1.Prokaryotic expression and purification of human hepatic stimulator substance.
Hai-Jun DU ; Hong-Liu SUN ; Li CHEN ; Wei AN
Acta Physiologica Sinica 2002;54(1):23-27
To explore the possibility of prokaryotic expression of human hepatic stimulator substance (hHSS), hHSS gene was inserted in the downstream of glutathion S-transferase (GST) in a pET-42a expression vector and recombinant GST-hHSS fusion protein was expressed under IPTG induction in BL-21(DE3) cells. The recombinant HSS was purified with His.Tag affinity chromatography, and its bioactivity was analyzed. The results showed that GST-hHSS fusion protein was expressed both as a soluble or a inclusive body in bacterial cytosol. The soluble GST-hHSS expression reached up to 30% of the whole soluble protein of bacteria as determined by densitometry. The cleavage of GST-hHSS fusion protein with Factor Xa produced two fragments of the protein, which sized 33 and 15 kD, respectively. The molecular weight of recombinant HSS protein was identical to theoretical deduction based on the DNA sequences. The protein homology of 15 kD hHSS could be efficiently eluted out after Factor Xa cleavage. It is further indicated that the recombinant hHSS is able to proliferate hepatoma cells of BEL-7402 in the preliminary experiments.
Cell Division
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drug effects
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Gene Expression
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Glutathione Transferase
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genetics
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Growth Substances
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genetics
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isolation & purification
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pharmacology
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Humans
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Peptides
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genetics
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isolation & purification
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pharmacology
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Recombinant Fusion Proteins
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genetics
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isolation & purification
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pharmacology
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Tumor Cells, Cultured
2.Protective effects of shark hepatic stimulator substance against acute hepatic injury induced by acetaminophen in mice.
Zheng-bing LÜ ; Qian LI ; Bo-ping YE ; Shan BIAN ; Ying WANG ; Qi-ping RUAN ; Wu-tong WU
Acta Pharmaceutica Sinica 2004;39(1):17-21
AIMTo investigate the protective effects of shark hepatic stimulator substance (sHSS) against acute hepatic injury induced by acetaminophen (AAP) in mice.
METHODSAcute hepatic injury model of Balb/c mice was induced by a single intraperitoneal injection of AAP (200 mg.kg-1, i.p.). Serum ALT and AST activities were analyzed. The changes of microstructure and ultrastructure of hepatocyte were observed under optical and electronic microscope. The hepatocyte apoptosis was analyzed by flow cytometer and the expression level of Fas mRNA was determined by RT-PCR.
RESULTSThe activities of serum ALT and AST were significantly decreased and both necrosis and inflammatory infiltration were improved in the mice treated with sHSS 3.0 and 1.5 mg.kg-1. sHSS (3.0 mg.kg-1) prevented the ultrastructural changes of hepatocytes caused by AAP, decreased the percentage of apoptotic cells, and downregulated the expression level of Fas mRNA.
CONCLUSIONsHSS protected hepatocytes from AAP-induced injury, which might be associated with its protection of the mitochondria and inhibition of apoptosis and expression of Fas mRNA in hepatocytes.
Acetaminophen ; Animals ; Apoptosis ; drug effects ; Chemical and Drug Induced Liver Injury ; etiology ; pathology ; Female ; Growth Substances ; isolation & purification ; pharmacology ; Mice ; Mice, Inbred BALB C ; Peptides ; isolation & purification ; pharmacology ; Protective Agents ; pharmacology ; RNA, Messenger ; genetics ; Random Allocation ; Sharks ; fas Receptor ; biosynthesis ; genetics
3.Effect of growth factors on the expression of proto-oncogenes c-fos and c-myc in FRTL-5 cell line.
