Regulation of estrogen receptor protein and messenger ribonucleic acid by estradiol and progesterone in rat uterus

The objective of this study was to examine the mechanisms of estrogen receptor (ER) processing and replenishment in the uterus of ovariectomized rats after estradiol and progesterone treatment. Uterine ER binding activity, ER protein and ER mRNA were measured by receptor binding exchange assay, Western blot and slot blot, respectively. The regulation of ER levels in rat uterus by estradiol and progesterone was very dramatic. Changes in ER protein were faithfully reflected by changes in binding activity. Estradiol caused receptor “processing” within 4 h of administration followed by recovery or “replenishment” of ER levels to the initial level by 20 h. The term “processing” has previously been used to describe the loss of ER binding activity in the early phase of estradiol-action, but it was never clear whether the ligand binding site was inactivated by processing or if the receptor molecule actually disappeared. This study shows that receptor “processing” constitutes disappearance of receptor protein and the later “replenishment” phase represents new ER protein rather than recycling of “processed” receptor. Progesterone-action, on the other hand, influenced only the “replenishment” phase by blocking recovery of ER protein. ER mRNA was suppressed by estradiol at 8 h, after the receptor was “processed” and “replenishment” already initiated. Progesterone, on the other hand, did not alter the steady state level of the message. Other mechanisms, such as regulation of translation rate of existing mRNA and changes in the rate of degradation of ER proteins are more likely involved in acute regulation of ER by these ovarian steroid hormones.     

Diol sulfonamides: A potent and novel class of inhibitors of human renin

The syntheses and pharmacological activity of a series of diol sulfonamides which function as inhibitors of human renin are described. The most potent compound in this series, compound 20 (SQ 33,800), is a subnanomolar inhibitor of human renin (IC₅₀ = 0.35 × 10⁻⁹ M).     

Regulation of collagenase production by steroids in uterine smooth muscle cells: An enzymatic and immunologic study

An enzyme-linked immunosorbent assay (ELISA) has been developed for rat collagenase. The assay is capable of measuring the enzyme from a variety of rat cell sources at concentrations of 10–;50 ng/ml, approximately 500–;1,000-fold more sensitive than radiolabelled collagen fibril assay systems. The assay is specific to collagenase from the rat: enzymes from human, tadpole, mouse, and bacterial sources failed to cross-react significantly with rat enzyme. The assay is reproducible and accurate, and is capable of detecting enzyme in the presence of serum or tissue inhibitors. Using the ELISA, we have examined the effect of a variety of hormones on the production of collagenase by rat myometrial smooth muscle cells in culture. Of all the reproductive hormones examined, only progesterone and its synthetic derivative medroxyprogesterone acetate were capable of inhibiting the production of the enzyme by these cells. The maximally effective concentration of progesterone was 1 x 10^?6M, and that of medroxyprogesterone acetate was 1 x 10^?7M. The effect of the steroid was selective: no effect on cell proliferation or on general protein synthesis was observed. In addition to the progestational steroids, the glucocorticoids were also capable of inhibiting the production of collagenase by the cells at similar nominal concentrations. However, the myometrial cells were found actively to metabolize progesterone but not hydrocortisone in culture. Thus, the effective inhibitory concentration of progesterone was approximately ten-fold lower than that of hydrocortisone. The results of this study support the concept that progesterone plays a major role in preventing the production of collagenase in the rat uterus.     

Genetic influences in self-reported symptoms of obstructive sleep apnoea and restless legs: a twin study.

