Design, synthesis and anticancer activity of 5-((2-(4-bromo/chloro benzoyl) benzofuran-5-yl) methyl)-2-((1-(substituted)-1H-1,2,3-triazol-4-yl)methoxy)benzaldehyde analogues.

Novel 5-((2-(4-bromo/chloro benzoyl) benzofuran-5-yl) methyl)-2-((1-(substituted)-1H-1,2,3-triazol-4-yl)methoxy)benzaldehyde analogues about twenty-one were synthesized all through standard chemical procedures. The structure of the compounds were confirmed by 1H NMR, 13C NMR and Mass spectral analysis after purification. All the compounds were screened for In Vitro lung and cervical cancer activity against A-549 and HeLa cell lines, respectively, by MTT assay protocol using various nanomolar (nM) concentrations. IC50 value were calculated from cell viability data. 2-(trifluoromethyl)benzyl substituted derivative presented outstanding activity against both the cell lines compared to standard drug doxorubicin. The methoxy, chloro, fluoro and formyl substituted analogues showed a moderate activity and whereas methyl substituted analogues activity was poor. The morphological deformation of both cell lines by best IC50 value analogues proved as potent inhibitors of cancer cells growth. Molecular docking studies were performed against extracellular signal-regulated kinase 2 and fibroblast growth factor receptor 2 these results are incredibly in agreement with the investigational data.

Development of artificial neural networks software for arsenic adsorption from an aqueous environment.

Arsenic contamination is a global problem, as it affects the health of millions of people. For this study, data-driven artificial neural network (ANN) software was developed to predict and validate the removal of As(V) from an aqueous solution using graphene oxide (GO) under various experimental conditions. A reliable model for wastewater treatment is essential in order to predict its overall performance and to provide an idea of how to control its operation. This model considered the adsorption process parameters (initial concentration, adsorbent dosage, pH, and residence time) as the input variables and arsenic removal as the only output. The ANN model predicted the adsorption efficiency with high accuracy for both training and testing datasets, when compared with the available response surface methodology (RSM) model. Based on the best model synaptic weights, user-friendly ANN software was created to predict and analyze arsenic removal as a function of adsorption process parameters. We developed various graphical user interfaces (GUI) for easy use of the developed model. Thus, a researcher can efficiently operate the software without an understanding of programming or artificial neural networks. Sensitivity analysis and quantitative estimation were carried out to study the function of adsorption process parameter variables on As(V) removal efficiency, using the GUI of the model. The model prediction shows that the adsorbent dosages, initial concentration, and pH are the most influential parameters. The efficiency was increased as the adsorbent dosages increased, decreasing with initial concentration and pH. The result show that the pH 2.0-5.0 is optimal for adsorbent efficiency (%).

Chemical characteristics of groundwater and assessment of groundwater quality in Varaha River Basin, Visakhapatnam District, Andhra Pradesh, India.

Study on chemical characteristics of groundwater and impacts of groundwater quality on human health, plant growth, and industrial sector is essential to control and improve the water quality in every part of the country. The area of the Varaha River Basin is chosen for the present study, where the Precambrian Eastern Ghats underlain the Recent sediments. Groundwater quality is of mostly brackish and very hard, caused by the sources of geogenic, anthropogenic, and marine origin. The resulting groundwater is characterized by Na(+) > Mg(2+) > Ca(2+) : [Formula: see text] > Cl(-) > [Formula: see text], Na(+) > Mg(2+) > Ca(2+) : [Formula: see text] > Cl(-) > [Formula: see text] > [Formula: see text], Na(+) > Mg(2+) > Ca(2+) : [Formula: see text] > Cl(-), and Na(+) > Mg(2+) > Ca(2+) : Cl(-) > [Formula: see text] > [Formula: see text] facies, following the topographical and water flow-path conditions. The genetic geochemical evolution of groundwater ([Formula: see text] and Cl(-)-[Formula: see text] types under major group of [Formula: see text]) and the hydrogeochemical signatures (Na(+)/Cl(-), >1 and [Formula: see text]/Cl(-), <1) indicate that the groundwater is of originally fresh quality, but is subsequently modified to brackish by the influences of anthropogenic and marine sources, which also supported by the statistical analysis. The concentrations of total dissolved solids (TDS), TH, Mg(2+), Na(+), K(+), [Formula: see text], Cl(-), [Formula: see text], and F(-) are above the recommended limits prescribed for drinking water in many locations. The quality of groundwater is of mostly moderate in comparison with the salinity hazard versus sodium hazard, the total salt concentration versus percent sodium, the residual sodium carbonate, and the magnesium hazard, but is of mostly suitable with respect to the permeability index for irrigation. The higher concentrations of TDS, TH, [Formula: see text], Cl(-), and [Formula: see text] in the groundwater cause the undesirable effects of incrustation and corrosion in many locations. Appropriate management measures are, therefore, suggested to improve the groundwater quality.

