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1.
Bacteriorhodopsin (bR) was regenerated from the cation-depleted blue membrane with pentaammineaquocobalt(III) tetrafluoroborate [( Co(NH3)5H2O]3+[BF4-]3). Illumination of the sample with orange light decreased the extinction at 568 nm concomitantly with a hypsochromic shift of the absorption maximum. The photocycle of this sample was inhibited, and the rate of proton pumping was reduced. Chymotryptic cleavage of the corresponding apomembrane into the two fragments C1 and C2 and their subsequent separation revealed that cobalt label is only attached to C1. The maximal incorporation of Co into this peptide was 0.3 Co/C1. After cleavage of C1 with cyanogen bromide and subsequent proteolysis with trypsin and chymotrypsin, this modification could be associated with peptides from cyanogen bromide fragments 6 and 9. The sequences were determined to be 101Val-Asp-Ala-Asp-Gln and 228Ala-Ile-Phe-Gly-Glu-Ala-Glu-Ala. These peptides contain the sequences Asp-Ala-Asp and Glu-Ala-Glu, respectively, which might be constituents of the same cation binding site. The observation that the incorporation of Co into bacteriorhodopsin is enhanced under illumination with orange light indicates that this site might be involved in the proton uptake. 相似文献
2.
Reinhard Lakes Klaus Kalmring Karl-Heinz Engelhard 《Journal of comparative physiology. A, Neuroethology, sensory, neural, and behavioral physiology》1990,166(4):553-563
Deafferentation experiments during postembryonic development show morphological and/or physiological changes of receptor fibers and of identified auditory interneurons in the CNS of the locusts Locusta migratoria and Schistocerca gregaria after unilateral ablation of one tympanic organ either in the larva or the adult animal.
相似文献
1. | In Locusta migratoria, 5 days after deafferentation, intact, contralateral receptor fibers had sprouted collaterals in the frontal acoustic neuropil of the metathoracic ganglion (Figs. 1, 2). Collateral sprouts were only rarely found in Schistocerca gregaria. |
2. | After about 20 days the deafferented auditory interneurons receive new inputs from the contralateral receptors (Figs. 3, 5, 7, 10). This largely restores their thresholds and intensity/response functions. Collaterals from the first order interneurons cross the midline to the contralateral neuropil (BSN1 neuron, Fig. 4), which is never seen in intact animals. By contrast, in the TN1 neuron no consistent morphological change due to the deafferentation could be found (Fig. 6). |
3. | Interneurons of higher order (AN1, TN3 neuron in locusts) regain their response pattern (Fig. 7) without morphological changes (Fig. 9). Bilateral recordings show that the deafferented interneurons respond more weakly to auditory stimuli than the intact neuron, but the response to vibration stimuli remains unchanged (TN3 neuron, Fig. 8). |
3.
Mating behavior in seaweed flies (Coelopa frigida) 总被引:1,自引:0,他引:1
4.
A new tissue isolation technique was used to create intact midgut epithelial wholemounts from three Trichoplusia ni (Lepidoptera: Noctuidae) larval instars. The protease, dispase, removed the basal lamina and associated connective tissue and allowed for high resolution light microscopy of entire epithelia. Columnar, goblet, differentiating, and stem cells were characterized by double fluorescent labelling of f-actin and nuclei. A comparison of cell populations by digital image analysis revealed significant regional and temporal changes in the density and number of differentiating and stem cells. Growth of the midgut epithelium from third to fourth instar, and from fourth to fifth instar, was accomplished by both cell differentiation and cell division. Cell division however, was greatly reduced from fourth to fifth instar with a concomitant sharp decrease in the stem cell population. 相似文献
5.
