对氨基苯甲酸酯同系物形成细胞色素P450代谢中间体络合物的定…

细胞色素Ｐ４５０（Ｐ４５０,Ｅｃ,１．１４．１４．１）是一种十分重要的催化氧化反应的酶,本文测定了１２个对氨基苯甲酸酯用系物与Ｐ４５０相互作用而形成Ｐ４５０代谢中间体络合物的活性,用半经验分子轨道法ＭＮＤＯ－ＰＭ３计算得到了这些同系物的分子轨道指数,并用逐步多元回归分析法导出了活性与分子轨道指数及正辛醇／水分配系数的对数值之间的定量结构与活性关系。结果表明,对苯氨基苯甲酸脂同系物形成Ｐ４５０代谢中     

对氨基二苯醚类化合物与细胞色素P450相互作用的定量与活性关系

对氨基二苯醚类化合物是结构色素Ｐ４５０（Ｐ４５０）的一种准不可逆抑制剂。本文测定了１７种这类化合物生成Ｐ４５０代谢中间体络合物的活性和抑制Ｐ４５０催化氧化氨基比林脱甲基的活性。用逐步多元回归分析法导出了这两种活性与Ｈａｎｓｃｈ理化参数或分子轨道指数之间的定量结构与活性关系（ＱＳＡＲ）。结果表明：这两种活性都与取代基的脂溶性参数之（Σπ）和间位的立体性参数Ｅｓ（３′）等Ｈａｎｓｃｈ理化参数或最低未占据分子轨道级（ＥＬＵＭＯ）和原子净电荷的绝对值之和（ΣＱ）等分子轨道指数相关;生成Ｐ４５０代谢中间体络合物的活性还与取代基的电性参数之和（Σσ）和邻位和立体性参数（Ｅｓ（２′）相关。两种活性之间存在很好的相关性。     

对氨基二苯醚类化合物与细胞色素P450相互作用的定量与活性关系

对氨基二苯醚类化合物是结构色素Ｐ４５０（Ｐ４５０）的一种准不可逆抑制剂。本文测定了１７种这类化合物生成Ｐ４５０代谢中间体络合物的活性和抑制Ｐ４５０催化氧化氨基比林脱甲基的活性。用逐步多元回归分析法导出了这两种活性与Ｈａｎｓｃｈ理化参数或分子轨道指数之间的定量结构与活性关系（ＱＳＡＲ）。结果表明：这两种活性都与取代基的脂溶性参数之和（Σπ）和间位的立体性参数Ｅｓ（３＇）等Ｈａｎｓｃｈ理化参数或最低未     

4—氨基二苯醚对体内外肝微粒体混合功能氧化酶催化活性的影响

小鼠每天腹腔注射４－氨基－２’,４’－二氯二苯醚（２’,４’－ｄｉＣｌ）及４－氨基－４’－甲基二苯醚（４’－ＣＨ３）４天,于末次给药２４ｈ后实验,发现：（１）以小鼠戊巴比妥睡眠时间作为体内Ｐ４５０活性指标,４’－ＣＨ３组Ｐ４５０活性高于生理盐水组,而２’,４’－ｄｉＣｌ组小鼠体内Ｐ４５０活性低于生理盐水组。（２）两处理组小鼠体外测得肝微粒体Ｐ４５０含量均显著高于生理盐小组（Ｐ＜０．０５）,低于苯巴     

