Establishment and characterization of a human cell line derived from a uterine papillary serous carcinoma with wild-type p53 function

Uterine papillary serous carcinoma is an uncommon histologic subtype of endometrial cancer that behaves aggressively and has a poor prognosis. We successfully established a uterine papillary serous carcinoma cell line. The population-doubling time was approximately 16 h. Although loss of p53 function is considered critical for the molecular pathogenesis of uterine papillary serous carcinoma, p53 was not only mutated but functionally active in this cell line. This newly established cell line should be useful for investigating the characteristics of uterine papillary serous carcinoma.     

Brz220 interacts with DWF4, a cytochrome P450 monooxygenase in brassinosteroid biosynthesis, and exerts biological activity

Arabidopsis thaliana (Arabidopsis) treated with the four stereoisomers of Brz220 (2RS, 4RS-1-[4-propyl-2-(4-trifluoromethylphenyl)-1, 3-dioxane-2-ylmethyl]-1H-1, 2, 4-triazole) showed a dwarf phenotype like brassinosteroid (BR) biosynthesis mutants that were rescued by treatment of BRs. The target sites of each Brz220 stereoisomer were investigated by treatment of Arabidopsis with BRs in the dark. The results suggest that the stereoisomers block the 22-hydroxylation step in BR biosynthesis. This step is catalyzed by DWF4, an Arabidopsis cytochrome P450 identified as a steroid 22-hydroxylase. The enzyme was expressed in E. coli, and the binding affinity of the stereoisomers to recombinant DWF4 was analyzed. The results indicate that in these stereoisomers there exists a positive correlation between binding affinity to DWF4 and inhibition of Arabidopsis hypocotyl growth. In this context, we concluded that DWF4 is the target site of Brz220 in Arabidopsis.     

Expression and biochemical characterization of beta-primeverosidase and application of beta-primeverosylamidine to affinity purification

Beta-primeverosidase (PD) is a family 1 glycosidase catalyzing the hydrolysis of beta-primeverosides (6-O-beta-D-xylopyranosyl-beta-D-glucopyranosides) to release a disaccharide primeverose. To investigate how PD recognizes the disaccharide moiety of beta-primeverosides, the recombinant PD was expressed by a baculovirus-insect cell system. The recombinant PD was secreted from High Five cells and was properly modified with N-glycosylation and correct cleavage at the N-terminal signal peptide. The recombinant PD exhibited high substrate specificity to beta-primeverosides in terms of the glycone moiety, consistently with the substrate specificity of native PD from Camellia sinensis. Next, beta-glycosylamidines were synthesized as substrate analog inhibitors. Beta-primeverosylamidine strongly inhibited PD activity, but beta-glucosylamidine did not. Hence beta-primeverosylamidine is an ideal chemical tool for probing disaccharide recognition in the active site of PD. An affinity adsorbent for PD was prepared using beta-primeverosylamidine as a ligand. Affinity chromatography gave large amounts of PD with high purity, permitting crystallographic study.     

Loss of Borealin/DasraB leads to defective cell proliferation, p53 accumulation and early embryonic lethality

Borealin/DasraB is a member of the chromosomal passenger protein complex (CPC) required for proper segregation of chromosomes during mitosis. In Drosophila melanogaster, inactivation of Borealin/DasraB results in polyploidy, delayed mitosis and abnormal tissue development, indicating its critical role for cell proliferation. However, the in vivo role of mammalian Borealin/DasraB remains unclear. Here, we analyzed the expression of Borealin/DasraB and found that borealin is widely expressed in embryonic tissues and later restricted to adult tissues which relies on rapid cell proliferation. To determine the role of borealin during mouse development, we generated borealin-null mice through targeted disruption. While heterozygous mice developed normally, disruption of both borealin alleles resulted in early embryonic lethality by 5.5 dpc (days postcoitus) due to mitotic defects and apoptosis in blastocyst cells that showed microtubule disorganization and no CPC enrichment. At 5.5 dpc, borealin-null embryos exhibited excessive apoptosis and elevated expression of p53. However, loss of p53 did not abrogate or delay embryonic lethality, revealing that Borealin/DasraB inactivation triggered impaired mitosis and apoptosis though p53-independent mechanisms. Our data show that Borealin/DasraB is essential for cell proliferation during early embryonic development, and its early embryonic lethality cannot be rescued by the loss of p53.     

Mosaic genealogy of the Mus musculus genome revealed by 21 nuclear genes from its three subspecies

Patterns of genetic variation provide insight into the evolutionary history of a species. Mouse (Mus musculus) is a good model for this purpose. Here we present the analysis of genealogies of the 21 nuclear loci and one mitochondrial DNA region in M. musculus based on our nucleotide sequences of nine inbred strains from three M. musculus subspecies (musculus, domesticus, and castaneus) and one M. spicilegus strain as an outgroup. The mitochondrial DNA gene genealogy of those strains confirmed the introgression pattern of one musculus strain. When all the nuclear DNA data were concatenated to produce a phylogenetic tree of nine strains, musculus and domesticus strains formed monophyletic clusters with each other, while the two castaneus strains were paraphyletic. When each DNA region was treated independently, the phylogenetic networks revealed an unnegligibly high level of subspecies admixture and the mosaic nature of their genome. Estimation of ancestral and derived population sizes and migration rates suggests the effects of ancestral polymorphism and gene flow on the pattern of genetic variation of the current subspecies. Gene genealogies of Fut4 and Dfy loci also suggested existence of the gene flow between M. musculus and M. spicilegus or other distant species.     

A new species of horseshoe bat of the genus Rhinolophus from China (Chiroptera: Rhinolophidae)

A new species of the Rhinolophus philippinensis group (Chiroptera: Rhinolophidae) is described from Guangdong, Guangxi, and Jiangxi Provinces in China. Rhinolophus huananus n. sp. is characterized by the horseshoe, as well as by external and cranial characteristics that separate it at the species level from the other members of the philippinensis group. One of the small species of the philippinensis group, R. huananus is intermediate in size between smaller R. siamensis and larger R. macrotis.     

A new detection method for arginine-specific ADP-ribosylation of protein -- a combinational use of anti-ADP-ribosylarginine antibody and ADP-ribosylarginine hydrolase

Arginine-specific ADP-ribosylation is one of the posttranslational modifications of proteins by transferring one ADP-ribose moiety of NAD to arginine residues of target proteins. This modification, catalyzed by ADP-ribosyltransferase (Art), is reversed by ADP-ribosylarginine hydrolase (AAH).

In this study, we describe a new method combining an anti-ADP-ribosylarginine antibody (ADP-R-Arg Ab) and AAH for detection of the target protein of ADP-ribosylation. We have raised ADP-R-Arg Ab with ADP-ribosylated histone and examined the reactivity of the antibody with proteins treated by Art and/or AAH, as well as in situ ADP-ribosylation system with mouse T cells. Our results indicate that the detection of ADP-ribosylated protein with ADP-R-Arg Ab and AAH is a useful tool to explore the target proteins of ADP-ribosylation. We applied the method to search endogenously ADP-ribosylated protein in the rat, and detected possible target proteins in the skeletal muscle, which has high Art activity.