OBJECTIVETo investigate telomerase activity and expression of hTR and hTERT in human neuroepithelial tumors for exploring new strategy for clinical diagnosis and treatment.

METHODSTelomerase activity was detected by modified TRAP method and the expression of hTR and hTERT was measured by RT-PCR method in 65 human neuroepithelial tumors, respectively.

RESULTSThe positive rates of telomerase and hTERT were 61.54% and 70.77% respectively in human neuroepithelial tumors, and the positive rate and their level of expression were correlated with the degree of malignancy of tumors positively.

CONCLUSIONSTelomerase activity and hTERT are significantly correlated with the degree of malignancyin human neuroepithelial tumors. hTERT may play a key role in the regulation of telomerase activity.

DNA-Binding Proteins ; Gene Expression Regulation, Enzymologic ; Gene Expression Regulation, Neoplastic ; Humans ; Neoplasms, Neuroepithelial ; enzymology ; genetics ; Telomerase ; biosynthesis ; genetics ; metabolism

OBJECTIVETo purify a low-temperature hydroxylamine oxidase (HAO) from a heterotrophic nitrifying bacterium Acinetobacter sp. Y16 and investigate the enzyme property.

METHODSA HAO was purified by an anion-exchange and gel-filtration chromatography from strain Y16. The purity and molecular mass were determined by RP-HPLC and SDS-PAGE. The HAO activity was detected by monitoring the reduction of potassium ferricyanide using hydroxylamine as substrate and ferricyanide as electron acceptor. The partial amino acid sequence was determined by mass spectrometry.

RESULTSThe low-temperature HAO with a molecular mass of 61 kDa was purified from strain Y16 by an anion-exchange and gel-filtration chromatography. The enzyme exhibited an ability to oxidize hydroxylamine in wide temperature range (4-40 °C) in vitro using hydroxylamine as substrate and ferricyanide as electron acceptor. It was stable in the temperature range of 4 to 15 °C and pH range of 6.0 to 8.5 with less than 30% change in its activity. The optimal temperature and pH were 15 °C and 7.5, respectively. Three peptides were determined by mass spectrometry which were shown to be not identical to other reported HAOs.

CONCLUSIONThis is the first study to purify a low-temperature HAO from a heterotrophic nitrifier Acinetobacter sp. It differs from other reported HAOs in molecular mass and enzyme properties. The findings of the present study have suggested that the strain Y16 passes through a hydroxylamine-oxidizing process catalyzed by a low-temperature HAO for ammonium removal.

Acinetobacter ; enzymology ; genetics ; metabolism ; Amino Acid Sequence ; Cold Temperature ; Gene Expression Regulation, Bacterial ; physiology ; Gene Expression Regulation, Enzymologic ; physiology ; Hydrogen-Ion Concentration ; Oxidoreductases ; genetics ; metabolism ; Substrate Specificity

This study was aimed to construct a plasmid expressing siRNA specific for the human telomerase reverse transcriptase (hTERT) gene and to evaluate the ability of small interference RNA(siRNA) for inhibiting telomerase activity in HeLa cells. 64 nucleotides, in which 19 nt were homologous with hTERT gene, were chemically synthesized, annealed and linked into pSUPER to get pSUP-hTE. Then pSUP-hTE was digested with enzyme. We obtained its fragmant concluding promoter and 64nt. So we cloned it into pEGFP-C1 for constructing pEGFP-hTE which contains neo gene and the enhanced green fluorescent protein (EGFP). Recombinant pEGFP-hTE was transfected to HeLa cells. These cells were screened with medium containing G418. When stable colonies appeared, G418-resistant cells were harvested and propagated. At the different cell generations, hTERT mRNA and protein expression, telomerase activity and cell growth activity were analyzed. Compared with control cells, HeLa cells transfected with pEGFP-hTE showed that hTERT mRNA level and hTERT protein expression decreased and telomerase activity reduced by 38%, but the cells growth activity displayed no changes. So pEGFP-hTE could specifically inhibit expression of hTERT and telomerase activity. These results suggested that siRNA targeting hTERT gene might provide a new strategy for cancer biotherapy.
Base Sequence ; Gene Expression Regulation, Enzymologic ; Gene Expression Regulation, Neoplastic ; HeLa Cells ; Humans ; Molecular Sequence Data ; Plasmids ; genetics ; RNA, Small Interfering ; genetics ; pharmacology ; Telomerase ; antagonists & inhibitors ; genetics ; Transfection

