This study examined the role of PI3K/Akt pathway in radiosensitization of DNA damage of cervical carcinoma cells. The 50% inhibition concentration (IC(50)) of cisplatin and docetaxel in HeLa cells was detected by Mono-nuclear cell direct cytotoxicity assay (MTT) in vitro. HeLa cells were treated by cisplatin/docetaxel of 10 percent of IC(20) alone or combined with LY294002 for 24 h, and then radiated by different doses of X-ray. The cell survival ratio was obtained by means of clone formation. One-hit multi-target model was fitted to the cell survival curve to calculate dose quasithreshold (Dq), mean lethal dose (D(0)), 2Gy survival fraction (SF(2)) and sensitization enhancement ratio (SER). The pAkt and total Akt expression was detected by Western blotting and DNA damage by neutro-comet electrophoresis. The HeLa cells were randomly divided into 7 groups in terms of different treatments: Control; radiation treatment (RT) group; LY294002+RT group; cisplatin+RT group; docetaxel+RT group; LY294002+cisplatin+RT group; LY294002+docetaxel+RT group. The apoptosis ratio of each group was measured by flow cytometry. The results showed that docetaxel and cisplatin significantly enhanced the phosphorylation of Akt in radiation-treated HeLa cells. The Dq, D(0) and SF2 in LY294002-contained groups were lower than those in docetaxel or cisplatin+RT group. The SER in the LY294002+docetaxel+RT group was 1.35 times that of the docetaxel+RT group, and that in the LY294002+cisplatin+RT group 1.26 times that of the cisplatin+RT group. The Comet electrophoresis showed that tail distance in the LY294002+cisplatin+RT group or LY294002+docetaxel+ RT group was longer than in the cisplatin+RT group or docetaxel+RT group. The apoptosis ratio in the LY294002+cisplatin+RT group or LY294002+docetaxel +RT group was higher than in the cisplatin+RT group or docetaxel+RT group. It was concluded that inhibiting PI3K/Akt pathway can increase the effect of docetaxel and cisplatin on the radiosensitivity of HeLa cells and DNA damage resulted from radiation.

OBJECTIVE CYP2D is one of the most abundant subfamily of CYPs in the brain, especially in the cerebellum. Brain CYP2D is responsible for the metabolism of endogenous neurotransmitters such as tyramine and serotonin. Our previous studies have shown brain CYP2D can be regulated by exogenous and endogenous substances with tissue- specificity. The purpose of this study is to examine the effects of cerebral CYP2D on the mice behavior and the regulatory mechanism of brain CYP2D by growth hormone. METHODS Mice received the stereotaxic injection with CYP2D inhibitor quinine in deep cerebellar nuclei of cerebellum. The animals were tested with rotarod apparatus, balance beam, water maze, elevated plus maze and open field. The changes in CYP2D22, PPARαand PPARγ in brain regions and liver were assayed in male growth hormone receptor knockout mice, SH-SY5Y cells and HepG2 cells. RESULTS The inhibition of cerebellum CYP2D significantly affected the spatial learning and exploring ability of mice. Compared with WT mice, CYP2D expression was lower in brain regions from GHR(-/- ) male mice; however, hepatic CYP2D level was similar. Pulsatile GH decreased PPARα mRNA level, and increased mRNA levels of CYP2D6 and PPARα in SH- SY5Y cells. In HepG2 cells, pulsatile GH resulted in decreases in PPARα and PPARγ mRNA levels, but not CYP2D6. PPARα inhibitor induced CYP2D6 mRNA and protein by 1.32-fold and 1.43-fold in SH-SY5Y cells. PPARγ inhibitor decreased CYP2D6 mRNA and protein by 74.76% and 40.93%. PPARα agonist decreased the level of CYP2D22 mRNA in liver and cerebellum, while PPARγ agonist rosiglitazone resulted in diametrically increases. The luciferase assay showed that PPARγ actived the CYP2D6 gene promoter while PPARα inhibited its function. Pulsatile GH declined the binding of PPARα with CYP2D6 promoter by 40%, promoted the binding of PPARγ with CYP2D6 promoter by approximate 60%. The levels of brain and liver PPARα expression in male GHR(-/- ) mice is obviously higher than those in WT mice. The level of PPARγ in male GHR(-/- ) mice was decreased in the frontal cortex and hippocampus, while remained stable in the cerebellum and striatum; meanwhile, PPARγ was increased in the liver. CONCLUSION Brain CYP2D may be involved in learning and memory functions of central system. Masculine GH secretion altered the PPARs expression and the binding of PPARs to CYP2D promoter, leading to the elevated brain CYP2D in a tissue- specific manner. Growth hormone may specifically alter the metabolic and synthetic of important endogenous substances in the central nervous system (such as serotonin) through the specific regulation of brain CYP2D expression.

OBJECTIVETo investigate the distribution of GAD67 and the co-localization with bNOS in the main olfactory bulb of GAD67-GFP knock-in mouse.

