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PubMed1992

In vitro kinetics of 8-methoxypsoralen penetration into human lymphoid cells.

Extracorporeal photochemotherapy (ECPC) requires ex vivo UVA irradiation of blood lymphocytes during the time of the theoretical peak 8-methoxsalen (8-MOP) concentration. The aims of this study were to determine the mechanism of cellular uptake of 8-MOP, its possible saturation and the time needed to reach maximal concentration (Tmax) in lymphoid cells. 8-MOP was measured by liquid chromatography in the supernatant of lymphoid cell suspensions incubated with a known amount of 8-MOP. The kinetics of cellular uptake were determined and showed that equilibrium had already been reached after 2 min and remained constant for at least 60 min. The uptake was independent of temperature (4, 25 and 37 degrees C) and was proportional to the 8-MOP concentration in the supernatant. This indicated that 8-MOP penetrated into lymphoid cells by passive diffusion, rather than by active transport or facilitated diffusion, and was thus a non-saturable process. In addition, intracellular metabolism was negligible. These findings demonstrated that the plasma and lymphocytic Tmax were reached simultaneously and statistical analysis showed them to be significantly correlated, thereby validating the standard ECPC protocol for drug ingestion and lymphocyte irradiation.

Humans↗
PubMed2004

Ca2+-dependent and -independent mechanisms of calmodulin nuclear translocation.

Translocation from the cytosol to the nucleus is a major response by calmodulin (CaM) to stimulation of cells by Ca2+. However, the mechanisms involved in this process are still controversial and both passive and facilitated diffusion have been put forward. We tested nuclear translocation mechanisms in electroporated HeLa cells, rat cortical neurons and glial cells using novel calmodulin and inhibitor peptide probes and confocal microscopy. Passive diffusion of calmodulin across the nuclear membrane was measured in conditions in which facilitated transport was blocked and was compared to that of a similarly sized fluorescein-labeled dextran. Wheat germ agglutinin, which blocks facilitated transport but not passive diffusion, inhibited the nuclear entry of both wild-type and Ca2+-binding-deficient mutant calmodulin both in low and elevated [Ca2+]. Ca2+-dependent nuclear translocation was prevented by a membrane-permeant CaM inhibitor, the mTrp peptide, which indicated that it was specific to Ca2+/CaM. Diffusion of free CaM and Ca2+/CaM was considerably slower than the observed nuclear translocation by facilitated transport. Our data show that the majority of CaM nuclear entry occurred by facilitated mechanisms in all cell types examined, in part by a Ca2+-independent and in part by a Ca2+-dependent translocation mechanism.

Active Transport, Cell Nucleus↗
PubMed1976

Ferric hydroxamate transport without subsequent iron utilization in Bacillus megaterium.

Iron transport and utilization were examined in Bacillus megaterium Ard1, a mutant that is resistant to the hydroxymate antibiotic A22765 and whose growth is inhibited by the structurally similar hydroxamate Desferal. Rapid, low-level uptake of Desferal-50Fe was observed; such uptake was temperature and energy independent. Gel filtration chromatography of the cytoplasmic fraction of protoplasts labeled with Desferal-55Fe for 30 to 120 s demonstrated only unchanged esferal-55Fe in the cytoplasm. Although B. megaterium Ard1 showed transport of Desferal-59Fe by a process that resembles facilitated diffusion, this organism was unable to transfer iron from this chelate to cellular macromolecules for metabolic use. High-level transport of the ferric hydroxamate schizokinen-59Fe by B. megaterium Ard1 was both temperature and energy dependent. Within 30 s, protoplasts labeled with schizokinen-55Fe contained iron associated with certain macromolecules and in an apparent "pool" of schizokinen-55Fe in the cytoplasmic fraction. Prior transport of Dseferal-55Fe by protoplasts of strain Ard1 did not interfere with subsequent transport and utilization of schizokinen-59Fe. These studies suggest that transport of ferric hydroxamates may occur by a facilitated diffusion-type process; transfer of iron to cellular macromolecules may drive high-level transport of the chelate and may be the step at which energy is required in the iron transport-assimilation process.

