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Biomedical subjects

R E Scott

Publications and source records attributed to R E Scott.

At least 127 records · Page 7Linked to original sources
PubMed1979

Plasma membrane vesiculation in 3T3 and SV3T3 cells. I. Morphological and biochemical characterization.

3T3 and SV3T3 mouse embryo cells and a variety of other monolayer cell lines can be induced to form and shed plasma membrane vesicles by exposure to sulphydryl blocking agents including formaldehyde and N-ethyl malemide. Morphological studies show that multiple vesicles are formed and released from individual cells and that the vesicle membrane is continuous with the plasma membrane of the cell. Vesicles measure from o.1 to 15 micrometer in diameter and are free of detectable contamination with cytoplasmic membranes and organelles. Vesicles also show a 10-fold enrichment in the plasma membrane marker enzyme 5'-nucleotidase and are devoid of detectable NADH-cytochrome C reductase and succinic dehydrogenase activity which are marker enzymes for endoplasmic reticulum and mitochondria, respectively. Vesicles have a high cholesterol: phospholipid ratio and show enrichment in sphingomyelin content. They contain receptors for Con A and WGA, approximately 20 size class polypeptides and intramembranous particles. These results suggest that vesicles are derived from and have the general characteristics of plasma membranes.

Cell Line↗
PubMed1979

Plasma membrane vesiculation in 3T3 and SV3T3 cells. II. Factors affecting the process of vesiculation.

Plasma membrane vesicles are shed from monolayer cell cultures during incubation in low concentrations of formaldehyde and other sulphydryl blocking reagents. In both 3T3 and SV3T3 cells disulphide reducing agents, including dithiothreitol and mercaptoethanol, potentiate formaldehyde-induced vesiculation. Plasma membrane vesiculation is shown to be a temperature-dependent phenomenon which occurs optimally between 22 and 37 degrees C and at pH 7.0 to 7.5. Membrane shedding is an energy-dependent phenomenon, requiring monovalent and divalent cations and slightly hypertonic medium. Plasma membrane vesiculation is not affected by pretreatment of cells with inhibitors of protein synthesis, i.e. cycloheximide, nor by agents which disrupt the cytoskeleton.

Antimetabolites↗
PubMed1978

Plasma membrane vesiculation: a cellular response to injury.

The shedding of plasma membrane vesicles has been shown to result from exposure of monolayer cell cultures to formaldehyde and other sulfhydryl blocking agents. Incubation of cells in concentrations of these agents as low as 5 to 10 mM for intervals as brief as fifteen minutes is effective (Scott, 1976). Plasma membrane vesiculation has been shown to be an energy-dependent process that requires Ca++ and physiological temperature. Following plasma membrane vesiculation, cell monolayers appear intact by phase microscopy and show only slight evidence of cell injury by electron microscopy. In view of these observations, the question has been raised whether plasma membrane vesiculation is compatible with continued cell growth and metabolism. The experiments described in this paper were designed to answer these questions. We pulse exposed 3T3 mouse embryo cells to concentrations of formaldehyde, between 2.5 and 250 mM, for intervals 15, 30 or 60 min. Cell momolayers were then washed in a variety of different media in an attempt to reverse the effect of formaldehyde on cells. Cell monolayers were thereafter assayed for the shedding of plasma membrane vesicles and for their ability to transport 2-deoxy-D-glucose. Cells were also replated in serum-containing medium and their ability to grow was assayed over a seven day interval. The results show an inverse relationship between the shedding of plasma membrane vesicles and the ability of the cells to transport nutrients and to grow. We interpret these data to suggest that the process of plasma membrane vesiculation results from a form of cell injury which blocks cellular metabolism and growth.

Biological Transport, Active↗
PubMed1978

Plasma membrane cyclic AMP-dependent protein phosphorylation system in L6 myoblasts.

