CDKN2B expression in adipose tissue of familial combined hyperlipidemia patients.

The purpose of this study was to determine the core biological processes perturbed in the subcutaneous adipose tissue of familial combined hyperlipidemia (FCHL) patients. Annotation of FCHL and control microarray datasets revealed a distinctive FCHL transcriptome, characterized by gene expression changes regulating five overlapping systems: the cytoskeleton, cell adhesion and extracellular matrix; vesicular trafficking; lipid homeostasis; and cell cycle and apoptosis. Expression values for the cell-cycle inhibitor CDKN2B were increased, replicating data from an independent FCHL cohort. In 3T3-L1 cells, CDKN2B knockdown induced C/EBPα expression and lipid accumulation. The minor allele at SNP site rs1063192 (C) was predicted to create a perfect seed for the human miRNA-323b-5p. A miR-323b-5p mimic significantly reduced endogenous CDKN2B protein levels and the activity of a CDKN2B 3'UTR luciferase reporter carrying the rs1063192 C allele. Although the allele displayed suggestive evidence of association with reduced CDKN2B mRNA in the MuTHER adipose tissue dataset, family studies suggest the association between increased CDKN2B expression and FCHL-lipid abnormalities is driven by factors external to this gene locus. In conclusion, from a comparative annotation analysis of two separate FCHL adipose tissue transcriptomes and a subsequent focus on CDKN2B, we propose that dysfunctional adipogenesis forms an integral part of FCHL pathogenesis.

Genetic and other sources of variation in the activity of serum paraoxonase/diazoxonase in humans: consequences for risk from exposure to diazinon.

Diazinon is the only organophosphorus insecticide that is currently approved for use in sheep dip in the UK. Reports that some individuals may be genetically more susceptible to possible chronic adverse health effects, due to variations in PON1 activity, are complicated by the reliability of activity measurements. In the present study, the influence of three polymorphisms of PON1 on serum diazoxonase activity was investigated in 85 healthy volunteers. Serum activity was assessed in as close to physiological conditions as possible (at pH 7.4, 150 mM NaCl and 37 degrees C with 50 microM diazoxon as substrate) and by quantifying pyrimidinol formation using high-performance liquid chromatography. PON1 genotypes were determined by the polymerase chain reaction and restriction enzyme digestion. For PON1 Q192R, individuals with the RR genotype had the highest serum diazoxonase activity, in contrast to some previous reports where activity was determined under less physiological conditions. Activity was slightly reduced in individuals with the QR genotype and activity was reduced even further in those with the QQ genotype. For PON1 L55 M, there was a significant decrease in mean enzyme activity from LL>LM>MM genotypes. The promoter polymorphism PON1 -108 C/T had only a slight effect on activity. Overall, intragenotype variation in PON1 activity was appreciably greater than the mean intergenotype differences. In conclusion, although there is a wide variation in activity in individuals both within and between genotypes, those individuals with a combination of Q and M alleles generally have a lower ability to detoxify diazoxon, which implies a potentially greater susceptibility to toxicity from diazinon.

Immunolocalization of cystinosin, the protein defective in cystinosis.

Cystinosis is an autosomal recessive disorder associated with excessive lysosomal cystine accumulation secondary to defective lysosomal cystine efflux. CTNS, the gene mutated in cystinosis, codes for the lysosomal membrane protein cystinosin. Antisera were raised in rabbits to a carboxy-terminal oligopeptide sequence from cystinosin. Antisera were screened by Western blotting and immunocytochemical analyses of transfected COS-7 cells expressing either human wild-type cystinosin, a wild-type cystinosin-green fluorescent protein (GFP) fusion protein, or a fusion protein of GFP and mutant human cystinosin with a carboxy-terminal deletion. In Western blots, bands corresponding to cystinosin or cystinosin-GFP were observed in transfected cells but no signal was detected in cells expressing the carboxy-terminal mutant; preimmune sera yielded negative results in all three cases. In transfected cells expressing wild-type cystinosin, immunoreactivity appeared in subcellular vesicles. In cells expressing the wild-type cystinosin-GFP fusion protein, immunoreactivity colocalized with GFP fluorescence. Previous studies demonstrated that GFP fluorescence from this construct colocalized with immunostaining for a known lysosomal membrane protein, i.e., lysosome-associated membrane protein 2. In immunohistochemical analyses, cystinosin localized to tubule epithelia in three normal human kidneys, with a pattern similar to that of lysosome-associated membrane protein 2; cystinosin immunoreactivity was absent in kidneys from patients with a CTNS deletion. For the first time, antisera have been raised that localize cystinosin in cells in vitro and in vivo.