A highly conserved pocket on PP2A‐B56 is required for hSgo1 binding and cohesion protection during mitosis

The shugoshin proteins are universal protectors of centromeric cohesin during mitosis and meiosis. The binding of human hSgo1 to the PP2A‐B56 phosphatase through a coiled‐coil (CC) region mediates cohesion protection during mitosis. Here we undertook a structure function analysis of the PP2A‐B56‐hSgo1 complex, revealing unanticipated aspects of complex formation and function. We establish that a highly conserved pocket on the B56 regulatory subunit is required for hSgo1 binding and cohesion protection during mitosis in human somatic cells. Consistent with this, we show that hSgo1 blocks the binding of PP2A‐B56 substrates containing a canonical B56 binding motif. We find that PP2A‐B56 bound to hSgo1 dephosphorylates Cdk1 sites on hSgo1 itself to modulate cohesin interactions. Collectively our work provides important insight into cohesion protection during mitosis.     

Development and Application of a Whole-Genome Simple Sequence Repeat Panel for High-Throughput Genotyping in Soybean

Among commonly applied molecular markers, simple sequence repeats (SSRs, or microsatellites) possess advantages such as a high level of polymorphism and codominant pattern of inheritance at individual loci. To facilitate systematic and rapid genetic mapping in soybean, we designed a genotyping panel comprised 304 SSR markers selected for allelic diversity and chromosomal location so as to provide wide coverage. Most primer pairs for the markers in the panel were redesigned to yield amplicons of 80–600 bp in multiplex polymerase chain reaction (PCR) and fluorescence-based sequencer analysis, and they were labelled with one of four different fluorescent dyes. Multiplex PCR with sets of six to eight primer pairs per reaction generated allelic data for 283 of the 304 SSR loci in three different mapping populations, with the loci mapping to the same positions as previously determined. Four SSRs on each chromosome were analysed for allelic diversity in 87 diverse soybean germplasms with four-plex PCR. These 80 loci showed an average allele number and polymorphic information content value of 14.8 and 0.78, respectively. The high level of polymorphism, ease of analysis, and high accuracy of the SSR genotyping panel should render it widely applicable to soybean genetics and breeding.     

Detection of Fas ligand in the bovine oviduct

Presence of a Fas-Fas ligand (FasL) system defines the immune-privileged status of certain tissues such as placenta. This study examined the fluids and tissue(s) of the bovine oviduct, where both spermatozoa and early embryos escape elimination by the female immune system, for the presence and the distribution of Fas and FasL, which might provide an explanation for the immune-privileged site of this organ. In the present study, the immunolocalisation of FasL and Fas, as well as the gene expression of FasL, were determined in the uterotubal junction (UTJ), isthmic (I) and ampullar (A) segments of the oviduct during oestrus and the luteal phase of the oestrous cycle. The degree of apoptosis of oviductal epithelium was examined by the TUNEL method. Oviductal fluid (ODF), collected chronically via indwelling catheters from the I or A segments during both non-luteal and luteal phases of the cycle, was analysed for the presence of FasL. The Fas immunostaining was scattered along the epithelium of all regions of the oviduct and cycle stages investigated, whereas FasL immunolabelling was more conspicuous in oestrous samples. This staining disappeared during the luteal phase, which was particularly evident in the sperm reservoir (UTJ and I). There were fewer TUNEL-positive cells than Fas- or FasL-positive cells in the oviductal epithelium, suggesting that tubal Fas and FasL are not directly involved in epithelial apoptosis. Western blot analyses detected FasL in ODF collected from both I and A, most conspicuously as a 24-27kDa band but also at a 40-45kDa band level. FasL mRNA was expressed in the epithelial cells from the sperm reservoir and A during both non-luteal and luteal phases. However, the level of expression differed significantly between segments during the luteal phase. The results provide novel evidence that the Fas-FasL system is present in the bovine oviduct and could be involved in mediating survival of spermatozoa and early embryos.     

Characterization of Cep135, a novel coiled-coil centrosomal protein involved in microtubule organization in mammalian cells.

