用Phast System自动电泳仪快速测定血清CK-MM亚型及初步临床应用

本文介绍了在Phast System全自动电泳仪上用等电聚焦法对血清CK-MM亚型的快速测定。灵敏度高,分辨率好,用1小时即可完成测定,特别适合临床需要。30例正常人血中CK-MM₁含量为44.3％,CK-MM₂为38.9％,CK-MM₃含量最低为16.7％,MM₃/MM₁比值小于1。而12例患者血中CK-MM亚型有明显变化,初步证实本法对早期诊断急性心肌梗塞(AMI)和溶栓治疗后判断是否出现再灌注有较高的临床价值。     

Gc、Pi蛋白亚型的快速微量电泳分析

用快速微量等电聚焦技术对190名北京地区汉族健康人血清Gc蛋白亚型、Pi蛋白亚型进行分型鉴定和基因频率调查.上样量为1.5μl,电泳和染色各0.5h.Gc^1F=0.4891,Gc^1S=0.2432,Gc²=0.2678.观察值与期望值吻合良好.(∑X²=1.404,0.7<P<0.8).Pi^M₁=0.7542,Pi^M₂=0.1808,Pi^M₃=0.0650,观察值与期望值吻合也良好,(∑X²=1.1233,0.7<P<0.8).     

全自动生化分析仪测定血清AST同工酶

应用天冬氨酸氨基转移酶抑制剂“AMANO-3”水解样品中的线粒体型天冬氨酸氨基转移酶同工酶（m-AST）,测定血清中剩余的胞浆型AST同工酶（c-AST）活性,进而与总AST活性相比较并计算出受抑制剂水解的m-AST同工酶活性．由于此方法采用蛋白水解反应破坏m-AST,因此测定方法可直接应用于生化自动分析仪．亦建立了一个适于日立7150分析仪的AST同工酶联合测定方法．其测定和计算出的m-AST同工酶批内CV为3.5％～7.9％;其结果(y)与AST同工酶电泳迁移率(x)相关．y=1.019x-0.489,r＝0.996（n＝30）．测定了113名健康人m-AST和c-AST,其m-AST参考值范围1.64～9.64U/L,x±s＝(5.641±2.013)U/L;c-AST参考值范围5.69～16.81U/L,x±s＝(5.641±2.013)U/L．     

均匀设计法优化烟管菌产漆酶培养基

应用均匀设计、二次多项式逐步回归分析对烟管菌(Bjerkandera adusta)WZFF.W-Y11产漆酶液态发酵培养基进行优化。结果表明,培养基组成为麸皮水解液1%、淀粉24.0 g/L、葡萄糖24.0 g/L、豆饼粉4.8 g/L、NH₄Cl 3.2g/L、KH₂PO₄ 3.2 g/L、MgSO₄·7H₂O 0.2 g/L、CuSO₄ ·5H₂O 0.006 g/L,起始pH6.5,在28℃、150r/min、250mL的摇瓶培养条件下可以稳定地获得9672U/L的漆酶活力。     

人心肌肌钙蛋白T的纯化和单克隆抗体的制备

从人左室心肌中成功纯化心肌肌钙蛋白T(cTnT). 经匀浆, 70℃加热处理, 咪唑盐酸透析, DEAE-纤维素层析, 100g心肌获取cTnT 5mg, 纯度为97.6%. 同时采用脾内免疫法, 免疫Balb/C小鼠, 经细胞融合, 筛选, 克隆化得5株稳定分泌抗人cTnT单克隆抗体(McAb)的杂交瘤细胞(G₃, G₈, G₁₀, A₅, A₇), 4株为IgM, 1株为IgG, 染色体数目92～110条. 腹水效价为3.2×10^-6～1. 6×10^-7.     

用Fura－2双波长荧光法测定神经细胞内游离钙

采用新型Ca²⁺荧光指示剂Fura-2建立双波长荧光法测定分离的大鼠神经细胞内游离钙浓度([Ca²⁺]_i).结果显示,在静息状态下,其[Ca²⁺]_i为109±12nmol/L.30mmol/L KCl可显著增加[Ca²⁺]_i,并且KCl的这种效应呈一定的剂量依赖关系,提示该法灵敏、可靠.     

