A single amino acid substitution in an MHC class I molecule allows heteroclitic recognition by lymphocytic choriomeningitis virus-specific cytotoxic T lymphocytes

Class I molecules of the MHC bind foreign and endogenous peptides allowing recognition by the TCR on CTL. The recognition and killing of cells infected with lymphocytic choriomeningitis virus (LCMV) depends on the recognition of LCMV peptides bound to class I MHC. Mutations in class I MHC molecules have enabled the delineation of regions in the class I molecule important for binding peptides and for interaction with the TCR. We have constructed a library of class I mutants using saturation mutagenesis and report a phenotypic change resulting from a single amino acid substitution that results in the heteroclitic (increased) killing of LCMV-infected cells. This amino acid change, asparagine to serine at position 30, is in a conserved region of the class I molecule contacting the alpha 3 domain. This mutation does not result in increased expression of the class I molecule on the cell surface, does not affect the binding of CD8, and does not affect allogeneic recognition. Cold target experiments show that this heteroclitic killing is due to increased recognition by CTL. These data point toward a critical function for this region of the class I molecule in the binding of peptides or their presentation to CTL.     

Conservation of binding site specificity of three yeast DNA binding proteins.

Sequence specific binding of protein extracts from 13 different yeast species to three oligonucleotide probes and two points mutants derived from Saccharomyces cerevisiae DNA binding proteins were tested using mobility shift assays. The probes were high affinity binding sites for GRF1/RAP1/ABF1 and CP1/CPF1. Most yeasts in the genus Saccharomyces showed specific binding to all three probes and also displayed similar sequence requirements when challenged by molar excesses of mutant probes. The affinities for the probes varied amongst the other yeasts tested, but in general, CPF1 binding activity was the most widespread, while the other two were more limited.     

Erythropoietin induces the tyrosine phosphorylation of its own receptor in human erythropoietin-responsive cells.

Using the human erythropoietin-responsive hematopoietic cell line UT-7, we showed that erythropoietin (Epo) rapidly and specifically induced the tyrosine phosphorylation of its own receptor (M(r) 75,000) and increased the tyrosine phosphorylation of other proteins of M(r) 140,000, 120,000, 95,000, 60,000, 57,000, and 42,000. Neither granulocyte-macrophage colony-stimulating factor, interleukin 3, interleukin 6, nor the kit ligand induced the phosphorylation of the M(r) 75,000 receptor protein, although these growth factors induced the phosphorylation of other proteins. Cross-linking experiments using 125I-Epo indicated that the UT-7 cells expressed three Epo receptor subunits, of M(r) 100,000, 85,000, and 75,000, among which only the M(r) 75,000 subunit was tyrosine-phosphorylated following activation with Epo.     

Monitoring DNA cytometric parameters during the course of chronic myelogenous leukemia.

The prognostic value of three DNA cytometric parameters--stemline ploidy (STL), stemline shoulder fraction (SSF) and "proliferative" fraction (PRF)--for the prediction of disease transformation and survival was examined for 20 patients with chronic myelogenous leukemia (CML) during the course of their disease and compared with two commonly used hematologic parameters (degree of leukocytosis and percentage of circulating leukemic progenitor cells). With disease progression, STL and SSF increased significantly, whereas PRF showed a steady decrease from diagnosis to blast crisis. The most significant part of these changes took place during the chronic phase, before the clinical onset of disease transformation. Hematologic parameters, in comparison, revealed significant changes later, shortly before blast crisis. The remaining duration of the chronic phase diminished from 25.5 months at the time of diagnosis, when the median STL was 2.0c, to 19.6 months for patients showing an STL of 2.1c, to 15.0 months with an STL of 2.2c and to 1.0 months for those with an STL of greater than or equal to 2.3c. Prognostically relevant limits for SSF and PRF were at 20%. When the SSF passed this limit or the PRF fell below it, the mean remaining chronic phase of these patients amounted to only 14.1 and 10.1 months. Interactive cytometry allows analysis of the DNA cytometric equivalent of changes in leukemic progenitor cells, which are well known from cytogenetic and cell kinetic studies. These three DNA cytometric parameters reflect the "natural history" of CML with the development of a cytogenetically hyperdiploid clone during disease progression in most patients and a simultaneous loss of proliferative potential on the level of myelobasts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunochemical localization of the C-terminal hexapeptide of histone H3 at the surface of chromatin subunits.

The C-terminal hexapeptide of histone H3 of chicken erythrocytes (residues 130-135) corresponding to the sequence Ile-Arg-Gly-Glu-Arg-Ala ( IRGERA ) was prepared by solid-phase peptide synthesis and, after coupling to bovine serum albumin, was used to elicit antibodies in rabbits. The antigenic activity of the synthetic peptide IRGERA was found to be very similar to that of the natural CN3 fragment (residues 121-135), and it inhibited the H3-anti H3 reaction in complement fixation, solid-phase radioimmunoassay, and enzyme-linked immunosorbent assay. Antibodies induced by IRGERA were found to bind equally well to IRGERA coupled to hemocyanin, to the intact H3 molecule, and to chromatin subunits (nucleosomes and core particles). The results demonstrate that the C-terminal hexapeptide of histone H3 is located at the surface of chromatin subunits and agree with current models proposed for the spatial organization of the chromatin core particle.     

Correlation between cell density,membrane fluidity,and the availability of transferrin receptors in friend erythroleukemic cells

Undifferentiated Friend erythroleukemic cells (FL cells) acquire membrane microviscosity ( ), in accord with the culture cell density. At low cell density poise, whereas at confluency it increases to poise. Concomitantly, the total number of available transferrin receptors per cell decreases by about 80% upon increase in cell density. Modulation of membrane microviscosity, by artificial alteration of the membrane cholesterol level, mediates similar modulations of the availability of the transferrin receptors. The correlation between the availability of the transferrin receptors and the membrane lipid fluidity may take part in the overt decrease in iron uptake by erythroid cells along the erythropoiesis pathway.     

Binding of ADP to beef-heart mitochondrial ATPase (F1)

1. ADP binding to beef-heart mitochondrial ATPase (F₁), in the absence of Mg²⁺, has been determined by separating the free ligand by ultrafiltration and determining it in the filtrate by a specially modified isotachophoretic procedure.2. Since during the binding experiments the ‘tightly’ bound ADP (but not the ATP) dissociates, it is necessary to take this into account in calculating the binding parameters.3. The binding data show that only one tight binding site (K_d about 0.5 μM) for ADP is present.4. It is not possible to calculate from the binding data alone the number of or the dissociation constants for the weak binding sites. It can be concluded, however, that the latter is not less than about 50 μM.