Exposure to power-frequency magnetic fields can induce activation of P38 mitogen-activated protein kinase / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To study the effects of 50 Hz power-frequency magnetic fields on signal transduction pathway of P38 mitogen-activated protein kinase (P38 MAPK), and explore the cellular signal transduction mechanism of the biological effects induced by power-frequency magnetic fields.</p><p><b>METHODS</b>Chinese hamster lung (CHL) cell line was exposed to power-frequency magnetic fields with two intensities(0.1 and 0.4 mT) for different exposure durations. The cytoplasmic protein was extracted. The phosphorylated(activated) and non-phosphorylated P38 MAPK and MKK3/MKK6 were measured by Western blotting analysis with their specific corresponding antibodies.</p><p><b>RESULTS</b>Power-frequency magnetic fields at 0.4 mT for 10 min could transitorily induce the activation of P38 MAPK and after 15 min the phosphorylation of P38 MAPK restored to control level, while 0.1 mT power-frequency magnetic fields could not induce the activation of P38 MAPK within 24 h. However, both 0.1 mT and 0.4 mT power-frequency magnetic fields could not phosphorylate(activate) the MKK3/MKK6, which is a general upstream kinase of P38 MAPK.</p><p><b>CONCLUSION</b>Power-frequency magnetic fields could transitorily activate the P38 MAPK, but not MKK3/MKK6. The activation mechanism of P38 MAPK needs to be further identified.</p>

Effects of power-frequency magnetic fields exposure on phosphorylation and enzymatic activity of stress-activated protein kinase and its upstream kinase / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To study the effects of 50 Hz power-frequency magnetic fields on signal transduction pathway of stress-activated protein kinase(SAPK), and explore the cellular signal transduction mechanism of the biological effects induced by power-frequency magnetic fields.</p><p><b>METHODS</b>Chinese hamster lung(CHL) cell line was exposed to power-frequency magnetic fields with two intensities for different exposure durations. The cytoplasmic protein was extracted and the phosphorylated portion of SAPK and SEK1/MKK4 was measured with Western blotting analysis. The SAPK enzymatic activity was measured by the solid-phase kinase assay in cells exposed to power-frequency magnetic fields for 15 min.</p><p><b>RESULTS</b>Both 0.4 mT and 0.8 mT power-frequency magnetic fields could enhance the phosphorylation of SAPK in a time-relative course manner, and reached the maximum extent at 15 min, with an increase of 20% and 17% respectively. The solid-phase kinase assay showed that the enzymatic activities of SAPK were also increased, which were 2.9 +/- 0.4 and 2.1 +/- 0.9 times of control respectively. However, the duration of SAPK phosphorylation induced by 0.8 mT was longer than that of 0.4 mT, while the duration and extent of SAPK dephosphorylation was remarkably shorter than that of 0.4 mT. The power-frequency magnetic fields under equal conditions could not phosphorylate(activate) the SEK1/MKK4.</p><p><b>CONCLUSION</b>Power-frequency magnetic fields could activate the SAPK, but not SEK1/MKK4. It is suggested that power-frequency magnetic fields may activate SAPK signal transduction pathway through a kinase other than SEK1/MKK4. The activation mechanism of SAPK of power-frequency magnetic fields needs to be identified in more detail.</p>

Researches on the polymorphism of cytochrome P450 2A6 / 中华医学遗传学杂志

Cytochrome P450 2A6(CYP2A6) is known as a major enzyme responsible for C-oxidation of nicotine and 7-hydroxylation of coumarin. The article reviews different alleles of CYP2A6 that have been discovered, their effect on CYP 2A6 activity and the relationship between genetic polymorphism of CYP2A6 and smoking behavior as well as susceptibility of lung and esophageal cancer in different individuals.

