Iodide transport in isolated cells of mouse submaxillary gland

A method has been developed to isolate cells from the submaxillary gland of mouse by treatment with pronase. Three fractions of cells have been isolated having almost equal iodide concentrating activity. The isolated cells show time dependent uphill transport of iodide. The transport is substrate-saturable, having aK _m value of 0.3 μM for iodide. The transport is sensitive to antithyroid drugs, metabolic inhibitors and to some extent to ouabain. Pseudohalide such as thiocyanate competes with the transport of iodide. Thyroid hormones or thyroid stimulating hormone have no significant effect on the iodide transport in these cells.     

Binding dynamics of isolated nucleoporin repeat regions to importin-beta

The nuclear pore complex, through the interaction of its proteins with transport receptors, controls the transport of large molecules into and out of the cell's nucleus. There is ample evidence for proteins with FG sequence repeats playing an essential role in this control. Previous studies have elucidated binding spots for FG sequence repeats on the surface of the transport receptor importin-beta by X-ray crystallography and mutational studies. Molecular dynamics simulations have been performed to characterize the interaction of FG sequence repeats with the transport receptor. Observed binding spots have been verified and novel sites discovered, suggesting that importin-beta features many more binding spots than suspected so far. The observed binding spots are in accord with several models of nucleocytoplasmic transport, and the large number of binding spots on importin-beta may be necessary for the pore complex to distinguish between importin-beta and inert proteins, and to allow for its passage through the pore.     

How a soluble enzyme can be forced to work as a transport system: description of an experimental design

The cellular transport systems which have been studied up to now have been found to be based on the functioning of specialized proteins anchored asymmetrically in cell membranes. In the present paper we show that a single soluble enzyme inserted at random in a gel slab can drive an uphill transport, provided that asymmetrical boundary conditions force the reversible reaction catalyzed by this enzyme to work forward on one face of the gel slab and backward on the other face. Experimentally, we have used a yeast alcohol dehydrogenase to induce an uphill transport of NADH. It cannot be excluded that comparable structurally symmetrical transport systems also exist in living cells. Such systems would be particularly well suited to preserving cell homeostasis with regard to small solutes.     

Unsedated animal models of pulmonary mucociliary transport

Measurements of the rate of mucociliary transport in the airways of the lower respiratory tract have been shown to be influenced by the techniques and protocols used. To avoid the effects associated with invasive techniques and anesthesia in animal models used to study the effect of maturation, drugs, disease, and inhaled pollutants on mucociliary transport we have developed unsedated dog and baboon models of mucociliary transport using radioaerosol techniques. As far as they have been tested these animal models of mucociliary transport react to drugs such as isoproterenol and atropine in the same manner as in man.     

Phosphate transport processes in eukaryotic cells

Conclusion Much more work has been done on P_i transport processes, even in the last five years, than we have been able to mention in the space available. We have restricted our discussion to studies on mechanisms of transport or transport regulation, identification of transport proteins and their essential amino acids, and isolation, purification, and reconstitution of P_i transport systems. Many valuable studies on the physiology of P_i transport and its regulation and P_i transport in nonepithelial cells have also been conducted. Transport of P_i into and out of organelles other than the mitochondrion is gaining well-deserved attention, as are transport processes in fungi and plants. It is hoped that in another five years many P_i transport processes will be understood in true molecular terms and that this will increase our knowledge of cellular bioenergetics and metabolism.     

Transport pathways of macromolecules between the nucleus and the cytoplasm.

Transport between the nucleus and cytoplasm involves both stationary components and mobile factors acting in concert to move macromolecules through the nuclear pore complex. Multiple transport pathways requiring both unique and shared components have been identified. In the past 18 months, new findings have shed light on the nature of some of the mobile components of these pathways. New receptor-cargo pairs for both import and export pathways have been identified extending the breadth of known transport pathways. Surprising findings on the role of Ran and energy in transport have changed our way of thinking about the mechanism of movement through the nuclear pore.     

Nucleocytoplasmic transport of macromolecules.