Toward gene therapy of primary ovarian failure: adenovirus expressing human FSH receptor corrects the Finnish C566T mutation.

Resistance ovarian syndrome is a heterogeneous disorder inherited as a Mendelian recessive trait and characterized by infertility, primary amenorrhea, normal karyotype and elevated serum FSH and LH levels. An inactivating mutation, C566T, in FSH receptor gene (FSHR) has been identified initially in Finland. We investigated if an adenovirus expressing a normal copy of human FSHR (Ad-hFSHR) has the ability to: (i) transfect granulosa cell lines, (ii) render the transfected cell lines responsive to FSH stimulation and (iii) transcomplement the malfunctioning form of human FSHR gene with C566T mutation. COS-7, JC-410, JC-410-P450-scc-luc and JC-410-StAR-luc cell lines were infected by Ad-hFSHR followed by treatment with FSH. Functional activity of the Ad-hFSHR was tested by measuring cyclic adenosine monophosphate (cAMP) or luciferase activity in response to FSH stimulation, and showed 2-4.6-fold increases in Ad-hFSHR transfected cells compared with untransfected or Ad-LacZ transfected cells, indicating that Ad-hFSHR is functionally active and expressing hFSHR. Generation of cAMP in cells expressing only mutated hFSHR-T566 showed minimal increase after FSH stimulation. Co-transfection of Ad-hFSHR in these cells carrying the malfunction form of human FSHR caused significant increases of 2.2-7.4-fold in FSH dependent cAMP generation (P = 0.0007). We concluded that adenovirus expressing a normal human FSHR can compensate the inactivating human FSHR-C566T mutation and restore FSH responsiveness.

Expression of decay accelerating factor in endometrial adenocarcinoma is inversely related to the stage of tumor.

PROBLEM: Decay accelerating factor (DAF) is implicated in protection of cell membrane from toxicity of complement. In this study, we investigated a hypothesis that DAF is up-regulated in the endometrial adenocarcinoma, which could increase potential of malignant cells to escape destruction by complement. METHODS: DAF density was evaluated in endometrial biopsies of patients with adenocarcinoma at various stages and compared with ten endometrial biopsies from non-malignant patients at the proliferative phase. RESULTS: DAF expression in normal proliferative endometrium varied between 1 and 30%. While DAF density in patients with stage I cancer was in the range 56-98% (mean 78%), stage III values varied from 28 to 16% (mean 21%), P < 0.05. DAF density in the well-differentiated Ishikawa cell line was two-fold higher than in metastatic cell line AN3CA. CONCLUSIONS: Our findings are consistent with a hypothesis that endometrial adenocarcinoma of early stage that is exposed to complement attack may up-regulate DAF to protect malignant cells from complement lysis.

Effects of an isoflavone-free soy diet on ovarian hormones in premenopausal women.

