A 22 kDa protein specific for yeast mitochondrial nucleoids is an unidentified putative ribosomal protein encoded in open reading frame YGL068W.

Mitochondrial-nucleoid (mt-nucleoid) proteins of the yeast Saccharomyces cerevisiae were separated by two-dimensional gel electrophoresis. Analysis of the N-terminal amino acid sequence showed that a 22 kDa protein which is unique in the mt-nucleoid fraction is an unidentified protein encoded in the open reading frame YGL068W and shows a homology with the ribosomal protein L7/L12 of bacteria. We named this protein Mnp1p (for the mitochondrial-nucleoid protein 1). Immunoblotting of each fraction with an anti-Mnp1p antibody during the mt-nucleoid isolation showed that Mnp1p is highly concentrated in the mt-nucleoid fraction. Immunofluorescence microscopy suggested that Mnp1p is localized to mitochondria in vivo, and a significant amount of Mnp1p is associated with the mt-nucleoids. On the other hand, Northern blotting showed that a large amount of large and small mitochondrial ribosomal RNAs was not associated with the mt-nucleoids and remained in the supernatant after the isolation of mt-nucleoids. The null mutation of MNP1 led to a respiratory-deficient phenotype, but the morphology of the mt-nucleoids in the transformants carrying the null mutation was normal. These results suggest that a significant amount of Mnp1p plays a role as a major component of the mt-nucleoids.

Gastrointestinal stromal tumor arising in the rectovaginal septum.

We report herein a rare case of malignant gastrointestinal stromal tumor (GIST) originated from the rectal wall, which presented as a tumor on the rectovaginal septum. A 54-year-old Japanese woman, gravida 4, para 3, was admitted complaining of anuresis and severe constipation. She had a history of hysterectomy and right salpingo-oophorectomy for uterine leiomyoma 11 years previously. Pelvic examination revealed an 8.5 x 7.5 x 7.5 cm hard mass in the rectovaginal space. The inferior border of the tumor was 2 cm from the vaginal introitus and 2 cm from the anus. Computed tomography and magnetic resonance imaging showed a well-circumscribed soft-tissue mass filling the rectovaginal space. Urinary bladder and rectum were markedly compressed and displaced. Colon fiberscopy revealed invasion of the tumor into the rectal mucosa. An abdominoperineal resection of the rectum with posterior vaginal wall resection and pelvic lymphadenectomy was performed. The resected specimen showed a rectal submucosal tumor that was 8 x 8 x 7 cm in size. The tumor was diagnosed as a malignant GIST. Immunohistochemical analysis confirmed this diagnosis. The patient is now healthy without evidence of recurrence at 13 months after surgery. Gynecologists should be aware of rectal GIST arising in the rectovaginal space as a differential diagnosis of vaginal submucosal tumor.

The clinical role of interleukin-6 and interleukin-6 soluble receptor in human follicular fluids.

In order to investigate the role of interleukin-6 (IL-6) and interleukin-6 soluble receptor (sR) in human ovulation, we evaluated the concentrations in human follicular fluid and analyzed the correlation of IL-6 and IL-6 sR with oocyte maturation. The oocytes were obtained from the follicular fluid of 45 women undergoing in vitro fertilization and embryo transfer. The concentrations of IL-6 and IL-6 sR in follicular fluid were measured by ELISA. In addition, granulosa cells obtained from the follicular fluid were cultured and treated with forskolin and 12- o-tetradecanoylphorbol 13-acetate for 24-48 h. The concentration of IL-6 was significantly higher in the follicular fluid than in the serum (P<0.01). In contrast, the concentration of IL-6 sR was significantly lower in the follicular fluid than in the serum (P<0.001). The concentrations of IL-6 and IL-6 sR were significantly higher in the follicular fluid containing mature oocytes than in fluid containing immature oocytes (P<0.05). The production of IL-6 was markedly increased over the basal level after 24 h of treatment with forskolin (P<0.001) and 48 h of treatment (P<0.01) with cultured granulosa cells. Our data suggest that IL-6 and IL-6 sR may play an important role in follicular growth and development in human preovulatory processes. It is possible that IL-6 in particular may be regulated by cAMP. IL-6 and IL-6 sR might also be valuable biochemical markers in the evaluation of oocyte maturation.

