A specific concanavalin A-mediated binding of bovine serum α-mannosidase to cultured human skin fibroblasts

A specific elevation of cell-associated α-mannosidase was observed in human skin fibroblasts cultured with concanavalin A for 12–72 hours. There was a latency of several hours before the increase of the enzyme activity occurred. When the cells were washed with α-methylmannoside, α-mannosidase activity was not increased. Other lysosomal enzymes including β-mannosidase showed a slight decrease in activity. It was concluded that the elevation of this enzyme activity was the result of a specific binding to the cell surface mediated by concanavalin A.     

Negative regulation of platelet clearance and of the macrophage phagocytic response by the transmembrane glycoprotein SHPS-1

SHPS-1 is a receptor-type glycoprotein that binds and activates the protein-tyrosine phosphatases SHP-1 and SHP-2, and thereby negatively modulates intracellular signaling initiated by various cell surface receptors coupled to tyrosine kinases. SHPS-1 also regulates intercellular communication in the neural and immune systems through its association with CD47 (integrin-associated protein) on adjacent cells. Furthermore, recent studies with fibroblasts derived from mice expressing an SHPS-1 mutant that lacks most of the cytoplasmic region suggested that the intact protein contributes to cytoskeletal function. Mice homozygous for this SHPS-1 mutation have now been shown to manifest thrombocytopenia. These animals did not exhibit a defect in megakaryocytopoiesis or in platelet production. However, platelets were cleared from the bloodstream more rapidly in the mutant mice than in wild-type animals. Furthermore, peritoneal macrophages from the mutant mice phagocytosed red blood cells more effectively than did those from wild-type mice; in addition, they exhibited an increase both in the rate of cell spreading and in the formation of filopodia-like structures at the cell periphery. These results indicate that SHPS-1 both contributes to the survival of circulating platelets and down-regulates the macrophage phagocytic response.     

Shelterin promotes tethering of late replication origins to telomeres for replication‐timing control

DNA replication initiates at many discrete loci on eukaryotic chromosomes, and individual replication origins are regulated under a spatiotemporal program. However, the underlying mechanisms of this regulation remain largely unknown. In the fission yeast Schizosaccharomyces pombe, the telomere‐binding protein Taz1, ortholog of human TRF1/TRF2, regulates a subset of late replication origins by binding to the telomere‐like sequence near the origins. Here, we showed using a lacO/LacI‐GFP system that Taz1‐dependent late origins were predominantly localized at the nuclear periphery throughout interphase, and were localized adjacent to the telomeres in the G1/S phase. The peripheral localization that depended on the nuclear membrane protein Bqt4 was not necessary for telomeric association and replication‐timing control of the replication origins. Interestingly, the shelterin components Rap1 and Poz1 were required for replication‐timing control and telomeric association of Taz1‐dependent late origins, and this requirement was bypassed by a minishelterin Tpz1‐Taz1 fusion protein. Our results suggest that Taz1 suppresses replication initiation through shelterin‐mediated telomeric association of the origins at the onset of S phase.     

Suramin interrupts androgen-inducible autocrine loop involving heparin binding growth factor in mouse mammary cancer (Shionogi carcinoma 115) cells

