Search PubMed⌕ Search

Biomedical subjects

D A Handley

Publications and source records attributed to D A Handley.

72 records · Page 4Linked to original sources
PubMed1983

Colloidal gold: a pluripotent receptor probe.

Colloidal gold is an electron-dense, lyophobic colloid that readily forms a stable electrostatic interaction with a variety of macromolecules. Monodispersed colloids ranging from 3-150 nm in diameter can be produced to provide the researcher with flexibility in selecting the optimally sized probe. Gold labeling of antibodies and lectins has been extensively used to study surface antigens and cell components. Recently, the use of gold labeling has been extended to study receptor-ligand binding, enzyme-substrate reactions, and transcellular pathways. Published applications include gold labeling of metabolites (low-density lipoproteins), enzymes (DNAase and RNAase, RNA polymerase, thrombin, collagenase, elastase), hormones (insulin, epidermal growth factor, glucagon), circulating plasma proteins (asialoglycoprotein, alpha 2-macroglobulin, factor VIII-von Willebrand factor), and endotoxins (tetanus toxin, cholera toxin). This broad spectrum of applications emphasizes the versatility and usefulness of colloidal gold as a probe in areas of cell biology related to receptors, endocytosis, transport, and functions of proteins.

Animals↗
PubMed1983

Sinusoidal endothelial endocytosis of low density lipoprotein-gold conjugates in perfused livers of ethinyl-estradiol treated rats.

We examined endocytosis of low-density lipoproteins (LDL) conjugated to colloidal gold by the sinusoidal endothelium in perfused livers of 17 alpha-ethinyl estradiol-treated rats. After 15 min of perfusion, the gold-LDL was randomly bound at the endothelial surface, but internalized only at coated pits. Uptake of the conjugate was to electron-lucid vacuoles. After 1 h of perfusion, there was a progressive accumulation of gold in organelles resembling lysosomes, with further accumulation seen at 2 h perfusion. Uptake of the conjugate was equivalent to the rate of 125I-LDL and competitively inhibited by a 20-fold excess of free LDL. These results suggest that a specific endocytotic pathway for LDL is present in the sinusoidal endothelium in the estrogen-hypolipidemic state.

Animals↗
PubMed1981

Oxidation of ruthenium red for use as an intercellular tracer.

When ruthenium red (RR) is combined with OsO4, an electron-opaque complex forms which readily binds to the cell surface coat. However, the RR-OsO4 complex is often excluded from intercellular spaces in many cell types, and thus is not dependable as a tracer of regions continuous with the extracellular space. Postfixation of erythrocytes agglutinated by the lectin helix (Helix promatia) and intact carotid artery endothelium with a freshly prepared mixture of 1% OsO4 containing 0.1% ruthenium red (RR) resulted in a dense surface deposit of these cells, but intercellular regions were penetrated to a minimal degree by the stain. When a similar mixture of RR-OsO4 was allowed to stand 3 h before use, RR is oxidized by OsO4 to yield a ruthenium compound that has a spectrophotometric absorbance maximum at 365 nm. This RR molecule has a reduced number of cationic sites due to binding with osmium dioxide OsO2=. Postfixation of agglutinated RBCs and carotid artery endothelium with this oxidized ruthenium-OsO4 mixture resulted in a 50-80% decrease in surface deposition but markedly enhanced penetration into intercellular regions. The enhanced penetration is attributed to decreased binding affinity of the oxidized ruthenium for anionic surface membrane components, permitting effective stain penetration of cell-to-cell contact rather than extensive surface deposition. These studies indicate that the ruthenium compound formed by OsO4 oxidation of ruthenium red may be a useful tracer for ultrastructural visualization of intercellular spaces and junctions.

Animals↗
PubMed1981

Colloidal gold--low density lipoprotein conjugates as membrane receptor probes.

We have developed a method for conjugating low density lipoproteins (LDL) with colloidal gold. Conjugation, complete after 1 min, occurs by electrostatic adsorption of the LDL to the negatively charged gold particle. Each conjugate consists of approximately eight biologically active LDL molecules clustered around a central 19-nm gold granule. Acidic (pH 4), alkaline (pH 9), or high ionic (600 milliosmolar NaCl) environments do not dissociate the conjugate. Colloidal gold is an electron-dense, nondegradable marker that is easily identified within the cell and serves as a valuable probe for studying receptor binding and endocytosis. By using a modified method of ruthenium red staining, the LDL molecules of the conjugate can be directly visualized when they are bound to the cell surface receptor. Receptor binding (4 degrees C) of the conjugate by cultured human fibroblasts reveals that the gold granule is positioned 18-21 nm from the coated pit region of the membrane. This distance, similar to the diameter of LDL, suggests concomitant internalization of the receptor during vesicular endocytosis and early lysosomal incorporation (10 min at 37 degrees C). Continued internalization (30-60 min at 37 degrees C) results in the formation of free pools of gold within the lysosome.

