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Summary Background and objective: CYP2C19 is a drug-metabolizing enzyme showing various genetic polymorphisms that may cause marked interindividual and interethnic variability in the disposition of its substrates. We assessed CYP2C19 genetic polymorphisms in a Korean population using a newly developed multiplex pyrosequencing method. Method: A multiplex pyrosequencing method to simultaneously detect CYP2C19*2, *3, and *17 alleles was designed. We established the frequency of these CYP2C19 alleles in 271 Korean subjects using the multiplex pyrosequencing method. Results: The results showed 100% concordance between single and multiplex pyrosequencing methods. We also validated the polymorphisms identified by pyrosequencing with direct sequencing method. The allele frequencies of CYP2C19*2, CYP2C19*3, and CYP2C19*17 were 0·284, 0·101 and 0·015 respectively. These frequencies are similar to that reported for other Asian populations including Japanese and Chinese but different from that of Caucasians and Africans. Conclusions: The multiplex pyrosequencing method to detect CYP2C19*2, CYP2C19*3, and CYP2C19*17 concurrently, seems to be a rapid and reliable genotyping method for the detection of important CYP2C19 genetic polymorphisms. Similar to studies conducted on other Asian populations, this study reported that in the Korean population tested, the CYP2C19*2 and CYP2C19*3 alleles were relatively frequently found, whereas the frequency of CYP2C19*17 was very low.

We assessed the association of CYP2B6, CYP3A5, and CYP2C19 polymorphisms with sibutramine pharmacokinetics. Forty six healthy male subjects were enrolled, and their CYP2B6 (*4 and *6), CYP3A5 (*3), and CYP2C19 (*2, and *3) genotypes were analyzed. After a single 15-mg dose of sibutramine was administered, plasma concentrations of sibutramine and its metabolites, M1 and M2, were measured. CYP2B6 and CYP3A5 polymorphisms did not affect the pharmacokinetics of sibutramine and its metabolites. However, the CYP2C19 genotype substantially influenced plasma levels of sibutramine and its metabolites. The mean area under the curve (AUC) of sibutramine in CYP2C19 intermediate metabolizers (IMs; *1/*2 or *1/*3) and poor metabolizers (PMs; *2/*2, *2/*3)) was 18.5 and 252.2% higher, respectively, than the AUC in extensive metabolizers (EMs, *1/*1) (P < 0.001). The AUC of M1 metabolite in IMs and PMs was 22.5 and 148.0% higher, respectively, than that of EMs (P < 0.001). Our findings indicate that the CYP2C19 genotype substantially affects the pharmacokinetics of sibutramine. Clinical Pharmacology & Therapeutics (2009) 86 5, 511–518. doi:10.1038/clpt.2009.145

Ecthyma gangrenosum is a well recognized cutaneous manifestation of Pseudomonas aeruginosa infections in immunocompromised patients. Most cases of ecthyma gangrenosum have been associated with concomitant septicaemia. However, ecthyma gangrenosum rarely develops due to Ps. aeruginosa in the absence of bacteraemia. We report a rare case of a nonsepticaemic form of ecthyma gangrenosum presenting as a large solitary necrotic ulcer in a patient with acute myelogenous leukaemia. A culture from the lesion revealed the presence of Ps. aeruginosa, but the results of repeated blood cultures were negative. Histological examination revealed numerous tiny eosinophilic bacilli in the dermis and panniculus with Gram's stain.

CYP2C19 is a drug-metabolizing enzyme showing various genetic polymorphisms that may cause marked interindividual and interethnic variability in the disposition of its substrates. We assessed CYP2C19 genetic polymorphisms in a Korean population using a newly developed multiplex pyrosequencing method.A multiplex pyrosequencing method to simultaneously detect CYP2C19*2, *3, and *17 alleles was designed. We established the frequency of these CYP2C19 alleles in 271 Korean subjects using the multiplex pyrosequencing method.The results showed 100% concordance between single and multiplex pyrosequencing methods. We also validated the polymorphisms identified by pyrosequencing with direct sequencing method. The allele frequencies of CYP2C19*2, CYP2C19*3, and CYP2C19*17 were 0·284, 0·101 and 0·015 respectively. These frequencies are similar to that reported for other Asian populations including Japanese and Chinese but different from that of Caucasians and Africans.The multiplex pyrosequencing method to detect CYP2C19*2, CYP2C19*3, and CYP2C19*17 concurrently, seems to be a rapid and reliable genotyping method for the detection of important CYP2C19 genetic polymorphisms. Similar to studies conducted on other Asian populations, this study reported that in the Korean population tested, the CYP2C19*2 and CYP2C19*3 alleles were relatively frequently found, whereas the frequency of CYP2C19*17 was very low.

