Detection of mitochondrial DNA deletion in the late-term placenta.
Explore the source record for details and available documents.
DNA Primers↗
Biomedical subjects
F Saji
Publications and source records attributed to F Saji.
Elevated nitric oxide concentration in the seminal plasma of infertile males: nitric oxide inhibits sperm motility.
PROBLEM: To evaluate the effect of nitric oxide in the seminal plasma on sperm motility. METHOD: Seminal plasma concentrations of NO2-, a stable end product of nitric oxide, of 108 males of infertile couples and 15 proven fertile donors were measured and compared with spermatogram parameters. Motile sperm was incubated with a nitric oxide-generating drug, sodium nitroprusside, for 6 hr in the absence or presence of oxyhemoglobin, an inhibitor of nitric oxide. RESULTS: The NO2- concentration in the seminal plasma was 6.58 +/- 0.5.6 microM in 26 infertile males with leukocytospermia, 5.51 +/- 0.25 microM in 82 infertile males without leukocytospermia, and 3.91 +/- 0.16 microM in 15 controls. There was a significant correlation between the NO2- concentration and sperm motility. Sodium nitroprusside reduced the sperm motility in a dose- and time-dependent manner and its reduction was completely inhibited by the addition of oxyhemoglobin. CONCLUSION: These findings indicate that nitric oxide concentration in the seminal plasma of infertile males is elevated and that nitric oxide is an inhibitor of sperm motion.
Cell Survival↗
Maintenance of imprinting of the insulin-like growth factor II gene (IGF2) and the small nuclear ribonucleoprotein polypeptide N gene (SNRPN) in the human uterus and leiomyoma.
The insulin-like growth factor II gene (IGF2) is thought to be involved in the growth of uterine smooth muscle tumors. We studied the allele-specific expression of IGF2 in 20 patients with uterine leiomyomas by analyzing restriction fragment length polymorphisms (RFLP), because IGF2 is a maternally imprinted gene and only the paternal allele is exclusively expressed in human somatic tissue. We also studied the allelic expression of the small nuclear ribonucleoprotein polypeptide N gene (SNRPN), which is reportedly maternally imprinted in humans, and compared the imprinting status with that of IGF2. Nine patients (45%) were heterozygous at the ApaI site of IGF2, nine (45%) were heterozygous at the possible AccII polymorphic site of SNRPN, and three (15%) showed polymorphism in both genes. The genomic DNA of 15 patients showed heterozygosity in either or both of these genes, and the mRNA of these was expressed monoallelically in myometrial tissues and leiomyomas of these patients. These results demonstrated that IGF2 and SNRPN imprinting is completely maintained in human uteri and leiomyomas and that increased expression of IGF2 is not due to biallelic expression.
Autoantigens↗
Plasma nitric oxide levels in pregnant patients with preeclampsia and essential hypertension.
Nitric oxide (NO) production may be an important causal factor in hypertensive disorders during pregnancy. The plasma concentrations of NO2-(+) NO3-, stable metabolites of NO, were measured in 70 nonpregnant women, 323 normotensive pregnant women, 23 pregnant patients with preeclampsia, and 7 pregnant patients with essential hypertension. The normotensive women had higher plasma concentrations (30.0 +/- 0.6 mumol/l) than nonpregnant women (18.3 +/- 1.0 mumol/l; p < 0.0001). The plasma concentrations in the patients with preeclampsia (45.6 +/- 2.3 mumol/l) were higher than in the normotensive women (30.3 +/- 1.0 mumol/l; p < 0.0001) and were correlated with the systolic blood pressure (r = 0.442; p < 0.05). However, pregnant patients with underlying essential hypertension had significantly lower plasma concentrations (19.1 +/- 3.0 mumol/l; p < 0.005). These findings suggest that NO contributes to maternal vasodilation, the maintenance of uterine quiescence, and the pathogenesis and clinical features of hypertensive disorders during pregnancy.
