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1.
G. S. Feng W. Dai S. L. Gupta G. Werner-Felmayer H. Wachter O. Takikawa M. W. Taylor 《Molecular & general genetics : MGG》1991,230(1-2):91-96
Summary Our previous observations indicated that mutants partially resistant to IFN-y cytotoxicity were defective in the induction of indoleamine 2,3-dioxygenase, (IDO). Two mutants highly resistant to IFN- were isolated following a second round of mutagenesis. The resistance to IFN- was inversely correlated with the inducibility of IDO in these mutants. Moreover, several other IFN- responsive genes, including those encoding 2-5A synthetase, GTP cyclohydrolase and HLA-DR, were also differentially altered in their expression upon INF- treatment. IFN-y receptor gene expression was not changed nor was the binding of the receptor to IFN-. Southern blot analysis failed to reveal any significant abnormality in the IDO gene structure in these mutants. We therefore suggest that these mutants are defective in the IFN- signaling pathway and will be useful in further analysis of the biochemical mechanism of IFN- activated gene expression in target cells. 相似文献
2.
Mitsuo Katano Hiroshi Yamamoto Mitsunari Nakamura Tatsuya Matsuo Takeharu Hisatsugu 《Biotherapy》1990,2(1):33-40
Interferon- (IFN-) and tumor growth inhibitory factor (TGIF) were inducedin vitro in the supernatant from mixed culture of human peripheral blood mononuclear cells (PBMC) and OK-432. TGIF activity was determined by growth inhibition of a human gastric adenocarcinoma cell line, MK-1 cells, and IFN- activity was measured by radioimmunoassay. The production of TGIF and IFN- was time-dependent, reaching its maximum around 48 hrs. Although there was no significant correlation between TGIF production and IFN- production, combination of a subthreshold concentration of recombinant IFN- (rIFN-) and TGIF induced significant growth inhibition of MK-1 cells. This fact indicates that the effects of rIFN- and TGIF are synergistic. The antiproliferative effect of these cytokines are highly species-specific, and their synergistic effects were also species-specific. rIFN--sensitive and -resistant clones were successfully established from the original MK-1 cell line; those clones are both sensitive to TGIF. Synergistic antiproliferative effects were found when the rIFN--sensitive clone, but not the resistant clone, was used as a target, suggesting that the synergistic effects require the target cells' sensitivity to IFN-. These results indicate that the synergistic effects of TGIF and IFN- may produce a clinical antitumor action in cancer patients receiving OK-432 administration. 相似文献
3.
Anaerobic dechlorination and degradation of hexachlorocyclohexane isomers by anaerobic and facultative anaerobic bacteria 总被引:2,自引:0,他引:2
Screening studies with strict and facultative anaerobic bacteria showed that Clostridium app. and several other representatives of Bacillaceae and Enterobacteriaceae actively degraded -hexachlorocyclohexane (-HCH) under anaerobic conditions. Representatives of Lactobacillaceae and Propronibacterium were inactive. With 36Cl-labelled -HCH a nearly complete dechlorination was shown to occur in 4–6 days by Clostridium butyricum, C. pasteurianum and Citrobacter freundii, while other facultative anaerobic species were less active.Aerobically grown facultative anaerobes also dechlorinated actively -HCH during subsequent anaerobic incubation with glucose, pyruvate or formate as substrates. The -, - and -HCH isomers were also, but more slowly, dechlorinated (>>-HCH). All species active in anaerobic degradation of -HCH formed -tetrachlorocyclohexene (TCH) as the main intermediate metabolite and no -pentachlorocyclohexene (PCH) or other isomers of TCH or PCH have been found. Small amounts of tri- and tetrachlorinated benzenes have been found too. The mechanism of dechlorination is discussed.Non-Common Abbreviations Used -HCH
-hexachlorocyclohexane
- -TCH
-2,3,4,5-tetrachlorocyclohexene
- -PCH
-1,2,3,4,5-pentachlorocyclohexene
- GLC
gas liquid chromatography 相似文献
4.
