Biomedical subjects
T Terao
Publications and source records attributed to T Terao.
Impact of urokinase-type plasminogen activator and its inhibitor type 1 on prognosis in cervical cancer of the uterus.
The present study was undertaken to assess the role of tumor-associated urokinase-type plasminogen activator (uPA) and its inhibitor type 1 (PAI-1) as a predictor for early relapse and poor prognosis in patients with stage II cervical cancer of the uterus. We have investigated the localization of uPA and PAI-1 immunohistochemically in formalin-fixed paraffin-embedded tissue sections. uPA and PAI-1 were analyzed antigenically, enzymologically, and zymographically in 28 patients with pelvic lymph node involvement and in 34 cases without nodal spread, as well as in 10 cases with normal cervix. In cancer tissues, strong staining for uPA was found in areas with invasive growth and degradation of surrounding normal tissue, while most tumor nests showed a mild or a moderate, evenly distributed PAI-1 staining. A significantly higher lymph node-positive rate was observed in patients having tumors with strong uPA and/or PAI-1 stainings than in those with tumors with weak stainings. In spite of significantly higher PAI-1 levels in the primary neoplastic tissues, uPA was found to be increased as well, both in antigen level and in activity. Most of PAI-1 obtained from cancer extracts is the latent form. These results suggest that cancer-associated increase in uPA seems not to be affected (or inhibited) by PAI-1 in areas where tumor cells are invading normal tissue. The overall survival and progression-free survival rate was worst in patients with the strong uPA staining confined to the tumor stromas and also with the strong PAI-1 staining at tumor nests, indicating that the greater localization of uPA in stromal cells than in malignant cells is a predictor of early relapse and poor prognosis in patients with cervical cancer of the uterus. Thus, the staining intensities and the localization of uPA and PAI-1 in tissue specimens appear to be predictors of increasing risk for lymph node metastasis, suggesting that some tumor cells recruit stromal cells to produce uPA and that PAI-1 may not act as a defense mechanism for tumor cell invasion and metastasis in the leading edge of tumor growth.
Endothelin-1 increased immunoreactive von Willebrand factor in endothelial cells and induced micro thrombosis in rats.
This study was performed (i) to investigate the interaction between ET-1 and endothelial cells and (ii) to study the role of ET-1 in in vivo thrombosis. Fura-2AM loaded human umbilical endothelial cell cultures were incubated with 0, 25, 50 and 100 pmol of ET-1 for 24 hours (n = 6) at 37 degrees C. Fura-2 released in the media was measured by spectroflurophotometer at wavelength of 350 nm excitation and 500 nm emission. We found significant (p < 0.01) and dose dependent decrease in Fura-2 release by the cells indicating increased intracellular calcium in HUVEC. Increased calcium by ET-1 was also confirmed at single cell level by fluorescence digital image analysis using Fura-2AM. 5 ml solution of ET-1 (100 pmol/ml) was injected within the venous lumen of umbilical cords (of normal pregnancy) clumped at both ends and incubated at a temperature 37 degrees C for 3 hours (n = 7). We found intensely stained immunoreactive von Willebrand factor (vWF) on the endothelial cells of ET treated umbilical cords when compared with sham control (Umbilical cords incubated with phosphate buffer saline; n = 7). Intravenous ET-1 infusions at a rate of 1 nmol/kg/hour for 2 hours (cases, n = 7) and 5% dextrose infusions (sham control, n = 7) were performed in rats. Aorta, kidney and liver tissues were obtained to perform immunostaining with polyclonal antibody to vWF and fibrinogen. ET-1 treated rat tissues showed intense staining for vWF and fibrinogen intravascularly at hte same site.(ABSTRACT TRUNCATED AT 250 WORDS)
Characterization of the cellular binding site for the urinary trypsin inhibitor.
