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M W Freeman

Publications and source records attributed to M W Freeman.

At least 37 records · Page 2Linked to original sources
PubMed1998

Decreased cholesterol efflux from fibroblasts of a patient without Tangier disease, but with markedly reduced high density lipoprotein cholesterol levels.

A 51-yr-old woman without clinical evidence of Tangier disease, but with an extremely low high density lipoprotein (HDL) cholesterol level, was studied. No defect in the major structural protein of HDL, apolipoprotein AI (apo AI), was detected. A preponderance of small HDL particles in the patient's plasma suggested defective uptake of cellular cholesterol. Efflux of [3H]cholesterol from patient fibroblasts to normal apo AI was decreased 50%. Cholesterol efflux to HDL was also decreased, but efflux to trypsin-modified HDL was not. The patient's cells partitioned more exogenously provided [3H]cholesterol into free cholesterol and synthesized greater amounts of phosphatidylcholine than did normal or Tangier fibroblasts. Her fibroblasts did not differ from normal fibroblasts in sterol synthesis rate, cellular cholesterol and cholesterol ester content, or incorporation of oleate into cholesterol ester. The data indicate the presence of a defect in apolipoprotein-dependent cellular cholesterol efflux that differs from that seen in Tangier disease. These findings are the first evidence that other low HDL cholesterol syndromes, besides Tangier disease, may also be associated with cholesterol efflux abnormalities. The identification of mutant genes responsible for apolipoprotein-mediated efflux abnormalities should provide valuable insights into cellular mechanisms involved in the reverse cholesterol transport pathway.

Apolipoprotein A-I↗
PubMed1997

Scavenger receptors in atherosclerosis.

Membership in the broad family of scavenger receptors has grown significantly. Two new scavenger receptor receptors have been cloned (SR-BI and SR-CI) and a previously isolated integral membrane protein (CD36) has been found to possess lipoprotein binding properties that entitle it to be included in the family. The breadth of ligand recognition of this class of receptors has also increased with the discovery that high-density lipoproteins and anionic phospholipids can bind to scavenger receptors. New studies linking these receptors to apoptotic and signal transduction pathways have further enlarged their potential contributions to normal immune function as well as the process of atherosclerosis. Clarifying the physiologic as well as pathophysiologic significance of these multifunctional proteins has proven challenging, but mouse homologous recombination technology will soon yield critical insights into the role scavenger receptors play in atherosclerosis and other macrophage-associated immune functions.

Animals↗
PubMed1995

Regulation of smooth muscle cell scavenger receptor expression in vivo by atherogenic diets and in vitro by cytokines.

Scavenger receptor (ScR)-mediated uptake of modified lipoproteins may contribute to the transformation of smooth muscle cells into lipid-laden foam cells during atherogenesis. This study examined the in vivo expression of ScRs in aortas, with or without balloon injury, taken from hypercholesterolemic or normocholesterolemic rabbits. Numerous intimal cells in the rabbit aortic lesions expressed ScRs as detected by immunocytochemical staining with a goat anti-rabbit ScR antibody. Single immunostaining for cell identification markers in serial sections, as well as double staining, confirmed the expression of ScRs by both intimal smooth muscle cells and macrophages. To explore potential inducers of ScR expression by smooth muscle cells in vivo, we studied the regulation of ScR expression in vitro by cytokines known to be present in atherosclerotic lesions. Tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma (IFN-gamma) increased ScR mRNA levels, protein expression, and AcLDL degradative activity in cultured rabbit aortic smooth muscle cells. The induction of ScR expression in intimal smooth muscle cells in vivo could be a useful marker of smooth muscle cell activation during atherogenesis and may contribute to foam cell formation by this cell type following balloon injury and/or hypercholesterolemia. Cytokines, such as TNF-alpha or IFN-gamma, may stimulate some of the phenotypic changes that characterize the alteration in gene expression of intimal smooth muscle cells in rabbit atherosclerotic lesions.

Animals↗
PubMed1995

Structure of the murine macrophage scavenger receptor gene and evaluation of sequences that regulate expression in the macrophage cell line, P388D.