Cell Membrane↗
PubMed1981

Hepatic binding and internalization of low density lipoprotein-gold conjugates in rats treated with 17 alpha-ethinylestradiol.

Receptor-mediated hepatic uptake of low density lipoproteins (LDL) conjugated to colloidal gold was studied by perfusion of livers from rats treated for 5 d with 17 alpha-ethinylestradiol. Estrogen treatment resulted in a marked decrease in serum lipid and lipoprotein concentrations. After 15 min of perfusion the conjugate was bound to the hepatic microvilli of both control and estrogen-treated rats; the estrogen-treated rats showed an 8- to 11-fold greater number of membrane-bound conjugates. The conjugates were bound to the membrane receptor by the LDL particle because the gold granules were regularly displaced from the membrane by 20 +/- 3.2 nm, the diameter of LDL. Internalization of the conjugate, evident by gold particles in multivesicular bodies, occurred at coated pits at the base of the microvillus where coated vesicles containing a single gold-LDL conjugate were released. After 1 h of perfusion, the livers from the estrogen-treated rats showed all phases of endocytosis and incorporation into multivesicular bodies of the conjugate. After 2 h of perfusion, there was congregation of gold-labeled lysosomes near the bile canaliculi. Gold-LDL conjugates were also observed to bind and be internalized by Kupffer cells and sinusoidal endothelium. These findings indicate that estrogen treatment induces hepatic receptors for LDL. The catabolic pathway of binding and endocytosis of the conjugate is similar to that seen in fibroblasts, although slower. Because gold-LDL conjugates were also present in the Kupffer and endothelial cells, the uptake of LDL by the liver involves the participation of more than a single cell type.

Animals↗
PubMed1981

The design and use of a simple device for rapid quench-freezing of biological samples.

The detailed design of a simple device for rapid quench-freezing of biological samples under reproducible conditions is presented. With spring-augmented descent, sample immersion velocity of 10 m s-1 into a cryogenic liquid is achieved. Biological samples, loaded in Balzers planchets, Denton holders, or a newly designed 'titanium envelope', are suitable for rapid-freezing with this device. Using 4 micrometers titanium foil, light weight (1 mg) streamlined holders can easily be made to enclose cell suspensions or tissue samples. The foil envelope is designed for efficient heat dissipation while protecting the sample from possible impact or flow distortions occurring from spring-augmented immersion. Human erythrocytes, quench-frozen in the titanium envelope, were prepared for electron microscopy by the freeze-substitution technique. Two opposing 25--30 micrometers surface zones were frozen in the apparent absence of ice. The extended depth of cryofixation is attributed to the advantages of thin foil in the titanium envelope design and the use of rapid-immersion technique.

Erythrocytes↗
PubMed1979

Butvar B-98 as a thin support film.

Support films prepared from butvar B-98 resin are mechanically stable, electron transparent, and possess minimum intrinsic structure. A simple procedure for routine preparation of support films using this resin is provided.

Microscopy, Electron↗
PubMedDate not supplied

Effects of oscillatory mechanical disturbance on macromolecular uptake by arterial wall.

Transport of 125I-albumin by isolated segments of canine common carotid arteries was studied in vitro at zero transmural pressure. Sinusoidal oscillatory variations in length (peak change 4%) for 15 minutes at frequencies of 5 and 10 Hz caused 40% increase in 125I-albumin uptake, and also a 30% increase in the apparent luminal surface area. Changes in the duration and frequency of oscillation indicate that the total number of oscillations (= frequency X duration) was the critical parameter in causing these effects. The increase in apparent luminal surface area was correlated with regional flattening of the internal elastic lamina and the overlying endothelial cells, as demonstrated by transmission and scanning electron microscopy. Endothelial vesicles were counted with the aid of ruthenium red as a postfixation extracellular marker. The ratio of unstained free vesicles to total vesicles averaged 0.083 in the control state and decreased slightly to 0.070 after oscillation. Although the decrease in free vesicle population indicated an acceleration of vesicle diffusion, our theoretical computations showed that the resulting increase in vesicle flux was negligible. The increase in 125I-albumin uptake by the artery following mechanical oscillation is mainly attributable to the increase in apparent luminal surface area.

Animals↗
PubMedDate not supplied

Synthesis and pharmacology of a novel class of long-lasting PAF receptor antagonists.

Several new charged PAF receptor antagonists were prepared, where the phosphate moiety has been replaced by a methylsulfonylcarbamoylpyridinium moiety, and evaluated for duration of inhibitory activity against PAF-induced bronchoconstriction and hemoconcentration in the guinea pig. One of these compounds (1d; SDZ 64-619) has shown potency and duration of inhibition in the range of CV-6209 (1c).

Adult↗