Publications
245 results found
Scott J, 1990, The genetics of apolipoprotein-B, Genetics and human nutrition, Editors: Randle, Bell, Scott, London, Publisher: John Libbey, Pages: 187-197, ISBN: 9780861962365
Scott J, 1989, Messenger RNA editing and modification., Curr Opin Cell Biol, Vol: 1, Pages: 1141-1147, ISSN: 0955-0674
Milne R, Théolis R, Maurice R, et al., 1989, The use of monoclonal antibodies to localize the low density lipoprotein receptor-binding domain of apolipoprotein B., J Biol Chem, Vol: 264, Pages: 19754-19760, ISSN: 0021-9258
Human apolipoprotein (apo) B-100 is composed of 4536 amino acids. It is thought that the binding of apoB to the low density lipoprotein (LDL) receptor involves an interaction between basic amino acids of the ligand and acidic residues of the receptor. Three alternative models have been proposed to describe this interaction: 1) a single region of apoB is involved in receptor binding; 2) groups of basic amino acids from throughout the apoB primary structure act in concert in apoB receptor binding; and 3) apoB contains multiple independent binding regions. We have found that monoclonal antibodies (Mabs) specific for a region that spans a thrombin cleavage site at apoB residue 3249 (T2/T3 junction) totally blocked LDL binding to the LDL receptor. Mabs specific for epitopes outside this region had either no or partial ability to block LDL binding. In order to define the region of apoB directly involved in the interaction with the LDL receptor we have tested 22 different Mabs for their ability to bind to LDL already fixed to the receptor. A Mab specific for an epitope situated between residues 2835 and 2922 could bind to its epitope on LDL fixed to its receptor whereas a second epitope between residues 2980 and 3084 is inaccessible on receptor-bound LDL. A series of epitopes near residue 3500 of apoB is totally inaccessible, and another situated between residues 4027 and 4081 is poorly accessible on receptor-bound LDL. In contrast, an epitope that is situated between residues 4154 and 4189 is fully exposed. Mabs specific for epitopes upstream and downstream of the region 3000-4000 can bind to receptor-bound LDL with a stoichiometry close to unity. Our results strongly suggest that the unique region of apoB directly involved in the LDL-receptor interaction is that of the T2/T3 junction.
Scott J, 1989, Lipoprotein(a). Thrombogenesis linked to atherogenesis at last?, Nature, Vol: 341, Pages: 22-23, ISSN: 0028-0836
Davies MS, Wallis SC, Driscoll DM, et al., 1989, Sequence requirements for apolipoprotein B RNA editing in transfected rat hepatoma cells., J Biol Chem, Vol: 264, Pages: 13395-13398, ISSN: 0021-9258
Apolipoprotein (apo) B mRNA undergoes a novel tissue-specific editing reaction, which replaces a genomically templated cytidine with uridine. This substitution converts codon 2153 from glutamine (CAA) in apo B100 mRNA to a stop codon (UAA) in apoB48 mRNA (Powell, L. M., Wallis, S. C., Pease, R. J., Edwards, Y. H., Knott, T. J., and Scott, J. (1987) Cell 50, 831-840). To examine sequences in the human apoB mRNA required for the editing reaction, a series of deletion mutants around the cytidine conversion site was prepared and transfected into a rat hepatoma cell line (McArdle 7777). This cell makes both apoB100 and apoB48. Editing was detected by a primer extension assay on cDNA that had been amplified by the polymerase chain reaction. RNAs of between 2385 and 26 nucleotides spanning the conversion site underwent similar levels of conversion. Editing was confirmed by cloning and sequencing of cDNA corresponding to the transfected RNAs. Conversion did not occur in transfected human hepatoblastoma (HepG2) or epithelial carcinoma (HeLa) cell lines, which do not make apoB48. These results verify that apoB48 is generated by a genuine tissue-specific RNA editing reaction and show that 26 nucleotides of apoB mRNA are sufficient for editing.