METHODSPolymerase chain reaction was applied to identify the genotype of GAD67-GFP knock-in mouse, the animals were sacrificed and frozen sections of olfactory bulb were prepared. The Nissl-staining was performed to show an framework of the neuron in the olfactory bulb. The distribution of GAD67 and co-localization with bNOS were detected by immunofluorescence technique.

RESULTSThe proportion of GAD67-positive cells among DAPI-positive cells were (42.98 ± 0.92)% in glomerular layer, (23.64 ± 0.84)% in mitral cell layer and (77.75 ± 0.84)% in granule cell layer; the bNOS-positive cells mainly existed in glomerular layer and mitral cell layer, very few in granule cell layer. No co-localization of GAD67 and bNOS in granule cell layer and mitral cell layer was found, but there was dispersed distribution in glomerular layer.

CONCLUSIONGAD67-positive neurons mainly appear in glomerular layer and granule cell layer, and the bNOS is mostly expressed in glomerular layer and mitral cell layer; while the co-localization of GAD67 and bNOS only occurs in glomerular layer of olfactory bulb.

Animals ; Gene Knock-In Techniques ; Glutamate Decarboxylase ; genetics ; metabolism ; Green Fluorescent Proteins ; genetics ; metabolism ; Mice ; Mice, Transgenic ; Neurons ; metabolism ; Nitric Oxide Synthase Type I ; metabolism ; Olfactory Bulb ; metabolism ; Tissue Distribution

This study examined the role of PI3K/Akt pathway in radiosensitization of DNA damage of cervical carcinoma cells.The 50% inhibition concentration(IC50)of cisplatin and docetaxel in HeLa cells was detected by Mono-nuclear cell direct cytotoxicity assay(MTT)in vitro.HeLa cells were treated by cisplatin/docetaxel of 10 percent of IC20 alone or combined with LY294002 for 24 h,and then radiated by different doses of X-ray.The cell survival ratio was obtained by means of clone formation.One-hit multi-target model was fitted to the cell survival curve to calculate dose quasithreshold (Dq),mean lethal dose(D0),2Gy survival fraction(SF2)and sensitization enhancement ratio(SER).The pAkt and total Akt expression was detected by Western blotting and DNA damage by neutro-comet electrophoresis.The HeLa cells were randomly divided into 7 groups in terms of different treatments: Control; radiation treatment(RT)group; LY294002+RT group; cisplatin+RT group; docetaxel+RT group; LY294002+cisplatin+RT group; LY294002+docetaxel+RT group.The apoptosis ratio of each group was measured by flow cytometry.The results showed that docetaxel and cisplatin significantly enhanced the phosphorylation of Akt in radiation-treated HeLa cells.The Dq,Do and SF2 in LY294002-contained groups were lower than those in docetaxel or cisplatin+RT group.The SER in the LY294002+docetaxel+RT group was 1.35 times that of the docetaxel+RT group,and that in the LY294002+cisplatin+RT group 1.26 times that of the cisplatin+RT group.The Comet electrophoresis showed that tail distance in the LY294002+cisplatin+RT group or LY294002+do-cetaxel+RT group was longer than in the cisplatin+RT group or docetaxel+RT group.The apoptosis ratio in the LY294002+cisplatin+RT group or LY294002+docetaxel+RT group was higher than in the cisplatin+RT group or docetaxel+RT group.It was concluded that inhibiting PI3K/Akt pathway can increase the effect of docetaxel and cisplatin on the radiosensitivity of HeLa cells and DNA damage resulted from radiation.

This study was aimed to establish one tube PCR reaction technique to determine ABO blood group genotypes. Salting-out method was adopted to extract genomic DNA; one tube polymerase chain reaction with GeneScan technique was used to identify ABO genotypes. The results showed that the ABO genotypes of 132 samples were in accordance with the phenotypes determined by serological technique. The frequencies of A, B and O were 0.205, 0.159 and 0.636 respectively. AA, AO, AB, BB, BO and OO genotypes were 8 (6.1%), 31 (23.5%), 7 (5.3%), 6 (4.5%), 23 (17.4%), and 57 (43.2%) respectively. It is concluded that one tube polymerase chain reaction with GeneScan technique can determine the genotypes of ABO blood group.
ABO Blood-Group System ; genetics ; Gene Frequency ; Genotype ; Humans ; Polymerase Chain Reaction ; methods ; Reproducibility of Results

0.05).In group Ⅱand Ⅲ,the carotid artery IMT was thicker and the amount of plagues were larger than those in group Ⅰ(P

