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Biomedical subjects

R Lawn

Publications and source records attributed to R Lawn.

14 recordsLinked to original sources
PubMed1998

Novel therapeutic strategy for atherosclerosis: ribozyme oligonucleotides against apolipoprotein(a) selectively inhibit apolipoprotein(a) but not plasminogen gene expression.

BACKGROUND: Because mechanisms of atherosclerosis by lipoprotein(a) [Lp(a)] have been postulated in the decrease in active transforming growth factor-beta conversion by decreased plasmin, selective decrease in apolipoprotein(a) [apo(a)] independent of plasminogen may have therapeutic values. Although antisense can decrease apo(a), its application may be difficult because of very high homology of apo(a) gene to plasminogen. Thus we used ribozyme strategy that actively cleaves targeted genes to selectively inhibit apo(a) expression. METHODS AND RESULTS: We constructed ribozyme oligonucleotides containing phosphorothioate DNA- and RNA-targeted kringle 4 of the apo(a) gene that showed 80% homology to plasminogen. Transfection of human apo(a) gene produced Lp(a) in medium of HepG2 cells, whereas Lp(a) could not be detected in control cells. Cotransfection of ribozyme and apo(a) gene resulted in the decrease in mRNA of apo(a) but not plasminogen. Moreover, marked decrease in Lp(a) was also observed in the medium transfected with ribozyme and apo(a) gene compared with apo(a) gene alone (P<0.01), whereas there was no significant change in plasminogen level between ribozyme-transfected and control cells. Incubation of human vascular smooth muscle cells (VSMC) with conditioned medium from apo(a)-transfected HepG2 cells resulted in a significant increase in VSMC number, whereas addition of conditioned medium from cells cotransfected with ribozyme oligonucleotides and apo(a) gene resulted in no VSMC growth (P<0.01). DNA-based control oligonucleotides and mismatched ribozyme oligonucleotides did not have an inhibitory effect on Lp(a) production. CONCLUSIONS: Overall, our data revealed that transfection of ribozyme against the apo(a) gene resulted in the selective inhibition of the apo(a) but not the plasminogen gene, providing novel therapeutic strategy for treatment of high Lp(a), a risk factor for atherosclerosis.

Aorta↗
PubMed1998

Apolipoprotein(a) gene enhancer resides within a LINE element.

Apolipoprotein(a), (apo(a)), is the distinguishing protein portion of the lipoprotein(a) particle, elevated plasma levels of which are a major risk factor for cardiovascular disease. A search for enhancer elements that control the transcription of the apo(a) gene led to the identification of an upstream element that contains target binding sites for members of the Ets and Sp1 nuclear protein families. The enhancer element functions in either orientation to confer a greater than 10-fold increase in the activity of the apo(a) minimal promoter in cultured hepatocyte cells. Unexpectedly, the enhancer element is located within a LINE retrotransposon element, suggesting that LINE elements may function as mobile regulatory elements to control the expression of nearby genes.

Apolipoproteins↗
PubMed1997

Apolipoproteins(a): a puzzling evolutionary story.

Human apolipoprotein(a), a risk factor for heart disease, has over 80% sequence identity to plasminogen. Plasminogen contains five distinct kringle domains plus a catalytic protease subunit. Human apo(a) consists of multiple copies (the number varies in individuals) of a domain resembling kringle 4, a single copy of a domain resembling kringle 5, and a protease-like domain. The recently cloned hedgehog version of apolipoprotein(a), which contains 31 nearly identical copies of plasminogen kringle 3 and lacks a protease domain, has prompted us to investigate the evolutionary history of the apolipoprotein(a) gene in mammals. Our analysis supports the nonfunctionality of the human apolipoprotein(a) protease domain, and a single (or multiple) duplication of plasminogen gene before mammal radiation, which originated apolipoprotein(a) in mammals.

Animals↗
PubMed1996

A quantitative immunoassay for the lysine-binding function of lipoprotein(a). Application to recombinant apo(a) and lipoprotein(a) in plasma.

