In this study, nattokinase gene was amplified by PCR using bacillus subtilis chromosomal DNA as template and cloned into expressed vector pBV220. After transforming recombinant plasmid into E.coli HB101, the recombinant strain was yielded. It was proved that expression products was secretive and expression protein was 12% of total cell protein by SDS-PAGE. Optimum culture time and inducing time was determined as 6h and 5h respectively. The plasmid stability studies showed that recombinant plasmid has excellent segregational stability but the structural stability was not good in the host cell.

Objective To identify the differential serum proteins in patients with hepatic injury resulting from coal-burning type of arsenism. Methods Six serum samples were collected from patients with liver injury resulting from coal-burning type of arsenism and healthy subjects(control gruop) in endemic arsenism area. Twodimensional gel electrophoresis(2-DE) was performed to separate serum proteins, after silver staining, the differential expression of proteins were analyzed and then identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS). Results The 2-DE map of serum protein patterns of patients and normal control were established successfully. The results showed that there were an average of (824 ± 31 ) spots and (782 ± 42) spots on 2-DE matching of the patients and control groups and the matching rate was 94.9%(782/824). From these two groups 49 differential protein spots were identified, of which over 3 times the difference in the expression of 30 protein spots were singled out and MALDI-TOF-MS analysis was carried out. Ten proteins were identified. Upregulated expression was observed in alpha-2-macroglobulin, B-cell receptor-associated protein, keratin 1,apolipoprotein A-I, and down-regulated expression was observed in haptoglobin, α2-heremans-schimid-glycoprotein,mitogen-activated protein kinase 4, zinc finger protein 323, ZAP-70 and SP40 in the patient group. Conclusions The well-resolved and reproducible 2-DE serum patterns of patients are established and some differentially expressed proteins are characterized. Whether these proteins of differential expression are serum markers for liver injury resulting from coal-burning type of arsenism need to be further verified.

To explore the action target of praziquantel (PZQ) and its underlying mechanism of action, adult male worms of Schistosoma japonicum were collected from the hepatic vein of Kunming mice infected at least 6 or more weeks previously with single-sex cercariae of S.japonicum by perfusion method. These worms were subjected to the action of calcium channel blockers (CCBs) or actin depolymerizing/stabilizing agents interfering with function of the calcium channel. The adult male worms in DMEM culture medium were co-cultivated with near-lethal dose of PZQ(14 μmol/L) overnight(16 hours).Then, the parasites were washed 3 times with sterile physiologic saline next morning after cultivation, re-suspended in new and drug-free medium and then observed under stereo-microscopy during the following 5 days. The experimental results showed that majority of adult male worms of S.japonicum were killed by the action of PZQ in a dose of 14 μmol/L in vitro under normal condition; while the worms pre-incubated with the actin depolymerizing agent cyto chalasin D (CyD) were able to survive in the condition containing 14 μmol/L of PZQ with a survival rate of 100%, and the worms pre-incubted with CCBs, such as nitrendipine and nifedipineu showed a survival rate of about 50% under the same condition. The results of this study suggest that the calcium channel of Schistosomes may be involved in the action target of PZQ and its underlying mechanism.

Objective To investigate the iodine nutritional status of targeted population in the high-risk areas of iodine deficiency disorders in Chongqing, so as to provide scientific evidence for establishing prevention and remedial measures. Methods Six towns were selected in Chengkou and Wuxi Counties to found suspected dementia patients born after first Jan, 1997. Two hundred children aged 8-10 years were investigated in every town. The thyroid volume, intelligence quotient(IQ) and urinary iodine of the children were examined. Forty women (pregnant and nursing women) were investigated in every town. The iodine content of salt from their home was measured. The thyroid volume was examined by palpation and B-uhrasound. IQ was measured by combined Raven Test in China(CRT-RC2). Urinary iodine was determined using the acid digest arsenic-cerium contacting method, and iodined salt was detected using direct titration method. Results Six suspected dementia patients were found in the local town hospital. Five eases were excluded. There was 1 case born in other place. The rates of goiter by palpation and B-ultrasound were 9.58%(92/960) and 8.89%(65/731), respectively. The median of urinary iodine of children and women was 319.15 μg/L and 248.42 μg/L, respectively. The mean of IQ of the children was 103.32. The coverage rate of iodine salt from residents was 98.82%(336/340). Conclusions The iodine nutrition of children was good and there is no newly occurred cretinism in Chengkou and Wuxi Counties. Goiter rate and median of urinary iodine aged 8-10 years and of women, coverage rate of iodine salt from resident has meet the standard set for basical elimination iodine deficiency disorders.

