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F S Cohen

Publications and source records attributed to F S Cohen.

At least 19 recordsLinked to original sources
PubMed2001

A quantitative model for membrane fusion based on low-energy intermediates.

The energetics of a fusion pathway is considered, starting from the contact site where two apposed membranes each locally protrude (as "nipples") toward each other. The equilibrium distance between the tips of the two nipples is determined by a balance of physical forces: repulsion caused by hydration and attraction generated by fusion proteins. The energy to create the initial stalk, caused by bending of cis monolayer leaflets, is much less when the stalk forms between nipples rather than parallel flat membranes. The stalk cannot, however, expand by bending deformations alone, because this would necessitate the creation of a hydrophobic void of prohibitively high energy. But small movements of the lipids out of the plane of their monolayers allow transformation of the stalk into a modified stalk. This intermediate, not previously considered, is a low-energy structure that can reconfigure into a fusion pore via an additional intermediate, the prepore. The lipids of this latter structure are oriented as in a fusion pore, but the bilayer is locally compressed. All membrane rearrangements occur in a discrete local region without creation of an extended hemifusion diaphragm. Importantly, all steps of the proposed pathway are energetically feasible.

Membrane Fusion↗
PubMed2001

Transanal hemorrhoidal dearterialization is an alternative to operative hemorrhoidectomy.

BACKGROUND: Transanal hemorrhoidal dearterialization (THD), a new approach for patients who would otherwise require an operative hemorrhoidectomy, accomplishes hemorrhoidal symptom relief with far less postoperative pain than an operative hemorrhoidectomy. METHODS: THD, an ambulatory procedure, employs a specially designed proctoscope coupled with a Doppler transducer to allow identification and suture ligation of the hemorrhoidal arteries. RESULTS: Sixty patients between ages 22 and 87 were treated. Bleeding was fully corrected in 88%, protrusion in 92%, and pain in 71%. Two patients (3%) failed to improve with THD. Complications included pain resulting in greater than 2 days loss of work in 5 patients, postoperative perirectal thromboses developed in 4 patients, and an anal fissure developed in 1 patient. CONCLUSIONS: THD was an effective alternative to operative hemorrhoidectomy. It may be the only option for patients where an operative hemorrhoidectomy is contraindicated because of incontinence.

Adult↗
PubMed2001

Characterization of cholesterol-sphingomyelin domains and their dynamics in bilayer membranes.

Lipids segregate with each other into small domains in biological membranes, which can facilitate the associations of particular proteins. The segregation of cholesterol and sphingomyelin (SPM) into domains known as rafts is thought to be especially important. The formation of rafts was studied by using planar bilayer membranes that contained rhodamine-phosphatidylethanolamine (rho-DOPE) as a fluorescent probe, and wide-field fluorescence microscopy was used to detect phase separation of the probe. A fluorescently labeled GM(1), known to preferentially partition into rafts, verified that rho-DOPE faithfully reported the rafts. SPM-cholesterol domains did not form at high temperatures but spontaneously formed when temperature was lowered to below the melting temperature of the SPM. Saturated acyl chains on SPMs therefore promote the formation of rafts. The domains were circular (resolution > or = 0.5 microm), quickly reassumed their circular shape after they were deformed, and merged with each other to create larger domains, all phenomena consistent with liquid-ordered (l(o)) rather than solid-ordered (s(o)) domains. A saturated phosphatidylcholine (PC), disteoryl-PC, could substitute for SPM to complex with cholesterol into a l(o)-domain. But in the presence of cholesterol, a saturated phosphatidylethanolamine or phosphatidylserine yielded s(o)-domains of irregular shape. Lipids with saturated acyl chains can therefore pack well among each other and with cholesterol to form l(o)-domains, but domain formation is dependent on the polar headgroup of the lipid. An individual raft always extended through both monolayers. Degrading cholesterol in one monolayer with cholesterol oxidase first caused the boundary of the raft to become irregular; then the raft gradually disappeared. The fluid nature of rafts, demonstrated in this study, may be important for permitting dynamic interactions between proteins localized within rafts.

Acylation↗
PubMed2001

Evolution of intermediates of influenza virus hemagglutinin-mediated fusion revealed by kinetic measurements of pore formation.