Bacillus megaterium↗
PubMed2003

Fps1p channel is the mediator of the major part of glycerol passive diffusion in Saccharomyces cerevisiae: artefacts and re-definitions.

Glycerol has been shown to cross the plasma membrane of Saccharomyces cerevisiae through (1) a H(+)/symport detected in cells grown on non-fermentable carbon sources, (2) the constitutively expressed Fps1p channel and (3) by passive diffusion. The Fps1p channel has been named a facilitator for mediating glycerol low affinity transport of the facilitated diffusion type. We present experimental evidence that this kinetic is an artefact created by glycerol kinase activity. Instead, the channel is shown to mediate the major part of glycerol's passive diffusion. This is not incompatible with Fps1p's major role in vivo, which has been previously shown to be the control of glycerol export under osmotic stress or in reaction to turgor changes. We also verified that FPS1 overexpression caused an increase in H(+)/symport V(max). Furthermore, yfl054c and fps1 mutants were equally affected by exogenously added ethanol, being the correspondent passive diffusion stimulated. For the first time, to our knowledge, a phenotype attributed to the functioning of YFL054c gene is presented. Glycerol passive diffusion is thus apparently channel-mediated. This is discussed according to glycerol's chemical properties, which contradict the widely spread concept of glycerol's liposoluble nature. The discussion considers the multiple roles that the intracellular levels of glycerol and its pathway regulation might play as a central key to metabolism control.

Artifacts↗
PubMed2002

The Yersinia pseudotuberculosis Yut protein, a new type of urea transporter homologous to eukaryotic channels and functionally interchangeable in vitro with the Helicobacter pylori UreI protein.

Urea uptake in eukaryotes and prokaryotes occurs via diffusion or active transport across the cell membrane. Facilitated diffusion of urea in both types of organisms requires a single-component channel. In bacteria, these transport systems allow rapid access of urease to its substrate, resulting in ammonia production, which is needed either for resistance to acidity or as a nitrogen source. In Yersinia pseudotuberculosis, a ureolytic enteropathogenic bacterium, a gene of unknown function (yut) located near the urease locus was found to encode a putative membrane protein with weak homology to single-component eukaryotic urea transporters. When expressed in Xenopus oocytes, Yut greatly increases cellular permeability to urea. Inactivation of yut in Y. pseudotuberculosis results in diminished apparent urease activity and reduced resistance to acidity in vitro when urea is present in the medium. In the mouse model, bacterial colonization of the intestine mucosa is delayed with the Yut-deficient mutant. Although structurally unrelated, Yut and the Helicobacter pylori UreI urea channel were shown to be functionally interchangeable in vitro and are sufficient to allow urea uptake in both bacteria, thereby confirming their function in the respective parent organisms. Homologues of Yut were found in other yersiniae, Actinobacillus pleuropneumoniae, Brucella melitensis, Pseudomonas aeruginosa and Staphylococcus aureus. The Y. pseudotuberculosis Yut protein is therefore the first member of a novel class of bacterial urea permeases related to eukaryotic transporters.

Amino Acid Sequence↗
PubMed1983

Role of the maternal organism in transplacental carcinogenesis.

The maternal organism plays a highly important role in transplacental carcinogenesis, since for carcinogens in the bloodstream of the mother to reach the fetus, they must cross several barriers, the first of which is the placenta. Some types of compounds require metabolic activation in the maternal organism, in the fetus and even sometimes in the placenta. Thus, four main pathways can be hypothesized by which substances exert a carcinogenic effect on the fetus. Most carcinogens can cross the placenta; data confirm that this process consists of simple diffusion or - in the case of high doses - facilitated diffusion. That carcinogens may be detoxified in the maternal organism is confirmed by experiments on activation of enzyme systems and on caesarean deliveries. Species and strain specificities are characteristic of transplacental carcinogenesis and are manifested in organotropism. Organotropism in transplacental carcinogenesis is determined by genetic predisposition, cell differentiation and proliferative activity in the target tissues. For indirect carcinogens, the level of metabolizing enzymes is also important.