The alpha-helical antibiotic peptide (P18: KWKLFKKIPKFLHLAKKF-NH2) designed from the cecropin A(1-8)-magainin 2 (1-12) hybrid displayed strong bactericidal and tumoricidal activity without inducing hemolysis. The effect of the Pro9 residue at central position of P18 on cell selectivity was investigated by Pro9 --Leu or Pro9 --Ser substitution. Either substitution markedly reduced the antibacterial activity of P18 and increased hemolysis, although it did not significantly affect cytotoxicity against human transformed tumor and normal fibroblast cells. These results suggest that a proline kink in alpha-helical antibiotic peptide P18 serves as a hinge region to facilitate ion channel formation on bacterial cell membranes and thus plays an important role in providing high selectivity against bacterial cells. Furthermore, to investigate the structure-antibiotic activity relationships of P18, a series of N- or C-terminal deletion and substitution analogs of P18 were synthesized. The C-terminal region of P18 was related to its antibiotic activity and alpha-helical conformation on lipid membranes rather than N-terminal one. Higher alpha-helicity of the peptides was involved in the hemolytic and antitumor activity rather than antibacterial activity. Except for [L9]-P18 and [S9]-P18, all the designed peptides containing a Pro residue showed potent antibacterial activity, although they did not induce a cytolytic effect against human erythrocyte and normal fibroblast cells at the concentration required to kill bacteria. In particular, P18 and some analogs (N-1, N-2, N-3, N-3L and N-4L) with potent bactericidal and tumoricidal activity and little or no normal cell toxicity may serve as an attractive candidate for the development of novel anti-infective or antitumor agents.

Citrullinemia is an autosomal recessive disease due to the mutations in the argininosuccinate synthetase (ASS) gene. Mutation analysis was performed on three Korean patients with citrullinemia. All of the three patients had the splicing mutation previously reported as IVS6-2AG mutation. Two had Gly324Ser mutation and the other patient had a 67-bp insertion mutation in exon 15. The IVS6-2AG mutation was reported to be found frequently in Japanese patients with citrullinemia, but Caucasian patients showed the extreme mutational heterogeneity. Although a limited number of Korean patients were studied, the IVS6-2AG mutation appears to be one of the most frequent mutant alleles in Korean patients with citrullinemia. The Gly324Ser mutation identified in two patients also suggests the possible high frequency of this mutation in Korean patients as well.

Nocodazole, a microtubule-disrupting agent, induced apoptosis in Rat-1 cells, as indicated by changes in cell morphology, DNA fragmentation, and eventual cell death. During nocodazole-induced apoptosis, normally flat cells became rounded in shape and detached from the extracellular matrix. These morphological changes appeared to be closely associated with degradation of focal adhesion proteins, including p130cas, p125(FAK) and paxillin. p130cas was also degraded in cells treated with staurosporine or etoposide, suggesting that degradation of focal adhesion proteins is a characteristic feature of apoptosis. Nocodazole-induced apoptosis was antagonized by Bcl-2: degradation of focal adhesion proteins was suppressed and cell viability was enhanced in bcl-2 over-expressing cells, even in the presence of nocodazole. Further study of the molecular mechanism of Bcl-2 activation should provide an understanding of the apoptosis induced by disruption of the microtubule network.

