Epidermal permeability barrier: transformation of lamellar granule-disks into intercellular sheets by a membrane fusion process.
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Animals↗
Biomedical subjects
D S Friend
Publications and source records attributed to D S Friend.
Selective enhancement of pentamidine uptake in the lung by aerosolization and delivery in liposomes.
We examined the tissue distribution of pentamidine, both as free drug and encapsulated in liposomes, after intravenous (iv) and aerosol administration in healthy rodents. After iv injection, drug levels in the lung were increased as much as 34-fold by administration in liposomes. Concurrently, peak liver uptake was increased 4-fold and renal deposition reduced 3-fold. Liposome-mediated lung delivery was highly dependent on liposome size. Decreasing liposome mean diameter from 2.5 to 0.4 micron reduced lung uptake of pentamidine 90-fold while affecting extrapulmonary organ deposition to a much lesser degree. Aerosol delivery of pentamidine produced high, sustained lung levels, with no evidence of drug clearance from the lung between 1 and 48 h after administration. Extrapulmonary drug levels produced by aerosol delivery were negligible throughout this period. There were no significant differences in the organ distribution of aerosolized free versus liposome-encapsulated drug, apparently because of sequestration of pentamidine within the lung. Comparison of drug levels in material recovered by bronchoalveolar lavage suggests that aerosol delivery of pentamidine produces substantially higher deposition in the alveolar space than does iv drug injection. Light and electron microscopic (EM) examination of lung, kidney, and liver after iv or aerosol administration of liposomes revealed no tissue abnormalities, although isolated platelet clumps were noted in pulmonary capillaries by EM examination.
Aerosols↗
Gonadal endodermal sinus (yolk sac) tumor with pure intestinal differentiation: a new histologic type.
We studied 3 cases of a variant of endodermal sinus tumor (EST) or yolk sac tumor (YST). Two tumors originated in the ovary and one in the testis. The patients had very high levels of serum alpha-fetoprotein (AFP). All three tumors had a characteristic histologic appearance and were composed of acinar structures lined by columnar epithelium with large, immature nuclei. Immunohistochemistry and electron microscopy, including freeze-fracture studies, confirmed that this unusual tumor is one with exclusive intestinal differentiation. We feel that this unique tumor is a pure EST (YST) with exclusive intestinal differentiation.
Adult↗
Alterations in hepatocyte plasma membrane in carbon tetrachloride poisoning. Freeze-fracture analysis of gap junction and electron spin resonance analysis of lipid fluidity.
The plasmalemma of the livers of rats treated with carbon tetrachloride (CCl4) were examined by freeze-fracture and electron spin resonance probe techniques. The rodents received by mouth either mineral oil alone (0 to 4.5 hours before sacrifice) or CCl4 in mineral oil (1:1) (2.5 ml of CCl4/kg, 0 to 3 hours before sacrifice). Rats were anesthetized with ether and livers were perfused in situ with saline either at ambient temperature or at 4 degrees C. After perfusion, livers were fixed in situ and processed for freeze-fracture and electron microscopy. Hepatocytes were isolated and incubated with 12-doxylstearic acid and subjected to electron spin resonance analysis. Electron microscopy revealed greater than a 2.5-fold increase in the individual mean gap junction size when rats were treated with mineral oil alone for 4.5 hours and the livers were processed at room temperature. The mean gap junction size in rats dosed with CCl4 for 0.5 hours before sacrifice equalled those of the group treated with mineral oil for 4.5 hours. Increases in gap junction size with CCl4 were progressive with time; by 3 hours, a 3.5-fold increase over controls was observed (p less than 0.05). When livers were perfused with iced saline, rats treated with mineral oil for 1.5 hours had a slight decrease (not significant) in mean gap junction size as compared to controls, while the size in rats treated for the same amount of time with CCl4 increased almost 5-fold over controls (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Lipids of the platelet membrane.
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Animals↗
Proton-induced fusion of oleic acid-phosphatidylethanolamine liposomes.
