1.Effect of porcine bone morphogenetic protein on healing of bone defect in the rabbit radius.
Nam Hyun KIM ; kyu Hyun YANG ; Hwan Mo LEE ; Sang Hwan OH
Yonsei Medical Journal 1992;33(1):54-63
Segmental long bone defects due to infection or trauma is a difficult problem to manage in patients. We studied the effect of porcine bone morphogenetic protein (pBMP) on healing of defects in the rabbit radius. Porcine BMP was separated and purified from the tibia and femur of pigs by repeated solubilization and precipitation of the protein with different concentrations of urea and GuHCl. The osteoinductive activity of pBMP was confirmed by bioassay using No. 615 mice. In rabbits, about a 15 mm length of radii were removed and 20 mg of pBMP was implanted in the defected area with fibrin sealant (FS), while only FS was implanted in controls. Union of the affected area was observed in 6 weeks in the experimental side. There was no definite evidence of bone bridging across the affected area in the controls. This suggests that pBMP has a bone forming activity in other species and the clinical use of pBMP in treating patients with segmental bone defects is promising.
Animal
;
Bone Morphogenetic Proteins
;
Growth Substances/*pharmacology
;
Osteogenesis/*drug effects
;
Proteins/*pharmacology
;
Rabbits
;
Radius/drug effects/pathology/radiography
;
Support, Non-U.S. Gov't
;
Swine
2.Induction of proliferation in resting B-cells by a factor released by activated mouse spleen cells.
Experimental & Molecular Medicine 1998;30(4):199-204
Mouse spleen cells activated in a mixed lymphocyte reaction release a soluble factor, which induces a significant proliferative response in fresh mouse spleen cells. This proliferation inducing factor (PIF) was found to be heat stable (90 degrees C for 45 min) and also resistant to trypsin or chymotrypsin treatment. By using a sizing HPLC column, the molecular weight of PIF appears to be 25 kDa. Mouse spleen cells treated with anti-thy-1 + complement lost Con-A induced proliferative responses but responded well to PIF. B cell depleted spleen cells obtained by negative selection panning, did not respond to PIF. These results indicate that B cells proliferated in response to PIF. Polymixin-B, which blocks the B cell proliferative response to LPS, did not inhibit PIF induced proliferation.
Animal
;
B-Lymphocytes/physiology*
;
B-Lymphocytes/drug effects
;
Bone Marrow/metabolism
;
Cell Division/physiology
;
Chromatography, High Pressure Liquid
;
Chymotrypsin/pharmacology
;
Dose-Response Relationship, Drug
;
Growth Substances/pharmacology*
;
Growth Substances/chemistry
;
Heat
;
Mice
;
Mice, Inbred BALB C
;
Mice, Inbred C57BL
;
Molecular Weight
;
Polymyxin B/pharmacology
;
Protein Denaturation
;
Spleen/metabolism*
;
Thymus Gland/metabolism
;
Trypsin/pharmacology
3.Ideal concentration of growth factors in rabbit's flexor tendon culture.
Ho Jung KANG ; Eung Shick KANG
Yonsei Medical Journal 1999;40(1):26-29
Growth factors have the ability to stimulate matrix synthesis and cell proliferation in rabbit flexor tendon. Maximal stimulation effects of growth factors have a wide variation. It depends upon the different anatomic sites of the tendon segment, the kinds of growth factor, the concentration of growth factors, and the time sequence. Since proliferation was an early component of intrinsic tendon healing, we investigated the short-term dose response to four different growth factors on in vitro rabbit's tendon culture. We evaluated the effects according to the various concentrations of recombinant human insulin-like growth factor 1 (IGF), recombinant human epidermal growth factor (EGF), fibroblast growth factor (FGF), and recombinant human platelet-derived growth factor-BB (PDGF). Fetal calf serum was the most potent stimulator of cell proliferation and protein synthesis in in vitro rabbit's tendon culture. Matrix synthesis and cell proliferation were stimulated dose-dependently by IGF between the doses of 50 and 150 ng/ml. The maximum mitogenic effect of EGF was observed at the concentration of 100 ng/ml (1.3 times more than the media-only control culture). The rabbit's tendon responded significantly dose-dependently to PDGF, whereas there was no significant response to FGF.
Animal
;
Cell Division/drug effects
;
Dose-Response Relationship, Drug
;
Growth Substances/pharmacology*
;
Organ Culture
;
Proteins/biosynthesis
;
Rabbits
;
Tendons/metabolism
;
Tendons/drug effects*
;
Tendons/cytology
4.Segment boundaries of the adult rat epididymis limit interstitial signaling by potential paracrine factors and segments lose differential gene expression after efferent duct ligation.