Complete response to radiation therapy in a patient with chemotherapy-resistant ovarian clear cell adenocarcinoma.

Recurrent ovarian cancer after front-line chemotherapy is incurable. In most institutions, chemotherapy is continued as salvage therapy after primary chemotherapy failure and despite the fact that long-term survival and complete responses are infrequent. Radiation therapy for patients with recurrent ovarian cancer has often been done with palliative intent. A patient with ovarian clear cell adenocarcinoma received irradiation with palliative intent to the whole pelvis after chemotherapy (paclitaxel, carboplatin, and irinotecan) produced no effect. Although she developed a rectovaginal fistula due to cancer invasion during radiation therapy. One year and half after the therapy, she is still alive with no evidence of disease. In an effort to maximize salvage potential and quality of life while minimizing toxicity, selected patients with ovarian cancer should be treated with radiation therapy directed to residual or recurrent sites.

The effect of epidermal growth factor on vascular endothelial growth factor secretion by endometrial stromal cells.

Endometrial stromal cells undergo morphological and functional changes to facilitate oocyte implantation under regulation of various hormones and growth factors. We studied physiological induction by epidermal growth factor (EGF) of vascular endothelial growth factor (VEGF) in these cells. In human endometrial stromal cells, the effect of EGF, genistein, tryphostin AG1478 (a tyrosine kinase inhibitor), and wortmannin (a phosphatidylinositol 3-kinase inhibitor) on production of VEGF was examined. Total RNA was extracted and VEGF mRNA expression was quantified by Northern analysis. EGF induced production of VEGF by stromal cells in a time-dependent manner; the effect became significant after 12 h and increased further between 24 and 48 h (P<0.05). Dose dependency was also significant (P<0.01). Genistein, tryphostin AG1478, and wortmannin partially suppressed the increase in production induced by EGF (P<0.01, P<0.01, P<0.01), respectively. Production of EGF by fertilized oocytes and trophoblasts has been reported in early pregnancy. VEGF is believed to be induced by EGF through mechanisms involving tyrosine kinase and phosphatidylinositol 3-kinase. The increase in VEGF may contribute to neovascularization that promotes proliferation of endometrium and placentation.

Identification of the YMN-1 antigen protein and biochemical analyses of protein components in the mitochondrial nucleoid fraction of the yeast Saccharomyces cerevisiae.

We analyzed the protein components contained in the mitochondrial nucleoid (mt-nucleoid) fraction of the yeast Saccharomyces cerevisiae. Immunoblotting with anti-Abf2p antibody demonstrated the association of Abf2p, a major mitochondrial DNA-binding protein, with the mt-nucleoids. In contrast, porin and cytochrome c oxidase subunit III (CoxIIIp) were not detected by immunoblotting in the mt-nucleoid fraction. The YMN-1 monoclonal antibody recognized a 48 kDa protein of the mt-nucleoid fraction. The N-terminal amino acid sequence of the protein and immunological evidence showed that the YMN-1 monoclonal antibody recognizes dihydrolipoyl transsuccinylase (KE2), which is one of the constituents of the alpha-ketoglutarate dehydrogenase complex (KGDC). alpha-Ketoglutarate dehydrogenase (KE1) and dihydrolipoyl dehydrogenase (E3), which are other subunits of KGDC, were also detected in the mt-nucleoid fraction. An enzyme assay of the mt-nucleoid fraction showed that cytochrome c oxidase and fumarase activity were barely detected in the fraction, but the specific activity of KGDC in the mt-nucleoid fraction was relatively high and was approximately 60% of the specific activity in the mitochondrial fraction. Three components of KGDC were detected in the DNA-binding protein fractions after DNA-cellulose column chromatography of mt-nucleoid proteins. These results suggested that a part of KGDC in the mitochondrial matrix is associated with mt-nucleoids in vivo.

c-Ets1 is a promising marker in epithelial ovarian cancer.

