Approaches used to examine the mechanism and regulation of hexose transport in rat myoblasts

This review discusses some of the approaches and general criteria that we have used to examine the properties of the hexose transport system in undifferentiated L6 rat myoblasts. These approaches include studying the kinetics of hexose transport in whole cells and plasma membrane vesicles, the effects of various inhibitors on hexose transport, the isolation and characterization of hexose transport mutants, and the use of cytochalasin B (CB) to identify the transport component(s). Transport kinetics indicated that two transport systems are present in these cells. 2-Deoxy-D-glucose is transported primarily by the high affinity system, whereas 3-O-methyl-D-glucose is transported by the low affinity system. Furthermore, these two transport systems are inactivated to different extents by CB. CB has a higher binding affinity for the low affinity hexose transport system. The inhibitory effect of various hexose analogues also revealed the presence of two hexose transport systems. The effects of various ionophores and energy uncouplers on hexose transport suggest that the high affinity system is an active transport process, whereas the low affinity system is of the facilitated diffusion type. The high affinity system is also sensitive to sulfhydryl reagents, whereas the low affinity system is not. Further evidence for the presence of two transport systems comes from the characterization of hexose transport mutants. Two of the mutants isolated are shown to be defective in the high affinity transport system, but not in the low affinity transport system. These mutants are also defective in the CB low affinity binding site. Based on our results a tentative working model for hexose transport in L6 rat myoblasts is presented.     

Life Cycle Inventories of Transport Services: Background Data for Freight Transport (10 pp)

Background, Goal and Scope The ecoinvent database is a reference work for life cycle inventory data covering the areas of energy, building materials, metals, chemicals, paper and cardboard, forestry, agriculture, detergents, transport services and waste treatment. Generic inventories are available for freight and passenger transport including air, rail, road, and water transport. The goal of freight transport modelling is to provide background data for transport services, which occur between nearly any two process steps of a product system. This paper presents and discusses the model structure, basic assumptions and results for selected freight transport services.Main Features Transport services are divided into several datasets referred to as transport components. In addition to vehicle operation (comprising vehicle travel and pre-combustion), infrastructure processes such as vehicle maintenance, manufacturing and disposal, as well as transport infrastructure construction, operation and disposal, are also modelled. In order to link the various transport components to the functional unit of one tonne kilometre (tkm), so-called demand factors are determined. In the case of transport infrastructure that is not exclusively used by freight transport, allocation is essential. The respective allocation parameters employed for line infrastructure construction/disposal and operation datasets (including land use) are yearly Gross-tonne kilometre performance (Gtkm) and kilometric vehicle/train performance. Results are presented for selected environmental exchanges related to gaseous emissions (climate change gases, nitrogen oxides, and hydrocarbons), heavy metal (zinc and cadmium) emissions to soil and air, as well as BOD (Biological Oxygen Demand), and land use. Particle emissions are further distinguished into fine (PM2.5) and coarse (diameter between 2.5 and 10 µm) particles. The results presented comprise both an intra- and inter-modal comparison.Results and Discussions A comparison of Swiss and European rail transport reveals considerably lower emissions from Swiss rail transport due to the almost exclusive use of hydropower as traction energy. For gaseous emissions, freight transport by water or rail exhibits considerably better performance than road transport (65-92% less gaseous emissions). As far as zinc and cadmium emissions to soil are concerned, water and rail transport produce less than 1% of the emissions resulting from road transport for either pollutant. For zinc and cadmium emissions to air, road transport has the highest emissions; however, the emissions due to water and rail transport range from 2 to 18% of the emission levels arising from road transport. Particle emissions show a more diverse pattern. Whilst fine particle emissions due to water and rail transport are considerably lower than road transport, rail transport with respect to coarse particles performs worse than road transport. Dominance analysis reveals the importance of infrastructure processes. For instance, the NMHC-emissions of infrastructure processes account for 40%, 30% and 50% of emissions for road, rail and barge transport, respectively. For the demand factor of infrastructure operation, a sensitivity analysis of the employed allocation factor was performed, revealing no sensitivity for gaseous emissions and particles. On the other hand, considerable changes in both emission levels and in the ranking of transport modes is observed for land occupation. Finally, we varied selected operation parameters for road transport, resulting in considerable reductions of CO2 and NOX emissions of up to 60%. In one extreme case (load factor: 100%), NOx emissions for vehicle operation of a lorry are lower than for inland water transport. Only as a result of the considerably higher NOx emissions occurring in infrastructure processes does road transport score worse than water transport, with the ranking remaining the same as for the generic data presented in ecoinvent 2000.Conclusions and Perspectives The provided datasets allow for a preliminary screening of the importance of transport processes within a product life cycle. In the cases for which transport processes are identified as sensitive for the overall outcome of certain product life cycle or for transport specific comparisons, the modular structure and transparent documentation of demand factors allows for an easy and transparent integration of more case-specific data for selected transport components.     