Plasma membranes can be isolated without disruption of cells by the plasma membrane vesiculation technique (Scott, R.E. (1976) Science 194, 743-745). A major advantage of this technique is that it avoids contamination of plasma membranes with intracellular membrane components. Using this method, we prepared plasma membranes from L6 myoblasts grown in tissue culture and studied the characteristics of the protein phosphorylation system. We found that these plasma membrane preparations contain protein kinase which is tightly bound to the membrane and cannot be removed by washing in EDTA or in high ionic strength salt solutions. This protein kinase activity can catalyze the phosphorylation of several exogenous substrates with decreasing efficiency as acceptors of phosphate: calf thymus histones f2b, protamine and caseine. Cyclic AMP causes a dose-dependent stimulation of protein kinase activity; the highest stimulation (4-fold) is achieved at concentration 10(-5) M cyclic AMP. Cyclic AMP-dependent stimulation can be completely inhibited by heat-stable protein kinase inhibitor isolated from rabbit skeletal muscle. On the other hand, cyclic GMP does not affect the activity of protein kinase. Plasma membrane-bound protein kinase also catalyzes the phosphorylation of endogenous membrane protein substrates and this is also stimulated by addition of cyclic AMP. Analysis of plasma membrane proteins by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed that specific polypeptides are phosphorylated by cyclic AMP-independent and by cyclic AMP-dependent protein kinase systems. The results of these studies demonstrate the presence of endogenous cyclic AMP-dependent and -independent protein phosphorylating systems (enzyme activity and substrates) in purified plasma membrane preparations. These data provide a basis for further investigations on the role of plasma membrane phosphorylation as a regulator of membrane functions including those that may control cellular differentiation.

Cell Membrane↗
PubMed1978

Plasma membrane phospholipid, cholesterol and fatty acyl composition of differentiated and undifferentiated L6 myoblasts.

The lipid composition of plasma membranes isolated from differentialted and undifferentiated L6 myoblasts have been compared. In general, the plasma membranes of differentiated L6 myoblasts have a higher cholesterol to phospholipid molar ratio than plasma membranes of undifferentiated cells. Differentiated L6 myoblasts have increased relative amounts of phosphatidylethanolamine and phosphatidylcholine ine in their plasma membrane and a decreased relative amount of sphingomyelin when compared with the plasma membranes of undifferentiated myoblasts. In addition, preliminary results show that differentiated L6 myoblasts plasma membrane phospholipid shows differences in the fatty acyl composition, specifically there appears to be relatively more 17:0 and 24:1 and less 16:1 and 18:1 than in plasma membrane phospholipids of undifferentiated L6 myoblasts. These observations indicate that significant changes in plasma membrane lipid composition occur during myoblast differentiation. The role that changes in lipid composition play in control of cellular differentiation, however, remains to be elucidated.

Cell Differentiation↗
PubMed1978

Differences in the phospholipid, cholesterol, and fatty acyl composition of 3T3 and SV3T3 plasma membranes.

An analysis of the phospholipid, cholesterol, and phospholipid fatty acyl composition of isolated plasma membranes of 3T3 and SV3T3 mouse mebryo cells has been performed. The results show that the plasma membrane of SV3T3 cells contain relatively less phosphatidylethanolamine and sphingomyelin and more cholesterol than 3T3 plasma membranes. The fatty acyl composition of individual phospholipid classes as determined by gas liquid chromatography also showed differences between 3T3 and SV3T3 plasma membranes. The plasma membranes of SV40 transformed 3T3 cells contain: (a) a higher percentage of 18:1 and less 20:3 and 20:4 in phosphatidylethanolamine; (b) a higher percentage of 18:1 in phosphatidylserine; and (c) a higher percentage of 18:2 and 20:4 in phosphatidylinositol.

Cell Membrane↗
PubMed1978

Undifferentiated and differentiated L6 myoblast plasma membranes. I: Comparison of the morphology of plasma membrane vesiculation and the factors influencing the vesiculation process.

