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Nucleocytoplasmic shuttling of JAZ, a new cargo protein for exportin-5.

Exportin-5 is a nuclear export receptor for certain classes of double-stranded RNA (dsRNA), including pre-micro-RNAs, viral hairpin RNAs, and some tRNAs. It can also export the RNA binding proteins ILF3 and elongation factor EF1A. However, the rules that determine which RNA binding proteins are exportin-5 cargoes remain unclear. JAZ possesses an unusual dsRNA binding domain consisting of multiple C2H2 zinc fingers. We found that JAZ binds to exportin-5 in a Ran-GTP- and dsRNA-dependent manner. Exportin-5 stimulates JAZ shuttling, and gene silencing of exportin-5 reduces shuttling. Recombinant exportin-5 also stimulates nuclear export of JAZ in permeabilized cells. JAZ also binds to ILF3, and surprisingly, this interaction is RNA independent, even though it requires the dsRNA binding domains of ILF3. Exportin-5, JAZ, and ILF3 can form a heteromeric complex with Ran-GTP and dsRNA, and JAZ increases ILF3 binding to exportin-5. JAZ does not contain a classical nuclear localization signal, and in digitonin-permeabilized cells, nuclear accumulation of JAZ does not require energy or cytosol. Nonetheless, low temperatures prevent JAZ import, suggesting that nuclear entry does not occur via simple diffusion. Together, these data suggest that JAZ is exported by exportin-5 but translocates back into nuclei by a facilitated diffusion mechanism.

Active Transport, Cell Nucleus↗

Diffusion-weighted MR of the brain: methodology and clinical application.

Clinical diffusion magnetic resonance (MR) imaging in humans started in the last decade with the demonstration of the capabilities of this technique of depicting the anatomy of the white matter fibre tracts in the brain. Two main approaches in terms of reconstruction and evaluation of the images obtained with application of diffusion sensitising gradients to an echo planar imaging sequence are possible. The first approach consists of reconstruction of images in which the effect of white matter anisotropy is averaged -- known as the isotropic or diffusion weighted images, which are usually evaluated subjectively for possible areas of increased or decreased signal, reflecting restricted and facilitated diffusion, respectively. The second approach implies reconstruction of image maps of the apparent diffusion coefficient (ADC), in which the T2 weighting of the echo planar diffusion sequence is cancelled out, and their objective, i.e. numerical, evaluation with regions of interest or histogram analysis. This second approach enables a quantitative and reproducible assessment of the diffusion changes not only in areas exhibiting signal abnormality in conventional MR images but also in areas of normal signal. A further level of image post-processing requires the acquisition of images after application of sensitising gradients along at least 6 different spatial orientations and consists of computation of the diffusion tensor and reconstruction of maps of the mean diffusivity (D) and of the white matter anisotropic properties, usually in terms of fractional anisotropy (FA). Diffusion-weighted imaging is complementary to conventional MR imaging in the evaluation of the acute ischaemic stroke. The combination of diffusion and perfusion MR imaging has the potential of providing all the information necessary for the diagnosis and management of the individual patient with acute ischaemic stroke. Diffusion-weighted MR, in particular quantitative evaluation based on the diffusion tensor, has a fundamental role in the assessment of brain maturation and of white matter diseases in the fetus, in the neonate and in the child. Diffusion MR imaging enables a better characterisation of the lesions demonstrated by conventional MR imaging, for instance in the hypoxic-ischaemic encephalopathy, in infections and in the inherited metabolic diseases, and is particularly important for the longitudinal evaluation of these conditions. Diffusion-weighted MR imaging has an established role in the differential diagnosis between brain abscess and cystic tumour and between epidermoid tumour and arachnoid cyst. On the other hand, the results obtained with diffusion MR in the characterisation of type and extension of glioma do not yet allow decision making in the individual patient. Diffusion is one of the most relevant MR techniques to have contributed to a better understanding of the pathophysiological mechanisms of multiple sclerosis (MS). In fact, it improves the specificity of MR in characterising the different pathological substrata underlying the rather uniform lesion appearance on the conventional images and enables detection of damage in the normal-appearing white and grey matter. In MS patients the ADC or D values in the normal-appearing white matter are increased as compared to control values, albeit to a lesser degree than in the lesions demonstrated by T2-weighted images. In addition, the D of the normal appearing grey matter is increased in MS patients and this change correlates with the cognitive deficit of these patients. Histogram analysis in MS patients shows that the peak of the brain D is decreased and right-shifted, reflecting an increase of its value, and the two features correlate with the patient's clinical disability. Ageing is associated to a mild but significant increase of the brain ADC or D which is predominantly due to changes in the white matter. Region of interest and histogram studies have demonstrated that D or ADC are increased in either the areas of leukoaraiosis or the normal-appearing white matter in patients with inherited cerebral autosomal dominant arteriopathy with subcortical infarcts and stroke or sporadic ischaemic leukoencephalopathy. Diffusion changes might be a more sensitive marker for progression of the disease than conventional imaging findings. In neurodegenerative diseases of the central nervous system such as Alzheimer's disease, Huntington's disease, hereditary ataxias and motor neuron disease, quantitative diffusion MR demonstrates the cortical and subcortical grey matter damage, which is reflected in a regional increase of D or ADC, but also reveals the concomitant white matter changes that are associated with an increase in D or ADC and decrease in FA. In all these diseases the diffusion changes are correlated to the clinical deficit and are potentially useful for early diagnosis and longitudinal evaluation, especially in the context of pharmacological trials.