Nucleocytoplasmic transport is a complex process that consists of the movement of numerous macromolecules back and forth across the nuclear envelope. All macromolecules that move in and out of the nucleus do so via nuclear pore complexes that form large proteinaceous channels in the nuclear envelope. In addition to nuclear pores, nuclear transport of macromolecules requires a number of soluble factors that are found both in the cytoplasm and in the nucleus. A combination of biochemical, genetic, and cell biological approaches have been used to identify and characterize the various components of the nuclear transport machinery. Recent studies have shown that both import to and export from the nucleus are mediated by signals found within the transport substrates. Several studies have demonstrated that these signals are recognized by soluble factors that target these substrates to the nuclear pore. Once substrates have been directed to the pore, most transport events depend on a cycle of GTP hydrolysis mediated by the small Ras-like GTPase, Ran, as well as other proteins that regulate the guanine nucleotide-bound state of Ran. Many of the essential factors have been identified, and the challenge that remains is to determine the exact mechanism by which transport occurs. This review attempts to present an integrated view of our current understanding of nuclear transport while highlighting the contributions that have been made through studies with genetic organisms such as the budding yeast, Saccharomyces cerevisiae.     

Modification of ion transport in lipid bilayer membranes in the presence of 2,4-dichlorophenoxyacetic acid. I. Enhancement of cationic conductance and changes of the kinetics of nonactin-mediated transport of potassium.

We have found that herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) has the ability to increase the rate of transport of positive ions of several kinds, and to inhibit transport of negatively charged tetraphenylborate ions in lipid bilayer membranes. It has been found that only the neutral form of 2,4-D is transport active, whereas the ionized from of 2,4-D does not modify transport of ions, and does not by itself permeate through lipid membranes. The results suggest that the enhancement of transport of positively charged ions such as tetraphenylarsonium + and nonactin-K+ is dominated by the increase of the ion translocation rate constant. It has been shown that the enhancement of nonactin-mediated transport of K+ by 2,4-D can be accounted for by a simple carrier model. We have observed that a 2,4-D concentration above 3 X 10(-4) M the potassium ion transport in phosphatidylcholine-cholesterol as well as in cholesterol-free glycerolmonooleate membranes is enhanced to such a degree that, depending upon the concentration of potassium ions, it becomes limited by the rate of recombination of K+ with nonactin, and/or by backdiffusion of unloaded nonactin molecules. Furthermore, the effect of 2,4-D is enhanced by ionic strength of aqueous solution. From the changes of kinetic parameters of nonactin-K+ transport, as well as from the changes of membranes conductance due to tetraphenylarsonium + ions, we have estimated the changes of the electrical potential of the membrane interior. We have found that the potential of the interior of the membrane becomes more negative in the presence of 2,4-D, and that its change is proportional to the aqueous concentration of 2,4-D. The effect of 2,4-D on ion transport has been attributed to a layer of 2,4-D molecules absorbed within the interfacial region, and having a dipole moment directed toward the aqueous medium. The results of kinetic studied of nonactin-K+ transport suggest that this layer is located on the hydrocarbon side of the interface.     

On the multiplicity of glucose analogues transport systems in rat intestine.

A study has been made to test if in intact epithelium of rat jejunum with in vivo and in vitro techniques, two transport systems for glucose and analogues, as those characterized in brush border membrane vesicles from guinea pig jejunum, are operative. The passive and mediated transport components of the D-galactose and methyl alpha-D-glucopyranoside intestinal absorption and the mutual inhibitions between both substrates at different relative concentrations have been measured. The effects of cytochalasin B and low temperature (20 degrees C) on the transport in vitro have also been observed. Cytochalasin B inhibits galactose and alpha-methylglucoside transport at 0.1 and 40 mM concentrations in similar percentage. Transport of 0.1 and 40 mM galactose is inhibited 61 and 77% respectively by low temperature (20 degrees C). The transport of galactose and alpha-methylglucoside could be explained by the assumption of just one transport system shared by both substrates, with a higher affinity for alpha-methylglucoside. Operation of two systems was not demanded by the results, due perhaps to species specificity or to the distorting action of the unstirred water layers.     

