Search PubMed⌕ Search

Biomedical subjects

K D Smith

Publications and source records attributed to K D Smith.

At least 55 records · Page 3Linked to original sources
PubMed1998

Suppression of peroxisomal membrane protein defects by peroxisomal ATP binding cassette (ABC) proteins.

X-Linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder characterized by reduced peroxisomal very long chain fatty acid (VLCFA) beta-oxidation. The X - ALD gene product (ALDP) is a peroxisomal transmembrane protein with an ATP binding cassette (ABC). ALDP and three other ABC proteins (PMP70, ALDR, P70R) localize to the peroxisomal membrane. The function of this family of peroxisomal membrane proteins is unknown. We used complementation studies to begin analysis of their role in VLCFA beta-oxidation and on the peroxisomal membrane. Expression of either ALDP or PMP70 restores VLCFA beta-oxidation in X-ALD fibroblasts, indicating overlapping functions. Their expression also restores peroxisome biogenesis in cells that are deficient in the peroxisomal membrane protein Pex2p. Thus it is likely that complex protein interactions are involved in the function and biogenesis of peroxisomal membranes that may contribute to disease heterogeneity.

ATP Binding Cassette Transporter, Subfamily D, Mem↗
PubMed1998

Sampling protocols for the detection of smokeless powder residues using capillary electrophoresis.

Present techniques for the detection of gunshot residue rely primarily metallic primer components such as lead, barium and antimony. With the advent of reformulated primers that reduce or eliminate these elements, new methods for the detection of organic residue compounds will be needed. Micellar electrokinetic capillary electrophoresis (MECE) is one technique that has been successfully applied to the analysis of these smokeless powder residue compounds. Protocols for the recovery of the organic residue components under a variety of sampling conditions were evaluated and improved for MECE analysis. The collection of residue samples where external contaminants such as grease or blood were present on the residue substrate were investigated using both tape lifts and solvent swab protocols. In addition, residue component recovery using supercritical fluid extraction techniques was preliminarily evaluated for samples contaminated with blood.

Blood Chemical Analysis↗
PubMed1997

A mouse model for X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal disorder with impaired beta-oxidation of very long chain fatty acids (VLCFAs) and reduced function of peroxisomal very long chain fatty acyl-CoA synthetase (VLCS) that leads to severe and progressive neurological disability. The X-ALD gene, identified by positional cloning, encodes a peroxisomal membrane protein (adrenoleukodystrophy protein; ALDP) that belongs to the ATP binding cassette transporter protein superfamily. Mutational analyses and functional studies of the X-ALD gene confirm that it and not VLCS is the gene responsible for X-ALD. Its role in the beta-oxidation of VLCFAs and its effect on the function of VLCS are unclear. The complex pathology of X-ALD and the extreme variability of its clinical phenotypes are also unexplained. To facilitate understanding of X-ALD pathophysiology, we developed an X-ALD mouse model by gene targeting. The X-ALD mouse exhibits reduced beta-oxidation of VLCFAs, resulting in significantly elevated levels of saturated VLCFAs in total lipids from all tissues measured and in cholesterol esters from adrenal glands. Lipid cleft inclusions were observed in adrenocortical cells of X-ALD mice under the electron microscope. No neurological involvement has been detected in X-ALD mice up to 6 months. We conclude that X-ALD mice exhibit biochemical defects equivalent to those found in human X-ALD and thus provide an experimental system for testing therapeutic intervention.

Adrenoleukodystrophy↗
PubMed1997

Cytotoxic CD8+ T cells recognize EBV antigen but poorly kill autologous EBV-infected B lymphoblasts: immunodominance is elicited by a peptide epitope that is presented at low levels in vitro.

