AIMTo observe the effects of Ca2+ -activated K+ channel of primary cultured fetal SD rat cortex neurons in the veratridine triggered neuronal damage.

METHODSThe patch clamp technique of cell-attach and inside-out mode for these two kinds of single channel recordings were used.

RESULTSExtracellular veratridine activated the Kca. In Ca2+ bath solution of cell-attach mode, Vp + 30 mV, when the concentration (micromol/L) of veratridine were 15,25,50 and 75, the open probabilities of the channel were 0.014 +/- 0.003, 0.085 +/- 0.010, 0.132 +/- 0.016 and 0.059 +/- 0.006 (P < 0.01) respectively. It appeared concentration-dependent within 50 micromol/L veratridine. In Ca2+ free bath solution of cell-attach mode, Vp = +50 mV, when the concentration (micromol/L) of veratridine were 15, 40,60 and 100, the open probabilities of the channel were 0.014 +/- 0.010, 0.113 +/- 0.006, 0.141 +/- 0.004 and 0.295 +/- 0.009 (P < 0.05) respectively. In the 6 cases of inside-out mode patch clamp, Vp = +40 mV, when the concentration of veratridine were 0, 25 micromol/L and 50 micromol/L, the open probabilities of the channel were 0.011 +/- 0.008, 0.010 +/- 0.010 and 0.012 +/- 0.007 (P > 0.05) respectively. There were no significant difference on open probabilities, average open/close times and amplitudes at different intracellular veratridine concentration.

CONCLUSIONVeratridine can affect the activation of the Kca channel through regulating the concentration of cytoplasmic free Ca2+. The opening of Kca activated by increase of intracellular Ca2+ during the early stage of anoxia may be a protection reaction of ischemic neurons.

Animals ; Animals, Newborn ; Calcium ; metabolism ; Cells, Cultured ; Neurons ; cytology ; drug effects ; physiology ; Patch-Clamp Techniques ; Potassium Channels, Calcium-Activated ; metabolism ; Rats ; Rats, Sprague-Dawley ; Veratridine ; pharmacology

OBJECTIVETo explore the action of pingxiao capsules (PXC) and its significance in the treatment of late stage mammary cancer (LSMC).

METHODSOne hundred and forty-two LSMC patients were randomized into four groups: the two single treated groups treated by endocrinotherapy (ET) alone (n = 27) and by chemotherapy alone (n=44) respectively, and the two PXC combined treated groups treated with PXC plus endocrinotherapy (n=27) or chemotherapy (n=44). The remission rate and progression time (TTP) of disease, the survival time and quality of life (QOL) of patients, and the adverse reaction were compared between the single treated groups and the combined treated groups.

RESULTSThe median progression time was obviously prolonged, and QOL improved in the combined treated groups than those in the single treated groups (P < 0.05), but no significant difference was found in the remission rate or adverse reaction between them.

CONCLUSIONPXC can improve QOL, prolong the progression time in patients of LSMC, and with less adverse reaction. It is worth spreading combination of PXC with chemo- or endocrino-therapy in clinical application for treatment of LSMC patients.

Adult ; Aged ; Antineoplastic Agents, Hormonal ; therapeutic use ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Breast Neoplasms ; drug therapy ; pathology ; Drug Therapy, Combination ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Humans ; Middle Aged ; Paclitaxel ; administration & dosage ; Phytotherapy ; Sepharose ; administration & dosage ; analogs & derivatives ; Tamoxifen ; therapeutic use

Objective To investigate the expression of macrophage migration inhibition factor (MIF) and cell cycle regulating factor Cyclin D1 in hepatocellular carcinoma tissue and the interaction between MIF and Cyclin D1 in hepatocellular carcinoma cell cycle controlling. Methods Using quantitative real-time PCR and Western blotting to detect mRNA and protein expression of MIF and Cyelin DI in HCC tissues and tumor adjacent tissues. Specific small interfering RNA(siRNA) targeting MIF gene was transfccted at doses of 50 nmol/L and 100 nmoL/L into HCC cell lines of PLC and HepG2 with lipofeetamine 2000 methods to knockdown the expression of M1F gene and to investigare the the interaction between M1F and Cyclin D1. Results MIF and Cyclin D1 protein and mRNA were overexpressed in HCC tumor tissues. The relative expression of MIF,Cyclin D1 protein and mRNA were 0.825±0.13,0.843± 0.104 and 7.31±1.85 folds、4.27±1.05 folds, compared with the tumor adjacent tissues (FMIF= 15.5, P<0.01;FCyclin D1=87.5,P <0.01). In MIF siRNA treated PLC and HepG2 cells, MIF mRNA down regulation 71.2%±7.2%, 87.4%±2.9% ,74.3%±8.9% and 88.4%±4.6% respectively (FPLC = 315.5 ,P < 0.01 ; FHepG2= 201.2 P < 0.01). While MIF protein expression were significandy reduced to 0.33±0.03,0.11±0.02, 0.81±0.08 and 0.36±0.02 in a dose-dependent manner (FPLC= 43.9, P <0.01 ;FHepG2 = 133.4 P <0.01). Cyclin D1 mRNA was significantly down-regnlated in MIF siRNA treated PLC and HepG2 cell lines when compared with control group(P <0.01). In 50 nmol/L and 100 nmol/L groups, Cyclin DI mRNA levels were respectively decreased by 68.2%±3% and 78.1%±1.4% in PLC cell, 65.8%±4.7% and 77.3%±2.6% in HepG2 cell (FPLC= 1569, P < 0.01 ; FHepG2= 480.4, P <0.01). Compared with control groups, Cyclin D1 protein levels significantly reduced to 0.28±0.06、0.15±0.03 and 0.44 ±0.04、0.13±0.02 in the PLC and HepG2 after M IF siRNA treatment(FPLC= 35.5, P < 0.01 ; FHepG2 = 114.7, P < 0.01). Conclusions MIF and Cyclin D1 mRNA and protein were overexpressed in HCC tumor tissues and participated in tumor cell cycle regulation. MIF may up-regnlate the expression of Cyclin DI via ERK signalling and precipitate in carcinogenesis of hepatocellular carcinoma.

