Objective To screen and identify the predominant T-and B-cell (T-B) combined antigenic epitopes in outer membrane protein GroEL of Helicobacter pylori (H.pylori).Methods The entire groEL gene of H.pylori strain NCTC11637 was amplified by PCR,and then a prokaryotic expression system for groEL gene was constructed.The expressed target recombinant protein rGroEL was analyzed by SDSPAGE and purified by Ni-NTA affinity chromatography.Laser confocal microscopy was applied to determine the distribution of GroEL in H.pyloristrains of NCTC11637 and SS1.The outer membrane protein (OMP) samples were extracted from the two strains using Triton X-114 method.GroEL in OMP samples was detected by Western blot assay.Special bioinformatic softwares were used to predict T-B combined antigenic epitopes on GroEL molecule,and phage display systems for every T-B combined antigenic epitope were established.Western blot assay and ELISA were used to detect the immunoreactivity of recombinant T-B combined antigenic epitope-containing phage PⅢ proteins and artificially synthesized T-B combined antigenic epitope peptides,respectively.Results The groEL genes from H.pylori strains of NCTC11637 and SS1 showed 97.68％ ～99.63％ identities in their nucleotide and amino acid sequences.The established prokaryotic expression system could efficiently express soluble rGroEL.GroEL located on the outer membrane of H.pylori strains of NCTC11637 and SS1.Among the six major predicted T-B combined epitopes (GroEL168,GroEL258,GroEL288,GroEL365,GroEL396 and GroEL438),GroEL168 showed the strongest positive immunoblotting signal.The results of ELISA showed that the immunoreactivity of GroEL168 was also the strongest (P＜0.01),followed by GroEL258 and GroEL288 (P＜0.05).Conclusion GroEL is an outer membrane protein of H.pylori.GroEL168 being the predominant T-B combined antigenic epitope of GroEL can be used to develop multiple antigenic peptide vaccines of H.pylori.

Executive impairment is one of the common sequelae of stroke, which seriously affects the quality of life of patients. Repeti-tive transcranial magnetic stimulation (rTMS), as a new type of electrophysiological technique, has been used in the clinical treatment of Post-Stroke Executive Impairment (PSEI). This paper summarized the survey of PSEI, the basic principle and mechanism of rTMS, clinical application of rTMS for PSEI and its safety. Clinical studies showed that high frequency stimulation, low frequency stimulation, and combi-nation with other therapeutic methods were effective in PSEI. However, there was no unified theory about the mechanism and the best treat-ment plan of rTMS for PSEI.

Objective To investigate clinical efficacy and safety of the memantine and donepezil in the treatment of moderate to severe Alzheimer's disease,in order to provide the basis for clinical treatment.Methods One hundred and twelve cases of moderate to severe Alzheimer's disease patients were given memantine plus donepezil(observation group) ,and single use of memantine treatment(control group), and the treatment for 24 weeks.Respectively before and after treatment, Mini Mental State Scale (MMSE), Alzheimer' s disease assessment scale (ADAS-cog), Alzheimer' s disease collaborative learning, daily life ability questionnaire (ADCS-ADL) ,and neuropsychological questionnaire (NPI) score, and adverse reactions were observed in the process of two kinds of therapy, the efficacy and safety of two kinds of treatment methods were evaluated.Results After treatment, the patients in observation group with MMSE, ADAS-cog, ADL and NPI evaluation results were (12.1 ± 2.1), (32.9 ± 8.3), (33.4 ± 5.0), (6.1 ± 3.1) scores, significantly improved compared with scoring (9.9±2.8), (46.2±7.6), (42.1±6.0), (10.5±2.9) scores before treatment,and the differences were statistically significant (t =2.138,-2.411,2.398, 2.107 respectively, P ＜ 0.05).The control group after treatment in patients with MMSE, ADAS-cog, ADL and NPI evaluation results were (12.3±2.6), (33.1 ±7.2), (35.1 ±6.6), (6.7 ± 2.9) scores, significantly improved compared with scoring 11.0 ± 2.5,44.9 ± 6.9,42.2 ±6.6,10.9 ± 3.5 before treatment, and the difference had statistical significanc (t =2.101,-2.033,2.105, 2.400 respectively, P＜0.05).After treatment, the difference of patients in the two groups of MMSE, NPI score had statistical significance(t =2.553,2.176, P＜0.05).The adverse reactions in two group respectively were 32.14％ (18/56) and 26.79％ (15/56), less difference was no statistical significance (P＞0.05).Vital signs checks,regular laboratory examination and ECG examination showed no obvious abnormalities.Conclusion Memantine combined with donepezil in the treatment of moderate and severe AD patients is superior to the singleeffect of memantine, and long-term use will not increase the risk of adverse reactions, which is safe and effective in the combined application.

