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D A Rees

Publications and source records attributed to D A Rees.

At least 37 records · Page 2Linked to original sources
PubMed1991

Desmosomal glycoprotein DGI, a component of intercellular desmosome junctions, is related to the cadherin family of cell adhesion molecules.

Among the variety of specialized intercellular junctions, those of the adherens type have the most obvious association with cytoskeletal elements. This may be with the actin microfilament system as in the zonula adherens or with intermediate filaments as in the macula adherens, or desmosome. In the former case, it is clear that transmembrane glycoproteins of the cadherin family are important adhesive components of the molecular assembly. We now show for desmosomes that a major glycoprotein component (desmosomal glycoprotein DGI) has extensive homology with the cadherins, defining an extended family, but also has unique features in its cytoplasmic domain that are likely to be relevant to the association with intermediate rather than actin filaments. A novel 282-residue extension contains repeats of approximately 29 amino acid residues predicted to have an antiparallel beta-sheet structure, followed by a glycine-rich sequence. As in the cadherins, the extracellular domain contains possible Ca2(+)-binding sequences and a potential protease processing site. The cell adhesion recognition region (His-Ala-Val) of the cadherins is modified to Arg-Ala-Leu.

Amino Acid Sequence↗
PubMed1989

The assembly of the major desmosome glycoproteins of Madin-Darby canine kidney cells.

Madin-Darby canine kidney (MDCK) cells are unable to form desmosomes when cultured in low-calcium medium [( Ca2+] less than 0.1 meq./l), but can be induced to do so by raising the calcium to physiological concentrations (1-2 meq./l). We have previously demonstrated that this block correlated with increased desmosomal protein turnover. Here we have immunoprecipitated the major desmosome glycoproteins [DGI (150 kDa) and DGII/III (120/100 kDa)] from non-ionic detergent-soluble and -insoluble fractions prepared from metabolically labelled MDCK cells cultured in standard or low-calcium medium. Pulse-chase studies showed that both DGI and DGII/III became unextractable in non-ionic detergent before their arrival at the cell surface, whether cells were grown in standard or low-calcium medium. The non-ionic detergent insolubility of these membrane components is therefore a separate step which precedes the formation of morphologically recognisable desmosomes.

Animals↗
PubMed1989

Myosin regulation and calcium transients in fibroblast shape change, attachment, and patching.

Following our study in Balb/c 3T3 cells and other cultured fibroblasts of the changes in myosin light chain phosphorylation associated with alterations in cell shape, attachment, and receptor patching, we have now determined the corresponding changes in cytoskeletal myosin distribution, and in the cellular calcium concentration, since this might, in part, mediate such responses. Immunofluorescence microscopy showed that myosin assembly into ordered forms such as actomyosin bundles and myosin sheath almost always correlated with previously shown high phosphorylation levels of myosin regulatory light chain, whereas diffuse distributions usually correlated with low or undetectable levels. An exception was observed in treatment to alter cellular cAMP levels when, in a biphasic response, assembly was correlated inversely with the phosphorylation states shown previously. Fluorescent indicators for intracellular calcium concentration, [Ca++]i, showed that myosin disassembly by trypsin or EGTA acting externally on the cells was preceded by a transient increase in [Ca++]i. For EGTA this was associated with transient recruitment of myosin into dorsal sheath structure as well as the transient enhancement of phosphorylation shown earlier. Blockage of EGTA-induced disassembly could be achieved by azide, which also caused an immediate increase in [Ca++]i and inhibited its subsequent decline. Trypsin-induced dephosphorylation did not appear to involve an eventual reduction of [Ca++]i. Therefore, in many but not all of the systems studied, correlated changes were observed in myosin assembly, [Ca++]i, and the myosin phosphorylation levels shown earlier.

Animals↗
PubMed1987

Vacuum ultraviolet circular dichroism of fibronectin. Dominant tyrosine effects.