OBJECTIVETo explore the effects of hydroquinone (HQ) on reactive oxygen species (ROS) generation, antioxydase activities and the expression of human 8-oxo-guanine DNA glycosylase (hOGG1) mRNA in human A549 lung adenocarcinoma cell strains.

METHODSA549 cells were treated with different concentrations of HQ. Cell survival was determined by methyl thiazolyl tetrazolium (MTT). Changes of ROS were detected by fluorescent probe. The contents of malonaldehyde and activities of antioxydase were determined through colorimetry. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to assess the level of hOGG1 mRNA.

RESULTSWith the increased concentration of HQ, the findings were as follows. (1) The absorbance value of A549 cell decreased. There was significant difference between 160 micromol/L (0.584+/-0.098) and 320 micromol/L (0.328+/-0.066) of HQ (q=5.56 and 9.07, P<0.05) with the control group (0.989+/-0.150), and the cell survival rate were less than 80%. (2) The ROS in A549 cell increased. 40 micromol/L (39.80+/-4.15) and 80 micromol/L (101.99+/-9.45) had statistical significance (q=10.74 and 30.32, P<0.05) with the control group (5.71+/-0.50). (3) It was found that the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) decreased and malonaldehyde (MDA) increased. Compared with the control group [(25.62+/-0.28) U/mg prot and (38.97+/-2.61) U/mg prot], the activities of SOD and GSH-Px had a significant decrease (q=12.17 and 8.78, P<0.05) in 80 micromol/L [(22.93+/-0.56) U/mg prot and (25.60+/-2.31) U/mg prot]. And MDA had a significant increase (q=10.90 and 15.49, P<0.05) in 40 micromol/L [(1.07+/-0.01) nmol/mg prot] and 80 micromol/L [(1.19+/-0.08) nmol/mg prot] as compared with the control group [(0.77+/-0.04) nmol/mg prot]. The decrease of SOD (r=-0.95, F=20.00, P=0.04) and GSH-Px activities (r=-0.99, F=115.48, P=0.01) and the increase of MDA contents (r=0.96, F=21.31, P=0.04) all had a dose-response relationship. (4) RT-PCR results showed that the expression of hOGG1 mRNA decreased. The significant difference was observed between the expression of hOGG1 mRNA in 80 micromol/L (0.478+/-0.017) (q=11.70, P<0.05) with the control group (0.715+/-0.038).

CONCLUSIONThis study suggests that HQ could induce oxidative damage and changes of the expression of hOGG1 mRNA in A549 cells.

Cell Line, Tumor ; DNA Glycosylases ; genetics ; Down-Regulation ; Gene Expression ; Gene Expression Regulation, Enzymologic ; drug effects ; Humans ; Hydroquinones ; toxicity ; RNA, Messenger ; genetics

OBJECTIVETo investigate whether telomerase activity, human telomerase RNA (HTR) and human telomerase reverse transcriptase (HTERT) expression were associated with tumor development in lung cancer and whether telomerase is regulated at gene level or transcriptional level.

METHODSExpression of HTR and HTERT was detected by reverse transcription-polymerase chain reaction (RT-PCR) in 68 human lung cancer and in 68 adjacent-neoplatic lung tissues. And telomerase activity was examined by a quantitative telomeric repeat amplification protocol (TRAP).

RESULTSIn 68 lung cancer tissues, telomerase activity, HTR and HTERT were expressed in 79%, 98.5% and 91.2% respectively, whereas all adjacent non-neoplastic lung tissues were telomerase negative. Most normal lung tissues expressed HTR (91.2%) and HTERT was detected in only 7 of 68 non-neoplastic tissues.