BACKGROUND:Human umbilical cord mesenchymal stem cel s are considered as novel seed cel s in bone tissue engineering. Cryopreservation is an effective method for storing cel s for a long time.
OBJECTIVE:To explore whether umbilical cord mesenchymal stem cel s of cryopreservation could be induced to differentiated into osteoblasts.
METHODS:Mesenchymal stem cel s were isolated from the Wharton’s jel y of human umbilical cord tissue by the tissue explant adherent method. Morphology of primitive cel s was observed by inverted microscopy. Immunophenotypes and cel cycle of umbilical cord mesenchymal stem cel s were measured using flow cytometry. After frozen storage for 6 months, the second passage of umbilical cord mesenchymal stem cel s was thawed and subcultured to passage 12. Upon induction with osteogenic inductive medium, the osteogenic ability of passage 12 of umbilical cord mesenchymal stem cel was evaluated by alkaline phosphatase activity, the immunofluorescent analysis of osteocalcin and bone sialoprotein and the assay of alizarin red staining separately.
RESULTS AND CONCLUSION:Primary umbilical cord mesenchymal stem cel s displayed a typical fibroblast-like morphology. Flow cytometry showed that the cultured cel s expressed high levels of the mesenchymal stem cel s surface markers CD73, CD105 and CD90, but did not express hematopoietic cel s surface markers CD34 and CD45. The survival rate of umbilical cord mesenchymal stem cel s after resuscitation was 90%. The cel cycle analysis indicated that 75%of the cel s of passage 8 were in G 0/G 1 phase and 25%in S+G 2 M phase. Passage 12 cel s treated with osteogenic inductive medium displayed a higher alkaline phosphatase activity compared with control cel s (P<0.01). Moreover, the cel s, induced in osteogenic inductive medium, were positive for osteocalcin and bone sialoprotein staining and formed the mineralized nodules. Umbilical cord mesenchymal stem cel s stil maintain their biological characteristics after cryopreservation, and can be induced into osteoblasts with osteogenic inductive medium.

Objective To determine the effect of apoptosis in different host cells induced by L. in- terrogans and the associated intracellular signaling pathway. Methods L. interrogans serogroup Icterohaem- orrhagiae serovar icterohaemorrhagiae strain Lai infected cell models in mouse mono-macrophage like cell J774A. 1, human EVC304 cells and A549 cells were established, respectively. Flow cytometry with fluores- cein labeling of FITC-Annexin V/PI was performed to examine the apoptosis or necrosis of the infected cells. Fluorometry as well as Western blot assay was applied to measure the activity of caspase-3, -8, -9 and the expression levels of apoptotic associated protein FADD in the infected cells , respectively . Results 36.70%-63.70% of the L. interrogans strain Lai infected J774A. 1 cells displayed obvious early apoptosis during the infection for 1-6 h, and then altered to later apoptosis / necrosis (53.68%) as the major injury pattern when infected for 12 h. 78.52% of the L. interrogans strain Lai infected A549 cells only showed later apoptosis / necrosis. However, no obvious apoptosis and / or necrosis could be found in the L. interrogans strain Lai infected EVC304 cells. The maximal activities of caspase-3 and -8 in the infected J774A. 1 cells were (1453.41±36.07) and (1402.15± 59.09) FU, respectively, which were the 16.38-fold and 29.99- fold of those the non-infected cells. The caspase-9 activity of the infected J774A. 1 cells slightly increased [(89.42±5.08) FU ], which significantly lower than those of caspase-3 and -8 (P <0.001). The FADD expression level of the infected J774A. 1 cells was gradually increased in an infection time-dependent man- ner. Conclusion There is a distinct diversity of apoptosis in different cells induced by L. interrogans, and FADD→caspase-8→caspase-3 is the major signaling pathway to mediate L. interrogans infection associated cell apoptosis.

Objective To observe how ox-LDL impacts the mRNA expressions of MMP-9 and LOX-1 of human umbilical endothelial cells (HUEVC) and how LOX-1 acts as in inflamation course.Methods HUVEC were incubated in vitro.mRNA Expressions of MMP-9 and LOX-1 were determined by reverse transcription polymerase chain reaction (RT-PCR).Results Compared to the control group(0.252±0.032;0.279 ±0.041 ),ox-LDL significantly increased the mRNA expressions of MMP-9 and LOX-1 (25 ng/group 0.486 ± 0.012,0.586 ± 0.02;50 ng/L group 0.668 ± 0.011,0.739 ± 0.014; 100 ng/group 0.817 ±0.030,0.872 ±0.003,P ＜0.01 ).Those expressions were increased by ox-LDL( 1.020 ±0.039)in a concentration-and time-dependent manner.MMP-9 mRNA(0.872 ±0.046) was reduced when LOX-1 was inhibited by polyinosinic acid ( P ＜ 0.01 ).Conclusions The mRNA expressions of MMP-9 and LOX-1 were induced by ox-LDL in HUVEC.Inhibition of LOX-1 may decrease the expression of MMP-9.Those data demonstrate that LOX-1 is involved in the process of ox-LDL-induced MMP-9 expression.

