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A McDowall

Publications and source records attributed to A McDowall.

18 recordsLinked to original sources
PubMed2000

The regulation of integrin function by Ca(2+).

Integrins are metalloproteins whose receptor function is dependent on the interplay between Mg(2+) and Ca(2+). Although the specificity of the putative divalent cation binding sites has been poorly understood, some issues are becoming clearer and this review will focus on the more recent information. The MIDAS motif is a unique Mg(2+)/Mn(2+) binding site located in the integrin alpha subunit I domain. Divalent cation bound at this site has a structural role in coordinating the binding of ligand to the I domain containing integrins. The I-like domain of the integrin beta subunit also has a MIDAS-like motif but much less is known about its cation binding preferences. The N-terminal region of the integrin alpha subunit has been modelled as a beta-propeller, containing three or four 'EF hand' type divalent cation binding motifs for which the function is ill defined. It seems certain that most integrins have a high affinity Ca(2+) site which is critical for alphabeta heterodimer formation, but the location of this site is unknown. Finally intracellular Ca(2+) fluxes activate the Ca(2+) requiring enzyme, calpain, which regulates cluster formation of leucocyte integrins.

Animals↗
PubMed2000

The second domain of intercellular adhesion molecule-1 (ICAM-1) maintains the structural integrity of the leucocyte function-associated antigen-1 (LFA-1) ligand-binding site in the first domain.

The first domain of intercellular adhesion molecule-1 (ICAM-1) binds to the leucocyte function-associated antigen-1 (LFA-1) I domain, which contains the principal ligand-binding site of this leucocyte integrin. Whether the function of the second domain is also to directly bind LFA-1 has been unclear. Our data show that mutation in the hydrophilic EF loop of ICAM-1 domain 2 resulted in impaired binding of the isolated I domain when compared with wild-type ICAM-1. LFA-1 on T-cells also binds with reduced affinity to this ICAM-1 mutant. A hybrid construct containing the first domain of vascular cell-adhesion molecule-1 joined to domains 2-5 of ICAM-1 was unable to bind to the I domain, showing that there is no direct interaction between the second domain of ICAM-1 and the I domain. This construct was also not bound by LFA-1 expressed in T-cells. Function-blocking monoclonal antibodies that map to domain 2 of ICAM-1, implicating this domain in ligand binding, were found to act indirectly. In summary our data suggest that the second domain of ICAM-1 has a role in maintaining the structure of the LFA-1 ligand-binding site in the first domain of ICAM-1 but does not appear to have a direct role in ligand binding.

Antibodies, Monoclonal↗
PubMed2000

A functional analysis of a natural variant of intercellular adhesion molecule-1 (ICAM-1Kilifi).

Intercellular adhesion molecule-1 (ICAM-1) is involved in a range of interactions both within the host and between the host and a number of pathogens. Recently we described a mutation within the coding region of the first N-terminal immunoglobulin-like domain of ICAM-1, present at high frequency within African populations, which increased the risk of cerebral malaria. To understand the mechanism by which such a polymorphism might be maintained despite counter-selection by malaria, we have carried out functional assays using both forms of ICAM-1 as soluble Fc chimeric fusion proteins. ICAM-1Kilifi has reduced avidity for LFA-1 compared with ICAM-1ref and binding to soluble fibrinogen was completely abolished with the Kilifi variant. In Plasmodium falciparum adhesion assays, ITO4-A4u binding to ICAM-1Kilifi was reduced compared with binding to the reference form. These results allow for the possibility of balanced selection between the reference and Kilifi forms of ICAM-1 through modulation of inflammatory responses and indicate the existence of differences within ICAM-1-binding P. falciparum isolates which may be relevant to pathogenesis.

Animals↗
PubMed1998

The I domain of integrin leukocyte function-associated antigen-1 is involved in a conformational change leading to high affinity binding to ligand intercellular adhesion molecule 1 (ICAM-1).

