Biomedical subjects

F Saji

Publications and source records attributed to F Saji.

At least 37 records · Page 2Linked to original sources

PubMed1998

Expression and immunolocalization of the oxytocin receptor in human lactating and non-lactating mammary glands.

The milk ejection reflex is mediated by the release of pituitary oxytocin and its interaction with specific receptors within the mammary gland. Although up-regulation of the oxytocin receptor during lactation has been shown for the rat mammary gland by ligand binding assay, investigation of the receptor expression in human breast at the molecular level has not yet been carried out in detail. Here we report the expression and immunolocalization of the oxytocin receptor in the human breast. It appears that the expression level of the receptor-specific mRNA is not significantly elevated during lactation and the protein remains at a relatively low level. However, this lack of increase may be only a dilution effect because of the high level of milk protein expression. Immunohistochemistry and immunoelectron microscopy using three anti-oxytocin receptor antibodies raised against different epitopes of the receptor indicated the presence of receptor immunoreactivity only to a very limited extent in the myoepithelial cells; more specific expression appeared to occur in the ductal/glandular epithelium in both the non-lactating as well as lactating breast. This finding was also confirmed in a New World monkey, the common marmoset (Callithrix jacchus). These results suggest that, at least for human and marmoset, in addition to--or even instead of--myoid cells, the ductal/glandular epithelium is also a target for oxytocin action, not only during lactation but also in the non-lactating breast. Thus, there may be other physiological effects of oxytocin besides direct myoid cell contraction in the breast.

Adult↗

PubMed1998

Secretory leukocyte protease inhibitor (SLP) concentrations in seminal plasma: SLPI restores sperm motility reduced by elastase.

In this study, we quantified secretory leukocyte protease inhibitor (SLPI) and elastase in ejaculates from normal donors and infertile patients with or without leukospermia and investigated the effect of SLPI on sperm motility reduced by elastase. Western blot analysis revealed that SLPI protein was detected in the seminal plasma. The SLPI titre in the seminal plasma with leukospermia was lower than that in the seminal plasma without leukospermia and in the seminal plasma of fertile donors. The elastase concentration in the seminal plasma with leukospermia was significantly higher than that in the seminal plasma without leukospermia. A significant correlation between SLPI and elastase concentrations in the seminal plasma (r = 0.36, P< 0.01) was observed. There was a positive correlation between SLPI titre in the seminal plasma and sperm motility (r = 0.51, P < 0.001). SLPI recovered the sperm motility reduced by elastase in a dose-dependent manner. Our results suggest that SLPI is a potential substance to treat infertile patients with leukospermia.

Adult↗

PubMed1998

Regulation of placental monocyte chemotactic and activating factor during pregnancy and chorioamnionitis.

A number of placental cytokines participate in the feto-maternal defence mechanism. Monocyte chemotactic and activating factor (MCAF) is one of these chemokines. We investigated the pattern of placental MCAF production, the localization of MCAF-producing cells in the placenta, and alterations in its expression in chorioamnionitis. The amounts of MCAF protein produced by the placenta increased during pregnancy irrespective of the presence of uterine contraction. MCAF mRNA was expressed at equivalent levels in the first and second trimester placenta, but at higher levels in the third trimester placenta. Chorioamnionitis in the third trimester placenta induced a 10-fold increase in MCAF protein production and a 3-fold increase in MCAF mRNA level. Immunohistochemical analysis of the placenta revealed the MCAF-producing cells to be trophoblasts. Stimulation of placental cells with lipopolysaccharide augmented MCAF production. These results indicate unique transcriptional and developmental regulation of MCAF mRNA and protein production during pregnancy and chorioamnionitis. Placental MCAF may be involved in the feto-maternal defence mechanism by activating feto-placental and maternal monocytes in chorioamnionitis.

Blotting, Western↗

PubMed1998

Lipocalin-type prostaglandin D synthase in human male reproductive organs and seminal plasma.

