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D A Rees

Publications and source records attributed to D A Rees.

At least 73 records · Page 4Linked to original sources
PubMed1981

Cooperativity of concanavalin A patching and its influence on cytoskeleton changes in fibroblast rounding and detachment.

We have studied the extent of concanavalin A (Con A) patching on spread fibroblasts as a function of increasing concentration of added Con A, and also the influence of this patching on subsequent cytoskeleton changes during exposure to trypsin and EGTA. Living cells do not patch at low concentrations of Con A (around 1 microgram ml-1) but progressively do so at higher levels (2-10 microgram ml-1), in very close parallel with a cooperative phase of binding characterized earlier. The proportion of visibly patched cells is at a maximum when this cooperative phase is complete and the correlation remains when cells are treated with azide of cytochalasin B to shift the concentration range over which both phenomena occur. All this evidence points to a common origin for cooperative binding and visible patching which is sensitive to the state of cellular actomyosin, and involves the mediation of actin-associated structures. As a consequence of Con A patching, cell attachment is preserved against the action of EGTA. On patched cells, this agent causes minor if any changes in cell shape and in the distributions of microtubules and 10-nm filaments, but actin stress fibres are dispersed. However, conditions appear to be retained for reassembly of the stress fibres because these can be caused to reappear within minutes after removal of EGTA when glucose, or Ca2+ or Mn2+ in the absence of glucose, is supplied in the medium. Trypsin caused only partial destruction of stress fibres in Con A-treated cells and did not promote detachment or dispersal of microtubules or 10-nm filament systems.

Animals↗
PubMed1980

Comparison of the cell cytoskeleton in migratory and stationary chick fibroblasts.

The organization of the principal cytoskeletal components (actin, tubulin and 10 nm filament protein) have been compared by immunofluorescence microscopy in two populations of chick heart fibroblasts, previously shown to be adapted respectively for rapid, directed migration or adhesion and growth. We find that neither microtubule nor 10 nm filament distributions after significantly during the conversion from the migratory to the stationary state but in contrast there are significant differences in the organization of actin. The stationary cells possess more numerous and thicker stress fibre bundles. The variety of patients observed in the migratory cells are documented and the possible roles of the different components of the cytoskeleton in cell locomotion are discussed.

Actins↗
PubMed1980

The 13C-NMR spectra of hyaluronate and chondroitin sulphates. Further evidence on an alkali-induced conformation change.

Complete assignments are given for the 13C NMR spectra of hyaluronate and chondroitin in deuterium oxide solution at 50.32 MHz. The assignments published earlier for chondroitin 4-sulphate and chondroitin 6-sulphate were largely confirmed but were found to need some revision in detail. Our conclusions for hyaluronate and the chondroitin sulphates were confirmed by off-resonance experiments based on the proton NMR assignments. The spectra for hyaluronate show line narrowing and chemical shift changes from neutral to alkaline solution which are consistent with, and clearer than, the effects reported earlier for the proton spectra. As before no such changes occur for the chondroitin sulphates. The suggested interpretation is in terms of a conformation change for hyaluronate which originates mainly in altered interaction energies across the 1 leads to 3 linkage with the results that motional freedom is enhanced above that of the parent hyaluronate and even above the chondroitin sulphates. This, and the evidence from a temperature effect, suggests that an additional potential energy minimum is made favourable in alkali so that the overall amplitudes of the bond oscillations are increased.

Carbohydrate Conformation↗
PubMed1980

Cytoskeleton changes in fibroblast adhesion and detachment.

The organization of the cytoskeleton in several anchorage-dependent fibroblast types has been compared with the pattern of adhesions to a glass substratum which will support either their growth or just their spreading. Components were stained separately for immunofluorescence microscopy using specific antisera against actin, tubulin, and gizzard 10-nm filament protein, and the adhesions were visualized by interference-reflexion microscopy. Of the cytoskeleton features, only stress fibres could be related to the pattern of focal adhesions; as shown before, each focal adhesion lies directly beneath a stress fibre, often near the terminus. Cells spread on fibronectin-treated glass in serum-free medium to arrest the development of focal adhesions, show correspondingly underdeveloped stress fibres. Actin geodesic domes, microtubules, and 10-nm filaments showed no relations with the adhesion pattern. During cell rounding leading to detachment with either EGTA or trypsin, stress fibres begin to disperse in advance of shape change whereas microtubules and 10-nm filaments seem to alter their distribution as a consequence of shape change. We therefore confirm that stress fibres are the cytoskeleton features most directly related to focal adhesions and are cytoskeleton targets for 2 agents which cause rounding and hence detachment. The sequences of events in dispersal of stress fibres by EGTA and by trypsin showed significant differences in detail. With trypsin, fibres higher in the cell and terminating at the cell edge were more sensitive than most basal fibres and, during disintegration, all types of fibre went through an intermediate 'beaded' structure. With EGTA, all stress fibres seemed to be similarly susceptible and the beaded stage was not seen. The implications of these differences for our understanding of the mechanisms of dispersal of stress fibres are discussed.

