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.

Animals↗

PubMed1994

Immunohistochemical characterization of a stage-specific antigen during oogenesis and spermatogenesis recognized with monoclonal antibody.

In order to detect and characterize novel molecules which function in oogenesis, immunohistochemical study using a monoclonal antibody (MAb) raised against oocytes of Xenopus laevis was carried out. The distribution of the detectable molecule with MAb X-80, which specifically reacts with Xenopus previtellogenic oocytes; stages I and II (Dumont, 1972), in particular was analyzed. The hamster, mouse, chick, quail, Caenorhabditis elegans and Lilium longiflorum were also examined. Interestingly MAb X-80 bound not only in Xenopus oocytes but in its secondary spermatocytes and spermatids. Furthermore, MAb X-80 gave a similar staining pattern in the Lilium and other, although the stage when the positive reaction is detectable is different in the male and female germ cells.

Animals↗

PubMed1994

Treatment of choice for unresectable small liver cancer: percutaneous ethanol injection therapy or transarterial chemoembolization therapy.

Early detection of hepatocellular carcinoma (HCC) has become easier with recent advances in imaging diagnosis, but the tumor is frequently unresectable due to underlying advanced liver cirrhosis. In this study, we evaluated the therapeutic effect of percutaneous ethanol injection therapy (PEIT) and transarterial chemoembolization therapy (TACE) for small liver cancers measuring 3 cm or less in diameter and discussed the treatment of choice for unresectable cases. The tumors were divided into two groups on the basis of size: 1.5 cm or less (group A) and 1.6-3 cm in diameter (group B). In group A, the estimated 1- and 3-year survival rates were both 82% for a total of 19 cases. The survival value determined for 10 patients treated with PEIT was slightly higher than that found for 9 patients treated with TACE. In group B, the overall 1-, 2-, and 3-year survival values for a total of 56 patients were estimated at 83%, 60%, and 35%, respectively. The survival rates for 41 patients treated with TACE were 82%, 53%, and 28% at 1, 2, and 3 years, respectively. PEIT was performed on only 6 patients, whose survival rate was equivalent to that of a surgical resection group. The 1-, 2-, and 3-year survival rates for 9 patients who underwent surgical resection were estimated to be 100%, 85%, and 68%, respectively. Based on these results, PEIT seems to be the treatment of first choice for patients with small liver cancers measuring less than 1.5 cm in diameter if the tumor is thought to be unresectable because of associated severe liver cirrhosis. On the other hand, tumors measuring 1.6-3 cm in diameter must first be treated with TACE using a long-acting Lipiodol-carcinostatic suspension, even if resectable. In addition to the tumor size, dynamic CT is also useful for prospective decision of the therapeutic strategy. If the mass is demonstrated to be a hypervascular lesion by dynamic CT, TACE must be selected as the treatment of first choice, even for small lesions measuring 1.5 cm or less in diameter.

Aged↗

PubMed1994

Role of myosin light-chain kinase and protein kinase C in pepsinogen secretion from guinea pig gastric chief cells in monolayer culture.

We evaluated the role of myosin light-chain kinase (MLCK) and protein kinase C (PKC) in pepsinogen secretion from guinea pig gastric chief cells using a monolayer culture system of chief cells and an enzyme immunoassay system for guinea pig pepsinogen. An MLCK inhibitor, 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9), significantly inhibited both the basal pepsinogen secretion and the secretion by carbamylcholine chloride (carbachol) or ionomycin without affecting intracellular free Ca2+ concentration ([Ca2+]i), but not by 12-O-tetradecanoylphorbol-13- acetate (TPA) or forskolin. A PKC inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), significantly reduced the pepsinogen secretion by carbachol or TPA, but not by forskolin or ionomycin, and did not affect the basal secretion and the [Ca2+]i elevated by carbachol or ionomycin. We concluded that: (1) MLCK plays an important role in basal and drug-stimulated pepsinogen secretion, (2) MLCK is involved in the Ca(2+)-dependent intracellular pathway but not in the cyclic adenosine monophosphate (cAMP) dependent pathway, (3) PKC is irrelevant to activation of MLCK, and (4) increases in cAMP and [Ca2+]i are independent of activation of PKC.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1994

Successful pregnancy in a patient with endometrial carcinoma treated with medroxyprogesterone acetate.

