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The ATP-binding cassette transporter ABCG2 is expressed in various organs, such as the small intestine, liver, and kidney, and influences the pharmacokinetics of drugs that are its substrates. ABCG2 is also expressed by cancer cells and mediates resistance to anticancer agents by promoting the efflux of these drugs. In the present study, we investigated the interactions between epidermal growth factor receptor tyrosine kinase inhibitors and ABCG2 by MTT assay, intracellular drug accumulation assay, and FACS. This study showed that four epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) (gefitinib, erlotinib, lapatinib, and afatinib) were transported from tumor cells as substrates of ABCG2. Q141K is a common single-nucleotide polymorphism of ABCG2 in Asians. We demonstrated that the extracellular efflux of gefitinib, erlotinib, and lapatinib was reduced by Q141K, whereas afatinib transport was not affected. In addition, all four EGFR TKIs inhibited the transport of other substrates by both wild-type and variant ABCG2 at 0.1 µM concentrations. Accordingly, epidermal growth factor receptor tyrosine kinase inhibitors may induce interactions with other drugs that are substrates of ABCG2, and single-nucleotide polymorphisms of ABCG2 may influence both the pharmacokinetics and efficacy of these anticancer agents.

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BACKGROUND/AIM: To investigate bioequivalence among generic and brand-name irinotecan products. MATERIALS AND METHODS: Products of Yakult and Daiichi-Sankyo (brand-name products), Sandoz, Nippon Kayaku, Taiho, and Sawai were compared with respect to their composition and antitumor activity. RESULTS: High-performance liquid chromatography demonstrated that related substances were within the acceptable range. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay revealed significant differences in cytotoxicity for four cancer cell lines among the products. The concentration of the active compound SN-38 was highest in Yakult's product (23.82 ng/ml) and lowest in Daiichi-Sankyo's product (8.96 ng/ml). MTT assay data were correlated with the SN-38 concentration, suggesting that it influenced differences in cytocidal activity among products. However, the SN-38 concentration was far lower than that of irinotecan (20 mg/ml), suggesting a negligible clinical effect. Metabolism of irinotecan to SN-38 or open-ring forms did not differ significantly among the products. CONCLUSION: The generic products showed equivalent efficacy and safety to the brand-name products.

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In Saccharomyces cerevisiae, genes involved in thiamin pyrophosphate (TPP) synthesis (THI genes) and the pyruvate decarboxylase structural gene PDC5 are transcriptionally induced in response to thiamin starvation. Three positive regulatory factors (Thi2p, Thi3p, and Pdc2p) are involved in the expression of THI genes, whereas only Pdc2p is required for the expression of PDC5. Thi2p and Pdc2p serve as transcriptional activators and each factor can interact with Thi3p. The target consensus DNA sequence of Thi2p has been deduced. When TPP is not bound to Thi3p, the interactions between the regulatory factors are increased and THI gene expression is upregulated. In this study, we demonstrated that Pdc2p interacts with the upstream region of THI genes and PDC5. The association of Pdc2p or Thi2p with THI gene promoters was enhanced by thiamin starvation, suggesting that Pdc2p and Thi2p assist each other in their recruitment to the THI promoters via interaction with Thi3p. It is highly likely that, under thiamin-deprived conditions, a ternary Thi2p/Thi3p/Pdc2p complex is formed and transactivates THI genes in yeast cells. On the other hand, the association of Pdc2p with PDC5 was unaffected by thiamin. We also identified a DNA element in the upstream region of PDC5, which can bind to Pdc2p and is required for the expression of PDC5.

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Yeast genes involved in thiamin pyrophosphate (TPP) synthesis are transcriptionally induced in response to thiamin starvation. In this system, three proteins (Thi2p, Thi3p, and Pdc2p) act as positive regulatory factors. Thi3p is a TPP-binding protein and upregulates THI genes expression when TPP is not bound. We found here that Pdc2p could transactivate gene expression and interact with Thi3p, both of which were enhanced by thiamin starvation. This enhancement of the transactivation activity was not observed in a thi3 strain. When the C-terminal region containing the deduced Thi3p-interacting domain was truncated, Pdc2p expressed striking transactivation activity in a Thi3p-independent fashion. We explored the hypothesis that Thi3p causes a conformational change in Pdc2p leading to full transactivation activity under favorable conditions.