Liposomes composed of oleic acid and phosphatidylethanolamine (3:7 mole ratio) aggregate, become destabilized, and fuse below pH 6.5 in 150 mM NaCl. Fusion is monitored by (i) the intermixing of internal aqueous contents of liposomes, utilizing the quenching of aminonaphthalene-3,6,8-trisulfonic acid (ANTS) by N,N'-p-xylylenebis(pyridinium bromide) (DPX) encapsulated in two separate populations of vesicles, (ii) a resonance energy transfer assay for the dilution of fluorescent phospholipids from labeled to unlabeled liposomes, (iii) irreversible changes in turbidity, and (iv) quick-freezing freeze-fracture electron microscopy. Destabilization is followed by the fluorescence increase caused by the leakage of coencapsulated ANTS/DPX or of calcein. Ca2+ and Mg2+ also induce fusion of these vesicles at 3 and 4 mM, respectively. The threshold for fusion is at a higher pH in the presence of low (subfusogenic) concentrations of these divalent cations. Vesicles composed of phosphatidylserine/phosphatidylethanolamine or of oleic acid/phosphatidylcholine (3:7 mole ratio) do not aggregate, destabilize, or fuse in the pH range 7-4, indicating that phosphatidylserine and phosphatidylcholine cannot be substituted for oleic acid and phosphatidylethanolamine, respectively, for proton-induced membrane fusion. Freeze-fracture replicas of oleic acid/phosphatidylethanolamine liposomes frozen within 1 s of stimulation with pH 5.3 display larger vesicles and vesicles undergoing fusion, with membrane ridges and areas of bilayer continuity between them. The construction of pH-sensitive liposomes is useful as a model for studying the molecular requirements for proton-induced membrane fusion in biological systems and for the cytoplasmic delivery of macromolecules.
Calcium↗
Localization of nucleoside triphosphatase activity to the inner nuclear envelope and associated heterochromatin.
Nuclear-envelope nucleoside triphosphatase activity (NTPase), an enzymatic activity thought to participate in RNA transport, was localized in rat liver in situ after brief perfusion with 3% paraformaldehyde. Reaction product was distributed along the nucleoplasmic side of the nuclear envelope (NE) in heterochromatin, was only occasionally found at nuclear pores, and nuclear deposition was selectively blocked by inhibitors of NE NTPase activity. Our results suggest that NTPases, which are active in the NE and which participate in RNA transport, are not specifically associated with nuclear-pore complexes.
Animals↗
Target cell lysis: ultrastructural and cytoskeletal alterations.
The cytoskeleton, with particular emphasis on filamentous actin (f-actin), was studied in target L cells treated with the cytotoxic lymphokine, alpha-lymphotoxin (LT), or with cytotoxic T lymphocytes. The f-actin distribution was also examined in cytotoxic T cells interacting with targets. LT-treated targets retracted their processes, rounded up, and subsequently underwent lysis. These changes were accompanied by loss of stress fibers and rearrangement of f-actin, although there was no appreciable change of total cytoskeletal protein or f-actin content. Thus, the arrangement of f-actin was altered, but not its cellular concentration. Similar morphologic changes were seen at the light microscopic level in targets affected by T cells. T lymphocytes interacting with targets showed marked polarization of f-actin adjacent to the effector target interface; some f-actin rearrangement was also noted in the underlying target cell cytoplasm. Ultrastructurally, the LT-affected targets showed a decrease of f-actin organization within microvilli and underneath the plasma membrane associated with loss of villi and cytoplasmic bleb formation. Furthermore, prominent bundles of intermediate filaments were apparent and clusters of vesicles were noted which may be an expression of plasma membrane repair. These findings suggest that target cell cytoskeletal changes may lead to the morphologic changes in LT- and T cell-mediated cytotoxicity and lysis.
Actins↗
Physicochemical characterization of large unilamellar phospholipid vesicles prepared by reverse-phase evaporation.
