Soluble protein and acid phosphatase exuded by ectomycorrhizal fungi and seedlings in response to excessive Cu and Cd

Fungi and their symbionts can alleviate heavy metal stress by exuding soluble proteins and enzymes. This study examined the role of soluble protein and acid phosphatase （APase） exuded by Xerocomus chrysenteron, an ectomycorrhizal fungus, and the seedlings of its symbiont, Chinese pine （Pinus tabulaeformis）, under conditions of excessive Cu and Cd. The growth type showed that this poorly studied ectomycorrhizal fungus was capable of tolerating high concentrations of Cu, and may be useful in phytoremediation. X. chrysenteron grew well at 80 mg/L Cu, and the EC50 for Cd was 17.82 mg/L. X. chrysenteron also showed enhanced exudation of soluble protein in both isolated and inoculated cultivations under the influence of Cu and Cd. Soluble protein exudation, however, differed under Cu and Cd stress in isolates. In mediums containing Cu, soluble protein exudation increased with concentration, but in mediums containing Cd the content of soluble protein increased to a comparable level at all concentrations. This study demonstrated that soluble protein was related to heavy metal tolerance, although the different ions played different roles. While APase activity in exudates of fungi and seedlings decreased under Cu and Cd stress in comparison to the control, the APase activity in seedlings was maintained by inoculation. Thus, X. chrysenteron facilitated the ability of plant to maintain a normal nutrient uptake, and therefore to protect it from heavy metal toxicity.

Relationship Between Soluble Protein,Chlorophyll and ATP in Drought-Resistant Sweet Potato Under Water Stress

Some indices concerning the metabolism of substance and energy in sweet potato leaves under water stress were studied. The results showed an obvious increase in soluble protein content. Compared with control, Chl a, Chl b, total Chl contents and the ratio of Chl a to Chl b all decreased to some extent. ATP content increased in some varieties and decreased in others, but the stronger the drought resistance of the variety , the higher the ATP content. The correlation coefficient(r)of the soluble protein content, ratio of Chl a to Chl b and ATP content as a percentage of the drought-resistant sweet potato control variety are 0. 8968, - 0. 8509 and 0. 8200, respectively, P<0. 01. So these indices can be used to evaluate the drought resistance of different sweet potato varieties.

The Relationship Between Developmental Accumulation of Leaf Soluble Proteins and Vernalization Response of Wheat(Triticum aestivum L.em. Thell)

The relationship between vernalization requirement and quantitative and qualitative changes in total leaf soluble proteins were determined in one spring （cv. Kohdasht） and two winter （cvs. Sardari and Norstar） cultivars of wheat （Triticum aestivum L.） exposed to 4℃. Plants were sampled on days 2, 14, 21 and 35 of exposure to 4℃. The final leaf number （FLN） was determined throughout the vernalization periods （0, 7, 14, 24, and 35 d） at 4℃. The final leaf number decreased until days 24 and 35 in Sardari and Norstar eultivars, respectively, indicating the vernalization saturation at these times. No clear changes were detected in the final leaf number of Kohdash cultivar, verifying no vernalization requirement for this spring wheat cultivar. Comparing with control, clear cold-induced 2-fold increases in proteins quantity occurred after 48 h following the 4℃-treatment in the leaves of the both winter wheat cultivars but, such response was not detected in the spring cultivar. However, the electrophoretic protein patterns showed between-cultivar and between-temperature treatment differences. With increasing exposure time to 4℃, the winter cultivars tended to produce more HMW polypeptides than the spring cultivar. Similar proteins were induced in both Sardari and Norstar winter wheat cultivars, however, the long vernalization requirement in Norstar resulted in high level and longer duration of expression of cold-induced proteins compared to Sardari with a short vernalization requirement. These observations indicate that vernalization response regulates the expression of low temperature （LT） tolerance proteins and determines the duration of expression of LT- induced proteins.

Analysis of SSLP and Soluble Protein Contents in Leaves of Mutants Induced by High Pressure in Rice (Oryza sativa)

Rice variety Yuexiangzhan and its mutants induced by high pressure were studied using microsatcllite markers and soluble protein content analyses. Eleven of the 88 microsatellite primer pairs showed evident polymorphisms repeatedly, and the polymorphic frequencies were 3.4-11.3% between the mutants and Yuexiangzhan. The polymorphic markers were randomly located on chromosomes. The more similar the plant types of the mutants like their original variety, the less polymorphic loci were detected. In addition, there was variation in the soluble protein contents among the leaves of mutants, and the contents were significantly lower than those of the original variety.

