Discrimination of psychrotolerant Bacillus cereus group based on MALDI-TOF MS analysis of ribosomal subunit proteins.

Psychrotolerant species of the Bacillus cereus group, Bacillus mycoides and Bacillus weihenstephanensis, can grow at ≥ 7 °C and are significant concerns for the food industry due to their ability to cause spoilage of refrigerated food. In addition to that, some strains of B. weihenstephanensis can produce emetic toxin, namely cereulide, which is known to cause vomiting. Therefore, rapid and simple methods to discriminate psychrotolerant B. cereus group species are crucial. Here, matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and the S10-spc-alpha operon gene encoded ribosomal protein mass spectrum (S10-GERMS) method were used to discriminate psychrotolerant species of the B. cereus group based on a set of four ribosomal subunit proteins (S10, S16, S20 and L30). A total of 36 strains of B. cereus group were cultured on LB agar, and analyzed by MALDI-TOF MS. The four biomarkers successfully discriminated 12 strains of psychrotolerant species from mesophilic species of the B. cereus group. Furthermore, the four biomarkers also classified some Bacillus thuringiensis strains. MALDI-TOF MS analysis using the S10-GERMS method allowed simple and rapid discrimination of psychrotolerant species of the B. cereus group from other mesophilic species. This method has a possibility to enable manufacturers and distributors of refrigerated foods to control psychrotolerant species of the B. cereus group effectively.

Induction of human P-glycoprotein in Caco-2 cells: development of a highly sensitive assay system for P-glycoprotein-mediated drug transport.

The aim of this work is to develop a highly sensitive assay system for P-gp-mediated transport by using two methods, induction of P-gp and short-term culture of Caco-2 cells. To induce P-gp in Caco-2 cells, cells were cultured in vinblastine-containing medium. The mRNA level of P-gp was approximately 7-fold higher in Caco-2 cells cultured with vinblastine (P-gp-induced Caco-2 cells) than in control cells. Western blot analysis showed a significant increase in P-gp expression. After cell differentiation, the mRNA level of P-gp was downregulated, however, P-gp-induced Caco-2 cells still possessed a 5.6-fold higher mRNA level of P-gp compared to control cells. Polarized transport of substrate drugs was greater in the monolayer of P-gp-induced cells than in that of control cells. Moreover, we found that P-gp expression in Caco-2 cells could be further enhanced by applying the higher concentration of vinblastine. Transport activity of P-gp in Caco-2 cells cultured with higher concentration of vinblastine was markedly higher than that in P-gp-induced Caco-2 cells and was comparable with that in MDR1-MDCKII cells. In conclusion, this study provided a stable and highly sensitive in vitro assay system that can identify compounds that are subject to P-gp-mediated efflux.

Simultaneous determination of single nucleotide polymorphisms of MDR1 genes by electrochemical DNA chip.

The on-chip genotyping system ("the electrochemical DNA chip") has been developed as a more cost-effective genotyping system and was applied to MDR1 genotyping in the present study, which is required for wide use in clinical application and for personalized medication based on genotype. The electrochemical DNA chip was optimized and applied to simultaneous genotyping of four MDR1 polymorphisms (T-129C, C1236T, G2677(A,T) and C3435T) using synthetic model oligonucleotide DNA and human genomic DNA. The electrochemical DNA chip successfully gave the T-129C, C1236T, G2677(A,T) and C3435T genotypes, which were completely consistent with those determined by direct sequencing. In conclusion, the electrochemical DNA chip is useful for simultaneous determination of some genotypes and haplotypes, and efficient genotyping using this system can support future genotype-phenotype studies at a large scale.

Chiral Phosphoric Acid Catalyzed Asymmetric Synthesis of 2-Substituted 2,3-Dihydro-4-quinolones by a Protecting-Group-Free Approach.

Chiral 2-substituted 2,3-dihydro-4-quinolones were synthesized based on the chiral phosphoric acid catalyzed intramolecular aza-Michael addition reaction using N-unprotected 2-aminophenyl vinyl ketones as substrates in good yields with high enantioselectivities.

Monocarboxylate transporter mediates uptake of lovastatin acid in rat cultured mesangial cells.

To clarify the uptake mechanism(s) for statins, we examined whether monocarboxylate transporter (MCT) contributed to the uptake of lovastatin acid by rat cultured mesangial cells. Expression of mRNAs for MCT1, 2, and 4 was confirmed in mesangial cells. The uptake of lovastatin acid by mesangial cells increased with decreasing extracellular pH. There was clear overshooting in lovastatin acid uptake by the ATP-depleted cells in the presence, but not in the absence, of an inwardly directed H(+)-gradient. The representative MCT substrates/inhibitors inhibited the lovastatin acid uptake. In particular, the inhibition of lovastatin acid uptake by L-lactic acid at the concentration of 80 mM reached 70%, and L-lactic acid and valproic acid inhibited the uptake competitively. On preloading of mesangial cells with L-lactic acid or valproic acid, the lovastatin acid uptake was significantly stimulated. The inhibition constant of L-lactic acid for the lovastatin acid uptake was 32 mM, and this value is comparable to the Michaelis constant (>20 mM) of L-lactic acid for MCT4 described elsewhere. These results demonstrate that lovastatin acid was largely taken up by mesangial cells via MCT, and suggest that MCT4 might contribute to lovastatin acid uptake in the cells.

