To explore the new method of discriminating Cistanche deserticola, Cynomorium songaricum and Orobanche pycnostachya by using PCR amplification of specific alleles. 30 samples of the different C. deserticola, 21 samples of C. songaricum and O. pycnostachya were collected. The total DNA of the samples were extracted, the ITS sequences from C. deserticola, C. songaricum and O. pycnostachya were amplified by PCR and sequenced unidirectionally. These sequences were aligned by using ClustulW. Specific primer was designed according to the ITS sequences of specific alleles, and PCR reaction system was optimized. Additionally, compare with the identification of specific PCR method and DNA sequence analysis method. The result showed that the 331 bp identification band for C. deserticola and the adulterants not amplified bands by a single PCR reaction, which showed good identification ability to the three species. PCR amplification of specific alleles can be used to identify C. deserticola, C. songaricum and O. pycnostachya successfully.
Alleles ; Cistanche ; classification ; genetics ; DNA Primers ; genetics ; DNA, Intergenic ; genetics ; DNA, Plant ; genetics ; Drug Contamination ; prevention & control ; Phylogeny ; Polymerase Chain Reaction ; methods

To explore a new method for identification of Mongolian patent medicine (MPM) by PCR amplification of specific alleles. Eight kinds of MPM were used to study the identification of "Digeda" raw materials. The total DNA of Lomatogonium rotatum and Corydalis bungeana samples were extracted through modified CTAB method, psbA-trnH sequence was amplified by PCR and sequenced directionally. Specific primer was designed. The DNA of 8 kinds of MPM also was extracted and purified by the commercial DNA purification kits. The rbcL and two pair of specific primers sequences were amplified. The specific amplified products were sequenced in forward directions. All specific sequences were aligned and were analyzed. The results indicated that L rotatum can be identified by specific primers from Digeda-4 Tang, Digeda-8 San, Digeda-4 San, and C. bungeana medicinal materials can be identified by specific primers from Li Dan Ba Wei San, Yi He Ha Ri-12 and A Ga Ri-35. PCR amplification of specific alleles can stably and accurately distinguish raw medicinal materials in MPM.
Alleles ; DNA Primers ; genetics ; DNA, Plant ; genetics ; Medicine, Mongolian Traditional ; Molecular Sequence Data ; Plants, Medicinal ; classification ; genetics ; Polymerase Chain Reaction ; methods

To explore the new method of discriminating Astragali Radix and Hedysari Radix by using PCR amplification of specific alleles, 30 samples of the different Astragali Radix materials and 28 samples of Hedysari Radix were collected. The total DNA of all samples were extracted, trnL-trnF sequence from Astragali Radix and Hedysari Radix was amplified by PCR and sequenced unidirectionally. These sequences were aligned by using Clustul W. Primer was designed and the PCR reaction systems including annealing temperature, dNTP, etc were optimized. All samples were amplified by PCR with specific primer, DNA from Astragali Radix would be amplified 136 bp, whereas PCR products from all of Hedysari Radix were 323 bp. This method can detect 10% of intentional Hedysari Radix DNA into Astragali Radix. PCR amplification of alleles can be used to identify Astragali Radix and Hedysari Radix successfully and is an efficient molecular marker for authentication of Astragali Radix and Hedysari Radix.
Alleles ; Astragalus Plant ; classification ; genetics ; DNA Barcoding, Taxonomic ; DNA, Plant ; genetics ; Polymerase Chain Reaction

Objective：This study was designed to establish and optimize the research methods for proteome,and to analyze the proteome components of human lung squamous carcinoma cell line NCI H520. Methods： A series of methods, including immobilized pH gradient two dimensional polyacrylamide gel electrophoresis(2DE), silver staining, PDQuest 2DE analysis software, peptide mass fingerprint based on matrix assisted laser desorption/ionization time of flying mass spectrometry (MALDI TOF MS) and SWISS PROT database searching, were used to separate and indentify the proteome of human lung squarmous carcinoma cell line NCI H520. Results： The good 2DE pattern including resolution and reproducibility was obtained. After silver staining, the 2DE image analysis by PDQuest 2DE software had detected average of 1146± 116 spots, and 851± 95 spots were matched. The average matching rate was 73.3％ . There had a good reproducibility of spot position in 2DE map, with average deviation in IEF direction of 1.52± 0.22 mm， while in SDS PAGE direction it was 1.97± 0.13 mm. Sixty spots were incised from silver staining gel randomly and digested in gel by TPCKtrypsin. Fifty four peptide mass fingerprints (PMF) maps were obtained by MALDI TOF MS. The typic peptide masses were searched in the SWISS PROT database by PeptIdent software. Forty four proteins were preliminarily identified. Some of them were cell cycle related proteins such as Cyclin H, some were signal transduction related proteins such as mitogen activated protein kinase, protein kinase C and receptor protein tyrosine kinase ERBB 2, some were oncogene related proteins such as Ras related protein RAB 36, etc. Conclusions： The main proteome research system including IPG 2DE, image analysis, MALDI TOF MS derived PMFs and database searching has been established. The data of NCI H520 obtained by above methods will be useful for the establishment of human lung squamous cell proteome database.

