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G W Butcher

Publications and source records attributed to G W Butcher.

At least 19 recordsLinked to original sources
PubMed1995

LMP2+ proteasomes are required for the presentation of specific antigens to cytotoxic T lymphocytes.

BACKGROUND: Major histocompatibility complex (MHC) class I molecules present short peptides generated by intracellular protein degradation to cytotoxic T lymphocytes (CTL). The multisubunit, non-lysosomal proteinases known as proteasomes have been implicated in the generation of these peptides. Two interferon-gamma (IFN-gamma)-inducible proteasome subunits, LMP2 and LMP7, are encoded within the MHC gene cluster in a region associated with antigen presentation. The incorporation of these LMP subunits into proteasomes may alter their activity so as to favour the generation of peptides able to bind to MHC class I molecules. It has been difficult, however, to demonstrate a specific requirement for LMP2 or LMP7 in the presentation of peptide epitopes to CTL. RESULTS: We describe a T-cell lymphoma, termed SP3, that displays a novel selective defect in MHC class I-restricted presentation of influenza virus antigens. Of the MHC-encoded genes implicated in the class I pathway, only LMP2 is underexpressed in SP3 cells. Expression of IFN-gamma in transfected SP3 cells simultaneously restores LMP2 expression and antigen presentation to CTL. Expression of antisense-LMP2 mRNA in these IFN-gamma-transfected cells selectively represses antigen recognition and the induction of surface class I MHC expression. Moreover, the expression of this antisense-LMP2 mRNA in L929 fibroblast cells, which constitutively express LMP2 and have no presentation defect, blocks the presentation of the same influenza virus antigens that SP3 cells are defective in presenting. CONCLUSIONS: Our results show that the LMP2 proteasome subunit can directly influence both MHC class I-restricted antigen presentation and class I surface expression.

Animals↗
PubMed1995

Alloreactive natural killer cells in the rat: complex genetics of major histocompatibility complex control.

A major role for the nonclassical major histocompatibility complex (MHC) class I region, i.e. RT1.C, in controlling rat natural killer (NK) cell alloreactivity has recently been established, and several findings suggested the existence of NK-triggering alloantigens coded for by this region. Here, we have extended our studies on the MHC control of NK cell cytotoxicity against concanavalin A-activated T cell blasts by comparing semi-syngeneic and fully allogeneic combinations, and we show the following: (a) The self MHC exerted a strong influence on the NK allorecognition repertoire. (b) When anti-F1 hybrid cytolytic activities of parental strain NK cells were measured, both recessively and non-recessively inherited susceptibility patterns emerged. (c) In most combinations parental strain cells were lysed by F1 hybrid NK cells, thus resembling the hybrid resistance phenomenon described in mice. The cytotoxicity was lower in strain combinations where NK susceptibility was inherited non-recessively, i.e. when parent anti-F1 reactivity was detected, than in recessive combinations. (d) LEW.1LM1 (RT1lm1) target cells, with a deletion in the RT1.C region that includes expressed class I genes, were more sensitive to lysis by MHC matched NK cells (PVG.1L(LEW), RT1l) than were parental LEW (RT1l) cells. The effect of the deletion was the opposite when MHC allogeneic (RT1c, RT1u) as well as semi-syngeneic (RT1l/c) NK cells were employed, i.e. sensitivity was decreased. We conclude that certain MHC-encoded antigens, depending on the haplotype combination of effector and target cells, may either trigger or inhibit rat NK cell cytotoxicity. Furthermore, the potential role of peptides bound to MHC class I molecules recognized by NK cells is discussed.

Alleles↗
PubMed1994

Selectivity of MHC-encoded peptide transporters from human, mouse and rat.