Expression of bioactive human interferon-gamma in transgenic rice cell suspension cultures 总被引:4,自引:0,他引:4
We investigated the possibility of producing the therapeutic recombinant cytokine, Interferon-gamma (IFN-), in transgenic rice cell (Oryza sativa, cultivar TNG67) suspension cultures. We tested expression of two vector constructs, each harboring an Amy3 leader peptide and a C-terminus His 6 tag fused to a human IFN- cDNA, one driven by a sucrose-starvation inducible promoter (rice Amy3 promoter) and the other by a constitutive maize ubiquitin promoter, in rice cell suspensions, introduced via Agrobacterium tumefaciens. There was a significant difference in the amounts of recombinant IFN- protein produced by the Ups and Amy cell lines, as cytosolic and secretory proteins respectively. Immunological analysis of IFN- recombinant protein conferred a dose-dependent anti-dengue virus activity in human A549 cells, similar to the commercial product. We discuss the attractive attributes of using rice cell suspension system for the expression of therapeutic recombinant IFN-. 相似文献
5.
Kohichi Sakamoto Hiroto Nakajima Jun Shimizu Tatsuo Katagiri Chiharu Kiyotaki Hiromi Fujiwara Toshiyuki Hamaoka 《Cancer immunology, immunotherapy : CII》1988,27(3):261-266
Summary The present study investigated the role of antigen-presenting cells (APC) in the activation of noncytolytic T cells against tumor antigens. The noncytolytic-type T cells exerted their antitumor effect by producing -interferon (IFN-) and by activating macrophages as the ultimate effectors. The production of IFN- by these noncytolytic T cells following the stimulation with tumor cells required the participation of Ia+ APC, since the depletion of APC from cultures of tumor-immunized spleen cells resulted in almost complete inhibition of the IFN- production. Both L3T4+ and Lyt-2+ subsets of T cells were capable of producing IFN-, and the requirement of APC for the production of IFN- was the case irrespective of whether noncytolytic T cells were of L3T4+ or Lyt-2+ phenotype. More importantly, it was demonstrated that the production of IFN- by L3T4+ and Lyt-2+ T cells was inhibited by addition of the respective anti-class II and anti-class I H-2 antibody to cultures. These results indicate that antitumor L3T4+ or Lyt-2+ noncytolytic T cells are activated for the IFN- production by recognizing tumor antigens in the context of self-class II or -class I H-2 molecules on APC.This work was supported by a Grant-in-Aid for the Special Project Cancer-Bioscience from the Ministry of Education, Science and Culture, Japan 相似文献
6.
Timothy M. Anderson Yukihiro Ibayashi E. Carmack Holmes Sidney H. Golub 《Cancer immunology, immunotherapy : CII》1987,25(1):65-68
Summary The purpose of these studies was to compare local and systemic human lymphokine activated killer (LAK) and natural killer (NK) cytotoxic activity and to determine its modulation by biologic agents. Local immunity may be an important component in limiting local tumor growth. Therefore, as a model for studying immune function in the local compartment, we assessed NK activity of lymphocytes present at the site of human tumors and in peripheral blood (PBL). We extracted tumor infiltrating lymphocytes (TIL) and PBL from patients with pulmonary tumors and compared NK activity and response to the biological modifiers gamma interferon (IFN-), indomethacin (INDO), and interleukin 2 (IL-2). We also studied TIL and PBL LAK activity using the NK-resistant M14 target cells and determined the TIL response to IL-2, plus IFN-. Titration of K562 targets in a 51Cr release assay revealed that untreated TIL have low cytotoxicity (4.32%) compared to untreated PBL (34.3%, P=<0.001). This low level of TIL NK activity was not affected by IFN-, INDO, or IL-2 at 1 h. However, at 3 days of culture, IL-2 with or without exogenous IFN- significantly increased TIL NK ctotoxicity (20.5%, P=0.02 without IFN- and 32.52 lytic units (LU), P=<0.02 with IFN-). Untreated TIL and PBL both had low cytotoxicity against M14 targets (1.08 LU and 1.26 LU), respectively. After 3 days culture with IL-2 plus IFN-, both TIL and PBL LAK cytotoxicity were increased (14.34 LU and 40.63 LU). We conclude that local NK and LAK activity is intrinsically low. However, this activity can be modulated by biologic agents, thus giving hope for the development of local antitumor effectors capable of in vivo tumor control. 相似文献
7.
