Biomedical subjects

M D Layne

Publications and source records attributed to M D Layne.

26 records · Page 2Linked to original sources

PubMed1998

Embryonic expression suggests an important role for CRP2/SmLIM in the developing cardiovascular system.

Proteins of the LIM family are critical regulators of development and differentiation in various cell types. We have described the cloning of cysteine-rich protein 2/smooth muscle LIM protein (CRP2/SmLIM), a LIM-only protein expressed in differentiated vascular smooth muscle cells. As a first step toward understanding the potential functions of CRP2/SmLIM, we analyzed its expression after gastrulation in developing mice and compared the expression of CRP2/SmLIM with that of the other 2 members of the CRP subclass, CRP1 and CRP3/MLP. In situ hybridization in whole-mount and sectioned embryos showed that CRP2/SmLIM was expressed in the sinus venosus and the 2 cardiac chambers at embryonic day 9. Vascular expression of CRP2/SmLIM was first seen at embryonic day 10. At subsequent time points, CRP2/SmLIM expression decreased in the heart but remained high in the vasculature. CRP1 was expressed both in vascular and nonvascular tissues containing smooth muscle cells, whereas CRP3/MLP was expressed only in tissues containing striated muscle. These patterns of expression were maintained in the adult animal and suggest an important role for this gene family in the development of smooth and striated muscle.

Age Factors↗

PubMed1998

Aortic carboxypeptidase-like protein, a novel protein with discoidin and carboxypeptidase-like domains, is up-regulated during vascular smooth muscle cell differentiation.

Phenotypic modulation of vascular smooth muscle cells plays an important role in the pathogenesis of arteriosclerosis. In a screen of proteins expressed in human aortic smooth muscle cells, we identified a novel gene product designated aortic carboxypeptidase-like protein (ACLP). The approximately 4-kilobase human cDNA and its mouse homologue encode 1158 and 1128 amino acid proteins, respectively, that are 85% identical. ACLP is a nonnuclear protein that contains a signal peptide, a lysine- and proline-rich 11-amino acid repeating motif, a discoidin-like domain, and a C-terminal domain with 39% identity to carboxypeptidase E. By Western blot analysis and in situ hybridization, we detected abundant ACLP expression in the adult aorta. ACLP was expressed predominantly in the smooth muscle cells of the adult mouse aorta but not in the adventitia or in several other tissues. In cultured mouse aortic smooth muscle cells, ACLP mRNA and protein were up-regulated 2-3-fold after serum starvation. Using a recently developed neural crest cell to smooth muscle cell in vitro differentiation system, we found that ACLP mRNA and protein were not expressed in neural crest cells but were up-regulated dramatically with the differentiation of these cells. These results indicate that ACLP may play a role in differentiated vascular smooth muscle cells.

Adult↗

PubMed1998

Molecular cloning, characterization, and promoter analysis of the mouse Crp2/SmLim gene. Preferential expression of its promoter in the vascular smooth muscle cells of transgenic mice.

Several members of the LIM protein family have important roles in development and differentiation. We recently isolated a rat cDNA encoding a new member of this family, CRP2/SmLIM, that contains two LIM domains and is expressed preferentially in vascular smooth muscle cells (VSMC). To study the molecular mechanisms that regulate VSMC-specific transcription of the Crp2/SmLim gene, we cloned the cDNA and gene of mouse Crp2/SmLim. Mouse Crp2/SmLim is a single copy gene of six exons and five introns spanning approximately 20 kilobases of genomic DNA. By 5'-rapid amplification of cDNA ends and S1 nuclease protection assay, we determined that the transcription start site is an A residue 80 base pairs 5' of the translation initiation codon. A TATA-like sequence is located 27 base pairs 5' of the transcription start site, and there are potential cis-acting elements (GATA, Sp1, AP-2, E box, CCAC box, and GArC motif) in the 5'-flanking sequence. In transient transfection assays in rat aortic smooth muscle cells in primary culture, 5 kilobases of the Crp2/SmLim 5'-flanking sequence generated a high level of luciferase reporter gene activity. By deletion analysis and gel mobility shift assay, we found that the region between bases -74 and -39 of this 5 kilobase DNA fragment binds Sp1 and confers basal promoter activity in the Crp2/SmLim gene. In vitro, the 5-kilobase fragment was active in multiple cell types. In vivo, however, the 5-kilobase fragment directed high level expression of the lacZ reporter gene preferentially in the VSMC of transgenic mice, indicating the presence of VSMC-specific element(s) in this fragment.