To develop a reverse dot blot assay for rapid detection of HBV genotypes.Specific oligonucleotides probes were desighed and immobilized on nylon membranes.The DNA sample to be tested was PCR-amplified with DIG labeling primers and then hybridized with the immobilized probes.This procedure for detecting HBV genotypes was simple,rapid and specificity.30 specimens in Chongqing area were collected and detected by this method,and results were evaluated using direct sequencing.Results showed that: This new method was applicable to precise detection HBV genotypes for specimen with copies up to 103,and the HBV genotyping results showed that genotype B was the predominant genotype in Chongqing area.

OBJECTIVETo observe the histological and ultrastructural changes of the skin and hair follicles following hair removal by alexandrite laser in Tibet mini-pigs.

METHODSTwelve healthy Tibet mini-pigs with dark hair were treated with alexandrite laser for hair removal. The skin specimens were taken immediately and at 1 h and 1, 3, 5, 10, 15, 30, 60 days after the laser treatment for observation under optical and transmission electron microscope.

RESULTSLaser hair removal resulted in extensive coagulation necrosis, carbonization and falling of the subcutaneous hair shafts, and some of the cells in the outer root sheath and hair bulb underwent degenerative and necrotic changes. One hour after laser treatment, the cells in the outer root sheath and bulb exhibited nuclear condensation, fragmentation and or karyolysis characteristic of cell apoptosis. The cell apoptosis reached the peak level on day 3 after the laser exposure, accompanied by endothelial degeneration in the hair papilla vessels, edema and lymphocyte infiltration in the dermal tissues. Tissue reaction and inflammation were relieved on day 5, and the dermal tissue and follicles recovered their normal structures on day 10. At 60 days after the treatment, the hair follicles decreased markedly but the structure of the residue follicles remained normal.

CONCLUSIONAlexandrite laser exposure results in selective destruction of the follicles by inducing direct coagulation and cell apoptosis to achieve permanent hair removal. Tibet mini-pigs with black hair can be used as the animal model of clinical laser hair removal.

Animals ; Hair Follicle ; radiation effects ; ultrastructure ; Hair Removal ; methods ; Lasers, Solid-State ; therapeutic use ; Microscopy, Electron, Transmission ; Swine ; Tibet

OBJECTIVETo investigate the effect of osteopontin (OPN) on the invasion and metastasis of human hapatocellular carcinoma (HCC).

METHODSHCC cell lines (HCC-LM3) were transfected with the chemically synthesized small interfering RNA (siRNA). Real-time PCR and Western blot were used to quantify the mRNA and OPN protein levels. The malignant phenotypes including cellular growth, colony formation and invasion capability of the HCC cells were analyzed.

RESULTSThe OPN mRNA and proteins levels were decreased by 75% and 80% in OPN siRNA treated cells. Colony formation and migratory capability were reduced in OPN siRNA treated cells (P < 0.05).

CONCLUSIONThe specific siRNA is able to reduce the OPN expression at both the mRNA and protein levels and significantly inhibits the invasiveness of HCC cells.

Carcinoma, Hepatocellular ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; Humans ; Liver Neoplasms ; genetics ; metabolism ; pathology ; Neoplasm Invasiveness ; prevention & control ; Neoplasm Metastasis ; prevention & control ; Osteopontin ; antagonists & inhibitors ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; Transfection

OBJECTIVETo investigate of the regulatory effect of Rho-kinase pathway activation on angiotensin II (Ang II)-induced contraction of human airway smooth muscle cells (HASMCs) in vitro.

