1.Study on the metabolism of Escherichia coli DH5alpha and its acetate-tolerant mutant DA19 based on key enzyme activity analysis.
Xiao-Yun ZHANG ; Yan-Jun ZHANG ; Zhi-Min LI ; Qin YE
Chinese Journal of Biotechnology 2007;23(5):896-901
The differences in metabolism in Escherichia coli DH5alpha and its acetate-tolerant mutant DA19 were analyzed based on the activity of key enzymes involved in central metabolism when both strains were continuously cultured in nitrogen source-limited defined media. The activity of glucose-6-phosphate dehydrogenase (G6PDH) and isocitrate dehydrogenase (ICDH) in DA19 increased as compared with those in DH5alpha, while acetate kinase (ACK) and phosphofructosekinase (PFK) decreased. These indicated that more carbon flux of DA19 entered the phosphopentose pathway (PPP) and less entered the glycolytic (EMP) pathway and acetic acid production (Ack-Pta) pathway. Therefore, the differences in activity of key enzyme coincided with increased cell yield based on consumed glucose (Y(X/G)) and decreased production of acetic acid and pyruvate of DA19. G6PDH and ICDH in DH5alpha were up-regulated by addition of adenine, while ACK and PFK were down-regulated. On the other hand, adenine had little effect on those in DA19. The enzymes except PFK in both strains were down-regulated by sodium acetate, especially the activity of ICDH in DH5alpha. These results suggested changed flux of central metabolic pathways were also consistent with the changes of growth properties and byproducts formation.
Acetate Kinase
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metabolism
;
Acetates
;
metabolism
;
Culture Techniques
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Escherichia coli
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enzymology
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genetics
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metabolism
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Glucosephosphate Dehydrogenase
;
metabolism
;
Mutation
2.Changes of Telomerase Activity by Protein Kinase C Modulators in Human Ovarian Cancer Cell Lines.
Soo Young HUR ; Joon Mo LEE ; Sung Eun NAMKOONG ; Jin Woo KIM
Journal of the Korean Cancer Association 2000;32(4):724-733
PURPOSE: This study was designed to find out whether protein kinase C (PKC) may affect telomerase activity in human ovarian cancers. MATERIALS AND METHODS: To determine whether PKC modulators influence PKC activities, NIH: OVCAR-3 and CUMO-2, cells were treated with PKC inhibitors, G 6976 and bisindolyl maleimide I, and PKC activator, 12-O-tetradecanoyl phorbol 13-acetate (TPA). Telomerase acti vity was determined by telomeric repeat amplification protocol (TRAP). Analysis of the expres sion of each telomerase subunits, human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT), was performed by RT-PCR. We also examined the alternative splicing of hTERT. RESULTS: G 6976 and bisindolylmaleimide I inhibited PKC activity. Telomerase activities appeared to be affected in a time-dependent manner by these two PKC inhibitors. PKC activities were increased in parallel with telomerase activity by TPA at the low dose (10 nM), but their activities were down-regulated at the high dose (1 micrometer). RT-PCR demonstrated the presence of hTR and hTERT mRNA before and after the treatment of PKC modulators, respectively, and showed the presence of one alternatively spliced transcript and full-length hTERT transcripts. CONCLUSION: These results showed that telomerase activity was affected by PKC and suggested PKC modulation may serve as an useful tool in the regulation of telomerase activity.
Alternative Splicing
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Cell Line*
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Humans*
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Ovarian Neoplasms*
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Protein Kinase C*
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Protein Kinases*
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RNA
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RNA, Messenger
;
Telomerase*
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Tetradecanoylphorbol Acetate
3.Expressional Regulation of Cancer Metastasis Related Gene, MMP-9, by Resveratrol.
Ju Hyung WOO ; Young Ho KIM ; Won Ki BAEK ; Seong Il SUH ; Min Ho SUH ; Jong Wook PARK ; Taeg Kyu KWON
Journal of Bacteriology and Virology 2003;33(3):235-243
Proteolytic degradation of the extracellular matrix and tumor metastasis correlate with the expression of endopeptidases known as matrix metalloproteinases (MMPs). Expression of MMPs is regulated by cytokines and signal transduction pathways including those activated by phorbol myristate acetate (PMA). We found that resveratrol, a phytoalexin present in grapes, significantly inhibits the PMA-induced increase of MMP-9 expression and activity. These effects of resveratrol were dose-dependent and correlated with the suppression of MMP-9 mRNA expression levels. PMA caused a 23-fold increase in MMP-9 promoter activity, which was suppressed by resveratrol. Transient transfection by MMP-9 constructs, in which specific transcriptional factors were mutagenized, indicated that the effects of PMA and resveratrol were mediated via AP1 and NFkB response elements. Resveratrol inhibited PMA-mediated activation of c-Jun Nterminal kinase (JNK) and protein kinase C (PKC)-delta activation. Therefore, we conclude that the inhibitory activities of resveratrol on MMP-9, JNK, and PKC-delta may have therapeutic potential for controlling growth and invasiveness of tumors.