Amino Acid Sequence↗

PubMed1998

In vitro system for differentiating pluripotent neural crest cells into smooth muscle cells.

The change in vascular smooth muscle cells (SMC) from a differentiated to a dedifferentiated state is the critical phenotypic response that promotes occlusive arteriosclerotic disease. Despite its importance, research into molecular mechanisms regulating smooth muscle differentiation has been hindered by the lack of an in vitro cell differentiation system. We identified culture conditions that promote efficient differentiation of Monc-1 pluripotent neural crest cells into SMC. Exclusive Monc-1 to SMC differentiation was indicated by cellular morphology and time-dependent induction of the SMC markers smooth muscle alpha-actin, smooth muscle myosin heavy chain, calponin, SM22alpha, and APEG-1. The activity of the SM22alpha promoter was low in Monc-1 cells. Differentiation of these cells into SMC caused a 20-30-fold increase in the activity of the wild-type SM22alpha promoter and that of a hybrid promoter containing three copies of the CArG element. By gel mobility shift analysis, we identified new DNA-protein complexes in nuclear extracts prepared from differentiated Monc-1 cells. One of the new complexes contained serum response factor. This Monc-1 to SMC model should facilitate the identification of nodal regulators of smooth muscle development and differentiation.

Animals↗

PubMed1998

Human EZF, a Krüppel-like zinc finger protein, is expressed in vascular endothelial cells and contains transcriptional activation and repression domains.

Members of the erythroid Krüppel-like factor (EKLF) multigene family contain three C-terminal zinc fingers, and they are typically expressed in a limited number of tissues. EKLF, the founding member, transactivates the beta-globin promoter by binding to the CACCC motif. EKLF is essential for expression of the beta-globin gene as demonstrated by gene deletion experiments in mice. Using a DNA probe from the zinc finger region of EKLF, we cloned a cDNA encoding a member of this family from a human vascular endothelial cell cDNA library. Sequence analysis indicated that our clone, hEZF, is the human homologue of the recently reported mouse EZF and GKLF. hEZF is a single-copy gene that maps to chromosome 9q31. By gel mobility shift analysis, purified recombinant hEZF protein bound specifically to a probe containing the CACCC core sequence. In co-transfection experiments, we found that sense but not antisense hEZF decreased the activity of a reporter plasmid containing the CACCC sequence upstream of the thymidine kinase promoter by 6-fold. In contrast, EKLF increased the activity of the reporter plasmid by 3-fold. By fusing hEZF to the DNA-binding domain of GAL4, we mapped a repression domain in hEZF to amino acids 181-388. We also found that amino acids 91-117 of hEZF confer an activation function on the GAL4 DNA-binding domain.

Amino Acid Sequence↗

PubMed1995

Animal cell mutants unable to take up biologically active glycerophospholipids.

