Yoichi Moriyama

Promotion of proliferation of murine BALB/c3T3 fibroblasts mediated by nitric oxide at lower concentrations

This study showed that nitric oxide (NO)‐generating S‐nitrosothiols, S‐nitroso‐N‐acetylpenicillamine (SNAP) and S‐nitrosoglutathione (GSNO), increased proliferation of BALB/c 3T3 (clone A31‐1‐1) fibroblasts in vitro. Treatment with SNAP at a relatively low concentration (0.005‐0.02 mM) induced an increase in cell number compared to control. SNAP (0.005‐0.02 mM) and GSNO (0.025‐0.05 mM) both showed an increase of [3H]thymidine incorporation in exponentially growing cells in a dose‐dependent manner. The maximal effect was observed at about 40 and 90% above control at 0.02 mM and 0.05 mM, respectively. The increase induced by 0.02 mM SNAP was abolished by the addition of 0.01mM oxyhemoglobin, a scavenger of NO. [3H]Thymidine incorporation in stationary cells was also increased by SNAP. In addition, 0.02mM SNAP produced a 1.8‐3.2‐fold increase of thymidine kinase activity in exponentially growing cells.

cAMP and cGMP changes associated with the differentiation of cultured chick embryo muscle cells

Abstract Changes in the intracellular concentration of cAMP and cGMP were examined in primary cultures of chick embryo skeletal muscle cells. The reciprocal changes, increase in cAMP and decrease in cGMP level, were observed during the early stage of myogenesis. Consequently, the ratio of cAMP to cGMP increased significantly during that period reaching the maximum level on day 3. The most marked change in the nucleotide ratio, an approx. 2-fold increase, was observed between the 2nd and 3rd day of culture which coincided with the period of cell fusion. After the 3rd day, the value of nucleotide ratio remained constant throughout the period examined. These findings pose the possibility that the intracellular levels of both cyclic nucleotides are involved in the regulation in myoblast differentiation.

Increased Hepatic Nicotinamide N-Methyltransferase Activity as a Marker of Cancer Cachexia in Mice Bearing Colon 26 Adenocarcinoma

When a cachexigenic subclone (clone 20) of murine colon 26 adenocarcinoma was transplanted into female BALB/c mice, hepatic NNMT activity continued to increase until death in proportion to progressive carcass weight loss, a marker of cancer cachexia. On the other hand, noncachexigenic subclone (clone 5)‐transplanted mice showed neither increase of NNMT activity nor carcass weight loss. Among cytostatic fluorinated pyrimidines, 5′‐dFUrd could inhibit the increase of NNMT activity and prevent weight loss in mice bearing clone 20. On the other hand, 2′‐dFUrd did not show these effects. 5‐FUra and Tegafur inhibited the increase of NNMT activity at higher concentrations. These findings suggest that the levels of hepatic NNMT activity are closely associated with the degree of weight loss, and they appear to be a useful marker of cancer cachexia.

Characterization of nicotinamide methyltransferase in livers of mice bearing Ehrlich ascites tumors: preferential increase of activity

There was a 2- to 7-fold increase in nicotinamide methyltransferase activity in the livers of mice and rats bearing seven different kinds of tumors compared with the respective control normal livers, while activity in the tumors themselves was hardly detectable. The activity in the liver started to increase markedly 3-7 days after i.p. transplantation of Ehrlich ascites tumors into the mice, maintaining a plateau up to death. Metabolic conversion of 14C-nicotinamide to 14C-N1-methylnicotinamide was 3-fold higher in the slices of the ascites tumor host liver than in the normal liver, but the conversion to other radioactive metabolites was not significantly different. Nicotinamide methyltransferase was finally purified 20,000-fold with a yield of 4% from the cytosolic fraction of the ascites tumor host liver by means of five purification steps. At every purification step, only one enzyme fraction was detected. The enzyme finally isolated exhibited a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a molecular weight of 26,000. As for the compounds investigated, including the substrates for methyltransferases other than nicotinamide methyltransferase, only quinoline could be the substrate for enzyme activity. It is suggested that the increase in enzyme activity in the tumor host liver probably derived from the endogenous enzyme preexisting in the liver before tumor transplantation.

