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1.
Characterization of recombinant human lymphotoxin (tumor necrosis factor-beta) produced by a mammalian cell line 总被引:1,自引:0,他引:1
Lymphotoxin (LT) was purified from serum-free conditioned media of a recombinant mammalian cell line transfected with human lymphotoxin cDNA. The purification scheme consisted of controlled pore glass chromatography, Q-Sepharose ion-exchange chromatography, and concanavalin A-Sepharose chromatography. The purified protein was found to be homogeneous by reverse-phase high-performance liquid chromatography and had an approximate specific activity of 130 X 10(6) units per milligram protein as determined by the L-929 cytotoxicity assay. Purified LT had an isoelectric point of approximately 6.85 and an apparent molecular weight of 50,000 by gel permeation high-pressure liquid chromatography. However, when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two distinct bands at approximate molecular sizes of 25 and 20 kDa were observed. Both the bands were immunoreactive by Western blot analysis and found to be associated with biological activity. The two forms of lymphotoxin differed from each other with respect to protein structure. Amino-terminal amino acid sequence analysis revealed that the 25-kDa LT sequence starts with Leu-Pro-Gly-residues whereas that of the 20-kDa LT begins with His-Leu-Ala; thus the latter form is truncated by 20 amino acid residues from the amino terminal. Two species of LT also differed from each other with respect to carbohydrate structure. Enzymatic removal of sialic acid reduced the molecular weight of 25 kDa by approximately 5 kDa whereas that of the 20-kDa LT was unchanged. A reduction in an apparent molecular size by approximately 4 kDa of both species of LT was observed on removal of N-linked oligosaccharides. Treatment with O-Glycanase had minimal effect on either form of LT. The recombinant lymphotoxin described here was found superior in its solubility behavior as compared to bacterial cell derived LT. Overall, mammalian cell line derived recombinant LT appears closer in its properties to natural LT than does bacterial cell derived recombinant LT. 相似文献
2.
Modifications of anionic-lipid domains preceding membrane fusion in guinea pig sperm 总被引:19,自引:15,他引:4 下载免费PDF全文
The relationship between anionic-lipid concentration and the functional properties of plasma-membrane domains was explored using the guinea-pig sperm membrane as a model, with polymyxin B (PXB) as a probe. Areas of plasmalemma specialized for fusion during the acrosome reaction had a higher affinity for the probe than adjacent nonfusigenic regions. In addition, capacitation--a process preceding acrosome:plasma-membrane fusion--markedly enlarged the area susceptible to PXB binding over the acrosomal cap. Protease treatment mimicked capacitation by increasing the acrosome-reaction incidence as well as PXB binding, at enzyme concentrations not affecting the surface coat nor altering filipin/sterol localization. Both proteolytic digestion and capacitation failed to augment PXB- or filipin-affinity in nonfusigenic zones, such as the post-acrosomal segment, including its particle-free maculae. Incubation of sperm in capacitating medium supplemented with 32P-labeled phosphate, followed by lipid extraction, thin-layer chromatography, and autoradiography, revealed a radioactive band comigrating with cardiolipin and phosphatidic acid. Vermiform protrusions elicited by PXB in the outer lamellae of cardiolipin- phosphatidylcholine liposomes resembled those seen in fusional regions of sperm membrane. We conclude that (a) differing concentrations of anionic lipids are found in adjacent domains of the sperm plasma membrane; (b) these domains mirror the functional regions of the membrane, with higher anionic-lipid concentrations localized over fusional zones; (c) the surface coat does not participate in the maintenance of such domains; (d) anionic-lipid synthesis may contribute to their formation; and (e) anionic-lipid concentrations increase as the membrane becomes fusionally competent, indicating that cellular modulation of lipid domains accompanies regulation of membrane function. 相似文献
3.
4.
