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Ribavirin is found to be absorbed in the intestine through the human concentrative nucleoside transporter 2 (hCNT2). Cellular uptake of ribavirin was strongly inhibited by purine nucleoside in an in vitro study. This study aims to examine the effects of dietary purine on the pharmacokinetics of orally administered ribavirin in vivo. Twenty healthy participants were enrolled in a randomized, 2-period crossover study. Participants were administered a single 600-mg oral dose of ribavirin after either a high-purine meal or a low-purine meal. Serial blood samples were collected predose and over 144 hours after dosing. Ribavirin concentrations were measured by liquid chromatography/tandem mass spectrometry. In comparison with corresponding plasma values of ribavirin following a high-purine meal, C(max), AUC(0-144) and AUC(0-infinity) of ribavirin following a low-purine meal were 136% (90% confidence internal [CI]: 120%-155%), 134% (90% CI: 118%-153%), and 139% (90% CI: 120%-159%), respectively. This study indicates that dietary purines have an effect on ribavirin absorption. Dosage regimens of ribavirin might need to be adjusted according to the purine content of the meal.

Mass spectrometry is the tool of choice for sequencing peptides and determining the sites of posttranslational modifications; however, this bottom-up approach lacks in providing global information about the modification states of proteins including the number and types of isoforms and their stoichiometry. Recently, various techniques and mass spectrometers, such as high-field Fourier Transform Ion Cyclotron Resonance (FTICR) mass spectrometers, have been developed to study intact proteins (top-down proteomics). While the protein molecular mass and the qualitative and quantitative information about protein isoforms can be revealed by FTICR-MS analysis, their primary structure (including the identification of modifications and their exact locations in the amino acid sequence) can directly be determined using the MS/MS capability offered by the FTICR mass spectrometer. The distinct advantage of top-down methods are that modifications can be determined for a specific protein isoform rather than for peptides belonging to one or several isoforms. In this chapter, we describe different top-down proteomic approaches enabled by high-field (7, 9.4, and 12 T) FTICR mass spectrometers, and their applicability to answer biological and biomedical questions. We also describe the use of the free flow electrophoresis (FFE) to separate proteins prior to top-down mass spectrometric characterization.

High-efficiency prefractionation of complex protein mixtures is critical for top-down proteomics, i.e., the analysis of intact proteins by MS. Free-flow electrophoresis (FFE) can be used for IEF to separate proteins within a pH gradient according to their pIs. In an FFE system, this separation is performed entirely in the liquid phase, without the need for particulate chromatographic media, gels, or membranes. Herein, we demonstrated the compatibility of IEF-FFE with ESI-Fourier transform ICR MS (ESI-FTICR-MS) for top-down experiments. We demonstrated that IEF-FFE of intact proteins were highly reproducible between FFE instruments, between laboratories, and between analyses. Applying native (0.2% hydroxypropylmethyl cellulose) IEF-FFE to an enzyme resulted in no decrease in enzyme activity; applying either native or denaturing (8 M urea) IEF-FFE to a four-protein mixture with different pIs resulted in isolation of each protein into separate fractions in a 96-well plate. After desalting, each protein was sequenced by top-down MS/MS. As an application of this technique, chicken erythrocyte histone H2A-IV and its major modified forms were enriched by IEF-FFE. Top-down analysis revealed Lys-5 to be a major acetylation site, in addition to N-terminal acetylation.

A multiplex PCR method incorporating primers flanking three variable-number tandem repeat (VNTR) loci (arbitrarily labeled TR1, TR2, and TR3) in the CT18 strain of Salmonella enterica serovar Typhi has been developed for molecular typing of S. enterica serovar Typhi clinical isolates from several Asian countries, including Singapore, Indonesia, India, Bangladesh, Malaysia, and Nepal. We have demonstrated that the multiplex PCR could be performed on crude cell lysates and that the VNTR banding profiles produced could be easily analyzed by visual inspection after conventional agarose gel electrophoresis. The assay was highly discriminative in identifying 49 distinct VNTR profiles among 59 individual isolates. A high level of VNTR profile heterogeneity was observed in isolates from within the same country and among countries. These VNTR profiles remained stable after the strains were passaged extensively under routine laboratory culture conditions. In contrast to the S. enterica serovar Typhi isolates, an absence of TR3 amplicons and a lack of length polymorphisms in TR1 and TR2 amplicons were observed for other S. enterica serovars, such as Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Enteritidis, and Salmonella enterica serovar Paratyphi A, B, and C. DNA sequencing of the amplified VNTR regions substantiated these results, suggesting the high stability of the multiplex PCR assay. The multiplex-PCR-based VNTR profiling developed in this study provides a simple, rapid, reproducible, and high-resolution molecular tool for the epidemiological analysis of S. enterica serovar Typhi strains.

