Sensitivity of yeast cells to reactive oxygen species generated in the extracellular space

Even when cytoplasmic scavenging activities are plentiful, yeast cells (S. cerevisiae) remain particularly sensitive towards reactive oxygen species generated in the extracellular space (either by the xanthine/xanthine oxidase reaction or by the redox cycling of menadione). A sharp reduction of the extent of cellular alterations when SOD and/or catalase were supplemented in the incubation buffer, points to a contribution of both O-.2 and H2O2 in the toxic process. Although oxygen metabolites as well as t-butylhydroperoxide (tBH), a highly toxic organic peroxide, may be directly responsible for cellular damage, their toxicity is largely reduced in the presence of Desferal. A role of metal ions in potentiating the toxicity points to the involvement of OH. radicals, actually produced in the medium. With tBH, metal cations would be rather active in promoting peroxidative chain reactions. In the case of an extracellular oxidative attack, it may be foreseen that the plasma membrane will form a preferential target. An increased permeability of the plasma membrane towards ionized molecules and uncharged polycarboxylic acids is indeed observed after an oxidative treatment. The loss of selective permeability is, as a rule, correlated with a drop in viability. Early alterations, disrupting the functional organization of the plasma membrane have been sought. The permease involved in the active transport of purine(s) has appeared to be an appropriate marker for checking its functional integrity. This transport function appears to be very sensitive to damage induced by O-.2 generators, particularly under conditions in which the resulting lethality is still kept low and in which the energization of active transport processes remains unimpaired.     

Isosmotic volume reabsorption in rat proximal tubule

A theoretical model incorporation both active and passive forces has been developed for fluid reabsorption from split oil droplets in rat intermediate and late proximal tubule. Of necessity, simplifying assumptions have been introduced; we have assumed that the epithelium can be treated as a single membrane and that the membrane "effective" HCO3 permeability is near zero. Based on this model with its underlying assumptions, the following conclusions are drawn. Regardless of the presence or absence of active NaCl transport, fluid reabsorption from the split oil droplet is isosmotic. The reabsorbate osmolarity can be affected by changes in tubular permeability parameters and applied forces but is not readily altered from an osmolarity essentially equal to that of plasma. In a split droplet, isosmotic flow need not be a special consequence of active Na transport, is not the result of a particular set of permeability properties, and is not merely a trivial consequence of a very high hydraulic conductivity; isosmotic flow can be obtained with hydraulic conductivity nearly an order of magnitude lower than that previously measured in the rat proximal convoluted tubule. Isosmotic reabsorption is, in part, the result of the interdependence of salt and water flows, their changing in parallel, and thus their ratio, the reabsorbate concentration being relatively invariant. Active NaCl transport can cause osmotic water flow by reducing the luminal fluid osmolarity. In the presence of passive forces the luminal fluid can be hypertonic to plasma, and active NaCl transport can still exert its osmotic effect on volume flow. There are two passive forces for volume flow: the Cl gradient and the difference in effective osmotic pressure; they have an approximately equivalent effect on volume flow. Experimentally, we have measured volume changes in a droplet made hyperosmotic by the addition of 50 mM NaCl; the experimental results are predicted reasonably well by our theoretical model.     

Effects of low-amplitude pulsed magnetic fields on cellular ion transport

Pulsed magnetic fields (PMFs) are widely used to treat difficult fractures of bone and other disorders of connective tissue. It is not clear how they interact with tissue metabolism, although it has been proposed that induced currents or electric fields impinging on cell membranes may modify their ion transport function. This hypothesis was tested by treating in vitro models for ion transport processes with short-term exposure to PMFs. No change occurred in active transport of potassium or calcium in human red cells or in calcium transport through an epithelial membrane. We considered less direct action on red cell membranes, that their permeability might be modified after PMF treatment, and also that PMFs might alter the extracellular ionic activity within connective tissue by interacting with its Donnan potential. Each of these studies proved negative, and we conclude that the PMF waveforms used here do not exert a general short-term effect on cellular ion transport.     

Energetics of active transport processes.