Cytokines
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Endopeptidases
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Extracellular Matrix
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Matrix Metalloproteinases
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Neoplasm Metastasis*
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Phosphotransferases
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Protein Kinase C
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Response Elements
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RNA, Messenger
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Signal Transduction
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Tetradecanoylphorbol Acetate
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Transfection
;
Vitis
4.The Effect of Doxycycline on PMA-Induced MUC5B Expression via MMP-9 and p38 in NCI-H292 Cells.
Chang Hoon BAE ; Seung Min CHEN ; Heung Man LEE ; Si Youn SONG ; Yong Dae KIM
Clinical and Experimental Otorhinolaryngology 2011;4(4):177-183
OBJECTIVES: Doxycycline is commonly used in medicine for its bacteriostatic antimicrobial properties. Recent studies have reported that doxycycline also has anti-inflammatory effects. Matrix metalloproteinase (MMP)-9 has been found to be involved in the physiological and pathological process of inflammatory airway disease. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, is known to stimulate the expression of MMP and mucin genes in the airway and intestinal epithelial cells. Therefore, the effects and signal pathways of doxycycline on PMA-induced MUC5B expression dependent MMP-9 in human airway epithelial cells were investigated. METHODS: In human NCI-H292 airway epithelial cells, MUC5B and MMP-9 mRNA expression, MUC5B protein expression, and MMP-9 protein activity after the treatment with PMA, MMP-9 or doxycycline were determined by reverse transcriptase-polymerase chain reaction, enzyme immunoassay, gelatin zymography, and Western blot analysis. RESULTS: PMA increased MMP-9 and MUC5B expression. MMP-9 increased MUC5B expression. Doxycycline inhibited PMA-induced MUC5B expression, and PMA-induced MMP-9 mRNA expression and protein activity. Doxycycline inhibited phosphorylation of p38 induced by PMA and MMP-9. CONCLUSION: The results of this study suggest that doxycycline inhibited PMA-induced MUC5B mRNA expression and protein production through the MMP-9 and p38 pathways in human NCI-H292 airway epithelial cells.
Blotting, Western
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Doxycycline
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Epithelial Cells
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Gelatin
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Humans
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Immunoenzyme Techniques
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Inflammation
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Matrix Metalloproteinase 9
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Mucins
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Phorbols
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Phosphorylation
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Protein Kinase C
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RNA, Messenger
;
Signal Transduction
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Tetradecanoylphorbol Acetate
;
Thiram
5.Effects of protein kinase C activity on CD44 expression and subcellular distribution in human erythrocytes.
Yi-Wen HAO ; Da-Ye CHENG ; Jin-Tao CHEN
Journal of Experimental Hematology 2009;17(4):933-937
The study was aimed to investigate the effects of protein kinase C (PKC) on standard type CD44 expression and subcellular distribution in human erythrocytes. PKC activity was detected by the incorporation of [gamma-(32)P]-ATP into exogenous substrate, phosphorylation of CD44 was determined by autoradiograph, distribution of CD44 was observed by indirect immunofluorescence, and expression of CD44 was analyzed by flow cytometry. The results showed that PKC activity reached the maximal level at 30 minutes after treatment with phorbol-myristate-acetate (PMA), and the peak of CD44 phosphorylation and CD44 expression appeared at the same time, which all increased significantly as compared with control group (p < 0.001). PKC activation resulted in CD44 aggregation on membrane and colocalization of PKC and CD44. Calphostin C could inhibit the above reaction resulted from PKC activation. It is concluded that PKC activation can up-regulate CD44 expression by phosphorylation, and result in the coherent migration and colocalization of CD44 and PKC in human erythrocytes.
