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Biomedical subjects

L B Margolis

Publications and source records attributed to L B Margolis.

At least 19 recordsLinked to original sources
PubMed1995

Regulation of intracellular pH by cell-cell adhesive interactions.

As was shown in our previous work, the intracellular pH (pHi) of cultured human fibroblasts depends on cell density. The pHi is low in single cells, higher in cells, forming small groups and maximal in a sparse monolayer. On the other hand, the pHi is low in areas of confluent monolayers. In the present work, we show that the effects of inhibitors of various pH-controlling mechanisms as well as inhibitors of key enzymes in signal transduction pathways depend on the local cell density. We have found that N-ethylmaleimide and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, known as inhibitors of V-type H+ ATPase, inhibit the elevation of pHi induced by cell-cell contact interactions; meanwhile Cd2+ ions, which inhibit H+ conductive pathway, cause an increase of pHi in a confluent monolayer. Our data revealed also that the Na+/H+ antiporter does not play an essential role in the pHi regulation by intercellular contacts. Inhibitors of phospholipase A2 (4-bromophenacyl-bromide), phospholipase C (neomycin) and protein kinase C (H-7) dramatically change the way the pHi is modulated by local cell density. It is suggested that cell-cell interactions regulate cell activities via modulation of pHi, which is under positive control from phospholipase A2 and under negative control from protein kinase C.

Acid-Base Equilibrium↗
PubMed1995

Syncytium formation in cultured human lymphoid tissue: fusion of implanted HIV glycoprotein 120/41-expressing cells with native CD4+ cells.

While glycoprotein gp120/41 clearly causes HIV-infected cells to form syncytia in monolayers and in suspension, there is unfortunately scant knowledge on syncytium formation in tissues. We implanted gp120/41-expressing cells labeled with fluorescent particles inside blocks of human lymphoid tissue kept in long-term histoculture. Observed by confocal microscopy, together with immunohistochemical and morphological analysis of implanted cells, more than one-third of these gp120/41-expressing cells fused with native CD4+ cells of the host tissue, yielding small (three to five nuclei) syncytia. Such widespread fusion of gp120/41-expressing cells in tissue in vitro, together with the finding of increased virulence of syncytium-inducing isolates of HIV, support the hypothesis that syncytium formation within lymph tissue of HIV-infected individuals contributes to AIDS pathogenesis. This system and the methods developed may provide a way to study HIV-infected cells inside the very tissue whose destruction may prevent immune system repopulation.

Antigens, CD20↗
PubMed1994

Organotypic growth and differentiation of human mammary gland in sponge-gel matrix supported histoculture.

Blocks of breast tissue obtained during radical mastectomies from 23 patients with mammary gland carcinomas were used for cultivation in native-state, gel-supported histocultures. We show that the human mammary gland can be successfully maintained in this system so that normal epithelial breast structures proliferate and undergo differentiation for several weeks and a well-developed system of ducts and lobules is formed. Using antibodies to individual keratins 17 and 8 we have shown for the first time that ducts and alveoles developing in vitro undergo differentiation into the lining epithelium and myoepithelium in the same way as mammary gland epithelium in vivo. Growth of epithelial structures in vitro is also accompanied by the development of continuous basal membrane.

Adult↗
PubMed1992

Hair shaft elongation, follicle growth, and spontaneous regression in long-term, gelatin sponge-supported histoculture of human scalp skin.

In order to better understand the molecular mechanisms of human hair growth control and to test hair growth-modulatory drugs, appropriate in vitro models are required. Here, we report the long-term growth, shaft elongation, and spontaneous regression of human hair follicles in histoculture of intact scalp skin. Human scalp skin with abundant hair follicles in various stages of the hair growth cycle was grown for up to 40 days in a gelatin sponge-supported histoculture system at the air/liquid interface. Isolated follicles placed in the gelatin-sponge matrix also supported hair shaft elongation, with the hair follicle cells remaining proliferative and viable for very long periods. Hair shaft elongation occurred mainly during the first 10 days of histoculture of both intact skin and isolated follicles. However, hair follicles were viable and follicle keratinocytes continued to incorporate [3H]thymidine for up to several weeks after shaft elongation had ceased as shown by fluorescent-dye double staining, measured by confocal laser scanning microscopy, and by histological autoradiography of [3H]thymidine incorporation, respectively. Hair follicles could continue their cycle in histoculture; for example, apparent spontaneous catagen induction was observed both histologically and by the actual regression of the hair follicle. In addition, vellus follicles were shown to be viable at day 40 after initiation of culture. In the histocultured human scalp we demonstrated the association of mast cells with anagen follicles and macrophages with catagen follicles, suggesting a role of these cells in the hair cycle. This histoculture technique should serve as a powerful tool for future hair research in the human system as well as a screening assay for compounds that can perturb the hair cycle.