Active sodium transport across epithelial membranes has been analyzed by means of linear nonequilibirium thermodynamics. In this formulation the rates of active sodium transport JNa and the associated metabolic reaction Jr are postulated to be linear functions of both the electrochemical potential difference of sodium--XNa and the affinity A (negative free energy) of the metabolic reaction of driving transport. Experimental studies in various epithelia demonstrate that both JNa and Jr (oxygen consumption) are indeed linear functions of XNa. Theoretical considerations and experimental studies in other systems suggest that likelihood of linearity in A as well. If so, A may be evaluated. Several observations indicate that the quantity A evaluated from the thermodynamic formalism does in fact reflect the substrate-product ratio of the metabolic reaction which supports transport. This is in contrast to measurements of mean cellular concentrations, which may not reflect conditions at the site of transport. Associated studies of isotope kinetics permit the distinction between effects on the permeability of the active and passive transport pathways. With these combined approaches, it may prove possible to characterize both the energetic and permeability factors which regulate transport. The formulation has been applied to an analysis of the mechanism of action of the hormone aldosterone.     

The flux-force relations across complex membranes with active transport

Equations are derived for the total material flux, and the total electric current flux, across a complex membrane system with active transport. The equations describe the fluxes as linear functions of forces across the system, and specifically of electrical potential, hydrostatic pressure, chemical potentials, and active transport rates. The equations can be simplified for experimental studies by making one or more of the forces equal to zero. The osmotic pressure difference across a membrane system is shown to be a function of the electrical potential and chemical potential differences and of the active transport rates. The transmembrane potential is shown to be the sum of a diffusion potential and an active transport potential. A simple equation is derived describing the current across a membrane as a linear function of the electrical potential and the active transport rate. Specific examples of the application of the equations to nerve membrane potentials are considered.     

Diffusion with memory in two cases of biological interest

The complexity of a biological structure, such as membrane where the transport process may carry solid particles which may obstruct some of the pores, diminishing their size and making the permeability dependent on the local structure of the medium, suggests the introduction of a space-dependent diffusion constant. In this note, the profile concentration of diffusing solutes inside a cell membrane has been calculated on the basis of the Fick diffusion equation modified by introducing a memory formalism (diffusion with memory). This approach has been employed to describe the concentration profile inside the membrane when a sudden change of the concentration in the medium bathing one of its face is applied for a limited interval of time. A further application of the method concerns the so-called concentration boundary layer that occurs at the membrane-aqueous medium interface, where the solute concentration depends, even at considerable depth, on the local structure of the interface. These profiles are compared to some recent experiments concerning the diffusion of ethanol in a layer close to a nephrophane membrane. This approach generalizes the diffusion models based on the Fick equation to more complex systems, where a space-independent diffusion coefficient could be inappropriate to take into account the large variety of diffusion processes in biological systems.     

Direct Cytoskeleton Forces Cause Membrane Softening in Red Blood Cells

Erythrocytes are flexible cells specialized in the systemic transport of oxygen in vertebrates. This physiological function is connected to their outstanding ability to deform in passing through narrow capillaries. In recent years, there has been an influx of experimental evidence of enhanced cell-shape fluctuations related to metabolically driven activity of the erythroid membrane skeleton. However, no direct observation of the active cytoskeleton forces has yet been reported to our knowledge. Here, we show experimental evidence of the presence of temporally correlated forces superposed over the thermal fluctuations of the erythrocyte membrane. These forces are ATP-dependent and drive enhanced flickering motions in human erythrocytes. Theoretical analyses provide support for a direct force exerted on the membrane by the cytoskeleton nodes as pulses of well-defined average duration. In addition, such metabolically regulated active forces cause global membrane softening, a mechanical attribute related to the functional erythroid deformability.     

