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Mycophenolic acid (MPA) is a selective inhibitor of inosine 5'-monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme of de novo synthesis of guanine nucleotides. The isoenzyme IMPDH2 predominates in activated lymphocytes, and its inhibition by MPA is part of standard immunosuppressive regimens. Yet, there are significant unexplained differences in efficacy and tolerability among patients. The objective of this study was to analyze whether frequent variants in the IMPDH2 gene lead to changes in IMPDH activity and to differences in responsiveness to MPA therapy. All 14 exons and intron-exon boundary regions of IMPDH2 were sequenced from genomic DNA probes from 100 healthy individuals. Two novel exonic single-nucleotide polymorphisms were identified in 1% and one intronic polymorphism (rs11706052) in 19% of the study population. Lymphocyte IMPDH activity and proliferation under three MPA concentrations (2.5, 10 and 25 micromol l(-1)) were compared in rs11706052 carriers and wild-type individuals. The presence of rs11706052 polymorphism reduced the antiproliferative effect of MPA on lymphocytes by approximately 50% compared with the IMPDH2 wild-type form at therapeutic relevant concentrations of 10 micromol l(-1) and 25 micromol l(-1). We conclude that a poorer response to MPA therapy can be explained in some individuals by the presence of the rs11706052 polymorphism.

Clusterin is a widely expressed secretory glycoprotein which is found in mammals as a disulfide-bonded alpha/beta heterodimer generated by cleavage of the single-chain precursor. In contrast, clusterin occurs in the chicken mainly as an intracellular single-chain form and is not observed in serum. The present report identifies chicken clusterin as a component of the eggshell. This extracellular clusterin originates in the uterine fluid, where it is a disulfide-bonded heterodimer derived from the precursor polypeptide by proteolytic cleavage at the same site as in mammals. Clusterin message expression in the oviduct was measured by real time RT-PCR, and levels were found to be highest in magnum and uterus. Western blotting using protein extracts of oviduct tissues indicated major clusterin production in the magnum, while immunostaining of the oviduct identified clusterin in the tubular glands of the uterus and the magnum. In addition, clusterin was detected in egg white by Western blotting. In the decalcified eggshell, immunofluorescence and colloidal-gold immunocytochemistry revealed that clusterin was predominantly localized in the palisade and mammillary layers, but also in the mantle and core of the inner and outer shell membranes. It has been suggested recently that clusterin acts as an extracellular chaperone. Thus clusterin could function in the uterine fluid to prevent the premature aggregation and precipitation of eggshell matrix components before and during their assembly into the rigid protein scaffold necessary for ordered mineralization.

Familial hypercholesterolemia (FH) is an autosomal-dominant inherited disorder characterized by high serum low-density lipoprotein (LDL)-cholesterol concentrations, xanthoma formation, and premature atherosclerosis. Homozygous individuals die of vascular disease as children or young adults; heterozygous persons are at high risk for premature cardiovascular death. Mutations in the LDL-receptor gene are responsible for FH. We studied 49 members of a consanguineous Syrian kindred containing 6 homozygous individuals from the same pedigree. Half of the homozygotes had giant xanthomas, while half did not, even though their LDL-cholesterol concentrations were elevated to similar degrees (> 14 mmol/l). Heterozygous FH individuals from this family were also clearly distinguishable with respect to xanthoma size. We performed DNA analysis and were successful in identifying a hitherto not described mutation in this family's LDL receptor. DNA sequence analysis of the LDL-receptor gene revealed a T to C substitution at nucleotide 1,999 in codon 646 of exon 14. We next conducted a segregation analysis, which suggests that a susceptibility gene may explain the formation of giant xanthomas in this family. We raise the hypothesis that the appearance of giant xanthomas in this FH family is controlled by a second gene acting in an autosomal-dominant or recessive fashion. Elucidation of this 'xanthoma' gene may shed additional light on LDL-cholesterol deposition.