Objective To purify AmpC beta-lactamase from Enterobacter cloacae and study its physical and chemical characteristics.Methods The bacteria were fragmentized by ultrasonication and AmpC beta-lactamase was purified by ion-exchange.The properties of the purified enzyme,such as molecular weight and isoelectric point were determined by electrophoresis.Results The purified AmpC beta-lactamase was obtained and its specificity to substrate was sufficient for classification of class C cephalosporinase.Conclusion AmpC can be successfully purified from Enterobacter cloacae and is effective for application.

BACKGROUND:It is reported that the cellsurface antigen may be damaged by the trypsin enzyme in some extent and the cellactivity may also be influenced, as a result of which, the subsequent separation and fol ow-up-test wil be affected. Accutase enzymes possess the activities of protease and col agen enzyme and need no special termination or cleaning at the end of digestion. Another special function of accutase enzyme is to protect cellsurface antigen and thus it has been widely applied in the stem celldigestion and culture.
OBJECTIVE:To compare the digestive effect of accutase enzymes and trypsin enzyme in the separation and original culture of spermatogonial stem cells.
METHODS:The testes of 5-7 days male Kunming mice (n=45) were col ected and primarily digested with col agenas. The suspension of digested tissue was divided into three parts with the same volume named accutases enzyme group, trypsin enzyme group and mixed enzyme group (trypsin enzyme and hyaluronidase). For the comparison of testis digestive status and the time required for the formation of single cells, the micrographs were taken at 1, 3 and 5 minutes respectively after enzyme digestion. The total number and the mortality of the single cells were estimated and compared. The leydig cells and sertoli cells were removed by differential adherent method and the remained spermatogenic cells were then treated with CD90.2 immune magnetic beads. The selected spermatogonial stem cells were subsequently labeled by glial cellline-derived neurotrophic factor receptor alpha-1 and sorted with flow cytometry. The number of spermatogonial stem cells positive for glial cellline-derived neurotrophic factor receptor alpha-1 in CD90.2+and CD90.2-cells was compared within different groups.
RESULTS AND CONCLUSION:The digestive capacities of different enzyme were different. Accutases enzyme obtained the single cellsuspension more quickly than trypsin enzyme and mixed enzyme, and had the least cellmasses and broken cellmembrane than the other two groups. After the differential attached treatment, the highest total number of CD90+spermatogonial stem cells and lowest cellmortality could be found in the accutases group, when compared with the other groups. The results of cellsorting by flow cytometry showed a higher rate (72.24%) of GFRα1+/CD90+cells in the accutases group than the trypsin group (51.16%) and mixed enzyme group (71.27%). The GFRα1+/CD90-number in the accutases group was lower (15.03%) than that of the trypsin group (18.8%) and mixed enzyme group (24.23%), respectively. The results indicate a better effect of accutases enzyme on the primary separation of spermatogonial stem cells than that of trypsin or mixed enzyme.

OBJECTIVE To investigate the disinfectant-resistant gene qacE△1-sul1 in Pseudomonas aeruginosa isolated from burned patients. METHODS GNS-448 and K-B tests were performed to detect the susceptibility of 19 kinds of antimicrobial agents against these strains. Genotype was analyzed by polymerase chain reaction(PCR) and verified by DNA sequencing. RESULTS The 32 strains isolated were all resistant to ampicillin,cefuroxime,cefoxitin,SMZ/TPM. The sensitive rates to amikacin and gentamicin were 68.0%,and 46.9%,respectively,the resistant rates to imipenem and meropenem were 68.8% and 59.4%,respectively. The positive rate of gene qacE△1-sul1 was 50.0%. CONCLUSIONS The resistance of P. aeruginosa isolated from burned patients is a serious issue.There is high positive percentage of qacE△1-sul1 gene in P. aeruginosa isolated from burned patients.