We previously sequenced the 4333-nucleotide cDNA of the COPA (HEP-COP) gene which encodes the human homologue of the alpha-subunit of the coatomer protein complex, involved in intracellular protein transport. Within the 3' untranslated region at nucleotides 4049-4333 was observed an Alu repeat containing conserved A and B block elements, and showing high homology to the human Alu-Sx subfamily consensus sequence. Upstream of the Alu repeat were noted a TATA box, a CAAT motif and two activating transcription factor (ATF)-like binding sites, which represent putative regulatory elements directing Alu transcription. In addition, the 25 and 35 N-terminal amino acid residues of COPA and its bovine homologue were identical to xenin-25 and proxenin, respectively. Xenin-25 is a gastrointestinal hormone that stimulates exocrine pancreatic secretion. This peptide is related to xenopsin, neurotensin and neuromedin N which are bioactive peptides derived from larger precursors via proteolytic cleavage by cathepsin E at processing sites determined by conserved C-terminal sequences, i.e. proline/valine-X-X-hydrophobic amino acid. Given the conformity of the C-terminal residues of xenin-25 (PWIL) and of its progenitor molecule, proxenin (VIQL), it is proposed that these peptides are generated by a similar mechanism of post-translational modification involving a parent precursor represented by the alpha-subunit of coatomer.

The traffic of proteins through the eukaryotic secretory pathway is achieved in part by nonclathrin-coated vesicles mediating transport between the Golgi network and the endoplasmic reticulum. These transit vesicles are coated with coat proteins (COP), which assemble to form a complex of seven polypeptides known as coatomer. From the Hep3B human hepatocellular carcinoma cell line, we have previously isolated and sequenced the cDNA of a novel gene, HEP-COP, whose predicted amino acid sequence, calculated relative molecular mass, and hydrophilicity are strikingly similar to the 160-kD alpha-subunit of the coatomer complex in yeast. Four synthetic peptides were designed for immunizing pairs of rabbits to generate polyclonal antisera. In Western blot experiments, these antibodies could specifically recognize protein bands of 160 kD, which were absent when control preimmune sera were used. Immunoblotting of subcellular components of Hep3B cells probed with one of the antisera revealed 160-kD protein bands predominantly in the microsomal and cytosolic fractions, but virtually none in the nuclear compartment. Indirect immunofluorescence of Hep3B cells using the same antibody exhibited fluorescent staining chiefly in the cytoplasm. Taken together with the cDNA data, the results of this immunological analysis of the putative HEP-COP protein support the suggestion that the latter is the human homolog of alpha-COP.

In eukaryotic cells, protein transport between the endoplasmic reticulum and Golgi compartments is mediated in part by non-clathrin-coated vesicular coat proteins (COP). Seven COP subunits have been recognized, and represent components of a complex known as coatomer. We have previously isolated the cDNA of the human homolog of alpha-COP, designated HEP-COP and given the official gene symbol COPA. Here we report the genomic organization of COPA, which contains 33 exons ranging in size from 67 to 611 bp. Mapped by PCR and cycle sequencing, all the exon-intron junctions conformed with the GT-AG rule, the 32 introns ranging from about 80 bp to 4 kbp, with the genomic DNA of COPA estimated to span approximately 37 kb. Southern blot analysis of genomic DNAs of nine eukaryotic species, from human to yeast, revealed identical signals totaling 36 kb each for man and monkey only. Using 5' RACE and primer extension analysis, the putative transcriptional start site was localized to 466 nucleotides upstream of the translation initiation codon. Comprising a 126-nucleotide 5' untranscribed genomic sequence and a 466-nucleotide 5' noncoding cDNA sequence, the 592-nucleotide 5' CpG island lacked TATA and CAAT boxes but displayed a high G+C content, was enriched for CpG dinucleotides, and contained a potential Sp1-binding site, i.e., features compatible with a housekeeping gene. COPA was mapped by fluorescence in situ hybridization to chromosome region 1q23-->q25.

