Expression Of Human Interleukin Research Articles

Cloning and Expression of Human Interleukin 2 (IL-2) in E. coli and its Antitumor Activity

Cloning and Expression of Human Interleukin 2 (IL-2) in E. coli and its Antitumor Activity

Expression of Human Interleukin 8 in Mice Alters Their Natural Behaviors.

ObjectiveTo examine the effects of human interleukin (IL) 8 expression on mouse behavior.MethodsA mouse line expressing human IL8 in the intervertebral discs (IVD) and cartilaginous tissues (hIL8+) was generated. Mouse spontaneous behaviors, including locomotion, climbing, rearing, grooming, eating, drinking, and immobility were recorded with a fully automatic, non-invasive platform.ResultsDistance traveled by the hIL8+ mice declined with age compared with control littermates, and male hIL8+ mice traveled a shorter distance than male controls and females of either genotype (p <0.05). The hIL8+ mice also spent less time in locomotion than control mice (p <0.01), and male hIL8+ mice spent the least amount of time and had lowest count in locomotion compared with the other 3 groups at 12 weeks of age or greater (p <0.05). The hIL8+ mice spent less time climbing than controls, and male mice spent less time climbing than female mice of the same genotype (p <0.01). The hIL8+ mice spent more time eating and less time drinking than controls, and all mice spent less time eating and more time drinking with increasing age. Finally, hIL8+ mice spent more time immobile than controls, and male hIL8+ mice spent more time immobile than any other group (p <0.05).ConclusionThe hIL8+ mice, especially hIL8+ males, showed reduced ambulation and climbing. Mice showed age-related decrease in eating and increase in drinking and grooming time that was also influenced by expression of hIL8. These changes in natural behaviors in control mice are consistent with functional decline with age. Effects of hIL8 superimposed on the natural aging process could involve systemic (e.g., on the brain) and local (e.g., in the spine and joint tissues) mechanisms. Future exploration of these mechanisms might be productive.

Construction of Foot-and-mouth disease virus 2A-based bicistronic expression vector and coexpression of two genes in goat mammary epithelial cells

Using animal mammary glands as bioreactors for producing commercially important proteins is a cutting-edge direction in the field of biotechnology development and application. Dairy goats are an important dairy livestock, with roughage-resistance, fast propagation, long lactation periods and high milk production per bodyweight; these characteristics make dairy goats ideal for use as mammary gland bioreactors. Foot-and-mouth disease virus 2A (FMDV 2A) is an efficient viral cleavage element that mediates proteolytic cleavage independent of the presence of other FMDV sequences. It is often incorporated into recombinant vectors to generate cleavage in the presence of heterologous sequences. To achieve specific co-expression of two heterologous genes in goat mammary gland epithelial (GMGE) cells, a mammary gland-specific bicistronic expression vector, pFIEβ, containing the β-casein 5′ flanking sequence and FMDV 2A, was successfully constructed and the specific expression of human interleukin 2 (hIL-2) and enhanced green fluorescent protein (EGFP) was conducted in primary GMGE cells. Another bicistronic expression vector, pFIEC, driven by the cytomegalovirus promoter, was constructed as a positive control. In cells transfected with pFIEβ and pFIEC, RT-PCR verified the existence of recombinant fusion mRNA of hIL-2 upstream of EGFP within the FMDV 2A cassette fragment and western blot analysis showed the existence of the fusion between hIL-2 and EGFP. It is concluded that FMDV 2A generated specific co-expression of multiple genes for the first time in primary GMGE cells driven by the β-casein promoter.

Expression of a murine homolog of apoptosis-inducing human IL-24/MDA-7 in murine tumors fails to induce apoptosis or produce anti-tumor effects

Expression of human interleukin (IL)-24 in tumors achieved anti-tumor effects through apoptosis. IL-24 also induced secretion of proinflammatory cytokines, suggesting the role in immunity. We showed that murine IL-24 transcripts started from the second initiation codon and that expressed mIL-24 in tumors failed to induce apoptosis. Proliferation of murine cells expressing mIL-24 was the same as that of the parent cells and inoculation of the mIL-24-expressing tumors into syngeneic mice did not produce anti-tumor effects. Secretory mIL-24 did not induce the expression of the IL-6, TNF-α or IFN-γ gene in spleen cells. Expression of mIL-24 receptor subunits, IL-22R and IL-20R1, was undetectable in spleen cells even though they were stimulated by anti-CD3, anti-CD40 antibody or concanavalin A. Transduction of murine tumors with adenoviruses expressing the human IL-24 gene however suppressed the viability and decreased the tumor growth. These data suggest that mIL-24 is functionally irrelevant to the human counterpart.

