantly high IgG titer was detectable. Isotyping studies revealed that the smBpF4-specific antibodies produced were predominantly IgG(1), proposing that immunization with smBpF4 triggered a Th2 immune response. Mice were immunized with smBpF4 and subsequently challenged with B. pseudomallei via the intraperitoneal route. Whilst control mice succumbed to the infection by day 9, smBpF4-immunized mice were protected against the lethal challenge and survived beyond 25 days post-infection. In conclusion, MprA is immunogenic in melioidosis patients whilst also eliciting a strong immune response upon bacterial challenge in mice and presents itself as a potential vaccine candidate for the treatment of melioidosis.
Recombinant elastase strain K overexpressed from E. coli KRX/pCon2(3) was purified to homogeneity by a combination of hydrophobic interaction chromatography and ion exchange chromatography, with a final yield of 48% and a 25-fold increase in specific activity. The purified protein had exhibited a first ever reported homodimer size of 65 kDa by SDS-PAGE and MALDI-TOF, a size which is totally distinct from that of typically reported 33 kDa monomer from P. aeruginosa. The organic solvent stability experiment had demonstrated a stability pattern which completely opposed the rules laid out in previous reports in which activity stability and enhancement were observed in hydrophilic organic solvents such as DMSO, methanol, ethanol and 1-propanol. The high stability and enhancement of the enzyme in hydrophilic solvents were explained from the view of alteration in secondary structures. Elastinolytic activation and stability were observed in 25 and 50% of methanol, respectively, despite slight reduction in α-helical structure caused upon the addition of the solvent. Further characterization experiments had postulated great stability and enhancement of elastase strain K in broad range of temperatures, pHs, metal ions, surfactants, denaturing agents and substrate specificity, indicating its potential application in detergent formulation.
Molecular-based techniques are becoming desirable as tools for identification of infectious diseases. Amongst the Burkholderia spp., there is a need to differentiate Burkholderia pseudomallei from Burkholderia cepacia, as misidentification could lead to false treatment of patients. In this study, conventional PCR assay targeting three genes was developed. Primers were designed for the amplification of Burkholderia genus-specific groEL gene, B. pseudomallei-specific mprA gene and B. cepacia-specific zmpA gene. The specificity and sensitivity of the assay was tested with 15 negative control strains and 71 Burkholderia spp. isolates including positive controls B. pseudomallei K96243 and ATCC B. cepacia strain. All B. pseudomallei strains were positive for groEL (139 bp) and mprA (162 bp), indicating a sensitivity of 100%. All B. cepacia strains produced amplicons for detection of groEL and zmpA (147 bp). Specificity using negative strains was 100%. In this study, a PCR assay specific for the detection of Burkholderia spp. and differentiation of the genus B. pseudomallei and B. cepacia was developed. The conventional assay has to be performed separately for each species due to the similar size of the PCR products amplified. This format may therefore be recommended for use as a diagnostic tool in laboratories where real-time PCR machines are not available. However, the real-time PCR was able to detect and differentiate the genus and species in single duplex assay.
Snake bite has been regarded as an important health problem in Myanmar since early 1960's. In the recent years, there has been growing interest in alternative therapies and therapeutic use of natural products, especially those derive from plants. In Myanmar and Indian traditional medicine, various plants have used as a remedy for treating snake bite. The present study was carried out to evaluate the effects of alcohol extract of Tamarind (Tamarindus indica Linn.) seed on some biologic properties of Russell's viper (Daboia russelli siamensis) venom (RVV). The Phospholipase A2 (PLA2) enzyme, coagulase enzyme and caseinolytic enzyme activities of Russell's viper venom (RVV) were reduced when mixed and incubated with the extract. When the RVV and the different amount of extracts were preincubated and injected intramuscularly into mice, all of them survived, but all the mice in the control group died. On the other hand, when RVV were injected first followed by the extract into mice, all of them died. If the extract was injected near the site where Russell's viper venom was injected, all the mice survived for more than 24 hours and the survival time prolonged but they all died within 96 hours. In conclusion, according to the results obtained, the extract neutralizes some biologic properties of the Russell's viper venom and prolonged the survival time if the extract was injected near the site where the Russell's viper venom was injected.