Monolayer cultures of a variety of cell lines can be induced to shed plasma membrane vesicles by exposure to 25 mM formaldehyde--2 mM dithiothreitol. An analysis of the process of membrane shedding can provide a method to probe the general characteristics of the plasma membrane in different cell populations. We have, therefore, analyzed membrane shedding in L6 myoblasts at different stages of differentiation. The results show that the morphology of membrane shedding is comparable in undifferentiated or differentiated L6 myoblasts, as are the factors that influence the process of membrane shedding. L6 myoblasts at various stages of differentiation have similar requirements for metabolic energy, physiological temperature, pH, and monovalent and divalent cations.

Cell Differentiation↗
PubMed1978

Undifferentiated and differentiated L6 myoblast plasma membranes. II: Comparison of the morphological and biochemical characteristics of isolated plasma membranes.

Plasma membrane vesicles isolated from undifferentiated and differentiated L6 myoblasts show a comparable phase and ultrastructural morphology. Both are agglutinated by Concanavalin A and wheat germ agglutinin, contain approx. 20 polypeptides and have a comparable intramembranous particle density and topography.

Agglutination↗
PubMed1978

Potential for immunization against herpesvirus infections with plasma membrane vesicles.

Virus-free, membrane antigen (MA)-positive plasma membrane vesicles produced from herpesvirus saimiri-infected owl monkey cells were used as immunogens in cotton-top marmosets. Neutralizing antibodies as well as antibodies to MA and HVS-induced LA were induced following immunization of marmosets with these vesicles. Preliminary findings demonstrated that immunized marmosets were significantly less susceptible to tumour induction by HVS than control animals.

Animals↗
PubMed1978

Principles and techniques of time management.

Managing time efficiently is, or should be, part of the credo of every manager from supervisor to chief executive. For managers to take advantage of every tip and technique available, they must be prepared to increase their self-discipline and adjust their work style each time they climb the organization ladder.

Administrative Personnel↗
PubMed1977

Isolation of virus-free Herpesvirus saimiri antigen-positive plasma membrane vesicles.

Experiments were undertaken to determine whether virus-free, antigen-positive plasma membrane vesicles could be isolated from owl monkey kidney cells infected with Herpesvirus saimiri. The results demonstrate that vesicles can be produced by using a vesiculation fluid containing 25 mM formaldehyde/2 mM dithiothreitol. Electron microscopy revealed that these vesicles were free of detectable virus particles. Vesicles prepared from the infected cells contained virus-induced membrane antigens as shown by membrane immunofluorescence and by inhibition of antibody-dependent lymphocyte cytotoxicity. Nonhuman primates immunized with vesicles produced antibodies to these membrane antigens, late cytoplasmic antigens, and neutralizing antibodies. Infectious virus was not demonstrated in these vesicles by cocultivation with owl monkey kidney cells or by the inoculation of cottontop marmosets. Furthermore, no DNA could be demonstrated in vesicles prepared from Herpesvirus-infected owl monkey kidney cells. The implication of these findings in relation to the question of a virus-free membrane vaccine against Herpesvirus infections is discussed.

Animals↗
PubMed1977

Plasma membrane intramembranous particle topography in 3T3 and SV3T3 cells: the effect of cytochalasin B.

The topography of plasma membrane intramembranous particles in contact-inhibited and transformed cells is a topic of current controversy. We have reported that particles are aggregated in 3T3 cells and that they are randomly distributed in SV3T3 cells. Other investigators have, however, proposed that these differences result from the effect of cryoprotectants and are blocked by aldehyde fixation. The experiments described in this paper re-examine PMP topography in large samples of 3T3 and SV3T3 cells prepared by a variety of preparative methods. The results confirm that 3T3 cells contain aggregated intramembranous particles and that native SV3T3 cells do not, regardless of whether cells are prepared in glycerol, sucrose, tissue culture medium or following prefixation in paraformaldehyde. Data presented in this paper also show that cytochalasin B induces dose- and time-dependent disaggregations of particles in contact-inhibited 3T3 cells. Particle disaggregation results from exposure of cells in situ for 1-3 h to 1-500 ng/ml cytochalasin B. Removal of the drug by washing with reincubation of cells in medium containing serum results in particle reaggregation within 1 to 3 h. These observations suggest that intramembranous particles in 3T3 cells are aggregated in the native state and that membrane-active drugs modulate their distribution by a mechanism possibly involving disruption of linkages between membrane components and the cytoskeleton.

