OBJECTIVETo explore low density lipoprotein (LDL) oxidation by macrophage myeloperoxidase (MPO) at molecular level.

METHODSUsing a mouse macrophage model, we examined the relationship between LDL oxidation and macrophage MPO by measuring macrophage MPO activity, LDL oxidation products, MPO gene expression and cellular orientation of LDL oxidation.

RESULTSMPO gene expression increased to its maximum gradually when the concentration of LDL was increased, and then maintained at that level. NaN(3) inhibied the elevation of MPO activity and LDL oxidation, which was LDL concentration-dependent. After the composition of macrophage membrane was roughly analyzed, it was determined that the contents of MPO and LDL in 5% sucrose were 7.667 and 21 times higher than those in 10% sucrose, respectively.

CONCLUSIONLDL is attached to the "microdomain" of the macrophage membrane in which LDL is oxidized by MPO.

Animals ; Lipoproteins, LDL ; metabolism ; Macrophages ; metabolism ; Mice ; Oxidation-Reduction ; Peroxidase ; genetics ; metabolism

BACKGROUNDMany studies have suggested that cigarette smoking and polymorphisms of resistin and glutathione peroxidase-1 (GPx-1) genes are closely correlated with the pathogenesis of nonalcoholic fatty liver disease (NAFLD). However, few reports have investigated these associations with respect to NAFLD susceptibility. We, therefore, examined the distribution of polymorphisms in GPx-1 and resistin genes in NAFLD patients and healthy controls and analyzed the relationship between these polymorphisms and smoking status.

METHODSNine hundred NAFLD patients and 900 healthy controls were selected, and the genetic polymorphisms of resistin gene promoter-420C/G and GPx-1 gene Pro198Leu were analyzed by polymorphism-polymerase chain reaction (PCR) in DNA extracted from peripheral blood leukocytes. Interactions between the two mutants and the gene-environment interaction with cigarette smoking were also analyzed.

RESULTSGenotype frequencies of -420C/G (GG) and Pro198Leu (LL) were significantly higher in NAFLD cases (49.56% and 50.11%, respectively) compared with healthy controls (23.67% and 24.22%, respectively) (P = 0.0069; P = 0.0072). Moreover, the risk of NAFLD with -420C/G (GG) was significantly higher than in controls (odds ratio [OR] =3.1685, 95% confidence interval (CI) =1.9366-5.2073). Individuals carrying Pro198Leu (LL) had a high risk of NAFLD (OR = 3.1424, 95% CI = 1.7951-5.2367). Combined analysis of the polymorphisms showed that the -420C/G (GG)/Pro198Leu (LL) genotype was significantly more common in the NAFLD group than in the control group (39.44% vs. 12.78%, respectively, P = 0.0054), while individuals with -420C/G (GG)/Pro198Leu (LL) had a high risk of NAFLD (OR = 5.0357, 95% CI = 3.1852-7.8106). Moreover, the cigarette smoking rate in the NAFLD group was significantly higher than in the control group (OR = 1.8990, P = 0.0083 in the smoking index (SI) ≤400 subgroup; OR = 5.0937, P = 0.0051 in the SI >400 subgroup), and statistical analysis suggested a positive interaction between cigarette smoking and -420C/G (GG) (γ = 5.6018 in the SI ≤400 subgroup; γ = 4.4770 in the SI >400 subgroup) and Pro198Leu (LL) (γ = 5.7715 in the SI ≤400 subgroup; γ = 4.5985 in the SI >400 subgroup) in increasing the risk of NAFLD.

CONCLUSIONNAFLD risk factors include -420C/G (GG), Pro198Leu (LL) and cigarette smoking, and these three factors have a significant additive effect on NAFLD risk.

