The effect of the recombinant human interleukin-2 Gene in potato (Solanum tuberosum cv. superior)

To examine the effect of the T-cell growth factor (human interleukin-2), we constructed a binary vector, pSSK-1, carrying the recombinant human interleukin-2 (rhlL-2) gene, and transferred it intoAsrobacterium tumefaciens. Using this construct, we then transformed potato explants(Solanum tuberosum cv. Superior), achieving 100% regeneration of shoots on a modified MS medium. Of the putative transformed shoots, 81% rooted and were selected on 200 ms/L kanamycin. Both Southern and northern analyses verified the transformation events. An ELISA test also indicated that the rhlL-2 protein was produced from rhlL-2-transformed potatoes. To determine whether this protein was biologically active in the potato cells, we performed a biological assay using the 11.-2 dependent cell line, CTLL-2. The suspension containing extract from the transformants showed significant proliferation of the 11.-2 dependent CTLL-2 cells, whereas cells did not proliferate in the nontransformed potato. We then grew the verified rhlL-2 transgenic potatoes in soil, and compared their performance with that of nontransgenic potatoes as well as those that had been transformed with GUS. Growth rates, as calculated from plant heights, were up to 50% higher than for either the nontrans-genic or the GUS-transformed potatoes. Similar patterns were found withArabidopsis thaliana plants treated in the same manner. All of these results suggest that rhlLo2 may function as a growth factor in potato.     

House dust mite, Dermatophagoides pteronissinus increases expression of MCP-1, IL-6, and IL-8 in human monocytic THP-1 cells

The house dust mite (Dermatophagoides pteronissinus) plays an important role in the pathogenesis of allergic diseases, including atopic dermatitis, and asthma. Monocyte chemotactic protein 1 (MCP-1/CCL2)/IL-6/IL-8 (CXCL8) plays a pivotal role in mediating the infiltration of various cells into the skin of atopic dermatitis and psoriasis. The aim of this study was to investigate the effect of D. pteronissinus extract (DpE) on expression of MCP-1/IL-6/IL-8 mRNA and protein and the signal transduction in the human monocytic cell line, THP-1. The mRNA and protein expression of MCP-1/CCL2, IL-6, and IL-8 were elevated by DpE in a time and dose-dependent manner in THP-1 cells. The increased expression of MCP-1, IL-6, and IL-8 was not affected by aprotinin (serine protease inhibitor) or E64 (cysteine protease inhibitor). We found that MCP-1 and IL-6 expression due to DpE was related to Src, protein kinase C δ (PKC δ), extracellular-signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) and IL-8 expression was involved in Src family tyrosine kinase, PKC δ, ERK. DpE increased the phosphorylation of ERK and p38 MAPK after 5 min and peaked at 30 min. The activation was significantly blocked by PP2, an inhibitor of Src family tyrosine kinase and rottlerin, an inhibitor of PKC δ (p < 0.01). DpE increases MCP-1, IL-6, and IL-8 expression and transduces its signal via Src family tyrosine kinase, PKC, and ERK in a protease-independent manner. This finding may contribute to the elucidation of the pathogenic mechanism triggered by DpE .     

Cytoplasmic and periplasmic expression of a highly basic protein,human interleukin 4, inEscherichia coli

Summary Human IL-4 (hIL-4) has been cloned from a human T cell line based on its homology to the murine IL-4 cDNA sequence [36]. We have compared cytoplasmic and extra-cytoplasmic expression of this basic protein inEscherichia coli using various combinations of promoters, replicons and host strains. Strains producing a cytoplasmic product were most successful at heterologous protein expression, producing up to 500 mg/l of an inactive aggregated form of the protein. The biological activity of the protein could be restored by refolding the protein with guanidine hydrochloride and glutathione giving a specific activity identical to that of IL-4 derived from CHO cell lines stably transformed with an hIL-4 expression plasmid. Strains designed to secrete human IL-4 into the periplasmic space produced far less protein (approximately 5 mg/l). However, a significant fraction of this protein was detected in the culture medium. This fraction appeared to be soluble after ultracentrifugation, and demonstrated high specific activity without refolding. Leakage of heterologous protein into the culture medium may be a viable way to recover biologically active products without relying on the denaturation and refolding in vitro that can, at times, yield incorrectly folded gene product.     

