Clinical observations indicate that a proportion of patients with schizophrenia experience cognitive impairment, which suggests that a neurodegenerative basis might be involved in the etiology of schizophrenia. Apolipoprotein E (ApoE), which has been confirmed to be genetically associated with Alzheimer's disease, is thus highlighted as a candidate gene for schizophrenia. Recently, novel functional polymorphisms in the ApoE transcriptional regulatory region have been found. To investigate whether these polymorphisms are associated with the risk of schizophrenia, we genotyped 144 patients with schizophrenia and 134 controls for two polymorphisms (-491A/T and -219G/T). No significant positive associations between both polymorphisms and schizophrenia were observed. Our findings exclude the regulatory region of the ApoE gene as a locus that might confer increased susceptibility to schizophrenia.

Apolipoproteins E↗

PubMed1998

Production of prostanoids via increased cyclo-oxygenase-2 expression in human amnion cells in response to low molecular weight hyaluronic acid fragment.

Increased concentrations of hyaluronic acid (HA) have been found in serum and at uterine cervix at term. In its native form, HA exists as a high molecular weight (MW) polymer, but during parturition a lower MW HA fragment accumulates. The aim of this study was to investigate the regulatory mechanisms responsible for increased amnion prostanoid production and cyclo-oxygenase (COX) expression in response to HA. Human term amnion cells in culture were exposed to native HA polymer (MW 2.2x106) and its fragment (MW 3.5x104). We have determined levels of prostanoids, prostaglandins E2 and F2alpha, in conditioned media using specific immunoassays. Expression of COX-1 and COX-2 was examined with Western blot. Results were analyzed for statistical significance with Mann-Whitney U-test. Human amnion cells treated with HA fragment (100 nmol/l) produced significantly more PGE2 (2.3+/-0.21 (mean+/-S.D.) pg/106 cells/24 h) than controls (0.34+/-0.03) or high MW HA-treated cells (1.2+/-0.21). Protein levels of COX-2, but not COX-1, were substantially increased in amnion cells treated with HA fragment. HA fragment-mediated prostanoid production is markedly diminished by pretreatment with indomethacin. Our results indicate that HA fragment, rather than physiologic native HA polymer, induces amnion cell-derived prostanoid production via increased COX-2 expression. COX-2-mediated prostanoid production is likely a key physiologic event in HA fragment-mediated cervical ripening and the labor onset.

Amnion↗

PubMed1998

Tardive dyskinesia and debrisoquine 4-hydroxylase (CYP2D6) genotype in Japanese schizophrenics.

Previous studies have shown that many neuroleptics are metabolized by debrisoquine 4-hydrolase (CYP2D6), which exhibits genetic polymorphisms. In Oriental populations, poor metabolizers (PMs) with a lack of CYP2D6 activity are rare, although the CYP2D6*10 allele, which is associated with decreased CYP2D6 activity, is commonly found. The authors examined the relationship between tardive dyskinesia (TD) and CYP2D6 polymorphisms, including the CYP2D6*10 allele. Subjects consisted of 100 Japanese schizophrenics. TD was evaluated using the Abnormal Involuntary Movement Scale (AIMS). Genotyping for the presence of the CYP2D6*3, CYP2D6*4 and CYP2D6*10 alleles was performed using allele-specific PCR amplification and endonuclease digestions. The frequency of the CYP2D6*10 allele was 0.52, and only one allele showed the PM genotype. There was a significant difference in the allelic distribution of CYP2D6*10 between subjects with and without TD. We also found significant genotypic and allelic associations with dichotomized total AIMS scores of 6 or more (moderate or severe abnormal movements) and with scores of less than 6 (mild or no movements). After these associations were adjusted for confounding variables (gender, age, duration of illness and neuroleptic dose) by regression analysis, the CYP2D6*10 genotype showed significant association with the total AIMS score, and a modest association with TD occurrence. These results indicate that the CYP2D6*10 genotype may play a role in the development of moderate or severe abnormal movements.

Alleles↗

PubMed1998

Urinary trypsin inhibitor suppresses vascular smooth muscle contraction by inhibition of Ca2+ influx.

