-
[show abstract]
[hide abstract]
ABSTRACT: According to the cDNA sequence of anti-neuroexcitation peptide of scorpion Buthus martensii Karsch, the putative mature anti-neuroexcitation peptide (ANEP) encoding DNA fragment was obtained by a PCR method, then was cloned into expression plasmid pET28a, fused with His tag at its 3' end. When expressed in E. coli BL21 (DE3), the expression of recombinant ANEP was 15% of total cellular proteins, while most recombinant ANEP products existed in the form of insoluble inclusion bodies. Coexpression of molecular chaperones or protein disulfide isomerase could not improve its solubility. The recombinant ANEP in the cell lysate was purified to homogeneity by metal chelating affinity chromatography and Superdex 30 chromatography. In bioassay with convulsive mice model induced by thiosemicarbazide, recombinant ANEP could apparently delay the convulsion seizure of model animals by 18% and showed anti-neuroexcitatory activity.
Preparative Biochemistry & Biotechnology 03/2001; 31(1):49-57. · 0.47 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To study the effects of recombinant human macrophage colony-stimulating factor (rhM-CSF) expressed in silkworm on cytokine productions and membrane molecule expressions of monocytes.
The rhM-CSF was added to the human peripheral blood monocyte cultures and 3 d later, the culture supernatants and cells were collected, respectively. TNF-alpha, IL-6, IL-8, and IFN-alpha levels in the supernatants were detected by biological activity test or ELISA and expressions of CD11b, CD16, HLA I, and HLA II on the cellular surface were examined by the method of alkaline phosphatase anti-alkaline phosphatase complex (APAAP).
The rhM-CSF promoted TNF-alpha, IL-6, and IL-8 inductions of monocytes and increased the percentages of CD11b, CD16, HLA I, and HLA II molecule expression on monocytes.
The rhM-CSF plays a role in monocyte function up-regulation and has a certain practical value in immunological therapy.
Acta Pharmacologica Sinica 10/2000; 21(9):797-801. · 1.95 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Ampicillin was used in in vitro renaturation process of recombinant human prourokinase and it can obviously improve the yield of the renaturation, although not as well as arginine. When ampicillin was used together with arginine or lysine, it decreased the yield of the renaturation comparing with the yield when arginine or lysine used alone.
Biochemistry and molecular biology international 09/1996; 39(6):1093-7.
-
[show abstract]
[hide abstract]
ABSTRACT: A Gly-Pro-Arg-Pro tetrapeptide, homologous to amino-terminal segment of the human fibrin alpha chain after the release of the fibrinopeptide A, was covalently coupled to peptide A of low molecular weight urokinase. The resulting derivative gained increased affinity for fibrin. In caseinolytic assay, fibrin can stimulate the derivative to activate plasminogen. The derivative had two-fold greater fibrinolytic potency than native low molecular weight urokinase and its affinity for fibrin clot was 3.9-fold higher than that of low molecular weight urokinase.
Biochemistry and molecular biology international 08/1996; 39(4):797-803.
-
[show abstract]
[hide abstract]
ABSTRACT: Two low molecular weight urokinase derivatives were obtained by covalent coupling of a synthetic Gly-Pro-Arg-Pro tetrapeptide to peptide A of low molecular weight urokinase and exchanging the native peptide A of low molecular weight urokinase with Gly-Pro-Arg-Pro-peptide A to obtain derivative I or direct conjugation of Gly-Pro-Arg-Pro tetrapeptide to low molecular weight urokinase to obtain derivative II. In caseinolytic assay, fibrin can stimulate the two derivatives to activate plasminogen. But the two derivatives showed different kinetic behaviors. The derivative I displayed immediate onset of lysis and derivative II displayed a lag phase.
Biochemistry and molecular biology international 07/1996; 39(3):455-60.
-
[show abstract]
[hide abstract]
ABSTRACT: A chimeric plasminogen activator, GPRP-u-PA (144-411), consisting of the Gly-Pro-Arg-Pro tetrapeptide fused to the N-terminal of a truncated urokinase-type plasminogen activator (comprising Leu 144 through Leu 411), was produced by expression of the corresponding chimeric cDNA in Escherichia coli cells. After renaturation, the chimera was purified to homogeneity with specific amidolytic activity of 100,000 IU/mg protein. The chimera showed 6-fold greater affinity for fibrin clots than native low molecular weight urokinase (LUK) and 1.5-fold greater affinity than a chemical conjugate, GPRP-LUK, generating via coupling Gly-Pro-Arg-Pro tetrapeptide to native low molecular weight urokinase. The chimera had 2 to 3 fold greater fibrinolytic potency than native LUK in vitro. Fibrinogen had no influence on fibrinolysis of the chimera. The chimera consumed much less fibrinogen than native LUK.