CONCLUSIONSThe relatively high frequency of telomerase activity in lung cancer whereas the detection of no telomerase activity in normal lung tissues suggested that telomerase may play an important role in tumorigenesis of lung cancer. Compared to HTR, HTERT expression was better associated with telomerase activity with a concordance of 88.9%. Telomerase activity may be regulated at transcription level or translation level.

DNA-Binding Proteins ; Gene Expression Regulation, Enzymologic ; Gene Expression Regulation, Neoplastic ; Humans ; Lung Neoplasms ; enzymology ; genetics ; RNA ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Telomerase ; genetics

The rhizome of Alpinia officinarum is a widely used Chinese herbal medicine. The essential oil in A. officinarum rhizome is mainly composed of 1, 8-cineole and other monoterpenes, as the major bioactive ingredients. In plants, monoterpenes are synthesized through the methylerythritol phosphate (MEP) pathway in the plastids, and 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is an enzyme catalyzing a committed step of the MEP pathway. In the present study, the full-length cDNA encoding DXR was cloned from the rhizome of A. officinarum, using homology-based RT-PCR and rapid amplification of cDNA ends (RACE) techniques. The new cDNA was designated as AoDXR and submitted to GenBank to be assigned with an accession number HQ874658. The full-length cDNA of AoDXR was 1 670 bp containing a 1 419 bp open reading frame encoding a polypeptide of 472 amino acids with a calculated molecular mass of 51.48 kDa and an isoelectric point of 6.15. Bioinformatic analyses revealed that AoDXR showed extensive homology with DXRs from other plant species and contained a conserved plastids transit peptide, a Pro-rich region and two highly conserved NADPH-binding motifs in its N-terminal region characterized by all plant DXRs. The phylogenetic analysis revealed that AoDXR belonged to angiosperm DXRs. The structural modeling of AoDXR showed that AoDXR had the typical V-shaped structure of DXR proteins. The tissue expression pattern analysis indicated that AoDXR expressed strongly in leaves, weak in rhizomes of A. officinarum. Exogenous methyl jasmonate (MeJA) could enhance the expression of AoDXR and the production of 1, 8-cineole in A. officinarum rhizomes. The cloning and characterization of AoDXR will be helpful to reveal the molecular regulation mechanism of monoterpene biosynthesis in A. officinarum and provides a candidate gene for metabolic engineering in improving the medicinal quality of A. officinarum rhizome.
Aldose-Ketose Isomerases ; genetics ; Alpinia ; chemistry ; enzymology ; genetics ; Amino Acid Sequence ; DNA, Complementary ; genetics ; Gene Expression Regulation, Enzymologic ; Gene Expression Regulation, Plant ; Molecular Sequence Data ; Monoterpenes ; metabolism ; Phylogeny

The degradation of arthrodial cartilage is a typical characteristic in the pathogenesis of osteoarthritis. matrix metalloproteinases (MMPs) are the primary enzymes involved in extracellular matrix degradation of cartilage. The mechanism of MMPs in extracellular matrix degradation of cartilage is becoming clear with the in-depth study about MMPs, such as activation, activity regulation, related signal transduction pathways and transcription factors. This artice reviewed the activation, expression and regulation of MMPs in the related theory and empirical study of osteoarthritis cartilage.
Cartilage ; enzymology ; Enzyme Activation ; Gene Expression Regulation, Enzymologic ; Humans ; Matrix Metalloproteinases ; genetics ; metabolism ; Multigene Family ; Osteoarthritis ; enzymology ; genetics ; Signal Transduction

