Objective To clone the gene encoding protein of EspA and Stx2B from EHEC OI57:H7 by DNA recombinant technology, construct prokaryotic expression vector pET-28a ( + )-espAstx2B, express fusion protein of EspA-Stx2B and to analyze the biological and immunological characteristics of the fusion protein. Methods the sequence encoding the protein of EspA and Stx2B was amplified by PCR from the enterohemorrhagic Escherichia coli strain. The amplified products were connected with linker by recombinant technology and cloned into pET-28a( + ) vector. The vector was then transferred to the host cells E. Coli BL21 strain (DE3). Following, the protein expression was induced by IPTG. The expression quantities and style of fusion protein was then determined by SDS-PAGE. Its immunoreactivity was analyzed by Western blot. Finally, BALB/c mice were injected with the preliminarily purified recombination protein EspA-Stx2B, then oral challenged these mice with EHEC O157-SMR2 and counteracted toxic substances with O157 ultrasonic supernatant. Results The determination of the sequence encoding of the espA-stx2B fusion gene has 100% of consistency with the sequence from GenBank Sakai strain and contrivable linker. This fusion protein EspA-Stx2B was expressed as inclusion body formation and the percentage is approximately 40%. Western blot suggested the fusion protein has excellent immunoreactivity. Titer of antiserum of the mice to EspA-Stx2B increased evidently. EspA-Stx2B could not decrease bacterial number attached to the intestinal tract of mice based on fecal shedding of Oi57 in mice. In the test of death of BAI,B/c causing by conteracting toxic substances with O157 ultrasonic supernatant, immunoprotection of EspA-Stx2B rate was 66.7%. Conclusion A recombinant plasmid that has high performance on expression of EspA-Stx2B prorein was successfully constructed in present study, and the fusion protein has excellent immunoreactivity and immunogenicity. EspA-Stx2B could not decrease bacteria] number attached to the intestinal tract of mice based on fecal shedding of O157 in mice, but evidently decrease the mortality rate of the mice. The antiEspA and anti-Stx2B had immunoprotection effect by different means. These results may provide the foundation for the further development on EHEC O157:H7 double subunit vaccine.

A in vitro platelet aggregation bioassay was developed for the quality control of XST capsules. The in vitro anti-platelet aggregation effect in rats was observed to detect the bioactivity of XST capsules. Panax notoginseng saponins and Xuesaitong lyophilizedpowder for injection were taken as standard control substances to determine the potency. According to the results, XST capsules showeda significant inhibitory effect on thrombin-induced platelet aggregation in a dose-dependent manner. The in vitro anti-platelet activity oflyophilized powder for injection was stabler than that of Panax notoginseng saponins, and so suitable to serve as a standard control substance. The biological potency of XST capsules compared with standard control substance was detected by using parallel line assay. According to the results, the established bioassay method had a good repeatability (RSD 2.92%). The sample test results could pass thereliability test(linear deviation P > 0.05, parallel deviation P > 0.05). This bioassay method could be used as one of the complementary quality control methods for XST capsules.
Animals ; Capsules ; pharmacology ; Drugs, Chinese Herbal ; pharmacology ; Male ; Panax notoginseng ; chemistry ; Platelet Aggregation ; drug effects ; Platelet Aggregation Inhibitors ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Saponins ; pharmacology

OBJECTIVETo study the effect of Feixianping (FXP) on collagen type I and II in rats with pulmonary fibrosis (PF).

METHODSSixty healthy male SD rats were randomly divided into 5 groups, the normal group (A), the model group (B), the positive control group (C) and the two FXP groups (D and E) treated respectively with high and low dose of FXP. Except those in Group A (they were not modeled and administered with normal saline), all rats were established into PF model by intra-tracheal instillation of bleomycin and administered with respective medicines starting from the 1st day after modeling. Rats were sacrificed in batches at 3 time points, the 7th, 14th, and 28th day for observing the pathological changes of lung under light microscope with HE staining and to identify collagen type I and III in lung tissue by immunohistochemical stain and image quantitative analysis.

