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Biomedical subjects

I Hunter

Publications and source records attributed to I Hunter.

At least 37 records · Page 2Linked to original sources
PubMed1994

Differential regulation of C-CAM isoforms in epithelial cells.

C-CAM is a Ca(2+)-independent cell adhesion molecule (CAM) that mediates intercellular adhesion of isolated rat hepatocytes. It is widely distributed in epithelia, where its presence both at lateral cell borders and on apical cell surfaces suggests that it may have diverse biological functions. Two major isoforms, C-CAM1 and C-CAM2, which differ in the lengths of their cytoplasmic domains, have been identified. The lack of suitable in vitro systems has so far prevented a detailed study of the physiological role of C-CAM in epithelia. We now report on the identification, biochemical characterization and functional analysis of C-CAM isoforms in the established epithelial cell line NBT II, derived from a chemically induced carcinoma of rat bladder. C-CAM in NBT II cells is a 110-115 kDa cell surface glycoprotein located predominantly at sites of cell-cell contact but also present on the apical cell surface. Northern blotting analysis revealed the presence of both C-CAM1 and C-CAM2, with the major transcripts for both isoforms present within the 4.0 kb size range. The dissociation of NBT II cell colonies by anti-C-CAM antibodies indicated that at least one function of C-CAM in these cells is to mediate intercellular adhesion. The maintenance of extensive cell-cell contacts and the expression of C-CAM at the contact sites in cells grown in low Ca2+ medium suggested that, like its counterpart in hepatocytes, C-CAM in NBT II cells may be a Ca(2+)-independent cell-cell adhesion molecule. The co-localization and coordinate reorganization of both C-CAM and actin by anti-C-CAM antibodies indicated that these two proteins were associated and suggested that interactions with the cytoskeleton may be important for the regulation of C-CAM function. The specific upregulation of C-CAM1 in cells induced to undergo epithelial to mesenchymal-like transitions (EMT) by the serum substitute Ultroser G suggested that C-CAM isoforms are important modulators of the adhesive properties of these cells.

Actins↗
PubMed1992

Laminin chain assembly by triple and double stranded coiled-coil structures.

We have previously provided evidence that laminin assembly occurs by the specific interaction of the alpha-helical domains of the A, B1, and B2 chains, located within the long arm of the molecule (Hunter, I., Schulthess, T., Bruch, M., Beck, K., and Engel, J. (1990) Eur. J. Biochem. 188, 205-211). Recent evidence for noncoordinate synthesis of the laminin chains, and in particular, the absence of the 400-kDa A chain from laminins produced by a number of cell types, has led us to examine the molecular mechanism of laminin assembly using the isolated A and B1-B2 chains of laminin fragment E8. E8A shows little tendency to self-associate, and when renatured from urea forms globular structures with little detectable alpha-helix. In contrast, E8B1-B2 renatures to form rod-like molecules, 30 nm in length. The rod-like structure, high alpha-helix content, and sharp thermal transition indicate that they are double stranded coiled coils. When mixed in equimolar amounts, E8A and E8B1-B2 renature to form molecules which are biochemically and ultrastructurally indistinguishable from native E8. If E8A and E8B1-B2 are renatured separately and mixed at a 1:1 molar ratio, they also form E8 molecules. These results suggest a mechanism of laminin assembly which involves the formation of a double coiled-coil B1-B2 intermediate with which the A chain subsequently interacts to form a triple coiled-coil laminin molecule. In addition, our results indicate that isoforms consisting of the B1 and B2 chains only would form stable "laminin-like" structures.

Circular Dichroism↗
PubMed1990

Evidence for a specific mechanism of laminin assembly.

The specificity of laminin chain assembly was investigated using fragments E8 and C8-9, derived from the long arm of the molecule, whose rod-like domain consists of the alpha-helical regions of the A, B1 and B2 chains. Urea-induced chain separation and unfolding were monitored by transverse urea/polyacrylamide gel electrophoresis (PAGE) and circular dichroism. Separation of the A and disulphide-linked B1-B2 chains occurred at 3.5-4.0 M urea and by 7.0 M urea all residual alpha-helicity was lost. Removal of urea by dialysis resulted in high recoveries (87-100%) of renatured protein which in its apparent molecular mass, alpha-helix content, chain composition, degree of association and ultrastructural appearance was indistinguishable from native E8. Reduction or reduction and alkylation of the chains did not lead to a decrease in their ability to reassemble specifically. Reformation of the single interchain disulphide, linking the B1 and B2 chains, clearly demonstrates that these chains are correctly aligned in parallel and in register in E8 renatured from its reduced chains. Renaturation of E8 from its reduced and alkylated chains precludes a role for disulphide formation in determining chain alignment but suggests rather than it is involved in the stabilisation of the correctly assembled molecule. These results, together with recent sequence data, provide evidence for the interaction of the alpha-helical regions of the A, B1 and B2 chains in the formation of a triple coiled-coil within the long arm of the molecule. The highly specific nature of this interaction suggests that it is the mechanism by which laminin is assembled in vivo.