Evidence that phosphatidylcholine-specific phospholipase C is a key molecule mediating insulin-induced enhancement of gene expression from human cytomegalovirus promoter in CHO cells.

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.

Expression of amplified cDNA and genomic sequences encoding human interleukin 2 in Chinese hamster ovary cells.

Expression of human interleukin 2 (IL-2) at high levels has been achieved in Chinese hamster ovary (CHO) cells by amplification of transfected sequences. Plasmids containing the human IL-2 cDNA or genomic DNA and mouse dihydrofolate reductase (DHFR) cDNA were transfected into DHFR-negative CHO cells. Transformants expressing DHFR were selected in media lacking nucleosides, and cells which amplified both DHFR and IL-2 genes were obtained by exposure to increasing methotrexate (MTX) concentrations. These cell lines constitutively expressed elevated levels of IL-2 at a concentration of 2 mg/liter. These cell lines continued to produce IL-2 stably through at least 1 month, even in the absence of MTX.

Expression of amplified cDNA sequences encoding human interleukin 2 in human cells.

Expression of human interleukin 2 (IL-2) at high levels has been achieved in human cells by cotransfection and subsequent coamplification of the transfected sequences. IL-2 production by these cells reached 2700 U/ml of culture medium. This productivity was higher than the 100U/ml attained by concanavalin A (ConA) stimulated human leukaemic Jurkat, T cells. A plasmid, pSDI, containing human IL-2 cDNA and mouse dihydrofolate reductase (DHFR) cDNA was con-structed, and cotransfected with plasmid pSV2neo at a 10:1 molar ratio, into Hela (human epitheloid carcinoma) cells by the DNA-calcium phosphate coprecipitation technique. Transformants producing IL-2 were easily obtained among antibiotic G418 resistant colonies. Cells resistant to increased levels of methotrexate (MTX) secreted a large amount of IL-2 as a result of coamplification of DHFR cDNA and IL-2 cDNA. Expression of IL-2 cDNA was stimulated by removing a G-C stretch which was present between Simian Virus 40 (SV40) early promoter and IL-2 cDNA. Plas...

Prostaglandins posttranscriptionally inhibit monocyte expression of interleukin 1 activity by increasing intracellular cyclic adenosine monophosphate.

The effect of prostaglandins and cyclic 3',5'-adenosine monophosphate (cAMP) on expression of human interleukin 1 (IL 1) activity was investigated in the promonocytic tumor cell line U937 and peripheral blood monocytes. After in vitro stimulation by bacteriotoxins, monocytes express IL 1 activity, as measured by the thymocyte costimulation assay. Although high doses of bacteriotoxins impaired expression of IL 1, this effect was reversed by indomethacin. When stimulated monocytes were cultured with exogenous prostaglandins, including PGE2 and PGI2, expression of IL 1 was reversibly inhibited. Interaction of U937 cells with PGE2 resulted in a transient increase in cellular cAMP concentration during the first hour of exposure. Other agents that cause an increase in levels of cellular cAMP, including theophylline, isobutylmethylxanthine, dibutyryl cAMP, or cholera toxin, also reversibly reduced expression of IL 1 by stimulated monocytes. The effect of these agents on levels of IL 1 mRNA was analyzed. TSS-stimulated increase in levels of IL 1-encoding mRNA was studied both by DNA-RNA hybridization analysis performed with an IL 1-beta cDNA probe and by injecting U937 polyadenylated mRNA into frog oocytes and then measuring expression of IL 1 activity in the oocyte supernatant. Agents that increased levels of cellular cAMP did not alter levels of IL 1 mRNA accumulation or global protein synthesis in TSS-stimulated U937 cells. IL 1 stimulates synthesis of prostaglandins that reach high levels during immune and inflammatory reactions. Our data suggest that prostaglandins participate in an autoregulatory pathway that posttranscriptionally reduces expression of IL 1 activity.

Cloning and expression of human interleukin 2 in Streptomyces lividans using the [formula omitted] consensus promoter

Streptomyces lividans was transformed with a plasmid containing the structural gene that specifies human interleukin 2. The expression of interleukin 2 in this plasmid is controlled by both the consensus promoter and a consensus ribosome binding site characteristic of Escherichia coli . We have detected production of active human interleukin 2 in liquid cultures of the transformed Streptomyces by both, biological assay and immuno-blotting analysis.