Effectors of amino acid transport processes in animal cell membranes

Various effectors, which act upon ion gradients, protein synthesis, membrane components or cellular functional groups, have been employed to provide insights into the nature of amino acid-membrane transport processes in animal cells. Such effectors, for example, include ions, hormones, metabolites and various organic reagents and their judicious use has allowed the following list of conclusions. Sodium ion has been found to stimulate amino acid transport in a wide variety of cell systems, although depending on the tissue and/or substrate, this ion may have no effect on such transport, or even inhibit it. Amino acid transport can be stimulated in some cell systems by other ions such as K+, Li+, H+ or Cl-. Both H+ and K+ have been found to be inhibitory in other systems. Amino acid transport is dependent in many cell systems upon an inwardly directed Na+ gradient and is stimulated by a membrane potential (negative cell interior). In some cell systems an inwardly directed Cl- and H+ gradient or an outwardly directed K+ gradient can energize transport. Structurally dissimilar effectors such as ouabain, Clostridium enterotoxin, aspirin and amiloride inhibit amino acid transport presumably through dissipation of the Na+ gradient. Inhibition by certain sugars or metabolic intermediates of the tricarboxylic acid cycle may compete with the substrate for the energy of the Na+ gradient or interact with the substrate at the carrier level either allosterically or at a common site. Stimulation of transport by other sugars or intermediates may result from their catabolism to furnish energy for transport. Insulin and glucagon stimulate transport of amino acids in a variety of cell systems by a mechanism which involves protein synthesis. Microtubules may be involved in the regulation of transport by insulin or glucagon. Some reports also suggest that insulin has a direct effect on membranes. In addition, a number of growth hormones and factors have stimulatory effects on amino acid transport which are also mediated by protein synthesis. Steroid hormones have been noted to enhance or diminish transport of amino acids depending on the nature of the hormone. These agents appear to function at the level of protein synthesis. While stimulation may involve increased carrier synthesis, inhibition probably involves synthesis of a labile protein which either decreases the rate of synthesis or increases the rate of degradation of a component of the transport system.(ABSTRACT TRUNCATED AT 400 WORDS)     

Problems and perspectives of the Na-pump reconstitution.

The review deals with basic criteria of reconstruction approach to the study of membrane functions of the cell. Possible methods of reconstruction for solution of the problems of active ion transport have been demonstrated on the example of Na, K-ATPase. The alternative ways of the study which open molecular mechanisms of work of a system of active transport of Na+ and K+ ions through the cell membrane have been analyzed.     

Impact of live cell imaging on coated vesicle research

The role of membrane traffic is to transfer cargo between distinct subcellular compartments. Each individual trafficking event involves the creation, transport and fusion of vesicular and tubular carriers that are formed and regulated via cytoplasmic coat protein complexes. The dynamic nature of this process is therefore highly suitable for studying using live cell imaging techniques. Although these approaches have raised further questions for the field, they have also been instrumental in providing essential new information, in particular relating to the morphology of transport carriers and the exchange kinetics of coat proteins and their regulators on membranes. Here, we present an overview of live cell-imaging experiments that have been used in the study of coated-vesicle transport, and provide specific examples of their impact on our understanding of coat function.     

Transport processes in Plasmodium falciparum-infected erythrocytes: potential as new drug targets

Plasmodium falciparum infection induces alterations in the transport properties of infected erythrocytes that have recently been defined using electrophysiological techniques. Mechanisms responsible for transport of substrates into intraerythrocytic parasites have also been clarified by studies of three substrate-specific (hexose, nucleoside and aquaglyceroporin) parasite plasma membrane transporters. These have been characterised functionally using the Xenopus laevis oocyte heterologous expression system. The same expression system is currently being used to define the function of parasite 'P' type ATPases responsible for intraparasitic [Ca(2+)] homeostasis. We review studies on these transport processes and examine their potential as novel drug targets.     

Na, Cl, and Water Transport by Rat Ileum in Vitro

Interrelationships between metabolism, NaCl transport, and water transport have been studied in an in vitro preparation of rat ileum. When glucose is present in the mucosal solution, Na and Cl both appear to be actively transported from mucosa to serosa while water absorption is passive and dependent on net solute transport. Removal of glucose from the mucosal solution or treatment with dinitrophenol, monoiodoacetate, or anoxia inhibits active salt transport and as a result, water absorption is also inhibited. The dependence of water absorption on metabolism can be explained as a secondary effect due to its dependence on active salt transport. The relationship between salt and water transport has been discussed in terms of a model system.     

