BACKGROUND: Matrix metalloproteinase (MMP) is a kind of protease family, its activity can be inhibited by tissue inhibitor of metalloproteinase (TIMP), especially by TIMP-3.OBJECTIVE: To fully isolate and purify natural TIMP-3, and to create enzyme-linked immunoassay of TIMP-3.DESIGN: Single-sample observation SETTING: Central Laboratory of Shenyang Medical College MATERIALS: Human placenta from Department of Obstetrics & Gynecology, Fengtian Hospital Affiliated to Shenyang Medical College (Informed consent was obtained from the relatives of patients) and MMP-1 from Research Institute. Fuji Chemical Industries, Ltd.METHODS: This experiment was conducted in the Central Laboratory of Shenyang Medical College between March 2001 and May 2002. Firstly,4 mol/L urea Tris-buffer solution (pH8.0) was used to prepare homogenate solution of placenta. Secondly, homogenate solution was performed chromatography through CM52 positive ion-exchange resin and Sephacryl S200 gel filtration. Thirdly, relative molecular weight and purity were detected by SDS- polyacrylamidedel gel electrophoresis (SDS-PAGE).Fourthly, Western blotting was used to identify the characters of purified protein. Fifthly, the inhibitory rate of TIMP-3 to MMP-1 was measured with immumofluorescence method.MAIN OUTCOME MEASURES: ① The relative molecular mass of protein showed by PAGE. ② Results of Western blot. ③ The inhibitory rate of TIMP-3 for MMP-1. ④ The recovery rate of TIMP-3 following purification.RESULTS: ①The isolated and purified TIMP-3 from human placenta consisted of non-glycosylated and glycosylated protein, with relative molecular mass of 24 000 and 27 000, respectively. ② The inhibitory concentration of non-glycosylated and glycosylated TIMP-3 was 1.1×1010 mol/L and 1.2×1010 mol/L respectively to MMP-1. The inhibitory concentration of placenta-derived TIMP-3 was significantly higher than that of recombinant TIMP-3 for MMP-1. ③ The recovery rate of TIMP-3 was 23.4% following two-step chromatography.CONCLUSION: Extracellular matrix of human placenta-derived TIMP-3 consists of non- glycosylated protein and glycosylated protein; Two kinds of purified TIMPs-3 have remarkable inhibitory concentration for MMP-1, and significantly higher in comparison with recombinant metalloproteinase inhibitory factor-3.

Editor's note: The research on effective constituents of traditional Chinese medicine is one of the crucial areas of basic research of traditional Chinese medicine. It is also a research hotspot which gains widespread attention and active application of projects of national natural science funds in the discipline of traditional Chinese medicine. In 2013, the National Natural Science Foundation of Chinese traditional medicine in the application code H2803 in the research field of effective constituents of traditional Chinese medicine , the direction of gener-al project , Youth Science Foundation and the regional Science Foundation project application 449 , which accept-ed 425 . In this paper , the general situation of the application and approved projects of national natural science funds in the research field of effective constituents of traditional Chinese medicine in 2013 has been introduced. The research strategies and plans of the approved projects have been summarized , and the problems of the ap-plications have been also analyzed .

Objective To determine the effect of age,body figure and risk framing in making treatment decision for female patients with breast cancer.Methods Two groups of healthy female:one group of 60 female ages 18-24 from undergraduate student and the other group of 60 female ages 35-60 from the university community.Healthy women imagined that they had been diagnosed with breast cancer and received information regarding lumpectomy versus mastectomy and recurrence rates.Participants indicated whether they would choose lumpectomy or mastectomy and reasons.Results The different concern levels of body appearance between younger and older women influences treatment selection of lumpectomy versus mastectomy.Facing the risk information,older women were unlikely to select the breast conserving operation.Conclusions The factors of age and the attention to body figure would effect the choice of breast conserving operation or mastectomy.Risk information on the selection of treatment would not affected directly,but the effects of risk information on younger and older female is different.Nurses should provide the age-appropriate information who recently have been diagnosed with breast cancer regarding treatment alternatives to ensure their active participation in the decision-making process,female who have different levels of investment in body figure also may have different concerns about treatment,and should be alert on empathetic of such concerns.