Erythrocyte Count
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Erythrocytes
;
enzymology
;
metabolism
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Humans
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Hyaluronan Receptors
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metabolism
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Membrane Proteins
;
metabolism
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Phosphorylation
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Protein Kinase C
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metabolism
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Tetradecanoylphorbol Acetate
;
analogs & derivatives
;
Up-Regulation
6.The effect of PKC phosphorylation sites mutation in JWA coding region on TPA-induced MCF-7 cell differentiation.
Jian YE ; Ai-ping LI ; Wen QIU ; Jin-hong ZHOU ; Jian-wei ZHOU
Chinese Journal of Industrial Hygiene and Occupational Diseases 2007;25(7):398-401
OBJECTIVETo investigate the effect of PKC phosphorylation sites mutation in JWA coding region on TPA-induced MCF-7 cell differentiation.
METHODSSite directed gene mutation was used to construct one or two PKC sites mutations in pEGFP-N1-JWA vectors, and transfected into MCF-7 cells by polyfect reagent, and cell differentiation was characterized by accumulation of lipid droplet as indicated by positive Oil-red-O staining of cells.
RESULTSAll these transfected cell lines, MCF-7-N1(transfected with pEGFP-N1 vector), MCF-7-JWA(transfected with pEGFP-N1-JWA vector), MCF-7-JWA-1(transfected with PKC site 1 mutation pEGFP-N1-JWA vector), MCF-7-JWA-2(transfected with PKC site 2 mutation pEGFP-N1-JWA vector), MCF-7-JWA-1+2 (transfected with both PKC site 1 and 2 mutation pEGFP-N1-JWA vector) were treated with 20 nmol/L TPA for 48 h, and the percentages of positive Oil-red-O staining of cells were 48%, 67%, 69%, 67% and 70% respectively. The percentages of cell differentiation in JWA containing vectors transfected cells treated with TPA were significantly higher those of MCF-7-N1 cells (vector only control). However there were no significant differences between mutated and unmutated cells.
CONCLUSIONJWA transfection enhanced MCF-7 cell differentiation induced by TPA significantly, and PKC sites mutation in JWA coding region has no obviously effect on TPA-induced MCF-7 cell differentiation.
Cell Differentiation ; drug effects ; Cell Line, Tumor ; Female ; Genetic Vectors ; Humans ; Mutagenesis, Site-Directed ; Phosphorylation ; Point Mutation ; Protein Kinase C ; genetics ; metabolism ; Tetradecanoylphorbol Acetate ; pharmacology ; Transfection
7.Effect of protein kinase C/transforming growth factor beta 1 pathway on activation of hepatic stellate cells.
Tao LI ; Xi-Sheng LENG ; Ji-Ye ZHU ; Yan-Tong GUO ; Yu-Hua WEI
Chinese Journal of Hepatology 2007;15(12):902-905
OBJECTIVETo investigate the effect of protein kinase C (PKC)/transforming growth factor beta 1 (TGF beta1) pathway on activation of hepatic stellate cells (HSC).
METHODSHSC rHSC-99 cell line was used in three groups in this study. Group A served as a control. In group B the HSC were incubated with PKC agonist PMA (0.5 micromol/L), and in group C the cells were incubated with PKC inhibitor calphostin C (100 nmol/L). The PKC activities were detected at different incubation time points (0, 3, 6, 12 and 24 h). Western blot and RT-PCR were used to detect the expression of TGF beta1, Smad 4, collagen type I, III and alpha-smooth muscle actin (alpha-SMA) at the 24 h point. Cell proliferation was assessed by MTT colorimetric assay.
RESULTSPMA increased the activity of PKC significantly, whereas calphostin C inhibited the activity of PKC. The increased activity of PKC promoted the HSC to express TGF beta1, Smad 4, collagen type I, III and alpha-SMA. In comparison with the controls, the expressions of TGF beta1, Smad 4, collagen type I, III and alpha-SMA increased 4.8, 13.1, 2.4, 1.8 and 1.3 fold respectively (P < 0.01). PKC promoted the proliferation of HSC. The above effects were inhibited by the inhibition of PKC activity.
CONCLUSIONChanging of PKC activity can regulate and control the expression of TGF beta1, which may play a role in regulating the activation of HSC.
Animals ; Cell Line ; Hepatic Stellate Cells ; metabolism ; Protein Kinase C ; metabolism ; Rats ; Signal Transduction ; Tetradecanoylphorbol Acetate ; Transforming Growth Factor beta1 ; metabolism
8.Epstein-Barr virus induces human nasopharyngeal epithelial cells to escape from the replicative senescence.
