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  • 1
    ISSN: 1573-0778
    Keywords: CHO cells ; coactivator ; recombinant protein production system ; transactivator
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract High level and inducible production of human interleukin 6 (hIL-6) was achieved using a novel expression system in Chinese hamster ovary (CHO) cells. In this system, the transcription of hIL-6 gene under the control of PhCMV*-1 promoter composed of tetracycline operator sequences and a minimal promoter is activated by a chimeric transactivator (tTA) composed of tetracycline repressor and transactivating domain of VP16 protein of herpes simplex virus. The transcription of tTA gene, which is also under the control of PhCMV*-1 promoter, is activated by itself via a positive feedback cycle. The expression of both genes is further enhanced by potentiating the VP16 transactivating domain of tTA transactivator with pX protein of hepatitis B virus. In the presence of tetracycline, the tTA transactivators can not bind to PhCMV*-1 promoter, therefore, the expression of hIL-6 and tTA gene is suppressed, and the pX will not activate basal transcription. In the absence of tetracycline, tTA transactivators bind to PhCMV*-1 promoter and activate efficient transcription of hIL-6 and tTA gene, and the transcription is further enhanced by pX via VP16 transactivating domain. Using this strategy, we isolated a clone (UX1) producing hIL-6 at a rate about 1425 ng/106 cells/day. Furthermore, the hIL-6 production is stringently regulated by tetracycline. This results suggested a novel strategy to establish highly efficient, inducible and cell type independent recombinant protein production system by using an artificial promoter to recruit transactivators and coactivators which can synergistically activate transcription.
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  • 2
    ISSN: 1573-0778
    Keywords: affinity improvement ; cancer specific antibody ; human monoclonal antibody ; hybrid antibody ; light chain shifting
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Human HB4C5 hybridoma cells produce a lung cancer-specific IgM human monoclonal antibody (mAb). HB4C5 human mAb cross-reacts with Candida cytochrome c (Cyt c) and carboxypeptidase (Cpase). Concanavalin A (ConA)-resistant variants of HB4C5 cells loss the original light chain followed by expression of various new light chains at a high incidence (light chain shifting) (Tachibana et al., 1996). HTD8 cells, one of the ConA-resistant variant subclones of HB4C5 cells, undergo the active light chain shifting and produce various sublines, each of which stably secretes new mAb consisting of a new light chain and a HB4C5 heavy chain. The new mAb exhibits altered antigen binding ability from that of the original antibody. We could expect that HTD8 cells can be used as ‘a light chain stem cell line’ to improve antigen binding ability and specificity of established human mAbs. A BD9D12 IgG human mAb recognizes lung cancer cells and cross-reacts with cytokeratin 8. Introduction of the heavy chain gene of BD9D12 mAb into HTD8 cells resulted in establishment of various sublines which secreted various kinds of hybrid antibodies consisting of different light chains derived from HTD8 subclones which underwent light chain shifting and a common IgG heavy chain derived from BD9D12. These hybrid antibodies exhibited different or improved reactivities to Cyt, Cpase, cytokeratin 8 and various cancer cells from those of parental mAb, demonstrating that light chain shifting can be applied to improve the affinity and specificity of human mAb.
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  • 3
    ISSN: 1573-0778
    Keywords: marine algae extract ; telomerase inhibiting activity ; telomere ; TRAP
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract We attempted to screen for telomerase inhibitingactivity in vitro from a total of 304 marine algae samples which were collected from various Japancoasts by nonradioisotope telomeric repeatamplification protocol (Non-RI TRAP) assay using humanleukemia MOLT-4 cells. Ten of the MeOH extracts andtwo of the PBS extracts from those algal samplesshowed telomerase inhibiting activity. In particular,the MeOH extract from a green alga, Caulerpasertularioides strongly inhibited telomerase activitywhen added to MOLT-4 cell culture at a level of 1.25%(v/v), suggesting that it may be possible to developa novel anti-cancer agent in view of its specificanti-telomeric property.