Major histocompatibility complex (MHC) class I molecules present peptides from degraded intracellular antigens to CD8+ T cells. These peptides are translocated in an ATP-dependent fashion into the lumen of the endoplasmic reticulum (ER) for binding to class I molecules by means of the MHC-encoded transporters associated with antigen processing, TAP1 and TAP2. These are members of a family of proteins containing an ATP-binding cassette and form heterodimers in the ER membrane. Defects in the genes encoding TAP1 or TAP2 account for impaired class I assembly and antigen presentation in several human and rodent cell lines. Whereas MHC class I molecules select peptides according to binding motifs, it is not clear to what extent the TAP1-TAP2 transporters have peptide sequence and length specificity. Previous studies of the rat MHC class I molecule RT1Aa, suggested a specific conveyance of peptides by rat TAP1-TAP2. Here we substitute the amino- and carboxy-terminal and the penultimate amino-acid residues of model peptides to show that these residues influence the efficiency of transport. Human TAP and rat TAPa translocated peptides with hydrophobic and basic C termini, whereas mouse TAP and rat TAPu preferred peptides with hydrophobic C termini. This pattern correlates with the predominant peptide binding profiles of mouse and human class I molecules.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Expression cloning of an equine T-lymphocyte glycoprotein CD2 cDNA. Structure-based analysis of conserved sequence elements.

An equine CD2 cDNA has been isolated by monoclonal antibody screening of a T-lymphocyte cDNA library. The cDNA contained an open reading frame of 1041 bp encoding a translated product of 347 amino acids. Northern blotting analysis revealed a single mRNA species expressed in spleen, thymus and activated peripheral lymphocytes. The predicted amino acid sequence has 50-65% identity with the human, rat and mouse CD2 sequences with greatest similarity shared with the human homologue. Evolutionarily conserved structural and functional domains in CD2 were identified by comparing the sequences of the equine, human, mouse and rat CD2 homologues in the context of the recently derived crystal structure of rat soluble CD2 [Jones, E. Y., Davis, S. J., Williams, A. F., Harlos, K. & Stuart, D. I. (1992) Nature 360, 232-239]. The key conserved features of the extracellular region included core residues necessary to preserve the structural integrity of the molecule, residues in the linker region likely to maintain the unique domain organization of CD2, an array of highly charged residues in the putative ligand-binding face of the molecule and glycosylation-signal distributions that render the putative ligand-binding GFCC'C" face of domain 1 relatively unhindered by glycosylation.

Amino Acid Sequence↗
PubMed1994

The distribution of Tap2 alleles among laboratory rat RT1 haplotypes.

We are reporting the cDNA sequences of Tap2 from two cima and two cimb rat strains. Comparison of the cDNA sequences shows that these alleles fall into two groups, which we refer to as Tap2-A and Tap2-B. We found that alleles from the Tap2-B group are more closely related to the mouse homologue than are Tap2-A alleles, and among the 48 nucleotides which differ between the Tap2-A and Tap2-B cDNAs, three affect restriction sites. We defined pairs of oligonucleotides which allow amplification of the regions bearing these restriction sites from genomic DNA or cDNA, and this technique has been successful for the genotyping of all of the 56 laboratory strains of Rattus norvegicus tested and for five cell lines tested so far. All 14 known RT1 standard haplotypes were tested, and 7 found to belong to the Tap2-B group, and 7 to Tap2-A. We also found that intron sizes among the alleles of the Tap2-B group fall into two subgroups, providing further insight into the phylogeny of these various haplotypes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

A mammalian homologue of Drosophila heterochromatin protein 1 (HP1) is a component of constitutive heterochromatin.

The Drosophila HP1 gene contains a highly conserved sequence, the chromobox, which can be used to isolate HP1-like genes from both mouse (M31 and M32) and man (HSM1) (Singh et al., 1991). Here we report that a monoclonal antibody (MoAb) raised against the M31 protein recognises a 26-kDa protein in murine and human nuclear extracts and localises to large masses of condensed chromatin within murine interphase nuclei, some of which are associated with the nucleoli. At metaphase, the MoAb binds to the centromeres of both human and murine chromosomes. The evolutionary conservation of this chromosomal localisation indicates that the M31 protein is likely to be important in the packaging of mammalian chromosomal DNA into constitutive heterochromatin.