Soy intakes have been associated with reduced rates of breast cancer in some Asian populations. The isoflavones daidzein and genistein and other components of soybeans may modulate endocrine function and lead to beneficial health effects. This study determined the effects of a soy diet containing minimum amounts of isoflavones on circulating levels of ovarian hormones and gonadotropins. Nine healthy, regularly cycling women consumed a constant soya-containing diet on a metabolic unit starting on day 2 of a menstrual cycle until day 2 of the next cycle. The soy diet was calculated to maintain constant body weight and included a 36-oz portion of soymilk that provided 334 kilocalories and less than 5 mg/day of total isoflavones. The energy distribution of the soy diet was 35.9% fat, 14.0% protein, and 49.8% carbohydrate whereas the home diets averaged 39% fat, 16.6% protein, and 42.5% carbohydrate. For the group, the soya diet provided more carbohydrate (P = 0.002) and less protein (P = 0.005) than the home diets. Daily consumption of the soya diet reduced daily circulating levels of 17beta-estradiol over the entire menstrual cycle by 20% (P < 0.01, paired t test, two-tailed) and progesterone by 33% (P < 0.0001) compared with levels during the home diet period, but had no effect on LH, FSH, or sex hormone-binding globulin. The decreases in follicular phase 17beta-estradiol during the soy diet can be accounted for by changes in energy intakes, nutrient density, and fiber intake, whereas changes in luteal phase 17beta-estradiol were most strongly associated with differences in fiber intake. Changes in progesterone levels were most strongly associated with changes in protein intake and much less with other nutrients. Isoflavones were not detectable in plasma and urine during either the soy or home diet periods. These results suggest that at least under the conditions of this study, a soy diet with low levels of isoflavones and low energy intake from protein can reduce circulating ovarian steroids without altering gonadotropins. Our results are consistent with previous studies showing decreased ovarian hormone levels and decreased risk of breast cancer in populations consuming soya diets and an inverse relationship between animal protein intake and breast cancer risk and, therefore, may have implications for breast cancer prevention.

Altered GLUT1 and GLUT3 gene expression and subcellular redistribution of GLUT4: protein in muscle from patients with acanthosis nigricans and severe insulin resistance.

Multiple isoforms of glucose transporters are found in muscle, the tissue that normally accounts for 85% of insulin-stimulated glucose uptake. Glucose uptake into muscle cells in the fasting state is mediated primarily by GLUT1 and GLUT3 glucose transporters, whereas postprandial (insulin-stimulated) and exercise-related increments in muscle glucose uptake are mediated primarily by GLUT4. To determine if glucose transporters are abnormally expressed in muscle from insulin-resistant subjects, muscle samples were obtained from 10 normal subjects and 6 obese, nondiabetic subjects with severe insulin resistance and acanthosis nigricans. Both GLUT4 total protein and mRNA were normal in the insulin-resistant subjects. Muscle GLUT3 protein and mRNA were lower than controls by 62% and 71%, respectively. GLUT1 mRNA was twice normal, whereas GLUT1 protein content was not significantly increased. GLUT4 protein was markedly redistributed to the muscle plasma membrane in subjects with severe insulin resistance compared with normals (92% v 40% GLUT4 in plasma membrane-enriched fractions, P <.001), whereas the percentage of GLUT1 and GLUT3 protein found in the plasma membrane-enriched fractions was not different from controls. These data document differences in the expression of genes for GLUT1 and GLUT3 in muscle from normal and insulin-resistant subjects. Further, insulin resistance with fasting hyperinsulinemia was associated with a redistribution of GLUT4 to the muscle cell surface with no change in total GLUT4 protein. These data suggest that glucose transporter gene expression and their basal distribution in human muscle are related to insulin resistance and could be determinants of whole body insulin responsiveness.

Troglitazone is a competitive inhibitor of 3beta-hydroxysteroid dehydrogenase enzyme in the ovary.

OBJECTIVE: Troglitazone is a potent inhibitor of progesterone release from porcine granulosa cells. This is associated with a marked increase in pregnenolone secretion, implicating inhibition of the 3beta-hydroxysteroid dehydrogenase enzyme. This study determined whether troglitazone is a direct inhibitor of 3beta-hydroxysteroid dehydrogenase activity. STUDY DESIGN: Homogenates of porcine granulosa cells underwent classic enzyme kinetic analysis through Lineweaver-Burke and Dixon plotting. Human ovarian homogenates were also assayed for the effects of troglitazone on 3beta-hydroxysteroid dehydrogenase enzyme activity. Enzyme kinetics data were analyzed by the HyperKinetics software program. Analysis of variance was used to determine statistical significance for human ovarian homogenate experiments. RESULTS: In porcine granulosa cells Lineweaver-Burke analysis found that troglitazone inhibition of 3beta-hydroxysteroid dehydrogenase enzyme activity was competitive in nature, with 5 microg/mL troglitazone increasing the apparent Michaelis constant from 1.3 to 4.3 micromol/L (no change in maximum velocity). Dixon plot analysis demonstrated that the inhibition constant for troglitazone of 3beta-hydroxysteroid dehydrogenase is approximately 6.5 microg/mL, which is in the same order of magnitude as its therapeutic concentration in blood. Troglitazone also significantly decreased the activity of 3beta-hydroxysteroid dehydrogenase in homogenates of human ovarian tissue. CONCLUSION: We conclude that troglitazone can inhibit steroidogenesis in the ovary by direct competitive inhibition of 3beta-hydroxysteroid dehydrogenase.