Prostaglandin D synthase (PGDS) activity was detected in human seminal plasma (0.05-1.83 nmol/min per milligram protein). The enzyme was purified from human seminal plasma by immunoaffinity chromatography and found to be 27 kDa in size and N-glycosylated, similar to PGDS in the cerebrospinal fluid. The N-terminal amino acid sequence of 16 residues of the seminal enzyme, APEAQVSVQPNFQQDK, was identical to that of the cerebrospinal fluid PGDS. Although PGDS activity and the content determined by the immunoassay each highly varied in the seminal plasma, the concentration was significantly (p < 0.001) lower in the oligozoospermic group (2.47 +/- 0.51 microg/ml) than in the normozoospermic group (9.75 +/- 1.49 microg/ml). Prostaglandin (PG) D2 was detected in the seminal plasma (5.00 +/- 0.65 ng/ml) with a positive correlation to the PGDS concentration (p < 0.05). PGD2 was converted to the J series of PGs in the seminal plasma with a half-life of 6.5 h. Northern blot analysis revealed that mRNA for PGDS was expressed in the testis, prostate, and epididymis. Through immunohistochemistry, PGDS was localized in Leydig cells of the testis and in epithelial cells of the prostate and ductus epididymidis.

Adult↗

PubMed1998

Effect of site directed mutagenesis in the CMGCC region of the alpha-subunit on immunoreactive human thyrotropin.

The cDNA of the common alpha-subunit of human glycoprotein hormone was mutated by site directed mutagenesis in the CMGCC region composed of cysteine-methionine-glycine-cysteine-cysteine (position 28-32). The cDNA of wild-type human thyrotropin (hTSH) beta-subunit and that of wild-type or mutant common alpha-subunits were co-transfected into COS-I cells. The concentration of hTSH determined by two immunoradiometric assay systems was detectable in culture media of COS-I cells transfected with wild-type (CMGCC) and a mutant (CRGCC) alpha-subunits but not four other mutants (YMGCC) (CMRCC) (CMACC) (CMDCC). The present data with the other studies on wild-type or mutant glycoprotein hormones support our hypothesis that an amino acid motif of "C-X-G-X-C" in the common alpha-(CMGCC in human) and beta-(CAGYC in human) subunits play an important role in biosynthesis of glycoprotein hormones in all species.

Amino Acid Substitution↗

PubMed1997

Administration of IL-12 induces a CD3+ CD4- CD8- B220+ lymphoid population capable of eliciting cytolysis against Fas-positive tumor cells.

The present study investigates the effect of IL-12 administration on the generation of lymphoid cells that exhibit cytotoxicity against tumor cells expressing Fas Ag. Systemic injection of rIL-12 into BALB/c or (B6C3)F1 mice bearing syngeneic CSA1M or OV-HM tumor induced complete tumor regression. CSA1M tumor cells expressed Fas Ag, and exposure of these cells to IFN-gamma enhanced Fas expression. In contrast, Fas Ag was hardly detected on OV-HM cells even after IFN-gamma exposure. Only CSA1M cells were lysed by anti-Fas mAb or cells expressing Fas ligand (FasL), indicating that Fas on CSA1M cells is functional in mediating cell death. An increase in the frequency of lymphoid cells characterized as CD3+ CD4- CD8- B220+ was observed in spleens from both CSA1M and OV-HM tumor-bearing mice after IL-12 treatment. A splenic population enriched in cells with these unique phenotypes exhibited considerable degrees of cytotoxicity against Fas+ CSA1M, but not against Fas- OV-HM tumor cells. The lysis of CSA1M cells was almost completely blocked by addition of Fas-Fc, a fusion protein between the extracellular domain of mouse Fas and the Cgamma1 domain of human Ig. Regressing CSA1M and OV-HM tumor masses after IL-12 treatment exhibited a massive lymphoid cell infiltration and expressed significant levels of FasL mRNA, suggesting the infiltration of FasL-expressing cells to tumor sites. These results indicate that IL-12 induces the expansion of lymphoid cells that exhibit FasL-mediated cytolytic activity and accumulate into regressing tumor masses.

Animals↗

PubMed1997

Biparental alleles of HLA-G are co-dominantly expressed in the placenta.

HLA-G is the only major histocompatibility complex molecule expressed in the human placenta and thus has been considered to be necessary for maintenance of pregnancy. We investigated whether HLA-G expression is regulated in a parent-of-origin allele-specific manner. Of six first trimester and three third trimester placentas, three first trimester and two third trimester placentas showed heterozygosity at the PstI polymorphic site in the 3'-untraslated region. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed biallelic expression of HLA-G in all the informative cases, indicating that HLA-G is not imprinted during the gestational period, at least at the transcriptional level. As HLA-G has been postulated to be polymorphic not only at the DNA sequence level but also at the peptide level, co-dominant expression of the gene suggests that each parental allele is involved in the allogenic response during pregnancy.

Alleles↗

PubMed1997

Serum vitamin K level and bone mineral density in post-menopausal women.