Beagle狗肾细胞及用于制备乙脑活疫苗的研究

Ｂｅａｇｌｅ乳狗（５－１０日龄）肾块用热消化法制成细胞批放液氮保存,检定合格后做下列试验：（１）复苏培养长满单层后更换维持液,其细胞能维持４天以上,克服了常规消化培养细胞维持时间短（３６小时）的缺陷。（２）传代１４－２株病毒时,１代病毒的滴度即达６．０４－６．２４ＬｏｇＰＦＵ／ｍｌ,１代后的各代（至１１代）病毒滴度无明显提高。（３）该细胞接种１４－２株病毒时不产生明显的破坏性病变（ＣＰＥ）,在收毒前采取冻溶１次比冻溶前的病毒滴度提高０．２７－０．５ＬｏｇＰＦＵ／ｍｌ。以ＢＤＫ细胞传１代的１４－２株病毒作生产毒种,收毒前冻溶１次等工艺法制备五批疫苗,全面检定结果均符合《乙脑活疫苗规程》的质量标准。其中病毒滴度为６．０９－６．２４ＬｏｇＰＦＵ／ｍｌ;免疫效价（ＩＤ５０＝ｍｌ）为１．９－４．０×１０－５,与相同滴度的原毒株１４－２ＰＨＫ苗对照无明显差异（ｔ＝０．９６８Ｐ＞０．０５）,表明其免疫原性与原毒株相似。     

4－氨基二苯醚对体内外肝微粒体混合功能氧化酶催化活性的影响

小鼠每天腹腔注射４－氨公－２’,４’－二氯二苯醚（２’,４’－ｄｉｃｌ）及４－氨基－４’－甲基二苯醚（４’－ＣＨ３）４天,于末次给药２４ｈ后实验,发现：（１）以小鼠戊巴比妥睡眠时间作为体内Ｐ４５０活性指标,４’－ＣＨ３组Ｐ４５０活性高于生理盐水组,而２’,４’－ｄｉＣｌ组小鼠体内Ｐ４５０活性低于生理盐水组。（２）两处理组小鼠体外测得肝微粒体Ｐ４５０含量均显著高于生理盐水组（Ｐ＜０．０５）,低于苯巴比妥组。（３）２’,４’－ｄｉＣｌ组小鼠由ｚｏｘａｚｏｌａｍｉｎｅ引起的瘫痪时间显著高于生理盐水组（Ｐ＜０．０１）,提示该化合物对体内Ｐ４４８活性亦有抑制作用。体外实验也发现,两者对ＢＮＦ诱导的大鼠肝微粒体Ｐ４４８的活性均有不同程度的抑制。     

磺胺类眼药水中金黄色葡萄球菌检验方法的比较试验

磺胺类眼药水中金黄色葡萄球菌检验方法的比较试验白秀艺（朝阳市药品检验所．１２２０００）磺服药物是有效的抑菌药物,其作用机制是对抗对胺基苯甲酸（ＰＡＢＡ）所参与的代谢作用。磺胺药物的分子结构和电荷分布与对氨基苯甲酸都很相似,这是它能和Ｐ人ＢＡ对抗的原因...     

芒果苷代谢产物的合成及黄嘌呤氧化酶抑制活性研究北大核心CSCD

以2,4,5-三甲氧基苯甲酸和2,5-二甲氧基苯甲酸为原料合成芒果苷的两个主要代谢产物1,3,6,7-四羟基氧杂蒽酮（2）和1,3,7-三羟基氧杂蒽酮（3）,同时获得四个合成中间体（4～7）,利用^1H NMR和^13C NMR鉴定结构。活性研究表明代谢产物2能浓度依赖性地抑制黄嘌呤氧化酶活性,其IC50为10.89 μM;代谢产物3及中间体5、7也有抑制作用,但弱于2,而中间体4、6和芒果苷则没有明显的抑制作用;灌胃给予小鼠等分子剂量的芒果苷及2后,均明显降低氧嗪酸钾诱导的高尿酸血症小鼠的血尿酸水平,两者效价相似,提示芒果苷的降尿酸作用与其代谢产物抑制黄嘌呤氧化酶活性的作用有关。     

大鼠肝生长发育过程中ACP、AKP、HSC70/HSP68和PCNA的变化(英文)