CD8+ T cells play a crucial role in surveillance against EBV-associated malignancies. EBV-specific T cells traditionally have been identified by their ability to kill autologous EBV-transformed B lymphoblastoid cell lines (LCL). Here we report CD8+ cloned and bulk T cells that specifically recognize EBV nuclear Ag EBNA-3C, but do not efficiently kill autologous or HLA-matched LCL. The low cytolysis of these T cells was due to the extremely low density of the antigenic epitope (LDFVRFMGV, EBNA-3C amino acids 285-293) on autologous LCL. The T cells efficiently killed target cells in the presence of < 1 pM synthetic EBNA-3C peptide and, therefore, recognize peptide/HLA complexes with high avidity. Donor T cells with this phenotype were stimulated by autologous LCL and dominated the in vitro EBV-specific response. This indicates that low abundance viral peptides can induce a dominant T cell response.

Amino Acid Sequence↗
PubMed1997

Alloreactive cytotoxic T lymphocytes focus on specific major histocompatibility complex-bound peptides.

Alloreactive T cells are often specific for individual peptides that are bound to allogeneic major histocompatibility complex (MHC) molecules. Other alloreactive T cells are reported to be peptide-independent or to recognize MHC conformational changes that are induced by multiple peptides. We tested 12 anti-HLA-B7 alloreactive cytotoxic T lymphocyte (CTL) clones that bind a restricted region of HLA-B7, including three CTL clones that were generated in a protocol designed to stimulate peptide-independent T cells. All 12 CTLs recognized multiple point mutations in the HLA-B7 peptide-binding groove. Eleven of the 12 CTLs recognized specific peptides that eluted in one or two fractions on high-performance liquid chromatography (HPLC). None of the CTLs promiscuously recognized 16 HLA-B7-binding synthetic peptides, although one CTL recognized minor by-products in one synthetic peptide preparation. CTL clone KID-9 cross-reacted with allogeneic HLA-B7 and HLA-B27 molecules and recognized a distinct peptide bound to each MHC molecule. CTL clone KD-11 recognized peptides that eluted in two HPLC fractions and recognized HLA-B7-transfected peptide antigen processing defective T2 cells. These results indicate that CTL allorecognition is peptide-specific whether the allogeneic MHC molecules are expressed on normal cells or antigen processing-deficient cells.

Antigen Presentation↗
PubMed1997

Alloreactive T cell recognition of MHC class I molecules: the T cell receptor interacts with limited regions of the MHC class I long alpha helices.

T cells recognize MHC-bound peptide, suggesting that the TCR contacts surface MHC residues adjacent to bound peptide, but the extent of MHC contact is not known. T cells also may recognize peptide-induced conformational changes, and alloreactive T cells may recognize surface MHC structures in addition to or independent of bound peptide. Alloreactive T cells are not intentionally biased to recognize particular MHC-bound peptides and should reveal general constraints for TCR binding. To map TCR binding sites, we tested 60 HLA-B7 site-specific mutations with 12 alloreactive CTL clones that express different TCRs. The alloreactive CTL clones recognize solvent-accessible residues that cluster between positions 62 to 80 and 150 to 170. Thus, TCRs contact largely overlapping MHC structures in the alpha1 and alpha2 domain long alpha helices. The dimensions and location of this site are consistent with recently reported crystallographic studies of two TCR/peptide-MHC class I complexes. In contrast to TCR, Abs recognize multiple discrete epitopes that encircle the peptide binding groove and potentially encompass the entire surface of the MHC molecule. Our data suggest that TCRs dock with a common discrete MHC site and that recent crystallographic models are likely to be generally applicable to T cell recognition of peptide-MHC class I complexes.

Amino Acid Sequence↗
PubMed1997

Investigation into the concanavalin A reactivity, fucosylation and oligosaccharide microheterogeneity of alpha 1-acid glycoprotein expressed in the sera of patients with rheumatoid arthritis.

alpha 1-Acid glycoprotein (AGP) exists as an heterogeneous population of glycosylated variants (glycoforms) in plasma. The concentration of AGP increases some 2-5 fold in certain pathophysiological states exemplified by the chronic inflammatory disease, rheumatoid arthritis (RA). Moreover, the expressed glycosylation pattern alters in such conditions, indicating functional significance that is likely to be related to the oligosaccharide heterogeneity. We have investigated the heterogeneity of AGP glycosylation using the technique of high pH anion-exchange chromatography (HPAEC). AGP was isolated from the blood of RA sufferers, partially separated by Concanavalin A (Con A) affinity chromatography into bound and non-bound fractions and was enzymatically deglycosylated. Chromatography on the pellicular HPAE resin at pH 13 separated the released oligosaccharides and allowed a comparison of profiles in terms of branching and fucosylation. Results demonstrate an abnormal RA AGP glycosylation, with a tendency towards tri- and tetra-antennary oligosaccharides and enhanced fucosylation, in addition to the possible existence of penta-sialylated RA AGP glycoforms.

