cDNA cloning and analysis of tissue-specific expression of mouse peroxisomal straight-chain acyl-CoA oxidase.

Straight-chain acyl-CoA oxidase is the first and rate limiting enzyme in the peroxisomal beta-oxidation pathway catalysing the desaturation of acyl-CoAs to 2-trans-enoyl-CoAs, thereby producing H2O2. To study peroxisomal beta-oxidation we cloned and characterized the cDNA of mouse peroxisomal acyl-CoA oxidase. It consists of 3778 bp, including a 1983-bp ORF encoding a polypeptide of 661 amino-acid residues. Like the rat and human homologue the C-terminus contains an SKL motif, an import signal present in several peroxisomal matrix proteins. Sequence analysis revealed high amino-acid homology with rat (96%) and human (87%) acyl-CoA oxidase in addition to minor homology ( approximately 40%) with other related proteins, such as rabbit trihydroxy-cholestanoyl-CoA oxidase, human branched chain acyl-CoA oxidase and rat trihydroxycoprostanoyl-CoA oxidase. Acyl-CoA oxidase mRNA and protein expression were most abundant in liver followed by kidney, brain and adipose tissue. During mouse brain development acyl-CoA oxidase mRNA expression was highest during the suckling period indicating that peroxisomal beta-oxidation is most critical during this developmental stage. Comparing tissue mRNA levels of peroxisome proliferator-activated receptor alpha and acyl-CoA oxidase, we noticed a constant relationship in all tissues investigated, except heart and adipose tissue in which much more, and respectively, much less, peroxisome proliferator-activated receptor alpha mRNA in proportion to acyl-CoA oxidase mRNA was found. Our data show that acyl-CoA oxidase is an evolutionary highly conserved enzyme with a distinct pattern of expression and indicate an important role in lipid metabolism.

A study on the short tandem repeat systems HumCD4, HumTH01 and HumFIBRA in population samples from Yemen and Egypt.

The short tandem repeat systems (STRs) HumCD4 (CD4), HumTH01 (TH01) and HumFIBRA (FGA) were amplified by the polymerase chain reaction (PCR) on blood samples from 100 unrelated Yemenians and 100 unrelated Egyptians. PCR products were separated on native horizontal discontinuous gel electrophoresis followed by silver staining. The distribution of observed phenotypes did not deviate from Hardy-Weinberg equilibrium. While significant differences between both Arab populations and an European population from Austria were found at all loci, differences between the Egyptian and the Yemenian samples were found only for CD4. In a number of verified Austrian families (TH01: 426 meioses, CD4: 275 meioses, FGA: 144 meioses) no mutations were found. The observation of a TH01 allele consisting of 4 repeats was confirmed by sequencing. Moreover we report the structure of a TH01 allele 6.3 observed in a Hungarian Caucasian population.

Inhibition of the transendothelial migration of human lymphocytes but not monocytes by phosphodiesterase inhibitors.

This study describes an in vitro model of peripheral blood mononuclear cell (PBMC) migration through human endothelial cells, held on polycarbonate inserts, which allows automatic differential counting of migrated cells as lymphocytes and monocytes. Using this system it was found that treatment of PBMC with the phosphodiesterase (PDE) inhibitors theophylline (at 1 and 10 micrograms/ml) and RO-20-1724 (at 1 microM) inhibited the migration of the lymphocyte component to 64.2+/-16.4%, 48.9+/-3.0% and 47.5+/-5.8% of the control values, respectively, while the migration of the monocytes component was largely unaffected. The PDE inhibitors needed to be present during the assay to inhibit migration, whereas pre-treatment of either the endothelium or the PBMC did not consistently effect lymphocyte migration. The drugs also inhibited the migration of lymphocytes through control inserts, either uncoated or coated with fibronectin, suggesting that some of the inhibition is an effect on lymphocyte motility rather than lymphocyte-endothelial interactions. Lymphocyte migration through fibronectin-coated filters was significantly enhanced compared with uncoated filters. Activation of the PBMC by anti-CD3 MoAb increased motility and migration by up to 300%. This migration appeared to be greatly inhibited by the PDE inhibitors, although the effect was complicated by problems of lymphocyte aggregation. This study provides a novel method of measuring mononuclear cell transendothelial migration, and suggests a possible role of PDE inhibitors in reducing this progress.

Mode of action of coumarin in immune cells.

In order to characterize further the mode of action of coumarin, binding studies were undertaken using human monocytes and radioactively labelled drug. Since coumarin is only a small compound and we wanted to exclude possible artefacts due to variations in size or conformation, the drug was produced by synthesis in the presence of radioactive 14C. Adding increasing amounts of a mixture of labelled and unlabelled drug to monocytes resulted in saturating conditions only at rather high concentrations. Performing Scatchard analysis demonstrated that binding sites for coumarin appeared to be present in relatively high numbers (7.5 x 10(8)/cell) but their affinity was rather low (K alpha approximately 2 x 10(2) M-1). Inhibition studies with 7-hydroxycoumarin revealed that an approximately four times higher molar concentration of the derivative was necessary to cause 50% displacement of coumarin from its binding site. These results indicate that binding of the drug to cells is characterized by high-capacity but low-affinity conditions. This would be compatible with the hypothesis that coumarin interacts with ubiquitous intracellular receptor proteins able to interact with aromatic hydrocarbons, which might form the basis for enzyme induction, and leads to the effects observed in vitro and in vivo.