By using monoclonal antibodies raised against isolated clam centrosomes, we have identified a novel 135-kD centrosomal protein (Cep135), present in a wide range of organisms. Cep135 is located at the centrosome throughout the cell cycle, and localization is independent of the microtubule network. It distributes throughout the centrosomal area in association with the electron-dense material surrounding centrioles. Sequence analysis of cDNA isolated from CHO cells predicted a protein of 1,145-amino acid residues with extensive alpha-helical domains. Expression of a series of deletion constructs revealed the presence of three independent centrosome-targeting domains. Overexpression of Cep135 resulted in the accumulation of unique whorl-like particles in both the centrosome and the cytoplasm. Although their size, shape, and number varied according to the level of protein expression, these whorls were composed of parallel dense lines arranged in a 6-nm space. Altered levels of Cep135 by protein overexpression and/or suppression of endogenous Cep135 by RNA interference caused disorganization of interphase and mitotic spindle microtubules. Thus, Cep135 may play an important role in the centrosomal function of organizing microtubules in mammalian cells.     

Age-related changes of elements in renal arteries of Thai and Japanese and the relationships among elements

To examine whether there were differences between reces in regard to age-related changes of elements and the relationships among elements in the arteries, the authors investigated both the renal arteries of Thai and Japanese. The Thai subjects consisted of 27 men and 11 women, ranging in age from 27 to 88 yr, whereas the Japanese subjects consisted of 19 men and 26 women, ranging in age from 61 to 99 yr. After the ordinary dissections at Chiang Mai University and Nara Medical University were completed, the bilateral renal arteries were resected and the element contents were determined by inductively coupled plasma-atomic emission spectrometry. In the Thai, a slight accumulation of calcium and phosphorus occurred in the fifties, but thereafter headly increased. In contrast, in the Japanese, an accumulation of calcium and phosphorus began to occur in the seventies and increased markedly in the eighties. The result reveraled that a higher accumulation of calcium and phosphorus occurred in the renal arteries of the Japanese in old age compared with those of the Thai. Regarding the relationships among elements, extremely or very significant direct correlations were found among the contents of calcium, phosphorus, magnesium, and sodium in both the renal arteries of the Thai and Japanese, except for magnesium and sodium contents in the renal arteries of the Thai.     

The planarian P2X homolog in the regulation of asexual reproduction

The growth in size of freshwater planarians in response to nutrient intake is limited by the eventual separation of tail and body fragments in a process called fission. The resulting tail fragment regenerates the entire body as an artificially amputated tail fragment would do, and the body fragment regenerates a tail, resulting in two whole planarians. This regenerative ability is supported by pluripotent somatic stem cells, called neoblasts, which are distributed throughout almost the entire body of the planarian. Neoblasts are the only planarian cells with the ability to continuously proliferate and give rise to all types of cells during regeneration, asexual reproduction, homeostasis, and growth. In order to investigate the molecular characteristics of neoblasts, we conducted an extensive search for neoblast-specific genes using the High Coverage Expression Profiling (HiCEP) method, and tested the function of the resulting candidates by RNAi. Disruption of the expression of one candidate gene, DjP2X-A (Dugesia japonica membrane protein P2X homologue), resulted in a unique phenotype. DjP2X-A RNAi leads to an increase of fission events upon feeding. We confirmed by immunohistochemistry that DjP2X-A is a membrane protein, and elucidated its role in regulating neoblast proliferation, thereby explaining its unique phenotype. We found that DjP2X-A decreases the burst of neoblast proliferation that normally occurs after feeding. We also found that DjP2X-A is required for normal proliferation in starved animals. We propose that DjP2X-A modulates stem cell proliferation in response to the nutritional condition.     

Sister group relationship of turtles to the bird-crocodilian clade revealed by nuclear DNA-coded proteins

The phylogenetic position of turtles is a currently controversial issue. Recent molecular studies rejected a traditional view that turtles are basal living reptiles (Hedges, S. B., and L. L. Poling. 1999. A molecular phylogeny. Science 83:998-1001; Kumazawa, Y., and M. Nishida. 1999. Complete mitochondrial DNA sequences of the green turtle and blue-tailed mole skink, statistical evidence for archosaurian affinity of turtles. Mol. Biol. Evol. 16:784-792). Instead, these studies grouped turtles with birds and crocodiles. The relationship among turtles, birds, and crocodiles remained unclear to date. To resolve this issue, we have cloned and sequenced two nuclear genes encoding the catalytic subunit of DNA polymerase alpha and glycinamide ribonucleotide synthetase-aminoimidazole ribonucleotide synthetase-glycinamide ribonucleotide formyltransferase from amniotes and an amphibian. The amino acid sequences of these proteins were subjected to a phylogenetic analysis based on the maximum likelihood method. The resulting tree showed that turtles are the sister group to a monophyletic cluster of archosaurs (birds and crocodiles). All other possible tree topologies were significantly rejected.