OBJECTIVE: Vitamin K is known to influence bone metabolism by facilitating the synthesis of osteocalcin (BGP). The bone mineral density decreases drastically after menopause. We investigated the relationship of bone mineral density, vitamin K levels and other biological parameters of bone metabolism in post-menopausal women. METHODS: Serum levels of vitamin K, BGP and other markers of bone metabolism were measured in 71 post-menopausal women (19 with reduced bone mineral density and 52 with normal bone density), and 24 women with climacteric symptoms receiving hormone replacement therapy (HRT), (6 with reduced bone mineral density and 18 with normal density). RESULTS: In the first group, women with reduced bone mineral density showed lower levels of vitamin K1 and K2 than those with normal bone mineral density. In the other group, the level of BGP decreased but levels of vitamin K showed no increase during HRT. CONCLUSION: The present findings suggested that vitamin K was related to post-menopausal bone mineral loss.

Bone Density↗

PubMed1997

Interleukin-8 level in maternal serum as a marker for screening of histological chorioamnionitis at term.

OBJECTIVE: To establish a clinical method for immediate diagnosis of histological chorioamnionitis, by maternal blood sampling at term. METHOD: The sera of 22 mothers with chorioamnionitis and 81 mothers without chorioamnionitis at term delivery were collected. The serum levels of cytokines including interleukin-1 alpha (IL-1 alpha), interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and interleukin-8 (IL-8) were titered and other conventional markers such as white blood cell and CRP were measured simultaneously. Chorioamnionitis was histopathologically confirmed after delivery. RESULT: The sera of mothers with histological chorioamnionitis showed a significant increase in IL-8 titer, but not in those of other cytokines or conventional markers, compared with those without chorioamnionitis. A positive correlation was observed between maternal and cord serum IL-8 levels. Maternal IL-8 showed the highest predictive value for diagnosis of histological chorioamnionitis. CONCLUSION: Measurement of maternal IL-8 is useful for rapid prenatal screening of histological chorioamnionitis at term.

Adult↗

PubMed1997

Loss of H19 imprinting and up-regulation of H19 and SNRPN in a case with malignant mixed Müllerian tumor of the uterus.

In several human cancers, it has been recently reported that abnormally altered status of genomic imprinting is related to oncogenesis. In this study, we investigated the expression of three imprinted genes in a case with malignant mixed Müllerian tumor of the uterus (MMMT). In the tumor, expression of H19 showed marked upregulation (6.3-fold) with biallelic expression compared with that in the corresponding normal myometrium. The 5'-promoter region of H19 was hypomethylated in the tumor, whereas it was hemimethylated in the myometrium. Expression of the small nuclear ribonucleoprotein polypeptide N gene (SNRPN) was also upregulated by 1.9-fold. However, the insulin-like growth factor II gene (IGF2) was expressed at low levels in both myometrium and MMMT. The overexpression of H19 is caused by reactivation of the repressed allele of H19 due to demethylation of CpG islands within its 5'-promoter region. Whether upregulation of SNRPN is caused by its biallelic expression remains undetermined because restriction fragment length polymorphisms (RFLP) sites were not informative in SNRPN and IGF2. In conclusion, H19 and SNRPN may play significant roles in the tumorigenesis of MMMT and H19 may have tumor-promoting activity in addition to its known tumor-suppressing activity, probably depending on the tissue and the local milieu.

Autoantigens↗

PubMed1997

The influence of peri-operative blood transfusion during radical hysterectomy on the prognosis of uterine cervical cancer.

To evaluate the clinical implications of peri-operative transfusion in the surgical treatment of uterine cervical cancer, a detailed analysis of the cumulative survival rate was performed using clinical data from 145 patients with FIGO stage Ib cervical cancer of the squamous cell type acquired between 1982 and 1989 at the Center for Adult Diseases. The cumulative survival rate was statistically analyzed using the Kaplan Meier method. Of the 145 patients with stage Ib cancer, 103 were considered to have undergone complete excision and received no further treatment post-operatively, and 42 were considered to have undergone incomplete excision because of the involvement of lymph nodes or the lymphovascular space. Of the 79 patients in the former group who received peri-operative transfusion, 69 received less than seven units (1400 mL) and 10 received more than eight units (1600 mL). The cumulative 5-year survival rate was 100% in the 24 patients who received no transfusions, 91.7% in those who received less than 1400 mL, and 90.0% in those who received more than 1600 mL. Similarly, of the 42 patients in the incomplete excision group, nine did not receive any peri-operative transfusion, 27 received less than 1400 mL, and six received more than 1600 mL. The 5-year survival rate in these groups was 88.9, 77.8 and 50.0%, respectively. The survival rate of our patients with stage Ib cervical cancer without peri-operative transfusion was significantly higher than that of those with transfusion, regardless of post-operative irradiation therapy status, indicating that peri-operative transfusion has an adverse influence on the prognosis in cervical cancer.

