Effects of ovarian hormone treatment on the gene expression of muscarinic acetylcholine receptors in the ovariectomized rat myometrium.

We investigate the effects of ovarian hormone on the gene expression of muscarinic acetylcholine receptors (M1-M5) in the myometrium using real-time PCR and evaluate the relationships between their expression and that of ovarian hormone receptors (ERα, ERß, and PgR). Wistar rats were sham operated (SO) or ovariectomized (OVX) and treated with vehicle, estradiol (E2), progesterone (P4), or both E2 and P4 for 2 days beginning on postoperative day 33. M1 and M4 mRNA expressions were not detected in the myometrium. M2 mRNA expression did not change significantly in the OVX and OVX+P4 groups compared to the SO group, but increased significantly in the OVX+E2 group and was normalized in the OVX+E2P4 group. M3 mRNA expression increased significantly in the OVX and OVX+P4 groups compared to the SO group, but was normalized in the OVX+E2 and OVX+E2P4 groups. M5 mRNA expression did not change significantly in all experimental groups. ERα mRNA expression increased significantly in the OVX, OVX+E2, and OVX+P4 groups compared to the SO group, but was normalized in the OVX+E2P4 group. The changes in ERß mRNA expression were similar to those of M3 mRNA expression in all experimental groups. In contrast, the changes in PgR mRNA expression did not correspond with that of M2, M3, or M5 mRNA expression in any of the experimental groups. Additionally, we evaluated the relationship between the expression of muscarinic acetylcholine receptors and ovarian hormone receptors in estrus cycle. M2 mRNA expression increased significantly in diestus and metaestrus compared in proestrus and estrus. M3 mRNA expression increased significantly in only diestrus compared in the other stages. In contrast, M5 mRNA expression did not change in estrus cycle. The changes in ERα mRNA expression appeared to be similar to those of M2 in estrus cycle, but no significant difference was found. The changes in ERß mRNA expression were similar to those of M3 mRNA expression. The change in PgR mRNA expression increased significantly in diestrus compared in metaestrus, but did not correspond with that of M2, M3, or M5 mRNA expression in estrus cycle. When acetylcholine sensitivity in the myometrium was compared between diestrus and estrus, the sensitivity is significantly lower in estrus than in diestrus. These results suggest that ovarian hormones influence the expression of M2 and M3 in the myometrium by regulating the expression of hormone receptors. E2 may upregulate M2 via ERα, but P4 may downregulate M2 by inhibiting ERα via PgR. E2 may downregulate M3 by inhibiting ERß, but P4 may not regulate the expression of M3 and ERß. M5 may be a constitutive muscarinic receptor in the myometrium because neither E2 nor P4 influence the expression of M5. The combination of E2 and P4 may contribute the reproduction by quieting down the acetylcholine-induced myometrial contraction.

Preparation of poly(methyl methacrylate) microcapsules by in situ polymerization on the surface of calcium carbonate particles.

Poly(methyl methacrylate) (PMMA) microcapsules were prepared by the in situ polymerization of methyl methacrylate (MMA) and N,N'-methylenebisacrylamide on the surface of calcium carbonate (CaCO(3)) particles, followed by the dissolution of the CaCO(3) core in ethylenediaminetetraacetic acid solution. The microcapsules were characterized using fluorescence microscopy, atomic force microscopy, scanning electron microscopy, and Fourier transform infrared spectroscopy. The average sizes of the CaCO(3) particles and PMMA capsules were 3.8±0.6 and 4.0±0.6 µm, respectively. A copolymer consisting of MMA and rhodamine B-bearing MMA was also used to prepare microcapsules for fluorescent microscopy observations. Fluorescein isothiocyanate-labeled bovine serum albumin was enclosed in the PMMA microcapsules and its release properties were studied.

Estrogen and selective estrogen receptor modulators regulate vascular endothelial growth factor and soluble vascular endothelial growth factor receptor 1 in human endometrial stromal cells.