On T cells the leukocyte integrin leukocyte function-associated antigen-1 (LFA-1) (CD11a/CD18) can be induced to bind its ligand intercellular adhesion molecule 1 (ICAM-1) (CD54) either by increasing the affinity of the receptor with Mg2+ and EGTA or by receptor clustering following activation with phorbol ester. The existence of these two adhesion-inducing pathways implies that alternative mechanisms might exist by which LFA-1 engages ICAM-1. The LFA-1 alpha subunit I domain contains a major binding site for ICAM-1. In this study we show that soluble LFA-1 I domain blocks ICAM-1 binding of the high affinity Mg2+-induced form of LFA-1 but not the phorbol ester-induced form. Under conditions of Mg2+-activation, the soluble I domain also prevents expression of an activation dependent epitope on LFA-1, implying that it inhibits a conformational change necessary for conversion to the high affinity form of this integrin. In addition, the binding of Mg2+-activated LFA-1 to ICAM-1 is blocked by peptides covering the alpha4-beta3 loop, the beta3-alpha5 loop, and the alpha5 helix of the I domain, whereas none of the peptides tested blocks phorbol ester-mediated adhesion. The blocking peptides localize to the same face of the crystal structure of the LFA-1 I domain and define an area that, during activation, may be involved in association of the I domain with another region of LFA-1, potentially the beta-propeller domain. This is the first evidence linking a structural domain of an integrin, in this case the I domain, with a particular activation mechanism.

Amino Acid Sequence↗
PubMed1998

LFA-1-mediated adhesion is regulated by cytoskeletal restraint and by a Ca2+-dependent protease, calpain.

The activity of integrins on leukocytes is kept under tight control to avoid inappropriate adhesion while these cells are circulating in blood or migrating through tissues. Using lymphocyte function-associated antigen-1 (LFA-1) on T cells as a model, we have investigated adhesion to ligand intercellular adhesion molecule-1 induced by the Ca2+ mobilizers, ionomycin, 2, 5-di-t-butylhydroquinone, and thapsigargin, and the well studied stimulators such as phorbol ester and cross-linking of the antigen-specific T cell receptor (TCR)-CD3 complex. We report here that after exposure of T cells to these agonists, integrin is released from cytoskeletal control by the Ca2+-induced activation of a calpain-like enzyme, and adhesive contact between cells is strengthened by means of the clustering of mobilized LFA-1 on the membrane. We propose that methods of leukocyte stimulation that cause Ca2+ fluxes induce LFA-1 adhesion by regulation of calpain activity. These findings suggest a mechanism whereby engagement of the TCR could promote adhesion strengthening at an early stage of interaction with an antigen-presenting cell.

Calcium↗
PubMed1997

Molecular identification of a novel fibrinogen binding site on the first domain of ICAM-1 regulating leukocyte-endothelium bridging.

Binding of fibrinogen to intercellular adhesion molecule 1 (ICAM-1) enhances leukocyte adhesion to endothelium by acting as a bridging molecule between the two cell types. Here, a panel of four monoclonal antibodies (mAbs) to ICAM-1 was used to dissect the structure-function requirements of this recognition. All four mAbs bound to ICAM-1 transfectants and immunoprecipitated and immunoblotted ICAM-1 from detergent-solubilized JY lymphocyte extracts. Functionally, mAbs 1G12 and 2D5 inhibited binding of 125I-fibrinogen to ICAM-1-transfectants and abrogated the enhancing effect of fibrinogen on mononuclear cell adhesion to endothelium and transendothelial migration. In contrast, mAbs 3D6 and 6E6 did not affect ICAM-1 recognition of fibrinogen. With respect to other ligands, mAbs 1G12 and 2D5 completely inhibited attachment of Plasmodium falciparum-infected erythrocytes to immobilized recombinant ICAM-1-Fc, whereas they had no effect on LFA-1-dependent T cell binding to ICAM-1-Fc. Conversely, mAbs 3D6 and 6E6 completely abolished LFA-1 binding to ICAM-1-Fc. Epitope assignment using ICAM-1 chimeras and receptor mutants revealed that the fibrinogen-blocking mAbs 1G12 and 2D5 reacted with domain 1 of ICAM-1, and their binding was disrupted by 97 and 70% by mutations of D26 and P70, respectively, whereas mAbs 3D6 and 6E6 bound to domain 2 of ICAM-1. By recognizing a site distinct from that of beta2 integrins Mac-1 or LFA-1, fibrinogen binding to ICAM-1 may provide an alternative pathway of intercellular adhesion and/or modulate integrin-dependent adherence during inflammation and vascular injury.

Antibodies, Monoclonal↗
PubMed1997

The human T-cell leukemia virus type 1 transactivator protein Tax colocalizes in unique nuclear structures with NF-kappaB proteins.

