Recombinant carboxyl-terminal fibrin-binding domain of human fibronectin expressed in mouse L cells

This research was originally published in the Journal of Biological Chemistry. K Ichihara-Tanaka, K Titani and K Sekiguchi. Recombinant carboxyl-terminal fibrin-binding domain of human fibronectin expressed in mouse L cells. J. Biol. Chem. 1990; 265: 401-407 © the American Society for Biochemistry and Molecular Biolog

Midkine as a factor to counteract the deposition of amyloid β-peptide plaques: in vitro analysis and examination in knockout mice

<p>Abstract</p> <p>Background</p> <p>Midkine is a heparin-binding cytokine involved in cell survival and various inflammatory processes. Midkine accumulates in senile plaques of patients with Alzheimer's disease, while it counteracts the cytotoxic effects of amyloid β-peptide and inhibits its oligomerization. The present study was conducted to understand the role of midkine upon plaque formation of amyloid β-peptide.</p> <p>Methods</p> <p>A surface plasmon assay was performed to determine the affinity of midkine for amyloid β-peptide. The deposition of amyloid β-peptide was compared in the brain of wild-type and midkine-deficient mice. An effect of midkine to microglias was examined by cell migration assay.</p> <p>Results</p> <p>Midkine bound to amyloid β-peptide with the affinity of 160 nM. The C-terminal half bound to the peptide more strongly than the N-terminal half, and heparin inhibited midkine from binding to the peptide. Pleiotrophin, which has about 50% sequence identity with midkine also bound to amyloid β-peptide. The deposition of amyloid β-peptide plaques in the cortex and hippocampus was more intense in 15-month-old midkine-deficient mice, compared to the corresponding wild-type mice. Midkine promoted migration of microglias in culture.</p> <p>Conclusions</p> <p>These results are consistent with the view that midkine attenuates the deposition of amyloid β-peptide plaques, and thus progression of Alzheimer's disease, by direct binding and also by promoting migration of microglias.</p

A novel cell adhesive protein engineered by insertion of the Arg-Gly-Asp-Ser tetrapeptide

This research was originally published in the Journal of Biological Chemistry. T Maeda, R Oyama, K Ichihara-Tanaka, F Kimizuka, I Kato, K Titani and K Sekiguchi. A novel cell adhesive protein engineered by insertion of the Arg-Gly-Asp-Ser tetrapeptide. J. Biol. Chem. 1989; 264: 15165-15168 © the American Society for Biochemistry and Molecular Biolog

Midkine inhibitors: application of a simple assay procedure to screening of inhibitory compounds

<p>Abstract</p> <p>Background</p> <p>Midkine is a heparin-binding cytokine and is involved in etiology of various diseases. Thus, midkine inhibitors are expected to be helpful in treatment of many diseases.</p> <p>Methods</p> <p>We developed a simple assay for midkine activity based on midkine-dependent migration of osteblastic cells. Midkine inhibitors were searched as materials that inhibit this midkine activity. To develop peptides that inhibit midkine activity, we constructed models in which C-terminal half of midkine interacted with α₄β₁-integrin. Low molecular weight compounds which are expected to bind to midkine with high affinity were searched by <it>in silico </it>screening with the aid of Presto-X2 program.</p> <p>Results</p> <p>Among peptides in putative binding sites of midkine and the integrin, a peptide derived from β₁-integrin and that derived from the first β sheet of the C-terminal half of midkine significantly inhibited midkine activity. Two low molecular weight compounds found by <it>in silico </it>screening exhibited no toxicity to target cells, but inhibited midkine activity. They are trifluoro compounds: one (PubChem 4603792) is 2-(2,6-dimethylpiperidin-1-yl)-4-thiophen-2-yl-6-(trifluoromethy)pyrimidine, and the other has a related structure.</p> <p>Conclusions</p> <p>The assay procedure is helpful in screening midkine inhibitors. All reagents described here might become mother material to develop clinically effective midkine inhibitors.</p