Culture Techniques↗
PubMed1992

Cell-cell contacts alter intracellular pH.

Intracellular pH, an important regulatory factor for many cellular activities, was shown to be modulated by cell adhesion to the solid substratum. In the present work we have shown that cell-cell contacts also affect intracellular pH. pH(i) depends on how many contacts the cell has established with the substratum and the neighboring cells. pH(i) is low in single cells, not contacting each other. It increased with the increase of cell density. pH(i) is again decreased in confluent (topoinhibited) monolayers. pH(i)-shifts triggered by cell-cell contacts seem to be mediated by Na+/H(+)-antiporter. Dependence of pH(i) on cell density could be simulated by different concentration of Arg-Gly-Asp--which is part of the site of extracellular matrix proteins involved in integrin binding. The dependence of pH(i) on cell-cell contacts is discussed in relation to the phenomena of topoinhibition.

Amino Acid Sequence↗
PubMed1991

Low-density lipoproteins interact with liposome-binding sites on the cell surface.

Under physiological conditions significant amounts of low-density lipoprotein LDL particles ar taken up by cells independently of specific high-affinity LDL receptors (apo-B receptors). Previously it was established that some cells contain surface sites capable of binding liposomes. We proposed that liposome-binding sites could contribute to LDL interaction with the cell surface via phospholipid molecules of LDL particles. To check this hypothesis we studied the competitive interaction of human LDL and DPPC liposomes with mouse embryo fibroblasts depleted of apo-B receptors by preliminary incubation with LDL. We have found that after removal of the liposome-binding sites from cell lamellae these areas of the cell surface lose their ability to bind LDL.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1991

Mechanism of cell protrusion formation in electrical field: the role of actin.

An intense alternating current electrical field that imposes membrane-applied force on the cell surface can induce formation of cell protrusions (Popov, S.V. and Margolis, L.B. (1988) J. Cell Sci. 90, 379-389). This technique has been used to investigate the role of actin in the cell protrusion formation. Platinum replicas of the cytoskeleton were prepared to characterize the organization of the cytoskeleton in external force-induced protrusions. Bundles of microfilaments were found in the processes. A specific inhibitor of actin polymerization, cytochalasin B, as well as inhibitors of ATP synthesis (sodium azide and carbonyl m-chlorophenylhydrazone) did not change the morphology of electrical field-generated protrusions, revealed by scanning electron microscopy. However, organization of the cytoskeleton inside the processes changed drastically using these inhibitors. The results of these experiments demonstrate that (i) Membrane-applied force is sufficient to produce native-like cell protrusions, even in conditions where activity of the cytoskeleton is inhibited; (ii) Actin microfilaments can be organized into bundles directly under the action of membrane-applied force. The significance of these observations to cell protrusion formation under normal physiological conditions is discussed.

Actin Cytoskeleton↗
PubMed1991

Local deformation of human red blood cells in high frequency electric field.

A method of local and general deformation of single erythrocytes by external forces in high-frequency electric field is described. The method allows the avoidance of any mechanical contact of the cell with electrodes. Under the action of the forces applied human erythrocytes change their shape and produce various membrane structures: long filopodia-like processes, retraction fibers and lamella-like structures. These structures are never formed by erythrocytes under normal conditions, but are typical for fibroblasts, macrophages and epithelium cells. By the method developed the elastic properties of spicules on the membranes of echinocytes were also studied. Deformation of echinocyte in high-frequency electric field leads to the smoothing out of spicules. However, after the electric field is turned off, echinocyte restores its initial forms including the number and localization of all initial spicules on the cell surface.

Biomechanical Phenomena↗
PubMed1991

Skin toxicity determined in vitro by three-dimensional, native-state histoculture.

We describe a gel-supported in vitro system for culturing skin samples in a three-dimensional native state. All the cell types of skin remain viable and maintain their native architecture for at least 10 days. The culture system is used for toxicity measurements by ascertaining cell viability using two fluorescent dyes: 2',7'-bis-(2-carboxyethyl)-5-(and -6)carboxyfluorescein acetoxymethyl ester, specific for living cells, and propidium iodide, specific for dead cells. Cell staining with the dyes is measured throughout the tissue block by confocal scanning fluorescence microscopy. The dose-response to three agents--ethanol, doxorubicin, and sodium hypochlorite--is shown and, in the case of sodium hypochlorite, compared to in vivo skin toxicity with a high correlation. We also demonstrate that the end point of [3H]thymidine incorporation measured by histological autoradiography can be used to measure toxicity. Our results with the [3H]thymidine end point demonstrate that the hair follicle cells are the most sensitive to doxorubicin. The native-state model for skin may be an effective replacement for animal systems and superior to the dispersed skin cell systems used previously. It can allow rapid, inexpensive measurements of the effect of manufactured products, drugs, and pollutants on skin.

