De novo filament formation by human hair keratins K85 and K35 follows a filament development pattern distinct from cytokeratin filament networks.

In human hair follicles, the hair-forming cells express 16 hair keratin genes depending on the differentiation stages. K85 and K35 are the first hair keratins expressed in cortical cells at the early stage of the differentiation. Two types of mutations in the gene encoding K85 are associated with ectodermal dysplasia of hair and nail type. Here, we transfected cultured SW-13 cells with human K85 and K35 genes and characterized filament formation. The K85-K35 pair formed short filaments in the cytoplasm, which gradually elongated and became thicker and entangled around the nucleus, indicating that K85-K35 promotes lateral association of short intermediate filaments (IFs) into bundles but cannot form IF networks in the cytoplasm. Of the K85 mutations related to ectodermal dysplasia of hair and nail type, a two-nucleotide (C1448 T1449 ) deletion (delCT) in the protein tail domain of K85 interfered with the K85-K35 filament formation and gave only aggregates, whereas a missense mutation (233A>G) that replaces Arg78 with His (R78H) in the head domain of K85 did not interfere with the filament formation. Transfection of cultured MCF-7 cells with all the hair keratin gene combinations, K85-K35, K85(R78H)-K35 and K85(delCT)-K35, as well as the individual hair keratin genes, formed well-developed cytoplasmic IF networks, probably by incorporating into the endogenous cytokeratin IF networks. Thus, the unique de novo assembly properties of the K85-K35 pair might play a key role in the early stage of hair formation.

Analysis of pregnancy outcome and decline of anti-Müllerian hormone after laparoscopic cystectomy for ovarian endometriomas.

AIM: Excision of ovarian endometrioma (OE) may induce the reduction of ovarian reserve. We evaluated pregnancy outcomes after laparoscopic cystectomy (LC), and the pre- and postoperative levels of anti-Müllerian hormone (AMH) to consider the ovarian reserve. METHODS: We enrolled 40 women with OE and 16 women with benign ovarian tumors who hoped to have a child and who underwent LC. To evaluate the ovarian reserve of 40 patients (OE group, n = 24; non-OE group, n = 16), we measured serum AMH levels before and after the surgery. RESULTS: In the 40 women who underwent LC for OE, the cumulative pregnancy rate was 50%. Prior to the cystectomy, serum AMH levels in the OE group, especially in patients over the age of 35, were significantly lower than those in the non-OE group. Rate of decline in serum AMH in the OE group was significant compared with that in the non-OE group 6 months after surgery. In patients over the age of 35 in the OE group, AMH levels 1 year after surgery decreased noticeably. CONCLUSION: LC for OE could be a preferred surgical approach, but effective therapeutic strategies will have to be developed to prevent damage to the ovarian reserve, especially for older patients.

Hepatocyte growth factor/Met system promotes endometrial and endometriotic stromal cell invasion via autocrine and paracrine pathways.

Endometrial stromal cells reportedly have a role in the initial invasion of endometrial tissue into the peritoneum. Hepatocyte growth factor (HGF), which is a ligand for the c-met protooncogene product (Met), stimulates proliferation and invasion of a large number of cells. In this study we investigated the role of the HGF/Met system in the pathogenesis of endometriosis. HGF concentrations in the peritoneal fluid of patients with endometriosis were significantly higher than in those without endometriosis and correlated positively with revised American Society of Reproductive Medicine scores. We showed that the peritoneum and endometriotic stromal cells may be major sources of HGF in peritoneal fluid. Endometrial and endometriotic stromal cells expressed the Met receptor, which was activated by endogenous and exogenous HGF. HGF enhanced stromal cell proliferation and invasion. We also demonstrated that the HGF-stimulated stromal cell invasion was due in part to the induction of urokinase-type plasminogen activator, a member of the extracellular proteolysis system. In conclusion, the HGF/Met system is involved in the pathogenesis of endometriosis by promoting stromal cell proliferation and invasion of shed endometria and endometrial lesions via autocrine and paracrine pathways.

Gonadotropin-releasing hormone agonist treatment reduced serum interleukin-6 concentrations in patients with ovarian endometriomas.

