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191.
We constructed biotinylated fusion proteins that linked to three detection tags to GAD65 at the N-terminus, and expressed them in an E. coli expression system. The Biotin14-GAD65 protein exhibited the strongest binding to both the GAD65 antibody and the streptavidin among the three constructs. We describe the optimal conditions using a Biotin14-GAD65-based immunoassay for the detection of GAD65 antibody. 相似文献
192.
Felicori LF Souza CT Velarde DT Magalhaes A Almeida AP Figueiredo S Richardson M Diniz CR Sanchez EF 《Protein expression and purification》2003,30(1):32-42
A kallikrein-like proteinase of Lachesis muta muta (bushmaster) venom, designated LV-Ka, was purified by gel filtration and anion exchange chromatographies. Physicochemical studies indicated that the purified enzyme is a 33 kDa monomeric glycoprotein, the Mr of which fell to 28 kDa after deglycosylation with PNGase F. Approximately 77% of the protein sequence was determined by sequencing the various fragments derived from digestions with endoproteases. The partial sequence obtained suggests that LV-Ka is of a similar size to other serine proteinases (i.e., approximately 234 amino acid residues). Sequence studies on the NH2-terminal region of the protein indicate that LV-Ka shares a high degree of sequence homology with the kallikrein-like enzymes EI and EII from Crotalus atrox, with crotalase from Crotalus adamanteus and significant homology with other serine proteinases from snake venoms and vertebrate serum enzymes. LV-Ka showed kallikrein-like activity, releasing bradikinin from kininogen as evidenced by guinea pig bioassay. In addition, intravenous injection of the proteinase (0.8 microg/g) was shown to lower blood pressure in experimental rats. In vitro, the isolated proteinase was shown to have neither fibrin(ogeno)lytic activity nor coagulant effect. LV-Ka was active upon the kallikrein substrates S-2266 and S-2302 (specific activity=13.0 and 31.5 U/mg, respectively; crude venom=0.25 and 6.0 U/mg) but had no proteolytic effect on dimethylcasein and insulin B chain. Its enzymatic activity was inhibited by NPGB and PMSF, indicating that the enzyme is a serine proteinase. Interestingly, one of the other reactions catalyzed by plasma kallikrein, the activation of plasminogen was one of the activities exhibited by LV-Ka. 相似文献
193.
Plant genome archaeology: evidence for conserved ancestral chromosome segments in dicotyledonous plant species 总被引:3,自引:0,他引:3
Dominguez I Graziano E Gebhardt C Barakat A Berry S Arús P Delseny M Barnes S 《Plant biotechnology journal》2003,1(2):91-99
We have developed genetic maps, based on expressed sequence tags (ESTs) that are homologous to Arabidopsis genes, in four dicotyledonous crop plant species from different families. A comparison of these maps with the physical map of Arabidopsis reveals common genome segments that appear to have been conserved throughout the evolution of the dicots. In the four crop species analysed these segments comprise between 16 and 33% of the Arabidopsis genome. Our findings extend the synteny patterns previously observed only within plant families, and indicate that structural and functional information from the model species will be, at least in part, applicable in crop plants with large genomes. 相似文献
194.
Bonamy C Labarre J Cazaubon L Jacob C Le Bohec F Reyes O Leblon G 《Journal of biotechnology》2003,104(1-3):301-309
IS1207 is the insertion most frequently found among the spontaneous mutations that abolish the activity of an Escherichia coli phage lambda cI gene integrated in the Corynebacterium Brevibacterium lactofermentum ATCC21086 genome. We examined the transposition of transposon-like structures composed of a selective kanamycin resistance gene (aph3), and one or two IS1207 sequences. One of these, the Tn5531 transposon, transposed efficiently in Corynebacterium glutamicum. A replicative and a non-replicative Tn5531 delivery vector were used in Tn5531 mutagenesis. As IS1207, transposon Tn5531 shows a high frequency of transposition and mutagenesis, and a low target specificity. These features make of Tn5531 an adequate choice for gene identification and gene tagging experiments. 相似文献
195.
196.
