Simple Carrier Research Articles

Persistent BK papovavirus infection of transformed human fetal brain cells. I. Episomal viral DNA in cloned lines deficient in T-antigen expression

After infection of permissive human fetal brain cells by BK human papovavirus (BKV), the vast majority of the cells were killed by the virus, but rare survivors were recovered after frequent medium changes. These surviving cells grew and formed visible colonies after 5 to 6 weeks and were thereafter established as permanent cell lines. These cells, designated as BK-HFB cells, were persistently infected and shed BKV. Morphologically, they were small polygonal cells and had transformed growth properties. Their plating efficiency on solid substrates or in semisolid medium was high, and they were tumorigenic in athymic nude mice. Cloning experiments in medium containing BKV antiserum revealed that BKV did not persist in the cultures in a simple carrier state. All cloned cell lines were initially T-antigen negative and virus-free. However, every clone began to release BKV and again became persistently infected within 3 weeks after removal of BKV antiserum. After rigorous antibody treatment, four of seven clones still released virus spontaneously upon removal of antiserum; three clones have remained virus-free and are apparently cured. Although these cloned cell lines are T- and V-antigen negative when grown in antiserum-containing medium, they retain "free" or episomal BKV genomes; integrated viral DNA was not detected in any of the clones. These free genomes are indistinguishable from prototype BKV DNA and are found in much larger amounts in virus-shedding cell lines.

The kinetic dissection of transport from metabolic trapping during substrate uptake by intact cells. Uridine uptake by quiescent and serum-activated nil 8 hamster cells and their murine sarcoma virus-transformed counterparts

1. 1. We present a theoretical analysis of the tandem processes of transport and metabolic trapping which together constitute uptake of a substrate by intact cells. 2. 2. Transport is assumed to occur by means of a simple carrier here analysed in its general form. Trapping is assumed to occur by a simple enzymic reaction. 3. 3. We show how to obtain the separate parameters of the two steps by analysing uptake data over a range of uptake times and substrate concentrations. 4. 4. We present uptake data for uridine and cytosine-β- d-arabinoside entering Nil 8 hamster fibroblasts, normal and murine sarcoma virus transformed, in the quiescent condition and after stimulation by added serum. We analyse the data in terms of the theory for tandem processes. 5. 5. Transport is characterised by a system having a high K m and a high V for entry. The data for cytosine-β- d-arabinoside suggest that the cytosine-β- d-arabinoside system is not far from a symmetric one. The data for uridine transport do not differ when quiescent serum-activated cells are compared. Transformed cells transport uridine at half the maximum velocity of normal cells, with or without added serum. 6. 6. Trapping of cytosine-β- d-arabinoside is insignificant. Trapping of uridine occurs by a system with both V and K m at least an order of magnitude smaller than are these parameters for transport. Trapping of uridine by non-transformed cells activated by serum, has twice the V of such cells in the quiescent state. 7. 7. In the virus-transformed cells, the control of uridine trapping by added serum lost, along with control of growth by this statement.

Effects of temperature on the transport of galactose in human erythrocytes.

The transport of galactose in human erythrocytes has been resolved recently into a mechanism which involves two asymmetric carriers operating in antiparallel fashion. The effects of temperature on this mediated transport system in the range of 0--25 degrees C show the following features. The Michaelis constants for zero-trans influx and efflux and for equilibrium-exchange efflux, do not vary with temperature. Arrhenius plots of the maximal velocities show breaks between 3 and 15 degrees C with activation energies two- to threefold larger below the break than above it. The relative contribution of the two types of carriers to the total transport rate is not affected by temperature. The kinetic properties of the prevalent type of carriers are analysed in terms of the simple carrier model as formulated by Lieb & Stein (1974). This analysis shows that the relative concentration of the unloaded carrier at the inner interface of the membrane increases upon cooling. The free energy of translocation of the unloaded carrier and the change in entropy involved in this step are significantly larger in the low temperature range (0--5 degrees C) than in the higher range (15--25 degrees C). The results are discussed briefly in terms of possible lipid-protein interaction and the physicochemical nature of the erythrocyte membrane.