The Tax protein of human T-cell leukemia virus type 1 (HTLV-1) is a potent activator of viral transcription. Tax also activates the expression of specific cellular genes involved in the control of T-lymphocyte growth via effects on cellular transcription factors, including members of the NF-kappaB/cRel family. Immunocytochemistry and electron microscopy were used to characterize the intracellular localization of Tax and identify cellular factors which are the potential targets for its transcriptional activity. These studies indicated that Tax localizes in discrete nuclear foci in T lymphocytes transformed by HTLV-1 and in cells transduced with Tax expression vectors. The Tax-containing foci are complex nuclear structures comprising a central core in which Tax colocalizes with splicing factor Sm. In addition to splicing factors Sm and SC-35, the Tax-containing nuclear structures also contain transcriptional components, including the largest subunit of RNA polymerase II and cyclin-dependent kinase CDK8. The inclusion of the two subunits of NF-kappaB, p50 and RelA, and the presence of the mRNA from a gene specifically activated by Tax through NF-kappaB binding sites suggest that these unique nuclear structures participate in Tax-mediated activation of gene expression via the NF-kappaB pathway.

Animals↗
PubMed1995

Analysis of the binding site on intercellular adhesion molecule 3 for the leukocyte integrin lymphocyte function-associated antigen 1.

Intercellular adhesion molecule 3 (ICAM-3, CD50) is a member of the immunoglobulin superfamily and is a constitutively expressed ligand for the leukocyte integrin LFA-1 (CD11a/CD18). ICAM-3 is expressed at high levels by all resting leukocyte populations and antigen presenting cells and is a major ligand for LFA-1 in the resting immune system. ICAM-3 is a signal transducer and may play a key role in initiating immune responses. Mutant ICAM-3 Fc-chimeric proteins were quantitatively analyzed for their ability to bind COS cells expressing human LFA-1. The LFA-1-binding site on ICAM-3 is located in the N-terminal 2 Ig domains. Domains 3-5 do not significantly contribute to adhesion. The binding site has been further resolved by rational targeting of 14 point mutations throughout domains 1 and 2, coupled with modeling studies. Within domain 1 a cluster of residues (Glu37, Leu66, Ser68, and Gln75), that are predicted to lie on the CC'FG face of the Ig fold, play a dominant role in LFA-1 binding.

Amino Acid Sequence↗
PubMed1994

Neutrophil apoptosis is associated with a reduction in CD16 (Fc gamma RIII) expression.

Resolution of inflammation involves removal of recruited neutrophils from inflamed sites via a noninflammatory mechanism, possibly involving neutrophil apoptosis and engulfment/phagocytosis by macrophages. In this study, we describe the reduction in surface expression (> 90%) of the neutrophil molecule Fc gamma RIII (CD16) during in vitro culture at 37 degrees C, which was found to be temporally associated with the appearance of neutrophils with apoptotic morphology during in vitro culture and inhibitable by granulocyte-macrophage colony-stimulating factor (GM-CSF), which postpones apoptosis in the neutrophil. By using dual fluorescence analysis, CD16 "low" expressing neutrophils showed reduced staining with the DNA-binding dye propidium iodide, suggesting that CD16 low expressing neutrophils were apoptotic. Separation of CD16 "high" and CD16 "low" expressing neutrophils by fluorescence-activated cell sorting revealed that morphologically apoptotic cells exhibited the CD16 low phenotype. We did not observe similar marked changes in expression of other neutrophil surface molecules (including other phosphatidylinositol (PI)-linked molecules), indicating that generalized loss of surface molecules does not occur during apoptosis. We believe this to be the first reported cell type-specific membrane alteration in a surface glycoprotein associated with apoptosis, suggesting that the program of cell death in the neutrophil, in addition to morphologic and nuclear changes, includes alterations in expression of surface receptors.

Apoptosis↗
PubMed1994

Involvement of the "I" domain of LFA-1 in selective binding to ligands ICAM-1 and ICAM-3.

