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  • 1
    Electronic Resource
    Electronic Resource
    Characterization of Cysteine Proteases Functioning in Degradation of Dynorphin in Neuroblastoma Cells: Evidence for the Presence of a Novel Enzyme with Strict Specificity Toward Paired Basic Residues (1989)
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 52 (1989), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Two dynorphin-degrading cysteine proteases, I and II, were extracted with Triton X-100 from neuroblastoma cell membrane, isolated from accompanying dynorphin-degrading trypsin-like enzyme by affinity chromatography on columns of soybean trypsin inhibitor-immobilized Sepharose and p-mercuribenzoate–Sepharose, and separated by ion-exchange chromatography on diethylaminoethyl (DEAE)-cellulose and TSK gel DEAE-5PW columns. Cysteine protease II was purified further by hydroxyapatite chromatography and gel filtration. The molecular weights of cysteine proteases I and II were estimated to be 100,000 and 70,000, respectively, by gel filtration. Both of the enzymes were inhibited by p-chloromercuribenzoate, N-ethylmaleimide, and high-molecular-weight kininogen, but not or only slightly inhibited by diisopropylphosphorofluoridate, antipain, leupeptin, E-64, calpain inhibitor, and phosphoramidon. Cysteine protease I cleaved dynorphin(1–17) at the Arg6-Arg7 bond with the optimum pH of 8.0, whereas II cleaved dynorphin(1–17) at the Lys11-Leu12 bond and the Leu12-Lys13 bond with the optimum pH values of 8.0 and 6.0, respectively. These bonds corresponded to those that had been proposed as the initial sites of degradation by neuroblastoma cell membrane. Cysteine protease I was further found to show strict specificity toward the Arg-Arg doublet, when susceptibilities of various peptides containing paired basic residues were examined as substrates for the enzyme.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1471-4159.1989.tb10898.x
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  • 2
    Electronic Resource
    Electronic Resource
    Optimization of cell culture conditions for production of biologically active proteins (1991)
    Springer
    Cytotechnology 5 (1991), S. 17-34 
    Details
    ISSN: 1573-0778
    Keywords: genetic engineering ; Namalwa cells ; perfusion culture ; scaling-up ; serum-free medium ; stable production
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract We investigated the basic technology of cell culture conditions for production of useful substances such as cytokines, and related proteins produced by Namalwa cells. Namalwa cells (Klein, 1972), human B lymphoblastoid cells, were used for large scale production of alpha-interferon (Klein, 1979). Namalwa KJM-1, a subline of Namalwa cells, adapted to serum- and albumin-free medium, can grow at a high density above 1 × 107 cells/ml in suspension mode by the use of a perfusion culture system, Biofermenter™, containing a cone-type cell-sedimentation column as cell separator (Sato, 1983). Several kinds of cytokine cDNA can be introduced and expressed in Namalwa KJM-1 cells (Miyaji, 1990a,b,c). Some of these were produced in large quantities by use of a gene amplification method with dhfr (Miyaji, 1990c), even though the Namalwa KJM-1 cells contained endogenous dhfr genes. For stable production of the target protein, Namalwa KJM-1 cells are very useful host cells, because they have no effective endogenous protease activity in the conditioned medium. Using Biofermenter with micro-silicone fibers and a dialysis system, the specific productivity of the target proteins was not depressed at a high cell density.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00573878
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  • 3
    Electronic Resource
    Electronic Resource
    Stable production of recombinant pro-urokinase by human lymphoblastoid Namalwa KJM-1 cells: Host-cell dependency of the expressed-protein stability (1993)
    Springer
    Cytotechnology 13 (1993), S. 79-88 
    Details
    ISSN: 1573-0778
    Keywords: Chinese hamster ovary cells ; Namalwa KJM-1 cells ; protease activity ; pro-urokinase ; serum-free medium ; stability of expressed-protein
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Human pro-urokinase (pro-UK) gene was engineered for expression in mammalian cells. The stability of recombinant pro-UKs produced by two kinds of cells, Chinese hamster ovary (CHO) and human lymphoblastoid Namalwa KJM-1 cells, were compared. The pro-UK expressed in CHO cells in serum-free medium was degraded by cysteine endopeptidase secreted by CHO cells. This endopeptidase was inhibited by pchloromercuribenzonate (PCMB) and leupeptin more efficiently than by aprotinin. On the other hand, the pro-UK expressed in Namalwa KJM-1 cells was not degraded, resulting in the stable production of pro-UK at a rate of 2–3 μg/106 cells/day by use of a gene amplification method with dihydrofolate reductase (DHFR) in serum-free medium. Thus, Namalwa KJM-1 cells showed the desired characteristics as a host cell for the production of recombinant proteins. The stability of recombinant proteins produced in heterologous systems may vary depending on the host cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00749934