Female ; Genetic Predisposition to Disease ; genetics ; Glutathione Peroxidase ; genetics ; Humans ; Male ; Non-alcoholic Fatty Liver Disease ; metabolism ; Polymorphism, Single Nucleotide ; genetics ; Promoter Regions, Genetic ; genetics ; Resistin ; genetics ; Smoking ; genetics

In order to find the characteristics of two members of gene family of squaleneexpoxidase (SE) , a quantitative real time PCR method was developed to analyze the expression of Eleutherococcus senticosus SE1 and SE2 gene from different growth periods and in different organs. The result indicated that all the expression of SE2 more than SE1 in the whole growth period and organs of E. senticosus. And in the whole growth period, expression of SE1 showed a low-high-low characteristic. Both expression of SE2 and growth period showed the same trend. The lowest content of the expression was in the roots. SE1 expression have been improved more than SE2 when treated with MeJA. The expression of E. senticosus SE1 and saponins content had significantly positive correlation (P < 0.05) and the correlation coefficients was 0. 858, while the correlation was not significant for SE2. That indicated that SE1 played a key enzyme gene in the biosynthesis of triterpenoidsaponins
Eleutherococcus ; chemistry ; enzymology ; genetics ; growth & development ; Gene Expression Regulation, Plant ; Peroxidase ; genetics ; metabolism ; Plant Proteins ; genetics ; metabolism ; Saponins ; analysis ; metabolism ; Transcriptome

OBJECTIVETo construct a Gpx1 and klk1 recombinant vector containing the kidney-specific promoter Ksp-cadherin.

METHODSHuman Gpx1, Klk1 and Ksp-cadherin cDNAs were amplified with PCR and inserted in a stepwise manner into the expressive vector pIRES-EGFP to construct the recombinant vector Ksp-cadherin-Gpx1-Klk1. The constructed vector was verified with restriction enzyme digestion and sequence analysis.

RESULTS AND CONCLUSIONThe recombinant expression vector Ksp-cadherin-Gpx1-Klk1 was constructed and identified successfully, which provides a potent tool for preparing transgenic animals to investigate gene therapy for ischemia-reperfusion injury in kidney transplantation.

Cadherins ; genetics ; Cloning, Molecular ; Genetic Therapy ; methods ; Genetic Vectors ; genetics ; Glutathione Peroxidase ; genetics ; Humans ; Kallikreins ; genetics ; Kidney ; metabolism ; Promoter Regions, Genetic ; genetics

OBJECTIVETo investigate the effects of escharectomy during shock stage on tissue high mobility group box-1 protein (HMGB1) expression and balance of pro-/anti-inflammatory cytokines, and to elucidate the potential mechanism underlying beneficial effect of early escharectomy after severe burns.

METHODSWistar rats inflicted by 30% full-thickness thermal injury were randomly divided into thermal injury group, 24 h escharectomy group and 72 h escharectomy group, in which escharectomy were performed at 24 and 72 h postburn, respectively. Gene expression of HMGB1, interleukin-10 (IL-10), and tumor necrosis factor-alpha (TNF-alpha) in liver and lungs was detected with reverse-transcription PCR, and protein levels of IL-10 and TNF-alpha in liver and lung tissues were measured by ELISA. The plasma AST and ALT contents, and pulmonary myeloperoxidase (MPO) activity were also assayed.

RESULTSThe mRNA expression of HMGB1 and TNF-alpha in liver and lungs was up-regulated on postburn day 2, with IL-10 over-expression on postburn day 8. In the 24 h escharectomy group, HMGB1 and TNF-alpha mRNA expression in liver and lungs was down-regulated on postburn day 4, and IL-10 expression returned to normal range on postburn day 8, while the down-regulation of HMGB1, TNF-alpha and IL-10 were not noted in the 72 h escharectomy group. There were two peaks in liver TNF-alpha protein levels appearing on postburn days 2 and 8, respectively, with an unexpected marked decrease on day 4 in thermal injury controls, yet liver TNF-alpha levels maintained in normal range in animals of 24 h and 72 h escharectomy groups. The ratios of TNF-alpha to IL-10 protein levels in liver tissue were significantly increased on postburn days 2 and 4 (P = 0.0001 and 0.002, respectively), while escharectomy during shock stage markedly reduced hepatic TNF-alpha to IL-10 ratios (P = 0.0008 and 0.040, respectively). No significant changes in TNF-alpha protein levels in lung tissue were observed. Additionally, plasma AST as well as ALT contents, and pulmonary MPO activity were markedly decreased on postburn days 4 and 8 in the 24 h escharectomy group compared to the 72 h escharectomy group or thermal injury controls (P < 0.05).