磷酸酶（ＡＣＰ、ＡＫＰ）在生物的机能分化中起重要作用,热休克蛋白（ＨＳＰｓ）是近几年发现的一类在胚胎发育、细胞生存中起重要作用的分子,无论是胚胎发育还是细胞结构和功能构建都和细胞增殖密切相关,增殖细胞核抗原（ＰＣＮＡ）是检测细胞增殖的良好指标。 本实验用组织化学、免疫组织化学、Ｗｅｓｔｅｒｎ印迹、酶的原位复性电泳、体视学分析等方法定性和定量分析了酸性磷酸酶（ＡＣＰ）（Ｆｉｇ．１＆２）、碱性磷酸酶（ＡＫＰ）（Ｆｉｇ．４＆５）、构成性热休克蛋白 ７０／诱导性热休克蛋白 ６８（ＨＳＣ７０／ＨＳＰ６８）（Ｆｉｇ．６）和ＰＣＮＡ（Ｆｉｇ．７＆８, Ｔａｂｌｅ１）在大鼠肝生长发育（从１４天胚胎到成体）过程中的动态变化。结果表明：（１）在大鼠肝生长发育过程中,ＡＣＰ有两个活性高峰期,其时段处于大鼠吃奶和吃饲料起始期（Ｆｉｇ．１＆２）;（２）在ＡＣＰ的第一个活性高峰期时,ＡＫＰ活性降低;而在ＡＣＰ的第二个活性高峰期时,正值ＡＫＰ的活性高峰期（Ｆｉｇ．３）;（３）ＡＣＰ活性高峰期也是ＰＣＮＡ含量高峰期;（４）ＨＳＣ７０／ＨＳＰ６８在刚断奶的幼鼠肝和成体肝中表达量较多,其他时段表达极少。根据上述结果推测：ＡＣＰ和ＰＣＮＡ通过调节细     

脂氧合酶对黄瓜叶片光合电子传递活性的影响

黄瓜（Ｃｕｃｕｍ ｉｓｓａｔｉｖｕｓＬ．）叶片的光合作用与电子传递活性随叶片衰老而降低,脂氧合酶（Ｌｏｘ）活性则相应增高。大豆Ｌｏｘ－１ 抑制黄瓜子叶分离的叶绿体ＰＳⅡ电子传递活性,没食子酸丙酯（ＰＧ）或槲皮酮（ＰＦ）能消除这种抑制作用。二氯苯二甲脲（ＤＣＭＵ）或２,３－二溴百里香醌（ＤＢＭＩＢ）存在时,大豆Ｌｏｘ－１ 对ＰＳⅡ电子传递活性有抑制作用,加入ＰＧ 使活性恢复到接近原有水平。二苯卡巴肼（ＤＰＣ）对经Ｌｏｘ－１ 处理的叶绿体ＰＳⅡ电子传递活性有明显恢复作用。Ｌｏｘ－１ 使Ｃｈｌａ室温荧光Ｆｍ 降低,ＤＰＣ则能轻微恢复Ｆｍ 。可见,ＰＳⅡ氧化侧、Ｑ与ＰＱ 位点都对Ｌｏｘ－１ 的作用敏感。Ｌｏｘ－１ 对叶绿体色素的漂白作用,以及活性氧对ＰＳⅡ电子传递活性的抑制,可能是Ｌｏｘ 影响光合膜功能的重要原因之一     

Biochemical background of toxic interaction between tiamulin and monensin

Tiamulin, a diterpene antibiotic, is used for treatment of pulmonary and gastrointestinal infections in swine and poultry. Combined administration of tiamulin and ionophores (e.g. monensin) to farm animals may lead to intoxication manifested in severe clinical symptoms. Tiamulin metabolite complex with cytochrome P450 has been suggested to be the basis of drug-interactions. However, the formation of metabolic intermediate complex is questionable. The effect of tiamulin-treatment on cytochrome P450 activities was investigated in rats. Ethylmorphine and aminopyrine N-demethylation activities as well as monensin metabolism (O-demethylation) increased in liver microsomes of tiamulin-treated (200 mg/kg) animals. CYP3A1 induction caused by tiamulin was confirmed by the results of Western blot analysis. To test metabolic intermediate complex formation as a result of tiamulin treatment, cytochrome P450 activities were also determined in the presence of potassium ferricyanide. The findings together with those of in vitro complex formation suggested that formation of metabolic intermediate complexes of tiamulin with cytochrome P450 could be excluded. On the other hand, the results of inhibition studies showed significant decrease of ethylmorphine or aminopyrine as well as monensin demethylation in the presence of tiamulin. Our results proved that tiamulin has dual effect on cytochromes P450. It is able to induce and directly inhibit CYP3A enzymes, which are predominantly responsible for monensin O-demethylation. The direct effect of tiamulin as an inhibitor might play a more important role in toxicity than its putative effect as a chemical inducer of CYP3A enzymes.     