Infrasound widely exists in nature, our living condition, productive and traffic environment. Gastrointestinal tract is relatively sensitive to infrasound. However, the effect of infrasound on gastrointestinal function is unclear. Therefore, the purpose of our study was to observe the effects of infrasound on gastric motility and gastric morphology and to assess the expression of nitric oxide synthase (NOS) in gastric antrum after exposure to infrasound of 8 Hz - 130 dB for 2 hours per day for 14 consecutive days. Gastric motility was assessed by gastric fluid-emptying rate. Gastric morphology was evaluated by HE. The expression of NOS was measured by tissue microarray technology. The results would contribute to understand the role of infrasound in gastroenterology, and help to explain the mechanism of infrasound on gastroenterology.
Animals ; Gastrointestinal Motility ; Gene Expression Regulation, Enzymologic ; Male ; Nitric Oxide Synthase ; metabolism ; Rats ; Sound ; adverse effects ; Stomach

Lead exposure is a known potential risk factor for neurodegenerative diseases such as Alzheimer's disease (AD). Exposure to lead during the critical phase of brain development has been linked with mental retardation and hypophrenia in later life. This study was aimed to investigate the effects of lead exposure of pregnant mice on the expressions of insulin-degrading enzyme (IDE) and nerve growth factor (NGF) in the hippocampus of their offspring. Blood samples were collected from the tail vein, and after anesthetizing the pups, the brain was excised on postnatal day 21. Lead concentrations were determined by graphite furnace atomic absorption spectrophotometry, and the expressions of IDE and NGF were determined by immunohistochemistry and Western blotting. Results showed that the reduction in IDE and NGF expression in the hippocampus of pups might be associated with impairment of learning and memory and dementia induced by maternal lead exposure during pregnancy and lactation.
Animals ; Down-Regulation ; Female ; Gene Expression Regulation, Developmental ; drug effects ; Gene Expression Regulation, Enzymologic ; drug effects ; Hippocampus ; drug effects ; growth & development ; metabolism ; Insulysin ; genetics ; metabolism ; Lead ; toxicity ; Mice ; Pregnancy ; Prenatal Exposure Delayed Effects ; chemically induced

Hydroxymethyltransferase (SHMT) and tryptophanase (TPase) are key enzymes in biosynthesis of L-tryptophan. We constructed three recombinant plasmids, including pET-SHMT, pET-TPase, and pET-ST for over-expression or co-expression of SHMT and TPase in Escherichia coli BL21 (DE3). The SDS-PAGE analysis showed that the recombinant proteins of 47 kDa and 50 kDa were expressed of pET-SHMT and pET-TPase, respectively. As compared to the host stain, the enzyme activity of SHMT and TPase was increased by 6.4 and 8.4 folds, respectively. Co-expression of both recombinant proteins, 47 kDa and 50 kDa, was also successful by using pET-ST and the enzyme activities were enhanced by 6.1 and 6.9 folds. We designed two pathways of dual-enzymatic synthesis of L-tryptophan by using these recombinant strains as source of SHMT and TPase. In the first pathway, the pET-SHMT carrying strain was used to catalyze synthesis of L-serine, which was further transformed into L-tryptophan by the pET-TPase expressing strain. These two steps sequentially took place in different bioreactors. In the second pathway, the pET-ST carrying strain, in which two enzymes were co-expressed, was used to catalyze simultaneously two steps in a single bioreactor. HPLC analysis indicated a high yield of 41.5 g/L of L-tryptophan was achieved in the first pathway, while a lower yield of 28.9 g/L was observed in the second pathway. In the first pathway, the calculated conversion rates for L-glycine and indole were 83.3% and 92.5%, respectively. In the second pathway, a comparable conversion rate, 82.7%, was achieved for L-glycine, while conversion of indole was much lower, only 82.9%.
Escherichia coli ; enzymology ; genetics ; metabolism ; Gene Expression Regulation, Bacterial ; physiology ; Gene Expression Regulation, Enzymologic ; physiology ; Genetic Vectors ; genetics ; Glycine Hydroxymethyltransferase ; biosynthesis ; genetics ; Plasmids ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Recombination, Genetic ; genetics ; Tryptophan ; biosynthesis ; Tryptophanase ; biosynthesis ; genetics