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R T Dean

Publications and source records attributed to R T Dean.

At least 145 records · Page 8Linked to original sources
PubMed1986

Degradation of cartilage by macrophages in culture: evidence for the involvement of an enzyme which is associated with the cell surface.

A cell culture system is described in which purified mononuclear phagocytes may be cultured with a cartilage substrate which is radiolabelled in its proteoglycan. Resident mouse peritoneal macrophages degraded this substrate, and did so more avidly if cultured in direct contact with it. There was no evidence for complete intralysosomal degradation of the proteoglycan of the cartilage. Lysates were found to contain considerable activity at pH 7, which was inhibited by the presence of 10% serum, or by boiling the lysate. Proximity of macrophages to the substrate did not induce selective release of the lysosomal marker enzyme hexosaminidase, and concentrated enzymes secreted from the macrophages after treatment with the lysosomotropic agent ammonium chloride were ineffective in degrading cartilage at neutral pH. The active enzyme in macrophage lysates at neutral pH was found to be sedimentable by 100,000 X g centrifugation for 1 hour, in absence of lysosomal protective agents. There is evidence for a cell membrane-associated process in the degradation of cartilage by these cells, which may be a proteolytic, endoglycosidic or free radical-mediated event.

Animals↗
PubMed1986

Oxidation induced proteolysis and its possible restriction by some secondary protein modifications.

The hydroxyl radical (.OH) in the presence of molecular oxygen (O2) fragmented BSA and formed distinct peptides as did hydrogen peroxide catalysed by copper. .OH in the absence of O2 caused protein crosslinking. The selective fragmentation was ascribed to cleavage at proline residues, a mechanism consistent with an increase in the proline/glutamate ratio. Other radicals, such as peroxyl and superoxide radicals caused no change in molecular weight of the protein. Peroxyl radicals and .OH were, however, able to cause some change in conformation of the protein which increased its susceptibility to hydrolysis and oxidation might thus be implicated in protein turnover. Mitochondrial monoamine oxidase (MAO) was also susceptible to free radical cleavage but lipids were able to compete with the protein for radical species. Peroxides also seemed to be involved in the cleavage of MAO and this was evidenced by a "post irradiation effect" and the ability of the hydroperoxy radical to cause degradation; in contrast to the case with BSA. In whole cells, depletion of the antioxidant GSH and inhibition of catalase increased endogenous protein degradation. Lens protein autolysis was increased under conditions where thiol autoxidation was permitted but glycosylation of protein caused a decrease in breakdown by extrinsic hydrolases. These results indicate that free radicals, possibly also associated with lipids may play a role in the regulation of protein turnover but that secondary modifications may promote the accumulation of damaged protein.

Animals↗
PubMed1985

An intracellular pool of the procoagulant thromboplastin in human monocytes.

Human monocytes cell extracts have been analysed by density gradient centrifugation in Percoll gradients. Two peaks of activity of the plasma membrane marker enzyme 5' nucleotidase were detected. The main peak was at a density of 1.040 and a secondary one at a lower density. Thromboplastin activity was recovered associated with the main peak of 5' nucleotidase activity in control cells. In NH4Cl treated cells, thromboplastin activity is found at both densities. In agreement with this, labelling of the cell surface with 125I shows a reduction in availability of proteins for labelling between control and NH4Cl treatment. The availability of thromboplastin in monocytes for attack by exogenous trypsin or for initiation of coagulation is also reduced by cultivation with amines. We suggest that the low density peak of 5'nucleotidase and thromboplastin represents an intracellular compartment.

5'-Nucleotidase↗
PubMed1985

Endogenous free radical generation may influence proteolysis in mitochondria.

Isolated Mitochondria were allowed to incorporate radioactive amino acids into protein and proteolysis was then measured. In State 4 free radical generation was manipulated by means of respiratory chain blockers and uncouplers. Conditions of enhanced radical flux resulted in accelerated protein breakdown. We suggest that radicals influence proteolysis in cells both directly (by fragmenting proteins) and indirectly (by rendering proteins more susceptible to proteinases).

Amino Acids↗
PubMed1985

Effect of additives on liposomes: an electric birefringence study.

Under the influence of an electric field, vesicles in suspension partially deform or orientate, rendering the medium optically birefringent. The amplitudes of the birefringence under fields of variable amplitude lead to evaluation of the anisotropy of the electrical delta alpha and optical delta G polarisabilities of the vesicles. By using pulsed fields, rates of establishment and decay of birefringence enable vesicle sizes d to be measured. Samples of 9:1 phosphatidylcholine-phosphatidylserine liposome suspension in water were studied in the presence of various additives, including sucrose, an antibiotic (streptomycin), a steroid (dexamethasone), an anaesthetic (lignocaine) and a fluidising agent (benzyl alcohol). The changes in the three parameters delta alpha, delta G and d were different for each additive and are thought to be indicative of the mode of interaction of each. Electric birefringence also appears to be a rapid means of detecting structure changes.

