Complex formation of Plk1 and INCENP required for metaphase-anaphase transition

Mitotic chromosomal dynamics is regulated by the coordinated activities of many mitotic kinases, such as cyclin-dependent kinase 1 (Cdk1), Aurora-B or Polo-like kinase 1 (Plk1), but the mechanisms of their coordination remain unknown. Here, we report that Cdk1 phosphorylates Thr 59 and Thr 388 on inner centromere protein (INCENP), which regulates the localization and kinase activity of Aurora-B from prophase to metaphase. INCENP depletion disrupts Plk1 localization specifically at the kinetochore. This phenotype is rescued by the exogenous expression of INCENP wild type and INCENP mutated at Thr 59 to Ala (T59A), but not at Thr 388 to Ala (T388A). The replacement of endogenous INCENP with T388A resulted in the delay of progression from metaphase to anaphase. We propose that INCENP phosphorylation by Cdk1 is necessary for the recruitment of Plk1 to the kinetochore, and that the complex formation of Plk1 and Aurora-B on INCENP may play crucial roles in the regulation of chromosomal dynamics.     

Rapid detection of mycoplasma contamination in cell cultures using sybr green-based real-time polymerase chain reaction

Summary We have developed a simple method for rapid detection of mycoplasma contamination in cell cultures using SYBR Green-based real-time polymerase chain reaction (PCR). To detect eight common contaminant mollicutes, including Mycoplasma (M. arginini, M. fermentans, M. orale, M. hyorhinis, M. hominis, M. salivarium, M. pirum) and Acholeplasma laidlawii, four primers were prepared based on the 23S rRNA regions. Using these primers and a minimum of 100 fg of mycoplasma genomic DNA, the 23S rRNA regions of these eight mycoplasma species were consistently amplified by real-time PCR. In contrast, no specific specific amplification product was observed using DNA templates prepared from various mammalian cell lines. Frozen and cultured samples of several cell lines were tested for mycoplasma contamination to evaluated the utility of this method. Of 25 samples that tested positive for mycoplasma by Hoechst staining, which requires two passages of cell cultures started from frozen samples, mycoplasma was detected by real-time PCR in 24 samples of cell extracts prepared directly from frozen samples. When cultured samples were used for this assay, the accuracy of the diagnoses was further improved. Thus, this technique, which is simple, rapid, and sensitive enough for practical application, in suitable for handling many samples and for routine screening for mycoplasma contamination of cell cultures.     

Defective smooth muscle development in qkI-deficient mice

The qkI gene encodes an RNA binding protein which was identified as a candidate for the classical neurologic mutation, qkv. Although qkI is involved in glial cell differentiation in mice, qkI homologues in other species play important roles in various developmental processes. Here, we show a novel function of qkI in smooth muscle cell differentiation during embryonic blood vessel formation. qkI null embryos died between embryonic day 9.5 and 10.5. Embryonic day 9.5 qkI null embryos showed a lack of large vitelline vessels in the yolk sacs, kinky neural tubes, pericardial effusion, open neural tubes and incomplete embryonic turning. Using X-gal and immunohistochemical staining, qkI is first shown to be expressed in endothelial cells and smooth muscle cells. Analyses of qkI null embryos in vivo and in vitro revealed that the vitelline artery was too thin to connect properly to the yolk sac, thereby preventing remodeling of the yolk sac vasculature, and that the vitelline vessel was deficient in smooth muscle cells. Addition of QKI and platelet-endothelial cell adhesion molecule-1 positive cells to an in vitro para-aortic splanchnopleural culture of qkI null embryos rescued the vascular remodeling deficit. These data suggest that QKI protein has a critical regulatory role in smooth muscle cell development, and that smooth muscle cells play an important role in inducing vascular remodeling.     

Aurora-B regulates the cleavage furrow-specific vimentin phosphorylation in the cytokinetic process

Aurora-B is an evolutionally conserved protein kinase that regulates several mitotic events including cytokinesis. We previously demonstrated the possible existence of a protein kinase that phosphorylates at least Ser-72 on vimentin, the most widely expressed intermediate filament protein, in the cleavage furrow-specific manner. Here we showed that vimentin-Ser-72 phosphorylation occurred specifically at the border of the Aurora-B-localized area from anaphase to telophase. Expression of a dominant-negative mutant of Aurora-B led to a reduction of this vimentin-Ser-72 phosphorylation. In vitro analyses revealed that Aurora-B phosphorylates vimentin at approximately 2 mol phosphate/mol of substrate for 30 min and that this phosphorylation dramatically inhibits vimentin filament formation. We further identified eight Aurora-B phosphorylation sites, including Ser-72 on vimentin, and then constructed the mutant vimentin in which these identified sites are changed into Ala. Cells expressing this mutant formed an unusually long bridge-like intermediate filament structure between unseparated daughter cells. We then identified important phosphorylation sites for the bridge phenotype. Our findings indicate that Aurora-B regulates the cleavage furrow-specific vimentin phosphorylation and controls vimentin filament segregation in cytokinetic process.     