Use of phosphomannose isomerase-based selection system for <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of tomato and potato

Two selection systems for Agrobacterium tumefaciens mediated transformation of tomato and potato were compared. In the tomato (Lycopersicon esculentum cv. Moneymaker), the highest transformation rate, 4.2 %, of cotyledon explants on mannose-selection medium was obtained when mannose/sucrose concentration in the regeneration medium was 5/15 g dm⁻³. The best transformation efficacy with the commonly used concentration of 100 mg dm⁻³ kanamycin as a selection agent was 9 %. In the potato (Solanum tuberosum cv. Bintje), the highest transformation frequency was 53.3 % when mannose concentration in the regeneration medium was 5 g dm⁻³ during the first 3 weeks after transformation and 10 g dm⁻³ afterwards. The optimum concentration of sucrose was 20 g dm⁻³. The transformation efficiency using kanamycin as a selection agent at a concentration 100 mg dm⁻³ was 33.3 % with potato. Our results demonstrate that the transformation efficiency using mannose selection is 1.6-fold higher for potato and about 2 times lower for tomato comparing with the ordinary protocol using kanamycin.     

Expression of hybrid <Emphasis Type="Italic">cry</Emphasis>3aM–<Emphasis Type="Italic">lic</Emphasis>BM2 genes in transgenic potatoes (<Emphasis Type="Italic">Solanum tuberosum</Emphasis>)

The full-modified Bacillus thuringiensis cry3a (cry3aM) gene was designed and synthesized for effective expression in plants. A plant expression vector pC29RBCS-leader-cry3aM–licBM2 was constructed for potato transformation. In this vector, the cry3aM sequence was fused in reading frame with a new reporter gene (licBM2) and a leader sequence for the rbcs gene. The reporter gene encoded thermostable lichenase and the leader sequence encoded a signal peptide for transporting protein product to chloroplasts. The vector contained the light-inducible promoter for rbcs gene isolated from Arabidopsis thaliana. Transgenic plants were obtained by Agrobacterium mediated transformation using microtuber explants. Transgenic plantlets were selected by kanamycin resistance and confirmed as transgenic by PCR with specific primers, evaluation of lichenase activity, and bioassay of Colorado potato beetle neonate larvae. Promoter activity assays under light induction (kinetic analysis) using lichenase activity and bioassay both showed high and stable expression of hybrid genes in transgenic plantlets. Furthermore, the presence of lichenase as a reporter protein in the composition of hybrid protein was shown to facilitate selection and analysis of the expression level of hybrid genes in transgenic plants.     

Transformation of homozygous diploid potato with an Agrobacterium tumefaciens binary vector system by adventitious shoot regeneration on leaf and stem segments

Transformed potato (Solanum tuberosum) plants were obtained from homozygous diploid potato by using a transformation procedure in combination with an adventitious shoot regeneration method. Leaf and stem explants were inoculated with an Agrobacterium tumefaciens strain which contained a binary vector (pVU 1011) carrying the neomycin phosphotransferase gene. Shoot regeneration most effectively on stem explants, occurred within six weeks directly from the explants without introducing a callus phase. A strong seasonal influence on transformation efficiencies was observed. Analysis of a number of randomly selected regenerated shoots for their ability to root and form shoots on kanamycin-containing medium shows that over 90% of the regenerated shoots obtained are transformed. In a number of shoots transformation was confirmed by a test for the presence and expression of the NPT-II gene.     

An oriP expression vector containing the HIV-1 Tat/TAR transactivation axis produces high levels of protein expression in mammalian cells

A mammalian gene expression vector based on cytomegalovirus (CMV)enhancer/promoter (CMVe/p) for the regulation of gene expression was further optimized by adding oriP elements derived from Epstein-Barr virus (EBV) and the Tat/TAR transactivation axisfrom human immunodeficiency virus type 1 (HIV-1). Using the Tat/TAR-oriP expression vector, a transient transfection system was optimized for an extended culture period to produce large amounts of secreted IL-2SA (an IL-2 mutein) in HKB11 cells. We observed a 4-fold increase in IL-2SA expression in cells transfected with vectors containing the HIV-1 transactivation axis (Tat/TAR) or oriP elements alone when compared to cells transfected with the control vector having a CMVe/p. Cells transfected with expression vectors equipped with both oriP and Tat/TAR showed an 18-fold increase in IL-2SA expression. This transient transfection system maintained high secretion of IL-2SA for a period of 10-day with no appreciable loss in expression. We demonstrate that during this 10-day culture period, it was possible to produce 1–100 mg of proteins using 500 μg of plasmid DNA. This revised version was published online in August 2006 with corrections to the Cover Date.     