A 32-year infertile Japanese woman suffered from endometrial cancer, which was treated with repeated endometrial curettage and medroxyprogesterone acetate for 6 months. The patient then became pregnant and was delivered of a live-born male infant at 37 weeks gestation by cesarean section for placenta previa. At cesarean section there were no abnormal findings in the uterine cavity or other pelvic organs.

Adenocarcinoma↗

PubMed1994

Assessment of dopamine metabolism in brain of patients with dementia by means of 18F-fluorodopa and PET.

By means of positron emission tomography (PET) and 18F-fluorodopa (FDOPA), a study was initiated to analyze the cerebral dopamine (DA) metabolism of 32 subjects including those with AD/SDAT and vascular dementia (VD, multi-infarct type). A semiautomated irregular ROI drawing routine to identify the striatum was developed that interactively defined the PET threshold pixels referring to the count histograms and location of the corresponding pixels. A comparative study by five examiners showed significant improvement in the area size definition and count linearity particularly for low contrast objects. The graphical plot was employed to calculate the FDOPA influx rate (Ki) for the ROI data with cerebellar radioactivity as an input function. The striatal Ki value was found to be relatively stable and did not show signs of a significant age-related change. The vascular patients had smaller Ki to the striatum than the aged control. Although the mean Ki of AD/SDAT was almost compatible with that of age-matched normals, their Ki was more scattered with higher and lower Ki cases. The multiple regression analysis revealed that the Ki could be predicted by age and the mini-mental state (MMS) performance (r2 = 0.590, p < 0.01 for AD/SDAT, r2 = 0.401, and p < 0.05 for VD). MMS was found to be a more dominant factor than age. We conclude that dopamine metabolism became disturbed as dementia became progressively severe.

Aged↗

PubMed1994

Metabolic disturbances in exo-focal brain areas after cortical stroke studied by positron emission tomography.

We have reported that exo-focal delayed neuronal damage was observed in the ipsilateral thalamus and the substantia nigra of the rat brain after occlusion of the middle cerebral artery (MCA). To determine if that phenomenon also occurs in humans, we measured cerebral metabolic rates for glucose (CMRGlc) in the remote brain areas at a chronic stage after cortical infarction using 2-[18F]fluoro-2-deoxy-D-glucose and position emission tomography (PET). The subjects studied were 11 patients who were affected by unilateral cerebral infarction in the cortex supplied by MCA. There were significant decreases of CMRGlc as compared with control values (p < 0.01), not only in the cerebral cortex directly damaged by the ischemic insult, but also in the ipsilateral thalamus and in the contralateral cerebellum, areas in which no lesions had been detected by MRI or CT scan. The present study indicates that different mechanisms may be responsible for multi-focal metabolic disturbances in the remote areas after stroke. The reduction of CMRGlc in the contralateral cerebellum may be explained by the crossed cerebellar diaschisis theory and in the ipsilateral thalamus as being due to retrograde degeneration associated with the infarcted cortex. We suggest that these multi-focal brain dysfunctions caused by neuronal network disturbances may exacerbate clinical symptoms at a chronic stage of stroke.

Adult↗

PubMed1994

Radial arm maze behavior in mice when a return to the home cage serves as the reinforcer.

