Pregnancy and the follicular phase are physiological states of elevated oestrogen levels and rises in uterine blood flow (UBF). The dramatic increase in utero-placental blood flow during gestation is required for normal fetal growth and development. Oestrogen exerts its vasodilatory effect by binding to its specific oestrogen receptors (ER) in target cells, resulting in increased expression and activity of endothelial nitric oxide synthase (eNOS) to relax vascular smooth muscle (VSM). However, the regulation of endothelial versus VSM ERα and ERβ expression in uterine arteries (UAs) during the ovarian cycle, pregnancy and with exogenous hormone replacement therapy (HRT) are currently unknown. ER mRNA and protein localization was determined by in situ hybridization (ISH) using 35 S-labelled riboprobes and immunohistochemistry (IHC), respectively. UA endothelial (UAendo), UA VSM, omental artery endothelium (OA endo), and OA VSM proteins were isolated and ERα and ERβ protein expression was determined by Western analysis. We observed by ISH and IHC that ERα and ERβ mRNA and protein were localized in both UAendo and UA VSM. Immunoblot data demonstrated ovarian hormone specific regulation of ERα and ERβ protein in UAendo and UA VSM. Compared to luteal phase sheep, both ERα and ERβ levels in UAendo were elevated in follicular phase sheep. Whereas ERβ was elevated by pregnancy in UAendo and UA VSM, ERα was not appreciably altered. eNOS was increased in UAendo from follicular and pregnant sheep. Ovariectomized ewes (OVEX) had substantially reduced UAendo ERβ, but not UAendo ERα or OAendo ERα and ERβ. In contrast, OVEX increased UA VSM ERα and ERβ and decreased OA VSM ERα and ERβ. Treatment with oestradiol-17β (E2β), but not progesterone or their combination, increased UAendo ERα levels. The reduced ERβ in UAendo from OVEX ewes was reversed by E 2 β and progesterone treatment. While ERα and eNOS were not elevated in any other reproductive or non-reproductive endothelia tested, ERβ was augmented by pregnancy in uterine, mammary, placenta, and coronary artery endothelia. ERα and ERβ mRNA and protein are expressed in UA endothelium with expression levels depending on the endocrine status of the animal, indicating UA endothelium is a target for oestrogen action in vivo, and that the two receptors appear to be differentially regulated in a spatial and temporal fashion with regard to the reproductive status or HRT.

A phenol aqueous emulsion allows the reassociation of DNA at temperatures from 6 to 68 °C. This phenol emulsion reassociation technique (PERT) also promotes the very rapid reassociation of DNA. E. coli and human DNAs at a concentration of 4 ng/mL reacted at room temperature with the PERT reassociate many thousand times faster than under the standard conditions of 0.18 M Na+, 60 °C. Solutions of DNA ranging in concentration from 6 X 10-5 to 6400 yug/mL have been successfully reassociated using the emulsion method. The greatest rate increases are seen at low DNA concentration. The half-time of reassociation does not decrease proportionally with an increase in DNA concentration when using the PERT. At 6400 Mg/mL the phenol emulsion rate of reassociation is only about 10 times faster than under the standard aqueous reference conditions of 0.12 M phosphate buffer (PB), 60 °C. The rate of DNA reassociation observed with the emulsion technique is at least dependent on: (a) the presence of an emulsion; (b) the type and concentration of ion present; (c) an appropriate temperature of incubation; (d) the proper pH; (e) the rate and manner of agitating the emulsion;

A method is described for the rapid preparation of plasmid DNAs of molecular weight up to 14 x lo6.This method involves the chromatography, at room temperature, of bacterial cleared lysates on hydroxyapatite in the presence of high concentrations of phosphate and urea. All detectable protein and RNA contamination of plasmid DNA is removed by this procedure and the conformation of the plasmid DNA is unaffected. Less than 0.5 % chromosomal DNA is present in the purified preparation and even this can be removed if necessary by a simple extention of the procedure to include a heat-denaturation step. The method is extremely rapid and amenable to large-scale plasmid preparation; 5 mg ColEl DNA have been purified within 40 min. The yield of plasmid DNA is similar to that obtained with the conventional dye-centrifugation technique, however the purity is greater.The advent of recombinant DNA technology has facilitated the routine isolation of specific nucleic acid sequences of both prokaryotes and eukaryotes. This technology relies heavily on the use of vectors and for most cloning experiments bacterial plasmids are the vectors of choice. Plasmid DNA rarely constitutes as much as 50% of the total DNA present in a bacterial cell, even under growth conditions most conducive to plasmid replication [l]. Purification of plasmid DNA therefore requires the selective removal of chromosomal DNA. A very effective initial pmification is achieved by the preparation of a cleared lysate [2]. Methods for the further purification of plasmid DNA take advantage of the closed-circular nature of the DNA [3,4]. The most widely used method relies on the greater density of the closed-circular DNA relative to sheared chromosomal DNA in ethidium bromide/caesium chloride density gradients [5]. While this technique provides a high degree of purification it suffers from a number of disadvantages: it is expensive in both centrifuge time and materials, has limited capacity, and complete removal of the ethidium bromide is both difficult and tedious.In this paper we describe a purification procedure which does not depend on CsCl centrifugation and which is rapid, inexpensive and yields DNA of high purity. Our method takes advantage of the selective and reversible retention by hydroxyapatite of small double-stranded DNA molecules under conditions known to prevent the binding of all RNA and protein [6].