Objective To investigate the diagnostic value of determination of the genotypes in codon 12 and 13 of K-ras oncogene in blood samples of patients with pancreatic carcinoma(PC).Methods Blood samples were obtained from 54 patients with pathologically confirmed PC,and 33 healthy controls.The DNAs were obtained in these samples.and then genotype of K-ras mutation was detected by using the PNA-clamping real-time quantitative PCR.Then the correlation between the K-ras genotypes of blood DNA and the clinical characteristics was analyzed.Results K-ras mutations were found in 74.1%(40/54)of patients with PC.There was no such mutation in control samples.The mutations of K-ras was associated with age,lymph node and vessel invasion.poorly differentiated tumor,CA19-9,while it was not associated with sex,tumor location,size of tumor,clinical staging and pathological type.Conclusions The one-step method was highly sensitive for detecting K-ras mutation in blood samples.Detection of circular blood cells harboring K-ras mutation suggested the tumor was highly invasive with poor prognosis.

To evaluate multi-slice computer tomography (MSCT) in mediastinal lymph node metastasis of T1 and T2 non-small cell lung cancer (NSCLC). Methods:A total of 32 patients with T1 and T2 NSCLC from February 2004 to October 2012 were selected. Preoperative MSCT assessment of mediastinal lymph nodes was performed on basis of the pathological results. Results:Lymph nodes with diameters of≥10 mm were evaluated, and the sensitivity and specificity of the MSCT mediastinal lymph node me-tastases were 82.4%and 92.4%, respectively. Lymph node size, primary tumor location, and visceral pleural invasion showed statistical significance in forecasting mediastinal lymph node metastases (P<0.05). Conclusion:MSCT can be used for the effective evaluation of mediastinal lymph node metastasis, lymph node size, and position of primary tumor. and visceral pleural invasion of the tumor had a higher risk of mediastinal lymph node metastasis.

Objective:To investigate the purification of antibacterial effective fraction of Syringae folium by macroporous resins. Methods:Static adsorption and desorption tests were carried out to screen the macroporous resins. The desorption experiment was per-formed on the selected D101 resin to optimize the separation process. The effects of resin amount, diameter length ratio, elution flow rate, elution solution concentration and volume were studied. Results:The optimal conditions were as follows:the elution solution was 55% ethanol, the adsorption flow rate was 1 BV·h-1 , the elution flow rate was 5 BV·h-1 , 6 BV 25% ethanol was used to eliminate impurity and 8 BV 55% ethanol was used to elute to obtain the effective fraction. Conclusion: The content of antibacterial effective component is above 65% after purified by D101 resin, indicating that the present method is suitable for large-scale preparation of anti-bacterial effective fraction of Syringae folium.

In order to improve the thermostability of the Penicillium expansum Lipase (PEL), the lipase encoding genes was mutated by site-directed mutagenesis. A recombinant vector pAO815-ep8-K55R which contain double mutant genes was constructed by overlap extension PCR using the cDNA of a random-mutant lipase ep8 (a single site mutant) as the template and two special primers were used to generate another mutation site K55R. The recombinant vector was transformed into Pichia pastoris GS115 by electroporation and the recombinant mutant GS-pAO815-ep8- K55R can secret double-mutant lipase PEL-ep8-K55R-GS into the medium when it was induced by Methanol. The yield of the double-mutant lipase is 508 u/mL, which is 81% that of the wild type lipase PEL-GS (627 u/mL) and 55% that of random-mutant PEL-ep8-GS (924 u/mL). The specific activity of double-mutant lipase is 2309.1 u/mg, which is similar to random-mutant lipase PEL-ep8-GS and the wild type lipase PEL-GS. The optimum temperature of the double-mutant lipase is same with the wild type lipase PEL-GS and random-mutant lipase PEL-ep8-GS. While the Tm of the double-mutant lipase is 41.0 degrees C, 2.3 degrees C higher than the wild type lipase PEL-GS and 0.8% higher than the random-mutant lipase PEL-ep8-GS, indicating that the double-mutant lipase PEL-ep8-K55R-GS has higher thermostability.
Electroporation ; Enzyme Stability ; Hot Temperature ; Lipase ; genetics ; metabolism ; Mutagenesis, Site-Directed ; Mutant Proteins ; metabolism ; Penicillium ; enzymology ; Pichia ; genetics ; metabolism ; Protein Engineering ; methods ; Recombinant Proteins ; biosynthesis ; genetics