Translesion Synthesis DNA Polymerase: A Novel DNA Polymerase / 生物化学与生物物理进展

although there are many repair pathways in cells, some lesions still escape repair inevitably and remain in genome. In cells, the molecular mechanism of translesion DNA synthesis has been one of the major unsolved problems in DNA repair for a long time. Recently, it was found that the members of a structurally related UmuC/DinB protein superfarnily have DNA polyrnerase function. Unlike the classical replicative DNA polymerases, these newly identified DNA polymerases can carry out translesion DNA synthesis in both error prone/mutagenic and/or error-free ways. It was also found that their functions are conserved from bacteria to human.

Cloning of a new human cytochrome P450 2A6 cDNA / 中国病理生理杂志

AIM:To clon human cytochrome P450 2A6 cDNA. METHODS:Using reverse transcription polymerase chain reaction(RT-PCR) and DNA recombinant technique, a full-length cDNA encoding cytochrome P450 2A6( CYP2A6 ) from human liver was cloned into pBluescript vector. The cDNA segment was identified by DNA sequencing.RESULTS: Comparing with the CYP2A6 sequence, the cloned CYP2A6 cDNA had two different bases, codon 8 CTG(Leu)→TTG(Leu), codon 479 GGC(Gly)→GTC(Val) and was quite different in their 3′end noncoding region. Comparing with CYP2A7 seqence reported by Fernandez-Salguero,the cloned CYP2A6 cDNA had some different in 5′ end coding sequence and several differencey in the 3′ end coding and noncoding sequence, but both codon 479 were GTC(Val).Comparing with the CYP2A7 seqence reported by Yamano,the cloned CYP2A6 cDNA had some difference in the coding sequence but the 3′ end no-coding area was the same. CONCLUSION: The cloned cDNA was a new cDNA of CYP2A6 which may be transcripted from a new allele of CYP2A6.

The antisense block of CROC -1 gene expression makes cells grow slower / 中国病理生理杂志

AIM:To establish FL- CROC -1 - cell line in which CROC -1 gene expression was blocked and study the role of CROC -1 gene in cell growth. METHODS:The appropriate length cDNA fragment of the recently identified human gene CROC -1 which encodes ubiquitin-conjugating enzyme like protein(Ubc-like protein) was cloned into the reconstructed eukaryotic expression vector pMAMneo-amp - by antisense strategy. The recombinant plasmid which can express CROC -1 antisense RNA was selected by restriction enzyme map analysis. The antisense expression recombinant plasmid pMAM-anti CROC -1 was then transfected into human amnion FL cells by a modified calcium phosphate-mediated transfection procedure and selected with MEM medium containing 400 ?g/mL geneticin. Finally ,the growth rate of the G418 resistant FL- CROC -1 - cell line was determined. RESULTS:When the antisense inhibition of CROC -1 gene expression was induced by dexamethasone, the growth rate of the FL- CROC -1 - cell line was obviously slower as compared with that of the control FL cell and FL-MAMneo cell which was established by transfection of plasmid pMAMneo-amp - ( P

Applications of mass spectrometry in proteomics / 中国病理生理杂志

As a crucial technique for proteome analysis, mass spectrometry (MS) has developed rapidly in the last two decades. MS has established itself as a powerful tool in life science by its high sensitivity, resolution, mass accuracy and the ability to be coupled with chromatogram and isotope labeling. This review discusses the principles and instrumentation developments of MS, especially focuses on the applications of MS in proteomic studies.

Analysis of the transcription factor binding sites in the promoter region of genes encoding the co-expressive proteins induced by N-methyl-N'-nitro-N-nitrosoguanidine / 中国病理生理杂志

AIM: To find out common transcription factor binding sites in the promoter regions of the encoding genes of the co-expressive proteins induced by N-methyl-N'-nitro-N- nitrosoguanidine (MNNG). METHODS: Using phylogenetic footprinting and TRANSFAC position weight matrix (PWM) searching program to predict the common transcription factor binding sites among the promoter regions of the genes encoding the co-expressive proteins. The predictive results were validated with electrophoresis mobility shift assay (EMSA). RESULTS: Eleven common transcription factor binding sites were predicted in the promoters of the co-expressive proteins, among them, besides the activator protein 1(AP1) which was previously identified to be activated in MNNG pretreated cells in this laboratory, the nuclear factor Y (NFY) and GATA binding factor (GATA) consensus oligonucleotides binding activity were found being increased in the nuclear extract of cells pre-treated with MNNG as demonstrated by EMSA. CONCLUSION: Phylogenetic footprinting can effectively decrease the false positive rate in predicting transcription factor binding sites. It is possible that NFY and GATA transcription factor binding sites are involved in the co-regulation of the MNNG induced co- expressive proteins. [