Male ddY mice were housed in a cage with an eight arm radial maze apparatus for 6 h a day. A water bottle was placed at the central platform. The end of each arm ran to the home cage through a guillotine door (G). Food was placed at the home cage. During the housing in the apparatus, one G was raised and the remaining seven G were lowered. The raised G was changed every 45 min in random order. Mice housed in this apparatus learned efficient strategy to return the home cage by trial and error. When they chose the arm in which the G was lowered on their way to the home cage from the platform, they returned to the platform, then chose a different arm until they were able to enter the home cage. The mice housed in this apparatus mastered the radial maze task on the 7th day. When scopolamine (SCO) was injected, SCO butylbromide had no effect on performance, but SCO hydrobromide (0.2 and 0.4 mg/kg) impaired working memory, dose dependently. These results show that the apparatus is useful for ease in estimating working memory in mice without the use of severe food or water deprivation.

Animals↗

PubMed1994

Production of a monoclonal antibody 14A20 that reacts with a 220-kDa protein in the islet cells.

To study the target antigens of the islet cell cytoplasmic antibodies (ICA), we tried to make a monoclonal islet cell antibody. We made a monoclonal antibody 14A20 (IgM, kappa) that reacted strongly with Wistar and BB rat islet cells and weakly with pancreatic acinar cells. The immunoreactivity of the 14A20 was also found to react extensively in neuroendocrine cells. Immunostaining showed a granular pattern in both neurons and islet cells. Chemical and enzymic studies revealed that the antigen has the property of a protein. We compared immunoreactivity of the monoclonal antibody 14A20 with anti-insulin, -glucagon or -somatostatin antibodies. The antigen recognized by the monoclonal antibody 14A20 was equally present in the alpha, beta, and delta cells of islet, which mimicked the immunoreactivity of the ICA in the pancreas. The recognized protein had a relative molecular weight of 220,000, indicating that it is different from the previously identified target antigen of the ICA.

Animals↗

PubMed1994

Cerebral oxygenation in childhood moyamoya disease investigated with near-infrared spectrophotometry.

In children with moyamoya disease, the time courses of hemoglobin concentrations before and after hyperventilation were examined using near-infrared spectrophotometry (NIRS) to determine brain tissue oxygenation. Five patients were studied: 1 was studied both before and after surgery and 4 were studied only after surgery. Nine hemispheres of these 5 patients were examined. The oxygenated hemoglobin concentration increased immediately after beginning hyperventilation. This parameter later decreased sharply to below the prehyperventilation level and remained at the reduced level for a prolonged period. These changes in oxygenated hemoglobin concentration were similar to those previously reported in arterial blood oxygen and CO2 tension. The magnitude of variance in oxygenated hemoglobin concentration (C-HV) was significantly greater in moyamoya disease patients than in healthy controls (P < .003, Wilcoxon test). The changes of NIRS after operation were markedly more sensitive than those of electroencephalography or single-photon emission computed tomography. NIRS could be applied to cerebral oxygenation as well as cerebral hemodynamics, which may be used to better define the time sequence and pathologic processes of various childhood encephalopathies.

Adolescent↗

PubMed1994

Hypobaric-hypoxic exposure and histochemical responses of soleus muscle fibers in the rat.

Hypoxia-acclimatized rats had a higher percentage of fast-twitch oxidative glycolytic fibers and a lower percentage of slow-twitch oxidative fibers in the soleus muscle than age-matched normoxic-rats. However, there was no difference in the fiber type composition of the soleus muscle between hypoxic- and normoxic-male rats after hypoxic-rats were bred at the sea level atmospheric pressure for 5 weeks. Therefore, it is suggested that a higher percentage of fast-twitch oxidative glycolytic fibers in the muscle is found only under hypoxic conditions and is due to an acute response to hypoxia.

Animals↗

PubMed1994

Alteration of p53 gene in ovarian carcinoma: clinicopathological correlation and prognostic significance.