Eukaryotic expression, purification and activity characterization of human soluble DSG2 extracellular domain protein

Objective:To construct a secretory eukaryotic expression vector of DSG2 fused with the Fc region of the human IgG,to validate its expression in 293T cells,and to purify the secretory protein with biological activity.Methods:The DSG2 extracellular domain fragment gene(DSG2ex),was amplified by PCR,and was inserted into the eukaryotic expression plasmid pCMV3-IgG1 to construct the recombinant eukaryotic expression plasmid-pCMV3-DSG2ex-IgG1.The successfully constructed eukaryotic expression plasmid was transfected into 293T cells to express and secrete DSG2 extracellular domain protein.The targeted protein was purified from the cell culture supernatant by Protein A affinity chromatography and confirmed by Western Blotting and ELISA.Results:The pCMV3-DSG2ex-IgG1 eukaryotic expression plasmid was successfully constructed.The highest protein expression level was obtained with 293T cells after 96 h of transfection.The relative molecular mass of the purified product was between 100 and 130 kDa was estimated by SDS-PAGE,which was consistent with the expectation.The yield of the purified protein reached 0.8 mg/ml with a purity over 90%.The purified DSG2 extracellular domain protein with IgG1 tag was recognized by IgG monoclonal antibodies by Western blotting.Moreover,the ELISA results showed that the prepared DSG2 extracellular domain protein had significant binding activity to human type 55 adenovirus Fiber Knob protein(HAdV-55).Conclusion:A simple and efficient method for eukaryotic expression and purification of human soluble DSG2 extracellular domain protein was successfully established,and biologically active DSG2 extracellular domain protein was purified,which laid the foundation for the later study of its protein function and anti-adenovirus drugs.

Trends of Superoxide Dismutase and Soluble Protein of Aquatic Plants in Lakes of Different Trophic Levels in the Middle and Lower Reaches of the Yangtze River, China

A limnological study was carried out to determine the responses of superoxide dismutase （SOD） activities and soluble protein （SP） contents of 11 common aquatic plants to eutrophication stress. Field investigation in 12 lakes in the middle and lower reaches of the Yangtze River was carried out from March to September 2004. Our results indicated that non- submersed （emergent and floating-leafed） plants and submersed plants showed different responses to eutrophication stress. Both SOD activities of the non-submersed and submersed plants were negatively correlated with their SP contents （P 〈 0.000 1）. SP contents of non-submersed plants were significantly correlated with all nitrogen variables in the water （P 〈 0.05）, whereas SP contents of submersed plants were only significantly correlated with carbon variables as well as ammonium and Secchi depth （SD） in water （P 〈 0.05）. Only SOD activities of submersed plants were decreased with decline of SD in water （P 〈 0.001）. Our results indicate that the decline of SOD activities of submersed plants were mainly caused by light limitation, this showed a coincidence with the decline of macrophytes in eutrophic lakes, which might imply that the antioxidant system of the submersed plants were impaired under eutrophication stress.

Restoration of Brain Acid Soluble Protein 1 Inhibits Proliferation and Migration of Thyroid Cancer Cells

Background： Brain acid soluble protein 1 （BASPI） is identified as a novel potential tumor suppressor in several cancers. However, its role in thyroid cancer has not been investigated yet. In the present study, the antitumor activities of BASP1 against the growth and migration of thyroid cancer cells were evaluated. Methods： BASP1 expression in thyroid cancer tissues and normal tissues were examined by immunohistochemical staining and the association between its expression and prognosis was analyzed, pcDNA-BASP 1 carrying full length of BASP1 cDNA was constructed to restore the expression of BASP1 in thyroid cancer cell lines （BHT- I 01 and KMH-2）. The cell proliferation in vitro and in vivo was evaluated by WST-1 assay and xenografl tumor models, respectively. Cell cycle distribution after transfection was analyzed using flow cytometry. Cell apoptosis after transfection was examined by annexin V/propidium iodide assay. The migration was examined using transwell assay. Results： BASP1 expression was abundant in normal tissues while it is significantly decreased in cancer tissues （P = 0.000）. pcDNA-BASP1 restored the expression of BASPI and significantly inhibited the growth of BHT-101 and KMH-2 cells as well as xenograft tumors in nude mice （P = 0.000）. pcDNA-BASPI induced G1 arrest and apoptosis in BHT-101 and KMH-2 cells. In addition, pcDNA-BASP1 significantly inhibited the cell migration. Conclusions： Downregulation of BASP1 expression may play a role in the tumorigenesis of thyroid cancer. Restoration of BASPI expression exerted extensive antitumor activities against growth and migration of thyroid cancer cells, which suggested that BASPI gene might act as a potential therapeutic agent for the treatment of thyroid cancer.

