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  • 1
    ISSN: 1432-1211
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract A procedure for the molecular identification of MHC class I products based on 1-D IEF and subsequent immunoblotting is described. Optimal conditions for 1-D IEF, the electrophoretic transfer of proteins out of denaturing, nonionic detergent-containing gels to nitrocellulose, and the requisite antibodies, both polyclonal and monoclonal, for the visualization of class I heavy chains have been established. Cross-reactivity of antibodies has enabled the biochemical analysis of class I heavy chains in the dog. The procedure reported here requires modest amounts of cells and allows a rapid molecular characterization of class I heavy chain polymorphisms in man and other species without the need for radiochemical methods.
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  • 2
    ISSN: 1432-1211
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The polymorphism of feline MHC antigens was examined using biochemical methods. The following observations were made: (1) feline class I and II antigens are polymorphic. Their biochemical features were established using rabbit and mouse reagents directed against human MHC products; they resemble those observed for other mammalian species; (2) the expression of class II antigens in unstimulated cat peripheral blood lymphocytes (PBLs) appears to be unusually high. Cat PBLs express far more class II than class I antigens, whereas in human Epstein-Barr virus-transformed lines, which are known to express relatively large amounts of class II antigens, the situation is reversed.
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  • 3
    ISSN: 1432-1211
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Class I antigens were isolated by immunoprecipitation from cell extracts prepared from mitogenically stimulated and internally radiolabeled peripheral blood lymphocytes (PLBs). The precipitating antibodies used are monomorphic and recognize a determinant on the heavy chain of HLA-A, B, C antigens regardless of their allelic specificities when complexed with β 2m, or determinants on β 2m itself. Comparison of class I molecules isolated from 25 different homozygous typing cels (HTC) and analyzed by two-dimensional (2-D) gel electrophoresis allowed the identification of those HLA-A,13 locus specificities most common in the European Caucasoid population. Class I antigens isolated from HTC that are HLA identical are biochemically indistinguishable also. Evidence was obtained for the expression of additional class I antigens besides the HLA-A, B, C locus products: for some haplotypes, up to six class I genes may be active in mitogenically activated PBLs. No differences in molecular weight and isoelectric point of the class I heavy chains were observed between the antigens recognized by W6/32, the anti-heavy chain reagent, and anti-β 2m reagents. The nature of the mitogenic stimulus, i. e., pokeweed mitogen or phytohemagglutinin, was irrelevant with respect to the class I antigens isolated by this method. Using the HTCs as reference, a panel of HLA-B27 positive heterozygous cells was analyzed. Two types of HLA-B27 antigens, distinct by CML typing were represented. These two forms differed also in their biochemical properties. In addition, we obtained evidence for the existence of an A2 variant. This finding was likewise confirmed by CML typing.
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    Immunogenetics 19 (1984), S. 95-107 
    ISSN: 1432-1211
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Dog peripheral blood lymphocytes, when cultured with 35S-methionine in the presence of tunicamycin, synthesize DLA molecules consisting of β 2-microglobulin and a heavy chain approximately 3000 daltons lower in apparent mol. wt. than observed in control cases. This difference in mol. wt. is consistent with the fact that a single N-linked carbohydrate side chain is present on the heavy chain of DLA class I antigens. There is no evidence of polymorphism in the DLA light chain (β 2m). Both glycosylated and nonglycosylated forms of the heavy chain, however, show microheterogeneity, which can be related to tissue-type. Analysis by two-dimensional electrophoresis shows that the biochemical heterogeneity in the DLA heavy chain is less than expected from DLA serology, and less than found in HLA class I antigens. The data are consistent with the fact that the products of only a single DLA class I locus are detected.
