Richard F. Baker

Sectioning techniques for electron microscopy using a conventional microtome.

Summary Conventional histological techniques have been modified so that is is possible consistently to cut 0.2 micron sections for use with the electron microscope. The material must be doubly embedded in strong collodion and hard paraffin. The face of the block to be cut must be small, and the tilt of the knife must be precisely adjusted. Procedures have been developed for partly or wholly removing the embedding media, and mounting the sections for the electron microscope. Micrographs of rat liver sections show that the principal artefacts are due to fixation rather than subsequent treatments.

The effect of calcium ionophore A23187 on the ATP level of human erythrocytes.

Incubation of red cells at 37° with the ionophore A23187 results in a loss of ATP that is dependent on the concentrations of A23187 and Ca2+ in the medium. ATP hydrolysis is greatest at micromolar concentrations of Ca2+ and decreases as Ca2+ in the medium is raised to millimolar levels. The ATP depletion is due to stimulation of calcium ATPase by A23187-mediated Ca2+ influx into the cell. The biphasic nature of Ca2+-stimulated ATP depletion in whole cells reflects the activity of Ca2+-ATPase in membrane preparations at varying Ca2+ concentrations. The ionophore can be removed by washing the cells with plasma or bovine serum albumin-containing medium and the ATP levels restored to normal by reincubating with 5 mM adenosine for 1 hr.

Denaturation of ferritin and its relationship with hemosiderin.

The fate of the ferritin molecules in vitro after degration with oxidizing agents and tryptic digestion has been determined using chemical, serological, and electron optical techniques. Oxidizing agents used were acetylphenylhydrazine, hydrogen peroxide, and molecular oxygen. The denaturation products of oxidized ferritin are compared with hemosiderin with respect to physicochemical, immunological, and ultrastructural characteristics. The conclusion is drawn that hemosiderin in vivo is a denaturation product of ferritin. The possible mechanism of the biosynthesis of hemosiderin in living tissue is discussed taking into account the above conclusion.

Restoration of the deformability of "irreversibly" sickled cells by procaine hydrochloride.

Summary Irreversibly sickled red cells may be produced in vitro by incubating deoxygenated sickle cells at 37°C. The deformability of oxygenated irreversibly sickled cells produced in this way may be restored by pre or post treatment with 0.09M procaine hydrochloride. The loss of deformability associated with metabolic depletion of normal erythrocytes in vitro is minimized in the presence of 0.006M procaine hydrochloride. Displacement of membrane-bound calcium by procaine is suggested as a possible mechanism for the observed effects.

Freeze-thawing as a preparatory technique for electron microscopy.

Small pieces of mouse kidney cortex were frozen by immersion in isopentane cooled to −160°C by liquid nitrogen. The tissue was warmed to −18°C and 10-μ sections were cut in a cryostat. Thawing was effected by immersion in buffered osmium tetroxide at +4°C. Fixation and embedding in methacrylate or Epon 812 followed. Ultrathin lead stained sections showed few vacuoles due to ice crystals. The probable sites of intra-cytoplasmic ice crystals were characterized by areas lacking fine structure and slightly denser than the surroundings. Brush border, mitochondria, cytoplasmic membranes, and ribosomes were well preserved except for mitochondrial swelling. Blebbing of the outer nuclear membrane and vacuolization of the chromatin were seen. Slow cooling at 2°C/minute to −18°C followed by section cutting and fixation as above produced ultrathin sections showing fine structure preservation nearly equivalent to that seen after fast freezing, except that intracellular ice crystal sites were not seen.

Fusion of human erythrocytes induced by uranyl acetate and rare earth metals

Incubation of human erythrocytes with either uranyl ions (UO22+) or rare earth metals (La3+, Nd3+, Sm3+, Eu3+, Tb3+, Dy3+ and Yb3+) at 37 degrees C for 30-45 min resulted in the fusion of erythrocytes. Redistribution of membrane-associated particles was observed using colloidal-iron charge labelling and freeze-fracture electron microscopy. The fusion of erythrocytes induced by these agents, unlike Ca2+, did not exhibit the absolute requirement for phosphate. Moreover, agglutination and fusion by these agents was observed in neuraminidase-treated erythrocytes in contrast to Ca2+- and phosphate-induced fusion. Inhibitors of intrinsic transglutaminase activity partially inhibited (35-45%) the fusion induced by UO22+ suggesting that cross-linking of membrane proteins results in protein-free areas of lipid where fusion may be initiated.

