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1.
Yanlu Zhang Liehua Wu Jingai Yu JianFeng Mei Yu Yi JianShu Chen 《Preparative biochemistry & biotechnology》2017,47(9):934-938
Human thymic stromal lymphopoietin (hTSLP) protein plays a central role in inflammation. Characterizing properties of hTSLP requires a recombinant overexpression system that produces correctly folded, active hTSLP. In this report, an efficient overexpression system for the production of hTSLP was developed. We constructed expression plasmids of the full-length hTslp gene with or without the signal peptide and transformed the plasmids into Escherichia coli. The design of the recombinant proteins included an N-terminal His-tag, which facilitated purification. An affinity gradient elution method was used to improve recovery and concentration levels of denatured hTSLP, with 90% purity observed following affinity chromatography. Refolding of the denatured hTSLP was tested using four different protein refolding approaches. The optimal refolding conditions involved stepwise buffer exchanges to reduce the urea concentration from 4 to 0?M in 50?mM Tris (pH 8.0), 1?mM EDTA, 50?mM NaCl, 10% glycerol, 400?mM L-Arg, 0.2?mM oxidized glutathione, and 2?mM reduced glutathione. The activity of the refolded recombinant hTSLP protein was measured by an ELISA assay. Interestingly, the presence of N-terminal signal peptide inhibited the overexpression of hTSLP in E. coli. The amount of recombinant hTSLP protein purified reached a level of 2.52?×?10?3?mg/L. 相似文献
2.
Elizabeth A. L. Martins Lyria Mori H. C. Birnboim Rogerio Meneghini 《Molecular and cellular biochemistry》1992,117(2):181-189
We have investigated the antioxidant properties of V79 Chinese hamster cells rendered resistant to menadione by chronic exposure to increasing concentrations of this quinone. MD1, a clone of resistant cells, was compared to the parental M8 cells; the former showed increased activity of catalase (3 fold), glutathione peroxidase (1.6 fold) and DT-diaphorase (2.6 fold), as well as an increase in glutathione (3.2 fold). Although one of the products of menadione metabolism is superoxide anion, no changes in total superoxide dismutase activity was observed in MD1 cells. MD1 menadione resistant cells were also resistant to killing by hydrogen peroxide and contained tandem duplication of chromosome 6. A similar duplication of chromosome 6 was seen in several independently derived menadione resistant clones and therefore seems closed linked to the establishment of the resistance. Upon removal of menadione from the medium, some of these properties of MD1 cells, viz., resistance to menadione, elevated glutathione levels, and glutathione peroxidase activity, were lost and the cells resembled M8 cells. However, resistance to H2O2, elevated catalase activity and the duplicated chromosome remained stable for more than 40 cell passages in the absence of menadione. The increase in catalase activity was correlated with an increase in catalase mRNA content and a 50% amplification of catalase gene, as determined, respectively, by Northern and Southern blot analysis. The role of the chromosome 6 duplication in resistance to oxidative stress remains to be established. It is not responsible directly for elevated catalase levels since the catalase gene is on chromosome 3.Abbreviations SDS
Sodium Dodecyl Sulphate
- SOD
Superoxide Dismutase
- PBS
Phosphate Buffered Saline (8.1 mM Na2HPO4, 1.47 mM KH2PO4, 2.68 mM KCl, 137 mM NaCl)
- CDTA
N,N,N,N-tetracetic-trans-1,2-diaminocyclohexane acid
- MOPS
Sulphonic-3-(N-morpholine)-propane acid
- SSC
150 mM Nacl, 15 mM sodium-citrate, pH 6.8 相似文献
3.