Terry T TURNER ; Daniel S JOHNSTON ; Scott A JELINSKY ; Jose L TOMSIG ; Joshua N FINGER
Asian Journal of Andrology 2007;9(4):565-573
The epididymis is divided into caput, corpus and cauda regions, organized into intraregional segments separated by connective tissue septa (CTS). In the adult rat and mouse these segments are highly differentiated. Regulation of these segments is by endocrine, lumicrine and paracrine factors, the relative importance of which remains under investigation. Here, the ability of the CTS to limit signaling in the interstitial compartment is reviewed as is the effect of 15 days of unilateral efferent duct ligation (EDL) on ipsilateral segmental transcriptional profiles. Inter-segmental microperifusions of epidermal growth factor (EGF), vascular endothelial growth factor (VEGFA) and fibroblast growth factor 2 (FGF2) increased phosphorylation of mitogen activated protein kinase (MAPK) in segments 1 and 2 of the rat epididymis and the effects of all factors were limited by the CTS separating the segments. Microarray analysis of segmental gene expression determined the effect of 15 days of unilateral EDL on the transcriptome-wide gene expression of rat segments 1-4. Over 11,000 genes were expressed in each of the four segments and over 2000 transcripts in segment 1 responded to deprivation of testicular lumicrine factors. Segments 1 and 2 of control tissues were the most transcriptionally different and EDL had its greatest effects there. In the absence of lumicrine factors, all four segments regressed to a transcriptionally undifferentiated state, consistent with the less differentiated histology. Deprivation of lumicrine factors could stimulate an individual gene's expression in some segments yet suppress it in others. Such results reveal a higher complexity of the regulation of rat epididymal segments than that is generally appreciated.
Animals
;
Ejaculatory Ducts
;
physiology
;
Epididymis
;
drug effects
;
physiology
;
Gene Expression Regulation
;
drug effects
;
Growth Substances
;
pharmacology
;
Male
;
Mice
;
Rats
;
Reverse Transcriptase Polymerase Chain Reaction
;
Signal Transduction
5.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
;
drug effects
;
Gene Expression
;
Glutathione Transferase
;
genetics
;
Growth Substances
;
genetics
;
isolation & purification
;
pharmacology
;
Humans
;
Peptides
;
genetics
;
isolation & purification
;
pharmacology
;
Recombinant Fusion Proteins
;
genetics
;
isolation & purification
;
pharmacology
;
Tumor Cells, Cultured
6.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
7.Characteristics of tenocyte adhesion to biologically-modified surface of polymer.
Tingwu QIN ; Zhiming YANG ; Huiqi XIE ; Hong LI ; Jian QIN ; Zezhi WU ; Shirong XU ; Shaoxi CAI
Journal of Biomedical Engineering 2002;19(4):633-638
In this study we examined the in vitro characteristics of tenocyte adhesion to biologically-modified surface of polymer. Polylactic-co-glycolic acid (PLGA) 85/15 films were prepared by a solvent-casting technique. Each film was adhered onto the bottom of a chamber. The film was precoated with poly-D-lysine (PDL), and then coated with serum-free F12 medium containing various concentrations of fibronectin (FN), type I collagen (CN I), and insulin-like growth factor1 (IGF-1). The monoclonal antibodies (to FN and to CN I) with various dilutions were used to inhibit attachment of tenocytes to surface precoated with FN or CN I. Human embryonic tendon cells (HETCs) and transformed human embryonic tendon cells (THETCs) were used as the seeding cells. The system used for the measurement of adhesion force was the micropipette aspiration experiment system. The micropipette was manipulated to aspirate a small portion of the tenocyte body by using a small aspiration pressure. Then the pipette was pulled away from the adhesion area by micromanipulation. The minimum force required to detach the tenocyte from the substrate was defined as the adhesion force. The results showed that modification of FN or CN I by precoating significantly enhanced attachment of tenocytes to surface of polymer (P < 0.05). As antibodies to FN or CN I were added to a polymer film precoated with FN or CN I, the adhesion force decreased significantly (P < 0.05). We concluded that the specific adhesion forces of tenocytes to extracellular matrix adhesion proteins (FN and CN I) had coordinated action and showed good dependence on their precoating concentrations, and were inhibited by the antibodies to these adhesion proteins. Films precoated with IGF-1 strongly accelerated the adhesion of tenocytes to polymer. These results indicate that the specific adhesion of tenocytes to polymer can be promoted by coating extracellular matrix adhesive proteins and insulin-like growth factor1. It is of great importance to construct tissue-engineered tendon.