Dominiecki ME Beatty GL Pan ZK Neeson P Paterson Y 《Cancer immunology, immunotherapy : CII》2005,54(5):477-488
Purpose: Many human tumors lose responsiveness to IFN-, providing a possible mechanism for the tumor to avoid immune recognition and destruction. Here we investigate the importance of tumor responsiveness to IFN- in the successful immunotherapy of TC1 tumors that were immortalized with human papillomavirus proteins E6 and E7. Methods: To investigate the role of IFN- in vivo, we constructed a variant of TC1, TC1.mugR, that is unresponsive to IFN- due to overexpression of a dominant negative IFN- receptor. Results: Using recombinant Listeria monocytogenes that express HPV-16 E7 (Lm-LLO-E7) to stimulate an antitumor response, we demonstrate that sensitivity to IFN- is required for therapeutic efficacy in that Lm-LLO-E7 induces regression of TC1 tumors but not TC1.mugR. In addition, we show that tumor sensitivity to IFN- is not required for inhibition of tumor angiogenesis by Lm-LLO-E7 or for trafficking of CD4+ and CD8+ T cells to the tumor. However, it is required for penetration of lymphocytes into the tumor mass in vivo. Conclusions: Our findings identify a role for IFN- in immunity to TC1 tumors and show that loss of tumor responsiveness to IFN- poses a challenge to antigen-based immunotherapy.Mary E. Dominiecki and Gregory L. Beatty contributed equally to this work. 相似文献
8.
Bulk populations of T-cell receptor (Tcr) -expressing splenocytes from different inbred strains of mice were examined for the diversity of Tcr proteins. Immunoprecipitations with anti-C1/2, anti-C4, and anti-V1 sera demonstrated that splenocytes from B10.BR, C57BL/6, and C57L strains of mice expressed the same array of Tcr proteins, namely V1-C2, V1-C4, and V2-C1, although the Tcr heterodimers observed for each of these strains were biochemically distinct. Examination of bulk splenic Tcr heterodimers from several other inbred strains of mice demonstrated that each of the strains could be categorized into one of three basic phenotypes. For several reasons, the differences observed between the strains appeared to be solely dependent on polymorphisms of the Tcrg loci. First, F1 mice co-expressed both parental Tcr phenotypes. Second, the distinguishing polymorphism between mice of phenotype 1 and phenotypes 2 or 3 was due to the presence of an N-linked glycosylation site within the Tcrg-C1 gene segment, previously described for BALB.B and C57BL/6 Tcrg-C1 genes. Finally, the V1-C4 polymorphism between mice of phenotype 3 and phenotypes 1 or 2 was due to differences in core protein size. Furthermore, the three defined Tcr chains were expressed independently of the major histocompatibility complex (MHC) haplotype. Although no striking qualitative differences in Tcr heterodimers were observed between strains (including those with autoimmune disorders), a quantitative difference in the relative amount of C4-encoded proteins was observed on Tcr splenocytes from both newborn euthymic and adult athymic mice when compared to adult Tcr splenocytes from euthymic mice. These results demonstrate that genetic polymorphisms exist among different mouse strains and suggest that selective developmental pressures may govern Tcr expression.
Offprint requests to: J. A. Bluestone 相似文献
9.
