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R H Butler

Publications and source records attributed to R H Butler.

At least 19 recordsLinked to original sources
PubMed2001

Allele- and temperature-dependency of in vitro HLA class I assembly.

Allelic variations of in vitro HLA class I assembly have been investigated in both the absence and the presence of binding peptides by flow cytometry using human leukocyte antigen (HLA) class I alpha chains isolated by alkali treatment from cultured HLA homozygous B cells and polystyrene beads coated with anti-HLA class I alpha chain antibodies specific to the C-terminal segment (anti-HLA class I beads). The specificity of assembly was temperature dependent, while the stability of the assembled complex depended on the bound peptide. The efficiency of assembly was allele dependent and primarily ruled by the binding affinity of alpha chains with beta(2)m. Thus, an allele hierarchy could be defined for the binding of HLA-B alpha chain with beta(2)-microglobulin: B7, B18 > B35, B62 > B27, B51. Allele and temperature dependency was found in HLA class I reassembly on acid treated B cells. The HLA class I proteins, reassembled with specific single peptides, could be efficiently transferred to anti-HLA class I beads. These findings would be used to produce microspheres coupled at high surface density with oriented single-peptide loaded HLA class I molecules and also to improve the preparation efficiency of HLA class I tetramers by the use of site-specific biotinylation.

Alleles↗
PubMed2001

Efficient MHC class I-independent amino-terminal trimming of epitope precursor peptides in the endoplasmic reticulum.

MHC class I ligands are produced mainly by proteasomal proteolysis, in conjunction with an unknown extent of trimming by peptidases. Trimming of precursor peptides in the endoplasmic reticulum, a process postulated to be class I dependent, may substantially enhance the efficiency of antigen presentation. However, monitoring of luminal peptide processing has not so far been possible. Here we show that several precursor peptides with amino-terminal extensions are rapidly converted to HLA-A2 ligands by one or several highly efficient metallo-peptidases found on the outer surface of, but also within, microsomes. Surprisingly, luminal trimming is fully active in HLA class I- or TAP-deficient microsomes and precedes peptide association with HLA class I molecules. Trimmed peptides are rapidly depleted from, and become undetectable in, microsomes lacking the restricting class I molecules.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Domain interactions affecting human DNA topoisomerase I catalysis and camptothecin sensitivity.

DNA topoisomerase I (Top1p) relaxes supercoiled DNA by the formation of a covalent intermediate in which the active site tyrosine is transiently bound to the severed DNA strand. The antineoplastic agent camptothecin (Cpt) specifically targets Top1p and several mutations have been isolated that render the enzyme Cpt resistant. The mutated residues, although located in different regions of the enzyme, may constitute part of the Cpt binding site. To begin identifying the structural features of DNA Top1p important for Cpt-induced cytotoxicity, we developed a novel yeast genetic screen to isolate catalytically active, yet Cpt-resistant enzymes from a pool of human top1 mutants. Among the mutations isolated were substitutions of Ser or Val for Gly363, which like the Gly363 to Cys mutation previously reported by us, suppressed the Cpt sensitivity of Top1p. In contrast, each amino-acid substitution differed in its ability to suppress the lethal phenotype and catalytic activity of a human top1 mutant top1T718A that resembles Cpt by stabilizing the covalent intermediate. Biochemical analyses and molecular modeling support a model where interactions between two conserved domains, a central "lip" region containing residue Gly363 and the residues around the active site tyrosine (Tyr723), directly affect the formation of the Cpt-binding site and enzyme catalysis.

Alanine↗
PubMed1996

Differential expression of heat shock protein (HSP70) mRNAs in rat cells.

Heat shock treatment in PC12 cells induces the synthesis of two HSP70 transcripts of 2.55 and 3.05 kb in size, while only a 70-kDa protein is shown by SDS-PAGE. Using reverse transcription-polymerase chain reaction strategy, two mRNA species were amplified from heat-shocked PC12 cells and sequenced. Both cDNAs revealed the same open reading frame encoding a single predicted 641-amino-acid polypeptide of about 70 kDa. The two 3' untranslated regions of the two mRNAs were completely different in both length and composition. Expression of the mRNA of the two corresponding genes and that of another rat HSP70 family member was investigated in PC12, Rat1 cells, and lung fibroblasts. Northern blot analysis revealed that the 2.55- and the 3.05-kb related gene transcripts are differentially expressed in the rat cell lines tested, while the third member of the subfamily is not induced. The single-copy nature of the three genes is also confirmed by Southern blot analysis.

Animals↗
PubMed1995

Differences in peptide-binding specificity of two ankylosing spondylitis-associated HLA-B27 subtypes.