Brain Diseases↗

Hexose transport in human adipocytes: factors influencing the response to insulin and kinetics of methylglucose and glucose transport.

Optimal experimental conditions were defined for measuring the initial uptake rate of the non-metabolizable sugar analogue 3-O-methylglucose in non-stimulated and insulin-stimulated human adipocytes. The permeability of the adipocyte plasma membrane for tracer methylglucose (100 mumol/l) was 2.9 X 10(-7) cm X s-1 at 37 degrees C and slightly lower at 20 degrees C. At 37 degrees C and pH 7.4 insulin (5 nmol/l) increased the permeability about twofold (range 1.5 to fivefold) with half maximal effect at about 100 pmol/l). At pH 7.0 the dose response curve for the insulin effect on the uptake rate of methylglucose was shifted about 2.5-fold to the right. The permeability to L-glucose due to simple diffusion was estimated as 3.0 X 10(-10) cm X s-1 suggesting that uptake of methylglucose occurs almost exclusively by facilitated diffusion. The Km for methylglucose equilibrium exchange in insulin stimulated cells was about 4.8 mmol/l. The initial uptake of tracer methylglucose in insulin-stimulated cells was inhibited by unlabelled methylglucose and by D-glucose with inhibition constants of about 3.8 and 7.7 mmol/l respectively. Uptake of tracer 2-deoxyglucose (50 mumol/l) in insulin-stimulated adipocytes was linear from 10 s to 5 min whereas the rate of uptake in the presence of 3 mmol/l of D-glucose was markedly decreased suggesting that deoxyglucose uptake after a few minutes is mainly limited by hexokinase in the presence of glucose.

Adipose Tissue↗

Sulphate uptake and metabolism in the chrysomonad, monochrysis lutheri.

The intracellular concentration of inorganic 35SO4 in Monochrysis lutheri cells exposed to 0.513 mM Na235SO4 for up to 6-hr remained constant at about 0.038 mM. The exchange rate of this 35SO4 with the external unlabelled sulphate was negligible compared to the rate of influx across the plasmalemma (0.032 mu moles/g cells/hr). The flux of free 35SO4 to organic 35S was 0.029 mu moles/g cells/hr. Assuming an internal electrical potential in the cells of -70 mV, this intracellular concentration of inorganic 35SO4 was well in excess of that obtainable by passive diffusion as calculated from the Nernst equation. These results indicate that sulphate is accumulated by an active mechanism rather than by facilitated diffusion. Sulphate uptake appears to occur via a carrier-mediated membrane transport system which conforms to Michaelis-Menten type saturation kinetics with a Km of 3.2 X 10(-5) M and Vmax of 7.9 X 10(-5) mu moles sulphate/hr/10(5) cells. Uptake was dependent on a source of energy since the metabolic inhibitor CCCP almost completely inhibited uptake under both light and dark conditions and DCMU caused a 50% decrease in uptake under light conditions. Under dark conditions, uptake remained at about 80% of that observed under light conditions and was little affected by DCMU, indicating that the energy for uptake could be supplied by either photosynthesis or respiration. A charge and size recognition site in the cell is implied by the finding that sulphate uptake was inhibited by chromate and selenate but not by tungstate, molybdate, nitrate or phosphate. Chromate did not inhibit photosynthesis. Cysteine and methionine added to the culture medium were apparently capable of exerting inhibition of sulphate uptake in both unstarved and sulphate-starved cells. Cycloheximide slightly inhibited sulphate uptake over an 8-hr period indicating, either a slow rate of entry of the inhibitor into the cells or a slow turnover of the protein(s) associated with sulphate transport.