THE FINE STRUCTURE OF STROMALYTIC FORMS PRODUCED BY OSMOTIC HEMOLYSIS OF RED BLOOD CELLS.

When human red blood cells are hemolyzed by hypotonic salt solutions, many of the cells give rise to long cylindrical tubes which range from 200 A to μ in diameter and may be several microns long. Negative staining of these preparations shows that the tubes are bounded by a membrane 40–60 A in width, are relatively impermeable to PTA, and show no periodic structures in either negative staining or in thin sections. Influenza Virus (PR8) does not attach to the tubes. Reasons are given for supposing that the tubes are not strictly identical to myelin forms, but probably contain a high percentage of hemoglobin.

Chemically induced fusion of fresh human erythrocytes.

Abstract The fusion of fresh human erythrocytes was shown to be induced by calcium and phosphate ions. Prior treatment of erythrocytes with phosphate ion was a pre-requisite for the calcium-induced fusion. ATP levels in cells incubated with phosphate and calcium decreased 46 fold while cell-associated calcium increased 70 fold during 1 hour of incubation at 37°C as compared to cells which were incubated with calcium in saline. Our results suggest that a phosphate complex formed bridges between adjacent erythrocytes causing agglutination followed by aggregation of membrane proteins leading to protein-free areas of lipids. Where these protein-free areas are in close contact fusion may occur.

Effect of cell shape, membrane deformability and phospholipid organization on phosphate-calcium-induced fusion of erythrocytes

Fusion of bovine and goat erythrocytes was studied using the phosphate-calcium protocol. Both bovine and goat red cells are resistant to fusion with phosphate and calcium, under conditions that promote fusion of normal human erythrocytes. Fusion resistance is not related to decreased (5%) membrane deformability of erythrocytes of these species, since chicken erythrocytes which are 40% less deformable than human erythrocytes undergo fusion with efficiency similar to human red blood cells. Incorporation of either phosphatidylcholine or phosphatidylserine into bovine erythrocytes mediated by lipid exchange/transfer protein, caused fusion of these erythrocytes. Fluorescence analysis of merocyanine 540 dye labeled erythrocytes, by flow cytometry, showed that the frequency of cells which exhibit dye binding was much less (35%) in dimyristoylphosphatidylcholine (DMPC) incorporated compared to untreated bovine erythrocytes (80%), indicating that incorporation of DMPC caused closed packing of lipids in the external leaflet of the bilayer. These studies show that fusion of bovine erythrocytes, mediated by phosphate and calcium, has a requirement for either specific phospholipids such as phosphatidylcholine, phosphatidylserine, or closed packing of lipids in the external leaflet of the bilayer.

Phosphate-calcium induced fusion of chicken erythrocytes

Abstract Phosphate-buffered saline (PBS) and calcium chloride in Hepes-buffered saline (HBS) have been used prior to incubation at 50 °C to induce fusion of fresh chicken erythrocytes. Prior treatment with PBS was found to be essential for the combined phosphate and calcium induced fusion to occur. Cells did not fuse if (i) calcium was introduced prior to the phosphate ion, or (ii) the chicken erythrocytes were suspended and incubated in a pre-mixed combination of phosphate and calcium. It was observed that the optimum incubation temperature was around 50 °C. At this temperature maximum fusion took place in 1 h without marked cell destruction. The calcium concentration for maximum fusion using 44 mM phosphate was 25 mM at pH 7. The work demonstrates that the polykaryocyte size may be decreased by vesiculation and therefore is not necessarily proportional to the number of fusing cells. Also, the number of nuclei seen in a polykaryocyte did not serve as an accurate indicator of the number of fusing cells. Membrane-associated protein aggregation was demonstrated by freeze-fracture of cells undergoing fusion.