MDR1, MRP1 and MRP2 genotypes and in vitro chemosensitivity in Japanese patients with colorectal adenocarcinomas.

In our previous paper, the chemosensitivity of human colorectal adenocarcinoma was evaluated against 12 anticancer drugs including 5-fluorouracil (5-FU), 7-ethyl-10-hydroxycamptothecin (SN-38), mitomycin C (MMC) and cisplatin (CDDP), and it was found that the anticancer drugs were effective against those with a relatively high growth rate. MMC was effective for those with a relatively high mRNA expression of the multidrug resistance-associated protein 2 (MRP2), whereas no correlation was found for the multidrug resistant transporter MDR1 and MRP1. In this study, 3 genotypes of MDR1, 4 genotypes of MRP1, and 6 genotypes of MRP2 were additionally evaluated, and it was suggested that MDR1 C3435T and MRP2 G1249A were related with the susceptibility to colorectal adenocarcinoma. The chemosensitivity against 5-FU, SN-38, MMC and CDDP was independent of MDR1 C3435T, MRP1 G2168A, and MRP2 C-24T (C3972T), possibly due to no association with the growth rate of and mRNA expression levels of MDR1, MRP1 and MRP2 in the adenocarcinoma, however, MDR1 C3435T tended to be accompanied with a higher expression of MDR1 mRNA.

Genetic polymorphisms associated with adverse events and elimination of methotrexate in childhood acute lymphoblastic leukemia and malignant lymphoma.

Methotrexate is administered in high doses to treat childhood acute lymphoblastic leukemia and malignant lymphoma. Hepatotoxicity and bone marrow suppression often limit its use, however. The objective of this study was to determine the genetic polymorphisms associated with the hepatotoxicity and elimination of methotrexate. Genetic polymorphisms of glutathione S-transferase (GST) genes including GSTT1 positive/null, GSTM1 positive/null, and GSTP1 A313G, and genes for reduced folate carrier 1 G80A (RFC1 G80A), methylenetetrahydrofolate reductase C677T (MTHFR C677T), and breast cancer resistant protein C421A (BCRP C421A) were determined for 26 patients by the polymerase chain reaction (PCR) method or by direct sequencing. A high frequency of hepatotoxicity (P = 0.035) was observed for patients with GSTM1 positive and RFC1 AA(80), and serum concentrations of methotrexate 48 h after the start of infusion were higher for patients with the TT(677) genotype of MTHFR (P = 0.028). In conclusion, GSTM1 positive/null and RFC1 G80A polymorphisms could be predictors for hepatotoxicity, and the MTHFR C677T polymorphism is associated with elimination of methotrexate.

Association of cumulative cyclosporine dose with its irreversible nephrotoxicity in Japanese patients with pediatric-onset autoimmune diseases.

Cyclosporine (CsA)-induced nephrotoxicity can become a major obstacle to continuous use. The aim of this study was to optimize CsA dose to avoid its irreversible nephrotoxicity. Twenty-three Japanese patients with pediatric-onset systemic lupus erythematosus or idiopathic nephrotic syndrome, who were maintained in a stable condition by oral dosing of CsA microemulsion, were enrolled in this study. The patients were stratified into 3 groups; those with no, reversible, and irreversible nephrotoxicity, according to periodically performed renal pathohistological examinations. A higher concentration of CsA in blood (p=0.002-0.011) and a longer duration of CsA treatment (p=0.002) were risk factors for irreversible nephrotoxicity, and the cumulative CsA dose, the product of the maintenance dose and duration of CsA treatment, was predictive of nephrotoxicity (p=0.036). The maximum target blood concentration at 2 h post-dose, C(2), to avoid CsA-induced irreversible nephrotoxicity was 700 ng/ml, although the cumulative CsA dose of 4850 mg/kg would result in a 50% probability of nephrotoxicity.

Prediction of systemic exposure to cyclosporine in Japanese pediatric patients.

The monitoring of the blood concentration at 2 h (C(2)) after the oral administration of a cyclosporine (CsA) microemulsion was reconfirmed to be useful for the prediction of systemic exposure, the area under the blood concentration-time curve from 0 to 4 h (AUC(0-4)), in a group of Japanese patients, consisting of 33 children aged 5-15 years and 19 young adults aged 16-27 years, with a greater correlation for C(2) (r = 0.927) than the trough concentration (r = 0.488). The dose-normalized AUC(0-4) was independent of gender or indications for CsA, while it depended on body size, i.e., the age (P = 0.065) and total body weight (P = 0.026). MDR1 C3435T had a weak, but insignificant effect (P = 0.072); it was about 22-31% lower in the patients with TT(3435). Co-administration of a steroid and further treatment with nifedipine had a more intensive effect (P = 0.018); co-administration resulted in a 51% increase in the dose-normalized AUC(0-4). A strong effect was also observed for the serum total cholesterol level (P = 0.001). Collectively, the discrepancies in the results on MDR1 C3435T among investigators might be due to variability in the age/total body weight, co-administration drugs or serum lipid level.