Wuhrer M Grimm C Zahringer U Dennis RD Berkefeld CM Idris MA Geyer R 《Glycobiology》2003,13(2):129-137
The acidic (glyco)lipids of the parasitic liver fluke Fasciola hepatica exhibited two different phosphate-containing species, designated AL-I and AL-II, which were analyzed by MALDI-TOF MS, ESI MS, NMR, methylation analysis, and combined GC-MS in conjunction with HF treatment. AL-I was structurally determined as 1-O-hexadecyl-sn-glycerol-3-phosphoinositol, an ether bond variant of lysophosphatidylinositol. The structure of AL-II was shown to be GlcNAcalpha1-HPO3-6Gal(1-1)ceramide. Ceramide analysis revealed as major components 2-hydroxyoctadecanoic acid [18:0(2-OH)] together with C18- and C20-phytosphingosines. AL-II was apparently highly antigenic and strongly recognized by both animal- and human-F. hepatica infection sera. Furthermore, inhibition ELISAs revealed that the unusual antigenic determinant GlcNAcalpha1-HPO3- phosphate might have a potential in the serodiagnosis of F. hepatica infections. 相似文献
197.
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199.
Utility of Green Fluorescent Nucleic Acid Dyes and Aluminum Oxide Membrane Filters for Rapid Epifluorescence Enumeration of Soil and Sediment Bacteria 总被引:14,自引:7,他引:7 下载免费PDF全文
High background fluorescence and unspecific staining hampered the epifluorescence enumeration of bacteria in 45% of the tested soil and sediment samples with 4′,6-diamidino-2-phenylindole (DAPI) and polycarbonate membrane filters. These problems of the determination of total cell counts can be circumvented by using green fluorescent high-affinity nucleic acid dyes and aluminum oxide membrane filters. Due to the bright staining of cells, we recommend SYBR Green II as dye. 相似文献
200.
Delphine S. Dupuis Christiane Palmier Francis C. Colpaert Petrus J. Pauwels 《Journal of neurochemistry》1998,70(3):1258-1268
Abstract: G protein activation mediated by serotonin 5-HT1A and 5-HT1B/D receptors in guinea pig brain was investigated by using quantitative autoradiography of agonist-stimulated [35S]GTPγS binding to brain sections. [35S]GTPγS binding was stimulated by the mixed 5-HT1A/5-HT1B/D agonist L694247 in brain structures enriched in 5-HT1A binding sites, i.e., hippocampus (+140 ± 14%), dorsal raphe (+70 ± 8%), lateral septum (+52 ± 12%), cingulate (+36 ± 8%), and entorhinal cortex (+34 ± 5%). L694247 caused little or no stimulation of [35S]GTPγS binding in brain regions with high densities of 5-HT1B/D binding sites (e.g., substantia nigra, striatum, central gray, and dorsal subiculum). The [35S]GTPγS binding response was antagonized by WAY100635 (10 µM) and methiothepin (10 µM). In contrast, the 5-HT1B inverse agonist SB224289 (10 µM) did not affect the L694247-mediated [35S]GTPγS binding response, and the mixed 5-HT1B/D antagonist GR127935 (10 µM) yielded a partial blockade. The distribution pattern of the [35S]GTPγS binding response and the antagonist profile suggest the L694247-mediated response in guinea pig brain to be mediated by 5-HT1A receptors. In addition to L694247, 8-hydroxy-2-(di-n-propylamino)tetralin, and flesinoxan also stimulated [35S]GTPγS binding; their maximal responses varied between 46 and 52% compared with L694247, irrespective of the brain structure being considered. Sumatriptan, rizatriptan, and zolmitriptan (10 µM) stimulated [35S]GTPγS binding in the hippocampus by 20–50%. Naratriptan, CP122638, and dihydroergotamine stimulated [35S]GTPγS binding to a similar level as L694247 in hippocampus, lateral septum, and dorsal raphe. It appears that under the present experimental conditions, G protein activation through 5-HT1A but not 5-HT1B/D receptors can be measured in guinea pig brain sections. 相似文献