Biochemical and Biophysical Research Communications 06/1996; 222(2):576-83. · 2.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A synthetic gene encoding human pro-urokinase (pro-UK) with E. coli-favored codon usage was cloned into plasmid pET-3d and expressed in E. coli BL21(DE3) LysS strain. The expressed products, which accumulated as inactive inclusion bodies, were denatured and renatured in vitro. A broad range of parameters such as pH, protein concentration, denaturant concentration, the use of cosolvent polyethylene glycol and presence of basic or acidic amino acid was examined. At optimal renaturation condition, pro-UK activity of more than 1000I.U was obtained from 1 milliliter cell culture.
Biochemical and Biophysical Research Communications 04/1996; 220(1):131-6. · 2.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The effects of PMA and IFN-gamma on regulation of growth and differentiation of human monoblastic leukemic cell U937 were examined. U937 cells were stimulated by different concentrations of PMA and IFN-gamma respectively and NBT reduction assay was used to detect the differentiation of the cells. The results showed that both PMA and IFN-gamma dose-dependently induced differentiation of U937 cells into mature macrophage-like cells. The data also revealed a time-course response in the differentiation induction. Moreover, the U937 cell growth was significantly inhibited by the treatment of PMA and IFN-gamma. These results suggest that PMA and IFN-gamma coupled the regulation of U937 cell growth and differentiation. It was found that tumor necrosis factor-alpha (TNF-alpha) was expressed by the stimulated U937 cells. The specific monoclonal antibody against TNF-alpha diminished the effects of PMA and IFN-gamma on the growth and differentiation of U937 cells. Thus the endogenous TNF-alpha may involved in the mechanism of the effects of PMA and IFN-gamma on the differentiation of U937 cells. The regulatory action of the endogenous TNF-alpha on U937 cells was not due to its cytotoxic effect.
Shi yan sheng wu xue bao 10/1994; 27(3):307-13.
-
[show abstract]
[hide abstract]
ABSTRACT: The DNA fragment corresponding to the tissue plasminogen activator (tPA) sequence 174-262 (Kringle-2 domain) has been synthesized by using the solid phase phosphotriester method. The Kringle-2 domain of human tPA was expressed in Escherichia coli by secretion into the periplasmic space using the Lpp-Lac promoter and PIN-III OmpA2 signal sequence. About two thirds of the expression product was secreted into the periplasmic space, and purified with ammonium sulfate fractionation, affinity chromatography on Lysine-Sepharose, and FPLC-Mono Q exchange chromatography. The amino acid composition observed from the Kringle-2 purified from E. coli is identical with that expected for the 174-262 fragment of human tPA. Radio binding assay shows that the recombinant Kringle-2 domain possesses the activity of fibrin binding.
Science in China. Series B, Chemistry, life sciences & earth sciences 07/1994; 37(6):667-76.
-
[show abstract]
[hide abstract]
ABSTRACT: A method is presented for efficient and large-scale isolation of plasmid DNAs from bacterial cells. Based on the cooperativity of heat and alkali actions, the method provides DNA preparations with high quality and yield (about 2 micrograms of DNA/ml culture), which are completely digestable by restriction enzymes and have a high transformation efficiency. Furthermore, the DNA preparations are extremely stable, and even through 4-year storage at -20 degrees C, the electrophorogram and transformation efficiency remain as high as before. The factors affecting the stability of various DNA samples are discussed.
DNA and Cell Biology 02/1994; 13(1):83-6. · 2.07 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The B-chain of urokinase (UK) was covalently linked by disulfide bond to the Fab fragment of an anti-human activated platelet monoclonal antibody (SZ-51). The UK-SZ-51 conjugate retained the original binding specificity of its parent antibody, and produced about a 5-fold enhancement in clot lysis in plasma over that of the urokinase in vitro. Whereas UK significantly decreased the concentration of fibrinogen in plasma clot assay supernatants, UK-SZ-51 did not.
Science in China. Series B, Chemistry, life sciences & earth sciences 01/1994; 36(12):1483-9.
-
[show abstract]
[hide abstract]
ABSTRACT: A human truncated macrophage colony-stimulating factor (M-CSF) cDNA encoding amino acid residues from 3 to 149 of the native M-CSF was isolated by using the polymerase chain reaction. When introduced into Saccharomyces cerevisiae by a general secretion vector pVT 102u/alpha, it directs the expression of the biologically active dimeric form of M-CSF. Through the 3 stages of purification, i.e. concentration by DEAE-cellulose column chromatography, hydrophobic chromatography on phenyl-sepharose and Mono Q fast protein liquid chromatography, the recombinant truncated M-CSF was purified as to exhibit a specific activity of 1.02 x 10(7) units/mg of protein. SDS-PAGE of this purified truncated M-CSF showed that its apparent molecular mass is 22 kDa under reducing conditions.