Jing YANG ; Faqing TANG ; Huanhua GU ; Xiyun DENG ; Xinxian WENG ; Min TANG ; Ya CAO
Chinese Medical Journal 2002;115(6):803-809
OBJECTIVETo observe the biological changes of primary human nasopharyngeal epithelial cells in the early stage of immortalization.
METHODSThe morphological changes of nasopharyngeal epithelial cells were observed by phase contrast microscopy, and the activity profile of senescence-associated beta-galactosidase (SA-beta-Gal) was detected by SA-beta-Gal staining. The expression of p16(INK4a) protein was tested by immunochemical assay, and the life span in vitro of nasopharyngeal epithelial cells was calculated as population doublings. In addition, the expression of Epstein-Barr (EB) virus latent membrane protein 1 (LMP1) was also detected by immunofluorescence staining.
RESULTSMorphologically, cells treated with EB virus and 12-o-tetradecanoyl-phorbol-13-acetate (TPA) formed multi-layer foci, and their cellular life span in vitro was extended (about 155 days of culture). A low percentage of cells (about 4.8%) expressed SA-beta-Gal activity at late primary culture, and did not always express p16(INK4a) protein in the progression of culture.
CONCLUSIONSNasopharyngeal epithelial cells treated with EB virus in cooperation with TPA can pass through the stage of senescence and enter the early stage of immortalization. Some changes of phenotype occur in these cells. Our results provide data for further studying the mechanism of immortalization and the establishment of a human nasopharyngeal epithelial cell line.
Cell Transformation, Viral ; Cellular Senescence ; Cyclin-Dependent Kinase Inhibitor p16 ; analysis ; Epithelial Cells ; physiology ; virology ; Herpesvirus 4, Human ; physiology ; Humans ; Nasopharynx ; cytology ; virology ; Tetradecanoylphorbol Acetate ; pharmacology
9.Arginine vasopressin stimulates proliferation of adult rat cardiac fibroblasts via protein kinase C-extracellular signal-regulated kinase 1/2 pathway.
Yan-Ping HE ; Lian-You ZHAO ; Qiang-Sun ZHENG ; Shao-Wei LIU ; Xiao-Yan ZHAO ; Xiao-Long LU ; Xiao-Lin NIU
Acta Physiologica Sinica 2008;60(3):333-340
Arginine vasopressin (AVP), a neurohormone and hemodynamic factor implicated in the pathophysiology of hypertension and congestive heart failure, can also act as a growth-stimulating factor. Our previous work demonstrated that AVP is a mitogen for neonatal rat cardiac fibroblasts (CFs). In the present study, we extended our investigations to adult rat CFs to explore whether AVP could induce adult rat CF proliferation and, if so, to identify the mechanism involved. Adult rat CFs were isolated, cultured and subjected to AVP treatment. DNA synthesis and cell cycle distribution were analyzed by [(3)H]-thymidine incorporation and flow cytometry. Cellular extracellular signal-regulated kinase 1/2 (ERK1/2) activity was measured by in vitro kinase assay using myelin basic protein (MBP) as a substrate. Protein expressions of total- and phospho-ERK1/2, p27(Kip1), cyclins D1, A, E were assessed by Western blot. The results showed that AVP stimulated DNA synthesis in adult rat CFs, and the effect was abolished by a V1 receptor antagonist, d(CH(2))(5)[Tyr(2)(Me), Arg(8)]-vasopressin (0.1 μmol/L), but not by a V2 receptor antagonist, desglycinamide-[d(CH(2))(5), D-Ile(2), Ile(4), Arg8]-vasopressin (0.1 μmol/L). AVP induced an activation of ERK1/2, which could be mimicked by the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA, 30 nmol/L, 5 min), but abolished by depletion of PKC via chronic PMA incubation (2.5 μmol/L, 24 h). In addition, AVP down-regulated protein expression of p27(Kip1), increased protein expressions of cyclins D1, A and E, and induced cell cycle progression from G(0)/G(1) into S stage. Inhibition of ERK1/2 activation by PD98059 (30 μmol/L) abolished the effect of AVP on DNA synthesis, protein expressions of p27(Kip1), cyclins D1, A and E as well as cell cycle progression. These results suggest that AVP is also a growth factor for adult rat CFs. The mitogenic effect of AVP is mediated via V1 receptors and PKC-ERK1/2 pathway. Moreover, AVP modulates the expressions of cell cycle regulatory proteins p27(Kip1) and cyclins D1, A and E, which lie downstream of ERK1/2 activation, and induces cell cycle progression in adult rat CFs.
