To improve the inulinase application in biotechnology, the characteristic of inulinase from Kluyveromyces marxianus YX01 was investigated. The inu gene of K. marxianus YX01 was transformed into Pichiapastoris GS115 host cells with molecular biology techniques. Then we achieved the heterologous expression of exo-inulinase whose molecular mass was about 86.0 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Furthermore, six His-tag was added to the inulinase and a two-step method was applied in the purification of inulinase, including concentration via dialysis by polyethylene glycol 20 000 and metal Ni-NTA Agarose affinity adsorption. The purification factor of purified protein was 3.6 and the recovery rate of enzyme activity was 33.1%. We characterized the purified inulinase. The optimum temperature was 60 degrees C and pH was 4.62. When inulin and sucrose were used as substrates, the K(m) and V(max) values were 80.53 g/L vs 4.49 g/(L x min) and 183.10 g/L vs 20.20 g/(L x min), respectively. In addition, metal ions including Mn2+, Ca2+, Cu2+, Zn2+ and Fe2+ exhibited different degrees of inhibition on the enzyme activity, and Cu2+, Zn2+ and Fe2+ exhibited the most significant inhibition. Our findings might lay a good foundation for industrial application of inulinase.
Glycoside Hydrolases ; chemistry ; genetics ; Industrial Microbiology ; Inulin ; Kluyveromyces ; enzymology ; genetics ; Pichia ; Sucrose ; Temperature

Objective To investigate the ultrasound diagnostic value for nipple discharge disease by comparison with fiberoptic ductoscopy. Methods Fiberoptic ductoscopy and ultrasonic inspection were performed on fifty-seven patients with spontaneous nipple discharge,and the results were analysed. Results In all patients, 55 cases (96. 49 % ) were abnormal in high-frequency ultrasound inspection:27 mammary duct expansion(47.36%),19 occupying lesion with mammary duct expansion or cyst (33.33%),6 cystic echo ( 10. 52% ) ,3 only solid echopattern (5.26%) ,and 2 nothing found (3.51% ). In ultrasonic inspection,color and pulse Doppler signal were found in 7 cases. On the other hand, 26 galactophoritis (45.61%), 12mammary duct ectasia (21.05 %), 17 intraductal papilloma (29.82%), 2 breast carcinoma (3.50%) were diagnosed by fiberoptic ductoscopy. In all 19 cases with occupying lesion,ultrasonography's sensitivity was 52. 63% ,specificity was 89. 47%, misdiagnosis rate was 10. 50%, rate of missed diagnosis was 47.36%.Conclusions To nipple discharge disease, high-frequency ultrasound has high sensitivity, easy to find abnormal indication,but is not accurate enough in the diagnosis of smaller occupying lesion.

The poor chemical stability and solubility, easy volatile and other shortcomings of TCM volatile oils can be improved after they are included by cyclodextrin. Therefore, cyclodextrin inclusion technology is widely used in the preparation process of volatile oil. This article reviewed the domain of cyclodextrin inclusion of volatile oils, the types of cyclodextrin, inclusion technologies, characterization for inclusion, components changes before and after inclusion, and dissolution characteristics of cyclodextrin inclusion.

Objective To investigate the factors affecting the stability of the volatile oil extracted fromYinqiaosan Decoction.Methods The main chemical compositions and the extraction repetitiveness of the compound volatile oil were determined by GC-MS, and the stability of multiple extracted volatile oil was studied. Absorbance of the compound volatile oil was used as the evaluation index, and the factors affecting the stability of the of the compound volatile oil were investigated, such as illumination, temperatures and pH values of volatile oil solution and metal ions.Results The results of the GC-MS chromatograph indicated that the main chemical compositions of the compound volatile oil extracted fromYinqiaosan Decoction twice were the same. The results of the stability of the volatile oil showed that the preservation temperature and illumination affected the stability of the volatile oil to a certain extent. The absorbance values of the compound volatile oil changed slowly when it was stored at a relatively low temperature (4℃) and shielded from light, and it was less stable when stored in normal temperature and under illumination. Meanwhile, the absorbance of the compound volatile oil changed quickly in acid or alkaline solutions and was in instability. The metal ions, such as Cu2+ and Fe3+, have chemical reactions with the compositions of the compound volatile oil and there was a big change in the UV-Vis spectrum of the compound volatile oils.Conclusion The compound volatile oil should be stored at a relatively low temperature (4℃) and shielded from light. At the same time, it should be stored avoiding acids, alkaline and the metal ions, such as Cu2+ and Fe3+, to guarantee its stability. This study provides a reference for the preservation conditions and the preparation conditions of the compound volatile oil extracted fromYinqiaosan Decoction.