Adult↗

PubMed1997

The role of N-terminal glycosylation in the human oxytocin receptor.

The human oxytocin receptor includes three N-glycosylation sites in its extracellular N-terminal domain. We have established permanent cell-lines in which the gene for the human oxytocin receptor (OTR) has been introduced into HeLa cells. These cells differ by the disruption of one or more of the N-terminal N-glycosylation sites by site-directed mutagenesis of the transfected OTR constructs. The binding capacity of each transfectant, calculated per mg membrane protein, was 5-17 times higher than that of human term myometrium. The pharmacological characteristics of the transfected wild-type OTR are very similar to those of native myometrial OTR. The mutation of N-glycosylation sites (Asn-X-Ser/Thr), namely OTR-D8N15N26 (Asn8-->Asp8), -N8D15N26(Asn15-->Asp15), -N8D15D26(Asn15-->Asp15, Asn26-->Asp26) and -D8N15D26 (Asn8-->Asp8, Asn26-->Asp26) appear to affect neither their dissociation constant (Kd), nor the affinities for various oxytocin related ligands. As a high level of cell surface binding was retained for each clone, receptor trafficking appears to be normal. This suggests that the full glycosylation of OTR observed in vivo is not essential for its activity. These results indicate also that these cell lines may prove very useful for pharmacological screening of oxytocin related products.

Gene Transfer Techniques↗

PubMed1997

Human decidual cell biosynthesis of leukemia inhibitory factor: regulation by decidual cytokines and steroid hormones.

The production of leukemia inhibitory factor (LIF) is suggested to be critical for the successful implantation of blastocysts into decidua, because LIF expression is essential for the implantation of mouse blastocytes. We investigated the regulation of LIF production by decidual cytokines and steroid hormones. Stimulation of decidual cells by interleukin-1, tumor necrosis factor alpha, or transforming growth factor beta augmented LIF production in a dose-dependent manner. Moreover, estradiol, a steroid hormone that increases during ovulation and early pregnancy, also enhanced LIF production in a dose-dependent manner. These responses were blocked by protein kinase C (PKC) inhibitor but not by other kinase inhibitors, suggesting an important role of PKC in decidual LIF production mediated by cytokines and estradiol. We also showed that stimulating decidual cells with LIF failed to stimulate DNA synthesis and prolactin production in these cells. In summary, LIF was mainly localized in the decidual glands and stroma, and its production was increased by cytokines and estradiol in a dose-dependent fashion; but stimulation of decidual cells by LIF did not influence their proliferation or their prolactin production.

Animals↗

PubMed1997

Increased interleukin-1 and interleukin-1 receptor antagonist levels in cervical mucus in the ovulatory phase in comparison with the follicular phase.

Interleukin-1 (IL-1) receptor antagonist (IL-1ra) levels in the cervical mucus of women in the ovulatory phase are significantly higher than those in the follicular phase. IL-1 titers of women in the ovulatory phase are also significantly higher than those in the follicular phase. A positive correlation between IL-1ra and IL-1 levels in the cervical mucus was observed. Immunohistochemistry using an anti-IL-1ra monoclonal antibody revealed positive staining in the epithelial cells of the endocervix. These results suggest that IL-1ra from cervical epithelial cells protects the reproductive system from the toxicity of IL-1 produced in the endocervix.

Adult↗

PubMed1997

Effect of different concentrations of amino acids in human serum and follicular fluid on the development of one-cell mouse embryos in vitro.

As a model for establishing an optimized medium for human in vitro fertilization (IVF), modified human tubal fluid (HTF) media containing amino acids at concentrations found in human serum and follicular fluid were prepared, and the effect of the media on development of random-bred (ICR) and F1 hybrid (CBF1) mice embryos was studied. The total concentrations of amino acids found in serum and follicular fluid were about one-third to one-half the concentrations present in two conventional media used in human IVF: Ham's F-10 and Eagle's minimal essential medium (MEM). When ICR mouse embryos were cultured in the HTF medium containing 21 amino acids at concentrations found in follicular fluid, the number of embryos developing to morulae at 72 h and to blastocysts at 96 h increased in comparison with those cultured in HTF medium. When HTF containing amino acids at concentrations found in serum was used, only induced morula formation at 72 h was enhanced. The number of hatching blastocysts at 96 h also increased when CBF1 mouse embryos were cultured with HTF supplemented with amino acids at concentrations found in follicular fluid. When ICR mouse embryos were cultured in modified HTF media containing concentrations of amino acids found in Ham's F-10 and MEM that contained higher concentrations of glutamine, embryo development was inhibited. The amount of ammonium produced during incubation for 3 days was significantly less when embryos were cultured in media containing concentrations of amino acids found in follicular fluid compared with when Ham's F-10 or MEM was the culture medium. Ammonium is produced by the breakdown of glutamine in the culture medium during incubation with or without embryos. These results suggest that the concentrations of amino acids found in follicular fluid are more effective and safer for embryo culture than those in other media currently in use.