Nine white-rot fungal strains were screened for biodecolourization of brilliant green, cresol red, crystal violet, congo red
and orange II. Dichomitus squalens, Phlebia fascicularia and P. floridensis decolourized all of the dyes on solid agar medium and possessed better decolourization ability than Phanerochaete chrysosporium when tested in nitrogen-limited broth medium. Journal of Industrial Microbiology & Biotechnology (2002) 28, 201–203 DOI: 10.1038/sj/jim/7000222
Received 12 July 2001/ Accepted in revised form 22 October 2001 相似文献
5.
Brian Becknell John David Spencer Ashley R. Carpenter Xi Chen Aspinder Singh Suzanne Ploeger Jennifer Kline Patrick Ellsworth Birong Li Ehrhardt Proksch Andrew L. Schwaderer David S. Hains Sheryl S. Justice Kirk M. McHugh 《PloS one》2013,8(10)
Beta defensins (BDs) are cationic peptides with antimicrobial activity that defend epithelial surfaces including the skin, gastrointestinal, and respiratory tracts. However, BD expression and function in the urinary tract are incompletely characterized. The purpose of this study was to describe Beta Defensin-1 (BD-1) expression in the lower urinary tract, regulation by cystitis, and antimicrobial activity toward uropathogenic Escherichia coli (UPEC) in vivo. Human DEFB1 and orthologous mouse Defb1 mRNA are detectable in bladder and ureter homogenates, and human BD-1 protein localizes to the urothelium. To determine the relevance of BD-1 to lower urinary tract defense in vivo, we evaluated clearance of UPEC by Defb1 knockout (Defb1
-/-) mice. At 6, 18, and 48 hours following transurethral UPEC inoculation, no significant differences were observed in bacterial burden in bladders or kidneys of Defb1
-/- and wild type C57BL/6 mice. In wild type mice, bladder Defb1 mRNA levels decreased as early as two hours post-infection and reached a nadir by six hours. RT-PCR profiling of BDs identified expression of Defb3 and Defb14 mRNA in murine bladder and ureter, which encode for mBD-3 and mBD-14 protein, respectively. MBD-14 protein expression was observed in bladder urothelium following UPEC infection, and both mBD-3 and mBD-14 displayed dose-dependent bactericidal activity toward UPEC in vitro. Thus, whereas mBD-1 deficiency does not alter bladder UPEC burden in vivo, we have identified mBD-3 and mBD-14 as potential mediators of mucosal immunity in the lower urinary tract. 相似文献
6.
A total of 220 cell envelope-associated proteins were successfully extracted and separated from Trichoderma reesei mycelia actively synthesizing and secreting proteins and from mycelia in which the secretion of proteins are low. Altogether 56 spots were examined by nanoelectrospray tandem mass spectrometry and amino acid sequence was obtained for 32 spots. From these, 20 spots were identified by Advanced BLAST searches against all databases available to BLAST. The most abundant protein in both types of mycelia was HEX1, the major protein in Woronin body, a structure unique to filamentous fungi. Other proteins identified were vacuolar protease A, enolase, glyceraldehyde-3-phosphate dehydrogenase, transaldolase, protein disulfide isomerase, mitochondrial outer membrane porin, diphosphate kinase and translation elongation factor beta. Partial short amino acid sequence obtained from some proteins did not allow them to be assigned to a specific protein in the database by BLAST search. In some cases, the tandem mass spectrometry spectra were too complicated to be able to assign an amino acid sequence with certainty. The number of spots (12) giving a clear signal but finding no match in the databases suggests that a majority of proteins associated with a filamentous fungal cell wall, are novel. Some technical problems related to protein isolation are also discussed. 相似文献
7.
