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Western blotting of autophagic markers LC3Ⅱ and p62 are widely used for estimating autophagic activity. To compare the regulation of various autophagy modulators on LC3Ⅱ and p62,HEK293 cells were treated separately with mTOR-dependent autophagy activator rapamycin or -independent autophagy activators trehalose, and autophagy inhibitors including 3-methyladenine (3-MA),bafilomycin A1 or E64d and pepstatin A that inhi-bited the initiation of autophagy,the fusion of autophagosome and lysosome,and the activities of lysosomal enzymes accordingly,and then LC3Ⅱ and p62 levels were assessed. Western blot results demonstrated that rapam-ycin enhanced the conversion of LC3I to LC3Ⅱ,promoted the degradation of p62 simultaneously,while trehalose merely increased the expression of LC3Ⅱ with no influence on the p62 level. Moreover,inhibition of autophagy commonly led to accumulation of LC3Ⅱ as well as blockage of p62 degradation in a concentration- and time-dependent manner. These results indicate that obvious differences exist in the regulation of LC3Ⅱ and p62 by various modulators although both are autophagic markers.
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Objective To study the effects of psychological stress on breast development and abnormal levels of related hormones or their receptors in pregnant rats .Methods Pregnant Wistar rats were randomly divided into control group and ex-perimental group.The experimental group rats were given unpredictable stress stimuli for 15 days, including noise, restraint, re-versal of day and night , swimming in cold water and tail pinch-induced pain.Gross changes of the breast were observed , the di-ameter and height of breast nipple was measured, and breast coefficient was calculated.DNA and RNA levels in the breast tis-sues were determined by optical density measurement.The estrogen (E2), progesterone (P), growth hormone (GH) and prolac-tin (PRL) levels in plasma and breast tissues were determined by radioimmunoassay.Bmax and Kd of estradiol and progesterone receptors were measured by radolig and bindng assay .Morphological changes of the breast tissues were examined by light micros-copy using HE staining.Results Compared with the control group, the nipple diameter and height, breast weight and breast co-efficient were significantly decreased in the experimental group (P<0.05, P<0.01), the levels of DNA, RNA and RNA/DNA ratio in the breast tissue were significantly decreased (P<0.05, P<0.01), the levels of plasma E2, P and GH were significant-ly decreased (P<0.05, P<0.01), the levels of E2 and GH in the breast tissue homogenate were decreased also (P<0.05, P<0.01), the Bmaxs of E2 receptor and P receptor were decreased, but their Kds increased significantly (P<0.05, P<0.01), and the number of lobules and diameter of acini were significantly decreased (P<0.05).Conclusions Psychological stress can lead to breast dysplasia and abnormal levels of estrogen and progesterone and their receptors in pregnant rats .
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The ompA gene of Chlamyia psittaci in cows was amplified by PCR with primers designed based on those reported in GenBank.The amplified ompA gene was inserted into the bacterial plasmid vector pGEX-4T-1 and then transformed into E.coli BL21(DE3) with IPTG induction. The gene was derived from plasmid pMD18-T vector and then sequenced.It was demonstrated that this recombinant fusion protein of approximately 68kD in molecular mass was highly expressed in inclusion body and more pure proteins would be produced after purification.The fusion protein specifically reacted with positive sera of bovine Chlamydia as demonstrated by Western blotting. These results indicate that this recombinant fusion protein shows good reactivity and could be used to develop the diagnostic kit for bovine Chlamydia and genetic engineering vaccine.
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Objective To establish a rat model of ulcerative colitis by immunological sensitization combined with local acetic acid irritation.Methods Thirty healthy 2-3-month-old Wistar rats were randomly divided into 3 groups:normal control group,model group and SASP-treated model group(n=10 rats per group).The ulcerative colitis in the model group and positive group was developed by immunological sensitization with rabbit colon mucosal proteins and local irritation with 5%acetic acid.The rats of the model and positive groups were fed with sulfasalazine,and rats of the control group fed with normal saline.The rats of positive control group were treated with sulfasalazine.Two weeks later,all rats were killed and colon mucosal and blood samples were collected.The concentrations of NO,NOS,MDA,SOD and GSH-Px in the colonic mucosal homogenate were measured by biochemistry,the contents of IL-4 and TNF-α were measured by RIA,and the serum concentration of IFN-γ was measured by ELISA.Results Compared with the normal control group,the concentration of MDA,NO,NOS in the colonic mucosa was increased,and SOD and GSH-Px levels significantly decreased in the model group.Compared with the model group,the concentrations of MDA,NO,and NOS in the colonic mucosal tissue were decreased,and the levels of SOD and GSH-Px decreased in the SASP-treated model group.The contents of IL-4 in the serum and colonic mucosal homogenate of model group were significantly lower, and the contents of TNF-α in the serum and colonic mucosal homogenate were significantly increased than those of the normal control group. The IL-4 levels in the serum and colonic mucosal homogenate was increased, and that of TNF and IFN-γ significantly decreased in the SASP-treated model group, compared with those of the normal control group.Conclusion The rat model of ulcerative colitis established by immunological sensitization combined with local acetic acid irritation, as described in the present study, can better simulate the characteristic chronic alterations in human ulcerative colitis,and may better serve studies and evaluation of treatment of this disease.
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<p><b>OBJECTIVE</b>To establish a chemical fingerprint method for reorganizing and validating angelica different processed products.</p><p><b>METHOD</b>A high-performance liquid chromatographic method was developed to establish the fingerprint. Principal component analysis, hierarchical cluster analysis and discriminate analysis were applied to study HPLC finger printing and chemical pattern reorganization.</p><p><b>RESULT</b>There were difference of characteristic peaks and its relative peak area of HPLC fingerprints between different processed products. Fish's discriminate functions were generated by using six selected predictor variables, the tested samples of different processed products were classified with 100% accuracy, and discriminate analysis plots for the five groups were well-resolved.</p><p><b>CONCLUSION</b>The developed HPLC finger print, combined with chemometrics, can accurately identify and validate angelica different processed products, the research provide theoretical basis for the processing mechanism and quality assess of angelica different processed products.</p>
Subject(s)
Angelica , Chemistry , Classification , China , Chromatography, High Pressure Liquid , Methods , Drugs, Chinese Herbal , Reference Standards , Food Handling , Plant Roots , Chemistry , Quality ControlABSTRACT
The paper reports the development of a quality evaluation method for Angelica different processed products. The data of high-performance liquid chromatography, water, total ash and extract were analyzed with SPSS Clementine 11.0 software. Discriminant analysis (DA) established the classification model and parameter for Angelica different processed products. Fish's discriminant functions of Angelica different processed products were generated using 8 predictor variables selected from 59 indexes. The correct rate of discriminating back substitution is 96.7%. Angelica different processed products can be accurately and reliably recognized and validated with DA of SPSS Clementine 11.0 software.