Sleep disorders, such as obstructive sleep apnoea (OSA) and restless legs syndrome (RLS), are very common. The relative importance of genetic and nongenetic (environmental) influences on the symptomatology of these conditions has not been well studied. This study uses the twin design to examine this by evaluating OSA and RLS symptoms in monozygotic (MZ) and dizygotic (DZ) twins. Six thousand six hundred unselected female twin pairs, identified from a national volunteer twin register, were asked to complete a medical questionnaire. This questionnaire included questions on OSA and RLS symptoms, as well as questions on subject demographics, past medical history, smoking history and menopausal status. Responses were obtained from 4503 individuals (68% response rate). A total of 1937 twin pairs were evaluable: 933 MZ pairs (mean [range] age 51 [20-76] years) and 1004 DZ pairs (age 51 [20-80] years). Concordance rates were higher for MZ than DZ twins for OSA and RLS symptoms. Multifactorial liability threshold modeling suggests that additive genetic effects combined with unique environmental factors provide the best model for OSA and RLS symptoms. Heritability was estimated to be 52% (95% confidence interval 36% to 68%) for disruptive snoring, 48% (37% to 58%) for daytime sleepiness, 54% (44% to 63%) for restless legs, and 60% (51% to 69%) for legs jerking. These estimates dropped only slightly after adjustment for potential confounding influences on the symptoms of snoring and daytime sleepiness. These results suggest a substantial genetic contribution to the symptomatology of OSA and RLS. More research is needed to identify the genes responsible, and may ultimately lead to new therapies.     

Histories and Stories from Chiapas: Border Identities in Southern Mexico.

Histories and Stories from Chiapas: Border Identities in Southern Mexico. R. Aída Hernández Castillo. Texas: University of Texas Press, 2001. 317 pp.     

Determination of two mebeverine metabolites, mebeverine alcohol and desmethylmebeverine alcohol, in human plasma by a dual stable isotope-based gas chromatographic-mass spectrometric method

A dual stable isotope-based GC-MS method was developed for the simultaneous determination of two metabolites of mebeverine, mebeverine alcohol and desmethylmebeverine alcohol, in human plasma. Plasma samples were treated with β-glucuronidase to cleave the glucuronide conjugates of both compounds prior to analysis. The treated plasma was prepared for analysis by solid-phase extraction using octadecylsilane cartridges. The isolated metabolites were derivatized and analyzed by GC-MS using selected-ion monitoring. Plots of peak-area ratio were linear with metabolite concentration from 2 to 200 ng/ml and the limit of detection for both metabolites was 0.5 ng/ml. The GC-MS methodology was applied to the analysis of plasma from human subjects following peroral administration of mebeverine. Pharmacokinetic parameters for both metabolites were determined and suggest that relative systemic mebeverine exposure may potentially be assessed using metabolite kinetics, if the latter subsequently are demonstrated to be linear with mebeverine dose.     

Protein Phosphorylation and Calcium Uptake into Rat Forebrain Synaptosomes: Modulation by the σ Ligand, 1,3-Ditolylguanidine

Abstract: The σ ligand 1,3-di- O -tolylguanidine (DTG) increased basal dynamin and decreased depolarization-stimulated phosphorylation of the synaptosomal protein synapsin Ib without having direct effects on protein kinases or protein phosphatases. DTG dose-dependently decreased the basal cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) and blocked the depolarization-dependent increases in [Ca²⁺]_i. These effects were inhibited by the σ antagonists rimcazole and BMY14802. The nitric oxide donors sodium nitroprusside (SNP) and 8-( p -chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate decreased basal [Ca²⁺]_i and the KCl-evoked rise in [Ca²⁺]_i to an extent similar to DTG. SNP, but not DTG, produced a rise in cyclic GMP levels, suggesting that the effect of DTG on [Ca²⁺]_i was not mediated via downstream regulation of cyclic GMP levels. DTG increased ⁴⁵Ca²⁺ uptake and efflux under basal conditions and inhibited the ⁴⁵Ca²⁺ uptake induced by depolarization with KCl. The KCl-evoked rise in [Ca²⁺]_i was inhibited by ω-conotoxin (ω-CgTx)-GVIA and -MVIIC but not nifedipine and ω-agatoxin-IVA. The effect of DTG on decreasing the KCl-evoked rise in [Ca²⁺]_i was additive with ω-CgTx-MVIIC but not with ω-CgTx-GVIA. These data suggest that DTG was producing some of its effects on synapsin I and dynamin phosphorylation and intrasynaptosomal Ca²⁺ levels via inhibition of N-type Ca²⁺ channels.     

