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Biomedical subjects

J Lawler

Publications and source records attributed to J Lawler.

At least 55 records · Page 3Linked to original sources
PubMed1998

CD36 mediates binding of soluble thrombospondin-1 but not cell adhesion and haptotaxis on immobilized thrombospondin-1.

In this study, we examined the binding of soluble TSP1 (and ox-LDL) to CD36-transfected cells and the mechanisms by which immobilized TSP1 mediated attachment and haptotaxis (cell migration towards a substratum-bound ligand) of these transfected cells. CD36 cDNA transfection of NIH 3T3 cells clearly induced a dramatic increase in binding of both soluble [125I]-TSP1 and [125I]-ox-LDL to the surface of CD36-transfected cells, indicating that there was a gain of function with CD36 transfection in NIH 3T3 cells. Despite this gain of function, mock- and CD36-transfected NIH 3T3 cells attached and migrated to a similar extent on immobilized TSP1. An anti-TSP1 oligoclonal antibody inhibited CD36-transfected cell attachment to TSP1 while function blocking anti-CD36 antibodies, alone or in combination with heparin, did not. A series of fusion proteins encompassing cell-recognition domains of TSP1 was then used to delineate mechanisms by which NIH 3T3 cells adhere to TSP1. Although CD36 binds soluble TSP1 through a CSVTCG sequence located within type 1 repeats, 18,19CD36-transfected NIH 3T3 cells did not attach to immobilized type 1 repeats while they did adhere to the N-terminal, type 3 repeats (in an RGD-dependent manner) and the C-terminal domain of TSP1. Conversely, Bowes melanoma cells attached to type 1 repeats and the N- and C-terminal domains of TSP1. However, CD36cDNA transfection of Bowes cells did not increase cell attachment to type 1 repeats compared to that observed with mock-transfected Bowes cells. Moreover, a function blocking anti-CSVTCG peptide antibody did not inhibit the attachment of mock- and CD36-transfected Bowes cells to type 1 repeats. It is suggested that CD36/TSP1 interaction does not occur upon cell-matrix adhesion and haptotaxis because TSP1 undergoes conformational changes that do not allow the exposure of the CD36 binding site.

3T3 Cells↗
PubMed1998

Immunolocalization of thrombospondin-1 in human atherosclerotic and restenotic arteries.

Experimental studies have implicated a functional role for the extracellular matrix glycoprotein thrombospondin-1 (TSP-1) in vascular smooth muscle cell proliferation and migration. We therefore sought to determine if TSP-1 might represent a specific component of the fibroproliferative tissue typically associated with restenosis lesions from human coronary and peripheral arteries. Positive immunostaining for TSP-1 was limited to hypocellular plaques typical of primary atherosclerosis; in contrast, such staining was nearly absent from the loose extracellular matrix of the fibroproliferative tissue typical of restenotic lesions. Only a small fraction of vascular smooth muscle cells in either primary or restenotic lesions demonstrated a cellular staining pattern for TSP-1, which was also observed in control studies performed in cell culture and in atherosclerotic rabbit arteries examined 3 days after experimental balloon angioplasty. Double-staining for TSP-1 and proliferating cell nuclear antigen in studies of human beings disclosed that only a small portion of proliferating cell nuclear antigen-positive cells also stained for TSP-1. The observations made in this series of specimens thus indicate that TSP-1 is not a major component of the extracellular matrix of human restenotic tissues, even when such specimens demonstrate evidence of hypercellularity or ongoing cellular proliferation. Because most restenosis specimens, however, were retrieved > or =1 month after the primary intervention, a functional role for TSP-1 in smooth muscle cell proliferation or migration at the early stages of lesion development is still possible.

Animals↗
PubMed1998

Characterization of cartilage oligomeric matrix protein (COMP) in human normal and pseudoachondroplasia musculoskeletal tissues.

Cartilage oligomeric matrix protein (COMP), the fifth member of the -thrombospondin gene family, is an extracellular matrix calcium-binding protein. The importance of COMP is underscored by the finding that mutations in COMP cause the human dwarfing condition, pseudoachondroplasia (PSACH). Here, we report the results of human tissue distribution and cell secretion studies of human COMP. COMP is expressed and secreted by cultured monolayer chondrocyte, tendon and ligament cells, and COMP secretion is not restricted to a differentiated chondrocyte phenotype. Whereas COMP is retained in the endoplasmic reticulum that accumulates within PSACH chondrocytes in vivo, COMP is not retained intracellularly in the dedifferentiated PSACH chondrocytes in cultures. These results lend further support to the hypothesis that retention of COMP is related to the terminal PSACH chondrocyte phenotype, processing of proteins related to extracellular matrix formation, and maintenance in cartilage.