A highly purified urinary trypsin inhibitor (UTI) inhibits not only tumor cell invasion in an in vitro assay but also production of experimental and spontaneous lung metastasis in an in vivo mouse model. UTI is present both in the lysate of tumor cells (human choriocarcinoma SMT-cc1 cells, human promyeloid leukemia U937 cells, and murine Lewis lung carcinoma 3LL cells) and human neutrophils. In each medium from tumor cells, most of the cell-associated UTI is on the cell surface. Cell-binding experiments employing cell enzyme-linked immunosorbent assay and flow cytometry indicated that tumor cells (SMT-cc1 and 3LL cells) have specific binding sites for UTI on their cell surface. UTI binds rapidly and with relatively high affinity to SMT-cc1 and 3LL cells. UTI is bound to a specific surface receptor that is incompletely saturated. U937 cells and neutrophils did not show any specific binding to UTI, since UTI receptors on the cell surface of U937 cells and neutrophils were completely saturated with endogenous UTI. UTI forms yielded cross-linked 150- and 80-kDa ligand receptor complexes with cultured SMT-cc1 cells, suggesting molecular masses of 110 and 40 kDa for the UTI receptors. Purification of the two UTI receptor proteins by ligand affinity chromatography (ligand-blotting analysis) yielded two bands when analyzed by SDS-polyacrylamide gel electrophoresis, corresponding in electrophoretic mobility to those calculated by cross-linking analysis. The results reported here showing that some tumor cells carry UTI receptors are in line with a possible role of surface-bound UTI in modulating plasmin activity to the close environment of the cell surface and in processes like prevention of tumor cell invasion and metastasis.
Inhibition of metastasis of Lewis lung carcinoma by a synthetic peptide within growth factor-like domain of urokinase in the experimental and spontaneous metastasis model.
Four synthetic peptides (residues 20-30 and 17-34) within the growth factor-like domain (GFD) of murine and human urokinase-type plasminogen activator (uPA) were examined to determine whether they inhibit production of experimental and spontaneous lung metastasis by murine Lewis lung carcinoma (3LL) cells. In an in vivo experimental metastasis assay, which determines mainly the later steps of the metastatic migration process (extravasation from the bloodstream and then growth into pulmonary tumor), none of the peptides introduced by i.v. single co-injection into syngeneic C57B1/6 mice inhibited pulmonary metastasis, when 3LL cells were pre-incubated with the peptides followed by i.v. co-injection of the peptide and cells. In addition, none of the peptides, when injected i.p. daily for 7 days after i.v. tumor cell inoculation, reduced the number of lung tumor colonies. In a second in vivo assay that measures metastasis from a primary tumor (spontaneous metastasis model), multiple i.p. injections of the mouse peptide 17-34 for 7 days after s.c. tumor cell inoculation significantly inhibited metastatic lung tumor colonization in a dose-dependent manner, whereas human peptide 17-34 had no effect. Mouse and human peptide 20-30 had no effect either. The inhibition of lung metastasis was not due to direct antitumor effects of mouse peptide 17-34. Our results indicate that occupation of uPA receptors on 3LL cells by the enzymatically inactive mouse peptide 17-34 or prevention of rebinding of uPA synthesized by tumor cells to their receptor specifically reduced tumor cell invasion and formation of metastasis and that uPA may regulate more efficiently the mechanism involved in the entry of tumor cells into vascular circulation than extravasation during the metastatic process.
Effects of urinary trypsin inhibitor on the invasion of reconstituted basement membranes by ovarian cancer cells.
Using the human ovarian cancer cell line HOC-1, we investigated the effects of urinary trypsin inhibitor (UTI) purified from human urine and its related synthetic peptides on the invasive potential of cancer cells in an in vitro assay. Invasiveness of tumor cells was determined using a modified Boyden chamber and a reconstituted basement membrane Matrigel. Three peptides (peptide 1, peptide 2, and peptide 3), representing sequences within UTI, were synthesized. HOC-1 cells showed detectable and reproducible levels of expression of surface urokinase-type plasminogen activator (uPA) and plasminogen/plasmin by cell ELISAs and enzyme assays. UTI was found to strongly inhibit plasmin and human leukocyte elastase (HLE). Peptide 2 and peptide 3 specifically inhibit HLE and plasmin activity respectively. Peptide 1 has essentially no inhibitory activity. Treatment with UTI and peptide 3 reduces the incidence of invasion, whereas peptide 1 and peptide 2 do not affect invasion. The inhibitory effect on cell invasion is dose-dependent. The proteolytic enzyme plasmin may be involved in human ovarian cancer invasion in extracellular matrix degradation, and the use of UTI and peptide 3 that inhibits plasmin specifically reduces invasion by tumor cells.
