To improve the growth enhancement activity of Vitreoscilla hemoglobin(VHb), Vitreoscilla hemoglobin gene(vgb) was mutated by error-prone PCR and then reconstituted by DNA shuffling. The shuffling library was constructed by inserting the shuffled genes into the downstream of vgb natural promoter and transforming them into E.coli DH5?. Mutated active VHb proteins were first screened in test tubes according to host cell pellets color and then in shake flasks according to host pellets wet weight .One active mutant protein, VHb′042506, was obtained after second screening. It could increased the host wet weight by 31.25% and 58.75% than that of the control which bearing natural VHb under microaerobic and extremely microaerobic conditions, respectively. Sequencing and alignment results showed that 11 nucleotides were mutated, thus resulted in 4 amino acids changes occurred in this mutant protein. CO difference spectrum test also indicated that it had higher specific absorption.

Objective To study the mutability of ultra sodium pyrosulfite intake on ultrastructure changes and spermatogonium mice testis.Methods Forty male Kunming mice were used.Experimental group had been exposed to ultra sodium pyrosulfite by fed for 10 days,and sodium pyrosulfite′s contaminated dose were 1% and 1‰.Mice were killed at 11~(th) day,and ultrastructure changes were observed under transmission electron microscopy(TEM) and the tests of sister chromosome exchanges(SCE) were made.Mmutation of ultra sodium pyrosulfite on spermatogonium of mice testis was judged.Results Compared with control group,there was a significant increase of SCE ratio in spermatogonium of testis in experimental groups(P

Objective To study the effect of hydrogen peroxide on microstructure of mice kidney and discuss the toxic effect on mice kidney.Methods Thirty healthy male mice of Kunming Genus were divided into 3 groups at random:control group and two experimental groups. Running water was fed to control group for 10 days while 0.3,3 g/L hydrogen peroxide running water readily prepared was fed to the experimental groups for 10 days. On the 10th day,the kidneys were taken out,and fixed in the fixation solutions,conventionally produced and stained.Finally,they were studied under the optical microscope.Results Experimental groups:in the kidney tissue cytoplasm of proximal convoluted tubule showed hydropic degeneration and vacuolation which depend on dose of hydrogen peroxide.Conclusion Toxic effect on mice kidney can be caused by hydrogen peroxide.

ObjectiveTo study the methods of how to protect facial nerve function following complete resection of acoustic neurinomas and the value of the techniques of F wave assisted electrophysiological monitoring intraoperatively.Methods Retrospectivelysummarizing theresultsof combining three electrophysiological monitoring techniques such as nasal muscle F wave recording,online EMG and triggered EMG to monitor 46 cases of microoperations for acoustic neurinomas intraoperatively during the period of Feb.2004 to Dec. 2008. Correlating every intraoperative monitoring index with their follow-up results of facial nerve function 1 day and 6 months after their operations.The tendency of the two continuous monitoring techniques between nasal F wave recording and online EMG of facial muscles has also been studied in this paper. Results Among 46 cases of acoustic neurinomas, 45(97.83 %) tumors have been totally resected, and 1 (2.17 %) tumor subtotally resected,lcase (2.17 %)died after operation,and 2ases occurred the leakage of cerebrospinal fluid(CSF) which have been cured through conservative treatment. The whole anatomic protection rate of facial nerve is 97.83 %,and their functional protection rates 6 months after operation are:HB Ⅰ - Ⅱ,75.56 %;Ⅲ-Ⅳ,22.22 % and Ⅴ-Ⅵ,2.22 %.The completely accordant rate between the intraoperative findings of nasal F wave recording and online EMG is 52.17 %, partially accordant rate is 45.65 %, and totally opposite rate is 2.17 % (x2 趋势= 6.113, P ＜0.05). The intraoperative monitoring indexes in nasal muscle F wave recording are correlated well with the facial nerve function in the 6th month' s follow-up (κ=0.429, P ＜0.001).In triggered EMG monitoring after tumors being resected,the stimulus threshold ratio and maximum amplitude ratio of facial nerve between leaving brain stem part and inner acoustic porus part are also correlated well with the facial nerve function 6 months after operation(κ=0.576, P ＜0.001; κ=0.595, P ＜0.001). ConclusionNasal muscle F wa recording cooperated well with online EMG and triggered EMG intraoperatively and correlates well with the postoperative facial nerve function, so they should be routinely applied together intraoperatively.