N(3, 4-dimethoxycinnamoyl) anthranilic acid (Tranilast) inhibits antibody-mediated hypersensitivity reactions, and is an effective drug for patients with bronchial asthma or allergic rhinitis. Interferon- (IFN-) production of ovalbumin (OA)-stimulated peripheral blood mononuclear cells (PBMCs) from hen's egg-sensitive patients with atopic dermatitis (AD) was significantly higher than those of healthy controls. Tranilast inhibited this IFN- production. Moreover, interleukin-2 (IL-2) production of OA-stimulated PBMCs from hen's egg-sensitive patients with AD was also inhibited by Tranilast. Our results suggest that Tranilast can be used to the patients with food sensitive AD.Abbreviations PBMCs
peripheral blood mononuclear cells
- OA
ovalbumin
- BSA
bovine serum albumin
- AD
atopic dermatitis
- IL-2
interleukin-2
- IFN-
interferon-
- Tranilast
N(3, 4-dimethoxycinnamoyl) anthranilic acid
- IL-4
interleukin 4
- IL-5
interleukin 5 相似文献
10.
Summary The -glucuronidase staining characteristics of isolated T cell populations and the T and T enriched fractions derived of them were studied. T lymphocytes were obtained from purified T lymphocytes by ox-IgG rosette sedimentation. The rosette-forming cells in the pellet were referred to as T lymphocytes, whereas the lymphocytes in the interface were referred to as T depleted or T lymphocytes. B cells were studied on rosetted mononuclear cells with either mouse erythrocytes or with Staphylococcus Aureus (Cowan I) bacteria, after a preceeding polyvalent anti-human Ig treatment of the cells. While B cells showed mostly no reactivity, T and T cells were respectively characterised by a dot-like and granular pattern of reactivity. These findings are in agreement with those observed by others after -naphthyl-acetate esterase or acid phosphatase staining. Within the T lymphocyte fraction, the T non-, non lymphocytes seemed to have a granular pattern of reactivity. The same staining pattern was found in non-B, non-T lymphocytes. 相似文献
11.
Sustained low-level expression of interferon-γ promotes tumor development: potential insights in tumor prevention and tumor immunotherapy 总被引:2,自引:0,他引:2
He YF Wang XH Zhang GM Chen HT Zhang H Feng ZH 《Cancer immunology, immunotherapy : CII》2005,54(9):891-897
Although the proinflammatory cytokine interferon- (IFN-) has been generally thought to enhance antitumor immune responses and be involved in antitumor mechanisms of many other immunotherapy molecules, it has also been reported that IFN- could promote tumor immune evasion. In this report, by using an ideal mouse model that expresses IFN- locally in muscle, we demonstrate that sustained low-level expression of IFN- promotes the development of several types of tumor including H22 hepatoma, MA782/5S mammary adenocarcinoma and B16 melanoma. However, transitory expression of IFN- does not have such an effect. On the other hand, sustained high-level expression of IFN- mediates significant antitumor effect on H22 hepatoma. Low level of IFN- upregulates expression of PD-L1, PD-L2, CTLA-4 and Foxp3, which may partly account for the tumor immune evasion promoted by IFN-. Furthermore, blockade of PD-L inhibits IFN-s tumor-promoting effect. Our findings provide a mechanistic link between chronic inflammation and cancer and would have potential implications for cancer prevention and also for the design of cytokine–based cancer immunotherapy. 相似文献
12.
de Pater-Huijsen FL van der Loos CM de Riemer MJ van der Zee JS Jansen HM Out TA 《The Histochemical journal》2000,32(1):3-11
An immunocytochemical staining method has been developed for simultaneous staining of both cell surface markers (CD4 and CD8) and intracellular cytokine proteins IFN-, IL-4 and IL-5. Cell surface molecules were visualized with alkaline phosphatase, which was developed by Fast Blue BB. Intracellular cytokine proteins were detected by amino-ethyl carbazole. We applied this technique to T cells from T-cell lines and T-cell clones, peripheral blood mononuclear cells and broncho-alveolar lavage fluid cells. Cells were used either unstimulated or stimulated for 4h with 1ng/ml PMA and 1g/ml ionomycin, which proved to be an optimal stimulus taking cytokine staining, cell recovery and cell viability into account. We studied peripheral blood mononuclear cells from healthy subjects and found that without in vitro stimulation on average 0.4% of the cells were IFN- positive cells. In unstimulated broncho-alveolar lavage fluid cells of the 2 allergic asthmatic subjects studied so far we found higher numbers of cytokine-positive cells (up to 22% of the lymphocytes being IL-4+ cells). By in vitro stimulation, the numbers of cytokine-positive peripheral blood mononuclear cells from the healthy subjects were increased to maximally 5% IFN-+ cells. In stimulated lavage fluid cells from allergic asthmatic subjects maximally 34% of the lymphocytes became IFN-+. We conclude that this method allows detection of intracellular cytokine proteins in both CD4+ and CD8+ T cells without the need for stimulating the cells in vitro. In vitro stimulation may change the cytokine profile detected. 相似文献
13.