Coxsackievirns A16(CVA16),together with enterovirus type 71(EV71),is responsible for most cases of hand,foot and mouth disease(HFMD) worldwide.Recent findings suggest that the recombination between CVA16 and EV71,and the co-circulation of these two viruses may have contributed to the increase of HFMD cases in China over the past few years.It is therefore important to further understand the virology,epidemlology,virus-host interactions and host pathogeuesis of CVA16.In this study,we describe the viral kinetics of CVAI6 in human rhabdomyosarcoma(RD) cells by analyzing the cytopathic effect(CPE),viral RNA replication,viral protein expression,viral RNA package and viral particle secretion in RD cells.We show that CVA16 appears to first attach,uncoat and enter into the host cell after adsorption for 1 h.Later on,CVA16 undergoes rapid replication from 3 to 6 h at MOI 1 and until 9 h at MOI 0.1.At MOI 0.1,CVA16 initiates a secondary infection as the virions were secreted before 9 h p.i.CPE was observed after 12 h p.i.,and viral antigen was first detected at 6 h p.i.at MOI 1 and at 9 h p.i.at MOI 0.1.Thus,our study provides important information for further investigation of CVA16 in order to better understand and ultimately control infections with this virus.

Objective To investigate rite effects of loag-term exposure to low-level sevoflurane on reproductive function in mice.Method F0ny male ICR mice,aged 60 d,weighlag 20-25 g,were randomly divided into 4 groups(n=10 each):control group received no sevoflunme(C);group S1-3 were exposed to 0.003%.0.01% and 0.03% sevoflurane 2 h per day for 5 consecutive days per week for 8 weeks respectively. The mice were then sacrificed at the end of the 8 weeks.The testes and epididymis were emoved and sampled for determination of the activities of total lactic dehydregenase(LDH)and lactic dehydrogenase-X(LDH-X),and the motility rate,amount,and aberration rate of sperm.Testicular uhrastructure were observed by transmission electron microscopy.Results The sperm motifity nne were significantly lower.the sperm aberration rate higher and the activity of LDH-X lower in group S3 than in group C(P<0.05),but there was no significant difference in the above parametem between group SI and group S2(P>0.05).The pathology changed of testes occurred only in group S3 among the 3 groups.Conclusion Long-term exposure to 0.03% sevoflurane can result in the abnormality of the reproductive function in male mice but exposure to≤0.01%sevoflurane dose not.

Objective To evaluate the effects of baseline and changes of peritoneal transport characteristics on the prognosis of maintaining peritoneal dialysis (PD) patients.Methods Five hundred and eight-six PD patients who started PD from September 11,2006 to October 30,2014 in a single center were included and followed up until March 30,2016.According to their baseline D/Pcr value in peritoneal equilibrium test (PET),the patients were divided into high transport (H) group (D/ Pcr 0.82-1.03),high average transport (HA) group (D/Pcr 0.65-0.81),low average transport (LA) group (D/Pcr 0.50-0.64) and low transport (L) group (D/Pcr 0.34-0.49).According to the changes of follow-up D/Pcr comparing with baseline D/Pcr,the patients were also divided into ascending group,descending group and no-change group.The patient and technical survival rates were estimated by Kaplan-Meier analysis.Cox proportional hazards analyses were used to analyze the risk factors for PD patient death and technical failure.Results There were 67 patients in L group,229 patients in LA group,252 patients in HA group,and 38 patients in H group.The patient survival rate in H group was significantly lower than those of L group (P=0.036),LA group (P=0.008) and HA group (P=0.041).There was no significant difference on technical survival rate among these 4 groups.According to the tendency of follow-up D/Pcr changes,there were 127 patients in ascending group,101 patients in descending group and 179 patients in no-change group.There was no significant difference on patient survival among these 3 groups (P=0.064).However in patients with a high transport rate (D/Pcr≥0.65),the patient survival was lower in descending group than those in ascending group (P=0.033) and nochange group (P=0.049).Age over 65 years old (HR=2.499),malnutrition during follow-up (HR=3.144),ultrafiltration less than 400 ml/d during follow-up (HR=1.863) and high sensitive C reactive protein≥ 10 mg/L (HR=4.526) were the independent risk factors for patient death (all P ＜ 0.05).Gender (HR=1.609),age over 65 years old (HR=1.929),ultrafiltration less than 400 ml/d during follow-up (HR=1.708),high sensitive C reactive protein ≥10 mg/L (HR=1.829),malnutrition (HR=1.876) and change of peritoneal transport function (HR=0.579) affect technical failure (all P ＜ 0.05).Conclusions The survival rate of PD patients with basal high peritoneal transit is relatively low,especially for patients with descending transport rate during follow-up.The concern on the peritoneal transport status is constructive for the prognosis of PD patients.