Animals↗
PubMed1993

Glycerol kinase of Escherichia coli is activated by interaction with the glycerol facilitator.

Glycerol transport is commonly cited as the only example of facilitated diffusion across the Escherichia coli cytoplasmic membrane. Two proteins, the glycerol facilitator and glycerol kinase, are involved in the entry of external glycerol into cellular metabolism. The glycerol facilitator is thought to act as a carrier or to form a selective pore in the cytoplasmic membrane, whereas the kinase traps the glycerol inside the cell as sn-glycerol-3-phosphate. We found that the kinetics of glycerol uptake in a facilitator-minus strain are significantly different from the kinetics of glycerol uptake in the wild type. Free glycerol was not observed inside wild-type cells transporting glycerol, and diffusion of glycerol across the cytoplasmic membrane was not the rate-limiting step for phosphorylation in facilitator-minus mutants. Therefore, the kinetics of glycerol phosphorylation are different, depending on the presence or absence of the facilitator protein. We conclude that there is an interaction between the glycerol facilitator protein and glycerol kinase that stimulates kinase activity, analogous to the hexokinase- and glycerol kinase-porin interactions in mitochondria.

Aquaporins↗
PubMed1996

Modeling membrane transport.

Many substrates cross cell membranes by processes other than passive diffusion. When the transport is carrier-mediated, e.g., facilitated diffusion, active transport, and exchange diffusion, the carrier modifies the conductance of the membrane and may either increase or decrease the flux of the substrate across the membrane. A common characteristic of all carrier-mediated transport is its saturability, as only a finite amount of carrier is available to bind with the substrate; even the simplest one-site carrier model exhibits saturation. Inclusion of carrier-mediated transport adds additional model parameters that describe the transporter. In addition, the model must account for both labeled (tracer) and unlabeled (mother) substrate, but this introduces no new parameters. There are many possible models for a membrane carrier. The applicability of these models must be examined for the specific substrate of interest. Many experiments aimed at measuring carrier parameters are carried out on isolated cells or cell fragments. Experiments in intact organs (either in vivo and in vitro) are also possible. Of particular note is the "bolus sweep" method described by Rickaby et al. (1981) and Malcorps et al. (1984). The increasing sophistication of experimental procedures, data collection techniques, and computers available to investigators continues to extend the depth to which we can probe biological systems. With this increased sophistication comes increased costs in time and equipment. It behooves us then to extract the maximum amount of information from each experimental procedure. Mathematical models assist in doing so, and sophistication in model analysis should parallel that in other phases of the experiment. Increased realism brings several advantages. Simplification of a model to increase its ease of usage and speed in routine data analysis is a desirable goal, and comparing a simplified model against a more realistic model under the conditions specific to a given experiment is one way to test the simplifying assumptions. Additionally, increased model realism can bring new insight into unknown aspects of the system. All models, no matter how realistic, are always "wrong" in that they are less complex than the real system. Failure of the model to explain observed results forces us to further refine the model and teaches us something more about the system.

Animals↗
PubMed2003

Porin OmpP2 of Haemophilus influenzae shows specificity for nicotinamide-derived nucleotide substrates.

Haemophilus influenzae has an absolute requirement for NAD (factor V) because it lacks all biosynthetic enzymes necessary for de novo synthesis of that cofactor. Therefore, growth in vitro requires the presence of NAD itself, NMN, or nicotinamide riboside (NR). To address uptake abilities of these compounds, we investigated outer membrane proteins. By analyzing ompP2 knockout mutants, we found that NAD and NMN uptake was prevented, whereas NR uptake was not. Through investigation of the properties of purified OmpP2 in artificial lipid membrane systems, the substrate specificity of OmpP2 for NAD and NMN was determined, with KS values of approximately 8 and 4mm, respectively, in 0.1 m KCl, whereas no interaction was detected for the nucleoside NR and other purine or pyrimidine nucleotide or nucleoside species. Based on our analysis, we assume that an intrinsic binding site within OmpP2 exists that facilitates diffusion of these compounds across the outer membrane, recognizing carbonyl and exposed phosphate groups. Because OmpP2 was formerly described as a general diffusion porin, an additional property of acting as a facilitator for nicotinamide-based nucleotide transport may have evolved to support and optimize utilization of the essential cofactor sources NAD and NMN in H. influenzae.

