Ontogeny of Hepatic Drug Transporters as Quantified by LC-MS/MS Proteomics.

Protein expression of major hepatic uptake and efflux drug transporters in human pediatric (n = 69) and adult (n = 41) livers was quantified by liquid chromatography / tandem mass spectroscopy (LC-MS/MS). Transporter protein expression of OCT1, OATP1B3, P-gp, and MRP3 was age-dependent. Particularly, significant differences were observed in transporter expression (P < 0.05) between the following age groups: neonates vs. adults (OCT1, OATP1B3, P-gp), neonates or infants vs. adolescents and/or adults (OCT1, OATP1B3, and P-gp), infants vs. children (OATP1B3 and P-gp), and adolescents vs. adults (MRP3). OCT1 showed the largest increase, of almost 5-fold, in protein expression with age. Ontogenic expression of OATP1B1 was confounded by genotype and was revealed only in livers harboring SLCO1B1*1A/*1A. In livers >1 year, tissues harboring SLCO1B1*14/*1A showed 2.5-fold higher (P < 0.05) protein expression than SLCO1B1*15/*1A. Integration of these ontogeny data in physiologically based pharmacokinetic (PBPK) models will be a crucial step in predicting hepatic drug disposition in children.

Age-Related Changes in MicroRNA Expression and Pharmacogenes in Human Liver.

Developmental changes in the liver can significantly impact drug disposition. Due to the emergence of microRNAs (miRNAs) as important regulators of drug disposition gene expression, we studied age-dependent changes in miRNA expression. Expression of 533 miRNAs was measured in 90 human liver tissues (fetal, pediatric [1-17 years], and adult [28-80 years]; n = 30 each). In all, 114 miRNAs were upregulated and 72 were downregulated from fetal to pediatric, and 2 and 3, respectively, from pediatric to adult. Among the developmentally changing miRNAs, 99 miRNA-mRNA interactions were predicted or experimentally validated (e.g., hsa-miR-125b-5p-CYP1A1; hsa-miR-34a-5p-HNF4A). In human liver samples (n = 10 each), analyzed by RNA-sequencing, significant negative correlations were observed between the expression of >1,000 miRNAs and mRNAs of drug disposition and regulatory genes. Our data suggest a mechanism for the marked changes in hepatic gene expression between the fetal and pediatric developmental periods, and support a role for these age-dependent miRNAs in regulating drug disposition.

Peptide dose, MHC affinity, and target self-antigen expression are critical for effective immunotherapy of nonobese diabetic mouse prediabetes.

Cross-reactive T cells that recognize both Tep69 (dominant nonobese diabetic (NOD) T cell epitope in ICA69 (islet cell autoantigen of 69 kDa)) and ABBOS (dominant NOD T cell epitope in BSA) are routinely generated during human and NOD mouse prediabetes. Here we analyzed how systemic administration of these mimicry peptides affects progressive autoimmunity in adoptively transferred and cyclophosphamide-accelerated NOD mouse diabetes. These models were chosen to approximate mid to late stage prediabetes, the typical status of probands in human intervention trials. Unexpectedly, high dose (100 microg) i.v. ABBOS prevented, while Tep69 exacerbated, disease in both study models. Peptide effects required cognate recognition of endogenous self-Ag, because both treatments were ineffective in ICA69null NOD congenic mice adoptively transferred with wild-type, diabetic splenocytes. The affinity of ABBOS for NOD I-A(g7) was orders of magnitude higher than that of Tep69. This explained 1) the expansion of the mimicry T cell pool following i.v. Tep69, 2) the long-term unresponsiveness of these cells after i.v. ABBOS, and 3) precipitation of the disease after low dose i.v. ABBOS. Disease precipitation and prevention in mid to late stage prediabetes are thus governed by affinity profiles and doses of therapeutic peptides. ABBOS or ABBOS analogues with even higher MHC affinity may be candidates for experimental intervention strategies in human prediabetes, but the dose translation from NOD mice to humans requires caution.

Structure, expression, and chromosomal localization of the mouse Atox1 gene.