Comparison of synthetic DNA templates with authentic cDNA templates in terms of quantification by real-time quantitative reverse transcription polymerase chain reaction.

Synthetic DNA templates were compared with authentic cDNA templates as standards for the real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). The single-stranded DNA template used here targeted the multidrug resistant transporter P-glycoprotein/MDR1. The double-stranded DNA template, targeting glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was synthesized using an exonuclease-free large fragment E. coli DNA polymerase I. The human colon carcinoma cell line Caco-2 and human duodenum biopsies were used to prepare the authentic cDNA templates. The standard lines were comparable for the synthetic DNA templates and authentic cDNA templates. Long-term cryopreservation at -80 degrees C resulted in the destabilization of the synthetic single-stranded DNA template compared with the authentic cDNA templates in the case of MDR1, whereas for GAPDH, the stability of the synthetic double-stranded DNA template was comparable with that of the authentic cDNA templates. Even for the synthetic DNA templates, repetitive freeze-thawing resulted in destabilization, especially at lower concentrations, and degradation products might have interfered with the RT-PCR's efficiency. The synthetic DNA templates are better than the authentic cDNA templates, but more than 5 cycles of repetitive freeze-thawing should be avoided.

Gene expression profiles of ABC transporters and cytochrome P450 3A in Caco-2 and human colorectal cancer cell lines.

PURPOSE: The mRNA levels of MDR1 (P-glycoprotein), multidrug resistance-associated proteins (MRP1, MRP2), cytochrome P450 3A (CYP3A) and villin in human colorectal cell lines (HCT-15, LoVo DLD-1, HCT-116 and SW620) were quantitatively compared with those in Caco-2 cells. METHODS: The mRNA levels were determined by real time quantitative polymerase chain reaction and expressed as the relative concen trations of MDR1 mRNA to glyceraldehyde-3-phosphate dehydro genase (GAPDH) mRNA. RESULTSl MDR1 mRNA was expressed in HCT-15 LoVo and DLD-1 cells at similar or lower level to Caco-2. The expression of MRP mRNA in the cell lines tested was comparable with Caco-2. MRP. mRNA was detected only in HCT-116 and SW620 at significantly lower level than Caco-2. CYP3A mRNA was detected in HCT-15 LoVo, DLD-1 and SW620 at similar level to Caco-2. CONCLUSIONS: HCT-15 LoVo and DLD-1 cells express proteins important for regulating the intestinal absorption of drugs, i.e., MDR1 MRP1 and CYP3A, whereas HCT-116 and SW620 cells were not acceptable for evaluation of absorption properties of drug candidates

Effects of polymorphisms of MDR1, MRP1, and MRP2 genes on their mRNA expression levels in duodenal enterocytes of healthy Japanese subjects.

In the present study, we examined whether polymorphisms in the ATP-binding cassette (ABC) transporter genes, MDR1, MRP1 and MRP2, were associated with their respective mRNA expression levels in duodenal enterocytes of 13 healthy Japanese volunteers. MDR1 genotypes of T-129C, G2677(A,T) and C3435T, MRP1 genotypes of G128C, C218T, G2168A and G3173A, and MRP2 genotypes of C-24T, G1249A, C2302T, C2366T and G4348A were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) or direct sequencing. Mutations T-129C, G2677(A,T) and C3435T of MDRI gene were found at allele frequencies of 2/26, 16/26 and 12/26, respectively. Mutations G2168A of the MRPI gene and C-24T of the MRP2 gene were also found at allele frequencies of 1/26 and 6/26, respectively, whereas other mutations were not detected in MRP1 and MRP2 genes. The relative concentrations (mean +/- S.E.) of MDR1 mRNA to villin mRNA were 0.38 +/- 0.15, 0.56 +/- 0.14 and 1.13 +/- 0.42 in the subjects with C/C3435, C/T(3435) and T/T(3435), respectively, which supported the lower serum concentrations of digoxin after single oral administration in the subjects with the mutant T-allele at position 3435. Genetic collaboration between positions 3435 and 2677 was suggested, and those in G/G2677, G/(A,T)(2677) and T/(A,T)(2677) were 0.16 +/- 0.05, 1.10 +/- 0.40, and 0.63 +/- 0.16, respectively (p = 0.107). However, there was no remarkable effect of the G2168A of the MRP1 gene or of C-24T of the MRP2 gene on the relative MRP1 or MRP2 mRNA concentrations, respectively.