The structure of the entire murine scavenger receptor gene was determined; it consists of eleven exons spanning more than 60 kilobases. Primer extension showed that transcription initiates at a cluster of sites unassociated with a TATAA element. DNA sequences adjacent to these transcription start sites are highly conserved in murine, human, and bovine genes. When transcriptional activity was tested using a luciferase reporter gene, a promoter fragment (-124 to +20) stimulated luciferase production in P388D1 macrophage-like cells but not in non-macrophage COS-7 or 3T3 cells. A longer promoter fragment (approximately 5 kb) stimulated luciferase activity a further 10-fold in P388D1 cells. However, using a series of fragments from -67 to -1500 bp, a 127 bp fragment (-67 to +50) was as active as a 1500 bp fragment in these assays. Mutation of a putative AP-1 element in the -67 to +50 promoter fragment reduced luciferase activity by 40%; mutation of a putative GATA factor element to TATA increased luciferase activity nearly 2-fold while mutation to AATA had no effect and deletion of the GATA sequence inhibited activity by about 50%. The results suggest that a scavenger receptor promoter fragment can confer cell-specific transcription and that the activity may be mediated in part by factors that recognize the AP-1 and GATA elements.

Amino Acid Sequence↗
PubMed1994

Macrophage scavenger receptors.

Macrophage scavenger receptors are integral membrane proteins whose ability to bind and degrade modified LDL has implicated them in the process of atherosclerotic foam cell formation. Their ability to bind non-lipoprotein ligands suggests that they participate in other macrophage-associated host defense activities. Studies utilizing cloned native and mutant forms of the scavenger receptor have provided insights into the structural basis for their function.

Amino Acid Sequence↗
PubMed1993

Surface expression of human CD14 in Chinese hamster ovary fibroblasts imparts macrophage-like responsiveness to bacterial endotoxin.

Cardiovascular collapse associated with Gram-negative septicemia is believed to result from the stimulation of phagocytes by bacterial lipopolysaccharide (endotoxin, LPS). It remains unclear how endotoxin activates phagocytes, but recent evidence suggests the involvement of the glycosyl phosphatidylinositol-linked myelocyte antigen, CD14. We report that transfection of human CD14 into Chinese hamster ovary fibroblasts transfers macrophage-like responsiveness to otherwise LPS-unresponsive cells. These data demonstrate that LPS-induced responsiveness can be transferred to a heterologous non-responder cell type by expression of a single leukocyte-specific gene product.

Animals↗
PubMed1993

Ascorbic acid oxidation product(s) protect human low density lipoprotein against atherogenic modification. Anti- rather than prooxidant activity of vitamin C in the presence of transition metal ions.

The oxidative modification of low density lipoprotein (LDL) has been proposed as an important causative event in the development of human atherosclerosis. As a corollary of this hypothesis, antioxidants that can prevent LDL oxidation may inhibit atherosclerosis. Oxidative modification of LDL in vitro, either induced by Cu2+ or mediated by cultured arterial wall cells in media containing trace amounts of transition metal ions, is strongly inhibited by vitamin C (L-ascorbic acid (AA)). AA, however, is known to act as a prooxidant rather than an antioxidant in the presence of transition metal ions. We observed that AA is oxidized rapidly when incubated with Cu2+ and LDL, leading to transient formation of dehydro-L-ascorbic acid (DHA). Although AA and DHA can no longer be detected after 3.5 h of incubation, LDL resists oxidative modification for at least 20 h, as assessed by anodic gel electrophoretic mobility. Remarkably, DHA protects LDL more effectively against both Cu(2+)-induced lipid peroxidation and shifts in electrophoretic mobility than does AA; indeed, AA per se, without oxidation to DHA, offers no protection. By inhibiting oxidative modification of LDL, AA and DHA prevent uptake of LDL by macrophages via the scavenger receptor pathway. When LDL is incubated with DHA followed by gel filtration, LDL remains protected against subsequent Cu(2+)-induced oxidative modification, suggestive of stable modification of LDL in the presence of DHA. In contrast, DHA is ineffective against a metal ion-independent type of oxidative stress, viz. aqueous peroxyl radicals; under these conditions, only AA is able to inhibit lipid peroxidation in LDL. Our data indicate that vitamin C protects LDL against atherogenic modification by two different mechanisms that may act in concert: (i) free radical scavenging by AA prevents aqueous oxidants from attacking and oxidizing LDL, and (ii) stable modification of LDL by DHA or decomposition product(s) thereof imparts increased resistance to metal ion-dependent oxidation.

