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.     

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.     

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.     

A new estimation method for plant cell viability by determining electron transport activity

A new method with which to estimate the viability of tissue-cultured plant cells was developed. In this method, electrons from the electron transport system of Coptis japonica cells were trapped by artificial electron acceptors, and the color of the reduced acceptor was monitored with a spectrophotometer through an optical fiber as surface-reflected light. Cell viability is represented by the amount of increased reflected light per unit time as electron transport activity (ETA). The electron transport activities of cultured Coptis japonica cells that had been effected in viability by the addition of different concentrations of a microbial broth, were related to the ability of the cells to proliferate. When various microbial broths were added to our Coptis japonica cultures, there was a negative correlation between electron transport activity and the amount of berberine released. During usual subculture, electron transport activity increased from the onset of culture, reached a maximum in the late log phase, then decresed rapidly.     

Application of spin-echo nuclear magnetic resonance to whole-cell systems. Membrane transport.

A new method for studying membrane transport is presented. High resolution n.m.r. is used to measure the distribution of small molecules between the intracellular and extracellular compartments. The method uses spin-echo techniques and relies on a difference in the magnetic susceptibility of the media inside and outside of cells. It also provides simultaneous information on the metabolic status of the cell. The method is illustrated by a study of alanine and lactate transport in the human erythrocyte.     

Stimulatory effect of lithium ion on proline transport by whole cells of Escherichia coli.

The effect of monovalent cations on proline transport in whole cells of Escherichia coli K-12 has been examined. Lithium ion added to the uptake medium stimulated proline transport severalfold and K+ and Na+ were slightly effective, whereas Rb+, Cs+, and NH4+ were completely without effect. The stimulatory effect of Li+ on proline transport was not due to an increase in osmolarity of the uptake medium, and d 5 mM p-chloromercuribenzene sulfonic acid completely blocked this effect of Li+ without having any effect on the basal rate of proline transport. The Arrhenius plots for Li+-stimulated transport showed a clear transition point at 35 degrees C in addition to 20 degrees C which was also detectable in the basal transport. Lithium ion stimulated proline transport synergistically in the presence of glucose and succinate as a carbon source. The addition of 2.5 mM KCN or 0.5 mM arsenate did not inhibit this synergistic effect, although the presence of these inhibitors inhibited completely the stimulation of proline transport induced by the addition of carbon source. Carbonylcyanide m-chlorophenylhydrazone and 2,4-dinitrophenol blocked both the basal and Li+-stimulated proline transport. When membrane potential of E. coli cells was measured by the dibenzyldimethylammonium uptake method, the incubation of Li+ with the cells did not affect the preexisting membrane potential. These results suggest that Li+ stimulates proline transport by intact cells of E. coli in a manner somewhat affecting membrane component(s) different from the transport carrier of proline. It is uncertain whether the effect of Li+ is directly involved in the mechanisms of energy coupling of proline transport.     

Modeling particle transport by bubbles for performance guidelines in airlift fermentors

A calculation method has been developed to model the statistical transport of biological particles in bubble-driven flows, with special reference to the biokinetics of environmental excursions experienced by individual cells, aggregated cells, or immobilization beads in airlift bioreactors. Interim developments on modeling the transport of such particles in concentric tube devices are reported. The calculation is driven by user-prescribed global parameters for the bioreactor geometry, bulk air flow rate, and particle parameters (size and slip speed). The algorithm calls on empirical data correlations for void fraction, bulk liquid flow rate, and bubble sizes and slip speeds, optimally selected from a large bibliographic database. The Monte Carlo algorithm concentrates on simulating particle transport in the bubbly riser flows.The packaged family of correlations and calculations represents, in effect, an expert system augmented by a transport simulation suited to characterizing the biokinetic response of cells cultured in airlift bioreactors.     

Molecular fluctuation in living cells

The concept of molecular fluctuation in living cells is introduced. Many apparently different experi-mental facts in living cells, including the velocity non-uniformity of organelle movement, the saltatory movement of transport vesicles in axoplasmic transport, the chromosome oscillation during metaphase in mitosis and the pauses in the chromosome movement during anaphase are explained using a unified viewpoint. A method of determination of average number of the attached motor protein molecules from the experimental data is also proposed.     