Using antisense nucleic acid technology to study the influence of POL? on genetic stability / 中国病理生理杂志

AIM: To establish cell line FL-POL? - and to study the role of POL?(polymerase kappa) on genetic stability. METHODS: A mammalian expression vector expressing antisense POL? gene fragment pMAMneo -amp -- POL? was constructed by cloning the 1 690-1 918 fragment of POL? gene into the mammalian expression vector pMAMneo-amp - in antisense orientation. FL cells were fransfected with this antisense RNA expressing vector and selected by G418. Based on the shuttle-plasmid pZ189, the mutation assay was made. RESULTS: The spontaneous mutation frequency of supF tRNA gene in the plasmid replicated in the FL-POL? - was 11.2?10 -4 , while it was 4.9?10 -4 and 3.7?10 -4 in the control cells FL and FL-M , respectively. CONCLUSION: POL? playes an important role in maintenance of genetic stability.

Nifedipine induced reversal of multidrug resistance can be deteriorated by S9 mix prepared from the transgenic cell line CHL-3A4 / 中国病理生理杂志

AIM:To demonstate the expression of human cytochrome P450 3A4 isozyme in the transgenic cell line CHL - 3A4 established in this laboratory. METHODS:Nifedipine (NIF) can induce the reversal of the brug resistance for adriamycin of multidrug resistant(MDR) cell K562r. The NIF is the specific substlate for CYP3A4. To determine the NIF oxidase activity of the transgenic CHL - 3A4 cells by comparing the biological effect of NIF, which preinculbated with or without CHL - 3A4 S9 mix. RESULTS: When the multidrug resistance cell K562r was cultured in medium with NIF (12. 5 ?g? L- 1 ), a calcium channel blocker and specific substrate for CYP3A, it's IC50 for adri- amycin declined from(6.47?0.60) mg ? L- 1 to (0. 89?0. 15) mg? L-1. When cells were cultured in NIF(exactly the same concentration ) pretreated with CHL - 3A4 S9 mix,no reversal of MDR was observed [IC50 for adriamycin is (6.10?0.50) mg? L-1 ] while if NIF was pretreated with CHL S9 or inactivated CHL - 3A4 S9 mix, its biological effect was not deteriorated [IC50 for adriamycin is (0. 32?0.90) and (0.32?0.04) mg?L-1, P

HPLC analysis of 5-methylcytosine contents in DNAs isolated from 5-azacytidine and MNNG treated cells / 中国病理生理杂志

The 5-methylcytosine (~mC) in DNAs from 5-azacytidine and MNNG treated FL, Wish and Veto-E6 ceUs were analysed by HPLC. In 2?10~(-6)mol/L 5-azaCR treated cells, the percentages of ~mC in total cytosine were all lowered significantly (P 0.05). These results were in good agreement with those obtained by radioactivity analysis of newly replicated DNA fragments from Hpa Ⅱ digest. These results further validate the idea that DNA hypomethylation as a general pathway in the initiation process of chemical carcinogenesis is based on the results obtained by a defectively designed experiment.

Regulation of cytochrome P450 genes by liver-enriched transcription factors / 中国病理生理杂志

Cytochrome P450 (CYP) is a complex gene superfamily of proteins that metabolizes a myriad of endogenous and exogenous substrates. Liver-enriched transcription factors (LETF) play a role in the constitutive and tissue-specific expression of hepatic genes. In this review, six families of LETF that play a role in the tissue-specific, developmental, sexual and temporal regulation of CYP are discussed.

