PubMed Health⌕ Search

Biomedical subjects

J McNeish

Publications and source records attributed to J McNeish.

12 recordsLinked to original sources
PubMed2000

The correlation of ATP-binding cassette 1 mRNA levels with cholesterol efflux from various cell lines.

Studies show that lipid-free apoA-I stimulates release of cholesterol and phospholipid from fibroblasts and macrophages. ATP-binding cassette 1 (ABC1) is implicated in this release and has been identified as the genetic defect in Tangier disease, evidence that ABC1 is critical to the biogenesis of high density lipoprotein. We quantified levels of ABC1 mRNA, protein, and cholesterol efflux from J774 mouse macrophages +/- exposure to a cAMP analog. Up-regulating ABC1 mRNA correlated to increased cholesterol efflux in a dose- and time-dependent manner. mRNA levels rose after 15 min of exposure while protein levels rose after 1 h, with increased efflux 2-4 h post-treatment. In contrast to cells from wild-type mice, peritoneal macrophages from the Abc1 -/- mouse showed a lower level of basal efflux and no increase with cAMP treatment. The stimulation of efflux exhibits specificity for apoA-I, high density lipoprotein, and other apolipoproteins as cholesterol acceptors, but not for small unilamellar vesicles, bile acid micelles, or cyclodextrin. We have studied a number of cell types and found that while other cell lines express ABC1 constitutively, only J774 and elicited mouse macrophages show a substantial increase of mRNA and efflux with cAMP treatment. ApoA-I-stimulated efflux was detected from the majority of cell lines examined, independent of treatment.

ATP Binding Cassette Transporter 1↗
PubMed2000

High density lipoprotein deficiency and foam cell accumulation in mice with targeted disruption of ATP-binding cassette transporter-1.

Recently, the human ATP-binding cassette transporter-1 (ABC1) gene has been demonstrated to be mutated in patients with Tangier disease. To investigate the role of the ABC1 protein in an experimental in vivo model, we used gene targeting in DBA-1J embryonic stem cells to produce an ABC1-deficient mouse. Expression of the murine Abc1 gene was ablated by using a nonisogenic targeting construct that deletes six exons coding for the first nucleotide-binding fold. Lipid profiles from Abc1 knockout (-/-) mice revealed an approximately 70% reduction in cholesterol, markedly reduced plasma phospholipids, and an almost complete lack of high density lipoproteins (HDL) when compared with wild-type littermates (+/+). Fractionation of lipoproteins by FPLC demonstrated dramatic alterations in HDL cholesterol (HDL-C), including the near absence of apolipoprotein AI. Low density lipoprotein (LDL) cholesterol (LDL-C) and apolipoprotein B were also significantly reduced in +/- and -/- compared with their littermate controls. The inactivation of the Abc1 gene led to an increase in the absorption of cholesterol in mice fed a chow or a high-fat and -cholesterol diet. Histopathologic examination of Abc1-/- mice at ages 7, 12, and 18 mo demonstrated a striking accumulation of lipid-laden macrophages and type II pneumocytes in the lungs. Taken together, these findings demonstrate that Abc1-/- mice display pathophysiologic hallmarks similar to human Tangier disease and highlight the capacity of ABC1 transporters to participate in the regulation of dietary cholesterol absorption.

ATP Binding Cassette Transporter 1↗
PubMed2000

Deficiency of the stress kinase p38alpha results in embryonic lethality: characterization of the kinase dependence of stress responses of enzyme-deficient embryonic stem cells.

