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Study on identifying genotypes of Echinococcus granulosus by microsatellite markers
Abstracts / Cell Biology International 32 (2008) S1eS67 and its induction was protective during fibrogenesis. Further elucidation of the molecular mechanisms involved might yield novel insights into potential roles of mUGBP during pulmonary fibrosis and help develop new therapeutic strategies. This study was supported by Grants 30400190 and 30670770 from National Natural Science Foundation of China.
STUDY ON IDENTIFYING GENOTYPES OF ECHINOCOCCUS GRANULOSUS BY MICROSATELLITE MARKERS Xiu Min Ma 1,2, Jian Bing Ding 1,2, Wulamu Mamuti 1,2, Xiao Mei Lu 2, Ren Yong Lin 2, Ya Lou Zhang 2, Ya Nan Wang 1, Akira Ito 3, Hao Wen 2 1 Department of Etiology, Basic Medical College 2 Medical Research Center, First Teaching Hospital, Xinjiang Medical University, Urumqi, 830054, Xinjiang, China 3 Department of Parasitology, Asahikawa Medical College, Asahikawa 078-8510, Japan This research used Sca, Emsk and C106 microsatellite sequence of Echinococcus granulosus as typing markers and identified the genotypes and heterozygosity of E.granulosus isolated from cystic echinococcosis (CE) patients in different areas of Xinjiang. Two different fluorescent dyes of FAM (carboxy fluorescein) and HEX (hexacho rofluorescein) were used as markers for three pairs of primers of microsatellite sequences. After amplification of satellites by PCR, amplified products were based on capillary electrophoresis by using 310 auto DNA analysis machine, and then basic number of amplified products was calculated by Genescan 2.1 software. Our result showed that 66 isolated strains from 44 CE patients were homozygote of E. granulosus, in which 65 isolated strains from 43 CE patients were identified as G1 genotype by PCR and microsatellite markers, and 1 isolated strain from 1 CE patient was identified as G6 genotype by PCR and microsatellite markers. The results of genotypes identified by microsatellite markers were consistent with the result of DNA sequence analysis. Mixing infection and heterozygote of G1 (sheep strain) and G6 (camel strain) genotypes were found in the dog intestines, but this research did not find E.granulosus heterozygote. It may indicate that E.granulosus was autofertilization, and therefore, few chance of heterozygote takes place in the patient. Microsatellite DNA markers can be used to finely and quickly identify the genotypes and heterozygosity of E.granulosus at genetic level, and possessed the important value in study of genetic polymophosim, epidemiology and pathogenicity of E.granulosus.
DETERMINATION OF PHENYLALANINE AND TYROSINE IN PERIPHERAL BLOOD AND ITS APPLICATION IN PHENYLKETONURIA Xi Ming Mo, Ai Guo Tang Department of Clinical Laboratory Science, Second Xiangya Hospital, Central South University, Changsha, China Several methods have been reported for determination of tyrosine (Tyr) or phenylalanine (Phe) in neonatal blood, but the simultaneous determination of Tyr and Phe in peripheral blood by reverse phase high-performance liquid chromatography (RP-HPLC) by no derivatization has not been reported. Here we describe a simple, accurate and reliable RP-HPLC assay for measurement of Tyr and Phe in peripheral blood of newborn infants and patients with phenylketonuria (PKU). Supernatant fluid of peripheral blood precipitated with perchloric acid was isocratically eluted using a base-deactivated C8 column with 5 % acetonitrile in water as the mobile phase. Untraviolet detector worked at 210nm. The retention time of tyrosine (Tyr) and phenylalanine (Phe) were 5.88min and 8.43min respectively. The effects of various aspects on operation and determination were examined to establish optimal assay conditions, such as precipitator, anticogulant, means of collecting and storing samples. Phenylalanine and tyrosine in finger blood of 102 healthy enfant were (67.7 15.4) mmol/L and (62.2 13.9) mmol/L respectively. The ratio of Phe/Tyr was 1.15 0.27. There was no obviously difference between males and females. Phenylalanine and tyrosine in heel blood of 32 healthy neonatal were (66.2 20.5) mmol/L and (59.518.8) mmol/L respectively. The ratio of Phe/Tyr was 1.12 0.24. The levels of
S61
phenylalanine and tyrosine in whole-blood which anticogulated by EDTA$K2 are consistent with those of anticogulating by heparin. The levels of phenylalanine and tyrosine in peripheral blood were significantly lower than those in plasma, but showed a good correlation. The means of collecting and storing sample affect the levels of phenylalanine and tyrosine in whole-blood.