We have isolated two mutant strains from the murine, macrophage-like cell line, RAW 264.7, that are resistant to the cytotoxic effects of the antineoplastic, platelet activating factor analogue, 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine (ET-18-OMe). The mutants were isolated using a single round of selection to ensure that resistance was due to a single gene defect. These mutants, RAW.R1 and RAW.R23, are approximately 20 times more resistant to ET-18-OMe (ID50 = 15-17 microM) than the parent strain (ID50 = 0.7-1.0 microM). Resistance to ET-18-OMe was due to a 90-95% reduction in the ability to take up and accumulate this compound. The uptake of other choline glycerophospholipids (e.g., platelet activating factor and 1-acyl-2-lyso-sn-glycero-3-phosphocholine) was also severely affected. This defect was not limited to choline glycerophospholipids; the uptake of an ethanolamine glycerophospholipid (1-alkyl-2-lyso-sn-glycero-3-phosphoethanolamine) was reduced by 80%. The uptake of palmitic acid, an amphipathic molecule bearing no phosphate-containing head group, was unaffected in the mutants. There was little metabolism of ET-18-OMe by either the wild-type or mutant cells. Binding of ET-18-OMe appeared to be normal in the mutants, but internalization of pre-bound ET-18-OMe was reduced. Uptake of non-lipid ligands such as horseradish peroxidase, lucifer yellow, and transferrin was normal in the mutants demonstrating that fluid-phase and receptor-mediated endocytosis is functional. The ability to generate mutants displaying a lesion that affects the uptake of both choline and ethanolamine phospholipids demonstrates that these species are internalized by RAW cells through one common primary route or through pathways that share a common factor. These mutants, and this approach to their isolation, offer a system with which to study and define the mechanisms of glycerophospholipid uptake into macrophages as well as other cell types.

Animals↗

PubMed1994

Superoxide production by macrophages stimulated in vivo with synthetic ether lipids.

The anticancer activity of synthetic ether lipids may depend in part upon their ability to activate cells of the monocyte/macrophage lineage. In the present study, we have sought to determine whether 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OMe) and related ether lipids enhance superoxide production by mouse peritoneal macrophages. Ether lipids were administered intraperitoneally to C57BL/6 mice 4 d after injection with thioglycollate broth. Elicited peritoneal macrophages were harvested and purified one day later, and superoxide production was detected by measuring the superoxide dismutase inhibitable reduction of cytochrome c. Low levels of superoxide were secreted by macrophages in the absence of phorbol 12-myristate 13-acetate (PMA). When PMA was added in vitro to macrophages from ET-18-OMe-treated mice, these cells secreted 194.2 nmol superoxide/mg protein in comparison to 53.5 nmol superoxide/mg protein for PMA-treated control cells. The in vitro treatment of the macrophages with ET-18-OMe was not effective in stimulating superoxide secretion. Macrophages harvested from mice treated with a series of ether lipids (with and without phosphorus) were examined, and superoxide secretion was found to vary with structure. AM-18-OEt and CP-7 were the most effective compounds, secreting 8.6 and 11.9 times more superoxide, respectively, than PMA-stimulated control cells. Moreover, direct cytotoxicity of the compounds for HL60 human promyelocytic leukemic cells did not necessarily correlate with the ability of each drug to increase macrophage superoxide production.

Animals↗

PubMed1993

Synthesis of phosphocholine and quaternary amine ether lipids and evaluation of in vitro antineoplastic activity.

The in vitro antineoplastic activity of many phosphorus-containing (e.g., phosphocholines) and non-phosphorus-containing (e.g., quaternary ammonium salts) ether lipids has been evaluated in the HL-60 promyelocytic cell line. These compounds are analogues of ET-18-OMe (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine). Structural modification of 1-(alkylamido)-, -(alkylthio)-, and -(alkyloxy)propyl backbones has provided further insight into the structure-activity relationships of these lipids. In this study, a long saturated C-1 chain and a three-carbon backbone with a single short C-2 substituent were preferred. At the positively charged nitrogen of phosphocholines, fewer than three substituents caused a significant loss of activity, and substituents larger than methyl decreased activity slightly. In the nonphosphorus compounds, many nitrogen heterocycles and also a sulfonium moiety were incorporated without changing the degree of activity; however, a thiazolium group decreased activity. The most active compound, 29 [N-[3-(hexadecyloxy)-2-methoxypropyl]-3-(hydroxymethyl)pyridinium bromide], was approximately twice as active as the reference standard, ET-18-OMe, in a trypan blue dye exclusion assay.

Antineoplastic Agents↗