Sisi Patricia Lolita Ameria Hye Sook Jung Hee Sook Kim Sang Soo Han Hak Sung Kim Jin Ho Lee 《Biotechnology letters》2015,37(8):1637-1644
Objective
To examine the role of a gene encoding flavin-containing monooxygenase (cFMO) from Corynebacterium glutamicum ATCC13032 when cloned and expressed in Escherichia coli for the production of indigo pigments.Results
The blue pigments produced by recombinant E. coli were identified as indigo and indirubin. The cFMO was purified as a fused form with maltose-binding protein (MBP). The enzyme was optimal at 25 °C and pH 8. From absorption spectrum analysis, the cFMO was classified as a flavoprotein. FMO activity was strongly inhibited by 1 mM Cu2+ and recovered by adding 1–10 mM EDTA. The enzyme catalyzed the oxidation of TMA, thiourea, and cysteamine, but not glutathione or cysteine. MBP-cFMO had an indole oxygenase activity through oxygenation of indole to indoxyl. The recombinant E. coli produced 685 mg indigo l?1 and 103 mg indirubin l?1 from 2.5 g l-tryptophan l?1.Conclusion
The results suggest the cFMO can be used for the microbial production of both indigo and indirubin.4.
Witcher Derrick R. Hood Elizabeth E. Peterson Dave Bailey Michele Bond Diane Kusnadi Ann Evangelista Roque Nikolov Zivko Wooge Cynthia Mehigh Richard Kappel William Register James Howard John A. 《Molecular breeding : new strategies in plant improvement》1998,4(4):301-312
We have generated transgenic maize seed containing -glucuronidase(GUS) for commercial production. While many other investigators have demonstrated the expression of GUS as a scoreable marker, this is one of the first cases where a detailed characterization of the transgenic plants and the protein were performed which are necessary to use this as a commercial source of GUS. The recombinant -glucuronidase was expressed at levels up to 0.7% of water-soluble protein from populations of dry seed, representing one of the highest levels of heterologous proteins reported for maize. Southern blot analysis revealed that one copy of the gene was present in the transformant with the highest level of expression. In seeds, the majority of recombinant protein was present in the embryo, and subcellular localization indicated that the protein was dispersed throughout the cytoplasm. The purified recombinant -glucuronidase (GUS) was compared to native -glucuronidase using SDS-PAGE and western blot analysis. The molecular mass of both the recombinant and native enzymes was 68 000 Da. N-terminal amino acid sequence of the recombinant protein was similar to the sequence predicted from the cloned Escherichia coli gene except that the initial methionine was cleaved from the recombinant GUS. The recombinant and native GUS proteins had isoelectric points (pI) from 4.8 to 5.0. The purified proteins were stable for 30 min at 25, 37, and 50 ° C. Kinetic analysis of the recombinant and native GUS enzymes using 4-methylumbelliferyl glucuronide (MUG) as the substrate was performed. Scatchard analysis of these data demonstrated that the recombinant enzyme had a Km of 0.20 mM and a Vmax of 0.29 mM MUG per hour, and the native enzyme had a Km and Vmax of 0.21 mM and 0.22 mM/h respectively. Using D-saccharic acid 1,4-lactone, which is an inhibitor of -glucuronidase, the Ki of the native and recombinant enzymes was determined to be 0.13 mM. Thus, these data demonstrate that recombinant GUS is functionally equivalent to native GUS. We have demonstrated the expression of high levels of GUS can be maintained in stable germlines and have used an efficient recovery system where the final protein product, GUS, has been successfully purified. We describe one of the first model systems for the commercial production of a foreign protein which relies on plants as the bioreactor. 相似文献
5.
Cytosolic prostaglandin (PG) E synthase was purified from human brain cortex. The N-terminal amino acid sequence, PMTLGYXNIRGL, was identical to that of the human mu-class glutathione transferase (GST) M2 subunit. Complementary DNAs for human GSTM2, GSTM3, and GSTM4 subunits were cloned, and recombinant proteins were expressed as homodimers in Escherichia coli. The recombinant GSTM2-2 and 3-3 catalyzed the conversion of PGH2 to PGE2 at the rates of 282 and 923 nmol/min/mg of protein, respectively, at the optimal pH of 8, whereas GSTM4-4 was inactive; although all three enzymes showed GST activity. The PGE synthase activity depended on thiols, such as glutathione, dithiothreitol, 2-mercaptoethanol, or L-cysteine. Michaelis-Menten constants and turnover numbers for PGH2 were 141 M and 10.8 min–1 for GSTM2-2 and 1.5 mM and 130 min–1 for GSTM3-3, respectively. GSTM2-2 and 3-3 may play crucial roles in temperature regulation, nociception, and sleep-wake regulation by producing PGE2 in the brain. 相似文献
6.