Dynamic Effects of Cerium on Syntheses of Soluble Protein and Taxol in Suspension Culture of Taxus Chinensis Var. Mairei Cells

The dynamic effects of Ce4+ on the syntheses of soluble protein and taxol in suspension cultures of Taxus chinensis var. mairei cells were studied. The phenomena of 'partition' and 'bifurcation' were observed in studying the dynamic effect of Ce4+ on soluble protein synthesis and cell activity. That is, Ce4+ of low concentration improves the soluble protein synthetic strength and cell activity, while Ce4+ of high concentration is harmful to protein synthesis and cell activity. In addition, Ce4+ of appropriate concentration enhances taxol synthesis.

Soluble expression of recomb inant cMyc,Klf4,Oct4,and Sox2 proteins in bacteria and transduction into living cells

AIM：To develop a new method to produce recombinant reprogramming proteins,c Myc,Klf4,Oct4,and Sox2,in soluble format with low cost for the generation of induced pluripotent stem cells（i PSCs）.METHODS：A short polypeptide sequence derived from the HIV trans-activator of transcription protein（TAT） and the nucleus localization signal（NLS） polypeptide were fused to the N terminus of the reprogramming proteins and they were constructed into p Cold-SUMO vector which can extremely improve the solubility of recombinant proteins.Then these vector plasmids were transformed into E.coli BL21（DE3） Chaperone competent cells for amplification.The solubility of these recombinant proteins was determined by SDS-PAGE and Coomassie brilliant blue staining.The recombinant proteins were purified by NiNTA resin and identified by Western blot.The transduction of these proteins into HEK 293 T cells were evaluated by immunofluorescence staining.RESULTS：These four reprogramming proteins could be produced in soluble format in p Cold-SUMO expression vector system with the assistance of chaperone proteins in bacteria.The proteins were purified successfully with a purity of over 70% with a relative high transduction rate into 293 cells.CONCLUSION：The results in the present study indicate the four important reprogramming proteins,c Myc,Klf4,Oct4,and Sox2,can be produced in soluble format in bacteria with low cost.Our new method thus might be expected to greatly contribute to the future study of i PSCs.

Study on The Method of Quantitative Analysis of Serum Ferritin and Soluble Transferrin Receptor with Protein Microarray Technology

Objective To establish and evaluate a protein serum ferritin （SF） and soluble transferrin receptor microarray method for combined measurement of （sTfR）. Methods Microarrayer was used to print both anti-SF antibodies I and anti-sTfR antibodies I on each protein microarray. Anti-SF antibodies II and anti-sTfR antibodies II were used as detection antibodies and goat antibodies coupled to Cy3 were used as antibodies Ill. The detection conditions of the quantitative analysis method for simultaneous measurement of SF and sTfR with protein microarray were optimized and evaluated. The protein microarray was compared with commercially available traditional tests with 26 serum samples. Results By comparison experiment, mouse monoclonal antibodies were chosen as the probes and contact printing was chosen as the printing method. The concentrations of SF and sTfR probes were 0.5 mg/mL and 0.5 mg/mL respectively, while those of SF and sTfR detection antibodies were 5 μg/mL and 0.36 μg/mL respectively. Intra- and inter-assay variability was between 3.26% and 18.38% for all tests. The regression coefficients comparing protein microarray with traditional test assays were better than 0.81 for SF and sTfR. Conclusion The present study has established a protein microarray method for combined measurement of SF and sTfR.