Scaling properties of the dynamical structure factor of percolating Heisenberg antiferromagnets.
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Early antipsychotic response to resumption of neuroleptics in drug-free chronic schizophrenic patients.
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Effects of herbimycin A and ST638 on Fc epsilon receptor-mediated histamine release and Ca2+ signals in rat basophilic leukemia (RBL-2H3) cells.
We examined the effect of the two protein tyrosine kinase inhibitors, alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamide (ST638) and herbimycin A, on the activation processes of rat basophilic leukemia (RBL-2H3) cells by cross-linking of IgE receptors. RBL-2H3 cells sensitized with DNP-specific monoclonal IgE antibody were stimulated with multivalent antigen (DNP conjugate of bovine serum albumin). Analysis of phosphotyrosine-containing proteins in their lysates by SDS-PAGE and immunoblotting revealed that these two inhibitors efficiently inhibited the tyrosine phosphorylation of several proteins (32, 42, 56, 66, 72, 92, 150 kDa) including phospholipase C-gamma 1. The inhibitors also caused parallel inhibitions of the histamine release, the formation of inositol 1,4,5-trisphosphate, and the increase in cytosolic calcium ion concentration at the late sustained phase. A digital imaging fluorescence microscopic analysis of antigen-dependent calcium signals in individual cells showed that these two tyrosine kinase inhibitors inhibited the calcium influx from the external medium more powerfully than the mobilization of calcium ion from internal stores. In contrast, the inhibitors did not affect the increase in the cytosolic calcium ion concentration or the histamine release induced by the calcium ionophore A23187. Taken together, our results suggest that tyrosine phosphorylation following antigen stimulation regulates phosphatidylinositol hydrolysis and the influx of extracellular calcium.
Sensitive determination of N-terminal prolyl peptides by high-performance liquid chromatography with laser-induced fluorescence detection.
Short-chain peptides with an N-terminal proline (Pro-Gly, Pro-Ile, Pro-Gly-Gly, Pro-Leu-Gly-NH2, and Pro-Thr-Pro-Ser-NH2, etc.) were determined by HPLC with laser-induced fluorescence (LIF) detection. The peptides were quantitatively labelled with 4-(N,N-dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) at 50 degrees C after 1 h in a 0.1 M borax (pH 9.3)-acetonitrile mixture. The rate of reaction decreases inversely with the molecular weight of the peptides. The mean value of fluorescent emission of the resulting DBD-peptides and DBD-peptide amides was 573 nm (excitation, 453 nm). The proline peptides, including bioactive peptides such as Pro-Leu-Gly-NH2 (release inhibitor of melanocyte-stimulating hormone), Pro-Thr-Pro-Ser-NH2 (IgA1 proteinase inhibitor) and Pro-Asp-Val-Asp-His-Val- Phe-Leu-Arg-Phe-NH2 [FMRF amide-like (Phe-Met-Arg-Phe-NH2) neuropeptide], were well separated by reversed-phase HPLC with water-acetonitrile containing 0.1% trifluoroacetic acid (TFA). The acetonitrile concentration in the mobile phase had a profound effect upon the retention times, and the capacity factors (k') were dependent on the hydrophobicity of the peptides. The structure of DBD-Pro-Leu-Gly-NH2 was identified by LC-atmospheric pressure chemical ionization MS. The chromatographic detection limits (S/N = 2) of the peptides with a 15-mW argon-ion laser at 488 nm were in the 6-28 fmol range. The detection limits were improved to 2-5 fmol with a microbore column. The detectability was two orders of magnitude higher than with a conventional fluorescence detector using xenon arc lamp.
Inhibition of the soluble and the tumor cell receptor-bound plasmin by urinary trypsin inhibitor and subsequent effects on tumor cell invasion and metastasis.