Arthritis, Rheumatoid↗
PubMed1997

Conformational changes in MHC class I molecules. Antibody, T-cell receptor, and NK cell recognition in an HLA-B7 model system.

In this article we review the role of MHC conformation, including peptide-induced MHC conformation, in forming antibody (Ab), T-cell receptor (TCR), and natural killer (NK) cell receptor epitopes. Abs recognize conformational major histocompatibility (MHC) epitopes that often are influenced by the identity of MHC-bound peptide. Diverse TCRs recognize a common docking site on peptide/MHC complexes and directly contact peptide. Human NK cell inhibitory receptors (KIR) appear to recognize limited regions of the HLA alpha (1) helix. DX9+ KIR specifically focus on HLA-B residues 82 and 83. However, NK cells recognize much broader regions of HLA class I molecules and are sensitive to bound peptides. Thus, several classes of lymphocyte receptors are peptide-specific. Peptide specificity could be the result of direct contact with the receptor, or to conformational shifts in MHC residues that interact with both receptor and bound peptide.

Animals↗
PubMed1997

Human leukocyte antigens and cytokine expression in cerebral inflammatory demyelinative lesions of X-linked adrenoleukodystrophy and multiple sclerosis.

The two most common forms of X-linked adrenoleukodystrophy (X-ALD) are the cerebral forms (CER) with an inflammatory demyelinating reaction that resembles multiple sclerosis, and adrenomyeloneuropathy (AMN) which involves primarily the spinal cord and in which the inflammatory reaction is mild or absent. We found no significant association between the childhood cerebral form (CCER) or AMN and the human leukocyte (HLA) class I and Class II antigens including the class II DR2 haplotypes associated with multiple sclerosis. Inflammatory cytokine (tumor necrosis factor-alpha, interleukin-1 beta, interleukin-4, interleukin-6 and interferon-gamma) gene expression was increased in multiple sclerosis brain lesions, as has been reported previously, but much less so in CER brain lesions. These findings suggest that the pathogenesis of the inflammatory response in X-ALD differs from that in multiple sclerosis.

Adrenoleukodystrophy↗
PubMed1997

The relative importance of the X-linked FCP locus and beta-globin haplotypes in determining haemoglobin F levels: a study of SS patients homozygous for beta S haplotypes.

Five factors have been hypothesized to influence the 20-fold variation in fetal haemoglobin (Hb F) levels in sickle cell anaemia (SS): age sex, alpha-globin gene number, beta-globin haplotype, and the X-linked F-cell production locus (FCP) that regulates the production of Hb F containing erythrocytes (F cells). We analysed the association of these factors with Hb F levels in 112 SS patients living in France who are homozygous for the three common African beta-globin haplotypes (Benin, Bantu or Central African Republic and Senegal). We found that: (1) FCP accounts for about 40% of the overall variation in Hb F levels, (2) when the FCP influence is removed, beta-globin haplotype is associated with 14% of the remaining Hb F variation, and (3) the other factors have little influence. Comparison with our previous study of SS individuals in Jamaica leads to the following conclusions: (1) the X-linked FCP locus is a major determinant of Hb F levels in SS disease, (2) factors linked to the beta-globin haplotype have only a small effect on the variation in Hb F levels, in either the homozygous or heterozygous state, and (3)approximately half of the variation in Hb F levels still remains to be explained.

Adolescent↗
PubMed1996

Invariant chain association with MHC class I: preference for HLA class I/beta 2-microglobulin heterodimers, specificity, and influence of the MHC peptide-binding groove.