Decreased ovarian hormones during a soya diet: implications for breast cancer prevention.

Ovarian hormones are biomarkers for breast cancer risk. Soybean consumption may be responsible in part for lower levels of ovarian hormones and decreased rates of breast cancer in women in Asia compared with Western populations. Soybeans contain a significant amount of the isoflavones daidzein and genistein, which are weak estrogens. The purpose of this study was to determine whether soya feeding decreases circulating levels of ovarian hormones and gonadotropins. Ten healthy, regularly cycling women consumed a constant soya-containing diet on a metabolic unit, starting on day 2 of a menstrual cycle until day 2 of the next cycle. Blood and urine samples were obtained daily for one menstrual cycle before and during soy feeding. The diet was calculated to maintain constant body weight, included 400 kilocalories from a 36-ounce portion of soymilk, and provided 113-207 mg/day (154.0+/-8.4 mg/day, mean +/- SE) of total isoflavones. For the group, the soya diet provided more carbohydrate and less protein than the home diets. Daily consumption of the soya diet reduced circulating levels of 17beta-estradiol by 25% (P<0.01, Wilcoxon signed rank test, two-tailed) and of progesterone by 45% (P<0.0001) compared with levels during the home diet period but had no effect on luteinizing hormone or follicle-stimulating hormone. Mean menstrual cycle length did not change during the soya diet; a slight decrease in mean luteal cycle length was marginally statistically significant (P = 0.06). Urinary excretion of isoflavones was 33.8+/-5.3 mg/day (mean +/- SE) and when expressed as percentage of intake, varied substantially (21.9+/-3.3% of intake; range, 9.1-36.7%) among the subjects. Mean daily serum levels of daidzein and genistein (free and conjugated forms) 15 h after soymilk were 2.89+/-0.53 microg/ml and 0.85+/-0.22 microg/ml, respectively, indicating systemic bioavailability of these substances. Secondary analyses by multiple regression showed that decreases in follicular and luteal phase 17beta-estradiol levels were positively associated with urinary isoflavone excretion, an association affected by age, and were inversely associated with decreases in protein intake. Decreases in progesterone levels during the soya diet were inversely associated with increases in intakes of genistein and were affected by the interaction of the intakes of daidzein with energy or with fiber. Consumption of an isoflavone-containing soya diet reduced levels of ovarian steroids in normal women over the entire menstrual cycle without affecting gonadotropins. This suggests that at least under the conditions of this study, soya-induced reductions of circulating ovarian steroids are not mediated by gonadotropins. Decreases in ovarian hormones are related to isoflavones contained in soy and also to energy intake and other components such as protein and fiber but not fat. Our results may explain decreased ovarian hormone levels and decreased risk of breast cancer in populations consuming soya diets and have implications for reducing breast cancer risk by dietary intervention.

Increased urinary excretion of 2-hydroxyestrone but not 16alpha-hydroxyestrone in premenopausal women during a soya diet containing isoflavones.

