A 2,4 -dichlorophenol degrading Pseudomonas strain GI241-1 was isolated from a soil sample. The dienelactone hydrolase gene, designated as dcpD which encodes dienelactone hydrolase involved in transforming cis-2-chloro-dienelactone into 2-chloromaleylacetic acid, was cloned from this bacterium strain. The gene cloning strategy was to construct genomic library after location of its neighbouring gene by Southem blot and to screen the aim transformant by dot blotting. Sequencing results showed that length of dcpD is 702bp. The sequence of dcpD and the deduced amino acid are different from the relative sequences registered in the GenBank.

Recent studies indicated that interleukin (IL)-17, growth-related oncogene (GRO)-α and IL-8 play an important role in the pathogenesis of nasal polyps. However, the effects of the increased amount of IL-17 and the production of GRO-α and IL-8 in human nasal polyp fibroblasts are not completely understood. This study aimed to determine the effects of the increased IL-17 on the changes of GRO-α and IL-8 expression in human nasal polyp fibroblasts and further investigate the mechanism of neutrophil infiltration in nasal polyps. Nasal polyp fibroblasts were isolated from six cases of human nasal polyps, and the cells were stimulated with five different concentrations of IL-17. Real-time fluorescence quantitative polymerase chain reaction (RT-PCR) was used to detect the mRNA expression of GRO-α and IL-8. The mRNA of GRO-α and IL-8 was expressed in unstimulated controls and remarkably increased by stimulation with IL-17. Moreover, the levels of GRO-α and IL-8 produced by fibroblasts were increased gradually with the increases in IL-17 concentrations. The present study showed that nasal fibroblasts can produce GRO-α and IL-8, and their production is remarkably enhanced by IL-17 stimulation, thereby clarifying the mechanism of the IL-17 mediated neutrophil infiltration in nasal polyps. These findings might provide a rationale for using IL-17 inhibitors as a treatment for nasal inflammatory diseases such as nasal polyps.

2,4-Dichlorophenol is toxic and biorefratory organic pollutant. A 2,4-dichlorophenol degrading bacterial strain GT241-1, identified as Pseudomonas sp., was isolated from soil samples which was collected from drainage area of several 2,4-dichlorophenol producing factories. Strain GT241-1 had strong 2,4-dichlorophenol degrading ability, it could decompose 91% 2, 4-dichlorophenol of 90 mg/L within 48 hours at 25 - 30 degrees C, and could utilize 2,4-dichlorophenol, 2,4-dichlorophenoxyacetic acid (2,4-D), benzoate and catechol as sole carbon and energy source. Southern blot showed that 2,4-dichlorophenol hydroxylase gene (dcpA) of strain GT241-1 locates on the about 10kb EcoR I/Xba I fragment. This fragment was recovered, linked to the vecter pUC19 and transformed into the E. coli DH5alpha. A aim transformant, Z539, was obtained by dot blotting from about 1200 transformants. PCR and the sequencing results shew that the whole dcpA gene is contained within the 10kb EcoR I /Xba I fragment of pZ539. This fragment was shortened to about 2.4kb by HindmIII. The shorted fragment was subcloned to vecter pRSET-B to get a transformant BS1-12. The subcloned fragment was sequenced. Sequencing results showed that the whole length of the subcloned fragment containing dcpA is 2389bp and the nucleotide span of coding region is from number 276 to number 2072 (1797 bp), with ATG and TAA as start and stop codon respectively. The sequence analysis of dcpA and the deduced amino acid encoded by dcpA showed that they are different from the relative sequences registered in the GenBank. The subcloned fragment carry the promoter of dcpA, this can deduce from the fact that the upflow length of dcpA coding region is 275bp, and further confirmed by the 2,4-dichlorophenol hydroxylase activity measurement results. The 2,4-dichlorophenol hydroxylase activity of transformant Z539 and BS1-12 were detected, the results showed these transformants have 2,4-dichlorophenol hydroxylase activity. By comparison, the activity of these transformants were lower than that of the strain GT241-1.
Amino Acid Sequence ; Bacterial Proteins ; genetics ; metabolism ; Biodegradation, Environmental ; Chlorophenols ; metabolism ; Cloning, Molecular ; Environmental Pollutants ; metabolism ; Mixed Function Oxygenases ; genetics ; metabolism ; Molecular Sequence Data ; Pseudomonas ; enzymology ; genetics ; isolation & purification ; Soil Microbiology