Sinisan is a widely used traditional Chinese medicine (TCM) in treating various diseases; however, the in vivo metabolic profile of its multiple components remains unknown. In this paper, ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was applied to identify the metabolites of Sinisan extract in rat plasma, urine, feces and bile after intragastric administration. Using MS(E) and mass defect filter techniques, 41 metabolites of 10 parent compounds (naringin, naringenin, hesperidin, neohesperidin, liquiritin, liquiritigenin, glycyrrhizic acid, glycyrrhetinic acid, saikosaponin a and saikosaponin d) were detected and tentatively identified. It was shown by our results that these compounds was metabolized to the forms of hydroxylation, glucuronidation, sulfation, glucuronidation with sulfation and glucuronidation with hydroxylation in vivo.
Administration, Oral ; Animals ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; metabolism ; pharmacokinetics ; Flavanones ; analysis ; metabolism ; pharmacokinetics ; Glucosides ; analysis ; metabolism ; pharmacokinetics ; Glycyrrhizic Acid ; analysis ; metabolism ; pharmacokinetics ; Hesperidin ; analogs & derivatives ; analysis ; metabolism ; pharmacokinetics ; Hydroxylation ; Male ; Plants, Medicinal ; chemistry ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

OBJECTIVETo investigate the therapeutic effect of recombinant human parathyroid hormone(1-34) [rhPTH(1-34)] on osteoporosis of ovariectomized rats.

METHODSThe model of osteoporosis was formed after 3 months of ovariectomy with 6-month age of 80 rats. Another 20 rats was control of sham operation. rhPTH(1-34) was subcutaneously injected once daily with 5, 10, 20, 40 micrograms/kg for 3 months. There were 10 rats in each group. The control of therapy included Salmon Calcitonin to 10 rats and Alendronate sodium to 10 rats. The bone weight of dry and ash, bone mineral density, bone biomechanical property, trabecular area, bone mineral deposition and serum alkaline phosphatase, Ca, P and urinary Pyridinoline/creatin (Pyd/Cr) were measured after the end of therapy.

RESULTSWhen administered to animals as a single subcutaneous injection once daily, rhPTH(1-34) increased obviously bone mass, bone biomechanical property and trabecular area, as well as bone deposition compared with the animals of control group. The bone architecture was ultimately improved by rhPTH(1-34) therapy.

CONCLUSIONSRats of ovariectomized-induced osteoporosis possess obvious effect of treatment with low dose of rhPTH(1-34) administered once daily.

Animals ; Female ; Osteoporosis ; drug therapy ; etiology ; Ovariectomy ; Rats ; Recombinant Proteins ; therapeutic use ; Teriparatide ; therapeutic use

Objective To establish a dual RT-PCR detection method for bovine viral diarrhea virus(BVDV)in bovine-derived samples. Methods The primers were designed and synthesized according to the published BVDV1 and BVDV2 genes containing highly conservative sequences in the 5' untranslated regions(5' UTR)to establish the dual RT-PCR method. The specificity,sensitivity,stability of this method were evaluated. Then 41 bovine-derived samples and 64 bovine plasma samples including bovine calf serum, deproteinized calf serum extract and one lienal polypeptide injection were detected with this method. Results There was no cross reaction with bovine parainfluenza virus type 3(BPIV3), classical swine fever virus(CSFV)and Japanese encephalitis virus(JEV)when samples were detected with the established dual RT-PCR method. The lowest concentration of template DNA for detection of BVDV1 and BVDV2 was 8.87 × 102copies and 6.31 × 102copies per microliter,respectively. Electrophoresis bands of about 151 bp and 303 bp were still amplified and detected after the BVDV1 and BVDV2 cDNA was stored at -30℃ for 12 months. The BVDV positive rate of 41 bovine-derived samples and 64 bovine plasma samples detected with this dual RT-PCR method was 14.6% and 29.7%, respectively. Conclusions The established dual RT-PCR method has the advantages of high efficiency, specificity,sensitivity and stability,and can be used for the detection of BVDV in bovine-derived samples.

Objective To establish a fluorescence quantitative PCR assay for polyomavirus and to apply this technique in the investigation of its infection rate in naked mole rats. Methods To compare the nucleic acid sequence of murine polyomavirus (Genbank:NC 001515) in NCBI and design primers and probes in its conserved region. To establish a fluorescence quantitavive PCR method for polyomavirus and evaluate the sensitivity and specificity of the method. To infect nine one-day old KM strain suckling mice, and to collect samples of the heart, liver, spleen, lung, kidney, brain, thymus, cecal contents and blood at 21 days after infection. The efficacy of the method was validated by detecting the virus in organs. 62 cecal samples from naked mole rats were tested by the established assay. Results There was obvious fluorescence signal when polyomavirus was used as the template and no fluorescence signal when simian virus 40, murine K virus, MVM and H-1 were used as templates. The detection limit of the assay was 100 copies/μL. Polyomavirus DNA was detected in the heart, liver, spleen, lung, kidney and cecal contents of the mice which were inoculated with polyomavirus. The polyomavirus DNA content was highest in the lung tissue. There was no detectable polyomavirus DNA in the brain, thymus and blood of the infected mice. Sixty-two cecal contents of naked mole rats were tested for polyomavirus and the results were negative. Conclusions The fluorescence quantitative PCR assay for polyomavirus established in this study can effectively detect polyomavirus DNA in animal tissues. The results of investigation of the natural infected polyomavirus of naked mole rats provide a reference for the formulation of microbiological criteria for experimental naked mole rats.