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Biomedical subjects

A Tohgo

Publications and source records attributed to A Tohgo.

At least 37 records · Page 2Linked to original sources
PubMed1995

Requirement of calmodulin-dependent protein kinase II in cyclic ADP-ribose-mediated intracellular Ca2+ mobilization.

Cyclic ADP-ribose (cADPR) is generated in pancreatic islets by glucose stimulation, serving as a second messenger for Ca2+ mobilization from the endoplasmic reticulum for insulin secretion (Takasawa, S., Nata, K., Yonekura, H., and Okamoto, H. (1993) Science 259, 370-373). In the present study, we observed that the addition of calmodulin (CaM) to rat islet microsomes sensitized and activated the cADPR-mediated Ca2+ release. Inhibitors for CaM-dependent protein kinase II (CaM kinase II) completely abolished the glucose-induced insulin secretion as well as the cADPR-mediated and CaM-activated Ca2+ mobilization. Western blot analysis revealed that the microsomes contain the alpha isoform of CaM kinase II but do not contain CaM. When the active 30-kDa chymotryptic fragment of CaM kinase II was added to the microsomes, fully activated cADPR-mediated Ca2+ release was observed in the absence of CaM. These results along with available evidence strongly suggest that CaM kinase II is required to phosphorylate and activate the ryanodine-like receptor, a Ca2+ channel for cADPR as an endogenous activator, for the cADPR-mediated Ca2+ release.

Adenosine Diphosphate Ribose↗
PubMed1995

The structure of the Aplysia kurodai gene encoding ADP-ribosyl cyclase, a second-messenger enzyme.

The complete nucleotide (nt) sequences of the cDNA and gene encoding the marine mollusk Aplysia kurodai (Ak) ADP-ribosyl cyclase (ADRC) which synthesizes cyclic ADP-ribose (cADP-ribose), a second messenger for Ca2+ mobilization from endoplasmic reticulum, were determined. Ak ADRC consists of 258 amino acids (aa) (29 kDa). It shares 86% aa sequence homology with that from A. californica, and 31-32% homology with the human, rat and mouse cluster of differentiation 38 (CD38) that has both ADRC and cADP-ribose hydrolase activities. The Ak ADRC-encoding gene (ADRC) spans approx. 7 kb and contains eight exons and seven introns. The transcription start point (tsp) determined by primer extension analysis and S1 mapping is 28 bp downstream from the TATA box. This gene is expressed specifically in the ovotestis, although the mammalian CD38-encoding gene is expressed in many kinds of tissues and cells. The 5'-flanking region contains several consensus sequences responsible for the germ-cell-specific expression of the mouse zona pellucida 3 (ZP3) and Drosophila melanogaster chorion genes. The existence of the consensus sequences located at nt -1649, -1161, -234 and -90 may account for the ovotestis-specific expression of the Ak ADRC gene.

ADP-ribosyl Cyclase↗
PubMed1995

Effect of combined administration of a synthetic low-toxicity lipid A derivative, DT-5461a, and indomethacin in various experimental tumor models of colon 26 carcinoma in mice.

We investigated the antitumor effects of a synthetic lipid A derivative, DT-5461a, in combination with indomethacin in three experimental tumor models (peritoneal carcinomatosis, liver tumor, and lung tumor models) of transplanted colon 26 carcinoma in mice. This carcinoma produces the immunosuppressive prostaglandin E2 (PGE2). Intravenous administration of DT-5461a alone resulted in little or no prolongation of survival time [increase in life span (ILS): -2%-22%]. When indomethacin was given in drinking water a slight or moderate increase in survival time was seen (ILS: 4%-45%). In contrast, the combination of DT-5461a and indomethacin induced an additive increase in life span (ILS: 16% to more than 193%). The strongest antitumor effect of this combined therapy was seen in the peritoneal carcinomatosis model; in this model, plasma PGE2 concentrations were considerably higher than in normal mice, and concentrations were further but transiently increased by DT-5461a administration. Following oral indomethacin administration, these elevated PGE2 concentrations were reduced to the level in untreated normal mice. Furthermore, intratumoral tumor necrosis factor (TNF) activity in the group receiving the combined therapy was significantly higher than that in the DT-5461a-treated group. No TNF production was induced by the administration of indomethacin alone. These results suggest that the antitumor effect of DT-5461a can be enhanced by combination with indomethacin, and that the inhibition of PGE2 production may have a role in this antitumor effect.

Animals↗
PubMed1995

Production of tumor necrosis factor induced by synthetic low-toxicity lipid A analog, DT-5461a, is mediated by LPS receptor sites and tyrosine kinase-MAP kinase signaling pathway in murine macrophages.

