AIM: To establish the method of determining procyanidin B2 content in grape seed extract by HPLC. METHODS: The determination was carried out with ZORBAX SB-C_ 18 column. The mobile phase consisted of acetonitrile-2% acetate buffer under the gradient elution condition, detection wavelength was at 280nm. RESULTS: There was a good linear relationship between the peak area and concentration in the range of 1~30 ?g/mL for procyanidin B2. The average of procyanidin B2 (n=5) was 99.29% and RSD=1.64%, respectively. CONCLUSION: The method is simple, accurate, reproducible and can be used for assay of procyanidin B2.

A themostable intracellular ? galactosidase from a thermophilic Bacillus stearothermophilus was purified by a combination of (NH 4) 2SO 4 fractionation,ion exchange (DEAE 22)and gel filtration (Sephades G 75).The optimum temperature and pH of the enzyme acivity were 60℃and pH6.4 respectively.The ? galatosidase activity exhibited thermosttability at 50 ℃.The enzyme was significaantly activated by alkali and alkali earth metal ions.The activity was inhibited by Zn 2+ 、 Fe 3+ 、 Cu 2+ Reducing agents enhanced ? galactosidase activity.Thiol binding agents drastically decreased the enzyme activity.The enzyme was specific for ? D glycosidic linkages,and the identity of the aglycone moiety also influenced enzyme activity.At 55℃the Km for O nitrophenyl ? D galactosidase(ONPG)and lactose were 2.63mmol/L and 4.39mmol/L, respectively,and Vmax for both substrates were 1.93?10 5 mmol.min 1 .mg 1 protein6.54?10 5 mmol.min 1 .mg 1 protein,respectively.The enzyme was inhibited by glucose (the products of lactose hydrolysis,ki 2.47mmol/L),but not by galactose.In addition,the enzyme possessed transgalactosylation activity.Galacto oligosaccharides,both tri and tetrasaccharide,were involved in the products during lactose hydrolysis.

AIM: To develop a rapid HPLC method for quality control of traditional Chinese medicinal ingredients consisted of citrus flavonoids, naringin, hesperidin, neohesperidin, sinensetin and nobiletin. METHODS:Gradient elution with non-salt mobile phase ( methanol and water only) HPLC method on a Kromasil column ( 100-5C18-250A, 4.6 mm ×250 mm, 5 μm, C18 reverse phase) with peaks identification through DAD full UV wavelength scan. UV 284 nm and 332 nm profiles were observed. RESULTS: Satisfactory resolution, linearity, 95%～ 102% of recovery and 1.88 ～ 2.93% of repeatability were obtained for those five citrus flavonoids. Content of 6 Citrus aurantium L. based TCM ingredients were analyzed and identified. CONCLUSION: Rapid HPLC test method on citrus flavonoids was developed and can be in LC-MS identification.

BACKGROUND: Cleistocalyx operculatus is a dried alsbastrum of myrtle. It is reported that cleistocalyx operculatus extracts can improve cardiac contraction through inhibiting the activity of Na+/K+-ATPase, and decrease rate of contraction. Do cleistocalyx operculatus extracts have the biological activity of antioxidation?OBJECTIVE: To observe the effects of cleistocalyx operculatus on oxidative injury of PC12 nerve cells induced by H2O2.DESIGN: Non-randomized controlled study.SETTING: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology.MATERIALS: The experiment was conducted at New World Institute of Biotechnology, State Key Laboratory of Bioreactor Engineering of East China University of Science and Technology, from May to November 2002.Eight adult male Kunming mice were selected. PC12 nerve cells were supplied by Shanghai Cell Institute of the Chinese Academy of Sciences.METHODS: Model of oxygenic injury of PC12 nerve cells was estabPC12 cells were cultured in 96-well plates. Cleistocalyx operculatus was diluted with RPMI1640 culture medium into five concentrations of 0.001,0.01, 0.1, 0.5 and 1 g/L with 3 wells in each concentration; each well had 2×103 cells. Blank control group, or non-drug culture medium group, was set. Under the standard condition, cells were cultured for 48 hours and ascells were inoculated in 96-well plate with the density of 2×103 for 24-hour wall adhering, and then divided into normal control group (normal cell without H2O2 or cleistocalyx operculatus extracts), 0, 0.01, 0.1, 0.5 and 1 g/L cleistocalyx operculatus. Cells in all groups except normal control group were treated with 200 μmol/L H2O2 at 37℃ for 3 hours, then cleistocalyx operculatus of various concentrations was added and survival rate was asfree radicals: PC12 cells with oxygen-derived free radicals were treated in the same way as done for cell survival rate assay and measured with CDCFH staining method.fect of cleistocalyx operculatus extracts on intracellular and extracellular oxygen-derived free radicals in PC12 nerve cells induced by oxidative injury.operculatus could protect nerve cells; however, at 0.055-1.00 g/L the effect on cell growth did not significantly differ from that of blank control extracts had no protective effect on the injury of PC12 nerve cells induced by H2O2. At 1.00 g/L, it had strong plerosis for oxidative injury of PC12 and extracellular oxygen-derived free radicals in PC12 nerve cells was increased; however, at 0.01 g/L concentration of cleistocalyx operculatus extracts, the level was lower than that in model group.dation of membrane lipid of hepatic microsome, but also protect against oxcleistocalyx operculatus extracts is related to its concentration. At 1.00 g/L,it has great capacity of oxidation plerosis, and at 0.01 g/L it can decrease the level of oxygen-derived free radicals inside and outside cells.

