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101.
Secretion of a heterologous protein from Bacillus subtilis with the aid of protease signal sequences. 总被引:7,自引:5,他引:2
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Secretion vectors based on the genes from Bacillus amyloliquefaciens P for alkaline protease (aprBamP) and neutral protease (nprBamP) were constructed. With both aprBamP and nprBamP, a unique restriction site was introduced 3' of the predicted signal coding region by using the technique of oligonucleotide-directed mutagenesis. The new sites enabled us to fuse a heterologous gene to the expression and secretion elements. We used the protein A gene (spa) from Staphylococcus aureus as a heterologous gene. Bacillus subtilis cells carrying the resulting apr-spa or npr-spa gene fusions synthesized the fusion protein. B. subtilis cells were also capable of removing the signal peptide from the fusion protein, as indicated by the appearance of processed protein A into the growth medium. In addition, these gene fusions allowed us to identify the signal processing site of both the APR-SPA and NPR-SPA proteins. 相似文献
102.
Structural similarity between the lepidoptera- and diptera-specific insecticidal endotoxin genes of Bacillus thuringiensis subsp. "kurstaki" and "israelensis" 总被引:30,自引:8,他引:22
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L Thorne F Garduno T Thompson D Decker M Zounes M Wild A M Walfield T J Pollock 《Journal of bacteriology》1986,166(3):801-811
103.
Localization of the ganglioside-binding site of fibronectin 总被引:10,自引:0,他引:10
L K Thompson P M Horowitz K L Bentley D D Thomas J F Alderete R J Klebe 《The Journal of biological chemistry》1986,261(11):5209-5214
It has been demonstrated via biological assays that fibronectin possesses a receptor for gangliosides that is involved in cell adhesion and restoration of the normal morphology of transformed cells. In this study, fluorescence polarization has been employed to monitor the binding of ganglioside oligosaccharide to fibronectin. Parameters involved in ganglioside oligosaccharide binding to fibronectin are described and compared to the interaction of heparin with fibronectin. A Kd of 1.4 X 10(-8) mol/liter has been calculated, and it is demonstrated that labeled ganglioside oligosaccharides can be eluted from fibronectin with either unlabeled ganglioside oligosaccharides or 4 M urea. Using the fluorescence polarization assay developed in this study for measurement of ganglioside binding to fibronectin, it is demonstrated that gangliosides bind to the 31,000-dalton amino terminal heparin-binding domain of fibronectin. A ganglioside-Sepharose affinity column has been constructed which specifically binds the 31,000-dalton amino terminal fragment of fibronectin. The localization of the ganglioside receptor to the amino terminal domain of fibronectin indicates that the ganglioside receptor is distinct from the putative fibronectin cell surface receptor which is located near the center of the fibronectin molecule. 相似文献
104.
A radiolabeled monoclonal antibody binding assay for cytoskeletal tubulin in cultured cells 总被引:1,自引:0,他引:1
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R L Ball D H Carney T Albrecht D J Asai W C Thompson 《The Journal of cell biology》1986,103(3):1033-1041
To detect changes in the extent of tubulin polymerization in cultured cells, we have developed a radioactive antibody binding assay that can be used to quantitate total cytoskeletal tubulin or specific antigenic subsets of polymerized tubulin. Fibroblastic cells, grown to confluence in multiwell plates, were permeabilized and extracted with 0.5% Triton X-100 in a microtubule-stabilizing buffer. These extracted cytoskeletons were then fixed and incubated with translationally radiolabeled monoclonal antitubulin antibody (Ab 1-1.1), an IgM antibody specific for the beta subunit of tubulin. Specific binding of Ab 1-1.1 to the cytoskeletons was saturable and of a single apparent affinity. All specific binding was blocked by preincubation of the radiolabeled antibody with excess purified brain tubulin. Specific Ab 1-1.1 binding appeared to represent binding to cytoskeletal tubulin inasmuch as: pretreatment of cells with colchicine decreased Ab 1-1.1 binding in a dose-dependent manner which correlated with the amount of polymerized tubulin visualized in parallel cultures by indirect immunofluorescence, taxol pretreatment alone caused an increase in Ab 1-1.1 binding and prevented in a dose-dependent manner the colchicine-induced decrease in antibody binding, in cells pretreated with colcemid and returned to fresh medium, Ab 1-1.1 binding decreased and recovered in parallel with the depolymerization and regrowth of microtubules in these cells, and comparison of maximal antibody binding per cell between primary mouse embryo, 3T3, and human foreskin fibroblasts correlated with immunofluorescence visualization of microtubules in these cells. Thus, this assay can be used to measure relative changes in the level of polymerized cytoskeletal tubulin. Moreover, by Scatchard-type analysis of the binding data it is possible to estimate the total number of antibody binding sites per cell. Therefore, depending on the stoichiometry of antibody binding, this type of assay may be used for quantitating total cytoskeletal tubulin, specific antigenic subsets of cytoskeletal tubulin, or other cytoskeletal proteins. 相似文献
105.