The synthetic low-toxicity lipid A analog DT-5461a induces endogenous TNF production in mice. The activity of TNF so induced is probably the main contributor to the antitumor effect of this compound. In the present study, we investigated the mechanism by which DT-5461a induces TNF production in murine macrophage RAW 264 cells. DT-5461a mimicked the ability of LPS to induce TNF production in a dose-dependent manner. DT-5461a at higher concentrations inhibited specific binding of [3H]LPS to the cells and reduced LPS-induced TNF production to the level induced by DT-5461a alone. In addition, DT-5461a, as well as LPS, induced tyrosine phosphorylation of MAP kinases, the early signal transduction pathway of this production. Herbimycin A, an inhibitor of tyrosine kinase, inhibited the LPS- and DT-5461a-induced tyrosine phosphorylation, expression of TNF mRNA, and subsequent TNF secretion. These results suggest that DT-5461a and LPS induce TNF production in murine macrophages through the common receptor sites and the similar early signaling pathway.

Animals↗
PubMed1995

Intratumoral production of tumor necrosis factor augmented by endogenous interferons results in potent antitumor effects of DT-5461, a synthetic lipid A analog.

We previously reported that DT-5461 exhibits potent antitumor effects on various murine syngeneic tumors, probably via activation of host immune systems. Of the various systemic administration routes, intravenous (i.v.) administration gave the best antitumor effects. When the total dose was fixed, multiple and intermittent applications resulted in greater therapeutic efficacy than single and daily applications, respectively. The therapeutically effective applications of DT-5461 induced endogenous tumor necrosis factor (TNF) activity in serum and tumor tissue. The TNF activity peaked at 1-2 h after the administration. Although TNF activity in the serum declined to an undetectable level by 4 h, intratumoral TNF activity persisted even at 16 h. TNF-alpha messenger RNA (mRNA) was clearly expressed in the tumor tissues as early as 0.5 h after the DT-5461 administration. DT-5461 also caused increases in interferon activity in tumor-bearing mice. In vivo treatment with anti-interferon-alpha/beta serum or anti-interferon-gamma serum, as well as with anti-TNF-alpha serum, significantly reduced the antitumor effect of DT-5461. DT-5461-induced endogenous TNF production was also inhibited by treatment with either of these anti-interferon antisera alone. These results suggest that intermittent i.v. administration is optimal for cancer treatment with DT-5461, and that the optimal application of DT-5461 causes a long-lasting production of intratumoral TNF-alpha that may play a crucial role in the antitumor mechanisms of this compound. Furthermore, endogenous interferons induced by DT-5461 are involved in the antitumor mechanisms of this compound, probably by regulating the intratumoral TNF induction.

Animals↗
PubMed1995

Assignment of CD38, the gene encoding human leukocyte antigen CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase), to chromosome 4p15.

CD38 has been used as a phenotype marker of lymphocyte differentiation. Recently, we have demonstrated that cyclic ADP-ribose can be synthesized and hydrolyzed by CD38 and acts as a second messenger in insulin secretion from pancreatic beta-cells. We have mapped the CD38 gene to human chromosome 4p15 by fluorescence in situ hybridization.

ADP-ribosyl Cyclase↗
PubMed1994

Essential cysteine residues for cyclic ADP-ribose synthesis and hydrolysis by CD38.

We have recently demonstrated that cyclic ADP-ribose (cADPR) serves as a second messenger for glucose-induced insulin secretion (Takasawa, S., Nata, K., Yonekura, H., and Okamoto, H. (1993) Science 259, 370-373) and that human leukocyte antigen CD38 has both ADP-ribosyl cyclase and cADPR hydrolase activities (Takasawa, S., Tohgo, A., Noguchi, N., Koguma, T., Nata, K., Sugimoto, T., Yonekura, H., and Okamoto, H. (1993) J. Biol. Chem. 268, 26052-26054). Although the amino acid sequence of Aplysia ADP-ribosyl cyclase exhibits a high degree of amino acid sequence identity with that of CD38, the Aplysia enzyme shows only ADP-ribosyl cyclase but not cADPR hydrolase. In the present study, we introduced site-directed mutations to CD38 and found that C119K- and/or C201E-CD38 exhibited only ADP-ribosyl cyclase activity. Furthermore, Aplysia ADP-ribosyl cyclase into which we introduced the mutations K95C and E176C, which correspond to residues 119 and 201 of human CD38, exhibited not only ADP-ribosyl cyclase activity but also cADPR hydrolase. These results indicate that cysteine residues 119 and 201 in CD38 have crucial roles in the synthesis and hydrolysis of cADPR.

ADP-ribosyl Cyclase↗
PubMed1994

Antitumor synthetic lipid A analog DT-5461a upregulates cytokine expression in a murine macrophage cell line through LPS pathway.