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The 3C-like (3CL) protease of the severe acute respiratory syndrome (SARS) coronavirus is a key enzyme for the virus maturation. We found for the first time that the mature SARS 3CL protease is subject to degradation at 188Arg/189Gln. Replacing Arg with Ile at position 188 rendered the protease resistant to proteolysis. The R188I mutant digested a conserved undecapeptide substrate with a K(m) of 33.8 microM and k(cat) of 4753 s(-1). Compared with the value reported for the mature protease containing a C-terminal His-tag, the relative activity of the mutant was nearly 10(6). Novel peptide-aldehyde derivatives containing a side-chain-protected C-terminal Gln efficiently inhibited the catalytic activity of the R188I mutant. The results indicated for the first time that the tetrapeptide sequence is enough for inhibitory activities of peptide-aldehyde derivatives.

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The physiological significance of thiaminase II, which catalyzes the hydrolysis of thiamin, has remained elusive for several decades. The C-terminal domains of THI20 family proteins (THI20/21/22) and the whole region of PET18 gene product of Saccharomyces cerevisiae are homologous to bacterial thiaminase II. On the other hand, the N-terminal domains of THI20 and THI21 encode 2-methyl-4-amino-5-hydroxymethylpyrimidine kinase and 2-methyl-4-amino-5-hydroxymethylpyrimidine phosphate kinase involved in the thiamin synthetic pathway. In this study, it was first indicated that the C-terminal domains of the THI20 family and PET18 are not required for de novo thiamin synthesis in S. cerevisiae, using a quadruple deletion strain expressing the N-terminal domain of THI20. Biochemical analysis using cell-free extracts and recombinant proteins demonstrated that yeast thiaminase II activity is exclusively encoded by THI20. It appeared that Thi20p has an affinity for the pyrimidine moiety of thiamin, and HMP produced by the thiaminase II activity is immediately phosphorylated. Thi20p was found to participate in the formation of thiamin from two synthetic antagonists, pyrithiamin and oxythiamin, by hydrolyzing both antagonists and phosphorylating HMP to give HMP pyrophosphate. Furthermore, 2-methyl-4-amino-5-aminomethylpyrimidine, a presumed naturally occurring thiamin precursor, was effectively converted to HMP by incubation with Thi20p. It is proposed that the thiaminase II activity of Thi20p is involved in the thiamin salvage pathway by catalyzing the hydrolysis of HMP precursors in S. cerevisiae.

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We cloned and analyzed the 5'-flanking region of the human thiamin pyrophosphokinase gene (hTPK1). Truncation analysis using transiently transfected HepG2 cells revealed the minimal region required for basal activity of the hTPK1 promoter, which was encoded in a sequence between -105 and +441 relative to the transcription start site. In an electrophoretic mobility shift assay using the nuclear extracts from HepG2 cells and the synthetic oligonucleotide containing the Sp1 site, specific DNA-protein complexes were identified. These findings indicate the importance of the Sp1 cis-element in regulating the hTPK1 gene expression.

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The expression of genes of Saccharomyces cerevisiae encoding the enzymes involved in the metabolism of thiamin (THI genes) is co-ordinately repressed by exogenous thiamin and induced in the absence of thiamin. In this yeast THI regulatory system acts mainly at the transcriptional level, thiamin pyrophosphate (TDP) seems to serve as a corepressor, and genetic studies have identified three positive regulatory factors (Thi2p, Thi3p and Pdc2p). We found in a DNA microarray analysis that the expression of THI genes increased 10- to 90-fold in response to thiamin deprivation, and likewise, the expression of THI2 and THI3 increased 17-fold and threefold, respectively. After transfer from repressing to inducing medium, the promoter activity of both THI2 and THI3 increased in parallel with that of PHO3, one of THI genes. The stimulation of THI3 promoter activity was diminished by deletion of THI3, indicative of the autoregulation of THI3. The THI genes were not induced when THI2 was expressed from the yeast GAL1 promoter in a thi3Delta strain or when THI3 was expressed in a thi2Delta strain, suggesting that Thi2p and Thi3p participate simultaneously in the induction. When mutant Thi3p proteins lacking TDP-binding activity were produced in the thi3Delta strain, THI genes were expressed even under thiamin-replete conditions. This result supports the hypothesis that Thi3p senses the intracellular signal of the THI regulatory system to exert transcriptional control. Furthermore, Thi2p and Thi3p were demonstrated to bind each other and this interaction was partially diminished by exogenous thiamin, suggesting that Thi2p and Thi3p stimulate the expression as a complex whose function is disturbed by TDP bound to Thi3p. We discuss the possibility that the induction of THI genes is triggered by the activation of the complex attributed to decrease in intracellular TDP and the elevated complex in the autoregulatory fashion further upregulates THI genes. This is the first report of the involvement of the TDP-binding motif in genetic regulation.