Asian diets high in soy are associated with lower risk for breast cancer compared with Western diets. Moreover, higher levels of two putative carcinogenic metabolites of 17beta-estradiol, 4- and 16alpha-hydroxyestrogen, and lower amounts of anticarcinogenic metabolites, 2-hydroxyestrogens, have been associated with greater breast cancer risk. In this study, we tested the hypothesis that consumption of a soya diet containing the weakly estrogenic isoflavones genistein and daidzein may alter the metabolism of 17beta-estradiol to 2- and 16alpha-hydroxylated products. Eight pre-menopausal women were placed on a soya-containing, constant diet in a metabolic unit. The diet provided 400 kilocalories from soymilk and 113-202 mg/day (158 +/- 26 mg/day, mean +/- SD) isoflavones daily for a complete menstrual cycle. After a washout period of 4 months, the subjects consumed the same diet, but with soymilk that contained <4.5 mg/day isoflavones ("isoflavone-free"). Urine samples were collected for 24 h daily for the entire cycle during each soya diet period for the analysis of daidzein, genistein, and 2- and 16alpha-hydroxyestrone. Subjects excreted measurable amounts of daidzein (11.6-39.2 mg/day) and genistein (2.9-18.2 mg/day) during the isoflavone-rich soya diet but not during the isoflavone-free soya diet. The diet rich in isoflavones increased the cycle mean daily urinary excretion of 2-hydroxyestrone (averaged over the entire cycle) from 11.6 +/- 2.06 to 17.0 +/- 2.96 nmol/12-h (P = 0.03), a 47% increase. However, the mean daily excretion of 16alpha-hydroxyestrone did not change (7.0 +/- 1.14 nmol/12-h during the isoflavone-free and 7.7 +/- 1.25 nmol/12-h during the isoflavone-rich diet; P = 0.36). The ratio of 2-hydroxyestrone to 16alpha-hydroxyestrone was higher during the isoflavone-rich soya diet (2.6 +/- 0.34) than during the isoflavone-free diet (2.0 +/- 0.32; P = 0.01), a 27% increase. These results suggest that soya isoflavones increase the metabolism of endogenous estrogens to the protective 2-hydroxylated estrogens in women, and this may play an important role in lowering 17beta-estradiol levels and the long-term risk for breast cancer.

Increased bioactive luteinizing hormone levels and bio/immuno ratio in women with hyperthecosis of the ovaries: possible role of hyperinsulinemia.

Unlike women with polycystic ovarian disease, women with hyperthecosis have normal or low immunoactive LH levels. They have severe insulin resistance with marked hyperinsulinemia. Bioactive LH levels have not been studied in these women. The purpose of this study was to investigate 1) whether there is an increase in bioactive LH levels in women with hyperthecosis of the ovaries and 2) whether hyperinsulinemia has an effect on LH secretion. Six women with hyperthecosis of the ovaries confirmed by histological examination were included in the study. Six normal women in the midproliferative phase of the cycle served as controls. All women were admitted to the Clinical Research Center at 0800 h, and blood samples were obtained every 15 min for 6 h. All samples were assayed for LH by RIA and bioassay. The PC Pulsar Program was used for pulse analysis of LH secretion. Patients with hyperthecosis had significantly higher (P < 0.002) bioactive LH levels (66.9 +/- 13 mIU/mL) than controls (29.3 +/- 6 mIU/mL). Immunoactive LH levels in hyperthecosis were not significantly different from those in control women. Significantly higher bio/immuno LH ratios (P < 0.001) were observed in women with hyperthecosis (6.2 +/- 0.9) than in normal control women (2.4 +/- 0.5). There was a significant positive correlation between insulin levels and the bio/immuno ratio of LH. Pulse amplitude and interpulse intervals for immunoactive LH in hyperthecosis patients were similar to those in control women. The pulse amplitude of bioactive LH was significantly higher (P < 0.01) in women with hyperthecosis compared to that in normal controls. Hyperinsulinemia induced during LH sampling resulted in increased bioactive LH levels with no change in immunoactive LH. These results indicate that 1) women with hyperthecosis of the ovaries have increased secretion of biologically active LH, and 2) hyperinsulinemia may enhance the secretion of the biologically active form of LH.

Increased expression of messenger ribonucleic acid encoding cytochrome P450 cholesterol side-chain cleavage and P450 17alpha-hydroxylase enzymes in ovarian hyperthecosis.

