Adult ; Brucellosis ; Humans ; Male ; Middle Aged ; Occupational Diseases

Action Potentials ; drug effects ; physiology ; Animals ; Female ; Guinea Pigs ; Heart Ventricles ; drug effects ; Papillary Muscles ; drug effects ; physiology ; Progesterone ; pharmacology

Pseudorabies virus (PRV) is a swine herpesvirus of the Alphaherpesvirinae subfamily and a pathogen of swine resulting in devastating disease and economic losses worldwide. Cre/loxP site-specific system has the character of site specific, time specific, tissue specific and high efficiency in recombination, which makes this system universal in vivo and in vitro recombination of bacteria, fungus, plants, insects and mammals. A recombinant PRV which contain a loxP site in TK locus by using Cre/LoxP recombinant system was construsted. A pair of primers were synthesized according to the pEGFP-C1 sequence published on GenBank, and were used to amplify the EGFP gene expression cassette with two loxP sites flanking each side. This target gene was cloned into pSKLR, the resulting transfer vector pSKLR-GFP-loxP was then cotransfected into 293T cells with PRV SH strain genomic DNA. The recombinant virus rPRV1 was selected and purified in TK-143 cells by choosing fluorescent expressing plaques. Cre expression vector pOG231 was cotransfected into 293T cells with rPRV1 genomic DNA. The second recombinant virus rPRV2 was obtained, which contains only one loxP site in TK locus. Sequencing results of rPRV2 TK gene indicated that 34bp loxP site was inserted into rPRV2 genome and there were 270bp deletion in TK gene. PCR amplifying different generations of rPRV2 TK gene showed that the mutant was stable when passages in RK-13 cells. TCID_ 50 assay indicated that rPRV2 grows well on RK-13 cells. The LD_ 50 test results on BALB/C mice suggested that the virulence of rPRV2 was reduced. As a conclusion, the report gene GFP expression cassette was removed successfully from rPRV1 genome and only one LoxP site was leaved in rPRV2 genome by using Cre/LoxP recombinant system.

Response Surface Methodology was applied to optimize the culture components for lipopeptide production by Bacillus natto TK-1. In the first step, two level factorial design of Plackett-Burman was used to evaluate the influence of six related factors. It showed that three factors playing the important roles in the medium, including peptone, yeast extract powder and CaCl_2. The path of steepest ascent was used to approach the optimal region of the fermentation conditions subsequently. In the third step, the concentrations of those three main factors were further optimized by using Box-Behnken and Response Surface Analysis. By solving the quadratic regression model equation, the optimal concentrations of the variables were determined as: peptone 1.73%, yeast extract powder 0.063 %, CaCl_2 1.385?10-4mol/L. Under the optimal culture conditions, the diameter of haemolysis zone increased 29.3 % than before. HPLC analysis showed the precise production of lipopeptide was 30.2% higher than preliminary culture. Furthermore, at three batches cultivation, the experiment values under the optimal conditions agreed with the predictive values. It showed that Response Surface Methodology was proper and a good choice for optimization.

Fermentation for Eicosapentaenoic Acid(EPA) production by Nitzschia laevis at various temperature between 10℃ and 30℃ was investigated and the dynamics characteristics during fermentation process were also analyzed.Based on the results,a varying temperature nursing method of two stage control strategy is proposed:During the first stage,which comprises the delay phase and the initial index phase,the temperature is maintained at 25℃;then the temperature is shifted to 20℃ and kept up till the end of the fermentation process.By this method,a EPA content of 6.0% and a yield of 291.60 mg/L have been gained.These are 24.07% and 18.81% higher than that of fixed temperature(25℃) fermentation,respectively.

This thesis aimed at the Bacillus natto TK-1 screened out from Natto.The lipopeptide was purified using Thin-Layer Chromatography(TLC),and investigated its anti-tumor activity.After acid precipitation and methanol extraction,the lipopeptide was separated on TLC.Then the authors get the monomer surfactin which molecular weight is 1036Da through the High performance liquid chromatography(HPLC),electro spray ionization-mass spectrometry(ESI-MS) and infrared(IR).MTT method was implied to testify the anti-tumor activity of the purified sample from TLC.The results indicated a concentration and time-dependent relationships.After 48h,their IC50 were 40 mg/L.The detection with inverted microscope fluorescence microscope displays that the surfactin will cause a series of Morphological changes to the cells.In TUNEL experiment,the authors noticed that surfactin has the ability to induce apoptosis,besides this inhibition shows an obvious time-dependent relationship.

The study was to develop cis-dichlorodiammineplatinum(DDP)-loaded formulations using a novel type of self-assembled compound composed of block copolymers synthesized by poly(?-glutamic acid)(?-PGA).For the potential of targeting liver cancer cells,D-galactose was conjugated on the prepared ?-PGA.In vitro,DDP can be released from the resulting conjugate in PBS:there was a burst release during the first 8 h,then followed by sustained release.DDP could be easily incorporated into poly(?-glutamic acid)-D-galactose esterifiable derivative through a covalent bond.The yield of DDP incorporation into the esterifiable derivative was 9.4%～10.2%.In vitro experiments conclusively established that the poly(?-glutamic acid)-D-galactose esterifiable derivative-Cisplatin Complex Compound(?-D+-DDP)was much less toxic to normal cell lines than DDP only.The surviving rate of cells treated with ?-D+-DDP compound is higher than those treated with free DDP.Also it has obvious antitumor efficiency on human liver tumor BEL-7402 cells.HE staining indicated that the ?-D+-DDP compound make the BEL-7402 apoptosis.These results indicated that the conjugation of DDP to the esterifiable derivative reduced its cytotoxicity activity,but retains its antitumor activity in vitro.In conclusion,the ?-D+-DDP compound could be used as a potential clinic antitumor drug.The ?-PGA obtained by fermentation can be used as a valuable drug carrier system.