枯草芽孢杆菌刺激小鼠肠上皮细胞分泌的IL-33在活化树突状细胞中发挥重要作用

【目的】枯草芽孢杆菌能有效诱导肠道黏膜免疫应答,但活化黏膜下树突状细胞(DC)的具体机制不完全清楚。【方法】本研究首先用不同浓度枯草芽孢杆菌刺激小鼠肠上皮CMT93细胞,用荧光定量PCR和ELISA检测细胞因子表达水平,然后将枯草芽孢杆菌刺激细胞的培养上清与小鼠骨髓源树突状细胞(BMDC)进行共孵育,用流式细胞术检测BMDC活化标志,最后用RNA干扰技术证明IL-33在活化BMDC中的作用。【结果】枯草芽孢杆菌能显著刺激CMT93细胞分泌IL-6、IL-33和IFN-γ等细胞因子,对刺激IL-33表达呈现剂量依赖性;枯草芽孢杆菌刺激CMT93细胞产生的细胞因子能活化BMDC,在RNA干扰IL-33基因表达和枯草芽孢杆菌刺激后,CMT93细胞培养上清活化BMDC的能力显著降低。【结论】本研究结果表明枯草芽孢杆菌刺激肠上皮细胞产生的IL-33在BMDC活化中具有重要作用。     

Transformation of sweet potato tissues with green-fluorescent protein gene

Summary The expression of the green-fluorescent protein (GFP) gene from Aequorea victoria (jellyfish) was analyzed by transient and stable expression in sweet potato Ipomoea batatas L. (Lam.) ev. Beauregard tissues by electroporation and particle bombardment. Leaf and petiole segments from in vitro-raised young plantlets were used for protoplast isolation and electroporation. Embyrogenic callus was also produced from leaf segments for particle bombardment experiments. A buffer solution containing 1×10⁶ protoplasts ml⁻¹ was mixed with plasmid DNA containing the GFP gene, and electroporated at 375 V cm⁻¹. Approximately 25–30% of electroporated mesophyll cell protoplasts subsequently cultured in KM8P medium regenerated cell walls after 48 h. Of these, 3% emitted bright green fluorescence when exposed to UV-blue light at 395 nm. Transformed cells continued to grow after embedding in KM8P medium solidifed with 1.2% SeaPlaque agarose. Stable expression of GFP was observed after 4 wk of culture in approximately 1.0% of the initial GFP positive cells (27.5 GFP positive micro callases out of 3024 cells which transiently expressed GFP 48 h after electroporation). In a separate experiment, 600–700 bright green spots were observed per plate 48 h after bombarding leaf segments or embryogenic cellus. In bombarded cultures, several stable GEP-expressing sectors were observed in leafderived embryogenic callus grown without selection for 4 wk. These results show that GFP gene expression can occur in various sweet potato tissues, and that it may be a useful sereenable marker to improve transformation efficiency and obtain transgenic sweet potato plants.     

PGE2 induces interleukin-8 derepression in human astrocytoma through coordinated DNA demethylation and histone hyperacetylation

We have recently reported that in astrocytoma cells the expression of interleukin-8 (IL-8) is upregulated by prostaglandin E2 (PGE2). Unfortunately, the exact mechanism by which this happens has not been clarified yet. Here, we have investigated whether IL-8 activation by PGE2 involves changes in DNA methylation and/or histone modifications in 46 astrocytoma specimens, two astrocytoma cell lines and normal astrocytic cells. The DNA methylation status of the IL-8 promoter was analyzed by bisulphite sequencing and by methylation DNA immunoprecipitation analysis. The involvement of DNA methyltransferases (DNMTs) and histone deacetylases (HDACs), as well as histone acetylation levels, was assayed by chromatin immunoprecipitation. IL-8 expression at promoter, mRNA and protein level was explored by transfection, real-time PCR and enzyme immunoassay experiments in cells untreated or treated with PGE2, 5-aza-2'-deoxycytidine (5-aza-dC) and HDAC inhibitors, alone or in combination. EMSA was performed with crude cell extracts or recombinant protein. We observed that PGE2 induced IL-8 activation through: (1) demethylation of the single CpG site 5 located at position -83 within the binding region for CEBP-β in the IL-8 promoter; (2) C/EBP-β and p300 co-activator recruitment; (3) H3 acetylation enhancement and (4) reduction in DNMT1, DNMT3a, HDAC2 and HDAC3 association to CpG site 5 region. Treatment with 5-aza-dC or HDAC inhibitors of class I HDACs strengthened either basal or PGE2-mediated IL-8 expression. These findings have elucidated an orchestrated mechanism triggered by PGE2 whereby concurrent association of site-specific demethylation and histone H3 hyperacetylation resulted in derepression of IL-8 gene expression in human astrocytoma.     