Actins↗
PubMed1979

Fibronectin distribution in epithelial and associated tissues of the rat.

Specific antiserum was used to investigate the distribution of the extracellular glycoprotein, fibronectin, in rat skin and tongue tissue by light and electron microscopy with immunofluorescence and immunoperoxidase techniques. We conclude that fibronectin is absent from stable, differentiated parts of tissues, such as the sebaceous glands or the matrix, medulla, cortex, and cuticles of the hair and the inner and outer root sheaths, or even in tissues in which there is some cell movement, such as the epidermis. It is, however, characteristic of sites at which cell division is occurring in contact with an extracellular scaffolding, such as basement membrane or loose connective tissue. Conspicuous examples were in the glassy membrane and connective tissue sheath associated with the follicular epithelium, the basement membrane underlying vascular endothelial cells, the connective tissues surrounding and investing nerve and muscle fibre bundles, and the dermal connective tissue where fibronectin was often associated closely with collagen fibres. At the basement membrane of the dermal/epidermal junction, fibronectin occurred at the plasma membrane of the basal cells and in the lamina lucida area. There was no correlation with specific areas of cell-substrate adhesion, such as the hemidesmosomes. The endoplasmic reticulum of fibroblasts stained strongly suggesting that these cells represent a major site of synthesis.

Animals↗
PubMed1979

Actomyosin organisation for adhesion, spreading, growth and movement in chick fibroblasts.

Examination of the actomyosin structures and their relation to adhesion, movement and growth in the first fibroblasts migrating from chick heart explants shows striking differences with fibroblasts adapted to grow in culture. The latter have focal adhesions which seem to immobilize them for anchorage-dependent growth, rather than facilitate their movement. The fibroblasts specialized for movement from the explants, though equally well spread, make contact with substratum through extensive areas of relatively unspecialized membrane, have less well developed stress fibres and a low growth rate.

Actomyosin↗
PubMed1979

Solution conformation of glycosaminoglycans: assignment of the 300-MHz 1H-magnetic resonance spectra of chondroitin 4-sulphate, chondroitin 6-sulphate and hyaluronate, and investigation of an alkali-induced conformation change.

Complete assignments are given for the 1H nuclear magnetic resonance (NMR) spectra at 300 MHz of chondroitin 4-sulphate, chondroitin 6-sulphate and hyaluronate in deuterium oxide solution, supported by spin decoupling and computer simulation. Coupling constants and chemical shifts are as expected from spectra of the model glycosides, methyl beta-D-glucopyranosiduronate, methyl 2-acetamido-2-deoxy-beta-D-glucopyranoside and methyl 2-acetamido-2-deoxy-beta-D-galactopyranoside, when allowance is made for systematic influences on chemical shifts of interglycosidic linkages and sulphate substitution. As reported elsewhere, addition of alkali causes the hyaluronate spectrum to sharpen considerably. This is taken to indicate that segmental motion is enhanced by disruption of some system of inter-residue bonding on ionisation of hydroxy groups. Concomitant changes in chemical shifts are seen mainly for H-2 of the glucuronate residue, and the CH3 and H-2 of the acetamidodeoxyglucose residue. Similar effects are not seen for chondroitin sulphates, either in line widths or chemical shifts. Comparison of the spectra of hyaluronate, chondroitin sulphates, and the model glycosides, indicates that proton chemical shifts are sensitive to the conformation differences between the polysaccharides in alkaline solution, but do not detect the differences in neutral solution that are known from NMR relaxation to be present. The altered configuration and/or substitution pattern of the acetamidodeoxyhexose residue in hyaluronate compared with chondroitin sulphates appears to have a critical influence on overall conformation in both alkaline and neutral solution.

Chondroitin↗
PubMed1979

1H-Nuclear magnetic-resonance studies on glycophorin and its carbohydrate-containing tryptic peptides.

The proton nuclear magnetic resonance (1H-NMR) spectra of glycophorin and its tryptic sialoglycopeptides were investigated. From the intensities of the assigned resonances it was concluded that all of the residues in the sialoglycopeptides are sufficiently mobile in conformation to give sharp resonances, while in glycophorin this is true for only approximately 80% of the peptide backbone. The resonances of the central sequence of some 20 of the hydrophobic residues are strongly broadened. This region is probably that of alpha-helical structure which is known to aggregate. The linewidths and intensities of the resonances are not, or only slightly, affected by changing the ionic strength, temperature or by carboxymethylation of the Met-81 residue in glycophorin. Glycophorin was found to bind about 100 mol sodium dodecylsulphate/mol protein as derived from studies on linebroadening of the latter's C-3 to C-11 methylene resonances. The bound dodecyl-sulphate probably increases the mobilities of the hydrophobic residues in the protein as these resonance intensities are increased by the binding. The carbohydrate chains in glycophorin were conformationally mobile; no evidence was found for tight carbohydrate-protein interactions. The relevance of flexible carbohydrate chains in membrane glycoproteins is discussed in relation to cell surface chemistry.