Traffic jams II: an update of diseases of intracellular transport

As more details emerge on the mechanisms that mediate and control intracellular transport, the molecular basis for variety of human diseases has been revealed. In turn, disease pathology and physiology shed light on the intricate controls that regulate intracellular transport to assure proper cellular and tissue function and homeostasis. We previously listed a number of diseases that are the result of defects in intracellular transport, or cause defects in intracellular transport. (Aridor M, Hannan LA. Traffic Jam: A compendium of human diseases that affect intracellular transport processes. Traffic 2000; 1: 836–851). This Toolbox updates the previous list to include additional disorders that were recently identified to be related to intracellular trafficking. In the time since we have published our first list there have been significant advances in understanding of the molecular basis of these defects. Such advances will pave the way to future effective therapeutics.     

The use of the logarithmic transformation in the calculation of the transport parameters of a system that obeys Michaelis–Menten kinetics

1. A logarithmic method is described for the calculation of the transport parameters, K(m) and V(max.)' of a biological system obeying Michaelis-Menten kinetics. 2. This logarithmic method leads to a way of estimating the transport parameters that has not apparently been used previously. It allows the separation of variance due to V(max.) from other variance, and so reduces the fiducial limits that can be placed on an estimation of K(m). 3. The results of studies on the transport of l-histidine and l-monoiodohistidine by rat intestinal sacs in vitro have been used to illustrate the application of the new method. Estimates of the transport parameters have also been made by two alternative procedures. The relative merits of the three methods are discussed.     

New movements in neurofilament transport, turnover and disease

Revealing the mechanisms by which neurofilament transport and turnover are regulated has proven difficult over the years but recent studies have given new insight into these processes. Mature neurofilament fibers may incorporate a fourth functional subunit, alpha-internexin, as new evidence suggests. Recent findings have made the role of phosphorylation in regulating neurofilament transport velocity controversial. Kinesin and dynein may transport neurofilaments in slow axonal transport as they have been found to associate with neurofilaments. Neurofilament transport and turnover rates may be reduced depending on the existing stationary neurofilament network. Finally, mutations in neurofilament light that have been linked to Charcot-Marie-Tooth disease as well as other neurofilament abnormalities in human disease are discussed.     

What we can learn from the effects of thiol reagents on transport proteins.

Many secondary membrane transport systems contain reactive sulfhydryl groups. In this review the applications of SH reagents for analyzing the role of sulfhydryl groups in membrane transport systems will be discussed. First an overview will be given of the more important reagents, that have been used to study SH-groups in membrane transport systems, and examples will be given of transport proteins in which the role of cysteines have been analyzed. An important application of SH-reagents to label transport proteins using various SH-reagents modified with fluorescent- or spin-label moieties will be discussed. Two general models are shown which have been proposed to explain the role of sulfhydryl groups in some membrane transport systems.     

The effect of dimethyl sulphoxide on electron transport in chloroplasts

The effect of DMSO (dimethyl sulphoxide) on electron transport in chloroplast membranes has been studied. It has been found that concentrations of DMSO up to 20% (v/v) do not inhibit electron transport in freshly isolated chloroplasts, but that higher concentrations start to cause inhibition. However, in chloroplasts that have been aged for 8 to 24 hours by storage at 4 degrees C, the addition of DMSO at concentrations up to 20% causes stimulation of electron transport. Possible mechanisms for this effect are discussed.     

Auxin Transport Inhibitors: IV. EVIDENCE OF A COMMON MODE OF ACTION FOR A PROPOSED CLASS OF AUXIN TRANSPORT INHIBITORS: THE PHYTOTROPINS

The more active members of a proposed class of auxin transport inhibitors have been shown to have the ability to inhibit the active movement of auxin at concentrations where they have little effect on auxin action and no significant auxin activity. They have also been shown to give rise to characteristic biphasic dose-response curves on cress root growth. Based on these physiological similarities and other common physiological properties, it is concluded that they may achieve their effects by a common mode of action which differs from that of other known auxin transport inhibitors. It is suggested that the name "phytotropins" be given to the class of auxin transport inhibitors now defined by a similar mode of action and common chemical properties.