Separation of cytidine diphosphate reductase from rat Yoshida ascites sarcoma.

CDP reductase was separated from the cytosol of rat Yoshida ascites sarcoma. The precipitate, which resulted from the acidification of the cytosol by acetic acid at pH 5.2, catalyzed specifically the reduction of CDP, whereas the concurrently resulted supernatant catalyzed those of UDP, ADP and GDP. The CDP reductase showed a single peak in the pattern of the enzyme activity in DEAE-cellulose and also in Sepharose 4B column chromatography with adequate recovery of the activity.

Characterization of a 54 kDa, α1Antitrypsin‐like Protein Isolated from Ascitic Fluid of an Endometrial Cancer Patient

A protein factor which stimulated [3H]thymidine uptake into free hepatocytes prepared from normal mouse liver was detected in the ascitic fluid of gynecological cancer patients. The factor was subsequently further purified from the ascitic fluid of an endometrial cancer patient by DEAE‐Sephacel, Sephadex G‐150 and Phenyl‐Sepharose CL‐4B column chromatographies, and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) showed a single protein band of 54,000 Da, designated tentatively as 54K ascitic protein (54K‐AP). 54K‐AP was similar to human Qt‐antitrypsin (α1‐AT) in terms of SDS‐PAGE and immunological behavior, but was slightly different in terms of amino acid sequence and isoelectric point. Although 54K‐AP inhibited the activities of bovine trypsin and a‐chymotrypsin as did human α1‐AT, 54K‐AP inhibited the plasminogen activator released from human endometrial cancer Ishikawa cells more efficiently than α1‐AT. Because, in contrast to normal serum, the serum from the endometrial cancer patients stimulated [3H]thymidine uptake into hepatocytes, the possibility arises that 54K‐AP could he produced hy the cancer host as a defence mechanism against the cancer.

Uridine diphosphate reductase of Ehrlich ascites tumor is insensitive to hydroxyurea

Uridine diphosphate (UDP) reductase was isolated in the supernatant fraction obtained after the acidification of the cytosol of Ehrlich ascites tumor cells, and was found insensitive to 10 mM hydroxyurea. However, cytidine diphosphate (CDP) reductase, being separated concurrently in the precipitate fraction, was readily inhibited. In the cytosol fraction of either Ehrlich ascites tumor, Yoshida ascites sarcoma or regenerating rat liver after partial hepatectomy, UDP reduction activity, in contrast to CDP reduction activity, is not sensitive to hydroxyurea.

Characterization of the purified cytosolic thymidine kinase from murine Ehrlich ascites tumor: Interconversion of two different relative molecular weight forms

Cytosolic thymidine kinase was purified 18,000‐fold of the homogenate from murine Ehrlich ascites tumor, using the [p‐aminophenyl 3′‐dTMP]‐CH‐Sepharose affinity column. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band of molecular weight 26,000. Two different forms, relative molecular weight 50,000 and 70,000, were found by gel filtration, depending on the existence of dithiothreitol, ATP and other nucleotides. These agents also stabilize and stimulate the enzyme activity. The existence of two forms was also manifested by DEAE‐Sephacel column chromatography, where the 50,000 form was eluted by 50 mM NaCl and the 70,000 form by 400 mM NaCl.

Nicotinamide methyltransferase activity and cell-death in the liver of the mouse bearing Ehrlich ascites tumor.

In order to develop new ways of tumor diagnosis and therapy, it is important to know the metabolic changes occurring while bearing the tumor, since these ways should be applied to the cancer patient, who is, after all, a tumor bearer. Various metabolic changes in the tumor host tissues, especially in the liver, have been observed to take place together with tumor-growth (Fujimura and Shimizu, 1977; Shimizu and Fujimura, 1978; 1984; Asanagi et al., 1988; Tanaka et al., 1991; Nakagawa et al., 1991; Fujimura et al., 1992a; 1992b; Hanazawa et al., 1994; Fujimura et al., 1995).