The SRY gene from buffalo (Bubalus bubalis) genome was amplified by the polymerase chain reaction ( PCR) with primers based on the sequence of Hostein SRY gene. The amplified fragment was 2005 bp include 5UTR ( 1 - 504bp) and 3'UTR(1196 - 2005bp). And the amplified fragment was cloned and sequenced. Sequence analysis showed that the coding region of SRY gene (505 - 1195bp) from buffalo was highly homologous with those of other bovine counterpart genes (96% homology) , especially in the HMG box region (99%homology). It was found that there were only signal on male buffalo genome on Southern blot,which indicate SRY gene are highly conservative on evolves.

Objective To screen protective antigen genes and construct the T7 phage display library from adult worms of Schistosoma japonicum.Methods Total RNA was extracted from adult worms of S.japonicum by Trizol reagent and mRNA was isolated from the total RNA.The ds cDNA was synthesized by reverse transcription using random primer.Directional EcoRⅠ/HindⅢ linkers were ligated into the ends of ds cDNA and the ds cDNA was digested with EcoRⅠand HindⅢ,which resulted in ds cDNA with EcoRⅠand HindⅢ adhering ends.The digested ds cDNA fragments longer than 300 bp in length were fractionated and ligated into T7 Select 10-3b vector.After packaging in vitro,the T7 Select 10-3b vector was transformed into BLT5403 to construct the T7 phage display cDNA library.Plaque assay and PCR were used to evaluate the library.Seven known objective genes of S.japonicum were screened by PCR to detect the representation of the library.Result Primary library capacity was 4.98?106 pfu,and the titer of amplified library was 3.85?1011 pfu/mL.The PCR identification result of 96 clones picked at random showed that recombination rate was 93.8%,in which 95.6% inserted cDNA fragments were longer than 300 bp in length.All the seven known objective genes of S.japonicum were amplified from the library.Conclusion The T7 phage display library from adult worms of Schistosoma japonicum was constructed.

Objective To look for the genes of Schistosoma japonicum related to the Schistosoma-resistance of Microtus fortis.Methods The fresh sera of Microtus fortis were used to screen a T7 phage display cDNA library from worms of Schistosoma japonicum established in our lab.The positive clones were sequenced and functionally analysed through bioinformatics.Results The specific phages binding to the sera of Microtus fortis were enriched 857-fold after three rounds of biopanning,and 58 positive clones picked at random were sequenced and 10 ESTs were obtained.BLASTn results showed that 7 ESTs had 99%-100% similarity to the genes of Shistosoma japonicum reported in GenBank and 1 EST had 82% similarity to a zinc finger protein encoden gene from Pan troglodytes.The results of these ESTs function prediction indicated most of them were involved in the regulation of gene expresion of Schistosoma japonicum.Conclusions Several target genes of Schistosoma japonicum related to the Schistosoma-resistance of Microtus fortis are obtained and those would lay foundation to expatiate the native resistance mechnism of Microtus fortis to Schistosoma japonicum.

Objective To construct a T7 phage display cDNA library from the lung of Microtus fortis for further screening the schistosomiasis-resistence-related genes of Microtus fortis. Methods mRNA was isolated from total RNA extracted from the lungs of Microtus fortis by TRIzol reagent, and was used to synthesize double strain cDNA by the reverse transcription. Then the double strain cDNA was given with EcoRⅠ and Hind Ⅲ adhering ends by ligation with the directional EcoRⅠ/Hind Ⅲ linkers and digestion with EcoRⅠ and Hind Ⅲ. The double strain cDNA fragments longer than 300 bp in length were fractionated by the Mini Column, and ligated into the T7 Select 10-3b vector with EcoRⅠ and Hind Ⅲ adhering ends. After packaging in vitro, the recombinant T7 Select 10-3b was transformed into BLT5403 to construct a T7 phage display cDNA library. Results The library constructed here contained 1.5?106 clones and the titer of the amplied library was 1.1?1012 pfu/ml. The PCR identification results of 100 clones picked at random showed that 91% clones were recombinant and 90% of recombinant clones contained cDNA fragments longer than 300 bp in length. Conclusion A T7 phage display cDNA library from the lung of Microtus fortis is successfully constructed.

OBJECTIVETo investigate the chemical constituents in leaves of Ilex centrochinensis and their antitumor bioactivity.

METHODVarious chromatography techniques such as column chromatography on silica gel, Sephadex LH-20 and preparative HPLC were used to isolate and purify the compounds and their structures were identified by spectral data and physicochemical properties. Their antitumor effect was tested by MTT method.

RESULTTen compounds were isolated and identified as 1,4-benzenediol (1), (2S)-5,4'-dihydroxy-7,3'-dimethoxyflavan(2), (2S)-5,4'-dihydroxy-7-methoxyflavan (3), kaempferol (4), quercetin (5), naringenin (6), ursolic acid (7), uvaol (8), oleanolic acid (9) and beta-sitosterols (10).

CONCLUSIONCompounds 1-5, 7, 8 were isolated from the species for the first time, among which compounds 1-3 were isolated from the Ilex genus for the first time. Compounds 2 and 3 showed strong cytotoxic activity against Huh7 cell lines with IC50 values of 8.98, 13.04 mg x L(-1), respectively. Compounds 7-9 exhibited weak cytotoxic activity against Caco-2 cell lines with IC50 values of 28.52, 38.28, 33.04 mg x L(-1), respectively.