Inhibition of T-Cell Proliferation Induced by a Cell-Free Salmonella typhimurium Extract Does Not Involve a Nitric Oxide-Mediated Mechanism

In a previous study, we observed that a cell-free Salmonella typhimurium extract induced suppression of mitogen-induced T-cell proliferation and that this suppression involved non-responsiveness of T-cells to interleukin-2 (IL-2) and augmentation of IL-2 receptor (IL-2R) expression. In this study, we found that inhibition of phytohemagglutinin (PHA)-stimulated murine spleen cell proliferation induced by a cell-free S. typhimurium extract was reversed by treatment with an anti-interferon-γ monoclonal antibody (anti-IFN-γ Ab), but not by interleukin-4 or N^G-monomethyl-l -arginine, which is known to inhibit nitric oxide (NO)-secretion from spleen cells in culture. However, IL-2R expression was augmented by treatment with the extract, although this was independent of an NO-mediated mechanism. Only anti-IFN-γ Ab treatment reduced the augmented IL-2R expression to a normal level. These results suggest that the suppression of T-cell proliferation induced by the Salmonella cell-free extract is associated with augmentation of IL-2R expression in an NO production-independent manner.     

Synthesis of a ricin toxin B subunit-rotavirus VP7 fusion protein in potato

A gene encoding the outer capsid glycoprotein (VP7) of simian rotavirus SA11, was genetically linked to the amino terminus of the ricin toxin B subunit (RTB) isolated from castor-oil plant (Ricinus communis) seeds. To assess fusion protein expression in plant cells, the VP7::RTB fussion gene was transferred into potato (Solanum tuberosum) cells by Agrobacterium tumefaciens-mediated transformation methods and transformed plants regenerated. The fusion gene was detected in transformed potato genomic DNA by polymerase chain reaction DNA amplification methods. Immunoblot analysis with anti-SA11 antiserum as the primary antibody verified the presence of VP7::RTB fusion protein in transformed potato tuber tissues. The plant-synthesized fusion protein bound RTB membrane receptors as measured by asialofetuin-enzyme-linked immunosorbent assay (ELISA). The ELISA results indicated that the VP7::RTB fusion protein was biologically active and made up approx 0.03% of total soluble transformed tuber protein. The biosynthesis of receptor binding VP7::RTB fusion protein in potato tissues demonstrates the feasibility of producing monomeric ricin toxin B subunit adjuvant-virus antigen fusion proteins in crop plants for enhanced immunity.     

IL-2 limits IL-12 enhanced lymphocyte proliferation during Leishmania amazonensis infection

C3H mice infected with Leishmania amazonensis develop persistent, localized lesions with high parasite loads. During infection, memory/effector CD44^hiCD4⁺ T cells proliferate and produce IL-2, but do not polarize to a known effector phenotype. Previous studies have demonstrated IL-12 is insufficient to skew these antigen-responsive T cells to a functional Th1 response. To determine the mechanism of this IL-12 unresponsiveness, we used an in vitro assay of repeated antigen activation. Memory/effector CD44^hiCD4⁺ T cells did not increase proliferation in response to either IL-2 or IL-12, although these cytokines upregulated CD25 expression. Neutralization of IL-2 enhanced CD4⁺ T cell proliferation in response to IL-12. This cross-regulation of IL-12 responsiveness by IL-2 was confirmed in vivo by treatment with anti-IL-2 antibodies and IL-12 during antigen challenge of previously infected mice. These results suggest that during chronic infection with L. amazonensis, IL-2 plays a dominant, immunosuppressive role independent of identifiable conventional T_reg cells.     

Eosinophils in the cerebrospinal fluid of mice infected with Angiostrongylus cantonensis are resistant to apoptosis