A themostable intracellular β-galactosidase from a thermophilic Bacillus stearothermophilus was purified by a combination of (NH4)2SO4 fractionation, ion-exchange (DEAE-22)and gel filtration (Sephades G-75). The optimum temperature and pH of the enzyme acivity were 60Cand pH6.4 respectively. The β-galatosidase activity exhibited thermosttability at 50 C. The enzyme was significaantly activated by alkali and alkali-earth metal ions. The activity was inhibited by Zn2+ 、 Fe3+ 、 Cu2+Reducing agents enhanced β- galactosidase activity. Thiol-binding agents drastically decreased the enzyme activity. The enzyme was specific for β-D glycosidic linkages,and the identity of the aglycone moiety also influenced enzyme activity. At 55Cthe Km for O-nitrophenyl-β-D-galactosidase (ONPG)and lactose were 2. 63mmol/L and 4.39mmol/L, respectively,and Vmax for both substrates were 1.93 × 10-5mmol. min-1 mg-1protein6.54 ×105 mmol. min-1. mg-1protein,respectively. The enzyme was inhibited by glucose (the products of lactose hydrolysis,ki 2.47mmol/L),but not by galactose. In addition,the enzyme possessed transgalactosylation activity. Galacto-oligosaccharides,both tri- and tetrasaccharide,were involved in the products during lactose hydrolysi

Objective To compare the clinical effect of total laparoscopic and open liver resection for tumors in segments Ⅶ and Ⅷ.Methods The clinical data of patients with tumors in segments Ⅶ and Ⅷ of the liver who met the inclusion criteria and received operation at Affiliated Hospital of Nantong University from January 2011 to January 2015 were retrospectively analyzed.Among these patients, there were 17 cases who received total laparoscopic liver resection (LLR group), and 25 cases who received open liver resection (OLR group).Results LLR group has obvious advantages in aspects of the level of serum alanine transaminase (ALT) on 1st and 3rd day postoperation, the time anal exsufflation, the drainage volume of abdominal cavity in 3 days after operation and the postoperative hospital stay than those in OLR group (respectively t =-3.075,-3.175,-2.499,-2.088,-2.419, all P ＜ 0.05).There were no significant differences in blood transfusion rate, the resection margin to the tumor, the postoperative morbidity and the total medical cost between the two groups (x2 =1.437, t =-1.244, x2 =0.209, t =1.079, all P ＞ 0.05).Though the mean operative time and intraoperative blood loss of LLR group compared with OLR group increased obviously (respectively t =3.360, 2.189, all P ＜ 0.05).During the postoperative follow-up, there were no significant differences in the postoperative recurrence rate and the long-term survival rate in patients with malignant tumors (respectively x2 =0.240, 0.000, all P ＞ 0.05).Conclusion The therapeutic effect of total laparoscopic and open liver resection are equal in segments Ⅷ and Ⅷ hepatectomy, while, LLR has advantages of less trauma.