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  • 4
    Electronic Resource
    Electronic Resource
    Stabile production of a thrombin resistant pro-urokinase derivative (PRO-UKS1) by Namalwa KJM-1 cells adapted to serum-free medium (1995)
    Springer
    Cytotechnology 19 (1995), S. 1-10 
    Details
    ISSN: 1573-0778
    Keywords: Namalwa KJM-1 cells ; pro-urokinase ; thrombin resistant ; pro-UKS1 ; stable production ; culture conditions ; serum-free medium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Pro-UKS1 was designed as a thrombin-resistant derivative of pro-urokinase (pro-UK) by introducing a glycosylation site using site-directed mutagenesis. An expression plasmid for pro-UKS1, pMo1UKS1SEd1-5, was constructed and introduced into Namalwa KJM-1 cells (Hosoiet al., 1988), and cells resistant to G418 and Methotrexate (MTX) were obtained. Amongst them, the highest pro-UKS1 producer (resistant to 500 nM of MTX), clone 41-8, was selected and further characterized. Clone 41-8 was cultured in serum-free ITPSGF medium (Hosoiet al., 1988). Under the conventional conditions, the concentration of pro-UKS1 reached 26 μg ml−1. Addition of glucose and tri-iodothyronine (T3) improved productivity, and the maximal productivity of pro-UKS1 was 67 μg ml−1 day−1. In this conditioned medium, content of pro-UKS1 was above 80% of total proteins.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00749750
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  • 5
    Electronic Resource
    Electronic Resource
    Optimization of cell culture conditions for G-CSF (granulocyte-colony stimulating factor) production by genetically engineered namalwa KJM-1 cells (1991)
    Springer
    Cytotechnology 7 (1991), S. 25-32 
    Details
    ISSN: 1573-0778
    Keywords: G-CSF ; high density culture ; Namalwa KJM-1 cells ; optimization ; productivity ; serum-free medium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract An expression vector for G-CSF, pASLB3-3, was constructed and introduced into Namalwa KJM-1 cells (Hosoi et al., 1988), and cells resistant to 100 nM of methotrexate (MTX) were obtained. Among them, the highest producer, clone SC57, was selected and the productivity of this clone was further characterized. The maximal production of G-CSF was at the most 1.8 μg/ml/day using a 25 cm2 tissue culture flask, even though the cell number was above 7×105 cells/ml. The limiting factors at high density were analyzed as the deficiency of nutrients, such as glucose, cysteine and serine, and pH control. The depression of specific G-CSF productivity per cell under the batch culture conditions was overcome by using a perfusion culture system, BiofermenterTM (Sato, 1983) with modifications of nutrients supplementation by a dialysis membrane and/or dissolved oxygen (DO) supplementation by microsilicone fibers. ITPSGF medium was modified to elevate concentrations of amino acids and glucose by 2.0- and 2.5-times, respectively. Under the control of pH at 7.4 and DO at 3 ppm, the specific G-CSF productivity was not depressed even at high cell density (above 1×107 cells/ml), and the amount of G-CSF reached 41 μg/ml. These results indicated the possibility of finding the optimum culture conditions for the production of recombinant proteins by Namalwa KJM-1 cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00135635
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  • 6
    Electronic Resource
    Electronic Resource
    Efficient expression of pro-urokinase by human lymphoblastoid Namalwa KJM-1 cells using Moloney retroviral promoter (1995)
    Springer
    Cytotechnology 18 (1995), S. 167-172 
    Details
    ISSN: 1573-0778
    Keywords: efficient gene expression ; Moloney retroviral promoter ; Namalwa KJM-1 ; pro-urokinase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract We have compared the level of expression of several enhancer/promoters in human lymphoblastoid Namalwa KJM-1 cells when fused to a common reporter gene. A cassette containing the pro-urokinase (pro-UK) coding sequence followed by the rabbit β-globin and simian virus 40 (SV40) 3′ nontranslated region was used for evaluation of the enhancer activity. Cells containing Moloney murine leukemia virus (Mo-MuLV) promoter had an average of 10–20 fold higher expression levels of pro-UK than those containing other promoters, such as SV40 early gene promoter, human cytomegalovirus (hCMV) major immediate-early gene promoter, Rous sarcoma virus (RSV) promoter and chicken β-actin gene promoter. The expression level of pro-UK under the control of Mo-MuLV promoter was 2–3 μg/106 cells/day and was constant for more than 6 months. Furthermore, the production of a high producer clone, obtained by using dhfr gene coamplification, reached 30–40 μg/106 cells/day. Thus, Mo-MuLV promoter showed the desired characteristics for efficient expression of foreign genes in Namalwa KJM-1 cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00767764
