ABSTRACT
Mycoplasma capricolum subsp. capripneumoniae (Mccp) is the cause of contagious caprine pleuropneumonia (CCPP) in goats. Inactivated vaccines and capsular polysaccharide (CPS) indirect hemagglutination reagents are available for prevention and serological detection, but high culture costs and complex antigen quantification have been plagued by production staff. In order to solve these problems in production practice, a sugar fermentation medium with an initial pH value of 7.8, which could improve the production of two antigens simultaneously, was screened out by changing the initial pH value based on previous Mccp metabolomics analysis. Since phenol red can be identified by UV absorption spectrum and cetyltrimethylammonium bromide (CTAB) can bind to anionic capsular polysaccharide, a UV spectrum measurement method for analyzing the culture stage reached by Mccp and a CTAB precipitation test for relative quantification of capsular polysaccharide antigen content in the fermentation broth were established. The UV spectrum observation method can guide the production of Mccp according to the growth curve of Mccp, which greatly reduces the monitoring time of the traditional CCU method and improves the accuracy of the original eye-observation method. The established CTAB precipitation test can complete the monitoring of CPS content within 5 hours, which greatly reduces the time required compared with the traditional differential technique, and its accuracy was verified by the phenol-sulfuric acid method. The optimized culture medium and the two correlation comparison methods established in this study can effectively reduce the production cost of Mccp and improve the production efficiency. The two assays have been used in the research at our laboratory, which provides experimental data for further improvement of the production process of CCPP inactivated vaccine and capsular polysaccharide as well as rapid quantification.
Subject(s)
Humans , Animals , Goats , Cetrimonium , Mycoplasma , PolysaccharidesABSTRACT
AIM: Changes of endoxin level, ATPase activities, intramitochondrial Ca2+ concentration, and gene expression of Na+-K+-ATPase isoforms in myocardium of rats with MIR and effect of verapamil were observed, in order to investigate mechanism of endoxin mediating intracellular calcium overload of myocytes. METHODS: Twenty four male Sprauge Dawley rats were randomized into 3 groups. Sham operation group: silk suture was threaded the left anterior descending coronary artery without ligature; MIR group (MIR): left anterior descending coronary artery was subjected to 30 min ligation followed by 45 min reperfusion; verapamil group: MIR model was given 5 mg/kg verapamil. Verapamil was injected via femoral vein 5 min before reperfusion. Left ventricle myocardium samples were processed immediately after reperfusion in order to measure the activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase, endoxin level, and intramitochondrial Ca2+ concentration. The levels of ?1, ?2, ?3 and ?1 isoforms of Na+-K+-ATPase were measured by immunohistochemical assay. RESULTS: After MIR, the level of endoxin in myocardium was substantially increased; the activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase in myocardial membrane were significantly decreased while the concentration of intramitochondrial Ca2+ was increased; the levels of the ?1, ?2, ?3 and ?1 isoforms of Na+-K+-ATPase were reduced markedly. Verapamil had only effect on reducing the concentration of intramitochondrial Ca2+. CONCLUSION: MIR increases endoxin secretion. The latter may depress the activity of Na+-K+-ATPase by changing the gene expression of ?1, ?2, ?3 and ?1 isoforms of Na+-K+-ATPase in myocardial membrane, inducing intramitochondrial Ca2+ overload.
ABSTRACT
AIM: To observe the effect of endoxin antagonist,anti-digoxin antiserum,on endoxin level,ATPase activities,intramitochondrial total calcium concentration and gene expression of sodium pump isoforms in myocardium of rats with myocardial ischemia reperfusion(MIR).METHODS: Fifty-six male Sprauge Dawley rats were randomly divided into 7 groups.Sham operation group: silk suture threading the left anterior descending coronary artery without ligature;MIR group: left anterior descending coronary artery was subjected to 30 min ligation followed by 45 min reperfusion;normal saline group: MIR model was given 5 mL/kg normal saline;verapamil group: MIR model was given 5 mg/kg verapamil;low dose antidigoxin antiserum group: MIR model was given 8.6 mg/kg antidigoxin antiserum;middle dose antidigoxin antiserum group: MIR model was given 17.3 mg/kg antidigoxin antiserum;high dose antidigoxin antiserum group: MIR model was given 34.5 mg/kg antidigoxin antiserum.All drugs were injected into vessel via femoral vein within 5 min before reperfusion,respectively.After reperfusion,left ventricle myocardium samples were processed immediately in order to measure the activity of Na+-K+-ATPase and Ca2+-Mg2+-ATPase,endoxin level,intramitochondrial total calcium concentration and the experssion of ?1,?2,?3 and ?1 isoforms of sodium pump on mRNA and protein levels by RT-PCR and Western blotting and immunohistochemical assay,respectively.RESULTS: After MIR,the level of endoxin in myocardium was obviously increased.The activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase in myocardial membrane were significantly decreased while intramitochondrial total calcium concentration increased.The gene expression of the ?1,?2,?3 and ?1 isoforms of sodium pump at both mRNA and protein levels were reduced markedly.Only the effect of verapamil on reducing intramitochondrial total calcium concentration was observed.Antidigoxin antiserum significantly reduced the level of endoxin in myocardium,restored the activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase,reduced intramitochondrial total calcium concentration,and up-regulated the expression of ?1,?2,?3 and ?1 isoforms of sodium pump at both mRNA and protein levels.CONCLUSION: MIR results in increase of endoxin secretion.The latter depresses the activity of Na+-K+-ATPase by down-regulating the gene expression of ?1,?2,?3 and ?1 isoforms of sodium pump in myocardial membrane,and also induces intramitochondrial calcium overload,thereby mediates MIR injury.