OBJECTIVE: To investigate whether the increased ovarian androgen synthesis in hyperthecosis is due to increased expression of the steroidogenic enzymes essential for androgen synthesis. DESIGN: Controlled study to investigate concentration of steroidogenic enzymes in the ovarian stroma of women with hyperthecosis of the ovaries. SETTING: Academic research environment. PATIENT(S): Three women with ovarian hyperthecosis and eight with normal ovulatory cycles. INTERVENTION(S): Ovarian stromal tissues were obtained from women with hyperthecosis and women with normal ovaries. Diagnosis of hyperthecosis was confirmed by histologic examination of the ovaries. Steroid levels were measured in the ovarian vein serum of one patient with hyperthecosis. MAIN OUTCOME MEASURE(S): Tissues were frozen immediately in liquid nitrogen and kept frozen until RNA was extracted. Total RNA was examined by Northern blot analysis using 32P-labeled complementary DNA (cDNA) probes encoding human P450scc and P450(17alpha) enzymes. RESULT(S): P450scc and P450(17alpha) messenger RNAs (mRNAs) were detected in the normal ovarian stroma and stromal hyperthecosis. Compared with normal ovarian stroma, P450scc mRNA was increased twofold and P450(17alpha) mRNA was increased threefold in stromal hyperthecosis. CONCLUSION(S): [1] Ovarian stroma is probably the site of androgen production in ovarian hyperthecosis. [2] Increased stromal androgen synthesis in hyperthecosis could be due to increased expression of the enzymes P450scc and P450(17alpha) in the ovarian stroma. [3] Markedly increased concentrations of 17alpha-hydroxyprogesterone in the ovarian vein serum indicate possible dysregulation of P450(17alpha) in ovarian hyperthecosis.

Troglitazone inhibits progesterone production in porcine granulosa cells.

Troglitazone (a thiazolidinedione that improves insulin resistance) lowers elevated androgen concentrations in women with polycystic ovarian syndrome. In this study, we assessed the direct effects of troglitazone on steroidogenesis in porcine granulosa cells. Troglitazone inhibited progesterone production in a dose- and time-dependent manner (earliest effects at 4 h, maximum at 24 h) without affecting cell viability. Progesterone production was also inhibited by troglitazone in the presence of 25-hydroxycholesterol, indicating that the drug does not affect intracellular cholesterol transport. Troglitazone also inhibited FSH- and forskolin-stimulated progesterone secretion. The reduced progesterone production was accompanied by marked elevations of pregnenolone concentrations, suggesting inhibition of 3beta-hydroxysteroid dehydrogenase (3beta-HSD). The activity of 3beta-HSD in troglitazone-treated granulosa cells was decreased by more than 60%, compared with controls after 24 h. Troglitazone did not affect aromatase activity in porcine granulosa cells. In summary, troglitazone has direct effects on porcine granulosa cell steroidogenesis. The drug specifically inhibits 3beta-HSD activity, resulting in impaired progesterone production. The clinical relevance of this direct in vitro effect on steroidogenesis needs further investigation.

Specific binding and growth-promoting activity of insulin in endometrial cancer cells in culture.

OBJECTIVE: Insulin is known to be mitogenic to a variety of cells in culture. The purpose of this study was to investigate the possible role of insulin in the growth and development of endometrial cancers. STUDY DESIGN: Specific binding and growth effects of insulin were studied in 5 different human endometrial cancer cell lines derived from cancers with different degrees of differentiation: HEC-1-A and HEC-1-B (from a moderately well-differentiated adenocarcinoma), RL95-2 (from a moderately well-differentiated adenosquamous carcinoma), KLE (from poorly differentiated carcinoma), and AN3 CA (from a metastatic undifferentiated endometrial carcinoma). The receptors were further characterized by competitive binding and chemical cross-linking studies. RESULTS: Binding studies with 125I-insulin revealed the presence of high-affinity binding sites for insulin on all the 5 cell lines. Binding of insulin was found to be highly specific. Competitive binding studies with 125I-insulin revealed that insulin was most effective in displacing the labeled hormone, whereas insulin-like growth factor-I and insulin-like growth factor-II competed for binding only at very high concentrations. Scatchard analysis of the binding data revealed that the association constant for the high-affinity binding sites ranged from 0.72 to 1.91 x 10(9) L/mol. Estrogen-receptor-negative cell lines HEC-1-A and HEC-1-B had the highest number of insulin receptors, whereas the estrogen-receptor-positive cell line RL95-2 had the least number of receptors. The effect of insulin on cell proliferation was studied by monitoring cell number and incorporating [3H]thymidine into deoxyribonucleic acid of the cells. Insulin stimulated cell growth of all the cell lines. CONCLUSIONS: The results of this study indicate the potential role of hyperinsulinemia in the growth and development of endometrial cancer.

