Cartilage link protein interacts with neurocan, which shows hyaluronan binding characteristics different from CD44 and TSG-6

The interaction of neurocan with hyaluronan was qualitatively characterized with alkaline phosphatase fusion proteins secreted by mammalian cells. The wild type neurocan hyaluronan binding domain fused to alkaline phosphatase bound to immobilized hyaluronan under physiological as well as moderately hypertonic conditions, whereas its ability to bind to immobilized chondroitin sulfate dropped rapidly with increasing salt concentration. Strong hyaluronan binding ability was still evident when in both link modules within the hyaluronan binding domain a basic amino acid was mutated, which is well conserved among link modules of hyaluronan binding proteins. A strong enhancement of the binding of neurocan to immobilized hyaluronan was observed after preincubation of the immobilized hyaluronan with cartilage link protein. Moreover, this preincubation mediated also the binding of a fusion protein representing only the immunoglobulin module of neurocan linked to alkaline phosphatase, which showed no binding to immobilized hyaluronan alone. The interaction of the neurocan immunoglobulin module with link protein could also be shown by overlay blot analysis. These observations suggest that the hyaluronan binding characteristics of paired link modules are different from those of single link modules, and that the reported temporal co-expression of cartilage link protein and of neurocan in developing brain implicates the possibility of a cooperative function of these molecules.     

Simulation of ATP metabolism in cardiac excitation–contraction coupling

To obtain insights into the mechanisms underlying the membrane excitation and contraction of cardiac myocytes, we developed a computer model of excitation–contraction coupling (Kyoto model: Jpn. J. Physiol. 53 (2003) 105). This model was further expanded by incorporating pivotal reactions of ATP metabolism; the model of mitochondrial oxidative phosphorylation by Korzeniewski and Zoladz (Biophys. Chem. 92 (2001) 17). The ATP-dependence of contraction, and creatine kinase and adenylate kinase were also incorporated. After minor modifications, the steady-state condition was well established for all the variables, including the membrane potential, contraction, and the ion and metabolite concentrations in sarcoplasmic reticulum, mitochondria and cytoplasm. Concentrations of major metabolites were close to the experimental data. Responses of the new model to anoxia were similar to experimental results of the P-31 NMR study in whole heart. This model serves as a prototype for developing a more comprehensive model of excitation–contraction–metabolism coupling.     

Rabbit antibody detection with RNA aptamers

Antibody-based detection systems are widely used, but in the cases of immunoprecipitations and enzyme-linked immunoassays, they can be laborious. These techniques require the preparation of at least two kinds of non-cross-reactive immunoglobulin Gs (IgGs), usually made from different species against the single target molecule. Aptamers composed of nucleic acids possess strict recognition ability for the target molecule's three-dimensional structure and, thus, are considered to act like IgG. In this study, experimental trials were designed to combine the advantages of IgG and aptamers. For this purpose, aptamers against rabbit IgG were identified by in vitro selection. One of the obtained aptamers had a dissociation constant lower than 15 pM to the rabbit IgG. It bound specifically to the constant region of the rabbit IgG, and no binding was observed with mouse or goat IgG. Moreover, this aptamer recognized only the native form of rabbit IgG and could not bind the sodium dodecyl sulfate (SDS)-denatured form. These features show the advantage of using the aptamer as a secondary probing agent rather than the usual secondary antibodies.     

Methods for reducing nonspecific interaction in antibody-antigen assay via atomic force microscopy

We developed a method to measure the rupture forces between antibody and antigen by atomic force microscopy (AFM). Previous studies have reported that in the measurement of antibody–antigen interaction using AFM, the specific intermolecular forces are often obscured by nonspecific adhesive binding forces between antibody immobilized cantilever and substrate surfaces on which antigen or nonantigen are fixed. Here, we examined whether detergent and nonreactive protein, which have been widely used to reduce nonspecific background signals in ordinary immunoassay and immunoblotting, could reduce the nonspecific forces in the AFM measurement. The results showed that, in the presence of both nonreactive protein and detergent, the rupture forces between anti-ferritin antibodies immobilized on a tip of cantilever and ferritin (antigen) on the substrate could be successfully measured, distinguishing from nonspecific adhesive forces. In addition, we found that approach/retraction velocity of the AFM cantilever was also important in the reduction of nonspecific adhesion. These insights will contribute to the detection of specific molecules at nanometer scale region and the investigation of intermolecular interaction by the use of AFM.     

