OBJECTIVETo develop a quantitative real-time polymerase chain reaction (PCR) for detecting Bartonella henselae.

METHODSAccording to the 16S-23S rRNA intervening sequences (IVS) specific for B. henselae, one pair of primers and one TaqMan-MGB probe were designed. A quantitative real-time PCR was developed with the primers, the probe, and the IVS, a standard template, in DNA sequence detection system (ABI 7900HT).

RESULTSThe standard curve was established with the standard template and the relationship between the value of threshold cycle (Ct) and the DNA copy number was linear (r = 0.997). The sensitivity of this quantitative real-time PCR was about 1000 times higher than that of a common PCR used to detect homologous DNA. By this quantitative real-time PCR, the DNA sample of B. henselae was positively detected but not from other rickettsial or bacterial DNA samples. The variation coefficients of intra- and inter-assay reproducibility were 0.2%-1.9%. Using the real-time quantitative PCR to detect samples from mice that were experimentally infected with B. henselae, the small amount of B. henselae DNA was detected in blood samples on days 2, 3, and 5 and large amount of B. henselae DNA was detected in spleen samples on days 1 and 2 after infection.

CONCLUSIONResults from our study suggested that this quantitative real-time PCR was highly specific, sensitive and with good repeatability for detection of B. henselae. It seemed quite useful for rapid detection of tiny DNA of B. henselae in various samples and laboratory diagnosis of bartonellosis caused by B. henselae.

Animals ; Bartonella Infections ; diagnosis ; Bartonella henselae ; genetics ; DNA, Bacterial ; analysis ; Mice ; Polymerase Chain Reaction ; methods ; Reproducibility of Results ; Sensitivity and Specificity

OBJECTIVETo study the expression levels of cdk5, p35 and p53 genes in arsenic trioxide (As2O3O)-induced neuron apoptosis and to explore the potential mechanism.

METHODSThe cultured primary rats' neurons were divided into 5 groups, which were exposed to 0, 1, 5, 10 micromol/L As2O3 and dimethyl sulfoxide (DMSO) for 8 h, respectively. The cell viability and cell apoptosis were detected by MTT colouration methods and flow cytometry, respectively. The real-time fluorescence quantitative PCR was used to measured the expression levels of cdk5, p35 and p53 genes.

RESULTSThe cell viability inhibition rates were 16.77%, 19.72% and 27.81% in 1, 5, 10 micromol/L As203 groups, respectively. Compared to the untreated group and DMSO group, the cell apoptosis rates were significantly increased in 5 and 10 micromol/L As2O3 groups (P < 0.05). The expression levels of cdk5, p35 and p53 genes increased with the exposure doses of AsO3. However, there were no significant differences in p35 gene expression between different dose subgroups (P > 0.05). There were significant differences in cdk5 and p53 gene expression between different dose subgroups (P < 0.05). The expression levels of cdk5 gene in 5 and 10 micromol/L As2O3 groups were significantly higher than those in untreated group and DMSO group (P < 0.05). The expression levels of p53 gene in 1, 5 and 10 micromol/L As2O3 groups were significantly higher than that in untreated group (P < 0.05). The expression level of p53 gene in 10 mciromol/L As2O3 group was significantly higher than that in DMSO group (P < 0.05).

CONCLUSIONCdk5, p35 and p53 genes may involve in the process of As2O3-induced neural cell apoptosis.

Animals ; Apoptosis ; drug effects ; Arsenicals ; Cells, Cultured ; Cyclin-Dependent Kinase 5 ; genetics ; metabolism ; Neurons ; drug effects ; metabolism ; Oxides ; toxicity ; Phosphotransferases ; genetics ; metabolism ; Rats ; Tumor Suppressor Protein p53 ; genetics ; metabolism

OBJECTIVETo develop a real-time quantitative polymerase chain reaction(PCR) assay for detecting Rickettsia rickettsii.

METHODSThe primers and TaqMan-MGB probe were designed according to the ompB gene of R. rickettsii. A DNA fragment of ompB gene amplified from R. rickettsii by PCR was used as a standard template for the development of the method.