Urinary trypsin inhibitor (UTI) and its precursor form inter-alpha trypsin inhibitor (ITI) are present in plasma. To determine the action of UTI on blood vessels, we performed isometric vascular muscle contraction tests, microcirculation studies and measurement of cytosolic free Ca2+ in vascular smooth muscle cells. An isometric vascular muscle contraction test showed that the contractions stimulated by endothelin-1 or norepinephrine were suppressed in the presence of UTI, and that the contractions were not inhibited in the presence of ITI. The microcirculation study showed that the contraction of mesenteric arterioles of WKY rats induced by norepinephrine were inhibited by treatment of UTI, and that they did not alter by treatment of ITI. Pre-incubation of UTI, but not ITI, with vascular smooth muscle cells inhibited the increase of cytosolic free Ca2+ induced by endothelin-1 or norepinephrine. Cell-binding study by biotinylated UTI showed that vascular smooth muscle cells have specific binding site for UTI, but not for ITI. We propose that circulating UTI converted from ITI has a regulatory effect on local vascular tone by regulation of Ca2+ influx into smooth muscle cells.

Animals↗

PubMed1998

A bifunctional hybrid molecule of the amino-terminal fragment of urokinase and domain II of bikunin efficiently inhibits tumor cell invasion and metastasis.

Urinary trypsin inhibitor (UTI) inhibits efficiently tumor cell invasion and the formation of metastasis. The anti-metastatic effect is dependent on the COOH-terminal domain II of UTI [UTI-(78-136)-peptide]. To develop a molecule that binds with high affinity to the urokinase (uPA) receptor (uPAR) on tumor cell surfaces, a bifunctional hybrid molecule [uPA-(1-134)-UTI-(78-136)] consisting of the uPAR-binding NH2-terminal fragment [UTI-(78-136)-peptide] of uPA at the NH2-terminus of UTI-(78-136)-peptide was produced in Escherichia coli by genetic engineering. The purified hybrid protein inhibited trypsin and plasmin 2-3-fold less effectively than UTI-(78-136)-peptide and was found to bind to human tumor cells via uPAR, which was confirmed by cell binding and competition experiments. Using a modified Boyden chamber and an artificial basement membrane, Matrigel, it was found that the hybrid protein is very effective at inhibiting invasion by uPAR-expressing human tumor cells. Sensitivities of tumor cells towards the anti-invasive effect of uPA-(1-134)-UTI-(78-136) correlated with the density of uPAR on human tumor cells. Furthermore, in the spontaneous metastasis model, the hybrid protein inhibited the formation of lung and/or lymphatic metastasis by human ovarian carcinoma and choriocarcinoma cells. The hybrid protein was much more effective than uPA-(1-134)-peptide, UTI-(78-136)-peptide, or UTI. We conclude that this approach extends the possibility of applying recombinant protein for therapeutic use in inhibition of human tumor cell metastasis.

Amino Acid Sequence↗

PubMed1998

Identification and characterization of the cell-associated binding protein for urinary trypsin inhibitor.

Urinary trypsin inhibitor (UTI) inhibits not only tumor cell invasion but also production of experimental and spontaneous metastasis. Cell-binding experiments indicated that human choriocarcinoma SMT-cc1 cells have specific binding sites for UTI on their cell surface. [Kobayashi et al., J. Biol. Chem. 269, 1994, 20,642-20,647]. UTI binding protein (UTIBP) was purified to homogeneity by a combination of UTI-coupled affinity beads, preparative polyacrylamide gel electrophoresis and reverse phase HPLC. This protein is very similar to a truncated form of human cartilage link protein (LP). LP was identified structurally by its apparent molecular mass with and without deglycosylation treatment: Immunologically by the reactivity with anti-UTIBP antibody, and functionally by its ability to bind the NH2-terminal domain of UTI. UTI and UTIBP are distributed uniformly in the cytoplasm and/or over the cell surface of tumor cells and fibroblasts. The level of staining for hyaluronic acid, UTIBP and UTI is much lower in sections digested with hyaluronidase. These results suggest that the cell membrane-derived UTI-associated binding protein is the LP of proteoglycan-hyaluronic acid aggregates, which interacts with hyaluronic acid. Cell-associated LP may play a role in modulating protease activity to the environment close to tumor and fibroblast cell surface.

Chromatography, High Pressure Liquid↗

PubMed1998

Association study of a functional catechol-O-methyltransferase gene polymorphism in Japanese schizophrenics.

Catechol-O-methyltransferase (COMT) is an enzyme which inactivates catecholamine neurotransmitters by methylation, and is considered a candidate for involvement in schizophrenia. A functional COMT gene polymorphism influencing the enzyme activities, the high activity (val-108) and the low activity allele (met-108), was recently confirmed. We investigated a genetic association between schizophrenia and the COMT gene polymorphism in 150 Japanese schizophrenics and controls. We detected the low activity met-108 allele more frequently in schizophrenics than in the controls, and found that subjects sharing the met-108 allele (val/met and met/met) are significantly more common in the patients than in the controls. The results suggest that the low activity met-108 allele may be involved in susceptibility for schizophrenia.