Adult↗
Investigation of the oxytocin receptor expression in human breast cancer tissue using newly established monoclonal antibodies.
The expression of the oxytocin (OT) receptor (OTR) in breast cancer was studied using newly established anti-OTR monoclonal antibodies. Immunoblotting indicated that the antibody 2F8 recognized a 70K OTR in the pregnant myometrium and breast cancer tissue. Among 57 breast cancer patients, we detected OTR immunoreactivity in 52 (91.2%) by immunohistochemistry using 2F8. Using another monoclonal antibody for different receptor domains, 1-2, the staining profile was identical in all positive samples. Of 52 OTR-positive samples, 28 were diffusely positive (> 80% of cancer cells were stained), and 24 were partially positive (< 80% cells were stained). The ratio of estrogen receptor-positive samples was slightly higher among those that were diffusely positive, but there was no apparent relationship between OTR expression and other clinical parameters. We also confirmed the expression of the OTR in positively stained samples by means of Northern blotting and RT-PCR at the transcription level. The OTR messenger RNA and RT-PCR product were the same size as those in the pregnant myometrium. We also determined the expression of the OTR using flow cytometry in four breast cancer cell lines (MCF-7, MDA-MB-231, MDA-MB-361, and MDA-MB-468). However, OT had no significant effect on their growth during a short period (7 days) of culture. These findings indicated that the OTR is expressed in breast cancer derived not from the myoepithelium but from the glandular or ductal epithelium; however, the biological function of OT in breast cancer remains to be determined.
Adult↗
Expression of oxytocin receptor in human pregnant myometrium.
Around the onset of labor, uterine sensitivity to oxytocin (OT) increases tremendously. Although this is considered to reflect OT receptor (OTR) augmentation in myometrium, neither spatial expression of OTR nor the level of the receptor message during the course of pregnancy have been investigated at the molecular level. We examined the localization and expression of the OTR in human myometrium by means of in situ hybridization, immunohistochemistry, and Northern and Western blotting. In the term pregnant myometrium, OTR expressing smooth muscle cells are observed diffusely and heterogeneously. Some of the smooth muscle cells were expressed high levels of the receptor at the messenger RNA and protein level, and they were surrounded with cells weakly positive for the OTR or negative. The level of OTR transcripts increased according to the course of pregnancy. The receptor messenger RNA level reached over 300-fold at parturition compared with the nonpregnant myometrium. In the myometrium at 32 weeks of gestation and not in labor, a relatively large amount (about 100-fold) of the receptor message was expressed. In the nonpregnant myometrium, significant amount of the receptor protein was revealed by Western blotting. We also found that the receptor protein was augmented at term and after the onset of labor. These findings indicated that the expression of OTR changes dynamically at the transcription and protein level during pregnancy and that its expression is heterogeneous in the term myometrium.
Amino Acid Sequence↗
The novel role of 3',5'-guanosine monophosphate (cGMP) on the differentiation of trophoblasts: comparison with the effects of 3',5'-adenosine monophosphate (cAMP).
We investigated the effects of 3',5'-guanosine monophosphate (cGMP) on the differentiation of human trophoblasts. Isolated cytotrophoblasts were cultured with 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP) or 8-bromoadenosine 3'5-cyclic monophosphate (8-Br-cAMP) and then stained immunocytochemically with anti-human chorionic gonadotropin (anti-hCG) antibody to identify hCG expression as an index of differentiation. Concurrently, morphological changes from cytotrophoblasts to syncytiotrophoblasts were analyzed. Both 8-Br-cGMP and 8-Br-cAMP enhanced the expression of hCG in cultured cytotrophoblasts with the differentiation of cytotrophoblasts to syncytiotrophoblasts dose-dependently. With regard to trophoblast proliferation, 8-Br-cAMP but not 8-Br-cGMP enhanced [3H]thymidine uptake by these cells. hCG, a trophoblast-specific glycoprotein hormone has been identified as a potent growth factor for trophoblasts, also increased [3H]thymidine uptake and the intracellular 3',5'-adenosine monophosphate (cAMP) concentration. However, in this study, hCG did not increase the concentration of intracellular cGMP. We also showed that sodium nitroprusside (SNP), which is a donor of nitric oxide (NO), enhanced intracellular cGMP concentration. These results suggest that cGMP enhances trophoblast differentiation without affecting their proliferation, while cAMP enhances both differentiation and proliferation. We conclude that an alternative pathway mediated through cGMP is responsible for the differentiation of trophoblasts. NO may be involved in trophoblast differentiation with an increase in cellular cGMP level.