THE MECHANISMS OF ALCOHOL-INDUCED MYOCARDIUM INJURY AND THE EFFECTS OF NIMODIPINE AND ERIGERON BREVISCAPUS Gui Lan Pan, Jing Hua Shi, Xiao Dong Chen The Department of Physiology, Baotou Medical College, Baotou, Inner Mongolia, 014010, China The changes of cardiac troponin I (cTnI), malondialdehyde (MDA), glutathione peroxidase (GSH-Px) contents in serum after acute alcohol administration, and the possible mechanisms of myocardium injury induced by alcohol and the protective effect of Nimodipine and Breviscapus were investigated. 40 Wistar rats were divided to 5 groups randomly. The rats in the control group were given physiological saline (1.5 ml/100g). The rats in the alcohol group were given 40% alcohol, 1.5 ml/100g by administration. The rats in the Nimodipine, Breviscapus and the united group were given corresponding medicine beforehand, and then given alcohol one hour later. All rats were administrated 14 days continuously. To detect the contents of cTnI, MDA, GSH-Px in the serum, blood was collected before alcohol administration, and 24 hours, 48 hours, 7 days and 14 days after. Compared to the control group, the content of cTnI in serum was increased significantly after alcohol administration and was higher than that before alcohol administration (p<0.05), Nimodipine or Breviscapus significantly decreased the increase of the cTnI content induced by alcohol. The changes of MDA content were similar to that of cTnI content. The content of GSH-Px in alcohol group was significantly decreased after 48 hours alcohol administration. However, in the Nimodipine, Breviscapus and the united group, the content of GSH-Px in serum was not obviously reduced. cTnI is one of the structural protein of myocardial cell. This experiment manifested that heavy alcohol consumption induced the increase of cTnI concentration in serum, which indicated that the myocardium was injured. The content of MDA was increased significantly in serum, indicating that oxidative damage might be one of the mechanisms of alcohol toxic effects. Our results show that administrated Nimodipine and Breviscapus can inhibit the increment of cTnI and MDA contents induced by alcohol administration, indicating that Nimodipine and Breviscapus have the protective effect on alcohol-induced myocardium injury.
ISOFLURANE MEDIATES RENAL ARTERY SMOOTH MUSCLE CONTRACTION THROUGH Ca2+-INDEPENDENT PKC SIGNALING Wan Min Pei 1, Jian Zhong Han 2, Li Li 1 1 Department of Anaesthesiology, the Second Xiang Ya Hospital 2 Department of Physiology, Basic Medical College, Central South University, Changsha, China To study the direct effect and potential mechanisms of isoflurane on renal artery smooth muscle, rabbit renal artery strips were treated with 3% saponin to permeate the cellular membrane. The calcium stored in endoplasmic reticulum (ER) was removed by caffeine. Then the renal artery strips were equilibrated in 10-7 M Ca2+-EGTA buffer. Steady tension was obtained when the strips were soaked in submaximal calcium concentration buffer. Then the submaximal calcium concentration EGTA buffer with isoflurane of various concentration or 10uM BIM was used, and the tension variety was collected and calculated by computer. 1 %; 3 % and 5 % isoflurane could all contract rabbit renal artery strips in the equilibrate buffer with a concentration-dependent manner. This effect of isoflurane on smooth muscle could be inhibited by BIM, an inhibitor for PKC, but not inhibited by Go¨6976, a Ca2+- dependent inhibitor for PKC. In conclusion, isoflurane could contract rabbit renal artery smooth muscle with a concentration-dependent manner, which may be mediated by Ca2+-independent PKC signaling.