Copper trafficking in eukaryotes involves small proteins termed metallochaperones, which mediate copper delivery to specific intracellular sites. Previous studies in yeast and human cell lines have suggested that Atox1 plays a critical role in copper delivery to the secretory pathway. In the present study, a mouse Atox1 (mAtox1) cDNA was cloned and shown to encode an open reading frame with 85% amino acid identity to human Atox1. RNA blot analysis revealed that mAtox1 was expressed as a single transcript in multiple tissues, and immunoblotting indicated that the relative abundance of mAtox1 mRNA directly correlated with mAtox1 protein. Analysis of the mAtox1 gene locus revealed a genomic structure with four exons encompassing a total of 14.5 kb. RFLP and haplotype analyses indicated that the mAtox1 locus was tightly linked to the Trhr and D15Bir7 loci on mouse chromosome 15. Taken together, these data reveal marked evolutionary conservation of Atox1 structure and provide a genomic organization and localization that will aid in the genetic deciphering of the molecular role of this protein in copper homeostasis.

Production of the islet cell antigen ICA69 (p69) with baculovirus expression system: analysis with a solid-phase time-resolved fluorescence method of sera from patients with IDDM and rheumatoid arthritis.

Islet cell antigen 69 (ICA69), previously implicated as an autoantigen in autoimmune insulin-dependent diabetes mellitus (IDDM), was produced using baculovirus-mediated expression in Spodopterafrugiperda (Sf9) insect cells. In these cells the protein was effectively expressed and ICA69 carrying C-terminal histidine-hexapeptide could be efficiently purified using immobilized metal chelate affinity chromatography. Screening of patient and control sera using this protein as an antigen in time-resolved fluoroimmunoassay (TR-FIA) identified 4/50 of patients with IDDM and 6/73 of patients with rheumatoid arthritis (RA) to be positive for ICA69 antibodies. The number of positives did not differ significantly between patients and control subjects but the level of binding was higher in sera from RA patients compared to that of control sera (P = 0.003). The results show that some subjects have specific autoreactive antibodies against the ICA69 protein produced with recombinant technology.

Characterization of Mayven, a novel actin-binding protein predominantly expressed in brain.

The cytoskeleton plays an important role in neuronal morphogenesis. We have identified and characterized a novel actin-binding protein, termed Mayven, predominantly expressed in brain. Mayven contains a BTB (broad complex, tramtrack, bric-a-brac)/POZ (poxvirus, zinc finger) domain-like structure in the predicted N terminus and "kelch repeats" in the predicted C-terminal domain. Mayven shares 63% identity (77% similarity) with the Drosophila ring canal ("kelch") protein. Somatic cell-hybrid analysis indicated that the human Mayven gene is located on chromosome 4q21.2, whereas the murine homolog gene is located on chromosome 8. The BTB/POZ domain of Mayven can self-dimerize in vitro, which might be important for its interaction with other BTB/POZ-containing proteins. Confocal microscopic studies of endogenous Mayven protein revealed a highly dynamic localization pattern of the protein. In U373-MG astrocytoma/glioblastoma cells, Mayven colocalized with actin filaments in stress fibers and in patchy cortical actin-rich regions of the cell margins. In primary rat hippocampal neurons, Mayven is highly expressed in the cell body and in neurite processes. Binding assays and far Western blotting analysis demonstrated association of Mayven with actin. This association is mediated through the "kelch repeats" within the C terminus of Mayven. Depolarization of primary hippocampal neurons with KCl enhanced the association of Mayven with actin. This increased association resulted in dynamic changes in Mayven distribution from uniform to punctate localization along neuronal processes. These results suggest that Mayven functions as an actin-binding protein that may be translocated along axonal processes and might be involved in the dynamic organization of the actin cytoskeleton in brain cells.

Possible involvement of proteasomes (prosomes) in AUUUA-mediated mRNA decay.

We have identified a cellular target for proteasomal endonuclease activity. Thus, 20 S proteasomes interact with the 3'-untranslated region of certain cytoplasmic mRNAs in vivo, and 20 S proteasomes isolated from Friend leukemia virus-infected mouse spleen cells were found to be associated with a mRNA fragment showing great homology to the 3'-untranslated region of tumor necrosis factor-beta mRNA that contains AUUUA sequences. We furthermore demonstrate that 20 S proteasomes destabilize oligoribonucleotides corresponding to the 3'-untranslated region of tumor necrosis factor-alpha, creating a specific cleavage pattern. The cleavage reaction is accelerated with increasing number of AUUUA motifs, and major cleavage sites are localized at the 5' side of the A residues. These results strongly suggest that 20 S proteasomes could be involved in the destabilization of cytokine mRNAs such as tumor necrosis factor mRNAs and other short-lived mRNAs containing AUUUA sequences.

Loss of self-tolerance to ICA69 in nonobese diabetic mice.