Kyung Hwa Chang Jong Hwa Park Youn Hyung Lee Jong Ho Kim Hyung Ok Chun Je Hak Kim In Sik Chung 《Biotechnology letters》2002,24(16):1353-1359
Recombinant human cyclooxygenase 2 (Cox 2) was expressed in stably transformed Drosophila melanogaster S2 cells, and was present primarily in the cellular fraction at a molecular weight of 70 to 74 kDa. Recombinant Cox 2 was purified using Ni2+-affinity fractionation to a specific activity of 24 800 U mg–1 protein. The peak level of recombinant Cox 2 production was 1.6 g (107 cells)–1, seven days after induction with 0.5 mM CuSO4. Supplementing the cultures with dimethylsulfoxide or sodium butyrate increased recombinant Cox 2 production by 170% and 86%, respectively. 相似文献
7.
Functional expression of recombinant tumstatin in stably transformed Drosophila melanogaster S2 cells 总被引:1,自引:0,他引:1
Recombinant tumstatin was expressed in stably transformed Drosophila melanogaster S2 cells and secreted into the medium with a molecular size of 29 kDa. Recombinant endostatin was also purified to homogeneity using a simple one-step Ni2+ affinity fractionation. Purified recombinant tumstatin inhibited endothelial cell proliferation in a dose-dependent manner. The concentration at half-maximum inhibition for recombinant tumstatin was approx. 0.7 g ml–1. A maximum production of 4.6 g recombinant tumstatin (107 cells)–1 was obtained in a T-flask culture of S2 cells, 7 d after induction with 0.5 mM CuSO4. 相似文献
8.
Yongqing Yan Chenhui Pan Yuling Du Danyang Li Wei Liu 《Physiology and Molecular Biology of Plants》2018,24(4):577-589
The effect of 0.5–1.5 mM salicylic acid (SA) on modulating reactive oxygen species metabolism and ascorbate–glutathione cycle in NaCl-stressed Nitraria tangutorum seedlings was investigated. The individual plant fresh weight (PFW) and plant dry weight (PDW) significantly increased under 100 mM NaCl while remained unchanged or decreased under 200–400 mM NaCl compared to the control. Superoxide anion (O 2 ·? ), hydrogen peroxide (H2O2), thiobarbituric acid reactive substances (TBARS), reduced ascorbate (AsA), dehydroascorbate (DHA), reduced glutathione (GSH) and oxidized glutathione (GSSG) increased whereas the ratios of AsA/DHA and GSH/GSSG decreased under varied NaCl treatments. Ascorbate peroxidase (APX) and glutathione reductase (GR) activities were enhanced while dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDHAR) activities remained unvaried under 100–400 mM NaCl stresses. In addition, exogenous SA further increased PFW, PDW and root/shoot ratio. SA effectively diminished O 2 ·? accumulation. H2O2 and TBARS decreased under 0.5 and 1.0 mM SA treatments compared to those without SA. 0.5 mM of SA increased while 1.0 and 1.5 mM SA decreased APX activities. DHAR activities were elevated by 0.5 and 1.0 mM SA but not by 1.5 mM SA. MDHAR and GR activities kept constant or significantly increased at varying SA concentrations. Under SA treatments, AsA and GSH contents further increased, DHA and GSSG levels remained unaltered, while the decreases in AsA/DHA and GSH/GSSG ratios were inhibited. The above results demonstrated that the enhanced tolerance of N. tangutorum seedlings conferred by SA could be attributed mainly to the elevated GR and DHAR activities as well as the increased AsA/DHA and GSH/GSSG ratios. 相似文献
9.