Bacterial Outer Membrane Proteins↗
PubMed2005

Characteristics of (+)-catechin and (-)-epicatechin transport across pig intestinal brush border membranes.

BACKGROUND/AIMS: (+)-Catechin and (-)-epicatechin are considered as disease preventive flavan-3-ols of foods like fruits, beverages and chocolate. We investigated mechanisms and kinetics of (+)-catechin and (-)-epicatechin uptake employing a validated in vitro model with isolated pig brush border membrane vesicles. METHODS: Vesicles were isolated from pig small intestine employing the divalent cation method. Characterization (marker enzymes, electron microscopy) confirmed their purity and function. Transport studies with (+)-catechin and (-)-epicatechin under predefined conditions [presence/absence of sodium, pH gradient, temperature (8-37 degrees C), various initial substrate concentrations (2-20 mmol/l)] revealed a measurable transport (HPLC analyses) across the brush border membrane for both substrates. RESULTS: Catechin transport was stimulated by an outwardly directed H(+) gradient (pH(i) 5.5/pH(o) 7.5). The presence of an inwardly directed Na(+) gradient did not result in a transient overshoot in (+)-catechin and (-)-epicatechin uptake. At 37 degrees C, subtraction of diffusion from the total transport rate showed saturation kinetics. CONCLUSION: Our in vitro study indicate that both (+)-catechin and (-)-epicatechin are transported across the basolateral membrane using a dual transport system consisting of free diffusion (dominant at low concentrations) and carrier-mediated facilitated diffusion.

Animals↗
PubMed1976

D-glucose uptake by a rat liver plasma membrane preparation.

1. Plasma membranes isolated from rat liver according to a modification of the method of Neville, D.M. ((1960) J. Biophys. Biochem. Cytol. 8, 413-422) were used as model to test current hypotheses on the mode of glucose uptake into the liver cell. Glucose uptake studies were performed by a filtration technique using labeled glucose analogues. 2. D-glucose Uptake by rat liver plasma membranes is characterized by features of simple diffusion, i.e. linearity of uptake, lack of stereospecificity, and by facilitated diffusion, i.e. temperature dependence, counterflow phenomenon and inhibition by phloretin. These findings confirm earlier studies on liver slices and perfused liver. 3. Binding studies on sonicated membranes provide evidence for a specific binding site or protein for D-glucose at the plasma membrane by isolating Tris-soluble membrane proteins which reveal a higher binding capacity than the unsonicated membrane. 4. These findings are interpreted as showing the presence of a "carrier" mediated transport system for D-glucose superimposed by free diffusion due to artificial disruption of the plasma membranes.

Animals↗
PubMed1997

Fructose transport mechanisms in humans.

BACKGROUND & AIMS: The possible mechanisms of fructose transport are diffusion, a disaccharidase-related transport system, and glucose-facilitated fructose transport. However, these mechanisms in the human small intestine have not been systematically examined. This study was designed to investigate the mechanisms of fructose transport in the human duodenojejunum. METHODS: A triple-lumen tube was fluoroscopically positioned in the duodenojejunum of 7 men. Nine carbohydrate-electrolyte solutions were perfused at the rate of 15 mL/min. Acarbose and lactulose were used to examine the disaccharidase-related transport system and glucose-facilitated fructose transport, respectively. RESULTS: Fructose absorption was greater (P < 0.05) from fructose-glucose (FruGlu) and fructose-glucose-acarbose (FruGluA) solutions than from fructose-mannitol (FruMann) and fructose-mannitol-acarbose (FruMannA) solutions, but there was no difference between FruGlu and FruGluA solutions. A sucrose solution produced greater (P < 0.05) sucrose absorption than a sucrose-acarbose solution. Lactulose absorption (0.016-0.039 mmol.h-1.cm-1) was observed from solutions containing glucose or sucrose. Water absorption was not different among sucrose, FruGlu, and glucose solutions. FruMann solution produced net water secretion. These data suggest that free fructose and glucose transport were not inhibited by acarbose and that the presence of glucose induced lactulose absorption and enhanced fructose absorption. CONCLUSIONS: Fructose is transported transcellularly by facilitated diffusion and paracellularly (based on lactulose transport) via glucose-activated solution drag. In the human small intestine, free fructose and glucose transport does not occur via the disaccharidase system.