RESULTS5 copies of ompB fragments of R. rickettsii were detected. The genomic DNA of R. rickettsii was detected by the developed quantitative PCR assay. However, the genomic DNA from another rickettsial or bacterial agent was not determined. Through this developed method, the positive results were obtained from the animals and cells, artificially infected with R. rickettsii.

CONCLUSIONThe real-time quantitative PCR assay seemed to be highly sensitive and specific which might be used to rapidly detect R. rickettsia DNA in various samples and to early diagnose patients infected by R. rickettsii.

DNA Primers ; Humans ; Polymerase Chain Reaction ; methods ; Rickettsia rickettsii ; genetics ; Rocky Mountain Spotted Fever ; diagnosis ; Sensitivity and Specificity

OBJECTIVETo explore the coincidence of lipid peroxidation and neurobehavioral function changes in coke oven workers.

METHODSOne hundred and thirty-four coke oven workers were divided into three groups: 35 in the oven-bottom group, 49 in the oven-side group and 50 in oven-top group. WHO recommended NCTB was performed on coke oven workers and 36 controls from material conservation department; The contents of total superoxide dismutases (T-SOD), glutathione (GSH) and malondialdehyde (MDA) in blood were determined by test kits.

RESULTSCompared with the controls, the coke oven workers showed lower levels of T-SOD and GSH (P < 0.01), significantly higher MDA levels in blood (P < 0.01), higher score on negative mood state, lower scores on positive mood state, and poorer performance in NCTB test (P < 0.05). Further analysis revealed that there was a weak positive correlation between neurobehavioral function changes and the level of lipid peroxidation with a coefficient lower than 0.25.

CONCLUSIONThe level of lipid peroxidation in coke oven workers' blood increased and coincided with neurobehavioral function impairment.

Adult ; Affect ; Anxiety ; Case-Control Studies ; Coke ; Fatigue ; Glutathione ; blood ; Humans ; Lipid Peroxidation ; Male ; Malondialdehyde ; blood ; Middle Aged ; Occupational Exposure ; adverse effects ; Superoxide Dismutase ; blood ; Young Adult

OBJECTIVETo discuss effective operative method for posterior edge separation of lumbar vertebral body.

METHODSTwelve cases of posterior edge separation of lumbar vertebral body were categorized as lateral type and central type, and were operated upon with limited-incision using lamina retractor. All patients were male and with an average age of 30 years (range, 23 to 40 years). The average history of disease was 17.9 months. In lateral type cases, separation bone was in the posterolateral lower edge of L4 vertebral body in 1 case, in the posterolateral upper edge of L5 vertebral body in 3 cases and in the posterolateral upper border of S1 vertebral body in 6 cases, all with ipsilateral disc herniation. Lateral type cases were treated with unilateral fenestration and removal of disc nucleus and posterior bony edge. Separation bone was in the central upper edge of S1 vertebral body with central disc herniation in 2 central type cases. Two central type cases were treated with bilateral fenestration and removal of disc nucleus and posterior bony edge.

RESULTSAll patients were followed up for 12 to 36 months, and the average follow-up was 22.5 months. According to Macnab postoperative evaluation criterion, the results were excellent in 10 cases, good in 2 cases.

CONCLUSIONLimited-incision for posterior edge separation of lumbar vertebral body has characteristic of less trauma and convenience. It is an effective method for removal posterior bony edge.

Adult ; Follow-Up Studies ; Humans ; Lumbar Vertebrae ; diagnostic imaging ; pathology ; surgery ; Male ; Postoperative Period ; Tomography, X-Ray Computed ; Treatment Outcome ; Young Adult

OBJECTIVETo study the associations of CYP1A1 gene polymorphisms with levels of urinary 1-hydroxypyrene among coke oven workers.

METHODS223 male workers from a coke plant (76, 82 and 65 workers in oven top group, oven-side group and oven-bottom group respectively) and 119 controls without occupational polycyclic aromatic hydrocarbons exposure were selected. The MspI gene polymorphism in CYP1A1 3' flanking region and the genotypes at I462V site in exon 7 of CYP1A1 were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and allele specific amplification (ASA).