OBJECTIVE To investigate the 16S rRNA methylase gene and aminoglycoside-modifying enzyme genes in Pseudomonas aeruginosa isolated from burned patients. METHODS GNS-448 and K-B tests were performed to detect the susceptibility to 19 kinds of antimicrobial agents against these strains. 16S rRNA methylase gene and aminoglycoside-modifying enzyme genes were amplified by polymerase chain reaction (PCR) and verified by DNA sequencing. RESULTS The 32 isolated strains were all resistant to ampicillin,cefuroxime,cefoxitin,SMZ-TMP,The sensitive rates to amikacin and gentamicin were 68% and 46.9%,respectively. The resistant rates to imipenem and meropenem were 68.8% and 59.4%,respectively. The 16S rRNA methylase gene and aminoglycoside-modifying enzyme genes including aac(6')-Ⅰb,aac(6')-Ⅱ,ant(3″)-Ⅰ,ant(2″)-Ⅰ and rmtB were found and positive rates were 9.4%,3.1%,28.1%,25.0% and 3.1%,respectively. A novel subtype of aac(6')-Ⅰb was reported firstly. CONCLUSIONS There are high positive percentage of 16S rRNA methylase gene and aminoglycoside-modifying enzyme genes in P. aeruginosa isolated from burned patients. P. aeruginosa resistance to aminoglycoside relates to the existence of 16S rRNA methylase gene and aminoglycoside-modifying enzyme genes.

Food Testing was the experimental and applied basic courses for Prevent Medicine.According as the problems in teaching,some advice on teaching content,teaching method,and laboratory construction and management was discussed in order to cultivate the innovative specialties and develop the students' experimental ability.

Physical and Chemical Analysis of Food is one of the important courses of hygiene quarantine speciality,food science and engineering speciality,food quality and safety speciality,and so on. The application of concept center method on the teaching of the course was discussed in the article. The prolegomenon of the course and the eleventh chapter of the course--Determination of Gene Modified Food had been worked as two examples.

Objective To investigate the differences of hand and foot morphology and genetic phenotypic characteristics in adults of Hui and Han nationality in Southwestern Henan.Methods The indicators of height,weight,hand and foot were measured by the morphological measurements,the hand and foot genetic phenotype classification was observed and performed the statistical analysis.Results The hand width,foot length and foot width of the Hui adult men and women in Southwestern Henan were(8.27±0.55,23.10±1.20,9.34±0.83)cm and(7.41±0.44,20.50±1.23,8.79±0.69) cm,respectively,while which of the Han adult men and women were(8.56±0.09,24.57±1.33,9.47±0.70)cm and(7.74±0.36,22.46±1.21,8.91±0.85) cm,respectively.The total number of both hands fingerprint ridge line in Hui adult men and women were(135.06 ± 19.87) and (125.50 ±20.44)respectively,and which in Han adult men and women were (144.46 ±14.08) and (129.20 ± 20.34)respectively,the difference was statistically significant(P＜0.05).The tPD,atd angle and a-b crest line number among the Hui and Han nationalities were 16.07± 6.46,(44.61±8.66)°,34.04±5.47 and 16.53±6.27,(43.19±9.52)°,36.73±4.22 respectively.And the handedness,fingernail form,thumb type,footedness,right type ratio of foot and toe length of the Hui and Han nationalities were 90.01％,38.52％,85.59％,70.47％,56.92％ and 89.33％,45.26％,70.91％,96.98％,74.89％,respectively,the difference between the Hui and Han nationalities was statistically significant(P＜0.05).Conclusion The national differences and gender differences exist in the multiple indicators of hand and foot morphology,finger and palm prints,and genetic phenotype among the Hui and Han adults in Southwestern Henan.