Biological chemistry Hoppe-Seyler 10/1993; 374(9):903-8.
-
[show abstract]
[hide abstract]
ABSTRACT: The expression of p53 gene has been found to be regulated during the induction of differentiation of U937 leukemic cells into mature macrophages by recombinant human granulocyte- macrophage colony stimulating factors (rhGM-CSF) We showed here that the increased expression of p53 seemed to be necessary for the differentiation of U937 cells induced by rh-GM-CSF. The inhibition of p53 expression by a p53 antisense oligodeoxynucleotide lead to the significant decrease of formation of mature macrophages from U 937 cells in the presence of rhGM-CSF. By contrast, the p53 sense oligodeoxynucleotide had no any effect. Furthermore, we have analysed the growth of U937 cells in the presence or absence of rhGM-CSF. The results showed that rhGM-CSF dramatically inhibited the growth of U 937 cells in the cultures. At the same time, the antisense inhibition experiment demonstrated that the inhibition of p53 expression partially diminished the growth-inhibitory effect of rhGM-CSF on U 937 cells. These results suggested that the p53 was required for the initiation of rhGM-CSF-induced differentiation of U 937 cells on one hand, and the inhibition of cell growth on the other hand. Thus we deduce that the increased expression of p53 induced by rhGM-CSF may be a coupling event of switch of U 937 cells from growth into differentiation.
Shi yan sheng wu xue bao 01/1993; 25(4):311-6.
-
[show abstract]
[hide abstract]
ABSTRACT: In view of the similarity of the charge distribution between fibrin A alpha 148-161 and A chain 149-157 of urokinase, the latter might compete with fibrin A alpha 148-161 when single chain pro-urokinase is converted to double chain urokinase. To test this, the stretch of urokinase A chain 135-157 was separated from the low molecular weight urokinase, a competitive binding between this stretch and fibrin to tPA kringle-2 was shown by radio-binding assay. The inhibition of the stretch on the fibrin stimulated activation of plasminogen was demonstrated in the caseinolytic system. The synthesized novapeptide urokinase A chain 149-157 (R-peptide) showed a significant inhibition on the activation of plasminogen in the presence of fibrin. By contrasting finely with R-peptide, a synthesized novapeptide in which Arg154 and Arg156 were replaced by Asp (D-peptide) did not show any inhibition effect on the fibrin stimulated activation of plasminogen by tPA. These results suggest that the positively charged residues in the stretch 149-157 of urokinase are crucial for the inhibition of fibrin binding with the kringle domain of urokinase.
Science in China. Series B, Chemistry, life sciences & earth sciences 09/1992; 35(8):966-73.
-
[show abstract]
[hide abstract]
ABSTRACT: Multiple homeobox genes are expressed in haematopoietic cell lineages and their expression is cell-type specific. Thus we hypothesized that certain homeobox genes may play an important role in the process of haematopoiesis. To prove that issue, normal murine bone marrow cells were stimulated with appropriate Colony Stimulating Factors in the presence of mouse homeobox gene (Hox 2.3) sense or antisense oligodeoxynucleotides and the effects on the haematopoietic colony formation were examined. Treatment of the cells to Hox 2.3 antisense oligodeoxynucleotides led to a selective inhibition of myeloid colony formation, both in size and in numbers, but without significant effect on erythroid and megakaryocytic haematopoiesis. Exposure to Hox 2.3 sense oligodeoxynucleotides (no-oligomers), had no such effect. It was further showed that inhibition of myelopoiesis by Hox 2.3 antisense oligodeoxynucleotides was dependent on the differentiation stage of target cells. These findings demonstrated that Hox 2.3 gene plays a critical role in regulating normal murine myelopoiesis.
Cellular and molecular biology 08/1992; 38(4):367-76. · 0.98 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Two arrowhead proteinase inhibitors (inhibitors A and B) were characterized and their primary structures were determined. Both inhibitors A and B are double-headed and multifunctional protease inhibitors. Inhibitor A inhibits an equimolar amount of trypsin and chymotrypsin simultaneously and weakly inhibits kallikrein. Inhibitor B inhibits two molecules of trypsin simultaneously and inhibits kallikrein more strongly than does inhibitor A. The amino acid sequences of inhibitors A and B were determined by sequencing the reduced and S-carboxamidomethylated proteins and their peptides produced by cyanogen bromide or proteolytic lysylendopeptidase or Staphylococcus aureus V8 protease cleavage. Inhibitors A and B consist of 150 amino acid residues with three disulfide bonds (Cys 43-Cys 89, Cys 110-Cys 119, and Cys 112-Cys 115) and share 90% sequence identity, with 13 different residues. Since the primary structures are totally different from those of all other serine protease inhibitors so far known, these inhibitors might be classified into a new protease inhibitor family.