The prevalence of atherosclerosis has increased significantly in the recent years due to sedentary lifestyle and high-fat diet. However, the association between saturated fat intake and the increased risk for atherosclerotic cardiovascular diseases remains heavily debated. Lauric acid belongs to the saturated fatty acid group and its unique medium chain fatty acid properties are proven to be beneficial to humans in many ways. Thus, the aim of this project is to investigate the effect of lauric acid on the expression of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) genes-ADAMTS-1, ADAMTS-4, and ADAMTS-5-in macrophages. These genes encode for proteases that participate in the extracellular matrix remodeling and they play important roles in the vulnerability of atherosclerotic plaque. Here, we show that the treatment of 20 µM of lauric acid successfully reduced both transcriptional and translational expressions of these genes in THP-1 differentiated macrophages after 24-h incubation. Further cell signaling experiments using a panel of kinase inhibitors and phosphorylated antibodies proved that lauric acid down-regulated ADAMTS-1 by reducing the activation of PI3K and JNK at Tyr458 and Tyr185, respectively. Finally, JNK1 siRNA knockdown assay confirmed that ADAMTS-1 was regulated through JNK pathway, and lauric acid interfered with this pathway to down-regulate ADAMTS-1 expression. Although preliminary, this present study indicates that lauric acid has the potential to stabilize atherosclerotic plaque and may prevent thrombosis by interfering with the ADAMTS-1 expression through PI3K/JNK pathways.
Burkholderia cepacia complex (Bcc) are opportunistic pathogens implicated with nosocomial infections, and high rates of morbidity and mortality, especially in individuals with cystic fibrosis (CF). B. cepacia are naturally resistant to different classes of antibiotics, and can subvert the host innate immune responses by producing quorum sensing (QS) controlled virulence factors and biofilms. It still remains a conundrum as to how exactly the bacterium survives the intracellular environment within the host cells of CF patients and immunocompromised individuals although the bacterium can invade human lung epithelial cells, neutrophils, and murine macrophages. The mechanisms associated with intracellular survival in the airway epithelial cells and the role of QS and virulence factors in B. cepacia infections in cystic fibrosis remain largely unclear. The current review focuses on understanding the role of QS-controlled virulence factors and biofilms, and provides additional impetus to understanding the potentials of QS-inhibitory strategies against B. cepacia.
Experimental evidence has implicated genotoxic Escherichia coli (E. coli) and enterotoxigenic Bacteroides fragilis (ETBF) in the development of colorectal cancer (CRC). However, evidence from epidemiological studies is sparse. We therefore assessed the association of serological markers of E. coli and ETBF exposure with odds of developing CRC in the European Prospective Investigation into Nutrition and Cancer (EPIC) study.Serum samples of incident CRC cases and matched controls (n = 442 pairs) were analyzed for immunoglobulin (Ig) A and G antibody responses to seven E. coli proteins and two isoforms of the ETBF toxin via multiplex serology. Multivariable-adjusted conditional logistic regression analyses were used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for the association of sero-positivity to E. coli and ETBF with CRC.The IgA-positivity of any of the tested E. coli antigens was associated with higher odds of developing CRC (OR: 1.42; 95% CI: 1.05-1.91). Dual-positivity for both IgA and IgG to E. coli and ETBF was associated with >1.7-fold higher odds of developing CRC, with a significant association only for IgG (OR: 1.75; 95% CI: 1.04, 2.94). This association was more pronounced when restricted to the proximal colon cancers (OR: 2.62; 95% CI: 1.09, 6.29) compared to those of the distal colon (OR: 1.24; 95% CI: 0.51, 3.00) (pheterogeneity = 0.095). Sero-positivity to E. coli and ETBF was associated with CRC development, suggesting that co-infection of these bacterial species may contribute to colorectal carcinogenesis. These findings warrant further exploration in larger prospective studies and within different population groups.
Galloway-Mowat syndrome (GAMOS) is an autosomal-recessive disease characterized by the combination of early-onset nephrotic syndrome (SRNS) and microcephaly with brain anomalies. Here we identified recessive mutations in OSGEP, TP53RK, TPRKB, and LAGE3, genes encoding the four subunits of the KEOPS complex, in 37 individuals from 32 families with GAMOS. CRISPR-Cas9 knockout in zebrafish and mice recapitulated the human phenotype of primary microcephaly and resulted in early lethality. Knockdown of OSGEP, TP53RK, or TPRKB inhibited cell proliferation, which human mutations did not rescue. Furthermore, knockdown of these genes impaired protein translation, caused endoplasmic reticulum stress, activated DNA-damage-response signaling, and ultimately induced apoptosis. Knockdown of OSGEP or TP53RK induced defects in the actin cytoskeleton and decreased the migration rate of human podocytes, an established intermediate phenotype of SRNS. We thus identified four new monogenic causes of GAMOS, describe a link between KEOPS function and human disease, and delineate potential pathogenic mechanisms.