Nitric oxide inhibits development of embryos and implantation in mice.

The objectives of this study were to evaluate the effects of a nitric oxide (NO) donor on embryo development in vitro and on implantation of embryos in vivo in mice. Mouse embryos (2-cell) were incubated in media containing different concentrations of diethylenetriamine/NO (DETA/NO), a nitric oxide donor, and development was monitored daily for 4 days. Specificity of NO effects was assessed by using DETA without NO or 48 h preincubated DETA/NO. In in-vivo studies, mated mice were continuously infused, subcutaneously, with various concentrations of DETA/NO or DETA through mini-osmotic pumps (from day 1 of pregnancy), and implantations in the uterus were assessed on day 6. None of the embryos progressed beyond 4-cell stage when exposed to 0.1 or 1.0 mM DETA/NO compared with 94.5% of control embryos that developed beyond the morula stage by day 4. Embryo development was unaffected by lower (0.001 and 0.01 mM) concentrations of DETA/NO, 48 h preincubated DETA/NO, or DETA only. Infusion of DETA/NO to mice caused inhibition of embryo implantation in a dose-dependent manner. No implantation sites were observed in mice infused with a daily dose of 20 micromol DETA/NO rate, compared with an implantation rate of 81.8% in control or DETA-treated mice. This study demonstrates for the first time that higher concentrations of NO inhibit both embryo development in vitro and implantation in vivo in mice.

Acanthosis nigricans as a risk factor for non-insulin dependent diabetes mellitus.

The prevalences of obesity and of non-insulin dependent diabetes mellitus (NIDDM) have increased in the United States population over the past two decades, and thus diabetes prevention has become a major concern of public health agencies such as the National Institutes of Health. Identification of individuals at risk for diabetes is an essential first step in designing and implementing intervention programs. Insulin resistance is the hallmark of the pathophysiology of NIDDM. Subjects with hyperinsulinemia, impaired glucose tolerance, or gestational diabetes are well accepted as being at high risk for diabetes. We propose that the easily identifiable skin lesion, acanthosis nigricans, is common in the major minority groups in the United States and that its presence is a surrogate for laboratory-determined hyperinsulinemia.

Hyperinsulinemia and acanthosis nigricans in African Americans.

Compared with the US white, non-Hispanic population, the African-American population has a nearly two-fold higher prevalence of noninsulin-dependent diabetes mellitus (NIDDM). Obesity, which usually precedes NIDDM, is associated with the skin lesion acanthosis nigricans in African Americans. This study was undertaken to determine what the relationship of acanthosis nigricans was to hyperinsulinemia, a major risk factor for NIDDM. Eighty-nine African-American subjects with acanthosis nigricans and 25 others without the skin lesion were evaluated using oral glucose tolerance testing and responsiveness to insulin. Noninsulin-dependent diabetes mellitus was present in 19 of the subjects with acanthosis nigricans. The prevalence of NIDDM in this group increased with increasing age, reaching 50% among those in their 40s. Fasting plasma insulin concentration was in direct proportion to the severity of the acanthosis nigricans involvement of the neck. These data suggest that among African Americans, this skin lesion is a marker for hyperinsulinemia and insulin resistance. Furthermore, the presence of acanthosis nigricans identifies a subset with a much higher prevalence of NIDDM than is present in African Americans in the general population.

Tumor necrosis factor alpha inhibits transcriptional activity of the porcine P-45011A insulin-like growth factor response element.

