On Red Blood Cells, Hemolysis and Resealed Ghosts

Hoffman, Joseph F.

doi:10.1007/978-1-4615-3030-5_1

Cited by 34 publications

(27 citation statements)

References 80 publications

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“…(49) In contrast, the ghosts obtained by Fl-E5-TAT mediated lysis remained permeable to dextrans under similar conditions. Interestingly, Fl-E5-TAT could not be washed from the membrane at either pH 4 or pH 7.…”

Section: Discussionmentioning

confidence: 96%

Modeling of the Endosomolytic Activity of HA2-TAT Peptides with Red Blood Cells and Ghosts

2010

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HA2-TAT is a peptide-based delivery agent that combines the pH-sensitive HA2 fusion peptide from Influenza and the cell-penetrating peptide TAT from HIV. This chimeric peptide is engineered to induce the cellular uptake of macromolecules into endosomes via the TAT moiety and to respond to the acidifying lumen of endosomes to cause membrane leakage and release of macromolecules into cells via the HA2 moiety. The question of how HA2 and TAT affect the properties of one another remains, however, unanswered and the behavior of the peptide inside endosomes is mostly uncharacterized. To address these issues, the binding and membrane leakage activity of a glutamic acid-enriched analogue E5-TAT was assessed with red blood cells and giant unilamellar vesicles as membrane models for endosomes. Hemolysis and microscopy assays reveal that E5-TAT binds to membranes in a pH-dependent manner and causes membrane leakage by inducing the formation of pores through which macromolecules can escape. The TAT moiety contributes to this activity by causing a shift in the pH response of E5 and by binding to negatively charged phospholipids. On the other hand, TAT binding to glycosaminoglycans reduces the lytic activity of E5-TAT. Addition of TAT to the C-terminus of E5 can therefore either increase or inhibit the activity of E5 depending on the cellular components present at the membrane. Taken together, these results suggest a model for the endosomolytic activity of the peptide and provide the basis for the molecular design of future delivery agents.Cell-penetrating peptides (CPPs) provide general and useful tools to deliver macromolecular cargos into live cells. A prototypical CPP is the TAT peptide derived from HIV TAT.(1,2) TAT has been used to deliver peptides, proteins, DNA, liposomes and nanoparticles. (3,4) Multiple mechanisms are involved in cellular penetration mediated by TAT. TAT has been shown to directly translocate across the lipid bilayer of the plasma membrane when it is both labeled with a small molecule and present above a threshold concentration.(5) On the other hand, TAT is internalized by endocytosis at low concentration or when conjugated to large hydrophilic molecules.(6) Multiple endocytic mechanisms have been shown to be involved in the uptake of TAT-conjugates. TAT-conjugates can for instance enter cells by clathrin or caveolin-dependent endocytosis. In addition, TAT and other CPPs have also been shown to induce macropinocytosis, an effect that leads to the increase in uptake of fluid-phase endocytosis markers.(6-8) Induction of macropinocytosis appears to be mediated by the interaction of the positively charged peptide with membrane-associated heparan sulfate proteoglycans.(9) However, recent evidence suggests that HSPG do not act as receptors but † This work was supported by Award Number R01GM087227 from the National Institute of General Medical Sciences. G.J. was partially supported by the NIH molecular biophysics training grant (T32GM065088) * To whom correspondence should be addressed. Phone: 979 ...

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Section: Discussionmentioning

confidence: 96%

Modeling of the Endosomolytic Activity of HA2-TAT Peptides with Red Blood Cells and Ghosts

2010

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“…Studies on synthetic membranes and on RBC ghosts have shown that lipid bilayers have the ability to reseal their ruptures spontaneously (Hoffman et al, 1960;Bodemann and Passow, 1972;Billah et al, 1977;Chang and Reese, 1990;Hoffman, 1992), presumably driven by the thermodynamic force that favors a continuous sheet of amphophilic lipid bilayers in water. However, this passive reseaIing is not likely to be enough for living cells to survive since it is too slow (time constant ranges from 30 minutes to hours [Billah et al, 1977]) unless the lesion is extremely small (Chang and Reese, 1990).…”

Section: A Dynamic View Of Plasma Membrane Resealingmentioning

confidence: 99%

Calcium-regulated exocytosis is required for cell membrane resealing.