The mitogen-activated protein (MAP) kinase p38 is a key component of stress response pathways and the target of cytokine-suppressing antiinflammatory drugs (CSAIDs). A genetic approach was employed to inactivate the gene encoding one p38 isoform, p38alpha. Mice null for the p38alpha allele die during embryonic development. p38alpha(1/)- embryonic stem (ES) cells grown in the presence of high neomycin concentrations demonstrated conversion of the wild-type allele to a targeted allele. p38alpha(-/)- ES cells lacked p38alpha protein and failed to activate MAP kinase-activated protein (MAPKAP) kinase 2 in response to chemical stress inducers. In contrast, p38alpha(1/+) ES cells and primary embryonic fibroblasts responded to stress stimuli and phosphorylated p38alpha, and activated MAPKAP kinase 2. After in vitro differentiation, both wild-type and p38alpha(-/)- ES cells yielded cells that expressed the interleukin 1 receptor (IL-1R). p38alpha(1/+) but not p38alpha(-/)- IL-1R-positive cells responded to IL-1 activation to produce IL-6. Comparison of chemical-induced apoptosis processes revealed no significant difference between the p38alpha(1/+) and p38alpha(-/)- ES cells. Therefore, these studies demonstrate that p38alpha is a major upstream activator of MAPKAP kinase 2 and a key component of the IL-1 signaling pathway. However, p38alpha does not serve an indispensable role in apoptosis.

Animals↗
PubMed2000

ABC1 promotes engulfment of apoptotic cells and transbilayer redistribution of phosphatidylserine.

ATP-binding-cassette transporter 1 (ABC1) has been implicated in processes related to membrane-lipid turnover. Here, using in vivo loss-of-function and in vitro gain-of-function models, we show that ABC1 promotes Ca2+-induced exposure of phosphatidylserine at the membrane, as determined by a prothrombinase assay, membrane microvesiculation and measurement of transbilayer redistribution of spin-labelled phospholipids. That ABC1 promotes engulfment of dead cells is shown by the impaired ability of ABC1-deficient macrophages to engulf apoptotic preys and by the acquisition of phagocytic behaviour by ABC1 transfectants. Release of membrane phospholipids and cholesterol to apo-AI, the protein core of the cholesterol-shuttling high-density lipoprotein (HDL) particle, is also ABC1-dependent. We propose that both the efficiency of apoptotic-cell engulfment and the efflux of cellular lipids depend on ABC1-induced perturbation of membrane phosphatidylserine turnover. Transient local exposure of anionic phospholipids in the outer membrane leaflet may be sufficient to alter the general properties of the membrane and thus influence discrete physiological functions.

ATP Binding Cassette Transporter 1↗
PubMed1997

Expression of human lecithin:cholesterol acyltransferase in transgenic mice: effects on cholesterol efflux, esterification, and transport.

Human lecithin:cholesterol acyltransferase (LCAT) is a key enzyme in the plasma metabolism of cholesterol and is postulated to participate in a physiologic process called reverse cholesterol transport. We have used transgenic mice expressing the human LCAT transgene to study the effect of increased plasma levels of LCAT in each of the proposed steps involved in the reverse cholesterol transport pathway. High density lipoprotein (HDL) from LCAT transgenic mice was 44% more efficient than control mouse HDL in the efflux of cholesterol from human skin fibroblasts. Esterification of cell-derived cholesterol was also markedly increased in mice expressing the human LCAT transgene. The rate of plasma clearance of HDL cholesteryl ester was virtually the same in both types of animals whereas the HDL cholesteryl ester transport rate was significantly increased in mice expressing the human LCAT transgene (152.3 +/- 16.9 micrograms/h vs. 203.1 +/- 30.9 micrograms/h in control and transgenic mice, respectively). Liver cholesteryl ester uptake was significantly increased in mice expressing human LCAT (58.0 +/- 1.4 micrograms/h/g liver vs. 77.9 +/- 1.7 micrograms/h/g liver in control and transgenic mice, respectively). These studies indicate that LCAT modulates the rate by which cholesterol is effluxed from cell membranes onto HDL, esterified, and transported to the liver.

Animals↗
PubMed1996

Human CD8 alpha expression in NK cells but not cytotoxic T cells of transgenic mice.