Islet cell antigen p69 (ICA69) is a target autoantigen in IDDM. Studies of T-cells from newly diabetic children suggested possible antigenic mimicry between human ICA69 (in particular the Tep69 T-cell epitope, aa 36-47) and the ABBOS region in bovine serum albumin (BSA; aa 152-169), one of several cow's milk proteins that evoke abnormal immunity in diabetes-prone hosts. We recently found the sequence of Tep69 regions to be identical in the four alternatively spliced human and rodent ICA69 isoforms. Immunization of nonobese diabetic (NOD) mice with BSA or ICA69 generates fully cross-reactive T-cell responses to both Tep69 and ABBOS as the immunodominant, naturally generated, and presented T-cell mimicry epitopes. Such responses are absent or weak in healthy strains of mice. NOD mouse recipients of adoptive spleen cell grafts from diabetic donors spontaneously generate easily detectable pools of T-cells specific for ICA69/BSA, as well as the unrelated GAD65. NOD mice injected neonatally with ABBOS or Tep69 show cross-tolerance, but ABBOS-induced tolerance is transient. Neonatal injection of Tep69 reduces disease incidence (23 vs. 68% IDDM, P < 0.02), while neonatal injection of ABBOS has little effect. In contrast, systemic immunization of young NOD females with ABBOS (but not Tep69) reduces the diabetes incidence and delays disease expression, with protected mice generating ABBOS-specific T-cell repertoires unable to recognize the Tep69 mimicry antigen. Our observations demonstrate a loss of self-tolerance to ICA69 in NOD mice, and they establish antigenic mimicry between the two T-cell epitopes in ICA69 and BSA. Further studies are necessary to understand the molecular basis of this mimicry and how either T-cell peptide can modify the disease course.

Molecular cloning of murine ICA69: diabetes-prone mice recognize the human autoimmune-epitope, Tep69, conserved in splice variants from both species.

The islet cell antigen ICA69 is an autoimmune target in most patients with insulin-dependent diabetes. Understanding its role in diabetic autoimmunity would be facilitated by an animal model. We therefore cloned mouse ICA69. The different splice variants now identified conserve Tep69, the single T cell epitope recognized by patient T cells. We show that diabetes-prone NOD mice had Tep69-specific, autoreactive T cell repertoires and thus provide a relevant model for the study of ICA69's role in diabetic autoimmunity.

Genomic organization and transcript analysis of ICAp69, a target antigen in diabetic autoimmunity.

Islet cell antigen p69 (ICAp69) is a target self-antigen in autoimmune (insulin-dependent) diabetes mellitus. Distributed over more than 100 kb on chromosome 6 (6{A1-A2}), the single murine genomic locus contains 14 coding exons, 39-271 bp in length. The identified human and mouse intron-exon junctions are identical, with intron sizes ranging from 94 bp to 24 kb and with conserved flanking region intron sequences. cDNA cloning identified alternatively spliced ICAp69 mRNA transcripts. The predominating alpha-transcripts lack exon 4, while beta-transcripts include this exon, which codes translation termination in all reading frames and a truncated molecule following in vitro expression. gamma-Transcripts show splice removal of exons 8-12, while delta-transcripts exclude exon 11. Transcripts use alternative polyadenylation signals including a less frequent ATTAAA sequence. 5'-Untranslated cDNA and genomic sequencing and long PCR analysis suggest the presence of more noncoding exons. All splice variants encode the conserved T-cell epitope (in exon 2) recognized by autoreactive T cells in diabetic children and diabetes-prone NOD mice.

T cell activation and anergy to islet cell antigen in type I diabetes.

Early exposure to cow milk proteins was linked to the development of type I diabetes by consistent epidemiology, and by feeding and tolerization studies in diabetes-prone rodents. Dietary BSA was suggested as the culprit because patients and relevant rodents have elevated anti-BSA Abs that precipitate the recently cloned protein, p69, from beta cell lysates. A total of 68 of 78 children with recent onset diabetes had BSA-reactive T cells at the time of diagnosis. Here we 1) map the fine specificity of these T cells, 2) delineate a homologous peptide sequence near the N-terminus of p69, and 3) demonstrate T cell recognition of this p69 sequence (T cell epitope p69, Tep69) by patient T cells. The Tep69 sequence is conserved in p69 of patients and diabetes-prone rodents. Whereas BSA triggers T cell proliferation, recombinant p69 and a synthetic Tep69 peptide induce early stages of T cell activation (IL-2R transcription) but insufficient IL-2 production and thus anergy. Exogenous IL-2 overrides anergy and allows proliferative expansion of p69-responsive T cells. In mixing experiments, p69 and Tep69 peptide prevented proliferative responses to BSA even at 100-fold smaller concentrations. These findings imply that high-affinity self-peptide triggers anergy, whereas low-affinity mimicry Ag triggers proliferative expansion of these T cells. This implies a disease model in which mimicry Ag would rescue autoreactive cells from ablation by self-Ag.

