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Papers by Daniel Friend
![Research paper thumbnail of Fusion of liposomes containing a novel cationic lipid, N-[2,3-(dioleyloxy)propyl]-N,N,N-trimethylammonium: induction by multivalent anions and asymmetric fusion with acidic phospholipid vesicles](https://attachments.academia-assets.com/49459713/thumbnails/1.jpg)
](Biochemistry, 1989
The fusion behavior of large unilamellar liposomes composed of N-[2,3-(dioleyloxy)propyl] -N,N,N-... more The fusion behavior of large unilamellar liposomes composed of N-[2,3-(dioleyloxy)propyl] -N,N,N-trimethylammonium (DOTMA) and either phosphatidylcholine (PC) or phosphatidylethanolamine (PE) has been investigated by a fluorescence resonance energy transfer assay for lipid mixing, dynamic light scattering, and electron microscopy. Polyvalent anions induced the fusion of DOTMA/PE (1:l) liposomes
Biochemistry, 1985
Liposomes composed of oleic acid and phosphatidylethanolamine (3:7 mole ratio) aggregate, become ... more 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.
Biochimica Et Biophysica Acta-biomembranes, 1996
Cationic liposomes mediate efficient transfection of mammalian cells, but the manner in which cel... more Cationic liposomes mediate efficient transfection of mammalian cells, but the manner in which cells internalize and process cationic liposome-DNA complexes has not been well characterized. We exposed several cell types, including human and murine erythroleukemia cells. African green monkey kidney cells (CV-1), isolated rat alveolar type II cells and alveolar macrophages to DNA-cationic liposome complexes containing N-(1-2,3-dioleyloxypropyl)-N,N,N-triethylammonium (DOTMA) and Dioleylphosphatidylethanolamine (DOPE). The morphology of liposome-cell interactions was assessed by electron microscopy. Liposome preparations were complexed to colloidal gold particles or to both plasmid DNA and gold particles. Cells treated with DOTMA liposome-DNA complexes demonstrated endocytosis of the liposome-DNA complexes in coated pits, which were seen in early endosomes, late endosomes, and lysosomes. In isolated alveolar type II cells, the gold-labelled DOTMA lipid apparently mixed with the contents of lamellar bodies. In most cells, gold particles were dispersed throughout the cytoplasmic matrix. In a small proportion of CV-1 and U937 cells, a membrane system resembling the endoplasmic reticulum developed within the nucleus. This novel structure was also present in nuclei after they were isolated from CV-1 cells and then mixed with DOTMA-containing liposomes. Membranes which form after exposure to DOTMA-containing liposomes were 10 nm in thickness as compared to the approx. 8 nm thickness of endogenous cellular membranes. Based on these morphologic observations, we propose that the main route of entry of cationic liposomes into cells is by endocytosis. In some instances, the endosomal compartment releases its cationic liposome-DNA contents into the cytoplasmic matrix. Occasionally, liposomes may enter the nucleus by fusion with the nuclear envelope, creating vesicular and reticular intranuclear membranes. It is not clear at present which, if any of these morphological observations correlates with transfection mediated by cationic liposomes.
Journal of Investigative Dermatology, 1977
The Anatomical Record, 1977
Tight junctions (zonulae occludentes) create a pericellular barrier to the diffusion of large mol... more Tight junctions (zonulae occludentes) create a pericellular barrier to the diffusion of large molecules in non-keratinizing mammalian epithelia. However, in cornifying epithelia such as the epidermis, the importance of tight-junctional elements versus secreted intercellular lipid for barrier function is uncertain. In an attempt to resolve this question, we compared membrane structure in the stratum granulosum and stratum corneum of epidermis, esophagus, and vagina of newborn and adult humans and mice under both normal and various experimental conditions. We incubated pieces of epidermis in organ culture and infused tissues with lanthanum or horseradish peroxidase in vivo and in vitro. All were processed for electron microscopy of freeze-fracture replicas or thin sections. Lanthanum seeped outward to the stratum granulosum in all tissues examined-further apical migration was halted by lamellar-body contents in skin. A similar pattern of intercellular lamellar lipid deposition and membrane structure occurred in all epithelia studied. Freeze-fracture replicas of these obstructive regions revealed occasional, incomplete junctional strands (particularly in moist epithelia) and abundant lamellar material, but complete zonulae occludentes were never encountered. A possible relationship between moisture and tight junction formation was further suggested by organ culture experiments during which brief incubations stimulated an increase in the number of junctional strands and diminished numbers of lamellar bodies. We conclude that, in the epithelia studied, the deposition of secreted lamellar body contents forms the barrier to water-soluble tracer loss: tight-junctional elements are either absent or too fragmentary to constitute an effective barrier.
