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121.
Socrates J. Tzartos Heleni V. Loutrari Fen Tang Anna Kokla Susan L. Walgrave Robert P. Milius† Bianca M. Conti-Tronconi 《Journal of neurochemistry》1990,54(1):51-61
Most anti-nicotinic acetylcholine receptor (AChR) antibodies in myasthenia gravis are directed against an immunodominant epitope or epitopes [main immunogenic region (MIR)] on the AChR alpha-subunit. Thirty-two synthetic peptides, corresponding to the complete Torpedo alpha-subunit sequence and to a segment of human muscle alpha-subunit, were used to map the epitopes for 11 monoclonal antibodies (mAbs) directed against the Torpedo and/or the human MIR and for a panel of anti-AChR mAbs directed against epitopes on the alpha-subunit other than the MIR. A main constituent loop of the MIR was localized within residues alpha 67-76. Residues 70 and 75, which are different in the Torpedo and human alpha-subunits, seem to be crucial in determining the binding profile for several mAbs whose binding to the peptides correlated very well with their binding pattern to native Torpedo and human AChRs. This strongly supports the identification of the peptide loop alpha 67-76 as the actual location of the MIR on the intact AChR molecule. Residues 75 and 76 were necessary for binding of some mAbs and irrelevant for others, in agreement with earlier suggestions that the MIR comprises overlapping epitopes. Structural predictions for the sequence segment alpha 67-76 indicate that this segment has a relatively high segmental mobility and a very strong turning potential centered around residues 68-71. The most stable structure predicted for this segment, in both the Torpedo and human alpha-subunits, is a hairpin loop, whose apex is a type I beta-turn and whose arms are beta-strands. This loop is highly hydrophilic, and its apex is negatively charged. All these structural properties have been proposed as characteristic of antibody binding sites. We also localized the epitopes for mAbs against non-MIR regions. Among these, the epitope for a monoclonal antibody (mAb 13) that noncompetitively inhibits channel function was localized within residues alpha 331-351. 相似文献
122.
Joseph G. Major Jr. Melinda E. Wales John E. Houghton Julie A. Maley Jeffrey N. Davidson James R. Wild 《Journal of molecular evolution》1989,28(5):442-450
Summary Aspartate transcarbamoylase (ATCase, EC 2.1.3.2) is the first unique enzyme common to de novo pyrimidine biosynthesis and
is involved in a variety of structural patterns in different organisms. InEscherichia coli, ATCase is a functionally independent, oligomeric enzyme; in hamster, it is part of a trifunctional protein complex, designated
CAD, that includes the preceding and subsequent enzymes of the biosynthetic pathway (carbamoyl phosphate synthetase and dihydroorotase).
The complete complementary DNA (cDNA) nucleotide sequence of the ATCase-encoding portion of the hamster CAD gene is reported
here. A comparison of the deduced amino acid sequences of the hamster andE. coli catalytic peptides revealed an overall 44% amino acid similarity, substantial conservation of predicted secondary structure,
and complete conservation of all the amino acids implicated in the active site of theE. coli enzyme. These observations led to the construction of a functional hybrid ATCase formed by intragenic fusion based on the
known tertiary structure of the bacterial enzyme. In this fusion, the amino terminal half (the “polar domain”) of the fusion
protein was provided by a hamster ATCase cDNA subclone, and the carboxyl terminal portion (the “equatorial domain”) was derived
from a clonedpyrBI operon ofE. coli K-12. The recombinant plasmid bearing the hybrid ATCase was shown to satisfy growth requirements of transformedE. coli pyrB
− cells. The functionality of thisE. coli-hamster hybrid enzyme confirms conservation of essential structure-function relationships between evolutionarily distant
and structurally divergent ATCases. 相似文献
123.
Daniel K. Yee Anna Pastuszko David Nelson David F. Wilson 《Journal of neurochemistry》1989,52(1):54-60
Incubation of synaptosomes from rat brain with DL-2-amino-5-phosphonovalerate (APV) stimulated an increased release of dopamine, and this effect was strictly dependent on the extrasynaptosomal calcium level. APV increased biosynthesis of dopamine from tyrosine by 30%, whereas monoamine oxidase activity was inhibited by 30%. When synaptosomes were incubated with radioactive dopamine, APV caused a large decrease in incorporation of label into 3,4-dihydroxyphenylacetic acid but greatly increased incorporation into norepinephrine and its N-methyl derivatives. Quantification of dopamine and its metabolites in synaptosomes, using electrochemical detection, indicated that the presence of APV resulted in changes in the absolute levels of the aforementioned dopamine metabolites similar to the changes in radiolabel incorporation. Omission of Ca2+ from the extrasynaptosomal medium greatly diminished the APV-induced changes in catecholamine metabolism. The metabolic changes appear to largely result from an increased intrasynaptosomal Ca2+ level due to the APV-induced increase in calcium permeability of the plasma membrane. 相似文献
124.