Quantitative analysis of gene expression in different tissues by template-calibrated RT-PCR and laser-induced fluorescence.

RT-PCR is widely used to study gene transcription in many biological systems. Despite the development of a variety of, at times complex, procedures, quantitation of RT-PCR remains difficult, particularly when comparing RNA from different tissues or very small samples. In the procedure described here, we calibrate input cDNA through incorporation of trace label. PCR product is generated from equal amounts of cDNA with fluoresceinated primers, size fractionated, and quantitated by laser-induced fluorescence in an automated DNA sequencer. Eliminating variation in input cDNA resulted in reliable noncompetitive PCR quantitation from templates equivalent to > or = 50 pg of total RNA. Using the example of beta-glucuronidase, a low-copy-number housekeeping gene, we have drawn a map of differential gene expression for this protein in various rat tissues.

Cloning of human and rat p69 cDNA, a candidate autoimmune target in type 1 diabetes.

Triggering of autoimmunity in insulin-dependent diabetes was linked to dietary bovine serum albumin (BSA). Anti-BSA antibodies from diabetes-prone rats precipitate a protein, p69, from islet cell lysates. We have used these antibodies to identify rat p69 cDNAs. Human p69 cDNA was identified by crosshybridization. The p69 coding regions show 87% nucleotide and 89% amino acid homology. Recombinant p69 is recognized by autoantibody and T cells from diabetic children.

ICA1 encoding p69, a protein linked to the development of type 1 diabetes, maps to human chromosome 7p22.

The development of type 1 (insulin dependent) diabetes mellitus (IDDM) requires a genetically susceptible host and exposure, early in life, to environmental trigger molecules that induce diabetic autoimmunity to insulin producing islet cells. We previously identified islet cell protein p69 as a candidate autoimmune target in IDDM. Here we describe a human genomic p69 fragment which allowed us to map the gene (ICA1) to chromosome 7p22.

Deletion of the entire cytochrome P450 CYP2D6 gene as a cause of impaired drug metabolism in poor metabolizers of the debrisoquine/sparteine polymorphism.

The debrisoquine/sparteine polymorphism is associated with a clinically important genetic deficiency of oxidative drug metabolism. From 5% to 10% of Caucasians designated as poor metabolizers (PMs) of the debrisoquine/sparteine polymorphism have a severely impaired capacity to metabolize more than 25 therapeutically used drugs. The impaired drug metabolism in PMs is due to the absence of cytochrome P450IID6 protein. The gene controlling the P450IID6 protein, CYP2D6, is located on the long arm of chromosome 22. A pseudogene CYP2D8P and a related gene CYP2D7 are located upstream from CYP2D6. This gene locus is highly polymorphic. After digestion of genomic DNA with XbaI endonuclease, restriction fragments of 11.5 kb and 44 kb represent mutant alleles of the cytochrome CYP2D6 gene locus associated with the PM phenotype. In order to elucidate the molecular mechanism of the mutant allele reflected by the XbaI 11.5-kb fragment, a genomic library was constructed from leukocyte DNA of one individual homozygous for this fragment and screened with the human IID6 cDNA. The CYP2D genes were isolated and characterized by restriction mapping and partial sequencing. We demonstrate that the mutant 11.5-kb allele results from a deletion involving the entire functional CYP2D6 gene. This result provides an explanation for the total absence of P450IID6 protein in the liver of these PMs.

In vitro reconstitution of messenger ribonucleoprotein particles from globin messenger RNA and cytosol proteins.

Deproteinized globin poly(A) + mRNAs reassociate readily in vitro with soluble RNA-binding proteins of the cytosol; reconstituted messenger ribonucleoprotein complexes are obtained which are very similar to native globin polyribosomal-mRNP as far as bouyant density in Cs2SO4 and the composition of proteins which can be crosslinked to the mRNA are concerned. Proteins thus identified bind specifically to mRNA and not to ribosomal RNA or any synthetic oligonucleotides, with one exception: a 78-kDa protein could be cross-linked to poly(A).