Purification and Partial Characterization of an Indomethacin Hydrolyzing Enzyme from Pig Liver

Purpose. Indomethacin is well known to be metabolized via O-demethylation and N-deacylation. In this paper we found an enzyme involved in the hydrolysis of amide-linkage of indomethacin and partially characterized it as well as its substrate specificity. Methods. An indomethacin hydrolyzing enzyme was purified to homogeneity from pig liver microsomes using columns of Q-Sepharose, Red-Sepharose and Blue-Sepharose. The enzyme activity was assayed by measuring of -chlorobenzoic acid liberated from indomethacin by HPLC. Results. The purified enzyme effectively hydrolyzed the amide linkage in indomethacin but not those in -naphthylacetate and -nitrophenylacetate, which are typical substrates for carboxylesterase. The subunit molecular mass of the enzyme was 65 kDa according SDS-polyacrylamide gel electrophoresis. The Michaelis constant (Km) and maximum velocity (Vmax) values for indomethacin were 67.8 µM and 9.02 nmol/min/mg protein, respectively. The amino acid sequence analysis of the enzyme after cyanogen bromide cleavage showed high homology with a mouse carboxylesterase isozyme designated as ES-male. The activity of indomethacin hydrolysis was relatively high in the pig, rabbit and human liver homogenate, but not in those from rat and mouse. On the other hand, purified human liver carboxylesterases pl 5.3 and 4.5, and pig liver carboxylesterases have no catalytic activity for indomethacin. Conclusions. These results indicate that the hydrolysis of amide-linkage of indomethacin in humans would be associated with an enzyme similar to the indomethacin hydrolyzing enzyme from pig liver microsomes described here.     

Overexpression and purification of enzymatically active recombinant integrase protein of rous sarcoma virus

The carboxy-terminal domain of polymerase gene of Rous sarcoma virus was cloned into an expression vector under the control oflac regulatory elements, resulting in the plasmid pMF1413. Upon isopropyl--D-thiogalactopyranoside induction, viral integration (IN) protein was expressed in large quantity inEscherichia coli. The expressed recombinant protein was prepurified by successive washing of the bacterial pellet with 0.1 M NaCl and detergents. Further purification was performed in high yield by standard chromatography methods. The purified enzyme revealed selective DNA cleaving activity on supercoiled plasmid with the LTR-LTR junction fragment. The reaction was metal ion dependent, with a preference for Mn²⁺ over Mg²⁺, and showed substrate specificity at 1 mM MnCl₂.     

The effects of urinastatin on the plasma levels of granulocyte elastase during open heart surgery under simple deep hypothermia

Changes in granulocyte elastase (GLE) and -gluculonidase (-gl) were observed during open heart surgeries which were performed under deep hypothermia with surface cooling. In addition, the effect of urinary trypsin inhibitor, urinastatin, on the activities of these enzymes was studied. The patients were divided into three groups, namely group U-I with intravenous injection of 6000u·kg^–1 of urinastatin before cooling, group U-II administered with an additional 6000u·kg^–1 after warming to 30°C, and an untreated group (Group C). The plasma level of GLE increased significantly in the three groups compared with the level before cooling respectively. In the group U-II, the GLE level after the warming was lower than that in the control group. The serum level of -gl increased significantly in the three groups at the end of rewarming (36°C). The release of GLE from lysosomes in granulocytes was inhibited in the group U-II. The insufficient inhibition of GLE release in the group U-I is probably due to relatively short half-life of urinastatin. Therefore double administration of 6000u·kg^–1, before and after the cooling, may be required to achieve the therapeutic effect. Consequently, urinastatin appears to be useful in open heart surgery under deep hypothermia with surface cooling.(Kawamura T, Shimoda Y, Wakusawa R: The effects of urinastatin on the plasma levels of granulocyte elastase during open heart surgery under simple deep hypothermia. J Anesth 6: 269–276, 1992)     

Time-dependent kinetics. V: Time course of drug levels during enzyme induction (one-compartment model)

Equations were derived to describe the time course of drug levels during auto- and heteroinduction under a variety of input conditions. These equations were based on a pharmacokinetic theory of induction which assumes that metabolic clearance increases exponentially to a maximum value and that the rate of this increase is governed by the degradation rate constant of the induced enzyme (k). Closed form solutions could be obtained only for intravenous single-dose (case I) and multiple-dose (case IV) administration. For each of the other cases, constant-rate intravenous infusion (case III), oral single-dose administration (case II), and multiple-dose administration (case V), an exact solution (not closed form) and an approximation (closed form) were derived. Two sets of equations were derived for each of the five cases to take into consideration the possibility of a latency term ().Plots of drug amount X(or concentration C) vs. time (t) were constructed. In case I, a log Xvs. tplot was convex, the slope increasing with time. In case II, Xincreased,reached a peak, and decayed as in case I. In case III ( > 5In 2V/Q) Creached a preinduction steady state before decreasing to a lower (induced) steady state. When =0, Creached a maximum before decreasing to the same induced steady state. The behavior of Cvs. tfor cases IV and V was similar to that for case III. Determination of parameters was attempted in case III. Nonlinear least-square fitting of generated data with 3–9% error yielded reasonable estimates of k.This work was supported by NIH Research Contracts N0l-NS-1-2282 and N01-NS-6-2341.Parts I, II, III, IV, and VI of this series can be found in theJournal of Pharmaceutical Sciences.     