To analyze the binding requirements of LFA-1 for its two most homologous ligands, ICAM-1 and ICAM-3, we compared the effects of various LFA-1 activation regimes and a panel of anti-LFA-1 mAbs in T cell binding assays to ICAM-1 or ICAM-3 coated on plastic. These studies demonstrated that T cell binding to ICAM-3 was inducible both from the exterior of the cell by Mn2+ and from the interior by an agonist of the "inside-out" signaling pathway. T cells bound both ICAM ligands with comparable avidity. A screen of 29 anti-LFA-1 mAbs led to the identification of two mAbs specific for the alpha subunit of LFA-1 which selectively blocked adhesion of T cells to ICAM-3 but not ICAM-1. These two mAbs, YTH81.5 and 122.2A5, exhibited identical blocking properties in a more defined adhesion assay using LFA-1 transfected COS cells binding to immobilized ligand. Blocking was not due to a steric interference between anti-LFA-1 mAbs and N-linked carbohydrate residues present on ICAM-3 but not ICAM-1. The epitopes of mAbs YTH81.5 and 122.2A5 were shown to map to the I domain of the LFA-1 alpha subunit. A third I domain mAb, MEM-83, has been previously reported to uniquely activate LFA-1 to bind ICAM-1 (Landis, R. C., R. I. Bennett, and N. Hogg. 1993. J. Cell Biol. 120:1519-1527). We now show that mAb MEM-83 is not able to stimulate binding of T cells to ICAM-3 over a wide concentration range. Failure to induce ICAM-3 binding by mAb MEM-83 was not due to a blockade of the ICAM-3 binding site on LFA-1. This study has demonstrated that two sets of functionally distinct mAbs recognizing epitopes in the I domain of LFA-1 are able to exert differential effects on the binding of LFA-1 to its ligands ICAM-1, and ICAM-3. These results suggest for the first time that LFA-1 is capable of binding these two highly homologous ligands in a selective manner and that the I domain plays a role in this process.

Antibodies, Monoclonal↗
PubMed1993

Cytoarchitecture of size-excluding compartments in living cells.

By fluorescence ratio imaging of large and small inert tracer particles in living cells, we have previously shown that particles 24 nm in radius are excluded from otherwise uncharacterized compartments in the distal and perinuclear cytoplasm (Luby-Phelps, K. and Taylor, D.L., 1988. Cell Motil. Cytoskel. 10, 28-37). In this study we examined the cytoarchitecture of these compartments. Whole-mount TEM showed that distal size-excluding compartments were devoid of membrane-bounded organelles and were filled with a dense cytomatrix consisting of numerous, long bundles of thin filaments interconnected by a more random meshwork of short thin filaments. The mean diameter of void spaces in the cytomatrix of distal excluding compartments was 31 nm, compared to 53 nm in adjacent non-excluding domains. The height of the distal excluding compartments was generally < or = 50% of the height in the adjacent non-excluding compartment. An electron-dense structure having the same projected outline as the perinuclear size-excluding compartment was visible by whole-mount TEM, but the cells were too thick and osmiophilic in this region to resolve any detail. Immunofluorescence localization of cytoskeletal proteins in distal excluding compartments indicated the presence of filament bundles containing F-actin nonmuscle filamin (ABP280) and alpha-actinin. F-actin and ABP280, but not alpha-actinin, were found also in between these filament bundles. Microtubules and vimentin generally were rare or absent from distal excluding domains. Staining of living cells with DMB-ceramide revealed that the perinuclear size-excluding compartment consisted of a compact, juxtanuclear domain coinciding with the trans-Golgi, surrounded by a more diffuse domain coinciding with a perinuclear concentration of endoplasmic reticulum. Intense immunofluorescence staining for vimentin was also observed in the perinuclear size-excluding compartment. We propose that the most likely mechanism for exclusion from distal compartments is molecular sieving by a meshwork of actin filament bundles interconnected by an F-actin/ABP280 gel network, while exclusion from the perinuclear compartment may be due to close apposition of cisternae in the trans-Golgi and a network or basket of vimentin filaments in the centrosomal region of the cell.

3T3 Cells↗
PubMed1992

The binding site on ICAM-1 for Plasmodium falciparum-infected erythrocytes overlaps, but is distinct from, the LFA-1-binding site.

The intercellular adhesion molecule-1 (ICAM-1, CD54) is one of three putative endothelial receptors that mediate in vitro cytoadherence of P. falciparum-infected erythrocytes. Since cytoadherence to postcapillary venular endothelium is thought to be a major factor in the virulence of P. falciparum malaria, we have examined the interaction between ICAM-1 and the P. falciparum-infected cell, and have compared it with the interaction to the physiological counter receptor, the leukocyte integrin LFA-1. Our results demonstrate that the malaria-binding site resides in the first two domains of the ICAM-1 molecule and overlaps, but is distinct from, the LFA-1 site.

Amino Acid Sequence↗
PubMed1992

Leukocyte integrin activation.

