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Query: UNIPROT:P06889 (Mol)
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Functional interactions between CD8-dependent cytotoxic T cells and their targets require physical contact between CD8 and a non-polymorphic determinant on the alpha 3 domain of the class I MHC molecule. We developed a cell-free assay to directly monitor this molecular interaction, specifically excluding the participation of other cellular proteins and lipids. This assay employed a soluble CD8 derivative and a plate-bound HLA-A2.1 derivative, alpha 3/MalE, in which the alpha 3 domain has been expressed independently of its neighboring polypeptide domains on the native class I MHC molecule and beta 2-microglobulin (beta 2-m). These proteins were produced using eukaryotic and prokaryotic expression systems, respectively. Our data demonstrated specific, saturable binding between soluble CD8 alpha (sCD8 alpha) and alpha 3/MalE, and the Kd of this interaction was determined to be 4.5 x 10(-7) M. Monoclonal antibodies (mAb) directed against either CD8 or the alpha 3 domain of class I MHC inhibited binding; mAb directed against other sites on class I MHC and beta 2-m did not. Our data suggest that the interaction between CD8 alpha and the alpha 3 domain of class I MHC does not require the participation of neighboring class I sequences or beta 2-m.
Mol Immunol 1995 Mar
PMID:Class I MHC alpha 3 domain can function as an independent structural unit to bind CD8 alpha. 772 72

The major histocompatibility complex (Mhc) is a multigene family found in vertebrates. Mhc genes code for heterodimeric cell-surface molecules involved in presentation of peptides to T-lymphocytes. There are two classes of Mhc, and in eutherian mammals four main families of class II genes have been recognized; DR, DQ, DP, and DN/DO. Each class II family contains genes that code for one or more alpha and beta chains. Do the class II genes of marsupial mammals belong to any of these eutherian mammal class II families? The results to date are conflicting. The expressed class II beta-chain genes could not be satisfactorily assigned to any eutherian class II gene family and were designated as new gene families, while, conversely, a partial sequence of an expressed alpha-chain gene was clearly very similar to the DNA gene of eutherian mammals. The aim of this study was to conduct a more thorough analysis of the alpha-chain genes in a marsupial by obtaining full-length sequences of all the expressed alpha-chain genes in the red-necked wallaby, Macropus rufogriseus. Two class II alpha-chain genes were isolated from a spleen-derived cDNA library, and both have the potential to code for fully functional MHC molecules. Phylogenetic analysis indicated they belonged to previously identified eutherian class II families and are designated as Maru-DRA and Maru-DNA. Northern blot data indicated processed transcript sizes of approximately 1.6 kb for Maru-DRA and approximately 2.5 kb for Maru-DNA and that the latter was expressed at a lower level than the former. The phylogeny shows that the DR, DQ, DP, and DN/DO gene families diverged prior to the divergence of the marsupial and eutherian mammal lineages.
Mol Biol Evol 1995 May
PMID:The expressed class II alpha-chain genes of the marsupial major histocompatibility complex belong to eutherian mammal gene families. 773 86

Several mutations within the gene coding for the cardiac beta myosin heavy chain (designed MYH7) have been shown to be responsible for Familial Hypertrophic Cardiomyopathy (FHC) in several families, and evidence of genetic heterogeneity has been reported. To investigate the MYH7 gene as the cause of the disease in a small family with FHC, inheritance of the disease and chromosome 14 q11-q12 markers haplotype were studied, exons coding for the head domain of the cardiac beta myosin heavy chain (beta MHC) were analysed for mutations by MDE gel electrophoresis, and sequenced. We report a mutation within exon eight of the MYH7 gene at a very conserved amino acid at position 232, which results in the conversion of an asparagine to serine. This residue Asn-232 is located in a MHC area that has been recently identified as a critical site for ATPase activity. According to recent results on the three-dimensional structure of the myosin head or subfragment-1 (S1), Asn-232 is located in an alpha-helix which forms part of the nucleotide binding pocket. Although this mutation affects an active site, it seems to be associated with a favourable prognosis and a weak penetrance in this family.
J Mol Cell Cardiol 1994 Sep
PMID:Identification of a mutation near a functional site of the beta cardiac myosin heavy chain gene in a family with hypertrophic cardiomyopathy. 781 66