The present study was undertaken to determine whether highly purified human urinary trypsin inhibitor (UTI) efficiently inhibits the soluble and the tumor cell receptor-bound plasmin. The ability of plasmin inhibitors to regulate invasion by tumor cells which express membrane-associated plasmin was also examined. UTI and two other plasmin inhibitors [alpha 2-anti-plasmin (alpha 2AP) and alpha 2-macroglobulin (alpha 2M)] were used. alpha 2AP and alpha 2M, as well as UTI, rapidly inactivate the soluble plasmin that is not bound to cells. Experiments were performed in vitro using cultures of ovarian cancer HOC-I cells and gestational choriocarcinoma SMT-ccl cells. HOC-I and SMT-ccl cells had plasmin(ogen) on their cell surface, and the plasmin activity was detected on their cell surface enzymologically and immunologically. Receptor-bound plasmin reacted effectively with UTI and was directly inactivated by UTI. In contrast, receptor-bound plasmin was not inhibited by alpha 2AP and alpha 2M. Using a modified Boyden chamber and an artificial basement membrane, Matrigel, it was found that UTI, but not alpha 2AP or alpha 2M, can inhibit HOC-I and SMT-ccl cells invasion in vitro. Furthermore, in the experimental lung metastasis model, UTI inhibited the formation of lung metastasis by Lewis lung carcinoma cells. The inhibition of tumor cell invasion was not due to direct antitumor effects of UTI. These results suggest that inhibition of receptor-bound plasmin by UTI is associated with significantly reduced tumor cell invasiveness in vitro and with a decreased number of metastasis in vivo.
Role of activated protein C in facilitating basement membrane invasion by tumor cells.
The present study was undertaken to investigate the role of plasminogen activator inhibitor type 1 (PAI-1) and activated protein C (APC) in the regulation of tumor cell invasion. PAI-1 was purified in active form from conditioned medium of human umbilical vein endothelial cells under denaturing conditions (4 M guanidine-HCl). The purified inhibitor reacts with urokinase-type plasminogen activator (uPA) and APC. Two selected human lines, HOC-I (ovarian cancer cells) and SMT-ccl (choriocarcinoma cells), preferentially invaded through reconstituted basement membranes in an in vitro invasion assay using a modified Boyden chamber. The present study determined the efficacy of these two agents (PAI-1 and APC) used alone or in combination in inhibiting or facilitating tumor cell invasion. Active PAI-1 inhibited the tumor cell surface receptor-bound uPA activity. In an in vitro invasion assay, active PAI-1 reduced tumor cell invasive potential in a dose-dependent manner. When SMT-ccl cells saturated with uPA-PAI-1 complexes were treated with a 50-fold molar excess of APC, PAI-1-APC complex was demonstrated in conditioned medium, indicating that PAI-1 was dissociated from receptor-bound uPA on tumor cells and that tumor cell-associated uPA restored its enzymatic activity. Although APC alone had no effect on tumor cell invasion, the addition of APC to the cells saturated with uPA-PAI-1 complexes showed regeneration of tumor cell surface receptor-bound uPA activity and produced substantial and efficient invading effects. These data suggest that PAI-1 activity may be neutralized by APC or that APC may promote tumor cell invasion via inactivation of PAI-1 by formation of a stable PAI-1-APC complex. These observations suggest that APC may play a critical role in the initiation of a hematogenous metastatic process (extravasation step).
Analysis of monoclonal antibodies reactive with meconium- and amniotic fluid-derived mucin.
The present study was undertaken to determine whether monoclonal antibodies (moABs TKH-2, MA54, MA61, B72.3, and CC49) directed toward O-linked mucin-type glycoprotein detect the NeuAc alpha 2-6GalNAc (sialyl Tn) epitope in meconium- and amniotic fluid-derived mucin. Fetal colonic mucosal cells express the sialyl Tn antigen, particularly in goblet cell mucin. The reactivities of these moABs with a perchloric acid extract of meconium (meconium extract) and different native and neuraminidase treated glycoproteins were examined by solid-phase enzyme-linked immunosorbent assay (ELISA). All the moABs react with the meconium supernatant and meconium extract. The reactivities of TKH-2, MA54, and MA61 are neuraminidase sensitive, and the reactivity of TKH-2 with meconium extract was specifically inhibited by ovine submaxillary mucin (OSM). A NeuAc alpha 2-6GalNAc epitope is the characteristic component in meconium. Mucin released from the fetal respiratory tract could, in part, provide an alternative source in the amniotic fluid. TKH-2 is the most sensitive antibody directed to sialyl Tn antigen in meconium supernatant. The likelihood of TKH-2 serving as the basis for a sensitive assay to detect sialyl Tn in meconium- and amniotic fluid-derived mucin is provided.
Application of accelerated testing to shelf-life prediction of commercial protein preparations.