Cells expressing wild-type influenza virus hemagglutinin (HA) or HA with a point mutation within the transmembrane domain (G520L) were bound to red blood cells and exposed to low pH for short times at suboptimal temperatures followed by reneutralization. This produced intermediate states of fusion. The ability of intermediate states to proceed on to fusion when temperature was raised was compared kinetically. In general, for wild-type HA, fusion occurred more quickly by directly lowering pH at 37 degrees C in the bound state than by raising temperature at the intermediate stage. When pH was lowered for 1-2 min, kinetics of fusion upon raising temperature of an intermediate slowed the longer the intermediate was maintained at neutral pH. But for a more sustained (10 min) acidification, kinetics was independent of the time the intermediate was held at neutral pH before triggering fusion by raising temperature. In contrast, generating intermediates in the same way with G520L yielded kinetics of fusion that did not depend on the time intermediates were maintained after reneutralization. For both HA and G520L, the extents of fusion did not depend on the temperature at which pH was lowered, but fusion from the intermediate was extremely sensitive to the temperature to which the cells were raised. The measured kinetics and temperature dependencies suggest that the rate-limiting step of fusion occurs subsequent to formation of any of the intermediates; the conformational change of HA into its final configuration may be the rate-limiting step.

Animals↗
PubMed2001

Tissue characterization using the continuous wavelet transform. Part I: Decomposition method.

In this paper, a novel decomposition of the RF ultrasound signal into its coherent and diffused components is proposed. This decomposition is based on thresholding the energy of the continuous wavelet transform of the RF signal using appropriate wavelets. The two components are modeled separately, and the model parameters are estimated. Previous work [1] required assumptions about the periodicity of the coherent scatterers in the tissue. These assumptions are not necessary in this work. The decomposition algorithm is tested on simulated RF images. The accuracy of the estimated parameters is presented as well as the performance of the algorithm in low coherent-to-diffuse components' energy ratios (SNR).

Algorithms↗
PubMed2001

Tissue characterization using the continuous wavelet transform. Part II: Application on breast RF data.

In the first part of this work [16], a wavelet-based decomposition algorithm of the RF echo into its coherent and diffuse components was introduced. In this paper, the proposed algorithm is used to estimate structural parameters of the breast tissue such as the number and energy of coherent scatterers, the energy of the diffuse scatterers, and the correlation between them. Based on these individual parameters, breast tissue characterization is performed. The database used consists of 155 breast scans from 42 patients. The results are presented in terms of empirical receiver operating characteristics (ROC) curves. The results of this study are discussed in relation to the tissue microstructure. Individual estimated parameters are able to differentiate reliably between normal and fibroadenoma or fibrocystic or cancerous tissue (area under the ROC Az > 0.93). Also, the differentiation between malignant and benign (normal, fibrocystic, and fibroadenoma) tissue was possible (Az > 0.89).

Algorithms↗
PubMed2000

Sterols and sphingolipids strongly affect the growth of fusion pores induced by the hemagglutinin of influenza virus.

Cells expressing the hemagglutinin (HA) of influenza virus were fused to planar phospholipid bilayer membranes to evaluate the effects of sterols and sphingolipids in the target bilayer membranes on properties of fusion pores. Typically, in the absence of sterol, flickering pores are observed, followed by a successful pore (i.e., a pore that fully opens). The incorporation of cholesterol into the lipid bilayer had a marked effect: it greatly decreased the number of flickers, and the first pore formed was usually successful. Similar effects were produced by the sterols epicholesterol and 5beta-cholestanol. In contrast, the sterols cholesteryl acetate, coprostanol, and stanolone did not affect pore flickering, and a successful pore was observed to follow the typical number of flickers. 5alpha-cholestanol gave intermediate results. From these results, it follows that the 3-OH of cholesterol is essential to reduce flickering, but it does not matter if the 3-OH is in an alpha or beta configuration. The double bond is also not critical for the actions of cholesterol nor is the fact that it is a flat molecule. The sphingolipids sphingomyelin, lactosyl cerebroside, and glucosyl cerebroside tended to inhibit full pore enlargement, prolonging the stage of pore flickering. If a sphingolipid and a sterol that strongly interact were both included in the planar membrane, the pattern of flickering was the same as if neither had been included in the bilayer. However, if a sphingolipid and sterol that do not interact with each other were included in the bilayer, the reduced flickering characteristic of the sterol was observed.

3T3 Cells↗
PubMed2000

Evidence that the transition of HIV-1 gp41 into a six-helix bundle, not the bundle configuration, induces membrane fusion.