The spectroscopic authenticity of a very intense negative band at about 183 nm reported previously from conventional circular dichroism (c.d.) studies of bovine plasma fibronectin has now been confirmed by vacuum ultraviolet c.d. measurements on two prototype spectrometers, one using a conventional light source and the other using synchrotron radiation. Closely similar spectra were obtained from both instruments, and from both solid films and solutions. The spectra show no obvious parentage in the known c.d. of the peptide backbone, but have marked similarities to the c.d. of N-acetyltyrosineamide, both in the strong band at 183 nm and in a characteristic positive band at 230 nm, It is concluded that the c.d. of fibronectin is dominated by contributions from tyrosine side-chains and that, as suggested previously, these may provide a sensitive probe for molecular organization and interactions.

Circular Dichroism↗
PubMed1987

Fibroblast adhesion to RGDS shows novel features compared with fibronectin.

As previously shown by others, the fibroblast attachment and spreading activity of fibronectin is mimicked by a short peptide (RGDS or longer) from the cell binding domain. Normal rat kidney fibroblasts showed similar attachment kinetics on either peptide GRGDSC or bovine plasma fibronectin and binding to either substratum was inhibited by peptide alone. We now demonstrate, however, considerable differences in biological activity between peptide and fibronectin. In particular, cells developed novel adhesion structures on peptide-coated substrata. Interference reflection microscopy showed a predominance of small round dark grey/black patches of adherent membrane ("spots") with relatively few focal adhesions, which occurred only at the outermost cell margins in contrast to their distribution in cells spread on fibronectin. The spots were resistant to detergent extraction and stained less strongly or not at all for vinculin. Electron microscopy in vertical thin section showed that the ventral surface of the cell was characterized by "point-contacts", corresponding in size to the spot structures seen by interference reflection microscopy, and which were only occasionally associated with microfilaments. Cells also required a higher substratum loading of peptide than fibronectin to promote spreading and proceeded to spread less rapidly and to a lesser extent, developing very few and extremely fine actin cables.

Actins↗
PubMed1987

Structure and assembly of desmosome junctions: biosynthesis, processing, and transport of the major protein and glycoprotein components in cultured epithelial cells.

Extracts of metabolically labeled cultured epithelial cells have been analyzed by immunoprecipitation followed by SDS-PAGE, using antisera to the major high molecular mass proteins and glycoproteins (greater than 100 kD) from desmosomes of bovine muzzle epidermis. For nonstratifying cells (Madin-Darby canine kidney [MDCK] and Madin-Darby bovine kidney), and A431 cells that have lost the ability to stratify through transformation, and a stratifying cell type (primary human keratinocytes) apparently similar polypeptides were immunoprecipitated with our antisera. These comprised three glycoproteins (DGI, DGII, and DGIII) and one major nonglycosylated protein (DPI). DPII, which has already been characterized by others in stratifying tissues, appeared to be absent or present in greatly reduced amounts in the nonstratifying cell types. The desmosome glycoproteins were further characterized in MDCK cells. Pulse-chase studies showed all three DGs were separate translation products. The two major glycoprotein families (DGI and DGII/III) were both found to be synthesized with co-translational addition of 2-4 high mannose cores later processed into complex type chains. However, they became endo-beta-N-acetylglucosaminidase H resistant at different times (DGII/III being slower). None of the DGs were found to have O-linked oligosaccharides unlike bovine muzzle DGI. Transport to the cell surface was rapid for all glycoproteins (60-120 min) as demonstrated by the rate at which they became sensitive to trypsin in intact cells. This also indicated that they were exposed at the outer cell surface. DGII/III, but not DGI, underwent a proteolytic processing step, losing 10 kD of carbohydrate-free peptide, during transport to the cell surface suggesting a possible regulatory mechanism in desmosome assembly.

Adenocarcinoma↗
PubMed1987

Structure and assembly of desmosome junctions: biosynthesis and turnover of the major desmosome components of Madin-Darby canine kidney cells in low calcium medium.

