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.

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

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 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

OBJECTIVETo study the protective effects of garlic oil (GO) on the peripheral nerve injuries induced by n-hexane.

METHODSMale Wistar rats were randomly divided into four groups (10 rats in each group): the control, the n-hexane treatment (2000 mg/kg), the low dose GO, and the high dose GO groups. The rats in the low and high doses of GO groups were pretreated with GO (40 and 80 mg/kg) before exposure to n-hexane (2000 mg/ kg), while the animals of the n-hexane treatment group were given normal saline and then 2000 mg/ kg n-hexane. The rats were exposed to GO and n-hexane 6 times a week for 10 weeks. The gait scores and staying time on the rotating rod for all rats were detected every two weeks. The rats were sacrificed at the end of ten weeks, then the levels of alcohol dehydrogenase (ADH), maleic dialdehyde (MDA), reduced glutathione (GSH), glutathione peroxidase(GSH-Px), total antioxidation capacity(T-AOC) and the ability of inhibition of *OH in livers were examined.

RESULTSThe gait scores increased significantly and the time staying on the rotating rod obviously decreased in rats of n-hexane treatment group, as compared with control group (P < 0.05 or P < 0.01). In the hepatic tissues of n-hexane group, the levels of MDA and ADH significantly increased, the activities of GSH-Px, T-AOC and the ability of inhibition of *OH obviously decreased, as compared to control group (P < 0.05 or P < 0.01). In 2 GO groups, the gait scores and the staying time on the rotating rod were significantly improved, the levels of MDA and ADH significantly decreased, the activities of GSH-Px, T-AOC and the ability of inhibition of *OH obviously increased, as compared with n-hexane group (P < 0.05 or P < 0.01 ).

CONCLUSIONADH could play an important role in the protective effects induced by garlic oil on the peripheral nerve injuries produced by n-hexane.

Alcohol Dehydrogenase ; metabolism ; Animals ; Garlic ; Hexanes ; toxicity ; Lipid Peroxidation ; drug effects ; Liver ; enzymology ; metabolism ; Male ; Neuroprotective Agents ; pharmacology ; Peripheral Nerve Injuries ; chemically induced ; metabolism ; Plant Oils ; pharmacology ; Rats ; Rats, Wistar

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 study the clinicopathologic features, diagnosis and differential diagnosis of epithelioid hemangioma.

METHODSThe morphologic features of 7 cases of epithelioid hemangioma of skin, bone and venous vessels were studied.

RESULTSThere were altogether 4 male and 3 female patients (median age = 34 years; age range from 14 to 54 years). The 3 skin cases presented as single or multiple erythematous to bluish nodules or papules, with or without itchiness. The 2 bone cases appeared as osteolytic expansile lesions on radiologic examination. The remaining 2 cases involved medium-sized venous structures and presented as small isolated nodules in soft tissue. Histologically, the lesions were characterized by the presence of exuberant endothelial proliferations with various degree of inflammatory reaction. The neoplastic endothelial cells were plump, eosinophilic and polygonal, forming vascular channels. Occasional solid sheet-like arrangement was demonstrated. Intracytoplasmic vacuoles were commonly identified, indicating formation of primary lumen. The surrounding stroma contained various number of eosinophils and lymphoplasmacytic cells. Immunohistochemical study showed that the tumor cells were positive for endothelial markers (CD31 and CD34) and negative for epithelial marker (cytokeratin). Follow-up information was available in 6 cases. The duration of follow-up ranged from 5 to 36 months (median = 14 months). There was no evidence of recurrence or distant metastasis.

CONCLUSIONSEpithelioid hemangioma is a rare benign curable lesion which can be multifocal, involving skin, soft tissue and bone. It needs to be distinguished from Kimura's disease and epithelioid hemangioendothelioma.

