Optimization of cell culture conditions for production of biologically active proteins

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.     

Efficient expression of pro-urokinase by human lymphoblastoid Namalwa KJM-1 cells using Moloney retroviral promoter

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 -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 -actin gene promoter. The expression level of pro-UK under the control of Mo-MuLV promoter was 2–3 g/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 g/10⁶ cells/day. Thus, Mo-MuLV promoter showed the desired characteristics for efficient expression of foreign genes in Namalwa KJM-1 cells.Abbreviations dhfr dihydrofolate reductase - G-CSF granulocyte colony-stimulating factor - hCMV human cytomegalovirus - LTR long terminal repeat - Mo-MuLV Moloney murine leukemia virus - MTX methotrexate - pro-UK pro-urokinase - RSV Rous sarcoma virus - SV40 simian virus 40 - T3 triiodo-thyronine - TRE thyroid-hormone responsive element     

Optimization of cell culture conditions for production of biologically active proteins

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.     

Stable production of recombinant pro-urokinase by human lymphoblastoid Namalwa KJM-1 cells: Host-cell dependency of the expressed-protein stability

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 g/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.Abbreviations ABTS 2,2-azino-di-(3-ethylbenzothiazoline) sulfonic acid diammonium salt - AMC 7-amino-4-methylcoumarin - CHO Chinese hamster ovary - DFP diisopropylfluorophosphate - DHFR dihydrofolate reductase - ELISA enzyme-linked immunosorbent assay - MCA 4-methylcoumaryl-7-amide - MTX methotrexate - NEAA non essential amino acid - NEM N-ethylmaleimide - PCMB p-chloromercuribenzonate - PMSF phenylmethanesulfonyl fluoride - pro-UK pro-urokinase     

Production of useful recombinant proteins using Namalwa KJM-1 cells adapted to serum-free medium]

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, human B lymphoblastoid cells, were used for large scale production of alpha-interferon. Namalwa KJM-1, a subline of Namalwa cells, adapted to serum- and albumin-free medium, can grow at a high density above 1 x 10(7) cells/ml in suspension manner by the use of a perfusion culture system, Biofermenter, containing a cone-type cell-sedimentation column as cell separator. Several kinds of cytokine cDNA can be introduced and expressed in Namalwa KJM-1 cells. Some of these were produced in large quantities by use of a gene amplification method with dhfr, even through 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.     

Effect of propranolol on cardiac cytokine expression after myocardial infarction in rats

The pro-inflammatory cytokines interleukin (IL)-1 and IL-6 have been shown to be upregulated in the myocardium after injury and after adrenergic receptor stimulation. Together with other cytokines, such as the transforming growth factor (TGF)-, the pro-inflammatory cytokines have been implicated in the initiation of tissue repair and wound healing after myocardial infarction (MI). In the present study, the effect of -adrenergic receptor blockade with propranolol (2 mg/kg·h s.c. by miniosmotic pumps) on cardiac cytokine expression and on wound healing was analyzed in rats from 6–72 h after MI. IL-1 and IL-6 gene expression strongly increased in the infarcted myocardium 6 h after MI and peaked after 12 h, while TGF-, progressively increased from 12 h onwards. Also, TGF-₂ increased after 12 h, peaked after 24 h and declined thereafter, while TGF-, was only elevated after 72 h. Treatment with propranolol had a negative chronotropic effect throughout the observation period of 72 h. It attenuated the initial elevation in LVEDP and increased cardiac output ultimately. Furthermore, propranolol attenuated IL-1 mRNA expression, but had not effect on the other cytokines. Moreover, MMP-9 gelatinolytic activity was markedly attenuated by propranolol indicating a delayed resorption of the necrotic tissue and, possibly, collagen turnover. Replacement by scar tissue, however, was not affected as indicated by normal collagen expression.     

