Kinetic studies of the applicability of the common site concept to the 3β-hydroxysteroid dehydrogenase: 5-ene-3-ketosteroid isomerase activities of human placental microsomes

Pregnenolone (3β-hydroxy-5-pregnen-20-one) and DHA (3β-hydroxy-5-androsten-17-one), substrates for 3β-hy-droxysteroid dehydrogenase (3β-HSD), with K_M values of 15–40 nM, were ineffective inhibitors of 5-ene-3-ketosteroid isomerase (isomerase), with K_I values >40 μM in each case. Progesterone and androstenedione (4-androstene-3, 17-dione), 3β-HSD inhibitors with K_I values of 5.0 μM and 0.8 μM respectively, were also relatively ineffective inhibitors of isomerase, with K_I values of 30 μM and 16.5 μM respectively. Exposure of microsomes to hydrogen peroxide, which significantly increases the K_M for pregnenolone as a 3β-HSD substrate, had no effect on the K_M for the isomerase substrate 5-pregnene-3, 20-dione.It is concluded that the data do not support the common site concept with regard to the conversion of pregnenolone to progesterone by human placental microsomes.     

Inhibition of 17β-hydroxysteroid dehydrogenase (17β-HSD) activities of human placenta by steroids and non-steroidal hormone agonists and antagonists

Various naturally occurring steroids, synthetic steroid derivatives and non-steroidal hormone agonists and antagonists were assayed as inhibitors of human placental 17β-HSD activities. Microsomal 17β-HSD was inhibited by C₁₈ -,C₁₉- and C₂₁-steroids. Soluble 17β-HSD was highly specific for C₁₈-steroids. In contrast to the soluble activity, the microsomal enzyme also had a strong affinity for ethinylestradiol (K_I=0.3 μM) and danazol (K_I=0.6 μM); anabolic steroids and norethisterone were weaker inhibitors. Of the non-steroids tested only diethylstilbestrol and o-demethyl CI-680 were inhibitors and they showed a greater affinity for soluble 17β-HSD.K_I-values for estradiol-17β, (0.8 μM), progesterone (27.0 μM) and 20α-dihydroprogesterone (1.5 μM) were comparable to reported tissue levels of these compounds, consistent with a possible competition in vivo among naturally occurring C₁₈-, C₁₉-, and C₂₁-steroids for the active site of microsomal 17β-HSD.     

Organotypic differentiation of trypsin-dissociated fetal rat intestine

These studies examined the potential for reorganization and differentiation of dissociated 18-day fetal rat intestine. Cultures of trypsin-dissociated fetal intestine were maintained in vitro for 1 week on a three-dimensional matrix, then transplanted into syngeneic hosts. When harvested after 4 weeks, these transplants consistently demonstrated organotypic differentiation. Spherical structures containing crypts with frequent mitotic figures and villi lined with columnar epithelium had formed. PAS staining demonstrated positive epithelial cell brush borders, goblet cells, and luminal contents. Significant levels of the microvillus membrane enzymes lactase, sucrase, maltase, and alkaline phosphatase were present in the luminal contents. Sucrase-isomaltase, an enzyme characteristic of postweaning small intestine, was demonstrated by immunoprecipitation and SDS-PAGE. Thus, both morphological and biochemical maturation occurred in the transplants.     

Inhibiton of human placental 17 beta-hydroxysteroid dehydrogenase by steroids and nonsteroidal alcohols: aspects of inhibitor structure and binding specificity

Inhibition of human placental 17β-hydroxysteroid dehydrogenase by C₁₈ and C₁₉ steroids and nonsteroidal alcohols was assayed at pH 9.0 with 17β-estradiol 3-methyl ether and NAD⁺ as reactants. The nonstaroidal alcohols tested were poor inhibitors. Cyclopentanol and cyclohexanol had K_i values greater than 5 mm. Nonaromatic C₁₈ and C₁₉ steroids with oxygen functions at both positions 3 and 17 gave no detectable inhibition or had K_i, values greater than or equal to 160 μm. 3μ-Hydroxy-5,16-androstadiene, 5-androsten-3β-ol, 1,3,5(10)-estratrien-3-ol, and 1,3,5(10),16-estratetraen-3-ol, steroids lacking a C(17) oxygen function, had K_i values of 1.8, 6.0, 0.04, and 0.17 μm, respectively, demonstrating that both C₁₈ and C₁₉ steroids can bind at the steroid site. Binding specificity is narrowed and binding affinity for nonaromatic steroids weakened by oxygen functions at C(17) or both C(3) and C(17). The structural implications of the specificity data for steroid recognition and complex formation and in vivo control of enzyme activity are discussed.     