Thompson Webb III 《Plant Ecology》1986,67(2):75-91
Current methods for estimating past climatic patterns from pollen data require that the vegetation be in dynamic equilibrium with the climate. Because climate varies continuously on all time scales, judgement about equilibrium conditions must be made separately for each frequency band (i.e. time scale) of climatic change. For equilibrium conditions to exist between vegetation and climatic changes at a particular time scale, the climatic response time of the vegetation must be small compared to the time scale of climatic variation to which it is responding. The time required for vegetation to respond completely to climatic forcing at a time scale of 104 yr is still unknown, but records of the vegetational response to climatic events of 500-to 1000-yr duration provide evidence for relatively short response times. Independent estimates for the possible patterns and timing of late-Quaternary climate changes suggest that much of the vegetational evidence previously interpreted as resulting from disequilibrium conditions can instead be interpreted as resulting from the individualistic response of plant taxa to the different regional patterns of temperature and precipitation change. The differences among taxa in their response to climate can lead a) to rates and direction of plant-population movements that differ among taxa and b) to fossil assemblages that differ from any modern assemblage. An example of late-Holocene vegetational change in southern Quebec illustrates how separate changes in summer and winter climates may explain the simultaneous expansion of spruce (Picea) populations southward and beech (Fagus) populations northward. 相似文献
106.
107.
P W Davenport W A Friedman F J Thompson O Franzén 《Journal of applied physiology》1986,60(6):1843-1848
It has long been recognized that humans can perceive respiratory loads. There have been several studies on the detection and psychophysical quantification of mechanical load perception. This investigation was designed to record cortical sensory neurogenic activity related to inspiratory mechanical loading in humans. Inspiration was periodically occluded in human subjects while the electroencephalographic (EEG) activity in the somatosensory region of the cerebral cortex was recorded. The onset of inspiratory mouth pressure (Pm) was used to initiate signal averaging of the EEG signals. Cortical evoked potentials elicited by inspiratory occlusions were observed when C3 and C alpha were referenced to CZ. This evoked potential was not observed with the control (unoccluded) breaths. There was considerable subject variability in the peak latencies that was related to the differences in the inspiratory drive, as measured by occlusion pressure (P0.1). The results of this study demonstrate that neurogenic activity can be recorded in the somatosensory region of the cortex that is related to inspiratory occlusions. The peak latencies are longer than analogous somatosensory evoked potentials elicited by stimulation of the hand and foot. It is hypothesized that a portion of this latency difference is related to the time required for the subject to generate sufficient inspiratory force to activate the afferents mediating the cortical response. 相似文献
108.
