Gummy stem blight, caused by Didymella bryoniae occurs exclusively on cucurbits. This fungus has been known not to produce its pycnidium in vitro unless irradiated. Through this study, we optimized cultural conditions for mass-production of pycnidiospore by Metal Halide Lamp irradiation. In brief, the mycelial was cultured at $26^{\circ}C$ on PDA, for 2 days under the darkness, and then the plate was illuminated with MH lamp continuously for 3-4 days at $26^{\circ}C$, a great number of pycnidia was simultaneously formed. Thus produced pycnidiospores were used as immunogen. From fusions of myeloma cell (v-653) with splenocytes from immunifed mice were car ried out. And, two hybridoma cell lines that recognized the immunogen Didymella bryoniae were obtained. One Monoclonal Antibody, Db1, recognized the supernatant and the other monoclonal antibody, Db15, recognized the spore. Two clones were selected which were used to produce ascite fluid two MAb Db1 and Db15, were immunotyped and identified as IgG1 and IgG2b, respectively. Titer of MAb Db1 and MAb Db15 was measured absorbance exceeded 0.5 even at a $10^{-5}$ dilution. The MAbs reacted positively with Didymella bryoniae but none reacted with other of fungi and CMV, CGMMV Sensitivity of MAb was precise enough to detect spore concentration as low as $10^{3}$ well by indirect ELISA characterization of the MAb Db1, Db15 antigen by heat and protease treatments show that the epitope recognized by the MAb Bb1, Db15 were a glycoprotein.
Kim, Kyung-A;Chung, Soo-Bong;Hawng, Eun-Young;Noh, Seung-Hyun;Song, Kwon-Ho;Kim, Hanna-Hyun;Kim, Cheorl-Ho;Park, Young-Guk
Journal of Periodontal and Implant Science
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제43권1호
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pp.24-29
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2013
Purpose: Matrix metalloproteinases (MMPs) are capable of degrading extracellular matrix, and they are inducible enzymes depending on an inflammatory environment such as periodontitis and bacterial infection in periodontal tissue. Gingival inflammation has been postulated to be correlated with the production of MMP-2 and MMP-9. The objective of this study was to quantify the expression and activity of MMP-9 and -2, and to determine the correlation between activity and expression of these MMPs in human gingival tissues with periodontitis. Methods: The gingival tissues of 13 patients were homogenized in $500{\mu}L$ of phosphate buffered saline with a protease inhibitor cocktail. The expression and activity of MMP-2 and -9 were measured by enzyme-linked immunosorbent assay and Western blot analysis, and quantified by a densitometer. For the correlation line, statistical analysis was performed using the Systat software package. Results: MMP-9 was highly expressed in all gingival tissue samples, whereas MMP-2 was underexpressed compared with MMP-9. MMP-9 activity increased together with the MMP-9 expression level, with a positive correlation (r=0.793, P=0.01). The correlation was not observed in MMP-2. Conclusions: The expression of MMP-2 and -9 might contribute to periodontal physiological and pathological processes, and the degree of MMP-9 expression and activity are predictive indicators relevant to the progression of periodontitis.
The purpose of this study was to evaluate the effect of natural clinoptilolite (NCLI) and modified clinoptilolite (MCLI) on broiler performance, gut morphology, intestinal length and weight, and gut digestive enzyme activity. A total of 240 d-old male chicks were randomly assigned to 3 treatments, each of which comprised 8 pens of 10 chicks per pen. Birds in the control group were fed the basal diet, while those in the experimental groups were fed diets supplemented with NCLI at 2% (NCLI group), or MCLI at 2% (MCLI group), respectively, for 42 d. Compared with the control, supplementation with NCLI or MCLI had no significant (p>0.05) effects on productive parameters from d 1 to 42. Supplementation with NCLI or MCLI had no influence on the relative length and weight of small intestine at d 1 to 21. But supplementation with NCLI or MCLI significantly reduced the relative weight of duodenum. Supplementation with MCLI and NCLI was associated with greater (p<0.05) villus height in the jejunal and ileal mucosa compared with those areas in the controls from d 1 to 42. However, supplementation with NCLI and MCLI had no significant (p>0.05) influence on the crypt depth in the jejunal and ileal mucosa compared with those in the controls. The addition of either NCLI or MCLI to the diet improved the activities of total protease, and amylase in the small intestinal contents. In conclusion, supplementation with NCLI or MCLI in diets improved intestinal morphology, increased the intestinal length and weigh and gut digestive enzyme activity.