The preparation and properties of cytoplasts from Ehrlich ascites tumor cells

A method is described in which cytochalasin B is used to fractionate Ehrlich ascites tumor cells into cytoplasts and (nucleated) karyoplasts. The plasma membrane and cytoplasm are selectively removed from these cells by this method such that the cytoplasts rarely contain membranous organelles (e.g., mitochondria) which are retained in the karyoplast during fractionation. ATP concentrations similar to those found in whole cells and glycolytic activity were measured in cytoplasts in the presence but not the absence of glycose. Cytoplasts also actively transport Na⁺, K⁺, and α-aminoisobutyric acid to steadystate distribution ratios similar to those found in whole cells. It was concluded that these cytoplasts are a simplified model system for the study of active transport in Ehrlich cells.     

Use of paramagnetic ions in the study of intercellular water transport by nuclear magnetic resonance with pulsed gradient ofthe magnetic field

A methodical approach to the studies of water transport in biological cells by NMR method with impulse gradient of the magnetic field is proposed. It allows to exclude experimentally water transport through the plasmalemma, without touching upon transport along intercellular contacts. The effect is achieved by preliminary introduction of paramagnetic ions into intercellular space.     

ETMB-RBF: Discrimination of Metal-Binding Sites in Electron Transporters Based on RBF Networks with PSSM Profiles and Significant Amino Acid Pairs

Background

Cellular respiration is the process by which cells obtain energy from glucose and is a very important biological process in living cell. As cells do cellular respiration, they need a pathway to store and transport electrons, the electron transport chain. The function of the electron transport chain is to produce a trans-membrane proton electrochemical gradient as a result of oxidation–reduction reactions. In these oxidation–reduction reactions in electron transport chains, metal ions play very important role as electron donor and acceptor. For example, Fe ions are in complex I and complex II, and Cu ions are in complex IV. Therefore, to identify metal-binding sites in electron transporters is an important issue in helping biologists better understand the workings of the electron transport chain.

Methods

We propose a method based on Position Specific Scoring Matrix (PSSM) profiles and significant amino acid pairs to identify metal-binding residues in electron transport proteins.

Results

We have selected a non-redundant set of 55 metal-binding electron transport proteins as our dataset. The proposed method can predict metal-binding sites in electron transport proteins with an average 10-fold cross-validation accuracy of 93.2% and 93.1% for metal-binding cysteine and histidine, respectively. Compared with the general metal-binding predictor from A. Passerini et al., the proposed method can improve over 9% of sensitivity, and 14% specificity on the independent dataset in identifying metal-binding cysteines. The proposed method can also improve almost 76% sensitivity with same specificity in metal-binding histidine, and MCC is also improved from 0.28 to 0.88.

Conclusions

We have developed a novel approach based on PSSM profiles and significant amino acid pairs for identifying metal-binding sites from electron transport proteins. The proposed approach achieved a significant improvement with independent test set of metal-binding electron transport proteins.     

Transmembrane transfer of sodium ions in cell membranes of normal and tumor cells

Changes of sodium transport in tumor cells and their normal analogs were studied. For the first time we used a direct method of investigation with sodium-selective electrodes which permitted the study of ion transport in dynamics. As a result of investigation of sodium transport on malignant transplanted cultures of fibroblasts (7L strain) and on their normal prototypes--embryonic fibroblasts of hamsters, and also on the cells of Ehrlich ascite tumor--it was shown that when transferred the tumor cells in 15% hypertonic solution significant active transport of ions from the cells into the external medium was recorded. This phenomenon was not found in the normal cells. The experiments showed that in the process of malignancy significant changes began in the cellular membranes connected with the disturbance of activation of some enzymes, particularly Na,K-ATPase.     

TATA-binding protein-associated factor 7 regulates polyamine transport activity and polyamine analog-induced apoptosis