CK-MB特异性单抗的制备方法及其应用

本研究拟建立肌酸激酶同工酶MB(CK-MB)特异性单克隆抗体(m Ab)的研制方法,对抗CK-MB单抗进行评价分类及性质鉴定,并初步建立CK-MB定量检测试剂。以CK-MB抗原免疫BALB/c小鼠,利用常规单抗制备技术,使用间接和捕获ELISA差异筛选法筛选单抗。利用肌酸激酶同工酶(CK-MM/BB/MB)抗原对所制备单抗的抗原识别表位进行鉴定,另通过免疫印迹法及合成CK-MM、CK-BB差异性的线性表位肽鉴定对所制备的单抗进行评价分类。使用双抗体夹心ELISA方法筛选检测CK-MB抗原的配对m Ab,并初步建立CK-MB定量检测试剂。使用74例临床标本初步评价该试剂与罗氏试剂的检测一致性。最终,我们成功筛选到22株稳定分泌抗CK-MB抗体的杂交瘤细胞株,这些单抗可以分为线性、偏构象的CK-MB和CK-MM或者CK-BB交叉的单抗以及与CK-MB特异反应的偏构象型单抗,并使用偏构象型单抗研制出CK-MB定量检测试剂,该试剂与罗氏试剂相关系数r达到0.930 9。综上所述,本研究建立了研制CK-MB偏构象型特异性单抗的筛选方法,通过对所筛选的单抗进行分析鉴定并建立了CK-MB定量检测试剂,与罗氏试剂检测结果符合率高。     

重组大肠杆菌产角质酶-CBM摇瓶发酵优化及分泌表达研究

在TB培养基的基础上,通过单因素分析和正交设计对重组大肠杆菌产角质酶-CBM发酵进行优化,得到最适培养基的组分为:甘油5 g/L,蛋白胨 16 g/L,MgSO₄·7H₂O 2.5 mmol/L,K₂HPO₄ 13.7 g/L,KH₂PO₄ 1.53 g/L,菌体生长至对数前中期时添加终浓度为1 g/L乳糖 和0.75 g/L 甘氨酸,30℃发酵48 h,角质酶-CBM产量可达63 U/ml,较TB培养(20 U/ml)提高了近3倍。考察了热激作用、渗透调节物质及温度两控制对角质酶-CBM分泌表达的影响,在添加Lactose和Glycine后,发现在添加终浓度为75 mmol/L的L-脯氨酸,37℃热激1 h或47℃热激0.5 h,变温至25℃发酵,角质酶-CBM产量可达90 U/ml,较TB恒温培养提高了近四倍。     

基因工程菌Pichia pastoris高密度培养条件研究*

基因工程菌pichiapastoris最佳种子培养基为添加 4mL/LPTMl的BMGY培养基 ;全合成高密度摇瓶培养基是甘油 4% ,(NH₄) ₂ SO₄1 0g/L ,CaSO₄0 .93g/L ,K₂ SO₄1 8.2g/L ,MgSO₄·7H₂O 1 4.9g/L ,0 1mol/L磷酸缓冲液 (pH =6     