Galactose transport in human erythrocytes. The transport mechanism is resolved into two simple asymmetric antiparallel carriers

The kinetic properties of the mediated transport of galactose in human erythrocytes are investigated at 20°C. Different methodological procedures are used to acquire a complete kinetic description of the system. Under zero- trans conditions the uptake of galactose is mediated by two distinctly different carriers (defined as α and β) having significantly different Michaelis parameters: αK = 12.7 mM and βK = 81.5 mM , but similar maximal velocities, approx. 40 mM · min −1 . The zero- trans efflux procedure reveals apparently one single carrier with K = 74.4 mM and V = 241 mM · min −1 . Under equilibrium-exchange conditions the galactose transport is mediated apparently by a single site with K = 146 mM and V = 521 mM · min −1 . The data for the α-carrier are analyzed in terms of the simple carrier model as formulated by Lieb and Stein (Biochim. Biophys. Acta (1974) 373, 178). Application of several rejection criteria for the simple carrier failed to indicate lack of fitness of the α-carrier to a simple asymmetric carrier. From the analysis of the kinetic data it is inferred that the transport of galactose across the human erythrocyte membrane is mediated by two simple asymmetric carriers operating in antiparallel fashion. Using this model and the data of zero- trans and equilibrium-exchange, it is shown that the predicted half-saturation constants for both uptake and efflux in infinite- cis conditions fully agree with the experimentally derived values. Further analysis of the kinetic data indicate that the translocation of the unloaded α-carrier is the rate-limiting step in galactose uptake. Under equilibrium-exchange conditions the unloaded carrier is asymmetrically distributed across the membrane so that its concentration is 8 times higher on the inner side of the membrane. Using the value of 3.3 · 10 5 hexose carriers per cell, the turnover number of galactose exchange is 6.5 · 10 4 molecules/carrier per min.

How the kinetic parameters of the simple carrier are affected by an applied voltage

The kinetic parameters ( K, R oo , R 12, R 21, and R ee ) of the simple carrier model are analysed as a function of applied voltage for various cases of charged substrates and carriers. If certain parameters are invariant with voltage, strong conclusions can be made as to the charge on the free carrier. In one particular case (where the parameter R 00 is invariant while R 12 and R 21 display a nonlinear dependence on the exponential of the voltage) it may be possible to identify the isomerisation of the carrier-substrate complex.

Testing the simple carrier using irreversible inhibitors

1. 1. We analyse the kinetics of irreversible inhibition of the simple carrier. We consider how the rate of inactivation is influenced by the concentrations of permeant on the two sides of the membrane. 2. 2. We consider various kinetic schemes for the simple carrier and show that these are all indistinguishable kinetically, using steady-state transport or inactivation data. We point out the advantages of using the simplest kinetic scheme and the possible pitfalls of using more complicated schemes, including the conventional carrier model. 3. 3. We show that in the absence of information on the transport properties of the system, irreversible inhibition data are ambiguous. Taken together with transport data, however, inactivation data provide new tests for the applicability of the simple carrier. 4. 4. The new tests show that for the simple carrier model to be applicable, the substrate dependencies of transport and of inactivation must be identical in comparable experimental situations. Further, the maximal rates of transport and of stimulation (or inhibition) of inactivation must obey a simple relationship, which we derive. We illustrate the use of these tests with published data on the glucose and choline transport systems of the human red blood cell.

An extended kinetic analysis of valinomycin-induced Rb-transport through monoglyceride membranes.

The time course of the current following a voltage jump, which is applied to monoglyceride bilayers in the presence of valinomycin, shows two relaxation times. This is basically in agreement with a simple carrier model which has been described in full detail formerly. Relaxation times and amplitudes allow a calculation of the rate constants of the transport model. The presented data supplement an analysis which was hitherto based only on the slower relaxation process and on information derived from the nonlinearity of current-voltage characteristics. The additional resolution of the faster relaxation time allowed an approximate determination of the voltage dependence of the translocation rate constant for carrier-ion-complex and provided evidence for a small voltage dependence of the interfacial reaction. The dependence of the relaxation parameters on the ion concentration in the aqueous phase was interpreted assuming a saturation of the ion concentration at the reaction plane at high bulk concentrations.

Testing and characterizing the simple carrier

1. 1. We treat the simple carrier at the level of steady-state analysis, making no assumptions as to equilibrium or symmetry and taking into account specifically the effect of electric fields and unstirred layers. 2. 2. We develop tests and rejection criteria applicable under these conditions. 3. 3. For those transport systems which pass the tests for the simple carrier, we show how to obtain from the experimental data (i) the permeabilities of any unstirred layers which may be present at the membrane surfaces and (ii) a complete characterization of the transport system itself in terms of only four independent parameters. 4. 4. The interpretation of these parameters in terms of molecular rate constants is discussed.