Journal of Biochemistry 05/1992; 111(4):537-45. · 2.37 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: In addition to the regulation of T cell growth, IL-2 exerts effects on the induction of certain lymphokines. We show here that IL-2 synergizes with 5 micrograms/ml of ConA to promote the production of IL-3 in mouse splenic T cell cultures. IL-3 was measured as CFU-GEMM-inducing activity on mouse bone marrow progenitor cells in the supernatant of the stimulated mouse splenic T cells (TCM). The resting T cells produced no CFU-GEMM-inducing activity, but could be induced to produce low level of CFU-GEMM-inducing activity in the presence of ConA. In vitro exposure to IL-2 markedly increased CFU-GEMM-inducing activity production (nearly up to 8-fold) by the ConA-activated T cells. Optimal stimulation was observed when 80 u/ml IL-2 was used for 48 h incubation. Anti-mouse IL-3 monoclonal antibody inhibited the CFU-GEMM inducing activity of TCM. Moreover, the TCM stimulated the proliferation of IL-3 dependent cell line FDC-P1. We also show that IL-2 and ConA-treated T cells expressed high level of IL-3 mRNA through dot blot analysis. These results confirmed the nature of CFU-GEMM-inducing activity of TCM as IL-3. The capacity of IL-2 to promote the production of IL-3 may represent an important mechanism by which it mediate the communication between the immune and hematopoietic systems.
Shi yan sheng wu xue bao 04/1992; 25(1):25-30.
-
[show abstract]
[hide abstract]
ABSTRACT: The Kringle-1 structure of plasminogen (PGK-1), the Kringle-2 structure of tissue plasminogen activator (PAK-2) and the Kringle structure of prourokinase (UKK) has been modeled on the basis of the three-dimensional structure of Kringle-1 of prothrombin (PTK-1) at 2.8 A resolution. The predicted three-dimensional structure of these Kringles shows that the binding site of PGK-1 is characterized by an apparent dipolar site, the polar parts of which are separated by a hydrophobic region. PAK-2 possesses the anionic center but has not a cationic binding center which might be provided by a guanidinium group from Arg-69 located adjacent to the Arg-71 position. UKK possesses neither the anionic binding center nor the cationic center which are probably the main reason for the poor fibrin specificity of urokinase.
Science in China. Series B, Chemistry, life sciences & earth sciences 03/1992; 35(2):176-82.
-
[show abstract]
[hide abstract]
ABSTRACT: Affinity chromatography was used to separate two components A and B of the crystalline arrowhead proteinase inhibitor. Both A and B are double-headed and multifunctional proteinase inhibitors. Inhibitor A is capable of inhibiting equimolarly trypsin and chymotrypsin simultaneously, and has a weak inhibitory activity toward kallikrein; whereas inhibitor B can inhibit two molecules of trypsin simultaneously, and shows rather higher inhibitory activity toward kallikrein than inhibitor A, but its inhibitory activity toward chymotrypsin is much weaker than that of inhibitor A. The results of chemical modification and the competitive binding of trypsin and chymotrypsin with inhibitor A showed that the two reactive sites of both inhibitors A and B are Lys and Arg residues. Among them the Lys reactive site is specific for inhibiting mainly trypsin, whereas the active domain composed of the Arg reactive site appears to be multifunctional and capable of inhibiting many different Ser proteinases. Based on the structural characteristics of inhibitors A and B, it was predicated that the two reactive sites should be located in the positions Lys-Ser (44-45) and Arg-Tyr-Lys (76-78), respectively. In inhibitor A, there exists another hydrophobic residue involved in inhibiting chymotrypsin. This residue might be situated in the reactive region composed of the Arg reactive site.
Science in China. Series B, Chemistry, life sciences & earth sciences 08/1991; 34(7):832-9.
-
[show abstract]
[hide abstract]
ABSTRACT: Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) is known as an inducer of proliferation and functional activation of myeloid cells. This study was carried out to characterize the effect of purified recombinant human GM-CSF (rhGM-CSF) on induction of TGF-alpha in macrophages. Using Northern blot analysis and immunoassays, we show here that rhGM-CSF markedly stimulates production of TGF-alpha messenger RNA and protein in normal tonsil macrophages. The findings are consistent with macrophages being a normal inducible source of TGF-alpha which may be an important mediator of various activities of GM-CSF both in hematopoietic and non-hematopoietic cells.
Cellular and molecular biology 02/1991; 37(4):413-9. · 0.98 Impact Factor