Two HLA-B27 subtypes, B*2702 and B*2705, both associated with ankylosing spondylitis, were tested for binding affinity with a panel of polyalanine model nonapeptides carrying Arg at position 2 (P2) and a series of different amino acids at position 9 (P9). The alpha chains were isolated from BTB(B*2705), C1R/B*2702 (a B*2702 transfectant cell line) and from the NW (B*2702) cell line that has a peculiar peptide presentation behavior. Peptide binding was measured by the HLA alpha chain refolding assay. The results obtained show that: 1) Peptides with basic residues (Arg and Lys) and also aliphatic (Leu) and aromatic (Phe and Tyr) peptides at P9 have a similar high affinity in the binding to B*2705; 2) B*2702 binds well to P9 aliphatic and aromatic peptides but only very weakly to P9 basic peptides. Since both B*2702 and B*2705 are associated with AS the presumed arthritogenic peptide is hypothesized to have an aromatic or aliphatic residue at position 9. Peptides with basic residues in this position would be excluded as candidates because of their low binding affinity with B*2702.

Amino Acid Sequence↗
PubMed1994

Exploring myelin basic protein for HLA class I-binding sequences.

In view of the increasing evidence of the involvement of CD8+ T cells in the pathogenesis of multiple sclerosis (MS), we have scanned the sequence of the myelin basic protein (MBP), using 162 overlapping nonapeptides, for HLA-class I binding sites. Peptide binding was measured using the recently reported HLA class I alpha-chain-refolding assay, and the following HLA allelic products were analyzed: HLA-A2 (*0201, *0204), B27 (*2705), B35, B51 and B62. A considerable number of binding peptides were distinguished for each of the allelic products tested. In addition, three interesting points emerged. The first was the identification of several binding peptides which did not contain the known anchor motifs. The second was the evidence that several peptides showed a promiscuous binding profile, being able to bind to different HLA class I molecules that were either allelic or non allelic. The third was that in several cases two consecutive peptides could bind to the same HLA molecule.

Amino Acid Sequence↗
PubMed1994

The peptide binding specificity of HLA-B27 subtypes.

Five HLA-B27 subtypes, B*2701, B*2703, B*2704, B*2705, and B*2706, were tested for direct binding with twenty-six synthetic nonapeptides carrying the primary anchor residue motifs (combination of amino residues at positions 2 and 9) relevant to B*2705. The peptide sequences were derived from human HSP89 alpha, P53 and MBP. The alpha chains were immunospecifically isolated from LH (B*2701), CH (B*2703), WE1 (B*2704), BTB (B*2705), and LIE (B*2706) cells and their peptide binding was measured by the HLA class I alpha chain refolding assay. The data obtained indicated that the B27 subtypes tested can bind a common set of peptides carrying several different anchor residue motifs. The motifs, R-K and R-R, reported for B*2705 and a new motif H-R were accepted by B*2703, B*2704, and B*2706, but not by B*2701. However, other motifs, including known B*2702 and/or B*2705 motifs, R-H, R-L, R-A, and R-F, and a new motif found here, R-G, were apparently accepted by all B27 subtypes tested. The observed cross-peptide binding in the B27 subgroup is compatible with the so-called arthritogenic peptide hypothesis in the pathogenesis of ankylosing spondylitis.

Amino Acid Sequence↗
PubMed1994

The peptide-binding specificity of HLA-B27 subtype (B*2705) analyzed by the use of polyalanine model peptides.

Model peptides have been used to quantitate the effect on HLA-B*2705 binding of the spacing between primary anchor residues, the type of amino acid accepted in the P9 anchor position, and the type of amino acid accepted in the "secondary anchor positions" P3 and P7. Peptide binding was measured by the HLA class I alpha-chain-refolding assay. The results obtained show that (a) Among the model peptides differing in the spacing between anchor residues, the nonamer with Arg in P2 and Lys in P9 (R2, K9) has the maximum binding with B*2705 molecule. The decamer, with an extra Ala inserted between Arg and Lys (R2, K10), has much lower binding, and still lower binding is observed for the octamer, where an Ala is removed (R2, K8). (b) Besides the "classic" Lys and Arg, several other aminoacids such as Tyr, Leu, Ala, and Gln can be accepted in P9, but with significant differences in binding affinity. (c) Different amino acids in P3 have an influence on peptide binding. Trp and Phe have a favorable influence, whereas Lys and Val appear to hinder the binding. Some variations are seen also for different amino acids in P7.