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  • 4
    ISSN: 1573-0778
    Keywords: Chinese hamster ovary cell ; human cytomegalovirus promoter ; insulin ; phosphatidylcholine-specific phospholipase C ; signal transduction
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The signal transduction from insulin to its receptors and Ras has been extensively studied, while little has been reported beyond these steps. We found that the expression of human interleukin 6 gene under the control of immediate early gene promoter of human cytomegalovirus was enhanced by insulin sitmulation in Chinese hamster ovary cells. The induction effect of insulin was not significantly affected by inhibitors or activators of conventional protein kinase C, cAMP dependent protein kinase and phosphoinositide -3 kinase, however, pre-incubation of the cells with D609, a specific inhibitors of phosphatidylcholine-specific phospholipase C completely abolished the induction effect. These results clearly demonstrate that phosphatidylcholine-specific phospholipase C is a key molecule mediating insulin-induced enhancement of hIL-6 expression from the human cytomegalovirus promoter in Chinese hamster ovary cells and strongly suggest that it plays an important role in the insulin signaling pathways. Abbreviations CHO – Chinese hamster ovary; hCMV promoter – immediate early gene promoter of human cytomegalovirus; hIL-6 – human interleukin 6; PC-PLC-phosphatidylcholine-specific phospholipase C; PI-3 kinase – phosphoinositide 3 kinase; PKA – cAMP dependent protein kinase; PKC – protein kinase C.
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  • 5
    ISSN: 1573-0778
    Keywords: human T cell ; immortalization ; oncogene
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract We have succeeded in immortalizing human lymphocytes derived from the peripheral blood of a healthy donor and of an atopic patient, and from the lymph node of a cancer patient by oncogene transfection (Alam et al., 1996). All immortalized human lymphocytes were shown to be CD3+ and CD19−, indicating that these immortalized human lymphocytes were all T cells. We established 317, 154 and 692 individual immortalized human T cell lines derived from the healthy donor, the atopic patient and the cancer patient, respectively. The ratios of CD4+ and CD8+ subpopulations within the set containing immortalized T cells derived from the healthy donor were shown to be varied depending on the combinations of transfected oncogenes used. However, CD8+ cells were found to be the dominant subpopulation of immortalized T cells derived from the atopic patient and the cancer patient. These immortalized T cells showed different proliferative responses in the presence of exogenous human IL–2 depending on their origin, and was consistent with the surface expression of the IL–2 receptor. Furthermore, the cytokine secretion patterns of these immortalized T cells stimulated with mitogen were investigated. The results showed that the immortalized T cells from the healthy donor is able to secrete various kinds of cytokines such as IL–2, IL–10, β-IFN and GM-CSF. However, immortalized T cells from the cancer patient was shown to only secrete IL–2 and GM-CSF. These results suggest that depending on the origin, the immortalized T cells came from different subsets or from cells in different activated states. Mixed lymphocytes reactions demonstrated that these immortalized T cells are able to proliferate in the presence of allogenic or xenogenic stimulator cells, suggesting that they maintain the ability to recognize specific antigens on the stimulator cells and can proliferate even after the immortalization. Furthermore, immortalized T cells derived from the healthy donor and the cancer patient strongly responded to K562 cells, suggesting that MHC-nonrestricted killer T cells were also immortalized. Abbreviations IL–2R – interleukin 2 receptor; MLR – mixed lymphocyte reaction
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  • 6
    ISSN: 1573-0778
    Keywords: diacylglycerol ; fermented milk ; food component ; interferon-β ; stress hormone
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract In today's 'modern' society, no one can escape from the stresses of daily life. Stress stimulates the secretion of stress hormones (e.g. cortisol or noradrenaline) which generally suppress the immune response system, thus rendering the body vulnerable to infectious diseases and cancer. Therefore finding anti-stress food components, which diminish and/or inhibit the stress related suppression of the immune response system would be helpful in maintaining and promoting the health of the human population. Here we established a screening system for anti-stress substances using the cultured human cell line MG-63. The production of interferon-β (IFN-β) by MG-63 cells super-induced by Poly (I): Poly (C) was shown to decrease in a dose dependent manner upon the addition of 0.01–10 μg/ml of cortisol or noradrenaline (NA). 1,2–Diacylglycerol (DG) was demonstrated to abrogate this suppression. Lipid from the fermented milk, kefir, also inhibited the influence of cortisol. Kefiran, a polysaccharide secreted from L. kafiranofasiens GKL-28 diminished the cortisol or NA influenced IFN-β production. But phosphatidylcholine had no significant effect in this system. These results suggest that DG, lipids from kefir and kefiran may be equated as anti-stress food component. Abbreviations DG – diacylglycerol; IFN-β – interferon-β; NA – noradrenaline; PC – phosphatidylcholine; Poly (I):Poly (C) – polyinosinic polycytidylic acid.