STUDY ON IDENTIFYING GENOTYPES OF ECHINOCOCCUS GRANULOSUS BY MICROSATELLITE MARKERS Xiu Min Ma 1,2, Jian Bing Ding 1,2, Wulamu Mamuti 1,2, Xiao Mei Lu 2, Ren Yong Lin 2, Ya Lou Zhang 2, Ya Nan Wang 1, Akira Ito 3, Hao Wen 2 1 Department of Etiology, Basic Medical College 2 Medical Research Center, First Teaching Hospital, Xinjiang Medical University, Urumqi, 830054, Xinjiang, China 3 Department of Parasitology, Asahikawa Medical College, Asahikawa 078-8510, Japan This research used Sca, Emsk and C106 microsatellite sequence of Echinococcus granulosus as typing markers and identified the genotypes and heterozygosity of E.granulosus isolated from cystic echinococcosis (CE) patients in different areas of Xinjiang. Two different fluorescent dyes of FAM (carboxy fluorescein) and HEX (hexacho rofluorescein) were used as markers for three pairs of primers of microsatellite sequences. After amplification of satellites by PCR, amplified products were based on capillary electrophoresis by using 310 auto DNA analysis machine, and then basic number of amplified products was calculated by Genescan 2.1 software. Our result showed that 66 isolated strains from 44 CE patients were homozygote of E. granulosus, in which 65 isolated strains from 43 CE patients were identified as G1 genotype by PCR and microsatellite markers, and 1 isolated strain from 1 CE patient was identified as G6 genotype by PCR and microsatellite markers. The results of genotypes identified by microsatellite markers were consistent with the result of DNA sequence analysis. Mixing infection and heterozygote of G1 (sheep strain) and G6 (camel strain) genotypes were found in the dog intestines, but this research did not find E.granulosus heterozygote. It may indicate that E.granulosus was autofertilization, and therefore, few chance of heterozygote takes place in the patient. Microsatellite DNA markers can be used to finely and quickly identify the genotypes and heterozygosity of E.granulosus at genetic level, and possessed the important value in study of genetic polymophosim, epidemiology and pathogenicity of E.granulosus.
DETERMINATION OF PHENYLALANINE AND TYROSINE IN PERIPHERAL BLOOD AND ITS APPLICATION IN PHENYLKETONURIA Xi Ming Mo, Ai Guo Tang Department of Clinical Laboratory Science, Second Xiangya Hospital, Central South University, Changsha, China Several methods have been reported for determination of tyrosine (Tyr) or phenylalanine (Phe) in neonatal blood, but the simultaneous determination of Tyr and Phe in peripheral blood by reverse phase high-performance liquid chromatography (RP-HPLC) by no derivatization has not been reported. Here we describe a simple, accurate and reliable RP-HPLC assay for measurement of Tyr and Phe in peripheral blood of newborn infants and patients with phenylketonuria (PKU). Supernatant fluid of peripheral blood precipitated with perchloric acid was isocratically eluted using a base-deactivated C8 column with 5 % acetonitrile in water as the mobile phase. Untraviolet detector worked at 210nm. The retention time of tyrosine (Tyr) and phenylalanine (Phe) were 5.88min and 8.43min respectively. The effects of various aspects on operation and determination were examined to establish optimal assay conditions, such as precipitator, anticogulant, means of collecting and storing samples. Phenylalanine and tyrosine in finger blood of 102 healthy enfant were (67.7 15.4) mmol/L and (62.2 13.9) mmol/L respectively. The ratio of Phe/Tyr was 1.15 0.27. There was no obviously difference between males and females. Phenylalanine and tyrosine in heel blood of 32 healthy neonatal were (66.2 20.5) mmol/L and (59.518.8) mmol/L respectively. The ratio of Phe/Tyr was 1.12 0.24. The levels of
S61
phenylalanine and tyrosine in whole-blood which anticogulated by EDTA$K2 are consistent with those of anticogulating by heparin. The levels of phenylalanine and tyrosine in peripheral blood were significantly lower than those in plasma, but showed a good correlation. The means of collecting and storing sample affect the levels of phenylalanine and tyrosine in whole-blood.
