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Biomedical subjects

T Terao

Publications and source records attributed to T Terao.

At least 19 recordsLinked to original sources

Cytosolic and intranuclear calcium signals in rat basophilic leukemia cells as revealed by a confocal fluorescence microscope.

A confocal fluorescence microscope with an argon-ion laser (488 nm) and a He-Cd laser (325 nm) was used to study spatial heterogeneity of the calcium signals in rat basophilic leukemia 2H3 cloned cell line (RBL-2H3). After stimulation with antigen (2,4-dinitrophenol-conjugated bovine serum albumin), fluo-3-fluorescence intensities increased in individual RBL-2H3 cells with different lag times. Time-dependent profiles of the fluo-3-fluorescence intensities resembled closely the patterns of the sequential fluorescence-ratio images of fura-2, which were used to measure the intracellular free-calcium concentration ([Ca2+]i) in individual RBL-2H3 cells using a conventional fluorescence microscope. The present results obtained using the confocal fluorescence microscope showed spatial heterogeneities of fluo-3-fluorescence intensities, suggesting the existence of spatial heterogeneity of [Ca2+]i in RBL-2H3 cells. That is, the results showed that calcium signals first occurred transiently at pseudopodia in RBL-2H3 cells, then the signals transferred to the central parts of the cells. In addition, from the fluorescence images of co-loaded Hoechst 33342 (bisbenzimide H 33342, a DNA-specific probe) which were produced by excitation with a He-Cd laser, it was found that the fluorescence images of the nucleus were quite similar to those of the calcium signals mentioned above. This suggested that the receptor-mediated calcium signals were transferred not only to the cytoplasm but also to the nucleus.

Animals

Inhibition of in vitro ovarian cancer cell invasion by modulation of urokinase-type plasminogen activator and cathepsin B.

HOC-I ovarian cancer cells express the single-chain form of the urokinase-type plasminogen activator (uPA) and cathepsin B (cath B) on their cell surface. The significance of the expression of cell surface uPA/cath B activity to the invasive potential was examined by preincubating with uPA/cath B-modulating agents in in vitro invasion assay. The anti-uPA monoclonal antibody 394 effectively inhibited invasion in a dose-dependent manner. On the contrary, anti-cath B antibody did not affect the invasive potential of the cells. E-64, a specific inhibitor for cysteine proteases, blocked invasion as effectively as monoclonal antibody 394. The data reveal that the uPA and cysteine proteases contribute significantly to the invasive capacity of the cells. We suggest that the cysteine proteases facilitate the action of uPA, possibly by activating proenzyme uPA produced by cancer cells. Evidence for the role of a cathepsin-uPA activation cascade in HOC-I cell invasion is provided.

Cathepsin B

Antitumor activity of a novel nucleoside, 2'-C-cyano-2'-deoxy-1-beta-D-arabinofuranosylcytosine (CNDAC) against murine and human tumors.

The antitumor effects of 2'-C-cyano-2'-deoxy-1-beta-D- arabinofuranosylcytosine (CN-DAC), a synthetic 1-beta-D-arabinofuranosyl-cytosine (ara-C) derivative, were examined and compared with that of ara-C in murine tumors and in various human tumors using three different chemosensitivity tests. CNDAC extended the life span of mice bearing P388 leukemia. CNDAC had a unique in vitro antitumor spectrum for human cancers different from that of ara-C. Compared with ara-C, CNDAC was more effective in 10 human tumors (2 lung, 4 stomach and 4 osteosarcoma), equal in 2 tumors (lung and fibrosarcoma) and less potent in 11 tumors (4 lung, 4 osteosarcoma, bladder, renal and epidermoid). Characteristically CNDAC showed excellent activities against tumors, refractory to ara-C, such as HT-1080 human fibrosarcoma implanted in chick embryos or athymic mice, although its cytotoxicity against HT-1080 was almost equal to that of ara-C. Thus, CNDAC is an interesting and promising agent that should be considered for further detailed preclinical evaluation.

Animals

Lithium chloride stimulates catecholamine synthesis and secretion in cultured bovine adrenal medullary cells.