Hwan Young YOON ; Seung Keun OH ; Ka Hee YI ; Bo Youn CHO ; Chang Soon KOH
Journal of Korean Medical Science 1995;10(3):155-163
This study was performed to prove the hypothesis that oncogene expressions would have the same patterns with those of cellular growth to growth factors in FRTL-5 cells. Ribonucleic acids of FRTL-5 were extracted at 15', 30', 60' and 120' after administration of growth factors to quiescent FRTL-5, and blotted to the nitrocellulose membrane. They were hybridized with radiolabelled c-fos, c-myc and beta-actin probes. Hybridized dot blots were autoradiographed and the amount of radioactivity was measured by densitometry. Densitometric readings were used as the indices of oncogene expressions. Expressions of c-fos and c-myc were more prominent in combined administrations of TSH (10 mU/ml) and IGF-I (100 ng/ml) or IgG of Graves' disease (Graves' IgG; 1 mg/ml) and IGF-I than in combined administration of TSH and Graves' IgG. IgG of primary myxedema suppressed oncogene expressions by TSH or Graves' IgG, but not by IGF-I. From the above results, it was suggested that expressions of c-fos and c-myc to growth factors would have similar patterns with those of cell growth to growth factors in FRTL-5, and the actions of TSH and Graves' IgG would be manifested through same signal transduction system, but IGF-I would be manifested by its own.
Animal
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Cell Division/drug effects/genetics
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Cell Line/cytology/physiology
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Gene Expression/drug effects/immunology
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Graves' Disease/immunology
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Growth Substances/genetics/*pharmacology
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Immunoglobulin G/pharmacology
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Insulin-Like Growth Factor I/pharmacology
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Myxedema/immunology
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Proto-Oncogene Proteins c-fos/*genetics
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Proto-Oncogene Proteins c-myc/*genetics
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RNA/analysis
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Rats
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Rats, Inbred F344
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Support, Non-U.S. Gov't
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Thyroid Gland/cytology
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Thyrotropin/pharmacology
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Time Factors
4.Protective effects of estrogens and caloric restriction during aging on various rat testis parameters.
Khaled HAMDEN ; Dorothee SILANDRE ; Christelle DELALANDE ; Abdelfattah ELFEKI ; Serge CARREAU
Asian Journal of Andrology 2008;10(6):837-845
AIMTo investigate the effects of 17beta-estradiol (E2), Peganum harmala extract (PHE) and caloric restriction (CR) on various testis parameters during aging.
METHODSTwelve month-old male rats were treated for 6 months with either E2 or PHE, or submitted to CR (40%).
RESULTSOur results show that estrogens and CR are able to protect the male gonad by preventing the decrease of testosterone and E2 levels as well as the decrease of aromatase and estrogen receptor gene expressions. Indeed, E2, PHE and CR treatments induced an increase in the superoxide dismutase activities and decreased the activity of testicular enzymes: gamma-glutamyl transferase, alkaline phosphatase, lactate deshydrogenase as well as the aspartate and lactate transaminases in aged animals. In addition, the testicular catalase and gluthatione peroxidase activities were enhanced in E2, PHE and CR-treated rats compared to untreated animals at 18 months of age. Moreover, the positive effects of estradiol, PHE and CR were further supported by a lower level of lipid peroxidation. Recovery of spermatogenesis was recorded in treated rats.
CONCLUSIONBesides a low caloric diet which is beneficial for spermatogenesis, a protective antioxydant role of estrogens is suggested. Estrogens delay testicular cell damage, which leads to functional senescence and, therefore, estrogens are helpful in protecting the reproductive functions from the adverse effects exerted by reactive oxygen species (ROS) produced in large quantities in the aged testis.
Aging ; physiology ; Animals ; Antioxidants ; metabolism ; Aromatase ; biosynthesis ; genetics ; Caloric Restriction ; Estradiol ; metabolism ; pharmacology ; Estrogens ; pharmacology ; Lipid Peroxidation ; drug effects ; Male ; Oxidative Stress ; drug effects ; Peganum ; chemistry ; Plant Extracts ; pharmacology ; RNA ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; Receptors, Estrogen ; biosynthesis ; genetics ; Testis ; drug effects ; enzymology ; growth & development ; Testosterone ; metabolism ; Thiobarbituric Acid Reactive Substances ; metabolism
5.Effects of interleukin-10 on chemokine KC gene expression by mouse peritoneal macrophages in response to Candida albicans.