THE MECHANISMS OF ALCOHOL-INDUCED MYOCARDIUM INJURY AND THE EFFECTS OF NIMODIPINE AND ERIGERON BREVISCAPUS Gui Lan Pan, Jing Hua Shi, Xiao Dong Chen The Department of Physiology, Baotou Medical College, Baotou, Inner Mongolia, 014010, China The changes of cardiac troponin I (cTnI), malondialdehyde (MDA), glutathione peroxidase (GSH-Px) contents in serum after acute alcohol administration, and the possible mechanisms of myocardium injury induced by alcohol and the protective effect of Nimodipine and Breviscapus were investigated. 40 Wistar rats were divided to 5 groups randomly. The rats in the control group were given physiological saline (1.5 ml/100g). The rats in the alcohol group were given 40% alcohol, 1.5 ml/100g by administration. The rats in the Nimodipine, Breviscapus and the united group were given corresponding medicine beforehand, and then given alcohol one hour later. All rats were administrated 14 days continuously. To detect the contents of cTnI, MDA, GSH-Px in the serum, blood was collected before alcohol administration, and 24 hours, 48 hours, 7 days and 14 days after. Compared to the control group, the content of cTnI in serum was increased significantly after alcohol administration and was higher than that before alcohol administration (p<0.05), Nimodipine or Breviscapus significantly decreased the increase of the cTnI content induced by alcohol. The changes of MDA content were similar to that of cTnI content. The content of GSH-Px in alcohol group was significantly decreased after 48 hours alcohol administration. However, in the Nimodipine, Breviscapus and the united group, the content of GSH-Px in serum was not obviously reduced. cTnI is one of the structural protein of myocardial cell. This experiment manifested that heavy alcohol consumption induced the increase of cTnI concentration in serum, which indicated that the myocardium was injured. The content of MDA was increased significantly in serum, indicating that oxidative damage might be one of the mechanisms of alcohol toxic effects. Our results show that administrated Nimodipine and Breviscapus can inhibit the increment of cTnI and MDA contents induced by alcohol administration, indicating that Nimodipine and Breviscapus have the protective effect on alcohol-induced myocardium injury.
ISOFLURANE MEDIATES RENAL ARTERY SMOOTH MUSCLE CONTRACTION THROUGH Ca2+-INDEPENDENT PKC SIGNALING Wan Min Pei 1, Jian Zhong Han 2, Li Li 1 1 Department of Anaesthesiology, the Second Xiang Ya Hospital 2 Department of Physiology, Basic Medical College, Central South University, Changsha, China To study the direct effect and potential mechanisms of isoflurane on renal artery smooth muscle, rabbit renal artery strips were treated with 3% saponin to permeate the cellular membrane. The calcium stored in endoplasmic reticulum (ER) was removed by caffeine. Then the renal artery strips were equilibrated in 10-7 M Ca2+-EGTA buffer. Steady tension was obtained when the strips were soaked in submaximal calcium concentration buffer. Then the submaximal calcium concentration EGTA buffer with isoflurane of various concentration or 10uM BIM was used, and the tension variety was collected and calculated by computer. 1 %; 3 % and 5 % isoflurane could all contract rabbit renal artery strips in the equilibrate buffer with a concentration-dependent manner. This effect of isoflurane on smooth muscle could be inhibited by BIM, an inhibitor for PKC, but not inhibited by Go¨6976, a Ca2+- dependent inhibitor for PKC. In conclusion, isoflurane could contract rabbit renal artery smooth muscle with a concentration-dependent manner, which may be mediated by Ca2+-independent PKC signaling.