Amino Acid Sequence↗
PubMed1994

HLA-A2-binding peptides cross-react not only within the A2 subgroup but also with other HLA-A-locus allelic products.

Seven A2-binding peptides were tested by the HLA class I alpha-chain refolding assay previously described for their direct binding to HLA class I alpha chains derived from a panel of 18 HLA-homozygous B-cell lines of various HLA specificities, including four A2 subtypes: A*0201, A*0204, A*0205, and A*0206. All but one test peptide possessed the major anchor residue motifs, L-V, L-L, or I-L, of A2(A*0201)/A2(A*0205)-binding peptides or the closely related motifs, I-V or V-V. This cell panel analysis confirmed the high A2 allele specificity of the test peptides, but also revealed the existence of a broad cross-binding within the A2 subgroup. Most peptides bound to the alpha chains of the A2 subtypes tested, although their binding patterns showed differences. Furthermore, the A2-binding peptides carrying the I-V or V-V motif were found to cross-react also outside of the A2 subtypes, probably with A24, A26, A28, and A29. Other A-locus allelic products, A1, A3, A11, A30, and A31, and the B-locus allelic products carried by the cells tested were essentially negative, although a few exceptions were seen.

Alleles↗
PubMed1993

5-Methylcytosine in genes with methylation-dependent regulation.

An asymmetric distribution of deoxy-5-methylcytidylic acid-inhibiting restriction sites (dcm-sites) takes place in ten human genes regulated by 5-methylcytosine. These genes are dcm-site enriched upstream and dcm-site poor downstream. Along them, there is a scattering of hypermethylatable introns and hypomethylatable exons with a common code: the 5mCpG dinucleotides characterize promoters; Gp5mCs characterize introns; Tp5mCs and Cp5mCs are in small concentrations in exons. Housekeeping genes contain more dcm-sites when compared with tissue-specific genes. This depends on the higher number of dcm-sites in their promoters and introns. In exons, the relatively lower number of dcm-sites is almost the same in both housekeeping and tissue-specific genes. Going from 5' to 3', the average frequency of occurrence of these sites per nucleotide units decreases in introns and increases in exons. This difference is highly discriminated for tissue-specific and less discriminated for housekeeping genes.

5-Methylcytosine↗
PubMed1993

HLA class I binding of synthetic nonamer peptides carrying major anchor residue motifs of HLA-B27 (B*2705)-binding peptides.

Eight nonamer peptides that comply with the major anchor residue motifs (the combination of amino acid residues at positions 2 and 9), R - K and R - R, of HLA-B27 (B*2705)-binding peptides were synthesized and tested for their direct binding to HLA class I alpha chains by the HLA class I alpha chain refolding assay previously described. One was a known B27 (B*2705)-binding heat shock protein peptide, HSP89 alpha (201-209), and the other seven were derived from the sequence of wild-type P53, a human tumor suppressor protein. A total of 36 HLA class I allospecificities were tested. HSP89 alpha (201-209) and two P53 peptides, P53 (362-370) and P53 (378-386), all possessing the motif R - K, bound strongly to B27 (B*2705) alpha chains. A weak binding was seen for P53 (272-280) and P53 (334-342), both showing the motif R - R. Most of these B27-binding peptides were found to bind to A3 alpha chains as well. In addition, P53 (173-181) and P53 (334-342), both with the R - R motif, showed substantial binding with A31 alpha chains. All the peptides carrying the motif R - K also showed weak binding with A31 alpha chains. The remaining two peptides, P53 (201-209) and P53 (282-290), with the motif R - R, did not show significant binding with any of the alpha chains tested. This study demonstrates both the specificity of peptide binding to a given HLA allelic product and the occurrence of cross-peptide-binding between the allelic products of different HLA loci.

Amino Acid Sequence↗
PubMed1993

Unfolded HLA class I alpha chains and their use in an assay of HLA class-I-peptide binding.

Unfolded HLA class I alpha chains were isolated from B-cell lysates by alkaline denaturation and subsequent gel filtration and used for the detection of HLA class-I-peptide binding. Binding to specific peptides in the presence of excess beta 2-microglobulin induced the unfolded alpha chains to refold and acquire a conformation that is specific to folded alpha chains. This conformational change was measured by a specific RIA that involves inhibition of the binding of 125I-labeled HLA-A2 alpha/beta dimers and rabbit anti-HLA-B7 serum absorbed with beta 2-microglobulin. This assay procedure does not require labeling of either test peptides or test class I proteins and does not seem to have specificity degeneracy. It is applicable to the detection of peptide binding by all HLA class I allelic proteins. Evaluation of the assay conditions and HLA allelic specificity of the peptide binding defined by the use of synthetic peptides are described here, including the technical details, specificity, and reproducibility.