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  • 7
    Electronic Resource
    Electronic Resource
    Springer
    Cytotechnology 32 (2000), S. 77-77 
    ISSN: 1573-0778
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
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  • 8
    ISSN: 1573-0778
    Keywords: fish iridovirus ; PCR diagnosis ; hybridization assay
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A new disease of epidemic proportions caused by fish viruses within the Iridoviridae family inflicts serious damage on red sea breams (Pagrus major) and striped jack (Caranx delicatissimus) populations grown in aquacultures in Japan. A partial segment of the fish iridoviral DNA was directly amplified using the polymerase chain reaction (PCR) with synthetic primers designed from well conserved nucleotide sequences between the frog virus 3 (Ranavirus) and the silkworm iridescent virus type 6. The deduced amino acid sequence from the nucleotide sequence of the PCR fragment demonstrates a high correlation with a partial sequence from the frog virus 3. Using the PCR method with specific primers, we could detect three of four different known types of fish iridoviruses in diseased fishes. To construct more reliable detection methods specific for this viral family, DNA fragments which can specifically hybridize with all of the four known iridoviridae viral DNAs were screened from the genomic library of one iridoviridae strain. The hybridization assay, using a specific fragment which contains regions which are highly homologous with a characterized partial sequence from the frog virus 3, proved to be a reliable diagnostic tool for fish iridoviral diseases.
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  • 9
    ISSN: 1573-0778
    Keywords: antigenicity ; β-lactoglobulin ; monoclonal antibody ; recombinant protein
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract We purified recombinant bovine β-lactoglobulin (rβ-LG) from the culture supernatant of transformed yeast and investigated whether rβ-LG maintained the functional ability and antigenicity of native β-LG. Immunostaining following gel electrophoresis and reversed-phase high-performance liquid chromatography confirmed that rβ-LG was purified homogeneously. rβ-LG showed almost the same retinol-binding ability as native β-LG purified from bovine milk. However, affinities of two anti-β-LG monoclonal antibodies (mAbs) to rβ-LG were different from those to native β-LG, although three other mAbs bound these two proteins equally. Since our panel of five mAbs has been previously shown to be able to detect structural changes occurring in β-LG, this variance in antigenicity can be attributed to conformational differences between rβ-LG and native β-LG. Then, we studied which step in the production and purification procedure was responsible for altering the antigenicity of rβ-LG. Bovine milk native β-LG was added to several steps in this procedure and purified in the same manner as rβ-LG. The results suggested that incubation in the yeast culture had adverse effects on maintaining the antigenicity of this recombinant protein. We conclude from these results that even if no difference between the native and recombinant proteins can be detected by functional analysis, some subtle conformational change which can be distinguished by mAbs may be incorporated into the recombinant protein during its production and ultimately cause a different immune reaction in vivo. Abbreviations β-LG, β-lactoglobulin; rβ-LG, recombinant β-LG; PBS, phosphate-buffered saline; PBS-Tween, PBS containing 0.05% Tween 20; ELISA, enzyme-linked immunosorbent assay.
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  • 10
    ISSN: 1573-0778
    Keywords: cytosolic phospholipase A2 ; human monoclonal antibody ; lung cancer ; molecular cloning ; tumor-associated antigen ; 14-3-3 protein
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The human monoclonal antibody AE6F4 specifically reacts with human lung cancer tissues but does not with normal tissues. This monoclonal antibody recognizes a cytosolic 31 kDa antigen in the cancer cells. In a previous study, we elucidated that the 31 kDa antigen belonged to a family of proteins collectively designated as 14-3-3 proteins, which were known as protein kinase-dependent activators of tyrosine/trytophan hydroxylases, or protein kinase C inhibitor proteins. Here we report molecular cloning of the 31 kDa antigen from the human lung adenocarcinoma cell line, A549. Sequencing analysis indicates that the cloned cDNA is identical to that of previously reported human placental cytosolic phospholipase A2 (cPLA2), which is also a member of the 14-3-3 protein family. Western analysis demonstrated that a 31 kDa recombinant cPLA2 expressed in monkey COS cells was recognized by the AE6F4 monoclonal antibody. Binding of the monoclonal antibody to the recombinant cPLA2 was abolished when treated with sodium periodate, suggesting that not only are carbohydrate chains associated with the cPLA2, but they also play a crucial role in antigen recognition by the monoclonal antibody.
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