Expression patterns of laminin chain mRNAs (A, B1, and B2) during mouse oocyte maturation were examined using the competitive reverse transcription-polymerase chain reaction (RT-PCR) method. Total and poly (A)-rich mRNAs isolated from various stages of maturing oocytes in vitro were subjected to RT-PCR and the precise amount of laminin chain-specific mRNA transcripts was estimated by adding externally known amounts of in vitro transcribed mutant cRNA transcripts as an internal control. The estimated copy numbers for A, B1, and B2 chain mRNAs in a single germinal vesicle-stage oocyte were 1.34 +/- 0.19 x 10(5), 6.95 +/- 0.32 x 10(6), and 2.0 +/- 0.56 x 10(5), respectively. Although notable changes of all laminin chain mRNA levels were not observed at any stage of meiotic maturation in total mRNA preparation, chain- and meiotic stage-dependent alterations of poly (A)-tailed mRNA quantities were observed in poly (A)-rich mRNA preparation. A potent RNA synthesis blocker, alpha-amanitin did not influence the changes of mRNA levels, implying the presence of posttranscriptional regulation mechanism in the expression of laminin chains during mouse oocyte maturation. Discrete and time-dependent deadenylation of A and B1 chain, but not B2 chain mRNA, was observed during oocyte maturation by a rapid amplification of cDNA ends (RACE)-PCR. In germinal vesicle (GV)-stage oocytes, only B1 chain was found to be present in a highly polyadenylated state and subsequent deadenylation was observed as meiosis progressed. The poly (A) tail modification was dependent on the initiation of meiotic resumption. Although all laminin chain mRNAs were found in fully grown and meiotically competent mouse oocytes, Western blot analysis detected the B1 chain polypeptide only in GV- and polar body (PB)-stage eggs. These results suggest that the expression of laminin B1 chain in mouse oocytes may be due to its large amount of mRNA transcripts and/or high level of polyadenylation state that is efficient for translational activation.

Nitric oxide (NO) is a messenger molecule of vascular endothelial cells, macrophages, and neurons. Here, we demonstrate that the activity of NO synthase increases transiently but dramatically in chick embryonic myoblasts that are competent for fusion. This activity requires Ca2+, calmodulin, and NADPH. In addition, the increase in NO synthase activity coincides with an increase in cellular cGMP level. Furthermore, NO generated by treatment with sodium nitroprusside induces precocious myoblast fusion, while treatment with NG-monomethyl-L-arginine, a competitive inhibitor of NO synthase, or methylene blue, an inhibitor of guanylate cyclase, delays fusion. These results provide the first evidence for a strong association of NO with myoblast fusion.

Myoblast surface proteins with binding activity toward the N-terminal 29-kDa fragment of fibronectin were identified by two different experimental techniques: one involves radioiodination of the cell surface proteins, followed by solubilization with Triton X-100 and affinity purification on a Sepharose column conjugated with the 29-kDa fragment, and the other involves cross-linking of the 29-kDa fragment to the cells metabolically labeled with [35S]methionine, followed by immunoprecipitation with anti-29-kDa IgG. Both approaches revealed that primary cultures of chick myoblasts contain the 66- and 48-kDa proteins that bind to the 29-kDa fragment. These binding proteins were then purified to apparent homogeneity by two successive chromatographies of the solubilized extracts of 12-day-old embryonic muscle on wheat germ agglutinin-agarose and 29-kDa fragment-Sepharose columns. However, the 48-kDa protein was found to be derived from contaminating fibroblasts upon immunoblot analysis of the myogenic cell lines, rat L8E63 and mouse C2A3, and cultured fibroblasts using the antibody raised against the 66-kDa protein. Anti-66-kDa IgG inhibited the binding of the 125I-29-kDa protein to the primary culture of myoblasts in a dose-dependent manner. On the other hand, the same antibody showed little or no effect on the initial binding of 125I-fibronectin to the cell surface, but dramatically inhibited its incorporation into deoxycholate-insoluble matrices. Furthermore, Fab fragments of anti-66-kDa IgG completely blocked the incorporation of fluoresceinated fibronectin into matrices but not its binding to the cell surface. These results suggest that fibronectin matrix assembly is mediated at least in part by the interaction of the 66-kDa protein with the N-terminal type I domain of fibronectin.

The changes of higher-order aberrations (HOAs) after bilateral lateral rectus muscle recession were evaluated. Forty eyes of 20 children were enrolled and their wavefront information was assessed until postoperative 3 months. Even though the root mean square (RMS) of total aberration was not changed, the RMS of HOA was transiently increased at postoperative 1 week and returned to baseline level after 1 month. Among individual Zernike coefficient, secondary astigmatism, quadrafoil, secondary coma, secondary trefoil, and pentafoil showed similar tendency with the RMS of HOA. However, coma, trefoil, and spherical aberration were not changed. Regarding recession amount, it did not correlate with any Zernike coefficient. In summary, our data imply that the HOAs are transiently increased after lateral rectus recession surgery. These results are in collusion with previous reports that strabismus surgery induced transient corneal astigmatism.