Many viral fusion proteins exhibit a six-helix bundle as a core structure. HIV Env-induced fusion was studied to resolve whether membrane merger was due to the transition into the bundle configuration or occurred after bundle formation. Suboptimal temperature was used to arrest fusion at an intermediate stage. When bundle formation was prevented by adding inhibitory peptides at this stage, membranes did not merge upon raising temperature. Inversely, when membrane merger was prevented by incorporating lysophosphatidylcholine (LPC) into cell membranes at the intermediate, the bundle did not form upon optimizing temperature. In the absence of LPC, the six-helix bundle did not form when the temperature of the intermediate was raised for times too short to promote fusion. Kinetic measures showed that after the temperature pulse, cells had not advanced further toward fusion. The latter results indicate that bundle formation is the rate-limiting step between the arrested intermediate and fusion. Electrical measures showed that the HIV Env-induced pore is initially large and grows rapidly. It is proposed that bundle formation and fusion are each contingent on the other and that movement of Env during its transition into the six-helix bundle directly induces the lipid rearrangements of membrane fusion. Because peptide inhibition showed that, at the intermediate stage, the heptad repeats of gp41 have become stably exposed, creation of the intermediate could be of importance in drug and/or vaccine development.

CD4-Positive T-Lymphocytes↗
PubMed2000

Dynamics of fusion pores connecting membranes of different tensions.

The energetics underlying the expansion of fusion pores connecting biological or lipid bilayer membranes is elucidated. The energetics necessary to deform membranes as the pore enlarges, in some combination with the action of the fusion proteins, must determine pore growth. The dynamics of pore growth is considered for the case of two homogeneous fusing membranes under different tensions. It is rigorously shown that pore growth can be quantitatively described by treating the pore as a quasiparticle that moves in a medium with a viscosity determined by that of the membranes. Motion is subject to tension, bending, and viscous forces. Pore dynamics and lipid flow through the pore were calculated using Lagrange's equations, with dissipation caused by intra- and intermonolayer friction. These calculations show that the energy barrier that restrains pore enlargement depends only on the sum of the tensions; a difference in tension between the fusing membranes is irrelevant. In contrast, lipid flux through the fusion pore depends on the tension difference but is independent of the sum. Thus pore growth is not affected by tension-driven lipid flux from one membrane to the other. The calculations of the present study explain how increases in tension through osmotic swelling of vesicles cause enlargement of pores between the vesicles and planar bilayer membranes. In a similar fashion, swelling of secretory granules after fusion in biological systems could promote pore enlargement during exocytosis. The calculations also show that pore expansion can be caused by pore lengthening; lengthening may be facilitated by fusion proteins.

Biophysical Phenomena↗
PubMed2000

A point mutation in the transmembrane domain of the hemagglutinin of influenza virus stabilizes a hemifusion intermediate that can transit to fusion.

A hemagglutinin (HA) of influenza virus having a single semiconserved Gly residue within the transmembrane domain mutated to Leu (G520L) was expressed on cells; these cells were bound to red blood cells. By decreasing pH at 23 degrees C rather than 37 degrees C, an intermediate with properties expected of hemifusion just as the membranes are about to transit to full fusion was captured. As evidence: 1) increasing temperature to 37 degrees C at neutral pH allowed fusion to proceed; 2) after achieving the intermediate, the two membranes did not separate from each other after proteolytic cleavage of G520L because cells treated with proteinase K could not fuse upon temperature increase but could fuse upon the addition of chlorpromazine; and 3) at the point of the intermediate, adding exogenous lipids known to promote or inhibit the creation of hemifusion did not significantly alter the lipid dye spread that occurred upon increasing temperature, implying that at the intermediate, contacting membrane leaflets had already merged. A stable intermediate of hemifusion that could transit to fusion was also generated for wild-type HA, but pH had to be reduced at the significantly lower temperature of 4 degrees C. The fusion pores generated by G520L did not enlarge, whereas those induced by wild-type HA did. The finding that a state of transitional hemifusion can be readily obtained via a point mutation without the need for unusually low temperature supports the hypothesis that hemifusion occurs before pore formation.

Animals↗
PubMed2000

The lipid-anchored ectodomain of influenza virus hemagglutinin (GPI-HA) is capable of inducing nonenlarging fusion pores.