Early cardiopulmonary resuscitation on serum levels of myeloperoxidase,soluble ST2,and hypersensitive C-reactive protein in acute myocardial infarction patients

BACKGROUND Prompt and effective cardiopulmonary resuscitation(CPR)can promote the recovery of spontaneous circulation to some extent and can save patients’lives.The minimum target of cardiac resuscitation is the restoration of spontaneous circulation(ROSC).However,owing to prolonged sudden cardiac arrest,there is relatively high mortality within 24 h after cardiac resuscitation.Moreover,severe cerebral anoxia can deteriorate the prognosis of patients.Therefore,it is important to adopt an effective clinical evaluation of acute myocardial infarct(AMI)patients’prognosis after cardiac resuscitation for the purpose of prevention and management.AIM To investigate early CPR effects on human myeloperoxidase(MPO),soluble ST2(sST2),and hypersensitive C-reactive protein(hs-CRP)levels in AMI patients.METHODS In total,54 patients with cardiac arrest caused by AMI in our hospital were selected as the observation group,and 50 other patients with AMI were selected as the control group.The differences in serum levels of MPO,sST2,and hs-CRP between the observation group and the control group were tested,and the differences in the serum levels of MPO,sST2,and hs-CRP in ROSC and non-ROSC patients,and in patients who died and in those who survived,were analyzed.RESULTS Serum levels of MPO,sST2,hs-CRP,lactic acid,creatine kinase isoenzyme(CKMB),and cardiac troponin I(cTnI)were significantly higher in the observation group than in the control group(P<0.05).Serum levels of MPO,sST2,hs-CRP,lactic acid,CK-MB,and cTnI in the observation group were lower after CPR than before CPR(P<0.05).In the observation group,MPO,sST2,hs-CRP,lactic acid,CK-MB,and cTnI serum levels were lower in ROSC patients than in non-ROSC patients(P<0.05).MPO,sST2,hs-CRP,and lactic acid serum levels of patients who died in the observation group were higher than those of patients who survived(P<0.05).The areas under receiver operating characteristic curve predicted by MPO,sST2,hs-CRP,lactic acid,CK-MB,and cTnI were 0.616,0.681,0.705,0.704,0.702,and 0.656,respectively(P<0.05).The areas under receiver operating characteristic curve for MPO,SST2,hs-CRP,and lactic acid to predict death were 0.724,0.800,0.689,and 0.691,respectively(P<0.05).Logistic regression analysis showed that MPO,sST2,and hs-CRP were the influencing factors of ROSC[odds ratios=1.667,1.589,and 1.409,P<0.05],while MPO,sST2,hs-CRP,and lactic acid were the influencing factors of death(odds ratios=1.624,1.525,1.451,and 1.365,P<0.05).CONCLUSION Serum levels of MPO,sST2,hs-CRP,and lactic acid have a certain value in predicting recovery and prognosis of patients with ROSC.

An ELISA Based on a Truncated Soluble ORF2 Protein for the Detection of PCV2 Antibodies in Domestic Pigs

Postweaning multisystemic wasting syndrome (PMWS) is an important swine disease that is closely associated with porcine circovirus type 2 (PCV2). The capsid protein (Cap protein) is a major structural protein that has at least three immunoreactive regions, and it can be a suitable candidate antigen for detecting the specific antibodies of a PCV2 infection. In the present study, an indirect enzyme-linked immunosorbent assay (TcELISA) based on a truncated soluble Cap protein produced in Escherichia coli (E.coli) was established and validated for the diagnostic PCV2 antibodies in swine. The TcELISA was validated by comparison with an indirect immunofluorescence assay (IIFA). The diagnostic sensitivity (DSN), specificity (DSP), and accuracy of the TcELISA were 88.6%, 90.7% and 89.4%, respectively. The agreement rate was 89.38% between results obtained with TcELISA and IIFA on 113 field sera. A cross-reactivity assay showed that the method was PCV2-specific by comparison with other sera of viral disease. Therefore ,the TcELISA will be helpful for the development of a reliable serology diagnostic test for large scale detection of PCV2 antibodies and for the evaluation of vaccine against PCV2 in swine.

Therapeutic effect of soluble worm protein acting as immune regulatory on colitis