3T3 Cells↗
PubMed1994

Cytoskeletal proteins of insect muscle: location of zeelins in Lethocerus flight and leg muscle.

Asynchronous insect flight muscles produce oscillatory contractions and can contract at high frequency because they are activated by stretch as well as by Ca2+. Stretch activation depends on the high stiffness of the fibres and the regular structure of the filament lattice. Cytoskeletal proteins may be important in stabilising the lattice. Two proteins, zeelin 1 (35 kDa) and zeelin 2 (23 kDa), have been isolated from the cytoskeletal fraction of Lethocerus flight muscle. Both zeelins have multiple isoforms of the same molecular mass and different charge. Zeelin 1 forms micelles and zeelin 2 forms filaments when renatured in low ionic strength solutions. Filaments of zeelin 2 are ribbons 10 nm wide and 3 nm thick. The position of zeelins in fibres from Lethocerus flight and leg muscle was determined by immunofluorescence and immunoelectron microscopy. Zeelin 1 is found in flight and leg fibres and zeelin 2 only in flight fibres. In flight myofibrils, both zeelins are in discrete regions of the A-band in each half sarcomere. Zeelin 1 is across the whole A-band in leg myofibrils. Zeelins are not in the Z-disc, as was thought previously, but migrate to the Z-disc in glycerinated fibres. Zeelins are associated with thick filaments and analysis of oblique sections showed that zeelin 1 is closer to the filament shaft than zeelin 2. The antibody labelling pattern is consistent with zeelin molecules associated with myosin near the end of the rod region. Alternatively, the position of zeelins may be determined by other A-band proteins. There are about 2.0 to 2.5 moles of myosin per mole of each zeelin. The function of these cytoskeletal proteins may be to maintain the ordered structure of the thick filament.

Animals↗
PubMed1993

Monoclonal antibodies to phosphatidylinositol 4-phosphate 5-kinase: distribution and intracellular localization of the C isoform.

We have raised a panel of monoclonal antibodies to PtdIns4P 5-kinase C purified from bovine brain [Divecha, Brooksbank and Irvine (1992) Biochem. J. 288, 637-642]. This panel includes antibodies which specifically recognize PtdIns4P 5-kinase C both in a native catalytically active condition, and/or when presented on Western blots. Some of the former antibodies will also inhibit PtdIns4P 5-kinase C activity. We have used the blotting antibodies to study the bovine tissue distribution of PtdIns4P 5-kinase C and its distribution in mammalian species. We have also studied its localization in Jurkat cells and found it to be predominantly bound to membranes, with only a minority localized to the cytoskeleton. Neither PtdIns4P 5-kinase activity nor PtdIns4P 5-kinase C, as detected by Western blotting, were increased in the cytoskeleton after stimulation of Jurkat cells with OKT3. These antibodies should prove to be extremely useful tools with which to study the regulation of PtdIns4P 5-kinase C.

Animals↗
PubMed1993

Differential effect of transporter Tap 2 gene introduction into RMA-S cells on viral antigen processing.

The protein products of the Tap (Transporter associated with antigen processing) 1 and 2 genes are presumed to deliver peptides across the endoplasmic reticulum (ER) for assembly with major histocompatibility complex (MHC) class I molecules. The antigen processing-defective cell line RMA-S (H-2b) has a premature stop in the Tap 2 gene and probably therefore fails to deliver peptides into the ER, which leads to a low level of cell surface MHC class I molecules. Transfection of a Tap 2 gene restores to RMA-S both MHC class I molecule expression and the ability to present influenza viral antigens. We investigated the ability of RMA-S cells transfected with a Tap 2 gene to process and present alloantigens, Sendai and Rauscher viral antigens to allogeneic and virus-specific cytotoxic T lymphocytes. We found that allogeneic peptides as well as Rauscher and Sendai viral peptides can be processed and presented by RMA-S but at reduced levels. Transfection of a Tap 2 gene of mouse (BALB/c, H-2d) or rat origin into RMA-S increased the presentation of Sendai viral antigens and partially restored the presentation of allogeneic antigens. The already low level of Rauscher viral peptides presented by RMA-S is not elevated by transfection of either Tap 2 gene into RMA-S. This indicates a differential effect of transfection of a Tap 2 gene of rat or allogeneic mouse origin into RMA-S on viral antigen processing.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Lymphoma in the BB/E rat: c-myc translocation identified.

