Human corneal GlcNac 6-O-sulfotransferase and mouse intestinal GlcNac 6-O-sulfotransferase both produce keratan sulfate

Human corneal N-acetylglucosamine 6-O-sulfotransferase (hCGn6ST) has been identified by the positional candidate approach as the gene responsible for macular corneal dystrophy (MCD). Because of its high homology to carbohydrate sulfotransferases and the presence of mutations of this gene in MCD patients who lack sulfated keratan sulfate in the cornea and serum, hCGn6ST protein is thought to be a sulfotransferase that catalyzes sulfation of GlcNAc in keratan sulfate. In this report, we analyzed the enzymatic activity of hCGn6ST by expressing it in cultured cells. A lysate prepared from HeLa cells transfected with an intact form of hCGn6ST cDNA or culture medium from cells transfected with a secreted form of hCGn6ST cDNA showed an activity of transferring sulfate to C-6 of GlcNAc of synthetic oligosaccharide substrates in vitro. When hCGn6ST was expressed together with human keratan sulfate Gal-6-sulfotransferase (hKSG6ST), HeLa cells produced highly sulfated carbohydrate detected by an anti-keratan sulfate antibody 5D4. These results indicate that hCGn6ST transfers sulfate to C-6 of GlcNAc in keratan sulfate. Amino acid substitutions in hCGn6ST identical to changes resulting from missense mutations found in MCD patients abolished enzymatic activity. Moreover, mouse intestinal GlcNAc 6-O-sulfotransferase had the same activity as hCGn6ST. This observation suggests that mouse intestinal GlcNAc 6-O-sulfotransferase is the orthologue of hCGn6ST and functions as a sulfotransferase to produce keratan sulfate in the cornea.     

Reduced adhesion of monocyte-derived macrophages from CD36-deficient patients to type I collagen

CD36 is an 88-kDa glycoprotein expressed on platelets and monocyte/macrophages (Mphi). CD36 is a multifunctional receptor for collagen, thrombospondin, oxidized low density lipoproteins (LDL), and long-chain fatty acids. The present study was performed to investigate whether CD36 can function as an adhesion molecule which is involved in mediating human macrophages (Mphi) adhesion to type I collagen in vitro. The Mphi of human CD36-deficient as well as normal control subjects were isolated and cultured on the multi-well plates coated with type I collagen, a natural ligand for CD36. Up to 2 h of incubation, the Mphi from CD36-deficient patients showed almost a approximately 55% decrease in adhesion to type I collagen in comparison to those from controls (P < 0.01). However, there was no significant difference in the adhesion thereafter. Furthermore, the addition of antibody against CD36 into the media of control Mphi significantly inhibited the adhesion by approximately 50% (P < 0.05). The addition of oxidized LDL (OxLDL) did not alter adhesion of Mphi from both CD36-deficient and controls. These data suggest that CD36 is involved in the adhesion of Mphi to type I collagen, especially in the early stage of adhesion.     

A specific role of phosphatidylinositol 3-kinase gamma. A regulation of autonomic Ca(2)+ oscillations in cardiac cells

Purinergic stimulation of cardiomyocytes turns on a Src family tyrosine kinase-dependent pathway that stimulates PLCgamma and generates IP(3), a breakdown product of phosphatidylinositol 4,5-bisphosphate (PIP2). This signaling pathway closely regulates cardiac cell autonomic activity (i.e., spontaneous cell Ca(2+) spiking). PIP2 is phosphorylated on 3' by phosphoinositide 3-kinases (PI3Ks) that belong to a broad family of kinase isoforms. The product of PI3K, phosphatidylinositol 3,4,5-trisphosphate, regulates activity of PLCgamma. PI3Ks have emerged as crucial regulators of many cell functions including cell division, cell migration, cell secretion, and, via PLCgamma, Ca(2+) homeostasis. However, although PI3Kalpha and -beta have been shown to mediate specific cell functions in nonhematopoietic cells, such a role has not been found yet for PI3Kgamma.We report that neonatal rat cardiac cells in culture express PI3Kalpha, -beta, and -gamma. The purinergic agonist predominantly activates PI3Kgamma. Both wortmannin and LY294002 prevent tyrosine phosphorylation, and membrane translocation of PLCgamma as well as IP(3) generation in ATP-stimulated cells. Furthermore, an anti-PI3Kgamma, but not an anti-PI3Kbeta, injected in the cells prevents the effect of ATP on cell Ca(2+) spiking. A dominant negative mutant of PI3Kgamma transfected in the cells also exerts the same action. The effect of ATP was observed on spontaneous Ca(2+) spiking of wild-type but not of PI3Kgamma(2/2) embryonic stem cell-derived cardiomyocytes. ATP activates the Btk tyrosine kinase, Tec, and induces its association with PLCgamma. A dominant negative mutant of Tec blocks the purinergic effect on cell Ca(2+) spiking. Tec is translocated to the T-tubes upon ATP stimulation of cardiac cells. Both an anti-PI3Kgamma antibody and a dominant negative mutant of PI3Kgamma injected or transfected into cells prevent the latter event.We conclude that PI3Kgamma activation is a crucial step in the purinergic regulation of cardiac cell spontaneous Ca(2+) spiking. Our data further suggest that Tec works in concert with a Src family kinase and PI3Kgamma to fully activate PLCgamma in ATP-stimulated cardiac cells. This cluster of kinases provides the cardiomyocyte with a tight regulation of IP(3) generation and thus cardiac autonomic activity.     