RESULTSLight-dyeing proliferative collagen fiber was presented in the slightly thickened alveolar wall in lung of modeled rats from the 14th day on, and the pathological changes became more distinct on the 28th day. The highest amount of collagen appeared in the group B, correspondingly, that in all the other groups was much lower (P < 0.05). Reduction of collagen type I and III revealed in both FXP treated groups, but better effect was shown in the high dose FXP group. The effect of FXP was superior to that of positive control on the 14 th day (P <0.05).

CONCLUSIONFXP can effectively reduce the abnormal proliferation of collagen in experimental rats with PF.

Animals ; Bleomycin ; Collagen Type I ; analysis ; Collagen Type III ; analysis ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Immunohistochemistry ; Male ; Pulmonary Fibrosis ; chemically induced ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley

AIM: To establish an experimental model of high intraocular pressure in mice by laser photocoagulation and to prepare for future research.
METHODS: Experimental model of high intraocular pressure was induced unilaterally in 44 C57BL/6 mice. The fellow eye served as a control. TONO-PEN AVIA Tonomter was used to measure intraocular pressure (IOP) to guarantee IOP value at 1, 2, 4, 8wk. Slit-lamp biomicroscopy was performed throughout the period and the structural changes were assessed histologically. And then, their eyes were enucleated, postfixed, cryoprotected, and embedded in optimal cutting temperature medium. After hematoxylin and eosin stain ( HE stain ) , cryosections of the retina were observed under light microscope. TdT-mediated biotin-dUTP nick end labeling ( TUNEL ) was performed on the retinal sections to determine apoptosis rate.
RESULTS: IOP of laser-treated eyes was significantly higher than that of control eyes from 1-8wk (P<0. 05). The highest IOP was 31mmHg, but only one eye. The IOP was mainly around 20mmHg. In laser-treated eyes, the angle of anterior chamber were narrow. Number of cells in the inner nuclear layer and retial gangllion cell layer was slightly lower than that in control eyes at 2wk, but by 4 and 8wk the number of cells was significantly lower than that in the control contralateral eyes.
CONCLUSION: The laser photocoagulation of limbus causes chronic elevation of IOP and this method may be a promising experimental model for the investigation of biological mechanisms of glaucomatous retinal ganglion cell damage.

Objective To observe the combined poisonous effects of fluoride and aluminum on sex hormone of male rats.Methods Sixteen weaned SD healthy male rats aged two week were selected and divided into control group,aluminum group,fluoride group,fluorine-aluminum group,four rats in each group.All rats in the experimental groups were fed with corn collected from the prevailng areas containing different fluorine contents respectively for 90 days.Serum testosterone(T)and estradiol(E2)were detected.Results Compared separatelv with the control group[(3.317±0.635)μg/L],serum T level of fluorine-aluminum group[(15.994±6.558)μg/L]was higher(P＜0.05),but aluminum[(8.134±3.134)μg/L]and fluorine[(1.868±0.367)μg/L]groups had no significant differences(P＞0.05).Compared separately with the control group[(0.319±0.072)nmol/L],E2 level of the fluorine group[(0.172±0.030)nmol/L]being lower(P＜0.05),and it was not significant differences(P＞0.05)in the control group when compared with aluminum group[(0.282±0.012)nmol/L],and fluorine-aluminum group[(0.265±0.047)nmol/L].Fluorine and aluminum interacted with each other(F=9.82,P＜0.05).Conclusion The combined poisonous effects of fluorine and aluminum may influence sex hormone levels of male rats.