Alleles↗

PubMed1998

Macrophage colony-stimulating factor as a marker for malignant germ cell tumors of the ovary.

We assessed the usefulness of macrophage colony-stimulating factor (M-CSF) as a serum marker for malignant germ cell tumors of the ovary. Serum levels of M-CSF were measured in 49 patients with malignant germ cell tumors and in 64 patients with mature benign cystic teratoma by an enzyme-linked immunosorbent assay. CA125 was measured by radioimmunoassay. The serum level of M-CSF was above normal (>1056 U/ml) in 44 (90%) of 49 patients with malignant germ cell tumors; the CA125 level was above the cutoff value (35 U/ml) in 34 patients (69%) (P < 0.05). The serum level of M-CSF was elevated in 20 (87%) of 23 patients with stage I disease and in all 16 patients with dysgerminoma. Only 7 (11%) of 64 patients with mature benign cystic teratoma had elevated levels of M-CSF. These results suggest that M-CSF is highly sensitive and specific for malignant germ cell tumors of the ovary, especially for dysgerminoma.

Biomarkers, Tumor↗

PubMed1998

The effect of bulk magnetic susceptibility on solid state NMR spectra of paramagnetic compounds

The effect of bulk magnetic susceptibility (BMS) on solid state NMR spectra of paramagnetic compounds was investigated theoretically and experimentally. The BMS shift was calculated for cylindrical and spherocylinderical containers with some ratios of the length L and the diameter D. The results show the best resolution can be obtained by using a long cylindrical sample container with L/D > 10 and by exciting only the region near the center of the container. The effect of the random orientations and distributions of crystallites in a powder sample was also calculated according to a model proposed by Schwerk et al. [J. Magn. Reson. A 119, 157 (1996)] with removing the Fermi contact term from their model. Static and the magic-angle spinning 13C NMR spectra were recorded on two paramagnetic compounds of Ln(C2D5SO4)3 . 8H2O where Ln = Pr, Yb. The modified theory predicts the BMS broadening of the experimental spectra very well. Copyright 1998 Academic Press.

Journal Article↗

PubMed1998

An active metabolite of carbamazepine, carbamazepine-10,11-epoxide, inhibits ion channel-mediated catecholamine secretion in cultured bovine adrenal medullary cells.

We have recently reported inhibitory effects of carbamazepine (CBZ) on ion channel-mediated secretion of catecholamines in bovine adrenal medullary cells. Here, we report the effects of carbamazepine-10,11-epoxide (CBZ-E), an active metabolite of CBZ, and carbamazepine-10,11-diol (CBZ-D), a non-active metabolite, on 22Na+ influx, 45Ca2+ influx and catecholamine secretion in cultured adrenal medullary cells. CBZ-E, but not CBZ-D inhibited 22Na+ influx, 45Ca2+ influx and catecholamine secretion induced by carbachol or veratridine with a half-maximal inhibitory concentration (IC50) of 0.26 or 0.68 microg/ml, respectively. CBZ-E also inhibited high K+-evoked 45Ca2+ influx and catecholamine secretion (IC50 = 0.3 microg/ml), but CBZ-D did not. These findings suggest that CBZ-E, but not CBZ-D, attenuates catecholamine secretion by inhibiting nicotinic acetylcholine receptor-associated ion channels, voltage-dependent Na+ channels and voltage-dependent Ca2+ channels in the cells. This inhibition of CBZ-E as well as CBZ may be related to the clinical effects in neuropsychiatric disorders.

Adrenal Medulla↗

PubMed1998

Internalization of urinary trypsin inhibitor in human uterine fibroblasts.

We have characterized the molecular species and internalization of urinary trypsin inhibitor (UTI) in human uterine fibroblasts. Link protein (LP) has previously been identified as one of the cell-associated UTI binding proteins. The truncated forms of UTI were readily detectable in the cells after incubating the cells with purified UTI. Immunoblotting analysis with a panel of domain-specific antibodies revealed that the UTI species lacked the amino-terminal domain of UTI, but contained the carboxyl-terminal domain. We have examined whether LP is involved in the UTI internalization in the cells. Internalization of 125I-labelled UTI was blocked by the intact UTI, but not by the carboxyl-terminal domain of UTI. Treatment with a polyclonal antibody to the UTI binding domain of LP partially inhibited UTI binding to the cells, but did not significantly prevent UTI internalization. In addition, preincubation of the cells with hyaluronidase reduced the UTI binding to the cells, but had no effect on the rate with which UTI was internalized. These data allow us to conclude that there are at least two different mechanisms for internalization of UTI. The major one is via unknown UTI receptors in a Ca2+, Mg2+-sensitive manner and another is via LP.