Availability of oncogene activated production system for mass production of light chain of human antibody in CHO cells

We previously established a ras-oncogene amplified Chinesehamster ovary (CHO) cell line, named ras clone I, as anuniversal host cell line for oncogene activated production(OAP) system to mass-produce recombinant protein by activationof the cytomegalovirus immediate early (CMV) promoter with ras protein. The light chain(C5) of human monoclonal antibody HB4C5 is expected tobe potentially useful for lung cancer targeting. We generated aC5 hyper-producing cell line by transfecting ras cloneI with the C5 gene expression plasmid regulated by theCMV promoter, of which productivity was 5.3 times greater thanthe hyper productive CHO cell line generated by using conventional CHO cells. Introduction of the adenovirus E1A geneinto the hyper-producing cell line derived from ras clone I resulted in further 9.5 times enhancement of the productivity,suggesting the synergistic effect of E1A and ras oncogenes on the recombinant protein production driven by the CMV promoter. In addition, intracellular accumulation of C5 andupregulation of BiP was found in hyper-producing cell lineswhich were introduced E1A and ras oncogene. This resultsuggests that excessive intracellular accumulation ofC5 protein, which might be caused by that the amount of produced C5 in ER is beyond the ability of CHO cells to secrete, might signal the BiP promoter. Our data imply that ras clone I is available as a general host cell for establishing the recombinant protein hyper-producing CHOcells by the OAP system, and suggest that further mass production of recombinant proteins in the OAP system can be possible by clarifying the accurate role of upregulated BiP protein.     

Maternal macho-1 is an intrinsic factor that makes cell response to the same FGF signal differ between mesenchyme and notochord induction in ascidian embryos

An extracellular signaling molecule acts on several types of cells, evoking characteristic and different responses depending on intrinsic factors in the signal-receiving cells. In ascidian embryos, notochord and mesenchyme are induced in the anterior and posterior margins, respectively, of the vegetal hemisphere by the same FGF signal emanating from endoderm precursors. The difference in the responsiveness depends on the inheritance of the posterior-vegetal egg cytoplasm. We show that macho-1, first identified as a localized muscle determinant, is also required for mesenchyme induction, and that it plays a role in making the cell response differ between notochord and mesenchyme induction. A zygotic event involving snail expression downstream of maternal macho-1 mediates the suppression of notochord induction in mesenchyme precursors.     

Decrease in expression of alpha 5 beta 1 integrin during neuronal differentiation of cortical progenitor cells

Neuronal differentiation of embryonic neural progenitor cells is regulated by both intrinsic and extrinsic signals. Since dynamic changes in cell shape typify neuronal differentiation, cell adhesion molecules could be relevant to this process. Although it has been reported that fibronectin-integrin interactions are important for the proliferation of neural progenitor cells, little is known about the contribution of integrins to neuronal differentiation. In order to address this shortfall, we examined integrin expression on cortical progenitor cells by using immunohistochemistry and FACS analysis of cells in which GFP expression was driven by regulatory (promoter) regions of the nestin gene (nestin-GFP(+)). We here report that high levels of nestin promoter activity correlated with high expression levels of alpha(5)beta(1) integrin (alpha(5)beta(1)(high) cells). FACS analysis of nestin-GFP(+) cortical cells revealed an additional subpopulation with reduced expression of alpha(5)beta(1) integrin (alpha(5)beta(1)(low) cells). The size of the alpha(5)beta(1)(low) subpopulation increased during cortical development. To investigate the correlation between integrin and neuronal differentiation, nestin-GFP(+) cortical progenitor cells were sorted into alpha(5)beta(1)(high) or alpha(5)beta(1)(low) populations, and each potential to differentiate was analyzed. We show that the nestin-GFP(+) alpha(5)beta(1)(high) population corresponded to broadly multipotential neural progenitor cells, whereas nestin-GFP(+) alpha(5)beta(1)(low) cells appeared to be committed to a neuronal fate. These findings suggest that alpha(5)beta(1) expression on cortical progenitor cells is developmentally regulated and its downregulation is involved in the process of neuronal differentiation.     

Synthesis and structural analysis of five novel oligosaccharides prepared by glucosyltransfer from beta-D-glucose 1-phosphate to isokestose and nystose using Thermoanaerobacter brockii kojibiose phosphorylase

Five novel oligosaccharides (tetra-, penta- and hexa-saccharides) were synthesized by glucosyltransfer from beta-D-glucose 1-phosphate to isokestose (O-beta-D-fructofuranosyl-(2-->1)-O-beta-D-fructofuranosyl-(2-->1)-alpha-D-glucopyranoside) or nystose (O-beta-D-fructofuranosyl-(2-->1)-O-beta-D-fructofuranosyl-(2-->1)-O-beta-D-fructofuranosyl-(2-->1)-alpha-D-glucopyranoside) using Thermoanaerobacter brockii kojibiose phosphorylase. The oligosaccharides were identified as 2(2-alpha-D-glucopyranosyl)(m)isokestose; [O-alpha-D-glucopyranosyl-(1-->2)](m)-O-[beta-D-fructofuranosyl-(2-->1)](2)-alpha-D-glucopyranoside: m=1, 2, and 3, and 2(2-alpha-D-glucopyranosyl)(n)nystose; [O-alpha-D-glucopyranosyl-(1-->2)](n)-O-[beta-D-fructofuranosyl-(2-->1)](3)-alpha-D-glucopyranoside: n=1 and 2 using gas liquid chromatography analysis of the methyl derivatives, and MALDI-TOF-MS and NMR measurements of the newly formed oligosaccharides. 1H, 13C NMR signals of each saccharide were assigned using 2D-NMR techniques, including COSY, HSQC, HSQC-TOCSY, HMBC, CH(2)-selected E-HSQC, and CH(2)-selected E-HSQC-TOCSY.