Objective To explore the value of F wave recording in evaluating facial nerve function and its pathological changes in the pressure-induced rat models of acoustic neurinoma. Methods 58 rats in different groups were conducted F wave recording and biotinylated dextran amine(BDA) retrograde tracing for their right facial nerve one week after establishing models. Their latencies, amplitudes and F/M rates were analyzed first. 72 hours after BDA was injected into right whisker muscle, the rats were infused with 4% polyoxymethylene, then pontines and facial nerves in the CPA cistern were obtained. Pontiues were cut into frozen sections for histochemical staining with avidin-horseradish peroxidase (HRP)-DAB and Nissl 's counterstaining, calculating the positive BDA neurons ratio(BDA+-N%)in facial nuclear. Facial nerves were cut and stained with toluidine blue for light-micrescope inspection, and/or stained for transmission electron microscope observation. Correlating F/M with BDA+-N% and the facial nerve pathological findings. Results F/M are 97.66 % and 97.48 % in normal and pseudo-operation group, respectively, when stimulus are 1.4 mA; while 77.13 %, 48.91% and 11.54 % in from small to large tumor model groups because F waves were delayed in latencies or increasinglylost (P <0.001). Similarly, BDA+-N% are 98.37 % and 97.96 % in the above two control groups, while 77.28 %, 48.28 % and 11.55 % in from small to large tumor model groups (P < 0.001). Thus F/M are positively correlated with their BDA+-N% in all groups (r =0.996,P <0.001). Facial nerve examinations under light and electron microscope show increasing pathological changes along with increasing "tumor" size. Conclusion The findings of F wave recording in facial nerve may reflect its functional status and pathological changes. Therefore, F wave detection may help electrophysiological monitoring during acoustic neurinoma resection and facial nerve function evaluation after surgery.

Objective To elucidate the prevalence and clinical characteristics of human metapneumovirus(hMPV)in hospital- ized children with respiratory infection.Methods A total of 452 hospitalized children with lower respiratory tract infection were observed from Aug 2004 to Jan 2005.Respiratory tract aspirates were collected from all patients within 48 hours after admis sion.The specimens were routinely tested for respiratory syncytial virus,influenza virus A and B,parainfluenza virus 1 to 3 and adenovirus by direct fluorescent assay(DFA).The 245 specimens negative by DFA were tested for hMPV by RT-PCR. PCR products of hMPV M gene from some patients were randomly selected for sequencing analysis.Results hMPV was identi- fied in 59(24.1%)of the 245 specimens tested,hMPV infection alone accounted for 13.1% of the infections in the 452 chil- dren under study,The prevalence of hMPV was higher than other respiratory viruses in winter.The mean age of hMPV-infec- ted children(n=59)was 27.7 months.There was no significant difference between age groups in terms of the prevalence of hMPV(P＞0.05).There were no statistically significant difference in demographics and clinical symptoms between hMPV in- fection and other common respiratory virus infection.Genotyping for the hMPV M gene from 23 Shanghai patients showed two distinct hMPV genotypes.Sequence analysis of these hMPV M genes showed 82.8%-100% homology to the registered se- quence in GenBank.There was no significant difference in clinical characteristics between the 2 genotypes.Conclusions hMPV plays an important pathogenic role in lower respiratory tract infection of children,hMPV prevailed in the winter of 2004.Clini- cally,hMPV infection can not be discriminated from the infection of other respiratory viruses.Clinical manifestation is similar between the two hMPV genotypes.

OBJECTIVETo investigate the underlying tumor susceptibility mechanisms and reasons for the high risk of cancer in Fanconi anemia (FA).

METHODSGene Set Enrichment Analysis (GSEA) was performed to compare gene expression profiles between 21 FA patients' bone marrow (BM) mononuclear cell (BMNC) and 11 normal controls in cancer related gene sets from NCBI GEO database, then core enriched genes were identified by further investigation. Through enrichment analyzing biological processes of gene ontology sets and structural genomic gene sets between FA expression profiles and control, more details related with its tumor susceptibility had been revealed.

RESULTSCompared with normal control, gene expression in FA group had significant been enriched in resistance to Bcl-2 inhibitor gene set, fibroblast growth factors signalling pathways, insulin and insulin-like growth factors (IGF) signalling pathways induced cancer genesis gene sets. The high level of D4S234E, SST, FGFs, IGFs, FGFRs and IGFBP expression provided an initiate environment for tumorgenesis and drug resistance. There were significant differences in biogenesis extracellular molecules and cytomembrane structure organizations between FA and control. Genes with promoter regions around transcription start sites containing either motif RRCAGGTGNCV or CCTNTMAGA were enriched and those former genes match annotation for tumorgenic transcription factor 3 (TCF3).

CONCLUSIONSThe high tumor susceptibility of FA patients may be closely related with the dramatic changes in cancer related growth factors and hormones environment. This study provides new insights into tumor susceptibility mechanism in FA patients.