We investigated the effects of tumor necrosis factor alpha (TNFalpha) on the transcriptional activity of the porcine P-45011A (P450scc) insulin-like growth factor response element (IGFRE). TNFalpha inhibited insulin-like growth factor-I (IGF-I)-stimulated P450scc mRNA concentrations in cultures of porcine granulosa cells. Transient transfection experiments in granulosa cells with deletion P450scc/luciferase constructs showed that TNFalpha inhibited the transcriptional activity of the IGFRE. IGF-I binding and IGF-I receptor mRNA concentrations in porcine granulosa cells were not inhibited by TNFalpha. Electrophoretic mobility shift assay with nuclear extract protein from porcine granulosa cells treated with IGF-I and TNFalpha showed that Sp1 and a second transcription factor, P2, bound to the IGFRE. While IGF-I treatment increased the binding activity of both factors, TNFalpha specifically inhibited the IGF-I-stimulated binding activity of P2. Transient transfection studies done in mouse fibroblasts overexpressing the IGF-I receptor (NWTb3) with the porcine IGFRE (three repeats) in an SV40/luciferase construct also showed TNFalpha inhibited IGF-I-stimulated reporter gene expression. We conclude that TNFalpha inhibits the transcriptional activity of the porcine P450scc IGFRE by preventing IGF-I-stimulated binding of P2.

Estrogen regulation of lactoferrin expression in human endometrium.

PROBLEM: Lactoferrin is an iron-binding glycoprotein that has been shown to be overexpressed in human endometrial carcinomas. The purpose of our present study is to investigate the possible role of estradiol in the expression of lactoferrin. METHOD OF STUDY: We investigated 1) serum levels of lactoferrin in five women during normal ovulatory cycles, 2) serum levels of lactoferrin during ten human menopausal gonadotropin induced cycles when estradiol levels are high, and 3) lactoferrin expression in five proliferative and five secretory phase endometrium by immunohistochemical studies. The serum concentrations of lactoferrin were measured by a peroxidase-based enzyme-linked immunosorbent assay. RESULTS: In normal ovulatory cycles, the mean serum lactoferrin concentration during the proliferative phase (0.4013 +/- 0.0242 micrograms/mL) was significantly higher (P < 0.02) than in the secretory phase (0.3468 +/- 0.0209 micrograms/mL). In induced cycles, there was gradual increase in lactoferrin levels with increasing estradiol concentrations. Peak lactoferrin levels in induced cycles (0.7495 +/- 0.1148 micrograms/mL) were significantly higher (P < 0.003) than the midcycle levels (0.423 +/- 0.0424 micrograms/mL) in normal cycles. Immunohistochemical analysis of the endometrium revealed greater expression of lactoferrin in proliferative endometrium (50.7 +/- 13%, range 28-72%) than in secretory endometrium (19.2 +/- 4%, range 7-31%). CONCLUSION: These results indicate that estradiol may play a role in the regulation of lactoferrin expression in human endometrium.

Specific receptors and growth effects of insulin and insulin-like growth factors in a human cell line derived from mixed mesodermal tumor of the uterus.

OBJECTIVE: Mixed mesodermal tumors of the uterus are highly malignant. The purpose of our present study was to investigate possible roles of insulin and insulin-like growth factors I and II (IGF-I and IGF-II) in the growth and development of these tumors. METHODS: Specific binding and growth effects of insulin, IGF-I, and IGF-II were studied in SK-UT-1 cells, derived from a human mixed mesodermal tumor of the uterus. The receptors were further characterized by competitive binding and chemical cross-linking studies. RESULTS: Binding studies with 125I-insulin, 125I-IGF-I, and 125I-IGF-II revealed the presence of specific binding sites for these three growth factors. Specificity studies revealed that the binding sites for IGF-I and IGF-II are distinct. Binding of IGF-II was much higher than insulin and IGF-I binding. Scatchard analysis of the binding data revealed that the cell line has a higher number of IGF-II receptor (100,000 sites per cell) than insulin (1400 sites per cell) and IGF-1 (1200 sites per cell) receptors. The effect of these growth factors on cell growth was studied by monitoring the cell number and incorporation of [3H] thymidine into the DNA of the cells. Insulin-like growth factor-II was potent in stimulating the growth of these cells, but IGF-I did not have any effect. Insulin stimulated DNA synthesis only at pharmacologic concentrations. CONCLUSION: These results indicate that IGF-II is mitogenic to mixed mesodermal tumors of the uterus. Insulin-like growth factor II may be involved in the growth regulation of mixed mesodermal tumors of the uterus.