Acarbose↗
PubMed1992

[Structural heterochromatin and X-chromosome inactivation].

Our previous studies on the expression of the G6PD and alpha-GAL genes from the X chromosome of inter-specific hybrids of voles of the Microtus genus have demonstrated an unusual pattern of X-inactivation in the parents. The observed phenomenon was explained as the presumable result of nonrandom inactivation of the X chromosomes with a heterochromatin block in crosses involving Microtus arvalis whose X lacks a heterochromatin region and also of random X inactivation when both parents had heterochromatin blocks on the Xs. Based on known models, we discuss here the possible mechanisms of the effect of heterochromatin on X-inactivation; we give preference to the model postulating binding of nonhistone protein to the inactivation centre as the key event. The hypothesis we offer suggests change in chromatin conformation in the inactivation centre during packaging of heterochromatic region of a chromosome; the protein molecules diffusing along the chromosome towards the heterochromatin region by the "facilitated diffusion" mechanism may happen to be in the region of the X-inactivation centre, which, being in a favorable state, binds specifically to it; as a consequence, the binding probability of protein to heterochromatin increases as compared to chromosome without heterochromatin block.

Animals↗
PubMed1983

Intestinal absorption. I - General principles of transintestinal transport.

The paper deals mainly with the principles and the basic mechanisms underlying the transport processes of molecules across biological membranes. The simple diffusion of electrolytes and non-electrolytes, the chemically-facilitated diffusion and the active transport are briefly discussed. These transport mechanisms are utilized also by substances moving through the intestinal epithelium even if this tissue has a rather complex structure.

Biological Transport, Active↗
PubMed1977

Neutral amino acid transport. Characterization of the A and L systems in isolated rat hepatocytes.

Hepatocytes isolated from adult rat liver by enzymatic dispersion were used to investigate amino acid transport. Steady state and influx experiments were carried out with alpha-amino[1-14C]isobutyric acid and [1-14C]cycloleucine in the presence and absence of sodium under various experimental conditions. Hepatocytes concentrated alpha-aminoisobutyric acid to a 3-fold higher degree than cycloleucine. At low external alpha-aminoisobutyric acid levels (2 to 5 mM), about 25% and 75% of entry were accounted for by nonsaturable and saturable processes, respectively. The nonsaturable component was sodium-independent, and had the properties of passive diffusion. The saturable transport was dependent on external sodium; the rate of transport reached its maximal value with sodium greater than or equal to 75 mM. Sodium increased the apparent Vmax of transport without changing the apparent Km. This component was largely dependent on energy supplies and was strongly reduced at pH less than or equal to 6.5. The value for activation energy (Ea approximately equal to 15 kcal/mol, calculated from the Arrhenius plot) favors a mediated active transport. The Na+-dependent influx of alpha-aminoisobutyric acid was competitively inhibited by N-methyl-alpha-aminoisobutyric acid (Ki approximately equal to 9.3 mM) and alanine (Ki approximately equal to 2 mM) to the extent of 70% and 100%, respectively. The N-methyl-alpha-aminoisobutyric acid-sensitive part of alpha-aminoisobutyric acid influx represents transport through the "A" system, whereas the N-methyl-alpha-aminoisobutyric acid-insensitive part of transport is believed to occur through the "ASC" system. No evidence was obtained to suggest that alpha-aminoisobutyric acid is transported by the "L" system. Cycloleucine transport was a composite phenomenon involving at least two saturable processes, one of which was sodium-dependent and inhibited by alpha-aminoisobutyric acid, and probably represents entry through the A and ASC systems. The sodium-independent component was completely and competitively inhibited by 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (Ki approximately equal to 2 mM). This component exhibited accelerative exchange-diffusion and was pH-insensitive, properties which suggest a facilitated diffusion process. However, the weak inhibition exerted by oligomycin and cyanide along with the concentrative effect observed indicated that uphill transport was also operative. These data are in good agreement with those reported for the L system. We conclude that, as in Ehrlich ascites tumor cells and in embryonic heart cells, the A, ASC, and L systems are operative in isolated hepatocytes for the transport of amino acids.