We previously showed that DT-5461a, a lipid A analog, has antitumor activity and relatively low toxicity in animals. To clarify its immunostimulatory effect we have studied the cytokine inducibility of DT-5461a and the mechanism involved in the cytokine expression in macrophages. While the activity is lower than that of LPS, DT-5461a enhanced production of TNF-alpha and GM-CSF in the murine macrophage cell line J774.1 in a dose-dependent fashion. Simultaneous addition of IL-4 inhibited DT-5461a and LPS-induced TNF-alpha production in a similar manner. Expression of cytokine mRNAs including TNF-alpha, IL-1 beta, IL-6, and GM-CSF was also enhanced by the treatment with DT-5461a. Treatment with cycloheximide indicated that the expression of TNF-alpha mRNA does not require new protein synthesis. Actinomycin D chase experiments revealed that cytokine mRNAs have rather long half-lives and we could not find significant changes in mRNA stability of cytokines in the J774.1 cells. The binding activity of a nuclear transcription factor NF-kappa B was enhanced by treatment with DT-5461a, suggesting enhanced transcription of cytokine genes. Specific binding of [3H]LPS to the cells was significantly inhibited by the addition of DT-5461a. These results indicate that DT-5461a enhances production of a variety of cytokines in macrophages through the LPS receptor sites and that this cytokine induction may occur primarily via transcriptional enhancement. These immunostimulatory effects may provide evidence for the mechanisms of the antitumor effect of this lipid A analog.

Animals↗
PubMed1994

Structural determination and characterization of a 40 kDa protein isolated from rat 40 S ribosomal subunit.

We have purified a 40 kDa protein from the rat 40 S ribosomal subunit and determined its primary structure by amino acid and cDNA sequencing. The amino acid sequence of the 40 kDa protein shared 29-37% homology with prokaryotic ribosomal protein S2 of eubacteria and chloroplasts, indicating that the protein is a eukaryotic counterpart to prokaryotic S2. Moreover, the amino acid sequence shared 99% identity with those deduced from cDNAs for 68 kDa laminin binding proteins of human, murine and bovine origins. The cDNAs are capable of encoding polypeptides with predicted molecular mass of 33,000 which lacked typical signal sequences, N-linked glycosylation sites and putative transmembrane domains. These results indicate that the cDNAs for 68 kDa laminin binding proteins actually code for the 40 kDa ribosomal protein.

Amino Acid Sequence↗
PubMed1994

Antitumor effect of DT-5461a, a synthetic low-toxicity lipid A analog, involves endogenous tumor necrosis factor induction subsequent to macrophage activation.

We previously showed that the synthetic lipid A derivative DT-5461a exhibited significant antitumor effects against various murine solid tumors, probably via activation of host immune systems. To clarify the participation of the macrophage-stimulating effect of DT-5461a in the antitumor mechanisms, we studied the ability of this compound to induce cytostatic macrophages and TNF production in murine systems. Cytostatic macrophages were induced by treatment with DT-5461a either in vitro or in vivo. DT-5461a also induced TNF production by resident peritoneal macrophages or spleen cells obtained from untreated mice. When spleen cells prepared from DT-5461a-treated mice were re-stimulated in vitro with DT-5461a, no TNF was produced by cells obtained at 1 day after the treatment. This may be due to transient refractoriness of macrophages to the compound, since the response to re-stimulation with DT-5461a recovered in cells obtained at 3 or 5 days after treatment. Moreover, while the serum TNF production and antitumor effects by DT-5461a decreased on daily administration, they were elicited by intermittent administration at intervals of 3 days or more. This suggests that the antitumor effects of DT-5461a depend on the TNF-producing activity of macrophages. These results indicate that DT-5461a possesses significant macrophage-stimulating activity, and that macrophages so activated mediate the DT-5461a-induced augmentation of host response against solid tumors.

Animals↗
PubMed1993

Synthesis and hydrolysis of cyclic ADP-ribose by human leukocyte antigen CD38 and inhibition of the hydrolysis by ATP.

Cyclic ADP-ribose (cADPR) has been recently shown to be generated in pancreatic beta-cells by glucose stimulation, serving as a second messenger for Ca2+ mobilization in the endoplasmic reticulum in the process of insulin secretion (Takasawa, S., Nata, K., Yonekura, H., and Okamoto, H. (1993) Science 259, 370-373). In the present study, we isolated a cDNA for CD38, which has been reported to be a human leukocyte antigen, from a human insulinoma and expressed the cDNA in COS-7 cells. CD38 expression was observed in the plasma membrane and the microsome fractions of the COS-7 cells. When we incubated the plasma membrane fraction with NAD+ and analyzed the reaction products by high pressure liquid chromatography, the formation of cADPR was observed in addition to the ADP-ribose (ADPR) formation. When the plasma membrane fraction was incubated with cADPR, cADPR was converted to ADPR stoichiometrically. These results suggest that CD38 has both cADPR-forming and -hydrolyzing activities. Moreover, we found that ATP (2-10 mM), generated in the glucose metabolism in beta-cells, inhibited the cADPR-hydrolyzing activity, resulting in the increased formation of cADPR. These findings indicate a role for CD38 in the synthesis and hydrolysis of cADPR in the process of insulin secretion in pancreatic beta-cells.