The Mechanism of Isotonic Water Transport

The mechanism by which active solute transport causes water transport in isotonic proportions across epithelial membranes has been investigated. The principle of the experiments was to measure the osmolarity of the transported fluid when the osmolarity of the bathing solution was varied over an eightfold range by varying the NaCl concentration or by adding impermeant non-electrolytes. An in vitro preparation of rabbit gall bladder was suspended in moist oxygen without an outer bathing solution, and the pure transported fluid was collected as it dripped off the serosal surface. Under all conditions the transported fluid was found to approximate an NaCl solution isotonic to whatever bathing solution used. This finding means that the mechanism of isotonic water transport in the gall bladder is neither the double membrane effect nor co-diffusion but rather local osmosis. In other words, active NaCl transport maintains a locally high concentration of solute in some restricted space in the vicinity of the cell membrane, and water follows NaCl in response to this local osmotic gradient. An equation has been derived enabling one to calculate whether the passive water permeability of an organ is high enough to account for complete osmotic equilibration of actively transported solute. By application of this equation, water transport associated with active NaCl transport in the gall bladder cannot go through the channels for water flow under passive conditions, since these channels are grossly too impermeable. Furthermore, solute-linked water transport fails to produce the streaming potentials expected for water flow through these passive channels. Hence solute-linked water transport does not occur in the passive channels but instead involves special structures in the cell membrane, which remain to be identified.     

Permeability of Pseudomonas aeruginosa outer membrane to hydrophilic solutes.

Pseudomonas aeruginosa is usually resistant to a wide variety of antibacterial agents, and it has been inferred, on the basis of indirect evidence, that this was due to the low permeability of its outer membrane. We determined the permeability of P. aeruginosa outer membrane directly, by measuring the rates of hydrolysis of cephacetrile, cephaloridine, and various phosphate esters by hydrolytic enzymes located in the periplasm. The permeability to these compounds was about 100-fold lower than in the outer membrane of Escherichia coli K-12. Also, we found that the apparent Km values for active transport of various carbon and energy source compounds were typically higher than 20 microM in P. aeruginosa, in contrast to E. coli in which the values are usually lower than 5 microM. These results also are consistent with the notion that the P. aeruginosa outer membrane indeed has a low permeability to most hydrophilic compounds and that this membrane acts as a rate limiting step in active transport processes with high Vmax values.     

Ionic Permeability of Insect Epithelia

In general, ionic regulation will depend on active transportby epithelia and also on the permeability properties of thesetissues. Passive permeability has recently been studied in thehindgut of the desert locust Schistocerca gregaria using electrophysiologicaland radiotracer techniques. Although locust rectum has low electricalresistance, cell membranes provide the major route for transepithelialionic diffusion; i.e., the locust rectum is a tight epithelium.Potassium permeability (P_K) is apparently regulated by luminalK and osmotic concentrations (local control), and also by thepeptide hormone CTSH (chloride transport-stimulating hormone).Transepithelial resistance declines when isolated recta areexposed to CTSH or its "second-messenger" cAMP (adenosine 3':5'-cyclicmonophosphate). Cyclic-AMP also stimulates K diffusion acrossrecta by 400%. Intracellular cable analysis indicatesthat cAMPlowers apical and basal membrane resistances (R_a and R_b, respectively)by {small tilde}80%; however different ionic permeabilitiesare affected at thelumen- and hemolymph-facing membranes: ThecAMP-induced decline in R_a, requires potassium whereas R_b isCl-dependent. The actions of cAMP on active transport and passivepermeability are complementary and would allow remarkably efficientcontrol over KC1 absorption in vivo. One hypothesis is as follows:CTSH elevates intracellular cAMP concentration by stimulatingadenyl cyclase. Cyclic-AMP enhances transepithelial Cl absorptionby stimulating a Cl pump in the apical membrane and also byincreasing the Cl permeability of the basal membrane. PassiveK absorption would also increase during cAMP stimulation sinceCl transport results in a more positive luminal potential, andbecause cAMP elevates transrectal P_K. The mechanisms by whichmembrane permeability is regulated in insects have not yet beenstudied, but these might involve the modulation of ion channelsby cAMP- or calmodulin-dependent phosphorylation, Ca or calmodulinbinding, methylation, or insertion of new channels into themembrane.     