c-Ets1 controls the expression of some genes involved in extracellular matrix remodeling. To elucidate the involvement of c-Ets1 in epithelial ovarian carcinogenesis, we investigated the role of the proto-oncogene c-ets1 in the regulation of physiological processes such as cell proliferation, differentiation, and tumor invasion. Using fluorescent immunohistochemistry, we analyzed serial frozen sections for c-Ets1 protein expression in 26 patients with ovarian epithelial carcinoma, 10 patients with benign cystadenoma of the ovary, and 10 premenopausal patients with normal ovaries. We analyzed the relationship between the percentage of c-Ets1 stained cells in a patient and characteristics of the patient including histological classification, clinical stage, histological grade, and clinical outcome. c-Ets1 was not detected in any cases of benign ovarian cystadenoma. Most of the c-Ets1 proteins were found in the cytoplasm and some in the nucleus of epithelial ovarian cancer tissues. Moreover, c-Ets1 was strongly expressed in the head portion of papillary cancer tissues that had invaded the stroma. c-Ets1 expression was significantly associated with clinical stage (p<0.01), histological grade (p<0.01), and clinical outcome (p<0.01). Survival data were available for all patients and univariate Cox regression analysis showed that c-Ets1 expression was significantly associated with a poor prognosis (p<0.05). Our results demonstrate that c-Ets1 expression in epithelial ovarian cancer correlates to the malignant potential of the tumor.

Expression of cathepsin L in normal endometrium and endometrial cancer.

OBJECTIVE: To evaluate the expression of cathepsin L in normal endometrium and endometrial adenocarcinoma. STUDY DESIGN: Tissue from eight cases of G1 and eight of G2 endometrioid adenocarcinoma, and 15 normal endometrial specimens were examined by immunohistochemistry. RESULTS: In the normal endometrium, cathepsin L was expressed in a few cell layers of the apical part of the glandular epithelium throughout the menstrual cycle. In the carcinomas, there was an inverse correlation between the grade of tumor and the cathepsin L expression. CONCLUSION: Cathepsin L expression may cease during endometrial carcinogenesis and its expression may be less important in tumor progression than it is in tumors of other tissues.

The effect of dexamethasone on expression of mitogen-induced cyclooxygenase-2 mRNA by amnion-derived (WISH) cells.

OBJECTIVES: It has been reported that prostaglandin E(2) (PGE(2)) is synthesized in the amnion and that this synthesis increases during labor. The purpose of this study was to clarify the mechanism for the expression of cyclooxygenase-2 (COX-2) mRNA and the PGE(2) synthesis of amnion-derived (WISH) cells. STUDY DESIGN: Cells were cultured and treated by 12-O-tetradecanoylphorbol-13-acetate (TPA) and dexamethasone (DEX). PGE(2) in the culture medium was measured by ELISA. Total RNA was extracted from the cells, and COX-2 mRNA expression was analyzed by Northern blot analysis. RESULTS: During the time course of PGE(2) production in response to TPA stimulation, the PGE(2) production could not be detected until incubation had continued for 2h, but this production appeared to continue after 4h of incubation. PGE(2) production was significantly increased by TPA and suppressed by treatment with TPA and DEX. During the time course of COX-2 mRNA expression in response to treatment with TPA, the COX-2 mRNA band was detected after 1.5h. The strongest expression of COX-2 mRNA was observed at 2h incubation. After pre-treatment with TPA for 1h, the TPA-induced COX-2 mRNA was suppressed by treatment with DEX for 1 or 2h incubation in a dose-dependent manner. CONCLUSIONS: These results suggest that COX-2 mRNA is induced by TPA which activate protein kinase C, and suppressed by DEX in WISH cells.

Atypical polypoid adenomyoma in a patient with hyperprolactinemia.

We report a case of an atypical polypoid adenomyoma in a patient with hyperprolactinemia. A 23-year-old Japanese woman was admitted complaining of atypical genital bleeding. Specula examination revealed a walnut-size polypoid mass extruding from the cervix. The patient was oligomenorrheac, and endocrine analysis showed hyperprolactinemia. Transvaginal ultrasonography and magnetic resonance imaging revealed an endometrial polypoid mass (4 x 3 x 3 cm) arising from the lower segment of the uterine corpus. The pathologic diagnosis of the tumor after polypectomy was atypical polypoid adenomyoma. It is suggested that ovarian dysfunction caused by hyperprolactinemia may be involved in the pathogenesis of atypical polypoid adenomyoma in the present case.

The effect of pregnancy on cytochrome P4501A2, xanthine oxidase, and N-acetyltransferase activities in humans.