GPI-linked hemagglutinin (GPI-HA) of influenza virus was thought to induce hemifusion without pore formation. Cells expressing either HA or GPI-HA were bound to red blood cells, and their fusion was compared by patch-clamp capacitance measurements and fluorescence microscopy. It is now shown that under more optimal fusion conditions than have been used previously, GPI-HA is also able to induce fusion pore formation before lipid dye spread, although with fewer pores formed than those induced by HA. The GPI-HA pores did not enlarge substantially, as determined by the inability of a small aqueous dye to pass through them. The presence of 1,1'-dioctadecyl-3, 3,3',3'-tetramethylindocarbocyanine perchlorate or octadecylrhodamine B in red blood cells significantly increased the probability of pore formation by GPI-HA; the dyes affected pore formation to a much lesser degree for HA. This greater sensitivity of pore formation to lipid composition suggests that lipids are a more abundant component of a GPI-HA fusion pore than of an HA pore. The finding that GPI-HA can induce pores indicates that the ectodomain of HA is responsible for all steps up to the initial membrane merger and that the transmembrane domain, although not absolutely required, ensures reliable pore formation and is essential for pore growth. GPI-HA is the minimal unit identified to date that supports fusion to the point of pore formation.

Animals↗
PubMed2000

Role of the cytoplasmic tail of ecotropic moloney murine leukemia virus Env protein in fusion pore formation.

Fusion between cells expressing envelope protein (Env) of Moloney murine leukemia virus and target cells were studied by use of video fluorescence microscopy and electrical capacitance measurements. When the full-length 632-amino-acid residue Env was expressed, fusion did not occur at all for 3T3 cells as target and only somewhat for XC6 cells. Expression of Env 616*-a construct of Env with the last 16 amino acid residues (617 to 632; the R peptide) deleted from its C terminus to match the proteolytically cleaved Env produced during viral budding-resulted in high levels of fusion. Env 601*, lacking the entire cytoplasmic tail (CT) (identified by hydrophobicity), also led to fusion. Truncation of an additional six residues (Env 595*) abolished fusion. The kinetics of forming fusion pores did not depend on whether cells were first prebound at 4 degrees C and the time until fusion measured after the temperature was raised to 37 degrees C or whether cells were first brought into contact at 37 degrees C and the time until fusion immediately measured. This similarity in kinetics indicates that binding is accomplished quickly compared to subsequent steps in fusion. The fusion pores formed by Env 601* and Env 616* had the same initial size and enlarged in similar manners. Thus, once the R peptide is removed, the CT is not needed for fusion and does not affect formed pores. However, residues 595 to 601 are required for fusion. It is suggested here that the ectodomain and membrane-spanning domain of Env are directly responsible for fusion and that the R peptide affects their configurations at some point during the fusion process, thereby indirectly controlling fusion.

3T3 Cells↗
PubMed1999

Tension of membranes expressing the hemagglutinin of influenza virus inhibits fusion.

The effects of membrane tension on fusion between cells expressing the hemagglutinin (HA) of influenza virus and red blood cells were studied by capacitance measurements. Inflation of an HA-expressing cell was achieved by applying a positive hydrostatic pressure to its interior through a patch-clamp pipette in the whole-cell configuration. Inflating cells to the maximum extent possible without lysis created a membrane tension and completely inhibited low-pH-induced fusion at room temperature. Fully inflated cells that were subsequently deflated to normal size resumed the ability to fuse in response to low pH. At the higher temperature of 32 degrees C, fusion conditions were sufficiently optimal that full inflation did not hinder fusion, and once formed, pores enlarged more rapidly than those of never inflated cells. It is suggested that under fusogenic conditions HA causes the formation of a dimple within the membrane in which it resides, and that membrane tension hinders fusion by preventing the formation of dimples. Because dimpling bends the bilayer portion of bound membranes so that they come into intimate contact, the damping of dimpling would suppress this initial step in the fusion process.

3T3 Cells↗
PubMed1999

Hemifusion between cells expressing hemagglutinin of influenza virus and planar membranes can precede the formation of fusion pores that subsequently fully enlarge.

The chronological relation between the establishment of lipid continuity and fusion pore formation has been investigated for fusion of cells expressing hemagglutinin (HA) of influenza virus to planar bilayer membranes. Self-quenching concentrations of lipid dye were placed in the planar membrane to monitor lipid mixing, and time-resolved admittance measurements were used to measure fusion pores. For rhodamine-PE, fusion pores always occurred before a detectable amount of dye moved into an HA-expressing cell. However, with DiI in the planar membrane, the relationship was reversed: the spread of dye preceded formation of small pores. In other words, by using DiI as probe, hemifusion was clearly observed to occur before pore formation. For hemifused cells, a small pore could form and subsequently fully enlarge. In contrast, for cells that express a glycosylphosphatidylinositol-anchored ectodomain of HA, hemifusion occurred, but no fully enlarged pores were observed. Therefore, the transmembrane domain of HA is required for the formation of fully enlarging pores. Thus, with the planar bilayer membranes as target, hemifusion can precede pore formation, and the occurrence of lipid dye spread does not preclude formation of pores that can enlarge fully.