C. M. Tu 《Archives of microbiology》1976,108(3):259-263
Of 147 microorganisms isolated from a loamy sand, 71 showed good growth with lindane (-1,2,3,4,5,6-hexachlorocyclohexane) and produced chloride in an aqueous medium. Thirteen soil microorganisms were selected to study the utilization of lindane. Lindane was metabolized by the microbes to -2,3,4,5,6-pentachloro-1-cyclohexene (-PCCH), -3,4,5,6-tetrachloro-1-cyclohexene (-TCCH), -3,4,5,6-tetrachloro-1-cyclohexene (-TCCH), -3,4,5,6-tetrachloro-1-cyclohexene (-TCCH), and pentachlorobenzene (PCB). Cells of Pseudomonas sp. No. 62 grown on lindane simultaneously adapted to -PCCH, -TCCH, -TCCH, -TCCH, PCB, 1,2,3,4-tetrachlorobenzene (1,2,3,4-TCB) and 1,2,4,5-tetrachlorobenzene (1,2,4,5-TCB). The bacteria degraded each of these chemicals at least partially as indicated by an increased rate of oxygen consumption.Abbreviations Lindane
-1,2,3,4,5,6-hexachlorocyclohexane
- -PCCH
-2,3,4,5,6-pentachloro-1-cyclohexene
- -TCCH
-3,4,5,6-tetrachloro-1-cyclohexene
- -TCCH
-3,4,5,6-tetrachloro-1-cyclohexene
- -TCCH
-3,4,5,6-tetrachloro-1-cyclohexene
- PCB
pentachlorobenzene
- 1,2,3,4-TCB
1,2,3,4-tetrachlorobenzene
- 1,2,3,5-TCB
1,2,3,5-tetrachlorobenzene
- 1,2,4,5-TCB
1,2,4,5-tetrachlorobenzene
- 1,2,3-tCB
1,2,3-trichlorobenzene
- 1,2,4-tCB
1,2,4-trichlorobenzene
- 1,3,5-tCB
1,3,5-trichlorobenzene
- 1,2-DCB
1,2-dichlorobenzene
- 1,3-DCB
1,3-dichlorobenzene
- 1,4-DCB
1,4-dichlorobenzene
- MCB
monochlorobenzene
Contribution No. 631, Research Institute, Agriculture Canada, University Sub Post Office, London, Ontario N6A 5B7 相似文献
14.