Inactivation of the tumour-suppressor gene p53 has been demonstrated in a variety of human tumours. We extracted DNA from paraffin-embedded tissues of 67 ovarian carcinoma samples (54 primary tumours, seven metastases and six tumours obtained after chemotherapy), and analysed allelic losses and mutations of the p53 gene using single-strand conformation polymorphism (SSCP) analysis of DNA fragments amplified by a polymerase chain reaction (PCR). Allelic loss was observed in 24 of 32 informative cases. The mutation was detected in 14 of 54 primary ovarian carcinomas: eight serous cystadenocarcinomas (SCA), 42%), five endometrioid adenocarcinomas (EA, 42%) and one mucinous cystadenocarcinoma (14%). The incidence of the alteration was higher in SCA and EA than in other histological types, but the difference was not statistically significant. The incidence of p53 gene abnormalities in ovarian carcinomas tended to be increased in patients with disease advanced (over FIGO stage II). Mutations were found in exons 5 and 7 only and consisted mainly of single nucleotide substitutions [9 or 14 (64%) in exon 7; 4 of 14 (29%) in exon 5]. In 13 of 14 cases, p53 gene mutations occurred concomitantly with losses of the normal allele. The status of the p53 gene in metastases and the tumours obtained after chemotherapy was identical to that in the primary tumours. The presence of p53 gene mutation did not correlate with histological grade, response to primary therapy and survival. These findings suggest that mutational alterations of the p53 gene are involved in the development of a significant proportion of some ovarian carcinomas (SCAs or EAs), especially in advanced stages. However, they may not be a marker predicting the biological behaviour or the outcome of the disease.

Adenocarcinoma, Clear Cell↗

PubMed1994

Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions.

Occludin is an integral membrane protein localizing at tight junctions (TJ) with four transmembrane domains and a long COOH-terminal cytoplasmic domain (domain E) consisting of 255 amino acids. Immunofluorescence and laser scan microscopy revealed that chick full-length occludin introduced into human and bovine epithelial cells was correctly delivered to and incorporated into preexisting TJ. Further transfection studies with various deletion mutants showed that the domain E, especially its COOH-terminal approximately 150 amino acids (domain E358/504), was necessary for the localization of occludin at TJ. Secondly, domain E was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase, and this fusion protein was shown to be specifically bound to a complex of ZO-1 (220 kD) and ZO-2 (160 kD) among various membrane peripheral proteins. In vitro binding analyses using glutathione-S-transferase fusion proteins of various deletion mutants of domain E narrowed down the sequence necessary for the ZO-1/ZO-2 association into the domain E358/504. Furthermore, this region directly associated with the recombinant ZO-1 produced in E. coli. We concluded that occludin itself can localize at TJ and directly associate with ZO-1. The coincidence of the sequence necessary for the ZO-1 association with that for the TJ localization suggests that the association with underlying cytoskeletons through ZO-1 is required for occludin to be localized at TJ.

Animals↗

PubMed1994

Characterization of multiple molecular forms of Mg(2+)-dependent protein phosphatase from Saccharomyces cerevisiae.

Three molecular species of Mg(2+)-dependent protein phosphatase (MPPs-1, -2, and -3) were isolated by DEAE cellulose column chromatography and gel filtration from an extract of Saccharomyces cerevisiae. MPP-1 was further purified 150-fold by chromatography using thio-phosphorylated myosin light chain-agarose. MPPs-1, -2, and -3 were distinct from the major acid and alkaline phosphatases, and their activities were not affected by okadaic acid, microcystin-LR or Ca2+, and calmodulin, resembling the enzymatic properties of type 2C protein phosphatase of mammalian cells. The apparent molecular masses of MPPs-1, -2, and -3 on gel filtration were 53, 112, and 128 kDa, respectively. It was demonstrated that MPP-1 is a globular protein of 53-55 kDa and that MPPs-2 and -3 are oligomeric proteins that dissociate upon sucrose density gradient centrifugation, generating catalytic proteins of about 50 kDa. Since the substrate specificities of MPPs-1, -2, and -3 differed from each other both before and after sucrose density gradient centrifugation, it was suggested that the catalytic proteins of these three enzymes are distinct molecular species.

Chromatography, DEAE-Cellulose↗