Skeletal muscle-derived progenitors capable of differentiating into cardiomyocytes proliferate through myostatin-independent TGF-beta family signaling

The existence of skeletal muscle-derived stem cells (MDSCs) has been suggested in mammals; however, the signaling pathways controlling MDSC proliferation remain largely unknown. Here we report the isolation of myosphere-derived progenitor cells (MDPCs) that can give rise to beating cardiomyocytes from adult skeletal muscle. We identified that follistatin, an antagonist of TGF-β family members, was predominantly expressed in MDPCs, whereas myostatin was mainly expressed in myogenic cells and mature skeletal muscle. Although follistatin enhanced the replicative growth of MDPCs through Smad2/3 inactivation and cell cycle progression, disruption of myostatin did not increase the MDPC proliferation. By contrast, inhibition of activin A (ActA) or growth differentiation factor 11 (GDF11) signaling dramatically increased MDPC proliferation via down-regulation of p21 and increases in the levels of cdk2/4 and cyclin D1. Thus, follistatin may be an effective progenitor-enhancing agent neutralizing ActA and GDF11 signaling to regulate the growth of MDPCs in skeletal muscle.     

Inhibition of tumor-stromal interaction through HGF/Met signaling by valproic acid

Hepatocyte growth factor (HGF), which is produced by surrounding stromal cells, including fibroblasts and endothelial cells, has been shown to be a significant factor responsible for cancer cell invasion mediated by tumor-stromal interactions. We found in this study that the anti-tumor agent valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, strongly inhibited tumor-stromal interaction. VPA inhibited HGF production in fibroblasts induced by epidermal growth factor (EGF), platelet-derived growth factor, basic fibroblast growth factor, phorbol 12-myristate 13-acetate (PMA) and prostaglandin E₂ without any appreciable cytotoxic effect. Other HDAC inhibitors, including butyric acid and trichostatin A (TSA), showed similar inhibitory effects on HGF production stimulated by various inducers. Up-regulations of HGF gene expression induced by PMA and EGF were also suppressed by VPA and TSA. Furthermore, VPA significantly inhibited HGF-induced invasion of HepG2 hepatocellular carcinoma cells. VPA, however, did not affect the increases in phosphorylation of MAPK and Akt in HGF-treated HepG2 cells. These results demonstrated that VPA inhibited two critical processes of tumor-stromal interaction, induction of fibroblastic HGF production and HGF-induced invasion of HepG2 cells, and suggest that those activities serve for other anti-tumor mechanisms of VPA besides causing proliferation arrest, differentiation, and/or apoptosis of tumor cells.     

Identification of two catalytic domains in a luciferase secreted by the copepod Gaussia princeps

Gaussia luciferase secreted by the copepod Gaussia princeps catalyzes the oxidation of coelenterazine to produce blue light. The primary structure of Gaussia luciferase deduced from the cDNA sequence shows two repeat sequences of 71 amino acid residues, suggesting the luciferase consists of two structural domains. Two domains in Gaussia luciferase were expressed independently in Escherichia coli cells, purified and characterized. We found that both domains have luminescence activity with coelenterazine, and the catalytic properties including luminescence spectrum, optimal pH, substrate specificity and luminescence stimulation by halogen ions (Cl⁻, Br⁻ and I⁻) are identical to intact Gaussia luciferase. Thus, Gaussia luciferase has two catalytic domains for the luminescence reaction.     

Combined insulin B:9-23 self-peptide and polyinosinic-polycytidylic acid accelerate insulitis but inhibit development of diabetes by increasing the proportion of CD4+Foxp3+ regulatory T cells in the islets in non-obese diabetic mice

Insulin peptide B:9-23 is a major autoantigen in type 1 diabetes. Combined treatment with B:9-23 peptide and polyinosinic-polycytidylic acid (poly I:C), but neither alone, induce insulitis in normal BALB/c mice. In contrast, the combined treatment accelerated insulitis, but prevented diabetes in NOD mice. Our immunofluorescence study with anti-CD4/anti-Foxp3 revealed that the proportion of Foxp3 positive CD4⁺CD25⁺ regulatory T cells (Tregs) was elevated in the islets of NOD mice treated with B:9-23 peptide and poly I:C, as compared to non-treated mice. Depletion of Tregs by anti-CD25 antibody hastened spontaneous development of diabetes in non-treated NOD mice, and abolished the protective effect of the combined treatment and conversely accelerated the onset of diabetes in the treated mice. These results indicate that poly I:C combined with B:9-23 peptide promotes infiltration of both pathogenic T cells and predominantly Tregs into the islets, thereby inhibiting progression from insulitis to overt diabetes in NOD mice.     

Expression of highly sulfated keratan sulfate synthesized in human glioblastoma cells

Keratan sulfate (KS) proteoglycan is expressed in the extracellular matrix or cell surface in numerous tissues, predominantly in those of the cornea, cartilage, and brain. However, its structure, function, and regulation remain poorly understood. Our investigation of KS expression in glioblastoma cell lines using Western-blot and flow cytometry with anti-KS antibody (5D4) revealed that LN229 glioblastoma cell highly expresses KS on a cell surface. Real-time PCR analysis showed that LN229 expresses a high level of keratan sulfate Gal-6-sulfotransferase. Results of this study also demonstrate that recombinant 5D4-reactive aggrecan is produced in LN229. Taken together, these results suggest that LN229 produces 5D4-reactive highly sulfated KS and is useful to investigate the KS structure and function in glioblastoma.