Increased gene expression of endothelin-1 and vasoactive intestinal contractor/endothelin-2 in the mammary gland of lactating mice

In an attempt to understand the roles of endothelin-1 (ET-1) and vasoactive intestinal contractor/endothelin-2 (VIC/ET-2), we have studied the genes for both peptides to be expressed in the mammary gland of lactating mice. We observed through real-time PCR analysis that ET-1 and VIC/ET-2 gene expression gradually increase after parturition and that ET-1 gene expression is significantly higher than that of VIC/ET-2. The distribution of ET-1 peptide was found to be localized mainly in the epithelial cells of the mammary gland at 14th day of lactation. ET-1 gene expression increases significantly, parallel to the increase in beta-casein gene expression, in epithelial cell lines (HC11) of mouse mammary gland after hormonal stimulation by addition of dexamethazone and prolactin. The observed increase in ET-1 expression in differentiated epithelial cells suggests physiological roles for ET-1, including milk production and secretion in the mammary gland of lactating mice.     

Biogenesis of the mitochondrial matrix enzyme, glutamate dehydrogenase, in rat liver cells. II. Significance of binding of glutamate dehydrogenase to microsomal membrane

1. Glutamate dehydrogenase and malate dehydrogenase solubilized from liver microsomes were able to rebind to microsomal vesicles while the corresponding dehydrogenases extracted from mitochondria showed no affinity for microsomes. 2. Competition was noticed between microsomal glutamate dehydrogenase and microsomal malate dehydrogenase in the binding to microsomal membranes. Mitochondrial malate dehydrogenase or bovine serum albumin did not inhibit the binding of microsomal glutamate dehydrogenase to microsomes. 3. Binding of microsomal glutamate dehydrogenase to microsomal membranes decreased when microsomes was preincubated with trypsin. 4. Rough microsomal glutamate dehydrogenase was more efficiently bound to rough microsomes than smooth microsomes. Conversely, smooth microsomal glutamate dehydrogenase had higher affinity for smooth microsomes than for rough microsomes. 5. A difference was noticed among the glutamate dehydrogenase isolated from rough and smooth microsomes, and from mitochondria, which suggested the possibility of minor post-translational modification of enzyme molecules in the transport from the site of synthesis to mitochondria.     

Environmentally benign beta-stereoselective glycosidations of glycosyl phosphites using a reusable heterogeneous solid acid, montmorillonite K-10

Environmentally benign and stereoselective beta-glycosidations of glycopyranosyl phosphites and alcohols using a reusable heterogeneous solid acid, montmorillonite K-10, as an activator have been developed. By these glycosidations, beta-gluco-, 2-deoxy-beta-gluco-, and beta-mannopyranosides were selectively produced in good to high yields.     

Identification of a novel beta-catenin-interacting protein

Cadherin is a well-known cell-cell adhesion molecule, and it binds to beta-catenin, which in turn binds to alpha-catenin. However, little is known about the regulatory mechanism underlying the cadherin-mediated cell-cell adhesion. Here we purified two novel beta-catenin-interacting proteins with molecular masses of 180 kDa (p180) and 150 kDa (p150) from bovine brain cytosol by using glutathione S-transferase (GST)-beta-catenin affinity column chromatography. Mass spectral analysis revealed p180 to be identical to KIAA0313 which has a putative Rap1 guanine nucleotide exchange factor (GEF) domain and p150 to be the same as KIAA0705 which has a high degree of sequence similarity to the synaptic scaffolding molecule (S-SCAM), which binds beta-catenin and KIAA0313 in the yeast two-hybrid system and overlay assay, respectively (Ide et al., Biochem. Biophys. Res. Commun. 256, 456-461, 1999; Ohtsuka et al., Biochem. Biophys. Res. Commun. 265, 38-44, 1999). beta-Catenin was coimmunoprecipitated with KIAA0313 in Madin-Darby canine kidney II (MDCKII) cells, bovine brain cytosol, and EL cells. KIAA0313 and beta-catenin were partly colocalized at sites of cell-cell contact in MDCKII cells. Taken together, our data suggest that KIAA0313 associates with beta-catenin through KIAA0705 in vivo at sites of cell-cell contact.