过氧化氢对培养心肌细胞损伤作用的研究

氧化应激时产生大量的自由基,造成心肌细胞的损伤.过氧化氢(H₂O₂)是有机体氧化代谢产物,同时是一种活性氧.应用不同浓度的H₂O₂,分别于不同作用时间,动态观察其对心肌细胞的损伤作用.从实验结果看到,低浓度的H₂O₂（＜0.1 mmol/L）作用2 h,使心肌细胞产生早期的生物化学的改变,如MDA产生堆积和细胞周期时相改变（G1期细胞增加,G2期细胞减少）,此时心肌酶基本无泄漏,心肌细胞的死亡率很低,HE形态学观察基本无改变;随着H₂O₂浓度的增加（1～5 mmol/L）和作用时间的延长,进一步诱导细胞损伤加剧,LDH释放和MDA积累明显升高,细胞死亡率也明显增加,已具有统计学意义.同时可观察到其病理形态学的坏死性改变;当10 mmol/L H₂O₂作用时,细胞大量死亡,形态学可见细胞极度收缩、脱落,形成大面积的细胞脱失区.因此,H₂O₂作为一种活性氧自由基,依其浓度和作用时间不同可造成不同程度的心肌细胞的损伤.辣根过氧化物酶作为一种自由基清除剂,可明显减少H₂O₂活性氧自由基对心肌细胞的损伤作用.     

聚焦色谱法测定肌型肌酸激酶亚型

报道了测定CK-MM亚型的聚焦色谱法,此法简单,快速,结果可靠,线性范围宽,最低检测限（8U／L）较正常参考值低,比国外报道的类似方法高6倍以上,分离度亦有改进.测定了20例健康人血清亚型分布,与文献报道结果相近.该法自动化程度高,已在急性心梗的诊断中实际应用.     

Experimental determination of activation potentials of CK-isoenzymes in human serum and their significance

The activation/inactivation of creatine kinase isoenzymes is dependent upon the redox potential of the activator employed. From comparison of the different activating effects with various oxidizing-reducing agents of known redox potentials, a method for determining the mid-point potentials, E_m⁷ (pH 7.0, 30°C) for activation of CK-isoenzymes has been developed. The E_m⁷ values for CK-MM and MB in human serum were respectively, ?0.08 V, ?0.31 V (± 0.005 SD). The relative stabilities of CK isoenzymes are a function of their E_m⁷ for activation.     

Differentiation of creatine kinase MB and IgA-linked BB isoenzymes on electrophoresis

IgA-linked creatine kinase (CK, EC 2.7.3.2) is a macro CK type 1 isoenzyme that has an identical electrophoretic mobility to CK-MB. Its presence has the potential of causing misdiagnosis of myocardial infarction. Mixing anti-CK-B antiserum with the sample prior to electrophoresis did not unequivocally distinguish between the two isoenzymes. Similarly, anti-human IgG and IgM antibodies were also ineffective. However, the IgA-linked isoenzyme band was removed by anti-human IgA antiserum. While anti-CK-M antibodies did not affect the electrophoretic mobility of IgA-linked CK-BB, the antibody eliminated both the CK-MB and CK-MM bands. Thus, specific anti-IgA and anti-CK-M antibodies may be used to establish the presence of the myocardial isoenzyme.     

Glutamine synthetase from mesophyll and bundle sheath maize cells: isoenzyme complements and different sensitivities to phosphinothricin

 Anion-exchange FPLC has been used to resolve the isoforms of glutamine synthetase (GS, EC 6.3.1.2) from Zea mays mesophyll (MC) and bundle sheath cells (BSC). Two different isoforms were detected in both types of photosynthetic cells. The predominantly active isoform was GS1 (61%) in MC and GS2 (67%) in BSC. The relative contribution of GS1 and GS2 to the overall GS activity in BSC in maize here reported resembles the proportion described for most C3 plants. Differences among these isoforms in terms of their susceptibility to phosphinothricin (PPT), an analogue of glutamate and known inhibitor of GS, were found. The GS1 isoenzyme from MC was the most sensitive form, being inhibited by 50% at approximately 2.0 μM DL-PPT, whereas the GS2 from BSC presented the highest tolerance to the inhibitor (I₅₀=30 μM). The transferase-to-semibiosynthetic activity ratio for the MC isoforms, which was higher than the ratio for the BSC isoforms, and the differences shown by the isoforms in susceptibility to PPT predict important differences in the biochemical properties and regulation of GS isoenzymes. In this regard, the cytoplasmic isoenzymes, and especially the one in MC, due to its relatively high contribution to mesophyll cell GS activity, could play a vital role in nitrogen metabolism in maize. Received: 1 December 1999 / Revised: 7 February 2000 / Accepted: 23 February 2000     