Diffusion and the limiting substrate in two-substrate immobilized enzyme systems

The effects of mass transport resistances on two-substrate immobilized enzyme systems are investigated theoretically. It is shown that the effects of mass transport resistances on the overall reaction rate are related mainly to the transport of the limiting substrate. In the absence of external mass transport resistances, the limiting substrate can be identified by knowing only the ratio of the bulk substrate concentrations, the permeability of the support to the two substrates, and the stoichiometry of the reaction. However, a combination of internal and external mass transport resistances may result in the other substrate becoming limiting. These effects are most significant when the mass transport resistances are high. Applications in the design of enzyme electrodes and chemical reactors are discussed.     

Microtubules and the capacity of the system for rapid axonal transport

Current information favors the view that microtubules are required for rapid axonal transport of proteins and organelles but are normally present in surplus. Different types of axons tolerate losses of between 35 and 65% of their microtubules during exposure to low temperatures or antimitotic drugs before transport is impaired. Greater losses of microtubules are associated with progressive and marked failure of transport. The normal surplus of microtubules may explain why adrenergic axons of rabbit peroneal nerve have spare transport capacity, which enables them to transport between two and three time as much material as they do ordinarily. Spare capacity for transport is diminished or absent when nerves are incubated at temperatures that lead to a partial loss of microtubules. These observations are considered in the light of the hypothesis that the local density of microtubules determines the maximal local concentration of material that can be carried by rapid transport along vertebrate axons.     

Negative interactions between amino acid and methylamine/ammonia transport systems of Saccharomyces cerevisiae.

The transport of methylamine (methylammonium ion) and ammonia (ammonium ion) is accomplished in Saccharomyces cerevisiae by means of a specific active transport system. L-Amino acids are noncompetitive inhibitors of methylamine transport. This inhibition is relieved or eliminated in mutant strains that have a reduced ability to transport amino acids. The inhibition of methylamine transport occurs immediately upon the addition of amino acids to the assay system and persists until the external amino acid pool is depleted. The degree of inhibition observed is a direct function of the rate of amino acid transport. Both methylamine and ammonia are capable of inhibiting amino acid transport. The inhibition of amino acid transport is eliminated in mutant strains that cannot transport methylamine and ammonia.     

Mechanisms for the facilitated diffusion of substrates across cell membranes

Two classes of theoretical mechanisms for protein-mediated, passive, transmembrane substrate transport (facilitated diffusion) are compared. The simple carrier describes a carrier protein that exposes substrate influx and efflux sites alternately but never both sites simultaneously. Two-site models for substrate transport describe carrier proteins containing influx and efflux sites simultaneously. Velocity equations describing transport by these mechanisms are derived. These equations take the same general form, being characterized by five experimental constants. Simple carrier-mediated transport is restricted to hyperbolic kinetics under all conditions. Two-site carrier-mediated transport may deviate from hyperbolic kinetics only under equilibrium exchange conditions. When both simple- and two-site carriers display hyperbolic kinetics under equilibrium exchange conditions, these models are indistinguishable by using steady-state transport data alone. Seven sugar transport systems are analyzed. Five of these systems are consistent with both models for sugar transport. Uridine, leucine, and cAMP transport by human red cells are consistent with both simple- and two-site models for transport. Human erythrocyte sugar transport can be modeled by simple- and two-site carrier mechanisms, allowing for compartmentalization of intracellular sugars. In this instance, resolution of the intrinsic properties of the human red cell sugar carrier at 20 degrees C requires the use of submillisecond transport measurements.     