Differential proteome profiles of human lung squamous carcinoma tissue compared to paired tumor-adjacent normal bronchial epithelial tissue were established and analyzed by means of immobilized pH gradient-based two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). The results showed that well-resolved, reproducible 2-DE patterns of human lung squamous carcinoma and adjacent normal bronchial epithelial tissues were obtained under the condition of 0.75-mg protein-load. The average deviation of spot position was 0.733+/-0.101 mm in IEF direction, and 0.925+/-0.207 mm in SDS-PAGE direction. For tumor tissue, a total of 1241+/-88 spots were detected, 987+/-65 spots were matched with an average matching rate of 79.5%. For control, a total of 1190+/-72 spots were detected, and 875+/-48 spots were matched with an average matching rate of 73.5%. A total of 864+/-34 spots were matched between tumors and controls. Forty-three differential proteins were characterized: some proteins were related to oncogenes, and others involved in the regulation of cell cycle and signal transduction. It is suggested that the differential proteomic approach is valuable for mass identification of differentially expressed proteins involved in lung carcinogenesis. These data will be used to establish human lung cancer proteome database to further study human lung squamous carcinoma.
Amino Acid Sequence ; Bronchi ; pathology ; Carcinoma, Squamous Cell ; genetics ; pathology ; Databases as Topic ; Electrophoresis, Gel, Two-Dimensional ; methods ; Electrophoresis, Polyacrylamide Gel ; Epithelial Cells ; pathology ; Gene Expression Regulation, Neoplastic ; Humans ; Image Processing, Computer-Assisted ; Isoelectric Focusing ; Lung Neoplasms ; genetics ; pathology ; Molecular Sequence Data ; Proteomics ; methods ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

OBJECTIVETo study the proto-oncogene RON mediated aggression of Raji cells and the inhibitory effects by monoclonal antibody Zt/f2 (2f2).

METHODSThe effects of RON ligand macrophage stimulating protein (MSP) (2.0 nmol/L) and inhibitory Zt/f2 (2F2) (2.0 nmol/L) antibody on proliferation of RON positive Raji cells after treatment for 24 and72 hours were detected by MTT method, colony formation units (CFU) of Raji cells by methylcellulose semi solid culture, Raji cells apoptosis and cell cycle analysis by AnnexinV/PI double staining, expression of RON, apoptosis-related proteins, and cyclins by Western blot.

RESULTS(1)Compared with the cell viability (1.0) and counts of CFU (103.6±7.0) in control group, Raji cells after MSP treatment had better viability (1.35±0.20) and CFU counts (133.7±10.4) (P<0.05), but worse viability (0.68±0.11) and CFU counts (66.3±6.1) after Zt/f2 (2F2) treatment (P<0.05). (2)Percentage of Raji cells apoptosis after Zt/f2 (2F2) antibody treatment (12.16±2.33)% was significantly increased than the control (2.89±1.03)% (P<0.05). The percentage of Raji cells arrested in G0/G1 phase was increased after Zt/f2 (2F2) antibody treatment as compared to the control [ (54.96 ±3.70)% vs (39.10±2.30)%, (P<0.05) ]. (3) High-level of RON phosphorylation and β-catenin expression activated by MSP could be inhibited significantly by Zt/f2 (2F2), which also up-regulated the expression of caspase-3, caspase-8, caspase-9 and PARP and down-regulated anti-apoptotic MCL-1 gene and inhibitor of apoptosis protein XIAP expression, accompanied with G1 phase protein changes accordingly.

CONCLUSIONMSP could aggravate Raji cells proliferation. Inversely, Zt/f2 (2F2) could inhibit proliferation and induce apoptosis by inhibition of RON phosphorylation and up-regulation of apoptosis related proteins.

Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Proto-Oncogenes ; Receptor Protein-Tyrosine Kinases ; metabolism

Objective To find a effective and generally applicable oral-nursing method to prevent microorganism infection for patient with oral endotracheal tube. Methods 60 patients with oral endotracheal tube were randomly divided into two groups . Intervention group (n=30) was performed with the modified oral care method :Oral-B toothbrush instead of cotton balls, mouth mirror instead of a tongue depressor , aspirator tube instead of suction tube and swab instead of washing method . Control group (n = 30) was performed with traditional oral care. The dental plaque clearance and the number of oral bacteria and bacteria in sputum were analyzed in the two groups. Results The number of dental plaque and the number of oral bacteria after oralnursing in intervention group were significantly lower than those in control group with traditional oral care (P＜0. 05, P ＜ 0. 05) . Conclusions Modified oral care method is effective in removing the dental plaque, reducing the number of bacteria and preventing infection.

OBJECTIVENeonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) which resulted from mutation in SLC25A13 gene can present transient intrahepatic cholestasis, low birth weight, growth retardation, hypoproteinemia and so on. This study aimed to identify the mutation type of NICCD patients by DNA sequencing.

METHODSTwenty children diagnosed as NICCD were consented to enroll in this study. PCR assays were performed to amplify the eighteen exons and its flanking sequences of SLC25A13 gene, which were defined as the upstream and downstream 50 bp from starting and ending site of the exons. Then the PCR products were purified and followed by automated DNA sequencing. The IVS16ins3kb mutation was detected by nested PCR and RT-PCR.

RESULTSSeven genetic variations of SLC25A13, termed as 851del4, 1638ins23, IVS16ins3kb, IVS6+5G>A, c.775C>T (p.Q259X), c.1505C>T (p.P502L) and c.1311C>T (p.C437C), were identified in the subjects, of which c.775C>T (p.Q259X), c.1505C>T (p.P502L) and c.1311C>T (p.C437C) were reported for the first time in NICCD patients. And a compound mutation of［1638ins23+IVS16ins3kb］was also identified. In 20 patients with NICCD, 6 patients were 851del4 homozygotes, 7 patients were compound heterozygotes, and 7 patients were heterozygotes of single mutation. 851del4 was the major mutation type (64%), followed by 1638ins23 (15%), IVS16ins3kb (12%) and IVS6+5G>A (6%).

CONCLUSIONS851del4 is the major mutation type in Chinese patients with NICCD.

Cholestasis, Intrahepatic ; genetics ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Mitochondrial Membrane Transport Proteins ; deficiency ; genetics ; Mutation ; Sequence Analysis, DNA

For constructing pcDNA-ORF5, pcDNA-ORF5-ORF6, pcDNA-ORF5/6, the ORF5 and ORF6 of porcine reproductive and respiratory syndrome virus (PRRSV) were amplified by RT-PCR, and transiently transfected into 293T cells by calciumphosphate co-precipitation. After 48 h, 293T cells were collected and surveyed by flow cytometry examination. The result indicated that the expression level of the E protein that mediated by the M protein was higher than that of the E protein expressed independently. Then pcDNA-ORF5, pcDNA-ORF5-ORF6, pcDNA-ORF5/6 were respectivly co-transfected into 293T cells with pHIT60 (include MuLV structural genes,namely gag and pol) and pHIT111 (retroviral genome, containing LacZ as a reporter). The supernatants were harvested 48 h post-transfection,and the analysis of the characteristic of the pseudotyping virions was performed by Western blot and infection test. The result indicated that the E proteins were expressed on the virions, and incorporated into the retroviral virions. Infection test were performed on Marc-145 and PAM, all the cells infected were Lac Z positive. These results indicated the pseudotype virions of MuLV-E and MuLV-E/M were infectious, and higher infectivity was achieved by MuLV-E/M.
Flow Cytometry ; Leukemia Virus, Murine ; genetics ; Plasmids ; Porcine respiratory and reproductive syndrome virus ; genetics ; Viral Envelope Proteins ; genetics ; physiology ; Viral Matrix Proteins ; genetics ; physiology ; Virion ; genetics

BACKGROUNDRheumatoid arthritis (RA) is characterized by inflammation of the synovial membrane, leading to invasion of synovial tissue into the adjacent cartilage matrix with degradation of articular cartilage and bone as a consequence. Dickkopf-1 (DKK-1) and osteoprotegerin (OPG) have been demonstrated to be key molecules involved in bone erosion and bone remodeling. The aim of this study was to explore the potential role of DKK-1 and OPG in different stage of RA.

METHODSThe protein levels of DKK-1 and OPG were detected by ELISA. The serum samples were collected from 300 patients with RA and 60 healthy controls. Of which, 150 RA patients were defined as early RA (disease duration < or = 1 year), and other 150 RA patients were defined as longlasting RA (disease duration > or = 5 years). At the time of serum sampling, various clinical and laboratory parameters were assessed. The correlations of DKK-1 or OPG and clinical/laboratory parameters were analyzed.

RESULTSThe serum level of DKK-1 was elevated in patients with longstanding RA compared with healthy controls, while no significant difference was observed between the two groups in the level of OPG. In contrast, in early RA patients, the circulating OPG was elevated, while there was no significant difference between the two groups in expression of DKK-1. The serum DKK-1 was correlated with Sharp score and DAS28 in longstanding RA patients. In early RA, age was the only parameter that was significantly related to serum OPG.

CONCLUSIONSThere was a cross-talk between DKK-1 and OPG, which involved in bone destruction in RA. In different stage of RA, DKK-1 and OPG may play different roles in the pathogenesis of RA.

Adult ; Arthritis, Rheumatoid ; blood ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Intercellular Signaling Peptides and Proteins ; blood ; Male ; Middle Aged ; Osteoprotegerin ; blood ; Time Factors