Identification of the polyamine transporter gene will be useful for modulating polyamine accumulation in cells and should be a good target for controlling cell proliferation. Polyamine transport activity in mammalian cells is critical for accumulation of the polyamine analog methylglyoxal bis(guanylhydrazone) (MGBG) that induces apoptosis, although a gene responsible for transport activity has not been identified. Using a retroviral gene trap screen, we generated MGBG-resistant Chinese hamster ovary (CHO) cells to identify genes involved in polyamine transport activity. One gene identified by the method encodes TATA-binding protein-associated factor 7 (TAF7), which functions not only as one of the TAFs, but also a coactivator for c-Jun. TAF7-deficient cells had decreased capacity for polyamine uptake (20% of CHO cells), decreased AP-1 activation, as well as resistance to MGBG-induced apoptosis. Stable expression of TAF7 in TAF7-deficient cells restored transport activity (55% of CHO cells), AP-1 gene transactivation (100% of CHO cells), and sensitivity to MGBG-induced apoptosis. Overexpression of TAF7 in CHO cells did not increase transport activity, suggesting that TAF7 may be involved in the maintenance of basal activity. c-Jun NH2-terminal kinase inhibitors blocked MGBG-induced apoptosis without alteration of polyamine transport. Decreased TAF7 expression, by RNA interference, in androgen-independent human prostate cancer LN-CaP104-R1 cells resulted in lower polyamine transport activity (25% of control) and resistance to MGBG-induced growth arrest. Taken together, these results reveal a physiological function of TAF7 as a basal regulator for mammalian polyamine transport activity and MGBG-induced apoptosis.     

Nonradioactive monitoring of organic and inorganic solute transport into single Xenopus oocytes by capillary zone electrophoresis.

Transport of organic and inorganic solutes into and out of cells requires specialized transport proteins. Given a sufficiently sensitive analytical method for measuring cellular solute concentrations, it should be possible to monitor solute transport across the plasma membrane at the level of single cells. We report a capillary zone electrophoresis approach that is generally applicable to monitor solute transport into Xenopus laevis oocytes, requires only nanoliters of sample, and involves no radioactive materials. The sensitivity of capillary electrophoresis with UV detection is typically on the order of 10(-5)-10(-6) M, resulting in the mass detection limits in the low femtomole range. We show that capillary zone electrophoresis serves as a simple technique to measure solute transport into oocytes. Studies of the mammalian oligopeptide transporter PepT1 and the Na(+)- and K(+)-coupled epithelial and neuronal glutamate transporter EAAC1 expressed in oocytes demonstrate that transport of the dipeptide Trp-Gly via PepT1 and transport of Na+ and K+ via EAAC1 across the oocyte plasma membrane can be monitored by measuring intracellular tryptophan absorption and by indirect UV detection of inorganic ions, respectively. The CZE method allowed the simultaneous detection of changes of intracellular Na+ and K+ concentrations in response to EAAC1-mediated Na+ cotransport and K+ countertransport. This is the first report of a capillary zone electrophoresis-based quantitative analysis of intracellular components of a single cell in response to transport activity.     

Fluid transport across cultured rat alveolar epithelial cells: a novel in vitro system

Previous studies have used fluid-instilled lungs to measure net alveolar fluid transport in intact animal and human lungs. However, intact lung studies have two limitations: the contribution of different distal lung epithelial cells cannot be studied separately, and the surface area for fluid absorption can only be approximated. Therefore, we developed a method to measure net vectorial fluid transport in cultured rat alveolar type II cells using an air-liquid interface. The cells were seeded on 0.4-microm microporous inserts in a Transwell system. At 96 h, the transmembrane electrical resistance reached a peak level (1,530 +/- 115 Omega.cm(2)) with morphological evidence of tight junctions. We measured net fluid transport by placing 150 microl of culture medium containing 0.5 microCi of (131)I-albumin on the apical side of the polarized cells. Protein permeability across the cell monolayer, as measured by labeled albumin, was 1.17 +/- 0.34% over 24 h. The change in concentration of (131)I-albumin in the apical fluid was used to determine the net fluid transported across the monolayer over 12 and 24 h. The net basal fluid transport was 0.84 microl.cm(-2).h(-1). cAMP stimulation with forskolin and IBMX increased fluid transport by 96%. Amiloride inhibited both the basal and stimulated fluid transport. Ouabain inhibited basal fluid transport by 93%. The cultured cells retained alveolar type II-like features based on morphologic studies, including ultrastructural imaging. In conclusion, this novel in vitro system can be used to measure net vectorial fluid transport across cultured, polarized alveolar epithelial cells.     