Phylogenetic relationship of zeins and coixins as determined by immunological cross-reactivity and Southern blot analysis

Zeins from Zea mays L cv. Maya and coixins from Coix lacryma-jobi L. cv. Adlay were fractionated to obtain -, -, and -zein and -, -, and -coixin. The -coixins were composed of 4 polypeptide classes of 27 kDa (C1), 25 kDa (C2), 17 kDa (C4) and 15 kDa (C5) with solubility properties very similar to those of the 22 kDa and 19 kDa -zeins. Like the -zeins, the C1 and C2 -coixins corresponded to 80% of total Coix prolamins. The fraction corresponding to -coixin contained only one protein band of 22 kDa (C3). This coixin fraction has solubility properties similar to those of -zein and represents 15% of the total coixin. The -zein fraction was composed of a major 17 kDa protein band, while the -coixin fraction consisted of a mixture of - and -coixins.Polyclonal antibodies raised against C1 recognized C1 and C2 and cross-reacted strongly with the 22 kDa -zein, as did C4 and C5 antisera. The antiserum against -coixin showed strong cross-reaction with -zein. The homology between coixins and zeins was further investigated by using Southern hybridization analyses. The genomic DNA of maize and Coix were digested with several restriction enzymes and probed with cDNA clones representing 19 and 22 kDa -zeins as well as the 28 and 16 kDa -zeins. The Coix genome showed complex cross-hybridization sequences with the 22 kDa -zein cDNA, while no cross-hybridization was observed with the 19 kDa cDNA clone. The cDNA clone representing the 28 kDa -zein cross-hybridized with only one band of Coix genomic DNA, in contrast to the three bands observed in maize. This same Coix sequence also cross-hybridized with the cDNA clone representing the 16 kDa -zein. The relevance of these findings are discussed in the context of the origin of zein and coixin genes.     

Regional chromosomal assignment of genes encoding the α and β subunits of human complement protein C8 to 1p32

Summary The genes encoding the and subunits of human complement protein C8 previously mapped to chromosome 1 have been further localised to 1p32 by in situ hybridisation using biotinylated 2.4-kb human cDNA clones encoding the and subunits of human complement protein C8 as probes.     

β-Amyloid and Cholinergic Neurons

It is generally accepted that the crucial events in the pathogeny of Alzheimer's disease (AD) are the increased accumulation of amyloidogenic peptides derived from amyloid precursor protein and the harmful actions of these peptides on neurons, which bring about neurodegeneration. The enhanced -amyloid accumulation is known to be caused by mutations of specific genes in patients who suffer from the familial (hereditary) form of AD but who represent just a minor group within the total population of AD patients. The reasons for -amyloid accumulation are not known in the much larger group of patients with the sporadic form of the disease. A biochemical feature common to either form of the disease is the preferential atrophy and degeneration of cholinergic neurons, which is probably responsible for much of the cognitive decline characteristic of the disease. We present an overview of recent investigations on the interactions between -amyloid and cholinergic neurons.     

Assignment of human β-galactosidase-A gene to 3p21.33 by fluorescence in situ hybridization

GM₁ gangliosidosis and Morquio syndrome type B (MPS IVB) are inherited lyosomal storage disorders associated with deficiency of -galactosidase-A (GAL_A) activity. A recombinant plasmid containing a biotinylated cDNA (2.4-kb insert) encoding human GAL_A was used to localize the enzyme locus by fluorescence in situ hybridization (FISH). The human GAL_A gene was assigned to 3p21.33 by FISH.     

Stabile production of a thrombin resistant pro-urokinase derivative (PRO-UKS1) by Namalwa KJM-1 cells adapted to serum-free medium

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 g ml^–1. Addition of glucose and tri-iodothyronine (T₃) improved productivity, and the maximal productivity of pro-UKS1 was 67 g ml^–1 day^–1. In this conditioned medium, content of pro-UKS1 was above 80% of total proteins.Abbreviations BSA bovine serum albumin - dhfr dihydrofolate reductase - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - kb kilobase pairs - kDa kilodaltons - MTX Methotrexate - PBS phosphate buffered saline - pro-UK pro-urokinase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - T₃ tri-iodothyronine - Tween-PBS phosphate buffered saline containing 0.05% Tween 80     

Stretch-induced paracrine hypertrophic stimuli increase TGF-β1 expression in cardiomyocytes