The androgen-dependent clonal cell line SC-3, derived from Shionogi carcinoma 115, secretes a fibroblast growth factor (FGF)-autocrine growth factor in response to androgen, which is able to bind to FGF receptors. In SC-3 cells, FGF receptor expression is upregulated by the SC-3-derived growth factor, providing a means of amplifying an autocrine loop of cell growth. In the present investigations, the effect of the polysulfonated naphthylurea suramin on this autocrine loop and its amplification in SC-3 cells were studied. Suramin inhibited androgen-dependent growth of SC-3 cells in a concentration-dependent fashion: ～50% inhibition was observed at 25 μM. [³H]Thymidine incorporation into the cells stimulated with partially purified SC-3-derived growth factor was inhibited by suramin in a similar way. Additionally, suramin inhibited acidic (a) or basic (b) FGF-induced cell proliferation, though relatively high concentrations were necessary to achieve the maximal inhibition. Pretreatment of SC-3 cells with suramin decreased cell surface ¹²⁵I-bFGF binding without altering dissociation constant (Kd) of the binding sites. When the cells were incubated with 250 μM suramin for 24 h, the maximum binding (Bmax) decreased to almost 50% of the control. Treatment with suramin also decreased the levels of FGF receptor-1 mRNA to a similar extent, whereas it appeared not to affect the levels of β-actin mRNA. Moreover, suramin completely blocked androgen- or bFGF-induced accumulation of FGF receptor-1 mRNA. The inhibitory effects of suramin on FGF receptor expression were reversed by simultaneous addition of high concentrations of bFGF. These results indicate that suramin exerts its potent antiproliferative action on SC-3 cells through inhibition of an androgen-inducible autocrine loop involving SC-3-derived growth factor and FGF receptor. © 1993 Wiley-Liss, Inc.     

Reduction of phosphodiesterase 3B gene expression in peroxisome proliferator-activated receptor gamma (+/-) mice independent of adipocyte size

Phosphodiesterase 3B (PDE3B) gene expression is generally reduced in large adipocytes of obese, insulin-resistant mice. This reduced gene expression is restored by peroxisome proliferator-activated receptor (PPAR) gamma ligands accompanied by a reduced fat cell size. To determine whether PDE3B gene expression is regulated by PPAR gamma itself, we analyzed lean PPAR gamma (+/-) mice with adipocyte size comparable to control PPAR gamma (+/+) mice. In adipocytes of PPAR gamma (+/-) mice, PDE3B mRNA and protein were both reduced to 63% of wild-type levels. Basal PDE activity tended to be decreased to 70% of wild-type levels, and, similarly, insulin-induced PDE activity was significantly decreased to 70%. Thus, PPAR gamma is required for PDE3B gene expression independent of adipocyte size.     

Development of a novel immobilization method for enzymes from hyperthermophiles

Peptide tags containing tyrosines (Y-tag) were introduced at the C-terminus of a hyperthermophilic enzyme, alkaline phosphatase from Pyrococcus furiosus (PfuAP). Immobilization of the recombinant PfuAPs onto water-in-oil-in-water (W/O/W) type microcapsules was performed by an in situ polymerization method. All the recombinant PfuAPs prepared in this study were quantitatively immobilized onto microcapsules. The PfuAP-immobilized microcapsules showed no significant loss of enzymatic activity until the 5th round of assays. This result implies that the recombinant PfuAPs were covalently immobilized onto microcapsules. Immobilized PfuAP tagged with a Y-tag having the sequence GGYYY exhibited approximately a twofold higher catalytic activity compared with the wild-type PfuAP. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genome-wide identification of in vivo binding sites of GlxR, a cyclic AMP receptor protein-type regulator in Corynebacterium glutamicum

Corynebacterium glutamicum GlxR is a cyclic AMP (cAMP) receptor protein-type regulator. Although over 200 GlxR-binding sites in the C. glutamicum genome are predicted in silico, studies on the physiological function of GlxR have been hindered by the severe growth defects of a glxR mutant. This study identified the GlxR regulon by chromatin immunoprecipitation in conjunction with microarray (ChIP-chip) analyses. In total, 209 regions were detected as in vivo GlxR-binding sites. In vitro binding assays and promoter-reporter assays demonstrated that GlxR directly activates expression of genes for aerobic respiration, ATP synthesis, and glycolysis and that it is required for expression of genes for cell separation and mechanosensitive channels. GlxR also directly represses a citrate uptake gene in the presence of citrate. Moreover, ChIP-chip analyses showed that GlxR was still able to interact with its target sites in a mutant with a deletion of cyaB, the sole adenylate cyclase gene in the genome, even though binding affinity was markedly decreased. Thus, GlxR is physiologically functional at the relatively low cAMP levels in the cyaB mutant, allowing the cyaB mutant to grow much better than the glxR mutant.