Biotin uptake by isolated rat intestinal cells

Isolated intestinal mucosa cells of rats were used to investigate the intestinal transport of biotin. This method utilizing a double-label isotope technique showed that uptake could not be saturated, even in a wide range of biotin concentrations (0.01-2 microM). A metabolic inhibitor (antimycin A) did not prevent cell uptake of biotin. The transport mechanism was independent of temperature (Q10 = 1.04). When excess biotin was added to the incubation medium, there was no efflux of the vitamin from intestinal cells. The results also showed that the cells did not concentrate the vitamin, regardless of its concentration in the incubation medium. The mechanism of biotin uptake by rat cells at physiological concentrations is thus a passive diffusion phenomenon.     

General method for the derivation and numerical solution of epithelial transport models

Summary A general method is presented for the formulation and numerical evaluation of mathematical models describing epithelial transport. The method is based on the principles of conservation of mass, and maintenance of electroneutrality within the cells and bathing solutions. It is therefore independent of the specific membrane transport mechanisms, and can be used to evaluate different models describing arbitrary transport processes (including passive, active and cotransport processes). Detailed numerical methods are presented that allow computation of steady-state and transient responses under open-circuit, current-clamp and voltage-clamp conditions, using a general-purpose laboratory minicomputer. To evaluate the utility of this approach, a specific model is presented that is consistent with the Koefoed-Johnson and Ussing hypothesis of sodium transport in tight epithelia (Acta Physiol. Scand. 42:298–308, 1958). This model considers passive transport of an arbitrary number of permeant solutes, active transport of sodium and potassium, and osmotically induced water transport across the apical and basolateral membranes. Results of the model are compared to published experimental measurements in rabbit urinary bladder epithelium.     

Energy-dependent, carrier-mediated extrusion of carboxyfluorescein from Saccharomyces cerevisiae allows rapid assessment of cell viability by flow cytometry.

Carboxyfluorescein diacetate is a nonfluorescent compound which can be used in combination with flow cytometry for vital staining of yeasts and bacteria. The basis of this method is the assumption that, once inside the cell, carboxyfluorescein diacetate is hydrolyzed by nonspecific esterases to produce the fluorescent carboxyfluorescein (cF). cF is retained by cells with intact membranes (viable cells) and lost by cells with damaged membranes. In this report, we show that Saccharomyces cerevisiae extrudes cF in an energy-dependent manner. This efflux was studied in detail, and several indications that a transport system is involved were found. Efflux of cF was stimulated by the addition of glucose and displayed Michaelis-Menten kinetics. A Km for cF transport of 0.25 mM could be determined. The transport of cF was inhibited by the plasma membrane H(+)-ATPase inhibitors N,N'-dicyclohexylcarbodiimide and diethylstilbestrol and by high concentrations of tetraphenylphosphonium ions. These treatments resulted in a dissipation of the proton motive force, whereas the intracellular ATP concentration remained high. Transport of cF is therefore most probably driven by the membrane potential and/or the pH gradient. The viability of S. cerevisiae was determined by a two-step procedure consisting of loading the cells with cF followed by incubation at 40 degrees C in the presence of glucose. Subsequently, the fluorescence intensity of the cells was analyzed by flow cytometry. The efflux experiments showed an excellent correlation between the viability of S. cerevisiae cells and the ability to translocate cF. This method should prove of general utility for the rapid assessment of yeast vitality and viability.     

Newly synthesized G protein of vesicular stomatitis virus is not transported to the Golgi complex in mitotic cells

Newly synthesized G protein of vesicular stomatitis virus is not transported to the surface of cultured mammalian cells during mitosis (Warren et al., 1983, J. Cell Biol. 97:1623-1628). To determine where intracellular transport is inhibited, we have examined the post-translational modifications of G protein, which are indicators of specific compartments on the transport pathway. G protein in mitotic cells had only endo H-sensitive oligosaccharides containing seven or eight mannose residues, but no terminal glucose, and was not fatty acylated. These modifications were indicative of processing only by enzymes of the endoplasmic reticulum (ER). Quantitative immunocytochemistry was used as an independent method to confirm that transport of G protein out of the ER was inhibited. The density of G protein in the ER cisternae was 2.5 times greater than in infected G1 cells treated similarly. Incubation of infected mitotic cells with cycloheximide, which inhibits protein synthesis without affecting transport, did not result in a decrease in the density of G protein in the ER cisternae, demonstrating that G protein cannot be chased out of the ER. These results suggest that intracellular transport stops at or before the first vesicle-mediated step on the pathway.