Differential gene expression of vascular smooth muscle cells. Detection by RNA arbitrarily primed polymerase chain reaction.

BACKGROUND: Vascular smooth muscle cells (VSMC) play an important role in the development of restenotic lesions. However, regulation of proliferation, migration, and matrix synthesis of these cells is still poorly understood. The aim of this study was to analyze gene expression of differently stimulated bovine VSMC. MATERIAL AND METHODS: RNA was isolated from stimulated bovine VSMC after different time periods. For stimulation we used growth factors (platelet-derived growth factors PDGF-AA, PDGF-BB, basic fibroblast growth factor) and a nitric oxide donating drug (sodium nitroprusside). Gene expression of stimulated and control cells was analyzed by non-radioactive RNA fingerprinting (RNA arbitrarily primed polymerase chain reaction, RAP-PCR) and standard gel electrophoresis. Polymorphic fragments were sequenced and further characterized. RESULTS: By RAP-PCR we detected changes in the RNA fingerprint pattern of stimulated cells compared with unstimulated cells. Sequences of five fragments out of 12 showed high homology to known human genes (serine-methyl-transferase, DUTT1, laminin B2, a newly cloned translational regulator (p97), and a human expressed sequence tag). For laminin B2 we could confirm an upregulation after stimulation with growth factors at 1 and 6 hours and after stimulation with SNP at 1 hour in comparison to controls. For p97 we could show a downregulation after stimulation with SNP, bFGF and PDGF-BB but not PDGF-AA. CONCLUSION: RAP-PCR is well suited for analysis of VSMC gene expression in vitro. The laminin B2 and p97 gene are differently expressed after growth factor stimulation in bovine VSMC.

Development of an automated microbial sensor system.

An automated whole cell biosensor system was developed by integration of immobilized microbial cells in a flow-through system with screen-printed flow-through electrodes as detectors. The detectors used were thick-film Pt-electrodes in a 3-electrode configuration constructed as sandwich flow-through cells with a volume of about 36 microliters polarized at -900 mV. The measuring principle was the determination of oxygen consumption due to the microbial metabolism. Fructose was used as model analyte. The microorganisms were immobilized on cellulose-acetate membranes and integrated into a newly created reaction chamber (membrane reactor). The microbial cells used were Rhodococcus erythropolis and Issatchenkia orientalis known to be suitable for the determination of biological oxygen demand.