ADP-ribosyl Cyclase↗
PubMed1992

The primary structure of rat rig/ribosomal protein S15 gene.

We have isolated rat rig/ribosomal protein S15 gene from a DNA library derived from a rat insulinoma and determined the complete nucleotide sequence. The rat rig/S15 gene is composed of four exons and three introns spanning 2 kbp and exhibits distinctive structural features unique for a ribosomal protein gene.

Amino Acid Sequence↗
PubMed1992

Structural determination of Saccharomyces cerevisiae rig gene and identification of its product as ribosomal protein S21.

rig was originally isolated from a rat insulinoma-derived cDNA library. The 145 amino acid sequence of the rig protein is invariant in mammalian cDNAs. In this paper, we have isolated the cDNA and genomic clones for yeast (Saccharomyces cerevisiae) rig, determined their nucleotide sequences, and identified the gene product. The gene and the mRNA encode a basic protein of 142 amino acids which has 61.3% amino acid identity with mammalian rig protein. On two-dimensional gel electrophoresis, the in vitro transcription/translation product of yeast rig cDNA co-migrated with yeast ribosomal protein S21. These results led to the conclusion that yeast rig ribosomal protein S21 and to the determination of the previously unknown primary structure of yeast S21 protein. Unlike most ribosomal protein genes of S. cerevisiae, the gene exists as a single copy in a haploid set of the yeast genome and has no intron, locating at chromosome VII or XV.

Amino Acid Sequence↗
PubMed1992

Significant antitumor effect of a synthetic lipid A analogue, DT-5461, on murine syngeneic tumor models.

The antitumor effect of a synthetic lipid A analogue, DT-5461, was investigated using syngeneic tumor models in mice. Intravenous injection of DT-5461 into mice transplanted with solid tumors of MethA fibrosarcoma, MH134 hepatoma, MM46 mammary carcinoma, Lewis lung carcinoma (3LL), and colon adenocarcinomas 26 and 38 resulted in significant reductions in the weight of all tumors except Colon 26, with marked hemorrhagic necrosis of tumor tissues. Efficacy was almost equal to that of an Escherichia coli-type synthetic lipid A (compound 506), and also to those of some chemotherapeutics including Adriamycin, mitomycin C, fluorouracil and cisplatin. Furthermore, DT-5461 was more effective than other immunotherapeutics, including picibanil (OK-432) and lentinan. However, its antitumor effects were inferior to those of Adriamycin or OK-432 against the malignant ascites caused by intraperitoneal inoculation with MethA or with MH134 cells; life span was not prolonged by either intraperitoneal or intravenous administration. In addition, although DT-5461 showed direct inhibitory effects on the in vitro growth of MethA or MH134, these were much weaker than those of Adriamycin. These findings clearly indicated that DT-5461 with systemic administration is a highly effective antitumor agent on solid tumors, and suggest that the antitumor effect of DT-5461 with potent necrotizing activity might derive from indirect mechanisms related to the activation of host immune systems and not to the weak direct cytotoxicity.

Animals↗
PubMed1990

Inhibitory effects of DN-9693 on platelet-enhanced tumor cell attachment to cultured endothelium.

The effects of DN-9693, a platelet aggregation inhibitor, on tumor cell attachment to endothelium were investigated to clarify its inhibitory action on metastasis. The role of platelets in the attachment of murine metastasizing tumor cells to endothelium was first examined in vitro. Morphological studies and quantitative isotope measurements revealed that tumor-cell-activated platelets significantly enhanced tumor cell attachment to the endothelium. This amplification is considered to depend on morphological and functional changes of endothelial cells: activated platelets may accelerate endothelial retraction, exposing more reactive subendothelium, and may increase the adhesiveness of endothelial cells. The platelet-enhanced tumor cell attachment to the endothelium was inhibited in the presence of DN-9693. The pretreatment of endothelial cells with the compound was also effective in inhibiting cell attachment. These data suggest that the inhibitory effects of DN-9693 on metastasis are in part mediated by the prevention of tumor cell attachment to the vascular endothelium.

3',5'-Cyclic-AMP Phosphodiesterases↗