Granulocyte-mediated airway edema in guinea pigs 总被引:2,自引:0,他引:2
Sheppard D.; Scypinski L.; Horn J.; Gordon T.; Thompson J. E. 《Journal of applied physiology》1986,60(4):1213-1220
To determine the role of polymorphonuclear leukocytes (PMNs) in the airway edema that accompanies airway inflammation, we studied the effects of a 1-h exposure to 2 ppm toluene diisocyanate (TDI) on tracheal extravasation of Evans blue dye and on the concentration of PMNs in the tracheal wall. Tracheal Evans blue content was significantly increased by TDI exposure (53.6 +/- 8.0 micrograms/g tracheal tissue (mean +/- SE) for animals exposed to TDI and 16.3 +/- 2.0 for animals exposed to air, P less than 0.0025) as were both the intravascular and extravascular concentration of PMNs in tracheal sections (intravascular PMNs were 28.0 +/- 8.4 X 10(3) cells/mm3 for TDI and 1.5 +/- 1.5 X 10(3) for air, P less than 0.025, extravascular PMNs were 10.9 +/- 4.5 X 10(3) for TDI and 0 for air, P less than 0.05). PMN depletion with vinblastine or with hydroxyurea abolished both the increase in tracheal Evans blue extravasation and the increase in the concentration of intravascular and extravascular PMNs in animals exposed to TDI. PMN depletion with hydroxyurea did not significantly inhibit the increase in tracheal Evans blue extravasation caused by intravenous histamine. Administration of donor PMNs to animals depleted of PMNs with hydroxyurea reconstituted the TDI-induced increase in tracheal Evans blue extravasation (80.4 +/- 17.3 micrograms/g tissue (mean +/- SE) in animals exposed to TDI vs. 21.3 +/- 2.9 in animals exposed to air, P less than 0.025) and in the intravascular concentration of PMNs in tracheal sections [18.5 +/- 3.4 X 10(3) cells/mm3 (mean +/- SE) in animals exposed to TDI vs. 1.3 +/- 1.3 X 10(3) in animals exposed to air, P less than 0.0025].(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
109.
A control system has been devised for the maintenance of stable ammonium concentrations throughout a fedbatch fermentation. The control system is based on an ammonia gas-sensing electrode that monitors a pH-adjusted effluent stream from the fermentor. To overcome the time lag between the fermentor and the electrode, feedback control included metered flows of ammonium to both the fermentor and the electrode vessel. The system was used to study the growth of Escherichia coli B (ATCC 11303) at controlled ammonium concentrations of 5 to 200mM. Apparent specific growth rates, biomass and protein production, and glucose yields were essentially constant from 5 to 170mM. Above 170mM ammonium growth was inhibited. As ammonium concentration decreased from 170 to 5mM, ammonium yields increased from 1 to 24 g cell dry wt/g ammonium utilized. The results demonstrate that control of ammonium concentrations at levels so low that ammonium would be exhausted in batch fermentations can significantly increase overall ammonium yields. 相似文献
110.
R E Brown F A Stephenson T Markello Y Barenholz T E Thompson 《Chemistry and physics of lipids》1985,38(1-2):79-93
A transfer protein specific for glycolipids has been isolated from bovine brain. As judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, the protein is 68% pure and has a molecular weight of 20 000. Three different assays were employed to study the protein's specificity and glycolipid binding properties. The protein transferred several different neutral glycosphingolipids and ganglioside GM1 equally well, but failed to accelerate phosphatidylcholine or sphingomyelin intervesicular movement. The protein's ability to interact with glycolipids was strongly influenced by the physical properties of the matrix phospholipid in which the glycolipids reside. Both the phase state of the phospholipid matrix and bilayer curvature affected glycolipid intervesicular transfer rates. Protein binding to phospholipid vesicles containing either tritium-labeled or pyrene-labeled glucosylceramide could not be demonstrated by density gradient centrifugation or fluorescence energy transfer measurements, respectively. A specific association of the transfer protein for pyrene-labeled glucosylceramide was found when the fluorescence emission of the pyrene excimer-to-monomer ratio was measured suggesting that a portion of the fluorescent glycolipid was being sequestered from the phospholipid vesicles and was binding to the freely soluble protein. 相似文献