Establishment of cell lines whose hREV3 gene expression was inhibited by transfection of antisense RNA expression plasmids and their biological characteristics / 中国病理生理杂志

AIM: to establish the cell lines whose hREV3 gene expression was blocked by antisense RNA and observe their characteristis of cell growth rate and N - methyl - N' - nitrosoguanidine (MNNG) sensitivi- ty . METHODS: With modified calcium phosphate - DNA coprecipitation method the eukaryocytic expression plasmid expressing antisense fragment of hREV3,pBK - RSV - hREV3- and pMAMneo - amp hREV3 were transfected into human embryo kidney cell line of HEK - 293. After G418 selection, cell lines of 293 - B - hREV3- and 293 - M hREV3- were established. By cell counting method, the cell growth rate and MNNG sensitivity of these cell lines were characterized. RESULTS: No change of cell growth rates of these cell lines was observed whether hREV3 gene expres- sion was blocked by either the persistent or induced expression of the antisense hREV3 RNA. While the sensitivity of these cell lines to MNNG was somewhat elevated, as compared with their parent cell line 293 and the cell lines trans- fected with vector DNAs. CONCLUSION: The gene product of hREV3 was not essential for the cell growth, but it may play a role in the DNA repair functions of the cells after exposure to DNA damaging agents.

Advances of ubiquitin-conjugating pathway in eukaryotes / 中国病理生理杂志

A Review] A large quantity of intracellular structural and regulatory proteins can be covalently attached to ubiquitin posttranscriptionally and thus modified. The ubiquitination of proteins serves as targeting signals, making ubiquitinated proteins distributed to different parts of the cells, changing the activities, the interaction between macromolecules and the half-life of proteins. Therefore, ubiquitin conjugation is implicated in numerous metabolic processes in eukaryotes. Ubiquitin conjugates its protein substrates via a cascade reaction. This paper is a review of recent advances in this area.

Expression of GST fusion proteins of human cytochrome P 2B6 and preparation of anti-cytochrome P2B6 polyclonal antibody / 中国病理生理杂志

AIM: To get large amounts of pure antigens to raise specific antibodies and to perform quantifications.METHODS: CYP2B6 (cytochrome P) cDNA fragments was ligated into BamHI restricted PGEX-3b to generate recombinants PGEX/2B6. We identified recombinants PGEX/2B6 by EcoRI digestion. The expression of fusion proteins were induced by adding isopropyl-thiogalactoside(IPTG). Several clones showed high-level expression of fusion proteins. Insoluble proteins was isolated from the bacteria and the fusion proteins was recovered and purified from a preparative (2mm) SDS-PAGE. The polyarcrylamide gel containing the fusion proteins glutathione S-transferase(GST-2B6) were used to immunize BALB/C mice from which polyclonal ascites fluid was prepared. The purified fusion proteins GST-1A1(GST fusion protein of CYP1A1 cDNA246～386aa expressed in this library ,purified by preparative SDS-PAGE), GST-2B6 were used to test the specificity of 2B6pAb. RESULTS:Fusion proteins constructed between GST and CYP2B6 was expressed in Escherichia coli DH5?. Mouse antibodies are raised against the fusion proteins GST-2B6. 2B6pAb was fond to be specific antibody.CONCLUSION:Recombinant PGEX/2B6 were constructed and purified fusion proteins GST-2B6, and specific 2B6pAb were obtained.

Expression of phenylalanine ammonia-lyase cDNA from rice in E. coli BL21DE3 / 中国病理生理杂志

AIM: To study the expression and its kinetics of rice phenylalanine ammonia-lyase gene encoding into E. coli as the basis of treatment for phenylketouria. METHODS: The phenylalanine ammonia-lyase-1-cDNA(rPAL-1-cDNA) from rice was recombined into E. coli high expression vector pET-28c and transformed into E. coli host strain BL21DE3. Engineering bacteria was then inducted by isopropyl-?-D-thiogalactoside (IPTG) for 1, 3, 5, 7 hours, in order to obtain high level expression. RESULTS: After induction, the expression level of fusion protein was 21.40%, 30.60%, 35.40%, 35.43% respectively. The fusion protein exhibited a band of 78 6 kD on SDS-PAGE analysis,but was not found in controls.The target protein was mainly existed in the form of inclusion body. CONCLUSION:Rice PAL gene expressing E. coli was established by gentic engineering technique.

