Cytokines have been proposed as inducers of fl-cell damage in human insulin-dependent diabetes mellitus via the generation of nitric oxide (NO). This concept is mostly based on data obtained in rodent pancreatic islets using heterologous cytokine preparations. The present study examined whether exposure of human pancreatic islets to different cytokines induces NO and impairs fl-cell function. Islets from 30 human pancreata were exposed for 6-144 h to the following human recombinant cytokines, alone or in combination: IFN-y (1,000 U/ml), TNF-a (1,000 U/ml), IL-6 (25 U/ml), and IL-1if (50 U/ml). After 48 h, none of the cytokines alone increased islet nitrite production, but IFN-'y induced a 20% decrease in glucose-induced insulin release. Combinations of cytokines, notably IL-1,8 plus IFN-y plus TNF-a, induced increased expression of inducible NO synthase mRNA after 6 h and resulted in a fivefold increase in medium nitrite accumulation after 48 h. These cytokines did not impair glucose metabolism or insulin release in response to 16.7 mM glucose, but there was an 80% decrease in islet insulin content. An exposure of 144 h to IL-1if plus IFN-'y plus TNF-a increased NO production and decreased both glucose-induced insulin release and insulin content. Inhibitors of NO generation, aminoguanidine or NG-nitro-L-arginine, blocked this cytokine-induced NO generation, but did not prevent the suppressive effect of IL-1,8 plus IFN-'y plus TNF-a on insulin release and content. In conclusion, isolated human islets are more resistant to the suppressive effects of cytokines and NO than isolated rodent islets. Moreover, the present study suggests that NO is not the major mediator of cytokine effects on human islets. (J. Clin. Invest. 1994. 93:1968-1974

ADP-ribosyltransferases including toxins secreted by Vibrio cholera, Pseudomonas aerurginosa, and other pathogenic bacteria inactivate the function of human target proteins by attaching ADP-ribose onto a critical amino acid residue. Cross-species polymerase chain reaction (PCR) and database mining identified the orthologs of these ADP-ribosylating toxins in humans and the mouse. The human genome contains four functional toxin-related ADP-ribosyltransferase genes (ARTs) and two related intron-containing pseudogenes; the mouse has six functional orthologs. The human and mouse ART genes map to chromosomal regions with conserved linkage synteny. The individual ART genes reveal highly restricted expression patterns, which are largely conserved in humans and the mouse. We confirmed the predicted extracellular location of the ART proteins by expressing recombinant ARTs in insect cells. Two human and four mouse ARTs contain the active site motif (R-S-EXE) typical of arginine-specific ADP-ribosyltransferases and exhibit the predicted enzyme activities. Two other human ARTs and their murine orthologues deviate in the active site motif and lack detectable enzyme activity. Conceivably, these ARTs may have acquired a new specificity or function. The position-sensitive iterative database search program PSI-BLAST connected the mammalian ARTs with most known bacterial ADP-ribosylating toxins. In contrast, no related open reading frames occur in the four completed genomes of lower eucaryotes (yeast, worm, fly, and mustard weed). Interestingly, these organisms also lack genes for ADP-ribosylhydrolases, the enzymes that reverse protein ADP-ribosylation. This suggests that the two enzyme families that catalyze reversible mono-ADP-ribosylation either were lost from the genomes of these nonchordata eucaryotes or were subject to horizontal gene transfer between kingdoms.Keywords: ADP-ribosylation; recombinant proteins; PSI-BLAST; orthologous genes; paralogous gene; cross-species PCR; database searches The purpose of this study was to identify all recognizable human and mouse members of the family of toxin-related mono-ADP-ribosyltransferases; to clone, sequence, and chromosomally map their genes and cDNAs; to express the gene products as recombinant proteins and assay their enzyme activities; and to assess structural and sequence similarities between bacterial and mammalian ADP-ribosyltransferases.ADP-ribosylation is an enzyme-catalyzed post-translational protein modification in which the ADP-ribose moiety is transferred from NAD+ to a specific amino acid in a Reprint requests to: Friedrich Koch-Nolte, Institute for Immunology, University Hospital, Martinistr. 52, D-20246 Hamburg, Germany, e-mail: nolte@uke.uni-hamburg.de; fax: 49-40-42803-4243.Abbreviations: ARH, ADP-ribosylhydrolase; GAPD, glyceraldehyde-3-phosphate dehydrogenase; GPI, glycosylphosphatidylinositol; HPRT, hypoxanthine phosphoribosyltransferase; mART, mono(ADP-ribosyl)transferase; NAD+, nicotine adenine dinucleotide; PBS, phosphate-buffered saline; pART, poly...

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