To explore the antagonistic effect of gingerols against the inflammation induced by lectin from Pinellia ternata. In this study, ELISA method was used to determine the effect of different extracts from gingerols on the release of inflammatory factor TNF-α from macrophages induced by lectin from P. ternata. The fluorescence probe was used to determine the effect of gingerols on the changes in ROS of macrophages induced by lectin from P. ternata. The western-blot method was applied to study the effect of gingerols on the increase in expression of cell receptor interacting protein RIP3 in macrophages induced by lectin from P. ternata. The scanning electron microscope (SEM) was used to study the effect of gingerols on morphological changes in macrophages induced by lectin from P. ternata. According to the results, gingerols can significantly inhibit the release of inflammatory factor from macrophages induced by lectin from P. ternata, ROS overproduction and increase in RIP3 expression. SEM results showed that gingerols can inhibit the cytomorphosis and necrocytosis induced by lectin from P. ternata. Fresh ginger's detoxication may be related to gingerols' effects in inhibiing release of inflammatory factor, ROS overproduction and increase in RIP3 expression caused by macrophages induced by lectin from P. ternata, which are mainly inflammatory development.
Animals ; Catechols ; pharmacology ; Cells, Cultured ; Drug Antagonism ; Fatty Alcohols ; pharmacology ; Ginger ; chemistry ; Lectins ; toxicity ; Macrophages ; drug effects ; metabolism ; Male ; Mice ; Mice, Inbred ICR ; Pinellia ; chemistry ; toxicity ; Reactive Oxygen Species ; metabolism ; Receptor-Interacting Protein Serine-Threonine Kinases ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

Adult ; Foot Diseases ; diagnostic imaging ; Humans ; Male ; Melorheostosis ; diagnostic imaging ; Radiography ; X-Rays

OBJECTIVETo provide some new evidences for the classification and identification of medicinal materials of Curcuma.

METHODThe optical microscope and electronic microsccopic scaning were used to characterize the leaves of Curcuma.

RESULT AND CONCLUSIONThere were no obvious histological and morphological differences among the leaves of Curcuma. But the differences in the hair distribution, stoma density and size , shape and size of epidermic cells could be considered to be the main features for the microscopic identification of leaves of Curcuma.

China ; Curcuma ; classification ; ultrastructure ; Microscopy, Electron, Scanning ; Pharmacognosy ; Plant Epidermis ; cytology ; ultrastructure ; Plant Leaves ; cytology ; ultrastructure ; Plants, Medicinal ; classification ; ultrastructure ; Species Specificity

Chemical modification was performed for improving the antioxidant activity of lead compound glycyrrhetinic acid (Ib). Two conjugated diene derivatives were prepared by reduction and dehydration reactions. Their in vitro antioxidant activities were studied using a cytochrome P450/NADPH reductase system from rat liver microsomes. The generation of microsomal free radicals was followed by oxidation of the DCFH-DA probe, while evaluating the capacity to inhibit reactive oxygen species (ROS) formation. The initial result showed that the two homo- and heterocyclic diene derivatives--18beta-olean-11,13(18)-diene-3beta, 30-diol (IV) and 18beta-olean-9 (11), 12-diene-3beta, 30-diol (V) exhibited strong antioxidant activities, at a concentration of 1.0 mg x mL(-1), they inhibited free radical (ROS) formation by 45% and 41%, respectively. In the same conditions, the lead compound (Ib) and the reference vitamin E inhibited ROS activity by 31% and 32%. Our results suggest that the elimination of the 11-keto group and the chemical reduction of 30-carboxylic group into hydroxyl function can increase the antioxidant activity of Ib significantly.
Animals ; Antioxidants ; chemical synthesis ; pharmacology ; Glycyrrhetinic Acid ; analogs & derivatives ; Male ; Microsomes, Liver ; metabolism ; Rats ; Reactive Oxygen Species ; metabolism ; Triterpenes ; chemical synthesis ; pharmacology

A simple and rapid method for preparation of plasmid DNA from overnight incubation was introduced. It does not require any additional reagents; the incubation mixture containing recombinant plasmid DNA was just mixed with H2O and phenol/chloroform/isoamyl alcohol in certain ratio. After vortexing and spinning of the mixture, the supernatant could be directly loaded onto agarose gel and analyzed using electrophoresis. The whole preparation requires only 3-5 minutes. So to quickly screen recombinant clones, this method is better compared with traditional methods.
Centrifugation, Density Gradient ; Chloroform ; chemistry ; Cloning, Molecular ; methods ; DNA, Bacterial ; genetics ; isolation & purification ; metabolism ; Deoxyribonuclease HindIII ; metabolism ; Electrophoresis ; Escherichia coli ; genetics ; Pentanols ; chemistry ; Phenol ; chemistry ; Plasmids ; chemistry ; genetics ; Reproducibility of Results ; Time Factors ; Water ; chemistry