Journal of Cell Biology, 1975
The structural basis of the permeability barrier in mammalian epidermis was examined by tracer an... more The structural basis of the permeability barrier in mammalian epidermis was examined by tracer and freeze-fracture techniques. Water-soluble tracers (horseradish peroxidase, lanthanum, ferritin) were injected into neonatal mice or into isolated upper epidermal sheets obtained with staphylococcal exfoliatin. Tracers percolated through the intercellular spaces to the upper stratum granulosum, where further egress was impeded by extruded contents of lamellar bodies. The lamellar contents initially remain segregated in pockets, then fuse to form broad sheets which fill intercellular regions of the stratum corneum, obscuring the outer leaflet of the plasma membrane. These striated intercellular regions are interrupted by periodic bulbous dilatations. When adequately preserved, the interstices of the stratum corneum are wider, by a factor of 5-10 times that previously appreciated. Freeze-fracture replicas of granular cell membranes revealed desmosomes, sparse plasma membrane particles, and accumulating intercellular lamellae, but no tight junctions. Fractured stratum corneum displayed large, smooth, multilaminated fracture faces. By freeze-substitution, proof was obtained that the fracture plane had diverted from the usual intramembranous route in the stratum granulosum to the intercellular space in the stratum corneum. We conclude that: (a) the primary barrier to water loss is formed in the stratum granulosum and is subserved by intercellular deposition of lamellar bodies, rather than occluding zonules; (b) a novel, intercellular freeze-fracture plane occurs within the stratum corneum; (c) intercellular regions of the stratum corneum comprise an expanded, structurally complex, presumably lipid-rich region which may play an important role in percutaneous transport.
Biochemistry, 1989
The fusion behavior of large unilamellar liposomes composed of N-[2,3-(dioleyloxy)propyl] -N,N,N-... more The fusion behavior of large unilamellar liposomes composed of N-[2,3-(dioleyloxy)propyl] -N,N,N-trimethylammonium (DOTMA) and either phosphatidylcholine (PC) or phosphatidylethanolamine (PE) has been investigated by a fluorescence resonance energy transfer assay for lipid mixing, dynamic light scattering, and electron microscopy. Polyvalent anions induced the fusion of DOTMA/PE (1:l) liposomes
Biochemistry, 1985
Liposomes composed of oleic acid and phosphatidylethanolamine (3:7 mole ratio) aggregate, become ... more 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.
Biochimica Et Biophysica Acta-biomembranes, 1996
Cationic liposomes mediate efficient transfection of mammalian cells, but the manner in which cel... more Cationic liposomes mediate efficient transfection of mammalian cells, but the manner in which cells internalize and process cationic liposome-DNA complexes has not been well characterized. We exposed several cell types, including human and murine erythroleukemia cells. African green monkey kidney cells (CV-1), isolated rat alveolar type II cells and alveolar macrophages to DNA-cationic liposome complexes containing N-(1-2,3-dioleyloxypropyl)-N,N,N-triethylammonium (DOTMA) and Dioleylphosphatidylethanolamine (DOPE). The morphology of liposome-cell interactions was assessed by electron microscopy. Liposome preparations were complexed to colloidal gold particles or to both plasmid DNA and gold particles. Cells treated with DOTMA liposome-DNA complexes demonstrated endocytosis of the liposome-DNA complexes in coated pits, which were seen in early endosomes, late endosomes, and lysosomes. In isolated alveolar type II cells, the gold-labelled DOTMA lipid apparently mixed with the contents of lamellar bodies. In most cells, gold particles were dispersed throughout the cytoplasmic matrix. In a small proportion of CV-1 and U937 cells, a membrane system resembling the endoplasmic reticulum developed within the nucleus. This novel structure was also present in nuclei after they were isolated from CV-1 cells and then mixed with DOTMA-containing liposomes. Membranes which form after exposure to DOTMA-containing liposomes were 10 nm in thickness as compared to the approx. 8 nm thickness of endogenous cellular membranes. Based on these morphologic observations, we propose that the main route of entry of cationic liposomes into cells is by endocytosis. In some instances, the endosomal compartment releases its cationic liposome-DNA contents into the cytoplasmic matrix. Occasionally, liposomes may enter the nucleus by fusion with the nuclear envelope, creating vesicular and reticular intranuclear membranes. It is not clear at present which, if any of these morphological observations correlates with transfection mediated by cationic liposomes.