In a prospective necropsy study involving 257 animals in the BB rat colony in Edinburgh, there was an increased incidence of lymphoma (average 10.9 per cent in all three subgroups: 8 per cent in diabetic, 20 per cent in non-diabetic diabetes-prone, and 3.2 per cent in diabetes-resistant rats). The incidence was significantly increased (P < 0.05) in the non-diabetic diabetes-prone subgroup. These results differ markedly from previous results and indicate that the relationship between lymphoma and diabetes is more complex than previously suggested. All the lymphomas bar one involved the ileocaecal nodes and were classified as immunoblastic lymphomas of B-cell origin. There was a striking resemblance both in tissue distribution and in histological classification to the lymphoma seen in the established B-cell lymphoma model, the LOUVAIN rat. Southern blot analysis carried out on the BB rat lymphomas revealed a translocation of variable length involving the c-myc oncogene. Such a translocation has not been demonstrated in the BB rat before.

Animals↗
PubMed1993

BB-DR/Edinburgh: a lymphopenic, non-diabetic subline of BB rats.

Diabetes-prone (DP) and diabetes-resistant (DR) sublines of the BB rat have been established in Edinburgh, U.K., separately from other existing colonies. In an examination of the lymphoid status of the two lines, BB-DP/Ed and BB-DR/Ed, it has been found that both lines have very low T-cell numbers, depressed B-lymphocyte numbers and a complete absence of peripheral CD8+ T cells, all features characteristic of the previously described genetic lymphopenia lesion. It was also noted that the peripheral T cells of both BB/Ed lines were larger than normal. The DP/Ed and DR/Ed lines were indistinguishable in all these respects, and furthermore, they were both shown to type as RT1u at the major histocompatibility complex (MHC). The genetic combination of lymphopenia and RT1u without expression of diabetes is not present in other extant BB lines and makes BB-DR/Ed a uniquely useful control strain for BB rat studies as well as a valuable genetic resource for the further genetic analysis of diabetes susceptibility in rats.

Animals↗
PubMed1992

Proteasome subunits encoded by the major histocompatibility complex are not essential for antigen presentation.

Major histocompatibility complex (MHC) class I molecules bind and deliver peptides derived from endogenously synthesized proteins to the cell surface for survey by cytotoxic T lymphocytes. It is believed that endogenous antigens are generally degraded in the cytosol, the resulting peptides being translocated into the endoplasmic reticulum where they bind to MHC class I molecules. Transporters containing an ATP-binding cassette encoded by the MHC class II region seem to be responsible for this transport. Genes coding for two subunits of the '20S' proteasome (a multicatalytic proteinase) have been found in the vicinity of the two transporter genes in the MHC class II region, indicating that the proteasome could be the unknown proteolytic entity in the cytosol involved in the generation of MHC class I-binding peptides. By introducing rat genes encoding the MHC-linked transporters into a human cell line lacking both transporter and proteasome subunit genes, we show here that the MHC-encoded proteasome subunit are not essential for stable MHC class I surface expression, or for processing and presentation of antigenic peptides from influenza virus and an intracellular protein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Effect of polymorphism of an MHC-linked transporter on the peptides assembled in a class I molecule.

Short antigenic peptides bound in the groove of class I major histocompatibility complex molecules enable T cells to detect intracellular pathogens. It has been assumed that structural features of the class I molecule alone select which peptides are bound. It is now demonstrated that a complex polymorphism in one of the major histocompatibility complex-encoded putative peptide-transporter genes is associated with an altered spectrum of bound peptides.

Alleles↗