OBJECTIVE: To determine whether serum interleukin (IL)-6 can be measured in patients with ovarian endometriomas and whether these measurements are useful in managing this disease. DESIGN: A controlled clinical study and an in vitro study. SETTING: Department of Obstetrics and Gynecology, Tottori University, Japan.Twenty-two patients with ovarian endometriomas. INTERVENTION(S): Laparoscopic cystectomy for ovarian endometriomas was performed. Gonadotropin-releasing hormone (GnRH) agonist was administered for 3 months in nine patients before laparoscopic surgery. Endometriotic stromal cells obtained from patients with endometriomas with or without GnRH agonist treatment were cultured. MAIN OUTCOME MEASURES(S): IL-6 concentrations in serum or supernatant of the cell culture were measured using ELISA. RESULTS: The serum concentration of IL-6 in patients with endometriomas was higher at the time of diagnosis than in those without endometriomas. Laparoscopic surgery significantly reduced serum levels of IL-6. Serum IL-6 concentrations also decreased after treatment with GnRH agonist. IL-6 production was attenuated in the endometriotic stromal cells obtained from patients with GnRH agonist treatment compared with patients without such treatment. CONCLUSION(S): GnRH agonist treatment may decrease IL-6 production in endometriotic cells. Measurement of serum IL-6 concentrations may be of value in managing patients with endometriomas.

Tumor necrosis factor-alpha-induced interleukin-8 (IL-8) expression in endometriotic stromal cells, probably through nuclear factor-kappa B activation: gonadotropin-releasing hormone agonist treatment reduced IL-8 expression.

Endometriosis, a common disease among women of reproductive age, is characterized by the presence of endometrial-like tissue outside the uterus. We previously reported that TNFalpha promoted proliferation of endometriotic stromal cells by inducing IL-8 gene and protein expression. We hypothesize that TNFalpha may induce IL-8 production in endometriotic cells through nuclear factor-kappa B (NF-kappa B) activation. Western blot analyses and electrophoretic mobility shift assays revealed that incubation with TNF alpha induced the expression of phosphorylated inhibitor kappa B (p-I kappa B) and activation of NF-kappa B in endometriotic stromal cells. The NF-kappa B inhibitor, N-tosyl-L-phenylalanine chloromethyl ketone, reduced TNFalpha-induced IL-8 gene and protein expression. The medical treatment of endometriosis with GnRH agonist (GnRHa) has been shown to induce hypoestrogenemia and reduce the observable number of endometriotic implants. We compare the expression of IL-8 gene and protein in endometriotic stromal cells of patients treated with GnRHa and those of patients without treatment before laparoscopic cystectomy for endometrioma. The addition of TNFalpha (0.1 ng/ml) significantly increased protein and gene expression of IL-8 in the cells of patients without GnRHa treatment, but this expression was not observed in the cells of patients with GnRHa. The addition of estradiol (E2; 10(-7) M) enhanced the expression of IL-8. However, in the cells of patients who received GnRHa treatment, TNFalpha and E2 did not show any significant effect. In endometriotic stromal cells without GnRHa treatment, TNFalpha and E2 increased the expression of p-I kappa B. In contrast, TNFalpha and E2 had no significant effect on the expression of p-I kappa B in cells that received GnRHa treatment. These findings demonstrate that NF-kappa B activation is critical for TNFalpha-induced IL-8 expression in endometriotic stromal cells. The current study showed for the first time that GnRHa treatment attenuated the expression of IL-8 by reducing TNFalpha-induced NF-kappa B activation.

Activation of mitogen-activated protein kinase pathway by keratinocyte growth factor or fibroblast growth factor-10 promotes cell proliferation in human endometrial carcinoma cells.

Fibroblast growth factors (FGFs) exert diverse effects resulting from their interaction with cognate receptors on target cells. Our current study was designed to examine the local production and action of two specific stromal-epithelial cell mediatory factors, keratinocyte growth factor (KGF) and FGF-10, in human endometrial carcinoma cells. The RT-PCR method was used to determine gene expression of KGF, FGF-10, and KGF receptor in human endometrial carcinoma cells (HEC-1) and human endometrial stromal cells. KGF mRNAs were expressed in both of these cell types. On the other hand, FGF-10 mRNA was detected only in the endometrial stromal cells, and KGF receptor mRNA was observed in the HEC-1 cells. The novel finding of the present study is that KGF is expressed in carcinoma cells and FGF-10 is expressed in human endometrial stromal cells. The distinct phosphorylation of ERK-1 and -2 (ERK1/2), which are members of the MAPK family, was observed when HEC-1 cells were treated with KGF or FGF-10. KGF and FGF-10 could induce the prompt phosphorylation of ERK1/2 and consequently stimulate DNA synthesis. KGF and FGF-10 did not activate the phosphorylation of Akt, protein kinase C, or signal transducer and activator of transcription-3. Blocking the MAPK pathway with the specific methyl ethyl ketone 1/2 inhibitor (U0126) completely neutralized the enhancement of cell proliferation induced by KGF and FGF-10. In addition, KGF and FGF-10 activated expressions of downstream nuclear transcription factors, such as Elk-1 and c-myc, but not c-fos. These results demonstrate for the first time that KGF and FGF-10 are capable of stimulating the growth of endometrial carcinoma cells via activating MAPK pathway through autocrine/paracrine fashion.

