Shinji Fujimura

Polymerization of the adenosine 5'-diphosphate-ribose moiety of nicotinamide-adenine dinucleotide by nuclear enzyme. I. Enzymatic reactions.

Abstract [8- 14 C]ATP was incorporated by a rat-liver nuclear preparation into acid-insoluble material in the presence of NMN. Experiments using variously labeled ATP and NMN or NAD revealed that the ADP-ribose moiety of NAD was incorporated into the acid-insoluble reaction product. NAD was formed from ATP in the presence of NMN and was an immediate precursor of the acid-insoluble reaction product. The reaction involving the incorporation of NAD had an optimum pH of 8 and required neither a divalent cation nor a sulfhydryl compound. The reaction was effectively inhibited by nicotinamide but not by isonicotinic acid hydrazide. The contribution of NADase in the nuclei towards this enzymatic activity was suggested.

Bone Morphogenetic Protein-2 Stimulates Differentiation of Cultured Spinal Ligament Cells from Patients with Ossification of the Posterior Longitudinal Ligament

Ossification of the posterior longitudinal ligament (OPLL) of the spine is characterized by heterotopic bone formation occurring in spinal ligament, causing severe compression myelopathy. In order to investigate the mechanism of OPLL development, we isolated spinal ligament cells from OPLL patients as well as non-OPLL patients, and established 10 OPLL cell lines and 7 non-OPLL cell lines, respectively. We analyzed the effects of bone morphogenetic protein-2 (BMP-2) on these cells with respect to alkaline phosphatase (AP) activity, DNA synthesis, and collagen production. BMP-2 caused a significant increase of AP activity in 4 OPLL cell lines, whereas the activity did not change in any non-OPLL cells. Among OPLL cells, BMP-2 stimulated DNA synthesis in four cell lines and procollagen type I carboxyl-terminal peptide (PICP) synthesis in five cell lines. Some non-OPLL cells also responded to BMP-2, as there was an increase of DNA synthesis in three cell lines and PICP synthesis in one cell line. These data collectively indicate that BMP-2 preferentially induces osteogenic differentiation in OPLL cells rather than in non-OPLL cells. OPLL cells, therefore, exhibit a different response to BMP-2 than non-OPLL cells, suggesting that the expression of BMP receptor(s) and/or the signal transduction initiated by BMP-2 in the spinal ligament cells of OPLL patients somewhat deviate from those in normal spinal ligament cells. Such abnormal characteristics of OPLL cells as described here provide some clues to the clarification of the pathogenesis of OPLL.

The polymerization of adenosine 5'-diphosphate-ribose moiety of NAD by nuclear enzyme. II. Properties of the reaction product.

Abstract The radioactive acid-insoluble material, Product I, which was formed by rat-liver nuclear preparation from labeled NAD or labeled ATP with nicotinamide mononucleotide, has been partially purified. It has a peak at 3 S with sucrose density-gradient centrifugation, and a density of around 1.57 with Cs 2 SO 4 equilibrium density-gradient centrifugation. Product I was converted into the acid-soluble form by snake-venom phosphodiesterase. Product I was not digested by micrococcal nuclease, spleen phosphodiesterase, potato phosphodiesterase, potato nucleotide pyrophosphatase, spleen NAD nucleosidase, or trypsin. The radioactive acid-soluble substance, Product II, which was released by venom-phosphodiesterase digestion, was isolated from a Dowex 1 formate column. Product II showed absorption maxima at 261, 259, and 258 mμ under alkaline neutral and acidic conditions, respectively. The molar ratio of adenine (as adenosine), ribose, phosphorus and periodate consumption was 1:2:2:1. All phosphates were converted to inorganic phosphate by acid-phosphatase digestion. Structures of Products I and II were proposed.

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.

Elevation of alkaline phosphatase activity induced by parathyroid hormone in osteoblast-like cells from the spinal hyperostotic mouse TWY (twy/twy)

We have examined the alkaline phosphatase (AP) activity of primary calvaria-derived osteoblast-like cells from the twy (tip-toe walking Yoshimura) and normal ICR control mouse. The twy mouse displays elevated osseous formation particularly in the spine, and the pathophysiological features resemble that of human ankylosing spinal hyperostosis. In the proliferative stage of cultured bone cells, parathyroid hormone (PTH) stimulation induced the elevation of AP activity of both twy and ICR mouse-derived cells. When they reached confluence, the AP activity of ICR mouse-derived cells ceased to increase with PTH stimulation. The twy mouse-derived cells, however, continued to respond to PTH, with the enzyme activity increasing even in the confluent, stationary stage. PTH stimulation also increased the intracellular cAMP content of twy mouse-derived cells but it did not influence that of ICR mouse-derived cells in the stationary stage. Moreover, stimulation with dibutyryl cAMP, but not with phorbol myristate acetate, increased the AP activity of both twy and ICR-derived bone cells irrespective of culture conditions, either in the proliferative or in the confluent stage. These data suggest that the protein kinase A-mediated pathway plays a pivotal role in bone cells with PTH stimulation, and that the uninhibited AP activity observed in twy mouse-derived bone cells might be due to some deviating process between the PTH ligand/receptor interaction and cAMP generation.