Hongchen Zheng Zhenxiao Yu Xiaoping Fu Shufang Li Jianyong Xu Hui Song Yanhe Ma 《Journal of industrial microbiology & biotechnology》2016,43(7):977-987
To improve the extracellular production of alkaline β-mannanase from alkaliphilic Bacillus sp. N16-5 in Escherichia coli, two truncated recombinant mannanases (32a-ManAR2 and 22b-ManAR2) were obtained. Compared with the full-length mannanases (32a-ManAR1 and 22b-ManAR1), the truncated mannanases not only showed higher secretion rate, but also exhibited higher thermostability and alkalistability. The K m value (11 mg/mL) of 32a-ManAR2 was higher than that (1.46 mg/mL) of 32a-ManAR1. The specific activity of 22b-ManAR2 was 2.7 times higher than that of 22b-ManAR1. 22b-ManAR2 showed the highest k cat/K m value of 602.7 ml/mg s. The parameters of induction for recombinant mannanase production of E. coli BL21 (pET32a-manAR2) and E. coli BL21 (pET22b-manAR2) were subsequently optimized. The yield of soluble mannanase was found to be enhanced with lower induction temperature (25 °C), lower IPTG concentration (0.01–0.05 mM), and Triton X-100 supplement (0.1 %) in a shake flask. Moreover, a one-time feeding strategy and Triton X-100 supplement were applied in production of 22b-ManAR2 in a 10 L fermentor. The productivity of the total soluble mannanase reached 9284.64 U/mL with the extracellular rate of 74 % at 46 h of fermentation, which was the highest productive level of alkaline β-mannanase in recombinant E. coli to date. 相似文献
10.
Koshiki Mino Tsuyoshi Yamanoue Katsuhiro Ohno Takaharu Sakiyama Naoki Eisaki Asahi Matsuyama Kazuhiro Nakanishi 《Biotechnology letters》2001,23(24):2051-2055
-(Pyrazol-1-yl)-l-alanine (-PA) was produced from l-serine and pyrazol using recombinant Escherichia coli cells expressing serine acetyltransferase and O-acetylserine sulfhydrylase-A. The amount of -PA increased with increasing l-serine concentrations up to 600 mM at 50 mM pyrazol while 100 mM pyrazol gave the highest -PA production with 50 mM l-serine. Under the optimized conditions, -PA accumulated in the broth at approximately 140 mM with a conversion of 90% with respect to the added amount of pyrazol. 相似文献
11.
Summary In previous studies, oxidants such as hydrogen peroxide (H2O2) or hydroperoxy fatty acids were shown to induce apoptosis in the CEM human T cell line as demonstrated by the cleavage of cellular DNA into a 180-base pair ladder. Oxidant-induced DNA fragmentation was detectable within 3 h and inhibitable by various antioxidants. In the present study, apoptosis is shown to also be induced by the addition of low doses (0.1–3 mM) of N-acetyl-L-cysteine (NAC), reduced glutathione (GSH) or cysteine. By contrast, higher concentrations (10 mM) of the same thiols displayed a paradoxical lack of toxicity. Thiol-induced apoptosis was completely prevented by the addition of BAPTA-AM, an intracellular calcium chelator, or by simultaneous treatment with 5 mM pyruvate which forms a thiazolidine complex with sulfhydryl compounds. Catalase or glutathione peroxidase, but not Superoxide dismutase, protected the cells from thiol-induced apoptosis demonstrating a role for H2O2. The ability of thiol compounds to either evoke or prevent oxidative stress implies a unique role for these agents in the control of apoptosis in lymphoid cells. 相似文献
12.
Germán I?iguez Juan José Castro Mirna Garcia Elena Kakarieka M. Cecilia Johnson Fernando Cassorla Verónica Mericq 《PloS one》2014,9(7)
Introduction
The human placenta expresses the IGF-I and IGF-IR proteins and their intracellular signal components (IRS-1, AKT and mTOR). The aim of this study was to assess the IGF-IR content and activation of downstream signaling molecules in placentas from newborns who were classified by gestational age and birth weight. We studied placentas from 25 term appropriate (T-AGA), 26 term small (T-SGA), 22 preterm AGA (PT-AGA), and 20 preterm SGA (PT-SGA) newborns. The total and phosphorylated IGF-IR, IRS-1, AKT, and mTOR contents were determined by Western Blot and normalized by actin or with their respective total content. The effect of IGF-I was determined by stimulating placental explants with recombinant IGF-I 10-8 mol/L for 15, 30, and 60 minutes.Results
The IGF-IR content was higher in T-SGA compared to T-AGA placentas, and the IRS-1 content was higher in PT-placentas compared with their respective T-placentas. The effect of IGF-I on the phosphorylated forms of IGF-IR was increased in T-SGA (150%) and PT-SGA (300%) compared with their respective AGA placentas. In addition, AKT serine phosphorylation was higher in PT-SGA compared to PT-AGA and T-SGA placentas (90% and 390% respectively).Conclusion
The higher protein content and response to IGF-I of IGF-IR, IRS-1, and AKT observed in SGA placentas may represent a compensatory mechanism in response to fetal growth restriction. 相似文献13.
