Comparison of prostate gene expression and tissue weight changes as monitors of antiandrogen activity in GNRH-inhibited rats

BACKGROUND: The Hershberger assay for antiandrogens and modifiers of steroid biosynthesis uses surgically‐castrated rats. We described an adaptation of the assay using the GnRH inhibitor Antarelix in place of surgical castration [Ashby J, Lefevre PA, Deghenghi R, Wallis N. Regulatory Toxicology and Pharmacology 34:188–203, 2001], and concomitantly described changes in expression of the androgen‐dependent prostatic genes PBP C3, TRPM‐2, and ODC as a possible complement to gravimetric analysis of the sex accessory tissues (SAT) [Nellemann C, Vinggaard AM, Dalgaard M, Hossaini A, Larsen J‐J. Toxicology 163:29–38, 2001]. METHODS: The present study describes the results of combining these two modifications into a single assay. During the course of these experiments it was shown that SD rats gave similar results to AP rats and that the higher stimulatory dose of testosterone propionate (TP) used in our experiments gave stronger assay responses to FLU than the lower dose of TP used by some earlier investigators. The potent antiandrogen flutamide (FLU) and the weak antiandrogen DDE were used to evaluate this modified assay. RESULTS: For all parameters studied (SAT weights and changes in expression of the 3 prostatic genes) FLU gave the expected positive results. The weak antiandrogen DDE gave variable and mainly non‐reproducible responses. Use of DDE as a weak antiandrogen accelerated assessment of the new assay. CONCLUSIONS: Possible reasons for this failure to detect DDE are discussed, and it is concluded that the modified assay is unsuitable for use in its present form. The use of gene expression analyses together with evaluation of SAT weights is a promising tool as an early and sensitive marker of antiandrogen action, but more work is needed on the choice of time frame as well as the selection of genes to monitor. Birth Defects Res B 68:344–354, 2003. © 2003 Wiley‐Liss, Inc.     

A targeted extracellular approach for recording long-term firing patterns of excitable cells: a practical guide

Excitable cells in many endocrine and neuronal systems display rhythms with periodicities on the order of many minutes. To observe firing patterns that represent the output of these rhythms requires a recording technique that can monitor electrophysiological activity for several hours without affecting cell behavior. A targeted extracellular approach (also known as loose-patch) accomplishes this objective. Because low resistance seals (<20 MΩ) do not influence the cell membrane and because the normal intracellular milieu is maintained, this approach is the least invasive method for monitoring the endogenous electrical activity of single cells. In this report, we detail our use of this technique to record the firing patterns of gonadotropin-releasing hormone (GnRH) neurons in brain slices continuously for several hours. Published: February 17, 2003 This publication makes use, with permission, of data and methodologies published in Nunemaker CS, DeFazio RA, Moenter SM. Estradiol-sensitive afferents modulate long-term episodic firing patterns of GnRH neurons.Endocrinology 2002; 143:2284–2292, Copyright 2002 by The Endocrine Society.     

Partial purification and characterization of mitochondrial DNA polymerase from Plasmodium falciparum

Mitochondria of chloroquine-resistant Plasmodium falciparum (K1 strain) were isolated from mature trophozoites by differential centrifugation. The mitochondrial marker enzyme cytochrome c reductase was employed to monitor the steps of mitochondria isolation. Partial purification of DNA polymerase from P. falciparum mitochondria was performed using fast protein liquid chromatography (FPLC). DNA polymerase of P. falciparum mitochondria was characterized as a gamma-like DNA polymerase based on its sensitivity to the inhibitors aphidicolin, N-ethylmaleimide and 9-beta-D-arabinofuranosyladenine-5'-triphosphate. In contrast, the enzyme was found to be strongly resistant to 2',3'-dideoxythymidine-5'-triphosphate (IC(50)>400 microM) and differed in this aspect from the human homologue, possibly indicating structural differences between human and P. falciparum DNA polymerase gamma. In addition, the DNA polymerase of parasite mitochondria was shown to be resistant (IC(50)>1 mM) to the nucleotide analogue (S)-1-[3-hydroxy-2-phosphonylmethoxypropyl]adenine diphosphate (HPMPApp).     