Nicotine Enhances the Cyclic AMP-Dependent Protein Kinase-Mediated Phosphorylation of α4 Subunits of Neuronal Nicotinic Receptors

Abstract: Studies determined whether α4β2 or α3β2 neuronal nicotinic receptors expressed in Xenopus oocytes are substrates for cyclic AMP-dependent protein kinase (PKA) and whether nicotine affects receptor phosphorylation. The cRNAs for the subunits were coinjected into oocytes, and cells were incubated for 24 h in the absence or presence of nicotine (50 n M for α4β2 and 500 n M for α3β2 receptors). Nicotine did not interfere with the isolation of the receptors. When receptors isolated from oocytes expressing α4β2 receptors were incubated with [γ-³²P]ATP and the catalytic subunit of PKA, separated by electrophoresis, and visualized by autoradiography, a labeled phosphoprotein with the predicted molecular size of the α4 subunit was present. Phosphorylation of α4 subunits of α4β2 receptors increased within the first 5 min of incubation with nicotine and persisted for 24 h. In contrast, receptors isolated from oocytes expressing α3β2 receptors did not exhibit a labeled phosphoprotein corresponding to the size of the α3 subunit. Results suggest that the PKA-mediated phosphorylation of α4 and not α3 subunits may explain the differential inactivation by nicotine of these receptors subtypes expressed in oocytes.     

Adventitious regeneration of Juglans nigra L. (eastern black walnut)

Somatic embryos and adventitious shoots were initiated from immature cotyledons 10–14 weeks after anthesis. Maximum embryogenesis occurred 12 weeks after anthesis and maximum shoot organogenesis occurred 14 weeks after anthesis. The best treatment for induction of somatic embryos and adventitious shoots from immature cotyledon explants was on agar-solidified WPM supplemented with 0.1 M 2,4-D and 5.0 M TDZ and incubated in light for the first four weeks. Rooting of adventitious shoots was best if they were quickdipped in 2.5 mM IBA and 1.25 mM NAA in 1% dimethyl formamide and 3.9% ethanol (120 Wood's Rooting Compound: water, by volume). Plantlets from rooted adventitious shoots were acclimatized to the greenhouse.Abbreviations BA Benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - DKW Driver and Kuniyuki (1984) walnut medium - IBA indolebutyric acid - LP Long and Preece medium described herein - NAA naphthaleneacetic acid - PPF photosynthetic photon flux - TDZ thidiazuron - WPM Woody Plant Medium of Lloyd and McCown (1980)     

Isolation of a cDNA encoding a growth-arrest associated gene and characterization of its regulation

We are interested in understanding the molecular events associated with the growth-arrest of vascular SMCs. We constructed a subtracted cDNA library enriched in nucleotide sequences associated with quiescent SMCs. This library was screened with similarly subtracted ³²P-labeled cDNAs to identify growth-arrest associated cDNA clones. Characterization of 19 of these cDNA clones revealed that 9 hybridized to mRNAs that exhibited a 2–3 fold increase in growth-arrested SMCs. In addition, two other cDNAs hybridized to a 5 Kb mRNA that was elevated approximately 10-fold in high density growth-arrested SMCs. Genomic Southern blot hybridization and DNA sequencing analysis indicated that these cDNAs encoded the same gene (LG7) and that this gene may be a member of a multigene family or that it may contain a sequence shared by other unrelated genes. Augmented expression of LG7 was associated with both high cell density and serum deprivation induced growth-arrest. LG7 mRNA expression was down-regulated when SMCs were incubated with FBS or with reagents that arrest cells in early S-phase. Additional analysis with cell cycle specific inhibitors indicated that LG7 mRNA levels were also low when cells were blocked at the G₂ phase of the cell cycle but blockage at mitosis resulted in an elevated level of LG7 mRNA. We further demonstrated that the expression of LG7 was dependent on the presence of a relatively labile protein since protein synthesis inhibitors specifically blocked the expression of this mRNA but not the mRNA expression of α₁(III) collagen or ferritin H-chain. Finally, we demonstrated that Bt₂cAMP was able to induce mRNA expression of LG7 within 2 h, suggesting that this gene may be directly regulated via the cyclic-AMP-dependent protein kinase pathway.