OBJECTIVEIn Corynebacterium crenatum, the adjacent D311 and D312 of N-acetyl-L-glutamate kinase (NAGK), as a key rate-limiting enzyme of L-arginine biosynthesis under substrate regulatory control by arginine, were initially replaced with two arginine residues to investigate the L-arginine feedback inhibition for NAGK.

METHODSNAGK enzyme expression was evaluated using a plasmid-based method. Homologous recombination was employed to eliminate the proB.

RESULTSThe IC50 and enzyme activity of NAGK M4, in which the D311R and D312R amino acid substitutions were combined with the previously reported E19R and H26E substitutions, were 3.7-fold and 14.6% higher, respectively, than those of the wild-type NAGK. NAGK M4 was successfully introduced into the C. crenatum MT genome without any genetic markers; the L-arginine yield of C. crenatum MT-M4 was 26.2% higher than that of C. crenatum MT. To further improve upon the L-arginine yield, we constructed the mutant C. crenatum MT-M4 proB. The optimum concentration of L-proline was also investigated in order to determine its contribution to L-arginine yield. After L-proline was added to the medium at 10 mmol/L, the L-arginine yield reached 16.5 g/L after 108 h of shake-flask fermentation, approximately 70.1% higher than the yield attained using C. crenatum MT.

CONCLUSIONFeedback inhibition of L-arginine on NAGK in C. crenatum is clearly alleviated by the M4 mutation of NAGK, and deletion of the proB in C. crenatum from MT to M4 results in a significant increase in arginine production.

Animals ; Arginine ; biosynthesis ; Corynebacterium ; genetics ; metabolism ; Escherichia coli ; Feedback, Physiological ; Gene Deletion ; Mutagenesis, Site-Directed ; Phosphotransferases (Carboxyl Group Acceptor) ; genetics ; Proline ; metabolism

Polythioesters newly emerged as a type of novel polymer and they have showed great potential for application in industries. In this study, genes of butyrate kinase (buk) and phosphotransbutyrylase (ptb) from Clostridium acetobutylicum, and poly (3-hydroxybutyrate) (PHB) synthase gene from Thiocapsa pfennigii were used for construction of a metabolic pathway to synthesize the polythioesters. When 3-mercaptopropionate and 3-hydroxybutyrate were fed, poly (3-mercaptopropoinate) [poly (3MP)] and poly(3-mercaptopropionate-co-3-hydroxybutyrate) [poly(3MP-co-3HB)] were synthesized by recombinant Escherichia coli JM109 (pBPP1) harboring the constructed metabolic pathway. Results indicated clearly that all these genes are necessary for the synthesis of poly(3MP) and poly(3MP-co-3HB).
3-Hydroxybutyric Acid ; chemistry ; 3-Mercaptopropionic Acid ; chemistry ; Acyltransferases ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Gas Chromatography-Mass Spectrometry ; Models, Biological ; Molecular Weight ; Phosphate Acetyltransferase ; genetics ; metabolism ; Phosphotransferases (Carboxyl Group Acceptor) ; genetics ; metabolism ; Plasmids ; Polymers ; chemistry ; metabolism ; Spectrophotometry, Infrared

OBJECTIVETo investigate the protective effects of protein kinase C (PKC) and mitogen-activated protein kinases (MAPKs) their and mechanisms in liver ischemic preconditioning.

METHODSIn rat models of liver ischemia-reperfusion (IR) and ischemic preconditioning (IP), the liver function was evaluated by examining serum alanine aminotransferase and aspartate aminotransferase levels, and the morphological changes of the liver cells were observed under microscope. PKC activator phorbol 12-myristate 13-acetate(PMA) and inhibitor chelerythrine(CHE), as well as MEK inhibitor PD98059, were utilized to analyze the phosphorylation of PKC and P44/42 MAPKs.

RESULTSCompared with the control rats, the liver function was best protected in rats of IP group, but not in those of IP group with PD98059 or CHE treatment. The rats in IR group showed improved liver function after PMA treatment. Similarly, the phosphorylation of PKC and P44/42 MAPKs was correlated with the liver function, and highly enhanced PKC and P44/42 MAPKs activity was observed in IP and IR+PMA groups, but decreased activity in IR and IP+CHE groups.

CONCLUSIONPhosphorylation of PKC and MAPKs plays a pivotal role in the preservation of the hepatocytes during IP.

Alanine Transaminase ; blood ; Alkaloids ; pharmacology ; Animals ; Aspartate Aminotransferases ; blood ; Benzophenanthridines ; pharmacology ; Enzyme Inhibitors ; pharmacology ; Flavonoids ; pharmacology ; Ischemic Preconditioning ; Liver ; blood supply ; enzymology ; Male ; Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; metabolism ; Phosphorylation ; drug effects ; Protein Kinase C ; antagonists & inhibitors ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; blood ; physiopathology ; Tetradecanoylphorbol Acetate ; pharmacology

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 ; metabolism ; Acetates ; metabolism ; Culture Techniques ; Escherichia coli ; enzymology ; genetics ; metabolism ; Glucosephosphate Dehydrogenase ; metabolism ; Mutation

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 ; Cell Line* ; Humans* ; Ovarian Neoplasms* ; Protein Kinase C* ; Protein Kinases* ; RNA ; RNA, Messenger ; Telomerase* ; Tetradecanoylphorbol Acetate

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 ; Endopeptidases ; Extracellular Matrix ; Matrix Metalloproteinases ; Neoplasm Metastasis* ; Phosphotransferases ; Protein Kinase C ; Response Elements ; RNA, Messenger ; Signal Transduction ; Tetradecanoylphorbol Acetate ; Transfection ; Vitis

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 ; Doxycycline ; Epithelial Cells ; Gelatin ; Humans ; Immunoenzyme Techniques ; Inflammation ; Matrix Metalloproteinase 9 ; Mucins ; Phorbols ; Phosphorylation ; Protein Kinase C ; RNA, Messenger ; Signal Transduction ; Tetradecanoylphorbol Acetate ; Thiram

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

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

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