It was pointed out by many researches that keeping the concentration of Ca2+ in cells could increase the survival rate of hypothermic preserved kidneys and the survival rate of transplants. In this study, changes of the concentration of calcium were detected within catoplasm, mitochondria, endoplasmic reticulum and nucleus in the isolated hypothermic storage cat kidney, Ca2+ be marked with calcium cytochemical probe (K2H2Sb2O7) and detected by X-ray microanalysis of microsections. After 24, 48 and 72 hours preservation, the p/b (peak/back) of calcium within cytoplasm and mitochondria increased significantly. There were no obvious changes within endoplasmic reticulum and nucleus. It demonstrates that the Ca2+ were released from calcium pool (except the endoplasmic reticulum, nucleus, mitochondria etc.) to cytoplasm during preservation; and mitochondria can uptake calcium from cytoplasm to some extent, while the calcium concentration of cytoplasm is higher than normal.
Animals ; Calcium ; metabolism ; Cats ; Cryopreservation ; Female ; In Vitro Techniques ; Kidney Cortex ; cytology ; metabolism ; Male ; Spectrometry, X-Ray Emission ; Time Factors

We transformed the fip-fve gene into Pichia pastoris GS115 for inducible and constitutive expression to obtain feasible bioactvie recombinant Fip-fve. The fip-fve gene was cloned from Flammulina velutipes fruting body by PCR and ligated to pPIC9 to construct inducible expression vector pPIC9-FIP-fve, and promotor pgap was used to replace the paox1 to construct constitutive expression vector pPIC9-PGAP-FIP-fve. These two vectors were used to transform P. pastoris by PEG method. The fip-fve was expressed after histamine-absence screening and yeast colony PCR. The inducible expression level reached 158.2 mg/L at the fourth day and the constitutive expression level was 46.3 mg/L and 29.5 mg/L using glucose and glycerol, respectively. The SDS-PAGE and Western blotting both proved the correctness of rFip-fve, and the hemagglutination test indicats the rFip-fve's bioactivity.
Electrophoresis, Polyacrylamide Gel ; Flammulina ; chemistry ; Fungal Proteins ; biosynthesis ; Genetic Vectors ; Pichia ; metabolism ; Polymerase Chain Reaction ; Promoter Regions, Genetic ; Recombinant Proteins ; biosynthesis

Objective o evaluate the association between IL-28B ( rs12979860 ) polymorphism andantiviraltherapeutic effectbydetecting the genotype of interleukin-28B( IL-28 B) in patients with hepatitis C ( HCV ) .Methods Of total 1153 HCV patients, 303 diagnosed with CHC had been treated with pegylated interferon plus ribavirin for 24-48 weeks.IL-28B ( rs12979860 ) was genotyped by two-color fluorescent TaqMan assay.Results Among 1153 patients, CC, CT and TT genotype frequencies of IL-28B rs12979860 are 83.26%, 16.22%and 0.52%respectively.The results of HCV genotypingof 580 in 1153 cases, the frequencies of 1b, 2a and their non-1b/2a type are 63.45%, 35.00%and 1.55%respectively;In 303 CHC patients with clear medical history, the proportion of SVR was71.98% in patients with CC genotype and 16.90%in those with either the CT or TT genotypes.Logistic regression model was adopted to analyze the association of rs12979860 with SVR while adjusting for age, gender, viral load and HCV GT factors.Populations carrying combined genotype ( CT +TT) are making it harder to get SVR compared with those with CC genotype (OR, 95%CI:11.10,5.35-23.04;P<0.000 1).The percentages of SVR in HVC patients with 1b and 2a genotypeare 48.02% and 81.19% respectively.there is a statistically significant difference between these subgroups (χ2 =30.639,P<0.000 1).Conclusion IL-28B rs12979860 genotype is closely related to SVR in CHCpatients.Patients with CC genotype have a higher virus sustained response rate than those carrying CT or TT genotype.The SNP , rs12979860, might be applied as a predictor of clinical antiviral efficacy in the furture.

OBJECTIVETo establish a new method for the identification of Schisandra sphenanthera and S. chinensis.

METHODRandom amplified polymorphic DNA-Sequence characterized applied region (RAPD-SCAR) method was applied to screen primers.

RESULTScreening from 100 primers, only 2 random primers, which can be used to identify S. sphenanthera and S. chinensis accurately with a good reproducibility. It worked to fit them into sequence characterized applied region.

CONCLUSIONRAPD-SCAR can be used to identify S. sphenanthera and S. chinensis accurately.

Base Sequence ; Molecular Sequence Data ; Polymerase Chain Reaction ; methods ; Random Amplified Polymorphic DNA Technique ; Schisandra ; genetics ; Sequence Analysis, DNA

Objective:To investigate the changes of brain energy metabolism and cognitive function in mice with specifically knocking out AMP-activated protein kinase α1 subunit ( AMPKα1) gene in the excitatory neurons by Cre-loxP recombination system. Methods:Sixteen 6-month-old mice with genotype AMPKα1 flox/flox/Camk2a-Cre/ERT2 obtained by hybrid breeding were randomly divided into AMPKα1 knockout group ( n=8) and AMPKα1 wild-type group ( n=8). Mice in the AMPKα1 knockout group were intraperitoneally injected 0.1 mL tamoxifen (20 mg/mL, dissolved in corn oil) daily for a consecutive 5 d to control AMPKα1 gene knockout in the excitatory neurons; and mice in the AMPKα1 wild-type group were intraperitoneally injected 0.1 mL corn oil daily for a consecutive 5 d. Seven d after that, Morris water maze and T maze experiments were employed to detect the spatial learning and memory abilities and spatial working memory of these mice; chemical exchange saturation transfer imaging (CEST) was used to observe the glucose metabolism in the hippocampus and cortex surrounding the hippocampus; Western blotting was used to detect the AMPKα1 and glutamate receptor 1 (GluR1) protein expressions in the hippocampus and cortex surrounding hippocampus of two groups. Results:(1) Morris water maze showed that, as compared with those in the AMPKα1 wild-type group, mice in the AMPKα1 knockout group had significantly prolonged escape latency ([13.90±3.72] s vs. [22.40±6.28] s; [11.95±3.86] s vs. [22.39±9.77] s]) on the 3 rd and 4 th d of experiment, statistically decreased times crossing the platform ([5.25±1.83] times vs. [1.75±1.28] times, P<0.05). (2) T-maze experiment showed that as compared with that of the AMPKα1 wild-type group, the free alternation rate in mice of the AMPKα1 knockout group was significantly decreased ([73.21±9.16]% vs. [48.21±11.29]%, P<0.05). (3) CEST showed that the glucose metabolism levels in the hippocampus and cortex surrounding the hippocampus of AMPKα1 knockout group were significantly lower than those in AMPKα1 wild-type group (1.51±0.81 vs. 2.77±0.67; 1.31±0.83 vs. 2.42±0.95, P<0.05). (4) Western blotting showed that the AMPKα1 and GluR1 protein expressions in the hippocampus and cortex surrounding the hippocampus of the AMPKα1 wild-type group were significantly higher than those of the AMPKα1 knockout group (AMPKα1: 0.70±0.05 vs. 0.49±0.03, 0.98±0.04 vs. 0.64±0.06; GluR1: 1.22±0.18 vs. 0.60±0.11, 0.96±0.08 vs. 0.79±0.04, P<0.05). Conclusion:Specifically knocking out AMPKα1 in excitatory neurons can result in abnormal glucose metabolism in the brain of mice, and thus cause cognitive dysfunction, whose mechanism may be related to excitatory synaptic disorder caused by energy metabolism disorder.