Stable cell lines lacking cytotoxic activity against specific target cells were derived from highly active murine CTL clones by the omission of antigen from the culture for several weeks. Several independent CTL clones cultured in the absence of antigen showed a gradual decline in cytotoxic activity, resulting in complete loss by 5 to 10 wk. Such noncytotoxic (NC) cells lacked the ability to form stable conjugates with specific target cells, but were able to kill all target cells tested in the presence of Con A. It was shown by subcloning at limiting dilution that all cells in the starting population were cytolytically active, and that all cells in the NC population derived from such a clone were cytolytically inactive against target cells bearing an appropriate antigen under normal assay conditions. By using the monoclonal antibody F23.1, which reacted with the antigen receptors of two of the CTL clones, it was shown that the NC cells derived from these clones continued to express the receptor at normal levels. Levels of expression of Thy-1.2, Lyt-2.2, and LFA-1 were also similar in all cytotoxic cell lines and their noncytotoxic derivatives. The F23.1 antibody induced an increase in cytoplasmic free Ca2+ in both CTL and NC cells, and NC cells lysed F23.1 hybridoma cells in the absence of Con A. When cells expressing appropriate target cell antigen were added back to cultures of NC cells, cytotoxic activity of appropriate specificity was fully recovered in 2 wk. These results indicate that expression of an apparently functional antigen receptor alone is insufficient for stable binding of CTL to specific target cells, and that other factors dependent upon antigen stimulation may be involved in the recognition process. A difference in affinity for antigen between CTL and NC cells is suggested as a possible explanation for these observations. 相似文献
6.
Construction of novel class I histocompatibility antigens by interspecies exon shuffling 总被引:1,自引:0,他引:1
V H Engelhard J R Yannelli G A Evans S F Walk M J Holterman 《Journal of immunology (Baltimore, Md. : 1950)》1985,134(6):4218-4225
Human and mouse class I histocompatibility antigens share considerable structural homology at both the protein and DNA sequence level. This homology has allowed the production of hybrid class I molecules by the reciprocal exchange of DNA sequences corresponding to equivalent domains of HLA-B7 and either H-2Ld or H-2Dd. It is shown that these genes give rise to protein products that are stably expressed on the surface of murine L cells after DNA-mediated gene transfer. These proteins express only those monoclonal antibody-defined H-2 determinants that are expected based on their genetic construction. The molecules have allowed the localization of a number of polymorphic and monomorphic HLA-specific epitopes. In all but one case, expression of an epitope on a domain does not appear to be influenced by the replacement of adjacent human domains with their murine equivalents, suggesting a considerable degree of structural independence of the domains. Cells expressing the hybrid molecules have also been tested as targets for a panel of HLA-B7-specific cytotoxic T cell clones. The results show that the polymorphic determinants recognized by these clones map to the alpha 1 and alpha 2 domains of the HLA-B7 molecule. No evidence for an influence of species-related amino acid sequence differences in the third extracellular domain on T cell recognition was seen. The results are discussed in light of the proposed domain structure of the class I proteins and the potential use of such molecules for further functional studies. 相似文献
7.
The maximal nonlethal dose of the 3a-1 fraction of papain, determined by use of 9-week-old white albino rabbits, was 10 mg per injection, administered intravenously. The immunosuppressive activity of the 3a-1 fraction of papain was studied by its inhibition of sheep red blood cell hemolysin, bacterial agglutinin, and horse serum precipitin. Immune suppression was observed when papain injections preceded antigen injections by 12 to 18 hr. The enzyme preparation was analyzed by use of cellulose acetate and starch-gel electrophoresis and by the micro-Kjeldahl method. Starch-gel electrophoresis of serum samples revealed qualitative alterations in the alpha(-1) region 7 hr after papain administration. Changes were also observed in the urinary aminopolysaccharide content. 相似文献
8.
