OBJECTIVETo express and purify the protein coded by the TRAF-type zinc finger domain of myasthenia gravis (MG)-related gene P9 (P9-ZFD) and to prepare P9-ZFD antiserum for detecting expression and subcellular distribution of P9-ZFD protein in the skeletal muscles of patient with MG.

METHODSThe cDNA encoding P9-ZFD was amplified by RT-PCR. The cloned P9-ZFD cDNA was ligated into pET24a, and the P9-ZFD recombinant protein was induced via E. coli. BL21 (DE3) and purified by histidine affinity chromatography. P9-ZFD antiserum was prepared and its titer and specificity were determined by ELISA and Western blot. Expression and subcellular distribution of P9-ZFD protein in the skeletal muscles of MG and control were studied.

RESULTSThe molecular weight of purified P9-ZFD protein was about 30 kD. Its purity was more than 95%. Antiserum specific for P9-ZFD was excellent. P9-ZFD protein is fully confined to the cytoplasm membrane of skeletal muscle cell of MG, obvious immunostaining was absent in the A, I, and Z bands of cytoplasm and no immunoreactivity was observed in the skeletal muscle cell of control.

CONCLUSIONP9-ZFD protein is expressed as a cytoplasm membrane-bound protein and has obvious distribution difference in the skeletal muscle cells of patient with MG and normal control.

Adult ; Cell Membrane ; metabolism ; Escherichia coli ; metabolism ; Female ; Humans ; Muscle Proteins ; biosynthesis ; genetics ; Muscle, Skeletal ; metabolism ; pathology ; Myasthenia Gravis ; metabolism ; Peptide Fragments ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection ; Zinc Fingers

In this study, ITS2 barcode was used to identify Bupleurum chinense and B. longiradiatum. The ITS2 regions of 48 samples were amplified and sequenced. The sequences obtained above were aligned and the K2P distances were calculated. We used three methods, BLAST1, nearest distance and phylogenetic tree (NJ-tree), to test the identification ability. The results showed that the maximum intraspecific genetic distance of B. chinense was 0.013, and the minimum interspecific genetic distance between B. chinense and B. longiradiatum was 0.049. The NJ-tree can easily identify B. chinense and B. longiradiatum. Therefore, the ITS2 barcode is suitable to identify B. chinense and B. longiradiatum.
Bupleurum ; classification ; genetics ; DNA Barcoding, Taxonomic ; methods ; DNA, Plant ; genetics ; DNA, Ribosomal Spacer ; genetics ; Drugs, Chinese Herbal ; chemistry ; classification ; Molecular Sequence Data ; Phylogeny ; Quality Control

Biometry ; Clinical Protocols ; Cryoultramicrotomy ; methods ; Diagnostic Techniques and Procedures ; Frozen Sections ; utilization ; Humans ; Orthopedics ; methods

Antigens, Surface ; blood ; Diagnosis, Differential ; Glutamate Carboxypeptidase II ; blood ; Humans ; Male ; Neoplasm Staging ; Prostate-Specific Antigen ; blood ; Prostatic Hyperplasia ; pathology ; Prostatic Neoplasms ; etiology ; metabolism ; pathology ; Racemases and Epimerases ; analysis

0.05).ConclusionThe neoadjuvant radiochemotherapy can improve the sphincter-saving rate,probably can improve the resection rate and reduce the recurrence rate for the middle-lower rectal carcinoma.

Objective To evaluate the possibility of methylation analysis of secreted frizzled-related protein gene 2 (SFRP2),hyperplastic polyposis protein gene (HPP1) and O~6-methylguanine-DNA methyhransferase gene (MGMT) in feces for screening colorectal cancer (CRC).Methods Total DNA was isolated from fecal samples of 52 patients with colorectal cancer and 35 patients with benign colorectal diseases and 24 normal volunteers,and the methylation status of SFRP2,HPP1 and MGMT was analyzed with methylation-specific polymerase chain reaction.Results SFRP2,HPP1 and MGMT were methylated in 94.2%,71.2%,48.1% of CRCs,respectively.At least one of the three genes was methylated in 96.2% of CRCs and 81.8% of precancerous lesions,respectively.In contrast,of the 24 normal controls,only one had methylated SFRP2.These results indicated 93.7% sensitivity,77.1% specificity,84.3% positive predicative value and 90.2% negative predictive value of the test for detecting CRC and precancerous lesions.Conclusions Methylation analysis of SFRP2,HPP1 and MGMT gene in stool is a high sensitive screening tool for CRC and precancerous lesions.Analysis of fecal DNA methylation is expected to serve as a new detection way for CRC or a new screening tool for individuals at risk of developing colorectal neoplasm.

AIM:ToinvestigatetheultrastructuralchangesofisletmicrovascularendothelialcellsinSTZ-in-duced type 1 diabetic mice .METHODS:BALB/c mice were randomly divided into diabetic group and control group .The expression of insulin and platelet-endothelial cell adhesion molecule-1 ( CD31) in islet microvessels was detected by immu-nohistochemical staining .The ultrastructural changes of islet βcells and islet microvessels were observed under transmis-sion electron microscope .RESULTS:Compared with control group , the number of islet βcells, ratio of βcells/αcells, average number of secretory granules in βcells and insulin expression area per islet in diabetic group were significantly de-creased (P<0.01).Besides, diabetic group had fewer microvessels with lower expression of CD 31 (P<0.01).Mito-chondria in islet microvascular endothelial cells and pericytes in diabetic group were swelling .The basement membrane of islet microvessels became thicker in diabetic group ( P<0.01 ) .CONCLUSION: Islet microvascular endothelial cells were impaired in type 1 diabetic mice .