Studies on the mechanism of photosystem II photoinhibition I. A two-step degradation of D1-protein 总被引:2,自引:0,他引:2
The role of D1-protein in photoinhibition was examined. Photoinhibition of spinach thylakoids at 20°C caused considerable degradation of D1-protein and a parallel loss of variable fluorescence, QB-independent electron flow and QB-dependent electron flow. The breakdown of D1-protein as well as the loss of variable fluorescence and QB-independent electron flow were largely prevented when thylakoids were photoinhibited at 0°C. The QB-dependent electron flow markedly decreased under the same conditions. This inactivation may represent the primary event in photoinhibition and could be the result of some modification at the QB-site of D1-protein. Evidence for this comes from fluorescence relaxation kinetics following photoinhibition at 0°C which indicate a partial inactivation of QA
--reoxidation. These results support the idea of D1-protein breakdown during photoinhibition as a two step process consisting of an initial inactivation at the QB-site of the protein followed by its degradation. The latter is accompanied by the loss of PS II-reaction centre function.Abbreviations Asc
ascorbate
- p-BQ
1, 4-benzoquinone
- DAD
diaminodurene
- DPC
diphenylcarbazide
- DQH2
duroquinole
- Fecy
ferricyanide
- MV
methylviologen
- QA
primary quinone acceptor of PS II
- QB
secondary quinone acceptor of PS II
- SiMo
silicomolybdate 相似文献
125.
Anna L. Trifillis Myong Won Kahng 《In vitro cellular & developmental biology. Plant》1990,26(5):441-446
Summary We have developed an in vitro model of human papillary collecting duct cells isolated from cadaver kidneys using methods similar
to those we previously reported for the isolation of human proximal tubule cells. To date we have isolated papillary collecting
duct cells from 100 normal human kidneys. Papillae were dissected and digested in Cellgro containing 400 U/ml collagenase.
Cells were plated on fibronectin-coated culture flasks at a density of 104 live cells/ml in Cellgro supplemented with insulin and 10% fetal bovine serum. Confluent monolayers, which were able to withstand
600 mOsm for 8 h, were obtained within 10 to 15 d. Cells of primary isolates and first passages exhibited epithelial cell ultrastructure
including cell junctions, microvilli, and cilia. A dark-brown reaction product was observed in these cells when stained by
the immunoperoxidase method with peroxidase-labeled peanut lectin (Arachis hypogaea), which binds specifically to human distal tubule and collecting duct cells. These cells were negative for Factor-VIII (a
marker for endothelial cells) and γ-glutamyltransferase (a marker for proximal tubule cells). High activities of the glycolytic
enzyme pyruvate kinase and arginine vasopressin-stimulated cAMP production in these cells are consistent with a distal nephron
origin. The results indicate that human collecting duct cells can be isolated and cultured to provide an in vitro system to
probe pathogenetic mechanisms of potential nephrotoxins.
Part of this work was presented at a Symposium of the Center for Alternatives to Animal Testing, April 4–5, 1989, Johns Hopkins
Medical Institutions, Baltimore, MD 21205.
This work was supported in part by grants R01-AI24179, PO1-A804393 for the Public Health Service, U.S. Department of Health
and Human Services, and by a grant from the National Kidney Foundation, Baltimore, MD affiliate. 相似文献
126.
127.