Mice lacking the IFN-γ receptor or fyn develop severe experimental autoimmune uveoretinitis characterized by different immune responses 总被引:1,自引:0,他引:1
Endogenous interferon (IFN)- negatively regulates experimental autoimmune uveoretinitis (EAU), a Th1-mediated disease. Although it is well known that IFN- exerts its effects by binding to the IFN- receptor (IFN-R), the role that IFN-R plays in the development of EAU has not been investigated. Fyn has been reported to inhibit Th2 differentiation. We aimed to investigate how endogenous IFN-R and fyn, which influence Th1/Th2 differentiation, participate in the development of EAU. Sex-matched 6- to 10-week-old C57BL/6 wild-type (WT), IFN-R knockout (GRKO) and fyn knockout (fyn KO) mice were compared. Mice were immunized subcutaneously with human interphotoreceptor retinoid-binding protein peptide 1–20 emulsified in Freunds complete adjuvant together with an intraperitoneal injection of Bordetella pertussis toxin. Three weeks later, mice were sacrificed, and their eyes and spleens were harvested for histopathologic analyses and examination of cellular immune responses, respectively. Cellular immune responses were evaluated by measuring the proliferative responses and cytokine production [interleukin (IL)-4, IL-5, IL-6, IL-13, IFN- and tumor necrosis factor (TNF)-] of splenocytes. The incidence of EAU was 40.0% in WT mice, 59.3% in GRKO mice and 78.6% in fyn KO mice. The average EAU score was 0.294 in WT mice, 0.917 in GRKO mice and 1.063 in fyn KO mice. Upon EAU induction, significant infiltration of eosinophils into the eyes was observed in GRKO and fyn KO mice compared to WT mice. Splenocytes from GRKO mice proliferated against the antigen and a mitogen more vigorously than those from WT and fyn KO mice. Stimulation of splenocytes with the antigen induced a higher production of IL-4, IL-6, IL-13 and IFN- in GRKO mice compared to WT and fyn KO mice. In contrast, IL-5 and TNF- were most abundantly produced by splenocytes from fyn KO mice compared to WT and GRKO mice. The incidence and mean severity of EAU were significantly higher in GRKO and fyn KO mice than in WT mice, suggesting that endogenous IFN-R and fyn negatively regulate the development of EAU. The different cytokine production patterns by the GRKO and fyn KO mice indicate that the negative regulatory mechanism mediated by IFN-R and fyn may differ. 相似文献
15.
We have isolated an unusual T cell receptor chain cDNA clone (7.1) from a library made from RNA derived from adult thymus of C57BL/Ka mice. This cDNA clone corresponds to the appropriately processed C1 constant region exons preceded by 1.5 kb of J-C1 intron. The 7.1 coding region is extremely homologous to the C1 gene of BALB/c mice, differing at the protein level by a single deletion (alanine 139) and a single substitution. This latter change eliminates the sole N-linked sugar attachment site, providing a basis for strain-specific glycosylation patterns. The J-C1 intronic region contains two DNA segments (termed J1 and J2) that are highly reminiscent of joining (J) segments; both have potentially functional recombination and donor splice sequences flanking an open reading frame. Northern analysis suggests that 7.1 may be derived from a large, variable region-containing precursor. 相似文献
16.
Proteolytic cleavage of recombinant human interferon- (IFN-) expressed in Chinese hamster ovary (CHO) cells during batch fermentation has been monitored by mass spectrometric peptide mapping. IFN- was purified from cell-free culture supernatant by immunoaffinity chromatography and cleaved by endoprotease Asp-N. Peptide fragments were resolved by reverse-phase HPLC and identified by a combination of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and automated N-terminal peptide sequencing. Using this approach, a peptide was identified as the C-terminal fragment of the IFN- polypeptide. Analysis of this peptide by MS indicated that the recombinant IFN- polypeptide secreted by CHO cells was truncated by at least ten amino acids, initially at Gln133-Met134. No full length (143 amino acids) polypeptide molecules were observed at any stages of the fermentation. Additional proteolytic cleavages at basic amino acids N-terminal of Gln133 occurred during the later stages of the culture resulting in a heterogeneous IFN- polypeptide population with 'ragged' C-termini. 相似文献
17.
Perales M Parisi G Fornasari MS Colaneri A Villarreal F González-Schain N Echave J Gómez-Casati D Braun HP Araya A Zabaleta E 《Plant molecular biology》2004,56(6):947-957
We report the identification by two hybrid screens of two novel similar proteins, called Arabidopsis thaliana gamma carbonic anhydrase like1 and 2 (AtCAL1 and AtCAL2), that interact specifically with putative Arabidopsis thaliana gamma Carbonic Anhydrase (AtCA) proteins in plant mitochondria. The interaction region that was located in the N-terminal 150 amino acids of mature AtCA and AtCA like proteins represents a new interaction domain. In vitro experiments indicate that these proteins are imported into mitochondria and are associated with mitochondrial complex I as AtCAs. All plant species analyzed contain both AtCA and AtCAL sequences indicating that these genes were conserved throughout plant evolution. Structural modeling of AtCAL sequences show a deviation of functionally important active site residues with respect to CAs but could form active interfaces in the interaction with AtCAs. We postulate a CA complex tightly associated to plant mitochondrial complex. 相似文献
18.