Objective:To investigate the enantiomer separation for atropine by capillary electrophoresis .Methods:Capillary elec-trophoresis was used with an elastic quartz capillary column (60 cm ×75 mm, effective length of 40 cm).The concentration of phos-phate buffer was 30 mmol· L-1 .The high and time of injection was 10 cm and 5 s, respectively.The detection wavelength was 225 nm.The best separation conditions were selected including the type and concentration of chiral resolving agent , pH of the buffer solu-tion, operating voltage and organic solvent.Results:The optimum conditions of separation were as follows:the pH of buffer solution was 7.0, the concentration of S-β-CDP was 10 mg· ml-1 , and the operating voltage was 12 kV.Conclusion: The method is simple and fast, which can be used to se parate the optical isomers of atrpo ine.

Objective To observe the impact of the five-emotion restrictive therapy on coronary heart disease. Methods109 hospitalized patients with coronary heart disease were randomly divided into a experimental of 56 patients and a control group of 53 patients according to length of stay. The control group was treated with the conventional therapy; the experimental group was imposed five-emotion restrictive psychological intervention on the basis of conventional therapy. The investigation was done using self-rating anxiety scale(SAS) and self-rating depression scale(SDS) on 1 day after admission and ld before discharge respectively. SAS and SDS scores were surveyed. At the same time Holter monitoring and normal ECG test were compared. Results Compared with control group, the experimental group 1 d before discharge of the SAS and SDS scores were (41.9±7.4) and (38.6±8.4), and the difference was statistically significant (P＜0.01) . The total effective rate of Holter monitoring and normal ECG was 64.15％ and 92.85％ in the control and experimental group, and the difference was significant (P＜0.01) . ConclusionThe pathological seven emotions have a certain affection on the incidence of coronary heart disease, treatment and prognosis of the course. Five-emotion restrictive psychological intervention can adjust timely psychological state of patients, eliminate negative emotions, and significantly help the incidence, treatment and prognosis of the disease.

Objective To investigate the epidemic status of endemic fluorosis in Shandong Province,Jining City,and to provide a basis for prevention and control of the disease.Methods Based on Shandong Provincial Project Technical Solutions for Endemic Fluorosis,Rencheng,Jinxiang,Yutai,Jiaxiang and Liangshan Counties in Jining were selected as monitoring sites.According to the illness situation of mild,moderate or serious districts,one village was selected as a major survey site from each county(district).There were a total of 15 such villages selected.Survey content included drinking water fluorine level; dental fluorosis of children,adults' clinical skeletal fluorosis and urinary fluorine levels; water and urinary fluoride content were determined by the method of fluoride ion selective electrode; dental fluorosis of children was diagnosed by Deans method and clinical diagnosis was based on the Diagnostic Criteria of Endemic Skeletal Fluorosis (WS 192-2008).Results Sixty-one water samples from 15 villages of five counties (districts) were tested.Fluoride levels of 9 out of the 61 samples were exceeded the national standard (＞ 1.0 mg/L),and the rate was 14.75％; 1 sample ＞ 2.0 mg/L,and the maximum water fluoride was 2.25 mg/L.Seven hundred and seventeen people's real time urinary fluoride was detected in the 15 villages,including 420 children and 297 adults,and the geometric mean were 1.53 and 1.69 mg/L,respectively.Clinical examination of 755 children aged 8 to 12 showed that the detection rate of dental fluorosis was 26.89％ (203/755); defect rate was 9.12％(29/755) and dental fluorosis index weres 0.65.The detection rate of clinical skeletal fluorosis of 11 565 adults was 4.76％(550/11 565),including 303 moderate or serious cases.Conclusions The situation of excessive water fluorine in outside environment in Jining City has been controlled at a certain degree; groups urinary fluoride level is closed to the normal upper limit; the prevalence of dental fluorosis or skeletal fluorosis has been suppressed at a certain degree,therefore,the results of control should be further consolidated and expanded,in order to completely eliminate the fluoride hazard.