In our previous work, DNase hypersensitivity mapping was used to identify an enhancer within the human CD8 alpha (hCD8 alpha) gene which allowed T cell-specific expression of a reporter construct in transiently transfected cell lines. To study the role of this intronic enhancer in vivo, transgenic mice were made using human CD8 genomic constructs. We found that while a 14 kb wild-type human CD8 alpha (WThCD8) genomic construct did not lead to expression in mature peripheral CD8+ T cells, this transgene was consistently expressed in small populations of T cells and B cells, and in a subset of mouse NK cells. While murine CD8 is not normally expressed on resting NK cells, expression of the human CD8 transgene on mouse NK cells is appropriate since CD8 is expressed on a subset of human NK cells. Deletion of the intronic enhancer resulted in a complete loss of transgene expression in most lines and a loss of expression only in NK cells in one line. Our results indicate, firstly, that cis-acting sequences within the 14 kb genomic fragment are sufficient for NK cell-specific expression. In addition, our results suggest that the enhancer may have dual roles in regulation of transgene expression. It may enhance general expression of the transgene and may also be required for NK cell-specific expression.

Animals↗
PubMed1996

Treatment with growth hormone and dexamethasone in mice transgenic for human islet amyloid polypeptide causes islet amyloidosis and beta-cell dysfunction.

Islet amyloid derived from islet amyloid polypeptide (IAPP) is a well-recognized feature of type II diabetes. However, the mechanism of islet amyloidogenesis is unknown. In vitro studies suggest that amino acid residues 20-29 in human, but not mouse, IAPP confer amyloidogenicity consistent with the absence of spontaneous islet amyloidosis in mice. Several clinical and in vitro studies suggest that increased synthetic rates of IAPP predispose to IAPP-amyloidosis. In the present study, we sought to test the hypothesis that pharmacological induction of insulin resistance in a mouse transgenic (TG) for human IAPP would induce islet amyloid and beta-cell dysfunction. TG and non-transgenic (N-TG) control mice were treated with both rat growth hormone (12 micrograms/day) and dexamethasone (0.24 mg/day) (dex/GH) or received no treatment for 4 weeks, after which animals were killed to examine islet morphology. Treatment with dex/GH caused hyperglycemia (7.3 +/- 0.4 vs. 5.2 +/- 0.1 mmol/l, TG vs. N-TG, P < 0.001) associated with a decreased plasma insulin concentration (595 +/- 51 vs. 996 +/- 100 pmol/l, TG vs. N-TG, P < 0.05) in TG versus control mice. Islet amyloid was induced in treated TG mice but not in control mice. Islet amyloid was identified in both intra- and extracellular deposits, the former being associated with evidence of beta-cell degeneration. We conclude that dex/GH treatment in mice TG for human IAPP induces IAPP-derived islet amyloid, hyperglycemia, and islet dysfunction. The present model recapitulates the islet morphology and phenotype of type II diabetes.

Amyloid↗
PubMed1991

legless insertional mutation: morphological, molecular, and genetic characterization.

Limb morphogenesis is an excellent model system to study pattern formation during vertebrate development. The legless (lgl) insertional mutation can serve as a tool to analyze specific events in limb development at the embryologic, genetic, and molecular levels. Hemizygous mice of this transgenic line are phenotypically normal, but homozygous mutants are inviable and exhibit limb, brain, and craniofacial malformations, as well as situs inversus. By morphological analysis of mutant hindlimb buds we show absence of a normal apical ectodermal ridge, a structure required for limb bud outgrowth, and an unusually high degree of mesenchymal and ectodermal cell death. Mutant embryos are extremely sensitive to retinoic acid, a known teratogen with a proposed role in limb development. The hindlimb malformations in legless mutants are less severe when bred into the BALB/c background, suggesting the involvement of other strain-specific genes. Molecular analysis of the disrupted region indicates two tightly linked insertion sites. Sequences flanking the transgene insertions have been cloned and mapped to chromosome 12, near the iv (situs inversus viscerum) locus. Consistent with this, complementation tests confirm allelism of lgl and iv and suggest that the transgene insertion may have disrupted more than one gene. Phylogenetically conserved sequences flanking the transgene insertions were identified and used to isolate candidate lgl and iv cDNAs.