METHODSCultured primary HASMCs were divided into control group, AngII group, AngII + irbesartan group and AngII + Y-27632 group with corresponding treatment. AngII-induced contraction of HASMCs was evaluated using collagen gel lattices and observed morphologically using immunofluorescence assay. Western Blotting was significantly performed to examine the protein expression of Rho-kinase signal pathway.

RESULTSAngII-induced HASMC contraction was inhibited by treatments with irbesartan and Y-27632 as shown by gel contraction assay (P<0.001). Y-27632 treatment produced a stronger inhibitory effect than irbesartan on the expression of phosphorylated moesin, a substrate of Rho kinase (P<0.05).

CONCLUSIONAngII induces the contraction of HASMCs partially as a result of activation of Rho-kinase pathway.

Amides ; pharmacology ; Angiotensin II ; pharmacology ; Asthma ; physiopathology ; Biphenyl Compounds ; pharmacology ; Bronchi ; cytology ; Humans ; Muscle Contraction ; drug effects ; Muscle, Smooth ; cytology ; Primary Cell Culture ; Pyridines ; pharmacology ; Signal Transduction ; drug effects ; Tetrazoles ; pharmacology ; rho-Associated Kinases ; metabolism

OBJECTIVESTo establish a method for simultaneous detection of HBV resistant mutations associated with three kinds of nucleoside analogues.

METHODSAccording to 981 HBV complete sequences in GenBank, two pairs of conserved primers labeled with digoxigenin were synthesized to amplify the region of HBV reverse transcriptase. To detect non-synonymous amino acid substitutions associated with lamivudine, adefovir and entecavir, 26 specific oligonucleotide probes covering ten different codon positions, I169T, V173L/G, L180M, A181T/V, T184G, S202I/G, M204V/I, Q215S, N236T and M250V/I/L were synthesized and immobilized on nylon membranes charged positively. The oligonucleotide probes immobilized on nylon membranes were then hybridized with PCR products labeled with digoxigenin to detect three drug-resistant mutations. In order to observe specificity and accuracy of probes, HBV wild-type, resistant reference strains and patients serums were assayed by reverse hybridization technique, respectively.

RESULTSThe specific probes of 10 codon positions related to HBV wild-type and resistant reference strains, including I169T, V173L, L180M, A181T, T184G, S202I, M204V, Q215S, N236T, M250V, were distinguished effectively by reverse hybridization method. The results results of 37 samples applicated the method were in accordance with that Of DNA sequencing.

CONCLUSIONReverse hybridization technique can be applied to detect HBV resistant mutations associated with Lamivudine, Adefovir and Entecavir rapidly and accurately.

Amino Acid Substitution ; Antiviral Agents ; pharmacology ; DNA, Viral ; genetics ; Drug Resistance, Viral ; drug effects ; genetics ; Hepatitis B virus ; drug effects ; genetics ; Hepatitis B, Chronic ; virology ; Humans ; Mutation ; Nucleic Acid Hybridization ; methods

OBJECTIVETo establish a detection method for HBV drug-resistant mutations related to lamivudine, adefovir and entecavir by optimization and assessment of reverse hybridization system.

METHOD26 degenerated probes covering 10 drug-resistant hotspots of 3 drugs were synthesized and immobilized on the same positively charged nylon membrane. PCR products labeled with digoxigenin were hybridized with corresponding probes. To improve the sensitivity and specificity, 4 reaction steps of reverse hybridization were optimized including the number of labeled digoxigenin, the energy intensity of UV cross-linking, hybridization and stringency wash conditions. To prove the feasibility, the specificity, sensitivity and accuracy of this system were assessed respectively.

RESULTSensitive and specific results are obtained by the optimization of the following 4 reaction steps: the primers labeled with 3 digoxigenin, energy intensity of UV cross-linking for 1500 x 0.1 mJ/cm², hybridization at 42 degrees C and stringency wash with 0.5 x SSC and 0.1% SDS solution at 44 degrees C for 30 min. In the assessment of system, the majority of probes have high specificity. The quantity of PCR product with a concentration of 10 ng/μl or above can be detected by this method. The concordant rate between reverse hybridization and direct sequencing is 93.9% in the clinical sample test.

CONCLUSIONThough the specificity of several probes needs to be improved further, it is a simple, rapid and sensitive method which can detect HBV resistant mutations related to lamivudine, adefovir and entecavir simultaneously. Due to the short distance between 180 and 181, likewise 202 and 204, the sequence of the same probe covers two codon positions, and hybridization will be interfered by each other. To avoid such interference, the possible solution is that probes are designed by arranging and combining various forms of two near codons.

DNA, Viral ; genetics ; Drug Resistance, Viral ; genetics ; Hepatitis B virus ; drug effects ; genetics ; Humans ; Hybridization, Genetic ; Mutation ; Nucleic Acid Hybridization ; methods ; Oligonucleotide Array Sequence Analysis ; Sensitivity and Specificity