Nonequilibrium voltage fluctuations in biological membranes. I. General framework of charge transport in discrete systems and related voltage noise

This paper continues our work on the theory of nonequilibrium voltage noise generated by electric transport processes in membranes. Introducing the membrane voltage as a further variable, a system of kinetic equations linearized in voltage is derived by which generally the time-dependent behaviour of charge-transport processes under varying voltage can be discussed. Using these equations, the treatment of voltage noise can be based on the usual master equation approach to steady-state fluctuations of scalar quantities. Thus, a general theoretical approach to nonequilibrium voltage noise is presented, completing our approach to current fluctuations which had been developed some years ago. It is explicitly shown that at equilibrium the approach yields agreement with the Nyquist relation, while at nonequilibrium this relation is not valid. A further general property of voltage noise is the reduction of low-frequency noise with increasing number of transport units as a consequence of the interactions via the electric field. In a second paper, the approach will be applied for a number of special transport mechanisms, such as ionic channels, carriers or electrogenic pumps.     

The Effect of Ca and Antidiuretic Hormone on Na Transport across Frog Skin : II. Sites and mechanisms of action

A method has been developed for determining unidirectional Na fluxes across the two faces of the transporting cells in the frog skin. The method has been used to investigate the location of the sites at which Ca and anti-diuretic hormone act to alter the rate of active Na transport across the skin. The results have indicated that the primary effect of both agents is on the Na permeability of the outward facing membrane of the cells. Ca decreases and the hormone increases permeability of this barrier. Neither agent appears to have a direct effect on the active transport system itself assuming that it is located at the inner membrane of the cells. The rate of active Na transport is altered as a result of changes in the size of the Na pool in the cells which occur because of changes in the rate of Na entry through the outer membrane. Thus, the results indicate that the Na permeability of the outer membrane plays an important role in controlling the rate of net active Na transport across the skin.     

Interplay of magnetic interactions and active movements in the formation of magnetosome chains

Magnetotactic bacteria assemble chains of magnetosomes, organelles that contain magnetic nano-crystals. A number of genetic factors involved in the controlled biomineralization of these crystals and the assembly of magnetosome chains have been identified in recent years, but how the specific biological regulation is coordinated with general physical processes such as diffusion and magnetic interactions remains unresolved. Here, these questions are addressed by simulations of different scenarios for magnetosome chain formation, in which various physical processes and interactions are either switched on or off. The simulation results indicate that purely physical processes of magnetosome diffusion, guided by their magnetic interactions, are not sufficient for the robust chain formation observed experimentally and suggest that biologically encoded active movements of magnetosomes may be required. Not surprisingly, the chain pattern is most resembling experimental results when both magnetic interactions and active movement are coordinated. We estimate that the force such active transport has to generate is compatible with forces generated by the polymerization or depolymerization of cytoskeletal filaments. The simulations suggest that the pleiotropic phenotypes of mamK deletion strains may be due to a defect in active motility of magnetosomes and that crystal formation in magneteosome vesicles is coupled to the activation of their active motility in M. gryphiswaldense, but not in M. magneticum.     

Foundations of vectorial metabolism and osmochemistry

Chemical transformations, like osmotic translocations, are transport processes when looked at in detail. In chemiosmotic systems, the pathways of specific ligand conduction are spatially orientated through osmoenzymes and porters in which the actions of chemical group, electron and solute transfer occur as vectorial (or higher tensorial order) diffusion processes down gradients of total potential energy that represent real spatially-directed fields of force. Thus, it has been possible to describe classical bag-of-enzymes biochemistry as well as membrane biochemistry in terms of transport. But it would not have been possible to explain biological transport in terms of classical transformational biochemistry or chemistry. The recognition of this conceptual asymmetry in favour of transport has seemed to be upsetting to some biochemists and chemists; and they have resisted the shift towards thinking primarily in terms of the vectorial forces and co-linear displacements of ligands in place of their much less informative scalar products that correspond to the conventional scalar energies. Nevertheless, considerable progress has been made in establishing vectorial metabolism and osmochemistry as acceptable biochemical disciplines embracing transport and metabolism, and bioenergetics has been fundamentally transformed as a result.     