Properties of large unilamellar vesicles (LUV), composed of phosphatidylcholine and prepared by reverse-phase evaporation and subsequent extrusion through Unipore polycarbonate membranes, have been investigated and compared with those of small unilamellar vesicles (SUV) and of multilamellar vesicles (MLV). The unilamellar nature of the LUV is shown by 1H-NMR using Pr3+ as a shift reagent. The gel to liquid-crystalline phase transition of LUV composed of dipalmitoylphosphatidylcholine (DPPC) monitored by differential scanning calorimetry, fluorescence polarization of diphenylhexatriene and 90 degrees light scattering, occurs at a slight lower temperature (40.8 degrees C) than that of MLV (42 degrees C) and is broadened by about 50%. The phase transition of SUV is shifted to considerably lower temperatures (mid-point, 38 degrees C) and extends over a wide temperature range. In LUV a well-defined pretransition is not observed. The permeability of LUV (DPPC) monitored by leakage of carboxyfluorescein, increases sharply at the phase transition temperature, and the extent of release is greater than that from MLV. Leakage from SUV occurs in a wide temperature range. Freeze-fracture electron microscopy of LUV (DPPC) reveals vesicles of 0.1-0.2 micron diameter with mostly smooth fracture faces. At temperatures below the phase transition, the larger vesicles in the population have angled faces, as do extruded MLV. A banded pattern, seen in MLV at temperatures between the pretransition and the main transition, is not observed in the smaller LUV, although the larger vesicles reveal a dimpled appearance.
Animals↗
Liposomes containing colloidal gold are a useful probe of liposome-cell interactions.
A method is described for the preparation of liposomes containing colloidal gold as an electron-dense marker to trace liposome-cell interactions. Since gold sols would precipitate at the high concentrations necessary for loading a large proportion of liposomes, gold sols were formed within preformed liposomes which had encapsulated gold chloride. The optimal conditions for encapsulating the marker were ascertained for liposomes prepared by the method of reverse-phase evaporation. Gold sols formed rapidly at ambient temperature and without organic solvent, and produced homogeneous populations of gold granules inside liposomes. Most vesicles contained the marker, allowing us to determine unambiguously the intracellular fate of liposomes and their contents. The in vitro experiments showed that gold-liposomes were internalized by African green monkey kidney cells in a manner similar to receptor-mediated endocytosis of well-characterized ligands. Preliminary in vivo studies also indicated that liposomes were endocytosed by Kupffer cells via the coated vesicle pathway.
Animals↗
Isoprene synthesis in isolated embryonic Drosophila cells. I. Sterol-deficient eukaryotic cells.
Since insects are cholesterol auxotrophs, we analyzed the apparent paradox presented by an established cell line (Kc cells) from Drosophila embryos which grew in media which contained less than 0.05 micrograms/ml of sterols. Fresh Drosophila embryos contained 3.7 micrograms of 3 beta-hydroxysterols/mg of protein; however, Kc cells had maximally 0.50 micrograms of 3 beta-hydroxysterols/mg of protein. Kc cells, grown in media which contained cholesterol, showed the presence of sterol in their plasma and intracellular membranes. Kc cells did not synthesize sterols or any apparent replacement lipophilic molecule. However, two major compounds which comigrated with ubiquinone and dolichol were synthesized from radioactive mevalonate and acetate. Cholesterol incorporation into Kc cell membranes did not significantly alter total phospholipid head or acyl group composition. Similar observations were obtained with Schneider's Drosophila cell line I and a mosquito (Aedes albopictus) cell line. Our results (a) challenged current concepts that sterols or related replacement isopentenoid molecules were required for eukaryotic membrane structure, (b) demonstrated that marked alterations in eukaryotic membrane sterol composition was insufficient to change total phospholipid head and/or acyl group composition, and (c) set the stage for the use of a eukaryotic cell to examine the regulation of 3-hydroxy,3-methylglutaryl coenzyme A reductase activity independent of a requirement for sterol synthesis.
Animals↗
Development of membrane differentiations in the guinea pig spermatid during spermiogenesis.
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Acrosome↗
The Sertoli cell junctional complex: structure and permeability to filipin in the neonatal and adult guinea pig.