Driscoll DM, Wynne JK, Wallis SC, et al., 1989, An in vitro system for the editing of apolipoprotein B mRNA., Cell, Vol: 58, Pages: 519-525, ISSN: 0092-8674
A novel form of RNA editing generates two forms of apolipoprotein B (apo-B) mRNA by converting C at nucleotide 6666 to U or a U-like base. We have established an in vitro system for the editing of apo-B mRNA using synthetic RNAs and S100 extracts from rat hepatoma cells. Editing was detected by a sensitive primer extension assay and confirmed by DNA sequencing. The in vitro editing activity is specific and sensitive to proteinase K. Apo-B100 RNAs were synthesized in vitro from deletion mutants spanning nucleotide 6666. Synthetic RNAs containing 2383, 483, and 55 nucleotides of apo-B mRNA sequence were edited in vitro with similar efficiency, but an RNA containing 26 nucleotides was not edited.
Pullinger CR, North JD, Teng BB, et al., 1989, The apolipoprotein B gene is constitutively expressed in HepG2 cells: regulation of secretion by oleic acid, albumin, and insulin, and measurement of the mRNA half-life., J Lipid Res, Vol: 30, Pages: 1065-1077, ISSN: 0022-2275
The objective of this study was to establish conditions whereby apoB secreted from HepG2 cells could be regulated over a wide range, and to determine whether changes of output were correlated with the level of apoB mRNA. The presence of oleate (complexed to 3% albumin at a molar ratio of 1.7:1) resulted in a 3.5-fold stimulation of apoB secretion that was apparent after only 3 h. Insulin halved the rate of apoB output and the inhibition was detectable within the physiological insulin range, but was not apparent until 12-16 h. Albumin in the culture medium had a dose-dependent inhibitory effect on apoB production. Overall, apoB secretion from HepG2 cells was modulated over a 7-fold range. However, when apoB mRNA was assayed by slot-blot hybridization, no change was detectable under any of the conditions that modulated apoB output. Quantitative solution hybridization was used to confirm that oleate did not affect the level of apoB mRNA. Kinetic analysis of the decay of [3H]uridine-labeled apoB mRNA showed that the half-life of apoB mRNA was 16 h. We conclude from these studies that the apoB gene is constitutively expressed in HepG2 cells and that the mechanism of acute regulation of apoB production by these cells must involve co- or post-translational processes.
Rajput-Williams J, Eyre J, Nanjee MN, et al., 1989, Plasma lipoprotein lipids in relation to the MspI polymorphism of the apolipoprotein AII gene in Caucasian men. Lack of association with plasma triglyceride concentration., Atherosclerosis, Vol: 77, Pages: 31-36, ISSN: 0021-9150
Digestion of the human apolipoprotein (apo) A-II gene with the endonuclease MspI produces fragments of 3.0 or 3.7 kb, reflecting the presence or absence of a polymorphic site within an Alu sequence 3' to the gene. Patients with hypertriglyceridemia have been shown to have an increased prevalence of the 3.0 kb allele. To explore this observation further, plasma lipoprotein lipids were studied in a random sample of fasted middle-aged Caucasian men, of which 59 were 3.0 kb homozygotes, 24 were heterozygotes, and 2 were 3.7 kb homozygotes. After adjusting for the effects of age, height, weight, alcohol intake and cigarette consumption by covariance analysis, no statistically significant associations were present between genotype and the concentrations of triglyceride in whole plasma or the d less than 1.019 g/ml fraction of plasma (i.e., VLDL + IDL). Nor were the cholesterol concentrations in VLDL + IDL, low density lipoprotein (LDL, d = 1.019-1.063 g/ml), high density lipoprotein (HDL), HDL2 or HDL3 related to genotype. In an independent comparison of eight 3.0 kb homozygotes and eight 3.7 kb homozygotes (all Caucasians) drawn from a different community, genotype was unrelated to the triglyceride or cholesterol concentrations in VLDL (d less than 1.006 g/ml), IDL + LDL (d = 1.006-1.063 g/ml) or HDL, after adjustment for the effects of covariates. These results suggest that the MspI polymorphism of the apo A-II gene is not associated with genetic variation that significantly affects triglyceride transport in the majority of men.(ABSTRACT TRUNCATED AT 250 WORDS)
Scott J, 1989, Lipoprotein receptors. Unravelling atherosclerosis., Nature, Vol: 338, Pages: 118-119, ISSN: 0028-0836