Biological Transport, Active↗

Photo-cross-linked decyl methacrylate films for electrochemical and optical polyion probes.

Potentiometric and optical polyion probes based on photo-cross-linked thin films of decyl methacrylate (DMA) are described, and the effects of film composition on the response toward heparin are examined in detail. In accordance with existing theory governing potentiometric polyion response, lowering the amounts of plasticizer and tridodecylmethylammonium chloride ion exchanger within the film enhances its sensitivity toward heparin. Varying the cross-linker content of a DMA-based film, however, provides an additional mechanism to regulate its physical structure and, hence, the observed potentiometric polyion response. Films with low hexanedioldimethacrylate cross-linker content yield optimal potentiometric heparin detection limits (0.04 microM), suggesting a lower diffusion coefficient within such films, apparently due to interactions between adjacent pendant decyl groups. Increasing crosslinker content interrupts these interactions and facilitates diffusion. This knowledge is applied to optimize optical heparin sensing via DMA films covalently attached to glass substrates. When used in a limited volume/fixed exposure time measurement mode, such optically sensitive films can detect clinically relevant levels of heparin (0.5-5 units/mL) in undiluted human plasma.

Cross-Linking Reagents↗

Neurotransmitters and lymphatic-vascular transfer of prostaglandin F2 alpha stimulate ovarian oxytocin output in sheep.

The mechanisms of lymphatic-vascular transfer across the ovarian vascular pedicle were studied in anaesthetized sheep 8-15 days after ovulation. [3H]Prostaglandin F2 alpha (PGF2 alpha), [14C]mannitol and [36Cl]Na were infused continuously into either a uterine lymphatic or a uterine vein and the kinetics of transfer into the adjacent utero-ovarian vein or ovarian plasma were studied. Transfer occurred according to the sequence [36Cl] greater than [14C] greater than [3H] indicating that PGF2 alpha is not transferred by rapid diffusion, as with [36Cl]Na, nor by a paracellular route, as with [14C]mannitol, but by a slower process probably involving facilitated diffusion. Transfer into the adjacent utero-ovarian vein or ovarian blood was greater when compounds were infused into a uterine lymphatic than into a uterine vein. Substantially more [3H]PGF2 alpha occurred in the adjacent corpus luteum than either of the other compounds after a lymphatic infusion. Intra-lymphatic infusion of PGF2 alpha stimulated the release of ovarian oxytocin but the effect was not confined to the adjacent ovary. Intravenous (jugular) infusion of PGF2 alpha failed to stimulate ovarian oxytocin secretion whereas close-arterial infusion into the ovaries was effective, and the possibility was investigated that any systemic effect of PGF2 alpha was mediated through neural mechanisms. Noradrenaline and acetylcholine were both effective in causing the release of ovarian oxytocin when infused close-arterially into the ovary. With infusions of acetylcholine, ovarian oxytocin secretion rate was increased over fivefold without any change in posterior pituitary release. Noradrenaline and acetylcholine produced a concomitant fall in ovarian blood flow, and neurotransmitter-induced ischaemia may have played a role in ovarian oxytocin release. The finding that PGF2 alpha infused into a uterine lymphatic stimulates ovarian secretion of oxytocin, and that the effect is bilateral whereas PGF2 alpha accumulation in ovarian tissue is unilateral, implies that its mechanism of action may not be solely directed at the luteal cell.

Acetylcholine↗

Gastric PCO2 tonometry is independent of carbonic anhydrase inhibition.

Tonometric measurement of an elevated intragastric Pco2 and a decreased calculated gastric intramucosal pH can be used to detect gastric mucosal ischemia, provided that intraluminal production of CO2 through acid buffering by bicarbonate is avoided by adequate acid secretion suppression. If the diffusion rate is known, steady state Pco2 can be calculated when measurement intervals are used that are shorter than needed for complete equilibration. The CO2 diffusion might be influenced by the choice of acid-suppressive drugs, since some of them inhibit gastric carbonic anhydrase (CA) and CA facilitates diffusion of CO2/bicarbonate over the gastrointestinal mucosa. We therefore performed gastric Pco2 tonometry, using acid-suppressive regimens with and without CA inhibition. The diffusion rate of CO2 in a gastric tonometer was studied in healthy volunteers, following intravenously administered ranitidine (group I, N = 8) or ranitidine plus pirenzepine (group II, N = 12), a muscarinic antagonist with CA inhibiting capacities. Measurement intervals were 10, 20, 30 and 60 min. Neither the diffusion rate of CO2 (k = 0.13 +/- 0.02/min in group I and 0.11 +/- 0.02/min in group II), nor the steady-state Pco2 (38 +/- 3 mm Hg in group I and 40 +/- 4 mm Hg in group II), nor the gastric-blood differences in Pco2 and pH differed between groups. These results indicate that diffusion of CO2 into the tonometer balloon is independent of CA and thus of the type of gastric acid secretion inhibition.