The development and maintenance of the Sertoli cell junctional complex were investigated in prepubertal and adult guinea pigs. To correlate the structure of the blood-testis barrier with its permeability, the polyene antibiotic filipin (a cholesterol-binding agent of low molecular weight: 570.70) was added to the fixative as a tracer visible in freeze-fracture replicas. Discontinuous zonules, intermediate junctions (i.e., adhering fasciae) and gap junctions all proved permeable to filipin in the two age groups. Only the continuous occluding zonules characteristic of the adult guinea pig's testis were impermeable to the tracer. In pubertal animals, the establishment of the blood-testis barrier coincided with the completion of the junctional strands in occluding zonules. The formation of occluding zonules was similar in the newborn and the adult. In the adult, the Sertoli cell junctional complexes contained three types of cell junctions: occluding, adhering, and gap junctions. The sequence of occluding and adhering junctions from the base to the apex of the epithelium was the reverse of that demonstrated in most epithelia. The impermeable continuous occluding zonules at the base showed parallel patterns of uninterrupted junctional strands, whereas the permeable discontinuous zonules found higher in the epithelium showed a meandering pattern of broken strands. Our observations indicate that (1) Sertoli cell junctional complexes form near the young germinal cells at the base of the seminiferous epithelium and break down near the older germinal cells toward the apex; (2) the various patterns and orientations of the junctional strands reflect, respectively, the different stages of disintegration of the occluding zonules and the conformation of the mature Sertoli cell to the irregular contours of the germinal cells; (3) there is no relationship between permeability and junctional strand orientation; and (4) the cellular contacts between Sertoli cells and germinal cells situated below the blood-testis barrier may represent the early stages of formation of junctional elements which ultimately become incorporated into the Sertoli cell junctional complex.
Animals↗
Endocytosis of liposomes and intracellular fate of encapsulated molecules: encounter with a low pH compartment after internalization in coated vesicles.
We have compared the intracellular fate of several fluorescent probes and colloidal gold entrapped in negatively charged liposomes. Weakly acidic molecules (carboxyfluorescein) appear in the cytoplasm of CV-1 cells in 30 min; agents that raise lysosomal pH block this process. Highly charged molecules (calcein) and large molecules (FITC-dextran: 18 kd) remain confined to extra-or intracellular vesicles. Thin section electron micrographs show gold-containing liposomes bound to coated pits, in intracellular coated and uncoated vesicles, and in secondary lysosomes, including dense bodies. Free gold was not observed in the cytoplasm. We conclude that negatively charged liposomes are endocytosed and processed intracellularly by the coated vesicle pathway, and acidification of the endocytic vesicle, rather than liposome fusion, permits escape of certain molecules to the cytoplasm.
Ammonium Chloride↗
Changes in mitochondrial structure and function in 9l rat brain tumor cells treated in vitro with alpha-difluoromethylornithine, a polyamine biosynthesis inhibitor.
Mitochondrial structure and function were studied in 9L rat brain tumor cells depleted of polyamines by alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ornithine decarboxylase. Cells treated with methylglyoxal bis(guanylhydrazone), a reversible inhibitor of S-adenosylmethionine decarboxylase, were used for comparison because this polyamine biosynthesis inhibitor is known to cause structural and functional disruption of mitochondria. A significant increase in mitochondrial size, measured quantitatively, was found in alpha-difluoromethylornithine-treated cells (10 mM for 72 hr) compared with untreated cells (P < 0.001). This increase in mitochondrial size was reversed when putrescine was added to the cultures for 24 hr after alpha-difluoromethylornithine treatment. Putrescine alone had no effect on the size of mitochondria. Treatment of cells with methylglyoxal bis(guanylhydrazone) (80 muM for 48 hr) caused only a slight increase in mitochondrial size compared with mitochondria in untreated cells (P < 0.05) and failed to produce the dramatic ultrastructural changes reported in other cell lines. Ultrastructural examination revealed an increase in cytoplasmic and membrane-associated ribosomes in alpha-difluoromethylornithine-treated cells, an increase in cytoplasmic ribosomes in methylglyoxal bis(guanylhydrazone)-treated cells, and an increase in membrane-bound ribosomes in putrescine-treated cells. In cells treated first with alpha-difluoromethylornithine and then with putrescine, the distribution of ribosomes was normal. The distributions of ribosomes were not quantitatively assessed. Pyruvate utilization, a measure of mitochondrial function, was decreased in cells treated with 10 mM alpha-difluoromethylornithine for 72 hr, compared with untreated cells. Restoration of intracellular polyamine levels by the addition of putrescine 24 hr before analysis reversed this phenomenon. Putrescine treatment alone did not affect pyruvate utilization. Pyruvate utilization in methylglyoxal bis(guanylhydrazone)-treated cells was depressed to a greater extent than that in alpha-difluoromethylornithine-treated cells.