Cardiac hypertrophy refers to the abnormal growth of cardiomyocytes, and is often caused by valvular heart disease and hypertension. It involves the activation of growth, including increased protein synthesis and changes in gene expression. Transforming growth factor-₁ (TGF-₁) may play a central role in protecting the heart during the hypertrophic response by helping to restore normal functions of the affected myocardium. We tested the hypothesis that cardiomyocytes respond to stretch-induced paracrine hypertrophic stimuli with increased expression of TGF-₁. To that purpose, we investigated whether angiotensin II (AII), endothelin-1 (ET-1) and TGF-, secreted by stretched cardiac and vascular cells, are involved in the paracrine mechanisms of stretch-induced changes of TGF-₁ mRNA expression in stationary (i.e. non-stretched) cardiomyocytes.Our results indicated that TGF-₁ mRNA expression in stationary cardiomyocytes was increased by AII release from cardiomyocytes that had been stretched for 30–60 min. Furthermore, it is likely that ET-1 and TGF- were released by stretched cardiac fibroblasts and endothelial cells to induce TGF-₁ mRNA expression in stationary cardiomyocytes. Stretched vascular smooth muscle cells did not influence TGF-₁ mRNA expression in stationary cardiomyocytes. These results indicate that AII, ET-1 and TGF-, released by cardiac cell types, act as paracrine mediators of TGF-₁ mRNA expression in cardiomyocytes. Therefore, we conclude that in stretched myocardium the cardiomyocytes, cardiac fibroblasts and endothelial cells take part in intercellular interactions contributing to cardiomyocyte hypertrophy.     

High-Level Expression and Purification of a Recombinant Human α-1,3-Fucosyltransferase in Baculovirus-Infected Insect Cells

A human α-1,3-fucosyltransferase (Fuc-TVII) was expressed by recombinant baculovirus-infected insect Sf9 cells as a secretory fusion protein. The fusion protein consisted of the human granulocyte colony-stimulating factor signal peptide followed by an IgG-binding domain of protein A, a Fuc-TVI-derived peptide, and the putative catalytic domain of Fuc-TVII. The signal peptide was correctly cleaved and the recombinant Fuc-TVII was secreted into the culture medium at a concentration of 10 μg/ml. The recombinant Fuc-TVII could be highly purified in a single-step purification procedure, i.e., IgG–Sepharose column chromatography. The enzymatic properties of the Sf9-produced Fuc-TVII were compared with the properties of that expressed by a human B-cell line, Namalwa KJM-1, transfected with an episomal plasmid carrying the fusion Fuc-TVII cDNA. Both recombinant proteins showed α-1,3-fucosyltransferase activity toward a type II oligosaccharide with a terminal α-2,3-linked sialic acid among various acceptors. The apparentK_mvalues of Sf9-produced Fuc-TVII for GDP-fucose and its acceptor substrate were slightly lower than those of the Fuc-TVII produced by Namalwa KJM-1 cells. Sf9-produced Fuc-TVII has N-linked carbohydrate chains whose molecular weights are lower than those linked to Namalwa KJM-1-produced Fuc-TVII. This difference in carbohydrate structure hardly affects the thermal stability of Fuc-TVII. The baculovirus expression system is available for high-level expression of stable and enzymatically active secretory Fuc-TVII.     

Assignment of the gene encoding the catalytic subunit Cβ of CAMP-dependent protein kinase to the p36 band on chromosome

Summary A cDNA for the human catalytic subunit (C) of cAMP-dependent protein kinase (PKA) has been cloned from a testis cDNA library. In the present study, we have determined the chromosomal localization of this gene using a cDNA for C as a probe. Southern blot analysis of genomic DNA from human/mouse cell hybrids revealed that the presence or absence of a 20-kbXbaI fragment, which hybridized with the C probe, was concordant with the presence of human chromosome 1.In situ hybridization to metaphase chromosome confirmed the somatic cell hybrid data and regionally mapped the C gene of PKA to the p36 band on chromosome 1.     