RESULTSThe urinary 1-hydroxypyrene of coke oven workers in oven-top, oven-side and oven-bottom (3.77+/-0.64, 3.57+/-0.49, 3.26+/-0.80 micromol/mol Cr) were significantly higher than controls (2.80+/-1.02 micromol/mol Cr) (P<0.01). The urinary 1-hydroxypyrene was not significantly different among MspI genotypes in CYP1A1 3' flanking region (P>0.05). In oven-top group and oven-side group, the subjects with Val/Val genotype in exon 7 of CYP1A1 had significantly higher urinary 1-hydroxypyrene levels than those with Ile/Val or Ile/Ile genotype, and urinary 1-hydroxypyrene of Ile-Val genotype were also significantly higher than Ile/Ile genotype (P<0.01). Multivariate logistic regression analysis showed that the coke oven workers (OR in oven top group, oven-side group and oven-bottom group was 24.926, 4.226 and 6.729 respectively) and subjects with m2/m2 genotype in CYP1A1 3' flanking region (OR=4.031) or with Val/Val or Ile/Val genotype in exon 7 of CYP1A1 (OR were 5.524 and 3.811) had elevated urinary 1-hydroxypyrene (greater than 95 percentile of control group, 3.876 micromol/mol Cr).

CONCLUSIONBAP concentration of work environment contributes to the elevated urinary 1-hydroxypyrene levels, and the exposed BAP levels were regulated by the CYP1A1 MspI and I462V genotypes. Genetic polymorphism of CYP1A1 gene could be a susceptible biomarker in coke oven workers which was involved in the individual susceptibility on metabolism of PAHs.

Adult ; Benzo(a)pyrene ; adverse effects ; Coke ; Cytochrome P-450 CYP1A1 ; genetics ; Humans ; Male ; Middle Aged ; Occupational Exposure ; adverse effects ; Polymorphism, Genetic ; Pyrenes ; pharmacokinetics ; Urine ; chemistry

OBJECTIVETo investigate the changes of mitochondria membrane potential and cytoplasma cytochrome C as the mechanism of neuron apoptosis induced by B(a)P.

METHODSPrimary neurons were dissociated from cerebral cortex of 1 - 3 days old SD rats and cultured with DMEM incubator at 37 degrees C. After 5 days' cultivation, the neurons were added S9 and B(a)P, and the concentrations of treated B(a)P were 0, 10, 20 and 40 micromol/L respectively. After administering of B(a)P, the neurons were cultivated for 40 hours. Apoptosis rate was measured by flow cytometry using Annexin V-FITC and propidium iodide (PI) staining, and the changes in mitochondrial potential (DeltaPsim) were tested with Rhodamine fluorescence (R2123) technique. Preparation of cytosolic extracts by centrifugation. Western blotting analysis was used to evaluate the level of cytochrome C of cytoplasm.

RESULTSThe apoptotic rate of neuron increased in both the middle dose group and the high dose group compared with controls, and had a dose-response tendency with the concentration of B(a)P. Moreover mitochondrial potential decreased in a dose dependent manner. There was a negative correlation between DeltaPsim and the apoptotic rate of neurons (r = -0.763, P < 0.05); Western blotting analysis showed cytoplasmic cytochrome C level increased significantly, which was positively related with neuron apoptosis (r = 0.831, P < 0.01).

CONCLUSIONLoss of mitochondria membrane potential and increase of cytoplasma cytochrome C may be the main cause of neuron apoptosis induced by B(a)P.

Animals ; Apoptosis ; drug effects ; Benzo(a)pyrene ; toxicity ; Cells, Cultured ; Cytochromes c ; metabolism ; Membrane Potential, Mitochondrial ; Mitochondria ; drug effects ; metabolism ; Neurons ; cytology ; drug effects ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo study the effects of benzo[a]pyrene (B[a]P) on capability of learning and memory and the content of amino acid neurotransmitters in hippocampus of rats.