Objective To investigate the alterations of invariant nature killer T( iNKT) cells in peripheral blood samples from patients with rheumatoid arthritis ( RA) and to clarify the correlation between the percentage of iNKT cells and the ratio of IFN-γ/IL-4 in order to further understand the significance of iNKT cells in the development of RA.Methods Peripheral blood mononuclear cells ( PBMCs) were isola-ted from 70 patients with RA and 40 healthy subjects.Among them, thirty patients in the stage of inactive RA were involved in a follow-up study.Fluorescence activated cell sorting ( FACS) was used to detect the percentage of iNKT cells.PBMCs were cultured in vitro for analysis of cytokine production.The dynamic changes of iNKT cells in percentages were analyzed by FACS.MILLIPLEX MAP Human Cytokine/Chemo-kine kit was used to measure the secretion of IFN-γand IL-4 in serum samples and culture media of PBMCs. The expression of IFN-γand IL-4 in iNKT cells at mRNA level were analyzed by RT-PCR.Results Com-pared with the healthy subjects, the patients with active RA showed the delayed proliferation of iNKT cells and the decreased percentages and proliferation rates of iNKT cells (P<0.05).The percentages and prolif-eration rates of iNKT cells in patients with active RA were significantly lower than those in patients with inac-tive RA (P<0.05).No statistical significant differences with iNKT cells were found between healthy sub-jects and patients with inactive RA (P>0.05).The ratios of IFN-γ/IL-4 in serum samples and culture media of PBMCs were increased in patients with active RA as compared with those in patients with inactive RA and healthy subjects (P<0.05).No statistical significant differences with the ratios of IFN-γ/IL-4 were observed between healthy subjects and patients with inactive RA (P>0.05).Compared with healthy subjects and patients with inactive RA, patients with active RA showed increased transcriptional level of IFN-γand decreased transcriptional level of IL-4.No significant differences with the expression of IFN-γand IL-4 in iNKT cells at mRNA level were observed between healthy subjects and patients with inactive RA.The per-centage of iNKT cells was negatively related to the IFN-γ/IL-4 ratio in patients with RA (P<0.05).Con-clusion Decreased percentage and impaired function of iNKT cells were detected in patients with RA. iNKT cells were closely related to the development and disease activity of RA.

Objective:To establish experimental teaching model for slippery pulse by mini-pig.Methods:The model of slippery pulse was established by driping low molecule dextran in vein.The normal and slippery pulse were extracted from axil artery of mini-pig by two experienced traditional Chinese physician through double blind method.Meanwile,the correlative parameters of pulse graph of axil artery such as MSAB,MSBC,HFF,HE/HB and TW were extracted through optimizational extraction method by using NX-8 multifunctional sphygmograph.Results:The pulse rate of slippery pulse of mini-pig was slightly fast than that of normal pulse.The rhythm of slippery pulse was regularity,the nger sensation was powerful,and the pulse syate was smooth.Compared with the normal pluse,the pulse graph of slippery pluse displayed a steep ascend ramus,high and narrow B wave,tiny D wave,lower E valley and obvious F wave.HB,MSAB and MSBC increased(P

3-ketosteroid-9alpha-hydroxylase (KSH), a key enzyme in the microbial steroid degradation, is highly valuable for the production of some steroid drugs. Degenerate primers were designed by comparing the ksh from Rhodococcus erythropolis SQ1 and its homologous sequences in the reported genome of Mycobacteria. Subsequently, a gene fragment of KSH was cloned from Mycobacterium sp. NwIB-01, a sterol-transforming bacterium isolated from soil in our lab. According to the conservative sequence, the full-length 1188 bp gene encoding ksh (designated as M.S.-ksh) was obtained by chromosome walking, which showed 85% identity with the ksh of M. smegmatis mc(2)155. The heterologous expression of KSH was achieved in Escherichia coli BL21(DE3) using the pET-32a-c(+) vector system. The expressed KSH protein was mostly in soluble form after IPTG induction at 30 degreesC and accounted for more than 30% of total bacterial proteins according to SDS-PAGE electrophoresis. The molecular mass of KSH was about 45 kD, which was exactly the size predicted. After Ni2+ affinity chromatography, the purity of the target protein was more than 90%. Our work will definitely contribute to the industrial production of some steroid drugs by developing KSH genetically engineered bacteria.
Bacterial Proteins ; biosynthesis ; genetics ; isolation & purification ; Base Sequence ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Mixed Function Oxygenases ; biosynthesis ; genetics ; isolation & purification ; Molecular Sequence Data ; Mycobacterium ; enzymology ; Protein Engineering ; methods ; Soil Microbiology ; Steroids ; biosynthesis

Enzymes play such a pivotal role in cellular metabolism that enzyme assays are important for bio-engineering, disease diagnoses and drug discovery. Among the reported methods, fluoremetry has attracted more and more attention due to its high sensitivity and possibility of continuous dynamic monitoring. The recent progresses and applications in enzyme assays using fluorescent probes were reviewed. Different methods were classified into direct fluorescence detection and indirect fluorescence detection according to their labeled substrates and detection mechanisms. Our writing purpose is to provide the readers with a flavor of the kinds of tools and strategies available in enzyme assays with fluorescent probes. Also, the research situation and prospects were disucssed
Animals ; Enzyme Assays ; methods ; trends ; Fluorescent Dyes ; Fluorometry ; methods ; Humans ; Microscopy, Fluorescence