To understand the structure of the soluble region of Enterovirus 68 3A protein, we construct a prokaryotic expression vector expressing the soluble region of EV-D68 3A protein, and identify the forms of expression product after purification. The EV-D68 3A(1-61) gene was amplified by PCR and then cloned into the expression vector pET-28a-His-SUMO. The recombinant plasmid was transformed into Escherichia coli BL21 induced by IPTG to express the fusion protein His-SUMO-3A(1-61). The recombinant protein was purified by Ni-NTA Agarose and cleaved by ULP Protease to remove His-SUMO tag. After that, the target protein 3A(1-61) was purified by a series of purification methods such as Ni-NTA, anion exchange chromatography and gel filtration chromato- graphy. Chemical cross-linking reaction assay was taken to determine the multiple polymerization state of the 3A soluble region. A prokaryotic expression vector pET28a-His-SUMO-3A(1-61) expressing the solution region of EV-D68 3A was successfully constructed and plenty of highly pure target proteins were obtained by multiple purification steps . The total protein amount was about 5 mg obtained from 1L Escherichia coli BL21 with purity > 95%. At the same time, those results determined the homomultimer form of soluble 3A construct. These data demonstrated that the expression and purification system of the soluble region of 3A were successfully set up and provide some basic konwledge for the research about 3A crystal structure and the development of antiviral drugs targeted at 3A to block viral replication.
Amino Acid Sequence ; Enterovirus D, Human ; chemistry ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Models, Molecular ; Molecular Sequence Data ; Protein Structure, Tertiary ; Sequence Alignment ; Viral Proteins ; chemistry ; genetics ; metabolism

Objective To explore the relationship between substance P(SP),somatostatin(SS) expression and change of morphology structure in jejunum of arsenism rats.Methods Acoording to sex and body mass,forty five clean grade SD rats were divided into control(0.0 mg.kg-1.d-1),low-dose arsenic(0.4 mg.kg-1.d-1) and high-dose arsenic(10.0 mg.kg-1.d-1) groups,n =15.The rats in low-and high-dose groups were treated with As2O3(2,50 mg/L) through drinking water for 4 months,respectively.Morphology changes of jejunum were observed by histological technique-HE staining and SABC immunohistochemistry.SP and SS positive cells in the jejunum were observed and counted,and its average gray value was analyzed with image analysis software (Biomias).Results Some jejunal villi were irregular in arsenism rats; with some brush border loss and irregular; goblet cells increased; infiltration of inflammatory cells in the lamina propria; and vacuoles in some intestinal gland cells.The differences of SP and SS positive cells between groups were statistically significant (F =608.54,227.59,all P ＜0.05).Compared with the control group (0.94 + 0.21,1.14 + 0.14),SP and SS positive cells in low-and highdose arsenic groups(1.85 + 0.25,1.83 + 0.24 and 4.24 + 0.33,3.31 ± 0.41) were significantly higher(all P ＜0.05),and high-dose arsenic group was significantly higher than the low-dose arsenic group(all P ＜ 0.05).The differences of average gray values of SP and SS positive cells between groups were statistically significant(F =68.43,26.57,all P ＜ 0.05).Compared with the control group(133.76 ± 3.61,137.57 ± 5.49),SP and SS positive cells in low-and high-dose arsenic groups(125.13 + 2.35,131.28 ± 5.66 and 118.30 ± 4.58,124.03 ± 3.94) were significantly lower(all P ＜ 0.05),and high-dose arsenic group was significantly lower than the low-dose arsenic group (all P ＜ 0.05).Conclusions Up-regulation of SP,SS may be related to jejunal mucosal injury and morphology structure in arsenic poisoning rats.