Benzyl Alcohol↗
PubMed1984

Electric birefringence as a means of studying the effect of anaesthetics on liposomes.

Birefringence can be induced in liposome suspensions using electric fields. The fields interact predominantly with anisotropic electrical polarisabilities which give rise to induced dipole moments. Using pulsed electric fields, the optical and electrical polarisabilities and the geometrical size of the liposomes can be measured simultaneously. These parameters have been found to be very sensitive to the presence of small amounts of fluidising additives of polar and ionic nature. Illustrative data are presented for the influence of the amines ammonium chloride, methyl ammonium chloride and lignocaine and of benzyl alcohol on phosphatidylcholine/serine liposomes. Structural changes in the vesicle membranes were detected, which appeared to correlate with the biological functions, thus indicating that electric birefringence is a rapid and useful method for studying interactive phenomena in lipid membrane systems.

Anesthetics↗
PubMed1984

An abnormality in intracellular protein degradation in fibroblasts from patients with I-cell disease.

Degradation of endogenous proteins in five normal and five I-cell (mucolipidosis II) storage disease human fibroblasts was compared. In growing cultures (low density) long half-life proteins were degraded normally in each group of cells. However, the enhancement of proteolysis when confluence is reached, which we have characterised previously as being a lysosomal function, was less in the I-cell fibroblasts. The lysosomotropic agents ammonium chloride, pepstatin and Z-Phe-Ala-diazomethylketone, inhibited similarly proteolysis in growing and confluent cultures of both cell types. Leupeptin depressed proteolysis in both growth states for both cell types, but, whereas it failed to abolish the enhancement of degradation in confluent normal cells, surprisingly it depressed degradation in confluent I-cell fibroblasts to a lower rate than in growing I-cell fibroblasts. In spite of this, the inhibitory effects of ammonium chloride and leupeptin were not additive in either normal or I-cell fibroblasts, indicating that they act upon the same proteolytic mechanism(s). Non-lysosomal mechanisms may degrade short half-life proteins, and it seemed that turnover of such proteins was slower in I-cell fibroblasts. It is suggested that mutual regulation of participating proteolytic pathways may be responsible for the dysfunction of intracellular protein degradation in I-cell fibroblasts.

Ammonium Chloride↗
PubMed1984

Distinct proteolytic mechanisms in serum-sufficient and serum-restricted fibroblasts. Transformed 3T3 cells fail to regulate proteolysis in relation to culture density only during serum-sufficiency.

Thymidine incorporation (reflecting cell division), degradation of long-half-life proteins and protein synthesis were compared in normal Swiss mouse 3T3 fibroblasts and their counterparts transformed by simian virus 40 at both high and low culture densities (no. of cells/cm2). Normal cells maintained faster proteolysis at high culture density than at low. Degradation was in all conditions enhanced by serum deprivation (1% serum). In serum-sufficient (10%) conditions, there was an inverse correlation between degradation and cell division, but in serum-restricted conditions proteolysis increased substantially as culture density was increased, without change in cell division. Protein synthesis generally changed in a converse sense to protein degradation. In serum-sufficient conditions, transformed 3T3 cells failed to regulate proteolysis in response to culture density. However, in serum-restricted conditions they can regulate proteolysis as do normal cells. Transformed 3T3 cells regulate protein synthesis and thymidine incorporation very poorly in response to culture density in both conditions studied. The failure of regulation of both protein synthesis and degradation may contribute to the exaggerated growth of transformed cells in serum-sufficient conditions. The retention by such cells of regulation of proteolysis during serum restriction may also aid their survival. Studies with several lysosomotropic agents indicated that lysosomes contribute to proteolysis in all conditions studied, but also that its regulation in serum restriction is distinct from that in serum sufficiency, and may involve primarily a non-lysosomal mechanism.

Animals↗
PubMed1984

Effects of exogenous amines on mammalian cells, with particular reference to membrane flow.

