Environmental factors affecting black/white coloration of the silken girdle in the swallowtail butterfly, Atrophaneura alcinous (Lepidoptera: Papilionidae)

The silken girdles of pupae of the swallowtail butterfly Atrophaneura alcinous show black and white color diphenism. Field observations revealed that all pupae observed on non-food plants and the leaves and stems of the larval food plant Aristolochia debilis were classified as a silken girdle of a black type, while a large portion of pupae pupating on the twigs and trunks of cherry trees in close proximity to A. debilis were classified as a silken girdle of a black type. Additionally, all pupae observed on the surfaces of artificial objects in areas where there are no surrounding plants or trees were classified as a silken girdle of a white type. We demonstrated the effect of day length and the texture, light, plant odor and humidity of pupation sites on the coloration of the silken girdle in A. alcinous. Regardless of long-day or short-day day length conditions, light conditions of constant light or dark, or the presence of a plant odor of A. debilis as environmental cues, all larvae placed at over 80% relative humidity (R.H.) developed into pupae with a silken girdle of a black type. However, all larvae developed into pupae with a silken girdle of a white type when R.H. was below 75%. Furthermore, when pupae with a silken girdle of a white type were transferred to conditions of 90% R.H. within 24 hr of pupation, the white color of the silken girdle changed into a black type within 24 hr of the transfer. The present data suggest that the induction of a black coloration of the silken girdle in A. alcinous requires a R.H. of approximately 80% or more as an environmental factor.     

Gene knockout analysis of two gamma-tubulin isoforms in mice

Gamma-tubulin regulates the nucleation of microtubules, but knowledge of its functions in vivo is still fragmentary. Here, we report the identification of two closely related gamma-tubulin isoforms, TUBG1 and TUBG2, in mice, and the generation of TUBG1- and TUBG2-deficient mice. TUBG1 was expressed ubiquitously, whereas TUBG2 was primarily detected in the brain. The development of TUBG1-deficient (Tubg1-/-) embryos stopped at the morula/blastocyst stages due to a characteristic mitotic arrest: the mitotic spindle was highly disorganized, and disorganized spindles showed one or two pole-like foci of bundled MTs that were surrounded by condensed chromosomes. TUBG2 was expressed in blastocysts, but could not rescue the TUBG1 deficiency. By contrast, TUBG2-deficient (Tubg2-/-) mice were born, grew, and intercrossed normally. In the brain of wild-type mice, TUBG2 was expressed in approximately the same amount as TUBG1, but no histological abnormalities were found in the Tubg2-/- brain. These findings indicated that TUBG1 and TUBG2 are not functionally equivalent in vivo, that TUBG1 corresponds to conventional gamma-tubulin, and that TUBG2 may have some unidentified function in the brain.     

Vascular smooth muscle cell polyploidization involves changes in chromosome passenger proteins and an endomitotic cell cycle

Vascular smooth muscle cell polyploidization occurs during normal development and is enhanced under physiologic stress, but the mechanism of this cell cycle has not been explored. We show via time-lapse video imaging and immunofluorescence analyses that primary vascular smooth muscle cells (VSMC) undergo an endomitotic-type cell cycle, including a normal progression through part of mitosis. Mononuclear polyploid cells are generated by defects in sister chromatid separation and/or segregation, and cellular binucleation occurs by reversal of cytokinesis. To obtain further leads to regulators involved, we examined the chromosomal passenger proteins, Aurora B, inner centromere protein and Survivin, and concluded that Aurora B and inner centromere protein are normally colocalized in centromeres, the midzone, and the midbody during mitosis. Survivin, however, is dim and diffused; it does not colocalize with either Aurora B or inner centromere protein in VSMC, which could account for defects in sister chromatid separation and/or segregation and reversal of cytokinesis. In accordance with the reported dependency of Aurora B activity on Survivin, the Aurora B substrate, vimentin, is not phosphorylated during cytokinesis. Finally, the data show that ectopically expressed Survivin inhibits polyploidization in vascular smooth muscle cells. Hence, aberrant chromosome passenger protein activity and endomitosis are associated with VSMC polyploidization.     

Analysis of telomeric single-strand overhang length in human endometrial cancers

The 3' single-strand telomeric overhang (3'-OH) is a key component of telomere structure. Although telomere length has been well analyzed in a variety of human cancers, no information is available on the 3'-OH length in cancers. In the present study, we examined the 3'-OH length in normal and malignant endometria using telomere-oligonucleotide ligation assay. Although 3'-OH lengths varied among patients, 3'-OH length observed in endometrial cancers was significantly shorter than that found in samples derived from normal endometria (P < 0.001: Student's t-test), suggesting that erosion of 3'-OH length induces impaired telomeric integrity and genomic instability, leading to carcinogenesis. Interestingly, we found that the most aggressive subtypes of endometrial cancers harbored significantly longer 3'-OH length than those with non-aggressive subtypes (P < 0.001: Sheffe's test), suggesting that cancer cells with long 3'-OH length have growth advantage due to their stabilized telomere ends. In contrast, we failed to observe an association between overall telomere length and any clinicopathological characteristics of endometrial cancers. These findings suggest that erosion of 3'-OH length, rather than overall telomere length, play roles in endometrial carcinogenesis. Furthermore, long 3'-OH may serve as a molecular marker for aggressive phenotype of tumors.     