Scott J, 1989, The molecular and cell biology of apolipoprotein-B., Mol Biol Med, Vol: 6, Pages: 65-80, ISSN: 0735-1313
Jones T, Rajput-Williams J, Knott TJ, et al., 1989, An MspI RFLP in the APOB promoter., Nucleic Acids Res, Vol: 17, ISSN: 0305-1048
Powell LM, Davidson NO, Scott J, 1989, Apolipoprotein B: a novel mechanism for differential gene expression, Platelets and vascular occlusion, Editors: Patrono, FitzGerald, New York, Publisher: Raven Pr, Pages: 43-52, ISBN: 9780881674200
Rajput-Williams J, Knott TJ, Wallis SC, et al., 1988, Variation of apolipoprotein-B gene is associated with obesity, high blood cholesterol levels, and increased risk of coronary heart disease., Lancet, Vol: 2, Pages: 1442-1446, ISSN: 0140-6736
A random sample of 290 white men was examined for association between restriction fragment length polymorphism (RFLP) haplotypes (closely linked RFLPs on a single chromosome) of the apolipoprotein-B gene and serum levels of cholesterol, triglyceride, and high-density lipoprotein, obesity, smoking, alcohol consumption, and coronary heart disease. Haplotype or single RFLP frequencies differed significantly for obesity (p less than 0.005), serum cholesterol (p less than 0.005), and coronary heart disease (p less than 0.05), but for no other variable. Obesity was associated with haplotypes involving minimum PvuII and XbaI RFLPs are likely to be in linkage disequilibrium with nearby functional variation predisposing to obesity. Significant variation in serum cholesterol levels was associated with three functional alleles defined by MspI and EcoRI RFLP pairs (p less than 0.03). These RFLPs correspond to charged aminoacid variants at positions 3611 (arginine to glutamine) and 4154 (glutamic acid to lysine), which lie near the low-density-lipoprotein (LDL) receptor binding region of apolipoprotein-B. The three alleles showed stratification of serum cholesterol between low, normal, and high levels. Coronary heart disease was associated with minimum haplotypes involving XbaI and MspI RFLPs. Together these results suggest that inherited variations of the apolipoprotein-B gene, probably in the form of charged aminoacid substitutions, influence circulating cholesterol concentration, and that these and other functional variants of the apolipoprotein-B gene affect susceptibility to coronary heart disease and obesity.
TALMUD PJ, LLOYD JK, MULLER DPR, et al., 1988, GENETIC-EVIDENCE FROM 2 FAMILIES THAT THE APOLIPOPROTEIN-B GENE IS NOT INVOLVED IN ABETALIPOPROTEINEMIA, JOURNAL OF CLINICAL INVESTIGATION, Vol: 82, Pages: 1803-1806, ISSN: 0021-9738
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- Citations: 66
Davidson NO, Powell LM, Wallis SC, et al., 1988, Thyroid hormone modulates the introduction of a stop codon in rat liver apolipoprotein B messenger RNA., J Biol Chem, Vol: 263, Pages: 13482-13485, ISSN: 0021-9258
Apolipoprotein B (apoB) biosynthesis by rat liver was studied following thyroid hormone (3,5,3'-triiodo-L-thyronine) administration to hypothyroid rats. Pharmacologic doses of 3,5,3'-triiodo-L-thyronine caused suppression of apoB100 synthesis but did not affect apoB48 levels. There was no detectable apoB100 synthesis in hyperthyroid rats. To examine whether these results were mediated by the previously demonstrated mechanism of RNA modification (Powell, L. M., Wallis, S. C., Pease, R. J., Edwards, Y. H., Knott, T. J., and Scott, J. (1987) Cell 50, 831-840), the DNA sequence corresponding to the C-terminal end of rat apoB48 was determined from rat liver cDNA clones. Rat cDNAs contained a stop codon at an identical position to that found in human and rabbit apoB48 intestinal cDNA. To quantitate the relative amounts of apoB100 and apoB48 message, cDNA was synthesized from hepatic and intestinal apoB RNA and a 207-base pair fragment amplified using the polymerase chain reaction. The products were then differentially hybridized with oligonucleotides specific for apoB100 (containing CAA) or apoB48 (TAA). Control and hypothyroid liver contained approximately equal amounts of CAA and TAA, while hyperthyroid liver contained greater than 90% TAA. All gut samples contained 94-98% TAA. Genomic DNA from rat liver contained only CAA. The results demonstrate that apoB mRNA modification can be hormonally modulated in the adult rat by induction of a mechanism involving substitution of a stop codon into hepatic apoB100 mRNA.