CONCLUSIONSEscharectomy during burn shock stage could inhibit the over-expression of both early and late inflammatory mediators, and maintain the balance of pro-/anti-inflammatory response, thereby improving multiple organ functions in rats following severe burns.

Alanine Transaminase ; metabolism ; Animals ; Aspartate Aminotransferases ; metabolism ; Burns ; complications ; surgery ; HMGB1 Protein ; genetics ; metabolism ; Interleukin-10 ; genetics ; metabolism ; Liver ; enzymology ; metabolism ; Lung ; enzymology ; metabolism ; Male ; Peroxidase ; metabolism ; Rats ; Rats, Wistar ; Shock, Traumatic ; etiology ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

OBJECTIVETo investigate the mRNA expressions of RASSF1A, Galectin-3 and TPO in papillary thyroid carcinoma and some other thyroid benign lesions, and evaluate their diagnostic significance.

METHODSReverse transcription polymerase chain reaction (RT-PCR) was used to detect the mRNA expression of RASSF1A, galectin-3 and TPO in the samples from 73 cases, including 23 cases with papillary thyroid cancer, 16 with nodular goiter, 29 with thyroid adenoma and 5 with Hashimoto's disease.

RESULTSA statistically significant difference in the mRNA expression of RASSF1A, Galectin-3 and TPO was observed between papillary thyroid carcinoma and follicular benign lesions (P<0.05). However, there was no significant difference among various kinds of benign lesions (P>0.05). A negative correlation of the expression of RASSF1A and Galectin-3 mRNA was found between thyroid benign lesions and malignant ones (P = 0.000). While the mRNA expression of RASSF1A and TPO was positively correlated between benign and malignant lesions (P = 0.028).

CONCLUSIONLoss of expression of RASSF1A and TPO mRNA but high expression of Galectin-3 mRNA in papillary thyroid carcinoma are common. Therefore, the products of these three genes may be closely related to the development of thyroid papillary carcinoma, and may be used as useful markers in differential diagnosis of papillary thyroid carcinoma from the benign lesions. The results are more reliable if this detection method is used in combination with other techniques.

Adolescent ; Adult ; Aged ; Autoantigens ; genetics ; metabolism ; Biomarkers, Tumor ; metabolism ; Carcinoma, Papillary ; genetics ; metabolism ; pathology ; Diagnosis, Differential ; Female ; Galectin 3 ; genetics ; metabolism ; Goiter, Nodular ; genetics ; metabolism ; pathology ; Hashimoto Disease ; genetics ; metabolism ; pathology ; Humans ; Iodide Peroxidase ; genetics ; metabolism ; Iron-Binding Proteins ; genetics ; metabolism ; Male ; Middle Aged ; RNA, Messenger ; metabolism ; Thyroid Neoplasms ; genetics ; metabolism ; pathology ; Tumor Suppressor Proteins ; genetics ; metabolism ; Young Adult

BACKGROUND AND OBJECTIVELung cancer harms people's health or even lives severely. Especially, the therapy of non-small cell lung cancer (NSCLC) has not been obviously improved for many years. The aim of this study is to transfer the human sodium/iodide symporter (hNIS) and the human thyroperoxidase (hTPO) genes into H460 lung cancer cell line, and to study the uptake ability of iodide after co-transfected hTPO and hNIS gene in cell lines.