Cytochrome P-450 ligands: metyrapone revisited

Expressing metyrapone interactions with ferrous cytochrome P-450 as ligand saturation by cytochrome, rather than the more conventional cytochrome saturation by ligand, an extinction coefficient of 68.5 +/- 1.8 mM-1 cm-1 for the metyrapone complex of dithionite-reduced rat hepatic microsomal cytochrome P-450 was derived. Utilizing this new extinction coefficient, the increased cytochrome P-450 present after phenobarbital induction was almost exclusively that which is able to both bind to metyrapone and form a metabolic-intermediate complex from norbenzphetamine. However, it was not the only subpopulation present in microsomes that was able to bind metyrapone, nor the only one capable of forming a metabolic intermediate complex from norbenzphetamine. Thus, neither technique alone can be used to quantitate the "phenobarbital-induced form" of cytochrome P-450.     

Fluorescence energy transfer measurements of the complexes of aflatoxin B1 and cytochromes P-450

The distances between the heme of cytochrome P-450 and the substrate, aflatoxin B1, in the complex of aflatoxin B1 and each of two species of cytochrome P-450 were determined by fluorescence energy transfer measurements. Cytochromes P-450 used were cytochrome P-450 I-d and cytochrome P-450 II-a prepared from hepatic microsomes of polychlorinated biphenyl-treated rats; the main metabolic products of aflatoxin B1 were aflatoxin Q1 and aflatoxin M1, respectively. The distances between the heme and the substrate were calculated to be 6.9nm and 4.7nm in cytochrome P-450 I-d and cytochrome P-450 II-a, respectively. The results suggest that the difference in the metabolic products of aflatoxin B1 is due to the difference in the conformation of the enzyme-substrate complexes.     

A molecular orbital study of the metabolic pathway with hydroquinone intermediate from benzene as carcinogen

A possible metabolic activation pathway of benzenein vivo is the nonenzymatic oxidation of hydroquinone producedvia the cytochrome P-450-mediate two-step oxidation of benzene. The mechanism of the further oxidation of hydroquinone and the nature of the most reactive intermediate have not yet been clarified, although it is speculated that the intermediate isp-benzoquinone and/orp-benzosemiquinone. The theoretical result of using molecular orbital calculations (ab initio and CNDO/2 methods) indicates that although the mechanism of the nonenzymatic oxidation of hydroquinone cannot yet be determined, the intermediate is thep-benzosemiquinone anion radical. It is also suggested that active-oxygen species such as hydroxyl radical, which accelerates the nonenzymatic oxidation, play an important role in the metabolic pathway in question.     

Coenzyme Q: a molecular orbital study.

The molecular structure and electronic properties of coenzyme Q, also known as ubiquinone, have been studied with the methods of semiempirical molecular orbital theory. It is found that the preferred conformation of the isoprene unit is such that formation of the chroman is favorable. The methoxy groups then assume a nonplanar configuration. Orbital energies, dipole moments, and spectra for the coenzyme Q₁ molecule and some of its chroman intermediates are also given in the calculations. The possibility of charge-transfer complex formation of chroman intermediate with inorganic phosphate is discussed.     