Adult↗

Diffusive transfer of water and glucose across the chorionic plate of the isolated human term placenta.

This study investigated systematically the diffusive transfer of water and glucose across the chorionic plate of the human placenta. Isolated sections of human term placentae were perfused at the fetal side (open loop) with modified Ringer's solution (n=31). An artificial amniotic compartment was created on top of the chorionic plate. 3H- and 14C-labelled tracer pairs were added (donor side) to the fetal perfusion fluid or to the 'amniotic' fluid. Transfer fractions (TF, ratio of acceptor side to donor side radioactivity) were calculated as percentages. TF of water and L-glucose from perfusion fluid into the 'amniotic' fluid were 3.9+/-0.5 per cent (mean+/-SEM) and 1.2+/-0.3 per cent after 60 min and significantly different (n=6). In each sample of the following experiments the transfer fraction of the D-hexose was larger than that of the L-isomer. At 60 min, the TF were 1.6+/-0.2 and 1.1+/-0.2 per cent (D-glucose/L-glucose; fetal to amniotic compartment, n=8), from amniotic compartment to fetal perfusate 0.6+/-0.1 and 0.4+/-0.1 per cent (D-glucose/L-glucose, n=11), and 0.8+/-0.1 and 0.6+/-0.1 per cent (3-O-methyl-D-glucose/L-glucose, n=6). The difference between the latter TF lost its significance after cytochalasin B (0.1-0.2 mmol/l) had been added to the amniotic compartment. It is concluded that a limited diffusive pathway across the chorionic plate of the human placenta exists and that the transfer of D-glucose depends in part on facilitated diffusion.

3-O-Methylglucose↗

Molecular biology of mammalian glucose transporters.

The oxidation of glucose represents a major source of metabolic energy for mammalian cells. However, because the plasma membrane is impermeable to polar molecules such as glucose, the cellular uptake of this important nutrient is accomplished by membrane-associated carrier proteins that bind and transfer it across the lipid bilayer. Two classes of glucose carriers have been described in mammalian cells: the Na(+)-glucose cotransporter and the facilitative glucose transporter. The Na(+)-glucose cotransporter transports glucose against its concentration gradient by coupling its uptake with the uptake of Na+ that is being transported down its concentration gradient. Facilitative glucose carriers accelerate the transport of glucose down its concentration gradient by facilitative diffusion, a form of passive transport. cDNAs have been isolated from human tissues encoding a Na(+)-glucose-cotransporter protein and five functional facilitative glucose-transporter isoforms. The Na(+)-glucose cotransporter is expressed by absorptive epithelial cells of the small intestine and is involved in the dietary uptake of glucose. The same or a related protein may be responsible for the reabsorption of glucose by the kidney. Facilitative glucose carriers are expressed by most if not all cells. The facilitative glucose-transporter isoforms have distinct tissue distributions and biochemical properties and contribute to the precise disposal of glucose under varying physiological conditions. The GLUT1 (erythrocyte) and GLUT3 (brain) facilitative glucose-transporter isoforms may be responsible for basal or constitutive glucose uptake. The GLUT2 (liver) isoform mediates the bidirectional transport of glucose by the hepatocyte and is responsible, at least in part, for the movement of glucose out of absorptive epithelial cells into the circulation in the small intestine and kidney. This isoform may also comprise part of the glucose-sensing mechanism of the insulin-producing beta-cell. The subcellular localization of the GLUT4 (muscle/fat) isoform changes in response to insulin, and this isoform is responsible for most of the insulin-stimulated uptake of glucose that occurs in muscle and adipose tissue. The GLUT5 (small intestine) facilitative glucose-transporter isoform is expressed at highest levels in the small intestine and may be involved in the transcellular transport of glucose by absorptive epithelial cells. The exon-intron organizations of the human GLUT1, GLUT2, and GLUT4 genes have been determined. In addition, the chromosomal locations of the genes encoding the Na(+)-dependent and facilitative glucose carriers have been determined. Restriction-fragment-length polymorphisms have also been identified at several of these loci.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

Accurate scanning of the BssHII endonuclease in search for its DNA cleavage site.