Identification of CTL epitopes for tumor-specific responses is important for the development of immunotherapies to treat cancer patients. We have developed a strategy to identify potential CTL epitopes based on screening of sequences of target proteins for presence of specific motifs recognized by the most common HLA-A alleles, and identification of high affinity binding peptides using in vitro quantitative assays. A systematic analysis using the sequence of the product of the tumor-associated MAGE-1 gene has been carried out. All possible peptides of nine and ten residues, containing binding motifs for HLA-A1, -A2.1, A-3.2, -A11 and -A24 were synthesized and tested for binding using a quantitative assay. Out of 237 possible peptide/MHC combinations, 47 cases demonstrated good binding affinity (Kd < or = 500 nM). Several peptides were identified as good MHC binders for each one of the five HLA-A alleles studied (five for HLA-A1, 11 for HLA-A2.1, 10 for HLA-A3.2, 16 for HLA-A11 and five for HLA-A24. Furthermore, eight of these peptides were found to bind well to more than one HLA-A allele. These results have important implications for the development of immunotherapeutic vaccines to treat malignant melanoma.
Mol Immunol 1994 Dec
PMID:Identification of potential CTL epitopes of tumor-associated antigen MAGE-1 for five common HLA-A alleles. 782 68

Several recently proposed models for the in vivo biogenesis of class I MHC molecules focus on the retention of empty dimers as a postulated intermediate in the assembly of the complete complexes. The data presented in this study support a slightly different model of class I biogenesis, which includes a precursor population of H-2Db heavy chains (HCs) that is retained in the ER of murine cells prior to its association with beta-2 microglobulin (beta 2m). For this study the intracellular ratios of the subunits that comprise class I molecules have been manipulated to generate a transfected cell line which assembles only very small numbers of unstable H-2Db molecules. Immunoprecipitation experiments with this transfected cell line demonstrated that nascent beta 2m was assembled into complete H-2Db heterotrimers more rapidly than nascent H-2Db HCs by normal murine cells. These data were not consistent with the simultaneous retention of the two associated subunits (HC and beta 2m) in a pool of precursor molecules. However, a previously uncharacterized subset of immature H-2Db HCs, which were not associated with beta 2m, has been detected. These immature HCs exhibited several characteristics of a precursor to complete class I molecules and required a supply of endogenously synthesized peptides for their normal processing in vivo.
Mol Immunol 1995 Feb
PMID:Evidence for an early heavy chain intermediate in the assembly of H-2Db class I MHC molecules. 787 65

We investigated the use of PCR primers designed to conserved exons within nuclear DNA to amplify potentially variable regions such as introns or hypervariable exons from a wide range of species. We then explored various approaches to assay population-level variation in these PCR products. Primers designed to amplify regions within the histone H2AF, myoglobin, MHC DQA, and aldolase (ALD) genes gave clean amplifications in diverse mammals (DQA), and in birds, reptiles and mammals (aldolase, H2AF, myoglobin). The sequenced PCR products generally, but not always, confirmed that the correct locus had been amplified. Several primer sets produced smaller size fragments consistent with preferential amplification of intronless pseudogenes; this was confirmed by sequencing seal and reptile H2AF PCR products. Digestion with randomly selected four-base recognizing enzymes detected variation in some cases but not in others. In species/gene combinations with either low (e.g. seal H2AF, ALD-A) or high (e.g. skink ALD-1) nucleotide diversity it was more efficient to sequence a small number of distantly related individuals (e.g. one per geographic population) and from these data to identify informative or potentially informative restriction enzymes for 'targeted' digestion. We conclude that for studies of population-level variation, the optimal approach is to use a battery of primers for initial PCR of both mtDNA and scnDNA loci, select those that give clean amplifications, and sequence one sample from each population to (i) confirm gene identity, (ii) estimate the amount of variation and, (iii) search for diagnostic restriction sites. This will allow determination of the most efficient approach for a large-scale study.
Mol Ecol 1993 Dec
PMID:Rapid assessment of single-copy nuclear DNA variation in diverse species. 790 60