Alleles↗
PubMed1989

Analysis of possible repressor elements in the 5'-flanking region of the human beta-globin gene.

Human beta-globin gene expression is confined predominantly to the adult with little or no expression of this gene occurring during embryonic or fetal life. The lack of expression of this gene in embryonic and fetal erythroid tissue could be due to the absence of required positive regulatory factors in these cells or the presence of negative regulatory factors which prevent expression of the adult globin gene. To test the repressor model, we have used a gel electrophoretic mobility shift assay to identify regions in the human beta-globin gene which bind proteins found in K562 cells, a cell line that expresses embryonic and fetal globins but not adult beta-globin. DNA fragments comprising the entire human beta-globin gene were assayed using nuclear proteins from K562 cells, and four regions were found that bind proteins. These are located within the 5'-flanking region, within the first and second introns, and at the 3'-flanking region of the gene. Previous studies have suggested the presence of potential repressor sites 5' of exon 2. For this reason, we examined whether the lack of the binding regions in the 5'-flanking sequence allow expression of the human beta-globin gene in transgenic mice during embryonic life. beta-globin gene expression was confined to adult life, indicating that if a transcriptional repressor is responsible for inactivating this gene in embryonic tissue, it is not regulated solely by sequences upstream from -122 bp in the 5'-flanking region of the human beta-globin gene.

Animals↗
PubMed1989

Evidence for a complex regulatory array in the first intron of the human adenosine deaminase gene.

Adenosine deaminase (ADA) is expressed ubiquitously by diverse mammalian cells and tissues but at levels that vary according to tissue and species. In humans, the thymus exhibits levels of the enzyme up to 100-fold higher than most other tissues. Using transgenic mice, we identified human ADA gene regulatory domains. Up to 3.7 kb of 5'-flanking and first exon DNA from the human ADA gene failed to promote the expression of a chloramphenicol acetyl transferase (CAT) reporter gene in an efficient, reproducible, or tissue-appropriate manner in transgenic mice. However, when 12.8 kb of DNA from the first intron of the human ADA gene was placed 3' of CAT-coding and -processing sequences, transgenic mice reproducibly expressed CAT activity in most tissues, with profoundly high levels in the thymus. DNase I hypersensitivity studies demonstrated that among transgenic mouse tissues, human thymus, and a variety of human cell lines, a region of the intron 4-10 kb downstream of the first exon exhibited an array of hypersensitive sites that varied according to tissue and cell type. Deletion of this region from the gene construction eliminated high-level expression in transgenic mice. In transfection-transient expression assays, the 12.8-kb intron fragment exhibited enhancer activity in several cell types. A 1.3-kb fragment encompassing two of the hypersensitive sites exhibited some of these activities. The results of these studies suggest that the diverse pattern of human ADA gene expression is determined, in part, by a cluster of cis-regulatory elements contained within its large first intron.

Adenosine Deaminase↗
PubMed1988

Overlapping transcriptional units on the same strand within the murine beta-glucuronidase gene complex.

We have identified and partially characterized a complex transcriptional unit within the murine beta-glucuronidase gene complex on chromosome 5. On the same strand and within the first intron of the beta-glucuronidase structural gene, Gus-s, we observe an RNA polymerase II promoter motif. That sequences within this carefully defined region can promote RNA polymerase II transcription is supported by results of in vitro transcriptional runoff assays and by expression of a linked reporter gene in both cultured cells and transgenic mice. Results of RNA blot hybridization and S1 nuclease protection studies reveal a 2.2-kilobase processed liver transcript which is initiated just downstream of the promoter motif and sharing little, if any, sequence with the 2.7-kilobase beta-glucuronidase mRNA. Both RNA species are found in liver where beta-glucuronidase is known to be expressed in all cell types. To our knowledge, this is the first description of eukaryotic mRNAs from overlapping transcription units which share the same strand yet exhibit little, if any, sequence similarity. A possible regulatory relationship between these overlapping structural genes is discussed.

Animals↗