S Patyar R Joshi DS Prasad Byrav A Prakash B Medhi BK Das 《Journal of biomedical science》2010,17(1):21
Resistance to conventional anticancer therapies in patients with advanced solid tumors has prompted the need of alternative
cancer therapies. Moreover, the success of novel cancer therapies depends on their selectivity for cancer cells with limited
toxicity to normal tissues. Several decades after Coley's work a variety of natural and genetically modified non-pathogenic
bacterial species are being explored as potential antitumor agents, either to provide direct tumoricidal effects or to deliver
tumoricidal molecules. Live, attenuated or genetically modified non-pathogenic bacterial species are capable of multiplying
selectively in tumors and inhibiting their growth. Due to their selectivity for tumor tissues, these bacteria and their spores
also serve as ideal vectors for delivering therapeutic proteins to tumors. Bacterial toxins too have emerged as promising
cancer treatment strategy. The most potential and promising strategy is bacteria based gene-directed enzyme prodrug therapy.
Although it has shown successful results in vivo yet further investigation about the targeting mechanisms of the bacteria are required to make it a complete therapeutic approach
in cancer treatment. 相似文献
8.
Jim M Dunwell Mike J Wilkinson Stephen Nelson Sri Wening Andrew C Sitorus Devi Mienanti Yuzer Alfiko Adam E Croxford Caroline S Ford Brian P Forster Peter DS Caligari 《BMC plant biology》2010,10(1):1-25
Background
Studies on host-pathogen interactions in a range of pathosystems have revealed an array of mechanisms by which plants reduce the efficiency of pathogenesis. While R-gene mediated resistance confers highly effective defense responses against pathogen invasion, quantitative resistance is associated with intermediate levels of resistance that reduces disease progress. To test the hypothesis that specific loci affect distinct stages of fungal pathogenesis, a set of maize introgression lines was used for mapping and characterization of quantitative trait loci (QTL) conditioning resistance to Setosphaeria turcica, the causal agent of northern leaf blight (NLB). To better understand the nature of quantitative resistance, the identified QTL were further tested for three secondary hypotheses: (1) that disease QTL differ by host developmental stage; (2) that their performance changes across environments; and (3) that they condition broad-spectrum resistance.Results
Among a set of 82 introgression lines, seven lines were confirmed as more resistant or susceptible than B73. Two NLB QTL were validated in BC4F2 segregating populations and advanced introgression lines. These loci, designated qNLB1.02 and qNLB1.06, were investigated in detail by comparing the introgression lines with B73 for a series of macroscopic and microscopic disease components targeting different stages of NLB development. Repeated greenhouse and field trials revealed that qNLB1.06 Tx303 (the Tx303 allele at bin 1.06) reduces the efficiency of fungal penetration, while qNLB1.02 B73 (the B73 allele at bin 1.02) enhances the accumulation of callose and phenolics surrounding infection sites, reduces hyphal growth into the vascular bundle and impairs the subsequent necrotrophic colonization in the leaves. The QTL were equally effective in both juvenile and adult plants; qNLB1.06 Tx303 showed greater effectiveness in the field than in the greenhouse. In addition to NLB resistance, qNLB1.02 B73 was associated with resistance to Stewart's wilt and common rust, while qNLB1.06 Tx303 conferred resistance to Stewart's wilt. The non-specific resistance may be attributed to pleiotropy or linkage.Conclusions
Our research has led to successful identification of two reliably-expressed QTL that can potentially be utilized to protect maize from S. turcica in different environments. This approach to identifying and dissecting quantitative resistance in plants will facilitate the application of quantitative resistance in crop protection. 相似文献9.
Cytochrome P-448 (mol wt 55,000 Daltons) from rabbit liver was purified to a specific content of 16.6 nmol/mg. Mice were immunised with this preparation, their spleens removed and dissociated lymphocytes hybridised with myeloma cells. Four monoclonal antibodies against cytochrome P-448 were raised and partially characterised. All four antibodies interacted with cytochrome P-448 in intact microsomal fractions and selectively immunoadsorbed cytochrome P-448 from solubilised microsomal preparations. One of the antibodies inhibited benzo[a] pyrene hydroxylase activity in a reconstituted system, one had no effect on activity and two increased activity. The possible applications of such antibodies are discussed. 相似文献