Biomedical subjects
Publications and source records attributed to C Sardet.
We have examined the content and permeability of chloride in sea urchin eggs. After fertilization there is a large increase in the permeability to chloride. We discuss the mechanism underlying this permeability change and the generalized increase in ion permeability observed after fertilization.
Various species of teleostean fishes were adapted to fresh or salt water and their gill surface epithelium was examined using several techniques of electron microscopy. In both fresh and salt water the branchial epithelium is mostly covered by flat respiratory cells. They are characterized by unusual outer membrane fracture faces containing intramembranous particles and pits in various stages of ordered aggregation. Freeze fracture studies showed that the tight junctions between respiratory cells are made of several interconnecting strands, probably representing high resistance junctions. The organization of intramembranous elements and the morphological characteristics of the junctions do not vary in relation to the external salinity. Towards the base of the secondary gill lamellae, the layer of respiratory cells is interrupted by mitochondria-rich cells ("chloride cells"), also linked to respiratory cells by multistranded junctions. There is a fundamental reorganization of the chloride cells associated with salt water adaptation. In salt water young adjacent chloride cells send interdigitations into preexisting chloride cells. The apex of the seawater chloride cell is therefore part of a mosaic of sister cells linked to surrounding respiratory cells by multistranded junctions. The chloride cells are linked to each other by shallow junctions made of only one strand and permeable to lanthanum. It is therefore suggested that salt water adaptation triggers a cellular reorganization of the epithelium in such a way that leaky junctions (a low resistance pathway) appear at the apex of the chloride cells. Chloride cells are characterized by an extensive tubular reticulum which is an extension of the basolateral plasma membrane. It is made of repeating units and is the site of numerous ion pumps. The presence of shallow junctions in sea water-adapted fish makes it possible for the reticulum to contact the external milieu. In contrast in the freshwater-adapted fish the chloride cell's tubular reticulum is separated by deep apical junctions from the external environment. Based on these observations we discuss how solutes could transfer across the epithelium.
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When Pisaster, Asterias, or Thyone sperm are treated with the ionophore A23187 or X537A, an acrosomal reaction similar but not identical to a normal acrosomal reaction is induced in all the sperm. Based upon the response of the sperm, the acrosomal reaction consists of a series of temporally related steps. These include the fusion of the acrosomal vacuole with the cell surface, the polymerization of the actin, the alignment of the actin filaments, an increase in volume, an increase in the limiting membrane, and changes in the shape of the nucleus. In this report, we have concentrated on the first two steps in this sequence. Although fusion of the acrosomal vacuole with the cell surface requires Ca++, we found that the polymerization of actin instead appears to be dependent upon an increase in intracellular pH. This conclusion was reached by applying to sperm A23187, X537A, or nigericin, ionophores which all carry H+ at high affinity, yet vary in their affinity for other cations. When sperm are suspended in isotonic NaCl, isotonic KCl, calcium-free seawater, or seawater, all at pH 8.0, and the ionophore is added, the actin polymerizes explosively and an efflux of H+ from the cell occurs. However, if the pH, of the external medium is maintained at 6.5, the presumed intracellular pH, no effect is observed. And, finally, if egg jelly is added to sperm (the natural stimulus for the acrosomal reaction) at pH 8.0, H+ is also released. On the basis of these observations and those presented in earlier papers in this series, we conclude that a rise in intracellular pH induces the actin to disassociate from its binding proteins. Now it can polymerize.
The formation of the acrosomal process requires an increase of 40-150% in the plasma membrane limiting the cell body. In freeze fracture replicas and thin sections this new membrane behaves as a typical unit membrane. Since in sperm there are no vesicles or surface projections which could account for the increase in surface, we suggest that the new membrane is generated from precursors stored with the actin in the periacrosomal region. Consistent with this suggestion is the observation that compounds such as didansyl cysteine and N phenyl-1-napthylamine which fluoresce when they enter a hydrophobic environment stain the periacrosomal material.
Freeze fracture of euryhaline fish gill epithelium reveals large smooth surfaces with intramembrane particles or corresponding pits often aggregated into hexagonal arrays. They belong to the outer plasma membranes of respiratory cells, the main surface of contact between the internal and external milieu of the fish. Possible functions for the arrays are discussed in relation to the fine structure of the respiratory cell.