Biophysical characterization of recombinant proteins expressing the leucine zipper-like domain of the human immunodeficiency virus type 1 transmembrane protein gp41. 总被引:2,自引:2,他引:0
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Envelope oligomerization is thought to serve several crucial functions during the life cycle of human immunodeficiency virus type 1 (HIV-1). We recently reported that virus entry requires coiled-coil formation of the leucine zipper-like domain of the HIV-1 transmembrane envelope glycoprotein gp41 (C. Wild, T. Oas, C. McDanal, D. Bolognesi, and T. Matthews, Proc. Natl. Acad. Sci. USA 89:10537-10541, 1992; C. Wild, J. W. Dubay, T. Greenwell, T. Baird, Jr., T. G. Oas, C. McDanal, E. Hunter, and T. Matthews, Proc. Natl. Acad. Sci. USA 91:12676-12680, 1994). To determine the oligomeric state mediated by this region of the envelope, we have expressed the zipper motif as a fusion partner with the monomeric maltose-binding protein of Escherichia coli. The biophysical properties of this protein were characterized by velocity and equilibrium sedimentation, size exclusion chromatography, light scattering, and chemical cross-linking analyses. Results indicate that the leucine zipper sequence from HIV-1 is capable of multimerizing much larger and otherwise monomeric proteins into extremely stable tetramers. Recombinant proteins containing an alanine or a serine substitution at a critical isoleucine residue within the zipper region were also generated and similarly analyzed. The alanine- and serine-substituted proteins behaved as tetrameric and monomeric species, respectively, consistent with the influence of these same substitutions on the helical coiled-coil structure of synthetic peptide models. On the basis of these findings, we propose that the fusogenic gp4l structure involves tetramerization of the leucine zipper domain which is situated approximately 30 residues from the N-terminal fusion peptide sequence. 相似文献
128.
Identification of two amino acids in the hemagglutinin glycoprotein of measles virus (MV) that govern hemadsorption, HeLa cell fusion, and CD46 downregulation: phenotypic markers that differentiate vaccine and wild-type MV strains. 总被引:9,自引:9,他引:0
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V Lecouturier J Fayolle M Caballero J Carabaa M L Celma R Fernandez-Muoz T F Wild R Buckland 《Journal of virology》1996,70(7):4200-4204
We have used site-directed mutagenesis of the hemagglutinin (H) glycoprotein of measles virus (MV) to investigate the molecular basis for the phenotypic differences observed between MV vaccine strains and recently isolated wild-type MV strains. The former downregulate CD46, the putative cellular receptor of MV, are positive for hemadsorption, and are fusogenic in HeLa cells, whereas the latter are negative for these phenotypic markers. CD46 downregulation in particular, could have profound consequences for the immunopathology of MV infection, as this molecule protects the cell from complement lysis. Mutagenesis of two amino acids, valine and tyrosine at positions 451 and 481, respectively, in the H protein from the vaccine-like Hallé MV strain to their counterparts, glutamate and asparagine, in the H protein from the wild-type Ma93F MV strain (creating the V451E/Y481N double mutation) abrogated CD46 downregulation, HeLa cell fusion, and hemadsorption. The converse double mutagenesis of the Ma93F H protein (E451V/N481Y) transferred the CD46-downregulating, fusogenic, and hemadsorption functions to this protein. The data provide the first mapping study of the functional domains of MV H. The consequences of these results for MV vaccine design and the role of CD46 in MV infection are discussed. 相似文献
129.
Anna Jachner 《Hydrobiologia》1996,325(2):151-155
The response of bleak (Alburnus alburnus (L.)) to alarm substance (from skin extract of conspecifics) was quantified, using a video camera, in laboratory experiments across two light and one dark periods (18 hrs). In the light alarm substance induced hiding in refuges (vegetation) and formation of aggregations. There was no such reaction during darkness or during a second light period. 相似文献
130.
Butomus umbellatus L. is a plant species typical of littoral communities of river and stream shores. It can form continuous stands in shallow reservoirs with fluctuating water level. Their expansion is promoted by: (a) intensive vegetative reproduction of plants, (b) crowded sprouting from rhizome fragments on emerged pond bottom, (c) shallow water layer in the year following summer drainage. Expansion of B. umbellatus depends on ploidy level: two cytotypes were found in the Czech and Slovak Republics, differing in their reproductive ability. Seed production of triploids is strongly limited (they are self-incompatible within clones), while diploids can be fully fertile. Nevertheless, even in diploids, the efficiency of seed reproduction under natural conditions is low. Triploids spread by intensive vegetative reproduction, which is decisive for clonal growth of populations and their regeneration after scraping of bottom surface. During seasonal development, maximum of aboveground biomass is produced in early summer, while underground biomass increases till autumn. Growth of the plants is limited by cutting before maximum underground biomass is attained, or by duck grazing. 相似文献