Adolescent ; Adult ; Angiolymphoid Hyperplasia with Eosinophilia ; pathology ; Antigens, CD34 ; metabolism ; Bone Neoplasms ; metabolism ; pathology ; surgery ; Diagnosis, Differential ; Female ; Follow-Up Studies ; Hemangioendothelioma, Epithelioid ; pathology ; Hemangioma ; metabolism ; pathology ; surgery ; Humans ; Male ; Middle Aged ; Platelet Endothelial Cell Adhesion Molecule-1 ; metabolism ; Skin Neoplasms ; metabolism ; pathology ; surgery

OBJECTIVETo elucidate the effect of angiotensin II (AngII) on the expression of tissue factor (TF) by monocytes and its mechanisms.

METHODSMonocytes were isolated from healthy volunteers by Ficoll-Hypaque gradient and Percoll, and cultured in RPMI-1640. Procoagulant activity (PCA) was determined by one-stage clotting method, TF antigen by ELISA. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the TF gene mRNA. The levels of IkappaBalpha was detected by Western blot. Electrophoretic mobility shift assays (EMSA) were performed to evaluate the activity of NF-kappaB.

RESULTSAngII (10(-9) - 10(-7) mol/L) significantly increased monocyte PCA, TF antigen and TF mRNA expression in a dose and time dependent manner. Losartan (10(-6) - 10(-5) mol/L) significantly inhibited the effects of AngII on TF activity, antigen and mRNA expression in a dose-dependent effects. Staurosporine (2.5 x 10(-7) mol/L) and genistein (4 x 10(-5) mol/L) lowered TF level of monocytes (P < 0.05). Western blot analysis revealed that after exposure to AngII (10(-7) mol/L), IkappaBalpha level decreased at 15 min, reached nadir at 60 min, and recovered at 180 min. EMSA showed NF-kappaB binding activity increased at 15 min, reached peak at 60 min, and recovered at 180 min. Pyrrolidine dithiocarbamate (PDTC, 10(-4) mol/L), an inhibitor of NF-kappaB, or AT1R antagonist losartan (10(-5)mol/L) inhibited AngII-induced NF-kappaB translocation.

CONCLUSIONSAngII could induce the expression of TF in human monocytes, and this effect was mediated by AT1R. The PKC pathway played the most important role in AngII-induced TF expression. The activation of NF-kappaB was involved in TF expression in monocytes.

Angiotensin II ; pharmacology ; Gene Expression Regulation ; drug effects ; Genistein ; pharmacology ; Humans ; Losartan ; pharmacology ; Monocytes ; drug effects ; metabolism ; NF-kappa B ; metabolism ; Protein Kinase C ; physiology ; RNA, Messenger ; analysis ; Receptor, Angiotensin, Type 1 ; physiology ; Staurosporine ; pharmacology ; Thromboplastin ; genetics

OBJECTIVETo investigate the effects and mechanisms of Curcumol beta-cyclodextrin Compound (CbetaC) on the proliferation and apoptosis of esophageal carcinoma cell line TE-1.

METHODSThe CbetaC was prepared by saturated solution and confirmed by infrared absorption spectroscopy. The effects of CbetaC (at 25, 50, 100 mg/L) on the proliferation of human esophageal carcinoma cell line TE-1 in vitro was analyzed by MTT assay. The cell cycles and apoptosis were detected by flow cytometer. The relative expression of survivin mRNA was detected by real-time fluorescent quantitative PCR and calculated by the 2(-deltaCt) method. The protein expression of survivin was measured by Western blot.

RESULTSCompared with the control group, results of MTT showed that CbetaC at each dose significantly inhibited the proliferation of TE-1 cells in a dose-dependent manner (P < 0.05). The results of flow cytometry showed that CbetaC resulted in the cell cycle arrest at G0/G1 and G2/M phase, and promoted the cell apoptosis. Besides, when compared with the control group, the protein and mRNA expressions of survivin obviously decreased in each CbetaC group (P < 0.05).

CONCLUSIONSCbetaC could inhibit the proliferation of esophageal carcinoma cell TE-1 and promote the apoptosis. Its inhibition on the survivin expression was correlated with its inhibition on the malignant phenotypes of esophageal carcinoma cells.

Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Esophageal Neoplasms ; pathology ; Humans ; Sesquiterpenes ; pharmacology ; beta-Cyclodextrins ; pharmacology