Ethylene production in leaf petiole and laminae tissues was stimulated in tomato (Lycopersicon esculentum Mill. cv. UCT5) plants exposed to salinity-stress. At the highest salinity level (250 mM NaCl), rates of ethylene production more than doubled over those observed in non-stressed plants. Correspondingly, petiolar epinasty increased with increasing levels of stress impositions. Both responses were suppressed when either 1 mM -aminooxyacetic acid (AOA), or 100 M Co2+ was simultaneously applied. Co2+, but not AOA, had a pronounced effect on ethylene production resulting from the application of a saturating dose (2 mM) of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene. This result suggests that ethylene production is dependent upon the activity of ethylene forming enzyme (EFE). The magnitude of ethylene stimulation in leaf petioles was related to the salinity level imposed and to the induction of petiole epinasty. In the absence of stress impositions, epinastic responsiveness to ethylene or its precursor, ACC, might provide a simple, indirect criteria to adjudge salt-sensitivity among plants.Research supported by AID contract II, NEB-1070-A-00-2074-00. 相似文献
14.
Glutathione reductase activity was detected and characterized in heterocysts and vegetative cells of the cyanobacterium Nostoc muscorum. The activity of the enzyme varied between 50 and 150 nmol reduced glutathione· min-1·mg protein-1, and the apparent Km for NADPH was 0.125 and 0.200 mM for heterocysts and vegetative cells, respectively. The enzyme was found to be sensitive to Zn+2 ions, however, preincubation with oxidized glutathione rendered its resistance to Zn+2 inhibition. Nostoc muscorum filaments were found to contain 0.6–0.7mM glutathione, and it is suggested that glutathione reductase can regenerate reduced glutathione in both cell types. The combined activity of glutathione reductase and isocitrate dehydrogenase in heterocysts was as high as 18 nmol reduced glutathione·min-1·mg protein-1. A relatively high superoxide dismutase activity was found in the two cell types; 34.2 and 64.3 enzyme units·min-1·mg protein-1 in heterocysts and vegetative cells, respectively.We suggest that glutathione reductase plays a role in the protection mechanism which removes oxygen radicals in the N2-fixing cyanobacterium Nostoc muscorum.Abbreviations DTNB
5-5-dithiobis-(2-nitrobenzoic acid)
- EDTA
ethylenediaminetetra-acetic acid
- GR
glutathione reductase (EC1.6.4.2)
- GSH
reduced glutathione
- GSSG
oxidized glutathione
- OPT
O-phtaldialdehyde
- SOD
superoxide dismutase (EC 1.15.1.1) 相似文献
15.
Insulin-like growth factor I enhances the formation of type I collagen in hydrocortisone-treated human osteoblasts 总被引:2,自引:0,他引:2
Kenneth B. Jonsson Sverker Ljunghall Olle Karlström Anna G. Johansson Hans Mallmin Östen Ljunggren 《Bioscience reports》1993,13(5):297-302
We have studied the effect of insulin-like growth factor I (IGF-I) on the formation of osteocalcin and type I collagen in isolated human osteoblasts. IGF-I at and above 0.1 nM stimulated the formation of type I collagen as measured by the type I procollagen carboxyterminal peptide (PICP), in human osteoblasts, incubated for 72 hrs in serumfree conditions. The secretion of osteocalcin was not affected by IGF-I while 1,25(OH)2 vitamin D3 significantly enhanced the formation of osteocalcin. When human osteoblast-like cells were incubated with hydrocortisone (1 M), a significant decrease in the release of both PICP and osteocalcin was seen. Addition of IGF-I to human osteoblasts also treated with hydrocortisone normalized the PICP-formation but did not affect the suppressed osteocalcin-formation. These data indicate that IGF-I reverses selective effects of hydrocortisone on bone. 相似文献
16.