Objective: To investigate the anti-inflammatory effect of the protein derived from the soluble factor of Heligmosomoides polygyrus(H. polygyrus) excretory-secretory in a colitis model.Methods: Colitis was induced by providing drinking water containing 3% dextran sodium sulfate(DSS) for a week. DSS was administrated in a cycle protocol, each cycle consisted of 7 days of 3% DSS in the drinking water and followed by 7 days of regular water. This study consisted of five treatment groups, including Groups A(control)received untreated water, B(DSS only, without excretory-secretory), and C–E injected(i.p.) with excretory-secretory protein(H. polygyrus excretory-secretory total, excretorysecretory 28 k Da and excretory-secretory 55 k Da, respectively). Mice received injection every week. The injection of excretory-secretory was started from the 6th weeks and continued until 11 weeks. At the end of 11 weeks of the experiment, mice were sacrificed,colon tissue was removed and then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, flow cytometry, real-time PCR and histology examination.Results: Mice received H. polygyrus excretory-secretory 55 k Da reduced mono-nuclear cell infiltrations. H. polygyrus excretory-secretory 55 k Da induced the down-regulation of m RNA interferong expression. There were significant differences in the expression of m RNA interferon in the colon of mice after the administration of the excretory-secretory55 k Da protein fraction compared with other groups(P < 0.001), whereas m RNA transforming growth factorb expression up regulated in the colon of mice after the administration of the excretory-secretory 55 k Da protein fraction compared with total excretory-secretory group(P < 0.05). The treatment of colitis in mice with excretorysecretory 55 k Da protein fractions modulated interleukin-10(IL-10) expression,whereas excretory-secretory total and excretory-secretory 28 k Da protein fractions insufficient promoted IL-10 expression. Excretory-secretory 55 k Da proteins fraction promoted IL-10 expression via Foxp3-independent pathways.Conclusions: Excretory-secretory 55 k Da protein could reduce inflammation and have potential therapy. H. polygyrus excretory-secretory 55 k Da was the soluble factor that may help in the development of novel treatments to cure colitis.

Efficient and Soluble Expression of α Protein of Clostridium perfringens and Preparation of Genetic Engineering Subunit Vaccine

[Objective] The paper was to develop genetic engineering vaccine that can express α exotoxin antigen protein efficiently without destroying its immunogenicity for preventing and controlling the diseases caused by Clostridium perfringens. [Method] Efficiently expressed soluble recombinant α protein was obtained from Escherichia coli expression system by optimizing codon,removing signal peptide,selecting sequences with better hydrophilicity and antigenicity,and optimizing expression conditions. [Result] Mice obtained higher serum antibody level when immunized by α protein,and the immune protection rates against type A,type B,type C and type D C. perfringens were 100%,90%,85% and 90%,respectively. The antibody titer of mice within 7-14 d after the third immunization reached the peak. [Conclusion]The α protein has good immunogenicity,and can be further used to develop genetic engineering subunit vaccines for preventing C. perfringens.

Efficient and Soluble Expression of N Protein of Peste Des Petits Ruminants Virus and Development of Indirect ELISA

[ Objective] The paper was to get effective soluble N protein to establish indirect ELISA method for Peste des petits ruminants （PPR）. [ Method] Soluble N protein with high expression was obtained from Escherichia coli expression system through codon optimization and optimization of expression conditions, and indirect ELISA detection method based on N protein was further established. [ Result] The assay had no cross reaction with other sheep pathogens. The intra- and inter-batch variation coefficients were less than 9%, indicating the method had good repeatability. Furthermore, totally 480 clinical serum samples were detec- ted by the assay, and the agreement rate with commercial ELISA kit （IDVET） was 98.33%. [ Conclusion] The study laid a foundation for further development of mature PPRV antibody detection kits.

Effects of NaOH Treatment on Fiber, Crude Protein and Soluble Sugar Content of Corn Straw

[Objective] The paper was to study the effects of different levels of NaOH on fiber, crude protein and soluble sugar content of cornstraw. [Method] Four treatments were designed in the test, namely control group, group 1, group2 and group 3, which were added with 0, 2%, 3%and 4% NaOH according to the dry weight of corn straw, and the moisture of corn straw after treatment was 45%. After being treated at room tem-perature for 48 h, sensory evaluation and nutrient composition analysis were performed for each group, and the contents of neutral detergent fiber（NDF）, acid detergent fiber （ADF）, crude protein （CP） and soluble sugar （SS） were measured. [Result] With the increasing addition level of NaOH,the content of NDF and ADF decreased gradually, and significant differences were observed between group 3 （adding 4% NaOH） and control group（P〈0.05）. There was no significant difference in CP content among treatment groups（P〉0.05）; the SS content gradually increased, and there weresignificant differences between treatment groups and control group （P〈0.05）. [Conclusion] 4% NaOH reduced the fiber content and increased thesoluble sugar content of corn straw.