Localization of Transforming Growth Factor β in Association with Neuromuscular Junctions in Adult Human Muscle

Transforming growth factors- 1, 2, and 3 are known for their regulatory function in embryogenesis, fibrogenesis, and tissue repair of different cell types. A trophic function of TGF- subclasses for motoneurons has been shown in vitro. TGF- 1 is a potent survival factor for cultured embryonic rat motoneurons. In addition, TGF- 1 stimulates proliferation of rat Schwann cells. Recently, TGF- 2 has been reported to be associated with the subsynaptic nuclei of mature rat neuromuscular junctions. In this study, we investigated the expression of TGF- 1, 2, and 3 at neuromuscular junctions in skeletal muscle of 11 adults without neuromuscular disease. On muscle biopsies, neuromuscular junctions were depicted by acetylcholine esterase reaction and acetylcholine receptor antibodies. TGF- 1, 2, and 3 were stained immunohistochemically with monoclonal antibodies. Some muscle fibers showed low levels of inhomogeneous immunoreactivity for both TGF- 1 and TGF- 3. Intense immunoreactivity of TGF- 1 and 3 was shown at the postsynaptic area of neuromuscular junctions. TGF- 2 was expressed in the same subcellular distribution, but less strongly. In conclusion, the colocalization of TGF- with neuromuscular junctions may suggest a significant function in neuromuscular communication.     

Evidence for the direct involvement ofβ-hemolysin inAeromonas hydrophila enteropathogenicity

Culture filtrates of 19 of 21 (90%) -hemolytic isolates ofAeromonas hydrophila caused fluid accumulation in permanently ligated rabbit ileal loops, whereas no fluid was accumulated with filtrates of eight non--hemolytic isolates. Antiserum to purified -hemolysin neutralized the ileal loop activity of culture filtrates from four of four -hemolytic isolates, and treatment at 56°C for 10 min eliminated the loop activity of six additional isolates. These results support the conclusion that -hemolysin alone causes significant changes in intestinal permeability and that it is a more common pathogenic mechanism than the heat-stable cytotonic enterotoxin. Electrophoretic and serological assays showed evidence for production of only one species of -hemolysin byA. hydrophila.     

Protection against acute paraquat toxicity by ambroxol

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 g/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, Biofermenter^TM (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 g/ml. These results indicated the possibility of finding the optimum culture conditions for the production of recombinant proteins by Namalwa KJM-1 cells.Abbreviations ABTS 2,2-Azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid - BSA Bovine Serum Albumin - BSA-PBS Phosphate-buffered Saline without Ca²⁺ and Mg²⁺ containing Bovine Serum Albumin - dhfr Dihydrofolate Reductase - DO Dissolved Oxygen - G-CSF Granulocyte Colony-stimulating Factor - HEPES 4-(2-Hydroxyethyl)-1-piperazineethansulfonic Acid - IFN Interferon - MTX Methotrexate - PBS(-) Phosphate-buffered saline without Ca²⁺ and Mg²⁺ - Tween-PBS Phosphate-buffered saline without Ca²⁺ and Mg²⁺ containing 0.05% of Tween 20     

Analysis of T cell receptor Vβ gene usage during the course of disease in patients with chronic hepatitis B

The T cell receptor (TCR) is a heterodimeric molecule expressed on the surface of T cells and recognizes foreign peptides presented by the major histocompatibility complex on the surface of antigen-presenting cells or virusinfected cells. Analysis of TCR usage by T cells which recognize hepatitis B virus (HBV) provides further insight into the participation of T cell populations during the course of disease. In this study, we examined the T-cell-proliferative response and the TCR V gene usage of peripheral blood mononuclear cells in 3 patients with clinical evidence typical of chronic hepatitis B. All 3 patients had significant T-cell proliferative responses against HBV core antigen (HBcAg) during the remission stage, while no responses were detected during the acute exacerbation stage. In addition, the TCR V₇ gene was utilized more frequently in T cells recognizing HBcAg during remission, while TCR V₁ and V₂ were utilized at a higher percentage during acute exacerbation. On the contrary, the T cell proliferative response against HBV surface antigen was undetectable and no specific V gene was utilized more frequently by all 3 patients, regardless of disease state. Our longitudinal studies, although based on a small sample of patients, demonstrate that the population of HBcAg-activated T cells alters during the course of disease in chronic hepatitis B patients.