Hita Oscar Gallego Piedad Villalobos Nieves Lanas Itziar Blazquez Antonio Martin Juan Pedro Fernandez Javier Martin Luisa Guerra Hilario 《Plant Cell, Tissue and Organ Culture》2003,72(1):13-18
A number of medium constituents were evaluated in an attempt to improve somatic embryo production in Medicago arborea ssp. arborea, using cotyledons, petioles and leaves as explants. Two culture steps were applied: in the first stage (2 months), Murashige–Skoog (MS) medium was used, containing 2,4 dichlorophenoxyacetic acid (9M 2,4-D) and kinetin (9 M KIN) together with different nitrogen sources (alanine, glutamine, proline or tryptophan (2.5 and 5 mM); casein hydrolysate (100, 500 and 1000 mg l–1; nitrate (4.69 and 9.39 mM) or casein hydrolysate (100 mg l–1) and nitrate (4.69 mM)), polyalcohols (mannitol at 164 and 328 mM or sorbitol at 219 and 438 mM), sucrose (43.8 and 175.4 mM) or calcium (1.5 and 6 mM). In the second stage (3 months of cultivation), calli were transferred to a kinetin-free MS medium with 2,4-D (2.25 M) only. The inclusion of proline (2.5 mM) was the most effective treatment for the induction of somatic embryos, with the petiole being the best explant. Treatment with casein hydrolysate (100 mg l–1) also improved the embryonic efficiency. The rest of the treatments neither affect nor inhibit the embryonic response.A special treatment with sorbitol (219 mM) in the second stage of cultivation produced a slight increase in embryogenesis, but less than that obtained with proline. 相似文献
17.
Georgia Tanou Athanassios Molassiotis Grigorios Diamantidis 《Journal of plant physiology》2009,166(17):1904-1913
We tested whether pre-treatments of roots with H2O2 (10 mM for 8 h) or sodium nitroprusside (SNP; 100 μM for 48 h), a donor of NO, could induce prime antioxidant defense responses in the leaves of citrus plants grown in the absence or presence of 150 mM NaCl for 16 d. Both root pre-treatments increased leaf superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) activities, and induced related-isoform(s) expression under non-NaCl-stress conditions. When followed by salinity, certain enzymatic activities also exhibited an up-regulation in response to H2O2 or SNP pre-exposure. An NaCl-stress-provoked decrease in the ascorbate redox state was partially prevented by both pre-treatments, whereas the glutathione redox state under normal and NaCl-stress conditions was increased by SNP. Real-time imaging of NO production was found in vascular tissues and epidermal cells. Furthermore, NaCl-induced inhibition in OH scavenging activity and promotion of OH-mediated DNA strand cleavage was partially prevented by SNP. Moreover, NaCl-dependent protein oxidation (carbonylation) was totally reversed by both pre-treatments as revealed by quantitative assay and protein blotting analysis. These results provide strong evidence that H2O2 and NO elicit long-lasting systemic primer-like antioxidant activity in citrus plants under physiological and NaCl-stress conditions. 相似文献
18.