OBJECTIVE: To study the change of DNA degradation in nucleolus of mice organs and its relationship with the postmortem interval, and to investigate a new accurate method to estimate the postmortem interval. METHODS: Eight parameters of cell nuclei were chosen, including the head DNA level, the tail DNA level, the head radius, the tail length, the tail moment, the Olive moment, the head area and the tail area. The changes of DNA degradation were analyzed in skeletal muscle, myocardium, liver, kidney and brain in mice at different intervals (0-72 h postmortem) by using single-cell gel electrophoresis and fluorescent microscope connected with auto-analysis-image system. RESULTS: The tail DNA level, the tail length, the tail moment, the Olive moment and the tail area showed an increasing tendency. The head DNA level, the head radius and the head area showed a decreasing tendency within 72h postmortem in mice. A quadratic regression equation (P < 0.001) and multiple regression equation of DNA degradation tendency were established (P < 0.000 1). CONCLUSION The regression equations established can be used as a new method for estimating postmortem interval in forensic practice.
Animals ; Cell Nucleus/metabolism* ; Comet Assay/methods* ; DNA/metabolism* ; Female ; Forensic Pathology/methods* ; Image Processing, Computer-Assisted/methods* ; Kidney/metabolism* ; Liver/metabolism* ; Male ; Mice ; Muscle, Skeletal/metabolism* ; Myocytes, Cardiac/metabolism* ; Postmortem Changes ; Time Factors

To investigate the cell-killing effect and its possible mechanism of rClone30-hDR5 in combination with TRAIL on human hepatic carcinoma (HCC) cell line, first of all, recombinant plasmid pee12.4-hDR5 was introduced into HepG2 cells by liposome transfection. After five rounds of screening by flow cytometry, HepG2 cells expressing high levels of DR5 on cell surface were isolated. The cytotoxicity of TRAIL to selected cells was higher than that of TRAIL to HepG2 cells by MTT method (P < 0.01). The result suggested that the cloned hDR5 gene had biological activity. MTT assay showed that, rClone30- hDR5 in combination with TRAIL more efficiently inhibited the tumor growth of HepG2 cells compared to rClone30-hDR5 or TRAIL in vitro. The results of Annexin V-FITC/PI staining and Quantitative Real-time PCR indicated that rClone30-hDR5 in combination with TRAIL significantly increased the mRNA levels of caspase 3 and caspase 8, and induced the apoptosis of tumor cells. HepG2 cells were infected with rClone30-hDR5 or rClone30 at MOI of 1. The expression of hDR5 on tumor surface increased significantly by rClone30-hDR5 compared to that by rClone30, which contributed to the sensitivity to TRAIL. In conclusion, rClone30-hDR5 in combination with TRAIL has potential application value in cancer treatment.
Apoptosis ; Carcinoma, Hepatocellular ; pathology ; Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Drug Synergism ; Hep G2 Cells ; Humans ; Liver Neoplasms ; pathology ; Real-Time Polymerase Chain Reaction ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; pharmacology ; TNF-Related Apoptosis-Inducing Ligand ; pharmacology ; Transfection

OBJECTIVETo study the cytotoxic effect of allogenetic natural killer (NK) cells in vitro on human CD34+ acute myelogenous leukemia cells.

METHODSCD34 expression on acute myelogenous leukemia KG1a cells was detected by flow cytometry. KG1a cells were co-cultured at different effector-to-target (E:T) ratios with NK cells isolated from 5 healthy individuals using magnetic cell sorting. Lactate dehydrogenase (LDH) release assay was employed to examine the cytolysis of KG1a cells in the co-culture, and the inhibition rate of the KG1a cell colony formation in methylcellulose was determined with K562 cells sensitive to NK cells as the control.

RESULTSA expression rate as much as (98.0-/+1.1)% was detected for CD34 antigen on KG1a cells, and the isolated NK cells (CD3(-)CD16+CD56+ cells) had a purity of (93.2-/+3.7)% after magnetic cell sorting. Allogenetic NK cells exhibited obvious cytotoxicity and colony inhibition in vitro against KG1a cells at different E:T ratios, and the effects were significantly enhanced as the E:T ratios increased (P<0.05). At the same E:T ratio, the cytotoxicity and colony inhibition rate of allogenetic NK cells against KG1a cells was lower than those against K562 cells (P<0.05).