关于两种全自动生化分析仪校准周期的对比研究

目的:对日立H7180、奥林帕斯Au640全自动生化分析仪校准周期的对比分析。方法:相同条件下,两生化分析仪进行定标校准后,每隔2小时测定高、中、低3种浓度的质控血清各一次直到24 h,后每间隔24 h测定一次,共测定30 d,检测项目均重复测定4次取平均值。从校准完成后开始计时到累积变异系数(CV)大于1/6 CLIA'88允许误差时终止,确定该项目的校准周期。通过绘制校准周期图获得两种全自动生化分析仪不同检测项目的校准周期,并对结果进行对比分析。结果:以碱性磷酸酶(ALP)为例绘制校准周期图,进行对比分析。各检测项目在日立H7180中,低水平质控CV最先达到校准要求,高水平质控CV最后达到校准要求;而在奥林帕斯Au640中,中水平质控CV最先达到校准要求,高水平质控CV最后达到校准要求。结论:日立H7180和奥林帕斯Au640全自动生化分析仪,其校准周期基本一致,个别项目存在差异。     

科学确立奥林帕斯Au640 全自动生化分析仪校准周期

目的:利用校准周期图科学确立奥林帕斯Au640全自动生化分析仪校准周期。方法:生化分析仪定标校准后,每隔2小时分别测定三种浓度水平的质控血清一次,24小时后每天测定一次,总计测定30天。所有检测项目均重复测定四次取平均值。以积变异系数(CV)大于1/6 CLIA’88为评价标准,绘制校准周期图。结果:碱性磷酸酶(ALP)L1校准周期最短每12 h一次。酶类项目较稳定,校准周期可达30天以上,低水平质控血清必不可少,这样有利于测得更为准确的校准周期。结论:科学、合理地确立各检测项目的校准周期,对生化分析仪检测系统进行定期校准,既能够避免不必要的校准、又能够节省检验工作的时间和成本,保证生化检验项目检测结果可靠性。     

Structural organisation of prolamellar bodies (PLB) isolated from Zea mays. Parallel TEM, SAXS and absorption spectra measurements on samples subjected to freeze-thaw, reduced pH and high-salt perturbation

Well-organised PLB gives rise to a X-ray diffraction pattern overlaid by a scattering pattern arising from individual tubules within less well-organised regions of the lattice. TEM and SAXS measurements were used to characterise the structural changes in PLB subjected to perturbation by freeze-thaw, exposure to pH 6.5, or resuspension in high-salt media. Comparison of SAXS patterns measured, before and after structural perturbation allows the separation of the contributions from ordered and disordered PLB. The diffraction pattern is shown to be based on a diamond cubic (Fd3m) lattice of unit cell a = 78 nm. Freeze-thaw and high-salt disruption lead to the breakdown of ordered PLB into disordered tubules of similar dimensions to those making up the original PLB lattice. Their scattering patterns suggest that they are approximately 26 nm in diameter with a central lumen about 16 nm in diameter. The tubules formed at pH 6.5 are appreciably narrower, probably reflecting changes in the pattern of ionisation of charged groups at the membrane surface. Absorption spectra of PLB in media containing different concentrations of salts indicated that the structural and spectral changes are related. NADPH, have a significant role in the protection of POR-PChlide₆₅₀ but to have only a relatively small effect on the preservation of PLB organisation indicating that the retention of POR-PChlide₆₅₀ in isolated PLB preparations is a poor guide to their structural integrity.     

Membrane associations between subsurface cisternae of endoplasmic reticulum and the plasma membrane of rat Sertoli cells

Close membrane associations between the endoplasmic reticulum and the plasma membrane (ER-PM) occur in specialized regions of the rat Sertoli cell cytoplasm. They are characterized, in freeze fracture replicas, as mesa-like modifications of E membrane fracture faces or as corresponding discoid depressions on P membrane fracture faces. When these structures lie along transitional regions in the membrane fracture plane, they are seen to be complementary, and the space between them to be greatly reduced. These specialized close membrane associations may represent adhesive sites between the endoplasmic reticulum and the plasma membrane. However, their resemblance to vascular endothelial fenestrae which are known to be sites of increased membrane permeability may suggest other functional roles.     

Gap junctions between sertoli and germ cells of rat seminiferous tubules

Ultrastructural observations of rat seminiferous tubules show clearly the presence of plasma membrane junctions between Sertoli and germ cells in the basal and adluminal compartments. Results obtained from the freeze fracture and thin section techniques were correlated in order to elucidate the nature of these intercellular junctions. We suggest that these intercellular membrane specializations are gap junctions which occur within regions of plasma membrane that also exhibit adherens-like modifications.     

Interactions of Bovine Brain Tubulin with Pyridostigmine Bromide and N,N′-Diethyl-m-Toluamide

Pyridostigmine bromide (PB), an inhibitor of acetylcholinesterase, has been used as a prophylactic for nerve gas poisoning. N,N-diethyl-m-toluamide (DEET) is the active ingredient in most insect repellents and is thought to interact synergistically with PB. Since PB can inhibit the binding of organophosphates to tubulin and since organophosphates inhibit microtubule assembly, we decided to examine the effects of PB and DEET on microtubule assembly as well as their interactions with tubulin, the subunit protein of microtubules. We found that PB binds to tubulin with an apparent K _d of about 60 M. PB also inhibits microtubule assembly in vitro, although at higher concentrations PB induces formation of tubulin aggregates of high absorbance. Like PB, DEET is a weak inhibitor of microtubule assembly and also induces formation of tubulin aggregates. Many tubulin ligands stabilize the conformation of tubulin as measured by exposure of sulfhydryl groups and hydrophobic areas and stabilization of colchicine binding. PB appears to have very little effect on tubulin conformation, and DEET appears to have no effect. Neither compound interferes with colchicine binding to tubulin. Our results raise the possibility that PB and DEET may exert some of their effects in vivo by interfering with microtubule assembly or function, although high intracellular levels of these compounds would be required.