Silva AR Silva CG Ruschel C Helegda C Wyse AT Wannmacher CM Wajner M Dutra-Filho CS 《Neurochemical research》2001,26(12):1277-1283
In the present study we investigated the effects of L-pyroglutamic acid (PGA), which predominantly accumulates in the inherited metabolic diseases glutathione synthetase deficiency (GSD) and -glutamylcysteine synthetase deficiency (GCSD), on some in vitro parameters of energy metabolism and lipid biosynthesis. We evaluated the rates of CO2 production and lipid synthesis from [U-14C]acetate, as well as ATP levels and the activities of creatine kinase and of the respiratory chain complexes I-IV in cerebral cortex of young rats in the presence of PGA at final concentrations ranging from 0.5 to 3 mM. PGA significantly reduced brain CO2 production by 50% at the concentrations of 0.5 to 3 mM, lipid biosynthesis by 20% at concentrations of 0.5 to 3 mM and ATP levels by 52% at the concentration of 3 mM. Regarding the enzyme activities, PGA significantly decreased NADH:cytochrome c oxireductase (complex I plus CoQ plus complex III) by 40% at concentrations of 0.5–3.0 mM and cytochrome c oxidase activity by 22–30% at the concentration of 3.0 mM, without affecting the activities of succinate dehydrogenase, succinate:DCPIP oxireductase (complex II), succinate:cytochrome c oxireductase (complex II plus CoQ plus complex III) or creatine kinase. The results strongly indicate that PGA impairs brain energy production. If these effects also occur in humans, it is possible that they may contribute to the neuropathology of patients affected by these diseases. 相似文献
19.
Joseph A. M. J. L. Janssen Leo J. Hofland Christian J. Strasburger Elisabeth S. R. van den Dungen Mario Thevis 《PloS one》2016,11(3)
Aims
To compare full-length mechano growth factor (full-length MGF) with human recombinant insulin-like growth factor-I (IGF-I) and human recombinant insulin (HI) in their ability to activate the human IGF-I receptor (IGF-IR), the human insulin receptor (IR-A) and the human insulin receptor-B (IR-B), respectively. In addition, we tested the stimulatory activity of human MGF and its stabilized analog Goldspink-MGF on the IGF-IR.Methods
The effects of full-length MGF, IGF-I, human mechano growth factor (MGF), Goldspink-MGF and HI were compared using kinase specific receptor activation (KIRA) bioassays specific for IGF-I, IR-A or IR-B, respectively. These assays quantify activity by measuring auto-phosphorylation of the receptor upon ligand binding.Results
IGF-IR: At high equimolar concentrations maximal IGF-IR stimulating effects generated by full-length MGF were similar to that of IGF-I (89-fold vs. 77-fold, respectively). However, EC50 values of IGF-I and full-length MGF for the IGF-I receptor were 0.86 nmol/L (95% CI 0.69–1.07) and 7.83 nmol/L (95% CI: 4.87–12.58), respectively. No IGF-IR activation was observed by human MGF and Goldspink-MGF, respectively. IR-A/IR-B: At high equimolar concentrations similar maximal IR-A stimulating effects were observed for full -length MGF and HI, but maximal IR-B stimulation achieved by full -length MGF was stronger than that by HI (292-fold vs. 98-fold). EC50 values of HI and full-length MGF for the IR-A were 1.13 nmol/L (95% CI 0.69–1.84) and 73.11 nmol/L (42.87–124.69), respectively; for IR-B these values were 1.28 nmol/L (95% CI 0.64–2.57) and 35.10 nmol/L (95% 17.52–70.33), respectively.Conclusions
Full-length MGF directly stimulates the IGF-IR. Despite a higher EC50 concentration, at high equimolar concentrations full-length MGF showed a similar maximal potency to activate the IGF-IR as compared to IGF-I. Further research is needed to understand the actions of full-length MGF in vivo and to define the physiological relevance of our in vitro findings. 相似文献20.
Robert K. Bright Michael H. Shearer Ronald C. Kennedy 《Cancer immunology, immunotherapy : CII》1995,40(3):206-211
Baculovirus-derived recombinant simian virus 40 (SV40) large tumor antigen (T-Ag) was used to immunize BALB/c mice to examine the lymphokines produced following immunization. Specifically, we examined production of interleukin-2 (IL-2), IL-4, IL-5 and interferon (IFN) from immune lymphocytes cultured with decreasing concentrations of recombinant SV40 T-Ag. We identified elevated levels of IFN and IL-2 by enzyme-linked immunosorbent assay and a murine CTLL-2 proliferation biossay respectively. We were unable to detect either IL-4 or IL-5. These data indicate the previously reported tumor immunity induced by recombinant SV40 T-Ag immunization most likely reflects a TH1-like immune response based on thein vitro production of both IFN and IL-2 by immune lymphocytes. 相似文献