We examined the effects of lithium treatment on the synthesis and secretion of catecholamines in cultured bovine adrenal medullary cells. The treatment of cells with lithium (0.5-4 mmol/L) for 7 days caused an increase in basal and carbachol-stimulated synthesis of 14C-catecholamines from [14C]-tyrosine but not from [14C]-DOPA. Lithium treatment (4 mmol/L, 7 days) increased the activity of tyrosine hydroxylase in the cells. Lithium treatment (2-4 mmol/L, 7 days) also enhanced the secretion of catecholamines caused by carbachol, although the carbachol-induced influx of 45Ca2+ was reduced. Lithium (4 mmol/L, 7 days) potentiated the secretion of catecholamines evoked by the Ca2+ (1 mumol/L) from cells that were permeabilized by digitonin. The activity of protein kinase C in a soluble fraction was increased in lithium-treated cells (4 mmol/L, 7 days). These results demonstrate that lithium treatment increases the synthesis and secretion of catecholamines and the activity of protein kinase C in cultured adrenal medullary cells.

Adrenal Medulla

Veratridine causes the Ca(2+)-dependent increase in diacylglycerol formation and translocation of protein kinase C to membranes in cultured bovine adrenal medullary cells.

Our previous studies suggested that protein kinase C is involved in the veratridine (an activator of voltage-dependent Na+ channels)-induced phosphorylation and activation of tyrosine hydroxylase as well as the synthesis of catecholamines in adrenal medulla (Uezono et al. 1989). In the present study, we investigated whether treatment of cultured bovine adrenal medullary cells with veratridine causes the accumulation of diacylglycerol, a physiological activator of protein kinase C and the translocation of protein kinase C from cytosol to membrane, a process required for protein kinase C activation. Veratridine (100 mumol/l) increased diacylglycerol level about 2.2 fold in a monophasic manner, with peaking at 5 min and declining toward the basal level within 20 min. Veratridine also increased membrane protein kinase C from 15.6% to 26.9% of total protein kinase C in a time-course similar to that of diacylglycerol accumulation. Both stimulatory effects of veratridine were inhibited by tetrodotoxin and not observed in Ca(2+)-free, EGTA-containing medium. Amiloride, an inhibitor of Na+/Ca2+ and Na+/H+ exchange, did not alter veratridine-induced events. These results suggest that veratridine-induced Ca2+ influx contributes to the accumulation of diacylglycerol and the activation of protein kinase C in adrenal medullary cells.

Adrenal Medulla

Characterization and clinical evaluation of tumor-associated antigen CA54/61 identified by monoclonal antibodies MA54 and MA61 in epithelial ovarian cancer.

Monoclonal antibodies (moABs) MA54 and MA61, directed toward the O-linked mucin-type glycoprotein, have been established and showed highly specific reactivity with human ovarian cancer. Fetal intestinal and colonic mucosal cells expressed this antigen and meconium staining was also frequently positive. To investigate the characteristic of an epitopic carbohydrate recognized by these moABs, the reactivity of each moAB with meconium extract was monitored by solid-phase enzyme-linked immunosorbent assay with mono-, di-, and oligosaccharides. MA54 and MA61 react with meconium extract and the reactivities of these moABs are neuraminidase sensitive. Ovine submaxillary mucin had a strong inhibitory activity toward the reaction between meconium extract and MA54 as well as MA61, suggesting that these moABs recognize NeuAc 2-6GalNAc epitope in meconium. The second aim of this study is to investigate the possible application of moABs to diagnose ovarian cancer and to compare these levels with those of the CA125 antigen. While serum CA54/61 antigen levels were elevated in 44.4% of ovarian cancer cases and serum CA125 antigen levels were elevated in 86.7% of the same population, the use of both assays indicated a sensitivity of detection of 97.8% (44 of 45 patients) in the population studied.

Antibodies, Monoclonal

Clinical significance of urokinase-type plasminogen activator (uPA) in invasive cervical cancer of the uterus.

The relationship between the level of urokinase-type plasminogen activator (uPA) and pelvic lymph node metastasis was investigated in 20 patients with invasive cervical cancer of the uterus. Frozen sections from all surgical specimens were stained immunohistochemically by alkaline phosphatase anti-alkaline phosphatase method to detect uPA in cancer tissue. The concentration of uPA, determined immunologically, and the fibrinolytic activity were also examined in supernatants of homogenates of some cancer tissues. uPA was detected immunohistochemically in cancer cells of all specimens. A significant correlation was found between the extent of immunohistochemical staining for uPA and the concentration of uPA determined immunologically in cancer tissues (P less than 0.05). A positive correlation was also found between semiquantitative values determined by immunohistochemical staining for uPA and lymph node metastasis (P less than 0.05). Fibrinolytic activity in cancer tissues was confirmed by casein-plaque assay. These findings indicate that the pro-uPA/uPA contents in cervical cancer tissues are clinically useful for predicting the metastatic potential of these cancers to the pelvic lymph nodes.