Characterization of mouse lymphoma cells with altered nucleoside transport

A mutant clone (NT-1) of a T-cell lymphoma was selected for its ability to grow in HAT medium (hypoxanthine, aminopterin and thymidine) in the presence of the nucleoside transport inhibitor P-nitrobenzyl-6-mercaptoinosine (NBMI). NT-1 cells contain half the number of NBMI binding sites present on the parental S49 cells and are partially able to transport nucleosides in the presence of the transport inhibitor (NBMI). These observations suggest that the mutant cells are heterozygous for nucleoside transport proteins and contain two types of transport proteins: the first protein can both bind and is inhibited by NBMI similar to the wild type phenotype, and the second is an altered protein. The altered transport protein apparently lost its NBMI binding sites without a parallel loss of nucleoside transport ability suggesting that the nucleoside transported sites are separate from the binding sites of the transport inhibitor.     

Analysis of protein-mediated 3-O-methylglucose transport in rat erythrocytes: rejection of the alternating conformation carrier model for sugar transport

3-O-Methylglucose (3OMG) transport in rat erythrocytes (RBCs) is mediated by a low-capacity, facilitated diffusion-type process. This study examines whether the characteristics of sugar transport in rat RBCs are consistent with the predictions of two diametric, theoretical mechanisms for sugar transport. The one-site carrier describes a transport mechanism in which sugar influx and efflux substrate binding sites are mutually exclusive. The two-site carrier describes a transport mechanism in which sugar influx and efflux substrate binding sites can exist simultaneously but may interact in a cooperative fashion when occupied by substrate. Michaelis and velocity parameters for saturable 3OMG transport in rat erythrocytes at 24 degrees C were obtained from initial rate measurements of 3OMG transport. The results are incompatible with the predictions of the one-site carrier but are consistent with the predictions of a symmetric two-site carrier, displaying negligible cooperativity between substrate binding sites. This allows reduction of the two-site carrier transport equations to a form containing fewer constants than the one-site carrier equations without limiting their predictive success. While the available evidence does not prove that rat erythrocyte sugar transport is mediated by a two-site mechanism, we conclude that adoption of the formally more complex one-site model for sugar transport in rat erythrocytes is unnecessary and unwarranted. Counterflow experiments have also been performed in which the time course of radiolabeled 3OMG uptake is measured in cells containing saturating levels of 3OMG. The results of these experiments are consistent with the hypothesis [Naftalin et al. (1985) Biochim. Biophys. Acta 820, 235-249] that exchange of sugar between intracellular compartments (cell water and hemoglobin) can be rate limiting for transport under certain conditions.     

Alzheimer's disease: an intracellular movement disorder?

Axonal transport is essential for maintaining the structure and function of nerve cells. Deficient axonal transport has been implicated in several neurodegenerative diseases, including Alzheimer's disease (AD). In addition to a disturbed cytoskeleton and other abnormalities observed in AD that are suggestive of axonal transport deficits, several AD-related proteins are implicated in the regulation of axonal transport. A recent study has demonstrated that the axonal transport deficit occurs early in the course of AD, preceding amyloid pathology substantially in mouse models of AD; more importantly, the study showed that reduced axonal transport leads to increased amyloid beta production and deposition. These data place axonal transport deficits at a central point in the pathogenesis of AD.     