Transport across biological membranes: a rigorous test for the carrier hypothesis.

This paper presents several equations derived from an asymmetrical nonconcentrative carrier mechanism for biological transport. These equations inter-relate the kinetic constants derived from different types of kinetic experiments, and act as criteria for the validity of the mechanism. Because they require a precise numeral relationship between rate constants, these equations can act as efficient discriminators for transport systems which are not mediated by a simple carrier mechanism, while they can provide presumptive evidence for a carrier mechanism when the observed kinetic constants do fulfill the predictions.

The asymmetry of the facilitated transfer system for hexoses in human red cells and the simple kinetics of a two component model.

1. 4, 6-O-Ethylidene-alpha-D-glucopyranose (ethylidene glucose) has been used to study the competitive inhibition of glucose exchange fluxes when the reagent was (i) inside the cells and (ii) on the outside.2. 50% inhibition of glucose exchange at 20 mM and 16 degrees C required 200 mM ethylidene glucose when on the inside in contrast to 25-30 mM when on the outside.3. The inhibitions at different inhibitor/glucose concentration ratios were measured and analysis of the data suggested that the half-saturation constant for ethylidene glucose was 6 times that for glucose inside the cell as against 1.5 outside. The analysis, however, suggested an asymmetry in respect to the affinities for glucose of approximately ten-fold and this would make the asymmetry towards ethylidene glucose forty-fold.4. Such asymmetries make it necessary to consider a transfer mechanism for sugars with different components on the outer and inner membrane interfaces and simple kinetics for a two component system have been developed and used for analysing the experimental data quantitatively.5. The kinetic similarities to and difference from the kinetics of a simple mobile carrier and those of some more recent models are briefly discussed.

Processing small delicate biological specimens for scanning electron microscopy.

SUMMARYTo overcome the problems of collecting and handling delicate cells while dehydrating them for scanning electron microscopy, a method is suggested whereby cells are collected on solvent resistant Millipore filters. The schedule used for successfully processing a wide range of algae, protozoa and some other organisms is outlined as is the construction of a simple carrier to hold the filter in the critical point drying apparatus.

Carrier-mediated transport across the erythrocyte membrane: a rigorous test for the simple carrier model.

Carrier-mediated transport across the erythrocyte membrane: a rigorous test for the simple carrier model.

The transport of L‐leucine in human erythrocytes: a new kinetic analysis

1. Data on the transport of L-leucine into human erythrocytes at 25 degrees C shows (a) that the carrier re-orientation process is rate determining, (b) that the binding of leucine to the carrier increases the rate of carrier re-orientation and (c) that the carrier is, at equilibrium, about equally distributed between the membrane surfaces at 25 degrees C.2. These conclusions are reached through a new kinetic analysis of a simple carrier system, which involves no prior assumptions about the relative magnitudes of the rate constants, yet leads to usable kinetic equations. These equations allow the determination of the rate determining step, the calculation of the effect of bound substrate on the rate of carrier re-orientation and, in some cases, an estimate to be made of the equilibrium distribution of the carrier between the inner and outer membrane surfaces.

The temperature dependence of the transport of L-leucine in human erythrocytes.

1. Experiments are reported on the entry, exit and exchange fluxes of L-leucine in human erythrocytes in the temperature range 0-37 degrees C. The kinetics of entry and exit become quite different at low temperatures, indicating an asymmetry in the transport mechanism.2. The data is analysed in terms of a simple carrier mechanism. This shows that carrier re-orientation is rate determining at all temperatures studied, and suggests, although does not prove, that the origin of the asymmetry of the carrier may be an unequal equilibrium distribution of the carriers between inward and outward orientations.3. The activation energy increases sharply on going to low temperatures. This increase is the sum of two components. One arises from differences between the activation energies for inward and outward carrier re-orientations, and the other from changes, with temperature, in the activation energies of the individual carrier re-orientation steps.