METHODSThirty-two healthy, male SD rats were randomly divided into 4 groups according to their weights after intubated into ventricles: the solvent control group, 2.5, 5.0 and 10.0 mmol/L groups. 10 microl of B[a]P olive oil solutions, of different concentrations 2.5, 5.0 and 10.0 mmol/L, were injected into rats' lateral ventricles, respectively. Rats in the solvent control group were injected into the same volume of olive oil as that in B[a]P group. Rats' capability of learning and memory was tested by Morris water maze. The content of amino acid neurotransmitters in rats' hippocampus were determined by high performance liquid chromatogram with a fluorescence detector.

RESULTSCompared with the controls, the performances of learning and memory of rats decreased significantly in B[a]P treated groups (P<0.01). Levels of glutamate (Glu) were lower significantly in treated groups than that in controls (P<0.01). No significant differences were found in contents of aspartic acid (Asp), glycine (Gly) and aminobutyric acid (GABA) among the four groups.

CONCLUSIONB[a]P can damage rats' spatial learning and memory, and which could be related to decreased contents of excitatory amino acids in hippocampus.

Amino Acids ; metabolism ; Animals ; Benzo(a)pyrene ; toxicity ; Hippocampus ; drug effects ; metabolism ; Male ; Maze Learning ; drug effects ; Memory ; drug effects ; Neurotransmitter Agents ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo Evaluate the effects of different oxygen therapies on the rats with acute nitrogen asphyxia and to study the best oxygen therapic protocol for patients with acute nitrogen asphyxia on the spot.

METHODSSixty healthy male Wistar rats were divided into 5 groups: control, exposure to nitrogen, 33% oxygen treatment, 50% oxygen treatment and hyperbaric oxygen treatment groups. The behavioral performance, arterial oxygen pressure (PO2), carbon dioxide partial pressure (PCO2) and oxygen saturation (SPO2), biochemical changes in liver and kidney function and myocardial enzymes in 5 groups were measured.

RESULTSThe rats exposed to nitrogen firstly were excited then inactive symptoms, but consciousness was recovered after oxygen therapy. The PO2 and SPO2 in nitrogen exposure group were (79.67 +/- 9.12) and (94.92 +/- 2.78) mm Hg, respectively, which were significantly lower than those in control group (P<0.01). The PO2 and SPO2 of 3 oxygen treatment groups were (94.75 +/- 7.24), (94.92 +/- 8.98), (104.58 +/- 7.12)mm Hg and (97.17 +/- 0.83), (96.92 +/- 1.16), (97.42 +/- 0.67)mm Hg, respectively, which were significantly higher than those in nitrogen exposure group (P<0.05). The PO2 in hyperbaric oxygen treatment group was significantly higher than those in other 2 oxygen treatment groups (P<0.05). The SPO2 in hyperbaric oxygen treatment group was (51.42 +/- 6.60) mm Hg which was significantly higher than that [(44.58 +/- 3.42)mm Hg] in 50% oxygen treatment groups (P< 0.05). AST [(270.50 +/- 49.05 )U/L], ALT [(122.67 +/- 55.44 )U/L], BUN [(7.31 +/- 0.93 )mmol/L], Cr[(28.32 +/- 4.35) micromol/L], CK [(1808.42 +/- 582.05)U/L] and CtnI [(22.52 +/- 14.29 )ng/ml] in nitrogen exposure group were significantly higher than those in control group (P<0.05). AST [(165.25 +/- 30.87) U/L], HBDH [(350.83 +/- 103.00)U/L] and CtnI [(11.23 +/- 5.38) ng/ml] in hyperbaric oxygen treatment group were significantly lower than those in other 2 oxygen treatment groups (P<0.05).

CONCLUSIONTimely and effective oxygen therapy can significantly increase arterial pressure of oxygen and oxygen saturation in the rats with acute nitrogen asphyxia, and can improve liver function and cardiac damage. The hyperbaric oxygen chamber can significantly increase the therapeutic effects on rats with acute nitrogen asphyxiation.

