Gene, stimulus and cell-type specific regulation of activator protein-1 in mesangial cells by lipopolysaccharide and cytokines.

Activator protein-1 (AP-1) plays an important role in the regulation of gene expression in mesangial cells (MC) during the pathogenesis of glomerular inflammatory disease. The precise regulation of the AP-1 family by agents that are known to activate MC is, however, poorly understood. The action of platelet-derived growth factor (PDGF) and, for the first time, lipopolysaccharide (LPS), interleukin-6 (IL-6), interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) on AP-1 gene expression in MC was therefore studied. Whilst the expression of JunD was not affected by any of the mediators, the mRNA levels of c-fos and JunB were induced by LPS, IL-6, IFN-gamma, PDGF and TNF-alpha, and that of c-jun by LPS, IFN-gamma, PDGF and TNF-alpha. Electrophoretic mobility shift assays showed a time-dependent increase in AP-1 DNA binding activity with JunB representing the major mediator-inducible member involved in DNA-protein interactions. However, stimulus-specific changes in the kinetics and magnitude of AP-1 mRNA expression and DNA binding activity were identified and, additionally, the results showed the potential existence of cell-type-specific mechanisms in the regulation of the AP-1 family. These studies provide novel insights into the mediator-specific modulation of AP-1-regulated gene expression and the activation of MC in renal diseases.

Stimulus- and cell-type-specific regulation of CCAAT-enhancer binding protein isoforms in glomerular mesangial cells by lipopolysaccharide and cytokines.

Binding sites for the CCAAT-enhancer binding protein (C/EBP) family are present in the promoter regions of several genes that are known to be expressed by mesangial cells (MC) during the pathogenesis of glomerular inflammatory diseases. The precise regulation of the C/EBP family by agents that are known to activate MC is, however, poorly understood. We report here the action of interleukin-1 (IL)-1 and, for the first time, lipopolysaccharide (LPS), platelet-derived growth factor (PDGF), IL-6, interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) on the C/EBP expression profile and functional DNA binding activity in primary rat MC. Both cell-type- and stimulus-specific regulation of C/EBP mRNA expression and DNA binding activity were identified, with C/EBPalpha being induced by LPS, C/EBPbeta by LPS, IL-1, TNF-alpha and C/EBPdelta by LPS, IL-1, IFN-gamma, TNF-alpha and PDGF. Such differential regulation, particularly that of C/EBPbeta, may be responsible for the mediator-specific differences in the expression of C/EBP-regulated genes in MC. Additionally, the involvement of potential post-transcriptional mechanisms in the regulation of C/EBPdelta were identified. These studies provide novel insights into the stimulus-specific regulation of gene expression during renal diseases.

Phylogeny analysis of 25 apple rootstocks using RAPD markers and tactical gene tagging.

RAPD (random amplified polymorphic DNA) markers were used to fingerprint eight commercially available apple rootstocks (Nertchinsk, Northern Spy, Osman, Heyer 12, M.1, M.9, M.26 and MM.106), 10 winter hardy offsprings derived from the cross of Nertchinsk x M.9, six winter hardy offsprings derived from the cross of Nertchinsk x M.26 and one winter hardy offspring derived from each of the two crosses between Osman x Heyer 12 and Northern Spy x M.1. Phylogeny analysis using parsimony allowed us to draw the genetic relationship between these lines using only RAPD markers data. The resulting cladogram was compared to the true genetic relationship between these lines in order to assess the efficiency of RAPD markers in determining accurately the phylogenetic relationship. We also developed a DNA fingerprinting system based on 13 informative RAPD loci amplified by five RAPD primers that allowed the rapid identification of apple rootstocks.

Analysis by reverse-phase high-pressure liquid chromatography of phenylisothiocyanate-derivatized 1-aminocyclopropane-1-carboxylic acid in apple extracts.

A rapid and sensitive method for the determination of 1-aminocyclopropane-1-carboxylic acid (ACC) in apple tissues is described. This method is based on the derivatization of ACC with phenylisothiocyanate, and the subsequent separation and quantification of the resulting phenylthiocarbamyl-ACC by reverse-phase high-pressure liquid chromatography. Phenylthiocarbamylation of ACC (and other amino acids) in apple extracts is complete within 20 min at room temperature. After removing solvents and reagent, the phenylthiocarbamyl derivatives are separated on an octadecyl reverse-phase column, eluted with a mixture of acetonitrile and sodium acetate buffer at pH 4.6, and monitored with a uv detector set at 254 nm. An analysis of apple extract can thus be achieved in 23 min and detect quantities as low as 1 pmol. Assays have been done to compare the efficiency of this method with that of a method using an ion-exchange amino acid analyzer and with that of Lizada and Yang's method [(1979), Anal. Biochem. 100, 140-145]. The latter method proved to yield markedly less accurate results than the other two, but the derivatization-HPLC method was preferred because of simplicity of operation and a better separation of ACC.