Antineoplastic Agents ; chemistry ; pharmacology ; Caco-2 Cells ; Cell Line, Tumor ; Cell Survival ; drug effects ; Dose-Response Relationship, Drug ; Humans ; Ilex ; chemistry ; Inhibitory Concentration 50 ; Plant Extracts ; chemistry ; pharmacology ; Plant Leaves ; chemistry ; Plants, Medicinal ; chemistry

OBJECTIVETo develop a RP-HPLC method for simultaneous determination of orientin, isorientin, vitexin and isovitexin in Lophatherum gracile from different habitat and harvesting time.

METHODThe HPLC method was applied and the chromatographic column was a Waters XBridge C18 column (4.6 mm x 250 mm, 5 microm). The mobile phase consisted of methanol-0.05% acetic acid (35:65). The flow rate was 1.0 mL min(-1) and the detection wavelength was set at 340 nm. The column temperature was set at 25 degrees C.

RESULTFour components were isolated well, the linear relationships were excellent. The mean recoveries and RSD values of orientin, isorientin, vitexin and isovitexin were 103.2%, 2.1%; 101.6%, 2.7%; 98.4%, 2.3%; 99.2%, 1.8%, respectively.

CONCLUSIONThe HPLC method is simple, sensitive and reliable, and can be used for the quality control of the medicinal material.

Apigenin ; chemistry ; Chromatography, High Pressure Liquid ; methods ; Drugs, Chinese Herbal ; chemistry ; Flavonoids ; chemistry ; Glucosides ; chemistry ; Glycosides ; chemistry ; Poaceae ; chemistry ; Reproducibility of Results

OBJECTIVETo study the relation on overfilling with gutta-percha point or paste and acute periapical periodontitis.

METHODSCollected sixty cases of acute periapical periodontitis which had been filled with gutta-percha point and paste within 1 week, and took dental radiographs. The cases that dental radiographs showed only guttapercha point was overfilling were assigned to group A (34 cases), and the cases that dental radiographs showed only paste was overfilling were assigned to group B (26 cases). The cases that dental radiographs showed both gutta-percha point and paste were overfilling were excluded. Sixty cases were divided into light group and severe group according to clinical sign. Measured gutta-percha point length or paste areas over apex. Took out the ohturation material cornpletely, adjusted occlusion when necessary and changed root canal medicament every day until clinical sign disappeared completely. Recorded the time of clinical sign disappeared completely.

RESULTSIn group A, gutta-percha point length over apex averaged 1.01 mm on light cases, and 1.79 mm on severe cases. In group B, the paste areas over apex averaged 2.45 mm2 on light cases, and 8.26 mm2 on severe cases. Group A had 13 light cases and 21 severe cases, and group B had 18 light cases and 8 severe cases. In group A, the average time of clinical sign disappeared completely was 3.56 days, and in group B the average time was 6.19 days. The statistical test showed there were significant differences among these four couples.

CONCLUSIONThe more overfilling, the more severe clinical sign was. Clinical sign caused by gutta-percha point overfilling was more severe. The time of clinical sign which caused by gutta-percha point overfilling disappeared completely was shorter.

Gutta-Percha ; Humans ; Periapical Periodontitis ; Root Canal Filling Materials ; Root Canal Preparation

Objective To explore and assess the intervention mode consist of governments,professional organizations and volunteers for AIDS high -risk behaviors.Methods Commercial sex workers,gays,venereal outpatients and other target population were chosen to be under intervention measures by AIDS high -risk behaviors intervention team consist of government,CDC and volunteers.Then awareness rate,use rate of condom and care rate of infectious were calculated. Results A team of 313 volunteers were set up.The cadres,students and owners of public places were trained and it reached 42 000 person training times in 2012.The awareness rate of AIDS -prevention knowledge among commercial sex workers increased from 76.67% to 91.41% and the use of condom increased from 66.67% to 89.74%.The care rate of infections /patients increased from 0 to 100%.Conclusion This model plays an important role in the AIDS prevention and treatment.It is a good working mechanism to build the intervention mode consist of governments,professional organizations and volunteers.

Buffaloes ; Cells, Cultured ; In Vitro Techniques ; Primary Cell Culture ; Stem Cells ; Tretinoin

Currently, the systems for culturing buffalo spermatogonial stem cells (SSCs) in vitro are varied, and their effects are still inconclusive. In this study, we compared the effects of culture systems with undefined (foetal bovine serum) and defined (KnockOut Serum Replacement) materials on the in vitro culture of buffalo SSC-like cells. Significantly more DDX4- and UCHL1-positive cells (cultured for 2 days at passage 2) were observed in the defined materials culture system than in the undefined materials system (p < 0.01), and these cells were maintained for a longer period than those in the culture system with undefined materials (10 days vs. 6 days). Furthermore, NANOS2 (p < 0.05), DDX4 (p < 0.01) and UCHL1 (p < 0.05) were expressed at significantly higher levels in the culture system with defined materials than in that with undefined materials. Induction with retinoic acid was used to verify that the cultured cells maintained SSC characteristics, revealing an SCP3⁺ subset in the cells cultured in the defined materials system. The expression levels of Stra8 (p < 0.05) and Rec8 (p < 0.01) were significantly increased, and the expression levels of ZBTB16 (p < 0.01) and DDX4 (p < 0.05) were significantly decreased. These findings provided a clearer research platform for exploring the mechanism of buffalo SSCs in vitro.