Animals↗
PubMed1990

Structure and function of laminin: anatomy of a multidomain glycoprotein.

Laminin is a large (900 kDa) mosaic protein composed of many distinct domains with different structures and functions. Globular and rodlike domains are arranged in an extended four-armed, cruciform shape that is well suited for mediating between distant sites on cells and other components of the extracellular matrix. The alpha-helical coiled-coil domain of the long arm is involved in the specific assembly of the three chains (A, B1, B2, and possible variants) of laminin and is the only domain composed of multiple chains. It is terminated by a large globular domain composed of five homologous subdomains formed by the COOH-terminal part of the A chain. Sites for receptor-mediated cell attachment and promotion of neurite outgrowth reside in the terminal region of the long arm. A second cell attachment site, a cell signaling site with mitogenic action, binding sites for the closely associated glycoprotein nidogen/entactin, and regions involved in calcium-dependent aggregation are localized in the short arms. These domains, which to a large extent are composed of Cys-rich repeats with limited homology to EGF, are the most highly conserved regions in laminins of different origin. At present, most structural and functional data have been collected for a laminin expressed by a mouse tumor, which can be readily isolated in native form and dissected into functional fragments by limited proteolysis. Increasing information on laminins from different species and tissues demonstrates considerable variations of structure. Isoforms of laminin assembled from different chains are focally and transiently expressed and may serve distinct functions at early stages of development even before being laid down as major components of basement membranes.

Amino Acid Sequence↗
PubMed1989

Relationship of the final alpha-keratin compositions in dyskeratoses to those along the normal keratinization pathway: aetiological and diagnostic significance.

The final alpha-keratin compositions attained in the outer horny layer of 16 dyskeratoses have been compared with the series of compositions which is produced during normal epidermal differentiation. In each case, the abnormal outer horny layer composition corresponded with that of normal basal, spinous, granular or inner horny cells, with, in some cases, the addition of alpha-keratins characteristic of hyperproliferating/regenerating keratinocytes. The results have implications for the aetiology of these diseases. In addition, the distinction between the alpha-keratin patterns in certain of the conditions was sufficiently clear to allow the use of the technique as a non-invasive aid to diagnosis.

Epidermis↗
PubMed1987

Epidermal alpha-keratin is neutral-buffer-soluble and forms intermediate filaments under physiological conditions in vitro.

Undenatured bovine epidermal alpha-keratin has been solubilized in a low-ionic-strength buffer at physiological pH (5 mM Tris-HCl/25 mM 2-mercaptoethanol (pH 7.5). The particles in this buffer were multimeric, retaining their characteristic polypeptide chain composition and alpha-helical coiled-coil structure. They were shown by sucrose density gradient centrifugation to be in true solution and to have a narrow size distribution. Upon the addition to this solution of monovalent or divalent cations up to physiological concentrations, the alpha-keratin rapidly assembled into intermediate filaments which showed a high tendency to aggregate laterally and disassembled if returned to low-ionic-strength conditions. This behaviour closely resembles that of other intermediate filament proteins, but it is the first time that alpha-keratins have been shown to be neutral-buffer-soluble and to assemble from such solutions into intermediate filaments under physiological conditions in vitro. This is in direct contrast to the reported properties of alpha-keratins after urea denaturation and the system appears to be appropriate for studying aspects of alpha-keratin intermediate filament formation.

Animals↗
PubMed1987

Dissection of the bovine epidermal desmosome into cytoplasmic protein and membrane glycoprotein domains.

Epidermal desmosomes contain two main regions. The core consists of a pair of membranes, one on either side of a cross-striated intercellular space bisected by a denser midline. The cytoplasmic compartment comprises a dense plaque deposited on the cytoplasmic surface of each membrane and a diffuse layer occupying the zone between the plaque and attached alpha-keratin filaments. Analysis of isolated desmosomes by SDS-PAGE has shown the presence of four major protein (dpl-4) and three major glycoprotein (dgl-3) bands, which have been allocated to the cytoplasmic and core compartments, respectively. In the present paper, we report the use of urea to fractionate this complex structure, both in situ and following isolation with citrate buffer, pH2.6. Extraction of the living layers of bovine epidermis with 9M-urea, pH7.5, resulted in rapid removal of the dense desmosomal plaques, followed by separation and vesiculation of desmosomal membranes. The resistance of the plaque to urea increased abruptly at the transition between living epidermis and dead, dehydrated horny layer. A similar sequence of morphological changes accompanied the extraction of isolated desmosomes with urea. Analysis of residues and extracts of isolated desmosomes by SDS-PAGE confirmed the selectivity of 9 M-urea, pH7.5, for the cytoplasmic compartment. The four major desmosomal proteins, dpl-4 (Mr240, 215, 90 and 83 (X 10(3)), respectively) predominated in the extracts. Desmosomal membranes, both paired and vesiculated, consisted almost entirely of the three desmosomal glycoproteins dgl-3 (Mr150, 120 and 110 (X 10(3)), respectively). These results provide evidence that all three desmosomal glycoproteins are integral membrane proteins. The separation of desmosomal membranes by urea, which is not accompanied by additional loss of proteins, further suggests that desmosomal adhesion is based on interactions between membrane components with no separate extracellular molecules being involved. The dissection of the desmosome by urea into two topographically and biochemically distinct domains should facilitate further studies on the molecular basis of desmosomal adhesion and alpha-keratin filament binding.