OBJECTIVE: Our objective was to evaluate the activity of cytochrome P4501A2 (CYP1A2), xanthine oxidase (XO), and N-acetyltransferase 2 (NAT2) from early to late pregnancy and after delivery. METHODS: Twelve women were studied on three occasions during pregnancy (early, 8-16 weeks' gestation; middle, 20-28 weeks' gestation; and late, 32-39 weeks' gestation) and about 1 month after delivery. Caffeine was used as a metabolic probe. After the women ingested a can or a bottle of caffeine-containing soft drink, urine samples were collected for 12 hours. The caffeine metabolites measured were 5-acetylamino-6-amino-3-methyluracil (AAMU), 1-methylxanthine (1X), 1-methyl-uric acid (1U), 1,7-dimethyl-uric acid (17U), and 1,7-dimethylxanthine (17X). The hepatic enzyme activities were estimated by the urinary caffeine metabolic ratios as follows: CYP1A2 = (AAMU + 1X + 1U)/17U; XO = 1U/(1X + 1U); NAT2 = AAMU/(AAMU + 1X + 1U). RESULTS: Statistically significant differences were found in CYP1A2 (P < .0001) and NAT2 (P < .01). The mean metabolic ratios for CYP1A2 during pregnancy (6.80, 5.18, and 4.97 for the early phase, middle phase, and late phase, respectively) were significantly lower than the ratio after delivery (10.39). The mean metabolic ratio for NAT2 in the early phase (0.57) was significantly lower than after delivery (0.66). There was no significant difference in metabolic ratios for XO during pregnancy and after delivery. CONCLUSION: The data demonstrate that pregnancy influences CYP1A2 and NAT2 activity. CYP1A2 activity decreases not only in late pregnancy but also in early and middle pregnancy.

Effect of endotoxin-induced reactive oxygen species on sperm motility.

OBJECTIVE: To define the mechanism of infection-induced damage of sperm. DESIGN: The effect of lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) on sperm motility and its modification by scavengers were investigated. SETTING: Research laboratory of a university hospital. PATIENT(S): Normozoospermic semen samples were obtained from 37 healthy volunteers. INTERVENTION(S): The sperms were incubated in the presence of LPS with or without scavengers. MAIN OUTCOME MEASURE(S): Sperm motility was evaluated by a sperm quality analyzer (SQAIIB). ROS formation in semen samples was measured by a Berthold luminometer (LB953). RESULT(S): Motility of spermatozoa was decreased in the LPS-treated samples compared with that in the control groups. ROS was significantly higher in the LPS-treated groups than in the control groups. The addition of ROS scavengers restored the motility index and suppressed ROS production in the LPS-treated semen samples. CONCLUSION(S): These data suggest that endotoxin-induced excessive production of ROS is responsible for the decrease in sperm motility and that antioxidant therapy may be a therapeutic option for infertile men with bacterial genital tract infection.

Expression and regulation of growth-regulated oncogene alpha in human endometrial stromal cells.

Growth-regulated oncogene alpha (GROalpha), a potent chemoattractant for neutrophils, has previously been detected in the endometrial stromal cells (ESC) of human endometrium. In this study, the mRNA expression of GROalpha in the endometrium was evaluated by reverse transcription-polymerase chain reaction analysis, while the localization of GROalpha protein was studied by immunohistochemistry and the concentrations of GROalpha were measured using an enzyme-linked immunosorbent assay (ELISA). The effects of known modulators of endometrial function on the production of GROalpha by ESC were also examined by ELISA and Northern blot analysis. The expression of both GROalpha mRNA and GROalpha protein was detected in the cycling endometrium. GROalpha protein was localized mainly in the stroma, and endometrial tissues in the secretory phase contained higher amounts of GROalpha protein than did those in the proliferative phase. The production of GROalpha by ESC was enhanced by in-vitro decidualization. Lipopolysaccharide, tumour necrosis factor-alpha and interleukin-1beta also stimulated the expression of GROalpha mRNA and protein by ESC. These results suggest that the production of GROalpha by ESC is regulated by ovarian steroid hormones as well as by inflammatory mediators. The modulation of GROalpha concentrations in the local environment may contribute to normal and pathological processes in the uterus by regulating leukocyte trafficking in the endometrium.