Amino Acids↗
PubMed1977

Intestinal absorption of sugars and amino acids in the earthworm.

Absorption of glucose, galactose, fructose and leucine from luminal to coelomic compartments across the intestinal wall of Scherotheca sp. seems to be a simple diffusion process and not a product of active transport or facilitated diffusion. A concentration gradient never developed from identical initial concentrations of the 14C-labelled substrates on either side of in vitro everted intestinal sacs. The rates of net passage of glucose and galactose were in linear function with the respective sugar concentration gradient. A competition between these two sugars was not observed. The diffusion rate of glucose was higher from mucosal to serosal than in reverse direction. Intestinal tissues synthesized glycogen from glucose in the medium, this process being strongly inhibited by dinitrophenol.

Amino Acids↗
PubMed1991

Computed myocardial PO2 histograms: effects of various geometrical and functional conditions.

A model of myocardial oxygenation was developed that allows calculation of Po2 histograms under varying conditions. The model consists of parallel tissue cylinders with varying radii, simulating the heterogeneity of capillary spacing, in agreement with our previous experimental results. The facilitated diffusion of O2 by myoglobin, an additional resistance to diffusion at the capillary level, and the Michaelis-Menten type of O2 consumption were also incorporated. The shape of the histograms depends on input data. When no additional barrier to O2 transport is included, the histograms resemble those obtained with Po2 surface electrodes, and they are strongly dependent on heterogeneity in capillary spacing and capillary blood flow. On the other hand, an inclusion of an additional capillary barrier combined with the Michaelis-Menten type of O2 consumption can generate Po2 histograms similar to those derived from myoglobin cryospectroscopy. In this case, the Po2 histograms are relatively independent of heterogeneity of capillary spacing and blood flow. The facilitation of O2 diffusion by myoglobin has only a modest effect on the form of the histograms in all situations considered.

Animals↗
PubMed1988

Molecular mechanisms involved in the transport of antibiotics into bacteria.

Many clinically useful antibacterial drugs have intracellular target sites. Therefore, in order to reach their targets, these compounds must be able to cross bacterial outer and cytoplasmic membranes. Considerable information is available on the mechanisms by which antibiotics cross bacterial membranes and, in many cases, it is now possible to define the molecular basis of their uptake. Passage of drugs across the outer membrane of Gram-negative bacteria can occur by diffusion through porin channels (e.g. beta-lactams and tetracyclines), by facilitated diffusion using specific carriers (e.g. albomycin), or by self-promoted uptake (e.g. aminoglycosides and polymyxins). Transfer of antibiotics across the bacterial cytoplasmic membrane is usually mediated by active, carrier-mediated, transport systems normally operating to transport essential solutes into the cell. For example, the antibiotic streptozotocin bears sufficient structural resemblance to N-acetyl-D-glucosamine to be transported by the phosphoenolpyruvate:phosphotransferase system, and D-cycloserine is recognized by the D-alanine, proton motive force dependent transport system. However, in some cases (e.g. tetracycline) although carrier-mediated transport is implied by the observation that drug uptake is energy dependent, the nature of the membrane carrier(s) responsible is unknown. Knowledge acquired from studies on bacterial peptide transport has been successfully used to deliver (or smuggle) amino acid mimetics disguised as peptides into the bacterial cell. These amino acid mimetics, although often poorly transported in their own right, are frequently potent inhibitors of bacterial peptidoglycan or lipopolysaccharide synthesis once they have gained access to the interior of the cell.

Anti-Bacterial Agents↗