ObjectiveTo establish the reference intervals for ALT,AST,GGT and LDH among the Han nationality in Beijing.MethodsThe document C28-P3 issued by CLSI was a guideline about how to define,establish,and verify reference intervals in the clinical laboratory.IFCC had established multicenter enzymes reference intervals based on the guideline.Exclusion criteria were designed for screening candidate reference individual according to the document C28-P3 and the multicenter study's experience.Blood specimens were collected from 315 healthy individuals aged 20 to 60 years old,including 132 males and 183 females.Reference materials were used to ensure the accuracy of the test results of the four liver enzymes.The methods which used to test the four liver enzymes could be traced to the IFCC enzymes reference measure procedure,the reagent of ALT and AST included pyridoxal phosphate.Results There was statistically difference between males and females of the referenceranges forALT, ASTand GGT.Therefore,gender-specific reference intervals were established as ALT:8.2 -50.8 U/L (F),12.7 -71.8 U/L (M) ; AST:15.0 -36.7 U/L ( F),16.6 -51.1 U/L (M) ;GGT:9.0 -37.3 U/L (F),12.0 -50.9 U/L (M).For LDH,the reference interval was 127 -224 U/L,as no significant gender difference was found.ConclusionsThe reference intervals for the four liver enzymes based on the population of the Han nationality in Beijing are established.The upper reference limit for ALT in Beijing Han population is higher than that from other similar studies.

Field avian infectious bronchitis virus (IBV) designated as JS/9 5/03, which was isolated from Jiangsu province of china, was cultivated in chicken emb ryo. It's single strain RNA was extracted from purified virus and worked as temp late of reverse transcription polymerase chain reaction (RT-PCR), a pair of pri mer designed according to megalign results of published IBV sequences in Genbank was used to amplify the neucleocapsid gene, the RT-PCR product was sequenced d irectly. Sequence analysis revealed that the sequence of JS/95/03 is most homolo gized with that of M41 strain.

Aim: Clone and characterize of the 5′- flanking region of the nitrate reductase (NR) gene derived from Dunaliella salina(D. salina). Methods : The genomic DNA from D. salina was respectively digested with BamHI, EcoRI, HindIII, Pst I, Sal I and Xba I. A genomic walking cassette was ligated to the ends of the digested DNA fragments, and then genomic walking libraries comprising BL, EL, HL, PL, SL and XL were constsucted. The 5′- flanking region of the NR gene from genomic walking libraries of D. salina was amplified by LA-PCR. The DNA sequences were analyzed with the software - Promoter Predictions. Isolated 5′-flanking regions fused to the GUS gene were tested for transient expression in the alga. Results: A single specific PCR product of about 1200bp in length from the HL library was generated. Also, several conserved motifs, such as CAAT-box, GAGA-box were found, which are related to regulation of transcription, and the putative binding sites of transcriptional factors such as EBP, EFII, NF-E1 and LV. BLAST showed that the DNA sequences shared high homology with 5′-upstream region of the NR gene from Dunaliella viridis. The isolated 5′-flanking regions were able to strongly drive GUS reporter gene expression, suggesting that it contains the promoter elements necessary for the transcription of the NR gene. The expression pattern of the GUS gene and the NR gene were similar, both ware induced by nitrate and repressed by ammonium. Conclusion: The cloned 5′- flanking sequences of NR gene derived from D. salina might be a specific promoter with the ability to“switch on or off” an expression of the heterologous gene in transgenic D. salina.

We determined molecular characteristics of Listeria monocytogenes foodborne isolates in Hangzhou and investigated the characterization of local strains.Multi-locus sequence typing(MLST) and pulsed-field gel electrophoresis(PFGE) were applied to identify molecular types of Listeria monocytogenes isolates.Results showed that a total of 133 strains of 6 serotypes were divided into 19 MLST types including a new type ST767.ST9 and ST121 were the major ST types.There were 33 and 45 PFGE patterns characterized by AscⅠ and ApaⅠ.The molecular types of Listeria monocytogenes strains were widely distributed in Hangzhou.It is indicated that the major clusters were Lineage Ⅰ and Lineage Ⅱ which will cause listeriosis.The contamination of Listeria monocytogenes in food is serious in Hangzhou and the surveillance and management should be strengthened to prevent the food borne diseases.