Involvement of Insulin-Like Growth Factor I in Development of Ossification of the Posterior Longitudinal Ligament of the Spine

In order to investigate the pathogenesis of ossification of the posterior longitudinal ligament (OPLL) of the spine, we examined the distribution of insulin-like growth factor I (IGF-I) in the posterior longitudinal ligaments of OPLL patients, and analyzed the effects of IGF-I on the cultured spinal ligament cells. For that purpose we established eight varieties of OPLL and non-OPLL cell lines obtained from spinal ligaments of corresponding patients, respectively. In contrast to non-OPLL cases, all the OPLL cases were histologically shown to contain round-shaped cartilage-like cells in the transitional region from preossifying to ossifying ligaments, and these cells were strongly stained with an antibody for IGF-I. In the vicinity of preossifying cartilaginous tissues, ligament cells also had a rod-like appearance and were positive for IGF-I immunohistochemically. The effects of IGF-I on cultured spinal ligament cells were assayed by alkaline phosphatase (AP) activity, DNA synthesis, and the amounts of collagen produced. The number of OPLL cell lines that increased AP activity, responding to IGF-I irrespective of 1,25(OH)2D3, was significantly larger than that of non-OPLL cell lines, although IGF-I stimulated DNA and procollagen type I carboxyl-terminal peptide synthesis in most of both OPLL and non-OPLL cell lines. These data demonstrate the dominant expression of IGF-I in the posterior longitudinal ligaments of OPLL patients, and suggest that IGF-I preferentially induces osteogenic differentiation in OPLL cells rather than in non-OPLL cells. IGF-I, therefore, may be involved in the local ossification process of spinal ligaments observed in OPLL patients.

Purification and characterization of N1-methylnicotinamide oxidases I and II separated from rat liver

N1-Methylnicotinamide (1-CH3Nmd) oxidases I and II of the livers of Wistar strain male rats were separated by DEAE-cellulose column chromatography and purified by AcA 34 gel filtration, Affi-Gel blue column chromatography, and sucrose density gradient centrifugation. These two enzymes were purified 568- and 82-fold with yields of 1.4 and 0.1% of the total activity in the liver homogenate, respectively. They showed the same molecular weight of 300,000 by AcA 34 gel filtration and a single protein band with a molecular weight of 150,000 in sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. The difference in antigenicities of these two enzymes was observed with double immunodiffusion and immunoprecipitation using antibody produced in rabbits against each enzyme. Furthermore, these two enzymes were found to be different from each other in the heat stability, optimum pH values, Km values for 1-CH3Nmd, and response to various inhibitors.

Studies on the abnormal excretion of pyrimidine nucleosides in the urine of Yoshida ascites sarcoma-bearing rats. Increased excretion of deoxycytidine, pseudouridine and cytidine

Abstract Yoshida ascites sarcoma-bearing rats excreted significantly higher quantities of deoxycytidine and pseudouridine in urine than normal rats, with a peak 5 days after transplantation of the tumor cells, and excretion of cytidine peaking 3 or 4 days later. The contribution by the injected [14C]orotic acid to labeling of urinary deoxycytidine was 32 and 3 times higher than that by [14C]uridine or [14C]cytidine, respectively. Urinary pseudouridine was also labeled 5–6 times greater by [14C]orotic acid than by [14C]uridine or [14C]cytidine. The labeling in pseudouridine was as high as that in deoxycytidine by either [14C]orotic acid or [14C]cytidine and was about 10 times higher by [14C]uridine. Neither [14C]uracil nor [3H]thymidine resulted in any labeling of either nucleoside. [6-3H]Uridine resulted in radioactivity of urinary pseudouridine, whereas [5-3H]uridine did not. The extent of labeling by the injected [14C]orotic acid of urinary deoxycytidine and pseudouridine was almost constant, at least for several days around maximal excretion of nucleosides; this was true for each injection made 1, 3 or 5 days after tumor transplantation. This study suggests that an increase of urinary deoxycytidine and pseudouridine could be derived from not only the tumor cells but also from the host liver and that urinary pseudouridine could be synthesized by rearranging the ribose in a uridine molecule, i.e., by transferring the ribose from the nitrogen 1 position of uracil to the carbon 5 position.

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.

Specific recognition of cytosolic thymidine kinase in the human lung tumor by monoclonal antibodies raised against recombinant human thymidine kinase

Anti-TK monoclonal antibodies (mAbs) were raised against recombinant human cytosolic thymidine kinase (rhTK) and characterized by Western immunoblotting, enzyme-linked immunosorbent assay (ELISA) and immunostaining of tumor cells. Twenty-three clones of TK mAbs were characterized to recognize specifically not only rhTK produced by Escherichia coli but also TK subunit of 25 kDa in human lung cancer. The anti-TK mAbs reacted specifically with cytosolic TK but not with mitochondrial TK. Only one clone of the mAbs inhibited the catalytic activity of TK. By solid phase sandwich enzyme immunoassay using these mAbs, we could quantitate the cytosolic TK content in tissues. Immunohistochemical staining analysis using one of the TK mAbs showed that human lung adenocarcinoma and squamous cell carcinoma exhibited much higher staining intensity than stromal cells. These mAbs are useful for biochemical studies on the regulation of human TK in proliferating cells such as tumor cells and for diagnosis of highly proliferating tumors.