Polo-like kinase (PLK) expression in endometrial carcinoma.

Polo-like kinase (PLK) is a cell cycle-regulated, cyclin-independent serine/threonine protein kinase. Recent reports have shown a critical role for PLK during tumorigenesis. To explore whether PLK plays a general role as a tumor marker of endometrial carcinomas, we examined the expression of PLK mRNA and protein in endometrial carcinomas and normal endometrium, and analyzed the relationship between PLK protein expression and malignant potential. We found that PLK mRNA was expressed in all specimens from endometrial carcinoma patients using RT-PCR methods, although some specimens from normal endometria were negative. Immunohistochemically, most of the PLK was found in the cytoplasm (around the nucleus), and partly in the nucleus of endometrial carcinoma glands and also secreted tissues from endometrial carcinoma glands. PLK was expressed at the basement membrane of carcinoma glands and partly expressed in the head portion of papillary carcinoma tissues. There was a significant correlation between percentages of PLK-positive cells and histological grade of endometrial carcinoma (P<0.0001). However, the expression of proliferating cell nuclear antigen and Ki-67 was independent of PLK expression. Moreover, we noted that PLK is strongly expressed in invading carcinoma cells. PLK expression could reflect the degree of malignancy and proliferation in endometrial carcinoma. Thus, in addition to being of diagnostic value, modulation of PLK activity in the tumors by chemotherapeutic agents or gene therapy may prove to be of therapeutic value.

Expression of epithelial neutrophil-activating peptide 78 in cultured human endometrial stromal cells.

It has been demonstrated that human endometrial stromal cells (ESC) produce a variety of chemokines in vivo and in vitro. To evaluate the expression of epithelial neutrophil-activating peptide 78 (ENA-78) in the endometrium, concentrations of ENA-78 in cyclic endometrial tissues were measured using enzyme-linked immunosorbent assay. The expression of ENA-78 was also detected in cyclic endometrium by immunohistochemistry. Endometrial tissues in the secretory phase contained higher amounts of ENA-78 protein than did those in the proliferative phase. Immunofluorescence staining revealed that ENA-78 protein was localized mainly in the stroma of endometrium. In addition, to evaluate the involvement of inflammatory mediators and ovarian steroid hormones in the production of ENA-78 by ESC was evaluated by in-vitro studies. Unstimulated ESC constitutively secreted ENA-78. Progesterone, lipopolysaccharide, tumour necrosis factor-alpha, and interleukin-1beta significantly stimulated the expression of ENA-78 by ESC. It is suggested that the production of ENA-78 by ESC is regulated by progesterone as well as by the inflammatory mediators. The modulation of ENA-78 concentration in the local environment by these mediators may contribute to the normal and pathological processes of human reproduction through regulation of leukocyte trafficking into the endometrium.

Effects of platelet-activating factor on cytokine production by human uterine cervical fibroblasts.

Platelet-activating factor (PAF), a lipid that acts as a potent proinflammatory mediator, is involved in several reproductive processes including parturition. To investigate the effects of PAF on expression of various cytokines by cultured human uterine cervical fibroblasts obtained at term prior to labour, Northern blot analyses and enzyme-linked immunosorbent assays were performed. C-PAF, a stable analogue of PAF, increased expression of interleukin-6 and -8 mRNA in a dose-dependent manner (10(-10) to 10(-8) mol/l of C-PAF), and the expression peaked within 4 h. The corresponding protein concentrations were increased in culture media. Monocyte chemoattractant protein-1 mRNA showed marked induction by 10(-8) mol/l of C-PAF; this peaked by 4 h and was followed by an increase in the protein concentration. Another cytokine, RANTES (regulated upon activation, normal T cell expressed and secreted) showed marked mRNA induction by 10(-8) mol/l of C-PAF, and continued to increase in a time-dependent manner until 24 h. The protein concentration was correspondingly increased in the medium. The PAF-induced cytokine production was abolished by co-incubation with WEB 2170, a specific PAF receptor antagonist. PAF may stimulate local production of cytokines which may induce migration of leukocytes and accelerate collagenolysis in the uterine cervix, thus contributing to cervical ripening during parturition.

Expression of receptor tyrosine kinase EphB4 and its ligand ephrin-B2 is associated with malignant potential in endometrial cancer.