Genome-wide association study revealed novel loci which aggravate asymptomatic hyperuricaemia into gout

Objective The first ever genome-wide association study (GWAS) of clinically defined gout cases and asymptomatic hyperuricaemia (AHUA) controls was performed to identify novel gout loci that aggravate AHUA into gout. Methods We carried out a GWAS of 945 clinically defined gout cases and 1003 AHUA controls followed by 2 replication studies. In total, 2860 gout cases and 3149 AHUA controls (all Japanese men) were analysed. We also compared the ORs for each locus in the present GWAS (gout vs AHUA) with those in the previous GWAS (gout vs normouricaemia). Results This new approach enabled us to identify two novel gout loci (rs7927466 of CNTN5 and rs9952962 of MIR302F) and one suggestive locus (rs12980365 of ZNF724) at the genome-wide significance level (p<5.0×10– 8). The present study also identified the loci of ABCG2, ALDH2 and SLC2A9. One of them, rs671 of ALDH2, was identified as a gout locus by GWAS for the first time. Comparing ORs for each locus in the present versus the previous GWAS revealed three ‘gout vs AHUA GWAS’-specific loci (CNTN5, MIR302F and ZNF724) to be clearly associated with mechanisms of gout development which distinctly differ from the known gout risk loci that basically elevate serum uric acid level. Conclusions This meta-analysis is the first to reveal the loci associated with crystal-induced inflammation, the last step in gout development that aggravates AHUA into gout. Our findings should help to elucidate the molecular mechanisms of gout development and assist the prevention of gout attacks in high-risk AHUA individuals

Recombinant carboxyl-terminal fibrin-binding domain of human fibronectin expressed in mouse L cells

The carboxyl-terminal fibrin-binding domain, Fib2, of human fibronectin was expressed in mouse L cells as a fusion protein with the signal sequence of human protein C inhibitor. The recombinant Fib2 (rFib2) protein synthesized by transfected cells retained the ability to form dimers with each other or with mouse fibronectin subunits and was secreted to the medium after extensive glycosylation. Only a small fraction of the secreted protein was incorporated into the pericellular matrix. Interestingly, the secreted rFib2 protein displayed a remarkable heterogeneity upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, giving rise to a broad band corresponding to Mr of 60,000-90,000. The heterogeneity was eliminated mostly by treatment with neuraminidase and further by treatment with endo-alpha-N-acetylgalactosaminidase. Treatment with peptide:N-glycosidase F did not alter the heterogeneity of the protein, indicating that differential sialylation of O-linked, but not N-linked, glycans is largely responsible for the apparent heterogeneity. The presence of O-linked but absence of N-linked glycans was further supported by the observations that peanut agglutinin specifically bound to the desialylated rFib2 protein, whereas neither concanavalin A nor lentil lectin bound to the protein irrespective of prior neuraminidase treatment. Since the apparent heterogeneity of the rFib2 protein was only observable with the secreted, but not the cytoplasmic form, sialylation of O-linked glycans may be essential for, or regulate as a rate-limiting step, the transit of the recombinant protein to the extracellular space.This research was originally published in the Journal of Biological Chemistry. K Ichihara-Tanaka, K Titani and K Sekiguchi. Recombinant carboxyl-terminal fibrin-binding domain of human fibronectin expressed in mouse L cells. J. Biol. Chem. 1990; 265: 401-407 © the American Society for Biochemistry and Molecular Biolog

A novel cell adhesive protein engineered by insertion of the Arg-Gly-Asp-Ser tetrapeptide

This research was originally published in the Journal of Biological Chemistry. T Maeda, R Oyama, K Ichihara-Tanaka, F Kimizuka, I Kato, K Titani and K Sekiguchi. A novel cell adhesive protein engineered by insertion of the Arg-Gly-Asp-Ser tetrapeptide. J. Biol. Chem. 1989; 264: 15165-15168 © the American Society for Biochemistry and Molecular Biolog