4-Aminophenoxyacetic acids as a novel class of reversible cathepsin K inhibitors

We have designed and synthesized a novel series of 3-biphenylamino acid amides as cathepsin K inhibitors based on compound I. In these inhibitors, we have discovered 4-aminophenoxyacetic acids 43 and 47 with good IC(50) values, although lipophilic groups are favorable for the hydrophobic S1' pocket.     

Phylogeography and genetic structure of Hibiscus tiliaceus--speciation of a pantropical plant with sea-drifted seeds

Phylogenetic relationships and the spatial genetic structure of a pantropical plant with sea-drifted seeds, Hibiscus tiliaceus L., and its allied species were investigated. The combined distribution range of these species is over almost the entire littoral area of the tropics worldwide, which might result from the dispersal of their sea-drifted seeds and from recurrent speciation in local populations. A phylogenetic tree constructed using the nucleotide sequences of a c. 7500-bp portion of chloroplast DNA suggested the possibility that recurrent speciation from H. tiliaceus has given rise to all of its allied species. Three major sequence haplotypes of H. tiliaceus had wide and overlapping distributions throughout the Pacific, Atlantic and Indian Ocean regions. This distribution pattern was also confirmed by PCR-SSCP (polymerase chain reaction amplification with single-strand conformation polymorphism) and PCR-SSP (PCR amplification with sequence specific primers) analyses performed on more than 1100 samples from 65 populations worldwide. Statistical analysis using F(ST) and analysis of molecular variance did not show significant genetic differentiation among the H. tiliaceus populations in the three oceanic regions. The results reported here suggested substantial gene flow occurred between populations in the different oceanic regions due to sea-drifted seeds. A strong genetic difference between the Pacific and Atlantic populations of Hibiscus pernambucensis Arruda was observed, which indicates that gene flow in this species between the two regions has been prevented. The wide and dominant distribution of a haplotype shared by H. pernambucensis and H. tiliaceus in the Atlantic region suggests significant introgression between the two species in this region.     

A periplasmic,pyridoxal-5′-phosphate-dependent amino acid racemase in <Emphasis Type="Italic">Pseudomonas taetrolens</Emphasis>

The pyridoxal-5′-phosphate (PLP)-dependent amino acid racemases occur in almost every bacterium but may differ considerably with respect to substrate specificity. We here isolated the cloned broad substrate specificity racemase ArgR of Pseudomonas taetrolens from Escherichia coli by classical procedures. The racemase was biochemically characterized and amongst other aspects it was confirmed that it is mostly active with lysine, arginine and ornithine, but merely weakly active with alanine, whereas the alanine racemase of the same organism studied in comparison acts on alanine only. Unexpectedly, sequencing the amino-terminal end of ArgR revealed processing of the protein, with a signal peptide cleaved off. Subsequent localization studies demonstrated that in both P. taetrolens and E. coli ArgR activity was almost exclusively present in the periplasm, a feature so far unknown for any amino acid racemase. An ArgR-derivative carrying a carboxy-terminal His-tag was made and this was demonstrated to localize even in an E. coli mutant devoid of the twin-arginine translocation (Tat) pathway in the periplasm. These data indicate that ArgR is synthesized as a prepeptide and translocated in a Tat-independent manner. We therefore propose that ArgR translocation depends on the Sec system and a post-translocational insertion of PLP occurs. As further experiments showed, ArgR is necessary for the catabolism of d-arginine and d-lysine by P. taetrolens.     

Application of random amplified polymorphic DNA (RAPD) analysis coupled with microchip electrophoresis for high-resolution identification of <Emphasis Type="Italic">Monascus</Emphasis> strains

Monascus fungi are commonly used for a variety of food products in Asia, and are also known to produce some biologically active compounds. Since the use of Monascus is expected to increase in food industries, strain-level identification and management of Monascus will be needed in the near future. In the present study, random amplified polymorphic DNA (RAPD) analysis coupled with microchip electrophoresis was applied for this purpose. Evaluations of the analysis stability revealed that reproducible results could be obtained, although template DNA fragmentation could influence the resulting RAPD pattern. RAPD analysis using 15 Monascus strains consisting of four species, M. ruber, M. pilosus, M. purpureus, and M. kaoliang showed that each strain generated a unique RAPD pattern, which allows strain-level identification of Monascus. In addition, the phylogenetic tree constructed from RAPD patterns reflected M. ruber–M. pilosus and M. purpureus–M. kaoliang clusters inferred from both ITS and β-tubulin gene sequences, which indicated that the RAPD pattern could reflect their phylogenetic traits to a certain extent. On the other hand, RAPD analysis did not support the monophyletic clustering of the four Monascus species used in this study, which suggests the necessity of reexamination of species boundaries in Monascus.     