Two distinct states of sugar transport system in cultures of BHK cells

The sugar transport of growing and quiescent cultures of BHK-21 cells is studied by the equilibrium exchange method. Two distinct components of sugar transport can be detected. One component displays fast transport rates and is evident in cells at low cell density. The other displays slow transport properties and is typical of quiescent cells. In the course of increase in cell density or following serum-activation of quiescent cells, these two components are present in the same cell-culture. The two components of transport are interpreted as resulting from the presence of two types of cells, one in a “fast” and the other in a “slow” transport state. The transition in each cell from one state of transport into the other appears to be a discrete and sudden event. The gradual change in the cell population results from a change in the number of cells in each state. Cells in the fast transport state show a saturable and a non saturable component of sugar transport. Cells in the slow transport state display only a non saturable component.     

Continuous monitoring of transport by fluorescence on cells and vesicles

Fluorescence techniques are gaining wider applicability in the field of membrane transport due to their high temporal resolution, modest demand for biological material and the kinetic information which is made available by fluorescence tracings. The development of novel fluorescent substrates for particular transport systems and of novel fluorescent indicators for permeant ions, have opened the way for studying transport kinetics and regulation of transport in a variety of cellular and vesicular systems. The various methods of continuous monitoring of transport by fluorescence (CMTF) which are presently in use, are reviewed with emphasis on both analytical and applicative properties.     

Periplasmic binding protein-dependent transport systems: the membrane-associated components

Periplasmic binding protein-dependent transport systems are multicomponent, consisting of several inner membrane-associated proteins and a periplasmic component. The membrane-associated components of different systems are related in organization and function suggesting that, despite different substrate specificities, each transport system functions by a common mechanism. Current understanding of these components is reviewed. The nature of energy coupling to periplasmic transport systems has long been debated. Recent data now demonstrate that ATP hydrolysis is the primary source of energy for transport. The ATP-binding transport components are the best characterized of a family of closely related ATP-binding proteins believed to couple ATP hydrolysis to a variety of different biological processes. Intriguingly, systems closely related to periplasmic binding protein-dependent transport systems have recently been identified in several Gram-positive organisms (which lack a periplasm) and in eukaryotic cells. This class of transport system appears to be widespread in nature, serving a variety of important and diverse functions.     

The relationship between nuclear-cytoplasmic transport and chromosome segregation

Chromosomes/chromatids transport to cell division poles (chromosome segregation) and nuclear-cytoplasmic transport give way to each other during cell cycle. Disassembly and reassembly of the nuclear envelope during input or output mitosis are the critical time periods for changing one kind of transport system for the other one. The interest in the problem of relationships between nuclear-cytoplasmic transport and chromosome segregation came into view after revealing some factors involved in both transport systems. Among these factors are GTPase Ran, nuclear transport receptors and associated molecules, kinetochore proteins and proteins of nuclear pore complexes (nucleoprin, for example), spindle microtubules, microtubule motor proteins, and signal molecules. The role of all of these factors in the transport systems is not known. A decision of this problem will make it possible to determine mechanisms of both transport systems and to understand the character of evolutionary relationships between these systems.     

Low molecular weight GTP-binding proteins are associated with neuronal organelles involved in rapid axonal transport and exocytosis

Recent evidence suggests that low molecular weight GTP-binding proteins may play important roles in a variety of membrane transport processes. In order to address the question of whether these proteins are involved in transport processes in the nerve axon, we have assessed their presence in rapid transport membranes from rabbit optic nerve. We report the characterization of a group of low molecular weight GTP-binding proteins which are constituents of rapid transport vesicles. Although these proteins are components of rapid transport vesicles, they are apparently not major rapidly transported species. They are localized in cytosolic as well as in membrane fractions of axons, and the membrane-associated form behaves as an integral membrane protein(s). These proteins are also found in association with a variety of vesicular and organellar components of neurons including coated vesicles, synaptic vesicles, synaptic plasma membranes, and mitochondria. We discuss the possible roles of these proteins in rapid axonal transport and exocytosis.     

Transport of C4-dicarboxylic acids in salmonella typhimurium.