We have examined the expression and regulation of the two estrogen receptor (ER alpha and ER beta) genes in the rat ovary, using Northern blotting, RT-PCR, and in situ hybridization histochemistry. Northern blotting results show that the ovary expresses both ER alpha and ER beta genes as single (approximately 6.5-kb) and multiple (ranging from approximately 1.0-kb to approximately 10.0-kb) transcripts, respectively. ER alpha mRNA is expressed at a level lower than ER beta mRNA in immature rat ovaries. This relationship appears unchanged between sexually mature adult rats and immature rats. In sexually mature adult rats undergoing endogenous hormonal changes, whole ovarian content of ER beta mRNA, as determined by RT-PCR, remained more or less constant with the exception of the evening of proestrus when ER beta mRNA levels were decreased. Examination of ER beta mRNA expression at the cellular level, by in situ hybridization, showed that ER beta mRNA is expressed preferentially in granulosa cells of small, growing, and preovulatory follicles, although weak expression of ER beta mRNA was observed in a subset of corpora lutea, and that the decrease in ER beta mRNA during proestrous evening is attributable, at least in part, to down-regulation of ER beta mRNA in the preovulatory follicles. This type of expression and regulation was not typical for ER alpha mRNA in the ovary. Although whole ovarian content of ER alpha mRNA was clearly detected by RT-PCR, no apparent modulation of ER alpha mRNA levels was observed during the estrous cycle. Examination of ER alpha mRNA expression at the cellular level, by in situ hybridization, showed that ER alpha mRNA is expressed at a low level throughout the ovary with no particular cellular localization. To further examine the potential role of the preovulatory pituitary gonadotropins in regulating ER beta mRNA expression in the ovary, we used immature rats treated with gonadotropins. In rats undergoing exogenous hormonal challenges, whole ovarian content of ER beta mRNA, as determined by RT-PCR, remained more or less unchanged after an injection of PMSG. In contrast, a subsequent injection of human CG (hCG) resulted in a substantial decrease in whole ovarian content of ER beta mRNA. In situ hybridization for ER beta mRNA shows that small, growing, and preovulatory follicles express ER beta mRNA in the granulosa cells. The preovulatory follicles contain ER beta mRNA at a level lower than that observed for small and growing follicles. In addition, there is an abrupt decrease in ER beta mRNA expression in the preovulatory follicles after hCG injection. The inhibitory effect of hCG on ER beta mRNA expression was also observed in cultured granulosa cells. Moreover, agents stimulating LH/CG receptor-associated intracellular signaling pathways (forskolin and a phorbol ester) readily mimicked the effect of hCG in down-regulating ER beta mRNA in cultured granulosa cells. Taken together, our results demonstrate that 1) the ovary expresses both ER alpha and ER beta genes, although ER beta is ...

PRL has been shown to stimulate mRNA expression of both ERalpha and ERbeta in the rat corpus luteum and decidua of pregnancy. To investigate whether PRL may stimulate ER expression at the level of transcription and which transcription factors may mediate this stimulation, we have cloned the 5'-flanking regions of both rat ER genes. A constitutively active PRL receptor (PRL-R(CA)) stimulated both ERalpha and ERbeta promoter activity, indicating that PRL is acting to stimulate ER transcription. Putative signal transducer and activator of transcription (Stat)5 response elements were identified at -189 in the ERalpha promoter and at -330 in the ERbeta promoter. Mutation of these response elements or overexpression of dominant negative Stat5 prevented stimulation of ERalpha and ERbeta promoter activity, indicating that PRL regulation of ER expression requires both intact Stat5 binding sites as well as functional Stat5. Interestingly, either Stat5a or Stat5b could stimulate ERalpha transcription while stimulation of ERbeta occurred only in the presence of Stat5b. Through mutational analysis, a single nucleotide difference between the ERalpha and ERbeta Stat5 response elements was shown to be responsible for the lack of Stat5a-mediated stimulation of ERbeta. These findings indicate that PRL stimulation of ER expression occurs at the level of transcription and that PRL regulation of ERalpha can be mediated by either Stat5a or Stat5b, while regulation of ERbeta appears to be mediated only by Stat5b.