Basic Helix-Loop-Helix Transcription Factors ; genetics ; Case-Control Studies ; Fanconi Anemia ; genetics ; Female ; Gene Expression ; Genes, Neoplasm ; Genetic Predisposition to Disease ; Humans ; Insulin-Like Growth Factor Binding Proteins ; genetics ; Male ; Neoplasms ; genetics ; Promoter Regions, Genetic ; Signal Transduction ; genetics ; Somatomedins ; genetics ; Transcription Initiation Site ; Transcriptome

Following Escherichia coli lysis with alkali, cetyltrimethylammonium bromide (CTAB) was directly titrated into the supernatant. An easy and feasible technology for plasmid purification was established with the optimized proportion between the quantity of CTAB and plasmid, combined with the specific solution for DNA release and TritonX-114 for endotoxin removal. Quality detection showed that the purified plasmid was free of contamination of host RNA. The host bacterial genomic DNA, endotoxin and bacterial protein were less than 10 microg, 50 EU and 10 microg per mg plasmids, respectively. The ratio of OD260/OD280 was between 1.75-1.85. Eighty percent of the prepared plasmids were presented in the supercoiled form. The plasmid purified with this technology can satisfy all criteria stipulated by FDA. The main advantages of the technology include the avoidance of animal-derived enzymes such as ribonucleases A, Proteinase K and toxic reagents like chloroform and phenol. In addition, the technology has low cost and no pollution.
Cetrimonium Compounds ; chemistry ; Chemical Precipitation ; DNA ; genetics ; isolation & purification ; DNA, Bacterial ; genetics ; isolation & purification ; Escherichia coli ; chemistry ; genetics ; Plasmids ; genetics ; isolation & purification

Objective To explore the effect of action verb processing on the excitability of the motor cortex and any effect on the upper limb motor function of stroke survivors.Methods Experiment 1:The motor evoked potential by transcranial magnetic stimulation (TMS-MEP) was measured while 18 healthy subjects were processing concrete action verbs or abstract verbs.Experiment 2:Eight hemiplegic stroke survivors were asked to read silently text describing concrete hand actions,and then repeat and explain the meaning of the action verbs used in the text.This was repeated for 30 minutes per day,5 days per week for 3 weeks.In the 2nd week the same training was conducted except that the text was about abstract verbs.The modified Ashworth scale (MAS),Fugl-Meyer assessment (FMA),the Hong Kong version of the functional test for a hemiplegic upper extremity (FTHUE-HK) and a simple test for evaluating hand function (STEF) were used to assess upper limb motor function before and after the training each week.Results Experiment 1:Processing the concrete action verbs induced significantly greater MEP than processing the abstract verbs.Experiment 2:The average FMA and STEF ratings improved significantly after the training each week.Training with the concrete action verbs resulted in significantly better FMA and STEF scores than with the abstract verbs.The average MAS score increased significantly week by week.There was significantly more improvement in the average FTHUE-HK rating after the 1st and 3rd week of training than after the 2nd week.Conclusion Action verb processing induces greater motor cortex excitation than abstract verb processing among healthy subjects and better improves the upper limb motor function of stroke survivors.

OBJECTIVEThis paper aims to investigate the anti-tumor mechanism of inactivated Sendai virus (Hemagglutinating virus of Japan envelope, HVJ-E) for murine melanoma (B16F10).

METHODSThe murine dendritic cells (DCs) were treated with HVJ-E, and then the cytokines secreted from DCs and costimulation-related molecules on DCs were measured. Meanwhile, the expression of β-catenin in HVJ-E treated murine melanoma cells was detected. In addition, HVJ-E was intratumorally injected into the melanoma on C57BL/6 mice, and the immune cells, CTL response and tumor volume were analyzed.

RESULTSHVJ-E injected into B16F10 melanoma obviously inhibited the growth of the tumor and prolonged the survival time of the tumor-bearing mice. Profiles of cytokines secreted by dendritic cells (DCs) after HVJ-E stimulation showed that the number of cytokines released was significantly higher than that elicited by PBS (1P<0.05). The co-stimulation-related molecules on DCs were comparable to those stimulated by LPS. Immunohistochemical examinations demonstrated the repression of β-catenin in B16F10 melanoma cells after HVJ-E treatment. Meanwhile, real-time reverse transcription PCR revealed that HVJ-E induced a remarkable infiltration of CD11c positive cells, chemokine ligand 10 (CXCL10) molecules, interleukin-2 (IL-2) molecule, CD4(+) and CD8(+) T cells into HVJ-E injected tumors. Furthermore, the mRNA expression level of β-catenin in the HVJ-E injected tumors was also down-regulated. In addition, B16F10-specific CTLs were induced significantly after HVJ-E was injected into the tumor-bearing mice.

CONCLUSIONThis is the first report to show the effective inhibition of melanoma tumors by HVJ-E alone and the mechanism through which it induces antitumor immune responses and regulates important signal pathways for melanoma invasion. Therefore, HVJ-E shows its prospect as a novel therapeutic for melanoma therapy.

Animals ; Cell Line, Tumor ; Cytokines ; genetics ; metabolism ; Dendritic Cells ; immunology ; physiology ; virology ; Down-Regulation ; Gene Expression Regulation, Neoplastic ; Melanoma ; immunology ; pathology ; virology ; Mice ; Mice, Inbred C57BL ; Neoplasms, Experimental ; Real-Time Polymerase Chain Reaction ; Reverse Transcriptase Polymerase Chain Reaction ; Sendai virus ; physiology ; Virus Inactivation ; Virus Replication ; beta Catenin ; genetics ; metabolism