Animals
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Antidiuretic Hormone Receptor Antagonists
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pharmacology
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Arginine Vasopressin
;
pharmacology
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Cell Cycle
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Cell Cycle Proteins
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metabolism
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Cell Proliferation
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Fibroblasts
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cytology
;
drug effects
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Mitogen-Activated Protein Kinase 3
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metabolism
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Myocardium
;
cytology
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Phosphorylation
;
Protein Kinase C
;
metabolism
;
Rats
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Signal Transduction
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Tetradecanoylphorbol Acetate
;
pharmacology
10.Cardioprotective Effect of Ischemic Preconditioning: Relationship between Intracellular Glycogen and Protein Kinase C.
Ho Dirk KIM ; Hyun KIM ; Bong Jin RAH ; Myeong Ho KIM ; Chul Woo KIM ; Hye Won KIM ; Dae Joong KIM ; Young Bae KIM
Korean Circulation Journal 2001;31(1):5-15
BACKGROUND: Recent studies suggest that the cardioprotective effect of ischemic preconditioning (IPC) is related to intracellular glycogen content in rat hearts, however, controversies still remain. METHODS: To test this hypothesis, isolated Langendorff-perfused rabbit hearts were subjected to 45 min global ischemia followed by 120 min reperfusion with IPC (n=0) or without IPC (ischemic control, n=). IPC was induced by one cycle of 5 min global ischemia and 10 min reperfusion. In the glucose (G)-free preconditioned group (n=0), G depletion-repletion was induced by perfusion with G-free Tyrode solution for 5 min and then G-containing Tyrode solution for 10 min followed by 45 min ischemia and 120 min reperfusion. For glycogen depletion or loading, hearts were treated with sodium acetate (NA, 5 mM, n=) or insulin (Ins, 1 unit/L, n=) for 15 min before 45 min ischemia. Left ventricular function and coronary flow (CF) were continuously recorded during experiments. Myocardial cytosolic and membrane protein kinase C (PKC) activities were measured by 32P-gamma-ATP incorporation into PKC-specific pepetide; glycogen content in the cardiac myocytes was determined by spectrophotometry with amyloglucosidase; expression of PKC isozymes was determined by Western blot with monoclonal antibodies. Infarct size was determined by staining with tetrazolium salt and planimetry. Data were analyzed by ANOVA and Tukey's post-hoc test. RESULTS: IPC or G-free preconditioning enhanced LV functional recovery; NA did not influence on functional recovery but Ins depressed it. Infarct size was significantly reduced by IPC, G-free preconditioning, and NA treatment (35.3+/-2.1% in the ischemic control, 18.7+/-1.2% in the IPC, 22.1+/-1.2% in the G-free preconditioned, 16.3+/-1.2% in the NA-treated group, and 32.8+/-1.6% in the Ins-treated group, p<0.05). Membrane PKC activities significantly increased by IPC, IPC and 45 min ischemia, G-free preconditioning, and G-free preconditioning and 45 min ischemia; especially, expression of membrane PKC-epsilon increased by IPC and G-free preconditioning. Glycogen content decreased by 45 min ischemia, IPC, G-free preconditioning, and by NA treatment, but increased by Ins treatment. CONCLUSION: These results suggest that in rabbit heart, intracellular glycogen may not significantly be related with the cardioprotective effect of IPC; G-free preconditioning could not improve post-ischemic contractile dysfunction but it has an infarct size-limiting effect; this cardioprotective effect may be related in part to activation of PKC, especially epsilon isozyme.
Animals
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Antibodies, Monoclonal
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Blotting, Western
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Cytosol
;
Glucan 1,4-alpha-Glucosidase
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Glucose
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Glycogen*
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Heart
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Insulin
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Ischemia
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Ischemic Preconditioning*
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Isoenzymes
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Membrane Proteins
;
Membranes
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Myocytes, Cardiac
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Perfusion
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Phosphotransferases
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Protein Kinase C*
;
Protein Kinases*
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Rats
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Reperfusion
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Sodium Acetate
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Spectrophotometry
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Ventricular Function, Left