We have reviewed the evidence that amines accumulate in intracellular vesicles of low pH, such as lysosomes and endosomes. There is consequent elevation of intravesicular pH, and inhibition of receptor-ligand dissociation often results from this pH change. We have argued that the capacity for fusion of such vesicles is also reduced by the high pH. We suggest that the variety of effects of amines on membrane flow and macromolecular transport we describe are at least partly due to such reduced fusion (Figs. 1 and 2). We propose that an internal low pH may facilitate heterologous vesicle-vesicle and vesicle-plasma membrane fusion. There is some evidence that clathrin can accelerate phospholipid vesicle fusion in vitro at low pH (Blumenthal et al., 1983) but no direct evidence on the role of intravesicular pH. This idea is consistent not only with the preceding discussion, but also with the fact that the intracellular membrane-bound compartments least involved in fusion events (e.g. mitochondria) are of neutral or alkaline internal pH. Membrane fusion is certainly required for the formation of vesicles at the periphery of the Golgi apparatus, and possibly earlier in the transport and processing of biosynthetic products in the Golgi (Bergeron et al., 1982). Thus the accumulation of amines in the Golgi may be responsible for several effects on the flow of macromolecules along their translocation pathways. The status of the plasma membrane in this view is complex. It might be argued that the pH dictating the fusion step in endocytosis is that of the extracellular fluid, in which case the inhibitory effects of amines on this process are not explained. However, the rapidity of acidification of the newly formed endocytic vesicles allows the possibility that plasma membrane invaginations might temporarily sequester areas which are of lower pH than that of the bulk extracellular fluid even before fusion, since the proton pumping enzyme(s) are probably present on the plasma membrane. Were this the case, then an acid pH could again be a factor determining membrane fusion at the plasma membrane. The inhibition of endocytosis by weak bases thus may again reflect elevation of pH in a sequestered compartment. From the data on the dependence of response on the concentration of amines, we anticipate that most responses involving membrane flow will be biphasic, with inhibitory effects at low amine concentration, giving way to stimulatory ones at higher concentrations. We suggest that the reported dichotomy between different amines in intracellular membrane fusion systems (D'Arcy Hart, 1982) may result from this concentration dependence.(ABSTRACT TRUNCATED AT 400 WORDS)

Amines↗
PubMed1984

Cell surface events which may initiate lysosomal enzyme secretion by human monocytes.

Freshly isolated and subsequently matured human monocytes secreted lysosomal hexosaminidase in response to exposure to IgG-Sepharose, but not certain derivatized control Sepharoses. The cells bound selectively to the surface of IgG-Sepharose (and not the control Sepharoses) but because of the large size of the particles, could not ingest them. Since the soluble IgG was covalently linked to the Sepharose and free soluble IgG was not an inducer of secretion, the secretion was thus induced directly at the cell surface. Zymosan, a yeast cell wall particle which contains a mannan, was also able to induce secretion in the various monocyte stages under study. It could even bind to the cell surface of fresh monocytes which lacked the receptor for mannose-terminated glycoproteins, and induce secretion in these cells. The mannose receptor appeared as monocytes matured, and the Ir number on the surface was increased by the action of lymphokines. Although zymosan-induced secretion could be inhibited by mannose and certain other sugars, these seemed to have some complex metabolic effects in human monocytes (which previous work with mouse macrophages has not revealed). Thus, it was not possible to demonstrate whether zymosan could initiate secretion directly by interaction at the monocyte surface mannose glycoprotein receptor.

Cell Membrane↗
PubMed1984

Oxygen-centred free radicals can efficiently degrade the polypeptide of proteoglycans in whole cartilage.

Bovine nasal cartilage slices, biosynthetically labelled in their proteoglycan with 35SO4, were used as substrate for the attack of free radicals generated on exposure to a Co60 source (which allows study of single radical species), and by chemical and enzymatic means. Systems generating hydroxyl (OH.) and superoxide (O2.-) radicals degraded the proteoglycan efficiently, while the hydroperoxy radical (HO2.) was less efficient; addition of appropriate radical scavengers inhibited degradation. The radioactive products were heterogeneous in molecular size, but with doses up to 3600 Gy were the same size range as intact chondroitin sulphate. The contained free amino groups, and more were liberated by aminopeptidase M digestion, implying that at least a small peptide was present. Thus a major site of radical attack may be the polypeptide chain. We suggest that free-radical fragmentation of polypeptides may be important both in extracellular catabolism and in intracellular proteolysis.

Animals↗
PubMed1984

Accelerated fluid endocytosis and re-exocytosis by lysosomal storage disease fibroblasts.

Fluid-phase endocytosis was significantly faster in three lines of human lysosomal storage disease fibroblasts (two I cells and one Sandhoff's) than in three normals. Re-exocytosis of fluid showed similar kinetics in all lines when release was expressed as a percentage of the radioactivity associated with the cells at the commencement of the measurement. This indicates a greater absolute exocytosis of radioactive sucrose from the storage disease cells. The data imply that the interactions of lysosomes with other cellular membranes are perturbed in the storage diseases.

Cell Line↗
PubMed1984

Regulation of procoagulant factors in mononuclear phagocytes.

Mononuclear phagocytes can be activated to develop enhanced expression of a wide range of macromolecules and mediators. Amongst these are several procoagulants, most notably thromboplastin. A variety of stimuli (particles, ligands for cell surface receptors, and some pharmacological agents) enhance procoagulant expression. We indicate that such enhancement may contribute to intra-and extravascular coagulation in several types of infections. We also point to the paucity of knowledge on the biosynthesis and intracellular transport of these procoagulants and speculate on the nature of the mechanisms involved.

Animals↗