8-Bromo Cyclic Adenosine Monophosphate↗
Leukemia inhibitory factor (LIF) enhances trophoblast differentiation mediated by human chorionic gonadotropin (hCG).
We investigated the effect of LIF on the differentiation of trophoblasts. Isolated cytotrophoblasts were cultured with and without LIF and cell smears were immunocytochemically analyzed, using anti-hCG antibody. The percentage of differentiated trophoblasts stimulated by 10ng/ml of LIF was about 2.5-fold that in the control culture. The effect of LIF in inducing the differentiation of cytotrophoblasts to syncytiotrophoblasts was dose-dependent. The same effect was shown when hCG was added to the medium. This enhancing effect of LIF on trophoblast differentiation was blocked by adding anti-hCG antibody to the culture system. These results indicate that LIF enhanced trophoblast differentiation by stimulating hCG production in trophoblasts, and not by exerting a direct effect on the trophoblasts.
Abortion, Legal↗
Apoptosis of cultured mouse luteal cells induced by tumor necrosis factor-alpha and interferon-gamma.
BACKGROUND: Macrophages and T lymphocytes have been identified in the regressing corpus luteum, and they are thought to participate in structural luteolysis (destruction and removal of luteal cells). Since these cells produce cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), we investigated the effects of these two cytokines on death of luteal cells in vitro. METHODS: Mouse luteal cells were cultured in serum-free medium with TNF-alpha at 0, 500, 1,000, 3,000, or 5,000 U/ml in the presence or absence of IFN-gamma at 1,000 U/ml for 3 or 6 days. Then, for estimation of the actions of these cytokines on induction of luteal cell death, we determined the number of viable cells, the percentage of fragmented DNA in total DNA extracted from cultured cells, and the percentage of cells with fragmented DNA in their nuclei by the trypan blue exclusion test, the sensitive micromethod for DNA assay, and the in situ DNA 3' end labeling method, respectively. DNA fragmentation was also analysed by agarose gel electrophoresis, and cultured cells were examined by electron microscopy. RESULTS: On day 3 of culture, IFN-gamma alone at 1,000 U/ml or TNF-alpha alone at 500-5,000 U/ml did not decrease the number of viable cells, but a combination of IFN-gamma (1,000 U/ml) and TNF-alpha (5,000 U/ml) did. On day 6, IFN-gamma alone at 1,000 U/ml or TNF-alpha alone at 500, 1,000 and 3,000 U/ml did not decrease the number of viable cells, whereas TNF-alpha alone at 5,000 U/ml did, and combinations of IFN-gamma and TNF-alpha at 1,000, 3,000, and 5,000 U/ml decreased the number of viable cells in proportion to the concentration of TNF-alpha. On days 3-6 of culture, combinations of IFN-gamma and TNF-alpha that decreased the number of viable cells also increased the percentages of fragmented DNA in total DNA of cultured luteal cells and the percentages of luteal cells with fragmented DNA in their nuclei. Agarose gel electrophoresis of fragmented DNA showed a ladder-like pattern, and electron microscopic examination showed luteal cells with the characteristics of apoptosis. CONCLUSIONS: The presence of IFN-gamma modulates the ability of TNF-alpha to induce a reduction in the number of viable cells, although TNF-alpha alone at high concentrations can induce a reduction in the number of viable cells.