Amino Acids↗

PubMed1997

The desensitization of oxytocin receptors in human myometrial cells is accompanied by down-regulation of oxytocin receptor messenger RNA.

We have recently provided evidence for the desensitization of oxytocin receptors in human myometrial cells. In the present study, we have investigated the possible mechanisms by which oxytocin (OT) might regulate OT receptor density. The steady state level of OT binding in cultured myometrial cells was 220 x 10(3) binding sites/ cell, but this was time-dependently reduced to 27 x 10(3) sites/cell by exposure to OT for up to 20 h. Similarly, OT exposure decreased the binding of OT to cell membranes. In contrast, Western blotting data showed that the total amount of OT receptor protein was not affected by OT treatment for up to 48 h. Flow cytometry experiments demonstrated that OT receptors are not internalized during prolonged exposure of the cells to OT. However, RNase protection assays and Northern analysis showed that OT receptor mRNA was reduced by OT treatment to reach a new low steady state level with a time course similar to that of the disappearance of cell surface OT binding sites. Possible mechanisms involved in mRNA down-regulation include transcriptional suppression and destabilization of mRNA by RNA binding proteins.

Blotting, Northern↗

PubMed1997

Chorioamnionitis reduces placental endocrine functions: the role of bacterial lipopolysaccharide and superoxide anion.

Chorioamnionitis has been shown to be one of the most important factors in inducing preterm delivery. The present study was undertaken to examine the effects of chorioamnionitis on placental endocrine functions. Preterm placentas with histologic chorioamnionitis produced smaller amounts of human chorionic gonadotropin (hCG) and human placental lactogen (hPL) than those without chorioamnionitis (P < 0.001). To examine the mechanism involved in the suppression of placental endocrine functions induced by chorioamnionitis, we initially confirmed the expression of lipopolysaccharide (LPS) receptor, i.e. the CD14 molecule, on trophoblasts by Northern blot analysis and immunohistochemistry. We then stimulated purified trophoblasts with LPS, which is the major agent which induces inflammatory responses in the host via the LPS receptor. The trophoblasts stimulated with LPS produced reduced amounts of hCG, hPL, and progesterone in a time- and dose-dependent fashion in spite of the induced manganese-superoxide dismutase (SOD) synthesis. Stimulation of trophoblasts with hypoxanthine and xanthine oxidase resulted in suppressed hCG production, while the simultaneous addition of SOD into the culture medium reversed the suppression of hCG production. LPS in the placenta with chorioamnionitis might directly stimulate trophoblasts through the LPS receptor (CD14), thus reducing placental endocrine functions. Superoxide anions which exogenously act on trophoblasts might be generated by simultaneous stimulation of neutrophils and monocytes at the feto-maternal interface by LPS, and additively reduce placental endocrine functions.

Adult↗

PubMed1996

Structure and expression of the mouse oxytocin receptor gene.

To determine the structure of the mouse oxytocin receptor (OTR) gene, we have screened a mouse genomic library and confirmed cDNA sequence with rapid amplification of cDNA ends. Southern blot using human OTR cDNA probes indicated that the mouse genome has a single copy of the gene. The predicted amino acid sequence is 91% identical to human OTR. The gene contains 4 exons and 3 introns. Exons 1 and 2 contain the 5' untranslated region, with exons 3 and 4 encoding the amino acids of the receptor. Intron 3 interrupts the coding at the same location, after transmembrane domain 6, as in OTR genes of other species. The promoter region lacks an apparent TATA box but contains multiple putative interleukin-response elements and estrogen responsive elements. Expression of OTR gene in the uterus during pregnancy reached maximum at day 20 gestation. The information obtained from the mouse OTR gene facilitates further comparative and biochemical analysis for protein structure-function relationships and gene regulatory mechanisms.

Amino Acid Sequence↗