Glycopeptides↗
PubMed1979

Mechanisms of cellular adhesion. IV. Role of serum glycoproteins in fibroblast spreading on glass.

We have investigated the exogenous factors required for the transition from the round shape of suspended fibroblasts to the characteristic spread shape on serum-coated glass. Following the evidence of others that the transition is facilitated by adsorbed component(s) related to CIG/LETS (cold-insoluble globulin/large external transformation-sensitive) proteins, we have isolated 2 such preparations from chick serum. Their influence has been investigated on fibroblast adhesion, spreading and growth and they have been characterized by gel electrophoresis, immunological cross-reactivity, amino acid and carbohydrate residue analysis, sedimentation velocity behaviour and circular dichroism spectroscopy. The preparations have molecular weights of 225/215,000 and 140,000 Daltons respectively and are closely similar in composition and secondary structure. The 225/215 000 Dalton doublet is probably a product of limited proteolysis which almost certainly occurred in the avian circulation. For cells seeded on glass precoated in different ways and in different supplemented media we could detect no change in the extent of attachment but there were profound influences on cell shape following this initial adhesion. We confirm that prior adsorption of either CIG-related preparation to glass does indeed promote fibroblast spreading in the absence of other serum components and that CIG is the sole serum component with this type of activity. We now add 2 important qualifications: (i) the presence of substrate-adsorbed serum CIG is not essential for spreading when other serum components are present in the medium; and (ii) the adhesive organization shown by interference reflexion microscopy is incompletely formed unless the additional serum components are present in the medium. We therefore conclude that 16C fibroblasts have the ability when given the stimulus of soluble serum components other than CIG, but not otherwise, to synthesize all the components necessary for the highly organized contacts with glass, including endogenous CIG/LETS proteins.

Amino Acids↗
PubMed1979

Co-operativity of lectin binding to fibroblasts and its relation to cellular actomyosin.

We have investigated the binding of 125I-concanavalin A (125I-Con A) and 125I-succinyl concanavalin A (125I-s-Con A) to rat fibroblasts (16C line) as a function of the concentration of added lectin, and the alterations to this binding behaviour caused by drugs which modify the cytoskeleton. The changes in cell behaviour which occur at different levels of binding have also been studied. As shown previously for some other systems, the binding of Con A is complex and partly co-operative. Three phases can be distinguished in our system: (i) pre-nucleation binding, (ii) binding which shows a small positive slope in a Scatchard plot and a Hill coefficient greater than unity, and which therefore is incipiently co-operative, and (iii) post-co-operative binding. The co-operative phase of binding is paralleled by progressive inhibition of EGTA-mediated cell detachment from substrata, with inhibition being complete when this phase of binding is complete. Likewise, the phagocytosis of latex spheres is progressively inhibited up to a threshold which coincides with the completion of co-operative binding. Thirdly, cells pretreated with Con A round up with colchicine (10(-5) M) if co-operative finding is complete, but adopt broad epithelial shapes if it is not. s-Con A does not show cooperative binding, and correspondingly does not inhibit EGTA-mediated cell detachment, or show a distinct threshold in the inhibition of phagocytosis, or promote the 2 types of shape change with colchicine. The pattern of Con A binding is drastically altered by pretreatment of cells with cytochalasin B or azide. The Scatchard and Hill plots show that the co-operative phase remains and is complete at about the same level of binding, but that it is more readily nucleated and takes place against a changed number and/or distribution of receptors. Pretreatment of cells with colchicine causes changes in the pattern of binding which are different from those observed with cytochalasin B or azide and are more difficult to interpret. We conclude that a reciprocal relationship exists between the cellular actomyosin and the state of cell surface receptors. Perturbation of actomyosin by cytochalasin B or azide can enhance the freedom of some receptors to participate in a co-operative rearrangement which facilitates the binding of further molecules of lectin. Vice versa, the co-operative event has a feedback influence on the cellular actomyosin to cause alterations of cellular response.

Actomyosin↗
PubMed1979

The behaviour of fibroblasts migrating from chick heart explants: changes in adhesion, locomotion and growth, and in the distribution of actomyosin and fibronectin.

Fibroblasts migrating from heart explants of chick embryos at first have a high rate of locomotion but lack focal contacts or adhesions and also lack substantial actin-containing bundles. A meshwork of 7-nm filaments is present particularly in submembranous regions and is proposed to be directed towards efficient locomotion whilst maintaining a high degree of spreading. Also during the first 48 h there is little production of extracellular fibronectin and the growth rate is low. Later, these fibroblasts develop focal contacts and focal adhesions together with actomyosin bundles, with a parallel increase in fibronectin expression. We propose that progressive immobilization by the development of focal adhesions and actomyosin structures occurs to set these cells up for growth.

Actomyosin↗