Creatine kinase-MB isoenzyme adaptations in stressed human skeletal muscle of marathon runners

The creatine kinase (CK) isoenzyme composition was determined in serial gastrocnemius muscle biopsies obtained from 12 male marathon runners. The mean muscle CK-MB composition significantly increased after chronic exercise (training) from 5.3% (pretraining) to 7.7% (premarathon) as well as after acute exercise (postmarathon) to 10.5% of the total CK activity (P less than 0.05). However, no significant differences in total CK activities were detected. Additionally, mitochondrial CK and CK-BB isoenzymes were present in muscle homogenates. A significant correlation was observed in the increase in mean serum total CK (3,322 U/l) and CK-MB (174 U/l) activities 24 h after the race (r = 0.98, P less than 0.05). These results show that gastrocnemius muscle adapts to long-distance training and racing with increased CK-MB activities and imply that skeletal muscle is the major source of elevated serum CK-MB activities in marathon runners.     

Effect of Resveratrol on Antioxidant Enzyme Activities in the Brain of Healthy Rat

We have studied the effect of resveratrol on lipoperoxidation and antioxidant enzyme activity level in the brain of healthy rats. When intraperitoneally administered, resveratrol significantly and dose dependently decreased brain malondialdehyde level. Resveratrol also increased in a dose-dependent way brain superoxide dismutase, catalase and peroxidase activities. Optimal effect on antioxidant enzyme and lipoperoxidation products were obtained with resveratrol concentration of 12.5 mg/kg body wt. Native polyacrylamide gel electrophoresis analysis of antioxidant isoenzymes revealed that resveratrol up regulated at least two acidic superoxide dismutase isoforms called A₁ and A₂, two basic isoforms called B₁ and B₂. Resveratrol also up regulated two catalase isoforms and a broad peroxidase band corresponding to several isoforms. All these findings suggest that resveratrol is able to cross the blood brain barrier and exerts potent antioxidant features. Resveratrol also exerts neuroprotective properties by up regulating several detoxifying enzymes, most of which are iron proteins.     

Macro CK type 1 as a major component of serum creatine kinase activity in pregnant sheep

Electrophoretic separation of creatine kinase (ATP: creatine phosphotransferase, E.C. 2.7.3.2) isoenzymes in agarose gel was made in the serum of 93 sheep of three Slovenian domestic breeds (Solcavska, Pramenka and Bovska breeds). 44 sheep were pregnant between 112 and 135 days at the time of the blood collection. The median value of serum total CK activity for all animals investigated was 82 U/l. After direct immunoinhibition of CK with anti-M-CK monoclonal antibodies the total CK activity remained the same (88 U/l, P = 0.354). There were significant differences among breeds in CK activity for the Solcavska (101 U/l), Bovska (89 U/l) and Pramenka breeds (73 U/l), respectively (Kruskal–Wallis one way analysis of variance, P < 0.01), and between pregnant (105 U/l) and non-pregnant animals (76 U/l), irrespective of the breed (Mann–Whitney rank sum test, P < 0.05). According to electrophoresis, all non-pregnant sheep had exclusively free CK-BB serum bands activity. In all pregnant sheep coupled dimeric BB variant appeared as macro-CK type 1 in the range between 80% and 100% of total CK activity. The present study confirms the existence of an elimination mechanism for CK from the plasma abundance free CK-BB enzyme.     