Apo(a), the unique apoprotein of lipoprotein(a) (Lp[a]), can express lysine-binding sites(s) (LBS). However, the LBS activity of Lp(a) is variable, and this heterogeneity may influence its pathogenetic properties. An LBS-Lp(a) immunoassay has been developed to quantitatively assess the LBS function of Lp(a). Lp(a) within a sample is captured with an immobilized monoclonal antibody specific for apo(a), and the captured Lp(a) is reacted with an antibody specific for functional LBS. The binding of this LBS-specific antibody is then quantified by using an alkaline phosphatase-conjugated disclosing antibody. The critical LBS-specific antibody was raised to kringle 4 of plasminogen. When applied to plasma samples, the LBS activity of Lp(a) ranged from 0% to 100% of an isolated reference Lp(a); the signal corresponded to the percent retention of Lp(a) on a lysine-Sepharose but did not correlate well with total Lp(a) levels in plasma. Mutation of residues in the putative LBS in the carboxy-terminal kringle 4 repeat (K4-37) in an eight-kringle apo(a) construct resulted in marked but not complete loss of activity in the LBS-Lp(a) immunoassay. These data suggest that this kringle is the major but not the sole source of LBS activity in apo(a). The LBS-Lp(a) immunoassay should prove to be a useful tool in establishing the role of the LBS in the pathogenicity of Lp(a).

Amino Acid Sequence↗
PubMed1995

Targeting gene expression to the vascular wall in transgenic mice using the murine preproendothelin-1 promoter.

To develop a system for overexpressing genes in the vascular wall, we created transgenic mice using the reporter gene luciferase and the murine preproendothelin-1 promoter. In vitro analysis suggested that the murine 5'-flanking region contained endothelial-specific elements in a 5.9-kb fragment. Five transgenic mice colonies established from independent founders all exhibited the highest level of luciferase activity in the aorta with up to 8,540 light units per microgram of protein. Immunohistochemistry with anti-luciferase antisera revealed high levels of expression in the endothelial cells of both large and small arteries and lower levels of expression in veins and capillaries. Significant expression was also seen in arterial smooth muscle cells and in select epithelial surfaces which is consistent with the known distribution of endothelin-1 in mammals. The further demonstrate the targeting capability of this system, we overexpressed the lipid-peroxidating enzyme, human 15-lipoxygenase, in the vessel wall of transgenic mice. As with luciferase, expression of active enzyme and immunohistochemical localization in vascular cells were documented in transgenic animals. Hence, this new system can be used to direct expression of molecules to the vascular wall for the purpose of examining the biological significance of either overexpression or inhibition of select proteins.

Animals↗
PubMed1990

Restriction site polymorphism at the LPA (Lp(a) apolipoprotein; apolipoprotein (a)) locus.

A restriction site polymorphism in the Lp(a) apolipoprotein gene (the LPA gene) is reported. The basis for the polymorphism is presence or absence of an MspI restriction site that appears to be 3' to the last kringle IV structure of the gene. The "1" gene (presence of the restriction site) has a frequency of 0.316 and the "2" gene (absence of the restriction site) has a frequency of 0.684. Both members of each of 67 monozygotic (MZ) twin pairs had the same genotype and there was Mendelian segregation of the DNA variants in 40 families with a total of 75 children. There was a lower proportion of people with genotype 1-1 in the top quartile than in the 3 bottom quartiles of the population distribution of Lp(a) lipoprotein levels but the difference did not reach statistical significance.

Apolipoproteins↗
PubMed1989

DNA polymorphism at the locus for human cholesteryl ester transfer protein (CETP) is associated with high density lipoprotein cholesterol and apolipoprotein levels.

Cholesteryl ester transfer protein (CETP) is a protein involved in "reverse cholesterol transport" and it could play an important role in facilitating the removal of cholesteryl esters from peripheral tissues for transport to the liver or for transfer of cholesterol between plasma lipoprotein particles. Both functions may be relevant to susceptibility or resistance to atherosclerotic disease. We have studied 149 and 146 unrelated persons, respectively, for the A and B polymorphism at the CETP locus detectable with the restriction enzyme TaqI. The B system is by far the more polymorphic. A search for association with risk or "anti-risk" factor levels was conducted with the following quantitative parameters: total cholesterol, HDL cholesterol, triglycerides, apolipoprotein AI (apoA-I), apolipoprotein B (apoB) and Lp(a) lipoprotein levels. Highly significant differences in apoA-I concentration were found between the two categories of homozygotes in the B polymorphism. The association observed remained significant after multiplying the p value by the number of quantitative parameters used for the association tests. There was a dosage effect on the apoA-I level of genes in the B polymorphism. We conclude that the associations observed are likely to reflect true biological phenomena. The effect of CETP genes appeared to be limited to non-smokers.