大鼠颌下腺促性腺激素释放激素及其mRNA的免疫组织化学与原位杂交研究

用免疫组织化学及原位杂交法,研究了促性腺激素释放激素及其ｍＲＮＡ在大鼠颌下腺的分布。结果显示,大鼠颌下腺的浆液性腺泡的上皮细胞,各级导管的上皮细胞及副交感神经节细胞均呈促性腺激素释放激素免疫反应阳性,阳性反应物质分布在胞质,胞核呈阴性反应。颌下腺的浆液性腺泡上皮细胞,各级导管上皮细胞同样被检测到很强的促性腺激素释放激素ｍＲＮＡ杂交信号。以上结果提示,大鼠颌下腺能自身合成促性腺激素释放激素,促性腺激素释放激素对消化功能可能有重要调节作用。     

Isolation and characterization of mutants resistant to amino acid analogues obtained from Saccharomyces cerevisiae strains

Mutants resistant to the amino acid analogues dl-thiaisoleucine, dl-4-azaleucine, 5,5,5-trifluoro-dl-leucine and l-O-methylthreonine, were isolated from Saccharomyces cerevisiae wine yeast strains. The fermentative production of secondary metabolites by the mutants was tested in grape must. Higher alcohols, acetaldehyde and acetic acid concentration varied depending on strain and analogue. Most of the mutants produced increased amounts of amyl alcohol. A remarkable variability in the level of n-propanol, isobutanol, acetaldehyde and acetic acid was observed. In practical application, the use of mutants resistant to amino acid analogues can improve the quality of wines by reducing or increasing the presence of some secondary compounds.     

STUDIES OF NITRATE TRANSPORT BY MARINE PHYTOPLANKTON USING 36Cl-ClO3− AS A TRANSPORT ANALOGUE. I. PHYSIOLOGICAL FINDINGS1

Transport of a nitrate analogue, ³⁶Cl-ClO₃⁻, was examined in two diatoms, Skeletonema costatum (Greve.) Cleve and Nitzschia closterium (Ehrenb) W. Sm. A dinoflagellate, Gonyaulax polyedra did not transport ClO₃⁻. Transport of ³⁶Cl-ClO₃⁻ by diatoms appeared to be active and showed saturation kinetics. The data were fitted by Michaelis-Menten equation at all but the lowest chlorate concentrations (where plots of S vs. v showed a slight concave bend). Affinity of cells for nitrate was considerably higher than for chlorate. The K_i for nitrate inhibition of chlorate transport was calculated assuming competitive inhibition. Light had little or no effect on chlorate transport. Pulse-chase experiments demonstrated that (1) ClO₃⁻ (hence NO₃⁻) was stored in two intracellular compartments of equal size, (2) internal ClO₃⁻ was exchangeable with external ClO₃⁻ (rates of efflux and influx were measured), and (3) efflux of intracellular ClO₃⁻ showed transient states following a chase of ClO₃⁻ or NO₃⁻ which stabilized after 10–20 min. Transport of chlorate was a function of growth phase.     