Objective A rat model of chronic post-thoracotomy pain is used to study whether acute pain and pre-operative diffused noxious inhibitory controls(DNIC) can predict chronic pain and how DNIC changes when pain maintains.Methods Rats were randomly divided into three groups:naive group,sham group and model group.DNIC was constantly assessed in individual rats,along with each animal's mechanical hyperalgesia and cold allodynia after thoracotomy.Results In model group,the incidence of chronic post-thoracotomy pain was 55%(11 of 20),which was named CPTP group,and the other 9 rats without chronic pain was defined as non-CPTP group.The pre-operative DNIC was significantly weaker in CPTP group with lower mechanical threshold on 6 days after surgery and higher cold sensitivity on 6 days after surgery comparing with non-CPTP group.In the acute pain phase (day 3),DNIC was decreased in both CPTP group and non-CPTP group as compared with pre-operative period.Besides,DNIC was recovered in non-CPTP group while kept impaired in CPTP group on 21 days after surgery.Conclusions Pre-operatively assessed DNIC efficiency and acute post-operative pain intensity were two independent predictors for CPTP.DNIC was decreased both in acute pain and chronic state,while returned to normal when pain sense was normal.

Objective To construct the has-miR 146a eukaryotic overexpression vector pmR 146a and to explore its effect on the expression of c-Myc gene in HepG2.2.15 cells.Methods The has-miR-146a precursor gene fragment pre-has-miR-146a was amplified by PCR,then connected to the pmR-mCherry plasmid vector after double enzyme digestion,the accuracy of recombinant vector was verified by colony PCR,double enzyme digestion and sequencing;then the recombinant vector was transfected into HepG2.2.15 cells as the experimental group,meanwhile the empty vector group (transfecting pmR-mCherry empty plasmid group) and blank group(transfecting reagent lip2000+PBS),then the fluorescent protein expression amount was observed under the fluorescence microscopy at 24,48 h;the expression of has miR-146a was evaluated by qPCR;at 24,48 h after transfection,the expression levels of c-Myc gene mRNA were detected by qPCR,and the c-Myc protein expression level after 48 h was detected by Western blot.Results The colony PCR,double enzyme digestion and sequencing verified that the pre-has-miR-146a gene fragment was inserted into the pmR-mCherry vector;at 24,48 h after transfection in the experimental group and empty vector group,intracellular strong fluorescence was seen by fluorescent microscope,the transfection efficiency was at 50％-60％ contrasting without fluorescence;the has-miR-146a expression level in the experimental group was significantly higher than that in the empty vector group and blank group (P＜0.01);the c-Myc mRNA expression at 24,48 h after tranfection was significantly lower than that in the empty vector group and blank group (P＜0.05);the protein expression amount at 48 h after transfection was lower than that in the empty vector group and blank group (P＜0.01).Conclusion The pmR-146a eukaryotic overexpression vector is successfully constructed,this recombinant vector can express miR-146a stably;miR-146a can down-regulate c-Myc cancer gene expression,which can serve as one of potential targets for treating hepatocellular carcinoma.

Objective To investigate the effects of contrast dose and region of interest (ROI) depth on quantitative analysis of liver by contrast-enhanced ultrasound (CEUS) during clinical application.Methods After bolus injection of contrast agent,the change of quantitative parameters [including echo mean(EM),rise time(RT),peak intensity(PI),mean transit time(MTT),area under the curve(AUC),time from peak to one half(TPH),wash in slope(WIS),time to peak(TTP)] of time-intensity curves were analyzed based on groups from different doses (1.0 ml and 1.6 ml) and different depth (＜30 mm,30-60 mm,and ≥60 mm).Results MTT and TPH were increased with dose increasing from 1.0 ml to 1.6 ml (P＜0.05).With the dose 1.0 ml,TPH,WIS,PI,AUC and MTT showed significant difference when the depth of the ROI changes (P ＜0.05),with the depth increased,TPH,WIS,PI,and AUC all decreases and MTT increases.For all the other parameters,no significant changes were found (P ＞0.05).Conclusions CEUS and its imaging process can directly influence the accuracy of the parameters from the quantitative analysis.Standardization of contrast agent with predefined dose and depth can potentially facilitate future clinical studies in liver CEUS.