Adult↗
PubMed1992

Expression cloning of a common receptor for parathyroid hormone and parathyroid hormone-related peptide from rat osteoblast-like cells: a single receptor stimulates intracellular accumulation of both cAMP and inositol trisphosphates and increases intracellular free calcium.

Parathyroid hormone (PTH), a major regulator of mineral ion metabolism, and PTH-related peptide (PTHrP), which causes hypercalcemia in some cancer patients, stimulate multiple signals (cAMP, inositol phosphates, and calcium) probably by activating common receptors in bone and kidney. Using expression cloning, we have isolated a cDNA clone encoding rat bone PTH/PTHrP receptor from rat osteosarcoma (ROS 17/2.8) cells. The rat bone PTH/PTHrP receptor is 78% identical to the opossum kidney receptor; this identity indicates striking conservation of this receptor across distant mammalian species. Additionally, the rat bone PTH/PTHrP receptor has significant homology to the secretin and calcitonin receptors but not to any other G protein-linked receptor. When expressed in COS cells, a single cDNA clone, expressing either rat bone or opossum kidney PTH/PTHrP receptor, mediates PTH and PTHrP stimulation of both adenylate cyclase and phospholipase C. These properties could explain the diversity of PTH action without the need to postulate other receptor subtypes.

Amino Acid Sequence↗
PubMed1992

Rabbit aortic smooth muscle cells express inducible macrophage scavenger receptor messenger RNA that is absent from endothelial cells.

Scavenger receptors mediate uptake of modified low density lipoproteins by macrophages. The accumulation of lipids via this process is thought to lead to foam cell formation in developing atherosclerotic plaques. Smooth muscle cells, which can also be converted to foam cells in vivo, have not been shown to express the same scavenger receptor previously cloned in macrophages. We report the cloning of two cDNAs that encode type I and type II scavenger receptors isolated from rabbit smooth muscle cells. The deduced protein sequences of these isolates are highly homologous to the scavenger receptors previously isolated from macrophages. Treatment of smooth muscle cells with phorbol esters induced a marked increase in scavenger receptor mRNA and a fivefold increase in receptor degradation activity. Rabbit venous endothelial cells in primary culture and a bovine aortic endothelial cell line had no detectable scavenger receptor mRNA, despite having scavenger receptor degradation activity. The latter finding suggests that endothelial cells may possess a scavenger receptor which is structurally distinct from that found in macrophages and smooth muscle cells. The isolation of cDNAs encoding the rabbit scavenger receptor should prove useful for in vitro and in vivo studies that employ the rabbit as a model of human atherosclerosis.

Amino Acid Sequence↗
PubMed1990

Thymosin beta 4 is expressed in ROS 17/2.8 osteosarcoma cells in a regulated manner.

The differential expression of mRNAs between the closely related rat osteosarcoma cell lines ROS 17/2.8 and ROS 25/1 was used to identify genes whose expression is associated with the osteoblast phenotype. Thymosin beta 4 cDNA was cloned from an ROS 17/2.8 complimentary DAN library on the basis of its differential hybridization with radiolabeled cDNA prepared from ROS 17/2.8 and ROS 25/1 cells. Northern blot analysis confirmed that thymosin beta 4, hitherto a putative immunodulatory hormone, was indeed differentially expressed. Steady state mRNA levels were severalfold higher in ROS 17/2.8 cells exhibiting an osteoblast-like phenotype, compared with the less osteoblast-like ROS 25/1. Thymosin beta 4 transcripts were also detected in rat UMR 106 osteosarcoma cells and in intact neonatal and fetal rat calvaria. Sequence analysis of the cDNA indicated that thymosin beta 4 transcripts may arise by processing at a more distal polyadenylation signal. Treatment of ROS 17/2.8 cells with dexamethasone increased, while addition of 1,25-dihydroxyvitamin D3 decreased thymosin beta 4 mRNA. The phenotype-dependent expression in the ROS cells and the response to steroid hormone suggest that thymosin beta 4 expression contributes to the osteoblast phenotype.