Achondroplasia↗
PubMed1998

Retention of cartilage oligomeric matrix protein (COMP) and cell death in redifferentiated pseudoachondroplasia chondrocytes.

Cartilage oligomeric matrix protein (COMP) is a large extracellular glycoprotein that is found in the territorial matrix surrounding chondrocytes. Two skeletal dysplasias, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1) are caused by mutations in the calcium binding domains of COMP. In this study, we identified two PSACH mutations and assessed the effect of these mutations on redifferentiated chondrocyte structure and function. We confirmed, in vitro, that COMP is retained in enormous cisternae of the rough endoplasmic reticulum (rER) and relatively absent in the PSACH matrix. The rER accumulation may compromise chondrocyte function, leading to chondrocyte death. Moreover, while COMP appears to be deficient in the PSACH matrix, the matrix appeared to be normal but the over-all quantity was reduced. These results suggest that the abnormality in linear growth in PSACH may result from decreased chondrocyte numbers which would also affect the amount of matrix produced.

Achondroplasia↗
PubMed1998

A molecular basis for retinol stimulation of vesicle budding in vivo and in vitro.

Retinol stimulates the formation of transition vesicles in situ and in all free systems based on rat liver. The stimulation is on vesicle formation from transitional endoplasmic reticulum and not on vesicle fusion with donor membranes. Vesicle budding in the cell free system requires a nucleoside triphosphate and is sensitive to inhibition by thiol reagents. In this report we develop and test a model whereby a retinol-modulated NADH:protein disulfide reductase (NADH oxidase) with protein disulfide-thiol interchange activity is implicated in the vesicle budding mechanism. The protein has the ability to restore activity to scrambled, inactive RNase A and is stimulated or inhibited by retinol depending on the redox environment. Under reducing conditions and in the presence of a chemical reductant such as GSH, the partial reaction stimulated by retinol appears to be the oxidation of membrane thiols. This is the first report of an enzymatic mechanism to explain specific retinol effects both in vivo and in vitro on membrane trafficking not given by retinoic acid.

Animals↗
PubMed1998

The sulfonylurea-inhibited NADH oxidase activity of HeLa cell plasma membranes has properties of a protein disulfide-thiol oxidoreductase with protein disulfide-thiol interchange activity.

Plasma membrane vesicles of HeLa cells are characterized by a drug-responsive oxidation of NADH. The NADH oxidation takes place in an argon or nitrogen atmosphere and in samples purged of oxygen. Direct assay of protein thiols by reaction with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB; Ellman's reagent), suggests that protein disulfides may be the natural electron acceptors for NADH oxidation by the plasma membrane vesicles. In the presence of NADH, protein disulfides of the membranes were reduced with a concomitant stoichiometric increase in protein thiols. The increase in protein thiols was inhibited in parallel to the inhibition of NADH oxidation by the antitumor sulfonylurea LY181984 with an EC50 of ca. 30 nM. LY 181984, with an EC50 of 30 nM, also inhibited a protein disulfide-thiol interchange activity based on the restoration of activity to inactive (scrambled) RNase and thiol oxidation. The findings suggest that thiol oxidation, NADH-dependent disulfide reduction (NADH oxidation), and protein disulfide-thiol interchange in the absence of NADH all may be manifestations of the same sulfonylurea binding protein of the HeLa plasma membrane. A surface location of the thiols involved was demonstrated using detergents and the impermeant thiol reagent p-chloromercuriphenylsulfonic acid (PCMPS). The surface location precludes a physiological role of the protein in NADH oxidation. Rather, it may carry out some other role more closely related to a function in growth, such as protein disulfide-thiol interchange coupled to cell enlargement.

Cell Membrane↗
PubMed1998

Phenomenologies as research methodologies for nursing: from philosophy to researching practice.