OBJECTIVEIn oder to treat periodontitis by using tissue engineering and gene engineering technology, the article established an transient expression system of bone marrow stromal cells (BMSC) modified by osteoprotegerin (OPG) gene and detected its expression using eukaryotic secreted expression pSecTag2/B-OPG plasmid.

METHODSBy solation and culture of BMSC in vitro, the identified recombined plasmid was transiently transfected into BMSC by Lipofectamine 2000 and OPG expression in BMSC was determined by RT-PCR and Western blot in 6 weeks.

RESULTSThe fragments of the recombinant plasmid digested with Hind III, EcoR I and BamH I and examined by 10 g/L agarose electrophoresis, were consistent with predicted size. The sequence of OPG was identical to the sequence provided by GeneBank [gi:33878056]. OPG transcribing in BMSC was confirmed by RT-PCR and OPG sustainable expressing in BMSC was detected by Western blot in 39 days.

CONCLUSIONThe transiently expression system of BMSC modified by OPG gene was successfully established.

Humans ; Mesenchymal Stromal Cells ; Osteoprotegerin ; Tissue Engineering ; Transfection

OBJECTIVETo investigate the cardiac effect of interleukin-2 (IL-2) and to explore the underlying mechanism.

METHODSThe video tracking system and spectrofluorometric method were used to measure the cell contraction and intracellular calcium. Fura-2/AM was used as a calcium fluorescence probe. Langendorff perfusion technique was used to determine the effect of IL-2 on the intact heart.

RESULTSCompared with the control group, IL-2 5 U/ml, 50 U/ml significantly decreased cell contraction amplitude [(74.95+/-4.79) vs (98.09+/-5.02)%, (64.30+/-5.24) vs (97.38+/-4.05)%], peak velocity of cell shortening [(70.23+/-4.85)% vs (98.09+/-5.46)%, (61.15+/-5.20)% vs (97.38+/-6.85)%], peak velocity of cell relengthening [(71.22+/-4.79)% vs (98.32+/-6.08)%, (68.16+/-5.24)% vs (97.55+/-5.00)%] and end- diastolic cell length [(88.28+/-5.84)% vs (97.95+/-5.52)%, (84.18+/-6.52)% vs (98.94+/-6.76)%]. IL-2 (5 U/ml, 50 U/ml) also markedly inhibited intracellular calcium transient [(74.94+/-4.90)% vs (98.09+/-3.74)%,(71.00+/-5.28)% vs (97.38+/-5.52)%], and elevated end-diastolic calcium level of ventricular myocytes [(113.91+/-5.93)% vs (100.10+/-3.02)%, (119.09+/-7.12)% vs (100.52+/-6.00)%], which were attenuated by the opioid receptor antagonist naloxone (Nal,10 nmol/L). In the isolated perfused rat heart,when compared with the control group, IL-2 50 U/ml markedly decreased left ventricular developed pressure [(79.91+/-2.18) vs (93.84+/-2.94)mmHg], maximal rate of rise of left ventricular pressure [(2370.7358.29) vs (2591.50+/-62.81)mmHg] maximal rate of fall of left ventricular [-(1460.95+/-38.6) vs -(1634.24+/-54.05) mmHg/s] and heart rate [(217.35+/-10.56) vs (244.52+/-11.23) beats/min], but increased left ventricular end-diastolic pressure (11.44+/-1.02 vs 9.23+/-0.46). Pretreatment with Nal (10 nmol/L) antagonized the cardiac depression and left ventricular end-diastolic pressure elevation induced by IL-2.

CONCLUSIONThe cardiac effect of IL-2 is mediated by opioid receptors on the membrane of cardiomyocytes.

Animals ; Calcium ; metabolism ; Depression, Chemical ; In Vitro Techniques ; Interleukin-2 ; pharmacology ; Male ; Myocardial Contraction ; drug effects ; Naloxone ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Receptors, Opioid, kappa ; drug effects ; physiology

OBJECTIVETo investigate the mechanism of the negative inotropic effect of tumor necrosis factor-alpha (TNF-alpha) in cardiomyocytes.

METHODSThe spectrofluorometric method was used to verify the calcium handling of the single myocyte. The activities of Ca(2+)-ATPase of sarcoplasmic reticulum (SR) and the activities of Ca(2+)-ATPase and Na(+)/K(+)-ATPase of plasma membrane were measured with colorimetric methods.

RESULTSTNF-alpha at 20 U/ml and 200 U/ml depressed the contractility of ventricular papillary muscle to 91% and 76% of control (P<0.01) respectively, but had no effect on the amplitude of electrically induced calcium transient in single myocyte. TNF-alpha inhibited the responsiveness of SR Ca(2+)ATPase activity to ATP (0.1 - 4 mmol/L) and Ca(2+) (1 - 40 micromol/L). TNF-alpha did not alter the activities of Ca(2+)-ATPase and Na(+)/K(+)-ATPase of plasma membrane compared with control group.

CONCLUSIONTNF-alpha decreases the myocardial contractility, at least partly, by inhibiting the activity of SR Ca(2+)- ATPase.

Animals ; Calcium ; metabolism ; Calcium-Transporting ATPases ; metabolism ; Depression, Chemical ; In Vitro Techniques ; Male ; Myocardial Contraction ; drug effects ; Myocardium ; metabolism ; Rats ; Rats, Sprague-Dawley ; Sodium-Potassium-Exchanging ATPase ; metabolism ; Tumor Necrosis Factor-alpha ; pharmacology