Objective To study the molecular mechanism of renal injury of chronic arsenic poisoning rats induced by the expression of cysteine caspase-8 and P53 in renal proximal tubular epithelial cells.Methods Sixty healthy SD rats were divided into three groups,high-,low-dose group,and control group,n =20 in each group.The rats in high and low dose groups were treated with As203 through drinking water,10.0 and 0.4 mg/kg,respectively.The control rats were given distilled water.Four months later,serum and urinary arsenic level was determined,and kidney specimens were taken.The expression of cysteine caspase-8 and P53 in renal proximal tubular epithelial cells was detected by histological technique-HE staining and SABC immunohistochemistry.In addition,cell number counting and image analyses were used in the study.Results The number of caspase-8 positive cells of renal proximal tubule in control group,low-and high-dose group was 3.33±1.32,31.14±8.02 and 46.50±7.20 cell number/visual fields,respectively,which was increased with dose increasing(all P ＜0.05);the average gray value was 151.34±6.40,133.58±4.63 and 128.34±16.28,respectively,decreased with dose increasing(all P ＜0.05).The number of P53 positive cells was 3.17±1.59,26.29±4.23 and 47.00±6.22 cell number/visual fields,respectively,increased with dose increasing (all P ＜ 0.05) ; the average gray value was 142.54±8.06,121.48±5.68 and 101.89±6.35,respectively,decreased with dose increasing (all P ＜ 0.05).Conclusion The increase of caspase-8 and P53 positive cells is one of the molecular mechanisms of renal injury induced by arsenic poisoning.

The aim of this study was to detect the localization and level of tyrosine phosphorylated proteins during in vitro capacitation of guinea pig sperm. Sperm from mature guinea pigs were incubated in modified TALP under 5% CO_2 in air at 37 ℃. The capacitation effect was assessed by chlortetracycline (CTC) staining. Western blotting and indirect immunofluorescence were used to analyze the level and localization of tyrosine phosphorylation. The results showed that guinea pig sperm underwent a time-dependent increase in protein tyrosine phosphorylation during the in vitro capacitation and the percentage of protein tyrosine phosphorylated sperm increased from 36% to 92% from the beginning of incubation to 7h incubation. Also, there was a shift in the site of phosphotyrosine-specific fluorescence from the head of sperm to both the head and the flagellum of sperm. Moreover, there were three proteins phosphorylated in this experiment. After 0 to 0.5h incubation, the protein of 40kDa was detected by anti-phosphotyrosine monoclonal antibody, and the intensity of this protein increased in the following incubation. Then, after 1h incubation, another protein of 80kDa was found and the level of this protein reached the highest point at 3h. Also, in 3h incubation, a protein of 45kDa was detected and the intensity of this protein increased in the following incubation.

The paper is to report the development of an optic-fiber sensing technology method to analyze metronidazole tablets rapidly. In this fiber-optic sensing system, the light from source delivering to probe can be dipped into simple-handling sample solution, absorbed by the solution and reflected to the fiber-optic and detected in the detection system at last. Then the drug content can be shown in the screen from the ultraviolet absorption spectra and the consistency between that obtained by this method and that in China Pharmacopoeia can be compared. With regard to data processing, a new method is explored to identify the authenticity of drugs using the similarity between the sample map and the standard pattern by full ultraviolet spectrum. The results indicate that ultraviolet spectra of tablets can be obtained from this technology and the determination results showed no significant difference as compared with the method in China Pharmacopoeia (P > 0.05), and the similarity can be a parameter to identify the authenticity of drugs.

Objective To describe the immunological characteristics of refractory primary biliary cirrhosis compared with the typical patients for more than 1 year's administration of UDCA.Methods Sixty patients treated with UDCA for more than 1 year in our clinic were enrolled into this study.According to the response to UDCA by Paris criteria,patients were divided into refractory group (23 patients) and typical groups (37 patients).The recent peripheral lymphocyte subsets and cytokines of the two groups were tested and analyzed.One-way ANOVA and t test were used for statistical analysis.Results ① One-year treatment after diagnosis,there were no differences between the two groups in the distribution of peripheral lymphocytic subsets,meanwhile,the two groups had higher percentage of B cells,CD4+T cells,CD4+CD28+T cells and CD8+ CD28-T cells than healthy controls respectively.② The serum levels of IL-6 [(0.8±0.9) pg/ml vs (0.3±0.4) pg/ml] and HGF were higher in the refractory group than other groups.Conclusion During the plateau phase,refractory PBC patients have higher serum levels of IL-6 and HGF,which probably suggest that the refractory PBC patients may have severe immunologic disturbance in vivo.

