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Biomedical subjects

R Blumenthal

Publications and source records attributed to R Blumenthal.

198 records · Page 11Linked to original sources
PubMed1969

Flux ratio and driving forces in a model of active transport.

In order to analyze the energetics of active transport, a hypothetical carrier model is considered in which the active transport process is reduced to a minimal number of elementary steps. The relation between the following three quantities is examined: The affinity of the reaction driving the active transport, the ratio of isotope fluxes between identical solutions ("short-circuit"), and the maximal chemical potential difference which the active transport system can maintain. The interdependence of isotopeinteraction and the degree of coupling between transport and chemical reaction is shown explicitly: when the transport and chemical reaction are completely coupled, there is marked isotope interaction. In general, the logarithm of the short-circuit flux ratio (multiplied by RT) and the maximal chemical potential are not equal. The two quantities are approximately equal, when coupling between metabolism and transport is very loose, or when the reaction step is much faster than the transfer of the adsorbed solute across the barrier. Without prior knowledge of the kinetic parameters of the carrier, the maximal potential and the dependence of the metabolic reaction on solute flow have to be measured in order to derive the affinity of the driving reaction. Measurement of the flux ratio in the same system will then yield independent information on the carrier mechanism.

Biological Transport, Active↗
PubMedDate not supplied

Barriers to seeking treatment for major depression.

The purpose of this study was to examine the reasons people with an episode of Major Depression do or do not seek treatment for the episode. We interviewed 101 persons who met criteria for an RDC Major Depressive episode that lasted at least 4 weeks within 3 years of the interview date, exploring in detail the reasons they gave for seeking or not seeking treatment. GAS ratings indicated that all subjects were moderately impaired at the time of the episode. We found that 55% of the subjects did not seek treatment for this episode, while 45% did. Significant predictors of treatment seeking included a history of prior treatment, higher education, and greater episode length. Non-seekers felt they could handle the episode themselves, did not consider it serious or did not recognize it as an illness. Seekers on the other hand felt the episode was too painful and lasted too long and caused significant disruption in their interpersonal relationships and role functioning. We discuss the implications of these findings in terms of the importance of continued educational efforts to encourage treatment seeking as well as the need for further research to explore the manner in which people decide that affective signs and symptoms have reached a threshold that leads to treatment seeking.

Adult↗
PubMedDate not supplied

Cooperativity in viral fusion.

A simple model for membrane fusion mediated by vial spike glycoproteins is presented. The viral proteins are considered to be allosteric proteins that undergo concerted conformational transitions when they bind the ligand. The ligand in this case is H+. The effect of the conformational transition is to bring membranes together and induce their fusion. An equation is derived for the dependence of fusion rates on ligand concentration, for a given dissociation constant (Kd), equilibrium constant for the conformational change (L), and number of cooperating subunits (n). Curves generated by this equation provide a reasonable fit to data on the rates of fusion of Vesicular Stomatitis virus with cells for a pKd of 6.3, L = 1000 and n = 6.

Animals↗
PubMedDate not supplied

The influenza haemagglutinin-induced fusion cascade: effects of target membrane permeability changes.

To define the stages in influenza haemagglutinin (HA)-mediated fusion the kinetics of fusion between cell pairs consisting of single influenza HA-expressing cells and single erythrocytes (RBC) which had been labelled with both a fluorescent lipid (Dil) in the membrane and a fluorescent solute (calcein) in the aqueous space have been monitored. It is shown that release of solute from the target cell occurs, following the formation of the hemi-fusion diaphragm. These results are discussed in terms of a model in which fusion peptide insertion into the target membrane induces lipid stalks, which results in the formation of a hemifusion diaphragm and a fusion pore. Bilayer expansion due to overproduction of these stalks can give rise to collateral damage of target membranes.

3T3 Cells↗
PubMedDate not supplied

The role of the target membrane structure in fusion with Sendai virus.