Animals↗
PubMed1977

Plasma membrane vesiculation: correlation between macrophage spreading and the shedding of cell surface vesicles.

The data presented in this paper establish a correlation between the extent of macrophage spreading and the ability of macrophages to shed plasma membrane vesicles following incubation in a membrane vesiculant. Maximal macrophage spreading was observed following incubation in buffers containing Mn2+ or dithiothreitol. Scanning electron microscopy showed that such macrophages have a uniformly flat surface morphology. Exposure of these macrophage monolayers to a vesiculant containing 25 mM formaldehyde-2 mM dithiothreitol induced plasma membrane vesiculation. Scanning electron microscopy further demonstrated that vesicles were formed and released randomly over the cell surface and that vesicles varied in size from 1 to 15 micrometer. diameter. A variety of chemicals that share the common ability to bind to free sulfhydryl groups have been shown to act as membrane vesiculants as a result of their apparent ability to induce a unique form of cell injury which results in the shedding of cell surface vesicles. The differential effect of vesiculants on cells grown in suspension and as monolayer is interpreted to suggest that reorganization of the cellular cytoskeleton associated with cell spreading may underlie the ability of membrane vesiculants to induce plasma membrane vesiculation in highly spread and adherent macrophage monolayers.

Animals↗
PubMed1976

Plasma membrane vesiculation: a new technique for isolation of plasma membranes.

Monolayer cell cultures of macrophages, monocytes, myoblasts, and density-inhibited and transformed fibroblasts form and release cell surface membrane vesicles following exposure to formaldehyde, related low-molecular-weight aldehydes, and disulfide blocking agents. Vesicles have a unique composition of proteins and lipids. They show enrichment of cholesterol and sphingomyelin content and a seven-to tenfold enrichment of 5'-nucleotidase activity. Vesicles also contain intramembranous particles and show a trilamellar unit membrane and no ultrastructural evidence of contamination with other cytoplasmic organelles. The technique is proposed as a novel method for isolating plasma membrane vesicles from cells in culture.

Cell Fractionation↗
PubMed1976

Membrane pathology of normal and malignant cells--a review.

The plasma membrane has been shown to play a critical role in the regulation of cell growth and proliferation, and pathologic change in the plasma membrane has been detected in neoplastic cells. This article reviews the structure and functions of biological membranes and discusses the differences that have been reported in the plasma membranes of normal and malignant cells that may be associated with neoplastic transformation.

Animals↗
PubMed1976

Synergistic activity of carbenicillin and gentamicin in experimental Pseudomonas bacteremia in neutropenic rats.

Rats made neutropenic with cyclophosphamide were infected intraperitoneally with Pseudomonas aeruginosa. The challenge organism was killed synergistically in vitro by the combination of gentamicin and carbenicillin. Untreated neutropenic rats infected with 3 x 10(6)Pseudomonas died between days 2 and 7, and the overall mortality was 70%. Groups of infected neutropenic rats were treated intramuscularly with 1.5 or 6 mg of gentamicin per kg per dose, 100 or 400 mg of carbenicillin per kg per dose, or 1.5 mg of gentamicin and 100 mg of carbenicillin per kg per dose. Treatment was begun at 2 h postinfection and was continued every 8 h for about 72 h. Cultures of blood and peritoneal washings were performed in control and treated rats at 1, 4, 24, 48, and 72 h postinfection. Gentamicin at either dose level was ineffective in preventing death, but mortality was significantly reduced by high-dose carbenicillin and low-dose combination therapy. In addition, the latter regimens sterilized the peritoneal fluid and blood. Carbenicillin and gentamicin showed in vivo synergy in the treatment of neutropenic Pseudomonas-infected rats.

Agranulocytosis↗