Journal of Investigative Dermatology, 1977
The Anatomical Record, 1977
Tight junctions (zonulae occludentes) create a pericellular barrier to the diffusion of large mol... more Tight junctions (zonulae occludentes) create a pericellular barrier to the diffusion of large molecules in non-keratinizing mammalian epithelia. However, in cornifying epithelia such as the epidermis, the importance of tight-junctional elements versus secreted intercellular lipid for barrier function is uncertain. In an attempt to resolve this question, we compared membrane structure in the stratum granulosum and stratum corneum of epidermis, esophagus, and vagina of newborn and adult humans and mice under both normal and various experimental conditions. We incubated pieces of epidermis in organ culture and infused tissues with lanthanum or horseradish peroxidase in vivo and in vitro. All were processed for electron microscopy of freeze-fracture replicas or thin sections. Lanthanum seeped outward to the stratum granulosum in all tissues examined-further apical migration was halted by lamellar-body contents in skin. A similar pattern of intercellular lamellar lipid deposition and membrane structure occurred in all epithelia studied. Freeze-fracture replicas of these obstructive regions revealed occasional, incomplete junctional strands (particularly in moist epithelia) and abundant lamellar material, but complete zonulae occludentes were never encountered. A possible relationship between moisture and tight junction formation was further suggested by organ culture experiments during which brief incubations stimulated an increase in the number of junctional strands and diminished numbers of lamellar bodies. We conclude that, in the epithelia studied, the deposition of secreted lamellar body contents forms the barrier to water-soluble tracer loss: tight-junctional elements are either absent or too fragmentary to constitute an effective barrier.
Journal of Cell Biology, 1975
The structural basis of the permeability barrier in mammalian epidermis was examined by tracer an... more The structural basis of the permeability barrier in mammalian epidermis was examined by tracer and freeze-fracture techniques. Water-soluble tracers (horseradish peroxidase, lanthanum, ferritin) were injected into neonatal mice or into isolated upper epidermal sheets obtained with staphylococcal exfoliatin. Tracers percolated through the intercellular spaces to the upper stratum granulosum, where further egress was impeded by extruded contents of lamellar bodies. The lamellar contents initially remain segregated in pockets, then fuse to form broad sheets which fill intercellular regions of the stratum corneum, obscuring the outer leaflet of the plasma membrane. These striated intercellular regions are interrupted by periodic bulbous dilatations. When adequately preserved, the interstices of the stratum corneum are wider, by a factor of 5-10 times that previously appreciated. Freeze-fracture replicas of granular cell membranes revealed desmosomes, sparse plasma membrane particles, and accumulating intercellular lamellae, but no tight junctions. Fractured stratum corneum displayed large, smooth, multilaminated fracture faces. By freeze-substitution, proof was obtained that the fracture plane had diverted from the usual intramembranous route in the stratum granulosum to the intercellular space in the stratum corneum. We conclude that: (a) the primary barrier to water loss is formed in the stratum granulosum and is subserved by intercellular deposition of lamellar bodies, rather than occluding zonules; (b) a novel, intercellular freeze-fracture plane occurs within the stratum corneum; (c) intercellular regions of the stratum corneum comprise an expanded, structurally complex, presumably lipid-rich region which may play an important role in percutaneous transport.