The present experiments were performed to confirm the hypothesis that xanthine oxidase[XOD], as a source and mechanism of oxygen radical production, plays an important role in the genesis of the reperfusion injury of ischemic myocardium. The experimental ischemic-reperfusion injury was induced in isolated, Langendorff preparations of rat hearts by 60 min. Of global ischemia with aortic clamping followed by 20 min. of reperfusion with oxygenated Krebs-Henseleit solution[pH 7.4, 37*C]. The results were as follows: 1. The releases of creatine phosphokinase and a lipid peroxidation product, malondialdehyde[MDA] into the coronary effluent were abruptly increased upon reperfusion of ischemic hearts. The increases of the enzyme and MDA were suppressed significantly in the hearts removed from rats pretreated with allopurinol, a specific XOD inhibitor[20mg/kg, oral, 24 hrs and 2 hrs before study]. This effect of allopurinol was comparable to that of oxygen radical scavengers, superoxide dismutase[5, 000U] and catalase[12, 500 U]. 2. The increased SOD-inhibitable reduction of ferricytochrome C, which was infused to the hearts starting with reperfusion, was significantly suppressed in allopurinol pretreated hearts. 3. Activities of myocardial XOD were compared in the normal control hearts and the ischemic ones. Total enzyme activities were not different in both hearts. However, comparing with the control, the ischemic ones showed higher activity in 0-form and lower activities in D-form and D/O-form. 4. In the ischemic hearts, phenylmethylsulfonyl fluoride, a serine protease inhibitor, prevented significantly the increase of 0-form and the decreases of D and D/O-form, while thiol reagents did not affect the changes of the enzyme. 5. The increase of 0-form and the decreases of D and D/0-form were not significant in both calcium-free perfused and pimozide, a calmodulin inhibitor, treated ischemic hearts. 6. The SOD-inhibitable reduction of ferricytochrome C were suppressed by PMSF and pimozide treatment as well as by calcium-free perfusion. It is suggested from these results that in the ischemic rat myocardium, xanthine oxidase is converted to oxygen radical producing 0-form by calcium, calmodulin-dependent proteolysis and plays a contributing role in the genesis of ischemic-reperfusion injury by producing oxygen free radicals.
In oriental medicine, Radix Polygalae(RP) has been to treat tremors et al. But the mechanism how to decrease tremors was not known. The purpose of this study was to investigate the effect of RP on neurodegenerative disease. We used RP to execute the study of this defense mechanism on dopamine-induced cell death in human SH-SY5Y dopaminergic neuroblastoma cells. MTT assay was used to know the cytotoxicity of dopamine and the defense mechanism. As a result of this experiment, dopamine had cytotoxicity in human SH-SY5Y cells, but when it treated with RP, the cell survival rate increased. This suppressed the cell apoptosis, activation of caspase-3 protease, production of ROS, and repair of membrane potential change. In conclusion, RP has the protective effect on dopamine-induced cell death in human SH-SY5Y dopaminergic neuroblastoma cells, so this could be an effective agent on the neurodegenerative disease like Parkinsonism.
The chitinase-producing strain TKU008 was isolated from soil in Taiwan, and it was identified as a new species of Pseudomonas. The culture condition suitable for production of chitinase was found to be shaking at $30^{\circ}C$ for 4 days in 100 ml of medium containing 1% shrimp and crab shell powder, 0.1% $K_2HPO_4$, and 0.05% $MgSO_4{\cdot}7H_2O$ (pH 7). The TKU008 chitinase was suppressed by the simultaneously existing protease, which also showed the maximum activity at the fourth day of incubation. The molecular mass of the chitinase was estimated to be 40 kDa by SDS-PAGE. The optimum pH, optimum temperature, pH stability, and thermal stability of the chitinase were pH 7, $50^{\circ}C$, pH 6-7, and <$50^{\circ}C$, respectively. The chitinase was completely inhibited by $Mn^{2+}$ and $Cu^{2+}$. The results of peptide mass mapping showed that 11 tryptic peptides of the chitinase were identical to the chitinase CW from Bacillus cereus (GenBank Accession No. gi 45827175) with a 32% sequence coverage.
Rama Rao, S.V.;Raju, M.V.L.N.;Reddy, M.R.;Panda, A.K.
Asian-Australasian Journal of Animal Sciences
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제17권6호
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pp.836-842
/
2004
An experiment was conducted to study the performance of broilers chicks (2 to 42 d of age) fed diets containing pearl millet (PM, Pennisetum typhoides), foxtail millet (FOM, Setaria italica) or finger millet (FIM, Elusine coracana) totally replacing (w/w) yellow maize (YM) with and with out supplementing non-starch polysaccharide (NSP) hydrolysing enzymes at the rate of 0.5 g/kg diet. Enzyme preparation contained amylase 2,400 units, hemi-cellulase 5,400 units, cellulase 12,000 units, protease 2,400 units and beta-glucanase 106 units/g. Each diet was fed to eight replicates (five female Vencob broilers/replicate) housed in stainless steel battery brooders. The estimated metabolizable energy (ME) contents of YM, PM, FOM and FIM were FM (PM) were about 3,389, 2,736, 3,303 and 2,846 kcal/kg, respectively. Total replacement of YM with FOM did not influence the body weight gain, ready to cook yield, relative weights of giblet, liver, intestine, lymphoid organs (bursa and spleen) and length of intestine, antibody titers and livability at 42 d of age. But the food efficiency decreased significantly in FOM fed broilers compared those fed YM. Further, the fat content in thigh muscle reduced with FOM fed groups compared to those fed YM. The performance of broilers decreased significantly in PM and FIM fed broilers compared to those fed YM. The relative weights of giblet, gizzard and liver increased in FIM fed groups compared to those fed YM as the principal source of energy in broilers. Incorporation of NSP hydrolysing enzymes in commercial broiler diets improved the efficiency of feed utilization during starter phase but not at 42 d of age. The results thus indicate that yellow maize can be replaced in toto on weight basis in commercial broiler diets without affecting the performance. Supplementation of NSP hydrolysing enzymes was beneficial in enhancing feed utilization during the starter phase.