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  • 7
    Electronic Resource
    Electronic Resource
    Modulation of oligosaccharide structure of a pro-urokinase derivative (pro-UKΔGS1) by changing culture conditions of a lymphoblastoid cell line Namalwa KJM-1 adapted to serum-free medium (1995)
    Springer
    Cytotechnology 19 (1995), S. 125-135 
    Details
    ISSN: 1573-0778
    Keywords: culture conditions ; oligosaccharide structure ; modulation ; Namalwa KJM-1 ; pro-urokinase derivative
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Pro-UKΔGS1 was designed as a long-life and thrombin-resistant derivative of pro-urokinase (pro-UK) by deleting the growth factor domain of pro-UK and introducing a glycosylation site near the thrombin cleaving site for thrombin-resistance using site-directed mutagenesis. An expression plasmid for pro-UKDGS1, pIH1UKΔGS1SEd1–5 was constructed and introduced into Namalwa KJM-1, a lymphoblastoid cell line adapted to serum-free medium, and cells resistant to G418 and Methotrexate (MTX) were obtained. Amongst them, the highest pro-UKΔGS1 producer (resistant to 200 nM of MTX), clone 2–9, was selected and used for further studies. Under the conventional conditions, i.e. at 37°C in serum-free ITPSGF medium (based on RPMI-1640 medium), the oligosaccharide structure of pro-UKΔGS1 produced by clone 2–9 mainly consisted of fucose (Fuc)-containing biantennary complex-type oligosaccharide. Addition of dexamethasone (Dex), changed the carbohydrate contents in the media, and a shift down of incubation temperature caused a change in oligosaccharide structure of pro-UKΔGS1 from mainly Fuc-containing biantennary to mainly Fuc-containing tri-and tetraantennary complex-type oligosaccharide. The modulated pro-UKΔGS1 showed superiorin vivo activity for a canine femoral thrombosis formed by inserting a copper-coil.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00749767
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  • 8
    Electronic Resource
    Electronic Resource
    Multiphoton processes in a solid in a doubly rotating system by NMR (1986)
    New York, NY : Wiley-Blackwell
    International Journal of Quantum Chemistry 30 (1986), S. 647-655 
    Details
    ISSN: 0020-7608
    Keywords: Computational Chemistry and Molecular Modeling ; Atomic, Molecular and Optical Physics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: The transition probability of multiphoton processes strongly depends on the angle between the directions of the spin quantization and the magnetic field to induce the transitions. The rotary saturation method in NMR is an excellent one to observe the multiphoton processes in solids. In this paper, the experimental observation of the multiphoton transitions between the nuclear spin Zeeman levels of F nuclei in a CaF2 single crystal by the rotary saturation method is reported. Results are discussed by using a second quantization formalism and the nth order time-dependent perturbation theory in the doubly rotating frame.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/qua.560300757
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  • 9
    Electronic Resource
    Electronic Resource
    Lattice relaxation and electric-field-gradient calculations in impurity-doped single ionic crystals (1980)
    New York, NY : Wiley-Blackwell
    International Journal of Quantum Chemistry 18 (1980), S. 633-640 
    Details
    ISSN: 0020-7608
    Keywords: Computational Chemistry and Molecular Modeling ; Atomic, Molecular and Optical Physics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: The lattice-relaxation parameters in several ionic crystals doped with monovalent impurity ions are calculated by energy minimization, taking into account the Coulomb, overlap-repulsive, and three-body potential energies. The displaced 256 ions around one substitutional impurity ion are taken into consideration and the results are utilized to calculate the electric-field gradients at sites similar to the (1,0,0), (1,1,0), and (1,1,1) sites relative to the impurity at (0,0,0), by using two different models for three-body potentials.
    Additional Material: 2 Tab.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/qua.560180238
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