OBJECTIVE: To determine whether 17beta-estradiol (E(2)) and selective estrogen receptor modulators can regulate vascular endothelial growth factor (VEGF) and soluble VEGF receptor 1 (sVEGFR-1) as a VEGF antagonist in human endometrial stromal cells (ESCs). DESIGN: In vitro experiment. SETTING: Research laboratory at Kansai Medical University. PATIENT(S): Sixteen patients undergoing hysterectomy for benign reasons. INTERVENTION(S): The ESCs were cultured with E(2), 4-hydroxytamoxifen (OHT), and raloxifene. MAIN OUTCOME MEASURE(S): The VEGF and sVEGFR-1 messenger RNA (mRNA) levels in ESCs were determined using quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Free (unbound) VEGF and sVEGFR-1 protein levels from ESCs were measured using ELISA kits. RESULT(S): The E(2) significantly induced VEGF mRNA levels, whereas E2 caused a significant decrease in sVEGFR-1 messenger RNA (mRNA) levels. The E(2) or OHT significantly increased the VEGF production levels and attenuated the sVEGFR-1 production compared with control, but raloxifene had no significant effect. The decrease in levels of free VEGF was proportional to the increase in sVEGFR-1 levels in the culture media of ESCs. CONCLUSION(S): The E(2) or OHT stimulates VEGF production and concurrently attenuates sVEGFR-1 production in ESCs. This consequential increase in VEGF:sVEGFR-1 ratio might enhance the biological effects of VEGF on the angiogenic environment in human endometrium.

Role of protein kinase Cbeta in rhythmic contractions of human pregnant myometrium.

To assess the role of protein kinase Cbeta (PKCbeta) in human myometrial contractions during pregnancy, we evaluated the effect of a PKCbeta inhibitor (LY333531) on the pregnant and nonpregnant myometrial contractions and compared the level of PKCbeta in the pregnant myometrium with that in the nonpregnant myometrium. The effects of LY333531 on the myometrial contractions were examined by measuring contractile activity (frequency and amplitude). PKCbeta in human myometrium was assessed at mRNA level using real-time PCR method. The characteristics of contractile activity were different between the pregnant and the nonpregnant myometrium. The amplitude of rhythmic contractions in the preterm and term myometrium was increased 2- to 2.5-fold when compared with that in the nonpregnant myometrium, but the frequency of rhythmic contractions was decreased by about half. LY333531 (10(-6) M) reduced the increased amplitude in the preterm and term myometrium by about 50%, and the inhibitory effects of LY333531 in the pregnant myometrium were significantly greater than that in the nonpregnant myometrium (about 50 vs 25%). However, the frequency in the pregnant and nonpregnant myometrium was not influenced by LY333531. Real-time PCR revealed a significant, five- to sevenfold increase in the expression of PKCbeta mRNA in the preterm and term myometrium when compared with the nonpregnant myometrium. These findings suggest that the increased amplitude of human myometrial contractions during pregnancy is related to the increased level of PKCbeta. A PKCbeta inhibitor may reduce preterm uterine contractions and prevent preterm delivery.

Cigarette smoke extract enhances oxytocin-induced rhythmic contractions of rat and human preterm myometrium.

Although smoking during pregnancy is a major risk factor for preterm delivery, the underlying mechanism by which smoking stimulates uterine contractions is still poorly understood. In the present study, we tried to clarify the effects of smoking on myometrial contractility induced by oxytocin (OT) using cigarette smoke extract (CSE). Myometrial strips, which were taken from the rat on day 16 of pregnancy, and from human preterm and term delivery groups, were incubated overnight with several doses of CSE at 37 degrees C under non-hormonal conditions. The uterine contractile sensitivity and activity (force and frequency) upon exposure to OT were investigated. Furthermore, the expression levels of oxytocin receptor (OTR) mRNA in the myometrial strips were investigated by real-time PCR. Contractile sensitivity to OT in the rat CSE (10(-7) pieces/ml) group was found to be significantly higher than in the control group (P < 0.05). Contractile activity did not differ between the CSE and control groups. The expression levels of rat OTR mRNA in the CSE (10(-7) pieces/ml) group were significantly higher than in the control group (P < 0.01). Similarly, in preterm myometrial strips, the expression levels of human OTR mRNA in the CSE (10(-7) pieces/ml) group were significantly higher than in the control group (P < 0.05). These findings suggest that CSE directly increases the contractile sensitivity of preterm myometrium in response to OT by upregulating the expression of OTR mRNA and thereby increases the risk of preterm delivery in women, who smoke during pregnancy.

Primary relapse of acute lymphoblastic leukemia in a cervical smear: a case report.