Carcinoma, Squamous Cell

Effect of water mobility on drug hydrolysis rates in gelatin gels.

The stability of drugs incorporated in gelatin gels was studied, with a focus on the water mobility in the gels. Trichlormethiazide hydrolysis and kanamycin-catalyzed flomoxef hydrolysis in gelatin gels were chosen as models for apparent first-order and second-order hydrolysis, respectively. The mobility of water in gelatin gels was determined by NMR, ESR, and dielectric relaxation spectroscopies. The amount of bound water in the gels was determined from dielectric relaxation spectra. Spin-lattice relaxation time of water determined by 17O NMR and rotational correlation time of an ESR probe determined by an ESR probing method were useful in determining the microviscosity of the gels. The hydrolysis rate of trichlormethiazide in the gels was found to depend on the amount of free water available for the reaction, while that of flomoxef depended on the microviscosity of the gels, which reflected the mobility of water molecules. Thus the dependence of hydrolysis rates on the water mobility was influenced by the hydrolysis mechanism.

Cephalosporins

Identification of metallothionein in cultured cells by immunoblotting and immunofluorescence using a new monoclonal antibody.

A hybridoma clone (MT45-5-3) producing an IgG-class monoclonal antibody specific for metallothioneins (MTs) was established. The monoclonal antibody (MT45) cross-reacted with mouse, rat and rabbit Cd(2+)-induced MTs 1 and 2 and Zn(2+)-induced MT 2 as assessed by enzyme-linked immunosorbent assay. When the antibody was used to detect the MTs transferred to nylon membranes after SDS-polyacrylamide gel electrophoresis, the antibody reacted with cultured human cell MTs as well as rat, mouse and rabbit MTs 1 and 2 even after carboxymethylation. The antibody could be used for the indirect immunofluorescence test for Cd(2+)-induced MTs in cultured human and mouse cells.

Animals

Elastase activity of endocervical mucus in normal pregnancy.

Elastase activity of 294 samples of endocervical mucus taken from 125 normal pregnant women during the visits to antenatal clinic were measured. Activity of the enzyme increased gradually with the advance of gestational age. Steep rising of the enzyme was seen between 34-35 weeks of gestation and the peak was achieved at 37 week since then the enzyme activity sustained at relatively high level until deliveries. Average activity of the enzyme after 34 weeks of gestation showed significant difference over that of earlier period. To use delivery date as a referent point, the peak was reached in the last fourth week of pregnancies and then sustained until deliveries. Significantly higher average activity was also seen in these last 4 weeks of gestation. These correlated very well to the fact that cervical maturation progresses most rapidly during last month of pregnancy. Immunohistochemical staining for elastase demonstrated elastase-containing granules in polymorphonuclear leukocytes. These cells found in cervical tissue taken from immediately postpartal women were much more than in those of early pregnant women. These findings may be the evidences support roles of granulocyte elastase in the process of cervical maturation preceding spontaneous labors in normal pregnancies.

Cervix Mucus

Relationship of serum levels of pro-type I collagen peptide, pro-type III collagen peptide and type IV 7S collagen with cervical maturation.

The serum levels of pro-type I collagen peptide (PIP), pro-type III collagen peptide (PIIIP) and type IV 7S collagen (7S collagen) were measured in 41 women in various gestational weeks of pregnancy (5-41 weeks). The PIP level did not change during pregnancy, being 450 +/- 380 mg/ml before week 37 and 448 +/- 280 mg/ml after week 37. The PIIIP level also did not change being 0.55 +/- 0.21 and 0.59 +/- 0.22 U/ml before and after 37 weeks. The PIIIP level increased slightly but not significantly after week 37. The 7S collagen level, however, increased significantly (p less than 0.01), being 5.1 +/- 0.8 and 6.6 +/- 0.8 mg/ml before and after week 37. The correlation coefficients with the Bishop score were 0.22 for PIP, 0.42 for PIIIP, and 0.72 for 7S collagen. These data suggest that serum 7S collagen closely reflects collagen degradation in the cervix and so should be a good marker of cervical maturation.