Calcium↗

PubMed1998

Urinary trypsin inhibitor reduces the release of histamine from rat peritoneal mast cells.

We determined the ability of urinary trypsin inhibitor (UTI), which is a Kunitz-type protease inhibitor present in serum and in urine, to inhibit rat peritoneal mast cell (RPMC) mediator release induced by several stimuli. UTI attenuated the immunoglobulin E-mediated release of both preformed (histamine) and newly formed (leukotriene C4) mediators from RPMCs. Inhibition (21%+/-5%) of the anti-IgE-triggered release of histamine was observed after a 30-minute incubation of RPMCs with UTI (5 micromol/L). To investigate the specificity of the UTI effect, we studied the stimulatory activity of phorbol ester (phorbol 12-myristate 13-acetate (PMA)) or calcium ionophore A23187 in control and UTI-treated mast cells. The efficacy of UTI as an inhibitor was dependent on the nature of the stimulus, because histamine release induced by PMA-mediated or calcium ionophore A23187-mediated processes was not inhibited by UTI. A series of structurally distinct protease inhibitors did not inhibit IgE-induced release of mediators from RPMCs. The Kunitz-type protease inhibitors are important in the regulation of RPMC function. In parallel with the UTI-related decrease in anti-IgE stimulatory activity on mediator release, increased microviscosity of membrane lipids could be observed by two independent experiments on fluorescence polarization with diphenylhexatriene (DPH) and on the fluorescence probe fluorescein isothiocyanate-concanavalin A. UTI reduces mediator release by a mechanism-possibly an interruption of the coupling of receptor and effector systems-because UTI acts as an agent to decrease biologic lipid membrane fluidity.

Animals↗

PubMed1998

Thrombomodulin release from umbilical endothelial cells initiated by preeclampsia plasma-induced neutrophil activation.

OBJECTIVE: To assess the capacity of neutrophils to release thrombomodulin from umbilical vein endothelial cells in vitro in response to plasma from normal gravidas and those with preeclampsia. METHODS: We collected venous plasma samples from 66 pregnant women: 18 women with severe preeclampsia, 18 women with mild preeclampsia, and 30 normotensive healthy women with singleton pregnancies in the third trimester. Neutrophils were isolated from venous blood obtained from healthy adult volunteers. Endothelial cells from normal umbilical veins were digested by collagenase and cultured. The effect of preeclampsia plasma on thrombomodulin release from endothelial cells was tested after incubating cultured cells with plasma from severe preeclampsia. Thrombomodulin and elastase levels were measured with specific immunoassays. RESULTS: Plasma thrombomodulin levels (mean +/- standard error of the mean) were elevated in pregnant women with severe preeclampsia (62.5+/-7.9 compared with 40.1+/-3.3 [matched control] ng/mL; P = .02), and elastase levels correlated directly with thrombomodulin (r = .49; P < .05). Both coincubation of endothelial cells and neutrophils with preeclampsia plasma (16.3+/-2.0 compared with 9.4+/-1.2 [normotensive plasma] ng/mL; P < .05) and incubation of endothelial cells with neutrophils pretreated with preeclampsia plasma (13.9+/-1.8 compared with 9.6+/-0.9 [normotensive plasma] ng/mL; P < .05) significantly promoted thrombomodulin release from endothelial cells. Thrombomodulin can be released by the concerted action of preeclampsia plasma-stimulated neutrophils and endothelial cells. CONCLUSION: In patients with severe preeclampsia or eclampsia, neutrophil activation occurs and the activated neutrophils induce vascular endothelial damage.

Adult↗

PubMed1998

Solid-state photodimerization of 9-methylanthracene as studied by solid-state 13C NMR.

The locus of the photodimerization reaction of 9-methylanthracene in the crystal was examined by high-resolution solid-state 13C NMR techniques. Examination of the 13C spectra of the products showed that only the trans dimer is formed by the solid state photodimerization, while both trans and cis dimers are formed by the photodimerization in benzene solution. The 1H T1 relaxation curves for the monomer and the dimer at various exposure times were separately observed via well-resolved 13C peaks. The two T1 curves exhibit characteristic features for relaxation of a weakly coupled two-spin system. By analyzing the T1 curves, the spin diffusion rates between the monomer and the dimer and the fraction of the dimer were obtained for various exposure times. From the result, the maximum domain size of the minor component during the photodimerization process was estimated to be ca. 0.3 microm. The heterogeneous domain structure generated by the photodimerization indicates that the reaction takes place at defects of the crystal in the monomer.

Anthracenes↗