METHODSThrough cloning, recombination, packaging and amplifying, the recombinant adenosine virus (AdTPO) was constructed. Then the protein expression of AdTPO was tested by Western blot. After transfected hNIS gene into human lung cancer cell line H460 through liposome, stably expressing hNIS gene cell lines (hNIS-H460) selected by G418 antibiotics was determined as hNIS-H460 group. Using AdTPO, hTPO gene was transducted into hNIS-H460, as AdTPO-hNIS-H460 group. H460 cell without hNIS gene was applied as control group (H460). Then, we investigated the 125I uptake assay of the above cells.

RESULTSWe were successful in co-transfecting hNIS and hTPO gene into human lung cell lines H460, and were obtained hNIS and hTPO gene lung cancer cell lines (hNIS-H460 and AdTPO-hNIS-H460). In AdTPO-hNIS-H460, hNIS-H460 and H460, the uptake ability of 125I was (59 637.67 +/- 1 281.13), (48 622.17 +/- 2 242.28) and (1 440.17 +/- 372.86) counts x min(-1). The uptake ability of 125I was 41 fold higher in AdTPO-hNIS-H460 than in blank control H460 (P < 0.01), and 34 fold higher in hNIS-460 than in blank control H460 (P < 0.01), and 1.2 fold higher in AdTPO-hNIS-H460 than in hNIS-H460 (P < 0.01).

CONCLUSIONThe uptake ability of 125I could increase by co-transfected hNIS and hTPO genes into human lung cancer cell lines H460.

Adenoviridae ; genetics ; Cell Line, Tumor ; Genetic Therapy ; Humans ; Iodide Peroxidase ; genetics ; Iodine Radioisotopes ; pharmacokinetics ; therapeutic use ; Lung Neoplasms ; metabolism ; therapy ; Symporters ; genetics ; Transfection

AIM: To investigate the antioxidant and anti-inflammatory effects of Shengmai San (SMS) and its ethyl acetate extract (SEa), n-butanol extract (SBu), and aqueous extract (SWe), and clarify the material base of SMS and the roles played by its fractions. METHODS: A mouse model of transient forebrain ischemia/reperfusion (I/R) by means of common carotid artery occlusion (CCAO) was used to investigate the effects of SMS and its three fractions. Histopathological damage, blood-brain barrier disruption, and antioxidant and inflammation-related parameters, including malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GPx), myeloperoxidase (MPO), nitric oxide (NO), tumor necrosis factor-α (TNF-α) were measured. The chemical constituents of each fraction were identified by LC-MS. RESULTS: Eighteen lignans in SEa, and thirteen steroidal glycosides and ginsenosides in SBu were determined. SMS significantly inhibited I/R induced formation of histological injury and cerebral MPO activity. SMS showed the strongest antioxidant and anti-inflammatory effects against the I/R-caused injuries. SEa showed higher antioxidant activity than the other two fractions and SBu has a slightly stronger inhibition on the productions of NO and TNF-α. CONCLUSION SMS as a whole had the most effective protection against cerebral I/R-caused injuries compared with its fractions, which inferred that it contains different groups of compounds that contribute together to its protective effect.
Animals ; Chromatography, High Pressure Liquid ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; chemistry ; Glutathione Peroxidase ; genetics ; metabolism ; Humans ; Male ; Malondialdehyde ; metabolism ; Nitric Acid ; metabolism ; Oxidative Stress ; drug effects ; Peroxidase ; genetics ; metabolism ; Protective Agents ; administration & dosage ; chemistry ; Rats ; Reperfusion Injury ; drug therapy ; genetics ; metabolism ; prevention & control ; Superoxide Dismutase ; genetics ; metabolism