CONCLUSIONAllogenetic NK cells exhibit obvious cytotoxicity and colony formation against CD34+ acute myelogenous leukemia cells.

Antigens, CD34 ; immunology ; Coculture Techniques ; Cytotoxicity, Immunologic ; Flow Cytometry ; Humans ; K562 Cells ; Killer Cells, Natural ; immunology ; Leukemia, Myeloid, Acute ; immunology

Recent studies indicated that interleukin (IL)-17, growth-related oncogene (GRO)-α and IL-8 play an important role in the pathogenesis of nasal polyps. However, the effects of the increased amount of IL-17 and the production of GRO-α and IL-8 in human nasal polyp fibroblasts are not completely understood. This study aimed to determine the effects of the increased IL-17 on the changes of GRO-α and IL-8 expression in human nasal polyp fibroblasts and further investigate the mechanism of neutrophil infiltration in nasal polyps. Nasal polyp fibroblasts were isolated from six cases of human nasal polyps, and the cells were stimulated with five different concentrations of IL-17. Real-time fluorescence quantitative polymerase chain reaction (RT-PCR) was used to detect the mRNA expression of GRO-α and IL-8. The mRNA of GRO-α and IL-8 was expressed in unstimulated controls and remarkably increased by stimulation with IL-17. Moreover, the levels of GRO-α and IL-8 produced by fibroblasts were increased gradually with the increases in IL-17 concentrations. The present study showed that nasal fibroblasts can produce GRO-α and IL-8, and their production is remarkably enhanced by IL-17 stimulation, thereby clarifying the mechanism of the IL-17 mediated neutrophil infiltration in nasal polyps. These findings might provide a rationale for using IL-17 inhibitors as a treatment for nasal inflammatory diseases such as nasal polyps.
Adult ; Cells, Cultured ; Chemokine CXCL1 ; biosynthesis ; Female ; Fibroblasts ; metabolism ; pathology ; Humans ; Interleukin-17 ; pharmacology ; Interleukin-8 ; biosynthesis ; Male ; Middle Aged ; Nasal Polyps ; metabolism ; pathology ; Neutrophil Infiltration ; drug effects ; RNA, Messenger ; biosynthesis

OBJECTIVETo explore the association between polycyclic aromatic hydrocarbons (PAHs) exposure and telomere length (TL), so as to investigate the effective biomarkers to evaluate the genetic damage in peripheral blood of workers exposed to PAHs.

METHODSThe exposure group consisted of 145 coke-oven workers (including 30 top-oven workers, 76 side-oven workers and 39 bottom-oven workers), and the non-exposure control group comprised 68 medical staffs. At 6 hours after the weekend duty shift, the samples of urine and 1 ml venous blood were collected from each subject. Airborne benzene-soluble matter (BSM) and particulate-phase B(a)P in the working environment of coke-oven and controls were sampled and analyzed. The concentration of urinary 1-hydroxypyrene (1-OHPyr) was determined. A real-time PCR method was used to determine the relative telomere length (RTL) of genomic DNA in peripheral blood. The relationship between the RTL and external exposure of PAHs, the potential factors which might have influence on TL were analyzed.

RESULTSThe medians of air BSM and particulate-phase B(a)P were higher in coke-oven (BSM: 328.6 µg/m(3); B(a)P: 926.9 ng/m(3)) than those in control working environment (BSM:97.8 µg/m(3); B(a)P: 49.1 ng/m(3)). The level of 1-OHPyr among coke-oven workers was significantly higher than that of non-exposed group (12.2 µmol/mol Cr vs 0.7 µmol/mol Cr; t = 26.971, P < 0.01). RTL in coke-oven workers were significantly shorter than those of controls (1.10 ± 0.75 vs 1.43 ± 1.06; t = 2.263, P = 0.026), and after adjusting for cigarettes per day and urinary 1-OHPyr, the significant difference was still observed (F(adju) = 5.496, P(adju) = 0.020). Stratification analysis found that RTL among the male and non-drinking groups in coke-oven workers were shorter than those the same sex and alcohol using status in controls (1.08 ± 0.73 vs 1.51 ± 1.10, F = 9.212, P = 0.003; 0.96 ± 0.38 vs 1.26 ± 0.46, F = 6.484, P = 0.012). Significant correlation between RTL and age was found (r = -0.284, P = 0.019) in non-exposure group.