Animals↗
PubMed1987

Microplication patterns on human buccal epithelia following radiotherapy: a scanning electron microscopic analysis.

Surface topographical detail in human buccal smears of normal and irradiated tissue have been studied with scanning electron microscopy. Cells collected by buccal smearing show patterns of microplicae and microvilli, although such detail is occasionally masked by mucus and to a lesser extent by debris and bacteria. Buccal smearing permits collection of numerous specimens with little discomfort to the patient, but requires caution in identification of superficial and deep cell layers and of upper and lower surfaces. The analysis of specimens derived in this way shows that variations in the pattern of surface microplicae reflect the radiotherapeutic regimes of the patients from which they were obtained. Straight parallel microplicae characteristic of normal control and pre-irradiated specimens were absent in specimens collected during early irradiation treatments (between 24-48 Gy), but instead were present as short discontinuous ridges and microvillous projections. At the highest levels of radiation treatment (54-60 Gy) cells exhibited small areas devoid of any surface projections in addition to a range of normal and early irradiated microplicae patterns. From these results it is suggested that the patterns of microplications and microvilli on cell surfaces can be used to obtain information on the state of the stratified squamous epithelium from which they come.

Epithelium↗
PubMed1986

Responses of 'mature' human B lymphocyte lines to inducers of maturation and activation.

Four human B cell lines with a mature phenotype (immunoglobulin secretion and expression of membrane markers associated with maturation) were cultured in the presence of phorbol ester (PMA), dimethyl sulphoxide (DMSO) and two conditioned media. PMA and DMSO led to changes in phenotype which suggested the cells were being activated, whilst the conditioned media resulted in increased immunoglobulin secretion, accompanied by phenotypic changes more consistent with maturation towards the plasma cell stage. The four cell lines, which had different origins (EBV-transformed normal B cell, Burkitt's lymphoma, prolymphocytic leukaemia and multiple myeloma) responded differently to the culture stimuli. These differences suggest that the changes associated with transformation affect the way in which these cells respond to agents which stimulate activation and maturation.

B-Lymphocytes↗
PubMed1985

Determination of a behavioral transfer function: White-noise analysis of session-to-session response-ratio dynamics on concurrent VI VI schedules.

Six pigeons were exposed to concurrent variable-interval schedules in which the programmed reinforcer ratios changed from session to session according to a pseudorandom binary sequence. This procedure corresponded to the stochastic identification paradigm ("white-noise experiment") of systems theory and enabled the relation between log response ratios in the current session and log reinforcer ratios in all previous sessions to be determined. Such dynamic relations are called linear transfer functions. Both nonparametric and parametric representations of these, in the form of "impulse-response functions," were determined for each bird. The session-to-session response ratios resulting from the session-to-session pseudorandom binary variations in reinforcer ratios were well predicted by the impulse-response functions identified for each pigeon. The impulse-response functions were well fitted by a second-order dynamic model involving only two parameters: a time constant and a gain. The mean time constant was 0.67 sessions, implying that the effects of abrupt changes in log reinforcer ratios should be 96% complete within about five sessions. The mean gain was 0.53, which was surprisingly low inasmuch as it should equal the sensitivity to reinforcement ratio observed under steady-state conditions. The same six pigeons were subjected to a similar experiment 10 months following the first. Despite individual differences in impulse-response functions between birds within each experiment, the impulse-response functions determined from the two experiments were essentially the same.

Journal Article↗
PubMed1983

A novel combination of techniques for the assay of lactate dehydrogenase isoenzymes in plasma and red blood cell lysates.

A method is presented for the preparation and assay of lactate dehydrogenase isoenzymes 1 and 2 as pure fractions. The total method involves the use of heat treatment to destroy the heat-labile fractions (isoenzymes 3, 4 and 5); fractionation using an ion-exchange resin; and immunochemical blocking of the muscle subunit, to remove any contamination of the fraction containing isoenzyme 1 with isoenzyme 2 and vice versa. Finally, the enzyme activities in the various fractions are measured using a standard kinetic assay procedure. Estimates can be obtained of all five isoenzymes if the heat treatment step is omitted.

Adult↗
PubMed1982

Independence of response force and reinforcement rate on concurrent variable-interval schedule performance.

Five pigeons were trained over 43 experimental conditions on a variety of concurrent variable-interval schedules on which the forces required on the response keys were varied. The results were well described by the generalized matching law with log reinforcement ratios and log force ratios exerting independent (noninteractive) effects on preference. A further analysis using the Akaike criterion, an information-theoretic measure of the efficiency of a model, showed that overall reinforcement rate and overall force requirement did not affect preference. Unlike reinforcement rate changes, force requirement increases did not change the response rate on the alternate key, and an extension of Herrnstein's absolute response rate function for force variation on a single variable-interval schedule is suggested.

Journal Article↗