Fusion between membranes of Sendai virus and liposomes or human erythrocytes ghosts was studied using an assay for lipid mixing based on the relief of self-quenching of octadecylrhodamine (R18) fluorescence. We considered only viral fusion that reflects the biological activity of the viral spike glycoproteins. The liposomes were made of phosphatidylcholine, and the effects of including cholesterol, the sialoglycolipid GD1a, and/or the sialoglycoprotein glycophorin as receptors were tested. Binding of Sendai virus to those liposomes at 37 degrees C was very weak. Fusion with the erythrocyte membranes occurred at a 30-fold faster rate than with the liposomes. Experiments with biological and liposomal targets of different size indicated that size did not account for differences in fusion efficiency.

Erythrocyte Membrane↗
PubMedDate not supplied

Role of target membrane structure in fusion with influenza virus: effect of modulating erythrocyte transbilayer phospholipid distribution.

To study the role of the target membrane in influenza virus fusion we chose erythrocyte membranes whose phospholipid arrangement can readily be modified. The phospholipids of normal erythrocytes are arranged asymmetrically across the plasma membrane; phosphatidylcholine (PC) and sphingomyelin are predominantly on the outer surface, whereas others such as phosphatidylserine (PS) and phosphatidylethanolamine (PE) are predominantly restricted to the inner leaflet. However, erythrocytes can be lyzed and resealed under conditions where the asymmetric distribution of phospholipids is lost or retained. Low pH-induced fusion of the A/PR 8 strain of influenza virus, monitored spectrofluorometrically by the octadecylrhodamine dequenching assay, was more rapid with lipid-symmetric erythrocyte ghosts than with lipid-asymmetric ghosts or intact erythrocytes. Neither conversion of PS in the lipid-symmetric ghost membrane to PE by means of the enzyme PS decarboxylaze, nor incorporation of spin-labeled phospholipid analogs with PS, PC or PE headgroups into the outer leaflet of lipid-asymmetric erythrocytes altered rates or extents of fusion of A/PR 8 with the modified target. These results indicate that effects on influenza virus fusion are not associated with any particular phospholipid headgroup, but rather related to the packing characteristics of the target membrane.

Calcium↗
PubMedDate not supplied

A trans-dominant mutation in human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp41 inhibits membrane fusion when expressed in target cells.

A recombinant vaccinia virus was used to express a mutation in the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120-gp41. In this mutant protein, the second amino acid in the N-terminal region of gp41 has been converted from a hydrophobic valine residue to the polar glutamate. When recombinant vaccinia viruses encoding wild-type HIV-1 envelope glycoprotein infect a lymphocyte cell line lacking CD4, the cells express the HIV-1 envelope glycoprotein gp120-gp41 and are able to fuse with a CD4(4) T lymphocyte cell line. Cells expressing the mutant envelope glycoprotein are unable to fuse with CD4(4) T lymphocytes. When both viruses infect CD4- cells simultaneously, there is an inhibition of fusion to CD4+ cells with an increasing fraction of the virus encoding the mutated envelope glycoprotein. Interestingly, when the opposing, or CD4+ target cells are infected with the mutation-expressing virus, while CD4- cells are infected with wild-type envelope-expressing virus, a similar inhibition of fusion is observed. This suggests that the mutated envelope glycoprotein does not need to reside in the same membrane as the wild-type protein it inhibits.

Antibodies, Monoclonal↗
PubMedDate not supplied

Transient domains induced by influenza haemagglutinin during membrane fusion.