Individual residues derived from antigenic peptides are believed to control certain mAb epitopes on MHC class I molecules. To further evaluate this influence we studied the antibody recognition of H-2Kb complexed with the antigenic peptides OVA257-264, OVA55-62 and their reciprocally substituted analogs. The mAb 20.8.4, Y-3, EH144 and AF6 reacted strongly with Kb complexed to both OVA peptides and their analogs. However, mAb 28.8.6 bound to Kb/OVA257-264 but not Kb/OVA55-62 complexes. Recognition of Kb/OVA55-62 by mAb 28.8.6 was restored by a single OVA55-62-->OVA257-264 substitution at position 1 (OVA55-62P1K-->S). Recognition by mAb 5F1 was also peptide-dependent in that Kb complexed to either the P4 substituted OVA257-264-->OVA55-62 peptide (OVA257-264P4N-->R) or the P1 substituted OVA55-62-->OVA257-264 peptide (OVA55-62P1K-->S) was not permissive for 5F1 recognition even though Kb complexed to the parental peptides OVA55-62 (P4R) and OVA257-264 (P1S) reacted with this mAb. These data demonstrate that the same peptide residues located within defined positions can exert a negative influence on mAb recognition in one sequence context but be permissive for antibody binding in another context. These findings might be explained by conformational effects on class I heavy chain structure, the extended structure of the peptide or altered orientation of specific peptide side chains located at the same position but within different sequence contexts. Therefore the sequence context of defined amino acids within peptide antigen can strongly influence the fine structural contributions of these residues to MHC-peptide conformation.
Mol Immunol 1994 Oct
PMID:The structural influence of individual residues located within peptide antigen depends upon their sequence context. 793 98

Pulmonary dendritic cells (DC) are potent antigen-presenting cells that are thought to play a critical role in the initiation of immune responses within the lung. Because the lung is both a site of entry into the body for microbial pathogens and the organ of gas exchange, pulmonary immune responses must be meticulously regulated to achieve a balance between host defense and respiration. The initial interaction of DC with T cells in the lung is an excellent point at which to control local immune responses. Studies of the regulation of DC accessory cell function have been greatly hampered by difficulties in obtaining pure populations of pulmonary DC that have not been subjected to prolonged incubations during which the DC may undergo functional alteration. We now describe a method for isolating pulmonary DC from the rat that yields 1 x 10(5) cells/rat with > 90% purity. These cells are potent accessory cells, inducing T cell proliferation in a mixed leukocyte reaction (MLR) at a stimulator-to-responder ratio of 1:1,000. This method, which involves flow cytometric separation of nonphagocytic cells that stain brightly for class II MHC (OX6) from a population of low-density pulmonary interstitial cells, avoids extended incubations at 37 degrees C and thus allows study of a relatively pure population of cells that have functional capacities resembling those of naive cells from the normal lung. With these cells, we demonstrate that the functional capacity of pulmonary DC as stimulator cells in an MLR is significantly increased by exposure to the cytokines interleukin-1 or granulocyte/macrophage colony-stimulating factor (GM-CSF) and by culture with interstitial, but not alveolar, macrophages. Furthermore, DC are heterogeneous with respect to the cell surface expression of receptor for GM-CSF, and this expression is subject to modulation in cell culture. From these studies, we conclude that the immunostimulatory capacity of pulmonary DC is a function of local interactions with cytokines and other parenchymal cells. This suggests that DC function may be an important regulatory point for the local control of pulmonary immune responses.
Am J Respir Cell Mol Biol 1994 Dec
PMID:Regulation of the immunostimulatory activity of rat pulmonary interstitial dendritic cells by cell-cell interactions and cytokines. 794 97

MHC class II immuno-deficiency is a rare autosomal recessive disease due to a defect in transacting genes, which control the expression of the entire family of MHC alpha and beta class II genes. Previous analyses classified cells from eight MHC class II-deficient patients and four experimental mutant cell lines into four complementation groups, pointing to the existence of a large number of regulatory genes. We conducted fusion experiments with cell lines from two-thirds of all known patients and found that two complementation groups accounted for 20 of the 22 cases studied. These two complementation groups correspond closely to two ethnic groups: most patients of north African origin were classified into one group, while all patients originating from Spain were classified into a second main group. This suggests the existence of restricted number of ancestor mutations leading to this disease.
Hum Mol Genet 1994 Jun
PMID:Two complementation groups account for most cases of inherited MHC class II deficiency. 795 Dec 44

A physical chemical model of T cell stimulation by class I-peptide complexes was developed and used to analyse in vitro studies of gamma-interferon release as a function of the number of peptide and MHC molecules. The analysis provided reasonable estimates of well identified parameters, including equilibrium constants and the minimum number of T cell receptor-class I-peptide ternary complexes on a presenting cell required to activate T cells. The latter number was estimated as 3-5 per T cell. This is in distinct contrast to estimates in the literature of the number of peptide-MHC complexes required for activity, which is necessarily larger. The analysis also predicted that activity is potentiated by interaction between class I molecules, even if one member of the pair is not bound by antigen. The analytical approach used in this paper may be applicable to other activation systems.
Mol Immunol 1994 Nov
PMID:Minimal requirements for peptide mediated activation of CD8+ CTL. 796 89


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