C₄-Dicarboxylic acids are transported into Salmonella typhimurium by stereospecific systems of both high and low affinity. Succinate and l-malate are accumulated in a tricarboxylic acid cycle mutant as was d(+)-malate in induced wild-type cells. Accumulated dicarboxylates are exchangeable with exogenous dicarboxylates. The trichloroacetic acid cycle dicarboxylates are the best inducers of their own transport. Specific mutants devoid of dicarboxylate transport activity (dct) were isolated and differed from tricarboxylate transport mutants (tct) with respect to growth and transport. A mutant devoid of α-ketoglutarate dehydrogenase was unable to transport dicarboxylic acids but citrate transport remained unaffected. Tricarboxylic acid cycle mutants were markedly dependent on an exogenous energy source for the transport of succinate, proline, or leucine. Dicarboxylate transport was largely inhibited by various metabolic inhibitors but could only be inhibited by N,N'-dicyclohexylcarbodiimide anaerobically. ATPase mutants were unimpaired in their ability to transport succinate or proline aerobically.     

Secondary active transport

Secondary active transport is defined as the transport of a solute in the direction of its increasing electrochemical potential coupled to the facilitated diffusion of a second solute (usually an ion) in the direction of its decreasing electrochemical potential. The coupling agents are membrane proteins (carriers), each of which catalyzes simultaneously the facilitated diffusion of the driving ion and the active transport of a given solute. The review starts with some considerations on the energetics followed by a presentation of the kinetics of secondary active transport. Examples of information which may be gained by such studies are discussed. In the second part, some examples of secondary transport are given; we also describe the characteristics of the corresponding carriers. The various transport systems presented are: the D-glucose/Na+ symport in brush-border membranes, the lactose/H+ symport in E. coli, the Na+/H+ antiport, the different transport systems in the inner mitochondrial membrane.     

Slow axonal transport: the subunit transport model

A central problem concerning slow transport of cytoskeletal proteins along nerve axons is where they are assembled and the form in which they are transported. The polymer and subunit transport models are the two major hypotheses. Recent developments using molecular and cellular biophysics, molecular cell biology and gene technology have enabled visualization of moving forms of cytoskeletal proteins during their transport. Here, we argue that these studies support the subunit transport theory.     

Transport changes associated with growth control and malignant transformation.

We can distinguish two classes of membrane transport changes in cultured cells: (a) growth-rate contingent changes are those which occur in coordination with the onset of density-dependent inhibition of growth; (b) transformation-specific changes are those which occur when cells become transformed, and which can be detected even when normal and transformed cells are growing at the same rate. Growth-rate contingent changes include the density-dependent changes in phosphate, nucleoside, glucose, amino acid, and potassium transport. Only one transformation-specific transport change has been found in Rous-transformed chicken embryo fibroblasts: an increased rate of hexose transport. The variation in potassium transport are associated with variations in the number of ouabain binding sites in the membrane. The molecular basis for changes in the rate of hexose transport is unknown, although gross changes in membrane bilayer composition and "fluidity" seem not to be involved. In analyzing the regulation of hexose transport activity, we find that decreased cAMP may play a role in the transformation-specific increase in hexose transport, but that fibrinolytic activity is not necessary.     

Strategies to identify transport systems in plants

Since the first molecular structures of plant transporters were discovered over a decade ago, considerable advances have been made in the study of plant membrane transport, but we still do not understand transport regulation. The genes encoding the transport systems in the various cell membranes are still to be identified, as are the physiological roles of most transport systems. A wide variety of complementary strategies are now available to study transport systems in plants, including forward and reverse genetics, proteomics, and in silico exploitation of the huge amount of information contained in the completely known genomic sequence of Arabidopsis.     

The molecular control of transport vesicle fusion

The fusion of transport vesicles with the appropriate target membrane in constitutive transport is a complex and well-controlled process. Many of the molecular details of the reactions that result in this control are being revealed through the use of cell-free assays of protein transport as well as by the study of the molecular genetics of secretion in yeast. Kinetic analyses have indicated that several structural intermediates are formed after transport vesicles attach to their destination, but before they fuse with the appropriate membrane. Proteins that mediate the formation and processing of these intermediates have been identified. Included among these are small molecular weight GTP-binding proteins. This intricate set of reactions may ensure the fidelity of transport and guard the integrity of the organelles along the transport pathway.