Objective To evaluate the feasibility of MRI-guided vacuum-assisted breast biopsy in domestic clinical prac tice.Methods A total of 45 patients with 45 suspicious breast lesions were examined with MRI-guided vacuum assisted bi opsy.The complications were observed.And the followed-up was performed.Results In 45 suspected breast lesions,43 lesions (43/45,95.56％) were successfully sampled under MRI-guided vacuum-assisted biopsy and the pathologic diagnosis was obtained.For another 2 lesions (2/45,4.44％),the pathology diagnosis were not obtained.The time to perform MRI-guided biopsy was (45.87±8.41)min for one lesion.The 5 malignant lesions (5/45,11.11％) confirmed by biopsy pathology were consistent with postoperative pathological results.For the 38 benign lesions (38/45,84.44％) confirmed by biopsy pathology,there was no malignant performance found in MRI follow-up for 6 months.Because of successful MR imaging-guided core biopsy findings,the clinic treatments were changed in 14 patients (14/45,31.11％).Five cases (5/45,11.11％) with malignant tumors were diagnosed early.The surgical biopsy was avoided in 9 benign cases (9/45,20.00 ％) who suspected as malignant.In all 45 patients,no complications occurred,such as hematoma and infection.Con clusion MRI-Guided vacuum-assisted breast biopsy is sufficient and safe for obtaining histologic proof of lesions,and plays an important role in guiding clinic treatment of some lesions visible only by MRI.

AIMTo study the bioactive components from Schisandra propinqua (Wall.) Baill var. intermidia A. C. Smith.

METHODSCompounds were separated with a combination of multichromatography. Their chemical structures were determined on the basis of spectral analysis and chemical evidence.

RESULTSSeven compounds were isolated from the leaves of Helicia nilagirica. The structures were elucidated as 6'-O-alpha-L-arabinofuranosylthalictoside (1), 6'-O-beta-D-apiofuranosylthalictoside (2), thalictoside (3), icariside D2 (4), prinsepiol (5), (+)-1-hydroxypinoresinol (6) and (+)-medioresinol (7).

CONCLUSIONTwo compounds are new nitro phenolic glycosides.

Disaccharides ; chemistry ; isolation & purification ; Molecular Conformation ; Molecular Structure ; Nitro Compounds ; chemistry ; isolation & purification ; Plant Stems ; chemistry ; Plants, Medicinal ; chemistry ; Schisandra ; chemistry

AIMTo study the bioactive components from Helicia nilagirica.

METHODSCompounds were separated with a combination of multi-chromatography. Their chemical structures were determined on the basis of spectral analysis and chemical evidence.

RESULTSTwo compounds were isolated from the leaves of Helicia nilagirica. Compound 1 was elucidated as 1-O-3-D-glucopyranosyl-(2S,3S,4R,8Z)-2-[(2'R)-2'-hyd roxylignocenoyl-amino]-8-octadecene-1, 3, 4-triol. Compound 2 was an analogue of 1.

CONCLUSIONThe two compounds are new cerebrosides.

Cerebrosides ; chemistry ; isolation & purification ; Glucosylceramides ; chemistry ; isolation & purification ; Molecular Conformation ; Molecular Structure ; Plant Leaves ; chemistry ; Plants, Medicinal ; chemistry ; Proteaceae ; chemistry

The seeds of Rabdosia rubescens were as the materials to research the impacts of different lead (Pb2+) concentrations(0, 135, 270, 540, 1 080 mg x L(-1)) on seed germination and seedling growth. The results show that: Low concentration of lead had no obvious effect on early germination of the seed, the germination vigor and germination speed were lightly higher but not significantly differed at the level of Pb concentration 135 mg x L(-1) with control group; Mid-high concentration of Pb solution (270-1 080 mg x L(-1)) significantly inhibited the seed germination and seedling growth, which reduced the seed germination rate, germination vigor, germination index, embryo root length and shoot length, growth index with increasing of Pb concentrations. There was a inhibitory effect on embryo shoot length and root length at mid-high lead concentrations stress, and stronger inhibitory effect on root , which was more sensitive than shoot to Pb stress(P < 0.05). Pb bioaccumulation coefficient (BC) was 0.76-2.59, increased with concentration of Pb; Pb enrichment in seedling mainly caused the growth inhibition. The fitting model predictive analyses show, the critical concentration of Pb, which causes the germination rate and biomass fresh weight reducing 10%, is 195.18, 101.65 mg x L(-1).
Germination ; drug effects ; Isodon ; drug effects ; growth & development ; Lead ; toxicity ; Seedlings ; growth & development ; Seeds ; growth & development ; Stress, Physiological