Uterine cervix and corpus are rarely the initial site of relapse in leukemia or lymphoma. We report herein a case of uterine cervical relapse with B-cell acute lymphoblastic leukemia (ALL). The patient, a 60-yr-old woman, had a history of ALL that had been in remission for 2 yr after chemotherapy. She presented with a chief complaint of genital bleeding. In a routine cervico-vaginal Papanicolau smear, abundant atypical lymphoid cells with round-to-oval nuclei, scant cytoplasm, and high nuclear to cytoplasmic ratios was observed. The nuclei of these cells had fine and dark chromatin and thickened nuclear membranes, with one or several nucleoli being visible. Biopsy under colposcope was performed, and a diagnosis of relapse of ALL was confirmed. The ongoing genital bleeding presented a problem with clinical management of the patient. It was decided to proceed with hysterectomy to end that problem and thereafter proceed with therapy directed against the leukemia. Our results suggest that in patients with known extrauterine cancer, the presence of malignancy in uterine cellular samples provides information regarding the extent of the neoplasm.

Expression of the endothelial cell differentiation gene 7 (EDG-7), a lysophosphatidic acid receptor, in ovarian tumor.

AIM: Lysophosphatidic acid (LPA) has received attention as a mitogen because the physiologically active lipid stimulates ovarian cancer cell growth by interacting with specific receptors, the endothelial cell differentiation gene (EDG) family. In the present study, we have investigated the expression of EDG-7 mRNA, part of the EDG family, in both human ovarian cancers and established human ovarian cancer cell lines. METHODS: RNA was extracted from six ovarian cancer cell lines and multiple cancerous and normal ovarian tissues. The expression of EDG-7 mRNA was measured using reverse transcription-polymerase chain reaction and northern blotting, using reduced glyceraldehyde-phosphate dehydrogenase and S26 as internal controls. RESULTS: Of the cell lines tested, EDG-7 mRNA was expressed most intensely in CRL-11731 and CRL-1572 and at a lesser but still substantial level in CRL-11732. The expression of EDG-7 mRNA was limited in MCAS, CRL-11730 and TYKnu. In the ovarian cancer tissues, EDG-7 mRNA was expressed most highly in endometrioid adenocarcinoma and serous cystadenocarcinoma. The expression of EDG-7 mRNA was limited in clear cell adenocarcinoma and undetectable in mucinous cystadenocarcinoma. CONCLUSIONS: The intense EDG-7 expression in ovarian cancers suggests that the relation between LPA and EDG-7 (an LPA receptor) is involved in cancer cell growth and proliferation in some histologic subtypes of ovarian cancer.

Interleukin-1 inhibits interleukin-15 production by progesterone during in vitro decidualization in human.

Interleukin (IL)-15 is a novel cytokine that plays important roles in uterine natural killer cell function and one of the candidate genes that is upregulated during the window of implantation for human endometrium. IL-15 expression and production by human endometrial stromal cells (ESCs) is elevated during in vitro decidualization by progesterone (P). In the present study, we evaluated the effects of IL-1beta, a proinflammatory cytokine, on IL-15 production in ESCs. By enzyme-linked immunosorbent assay (ELISA), IL-1beta had no effect on IL-15 production from ESCs in short-term culture (for 24 h), whereas IL-1beta stimulated production of IL-8. However, using ELISA and Northern blot analyses we found that IL-1beta significantly inhibited P-induced IL-15 production and mRNA expression in long-term culture (for 12 days) of ESCs in vitro (P<0.01). This inhibition was not due to IL-1beta-mediated cytotoxicity, as ESCs cultured in the presence of IL-1beta showed no evidence of significant change in their viability. These results suggest that ovarian steroid hormones and IL-1beta regulate IL-15 mRNA expression and protein production in long-term culture, and that IL-1beta plays a role as a negative regulator of IL-15 production during decidualization in human endometrium.

Energy gaps and roton structure above the nu=1/2 Laughlin state of a rotating dilute Bose-Einstein condensate.

Exact diagonalization study of a rotating dilute Bose-Einstein condensate reveals that as the first vortex enters the system the degeneracy of the low-energy yrast spectrum is lifted and a large energy gap emerges. As more vortices enter with faster rotation, the energy gap decreases towards zero, but eventually the spectrum exhibits a rotonlike structure above the nu=1/2 Laughlin state without having a phonon branch despite the short-range nature of the interaction.

Expression of 20alpha-hydroxysteroid dehydrogenase mRNA in human endometrium and decidua.