This paper is concerned with the popularity of phenomenologies and the tensions that arise from their use as research methodologies in nursing. Among these tensions are: the troublesome issues of adapting a fundamentally philosophical means of understanding human being(s) for use as a more pragmatic and robust research approach in a practice discipline; the various types of phenomenology and the confusions that surround these and other interpretive methodologies, particularly within different intellectual and cultural traditions; and the need for nursing to find a space in which it can give voice to aspects of its practice that are silenced in less existentially oriented methodologies. Although phenomenologies are currently popular and possibly fashionable in nursing, there are important issues in relation to their use in a methodological sense that remain to be addressed.

Culture↗
PubMed1998

Systemic lupus erythematosus in a naval aviator--its aeromedical implications.

Systemic lupus erythematosus (SLE) is a chronic multisystem autoimmune disease, characterized by immune dysregulation resulting in the production of antinuclear antibodies (ANA), generation of circulating immune complexes, and activation of the complement system. SLE is notable for its broad spectrum of clinical manifestations and a variable course characterized by exacerbations and remissions. The pathologic hallmark of the disease is recurrent, widespread, and diverse vascular lesions. This multisystem affliction is unpredictable and brings many aeromedical and occupational health issues that need careful consideration in the differential diagnosis of a patient with symmetrical joint pain or facial rash.

Adult↗
PubMed1997

Decorin inhibits cell attachment to thrombospondin-1 by binding to a KKTR-dependent cell adhesive site present within the N-terminal domain of thrombospondin-1.

Skin decorin (DCN) is an antiadhesive dermatan sulfate-rich proteoglycan that interacts with thrombospondin-1 (TSP) and inhibits fibroblast adhesion to TSP [Winnemöller et al., 1992]. Molecular mechanisms by which DCN interacts with TSP and inhibits cell adhesion to TSP are unknown. In the present study, we showed that skin DCN and bone DCN (chondroitin sulfate-rich proteoglycan) were quantitatively identical with respect to their ability to interact with TSP. Using a series of fusion proteins corresponding to the different structural domains of TSP, binding of [125I]DCN to TSP was found to be dependent of the N-terminal domain and, to a lesser extent, of the type 1 repeats and the C-terminal domain of TSP. In addition, heparan sulfate drastically inhibited [125I]DCN binding to solid-phase adsorbed TSP (80% inhibition), suggesting that DCN could bind to the N-terminal domain of TSP through interaction with heparin-binding sequences. To address this question, a series of synthetic peptides, overlapping heparin-binding sequences ARKGSGRR (residues 22-29), KKTR (residues 80-83) and RLRIAKGGVNDN (residues 178-189), were synthesized and tested for their ability to interact with DCN. [125I]DCN interacted only with peptides VDAVRTEKGFLLLASLRQMKKTRGT and KKTRGTLLALERKDHS containing the heparin-binding consensus sequence KKTR. These peptides contained glycosaminoglycan-dependent and -independent binding sites because [125I]DCN binding to VDAVRTEKGFLLLASLRQMKKTRGT and KKTRGTLLALERKDHS was partially reduced upon removal of the glycosaminoglycan chain (65% and 46% inhibition, respectively). [125I]DCN poorly bound to subpeptide MKKTRG and did not bind at all to subpeptides VDAVRTEKGFLLLASLRQ and TLLALERKDHS, suggesting that heparin-binding sequence MKKTRG constituted a DCN binding site when flanked with peptides VDAVRTEKGFLLLASLRQ and TLLALERKDHS. The sequence VDAVRTEKGFLLLASLRQMKKTRGTLLALERKDHS constitutes a cell adhesive active site in the N-terminal domain of TSP [Clezardin et al., 1997], and DCN inhibited the attachment of fibroblastic and osteoblastic cells to peptides VDAVRTEKGFLLLASLRQMKKTRGT and KKTRGTLLALERKDHS by about 50 and 80%, respectively. Although fibroblastic cells also attached to type 3 repeats and the C-terminal domain of TSP, DCN only inhibited cell attachment to the C-terminal domain. Overall, these data indicate that modulation by steric exclusion of cell adhesion to a KKTR-dependent cell adhesive site present within the N-terminal domain of TSP could explain the antiadhesive properties of DCN.

3T3 Cells↗
PubMed1997

Inhibition of sickle erythrocyte adhesion to immobilized thrombospondin by von Willebrand factor under dynamic flow conditions.