Hee Sun KIM ; Dong Hoon SHIN ; Sung Kwang KIM
Journal of Korean Medical Science 1999;14(5):480-486
Chemokine KC has been considered to be a murine homologue of human GRO/MGSA and was identified as chemoattractant for monocytes and neutrophils. This study examined the expression of KC mRNA in thioglycollate-elicited mouse peritoneal macrophages that were stimulated in vitro with Candida albicans (CA). Also examined were the inhibitory effects of IL-10 on the CA-induced expression of KC gene by Northern blot analysis. CA was found to induce chemokine gene expression in a gene-specific manner, CXC chemokine IP-10 mRNA expression was not detected in CA-stimulated macrophages. Maximum KC mRNA expression was observed approximately 2 hr after adding CA. The inhibitory action of IL-10 to CA-induced KC mRNA expression on mouse peritoneal macrophages was independent on concentration and stimulation time of IL-10 and was observed approximately one hour after adding IL-10 and CA simultaneously. IL-10 produced a decrease in the stability of KC mRNA, and CA-stimulated macrophages with cycloheximide blocked the suppressive effect of IL-10. These results suggest that CA also induces chemokine KC from macrophages, and IL-10 acts to destabilize CA-induced KC mRNA and de novo synthesis of an intermediate protein is a part of the IL-10 suppressive mechanism.
Animal
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Blotting, Northern
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Candida albicans/metabolism*
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Cells, Cultured
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Chemotactic Factors/genetics*
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Dactinomycin/pharmacology
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Dose-Response Relationship, Drug
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Gene Expression Regulation/drug effects*
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Growth Substances/genetics*
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Interleukin-10/pharmacology*
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Interleukin-10/metabolism
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Macrophages/physiology*
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Mice
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Mice, Inbred BALB C
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Nucleic Acid Synthesis Inhibitors/pharmacology
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RNA, Messenger/metabolism
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RNA, Messenger/drug effects
6.Angiotensin II stimulates proliferation of adventitial fibroblasts cultured from rat aortic explants.
Duk Kyung KIM ; Jeong Eun HUH ; Sang Hoon LEE ; Kyung Pyo HONG ; Jeong Euy PARK ; Jung Don SEO ; Won Ro LEE
Journal of Korean Medical Science 1999;14(5):487-496
It has been proposed that the local renin-angiotensin system is activated in the adventitia after vascular injury. However, the physiological role of Angiotensin II (Ang II) in the adventitia has not been studied at a cellular level. This study was designed to assess the role of Ang II in the growth response of cultured adventitial fibroblasts (AFs). Adventitial explants of the rat thoracic aorta showed outgrowth of AFs within 5-7 days. Ang II caused hyperplastic response of AF cultures. The Ang II-induced mitogenic response of AFs was mediated primarily by the AT1 receptor. Ang II caused a rapid induction of immediate early genes (c-fos, c-myc and jun B). Induction of c-fos expression was fully blocked by an AT1 receptor antagonist but not by an AT2 receptor antagonist. Epidermal growth factor (EGF), platelet-derived growth factor-BB (PDGF-BB) and basic fibroblast growth factor (bFGF) induced DNA synthesis in AFs. Co-stimulation of AFs with the growth factors and Ang II potentiated the incorporation of 3H-thymidine into DNA. Results from this study indicate that Ang II causes mitogenesis of AFs via AT1 receptor stimulation and potentiates the responses to other mitogens. These data suggest that the Ang II may play an important role in regulating AF function during vascular remodeling following arterial injury.
Angiotensin II/metabolism*
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Animal
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Aorta/pathology
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Blotting, Northern
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Cell Division
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Cells, Cultured
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DNA/biosynthesis
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Fibroblasts/pathology
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Fibroblasts/metabolism*
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Gene Expression/physiology
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Genes, Immediate-Early/genetics
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Growth Substances/metabolism*
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Hyperplasia/metabolism
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Losartan/pharmacology
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Male
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Proto-Oncogenes/genetics
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RNA/biosynthesis
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Rats
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Rats, Sprague-Dawley
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Receptors, Angiotensin/antagonists & inhibitors