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Plasmin modulators, aprotinin and anti-catalytic plasmin antibody, efficiently inhibit destruction of bovine vascular endothelial cells by choriocarcinoma cells.
The interaction of human gestational choriocarcinoma cell line, SMT-cc1, with bovine vascular endothelial cells, CPAE, was examined in an in vitro coculture model. SMT-cc1 cells have urokinase-type plasminogen activator (uPA) on their cell surface, and more than half of the cell-associated uPA is enzymatically inactive single-chain uPA (pro-uPA). uPA is bound to a specific surface receptor that is not completely saturated. Also, the plasmin activity is detected on their cell surface. We measured the ability of added SMT-cc1 cells to cause morphological changes in CPAE cells, leading to destruction of CPAE cells. SMT-cc1 cells that adhered to CPAE cell monolayers were capable of causing CPAE cell destruction, followed by detachment, within 6 hr after coculture. Nonspecific serine proteinase inhibitor, aprotinin, and anti-catalytic antibody against plasmin(ogen) effectively inhibited the destruction/detachment in a dose-dependent manner. SMT-cc1 cell-mediated CPAE cell destruction was suppressed for 6 hr in the presence of aprotinin, at a concentration of 20 micrograms/ml, or anti-plasmin antibody, at a concentration of > or = 10 micrograms/ml, suggesting that the destruction is closely related to the proteolytic enzyme, plasmin. We suggest that the destruction of endothelial cells by some tumor cells is associated with tumor cell-associated proteolytic activity, and the uPA-plasmin cascade plays an important role as a critical step during blood-borne metastasis.
Urinary trypsin inhibitor (UTI) and fragments derived from UTI by limited proteolysis efficiently inhibit tumor cell invasion.
We investigated the effects of purified human urinary trypsin inhibitor (UTI) and fragments derived from UTI by proteolysis on the invasive potential of ovarian cancer cells (HOC-I) and gestational choriocarcinoma cells (SMT-ccl) using an in vitro reconstituted basement membrane invasion assay. These cells express cell-associated plasmin and functional uPA receptors that are partially occupied by ligands. SMT-ccl cells, which express threefold higher levels of cell-associated plasmin activity than HOC-I cells, showed approximately twofold increase in their invasive potential. For the invasion assay, HOC-I cells were primed with exogenous plasminogen, but SMT-ccl cells were not. Human leukocyte elastase (HLE)-digested UTI (22 kDa fragment; UTI-22) inhibited plasmin practically with the same strength as native UTI. Trypsin-digested UTI (20 kDa fragment; UTI-20), however, did not inhibit plasmin significantly. Treatment of cells with UTI or UTI-22 reduced the incidence of tumor cell invasive capacity, whereas the inhibitory effect of UTI-20 was not remarkable. The inhibitory effect on tumor cell invasion was dose-dependent and non-toxic; moreover, it was not mediated by inhibition of the tumor cell chemotactic response or of cell attachment to matrigel. These results indicate that inhibition of the proteolytic enzyme plasmin specifically reduced the invasive capacity of tumor cells in vitro.
Interleukin-8 induces cervical ripening in rabbits.
OBJECTIVE: The purpose of this study was to determine whether cervical ripening can be induced in rabbits by interleukin-8. STUDY DESIGN: Nonpregnant and pregnant rabbits were treated for 5 days with vaginal suppositories containing 100 ng of interleukin-8. Collagen and glycosaminoglycan concentration in the cervices were assessed histologically by picrosirius red and alcian blue, and the mean optical density was calculated. The mean neutrophil count in five random fields was calculated from each biopsy specimen. RESULTS: Interleukin-8 induced softening and dilatation of the rabbit cervices. Water content was significantly increased (p < 0.0001 and p < 0.001, respectively). Cervical collagen concentration was found to be significantly decreased (p < 0.0004 and p < 0.0001, respectively). Glycosaminoglycan concentration was significantly increased in nonpregnant and pregnant cervices (p < 0.0009 and p < 0.1, respectively). The mean number of neutrophils was significantly increased (p < 0.0005 and p < 0.006, respectively). CONCLUSION: Interleukin-8 can induce cervical ripening in nonpregnant and pregnant rabbits.
Entrapment of proteins in poly(L-lactide) microspheres using reversed micelle solvent evaporation.
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