Adult ; China ; epidemiology ; Cross-Sectional Studies ; Diabetes Mellitus ; diagnosis ; epidemiology ; Female ; Humans ; Logistic Models ; Male ; Middle Aged ; Multivariate Analysis ; Risk Factors ; Rural Population ; Sensitivity and Specificity

Objective To study the effect of carnosol on chemokines ex-pression in tumor necrosis factor -α( TNF-α)-induced HT-29 cells and its mechanism.Methods Cells were divided into control group ( DMEM medium ) , TNF -αgroup ( 10 ng? mL-1 TNF -α) , low, medium, high-dose carnosol groups (10 ng? mL-1 TNF -α+10, 20 , 40 μmol? L-1 carnosol ) and positive control group ( 10 ng? mL-1 TNF-α+40 μmol? L-1 ammonium pyrrolidinedithiocarbamate ).Cell counting kit-8 ( CCK-8 ) were used for analysis of HT -29 viability. The expression of interleukin -8 ( IL-8 ) and monocyte chemoattractant protein-1 ( MCP-1 ) were examined by enzyme linked immunosorbent assay.The expression of nuclear factor -κB ( NF -κB ) p65 were evaluated by Western -blot.Results The optical densities were (1.21 ±0.04 ), (1.17 ±0.06 ), (1.16 ±0.04 ), (1.15 ±0.05), (1.15 ±0.05),(1.18 ±0.05) in control, TNF-α, low-dose, medium-dose, high-dose and positive control group , also , there were no significant differences on cell viability in six groups ( P>0.05 ) .The ex-pressions of IL-8 and MCP-1 were (4942.00 ±220.30 ), (2299.00 ±273.38 )pg? mL-1 in TNF-αgroup, which were significantly higher than those in control group [ ( 841.67 ±27.15 ) , ( 140.67 ±23.54 ) pg? mL-1 , P<0.05 ] . Among the low -dose, medium -dose, high -dose and positive control group , the expressions of IL -8 were ( 4136.33 ±253.00 ) , ( 3156.00 ±241.89 ) , ( 2585.67 ±185.04 ) , ( 2652.67 ±219.55 ) pg? mL-1 and MCP -1 were (1980.67 ±167.02), (1259.33 ±256.66), (851.00 ±161.16), (1452.00 ±256.66)pg? mL-1, which were significantly lower than those in TNF -αgroup.The expression of NF-κB p65 in low, medium, high-dose carnosol groups were significantly lower than those of TNF -αgroup ( P <0.05 ) .Conclusion Carnosol can inhibit the expression of IL-8 and MCP-1 in TNF-α-induced HT-29 cells, which may associate with inhibition of TNF-α-induced NF-κB pathway activation.

OBJECTIVETo observe the effect of Wenyang Yiqi Huoxue Recipe (WYHR) on the apoptosis of photoreceptor cells in retinal degeneration slow (RDS) mice, and to investigate its molecular mechanisms.

METHODSRDS mice were randomly divided into the model group and the Chinese medicine group,and C57BL/6J mice were selected as the normal control group. Each group consisted of 4 female mice and 2 male mice. Mice in the Chinese medicine group were administered with WYHR (10 mg/g) by gastrogavage since mating. Baby mice drunk WYHR decoction instead of drinking water once they were born. The offspring were administrated with low dose WYHR decoction by gastrogavage from the 7th postnatal day, and the dose was increased to that for adult mice from the 21st postnatal day. Physiological saline was administrated to mice in the model group and the normal control group by gastrogavage. At 18, 28 and 48 postnatal days, electroretinogram (ERG) was used to evaluate the retina functional variation, and the apoptotic rate of photoreceptor cells was determined by TUNEL staining. HE staining was performed. The number of photoreceptor cells of the outer nuclear layer was calculated. Furthermore, effect of WYHR on Rhodopsin and basic fibroblast growth factor (bFGF) expression was examined using immunochemical assay.

RESULTSCompared with the model group, a- and b-wave latency and amplitude, as well as the bFGF expression sharply increased in the Max-ERG of the Chinese medicine group (P < 0.05, P < 0.01) at 18 postnatal days. At 28 and 48 postnatal days, a- and b-wave latency and amplitude sharply increased, photoreceptor cell layer numbers of the outer nuclear layer obviously increased, the apoptosis rate of retinal photoreceptor cells obviously decreased, expressions of Rhodopsin and bFGF in the Chinese medicine group significantly increased (P < 0.05, P < 0.01).

CONCLUSIONWYHR could effectively inhibit the apoptosis of photoreceptor cells in RDS mice, which might be attributed to up-regulating bFGF expression.

Animals ; Apoptosis ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Female ; Fibroblast Growth Factor 2 ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Photoreceptor Cells, Vertebrate ; cytology ; drug effects ; Retinal Degeneration ; metabolism ; pathology