COLLINS DR, KNOTT TJ, PEASE RJ, et al., 1988, TRUNCATED VARIANTS OF APOLIPOPROTEIN-B CAUSE HYPOBETALIPOPROTEINEMIA, NUCLEIC ACIDS RESEARCH, Vol: 16, Pages: 8361-8375, ISSN: 0305-1048
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- Citations: 98
Middleton-Price HR, van den Berghe JA, Scott J, et al., 1988, Regional chromosomal localisation of APOA2 to 1q21-1q23., Hum Genet, Vol: 79, Pages: 283-285, ISSN: 0340-6717
Using in situ hybridisation, we have mapped APOA2 to the 1q21-1q23 region of chromosome 1. DNA hybridisation to somatic cell hybrids made from cells carrying a balanced translocation between X and 1 confirms the localisation as proximal to 1q23. This was further confirmed by the presence of two polymorphic alleles in a cell line carrying a deletion of 1q25-1q32.
Krul ES, Kleinman Y, Kinoshita M, et al., 1988, Regional specificities of monoclonal anti-human apolipoprotein B antibodies., J Lipid Res, Vol: 29, Pages: 937-947, ISSN: 0022-2275
The usefulness of monoclonal antibodies as probes of protein structure is directly related to knowledge of the structures and locations of the epitopes with which they interact. In this report we provide a detailed map of 13 epitopes on apoB-100 defined by our anti-apoB monoclonal antibodies based on current information on the amino acid sequence of apoB-100. To localize antibody specificities to smaller regions along the linear sequence of the apoB-100 molecule we used a) thrombin- and kallikrein-generated fragments of apoB-100; b) beta-galactosidase- apoB fusion proteins; c) heparin; and d) antibody versus antibody competition experiments. Most of the monoclonal antibodies elicited by immunization with LDL were directed towards epitopes within the first 1279 amino terminal (T4/K2 fragments) or last 1292 carboxyl terminal amino acid residues (T2/K4 fragments) of apoB-100. One epitope localized to the mid-portion of apoB-100 was elicited by immunization with VLDL (D7.2). Saturating amounts of heparin bound to LDL did not inhibit the binding of any of the monoclonal antibodies to their respective epitopes on apoB-100, indicating that none of the antibody determinants is situated close to any of the reported heparin binding sites on LDL apoB. We examined the expression of apoB epitopes on VLDL subfractions and LDL isolated from a normolipidemic donor. The apparent affinities with which the antibodies interacted with their respective epitopes on the VLDL subfractions and LDL uniformly increased as follows: LDL greater than VLDL3 greater than VLDL2 greater than VLDL1, suggesting that each of the major regions of apoB-100 is progressively more exposed as normal VLDL particles become smaller in size and epitopes are most exposed in LDL. Previous experiments utilizing hypertriglyceridemic VLDL subfractions yielded similar results, but the rank order of VLDL subfractions and LDL was not the same for all antibodies tested. Thus, differences in apoB epitope expression on VLDL
, 1988, Apolipoprotein-B and atherogenesis., Lancet, Vol: 1, Pages: 1141-1142, ISSN: 0140-6736
KESSLING AM, RAJPUTWILIAMS J, BAINTON D, et al., 1988, DNA POLYMORPHISMS OF THE APOLIPOPROTEIN-AII AND AI-CIII-AIV GENES - A STUDY IN MEN SELECTED FOR DIFFERENCES IN HIGH-DENSITY-LIPOPROTEIN CHOLESTEROL CONCENTRATION, AMERICAN JOURNAL OF HUMAN GENETICS, Vol: 42, Pages: 458-467, ISSN: 0002-9297
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- Citations: 56
Rorsman F, Bywater M, Knott TJ, et al., 1988, Structural characterization of the human platelet-derived growth factor A-chain cDNA and gene: alternative exon usage predicts two different precursor proteins., Mol Cell Biol, Vol: 8, Pages: 571-577, ISSN: 0270-7306
The human platelet-derived growth factor (PDGF) A-chain locus was characterized by restriction endonuclease analysis, and the nucleotide sequence of its exons was determined. Seven exons were identified, spanning approximately 22 kilobase pairs of genomic DNA. Alternative exon usage, identified by cDNA cloning, occurs in a human glioblastoma cell line and may give rise to two types of A-chain precursors with different C termini. The exon-intron arrangement was similar to that of the PDGF B-chain/sis locus and seemed to divide the precursor proteins into functional domains. Southern blot analysis of genomic DNA showed that a single PDGF A-chain gene was present in the human genome.