Palatability of Water-Soluble Extracts of Protein Sources and Replacement of Fishmeal by a Selected Mixture of Protein Sources for Juvenile Turbot(Scophthalmus maximus)

Poor palatability is a limiting factor for replacing fishmeal with other protein sources in aquaculture. The water-soluble molecules with low molecular weights are the major determinants of the palatability of diets. The present study was conducted to investigate the palatability of water-soluble extracts from single protein source(single extract pellets) and the mixture of these extracts with different proportions(blended extract pellets) in juvenile turbot(Scophthalmus maximus). Then according to the palatability of blended extract pellets, an optimal mixture proportion was selected, and a new protein source made from raw protein materials with the selected proportion was formulated to replace fishmeal. Summarily, the palatability of single extract pellets for turbot was descendent from fishmeal to pet-food grade poultry by-product meal, wheat gluten meal, soybean meal, peanut meal, meat and bone meal, and corn gluten meal. Subsequently, according to the palatability of single extract pellets, 52 kinds of blended extract pellets were designed to test their palatability. The results showed that the pellets presented remarkably different palatability, and the optimal one was diet 52(wheat gluten meal: pet-food grade poultry by-product meal: meat and bone meal: corn gluten meal = 1:6:1:2). The highest ingestion ratio(the number of pellets ingested/the number of pellets fed) was 0.73 ± 0.03, which was observed in Diet 52. Then five isonitrogenous(52% crude protein) and isocaloric(20 k J g^(-1) gross energy) diets were formulated by replacing 0(control), 35%, 50%, 65% and 80% of fishmeal with No.52 blending proportion. After a 10-weeks feeding trial, a consistent feed intake was found among all replacement treatments. Replacement level of fishmeal up to 35% did not significantly influence final body weight, specific growth rate, feed efficiency ratio, and protein efficiency ratio of turbot. Therefore, the water-soluble extracts of protein sources play an important role in improving the palatability of non-fishmeal protein sources in aquafeed.

Change of Water-Soluble-Protein, Urea-Soluble-Protein and Membrane Intrinsic Protein in Human Senile Cataract

Purpose:To analyze the change of water-soluble-protein(WSP),urea-soluble-protein(USP)and membrane intrinsic protein(MIP)in human senile catarct.Methods:The water-soluble-fractions(WSF)were prepared basically according to the method of Kibbelear,et al.But in this study,5mmol/LB-mercaptoethanol was added to the buffer solution.The urea-soluble-fractions(USF)were pre-pared basically according to the method of Kibbelear,et al.Lens fiber cell mem-branes were purified basically according to the method of Russell,et al.SDS-PAGE were performed according to the procedure of Laemmili,et al.using re-solving gel13%and3%stacking gel.Results:The WSPwas fractionated intoHM^+α^-,β1-3^-andγ-crystallin compo-nents.In nuclear cataractous lenses HM^+α^-and B-crystallin increase,while r-crystallin decrease.The USP from clear lenses contains mainlyαβchains of22KD,whereas in cataractous lenses,especially in nuclear cataractous lenses,the relative amount of the 28-and23KDpolypeptide(the components of β-crys-tallin)increased markedly.Lens fiber cell MIP,clear lens and cataract lens con-tained the main polypeptide of 27KD(MIP)and23KD(MP23).Conclusion:The water-insolube protein,whether in quantity or in quality,plays an important role in cataract formation.Eye Science 1995,11:124-127.

Expression of a fusion protein of human ciliary neurotrophic factor and soluble CNTF-Receptor and identification of its activity

Ciliary neurotrophic factor (CNTF) has pleiotropic actions on many neuronal populations as well as on glia. Signal transduction by CNTF requires that it bind first to CNTF-R, permitting the recruitment of gpl30 and LIF-R, forming a tripartite receptor complex. Ceils that only express gpl30 and LIF-R, but not CNTF-R are refractory to stimulation by CNTF. On many target ceils CNTF only acts in the presence of its specific agonistic soluble receptors. We engineered a soluble fusion protein by linking the COOH-terminus of sCNTF-R to the NH2-terminus of CNTF. Recombinant CNTF/sCNTF-R fusion protein (Hyper-CNTF) was sac-cessfully expressed in COS-7 cells. The apparent molecular mass of the Hyper-CNTF protein was estimated from western blots to be 75 kDa. Proliferation assays of tmnsfected BAF/3 cells in response to CNTF and Hy-per-CNTF were used to verify the activity of the cytokines. The proliferative results confirmed that CNTF required homodimerization of the gpl30, CNTF-R and LIF-R receptor subunlt whereas Hyper-CNTF required heterodimerization of the gpl30 and LIF-R receptor subunit. We concluded that the fusion protein Hyper-CNTF had superagonistic activity on target cells expressing gpl30 and LIF-R, but lacking membrane-beund CNTF-R.