Animals ; Asphyxia ; blood ; chemically induced ; Blood Gas Analysis ; Hyperbaric Oxygenation ; Male ; Nitrogen ; toxicity ; Oxygen Inhalation Therapy ; Rats ; Rats, Wistar

OBJECTIVETo study therapeutic effects by using different oxygen therapies in rats with acute carbon dioxide poisoning, to select the best oxygen therapy technology for patients with acute carbon dioxide poisoning on the spot.

METHODSSixty healthy male Sprague-Dawley rats were randomized into normal control group, carbon dioxide exposure group, hyperbaric oxygen treatment group (pressure 2 ATA, FiO(2)100%), high concentration of atmospheric oxygen treatment group (FiO(2)50%), low concentration of atmospheric oxygen treatment group (FiO(2)33%). After treated with different oxygen in rats with acute carbon dioxide poisoning, arterial pH, PO2 and PCO2 of rats were detected, in addition observe pathological changes of lung tissue and brain tissue.

RESULTSThe arterial pH (7.31 ± 0.06) and PO2 [(68.50 ± 15.02) mm Hg] of carbon dioxide exposure group were lower than those of control group [pH (7.42 ± 0.02) and PO2 (92.83 ± 8.27) mm Hg], PCO2 [(71.66 ± 12.10) mm Hg] was higher than that of control group [(48.25 ± 2.59) mm Hg] (P < 0.05); the arterial pH (hyperbaric oxygen treatment group 7.37 ± 0.02, high concentration of atmospheric oxygen treatment group 7.39 ± 0.03, low concentration of atmospheric oxygen treatment group 7.38 ± 0.02) and PO2 of oxygen treatment groups [hyperbaric oxygen treatment group, high concentration of atmospheric oxygen treatment group, low concentration of atmospheric oxygen treatment group were (82.25 ± 12.98), (84.75 ± 11.24), (83.75 ± 16.77) mm Hg, respectively] were higher than that of carbon dioxide exposure group, PCO2 [hyperbaric oxygen treatment group, high concentration of atmospheric oxygen treatment group, low concentration of atmospheric oxygen treatment group were (52.25 ± 4.95), (51.75 ± 4.82), (52.66 ± 5.61) mm Hg, respectively] was lower than that of carbon dioxide exposure group (P < 0.05); there was no significant difference of the arterial pH, PO2 and PCO2 between oxygen treatment groups and control group (P > 0.05); there was no significant difference of the arterial pH, PO2 and PCO2 among oxygen treatment groups (P > 0.05). There was large area of bleeding of lungs in rats with carbon dioxide poisoning, the bleeding of lungs in rats with high concentration of atmospheric oxygen treatment and low concentration of atmospheric oxygen treatment was better than the rats with carbon dioxide poisoning, there was no abnormal appearance of lungs in rats with hyperbaric oxygen treatment. The light microscope observation showed that there were diffuse bleeding and exudation of lungs in rats with carbon dioxide poisoning, the bleeding and exudation of lungs in rats with high concentration of atmospheric oxygen treatment and low concentration of atmospheric oxygen treatment were better than the rats with carbon dioxide poisoning, there were only minor bleeding and exudation of lungs in rats with hyperbaric oxygen treatment. There was no difference of brain in anatomy and microscopy among all groups, there were no significant bleeding, edema, cell degeneration and necrosis.

CONCLUSIONSLung pathology in acute carbon dioxide poisoning rats with hyperbaric oxygen treatment is better than the rats with high concentration of atmospheric oxygen treatment and low concentration of atmospheric oxygen treatment, there is no significant difference of effect between high concentration of atmospheric oxygen treatment group and low concentration of atmospheric oxygen treatment group, however, the results of blood gas analysis and lung pathology than the exposure group improved, so qualified medical unit for hyperbaric oxygen therapy as soon as possible, hyperbaric oxygen treatment facilities in the absence of circumstances, the emergency treatment of early oxygen is also a good measure.

Animals ; Carbon Dioxide ; poisoning ; Hyperbaric Oxygenation ; Lung ; pathology ; Male ; Oxygen Inhalation Therapy ; methods ; Rats ; Rats, Sprague-Dawley ; Treatment Outcome