3T3 Cells↗
PubMed1999

Lipid flow through fusion pores connecting membranes of different tensions.

When two membranes fuse, their components mix; this is usually described as a purely diffusional process. However, if the membranes are under different tensions, the material will spread predominantly by convection. We use standard fluid mechanics to rigorously calculate the steady-state convective flux of lipids. A fusion pore is modeled as a toroid shape, connecting two planar membranes. Each of the membrane monolayers is considered separately as incompressible viscous media with the same shear viscosity, etas. The two monolayers interact by sliding past each other, described by an intermonolayer viscosity, etar. Combining a continuity equation with an equation that balances the work provided by the tension difference, Deltasigma, against the energy dissipated by flow in the viscous membrane, yields expressions for lipid velocity, upsilon, and area of lipid flux, Phi. These expressions for upsilon and Phi depend on Deltasigma, etas, etar, and geometrical aspects of a toroidal pore, but the general features of the theory hold for any fusion pore that has a roughly hourglass shape. These expressions are readily applicable to data from any experiments that monitor movement of lipid dye between fused membranes under different tensions. Lipid velocity increases nonlinearly from a small value for small pore radii, rp, to a saturating value at large rp. As a result of velocity saturation, the flux increases linearly with pore radius for large pores. The calculated lipid flux is in agreement with available experimental data for both large and transient fusion pores.

Biomechanical Phenomena↗
PubMed1999

Relationship of membrane sidedness to the effects of the lipophosphoglycan of Leishmania donovani on the fusion of influenza virus.

Cells expressing the influenza hemagglutinin protein were fused to planar lipid bilayers containing the viral receptor GD1a at pH 5.0. An amphiphile known to alter membrane properties is lipophosphoglycan (LPG). This glycoconjugate was added from aqueous solution to either the cis or the trans monolayer to examine its effects on the fusion process. LPG markedly inhibited the formation of fusion pores when present in the cis monolayer but LPG in the trans monolayer had no effect on the parameters of pore formation or on the properties of the pores. The N-terminal segment of the HA2 subunit of the influenza hemagglutinin protein is important for membrane fusion. The effect of LPG on the conformation and membrane insertion of a synthetic 20-amino-acid peptide, corresponding to the influenza fusion peptide, was examined at pH 5.0 by attenuated total reflection Fourier transform infrared spectroscopy and by the fluorescence properties of the Trp residues of this peptide. It was found that cis LPG did not prevent insertion of the peptide into the membrane but it did alter the conformation of the membrane-inserted peptide from alpha-helix to beta-structure. The beta-structure was oriented along the bilayer normal. The effect of cis LPG on the conformation of the fusion peptide probably contributes to the observed inhibition of pore formation and lipid mixing. In contrast, trans LPG has no effect on the conformation or angle of membrane insertion of the peptide, nor does it affect pore formation by HA-expressing cells. The ineffectiveness of trans LPG, despite it having strong positive curvature-promoting properties, may be a consequence of the size of this amphiphile being too large to enter a fusion pore.

3T3 Cells↗
PubMed1999

Amino acid sequence requirements of the transmembrane and cytoplasmic domains of influenza virus hemagglutinin for viable membrane fusion.

The amino acid sequence requirements of the transmembrane (TM) domain and cytoplasmic tail (CT) of the hemagglutinin (HA) of influenza virus in membrane fusion have been investigated. Fusion properties of wild-type HA were compared with those of chimeras consisting of the ectodomain of HA and the TM domain and/or CT of polyimmunoglobulin receptor, a nonviral integral membrane protein. The presence of a CT was not required for fusion. But when a TM domain and CT were present, fusion activity was greater when they were derived from the same protein than derived from different proteins. In fact, the chimera with a TM domain of HA and truncated CT of polyimmunoglobulin receptor did not support full fusion, indicating that the two regions are not functionally independent. Despite the fact that there is wide latitude in the sequence of the TM domain that supports fusion, a point mutation of a semiconserved residue within the TM domain of HA inhibited fusion. The ability of a foreign TM domain to support fusion contradicts the hypothesis that a pore is composed solely of fusion proteins and supports the theory that the TM domain creates fusion pores after a stage of hemifusion has been achieved.

Amino Acid Sequence↗