Expression patterns of class I <Emphasis Type="Italic">KNOX</Emphasis> and <Emphasis Type="Italic">YABBY</Emphasis> genes in <Emphasis Type="Italic">Ruscus aculeatus</Emphasis> (Asparagaceae) with implications for phylloclade homology

STM (RaSTM) and YAB2 (RaYAB2) homologues were isolated from Ruscus aculeatus (Asparagaceae, monocots), and their expressions were analyzed by real-time polymerase chain reaction (PCR) to assess hypotheses on the evolutionary origin of the phylloclade in the Asparagaceae. In young shoot buds, RaSTM is expressed in the shoot apex, while RaYAB2 is expressed in the scale leaf subtending the shoot bud. This expression pattern is shared by other angiosperms, suggesting that the expression patterns of RaSTM and RaYAB2 are useful as molecular markers to identify the shoot and leaf, respectively. RaSTM and RaYAB2 are expressed concomitantly in phylloclade primordia. These results suggest that the phylloclade is not homologous to either the shoot or leaf, but that it has a double organ identity.     

Antitumor drugs possessing topoisomerase I inhibition: applicable separation methods

Separation methods for antitumor drugs capable of topoisomerase I inhibition were reviewed in this study. Camptothecin (CPT) its related analogues seemed to be promising anticancer drugs that exhibit topoisomerase I inhibition. This group of compounds contain a closed α-hydroxy-δ-lactone ring (lactone form) that can undergo reversible hydrolysis to form the open-ring form (carboxylate form). In vitro pharmacological study showed that the antitumor activity of the lactone form was higher than that of the carboxylate form. Thus a quantitative method to separate these two forms is important to evaluate the pharmacokinetics and pharmacodynamics of these compounds. Nevertheless, current separation methods are complicated by the pH-dependent instability of the lactone moiety. High-performance liquid chromatography (HPLC) coupled with fluorometric detection has been widely used for the quantitation of the drug as the intact lactone form or as the total lactone carboxylate forms in biological matrices. In this report we reviewed current applicable chromatographic techniques for further bioanalytical studies of CPT derivatives including sample preparations, HPLC columns, mobile phases and additives.     

Insecticidal activities of methyleugenol and β-asarone,from the herbal medicines Saishin and Sekishōkon,and other alkoxy-propenyl-benzene derivatives against the cigarette beetle <Emphasis Type="Italic">Lasioderma serricorne</Emphasis> (Coleoptera: Anobiidae)

The principal insecticidal compounds from the herbal medicines Saishin, the root of Asarum sieboldii Miquel, and Sekishōkon, the rhizome of Acorus gramineus Soland, were isolated by successive silica gel column chromatography and high performance liquid chromatography. The active components, which work against larvae of the cigarette beetle, Lasioderma serricorne (F.), were identified as methyleugenol (4-allyl-1,2-dimethoxybenzene) and β-asarone {1,2,4-trimethoxy-5-[(Z)-prop-1-enyl]benzene} by gas chromatography and gas chromatography/mass spectrometry analyses. These two compounds share some structural features, such as a benzene ring with o-dimethoxy groups and a propenyl group in the opposite position. Consequently, the insecticidal activities of 20 structurally related compounds were tested to evaluate their structure–activity relationship. We found myristicin (5-allyl-1-methoxy-2,3-methylenedioxybenzene) exhibited comparable insecticidal activity to methyleugenol and β-asarone, but the other tested compounds were less active. The lack of insecticidal activity of compounds with a hydrogen, hydroxy, or acetoxy substituent in place of one methoxy group indicates that o-dimethoxy groups are essential for insecticidal activity. The position and configuration of a double bond in the propenyl side chain affected the toxicity, but there was a lack of consistency in the structure–activity relationship for this.     

The nucleosome binding protein HMGN1 interacts with PCNA and facilitates its binding to chromatin

Proliferating cell nuclear antigen (PCNA) is a ubiquitous protein that interacts with multiple partners and regulates nuclear activities, including chromatin assembly, histone modifications, replication, and DNA damage repair. The role of specific partners in regulating PCNA activities is not fully understood. Here we identify the nucleosome binding protein HMGN1 as a new PCNA-interacting protein that enhances the binding of PCNA to chromatin but not to purified DNA. Two tetrapeptides in the conservative domain of HMGN1 contain amino acids necessary for the binding of HMGN1 to PCNA. Deletion of both tetrapeptides abolishes the HMGN1-PCNA interaction. PCNA preferentially binds to the linker DNA adjacent to an HMGN-containing nucleosome. In living cells, loss of HMGN1 decreases the rate of PCNA recruitment to damaged DNA sites. Our study identifies a new factor that facilitates the interaction of PCNA with chromatin and provides insights into mechanisms whereby nucleosome binding architectural proteins affect the cellular phenotype.     

Validation study of a method for assaying DE-310, a macromolecular carrier conjugate containing an anti-tumor camptothecin derivative, and the free drug in human plasma by HPLC and LC/MS/MS

DE-310 is a macromolecular carrier conjugate containing an anti-tumor camptothecin derivative, DX-8951, conjugated to a water-soluble polymer by means of a peptide spacer. New assay methods have been developed to determine the polymer-bonded DX-8951 conjugate, free DX-8951, and Glycyl-DX-8951 in human plasma. Solid-phase extraction was used to extract free DX-8951 and Glycyl-DX-8951 from plasma, and LC/MS/MS (Method I) was used to determine the amount of each analyte. Protein precipitation was used to extract Conjugated DX-8951, which was then digested with thermolysin. HPLC (Method II) was used to determine the productive compound (Phenylalanyl-Glycyl-DX-8951). The lower limit of quantitation of DX-8951 was 50 pg/ml, of Glycyl-DX-8951 was 80 pg/ml, and of Conjugated DX-8951 was 100 ng/ml (as DX-8951 equivalent). Both methods showed satisfactory sensitivity, precision, and accuracy.     

Ubiquitylation of ε-COP by PIRH2 and regulation of the secretion of PSA

Ubiquitylation appears to be involved in the membrane trafficking system including endocytosis, exocytosis, and ER-to-Golgi transport. We found that PIRH2, which was identified as an interacting protein for androgen receptor or p53, interacts with and ubiquitylates the ε-subunit of coatmer complex, ε-COP. PIRH2 promotes the ubiquitylation of ε-COP in vitro and in vivo and consequently promotes the degradation of ε-COP. The interaction between PIRH2 and ε-COP is affected by the presence of androgen, and PIRH2 in the presence of androgen promotes ubiquitylation of ε-COP in vivo. Furthermore, overexpression of the wild type of PIRH2 in prostate cancer cells causes downregulation of the secretion of prostate-specific antigen (PSA), a secretory protein in prostate epithelial cells and one of diagnostic markers for prostate cancer. Our results indicate that PIRH2 functions as a regulator for COP I complex.