OBJECTIVE: To establish the electricity plate digestion and inductively coupled plasma-mass spectrometry (ICP-MS) method for determination of 33 inorganic elements in human hair. METHODS: Lithium (6Li), Germanium (72Ge), Yttrium (89Y), Indium (115In), and Terbium (159Tb) were used as internal standards. The electric heating board digestion in a mixture of nitric acid and hydrogen peroxide was used as the pre-treatment of the hair. Thirty-three inorganic elements in human hair were analyzed by ICP-MS method. RESULTS: The detection limit of ICP-MS was 0.0001 microg/g(Th)-10.9 microg/g (Ca) and the limit of quantitation was 0.0005 microg/g (Th)-25 microg/g (Ca). The recovery rate of this method was 86%-113%. The RSD for the intra-day and inter-day were less than 9.2%. The method was not statistically different from microwave digestion method. CONCLUSION This method is highly efficient and accurate. It can be used for analysis of 33 inorganic elements in human hair.
Electricity ; Hair/chemistry* ; Humans ; Limit of Detection ; Mass Spectrometry/methods* ; Microwaves ; Reference Standards ; Sensitivity and Specificity ; Trace Elements/analysis*

OBJECTIVE: To determine the normal reference values of 33 elements, Ag, Al, As, Au, B, Ba, Be, Ca, Cd, Co, Cr, Cs, Cu, Fe, Ga, Hg, Li, Mg, Mn, Mo, Ni, Pb, Rb, Sb, Se, Sr, Th, Ti, Tl, U, V, Zn and Zr, in the blood and urine samples from the general population in Sanmen County of Zhejiang province, a typical coastal area of eastern China. METHODS: The 33 elements in 272 blood and 300 urine samples were determined by inductively coupled plasma-mass spectrometry (ICP-MS). The normality test of data was conducted using SPSS 17.0 Statistics. The data was compared with other reports. RESULTS: The normal reference values of the 33 elements in the blood and urine samples from the general population in Sanmen County were obtained, which of some elements were found to be similar with other reports, such as Co, Cu, Mn and Sr, while As, Cd, Hg and Pb were generally found to be higher than those previously reported. There was a wide variation between the reports from different countries in blood Ba. CONCLUSION The normal reference values of the 33 elements in the blood and urine samples from the general population in Sanmen County are established, and successfully applied to two poisoning cases.
Blood Chemical Analysis ; China ; Elements ; Environmental Monitoring ; Humans ; Urinalysis

OBJECTIVE: To establish the method for measurement of acetonitrile in blood and urine by head-space gas chromatography. METHODS: DB-ALC1 (30 m x 320 microm x 1.8 microm) and DB-ALC2 (30 m x 320 microm x 1.2 microm) capillary column were used to measure the acetonitrile in blood and urine with the isopropanol as internal standard reference. RESULTS: The limits of detection of acetonitrile in both blood and urine were 0.5 microg/mL, with a linear range of 5-1000 microg/mL (r = 0.999).The accuracy of this method was 93.2%-98.0%. The RSD for the intra-day and inter-day were less than 3.7%. CONCLUSION The method is capable for measurement analysis of acetonitrile in blood and urine.
Acetonitriles/urine* ; Chromatography, Gas/methods* ; Cyanides/urine* ; Forensic Toxicology/methods* ; Humans ; Reproducibility of Results ; Suicide, Attempted