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Regions of different proton density in the low density lipoprotein (LDL) particle from human serum have been determined by neutron scattering. From measurements in various H2O/D2O mixtures, the LDL particle appears to be quasi-spherical, with the centers of gravity of the hydrocarbon and polar regions coinciding. The average volume occupied by a particle was found to be 3.2 X 10-6 A-3, with the volume fraction occupied by the hydrocarbons being 60%. The ratius of gyration of the hydrocarbon region was 64 A, while that of the polar region was 100 A. consequently, the core of LDL is predominantly occupied by the hydrocarbon chains, while the outer shell is sparsely occupied by protein emerging from the lipid core.
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The membrane-penetrating segment of the surface MN-glycoprotein of the human erythrocyte is contained intact within the tryptic peptide T(is). We report here on the association of this peptide with hydrated phospholipid vesicles. Under these conditions 80 A intramembranous particles, as seen by freeze-etch electron microscopy, are produced that are similar in size to those seen in the native erythrocyte membrane. These particles increase in number as a linear function of T(is) concentration and a plot of particle number versus concentration is compatible with a micelle-like phenomenon; from this curve the critical concentration for the formation of particles is estimated to be approximately one mole of T(is) to 120 moles of lecithin. These data suggest that the membrane-penetrating peptide T(is) is being incorporated, monomerically and multimerically, within the hydrocarbon phase of lecithin bilayers. From these data it can be calculated that each intramembranous particle contains between 10 and 20 T(is) monomers. The peptide portion of each particle, therefore, has a molecular weight of 45,000-85,000. An exact analogy cannot be drawn at this time between the in vivo structure of erythrocyte intramembranous particles and the reconstituted particles described here, although an argument has been constructed to support this possibility. What is clear is that the reconstituted system promises to be useful for further examination of protein-lipid interactions in membranes.
Dietary cholesterol induces a hemolytic anemia in guinea pigs, accompanied by changes in the lipid composition of red cells and of plasma lipoproteins. This report presents a characterization of the lipoprotein species present in each main density class in both control and cholesterol-fed guinea pigs. Traces of a typical high density lipoprotein (HDL) were detected in control plasma. HDL from cholesterol-fed, anemic guinea pigs differed from control HDL in electron microscopic appearance and lipid and peptide composition. Long stacks of discs were observed in the electron microscope in addition to smaller, spherical particles characteristic of control HDL. Low density lipoproteins (LDL) from cholesterol-fed, anemic guinea pigs had two main populations, which were separated by gel chromatography. One population appeared in the electron microscope as large transparent discs and contained mainly unesterified cholesterol and phospholipids in a 2:1 molar ratio. The other population resembled control LDL in size and composition except for its high unesterified cholesterol content. Dietary cholesterol also altered the composition and decreased the electrophoretic mobility of very low density lipoproteins. Gel electrophoretic and immunochemical evidence indicates that a peptide (mol wt 35,000) appears in lipoproteins from cholesterol-fed, anemic guinea pigs that is undetectable in those of controls. Similarities between the cholesterol-induced lipoprotein abnormalities in guinea pigs and those reported in patients with obstructive jaundice, biliary cirrhosis, type III hyperlipoproteinemia, or familial lecithin:cholesterol acyltransferase deficiency are discussed.
When guinea pigs are fed cholesterol, the cholesterol content of their red cells increases progressively, a large number of cells become spurred, and a hemolytic anemia develops. Unesterified cholesterol is readily transferred from plasma, HDL, or LDL of cholesterol-fed, anemic guinea pigs to normal red cells in vitro. This transfer is reversible and is proportional to the concentration of unesterified cholesterol in the incubation medium. Red cells loaded in vitro with cholesterol develop spurs identical with those on red cells in the circulation of cholesterol-fed, anemic guinea pigs. Neither the cholesterol content nor the morphology of normal red cells is altered by incubation in control plasma or in concentrated control lipoproteins. Plasma infranates (d > 1.21 g/ml) of either group do not cause spurring of control red cells. We conclude: (a) that accumulation of cholesterol by guinea pig red cells in vitro requires an increased concentration of unesterified cholesterol in lipoprotein rather than an increased concentration of normal lipoproteins, and (b) that an increased cholesterol content in guinea pig red cell membranes is necessary for their abnormal morphology. The flux of cholesterol between cholesterol-loaded cells and plasma from cholesterol-fed guinea pigs is three times greater than that between control red cells and control plasma, and the fractional exchange rates are altered.
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