Cooperative effect of hydrophobic and electrostatic forces on alcohol-induced alpha-helix formation of alpha1-acid glycoprotein

Alpha1-acid glycoprotein (AGP) is a serum glycoprotein that mainly binds basic drugs. Previous reports have shown that AGP converts from a beta-sheet to an alpha-helix upon interaction with biomembranes. In the current studies, we found that alkanols, diols, and halogenols all induce this conformational change. Increased length and bulkiness of the hydrocarbon group and the presence of a halogen atom promoted this conversion, whereas the presence of a hydroxyl group inhibited it. Moreover, the effect was dependent on the hydrophobic and electrostatic properties of the alcohols. These results indicate that, in a membrane environment, hydrophobic and electrostatic factors cooperatively induce the transition of AGP from a beta-sheet to an alpha-helix.     

Identification of the amino acid residue of CYP27B1 responsible for binding of 25-hydroxyvitamin D3 whose mutation causes vitamin D-dependent rickets type 1

We previously reported the three-dimensional structure of human CYP27B1 (25-hydroxyvitamin D3 1alpha-hydroxylase) constructed by homology modeling. Using the three-dimensional model we studied the docking of the substrate, 25-hydroxyvitamin D3, into the substrate binding pocket of CYP27B1. In this study, we focused on the amino acid residues whose point mutations cause vitamin D-dependent rickets type 1, especially unconserved residues among mitochondrial CYPs such as Gln65 and Thr409. Recently, we successfully overexpressed mouse CYP27B1 by using a GroEL/ES co-expression system. In a mutation study of mouse CYP27B1 that included spectroscopic analysis, we concluded that in a 1alpha-hydroxylation process, Ser408 of mouse CYP27B1 corresponding to Thr409 of human CYP27B1 forms a hydrogen bond with the 25-hydroxyl group of 25-hydroxyvitamin D3. This is the first report that shows a critical amino acid residue recognizing the 25-hydroxyl group of the vitamin D3.     

Orally active CCR5 antagonists as anti-HIV-1 agents. Part 3: Synthesis and biological activities of 1-benzazepine derivatives containing a sulfoxide moiety

In order to develop orally active CCR5 antagonists, 1-propyl- or 1-isobutyl-1-benzazepine derivatives containing a sulfoxide moiety have been designed, synthesized, and evaluated for their biological activities. Sulfoxide compounds containing a 2-pyridyl group were first investigated, which led to discovering that the presence of a methylene group between the sulfoxide moiety and 2-pyridyl group was necessary for increased inhibitory activity in a binding assay. After further chemical modification, it was found that replacement of the pyridyl group with an imidazolyl or 1,2,4-triazolyl group enhanced activity in the binding assay and that S-sulfoxide compounds were more active than R-isomers. Particularly, compounds (S)-4r, (S)-4s, and (S)-4w exhibited highly potent CCR5 antagonistic activities (IC50=1.9, 1.7, 1.6 nM, respectively) and inhibitory effects (IC50=1.0, 2.8, 7.7 nM, respectively) in the HIV-1 envelope mediated membrane fusion assay, together with good pharmacokinetic properties in rats. In addition, we established the synthesis of (S)-4r and (S)-4w by asymmetric oxidation with titanium-(S)-(-)-1,1'-bi-2-naphthol complex.     

Quinolactacide, a new quinolone insecticide from Penicillium citrinum Thom F 1539

By a screening program searching for new pesticides from fungal sources, an insecticidal compound was isolated from Penicillium citrinum F 1539. The compound, named quinolactacide, was novel and showed 88% mortality against green peach aphids (Myzus persicae) at 250 ppm. Its structure was determined by spectroscopic techniques.     

Characterization of mesendoderm: a diverging point of the definitive endoderm and mesoderm in embryonic stem cell differentiation culture

Bipotent mesendoderm that can give rise to both endoderm and mesoderm is an established entity from C. elegans to zebrafish. Although previous studies in mouse embryo indicated the presence of bi-potent mesendoderm cells in the organizer region, characterization of mesendoderm and its differentiation processes are still unclear. As bi-potent mesendoderm is implicated as the major precursor of definitive endoderm, its identification is also essential for exploring the differentiation of definitive endoderm. In this study, we have established embryonic stem (ES) cell lines that carry GFP gene in the goosecoid (Gsc) gene locus and have investigated the differentiation course of mesendodermal cells using Gsc expression as a marker. Our results show that mesendoderm is represented as a Gsc-GFP+ E-cadherin(ECD)+ PDGFRalpha(alphaR)+ population and is selectively induced from ES cells under defined conditions containing either activin or nodal. Subsequently, it diverges to Gsc+ ECD+ alphaR- and Gsc+ ECD- alphaR+ intermediates that eventually differentiate into definitive endoderm and mesodermal lineages, respectively. The presence of mesendodermal cells in nascent Gsc+ ECD+ alphaR+ population was also confirmed by single cell analysis. Finally, we show that the defined culture condition and surface markers developed in this study are applicable for obtaining pure mesendodermal cells and their immediate progenies from genetically unmanipulated ES cells.