MHC class I molecules require the assembly of heavy chain with beta2-microglobulin (beta 2m) and peptide in order to present Ag on the cell surface. Endoplasmic reticulum resident proteins associate with class I molecules and aid assembly. Free class I heavy chains associate with calnexin, which may facilitate association with beta 2m. Invariant chain (Ii) also associates with MHC class I molecules, but its role in class I assembly is not clear. We report here that Ii strongly associates with HLA class I/beta 2m heterodimers, but weakly with free class I heavy chains in HLA-B7-transfected T2 cells. Ii/HLA class I complexes persist stably within the endoplasmic reticulum/cis-Golgi compartment in peptide-processing deficient cells, but are much less prominent in normally processing cells. Furthermore, Ii differentially associates with variant HLA-B7 molecules that have peptide-binding groove mutations, and the degree of association correlates with HLA-B7 variant cell surface expression. Ii also shows HLA class I molecule specificity, associating to a greater degree with HLA-B7 than HLA-A2. Together these observations suggest that Ii stabilizes particular HLA class I/beta 2m heterodimers until peptide is loaded, and that this association may enhance class I cell surface expression.

Antigens, CD↗
PubMed1996

Alloreactive anti-HLA-B7 cytolytic T cell clones use restricted T cell receptor genes.

Most alloreactive T cells specifically recognize peptides bound to donor MHC molecules. In addition to peptides, solvent accessible MHC residues also may stimulate alloreactive T cells. We studied T cell receptor (TCR) usage by 16 independent anti-HLA-B7 alloreactive cytolytic T lymphocyte (CTL) clones. Most or all of these CTL clones recognized unique peptides bound to HLA-B7. Despite the diversity of peptides recognized, 11 out of 15 CTL clones analyzed expressed TCR V(alpha) gene segment (AV) subgroups 1 and 3. Within AV subgroup 1, four of six clones expressed AV2; within AV subgroup 3, three clones used AV6. Ten of 14 CTL clones analyzed expressed V(beta) gene segment (BV) subgroups 4 and 1. In subgroup 4, BV14 was expressed by four of five alloreactive CTL clones. Similar AV and BV usage restriction was not found in mitogen-stimulated peripheral blood T cells from the major donor of the CTL clones. TCR A and TCR B junctional region sequences were quite diverse in length and sequence, although two CTL clones expressed nearly identical TCR B chains. We found no correlation between TCR AV or TCR BV usage and CTL recognition of 81 HLA-B7 variants. These results are consistent with models of TCR structure, in which very diverse TCR CDR3 regions contact very diverse peptides, and moderately diverse TCR CDR1 and CDR2 regions contact moderately diverse MHC alpha-helices.

Amino Acid Sequence↗
PubMed1996

The resolution of the neutral N-linked oligosaccharides of IgG by high pH anion-exchange chromatography.

The introduction of high pH anion-exchange chromatography (HPAEC) has represented a major development in the qualitative analysis of glycoprotein derived oligosaccharides. When coupled with pulsed amperometric detection, the technique permits the detection of picomole quantities of heterogeneous mixtures of oligosaccharide without the need for derivatisation. The applications of HPAEC have generally been limited to the analysis of sialylated oligosaccharides, however, it is now possible to analyse heterogenous mixtures of neutral oligosaccharides with the latest systems. We have used such a system to separate completely a panel of seven commercially available neutral N-linked oligosaccharides and found the influence of monosaccharide substitution on elution position to be identical to that for sialylated structures. A standard monosialylated N-linked oligosaccharide was modified by sequential digestion with specific exoglycosidases to produce a monogalactosylated, diantennary oligosaccharide which is commercially unavailable. This standard's elution position was confirmed by HPAEC. The technique was applied to the identification of neutral N-linked oligosaccharides released from human immunoglobulin G using the enzyme peptide-N-glycosidase F.

Carbohydrate Conformation↗
PubMed1996

Probing HLA-B7 conformational shifts induced by peptide-binding groove mutations and bound peptide with anti-HLA monoclonal antibodies.

