Kiyoshi Inageda

ACCUMULATION OF CYCLIC ADP-RIBOSE MEASURED BY A SPECIFIC RADIOIMMUNOASSAY IN DIFFERENTIATED HUMAN LEUKEMIC HL-60 CELLS WITH ALL-TRANS-RETINOIC ACID

Cyclic adenosine diphosphoribose (cADPR) is a novel candidate for the mediator of Ca2+ release from intracellular Ca2+ stores. The formation of this cyclic nucleotide is catalyzed by not only Aplysia ADP‐ribosyl cyclase but also an ecto‐form enzyme of NAD+ glycohydrolase (NADase), which was previously identified as all‐trans‐retinoic acid (RA)‐inducible CD38 in human leukemic HL‐60 cells. In the present study, we developed a radioimmunoassay specific for cADPR, by which more than 100 fmol of cADPR could be detected without any interference by other nucleotides. The possible involvement of CD38 in the formation of cellular cADPR was investigated with the radioimmunoassay method. A marked increase in cellular cADPR was accompanied by all‐trans‐RA‐induced differentiation of HL‐60 cells. Moreover, a high level of cellular cADPR was observed in other leukemic cell lines, in which CD38 mRNA was expressed. Thus, CD38, which was initially identified as an NADase, appeared to be responsible for the formation of cellular cADPR.

Oscillation of ADP-ribosyl cyclase activity during the cell cycle and function of cyclic ADP-ribose in a unicellular organism, Euglena gracilis.

In Euglena gracilis, the activity of ADP‐ribosyl cyclase, which produces cyclic ADP‐ribose, oscillated during the cell cycle in a synchronous culture induced by a light‐dark cycle, and a marked increase in the activity was observed in the G2 phase. Similarly, the ADP‐ribosyl cyclase activity rose extremely immediately before cell division started, when synchronous cell division was induced by adding cobalamin (which is an essential growth factor and participates in DNA synthesis in this organism) to its deficient culture. Further, cADPR in these cells showed a maximum level immediately before cell division started. A dose‐dependent Ca2+ release was observed when microsomes were incubated with cADPR.

Large-scale search of SNPs for type 2 DM susceptibility genes in a Japanese population☆

The etiology of type 2 diabetes (DM) is polygenic. We investigated here genes and polymorphisms that associate with DM in the Japanese population. Single-nucleotide polymorphisms (SNPs) of 398 derived from 120 candidate genes were examined for association with DM in a population-based case-control study. The study group consisted of 148 cases and 227 controls recruited from Funagata, Japan. No evident subpopulation structure was detected for the tested population. The association tests were conducted with standard allele positivity tables (chi(2) tests) between SNP genotype frequency and case-control status. The independent association of the SNPs from serum triglyceride levels and body mass index was examined by multiple logistic regression analysis. A value of P<0.01 was accepted as statistically significant. Six genes (met proto-oncogene, ATP-binding cassette transporter A1, fatty acid binding protein 2, LDL receptor defect C complementing, aldolase B, and sulfonylurea receptor) were shown to be associated with DM.

Mono-ADP-ribosylation of Gs by an eukaryotic arginine-specific ADP-ribosyltransferase stimulates the adenylate cyclase system

An arginine-specific ADP-ribosyltransferase, named ADP-ribosyltransferase A, was partially purified from human platelets using polyarginine as an ADP-ribose acceptor. When human platelet membranes were incubated with the transferase A in the presence of NAD+, Gs, a stimulatory guanine nucleotide-binding protein of the adenylate cyclase was specifically mono-ADP-ribosylated. ADP-ribose transfer to Gs by this enzyme was suppressed when membranes were pre-ADP-ribosylated by cholera toxin. Incubation of membranes with the transferase A resulted in activation of the adenylate cyclase system. This stimulatory effect of the transferase A on the adenylate cyclase system was inhibited by the presence of polyarginine. These results indicate a role of ADP-ribosyltransferase A in regulation of the adenylate cyclase system via endogenous mono-ADP-ribosylation of Gs.

Insulin modulates induction of glucose-regulated protein 78 during endoplasmic reticulum stress via augmentation of ATF4 expression in human neuroblastoma cells.

The effect of insulin on endoplasmic reticulum (ER) stress was investigated. Insulin protected cell death induced by ER stress and increased glucose‐regulated protein 78 (GRP78) mRNA and protein levels. Insulin also significantly increased activating transcription factor‐4 (ATF4) protein in the nucleus, which was inhibited by LY294002, a phosphatidylinositol 3‐kinase (PI‐3 kinase) inhibitor. The increase of ATF4 protein by insulin was not due to transcriptional or translational up‐regulation but to a post‐translational mechanism. Knockdown of ATF4 by siRNA significantly inhibited GRP78 induction by insulin. These results indicate that insulin modulated ER stress‐induced GRP78 expression occurs via ATF4 up‐regulation.

Structural Characterization of Cyclic ADP-Ribose by NMR Spectroscopy

Abstract Structure of cyclic adenosine diphosphoribose (cADPR) was reinvestigated by using 1H, 13C, and 31P NMR spectroscopy. The 1H-1H coupling constants and NOE data suggested that the adenosine and ribose moieties have a predominant C2′-endo conformation and an unusual flat conformation, respectively.