Two-step concerted mechanism for methane hydroxylation on the diiron active site of soluble methane monooxygenase

A new concerted mechanism is proposed for the conversion of methane to methanol on intermediate Q of soluble methane monooxygenase (sMMO), the active site of which is considered to involve an Fe2(mu-O)2 diamond core. A hybrid density functional theory (DFT) method is used for our mechanistic study on the important reactivity of the bare FeO+ complex and a diiron model of intermediate Q. The reaction pathway for the methane hydroxylation on the diiron complex is essentially identical to that for the gas-phase reaction by the bare FeO+ complex. Methane is highly activated on the dinuclear iron model through the formation of a methane complex, in which a coordinatively unsaturated iron plays a central role in the bonding interaction between the diiron model and substrate methane. A H atom abstraction via a four-centered transition state and a recombination of the OH and CH3 groups via a three-centered transition state successively occur on the dinuclear iron-oxo species, leading to the formation of a methanol complex that corresponds to intermediate T. These electronic processes take place in a concerted manner. Our mechanism for methane hydroxylation by sMMO is different from the radical mechanism that has been widely accepted for enzymatic hydrocarbon hydroxylation, especially by cytochrome P450.     

The cytochrome P-450cam binding surface as defined by site-directed mutagenesis and electrostatic modeling

Cytochrome P-450cam cationic surface charges at Lys 344, Arg 72, and Lys 392 have been altered by site-directed mutagenesis techniques. The residues at Lys 344 and Arg 72 were previously suggested as salt bridge contacts in the cytochrome b5-cytochrome P-450cam association complex and implicated in the physiological putidaredoxin-cytochrome P-450cam complex [Stayton, P. S., Poulos, T. L., & Sligar, S. G. (1989) Biochemistry 28, 8201-8205]. Mutations to neutralize the basic charge at Arg 72 (R72Q) and to both neutralize and reverse the charge at Lys 344 (K344Q, K344E) resulted in alteration of NADH oxidation rates in the reconstituted physiological electron-transfer system, which is rate limited by putidaredoxin-cytochrome P-450cam electron transfer. The steady-state Vmax values were apparently unperturbed, suggesting that the observed rate differences were largely attributable to Km effects. The Km values observed for the K344Q (24 microM) and K344E (32 microM) mutants are in the direction expected for neutralization and reversal of a salt bridge charge interaction. A control mutation at a basic surface charge located away from the proposed site of interaction, Lys 392 (K392Q), resulted in overall activities quantitated by NADH oxidation rates that are similar to that of wild-type cytochrome P-450cam. Calculation of the cytochrome P-450cam electrostatic field revealed a patch of positive potential at the modeled cytochrome b5 interaction site lying directly above the nearest proximal approach to the buried heme prosthetic group. These results provide experimental and theoretical evidence for the modeled cytochrome P-450cam binding site implicated in both cytochrome b5 and putidaredoxin association.(ABSTRACT TRUNCATED AT 250 WORDS)     

Natural hybridisation between Quercus petraea (Matt.) Liebl. and Quercus pubescens Willd. within an Italian stand as revealed by microsatellite fingerprinting

Interspecific gene flow is frequently reported in the genus Quercus . However, interfertile oak species often seem to remain distinct, even within areas of sympatry. This study employed molecular markers to verify, at a fine scale, the presence of interspecific gene flow in a natural population of Quercus petraea and Quercus pubescens . Within a delimited area of 6 ha, all adult trees belonging to the studied oak complex and seeds from a subsample of such trees were collected and analysed using molecular microsatellite markers. A low interspecific genetic differentiation and a high level of interspecific genetic admixture suggested past hybridisation. Paternity inference of seeds allowed the estimation of pollination frequencies from the three groups of pollen donors ( Q. petraea, Q. pubescens , intermediate). We also assayed pollen viability and germinability of each species group. We observed natural hybridisation between Q. petraea and Q. pubescens, with a predominant component in the direction Q. petraea → Q. pubescens : Q. pubescens displayed a higher level of heterospecific pollination by Q. petraea (25.8%) and intermediate morphotypes (14.7%), compared to Q. petraea acting as pollen receptor (with less than 5% heterospecific pollinations). Intermediate 'mother trees' were pollinated in similar proportions by Q. petraea (23.1%), Q. pubescens (37.8%) and intermediate morphotypes (39.1%). The asymmetrical introgression observed for the studied generation may be caused, among other factors, by the relative abundance of trees from each species group in the studied area.