Cytotoxic T cell responses in HLA-A2.1 transgenic mice. Recognition of HLA alloantigens and utilization of HLA-A2.1 as a restriction element 总被引:4,自引:0,他引:4
A X Le E J Bernhard M J Holterman S Strub P Parham E Lacy V H Engelhard 《Journal of immunology (Baltimore, Md. : 1950)》1989,142(4):1366-1371
Previous studies have indicated that the frequency of murine CTL precursors (CTLp) for human class I molecules is one to two orders of magnitude lower than that for murine class I alloantigens, and that this is due to species-specific structural differences between these molecules. Transgenic mice expressing the human class I MHC Ag HLA-A2.1 were used to examine changes in the frequency of class I HLA-specific precursors after T cell differentiation in an HLA-A2.1 positive environment. The HLA-A2.1 gene product was expressed at levels comparable to those of the endogenous H-2Db molecule in thymus, bone marrow, and spleen. By limiting dilution analysis, it was observed that the frequencies of CTLp in transgenic mice responding to the human alloantigens HLA-B7 or HLA-A2.2 were comparable to or lower than those in normal C57BL/6 mice, regardless of whether the Ag was presented on human or murine cells. Thus, expression of a human class I molecule in these animals did not result in an expansion of the number of CTLp specific for other human class I Ag. In addition, the frequency of HLA-A2.1-restricted, influenza specific CTLp was substantially lower than the frequency of H-2b restricted CTLp, indicating a poor utilization of HLA-A2.1 as a restricting element. Finally, the frequencies of CTLp for HLA-A2.1 expressed on syngeneic murine tumor cells were decreased significantly. Thus, expression of HLA-A2.1 in these animals appeared to induced tolerance to this Ag. Interestingly, however, these mice were not tolerant to the HLA-A2.1 molecule expressed on human cells. This indicates that the HLA-A2.1 associated epitopes expressed on murine and human cells differ and suggests that, under these circumstances, HLA-A2.1 acts as a restricting element for human nominal Ag. These results are discussed in the context of current models of T cell repertoire development. 相似文献
9.
M. Engelhard K. D. Kohl K. H. Müller B. Hess J. Heidemeier M. Fischer F. Parak 《European biophysics journal : EBJ》1990,19(1):11-18
Bacteriorhodopsin (bR), converted by deionization to the blue form was reconstituted to the active purple membrane by the addition of Fe2+ or Fe3+ ions. 57Fe Mossbauer spectra of these samples were measured at different pH values (pH 3.9, pH 5.0 and pH 7.0) and at temperatures ranging from 4 K to 300 K. The hyperfine parameters reveal two iron environments with oxygen atoms in the neighbourhood of iron. Iron type 1 is in the 3+ high spin state. It is bound to acid side chains of the protein and/or the phosphate groups of the lipids. Iron type 2 is in the 2+ high spin state and is linked to carboxy groups of the protein in a rather unspecific way. Dynamics as measured by Mossbauer spectroscopy show that the purple membrane becomes flexible only above 220 K. At the interface between membrane and bulk water the mobility is comparable to that of proteins with hydrophilic surfaces. The photocycle of Fe 3+-bR is slowed down compared to native bR. 3–5 Fe3+/bR are sufficient to inhibit the photocycle turnover by one order of magnitude. This specific effect is also found with Cr3+, though it is less pronounced. Mössbauer spectra of Fe3+-bR at 4 K reveal that iron nuclei are spin-coupled, indicating their close spatial proximity. It is proposed that iron trinuclear clusters interact with the proton uptake site of bR.
Offprint requests to: M. Engelhard 相似文献
10.
Previous studies have indicated that in transgenic mice expressing human class I MHC molecules, it is difficult to demonstrate a significant CTL response to a viral Ag in the context of the transgenic molecule. In this paper, a procedure is reported for the isolation of influenza-specific murine CTL restricted by the human class I molecule HLA-A2.1. The principal specificity of such CTL is for a fragment of the influenza M1 protein that has been previously shown to be immunodominant for human HLA-A2.1-restricted CTL. CTL of this specificity were also established through the use of peptide-pulsed rather than virus-infected stimulators. The dependence of murine CTL recognition upon peptide length and HLA-A2 structure was established to be similar to that previously reported for human CTL. However, the fine specificity of CTL maintained on virus-infected stimulators was somewhat different from that of CTL maintained with M1 peptide. This suggests that differences in surface density or peptide structure between peptide-pulsed and virus-infected stimulators may result in the outgrowth of T cells with different receptor structures. The immunodominance of the M1 peptide determinant in both mice and humans suggests that species-specific differences in TCR structure, Ag-processing systems, and self-tolerance are of less importance than limitations on the ability of antigenic peptides to bind to appropriate class I molecules. These results thus establish the utility of the transgenic system for the identification of human class I MHC-restricted T cell epitopes. 相似文献