降纤酶低分子肝素治疗短暂性脑缺血发作的研究

目的 观察降纤酶与低分子肝素治疗短暂性脑缺血发作的效果及副作用。方法 选择本院神经内科住院患者36例应用降纤酶10U加入加入250ml生理盐水中静脉滴注，隔日1次，共3次；低分子肝素0．5ml脐旁皮下注射，12h 1次，连用7—10d,同时常规给予复方丹参滴注，口服尼莫地平，维生素E，维生素C，停用低分了肝素后给予肠溶阿斯匹林75mg，每日1次口服。结果 治疗开始后TLA发作相继减少，停止发作时间分别为1d内9例，3d内15例，5d内12例。随访6个月—1年，1例2个月后复发，重新应用上药治愈。结论 降纤酶与低分子肝素治疗TLA安全有效、无明显副作用、不易复发。     

亚硝基谷胱甘肽对大鼠肝微粒体谷胱甘肽转移酶的激活及机制

目的　探索一氧化氮供体亚硝基谷胱甘肽(GSNO)能否在体外通过S 亚硝酰化机制激活大鼠肝微粒体谷胱甘肽转移酶 (mGST)。方法　微粒体粗提物与GSNO体外共孵育 ,测定mGST催化动力学改变 ,结合N 乙基马来酰亚胺 (NEM )再激活实验和二巯基苏醇 (DTT)逆转实验 ,以及酶蛋白游离巯基和酶S 亚硝酰化蛋白的改变 ,研究酶的激活机制。结果　GSNO在 0 .12 5～ 2mmol·L- 1浓度范围内呈浓度和时间 (3～ 15min)依赖性地激活mGST ,NEM对酶的再激活效应消失 ,DTT可以逆转上述激活作用 ,同时酶蛋白游离巯基浓度依赖性减少 ,而S 亚硝酰化蛋白浓度依赖性增多。结论　GSNO体外可激活大鼠肝mGST ,激活机制可能与mGST第 4 9位半胱氨酸 (Cys4 9)的巯基被亚硝酰化形成S 亚硝基硫醇结构有关。     

中药地龙中溶栓成分研究进展

对近年来国内外地龙中溶栓成分(蚓纤溶酶、蚓激酶、蚓胶原酶)的研究情况进行归纳，介绍了地龙中溶栓成分的性质、分离纯化技术及开发应用状况，并提出了目前研究工作的不足和今后的发展方向。     

抗体夹心酶联免疫吸附法测定重组E.coli L-门冬酰胺酶研究

用重组E.coliL-门冬酰胺酶免疫家兔，DEAE-纤维素柱层析纯化IgG，采用二步戊二醛交联法将辣根过氧化物酶标记抗体，建立抗体夹心酶联免疫吸附法，测定大鼠血浆中重组E.coliL-门冬酰胺酶浓度。本测定方法的线性范围为1～64U/L，灵敏度为0.4U/L。建立的抗体夹心酶联免疫吸附法测定大鼠血浆中重组E.coliL-门冬酰胺酶具有良好的精密度、回收率、灵敏度和特异性。     

Diabetic nephropathy in children and adolescents: a critical review with particular reference to angiotensin-converting enzyme inhibitors

Clinical diabetic nephropathy is a well-recognized cause of increased morbidity and mortality in patients with type 1 diabetes. The finding that microalbuminuria predicts progression to overt nephropathy has allowed early diagnosis and preventive interventions. Several studies have demonstrated that treatment with angiotensin-converting enzyme (ACE) inhibitors slows down the rate of decline of the glomerular filtration rate in type 1 diabetes patients with established proteinuria. The renoprotective properties of the ACE inhibitor captopril extend beyond its antihypertensive effects. ACE inhibitors represent the most appropriate class of antihypertensive drugs for treating type 1 diabetes patients because of their efficacy and safety. When microalbuminuria is detected and confirmed in a diabetic child or adolescent, and if it persists despite 6-12 months of improved metabolic control, treatment with ACE inhibitors should be started, even if the child is normotensive. Careful follow-up of renal function is essential.     

尖吻蝮蛇毒中一种新类凝血酶的分离纯化

用阴离子交换色谱和凝胶过滤色谱技术,从尖吻蝮蛇毒中纯化得到一个具有凝血活性的组分.经Superdex 75 HR10/30预装柱检测,纯度达99.91%.SDS-PAGE显示为单一条带,还原、非还原条件下分子量分别为59.25k、52.58k.该组分的凝血酶比活为41.5u/mg,精氨酸酯酶比活为14.29u/mg,但不激活血浆凝血因子ⅩⅢ.EDTA不能抑制其凝血活性,而苯甲磺酰氟则产生不可逆抑制作用.结果表明该酶是一种新尖吻蝮蛇毒类凝血酶.