Direct effects on the membrane potential due to "pumps" that transfer no net charge

The effects of active ionic transport are included in the derivation of a general expression for the zero current membrane potential. It is demonstrated that an active transport system that transfers no net charge (nonrheogenic) may, nevertheless, directly alter the membrane potential. This effect depends upon the exchange of matter within the membrane between the active and passive diffusion regimes. Furthermore, in the presence of such exchange, the transmembrane active fluxes measured by the usual techniques and the local pumped fluxes are not identical. Several common uses of the term “electrogenic pump” are thus shown to be inconsistent with each other. These inconsistencies persist when the derivation is extended to produce a Goldman equation modified to account for active transport; however, that equation is shown to be limited by less narrow constraints on membrane heterogeneity and internal electric field than those previously required. In particular, it is applicable to idealized mosaic membranes limited by these requirements.     

Foundations of Vectorial Metabolism and Osmochemistry

Chemical transformations, like osmotic translocations, are transport processes when looked at in detail. In chemiosmotic systems, the pathways of specific ligand conduction are spatially orientated through osmoenzymes and porters in which the actions of chemical group, electron and solute transfer occur as vectorial (or higher tensorial order) diffusion processes down gradients of total potential energy that represent real spatially directed fields of force. Thus, it has been possible to describe classical bag-of-enzymes biochemistry as well as membrane biochemistry in terms of transport. But it would not have been possible to explain biological transport in terms of classical transformational biochemistry or chemistry. The recognition of this conceptual asymmetry in favour of transport has seemed to be upsetting to some biochemists and chemists; and they have resisted the shift towards thinking primarily in terms of the vectorial forces and co-linear displacements of ligands in place of their much less informative scalar products that correspond to the conventional scalar energies. Nevertheless, considerable progress has been made in establishing vectorial metabolism and osmochemistry as acceptable biochemical disciplines embracing transport and metabolism, and bioenergetics has been fundamentally transformed as a result.     

Decreased membrane potassium permeability and transport in human chronic leukemic and tonsillar lymphocytes.

Human blood T-lymphocytes increase their potassium (K+) permeability and active K+ transport following lectin or antigen stimulation. We have studied the permeability and active transport of K+ by lymphocytes in chronic lymphocytic leukemia (CLL) to determine if their membrane K+ transport was similar to resting or lectin-stimulated normal blood lymphocytes. K+ transport was assessed both by the rate of isotopic 42K+ uptake and by the rate of change in cell K+ concentration after inhibition of the K+ transport system with ouabain. CLL lymphocytes had a marked decrease in membrane K+ permeability and active transport of K+ when compared to blood T lymphocytes. K+ transport in five subjects with CLL (10 mmol.1 cell water-1.h-1) was half that in normal blood T-lymphocytes (20 mmol.1 cell water-1 h-1). Phytohemagglutinin (PHA) treatment of CLL lymphocytes did not increase significantly their active K+ transport, whereas K+ transport by normal T-lymphocytes increased by 100%. Since there were 73% T-lymphocytes in normal blood and 14% in CLL blood, the difference in membrane K+ turnover could be related either to neoplasia or to the proposed B-lymphocyte origin of CLL. We studied human tonsillar lymphocytes which contained a mean of 34% T-cells. In five studies of tonsils, K+ transport was 14 mmol.1 cell water-1.h-1 and treatment with PHA increased K+ transport only 30%. The intermediate values of basal K+ transport and K+ transport in response to PHA in tonsillar lymphocytes were consistent with the proportion of T-lymphocytes present. These data suggest that B-lymphocytes have reduced membrane permeability and active transport of K+. Thus the marked decrease in CLL lymphocyte membrane K+ permeability and transport may be a reflection of its presumed B-cell origin, rather than a membrane alteration related to malignant transformation.     

Dense fibers protect mammalian sperm against damage

The relative tensile strengths of the sperm of seven mammalian species and sea urchins have been measured by determining the minimum shear necessary to kill them (assayed by lack of motility) when they are suspended in a viscous fluid. In general, long sperm are killed by smaller shears than short sperm. However, the longer sperm are not as fragile as would be expected from theoretical predictions. Their additional tensile strength correlates well with the size of their dense fibers; a theory that includes the dense fiber contributions accurately predicts the sperm tensile strength for most of the species in which this has been measured. This added strength may be necessary to protect sperm from shear forces encountered during epididymal transport and especially during ejaculation, as these forces are strong enough to kill long sperm if they are not strengthened.