To evaluate the pathophysiological function of specific molecules in the renal glomerulus, selective, sustained, and modifiable expression of such molecules will be required. Towards achieving this end, we devised a gene transfer system using the glomerular mesangial cell as a vector for gene delivery. A reporter gene which encodes bacterial j3-galactosidase was introduced into cultured rat mesangial cells, and the stable transfectants were transferred into the rat kidney via the renal artery, leading to selective entrapment within the glomeruli. In the normal kidney, the reporter cells populated into 57+13% of glomeruli site specifically, and the expression of f8-galactosidase was sustained for 4 wk and declined thereafter. Within the glomerulus, some of the reporter cells remained in the glomerular capillaries, while others repopulated the mesangial area and, in part, extended their cytoplasmic processes toward the surrounding capillaries. When the cells were transferred into glomeruli subjected to transient mesangiolysis induced by monoclonal antibody 1-22-3, in situ expression of 13-galactosidase was amplified 7-12-fold, and the enhanced level of expression continued for up to 8 wk. The mesangial cell vector system thus achieves site-specific delivery of an exogenous gene into the glomerulus and is amenable to in situ amplification and sustained expression by preconditioning of the target site. (J. Clin. Invest. 1994. 94:497-505.)

Quinolinium salts were incubated with partially purified aldehyde oxidase, and the products were separated by high-pressure liquid chromatography and fully characterized by u.v. spectroscopy, i.r. spectroscopy and mass spectrometry. Oxidation of N-methylquinolinium salts with either rabbit or guinea-pig liver aldehyde oxidase in vitro gave two isomeric products, N-methyl-4-quinolone and N-methyl-2-quinolone. Incubation of N-phenylquinolinium perchlorate similarly yielded two oxidation products, N-phenyl-4-quinolone and N-phenyl-2-quinolone. The ratio of 2- to 4-quinolone production was species-dependent, the proportion of 4-quinolone with the guinea-pig enzyme being greater than that obtained with the rabbit liver enzyme. Kinetic constants were determined spectrophotometrically for both the quinolinium salts and a number of related quaternary compounds. In general, quaternization facilitated oxidation of a substrate, but a number of exceptions were noted, e.g. N-methylisoquinolinium and N-methylphen-anthridinium. Km values varied with the nature of electron acceptor employed, and this difference was more marked for quaternary substrates than the unquaternized counterparts. The product ratio obtained from N-methylquinolinium salts was found to be constant under various conditions, including purification of the enzyme and the use of either induced or inhibited aldehyde oxidase, but a change in the ratio was found at high pH values and in the presence of a competing substrate, N-methylphenanthridinium. This may indicate that a quaternary substrate binds to aldehyde oxidase in two alternative positions.

Chromosomal gain on 1q23-24 is a cytogenetic finding found in approximately 30% of bladder tumors. Currently, no defined or candidate tumor-associated genes from this region have been identified. The objective of this study was to identify and quantitate the expression of putative cancer genes located at this chromosome locus in normal urothelium, superficial, and muscle invasive bladder tumors. We examined both normal and bladder cancer tissue specimens (N = 40-80 RNA, DNA, and protein) by semiquantitative RT/PCR, genomic PCR, and by Western blotting. The KIAA1096 gene is located at 1q23-24 with no overexpression or amplification in normal urothelium, but was significantly upregulated in 30% of tumors (P = 0.0001). There was a trend towards increased expression in invasive compared to superficial lesions (P = 0.06). A significant increase in gene copy was also found in a 38% of TCC of the bladder compared to normal bladder mucosa or peripheral blood lymphocytes. Immunohistochemistry (IHC) demonstrated KIAA1096 expression in nonmalignant bladder mucosa tissue but apparent upregulation in invasive transitional cell carcinoma. Two other genes, CH1 and RGS5, which are situated in the same region of chromosome 1q, demonstrated disparate patterns of expression. In summary, KIAA1096 is a gene situated at 1q23-24, which demonstrated a pattern of RNA and DNA expression consistent with the 38% expression of cytogenetic amplification noted on previous studies. This gene may, therefore, be a putative marker for this cytogenetic phenomenon and provide an opportunity to evaluate the clinical significance of previous cytogenetic findings.