Sickle red blood cell (RBC) adhesion to the blood vessel wall is hypothesized to be the initiating event in the periodic vaso-occlusive episodes that characterize sickle cell disease (SCD). Thrombospondin-1 (TSP) and von Willebrand factor (vWF) have each been implicated in the adhesion of sickle RBC to vascular endothelial cells (EC) and subendothelial matrices. To better understand the contributions of each of these adhesive glycoproteins, we examined the adhesion of sickle RBC to immobilized TSP and vWF using a parallel plate flow chamber. Under postcapillary venular shear stress (1 dyne/cm2), sickle RBC adhered preferentially to TSP. To explore potential interactive effects of vWF and TSP, we examined sickle RBC adhesion to mixtures of these proteins. Whether the proteins were first combined in solution or sequentially applied to the slide, the presence of vWF inhibited the binding of sickle RBC to TSP. The inhibition of adhesion by vWF was shown to be the result of specific and saturable binding of vWF to TSP. Furthermore, vWF in solution at normal plasma levels also inhibited RBC adhesion to immobilized TSP. These data indicate that sickle RBC adhesion in vivo may be significantly influenced by the relative concentrations of TSP and vWF in the vascular wall.

Anemia, Sickle Cell↗
PubMed1997

Identification of cell adhesive active sites in the N-terminal domain of thrombospondin-1.

Using a series of fusion proteins that span almost all of the thrombospondin-1 (TSP-1) molecule, we observed in this study that Chinese hamster ovary (CHO) K1 cells strongly attached to the N-terminus but not to the other domains of TSP-1 (e.g. the C-terminus, and type 1, type 2 and type 3 repeats). In addition, attachment to the N-terminus of CHO S745 cells defective in cell-surface glycosaminoglycans (GAGs) was decreased by 47% compared with that observed with CHO K1 cells, indicating the presence of GAG-dependent cell adhesive sites. With the aim of identifying these cell adhesive sites, a series of synthetic peptides, overlapping heparin-binding sequences ARKGSGRR (residues 22-29), MKKTRG (residues 79-84) and TRDLASIARLRIAKGVNDNF (residues 170-189), were synthesized and tested for their ability to support CHO cell attachment. Using both centrifugation and cell-attachment assays, MKKTRG-containing peptides promoted CHO K1 cell adhesion, while ARKGSGRR-containing peptides and peptide TRDLASIARLRIAKGVNDNF did not. CHO S745 cell attachment to MKKTRG-containing peptides was partially decreased. A 36% decrease in CHO K1 cell attachment to the N-terminus was also observed when the heparin-binding consensus sequence KKTR was mutated to QNTR. In addition, peptide MKKTRG partially inhibited (25% inhibition) CHO K1 cell attachment to the N-terminus. However, peptide MKKTRG was not sufficient to fully promote cell attachment to the N-terminus of TSP-1. Peptides VDAVRTEKGFLLLASLRQ and TLLALERKDHS also supported CHO K1 cell attachment in a GAG-dependent and -independent manner respectively. Moreover, CHO K1 cell attachment to MKKTRG was found to be markedly enhanced when flanked with the sequences VDAVRTEKGFLLLASLRQ and TLLALERKDHS. Peptide VDAVRTEKGFLLLASLRQMKKTRG nearly abolished (98% inhibition) CHO K1 cell attachment to the N-terminus, while peptides MKKTRG, MKKTRGTLLALERKDHS and VDAVRTEKGFLLLASLRQ had only a moderate inhibitory effect (25, 27 and 53% inhibition respectively). These data indicate that the sequence VDAVRTEKGFLLLASLRQMKKTRGTLLALERKDHS (residues 60-94) constitutes a GAG-dependent cell adhesive site in the N-terminus of TSP-1. Moreover, a GAG-independent site, encompassing residues 189-200 (FQGVLQNVRFVF), has been identified. These two adhesive sites supported the attachment of a wide variety of cells (human breast carcinoma, melanoma and osteosarcoma cells), and a high degree of sequence homology was found between TSP-1 and TSP-2 between residues 60 and 94 (48% identity) and 189-200 (67% identity), further suggesting the functional importance of these two cell adhesive sites in the N-terminus of TSP-1.