A 4333-bp novel human cDNA sequence designated HEP-COP was isolated from the Hep3B hepatocellular carcinoma cell line by the RACE technique. Within HEP-COP was identified an ORF of 3672 bp encoding a deduced 1224-amino-acid (aa) sequence which exhibited striking homology with the 1201-aa sequence of RET1P, the alpha-subunit of the coatomer complex (alpha-COP) in Saccharomyces cerevisiae which participates in membrane transport between the endoplasmic reticulum and Golgi apparatus. The aa homology was highest in their N-terminal regions which each contained six WD-40 repeat motifs [Van der Voorn and Ploegh, FEBS Lett. 307 (1992) 131-134], and both proteins were predicted to be hydrophilic with similar estimated molecular masses of 138 324 and 135 599 Da, respectively. Northern blot hybridization demonstrated that HEP-COP was expressed in a wide range of human adult and fetal tissues. RT-PCR analysis revealed no differential expression of HEP-COP in 14 human cancer cell lines, as compared with normal control cells. Considering the close similarities between HEP-COP and yeast alpha-COP, and the ubiquitous expression of HEP-COP implying an essential cellular role, it is likely that HEP-COP is the human homologue of alpha-COP.

The expression of the p53 tumor suppressor gene in ten human cell lines (nine cancers and one normal) was studied using reverse transcription, polymerase chain reaction (PCR) and direct sequencing. Using P53U and P53D primers for amplifying a 371-base pair (bp) target fragment spanning exons 7-10 of p53 cDNA, normal-sized PCR products were amplified from 9 cell lines but not from the Hep3B hepatocellular carcinoma (HCC) cell line. An additional larger band (504 bp) was observed for the Molt-4 T-lymphoblastic leukemia cell line. Employing P531 and P53D primers which flank a 76-bp p53 cDNA fragment, 76 bp as well as 209 bp products were generated by PCR of Molt-4 cDNA. Direct sequencing of the 504 bp and 209 bp bands confirmed the presence of a 133 bp insertion between exons 9 and 10 in the aberrant transcript. This insertion was homologous to a 130-bp sequence within the wild-type p53 intron 9, except for 2 point mutations and 3 base insertions. Sequencing of P53U/P53D PCR products of Molt-4 genomic DNA revealed an 8 bp deletion just downstream to the 133 bp insertion, creating a novel donor splicing site within intron 9. This site, coupled with an inherent acceptor splicing site just upstream to the 133 bp insertion, suggests that the 133 bp stretch represents an alternative exon. The occurrence of a termination signal within this alternative transcript is predicted to culminate in a truncated p53 translational product. The sequences of the 371 bp PCR products of Molt-4, HT-1080, SiHa, CaSki, HeLa and MRC-5 cell lines corresponded with the wild-type p53 cDNA. G-->T transversions at the third base of codon 249 of p53 were detected in Mahlavu and PLC/PRF/5 HCC lines, while a TAC to CAC mutation at codon 234 was observed in an allele of the Raji Burkitt lymphoma line.

Aberrations in the WT1 tumour suppressor gene have been documented in a fraction of Wilms' tumours (WTs). Encoding a protein with four zinc fingers, the WT1 gene is expressed in the developing kidney, gonads, uterus, spleen, mesothelium and brain. Using polymerase chain reaction (PCR), single strand conformation polymorphism (SSCP) analysis and direct DNA sequencing, we analysed 156 diverse tumours for abnormalities of zinc finger 3 (ZF3), a mutational hotspot in WT1. Only one sample (WT) exhibited PCR-SSCP mobility shift. A CGA to TGA nonsense mutation at codon 390 with arginine being substituted with a stop codon was detected and predicted to encode a faulty WT1 protein in this WT, out of 8 WTs studied. Our results are consistent with the presence of WT1 ZF3 mutations in a subset of WTs, but not in other tumours of urogenital nor of WT1-related origin.

Cells from 102 nasopharyngeal biopsies of patients suspected of Nasopharyngeal Carcinoma (NPC) and family members of NPC patients were cultured. Metaphases were successfully obtained from 74 of the biopsies of which 52 were subsequently histologically confirmed to be NPC. Cytogenetical analysis using Q-banding showed abnormalities in 15 cultures, and these included polyploidy, aneuploidy and marker chromosomes. Of the 15 abnormal cultures, 14 were from confirmed NPC patients and in five of these, a consistent 5q+ abnormally was seen involving 5q31. The only other abnormal chromosome changes seen was in a patient with olfactory neuroblastoma.