A double targets of high throughput screening model for xanthine oxidase inhibitors and superoxide anion scavengers was established. In the reaction system of xanthine oxidase, WST-1 works as the probe for the ultra oxygen anion generation, and product uric acid works as xanthine oxidase activity indicator. By using SpectraMax M5 continuous spectrum enzyme sign reflectoscope reflector, the changes of these indicators' concentration were observed and the influence factors of this reaction system to establish the high throughput screening model were studied. And the model is confirmed by positive drugs. In the reaction system, the final volume of reaction system is 50 μL and the concentrations of xanthine oxidase is 4 mU x mL(-1), xanthine 250 μmol x L(-1) and WST-1 100 μmol x L(-1), separately. The Z'-factor of model for xanthine oxidase inhibitors is 0.537 4, S/N is 47.519 9; the Z'-factor of model for superoxide anion scavengers is 0.507 4, S/N is 5.388 9. This model for xanthine oxidase inhibitors and superoxide anion scavengers has more common characteristics of the good stability, the fewer reagent types and quantity, the good repeatability, and so on. And it can be widely applied in high-throughput screening research.
Enzyme Inhibitors ; pharmacology ; Free Radical Scavengers ; pharmacology ; High-Throughput Screening Assays ; Superoxides ; Uric Acid ; Xanthine ; Xanthine Oxidase ; antagonists & inhibitors

Objective To evaluate the performance of nested PCR in the detection of different fungi in paraffin wax embedded tissues. Methods Forty-four tissue samples were resected from rats infected with Fonsecaea monophora, patients with chromoblastomycosis, sporotrichosis or penicilliposis marneffei followed by preparation of paraffin wax embedded tissue sections for pathological examination and DNA extraction. Nested PCR was performed by using specific primers targeting the ribosomal DNA of Fonsecaea, Sporothrix and Penicillium marneffei, respectively. The sensitivity and specificity of nested PCR were analyzed and compared with those of pathological examination. Results The nested PCR showed positive results in 8 of 20 samples from rats with chromoblastomycosis, 7 of 10 samples from patients with sporotrichosis and all of the 10 samples from patients with penicilliposis marneffei, but not in the control samples. In the detection of Fonsecaea,Sporothrix schenki and Penicillium marneffei, the sensitivity was 40% ,70% and 100%, respectively, and the specificity was consistently 100%, for the nested PCR. Pathological examination revealed fungal elements in 95%, 70% and 80% of the corresponding samples, respectively. Conclusion Detection of fungal DNA in paraffin wax embedded tissue by nested PCR can be applied to the diagnosis of deep mycosis, especially to the diagnosis of penicilliposis marneffei.

The chemical structures of P1 A was identified by complete acid hydrolysis, partial acid hydrolysis, periodate oxidation-Smith degradation, methylation analysis, IR and NMR. The results showed that P1 A had a backbone consisting rhamnose, mannose, glucose and galactose. The side chain possessed arabinose and xylose. 1-->, 1-->6 and non-reducing terminal linkages existed in polysaccharide P1A, but there are doubling amount of 1-->2 and 1-->4 linkages. Oxidable linkage of P1 A accounted for 45%, and inoxidable linkage of P1A accounted for 55%. Mannose, glucose and galactose were mainly linked by 1-->2 linkage. Rhamnose, arabinose and xylose were mainly linked by 1-->2 and 1-->4 linkages. PlA contained beta-Glc(1,6)-,beta-Gal(1,3)-,beta-Man(1,4)-beta-Rha,-Glc(1,4)-, Glc(1)-,-Gal(1,4)- and Man(1)-.
Acanthaceae ; chemistry ; Drugs, Chinese Herbal ; chemistry ; Magnetic Resonance Spectroscopy ; Molecular Structure ; Molecular Weight ; Polysaccharides ; chemistry

A GeXP based multiplex PCR assay was developed to simultaneously detect six different chicken respiratory viruses including H5, H7, H9 subtypes of avian influenza virus(AIV), new castle disease virus (NDV), infectious bronchitis virus(IBV) and infectious laryngotracheitis virus(ILTV). According to the conserved sequences of genes of each pathogen, seven pairs of specific primers were designed, and the reaction conditions were optimized. The specificity and accuracy of GeXP were examined using samples of single and mixed infections of virus. The sensitivity was evaluated by performing the assay on serial 10-fold dilutions of cloned plasmids. To further evaluate the reliability, thirty-four clinical samples were detected by GeXP. The corresponding specific fragments of genes were amplified. The detection limit of GeXP was 10(2) copies/microL when all of 7 pre-mixed plasmids containing target genes of six chicken respiratory viruses were present. In the detection of thirty-four clinical samples, the results of GeXP were accorded with the viral isolation completely. In conclusion, this GeXP assay is a rapid, specific, sensitive and high-throughput method for the detection of chicken respiratory virus infections. It can be applied in rapid differential diagnosis for clinical samples, and also provide an effective tool to prevent and control chicken respiratory diseases with similar clinical symptoms.
Animals ; Chickens ; Influenza A virus ; classification ; genetics ; isolation & purification ; physiology ; Influenza in Birds ; diagnosis ; virology ; Multiplex Polymerase Chain Reaction ; methods ; Poultry Diseases ; diagnosis ; virology ; Respiratory Tract Infections ; diagnosis ; veterinary ; virology

OBJECTIVETo observe the peripheral neurotoxicity of 1-bromopropane (1-BP) by developing an animal model of peripheral neuropathy through oral administration of 1-BP.