During low pH-induced fusion of influenza virus with erythrocytes we have observed differential dispersion of viral lipid and haemagglutinin (HA) into the erythrocyte membrane, and viral RNA into the erythrocyte using fluorescence video microscopy. The movement of both viral lipid and HA from virus to cell was restricted during the initial stages of fusion relative to free diffusion. This indicates the existence of relatively long-lived barriers to diffusion subsequent to fusion pore formation. Fluorescence anisotropy of phospholipid analogues incorporated into the viral membrane decreased when the pH was lowered to levels required for optimum fusion. This indicates that the restricted motion of viral membrane components was not due to rigidification of membrane lipids. The movement of HA from the fusion site was also assessed by photosensitized labelling by means of a fluorescent substrate (NBD-taurine) passing through the band 3 sialoglycoprotein (the erythrocyte anion transporter). We also examined the flow of lipid and aqueous markers during fusion of HA-expressing cells with labelled erythrocytes. During this cell-cell fusion, movement of lipid between fusing membranes begins before the fusion pore is wide enough to allow diffusion of aqueous molecules (M(r) > 500). The data indicate that HA is capable of creating domains in the membrane and controlling continuity of aqueous compartments which are bounded by such domains.

Cell Fusion↗
PubMedDate not supplied

Influence of the spectrin network on fusion of influenza virus with red blood cells.

We examined the influence of the physical state of the membrane skeleton on low pH fusion of influenza virus A/PR 8/34 with intact human red blood cells. Spectrin, the major component of the skeleton, is known to become denaturated at 50 degrees C. After heat treatment of erythrocytes at 50 degrees C we observed an enhanced kinetics of fusion monitored spectrofluorometrically by the octadecylrhodamine fluorescence dequenching assay, while the extent of fusion was not affected. The accelerated fusion of influenza virus after preincubation of red blood cells at 50 degrees C is not mediated by alterations of the lipid phase of the target. From ESR measurements using spin-labelled phospholipids we conclude that heat-induced alterations of the spectrin network did not affect either the phospholipid asymmetry or the fluidity of the exoplasmic and the cytoplasmic leaflets of the erythrocyte membrane. Moreover, as deduced from our previous investigations, the swelling behaviour of red blood cells could not be responsible for the observed effect. Possible mechanisms for the spectrin effect include a change in the ability of the target membrane to bend locally, and a change in the rate of formation and development of the fusion pore.

Erythrocyte Membrane↗
PubMedDate not supplied

What studies of fusion peptides tell us about viral envelope glycoprotein-mediated membrane fusion (review).

This review describes the numerous and innovative methods used to study the structure and function of viral fusion peptides. The systems studied include both intact fusion proteins and synthetic peptides interacting with model membranes. The strategies and methods include dissecting the fusion process into intermediate stages, comparing the effects of sequence mutations, electrophysiological patch clamp methods, hydrophobic photolabelling, video microscopy of the redistribution of both aqueous and lipophilic fluorescent probes between cells, standard optical spectroscopy of peptides in solution (circular dichroism and fluorescence) and attenuated total reflection-Fourier transform infrared spectroscopy of peptides bound to planar bilayers. Although the goal of a detailed picture of the fusion pore has not been achieved for any of the intermediate stages, important properties useful for constraining the development of models are emerging. For example, the presence of alpha-helical structure in at least part of the fusion peptide is strongly correlated with activity; whereas, beta-structure tends to be less prevalent, associated with non-native experimental conditions, and more related to vesicle aggregation than fusion. The specific angle of insertion of the peptides into the membrane plane is also found to be an important characteristic for the fusion process. A shallow penetration, extending only to the central aliphatic core region, is likely responsible for the destabilization of the lipids required for coalescence of the apposing membranes and fusion. The functional role of the fusion peptides (which tend to be either nonpolar or aliphatic) is then to bind to and dehydrate the outer bilayers at a localized site; and thus reduce the energy barrier for the formation of highly curved, lipidic 'stalk' intermediates. In addition, the importance of the formation of specific, 'higher-order' fusion peptide complexes has also been shown. Recent crystallographic structures of core domains of two more fusion proteins (in addition to influenza haemagglutinin) has greatly facilitated the development of prototypic models of the fusion site. This latter effort will undoubtedly benefit from the insights and constraints gained from the studies of fusion peptides.

Amino Acid Sequence↗