Alderton

Steinhardt

1995

The Journal of Cell Biology

266

234

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Abstract. Using confocal microscopy, we visualized exocytosis during membrane resealing in sea urchin eggs and embryos. Upon wounding by a laser beam, both eggs and embryos showed a rapid burst of localized Ca 2+-regulated exocytosis. The rate of exocytosis was correlated quantitatively with successfully resealing. In embryos, whose activated surfaces must first dock vesicles before fusion, exocytosis and membrane resealing were inhibited by neurotoxins that selectively cleave the SNARE complex proteins, synaptobrevin, SNAP-25, and syntaxin. In eggs, whose cortical vesicles are already docked, vesicles could be reversibly undocked with externally applied stachyose. If cortical vesicles were undocked both exocytosis and plasma membrane resealing were completely inhibited. When cortical vesicles were transiently undocked, exposure to tetanus toxin and botulinum neurotoxin type C1 rendered them no longer competent for resealing, although botulinum neurotoxin type A was still ineffective. Cortical vesicles transiently undocked in the presence of tetanus toxin were subsequently fusion incompetent although to a large extent they retained their ability to redock when stachyose was diluted. We conclude that addition of internal membranes by exocytosis is required and that a SNARE-like complex plays differential roles in vesicle docking and fusion for the repair of disrupted plasma membrane. CELt membranes are able to reseal after mechanical injury or disruption by microinjection, chemical permeabilization, or electroporation, but the mechanism of resealing has not been

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“…Free radical induced structural changes in G6PD deficient erythrocyte membrane are not fully understood (Hoffman, 1992) but despite its complex nature the process of haemolysis, falls under two categories: one which involves cell damage and the other which results from permeability changes in the cell membrane (Cruz-Silva et al, 2002). While some xenobiotics have the ability to produce haemolytic changes of both types (Knopik-Strocka and Bielawski, 2002) it is not known if quinine or t-BHP has the potential of affecting cell membrane permeability, in addition to their ability of causing oxidative cell damage.…”

Section: Resultsmentioning

confidence: 98%

Pycnogenol^® prevents haemolytic injury in G6PD deficient human erythrocytes

Sharma

Gulati

2003

Phytotherapy Research

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Glucose6 phosphate dehydrogenase (G6PD) deficiency is the most common X-linked disorder of human erythrocytes where cells have inadequate capacity to destroy peroxides and high susceptibility towards haemolytic changes. Pycnogenol is a proprietary dry extract of the French Maritime pine (Pinus pinaster) bark with high ability to scavenge free radicals. In the present study we have investigated if Pycnogenol can protect G6PD deficient erythrocytes against haemolytic cell damage. Venous blood samples were obtained from six subject of Mediterranean origin with known G6PD deficiency which was also confirmed with standard techniques. Erythrocyte haemolysis in the presence and absence of Pycnogenol was induced either with tert-butylhydroperoxide (t-BHP) or quinine and the haemoglobin release in the supernatant was determined by recording the optical density at 540 nm in a Shimadzu spectrophotometer. Our results have shown that Pycnogenol has protective action against a Xenobiotic chemical induced haemolysis in G6PD deficient human erythrocytes.

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On Red Blood Cells, Hemolysis and Resealed Ghosts

Cited by 34 publications

References 80 publications

Modeling of the Endosomolytic Activity of HA2-TAT Peptides with Red Blood Cells and Ghosts

Modeling of the Endosomolytic Activity of HA2-TAT Peptides with Red Blood Cells and Ghosts

Calcium-regulated exocytosis is required for cell membrane resealing.

Pycnogenol^® prevents haemolytic injury in G6PD deficient human erythrocytes

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On Red Blood Cells, Hemolysis and Resealed Ghosts

Cited by 34 publications

References 80 publications

Modeling of the Endosomolytic Activity of HA2-TAT Peptides with Red Blood Cells and Ghosts

Modeling of the Endosomolytic Activity of HA2-TAT Peptides with Red Blood Cells and Ghosts

Calcium-regulated exocytosis is required for cell membrane resealing.

Pycnogenol® prevents haemolytic injury in G6PD deficient human erythrocytes

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Product

Resources

About

Pycnogenol^® prevents haemolytic injury in G6PD deficient human erythrocytes