Biocompatible Materials
;
chemistry
;
Cell Adhesion
;
drug effects
;
physiology
;
Cells, Cultured
;
Extracellular Matrix Proteins
;
pharmacology
;
Growth Substances
;
pharmacology
;
Humans
;
Lactic Acid
;
chemistry
;
Polyglycolic Acid
;
chemistry
;
Polylysine
;
pharmacology
;
Polymers
;
chemistry
;
Tendons
;
cytology
;
embryology
;
physiology
;
Tissue Engineering
8.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
;
Cell Division/drug effects/genetics
;
Cell Line/cytology/physiology
;
Gene Expression/drug effects/immunology
;
Graves' Disease/immunology
;
Growth Substances/genetics/*pharmacology
;
Immunoglobulin G/pharmacology
;
Insulin-Like Growth Factor I/pharmacology
;
Myxedema/immunology
;
Proto-Oncogene Proteins c-fos/*genetics
;
Proto-Oncogene Proteins c-myc/*genetics
;
RNA/analysis
;
Rats
;
Rats, Inbred F344
;
Support, Non-U.S. Gov't
;
Thyroid Gland/cytology
;
Thyrotropin/pharmacology
;
Time Factors
9.Signaling pathway for 2,3,7,8-tetrachlorodibenzo- p-dioxin-induced TNF-alpha production in differentiated THP-1 human macrophages.
Hyeon Joo CHEON ; Young Seok WOO ; Ji Young LEE ; Hee Sook KIM ; Hyun Jin KIM ; Sungwon CHO ; Nam Hee WON ; Jeongwon SOHN
Experimental & Molecular Medicine 2007;39(4):524-534
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a prototypic halogenated aromatic hydrocarbon (HAH), is known as one of the most potent toxicants. At least a part of its toxic effects appears to be derived from its ability to induce TNF-alpha production. However, the signaling pathway of TCDD that leads to TNF-alpha expression has not been elucidated. In this study, we investigated the signaling mechanism of TCDD-induced TNF-alpha expression in PMA-differentiated THP-1 macrophages. TCDD induced both mRNA and protein expression of TNF-alpha in a dose- and time-dependent manner. Alpha-Naphthoflavone (NF), an aryl hydrocarbon receptor (AhR) inhibitor, prevented the TCDD-induced expression of TNF-alpha at both mRNA and protein levels. Genistein, a protein tyrosine kinase (PTK) inhibitor, and PD153035, an EGFR inhibitor, also blocked the increase of TNF-alpha expression by TCDD, indicating the role of EGFR in TCDD-induced TNF-alpha expression. On the other hand, PP2, a c-Src specific inhibitor, did not affect TCDD-induced TNF-alpha expression. EGFR phosphorylation was detected as early as 5 min after TCDD treatment. TCDD-induced EGFR activation was AhR-dependent since co-treatment with alpha-NF prevented it. ERK was found to be a downstream effector of EGFR activation in the signaling pathway leading to TNF-alpha production after TCDD stimulation. Activation of ERK was observed from 30 min after TCDD treatment. PD98059, an inhibitor of the MEK-ERK pathway, completely prevented the TNF-alpha mRNA and protein expression induced by TCDD, whereas inhibitors of JNK and p38 MAPK had no effect. PD153035, an EGFR inhibitor, as well as alpha-NF significantly reduced ERK phosphorylation, suggesting that ERK activation by TCDD was mediated by both EGFR and AhR. These results indicate that TNF-alpha production by TCDD in differentiated THP-1 macrophages is AhR-dependent and involves activation of EGFR and ERK, but not c-Src, JNK, nor p38 MAPK. A signaling pathway is proposed where TCDD induces sequential activation of AhR, EGFR and ERK, leading to the increased expression of TNF-alpha.
Animals
;
Benzoflavones/pharmacology
;
Cell Differentiation
;
Cell Line, Tumor
;
Enzyme Activation
;
Genistein/pharmacology
;
Hazardous Substances/*toxicity
;
Humans
;
MAP Kinase Signaling System/drug effects/physiology
;
Macrophages/*metabolism
;
Mice
;
Phosphorylation
;
Pyrimidines/pharmacology
;
Quinazolines/pharmacology
;
RNA, Messenger/metabolism
;
Receptor, Epidermal Growth Factor/antagonists & inhibitors/metabolism
;
Receptors, Aryl Hydrocarbon/antagonists & inhibitors
;
Signal Transduction
;
Tetrachlorodibenzodioxin/*toxicity
;
Tumor Necrosis Factor-alpha/*biosynthesis
;
src-Family Kinases/antagonists & inhibitors/metabolism
10.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
;
Blotting, Northern
;
Candida albicans/metabolism*
;
Cells, Cultured
;
Chemotactic Factors/genetics*
;
Dactinomycin/pharmacology
;
Dose-Response Relationship, Drug
;
Gene Expression Regulation/drug effects*
;
Growth Substances/genetics*
;
Interleukin-10/pharmacology*
;
Interleukin-10/metabolism
;
Macrophages/physiology*
;
Mice
;
Mice, Inbred BALB C
;
Nucleic Acid Synthesis Inhibitors/pharmacology
;
RNA, Messenger/metabolism
;
RNA, Messenger/drug effects