Animals↗
Gossypol-induced inhibition of guinea pig sperm capacitation in vitro.
The effect of gossypol acetate at various concentrations (10(-6) to 10(-4) M) on guinea pig sperm forward progressive movement, capacitation, and the acrosome reaction was explored in vitro. We found that 10(-4) M gossypol completely abolished the forward progressive motility of the sperm, and that this inhibition of motility was proportional to the concentration of gossypol used. Also, a dose-dependent decrease in acrosome reactions occurred with concentrations of the agent as low as 5.0 X 10(-6) M. However, we observed that such prevention of the acrosome reaction apparently happens at the capacitation stage rather than during the acrosome reaction itself. Inhibition of capacitation by gossypol was reversible--once the spermatozoa were capacitated in gossypol-free medium, the compound did not block the reaction.
Acrosome↗
Lectin binding to guinea-pig sperm zipper particles.
Zipper particles are morphologically distinct transmembrane specializations of sperm tails. In freeze-fracture replicas of the guinea-pig sperm, they appear as an interdigitating double row of intramembranous particles running longitudinally within the plasma membrane of the principal piece. In thin section, zipper particles appear as an increase in electron density both above and below the bilayer. Zipper particles have been observed on a variety of both mammalian and non-mammalian species, suggesting that they have been conserved to serve an essential sperm function. As a first step towards biochemically characterizing guinea-pig zipper particles and towards developing a zipper isolation procedure, we performed an in situ lectin-binding study. Examination of nine gold- or ferritin-conjugated lectins revealed that three lectins, concanavalin A, Ricinus communis agglutinin I and wheatgerm agglutinin, bound to zipper particles. These lectin binding results suggest the presence of N-linked oligosaccharides within zipper particles. The results of the lectin binding study were then used in conjunction with a detergent solubilization procedure to identify potential zipper components. Detergent solubilization involved two non-ionic detergents: digitonin, which solubilized most of the plasma membrane, but left approximately two thirds of the zipper particles attached to the cytoskeleton, and Triton X-100, which solubilized the remaining zipper particles while leaving most other sperm structures intact. Within sodium dodecyl sulphate/polyacrylamide gels of the Triton-X-100-soluble fraction potential zipper particle components with the same lectin-binding characteristics as in situ zipper particles were identified.
Animals↗
Fusion of phospholipid vesicles arrested by quick-freezing. The question of lipidic particles as intermediates in membrane fusion.
We have examined the early events in Ca2+-induced fusion of large (0.2 microns diameter) unilamellar cardiolipin/phosphatidylcholine and phosphatidylserine/phosphatidylethanolamine vesicles by quick-freezing freeze-fracture electron microscopy, eliminating the necessity of using glycerol as a cryoprotectant. Freeze-fracture replicas of vesicle suspensions frozen after 1-2 s of stimulation revealed that the majority of vesicles had already undergone membrane fusion, as evidenced by dumbbell-shaped structures and large vesicles. In the absence of glycerol, lipidic particles or the hexagonal HII phase, which have been proposed to be intermediate structures in membrane fusion, were not observed at the sites of fusion. Lipidic particles were evident in less than 5% of the cardiolipin/phosphatidylcholine vesicles after long-term incubation with Ca2+, and the addition of glycerol produced more vesicles displaying the particles. We have also shown that rapid fusion occurred within seconds of Ca2+ addition by the time-course of fluorescence emission produced by the intermixing of aqueous contents of two separate vesicle populations. These studies therefore have produced no evidence that lipidic particles are necessary intermediates for membrane fusion. On the contrary, they indicate that lipidic particles are structures obtained at equilibrium long after fusion has occurred and they become particularly prevalent in the presence of glycerol.
Animals↗