经皮椎弓根空心螺钉微创椎体间融合治疗腰椎间盘突出的临床效果分析

目的：探究经皮椎弓根空心螺钉微创椎体间融合治疗腰椎间盘突出的临床效果及安全性。方法：病例来源于我院2009 年12 月~2013 年12 月收治的确诊为腰椎间盘突出症的病患174 例,依据随机数字表法将其均分为观察组与对照组,每组87 例。其 中,观察组施行Quadrant微创通道经皮椎弓根空心螺钉椎间融合术,对照组施行经后入路开放性椎间融合术。评估和比较两组病 患术前和随访结束时的视觉模拟评分系统(VAS)疼痛评分与Oswestry 功能障碍指数(ODI)的变化及术后并发症的发生情况。结 果：观察组治疗前、出院时及随访一年时的VAS 评分与ODI指数与对照组比较差异均不显著(P>0.05)。观察组手术切口长度、术 后住院时间及术中出血量均明显优于对照组(P<0.01),而其手术所需时间明显长于对照组(P<0.01)。对照组患者术后出现20 例神 经根损伤(22.99%),3 例椎间隙感染(3.45%),其并发症总发生率为(26.44%),而观察组患者术后仅出现3 例神经根损伤,发生率为 3.45%,显著低于对照组(P<0.01)。结论：经皮椎弓根空心螺钉微创椎体间融合治疗的临床效果肯定,能减少对病患的创伤,控制术 后并发症的发生,具有较高的临床应用价值。     

Adenosine analogue–oligo-arginine conjugates (ARCs) serve as high-affinity inhibitors and fluorescence probes of type I cGMP-dependent protein kinase (PKGIα)

Introduction

Type I cGMP-dependent protein kinase (PKGIα) belongs to the family of cyclic nucleotide-dependent protein kinases and is one of the main effectors of cGMP. PKGIα is involved in regulation of cardiac contractility, vasorelaxation, and blood pressure; hence, the development of potent modulators of PKGIα would lead to advances in the treatment of a variety of cardiovascular diseases. Aim: Representatives of ARC-type compounds previously characterized as potent inhibitors and high-affinity fluorescent probes of PKA catalytic subunit (PKAc) were tested towards PKGIα to determine that ARCs could serve as activity regulators and sensors for the latter protein kinase both in vitro and in complex biological systems. Results: Structure–activity profiling of ARCs with PKGIα in vitro demonstrated both similarities as well as differences to corresponding profiling with PKAc, whereas ARC-903 and ARC-668 revealed low nanomolar displacement constants and inhibition IC₅₀ values with both cyclic nucleotide-dependent kinases. The ability of ARC-based fluorescent probes to penetrate cell plasma membrane was demonstrated in the smooth muscle tissue of rat cerebellum isolated arteries, and the compound with the highest affinity in vitro (ARC-903) showed also potential for in vivo applications, fully abolishing the PKG1α-induced vasodilation.     

The GnRH antagonist acyline prevented ovulation, but did not affect ovarian follicular development or gestational corpora lutea in the domestic cat

Two experiments were conducted to investigate the effects of the GnRH antagonist acyline (330 μg/kg, given sc) on ovarian follicular development and ovulation, as well as on pregnancy maintenance in domestic cats. In the first experiment, seven queens in proestrus (total of 24 proestrus periods), were randomly assigned to treatment with either acyline (ACY; n = 17) or a placebo (PLC; n = 7). All queens were mated with a fertile tomcat. In the ACY and PLC groups, cessation of estrus occurred (mean ± SEM) 7.0 ± 1.3 and 7.0 ± 1.7 d after treatment (P > 0.1), ovulation occurred in 2 of 17 and all seven estrus periods (P < 0.05), and pregnancy rates were 1 of 16 and 7 of 7 (P < 0.05), respectively. In the ACY and PLC groups, intervals from treatment to the onset of the ensuing proestrus were 18.4 ± 1.7 and 120 ± 17.2 d. In the second experiment, 14 pregnant queens were randomly allocated, according to their mating date, to treatment with acyline in early pregnancy (from 20 to 25 d, n = 3), mid pregnancy (from 26 to 45 d; n = 4), late pregnancy (> 45 d; n = 3), or injection of a placebo in early (n = 1), mid (n = 2), or late pregnancy (n = 1). Ultrasonographic assessments of the uterus were done every second day for 2 wk post treatment, and serum progesterone (P₄) concentrations were determined before treatment, and at 7 and 14 d after treatment. No pregnancies were prematurely terminated and post-treatment P₄ concentrations did not differ among treatment groups (P > 0.1). In conclusion, in the domestic cat, GnRH withdrawal by acyline prevented ovulation when given in early follicular phase (proestrus), but did not significantly affect luteal function during pregnancy.