Animals↗
Interleukin-1 receptor antagonist expression in epithelial cells of human endometrium.
OBJECTIVE: To examine the expression of interleukin-1 (IL-1) and IL-1 receptor antagonist (IL-1ra) in the human endometrium in the follicular and luteal phases. METHODS: The concentrations of IL-1alpha and IL-1beta in the culture supernatants of endometrial cells were determined by enzyme-linked immunosorbent assay. Transcription of the IL-1ra gene in the endometrium was investigated by reverse polymerase chain reaction (PCR). Human endometrium was immunohistochemically stained using a monoclonal antibody specific to IL-1ra. RESULTS: The concentrations of IL-1alpha and IL-1beta in the culture supernatants were 11 and 55 pg/ml, respectively, in the follicular phase, and 10 and 40 pg/ml, respectively, in the luteal phase. The concentration of IL-1ra was 465 pg/ml in the follicular phase and 1710 pg/ml in the luteal phase. Densitometric analysis of the reverse PCR products showed that the expression of IL-1ra mRNA was increased in endometrial cells in the luteal phase. Immunohistochemical staining revealed that epithelial cells were the main source of IL-1ra in human endometrium. CONCLUSIONS: Human endometrial cells produce IL-1 (mainly IL-1beta) and IL-1ra. The level of IL-1ra production in human endometrial epithelial cells was greater in the luteal phase than in the follicular phase due to the increased transcription of the IL-1ra gene.
Endometrium↗
Cytotoxic actions of cytokines on cultured mouse luteal cells are independent of nitric oxide.
We investigated the cytotoxic effects of various cytokines secreted by macrophages or T lymphocytes on luteal cells, and the role of nitric oxide (NO) produced by luteal cells in cytotoxic actions of cytokines. Mouse luteal cells were cultured in serum-free medium with interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) or interleukin-1 beta (IL-1 beta) alone, or with various combinations of these cytokines for 6 days. Cytotoxic actions of cytokines and NO production by luteal cells were evaluated by number of viable cells and the amount of nitrite and nitrate (stable metabolites of NO) in medium, respectively. IFN-gamma (1000 U/ml), TNF-alpha (3000 U/ml), or IL-1 beta (30 U/ml) alone, and the combination of TFN-alpha and IL-1 beta (10 U/ml) did not decrease number of viable cells and was without effects on NO production. The combination of IFN-gamma and IL-1 beta (10 U/ml) also did not decrease the number of viable cells, while it increased NO production a little but significantly. Combinations of INF-gamma and TNF-alpha, and IFN-gamma, TNF-alpha and IL-1 beta (10 U/ml) markedly decreased number of viable cells. The combination of IFN-gamma and TNF-alpha increased NO production a little but significantly, and the combination of three cytokines (IFN-gamma, TNF-alpha, and IL-1 beta) caused a greater increase in NO production. An NO synthase inhibitor, L-NG-monomethy-L-arginine (0.5 mM) or aminoguanidine (0.5 mM) abolished increases in NO production induced by combinations of IFN-gamma and TNF-alpha, and IFN-gamma, TNF-alpha and IL-1 beta completely without effects on number of viable cells. The present results indicate that combinations of cytokines including IFN-gamma and TNF-alpha induce death of cultured mouse luteal cells, and that the cytotoxic actions of these cytokines are independent of NO production by luteal cells.
3-Hydroxysteroid Dehydrogenases↗
Loss of imprinting in choriocarcinoma.
Explore the source record for details and available documents.
Alleles↗
Acrosome reaction induced in a limited population of human spermatozoa by progesterone (Ca(2+)-dependent) and ATP (Ca(2+)-independent).