Progesterone secreted from ovarian corpus luteum plays pivotal roles in endometrial differentiation, and local progesterone metabolism to regulate its concentration in endometrial tissues is essential for the successful implantation and maintenance of pregnancy. In this study, we evaluated the expression of mRNA for 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), a key enzyme which converts progesterone to a biologically inactive metabolite, in human endometrial tissues and cultured endometrial stromal cells as well as decidua and chorionic tissues of early pregnancy. The level of 20alpha-HSD mRNA expression in secretory phase endometrium was significantly higher than that in proliferative phase endometrium and chorionic tissues. The expression level in decidual tissue was also significantly higher than that in chorionic tissue. In cultured endometrial stromal cells, 20alpha-HSD mRNA expression was slightly enhanced at a lower progesterone concentration of 0.01 micromol/l, and an increase in its expression was significantly suppressed at higher concentrations of 1 micromol/l or greater. No effect on the gene expression was seen in cultured endometrial stromal cells with various concentrations of 17beta-estradiol. These results suggest that progesterone itself contributes to the regulation of local progesterone concentration through 20alpha-HSD levels in endometrial stromal cells at peri-implantation periods.

Smoking enhances oxytocin-induced rhythmic myometrial contraction.

Although smoking during pregnancy is one of the major risk factors of premature delivery, the underlying mechanism by which smoking causes premature delivery is unknown. In the present study, we examined the effects of smoking on uterine contractility induced by oxytocin and prostaglandin F(2alpha). Rats inhaled either cigarette smoke or room air from Day 14 to Day 16 of pregnancy through an inhalation apparatus for experimental animals (type "Hamburg II"). After the rats were killed on Day 17 of pregnancy, the uterine contractile sensitivity and activity on exposure to oxytocin or prostaglandin F2alpha were investigated. The expression levels of oxytocin-receptor mRNA and prostaglandin F(2alpha) receptor mRNA in the uterus were investigated by reverse transcription-polymerase chain reaction. The contractile activity was assessed as the contractile force and the frequency of rhythmic contractions of myometrial strips that were treated with oxytocin or prostaglandin F(2alpha). The contractile sensitivity to oxytocin was significantly higher in the smoking group than in the control group (P < 0.01). Although the contractile force of oxytocin-induced contractions did not differ between the smoking and control groups, the frequency of contractions was significantly higher in the smoking group than in the control group (P < 0.01). On the other hand, no significant differences were found in the contractile sensitivity and activity in response to prostaglandin F(2alpha) between the smoking and control groups. The expression of oxytocin-receptor mRNA in the myometrium was significantly increased in the smoking group compared with the control group (P < 0.01). However, no significant difference was found in the level of expression of prostaglandin F(2alpha)-receptor mRNA between the two groups. These results suggest that smoking during pregnancy increases the contractile sensitivity and activity of the myometrium in response to oxytocin by up-regulating the expression of oxytocin-receptor mRNA. The effects of smoking on the contractile sensitivity and activity of the myometrium in response to oxytocin may increase the risk of premature delivery in smokers.

The human transcription factor activation protein-2 gamma (AP-2gamma): gene structure, promoter, and expression in mammary carcinoma cell lines.

Human activation protein-2 gamma (hAP-2gamma) is a key developmental transcription factor. It has been implicated in mammary carcinogenesis through its regulation of HER-2/neu proto-oncogene and estrogen receptor gene The hAP-2gamma gene is located on human chromosome 20q13.2. We cloned this gene, deduced its genomic structure, and mapped and analyzed its promoter. The hAP-2gamma gene contains seven exons. Primer extension analysis and 5'-rapid amplification of complementary DNA ends studies show that there is a single transcription start site 232 nt upstream of the translational start codon. The promoter lacks canonical binding sites for basal transcription factors such as TATA and CCAAT boxes, but contains a cluster of CpG islands and may rely on an initiator element for transcription. Deletion analyses of the promoter and chloramphenicol acetyl transferase reporter gene assays indicate that the sequence between -746 and -575 is important for its expression in mammary carcinoma cell lines. The hAP-2gamma gene is marginally activated in these cells suggesting that increased transcription partly contributes to its abundance. Architecture of the gene and promoter strikingly resembles that of hAP-2alpha, which is located on a different chromosome, suggesting a cognate origin. hAP-2alpha and hAP-2gamma have some common and some distinct roles in cells, and are likely the remarkable results of gene duplication, translocation and functional divergence through evolution.