Animals↗
PubMed1991

An upward shift of intracellular pH rather than the final absolute pH value is critical for controlling gap junction permeability in tumor promoter-treated cells.

The tumor promoter TPA is found to inhibit gap junction permeability in monolayer cultures of hamster fibroblasts. This effect is associated with an increase in intracellular pH. Here we show that neither an increase in pHi alone nor TPA treatment under conditions preventing pHi-shift affect gap junction permeability. It is not the level of pHi reached, but rather the pHi-shift itself that is essential for the inhibition of gap junction permeability in the presence of TPA.

Animals↗
PubMed1991

Restoration of adhesive potentials of Ehrlich ascites carcinoma cells by modification of plasma membrane.

A novel technique for modulating the spreading of ascites cells has been developed. Plasma membranes of Ehrlich ascites carcinoma cells were modified in two different ways: 1) biotin residues were covalently coupled to membrane components; 2) biotinylated lipid was introduced into plasma membrane. Adhesion and spreading of modified cells on avidin-coated substrates were studied and compared to those of non-modified cells. Both types of membrane alteration were shown to induce specific (biotin-dependent) interaction with immobilized avidin with resultant cell spreading. Spread cells attained epithelioid-like morphology with the formation of wide thin lamellae, focal contacts with substrate, and circular actin bundles. The process of spreading was shown to be energy-dependent: it could be blocked by metabolic inhibitors and by low temperature. Formation of extended lamellae was prevented by preincubation of cells in the presence of cytochalasin B. The effects of metabolic poisons, low temperature, and microfilament--disruptive drugs were reversible and after the restoration of physiological conditions the cells resumed the spreading process. Immunoprecipitation of biotinylated cell lysates with antiserum to cytoplasmic domain of beta 1-integrin subunit revealed a major 110 kD avidin-binding component. We conclude that lack of spreading of ascites carcinoma cells may be explained by the lack of functionally active adhesion- and spreading-competent cell-surface receptors, but may not be attributed to the defects in intracellular function or organization. Intracellular machinery of cell spreading is preserved in these ascites cells and could be turned on by cell attachment to the substrate via artificial adhesive site incorporated into plasma membrane.

Animals↗
PubMed1991

A model for the study of the mechanism of a low pH-induced interaction of the virus fusion proteins and cell membranes.

A model is proposed for the study of molecular mechanisms of a low pH-induced interaction of fusion proteins of enveloped viruses and cell membranes. The model consists of large monolamellar liposomes containing ionophore nigericin in their membranes and ectodomains of fusion protein in their inner space. The process of interaction of the protein with the lipid bilayer is triggered by acidification of the liposomal constituents to the pH of fusion with the help of nigericin by adding citric acid to the outer medium. To visualize the protein structural reorganization, the tritium planigraphy was used. Comparison of the values of specific labelling of the proteins and distribution of radioactivity in individual amino acids in control (at neutral pH) and experimental liposome samples (at the pH of fusion) permits to realise the character of protein-membrane interaction. We have obtained the first results in the study of interaction of the bromelain-released soluble ectodomain of the HAXX molecule (BHA)--with the lipid membrane. The observed increase in the protein specific activity and selective increase in the specific activity of hydrophobic amino acids Ile, Phe and Tyr in experimental liposome samples as compared with the controls did not contradict to the conventional concept, that a hydrophobic N-terminus of HA2 subunit of hemagglutinin is responsible for its interaction with lipid membranes.

Hemagglutinin Glycoproteins, Influenza Virus↗
PubMed1991

Induction of cell processes by local force.

A method of applying local mechanical force to the plasma membrane of mouse embryo fibroblasts is described. The force is generated by local treatment of cells with an alternating current (a.c.) electrical field. The phenomenon of cell process formation under the action of this force was investigated. Inhibitors of actin polymerization did not prevent generation of processes in the electrical field. At the early stages of cell spreading, the processes could be induced at any part of the cell membrane. After cell polarization was completed, protrusions could be formed at the active edge of the cell, but not at its stable edge. Pre-existing protrusions (but not retraction fibers) could be elongated by the external force. The results of these experiments demonstrate that different areas of the cell membrane differ in their ability to form processes under the action of a membrane-applied force. The significance of these data to the structure of the cortical layer is discussed.

Actins↗
PubMed1990

[The cytostatic action of the K+/H+ antiporter nigericin].

A decrease in pH in Ehrlich ascites carcinoma cells to 6.3 leads to intracellular DNA synthesis inhibition. The inhibitory effect was achieved in the course of incubation of cells at pH 6.2 as well as by the addition of low-dose K+/H+ antiporter antibiotic nigericin. DNA synthesis inhibition by nigericin was not associated with significant decrease in ATP level or mitochondrial membrane potential. The effect was mediated by a decrease in intracellular pH rather than by a decrease in intracellular potassium level or elevation in that of intracellular sodium which accompanied nigericin treatment.

Adenosine Triphosphate↗