Amino Acid Sequence↗
PubMed1989

Mutations in signal sequence cleavage domain of preproparathyroid hormone alter protein translocation, signal sequence cleavage, and membrane-binding properties.

Signal sequences, known to mediate the targeting of nascent secreted proteins to membranes, share common structural domains: a positively charged amino-terminus, a hydrophobic core, and a signal cleavage domain. Mutations have been introduced into the cDNA encoding the signal sequence of the mammalian protein preproparathyroid hormone to analyze the roles played by the signal cleavage domain in secretion. Two mutant genes were constructed missing the entire six-residue propeptide sequence and several residues of the signal cleavage domain. The effects of these mutations on signal function were assessed after expression in clonal cell lines and in a transcription-linked translation system. Alterations in the signal cleavage domain resulted in reduced translocation and signal cleavage. Furthermore, in one mutant, the removal of the signal cleavage domain converted the signal into a membrane anchor sequence. The nonhydrophobic sequences at the end of the signal sequence thus crucially affect the translocation, cleavage, and membrane-binding properties of signal sequences.

Amino Acid Sequence↗
PubMed1988

Central nervous system lesions in pediatric patients: Gd-DTPA-enhanced MR imaging.

Twenty pediatric patients, aged 2-18 years, with known or suspected masses in the brain and/or spinal cord were studied with magnetic resonance imaging at 0.6T with and without use of 0.1 mmol/kg gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA). The multisection, multiecho imaging mode was utilized. Surgically proved mass lesions included astrocytoma (n = 6), medulloblastoma (n = 2), ependymoma (n = 1), craniopharyngioma (n = 1), oligodendroglioma (n = 1), germinoma (n = 1) and fibrosarcoma (n = 1). Presumptive diagnoses included astrocytoma (n = 3), arachnoid cyst (n = 1), tuberous sclerosis (n = 1), cryptic vascular malformation (n = 1), and normal (n = 1). There was dramatic enhancement in 11 of 20 patients, with improved definition of the presence and extent of lesions in six patients. No adverse effects were noted in any of the 20 patients. It is concluded that Gd-DTPA is useful in delineating the presence, extent, and number of certain lesions of the central nervous system in children.

Adolescent↗
PubMed1987

Consequences of amino-terminal deletions of preproparathyroid hormone signal sequence.

PTH is initially synthesized as a larger precursor, containing a 25 amino acid signal sequence. Modification of cDNA encoding the hormone precursor resulted in the synthesis of proteins whose signal sequences were shortened at their amino termini. The effects of these mutations were analyzed using a cell-free translation system and rat pituitary GH4 cells in culture. Removal of the first six amino acids of the signal sequence had no effect on the efficiency or kinetics of protein processing as measured in the two assay systems. Mutants lacking 10 or 13 amino acids were not processed efficiently in the cells, nor were they translocated across microsomes in the cell-free translation system. These studies suggest that a modest change in the hydrophobic domain of the signal sequence, which might not have been predicted to alter function, led to a dramatic decline in signal activity.

Amino Acid Sequence↗
PubMed1983

Nucleotide sequence of the human parathyroid hormone gene.

From a lambda phage gene library we have isolated phage containing the gene encoding human preproparathyroid hormone. The phage were isolated by using both the plaque-hybridization technique and the in vivo recombination-selection technique. The human preproparathyroid hormone gene contains two intervening sequences that separate the gene into a 5' noncoding domain, a "prepro" sequence domain, and a domain containing the parathyroid hormone sequence and the 3' noncoding region. The gene is approximately 4,200 base pairs long. Restriction endonuclease analysis of human leukocyte DNA shows that the haploid human genome contains one copy of the preproparathyroid hormone gene. A 14-base-pair sequence of alternating purines and pyrimidines that has the potential of adopting the Z-DNA conformation lies 134 base pairs upstream from the presumed site of initiation of transcription.

Amino Acid Sequence↗