Cervix Uteri

Plasma fibronectin receptor levels during pregnancy complicated by preeclampsia and abruptio placentae.

The level of human fibronectin receptor (FNR) in plasma was measured by enzyme-linked immunosorbent assay in samples from normal pregnant women in the 1st trimester (n = 5), 2nd trimester (n = 7), 3rd trimester (n = 23), normal postpartum women day 1 (n = 4), day 2 (n = 5), day 3 (n = 8), nonpregnant women (n = 18), 20 preeclamptic patients in the 3rd trimester, and 8 patients with abruptio placentae in the 3rd trimester. In normal pregnancy, the mean value of FNR was 1.4 +/- 0.4 micrograms/ml in the 1st, 1.4 +/- 0.2 micrograms/ml in the 2nd, and 1.9 +/- 0.3 micrograms/ml (p less than 0.05) in the 3rd trimester. FNR values increased with pregnancy. During the puerperium, its level decreased with time, being 1.4 +/- 0.5 micrograms/ml (p less than 0.01) on day 1, 1.0 +/- 0.3 micrograms/ml on day 2, and 0.8 +/- 0.2 micrograms/ml on day 3. The level in preeclamptic patients was 2.0 +/- 0.4 micrograms/ml, and that in abruptio placentae was 2.7 +/- 0.4 micrograms/ml. There were significant differences between the levels in abruptio placentae versus preeclampsia (p less than 0.05) and 3rd-trimester normal pregnant women (p less than 0.01). In the immunohistochemical study, the surface of normal decidual cells stained weakly for FNR, and the decidual cell membranes of the cases of preeclampsia stained moderately or strongly. Decidual cells and their extracellular matrix close to hematomas of abruptio placentae stained very strongly for FNR.(ABSTRACT TRUNCATED AT 250 WORDS)

Abruptio Placentae

Serum sialyl Tn as an independent predictor of poor prognosis in patients with epithelial ovarian cancer.

PURPOSE: Monoclonal antibody (moAB) TKH-2 directed to the tumor-associated O-linked sialyl 2-6-alpha-N-acetylgalactosaminyl (sialyl Tn; STN) epitope was generated by immunization with ovine submaxillary mucin (Kjeldsen et al, Cancer Res 48:2214-2220, 1988). We investigated whether circulating serum levels of STN antigen might influence the prognosis of patients with ovarian cancer. PATIENTS AND METHODS: Serum samples were obtained from 126 healthy nonpregnant women, 157 patients with benign gynecologic disease, and 89 patients with histologically proven epithelial ovarian cancer. Circulating serum STN-antigen concentrations (U/mL) were determined by a competitive radioimmunoassay kit (Otsuka Assay Laboratories, Tokushima, Japan) in a one-step procedure. RESULTS: Serum antigen levels were elevated in 48.3% of the patients. The levels of STN antigen were significantly higher in the sera of patients with cancer when compared with levels in benign and healthy controls (P less than .05). The 5-year survival rate for patients with STN-negative (serum STN levels less than 50.0 U/mL) versus STN-positive (greater than or equal to 50 U/mL) tumors was 76.9% versus 10.8%, respectively (P less than .05). The progression-free interval (PFI) at 5 years was 51.9% versus 5.4%, respectively (P less than .05). The overall survival probability and PFI were worse in patients with STN-positive sera. Multivariate regression analysis revealed that stage, residual tumor size, positive STN, performance status, and histologic grade were the five important variables for predicting overall survival. CONCLUSION: We conclude that a positive STN-antigen level in sera is an independent predictor of poor prognosis in ovarian cancer.

Adult

Amino acids and peptides. XXXIII. Synthesis of N-terminal epitope peptides of mammalian metallothioneins (MTs).

In order to determine the fine structure of the mammalian metallothionein (MT) epitope to a monoclonal anti-rat Zn-MT-II antibody (MT 189-14-7), N-terminal peptides of various lengths of mammalian metallothioneins (MTs) were synthesized by a conventional solution method using the newly developed beta-2-adamantylaspartate, and their immunological properties were examined. It was found that the N-terminal acetyl group was indispensable for the reaction with the monoclonal antibody and the N-terminally acetylated pentapeptide, Ac-Met-Asp-Pro-Asn-Cys-OH, was the smallest peptide which exhibited a significant reactivity with the antibody.

Amino Acid Sequence