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  • 5
    ISSN: 1432-1211
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The population of HLA-A2-positive individuals, currently considered serologically homogeneous, can be divided into three subtypes on the basis of antigen recognition by various HLA-A2-specific cytotoxic T lymphocytes (CTLs). When these three types of HLA-A2 antigens were analyzed biochemically, they were found to be distinct. Isoelectric focusing (IEF) of HLA antigens digested with neuraminidase (NANAse) suggested that the difference(s) reside in the polypeptide backbone of the HLA-A2 heavy chain. Biochemical analysis distinguishes three distinct categories of HLA-A2 antigens: (1) a major subtype, designated HLA-A2.I, (2) a minor subtype, designated HLA-A2.II, possessing a more basic isoelectric point (IEP) and (3) a minor HLA-A2 subtype more acidic in its IEP than HLA-A2.I, designated HLA-A2.III. A fourth HLA-A2 subtype could be defined by discordance between cell-mediated lympholysis (CML) typing and biochemical analysis. The latter HLA-A2 antigen was defined as a variant by CTL, but was biochemically indistinguishable from the major subtype HLA-A2.I.
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  • 6
    ISSN: 1432-1211
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The HLA-Cw1 and -Cw2 genes were identified in a genomic library and their products characterized by biochemical methods. The HLA-Cw and -Cw2 genes, upon transfection in mouse L cells, give rise to class I antigen heavy chains that associate with neither mouse nor human beta-2 microglobulin. They are indistinguishable in isoelectric point from polypeptides identified as HLA-Cw1 and -Cw2 in human cells. The nucleotide sequence of HLA-Cw1 and -Cw2 and their comparison with HLA-Cw3, the only other known HLA-C sequence, reveal a characteristic pattern of locus-specific amino acids. A comparison of 13 different human class I primary structures leads us to speculate that the most variable region in HLA class I antigens, positions 61–83, could assume an alpha helical structure of critical importance for class I antigen function. The locus specificity and the higher degree of intralocus conservation in the COON-terminal region, especially in the transmembrane and cytoplasmic domains, must reflect evolutionary ancestry rather than positive selection. In view of the pattern and types of substitutions observed for HLA-C locus products, their function as immune response gene products is questioned.
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  • 7
    ISSN: 1432-1211
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    Immunogenetics 28 (1988), S. 406-411 
    ISSN: 1432-1211
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract A genomic library was constructed from DNA of a domestic cat and screened with a human HLA-DRα probe at low stringency. Several positive clones were isolated, and the DNA sequence of one of these clones was determined. Comparison with class II α gene sequences from other species suggested that the feline gene is a DPA homologue (FLA-DPA) showing 84% similarity with HLA-DPα1 in the exon encoding the second domain. The FLA-DPA gene that was isolated is a pseudogene, as two frame-shift mutations are present: one in the exon encoding the second domain, causing premature termination of translation, and one in the exon encoding the transmembrane region. The latter mutation and the further deletion of two codons in the transmembrane exon show a remarkable resemblance to the same exon of the human pseudogene, HLA-DPA2. Hence, both pseudogenes evolved from the same ancestral gene. The inactivation of this DPA gene could therefore have occurred prior to the major mammalian divergence.
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  • 9
    ISSN: 1432-1211
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract A method is described for a biochemical comparison of mouse class I antigens utilizing antisera with a monomorphic pattern of reaction and high-resolution one-dimensional isoelectric focusing (1D-IEF). The most commonly occurring and studied H-2K and D alleles were identified in a comparison of over 40 mouse strains. By comparing H-2 mutant mouse strains, cell lines transfected with defined class I genes, or mice transgenic for a mouse class I gene and H-2 recombinant mouse strains, unambiguous identification of class I alleles was possible. The complex pattern presented by H-2-heterozygous mice was readily resolved into the contribution by the individual parental alleles. The H-2 b bm series of mutants was analyzed, and for those mutants where a charge difference was predicted based on their known sequence, a change in isoelectric point (IEP) was indeed observed. Based on analysis by IEF, the bm8 mutant may contain (an) amino acid substitution(s) in addition to those reported. The present method further appears useful in elucidating defects in class I antigen synthesis and post-translational modifications, as these cannot be easily characterized when using surface staining with monoclonal antibodies (mAbs).
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  • 10
    ISSN: 1432-1211
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The sequence of a feline class I pseudogene and its comparison with class I genes from other species is presented. The gene isolated is a pseudogene because of the presence of four stop codons and two frame shift mutations in the first-and second-domain encoding exons, as well as a mutation in a splice acceptor site in the third intron. By sequence comparison with the other class I sequences determined to date, theFLA pseudogene is most closely related to theHLA-A locus products (88% nucleotide identity).
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