Effects of garlic extract on cell growth, cell cycle progression and protein kinase C activity in mammalian cells / 中国病理生理杂志

The possible mechanism of tumor promotion inhibiting activity of garlicextract was studied. The garlic extract could inhibit the cell proliferation of human amnionFL cell in vitro, exerting no influence on the S phase DNA synthesis while exhibitingobvious inhibitory effect on the mitosis of the cells. At suitable concentration, garlic ex-tract could also inhibit the changes of protein kinase C activity induced by tumor promoterTPA, while at higher concentration garlic extract itself could induce changes of proteinkinase C activity mimic to TPA stimulation.

The effects of several tumor promoters on gap junction intercellular communication in NIH/3T3 cells / 中国病理生理杂志

The effects of tumor promoter TPA, MZR and PB on gap junction intercellular communication in NIH/3T3 cells were studied using the scrape-loading/dye transfer technique. All of these 3 agents were shown to cause a dose-dependent inhibition of the intercellular communication. The blockage of intercellular communication induced by TPA and MZR was inhibited in the presence of the protein kinase C inhibitor, staurosporine or palmitoyl carnitine, but not inhibited in the casexinduced by PB. The results suggest that the serape-loading/dye transfer technique may be used as a rapid screening assay to detect the tumor promoters and protein kinase C may be involved in the genesis of intercellular communication blockage induced by TPA ane MZR, but not by PB.

Endoplasmic reticulum stress is involved in N-methyl-N'-nitro-N-nitrosoguanidine, benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide and mitomycin-induced cellular response in FL cells / 中国病理生理杂志

AIM: To understand whether endoplasmic reticulum stress (ER-stress) is involved in DNA-damaging agent/carcinogen induced cell responses. METHODS: Three DNA-damaging agents/carcinogens different in the mode of action, ie, alkylating agent N-methyl-N’-nitro-N-nitrosoguanidine (MNNG), bulky adduct forming agent benzo[a] pyrene-7, 8-dihydrodiol-9, 10-epoxide (BPDE) and cross-linking agent mitomycin C (MMC) were selected. SDS-PAGE and immunoblotting were used to examine the protein levels of GRP78/BiP, GDADD153/CHOP and activation state of endoplasmic reticulum located caspase-12 in FL cells before and after MNNG, BPDE or MMC exposure. RESULTS: Immunoblotting showed that the protein level of endoplasmic reticulum specific proteins GRP78/BiP and GADD153/CHOP were significantly increased and endoplasmic reticulum located caspase-12 was activated in low concentration of MNNG (0.25 and 1 ?mol/L) and BPDE (5 and 50 nmol/L)-treated cells. MMC at all of the three concentration used (5, 50 and 500 ?mol/L) decreased the expression of GRP78/BiP, while it has no effects on CHOP and caspase-12. CONCLUSIONS: Both low concentration MNNG and BPDE could trigger the ER-stress in the exposed cells, while MMC could induce the down-regulation of the GRP78/BiP protein, which plays an important mediating role in the induction of ER-stress and may thus change the responsiveness against ER-stress inducers. It is suggested that ER-stress might partially mediate the cellular responses excited by exposure to some DNA-damaging agents/carcinogens.

Progress on research of the alternative splicing of human cytochrome P450 pre-mRNA / 中国病理生理杂志

Human genes typically contain multiple intron s, and in many cases the exons can be joined more than one way to generate multi ple mRNAs, encoding distinct protein isoforms. This process is called alternativ e splicing. The article summarized the human cytochrome P450 pre mRNA alternati v e splicing and their regulatory mechanism and impacts on biological functions.