Scott J, Selby MJ, Bell GI, et al., 1988, Apolipoprotein B: a novel mechanism for deriving two proteins from one gene, Hyperlipidaemia and atherosclerosis, Editors: Suckling, Groot, London, Publisher: Academic Pr, Pages: 47-64
Scott J, 1988, The human apolipoprotein genes, Oxford surveys on eukaryotic genes, Editors: Maclean, Publisher: Oxford University Press, USA, Pages: 168-197
They review findings from their own laboratories and give an up-to-date account of the work of other research groups.
Scott J, 1987, New horizons in lipoprotein research, Baillière's clinical endocrinology and metabolism, Editors: Shepherd, London, Publisher: Baillière Tindall, ISBN: 9780702012082
Scott J, 1987, Molecular genetics of common diseases., Br Med J (Clin Res Ed), Vol: 295, Pages: 769-771, ISSN: 0267-0623
Powell LM, Wallis SC, Pease RJ, et al., 1987, A novel form of tissue-specific RNA processing produces apolipoprotein-B48 in intestine., Cell, Vol: 50, Pages: 831-840, ISSN: 0092-8674
Evidence suggests that intestinal apo-B48 is colinear with the amino-terminal half of hepatic apo-B100. To investigate the mechanism of apo-B48 production, we examined cDNA clones from human and rabbit small intestine. All clones contained a single C----T base difference from the hepatic sequence, resulting in a translational stop at codon 2153. Amplification by the polymerase chain reaction of cDNA from human and rabbit small intestine, rabbit liver, and the human hepatoma cell line HepG2 showed that the stop codon was only present in intestinal mRNA. Enterocyte genomic DNA did not contain the stop codon. We suggest that a co- or posttranscriptional C----U change may result in the production of apo-B48, which represents the amino-terminal 2152 amino acids of apo-B100. This is the first example of tissue-specific modification of a single mRNA nucleotide resulting in two different proteins from the same primary transcript.
Ludwig EH, Blackhart BD, Pierotti VR, et al., 1987, DNA sequence of the human apolipoprotein B gene., DNA, Vol: 6, Pages: 363-372, ISSN: 0198-0238
The sequence of the human apolipoprotein B gene comprises 43 kb divided into 29 exons, one of which is unusually long and contains 7572 bp. Comparison of the gene sequence with four complete and three partial cDNA sequences published elsewhere reveals a total of 60 nucleotide substitutions and 39 amino acid substitutions and one small deletion in the signal peptide.
Betsholtz C, Bergh J, Bywater M, et al., 1987, Expression of multiple growth factors in a human lung cancer cell line., Int J Cancer, Vol: 39, Pages: 502-507, ISSN: 0020-7136
U-1810, a human large-cell lung cancer line, was found to express a PDGF-like growth factor. 35S-cysteine labelling and immunoprecipitation revealed the synthesis and secretion of a 31-kDa PDGF-like protein. Serum-free conditioned medium contained PDGF-receptor-competing and mitogenic activity when tested on human fibroblasts. Whereas the receptor-competing activity was fully neutralized by anti-PDGF antibodies, the mitogenic activity was only partially affected. We therefore probed U-1810 mRNA with a panel of growth-factor DNA clones. We found expression of the genes for PDGF A- and B-chains, TGF-alpha, TGF-beta and IGF-II but not EGF or IGF-I. U-1810 cells lacked specific binding sites for PDGF but showed specific binding of EGF and expressed EGF-receptor transcripts. Thus, U-1810 is an example of a human tumor cell line that expresses multiple growth factor genes; in the intact tumor the corresponding growth factors may operate in autocrine stimulation of the tumor cells as well as in paracrine growth reactions (i.e. stroma recruitment).
Scott J, 1987, Oncogenes in atherosclerosis., Nature, Vol: 325, Pages: 574-575, ISSN: 0028-0836
Scott J, Pease RJ, Powell LM, et al., 1987, Apolipoprotein B gene: plasma cholesterol and atherosclerosis., Biochem Soc Symp, Vol: 53, Pages: 155-163, ISSN: 0067-8694
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