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We studied six infants with thiamine-responsive congenital lactic acidosis and normal pyruvate dehydrogenase complex activity in vitro, through clinical and biochemical analysis. In addition to elevated lactate and pyruvate levels, the data revealed increased urinary excretion of alpha-ketoglutarate, alpha-ketoadipate, and branched chain ketoacids, indicating functional impairment of thiamine-requiring enzymes, such as pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, alpha-ketoadipate dehydrogenase, and branched chain amino acid dehydrogenase. The metabolism of thiamine has not been investigated in patients with thiamine-responsive congenital lactic acidosis. We evaluated two specific transport systems, THTR-1 (SLC19A2) and THTR-2 (SLC19A3), and a pyrophosphorylating enzyme of thiamine, thiamine pyrophosphokinase (hTPK 1), in addition to pyruvate dehydrogenase complex and alpha-ketoglutarate dehydrogenase complex activity; no abnormality was found. Although the clinical features of thiamine-responsive congenital lactic acidosis are heterogeneous and clinical responses to thiamine administration vary, we emphasize the importance of early diagnosis and initiation of thiamine therapy before the occurrence of permanent brain damage. Careful monitoring of lactate and pyruvate would be useful in determining thiamine dosage.

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Two redundant genes, THI20 and THI21, of Saccharomyces cerevisiae encode a 2-methyl-4-amino-5-hydroxymethylpyrimidine monophosphate (HMP-P) kinase required for thiamin biosynthesis. Using functional complementation analysis with an Escherichia coli mutant strain and a defined biochemical system containing partially purified proteins for the reconstitution of thiamin monophosphate synthesis, we demonstrate that both Thi20p and Thi21p proteins also have HMP kinase activity. Although each isoform independently can synthesize HMP pyrophosphate (HMP-PP) from HMP, there is a marked difference in efficiency between the two proteins. The thi20 deletion strain grows at the same rate as the parental strain in minimal medium without thiamin, but its ability to synthesize HMP-PP from HMP is significantly decreased. We discuss the possibility that HMP is not involved in the pathway of de novo thiamin synthesis in S. cerevisiae.

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Thiamin pyrophosphokinase catalyzes the pyrophosphorylation of thiamin to thiamin pyrophosphate in the presence of ATP and Mg2+. The kinetic properties of human thiamin pyrophosphokinase (hTPK1) were investigated using purified histidine-tagged recombinant protein. The plots of the initial velocity against MgATP concentrations gave a sigmoidal character when Mg2+/ATP was maintained at 1. However, the addition of an excess amount of Mg2+ resulted in the restoration of activity at lower concentrations of MgATP. A steady-state kinetics study led us to conclude that the kinase reaction obeys a ping-pong mechanism. Site-directed mutagenesis was also performed on hTPK1 to examine the contributions of eight strictly conserved residues in thiamin pyrophosphokinase on the kinetic properties. Mutations D71N, D73N, and D100N reduced kcat markedly, indicating that these aspartic acids play a crucial role in carrying out the catalytic process of hTPK1. A selective decrease in the kcat/Km(thiamin) value was observed in the D133N mutant, whereas the kcat/Km(ATP) values of T99A and R131G were significantly decreased. Interestingly, the replacement of Gln-96 with Glu caused an increase in the kcat/Km(thiamin) value (3.53-fold of the wild-type). It was therefore suggested that the residues Gln-96, Thr-99, Arg-131, and Asp-133 are conserved as functionally significant components for substrate recognition in thiamin pyrophosphokinase.

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Cancer chemoprevention by phytochemicals may be one of the most feasible approaches for cancer control. For example, phytochemicals obtained from vegetables, fruits, spices, teas, herbs and medicinal plants, such as carotenoids, phenolic compounds and terpenoids, have been proven to suppress experimental carcinogenesis in various organs. These candidates should be evaluated by intervention studies, before acceptance as cancer preventive agents for human application. Phytochemicals may also be useful to develop "designer foods" or "functional foods" for cancer prevention. We are now planning animal foods, such as meats, eggs and milk, which contain anti-carcinogenic phytochemicals. In prototype experiments, expression of genes for synthesis of phytochemicals, such as phytoene and limonene, has been successful in cultured animal cells.