High-throughput detection of multiple genetic polymorphisms influencing drug metabolism with mismatch primers in allele-specific polymerase chain reaction

Because of genetic polymorphisms of drug-metabolizing enzyme genes, the activities of the enzymes in humans vary widely and alter the metabolism of commonly used clinical agents. Severe adverse effects or resistance to therapy may result. We have developed a rapid and high-throughput genotyping method for detecting polymorphisms of the drug-metabolizing enzyme genes CYP2C9*3, CYP2C19*2, *3, CYP2D6*2, *4, *10, *14, *21, NAT2*5, *6, *7, and TPMT*3 using allele-specific polymerase chain reaction (PCR) with mismatch primers (ASPCR-MP) and CYP2D6*5, *36, and CYP2D6xN using stepdown PCR with detection by SYBR Green I. We analyzed genomic DNA from 139 Japanese volunteers. Identical genotyping results were obtained by using ASPCR-MP, stepdown PCR, and conventional PCR. We found that the methods clearly differentiate three specific profiles with no overlap in the signals. Moreover, both ASPCR-MP and stepdown PCR for genotyping took less than 3-4h. To our knowledge, this is the first report of successful simultaneous detection of multiple genetic polymorphisms with point mutations using ASPCR-MP or multiple genetic polymorphisms with large structural alterations using stepdown PCR. In conclusion, ASPCR-MP and stepdown PCR appear to be suitable for large clinical and epidemiological studies as methods that enable highly sensitive genotyping and yield a high-throughput.     

Okadaic acid induces apoptosis through double-stranded RNA-dependent protein kinase/eukaryotic initiation factor-2alpha pathway in human osteoblastic MG63 cells

Double-stranded RNA-dependent protein kinase (PKR) is a participant in the cellular antiviral response and phosphorylates the alpha-subunit of eukaryotic translation initiation factor 2alpha (eIF-2alpha) to block protein synthesis. Treatment of human osteosarcoma cell line MG63 cells with a serine and threonine protein phosphatase inhibitor, okadaic acid, at the concentration of 100 nM, but not at 20 nM, induced apoptosis. To investigate the functional relationship between phosphatases and apoptosis, we examined the phosphorylation levels of PKR and eIF-2alpha by Western blot analysis. During treatment of cells with it at the higher concentration (100 nM), okadaic acid increased the level of phosphorylated PKR in MG63 cells, this kinase phosphorylating eIF-2alpha. However, at the lower concentration (20 nM), okadaic acid did not affect the level of phosphorylated PKR. In the cells treated with 100 nM okadaic acid, activation of NF-kappaB also occurred. Even though inhibition of translation occurred simultaneously in MG63 cells, the expression of pro-apoptotic proteins Fas and Bax was not affected by 100 nM okadaic acid in these cells. We concluded that the inhibition of translation decreased anti-apoptotic protein expression, thus resulting in apoptosis. Our results also suggest that the inhibition of the protein phosphatase activity by okadaic acid induced apoptosis in MG63 cells through PKR and eIF-2alpha.     

Esterification of ferulic acid with polyols using a ferulic acid esterase from Aspergillus niger

Commercially available enzyme preparations were screened for enzymes that have a high ability to catalyze direct ester-synthesis of ferulic acid with glycerol. Only a preparation, Pectinase PL “Amano” produced by Aspergillus niger, feruloylated glycerol under the experimental conditions. The enzyme responsible for the esterification was purified and characterized. This enzyme, called FAE-PL, was found to be quite similar to an A. niger ferulic acid esterase (FAE-III) in terms of molecular mass, pH and temperature optima, substrate specificity on synthetic substrates, and the N-terminal amino acid sequence. FAE-PL highly catalyzed direct esterification of ferulic acid and sinapinic acid with glycerol. FAE-PL could feruloylate monomeric sugars including arabinose, fructose, galactose, glucose, and xylose. We determined the suitable conditions for direct esterification of ferulic acid with glycerol to be as follows: 1% ferulic acid in the presence of 85% glycerol and 5% dimethyl sulfoxide at pH 4.0 and 50 °C. Under these conditions, 81% of ferulic acid could be converted to 1-glyceryl ferulate, which was identified by ¹H-NMR. The ability of 1-glyceryl ferulate to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals was higher than that of the anti-oxidant butyl hydroxytoluene.     

Geranylgeranyl-pyrophosphate (GGPP) synthase is down-regulated during differentiation of osteoblastic cell line MC3T3-E1

Isoprenylation of geranylgeranyl-pyrophosphate (GGPP) is critical for activation of small GTPases. We examined the roles of GGPP synthase (GGPPS) during the differentiation induced by the cell-to-cell contact in osteoblastic cell line MC3T3-E1 cells. We found that (1) both mRNA and protein expression of GGPPS was reduced with decrement of its activity during the differentiation, (2) GGOH, which is converted to GGPP in the cells, inhibited differentiation. These results suggest that the decrement of GGPP is critical for the cell-to-cell contact-induced differentiation, in which the down-regulation of GGPPS might be involved.     

Six new chalcones from Angelica keiskei inducing adiponectin production in 3T3-L1 adipocytes

Angelica keiskei (Ashitaba in Japanese), a traditional herb in Japan, contains abundant prenylated chalcones. It has been reported that the chalcones from A. keiskei showed such bioactivities as anti-bacterial, anti-cancer and anti-diabetic effects. Xanthoangelol, 4-hydroxyderricin and six new chalcones were isolated in this study from an ethanol extract of A. keiskei by octadecyl silyl (ODS) and silica gel chromatography, and identified by 1D- and 2D-nuclear magnetic resonance (NMR) and high-resolution mass spectrometric analyses. The chalcones from A. keiskei markedly increased the expression of the adiponectin gene and the production of adiponectin in 3T3-L1 adipocytes. These results suggest that the chalcones from A. keiskei might be useful for preventing the metabolic syndrome.     

The Protein Phosphatase Inhibitors Okadaic Acid and Calyculin A Induce Apoptosis in Human Osteoblastic Cells

To determine whether protein phosphorylation and dephosphorylation can affect apoptosis in osteoblastic cells, we examined the effects of okadaic acid (OA) and calyculin A (CA) on cultured human osteoblastic cells Saos-2 and MG63, and mouse osteoblastic MC3T3-E1 cells. After reaching confluence, these cells were exposed to varying concentrations of OA or CA. OA and CA induced cell death in all three cell lines in a dose- and time-dependent manner. Marked nuclear condensation and fragmentation of chromatin were also observed in these cells by using the Hoechst 33342 stain. DNA ladder formation, a hallmark of apoptosis, was detected in Saos-2 and MG63 cells, but not in MC3T3-E1 cells by treatment of OA or CA. In the Saos-2 cells, OA- and CA-induced DNA ladder formation was dose-dependent with maximal effect at concentrations of 10 and 2 nM,respectively, and was time-dependent from 14 to 48 h. DNA ladder formation in response to OA and CA was revealed by using conventional ethidium bromide staining of electrophoresed DNA without using autoradiography. Beyond the maximal effects at the respective concentrations, however, cell death did not indicate DNA laddering, suggesting that phosphatase activity may be required for ladder formation. Our results indicate that apoptosis in the cultured osteoblastic cells is induced by moderate inhibition of PP-1 or PP-2A based on the known selectivity of okadaic acid and of calyculin A.