Neither stratifying (primary keratinocytes) nor simple (Madin-Darby canine kidney [MDCK] and Madin-Darby bovine kidney [MDBK]) epithelial cell types from desmosomes in low calcium medium (LCM; less than 0.1 mM), but they can be induced to do so by raising the calcium level to physiological concentrations (standard calcium medium [SCM], 2 mM). We have used polyclonal antisera to the major bovine epidermal desmosome components (greater than 100 kD) in a sensitive assay involving immunoprecipitation of the components from metabolically labeled MDCK cell monolayers to investigate the mechanism of calcium-induced desmosome formation. MDCK cells, whether cultured in LCM or SCM, were found to synthesize the desmosome protein, DPI and desmosome glycoproteins DGI and DGII/III with identical electrophoretic mobility, and also, where relevant, with similar carbohydrate addition/processing and proteolytic processing. The timings of these events and of transport of DGI to the cell surface were similar in low and high calcium. Although the rates of synthesis of the various desmosome components were also similar under both conditions, the glycoprotein turnover rates increased dramatically in cells cultured in LCM. The half-lives decreased by a factor of about 7 for DGI and 12 for DGII/III and, consistent with this, MDCK cells labeled for 48 h in SCM had three and six times the amount of DGI and DGII/III, respectively, as cells labeled for 48 h in LCM. The rate of turnover and the levels of DPI were changed in the same direction, but to much lesser extents. Possible mechanisms for the Ca2+-dependent control of desmosome formation are discussed in the light of this new evidence.

Animals↗
PubMed1986

Solid-state 13C NMR and X-ray diffraction of dermatan sulfate.

Dermatan sulfate in the solid state has been studied by 13C CP/MAS nmr and X-ray diffraction in order to establish the ring conformation of the L-iduronate moiety. The solid state nmr spectrum is similar to the solution spectrum obtained previously, indicating that a ring conformation at least approximating to 1C4 predominates in the solid state. X-ray powder diffraction data from the same sample indicate the presence of the 8-fold helix form previously observed by fiber diffraction, and interpreted in terms of a 4C1 ring form. A likely explanation of the results is that a distorted 1C4 L-iduronate ring conformation, not considered in the initial X-ray analysis, may emerge to provide a satisfactory interpretation of all available physical-chemical data.

Animals↗
PubMed1986

Myosin light chain phosphorylation in fibroblast shape change, detachment and patching.

The level of phosphorylation of myosin regulatory light chain in BALB/c 3T3 and certain other cultured substrate-attached fibroblasts has been shown to be altered by several agents which influence cell shape, attachment and/or surface receptors. This was investigated by metabolic labelling with [32P]orthophosphate, followed by exposure of the cells to the chosen conditions, rapid freezing to 'fix' phosphorylation levels, extraction and concentration in the presence of kinase and phosphatase inhibitors, and final analysis by two-dimensional gel electrophoresis. Gel patterns were interpreted by comparison with immunoprecipitates with antiserum to mouse nonmuscle myosin. Treatment of cells either with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or dibutyryl-cAMP suppressed light chain phosphorylation as predicted from the control mechanisms proposed previously from in vitro studies for Ca++ calmodulin and cAMP-dependent protein kinase respectively. Other effects were less easily explained: in BALB/c 3T3 cells, contrasting with previously reported behaviour of CHO cells, the cAMP-induced decline was small and transitory; and in at least one cell line (16C) the EGTA-induced decline was preceded by a strong pulse of enhanced phosphorylation. A striking and unexpected result was that azide, almost certainly acting on mitochondrial function, caused myosin light chain phosphorylation to be maintained over a long period even in the presence of EGTA which would otherwise bring about an immediate drop. The cleavage (by trypsin) or binding (by con A) of surface receptors was also shown to trigger the biochemical modulation of cellular myosin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