Alleles↗
PubMed1991

Global changes in gene expression in Escherichia coli K12 induced by bacteriophage Mu Gem protein.

We have studied the growth properties of some Mu lysogens with respect to the non-lysogenic strain and have observed that the division time in minimal medium was increased over 4-fold when the bacteria carried the prophage mutated in the gem gene (Mu gem3). Since this phage gene has previously been shown to be involved in modulation of expression of host genes, we have analysed the proteins extracted from lysogens and non-lysogens as a rapid assay of global gene expression. The pattern of proteins extracted showed marked quantitative variations between non-lysogens, lysogens for wild-type Mu and lysogens for phage Mu gem3. These effects were no longer as evident when the strains were grown in rich medium. This dramatic change in the physiological state of the lysogenic strain versus the non-lysogenic in particular growth conditions extends the concept of lysogeny. For many years, the prophage has been considered only as a potentially lethal factor, while here it also appears as a genetic element capable of profoundly modifying host biology.

Animals↗
PubMed1990

Mode of action of the schistosomicide hycanthone: site of DNA alkylation.

Condensation of hycanthone N-methylcarbamate (HNMC) with deoxyguanosine (dG) furnished a mixture of the N-1 and N2 adducts which were purified and characterized as their acetates. Condensation of HNMC with thymidine (T) gave the N-3 adduct in poor yield. Adenosine (A) and cytidine (C) did not react with HNMC. Incubation of schistosomes with either [3H]hycanthone (HC) or [3H]HNMC furnished DNA to which [3H]HC was covalently bound. The alkylated DNA was degraded enzymically and the radiolabeled nucleosides were separated using HPLC. Two major peaks were observed which coincided in retention time with the synthetic N-1 and N2 alkylated dG. Alkylated T was absent. Thus, the site of alkylation of DNA by either HC or HNMC is dG.

Alkylation↗
PubMed1985

Interleukin-1 production by a cloned line of human monocyte-like cells (CM-SM). Correlation with state of differentiation.

Interleukin-1 (IL-1) production by the human monocyte-like cloned cell line CM-SM has been investigated as a function of the state of cell differentiation. CM-SM cells were induced to differentiate along the monocyte-macrophage lineage by bacterial lipopolysaccharides (LPS) or by 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Cell differentiation was studied by various morphological, functional, cytochemical, and immunological variables, whereas IL-1 activity in the supernatants was measured by the lectin-primed thymocyte proliferation assay. Unstimulated CM-SM cells constitutively produced small amounts of IL-1, and most of the cells appeared relatively undifferentiated. LPS induced cell differentiation, but the effect was reversible, and the cells, in general, did not acquire a capacity for phagocytosis. IL-1 levels were increased about 10-fold over the controls. TPA induced further cell differentiation to macrophage-like cells capable of phagocytosis. IL-1 activity could not be measured directly in the supernatants owing to the synergistic effect of TPA in the assay system. Unequivocal removal of the phorbol was not achieved, but the data indicated that the 'real' levels of IL-1 in the TPA-induced cultures were not significantly higher than those from LPS-induced cultures.

Cell Differentiation↗
PubMed1984

Growth factors that stimulate human granulocyte-macrophage colony formation produced by the cell line CM-S.

CM-S is an autonomous cell line of human hemopoietic precursor cells inducible to monocyte-macrophage differentiation in response to appropriate inducing agents. CM-S cells produce factors that stimulate their own growth and proliferation, and are also capable of stimulating clonal proliferation of human, but not mouse, monocytic and granulocytic bone marrow progenitor cells in viscous medium. Preliminary purification steps have demonstrated at least two species, one of which (MW 30,000-50,000) retains both these activities, while the other (MW less than or equal to 10,000) apparently retains only the autostimulatory activity. CM-S cells could thus be a useful source for the purification of human colony stimulating factors (CSFs). CM-S cells also respond to factors present in human placenta conditioned medium, known to contain human CSF. This suggests that CM-S cells could provide a homogeneous target cell population for testing CSFs from other human sources.

Animals↗
PubMed1983

Constitutive production of Interleukin-1 by the human continuous cell line, CM-S.

Supernatants from the human continuous cell line, CM-S, have been tested for Interleukin-1 (IL-1) activity and found to constitutively elaborate this factor. The CM-S-produced IL-1 activity has been partially purified and shown to be similar to IL-1 produced by human and mouse peripheral blood macrophages. To the best of our knowledge this is the first continuous human cell line reported that produces this monokine.

Cell Line↗