The effects of lipids on the glycosylation of recombinant human interferon- expressed in a Chinese Hamster Ovary cell line were investigated in batch culture. Lipids form an essential part of the N-glycosylation pathway, and have been shown to improve cell viability. In control (serum-free) medium the proportion of fully-glycosylated interferon- deteriorated reproducibly with time in batch culture, but the lipoprotein supplement ExCyte was shown to minimise this trend. Partially substituting the bovine serum albumin content of the medium with a fatty-acid free preparation also improved interferon- glycosylation, possibly indicating that oxidised lipids carried on Cohn fraction V albumin may damage the glycosylation process.Abbreviations BSA
bovine serum albumin
- CHO
chinese hamster ovary
- DHFR
dihydrofolate reductase
- FCS
foetal calf serum
- IFN-
human interferon-gamma
-
q
IFN
specific interferon production rate
-
specific growth rate
- 2N
doubly-gycosylated
- 1N
singly-glycosylated
- ON
non-glycosylated 相似文献
19.
Monaco L Marc A Eon-Duval A Acerbis G Distefano G Lamotte D Engasser JM Soria M Jenkins N 《Cytotechnology》1996,22(1-3):197-203
The CHO cell line has achieved considerable commercial importance as a vehicle for the production of human therapeutic proteins, but is known to lack a functional copy of the gene coding for 2,6-sialyltransferase (EC 2.4.99.1). The cDNA for rat 2,6-ST was expressed in a recombinant CHO cell line making interferon-, using a novel in vitro amplification vector. The enzyme was expressed efficiently, and resulted in up to 60% of the total sialic acids on interferon- being linked in the 2,6-conformation. This sialic acid linkage distribution was more akin to that seen in natural human glycoproteins. In the most successful cell clones, expression of 2,6-sialyltransferase improved the overall level of sialylation by up to 56%, and had no adverse effects on cell growth, IFN- productivity or other aspects of IFN- glycosylation. These experiments demonstrate how the glycosylation machinery of rodent cells can be genetically manipulated to replicate human tissues.Abbreviations AT-III
antithrombin-III
- CHO
Chinese hamster ovary
- dhfr
dihydrofolate reductase
- EPO
erythropoietin
- IFN-
human interferon-
- NEO
neomycin
- NeuAc
N-acetylneuraminic acid
- NeuGc
N-glycolylneuraminic acid
- ST
sialyltransferase
- tPA
tissue plasminogen activator 相似文献
20.
T. H. J. Huisman F. Kutlar T. Nakatsuji A. Bruce-Tagoe Y. Kilinç M. N. Cauchi C. Romero Garcia 《Human genetics》1985,71(2):127-133
Summary The occurrence of the AT chain (i.e. A75 Ile Thr) in different populations was evaluated through a study of 4250 cord blood samples and blood samples from more than 350 SS1 patients. High frequencies were observed in Italy, Yugoslavia, Turkey, Holland, but also in Japan, Vietnam, and India. The chain is (nearly) absent in the Black population of Ghana and Kenya, and low frequencies were observed in China and Australian aborigines. Only a few adult SS patients (18 out of 357) were AT heterozygotes. The chromosomes with the AT globin gene were mapped through an evaluation of the presence of 10 different restriction sites. The AT chromosomes from different populations were closely related and had the same subhaplotypes of [--++-+] (Hinc II 5 to ; Xmn I 5 to G; Hind III in G and A; Hinc II in and 3 to ), quite different from the subhaplotypes seen for AT negative chromosomes.2 This suggests a common ancestor which may have originated in Southern Europe. An evaluation of the chain production by both chromosomes in SS patients and -thalassemia heterozygotes was possible for subjects with an AT heterozygosity. It was concluded that in -thalassemia trait, the chain synthesis is directed for about two-thirds by the thalassemic chromosome and for about onethird by the normal chromosome; the contribution by the normal chromosome decreases with a decrease in total chain production.This is contribution #0890 of the Department of Cell and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, USA 相似文献