Objective:To compare the anti-thrombin activity and the effects on the coagulation pathway between Whitmania pigra Whitman and Hirudinaria manillensis to provide scientific reference for the anticoagulation mechanism revelation for Hirudo. Methods:Anti-thrombin titration and chromogenic substrate assay-extraction-HPLC were applied to study the anti-thrombin activity of Whitmania pigra Whitman and Hirudinaria manillensis. APTT, PT and TT were determined by a clotting assay to compare the effects on the path-way of blood clotting. Results:The anticoagulation activity order measured by anti-thrombin titration was living Hirudinaria manillensis> dried Hirudinaria manillensis > > dried Whitmania pigra Whitman. The results of chromogenic substrate assay-extraction-HPLC in-dicated that the low dose of aqueous extract promoted the thrombin activity, while the high dose inhibited the thrombin activity. Hirudi-naria manillensis significantly inhibited the activity of thrombin, while Whitmania pigra Whitman showed weak anti-thrombin activity only at the higher dose. All leeches could prolong APTT, PT and TT. However, living Hirudinaria manillensis mainly affected TT, and dried Hirudinaria manillensis mainly affected APTT. Dried Whitmania pigra Whitman dramatically influenced APTT and TT. All the results indicated that the anticoagulant activity of Whitmania pigra Whitmanis was significantly higher than that of Hirudinaria manillen-sis. Conclusion:There are notable differences in the anti-thrombin activity and the effect on the pathway of blood clotting between Whitmania pigra Whitman and Hirudinaria manillensis.

Objective To establish a pre-column derivation HPLC-UV method for the content determination of hyodeoxycholic acid in artificial calculus bovis. Methods The hyodeoxycholic acid was derived by 2-bromo-2’-acetonaphthoneat using triethylamine as the catalyst in 60 ℃ water bath. After that, a HPLC-UV method was established to determine the content of hyodeoxycholic acid in artificial calculus bovis. Results When the derivatising time at 60 ℃ water bath was 50 min, the radio of the molar amount of derived reagents and hyodeoxycholic was over 20:1 and the radio of catalyst and hyodeoxycholic was over 15:1; the reaction was completed. The calibration curve was linear within the range of 0.1–2 μg for hyodeoxycholic acid (r=0.999 7), and the average recovery was 97.85% (RSD=1.6%). In this sample, the content of hyodeoxycholic is 4.12%. Conclusion The method is with high sensitivity, highly reproducible, reliable and accurate for the content determination of hyodeoxycholic acid in artificial calculus bovis.

Objective To analyze the assessment results of external quality control and network operation of urinary iodine in local laboratories of Shandong Province,to evaluate the ability of consistent analysis; and to provide a reliable laboratory quality assurance for epidemiological surveillance and control of iodine deficiency disorders (IDD) and reliable decision-making.Methods Z-scores of the assessment results of external quality control of urinary iodine in local laboratories of Shandong Province were analyzed from 2000-2013.Results The feedback rate of urinary iodine in local laboratories of ShandongProvince was 100.0％ from 2000-2013; the qualified rate was 100.0％(14/14),100.0％(14/14),92.9％(13/14),100.0％(14/14),92.9％(13/14),100.0％ (14/14),100.0％ (14/14),100.0％ (14/14),92.9％ (13/14),100.0％ (17/17),100.0％ (17/17),94.1％ (16/17),100.0％ (17/17),and 100.0％ (17/17),respectively.The total qualified rate of Z-scores between Zs in local laboratories was 100.0％ (214/214)and within Zs was 98.6％ (211/214).Conclusion The test abilities of urinary iodine in local laboratories of Shandong Province are steady; the overall results are satisfactory,but some laboratories need to improve.