Animals ; Capsules ; Drugs, Chinese Herbal ; therapeutic use ; Liver Cirrhosis ; drug therapy ; enzymology ; Male ; Phytotherapy ; Protein Kinase C ; biosynthesis ; genetics ; Rats ; Rats, Wistar

OBJECTIVEFluctuations in arterial oxygen are associated with development of severe retinopathy of prematurity (ROP) in humans. However, the causal relationship between oxygen variability and ROP remains unknown. The authors developed a rat model of retinal neovascularization by repeated fluctuations of inhaled oxygen between hypoxia and hyperoxia to investigate the mechanism of the development of retinal neovascularization, the regulation of vascular endothelial growth factor (VEGF) and KDR/Flk-1 (VEGFR-2) expression.

METHODSTwo hundred and eight newborn Sprague Dawley rats were randomly divided into oxygen and air groups. The oxygen concentration in the oxygen group was alternated between 50% and 10% every 24 hours for 14 days. The control group were kept in room air environment. VEGF and Flk-1 expression was observed at 14, 18 and 25 days after birth in both exposed group and control group by immunohistochemical staining and semiquantitative RT-PCR. The status of retinal vasculature on day 4 after oxygen exposure was also observed. The retinas were dissected and stained by using a histochemical method for detecting adenosine diphosphatase (ADPase) activity, digital images of the retinas were captured and the peripheral avascular retina were measured. HE staining on methacrylate sections of eyes was used for counting the number of nuclei extending from retinal area into vitreous to identify extraretinal neovascularization. Numeric data were expressed as the mean +/- standard deviation (SD). Statistical calculations were performed using the SAS 8.1 statistical package. Differences in measured variables between experimental and control groups were determined using comparison of the means using two-way analysis of variance (ANOVA) statistical calculations and T-test. AP value less than 0.05 was regarded as significant.

RESULTS(1) The animal model was successfully established: the avascular areas of retina of 18-day-old rats were larger than those of the control group and the numbers of nuclei extending from retinal area into vitreous in exposed group were significantly higher compared to the control (P < 0.05). (2) The expression of VEGF and Flk-1 on the 14(th) day in the oxygen group was significantly stronger than that of the control group (P < 0.05). In the oxygen group, VEGF and Flk-1 expression was the strongest in the retina on the 18(th) day, the result had significant difference as compared with the 14(th) and 25(th) day (P < 0.05), and they were also stronger than that of the control group (P < 0.05). The expression of VEGF and Flk-1 decreased on the 25(th) day and had no significant difference as compared with the control group (P > 0.05). (3) Both VEGF-mRNA and Flk-1-mRNA significantly increased on the 14(th) day and the 18th day (P < 0.05). On the 25(th) day, the amounts of VEGF-mRNA and Flk-1-mRNA were similar between the control and oxygen group (P > 0.05).

CONCLUSIONFluctuation in oxygen is associated with the development of retinal neovascularization in the retinopathy. Increased expressions of VEGF and Flk-1 in the oxgen fluctuations-induced neovascularized retina suggested that VEGF and Flk-1 might play a critical role in the pathogenesis of ROP. The results also indicated the positive feedback in the pathogenesis of ROP that the synergistic interaction of VEGF and Flk-1 in the retinal vascular proliferation. These findings provide insight into the effect of repeated oxygen fluctuation on the development of severe ROP in preterm infants.

Animals ; Disease Models, Animal ; Humans ; Hyperoxia ; pathology ; Hypoxia ; pathology ; Infant, Newborn ; Oxygen ; adverse effects ; Rats ; Rats, Sprague-Dawley ; Retinal Neovascularization ; pathology ; Retinopathy of Prematurity ; pathology ; Vascular Endothelial Growth Factor A ; metabolism ; Vascular Endothelial Growth Factor Receptor-2 ; metabolism