Arylalkylamine N-acetyltransferase (AANAT) and Hydroxyindole O-methyltransferase(HIOMT) are the key regulation enzymes in the melatonin biosynthesis pathway in mammals. The AANAT and HIOMT genes were constructed into a binary plant expression vector YXu55. Using leaf strips as the recipiences, we efficiently transformed tobacco (Nicotiana tabacum) variety qinyan 95 by the Agrobacterium mediated method. After gradient selection with gentamycin, a number of transgenic plants were regenerated. Southern blot and RT-PCR analyses showed that the AANAT-HIOMT genes were integrated into the genome of the transgenic plants and the target genes could express at the level of RNA transcription. By RP-HPLC, we measured the melatonin contents in transgenic plants. The results showed that the melatonin level in YXu55 (containing the gentamycin-resistance gene, the AANAT gene and HIOMT gene) transgenic plants were much higher than those in pZP122 (control containing only the gentamycin-resistance gene) transgenic plants and nontransgenic plants. The content of melatonin in pZP122 transgenic plants was nearly the same as that in nontransgenic plants. Physiological determination of antioxidative characteristics demonstrated that 1) the capacity of total antioxidation, 2) the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) and 3) the content of glutathione (GSH) were increased in YXu55 transgenic plants containing the AANAT-HIOMT genes as compared to the control plants (pZP122 or nontransgenic plants). At the same time, malonaldehyde (MDA) content did not appear remarkably difference between transgenic plants and nontransgenic plants. The above mentioned facts indicate enhancement of melatonin levels in YXu55 transgenic plants might help to reduce damage by oxidative stress.
Acetylserotonin O-Methyltransferase ; genetics ; Agrobacterium tumefaciens ; genetics ; Arylalkylamine N-Acetyltransferase ; genetics ; Catalase ; metabolism ; Gene Expression Regulation, Enzymologic ; Gene Expression Regulation, Plant ; Melatonin ; biosynthesis ; Peroxidase ; metabolism ; Plants, Genetically Modified ; enzymology ; genetics ; Superoxide Dismutase ; metabolism ; Tobacco ; enzymology ; genetics ; Transduction, Genetic ; methods

OBJECTIVETo investigate the effect of rosiglitazone on angiotensin II (Ang II)-induced mRNA and protein expressions of toll-like receptor 4 (TLR4) and myeloperoxidase (MPO) activity in RAW264.7 cells to explore its anti- inflammatory and anti-atherosclerotic mechanisms.

METHODSMurine RAW264.7 cells were pretreated with rosiglitazone at 2.5, 5, and 10 micromol/L prior to exposure to AngII (0.1 micromol/L). TLR4 mRNA level was analyzed by RT-PCR, and TLR4 protein expression by Western blotting. MPO activity in the cell supernatant was assayed by colorimetry. In another experiment, the cells were pretreated with a neutralizing anti-TLR4 antibody (1 mg/L) for 1 h prior to rosiglitazone (10 micromol/L) treatment for 1 h, and subsequently stimulated with AngII or LPS (100 micromol/L) for 24 h to observe the change of MPO activity.

RESULTSRosiglitazone downregulated AngII-induced mRNA and protein expressions of TLR4, and inhibited MPO activity in RAW264.7 cells in a concentration-dependent manner. The TLR4 blocker partially antagonized the effect of AngII on MPO activity, and the inhibitory effect was markedly enhanced by rosiglitazone. Rosiglitazone significantly inhibited LPS (a specific TLR4 ligand)-induced MPO activity in RAW264.7 cells.

CONCLUSIONRosiglitazone downregulates Ang II-induced TLR4 expression in RAW264.7 cells and inhibits MPO secretion possibly by interfering with TLR4 to relieve the inflammatory reaction, which may be one of its anti-atherosclerotic mechanisms.

Angiotensin II ; pharmacology ; Animals ; Anti-Inflammatory Agents, Non-Steroidal ; pharmacology ; Cell Line ; Macrophages ; cytology ; metabolism ; Mice ; Peroxidase ; metabolism ; Thiazolidinediones ; pharmacology ; Toll-Like Receptor 4 ; genetics ; metabolism