Adult↗
PubMed1989

"Variability gene" effect of cholesteryl ester transfer protein (CETP) genes.

Cholesteryl ester transfer protein (CETP) may have important roles in transfer of lipids from cells to serum lipoproteins or between circulating lipoprotein particles. Restriction fragment length polymorphisms (RFLPs) in DNA at the CETP locus have been detected. In the present study we have used RFLPs detectable with the restriction enzyme TaqI to examine if CETP influences serum lipid variability (as opposed to absolute lipid levels). We have compared within-pair difference in serum lipid and apolipoprotein levels in monozygotic twin pairs of various genotypes in the B polymorphism at the CETP locus and uncovered significant differences between genotypes. We conclude that the CETP locus has "variability genes" (as opposed to "level genes") with respect to total and LDL cholesterol variability. A person's total genetic risk for coronary heart disease may depend on his or her combination of "level genes" and "variability genes". The method of analysis applied may be the best available for the study of gene - environment interaction.

Apolipoproteins↗
PubMed1986

Human lecithin-cholesterol acyltransferase gene: complete gene sequence and sites of expression.

The human lecithin-cholesterol acyltransferase (LCAT) gene has been sequenced to completion. The gene is divided into six exons spanning approximately 4,200 bp. Exon five codes for amino acids homologous to the interfacial active site of several lipases, and also codes for an amphipathic alpha-helix resembling the carboxy terminus of apolipoprotein E. Blot hybridization data suggest that there is only one LCAT gene in humans. The 1550 base LCAT mRNA can be detected in liver and HepG2 (hepatocyte) cells, but not in small intestine, spleen, pancreas, placenta or adrenal tissue.

Base Sequence↗
PubMed1986

Cloning and expression of human lecithin-cholesterol acyltransferase cDNA.

cDNA and genomic cloning has been used to determine the mRNA and amino acid sequence of human plasma lecithin-cholesterol acyltransferase (LCATase; EC 2.3.1.43). The mature protein was found to contain 416 amino acid residues with a hydrophobic leader sequence of 24 amino acids. An unusual feature of the message is that the poly(A) signal AATAAA overlaps the COOH-terminal glutamic acid and stop codons, and the 3' untranslated region is only 23 bases. The protein itself is distinguished by a number of extended sequences of hydrophobic amino acids, one of which contains a hexapeptide identical with the interfacial binding segment of the active site of pancreatic lipase and is similar to the same site of lingual lipase. The cloned cDNA allows the expression of active LCATase by transfected tissue culture cells.

Amino Acid Sequence↗
PubMedDate not supplied

Cloning and sequencing of human cholesteryl ester transfer protein cDNA.

The transfer of insoluble cholesteryl esters among lipoprotein particles is a vital step in normal cholesterol homeostasis and may be involved in the development of atherosclerosis. Extrahepatic tissues lack the enzymes required for the degradation of sterols to the excretable form of bile acids. Cholesterol synthesized in these tissues in excess of that needed for the synthesis of cell membranes or steroid hormones must accordingly be returned through the plasma to the liver for catabolism. The series of reactions involved has been termed reverse cholesterol transport. Catalysed steps of this pathway are believed to include an efflux from peripheral cells, which generates a diffusion gradient between these membranes and extracellular fluid; esterification of this cholesterol by lecithin-cholesterol acyltransferase (LCAT) (phosphatidylcholine-sterol acyltransferase) acting on species of high-density lipoproteins; transfer of the cholesteryl esters formed (largely to low- and very low-density lipoproteins) (LDL and VLDL) by a cholesteryl ester transfer protein (CETP); and removal of these lipoproteins, together with their cholesteryl ester content, by the liver through receptor-mediated and nonspecific endocytosis. Of these steps, the CETP reaction is the least characterized. Several laboratories have reported the purification from human plasma of proteins active on cholesteryl ester transfer between lipoprotein particles and possibly between cells and plasma. However, the reported relative molecular mass (Mr), abundance and specificity of the purified activities have differed considerably. We have recently described the preparation of a highly active CETP of Mr 74,000 purified about 100,000-fold from human plasma, which may represent the functional component of earlier preparations. Using a partial amino-acid sequence from this purified protein, CETP complementary DNA derived from human liver DNA has been cloned and sequenced and the cloned DNA used to detect CETP messenger RNA in a number of human tissues.

Amino Acid Sequence↗