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Crigler-Najjar syndrome, type I (CN-I) is a potentially lethal disorder characterized by severe unconjugated hyperbilirubinemia resulting from a recessively inherited deficiency of hepatic UDP-glucuronosyl-transferase (UGT) activity toward bilirubin (B-UGT). Two forms of B-UGT exist in human liver. mRNAs for these two forms and that for another isoform with activity toward simple phenols (P-UGT) have unique 5' regions, but their 3' regions are identical. The three mRNA species are derived from a single locus; the unique 5' regions are encoded by single unique exons and the identical 3' regions consist of four consecutive exons that are shared by all three isoforms. In this paper, we determined genetic lesions in two CN-I patients with deficiency of hepatic B-UGT and P-UGT activities. In one patient, there was a C----T substitution in exon 4 (common region) predicting the substitution of a serine residue with a phenylalanine residue; this mutation was present in the identical region of B-UGT and P-UGT mRNAs. In the other patient, a C----T substitution in exon 2 (common region) of the B-UGT/P-UGT locus resulted in a premature stop codon. This exon (132 nt) was absent in heptic B-UGT and P-UGT mRNAs of this patient due to exon skipping during pre-mRNA processing. Sequence abnormality of three distinct mRNA species explains the abnormality of multiple UGT isoforms in these patients. Presence of identical abnormalities in the common regions of the three mRNAs is consistent with the finding that the common 3' regions of the two B-UGT mRNAs and the P-UGT mRNA are encoded by four shared exons.

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Primary objective. Elevation of the thrombotic responses mediated by a variety of carbon-derived nanoparticles was recently reported in the literature. In this paper our objective was to investigate whether metal nanoparticles (iron, copper, gold or cadmium sulfide [CdS]) impart such prothrombotic effects on human platelets. Secondly, we wanted to examine whether such effects were mediated through any specific platelet receptor. Experimental design. The size distributions and zeta potentials of characterized gold, copper, iron and CdS (rod & sphere) nanoparticles were measured using photon correlation spectroscopy and laser Doppler velocimetry. The effect of two classes of agonists, adenosine diphosphate (ADP) and epinephrine were studied. To study the effect of ADP, a suboptimal concentration was chosen below a critical concentration. Above the critical concentration, the aggregation assumed its standard hyperbolic shape (and de-aggregation disappeared). Pro-aggregatory action of a given agent can be understood with better sensitivity using a transition from deaggregation to aggregation at this suboptimal agonist level. For epinephrine at low concentration this criticality was absent, however the aggregatory profile showed a delayed response. Two classes of human subjects (a) normal and (b) individuals with acute coronary syndrome, who were under a therapeutic regime of clopidogrel were chosen, as clopidogrel is a specific inhibitor of the low affinity ADP receptor P2Y12. This enabled us to understand the pro-aggregatory effects of nanoparticles with only P2Y1 (high affinity ADP receptor) active. In another set of aggregation experiments, the inhibitor MRS2179 was used to specifically block the high affinity ADP receptor P2Y1. Methods. The threshold ADP concentration was determined using an ADP titration. Nanoparticle rich platelet suspensions were exposed to a previously determined sub-optimal ADP concentration. The experiment was repeated with iron, copper, gold and CdS nanoparticles (later with two different morphologies, rod and sphere). Results. The primary result was that the nanoparticles, composed of various materials and shape features, are likely to impart a pro-aggregatory response in platelets. That the pro-aggregatory effect is not solely a physical self-assembly process and has ADP dependence, is evident from the reversal of the said response by apyrase. The fact that the response was absent in the case of P2Y12 blocked subjects (CdS nanoparticles being an exception) suggests that the low-affinity P2Y12 receptor may be an important target for the nanoparticles. If on the other hand P2Y1 (the high affinity receptor) was blocked by the specific inhibitor MRS2179, nanoparticles could still induce higher aggregation in normal subjects. No significant nanoparticle induced proaggregatory effect was observed for epinephrine. Inference. It is inferred, that the said platelet effect is mediated through ADP receptors, the probable target being the low affinity purinergic receptor P2Y12. The indication is tha...

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