CONCLUSIONPAH-exposure has effect on TL of genomic DNA in peripheral blood, which is mainly observed in the male and non-drinking groups between PAH-exposed workers and controls. It indicates that TL of genomic DNA in peripheral blood might be an effective biomarker as PAH-induced genetic damage.

Adult ; Benzene ; Case-Control Studies ; Coke ; DNA Damage ; Female ; Humans ; Male ; Occupational Exposure ; adverse effects ; analysis ; Polycyclic Aromatic Hydrocarbons ; adverse effects ; analysis ; Pyrenes ; analysis ; Telomere ; drug effects ; genetics

OBJECTIVETo screen a set of gene markers related to metastasis and prognosis of breast cancer by comparison of gene expression profiles of primary breast cancers with distant metastasis to the cases without distant metastasis within 3 years follow-up, and to explore the clinical significance of those gene expression in prognostic molecular classification of breast cancer patients.

METHODS5 cases with distant metastasis and 5 cases without distant metastasis within 3 years follow-up were used as training cases to compare their gene expression profiles by Oligo microarray hybridization containing 21 329 human functional genes. K-mean supervised cluster was done for 10 training cases and additional 20 testing cases based on the set of differential genes. "Leave-one-out" was used to eliminate useless genes to obtain optimal gene set that was used for prognostic molecular classification of breast cancer patients.

RESULTSThe different genes screened out from gene expression profiling of primary breast cancers with and without distant metastasis could classify breast cancer patients into two sub-groups. All patients with distant metastasis were included in the "poor prognosis group" (7/10), whereas there were no case showing distant metastasis in the "good prognosis group" (0/20), with a statistically significant difference by exact probability test (P =0. 03). In the set of 104 optimal genes, all 5 genes involved in cell adhesion and migration were up-regulated in cases with distant metastasis, all 2 genes related to immune response of host were down-regulated, 11 genes related to cell growth and metabolism were up-regulated and 14 down-regulated, and 15 genes related to cell signal transduction were significantly changed.

CONCLUSIONA set of genes involved in cell adhesion and migration, cell growth and metabolism, immune response mechanism, cell signal transduction were screened out by comparing gene expression profiles of primary breast cancers with and without distant metastasis within 3 years follow-up, showing highlight in prognostic molecular classification of breast cancer patients and hopeful would benefit to choose patient-tailored therapy strategies.

Adult ; Aged ; Breast Neoplasms ; genetics ; pathology ; Carcinoma, Ductal, Breast ; genetics ; secondary ; Cell Adhesion ; genetics ; Cell Movement ; genetics ; Cell Proliferation ; Cluster Analysis ; Female ; Follow-Up Studies ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Humans ; Lung Neoplasms ; genetics ; secondary ; Lymphatic Metastasis ; Middle Aged ; Oligonucleotide Array Sequence Analysis ; Prognosis ; Signal Transduction ; genetics

With the continuous development of DNA extraction and testing technology, the DNA left at a crime scene plays a decisive role in the determination of criminal suspects in criminal investigation. But in the meanwhile, the anti-reconnaissance awareness of suspect is growing, which leads to a decrease of evidence left at scene during and after a crime. Therefore, in the process of evidence collection at scene, the finding and extraction of touch biological evidence, and the DNA detection are more and more important. At present, the proportion of touch evidence at the crime scene increases, which plays an increasingly important role in the detection of cases. However, with the characteristics of minute quantities, small size and secrecy, these touch evidence is difficult to be observed. What's more, various forms of pollution at the scene greatly accelerate the degradation rate of trace material, thus, the test and analysis of such material has become the emphasis and difficulty of the forensic evidence identification. This article reviews different kinds, collection and extraction methods of touch DNA, the factors that affect the detection and the problems may meet in the detection for providing an application prospect to the forensic practice.
Crime ; Criminals ; DNA/isolation & purification* ; DNA Fingerprinting ; Forensic Genetics/methods* ; Humans ; Touch