Dark conduction through anthracene crystals

Dark currents through anthracene crystals with silver paste electrodes were studied as functions of temperature, applied voltage, and time. Activation energies between 0.5 eV and 2.9 eV were measured, there being a definite correlation between log �0 and E (the compensation effect'). However, it was shown that the compensation effect arises from the calculation of �0 and E, and not from a true linear free-energy relationship. As simple carrier generation mechanisms were inadequate to explain the temperature-dependence results, a model involving transitions between two limiting mechanisms was invoked and developed. The voltage-dependence results are shown to arise by a transition from SCL currents at low voltages, to barrier-limited currents (BLC) at higher fields, where the Schottky effect controls the current-voltage response. Vindication of the choice of this model is obtained from the calculated values of the parameters �, the permittivity of anthracene; A, the Richardson constant; �, the surface energy barrier for hole injection from silver paste into anthracene; and WE(Ag), the work function for silver paste electrodes.

Chain Conveyors for the Transport of Mail in Bags

The paper describes the adaptation of industrial chain conveyors for the task of moving mail bags, consequent upon the development of a cheap and simple self-gripping carrier for use with such bags. Elements of system design which particularly affect the mail-handling application are surveyed, together with environmental, safety, and economic considerations. Comparison is made of the relative merits of chain and belt conveyors, the latter being the only other broadly applicable alternative. An indication is given of possible lines of future development in the field of chain conveyors. The paper includes an appendix which outlines an empirical method for estimating tractive resistance and driving power requirements.

The Kinetics of Selective Biological Transport: IV. Assessment of Three Carrier Systems Using the Erythrocyte-Monosaccharide Transport Data

An assessment is made of the possible validity of three transport mechanisms proposed to describe the movement of monosaccharides in human erythrocytes. These mechanisms consist of a simple carrier system and two mechanisms derived from it, one of which postulates that the free carrier moves more slowly than the complexed carrier, and the other that slow diffusion steps occur at each surface of the membrane. None of these schemes provides an adequate quantitative description of the data so that all must be discarded, leaving the question of the mechanism still open to speculation.

Net flow and tracer flow in lattice and carrier models

Solute flux in the absence of active transport is analyzed in systems with discontinuity of chemical potential. Permeability coefficients for net flow ω and self-diffusion ( ω χ ) are expressed in terms of kinetic coefficients for adsorption (α), desorption (β), and transfer between sites ( k). For a 1-row lattice, a composite lattice-liquid membrane, and an n-row lattice without constraints, w = w x , both decreasing with increase of concentration. Restriction to “single file” movement, while not affecting net flow, induces positive coupling of isotopic flows ( w > w x ), disappearing at low concentrations. Switch (γ) between sites of a unit permits concentration-dependent conversion to negative coupling (“counter-transport”), as predicted previously for a divalent carrier. A simple univalent carrier shows negative coupling, which disappears (1) at low concentrations; (2) when transport of free carrier is much faster than that of loaded carrier ( k c ⪢ k); or (3) for k ⪢ β. Near equilibrium, both for continuous and discontinuous systems, w w x and the ratio of the logarithm of the flux ratio to the force are, equivalently, indices of coupling of isotopic flows (“isotope interaction”). Discontinuity of chemical potential per se does not produce isotope interaction or abnormality of the flux ratio.

The "dimeriser" hypothesis for sugar permeation through red cell membrane: reinvestigation of original evidence.

Abstract The original evidence for the operation of a “dimeriser” site in the human erythrocyte surface, in connection with facilitation of the passage of simple sugars through the cell membrane, has been experimentally re-examined and considerably extended. The course of total sugar uptake was followed photometrically, in mixtures of 21 pairs among nine monosaccharides, as a function of the varying proportions of the two sugars at a constant total level. The observed behavior was found to adhere closely to the quantitative predictions computed by numerical integration of rate equations derived from the simple monovalent carrier model proposed by Widdas , on the basis of previously estimated affinity constants for the various sugars, and a common rate constant for all. The most crucial of Stein 's dissonant conclusions from similar experiments, involving a systematic deviation of the initial cell swelling rates from this pattern, are shown to be accountable by errors inherent in the methods by which these data were analyzed. It is concluded that, although this type of experimental approach does not definitively exclude the possibility that the transport sites may be divalent for the substrate sugars, it provides no basis whatever for advancing this hypothesis.

The Kinetics of Selective Biological Transport: II. Equations for Induced Uphill Transport of Sugars in Human Erythrocytes

Equations describing the movement of sugars during induced uphill transport were derived on the assumption of a simple carrier transport mechanism and subjected to experimental verification. Since there was good agreement between the experimental points and the theoretical curves, no changes in the original postulates were required.