We examined the kinetics of the acrosome reaction induced in human spermatozoa by progesterone (Ca(2+)-dependent) and adenosine 5'-triphosphate (ATP; Ca(2+)-independent). ATP and progesterone did not induce the acrosome reaction unless spermatozoa were incubated in a capacitation medium. ATP exhibited a constant induction of the acrosome reaction regardless of the incubation period, while progesterone began to induce the acrosome reaction after > or = 6 h of incubation. At 24 h of incubation, the percentages of spermatozoa in which the acrosome reaction had been induced by both progesterone and ATP were almost equal. To determine whether a limited population of human spermatozoa was reactive to both progesterone and ATP, we employed an MH61 bead binding method. When the acrosome-reacted spermatozoa were removed with MH61 beads, their percentage in the sperm suspension was decreased to < 3%. At 24 h of incubation, progesterone and ATP induced the acrosome reaction in 12.0 and 10.0% of spermatozoa, respectively. After MH61 bead binding was performed, the remaining spermatozoa did not react to the other activator (progesterone-->ATP, ATP-->progesterone). These findings indicate that only a limited population of human spermatozoa has the potential to undergo the acrosome reaction when stimulated by both progesterone and ATP.
Acrosome↗
Detection of monocyte chemotactic and activating factor (MCAF) and interleukin (IL)-6 in human seminal plasma and effect of leukospermia on these cytokine levels.
PROBLEM: To demonstrate whether monocyte chemotactic and activating factor (MCAF) and interleukin-6 (IL-6) are present in the seminal plasma, and whether these presence is modulated by leukospermia. METHODS: Semen samples from 53 men were obtained by masturbation and examined for the presence of MCAF and IL-6 by enzyme immunoassay (EIA). Semen samples were obtained from 28 infertile men without leukospermia, 16 infertile men with leukospermia, and nine proven-fertile men. The correlation between the amount of MCAF in the seminal plasma with some spermiogram parameters and other cytokines such as IL-6 and IL-8 was statistically evaluated. RESULTS: Immunoreactive MCAF was detected in the seminal plasmas of all 53 subjects. The MCAF titer in the seminal plasma of patients with leukospermia (11.19 +/- 2.75 micrograms/l) was significantly higher than that in the seminal plasma of the patients without leukospermia (3.24 +/- 0.53 micrograms/l) and the fertile men (2.78 +/- 0.35 micrograms/l) (P < 0.001). The IL-6 titer in the seminal plasma of the patients with leukospermia (21.05 +/- 4.49 ng/l) was also significantly higher than that in the seminal plasma of the patients without leukospermia (8.77 +/- 1.92 ng/l) and the fertile men (6.94 +/- 1.27 ng/l) (P < 0.01). There was a high degree of correlation among the levels of MCAF, IL-6 and IL-8 in the seminal plasma. CONCLUSIONS: These findings demonstrated the presence of MCAF and IL-6 in the seminal plasma, and that the levels of these cytokines were elevated in the seminal plasma of the infertile patients with leukospermia.
Chemokine CCL2↗
Regulation of classical HLA class I genes in human choriocarcinoma cells by nuclear proteins binding to MHC class I regulatory elements.
PROBLEM: The regulation of classical HLA class I genes in choriocarcinoma have been reported. METHODS: We determined whether four choriocarcinoma cell lines expressed classical HLA class I or HLA-G by a reverse transcription-polymerase chain reaction (RT-PCR) and studied the regulatory mechanism of classical class I using a gel mobility shift assay. RESULTS: NUC1 and SCH expressed classical class I but not HLA-G. GCH1 and Jar did neither. Nuclear protein binding to the class I regulatory element (CRE) was detected in NUC1 and SCH. Interferon-gamma augmented both classical class I expression and the DNA-protein complex in NUC1. The DNA-protein complex was not observed in GCH1, and Jar showed a CRE-binding protein with different electrophoretic mobility and binding affinity from that of SCH and NUC1. CONCLUSION: The CRE is one of the regulatory elements of classical HLA class I genes in choriocarcinoma cells.
