Structural studies of human brain-type creatine kinase complexed with the ADP-Mg2+-NO3- -creatine transition-state analogue complex

Creatine kinase is a member of the phosphagen kinase family, which catalyzes the reversible phosphoryl transfer reaction that occurs between ATP and creatine to produce ADP and phosphocreatine. Here, three structural aspects of human-brain-type-creatine-kinase (hBB-CK) were identified by X-ray crystallography: the ligand-free-form at 2.2 Å; the ADP-Mg²⁺, nitrate, and creatine complex (transition-state-analogue complex; TSAC); and the ADP-Mg²⁺-complex at 2.0 Å. The structures of ligand-bound hBB-CK revealed two different monomeric states in a single homodimer. One monomer is a closed form, either bound to TSAC or the ADP-Mg²⁺-complex, and the second monomer is an unliganded open form. These structural studies provide a detailed mechanism indicating that the binding of ADP-Mg²⁺ alone may trigger conformational changes in hBB-CK that were not observed with muscle-type-CK.     

Enhanced production of recombinant protein inEscherichia coli using silkworm hemolymph

The effect of silkworm hemolymph on the expression of recombinant protein inEscherichia coli was investigated. The addition of silkworm hemolymph to the culture medium increased the production of recombinant β-galactosidase inE. coli. The production was dependent on the concentration of the added silkworm hemolymph, which increased 2-, 5-, and 8-fold in media supplemented with 1,3, and 5% silkworm hemolymph, respectively. To identify the effective component, the silkworm hemolymph was fractionated by gel filtration column chromatography. A fraction, with a molecular weight of about 30 K was identified as the effective component.     

Purification of a fibrinolytic enzyme (myulchikinase) from pickled anchovy and its cytotoxicity to the tumor cell lines

A fibrinolytic enzyme, myulchikinase, from a Korean seasoning ingredient, myul-chi-jeot-gal, has been purified to electrophoretic homogeneity. The molecular mass of the myulchikinase was estimated to about 28 kDa by SDS-PAGE and gel filtration. Amino acid sequence of the NH2-terminal of myulchikinase showed significant homology with other fibrinolytic enzymes including trypsin from starfish, katsuwokinase, and rat pancreatic elastase II. The purified myulchikinase hydrolyzed various synthetic substrates with different substrate specificity and cytotoxic to the tumor cell lines.     

Particle-hemodynamics simulations and design options for surgical reconstruction of diseased carotid artery bifurcations

Based on the hypothesis that aggravating hemodynamic factors play a key role in the onset of arterial diseases, the methodology of "virtual prototyping" of branching blood vessels was applied to diseased external carotid artery (ECA) segments. The goals were to understand the underlying particle-hemodynamics and to provide various geometric design options for improved surgical reconstruction based on the minimization of critical hemodynamic wall parameters (HWPs). First, a representative carotid artery bifurcation (CAB) and then CABs with stenosed ECAs, i.e., a distally occluded ECA and an ECA stump, were analyzed based on transient three-dimensional blood flow solutions, employing a user-enhanced commercial finite volume code. Specifically, the HWPs, i.e., oscillatory shear index, wall shear stress angle gradient, near-wall residence time of monocytes, and near-wall helicity angle difference were evaluated to compare the merits of each design option, including a reconstructed near-optimal junction which generates the lowest HWP-values. The results provide physical insight to the biofluid dynamics of branching blood vessels and guide vascular surgeons as well as stent manufacturers towards interventions leading to high sustained patency rates.     

Growth and physiological properties of wild type and mutants of Halomonas subglaciescola DH-1 in saline environment

A halophilic bacterium was isolated from fermented seafood. The 16S rDNA sequence identity between the isolate and Halomonas subglaciescola AJ306801 was above 95%. The isolate that did not grow in the condition without NaCl or in the condition with other sodium (Na+) or chloride ions (Cl-) instead of NaCl was named H. subglaciescola DH-1. Two mutants capable of growing without NaCl were obtained by random mutagenesis, of which their total soluble protein profiles were compared with those of the wild type by two-dimensional electrophoresis. The external compatible solutes (betaine and choline) and cell extract of the wild type did not function as osmoprotectants, and these parameters within the mutants did not enhance their growth in the saline environment. In the proton translocation test, rapid acidification of the reactant was not detected for the wild type, but it was detected for the mutant in the condition without NaCl. From these results, we derived the hypothesis that NaCl may be absolutely required for the energy metabolism of H. subglaciescola DH-1 but not for its osmoregulation, and the mutants may have another modified proton translocation system that is independent of NaCl, except for those mutants with an NaCl-dependent system.     

Anti-angiogenic and anti-tumor invasive activities of tissue inhibitor of metalloproteinase-3 from shark,Scyliorhinus torazame

In order to investigate the anti-angiogenic activity of shark TIMP-3 (sTIMP-3) in endothelial cells, angiogenic assays including in vitro invasion assay, migration assay, zymogram assay and tube formation assay were performed. We observed that the overexpression of sTIMP-3 decreased the invasive capacity by about 70%, the migratory activity by about 50% and the production of gelatinase A in bovine aortic endothelial cells (BAECs). In addition, the overexpression of sTIMP-3 interfered with the formation of capillary-like network in endothelial cells. We also examined whether sTIMP-3 shows the anti-invasive activity in cancer cells. We found that the overexpression of sTIMP-3 diminished the invasive ability of the human fibrosarcoma HT1080 cells by about 40%. Also, the production of specific gelatinases was suppressed in the cancer cells. Therefore, we propose that sTIMP-3 acts as the inhibitor of angiogenesis in endothelial cells and the suppressor of tumor invasion in human fibrosarcoma HT1080 cells.     

Specific modulation of the anti-DNA autoantibody-nucleic acids interaction by the high affinity RNA aptamer

Anti-DNA autoantibodies are one of the frequently found autoantibodies in systemic lupus erythematosus patient sera. RNA aptamers for the monoclonal G6-9 anti-DNA autoantibody were selected from a random pool of RNA library. Binding affinity of the best aptamer is around 2nM, which is at least 100-fold higher than that of cognate DNA antigen to the autoantibody. Aptamer binds specifically to the G6-9 autoantibody but not to other similar autoantibodies. Minimal binding motif of the aptamer was mapped, providing a hint for a natural epitope of the autoantibody. DNA binding to the G6-9 autoantibody is shown to be efficiently inhibited by the aptamer. Such binding property of the RNA aptamer could be used not only as a modulator for the pathogenic anti-DNA autoantibody, but also as a useful biochemical reagent for elucidating a fine specificity of the autoantibody-nucleic acid interaction.     

Modulation of cardiac growth and development by HOP,an unusual homeodomain protein

We have discovered an unusual homeodomain protein, called HOP, which is comprised simply of a homeodomain. HOP is highly expressed in the developing heart where its expression is dependent on the cardiac-restricted homeodomain protein Nkx2.5. HOP does not bind DNA and acts as an antagonist of serum response factor (SRF), which regulates the opposing processes of proliferation and myogenesis. Mice homozygous for a HOP null allele segregate into two phenotypic classes characterized by an excess or deficiency of cardiac myocytes. We propose that HOP modulates SRF activity during heart development; its absence results in an imbalance between cardiomyocyte proliferation and differentiation with consequent abnormalities in cardiac morphogenesis.