METHODSForty male Wistar rats were randomly and equally divided into low-dose group (200 mg/kg), medium-dose group (400 mg/kg), high-dose group (800 mg/kg), and control group. The rats in the low-dose, medium-dose, and high-dose groups were orally given 1-BP (dissolved in corn oil), while the rats in the control group were orally given an equal volume of corn oil. The oral administration (0.2 ml/100 g BW) was performed once per day, 5 days per week, for 16 consecutive weeks. Neurobehavioral indices including gait score, hindlimb grip strength, and hindlimb landing foot splay were recorded periodically. Hematological and biochemical parameters were also measured during and after 1-BP exposure.

RESULTSThe gait scores were significantly higher in the high-dose group (after 8 ∼ 16 weeks of 1-BP exposure), medium-dose group (after 14 ∼ 16 weeks of 1-BP exposure), and low-dose group (after 15 ∼ 16 weeks of 1-BP exposure) than in the control group (P < 0.05, P < 0.01). Compared with the control group, the high-dose group showed significantly decreased hindlimb grip strength after 9, 12, and 14 weeks of 1-BP exposure (P < 0.05, P < 0.01), with the hindlimbs paralyzed after 16 weeks of 1-BP exposure. After 16 weeks of 1-BP exposure, the hindlimb grip strengths of rats in the medium-dose and low-dose groups were decreased to 72.6% and 91.2% of the control value (P < 0.01, P < 0.05). Compared with the control group, the high-dose group showed significantly increased hindlimb landing foot splay after 12, 14, and 16 weeks of 1-BP exposure, and the medium-dose group showed significantly increased hindlimb landing foot splay after 14 and 16 weeks of 1-BP exposure (P < 0.05, P < 0.01). The high-dose and medium-dose groups showed significantly higher serum alanine aminotransferase (ALT) activity than the control group after 8 weeks of 1-BP exposure, and so did the low-dose group after 16 weeks of 1-BP exposure (P < 0.01).

CONCLUSIONThe nervous system is sensitive to the toxic effect of 1-BP, and 1-BP exposure can induce peripheral neuropathy in rats.

Animals ; Disease Models, Animal ; Hydrocarbons, Brominated ; administration & dosage ; toxicity ; Male ; Peripheral Nervous System Diseases ; chemically induced ; physiopathology ; Rats ; Rats, Wistar

In this paper, we studied the relationship between the prostaglandin F(2alpha) (PGF(2alpha))-induced cardiac hypertrophy and calcineurin (CaN) signal transduction pathway in vivo and in vitro. Male Sprague-Dawley rats were given a single i.p. injection with monocrotaline (MCT) (60 mg/kg) and then given orally with celecoxib (20 mg/kg) or vehicle once a day for 14 d before (from d 1 to d 14) or after (from d 15 to d 28) right ventricular hypertrophy (RVH) was formed. Body weight (BW), right ventricular weight (RV), left ventricular with septum weight (LV), as well as lung weight were determined. RVH index (RVHI=RV/LV), RV/BW, and lung weight/BW were calculated and histological changes were observed with transmission electron microscope. PGF(2alpha) level, atrial natriuretic peptide (ANP) and CaN mRNA expressions, expression of CaN and its downstream effectors, NFAT(3) and GATA(4) protein were assayed by EIA kit, RT-PCR, and Western blotting, respectively. The cardiomyocyte hypertrophy in primary culture induced by PGF(2alpha) (0.1 micromol/L) was evaluated by measuring the cell diameter, protein content, and ANP mRNA as well as CaN mRNA expressions. It was found that 14 d or 28 d after MCT was given, the RVHI, RV/BW, and lung weight/BW were significantly increased by 47%, 53% and 118%, and by 64%, 94% and 156%, respectively; at the same time PGF(2alpha) levels in RV tissue were increased by 44% and by 51% with increasing RVHI, and elevated expressions of ANP and CaN mRNA, as well as CaN, NFAT(3) and GATA(4) proteins in a positive correlation manner. Furthermore, some histological injuries were found in RV tissue. Celecoxib, a cyclooxygenase inhibitor, obviously blunted the elevation of RVHI, RV/BW, and lung weight/BW no matter it was given before or after RVH. In vitro experiments showed that 0.1 micromol/L PGF(2alpha) significantly increased the cardiomyocyte diameter and protein content, and promoted ANP and CaN mRNA expressions, which was blocked by cyclosporin A, a CaN inhibitor. Our results indicate that PGF(2alpha) may be involved in cardiac hypertrophy induced by MCT in rats through CaN signal transduction pathway.
Animals ; Calcineurin ; genetics ; metabolism ; physiology ; Cells, Cultured ; Dinoprost ; metabolism ; physiology ; Hypertrophy, Right Ventricular ; chemically induced ; metabolism ; physiopathology ; Male ; Monocrotaline ; Myocytes, Cardiac ; metabolism ; pathology ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; physiology

OBJECTIVETo investigate the feasibility of life span extension of transformed human embryonic tendon cells (THETC) by reconstitution of the telomerase activity.