Interleukin-5 (IL-5) transgenic mice were used to assess the immunological features of CSF eosinophils from mice infected with Angiostrongylus cantonensis. CSF eosinophils were hypodense by day 14 post infection (p.i.). CSF eosinophils survived longer in vitro than peritoneal eosinophils collected from cadmium sulphate (CdSO₄) -treated normal IL-5 transgenic mice. Apoptosis was measured by Annexin V binding and the presence of a distinct laddering pattern of DNA fragmentation on agarose electrophoresis. Regardless of the presence or absence of Actinomycin D, CSF eosinophils collected from IL-5 transgenic mice from days 15–36 p.i. exhibited less apoptosis than peritoneal eosinophils collected from uninfected IL-5 transgenic mice. CSF eosinophils collected from A. cantonensis infected C57BL/6 mice at days 15–34 p.i. showed elongation of survival time and less apoptosis during in vitro cultivation. Reduced apoptosis was noted only in CSF eosinophils, but not in peritoneal eosinophils recovered from the same infected IL-5 transgenic mice. CPP32/Caspase 3 activity of cultured peritoneal eosinophils from both infected and uninfected IL-5 transgenic mice was higher than that of cultured CSF eosinophils. Stimulation with A23187 readily induced apoptosis of peritoneal eosinophils, but not CSF eosinophils or peritoneal eosinophils cultured with mouse recombinant IL-5. The latter cells were morphologically identical to hypodense eosinophils. RT-PCR analysis indicated that bcl-2 and bcl-x_L mRNA expression was higher in CSF eosinophils compared with peritoneal eosinophils and this expression in the latter cells was upregulated after culture with mouse recombinant IL-5. These results suggest that CSF eosinophils, shifting to hypodense status through an accumulation from peripheral blood, are resistant to apoptosis. These changes may explain the long-lasting, helminthotoxic and neurotoxic actions of CSF eosinophils in A. cantonensis infection.     

Copper decreases gene expression of TNF-<Emphasis Type="Italic">α</Emphasis>, IL-10, and of matrix metalloproteinases MMP-2 and MMP-9 in isolated perfused rat livers

Copper is known to induce oxidative stress in a number of models. It was shown that many pathophysiological events were associated with oxidative stress. Further, oxidative stress can increase gene expression of cytokines and of metalloproteinases. We previously found that copper toxic effects in isolated perfused rat livers were associated with significant oxidative stress (as assessed by lipid peroxidation, protein oxidation and oxidative DNA damage, particularly at concentration of 0.03 mM of Cu²⁺ in the perfusate). Here we investigated gene expression of tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10); matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) in frozen liver tissue samples by the real-time PCR assay. Compared to controls, copper at concentration of 0.01 mM did not affect gene expression of TNF-α, IL-10, MMP-2 and MMP-9, whereas copper at concentration of 0.03 mM significantly decreased gene expression of all the four TNF-α, IL-10, MMP-2 and MMP-9 by 69%, 81%, 43%, and 62%, respectively. These results suggest that copper-induced oxidative stress in the isolated rat liver can lead to the suppression of gene expression. Because TNF-α and metalloproteinases are involved also in liver regeneration, the suppression of these genes by copper may be one of the mechanisms by which acute intoxication of animals and humans with copper may impair regenerative capability of the liver.     

Comparison between crude Interleukin-2 and recombinant Interleukin-2 in maintaining killing activity of cultured lymphocytes

Peripheral blood lymphocytes, regional lymph node lymphocytes or malignant effusion lymphocytes from cancer patients were incubated with crude IL-2 (cIL-2) for 13 days. These effectors, which frequently expressed IL-2 receptor (IL-2R), proliferated well and possessed augmented killing activity against fresh autologous tumor cells and K562. However, when recombinant IL-2 (rIL-2) was added for the last 4 days of culture instead of cIL-2, IL-2R expression and killing activity against fresh autologous tumor cells decreased significantly (P<0.05). Phenotypic analysis indicated that cIL-2 significantly promoted the expansion of the cytotoxic population (CD8⁺ .11b^–)(P<0.05). The decreases in killing activity and IL-2R expression were restored by 0.004% PHA plus rIL-2, but not in the presence of rIFN-, rIL-1, rIL-l, rIL-4 or rIL-6. PHA-free cIL-2 maintained killing activity, but not IL-2R expression.We conclude that some factors in cIL-2 and a low dose of PHA-P are necessary for the maintenance of killing activity and IL-2R expression of cultured lymphocytes in the late phase of culture.     

Interleukin-18 gene polymorphism,but not interleukin-2 gene polymorphism,is associated with rheumatoid arthritis

To investigate whether polymorphisms of IL-2 and IL-18 genes are associated with rheumatoid arthritis (RA), polymorphisms of IL-2 and IL-18 genes were detected by polymerase-chain-reaction-based restriction analysis in the patients with RA and normal controls. The results for the IL-18 gene revealed a significant difference between the patients and the normal controls (p = 0.000003), but there was no significant difference for the IL-2 gene (p = 0.876). The IL-18 gene 105A allele was associated with RA in Chinese patients. Individuals possessing the 105A allele had a higher incidence of RA. A lack of association of IL-2 gene polymorphism between RA patients and healthy individuals was noted. The results of this study provide genetic evidence that IL-18-105A/C polymorphism may play an effective role in RA.