Identification of nicotinamide N-methyltransferase as a novel tumor marker for renal clear cell carcinoma.

PURPOSE: To explore the involvement of enzymes of drug metabolism in renal cell carcinoma we analyzed the gene expression profiles of tumor and nontumor tissues from the same patient by DNA macroarray. The enzyme nicotinamide N-methyltransferase was selected for further evaluation. MATERIALS AND METHODS: Nicotinamide N-methyltransferase mRNA expression was investigated in paired tissue samples from cancerous and noncancerous parts of the kidneys of 30 patients with clear cell renal cell carcinoma who underwent tumor nephrectomy. Measurements were performed by semiquantitative reverse transcriptase-polymerase chain reaction and quantitative real-time polymerase chain reaction. Paired tissue samples were also obtained from 1 patient with chromophobe renal cell carcinoma and from another with oncocytoma to compare the specificity of changes in nicotinamide N-methyltransferase expression among tumors that are related to different renal epithelial cell types. Western blot analysis and catalytic activity assay were also performed to study nicotinamide N-methyltransferase expression. Expression correlated with tumor characteristics. RESULTS: A marked increased expression in tumor tissue was found for nicotinamide N-methyltransferase, which is an enzyme involved in the biotransformation of many drugs and xenobiotic compounds. Differential gene expression measurements in tumor vs normal tissue revealed up-regulation in all clear cell renal cell carcinomas at between 3 and 294-fold (mean 41). In contrast, in chromophobe renal cell carcinoma and oncocytoma nicotinamide N-methyltransferase expression did not increase. In addition, nicotinamide N-methyltransferase expression significantly correlated inversely with tumor size. CONCLUSIONS: Our results indicate that a marked nicotinamide N-methyltransferase increase is a peculiar feature of clear cell renal cell carcinoma. Additional studies may establish the role of nicotinamide N-methyltransferase in tumor formation and progression.

Variability of biological effects of silicas: different degrees of activation of the fifth component of complement by amorphous silicas.

A biogenic and a pyrogenic amorphous silica were incubated in normal human plasma and compared on a per unit surface basis for their ability to split C5 molecules and yield small C5a peptides. Since C5a peptides induce selective chemotactic attraction of polymorphonuclear leukocytes (PMN), measurement of PMN-induced chemotaxis was used as an index of C5 activation. Though to a lesser extent than the crystalline forms, amorphous silicas can promote the cleavage of C5 protein and generation of C5a-like fragment. The biogenic silica, which differs from the pyrogenic variety in particle shape, level of contaminants, and degree of surface hydrophilicity, besides specific surface, induced a greater response. Both silicas activated C5 through a process which seems to involve multiple events similar to those induced by crystalline silica. C5 molecules are adsorbed and hydroxyl radicals are generated through Haber Weiss cycles catalyzed by the redox-active iron present at the particle surface either as trace impurities or chelated from plasma by silanol groups. In turn, these radicals convert native C5 to an oxidized C5-like form C5(H2O2). Finally, C5(H2O2) is cleaved by protease enzymatic action of plasma kallikrein activated by the same silica dusts, yielding a product, C5a(H2O2), having the same functional characteristic as C5a.

Identification and characterization of a second NMN adenylyltransferase gene in Saccharomyces cerevisiae.

The enzyme nicotinamide mononucleotide (NMN) adenylyltransferase (NMNAT) (EC 2.7.7.1) catalyzes the transfer of the adenylyl moiety of ATP to NMN to form NAD(+). On the basis of a remarkable structural similarity with previously described Saccharomyces cerevisiae NMNAT (yNMNAT-1), the YGR010-encoded protein was identified as a second isoform of yeast NMNAT (yNMNAT-2). The YGR010 gene was isolated, cloned into a T7-based vector, and successfully expressed in Escherichia coli BL21 cells, yielding high level of NMN adenylyltransferase activity. The purification procedure reported in this paper, consisting of two chromatographic steps, allowed the isolation of 3mg of electrophoretically homogeneous yNMNAT-2 from 1 liter of E. coli culture. Under SDS/PAGE, the recombinant protein resulted in a single polypeptide of 46 kDa, in agreement with the molecular mass of the hypothetical protein encoded by YGR010 gene. The N-terminal sequence of the purified recombinant yNMNAT-2 exactly corresponds to the predicted sequence. Molecular and kinetic properties of recombinant yNMNAT-2 are reported and compared with those already known for yNMNAT-1.

Structure of human NMN adenylyltransferase. A key nuclear enzyme for NAD homeostasis.

Nicotinamide mononucleotide adenylyltransferase (NMNAT), a member of the nucleotidyltransferase alpha/beta-phosphodiesterases superfamily, catalyzes a universal step (NMN + ATP = NAD + PP(i)) in NAD biosynthesis. Localized within the nucleus, the activity of the human enzyme is greatly altered in tumor cells, rendering it a promising target for cancer chemotherapy. By using a combination of single isomorphous replacement and density modification techniques, the human NMNAT structure was solved by x-ray crystallography to a 2.5-A resolution, revealing a hexamer that is composed of alpha/beta-topology subunits. The active site topology of the enzyme, analyzed through homology modeling and structural comparison with other NMNATs, yielded convincing evidence for a substrate-induced conformational change. We also observed remarkable structural conservation in the ATP-recognition motifs GXXXPX(T/H)XXH and SXTXXR, which we take to be the universal signature for NMNATs. Structural comparison of human and prokaryotic NMNATs may also lead to the rational design of highly selective antimicrobial drugs.