Effect of flavonoids on androgen and glucocorticoid receptors based on in vitro reporter gene assay

The effect of 32 flavonoids on androgen (AR) and glucocorticoid receptors (GR) was investigated using an MDA-kb2 human breast cancer cell line to predict potential AR and GR activities. Among them, 5-hydroxyflavone (7) had the highest AR antagonistic activity with an IC₅₀ value of 0.3 μM, whereas 6-methoxyflavone (11) had the highest induced luciferase activity with an EC₁₅₀ value of 0.7 μM. Genistein (2) and daizein (1) showed a sufficient increase of luciferase activities as their concentrations increased with EC₁₅₀ values of 4.4 and 10.1 μM, respectively. These findings provide evidence of a fundamental property of their structure–activity relationship with AR and/or GR.     

Primordial germ cells contain subpopulations that have greater ability to develop into pluripotential stem cells

Primordial germ cells (PGCs) are undifferentiated germ cells in embryos. We previously found that some mouse PGCs develop into pluripotential cells (EG cells) when cultured on a feeder layer expressing the membrane bound form of Steel factor with culture medium containing leukemia inhibitory factor and basic fibroblast growth factor. To understand the mechanisms of the conversion of PGCs into EG cells, we attempted to identify PGC subpopulations that have the ability to develop into EG cells. Using flow cytometry, we fractionated PGCs by the expression of the cell surface antigen integrin α6, as well as by the detection of side‐population (SP) cells in which stem cells are enriched in various tissues. PGCs with negative or low integrin α6 expression and with SP cell phenotype showed higher potential to convert to EG cells. Negative or low integrin α6 expression in PGCs was also correlated with lower expression of Ddx4, which is specifically expressed in PGCs after embryonic day 10.5. The results indicate that the primitive PGC population showing the SP cell phenotype among undifferentiated PGCs has a higher ability of being converted into EG cells. Thus, conversion of PGCs into pluripotential stem cells may be regulated by being influenced by the natural status of individual PGCs as well as the reprogramming process after starting culture.     

The ZW sex chromosomes of <Emphasis Type="Italic">Gekko hokouensis</Emphasis> (Gekkonidae,Squamata) represent highly conserved homology with those of avian species

Populations of the gecko lizard Gekko hokouensis (Gekkonidae, Squamata) on Okinawajima Island and a few other islands of the Ryukyu Archipelago, Japan, have the morphologically differentiated sex chromosomes, the acrocentric Z chromosome and the subtelocentric W chromosome, although the continental representative of this species reportedly shows no sex chromosome heteromorphism. To investigate the origin of sex chromosomes and the process of sex chromosomal differentiation in this species, we molecularly cloned the homologues of six chicken Z-linked genes and mapped them to the metaphase chromosomes of the Okinawajima sample. They were all localized to the Z and W chromosomes in the order ACO1/IREBP–RPS6–DMRT1–CHD1–GHR–ATP5A1, indicating that the origin of ZW chromosomes in G. hokouensis is the same as that in the class Aves, but is different from that in the suborder Ophidia. These results suggest that in reptiles the origin of sex chromosomes varies even within such a small clade as the order Squamata, employing a variety of genetic sex determination. ACO1/IREBP, RPS6, and DMRT1 were located on the Z long arm and the W short arm in the same order, suggesting that multiple rearrangements have occurred in this region of the W chromosome, where genetic differentiation between the Z and W chromosomes has been probably caused by the cessation of meiotic recombination.     

A possible role of lysophospholipids produced by calcium-independent phospholipase A2 in membrane-raft budding and fission

Phospholipase A₂ (PLA₂) not only plays a role in the membrane vesiculation system but also mediates membrane-raft budding and fission in artificial giant liposomes. This study aimed to demonstrate the same effects in living cells. Differentiated Caco-2 cells were cultured on filter membranes. MDCK cells were challenged with Influenza virus. The MDCK cultures were harvested for virus titration with a plaque assay. Alkaline phosphatase (ALP), a membrane-raft associated glycosylphosphatidylinositol (GPI)-anchored protein, was 70% released by adding 0.2 mmol/l lysophosphatidylcholine, which was abolished by treatment with a membrane-raft disrupter, methyl-β-cyclodextrin. Activation of calcium-independent PLA₂ (iPLA₂) by brefeldin A increased the apical release of ALP by approximately 1.5-fold (p < 0.01), which was blocked by PLA₂ inhibitor bromoenol lactone (BEL). BEL also reduced Influenza virus production into the media (< 10%) in the MDCK culture. These results suggest that cells utilize inverted corn-shaped lysophospholipids generated by PLA₂ to modulate plasma membrane structure and assist the budding of raft-associated plasma membrane particles, which virus utilizes for its budding. Brush borders are enriched with membrane-rafts and undergo rapid turnover; thus, PLA₂ may be involved in the regulatory mechanism in membrane dynamism. Further, iPLA₂ may provide a therapeutic target for viral infections.