Certain stages of the immune response require interaction of leukocytes with each other and with non-hematopoietic cells. One of the systems implicated in these interactions involves an integrin, LFA-1 (Lymphocyte Function Antigen-1), expressed by all leukocytes at their cell surface, and a molecule belonging to the immunoglobulin superfamily, ICAM-1 (Intercellular Adhesion Molecule-1). The avidity of LFA-1 for ICAM-1 is transient. It is modulated both by regulation of ICAM-1 expression and by activation of LFA-1 molecules constitutively expressed on leukocyte membranes. This activation, which induces a conformational change in the molecule, depends on the presence of divalent cations, notably Mg++. This has been demonstrated by using a specific monoclonal antibody, MAb 24. In addition to being a ligand for LFA-1, ICAM-1 is sometimes used as a cell receptor by pathogens such as Plasmodium falciparum, the causative organism of malaria. Very careful study of the binding site of this pathogen using specific antibodies, mutagenesis studies and the construction of a three-dimensional model of the molecule suggests some interesting therapeutic possibilities for the treatment of malaria.

CD3 Complex↗
PubMed1991

Cellular location of thymus-leukemia (TL) antigen as shown by immuno-cryoultramicrotomy.

Thymus-leukemia (TL) antigen is a class I molecule of the major histocompatibility complex that is expressed on the surface of mouse cortical thymocytes. Though not expected, it has been reported that TL antigen can be found on isolated mitochondria of TL+ cells. We used immuno-cryoultramicrotomy to look for TL on mitochondria in situ, thereby avoiding the plasma membrane contamination that occurs when isolating organelles. Establishing optimal fixation conditions was crucial, as mitochondrial structure was not preserved by the low concentrations of fixative needed for detection of antibody labeling. The plasma membranes of tissue culture and thymus cells were labeled well with anti-TL antibody and protein A-gold conjugate, while mitochondria within the cells were not labeled. Isolation of mitochondria on a one-step Ficoll gradient resulted in a purer organelle preparation than did isolation of mitochondria by centrifugation alone. Generally, mitchondria within this purer preparation were not labeled. Our data show that under conditions where contamination by plasma membrane is not a major concern, TL antigen cannot be detected on mitochondria.

Animals↗
PubMed1989

The structure of organelles of the endocytic pathway in hydrated cryosections of cultured cells.

The structure of cellular organelles, in particular those involved in endocytosis, was studied by electron microscopy with hydrated cryosections. In this technique no chemical treatment is used, and the native structure of organelles can be observed in sections viewed at temperatures below -140 degrees C, using a cold stage accessory on the electron microscope. The compartments of the endocytic pathway were prelabeled with gold markers in the living cell, facilitating the identification of different structures in the cryosections. The structure of most identifiable cellular organelles, including those involved in endocytosis, appeared very similar in the hydrated cryosections to that seen after conventional plastic and cryosections of chemically fixed cells. In particular, the internal membranes of the structure we refer to as the prelysosomal compartment (Griffiths et al., Cell 52, 329-341 (1988] could be clearly visualized in these sections indicating that the organization of these membranes is not a consequence of the chemical fixation process.

Animals↗
PubMed1988

Nucleolar and nuclear envelope proteins of the yeast Saccharomyces cerevisiae.

We have developed a fast and reliable purification protocol to obtain yeast nuclei in intact and pure form and in a reasonable yield. The purified nuclei appear homogeneous at the light and electron microscopic level, are highly enriched in the nuclear marker histone H2B and devoid of mitochondrial, vacuolar and cytosolic marker proteins. On sodium dodecyl sulfate (SDS)-polyacrylamide gels, the nuclear fraction contains unique proteins which distinguishes them from the major yeast subcellular fractions. Yeast nuclei were separated by detergent/salt extraction into soluble, insoluble and membrane fractions. Antibodies raised against subnuclear fractions lead to the identification of an integral nuclear membrane protein and a high-abundance 38-kDa protein which is located in the yeast nucleolus.

Molecular Weight↗
PubMed1984

On the preparation of cryosections for immunocytochemistry.

The key preparation steps in the Tokuyasu thawed frozen section technique for immunocytochemistry, namely freezing, sectioning, thawing, and drying, were studied. A spherical tissue culture cell was used as a model system. The frozen hydrated section technique indicated that glutaraldehyde-fixed, 2.1 M sucrose-infused pellets of cells were routinely vitrified by immersion in liquid nitrogen but water was crystallized when lower sucrose concentrations (0.6-1 M) were used. Quantitative mass measurements showed that the fixed cells are freely permeable to sucrose. The frozen hydrated sections were severely compressed but cell profiles regained their circular appearance upon thawing. The average section thickness of our frozen-hydrated sections was 110 nm: this was reduced to 30-50 nm upon thawing, washing, and air-drying. This change was accompanied by severe drying artifacts. By using the methyl cellulose drying technique, this collapse upon air-drying could be significantly reduced, but not completely prevented, giving an average thickness of 70 nm.

Animals↗