METHODS: ACE inhibitory proteins were isolated from P. cystidiosus based on the bioassay guided purification steps, i.e. ammonium sulphate precipitation, reverse phase high performance liquid chromatography and size exclusion chromatography. Active fraction was then analysed by LC-MS/MS and potential ACE inhibitory peptides identified were chemically synthesized. Effect of in vitro gastrointestinal digestions on the ACE inhibitory activity of the peptides and their inhibition patterns were evaluated.
RESULTS: Two potential ACE inhibitory peptides, AHEPVK and GPSMR were identified from P. cystidiosus with molecular masses of 679.53 and 546.36 Da, respectively. Both peptides exhibited potentially high ACE inhibitory activity with IC50 values of 62.8 and 277.5 μM, respectively. SEC chromatograms and BIOPEP analysis of these peptides revealed that the peptide sequence of the hexapeptide, AHEPVK, was stable throughout gastrointestinal digestion. The pentapeptide, GPSMR, was hydrolysed after digestion and it was predicted to release a dipeptide ACE inhibitor, GP, from its precursor. The Lineweaver-Burk plot of AHEPVK showed that this potent and stable ACE inhibitor has a competitive inhibitory effect against ACE.
CONCLUSION: The present study indicated that the peptides from P. cystidiosus could be potential ACE inhibitors. Although these peptides had lower ACE inhibitory activity compared to commercial antihypertensive drugs, they are derived from mushroom which could be easily obtained and should have no side effects. Further in vivo studies can be carried out to reveal the clear mechanism of ACE inhibition by these peptides.
RESULTS: Out of 270 grouper samples, 195 (72%) were detected with the presence of Vibrio spp. Vibrio communis showed highest prevalence in grouper (28%), followed by V. parahaemolyticus (25%), V. alginolyticus (19%), V. vulnificus (14%), V. rotiferianus (3%), Vibrio sp. (3%), V. campbellii (2%), V. mytili (2%), V. furnissii (2%), V. harveyi (1%), V. tubiashii (1%), V. fluvialis (0.3%) and V. diabolicus (0.3%). Assessment on the antibiotic susceptibility profiles of the Vibrio spp. revealed that majority of the isolates were susceptible to tetracycline, streptomycin, erythromycin and bacitracin, but resistance to ampicillin, penicillin G and vancomycin. The mean MAR index of the Vibrio isolates was 0.51, with 85% of the isolates showed MAR index value of higher than 0.2. Results indicate that the Vibrio spp. were continuously exposed to antibiotics. Furthermore, the plasmid profiles of Vibrio spp. showed that 38.7% of the isolates harbored plasmid with molecular weight of more than 10 kb, while 61.3% were without plasmid. During curing process, Vibrio spp. lost their plasmid, but remained resistant to ampicillin, penicillin G, bacitracin and vancomycin while a few isolates remained resistant to erythromycin, streptomycin and tetracycline. The results suggested that the resistance to antibiotics in isolated Vibrio spp. might be due to chromosomal and plasmid borne.
CONCLUSIONS: This study demonstrates the prevalence of Vibrio spp. in groupers and the distribution of multidrug resistance strains that could be of concern to the farmers in Malaysia. In addition, data from this study can be further used in fish disease management plan.
RESULTS: An earlier published work discovered a highly antigenic 12 kDa excretory-secretory (ES) protein of T. gondii which may potentially be used for the development of an antigen detection test for toxoplasmosis. However, the three-dimensional structure of the protein is unknown. Since epitope identification is important prior to designing of a specific antibody for an antigen-detection based diagnostic test, the structural elucidation of this protein is essential. In this study, we constructed a three dimensional model of the 12 kDa ES protein. The built structure possesses a thioredoxin backbone which consists of four α-helices flanking five β-strands at the center. Three potential epitopes (6-8 residues) which can be combined into one "single" epitope have been identified from the built structure as the most potential antibody binding site.
CONCLUSION: Together with specific antibody design, this work could contribute towards future development of an antigen detection test for toxoplasmosis.