Amino Acid Sequence↗
PubMed1997

In situ localization of thrombospondin-1 and thrombospondin-3 transcripts in the avian embryo.

Two novel cDNA probes to chicken thrombospondin-1 (TSP-1) and TSP-3 were used to determine by in situ hybridization the origins of these extracellular matrix proteins during embryogenesis. Both TSP-1 and TSP-3 are expressed in embryonic cartilage. TSP-1 expression is limited to early chondrocytes, in contrast to TSP-3 mRNAs that are found in older proliferative and hypertrophic chondrocytes. TSP-1 and TSP-3 are expressed consecutively during neurogenesis as well, with the TSP-1 probe hybridizing in proliferating neuroblasts, and TSP-3 expressed by neurons as they are actively extending processes. A TSP-1 hybridization signal reappears in subpopulations of neurons in the spinal cord and brain after the periods of active neurite extension and programmed cell death are complete. TSP-1 is also expressed in the spinal cord floor plate, in mesenchyme surrounding the developing paramesonephric duct, at the tips of growing lung bronchioles, in lens, and in corneal endothelium. These observations indicate that there is little overlap in the expression patterns of TSP-1 and TSP-3 and that they are expressed consecutively during the development of cartilage and neurons. The pattern of TSP-1 expression in avascular tissues and in the floor plate is consistent with possible roles for this glycoprotein in regulating angiogenesis and establishing morphogenetic gradients.

Animals↗
PubMed1997

Life and the laundromat: reflections on dirty linen and everyday private life.

This is a paper in which I reflect on, and draw issues from, an unplanned ethnographic experience in a London laundromat. The concept of 'everyday life' has a kind of simple, even benign, quality. However, there are many events in everyday life that are complex and complicating. Everyday life can be mundane--boring even. But it can also confront and trouble us, even when it concerns such apparently ordinary matters like doing the laundry. This paper is about how the ordinary matters of everyday life can become problematic and how our involvement in them can confront us with dilemmas that are unwanted yet require our attention and judgement as participants in social life.

Anthropology, Cultural↗
PubMed1997

Pap smear brochures, misogyny and language: a discourse analysis and feminist critique.

Text from a public health brochure on pap smears was analysed with particular reference to ways in which the language used conveys particular messages about women and their bodies. In the text, 'humans' were found to be excluded as such. Both the pap smear service provider and the women who are the recipients of this service--and at whom such brochures are targeted--are objectified and their characteristics of human existence (i.e. ontological capacities) were restricted. The language of the pamphlet invokes an image for women associated with vaginal (penile penetrative) sex. The discourse also is found to be didactic, biomedical and written in the voice of the service provider. Further, the encounter of pap smear events is contextualized as procedural such that not only is the woman 'done to' in the process of having a cervical smear test but the woman's and provider's experiences of the encounter are silenced. It is concluded that the texts may be viewed as misogynist and that such texts do not take account of the complexity of women's decisions to 'submit to' or comply with cervical cancer screening.

Attitude to Health↗
PubMed1997

A qualitative study of specialist nurse support for stroke patients and care-givers at home.

BACKGROUND: The involvement of five specialist nurses in providing a stroke support service was evaluated quantitatively in a recent randomized controlled trial. This complementary study used qualitative methods to evaluate trial outcomes more comprehensively. AIMS: To identify whether the nurses' intervention may have influenced the process of stroke recovery. METHOD: A purposefully selected subsample of 30 patients and 15 care-givers were interviewed within 1-3 months of their final quantitative assessment (12 months after recruitment to the randomized trial). Fifteen of the patients and eight of the care-givers had received visits from a specialist nurse. A semistructured interview was designed to include questions on perceptions of the recovery process and evaluation of services received. RESULTS: Some differences were evident between the accounts of control and intervention group subjects. The less tangible aspects of nurses' interventions--concern, attention, empathy and interest, when combined with sound professional knowledge, had identifiable value to the patients and care-givers. It appeared that the nurses had employed considerable sensitivity and skill in identifying and responding to particular needs at appropriate times. CONCLUSION: The qualitative evaluation offers a different picture to the quantitative results of the randomized controlled trial. In general, the findings of the qualitative study are more positive and encouraging than the quantitative results. The majority of patients and care-givers in the intervention group believed that they had benefited from the specialist nurse's visits.

Caregivers↗