New farnesane-type sesquiterpenes, hedychiols A and B 8,9-diacetate, and inhibitors of degranulation in RBL-2H3 cells from the rhizome of Hedychium coronarium.

Two new farnesane-type sesquiterpenes, hedychiols A and B 8,9-diacetate, were isolated from the methanolic extract of the fresh rhizome of Hedychium coronarium KOEN. cultivated in Japan. Their stereostructures were elucidated on the basis of chemical and physicochemical evidence. The inhibitory effects of isolated constituents on the release of beta-hexosaminidase from RBL-2H3 cells were examined, and hedychilactone A and coronarin D were found to show the inhibitory activity.

Labdane-type diterpenes with inhibitory effects on increase in vascular permeability and nitric oxide production from Hedychium coronarium.

The methanolic extract from the rhizome of Hedychium coronarium was found to inhibit the increase in vascular permeability induced by acetic acid in mice and nitric oxide production in lipopolysaccharide-activated mouse peritoneal macrophages. From the methanolic extract, three new labdane-type diterpenes, hedychilactones A, B, and C, were isolated together with six known diterpenes. The structures of hedychilactones were elucidated on the basis of chemical and physicochemical evidence. The diterpene constituents showed inhibitory effects on the increase in vascular permeability, nitric oxide production, and inducible nitric oxide synthase induction.

Induction of hepatocyte growth factor in stromal cells by tumor-derived basic fibroblast growth factor enhances growth and invasion of endometrial cancer.

Tumor progression is often regulated through interactions between carcinoma cells and host stromal cells. In this study of endometrial cancer, we investigated one mechanism potentially involved in hepatocyte growth factor (HGF)-mediated cancer-stromal interactions. Endometrial cancer cells (HEC-1 and ISHIKAWA) expressed the c-met receptor, but HGF did not. HGF, however, did stimulate the proliferation and invasion of these cells. The HGF gene was expressed in stromal cells, which had been separated from primary cultures of endometrial cancers, 6.4 times more than in isolated normal endometrial stromal cells. Immunohistochemical staining revealed immunoreactive HGF in cancer stromal cells, the staining intensity being more pronounced in cancer tissue than in normal endometrium. The conditioned medium from normal epithelial cells and cancer cell lines induced HGF production in normal stromal cells. We identified basic fibroblast growth factor as an HGF inducer derived from endometrial cancer cell lines. Basic fibroblast growth factor derived from tumor cells may induce HGF in endometrial stromal cells, whereas stromal cell-derived HGF leads to the invasive growth of carcinoma cells. These interactions, mediated by HGF and HGF inducers, may play a significant role in the progression of endometrial cancer.

Molecular cloning of monkey CYP2C43 cDNA and expression in yeast.

A cDNA clone designated as CYP2C43 was isolated from the rhesus monkey liver cDNA library. The first 16 amino acid residues at the N-terminal region of this cDNA product were identical with those of P450 CMLd which have been purified and characterized as S-mephenytoin 4'-hydroxylase in monkey liver. The respective nucleotide and deduced amino acid sequences of CYP2C43 were 83% and 77%, identical to those of monkey CYP2C20. Antibody against CYP2C9 detected a protein in the microsomes of yeast transformed CYP2C43 expression plasmid. The specific content of recombinant CYP2C43 was 78.0 pmol/mg protein and the yield was 4.23 nmol/l of the culture. CYP2C43 was able to metabolize S-mephenytoin stereo-selectively. The activity for S-mephenytoin in the microsomes reconstituted with or without cytochrome b(5) was found to be 96.2 or 23.7 pmol/min/nmol P450, respectively. CYP2C43, however, did not show any oxidative activity for tolbutamide. These results indicate that CYP2C43 is the second identified member of the monkey CYP2C subfamily and a cDNA clone encoding P450 CMLd in monkey.