Base Sequence↗
Clonal determination of uterine leiomyomas by analyzing differential inactivation of the X-chromosome-linked phosphoglycerokinase gene.
To investigate the clonality of uterine leiomyomas, we developed a PCR-based method involving the differential inactivation of the X-chromosome-linked phosphoglycerokinase (PGK) gene. Small DNA samples of 22 leiomyomas from 9 Japanese patients, showing heterozygosity at the BstXI site of the PGK gene, were digested with the methylation-sensitive restriction enzyme HpaII. Only the inactive (methylated) PGK gene allele was selectively amplified by PCR followed by digestion with BstXI and electrophoresis. All leiomyoma samples consisted of a single type of inactive allele, even though alleles were detected that were specific to each nodule. The results indicated that all leiomyoma nodules were unicellular in origin but independently generated in the uterus.
Adult↗
Soluble interleukin-6 (IL-6) receptor in the sera of pregnant women forms a complex with IL-6 and augments human chorionic gonadotropin production by normal human trophoblasts through binding to the IL-6 signal transducer.
To study the role of soluble interleukin-6 receptor (sIL-6R) during pregnancy, sIL-6R levels in the sera of pregnant women in the first, second, and third trimesters were determined and found to remain unchanged during pregnancy, but were significantly higher than those in nonpregnant women in the follicular, ovulatory, and luteal phases of the menstrual cycle (P < 0.001). IL-6 levels, however, in the sera of pregnant women at all trimesters showed no difference from those in nonpregnant women at any stage of the menstrual cycle. Recombinant sIL-6R (rsIL-6R) augmented hCG production by rIL-6-stimulated trophoblasts dose dependently, but failed to enhance hCG production by unstimulated trophoblasts. rIL-6- and rsIL-6R-induced hCG production was significantly blocked by anti-IL-6R antibody, PM1; antisignal transducing glycoprotein 130 (gp130) antibody, GPX7; or a tyrosine kinase inhibitor, genistein. Thus, sIL-6R in serum from pregnant women forms a complex with placental and decidual IL-6 in a manner similar to trophoblast membrane-bound IL-6R. These two discrete types of IL-6R and IL-6 complex might act cooperatively by binding to gp130 and subsequently evoking tyrosine kinase activity in the trophoblasts to produce hCG in vivo.
Abortion, Legal↗
Leukemia inhibitory factor produced at the fetomaternal interface stimulates chorionic gonadotropin production: its possible implication during pregnancy, including implantation period.
We investigated the role of leukemia inhibitory factor (LIF) at the implantation site of human embryos. The first trimester decidual tissue produced higher levels of LIF than chorionic tissue, but the decidua produced much smaller amounts of interleukin-6 (IL-6) than the chorion in vitro, as determined by enzyme-linked immunosorbent assay. The reverse transcription-polymerase chain reaction and immunohistochemical analysis revealed the expression and localization, on the trophoblasts, of glycoprotein 130 (gp130), an IL-6 signal transducer receptor component shared by the cytokines such as LIF and IL-6. Trophoblasts stimulated by recombinant LIF (rLIF) produced CG titer at the amount similar to that induced by rIL-6. Recombinant LIF-induced CG production was significantly blocked by anti-gp130 antibody but not by anti-IL-6 receptor antibody, whereas rIL-6-induced CG was completely blocked by both antibodies. Recombinant LIF- and rIL-6-induced CG productions were both significantly blocked by genistein, a tyrosine kinase inhibitor, suggesting an involvement of tyrosine kinase in gp130-mediated CG production. Since CG is capable of stimulating trophoblast growth and differentiation as well as placental metabolism, LIF produced at the fetomaternal interface are considered to stimulate the trophoblasts to produce CG, which may contribute to the maintenance of the placental functions and embryonal growth.
Antibodies↗