The purpose of the present study was to investigate the effects of exogenous non-starch polysaccharides (NSP) enzymes on performance, intestinal content viscosity and digestive enzyme activities of growing pigs fed a rough rice-based diet. A total of 60 crossbred barrows with an initial body weight of 35.16 kg (SD = 0.82) were blocked by body weight and randomly assigned to two treatments with three replications. Each group was fed the diet based on rice with or without exogenous NSP enzymes (2 g/kg of diet). During the 70 days of the feeding trial, all pigs were given free access to feed and water. At the end of the feeding trial, six pigs from each treatment were randomly selected and slaughtered to collect intestinal digesta, intestinal mucosa, and pancreas. The addition of NSP enzymes improved average daily gain (p<0.05) and feed:gain (p<0.05), and decreased viscosity of digesta in the jejunum (p<0.001) and ileum (p<0.01) of pigs. The supplementation of NSP enzymes increased activities of protease (p<0.01), trypsin (p<0.01) and ${\alpha}$-amylase (p<0.05) in duodenal contents. However, digestive enzymes in the pancreas, jejunal and ileal mucosa were unaffected by the supplemental NSP enzymes (p>0.10). The results indicate that the addition of NSP enzymes to rough rice-based diets improved performance of pigs, reduced viscosity and increased digestive activity in the small intestine.
Kim, Yoo-Kyeong;Yu, Dae-Yeul;Kang, Hyun-Ah;Yoon, Sun;Chung, Bong-Hyun
Journal of Microbiology and Biotechnology
/
제9권2호
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pp.196-200
/
1999
A recombinant human $\alpha_{s1}$-casein was expressed as a secretory product in the yeast Saccharomyces cerevisiae. Three different leader sequences derived from the mating factor $\alpha$l (MF$\alpha$l), inulinase, and human $\alpha_{s1}$-casein were used to direct the secretion of human $\alpha_{s1}$-casein into the extracellular medium. Among the three leader sequences tested, the native leader sequence of human $\alpha_{s1}$-casein was found to be the most efficient in the secretory expression of human $\alpha_{s1}$-casein, which implies that the native leader sequence of human $\alpha_{s1}$-casein might be used very efficiently for the secretory production of other heterologous proteins in yeast. The recombinant human $\alpha_{s1}$-casein was proteolytically cleaved as the culture proceeded. Therefore, an attempt was made to produce human $\alpha_{s1}$-casein using a S. cerevisiae mutant in which the YAP3 gene encoding yeast aspartic protease 3 (YAP3) was disrupted. After 72 h of culture, most of the human $\alpha_{s1}$-casein secreted by the wild type was cleaved, whereas more than 70% of the human $\alpha_{s1}$-casein secreted by yap3-disruptant remained intact. The results suggest that YAP3 might be involved in the internal cleavage of human $\alpha_{s1}$-casein expressed in yeast
BACKGROUND/OBJECTIVES: Due to its beneficial health effects, use of buckwheat has shown a continuous increase, and concerns regarding the allergic property of buckwheat have also increased. This study was conducted for evaluation of the hydrolytic effects of seven commercial proteases on buckwheat allergens and its allergenicity. MATERIALS/METHODS: Extracted buckwheat protein was hydrolyzed by seven proteolytic enzymes at individual optimum temperature and pH for four hours. Analysis was then performed using SDS-PAGE, immunoblotting, and competitive inhibition ELISA (ciELISA) with rabbit antiserum to buckwheat protein, and direct ELISA with pooled serum of 21 buckwheat-sensitive patients. RESULTS: Alkaline protease, classified as serine peptidase, was most effective in reducing allergenicity of buckwheat protein. It caused decomposition of the whole buckwheat protein, as shown on SDS-PAGE, and results of immunoblotting showed that the rabbit antiserum to buckwheat protein no longer recognized it as an antigen. Allergenicity showed a decrease of more than 50% when pooled serum of patients was used in ELISA. Two proteolytic enzymes from Aspergillus sp. could not hydrolyze buckwheat allergens effectively, and the allergenicity even appeared to increase. CONCLUSIONS: Serine-type peptidases appeared to show a relatively effective reduction of buckwheat allergenicity. However, the antigenicity measured using rabbit antiserum did not correspond to the allergenicity measured using sera from human patients. Production of less allergenic buckwheat protein may be possible using enzymatic hydrolysis.
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