To determine the influence of peptide-binding groove residues and MHC-bound peptide on HLA-B7 conformation, we investigated the binding sites of nine locus- or allele-specific mAbs using a panel of 82 HLA-B7 variants. The functional mAb epitopes encircle the HLA-B7 peptide-binding groove. Three mAbs are affected by mutations at solvent-accessible peptide-binding groove mutations. Mutations in peptide-binding groove residues 45, 63, and 150 affect multiple nonoverlapping mAb epitopes, probably by interaction with other MHC residues or bound peptide. However, 18 of 24 peptide-binding groove mutations do not affect mAb binding, indicating that the conformation of solvent-accessible HLA-B7 structures is largely dissociated from changes in the peptide-binding groove. To test whether bound peptides alter HLA-B7 conformation, we loaded HLA-B7 heavy chains on acid-stripped cells with beta2-microglobulin and 20 individual synthetic peptides. Two of eight mAbs are sensitive to HLA-B7-bound peptides. A likely interpretation of these data is that the conformational flexibility of HLA-B7 is due to peptide-induced conformational shifts in MHC side chains, rather than major shifts in the MHC main chain. These results suggest that HLA-B7 conformation is largely maintained in the context of different bound peptides and different peptide-binding grooves.

Antibodies, Monoclonal↗
PubMed1996

Peptide-dependent expression of HLA-B7 on antigen processing-deficient T2 cells.

Class I MHC Ag presentation and cell surface expression largely depend on peptide transport into the ER/cis-Golgi by TAP, the transporter associated with Ag processing. Despite this dependency, class I MHC molecules are expressed at low levels on the surface of TAP-deficient T2 cells. We studied the peptide dependency of HLA-B7 expression in transfected T2 cells. HLA-B7 expression was affected by mutations at 19 out of 23 peptide-binding groove residues, but not by nine mutations outside of the peptide-binding groove. T2 cell surface HLA-A2, -B7, and -B51 had similar stabilities, and approximately half of these class I molecules had a long t1/2 consistent with tight peptide binding. Using metabolically labeled T2 cells, HLA-A2-bound peptide eluted as five prominent peaks, but HLA-B7-bound peptide was not detected. In contrast, HLA-B7-eluted peptides were detected spectrophotometrically. These data suggest that HLA-A2 and HLA-B7 molecules utilize distinct TAP-independent peptide supply mechanisms to different degrees. Equivalent amounts of HLA-B7 from TAP- and TAP+ cells yielded similar amounts of peptide, which had the characteristic HLA-B7 peptide-binding motif. The dependency of HLA-B7 cell surface expression on peptide-binding groove residues, the stability of cell surface class I molecules, and the ability to detect HLA-B7-bound peptide indicate that the low level expression on T2 cells is largely peptide dependent. TAP-independent peptide Ag presentation may allow immune recognition of intracellular pathogens that interfere with TAP-dependent peptide transport.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Alloreactive cytotoxic T-lymphocyte-defined HLA-B7 subtypes differ in peptide antigen presentation.

We investigated T-cell-defined HLA-B7 subtypes using cDNA sequencing, analysis of bound peptides, and reactivity with a panel of alloreactive cytotoxic T-lymphocyte (CTL) clones. Three subtypes (HLA-B*0702, HLA-B*0703, and HLA-B*0705) differ in nucleotide and predicted amino acid sequence. CTL reactivity and pooled peptide sequencing show that these three HLA-B7 subtypes bind distinct but overlapping sets of peptides. In particular B*0702 expresses D pocket residue Asp 114 and binds peptides with P3 Arg, whereas B*0705 expresses D pocket residue Asn 114 and binds peptides with P3 Ala, Leu, and Met. Consistent with different peptide-binding specificities, three alloreactive CTL differentiate between cells expressing B*0702, B*0703, and B*0705 by detecting specific peptide/HLA-B7 complexes. In contrast, three other T-cell-defined HLA-B7 subtypes are identical to HLA-B*0702. The B*0702-expressing cell lines are differentiated by two of ten CTL clones. One CTL clone differentiates B*0702-expressing cells by their ability to present peptide antigen. Thus differences in peptide presentation can explain differential CTL recognition of cell lines expressing structurally identical and variant HLA-B7.

Amino Acid Sequence↗