METHODSTHETC were transfected by pGRN145 plasmid containing the human telomerase reverse transcriptase (hTERT) cNDA in vitro by molecular cloning technique. The biological characteristics of transfected cells were detected and compared by morphological observation, plate cloning efficiency, soft agar culture, growth curve of cells cultured in different conditions, immunohistochemistry, telomerase activity assay by telomeric repeat amplification protocol (TRAP).

RESULTSThe THETC transfected by pGRN145 plasmid (telT) could express the telomerase activity with extension of life span. The telT maintained the original characteristics of temperature-dependant and serum-dependant, as well as secretion of type I collagen normally and without tendency of malignant transformation.

CONCLUSIONSThe life span of THETC can be prolonged by reconstitution of telomerase activity, which provides the novel experimental methods to establish the standard cells line.

Cell Line ; Cell Survival ; Embryo, Mammalian ; Humans ; Plasmids ; genetics ; RNA-Directed DNA Polymerase ; Telomerase ; genetics ; metabolism ; Tendons ; cytology ; enzymology ; Transfection

OBJECTIVETo observe the effect of carbon disulfide (CS(2)) on the expression of matrix metalloproteinase (MMP)-2, MMP-9 in mouse embryo and uterus tissues and to explore the mechanism of embryo toxicity induced by CS(2).

METHODSAt the phases of follicular development and embryonic implantation which was subdivided into early-implantation phase and late-implantation phase, mice were intraperitoneally exposed to CS(2) (the dosage was 631.4 mg/kg, and the volume was 0.1ml/10 g body weight) for 2 consecutive days. All indicators were got at the ninth day in gestation, and the expression of MMP-2 and MMP-9 in embryo and uterus tissues was analyzed by gelatin zymography.

RESULTSThe number of implanted embryos significantly decreased after exposure at late-implantation phase (16.000 ± 12.166) compared with those of the control (30.700 ± 5.599, P < 0.05). Expression of MMP-2 and MMP-9 in embryos declined obviously at the three reproductive phases (P < 0.01), and the levels of MMP-2 and MMP-9 expression in embryos at the phases of late-implantation phase (0.6837 ± 0.0929, 0.7309 ± 0.0822) and follicular development (0.6222 ± 0.0997, 0.7520 ± 0.1068) were much lower than those of the control (1.0000 ± 0.0710, 1.0000 ± 0.0413, P < 0.01). Expression of MMP-2 and MMP-9 in uterus significantly increased at the phase of late-implantation (1.3153 ± 0.3032, 5.0210 ± 4.0307) compared with those of the control (1.0000 ± 0.1771, 1.0000 ± 0.0996, P < 0.01).

CONCLUSIONEmbryo toxicity of CS(2) is more obvious at the phase of late-implantation. Exposure to CS(2) disturbs expression of MMP-2 and MMP-9 in embryo and uterus tissues, which might be one of the important factors contributed to embryo toxicity induced by CS(2).

Animals ; Carbon Disulfide ; toxicity ; Embryo Implantation ; Embryo, Mammalian ; drug effects ; metabolism ; Female ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Mice ; Mice, Inbred Strains ; Pregnancy ; Uterus ; drug effects ; metabolism

OBJECTIVETo compare the difference in the clinical efficacy on cervical spondylosis between acupuncture at three lines of cervical Jiaji (EX-B 2) and oral administration of jingfukang granules.

METHODSThree hundred cases of cervical spondylosis were divided into an acupuncture group and a medication group, 150 cases in each one. In the acupuncture group, according to the different types of cervical spondylosis, acupuncture was applied at three lines of cervical Jiaji (EX-B 2), once a day. In the medication group, jingfukang granules were prescribed for oral administration, one bag each time, three times a day. The treatment of ten days made one session in the two groups and two sessions were required totally. Before and after two sessions of treatment, the clinical assessment scale for cervical spondylosis (CASCS) was adopted to evaluate the score of subjective symptoms, clinical physical signs and adaptability as well as the total score in the patients of the two groups and the efficacy was compared.

RESULTSThe patients' symptoms and physical signs were alleviated, the adaptability was improved and the score of each item and the total score were increased in the two groups after treatment (all P<0.01). The improvements in the acupuncture group were better than those in the medication group (all P<0.01). The curative and markedly effective rate was 90.7% (136/150) in the acupuncture group, better than 66.0% (99/150) in the medication group (P<0.01).

CONCLUSIONAcupuncture at three lines of cervical Jiaji (EX-B 2) achieves the significant clinical efficacy on cervical spondylosis. This therapy is superior to relieving symptoms and physical signs and recovering adaptability as compared with jingfukang granules.

Acupuncture Points ; Acupuncture Therapy ; Adult ; Female ; Humans ; Male ; Middle Aged ; Spondylosis ; therapy ; Treatment Outcome ; Young Adult