Creatine kinase isoenzyme activity during and after an ultra-distance (200 km) run

It is commonly assumed that creatine kinase (CK) activity in plasma is related to the state of an inflammatory response at 24-48 h, and also it has shown biphasic patterns after a marathon run. No information is available on CK isoenzymes after an ultra-marathon run. The purpose of the present study is to examine the CK isoenzymes after a 200 km ultra-marathon run and during the subsequent recovery. Blood samples were obtained during registration 1 2 h before the 200-km race and during the race at 100 km, 150 km and at the end of 200 km, as well as after a 24 h period of recovery. Thirty-two male ultra-distance runners participated in the study. Serum CPK showed a marked increase throughout the race and 24 h recovery period (p < 0.001). Serum CK during the race occurs mostly in the CK-MM isoform and only minutely in the CK-MB isoform and is unchanged in the CK-BB isoform. High-sensitivity C-reactive protein (hs-CRP), oestradiol, AST and ALT increased significantly from the pre-race value at 100 km and a further increase took place by the end of the 200 km run. The results of our study demonstrate a different release pattern of creatine kinase after an ultra-distance (200 km) run compared to the studies of marathon running and intense eccentric exercise, and changes in several biomarkers, indicative of muscle damage during the race, were much more pronounced during the latter half (100–200 km) of the race. However, the increases in plasma concentration of muscle enzymes may reflect not only structural damage, but also their rate of clearance.     

Expression of Peroxidases During Seedling Growth in <Emphasis Type="Italic">Ebenus Cretica</Emphasis> L. as Affected by Light and Temperature Treatments

Peroxidase (POD) activity and isoform patterns were investigated during seedling growth (up to 20 days) of Ebenus cretica L. Seeds germinated to approx. 100% after a 24-h imbibition. Seedling growth proceeded smoothly, in both light and dark conditions. No seedling growth was noticed at 4°C. A positive effect of light and increasing temperature (4, 10, 16, 22 and 28°C) on seedlings growth, lignin content and POD activity was observed. Lignin content was 2.5 times higher in seedlings grown under light than in seedlings grown under darkness. Seedlingsȁ9 POD activity was higher in acid pH (5.5) in comparison to neutral pH (7.0). These activities were higher in seedlings grown under darkness than in those grown under light; since additional POD isoforms were expressed in dark conditions. The increase in POD activity was accompanied with the appearance of new POD isoforms correlated with the growth of the seedlings. Four soluble anionic POD isoforms (named A₁, A₂, A₃ and A₄) and three soluble cationic POD isoforms (named C₁, C₂ and C₃) were displayed depending on the treatment and the course of growth. POD isoforms were detected in gel after PAGE. The fast migrating (A₄) isoform, which appeared in the dark-grown seedlings as well as on day 20 at 28°C in the temperature treatment, was separated by DEAE–Sepharose column chromatography. A slow migrating C₁ isoform slightly appeared in both 4 and 10°C temperature treatments and could be related to the low temperature treatments, while A₁, A₂, A₃ and C₂ to the growth stage of seedlings. The expression of seven POD isoforms during seedling growth seems to be related to different developmental events of growth and could be used as useful biochemical markers in the analysis of metabolic regulation in seedling growth of Ebenus cretica.     

The effect of estrogen on muscle damage biomarkers following prolonged aerobic exercise in eumenorrheic women

This study assessed the influence of estrogen (E₂) on muscle damage biomarkers [skeletal muscle - creatine kinase (CK); cardiac muscle - CK-MB] responses to prolonged aerobic exercise. Eumenorrheic women (n=10) who were physically active completed two 60-minute treadmill running sessions at ∼60-65% maximal intensity during low E₂ (midfollicular menstrual phase) and high E₂ (midluteal menstrual phase) hormonal conditions. Blood samples were collected prior to exercise (following supine rest), immediately post-, 30 min post-, and 24 hours post-exercise to determine changes in muscle biomarkers. Resting blood samples confirmed appropriate E₂ hormonal levels Total CK concentrations increased following exercise and at 24 hours post-exercise were higher in the midfollicular low E₂ phase (p<0.001). However, CK-MB concentrations were unaffected by E₂ level or exercise (p=0.442) resulting in the ratio of CK-MB to total CK being consistently low in subject responses (i.e., indicative of skeletal muscle damage). Elevated E₂ levels reduce the CK responses of skeletal muscle, but had no effect on CK-MB responses following prolonged aerobic exercise. These findings support earlier work showing elevated E₂ is protective of skeletal muscle from exercise-induced damage associated with prolonged aerobic exercise.