The protein kinases includes many oncogenes and growth-factor receptors, as well as genes that are involved in cell cycle regulation. EphB4 receptors are a subfamily of receptor tyrosine kinases that are activated by ephrin-B2 ligands and are thought to play an important role in the development and oncogenesis of various tissues. However, very little experimental evidence exists to support this hypothesis. To elucidate the involvement of EphB4 and ephrin-B2 in endometrial carcinogenesis, we used fluorescent immunohistochemistry to analyze serial frozen sections of 20 endometrial carcinomas and 20 normal endometria for EphB4 and ephrin-B2 protein expression. We analyzed the relationship between the patient's characteristics and the percentages of EphB4- and ephrin-B2-stained cells. EphB4 expression was significantly associated with histological grade (p < 0.001) and certain clinical stages. Ephrin-B2 Expression was significantly associated with the presence of invasion to > 1/2 myometrium (p = 0.002). Our results demonstrate that increased EphB4 and ephrin-B2 expression may reflect or induce in endometrial carcinomas increased potential for growth and tumorigenicity. Furthermore, these results suggest that EphB4 receptor kinase may modulate the biological behavior of endometrial carcinomas through autocrine and/or paracrine activation, which is caused by ephrin-B2 ligands that are expressed in the same or neighbouring cells by immunohistochemistry.

Id1 expression is associated with histological grade and invasive behavior in endometrial carcinoma.

Basic helix-loop-helix (bHLH) DNA-binding proteins have been reported to regulate tissue-specific transcription of cellular differentiation within multiple cell lineages. The Id family of helix-loop-helix proteins does not possess a basic DNA-binding domain and functions as a negative regulator of bHLH proteins by forming high-affinity heterodimers with bHLH proteins. Id proteins were originally characterized as inhibitors of DNA binding and cell differentiation. Thus, overexpression of Id proteins correlates with cell proliferation and arrested differentiation in many cell lineages. To elucidate the involvement of Id1 in endometrial carcinogenesis, we analyzed serial frozen sections for Id1 protein expression in 20 cases of endometrial carcinoma and 20 cases of normal endometria by fluorescent immunohistochemistry. We analyzed the relationship between the percentages of Id1-stained cells and the patient's characteristics, including histological grade, clinical stage, presence of invasion to >1/2 myometrium, and clinical outcome. In normal endometria, Id1 was not detected in either the proliferative or the secretory phase. There was, however, abundant Id1 immunoreactivity in the endometrial carcinoma cells. Moreover, Id1 was strongly expressed in the inflammatory cells. Scoring on the basis of the percentage of positive cells indicated that Id1 expression is significantly associated with histological grade (P<0.05) and the presence of invasion to >1/2 myometrium (P<0.05). Our results demonstrate that increased Id1 expression in endometrial carcinoma correlates with the malignant potential of this tumor.

Effects of interleukin-4 on the in-vitro production of cytokines by human endometrial stromal cells.

A T helper (Th)1/Th2 model has been applied to as a system regulating the cytokine network during pregnancy. To evaluate the effects of interleukin (IL)-4, a Th2 cytokine, on the cytokine production by endometrial stromal cells (ESC), an enzyme-linked immunosorbent assay was used to measure the concentrations of IL-6, IL-8, monocyte chemoattractant protein-1 (MCP-1), and macrophage colony-stimulating factor (M-CSF) in the culture media of ESC and of an endometrial stromal sarcoma cell line, MaMi, following the addition of recombinant human IL-4. The expression of mRNAs for IL-6 and IL-8 in ESC after stimulation with IL-4 was also evaluated by Northern blot analysis. Increases in the concentrations of IL-6, IL-8, MCP-1, and M-CSF in the culture media of ESC and MaMi cells were observed on the addition of increasing amounts of IL-4. This cytokine also stimulated the transcription of IL-6 and IL-8 in ESC in a dose-dependent manner. It is suggested that IL-4 secreted by the maternal decidual tissue as well as by the developing embryo may stimulate the production of IL-6, IL-8, MCP-1, and M-CSF by ESC. The increased concentration of these cytokines in the local environment may contribute to the maintenance of early pregnancy by modulating the immune reaction at the feto-maternal interface.