A facilitated diffusion mechanism has been proposed to account for the kinetic efficiency with which restriction endonucleases are able to locate DNA recognition sites. Such a mechanism involves the initial formation of a nonspecific complex upon collision of the protein with the DNA, with the subsequent diffusion of the protein along the DNA helix until either a recognition site is located or the protein dissociates into solution. Protein translocation may be facilitated by either sliding along the DNA, hopping to nearby sites, or intersegment transfer over larger distances. Previous analyses of the manner in which restriction enzymes cleave DNA substrates did rule out the latter mechanism. To discriminate between protein sliding or scanning and protein hopping, we designed a unique DNA template with three overlapping, mutually exclusive recognition sites for the BssHII endonuclease. Analysis of the cleavage pattern demonstrated efficient usage of both external sites, whereas the centrally located site was not efficiently cleaved. These results confirm that linear diffusion of the BssHII enzyme occurs by scanning along the DNA. Furthermore, the scanning enzyme was found to stop and cleave at the first site encountered. Thus, a sliding restriction endonuclease recognizes cleavage sites with high fidelity, without skipping of potential sites.

Base Sequence↗

A novel rapid-reaction spectrophotometric method for monitoring monovalent anion exchange by human erythrocyte band 3.

Thiocyanate (SCN-) uptake into human erythrocytes and resealed ghosts was measured by monitoring the intracellular reaction of SCN- with methemoglobin using a dual wavelength stopped-flow apparatus. The cellular reaction was considerably slower than the reaction of SCN- with methemoglobin in solution, indicating that SCN- diffusion and not chemical reaction was rate limiting. This view was confirmed by showing that the uptake rate followed saturation kinetics (K(m) approximately 9 mM), thus indicating that SCN- transport involves a facilitated diffusion process. Addition of DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonate) totally inhibited SCN- uptake, thus identifying band 3 as the sole facilitator. Substitution of iodide or sulfate for trans chloride, slowed SCN- uptake by 4-fold and 35-fold respectively. Reducing the trans chloride concentration from 150 to 2 mM decreased the extent of the reaction, and slowed the observed rate by about 2-fold. These results define a new approach for the continuous monitoring of monovalent anion exchange by human erythrocyte band 3.

Anion Exchange Protein 1, Erythrocyte↗

Comparison of methods for local delivery of tissue factor pathway inhibitor to balloon-injured arteries in rabbits.

BACKGROUND: Prolonged intravenous infusions of recombinant tissue factor pathway inhibitor (rTFPI) have been shown to attenuate markedly neointimal formation and stenosis after balloon-induced injury to the carotid arteries in minipigs. DESIGN: Because local delivery of rTFPI to the injury site would be clinically advantageous, we designed this study to compare the local delivery and retention of rTFPI in balloon-injured arteries using three catheter-based systems. METHODS: Similar amounts (range 3-4.5 mg) of a mixture of 125I-labeled and unlabeled rTFPI were delivered by either passive diffusion at moderate pressure (5 x 10(5) Pa with the LocalMed InfusaSleeve, or 4 x 10(5) Pa with the SciMed Dispatch device), or facilitated diffusion combining lower pressure (2 x 10(5) Pa) and electrical current (3.5 mA/cm2; e-MED, iontophoresis) to balloon-injured carotid arteries in anesthetized rabbits. RESULTS: Comparable amounts of rTFPI were retained on the injured vessels immediately after delivery (t = 0) with the LocalMed (628 +/- 68 micrograms/g per cm2, n = 4), SciMed (522 +/- 167 micrograms/g per cm2, n = 4), and e-MED (497 +/- 142 micrograms/g per cm2, n = 4) catheters (NS). However, rTFPI was decreased by 37% after 24 h compared with t = 0 (P < 0.02) in the e-MED group, but was increased 1.5-fold (P = 0.02) and 1.3-fold in the SciMed and LocalMed groups, respectively, presumably because of redistribution of rTFPI from remote endothelial or perivascular sites. Retention of rTFPI was six to nine times higher for injured compared with non-injured arteries, and persisted for at least 48 h after delivery with the LocalMed catheter. CONCLUSIONS: Sustained, marked retention of rTFPI delivered locally at the site of balloon-induced arterial injury appears to result from catheter-based systems that use passive diffusion at moderate pressure.

Angioplasty, Balloon↗

Enantioselective uptake of BOF-4272, a xanthine oxidase inhibitor with a chiral sulfoxide, by isolated rat hepatocytes.

The transport mechanisms of the enantiomers of BOF-4272, a new drug for the treatment of hyperuricemia, were studied using freshly prepared rat hepatocytes. BOF-4272 consists of S(-) and R(+) enantiomers due to a chiral center in the sulfoxide moiety. The uptake of these BOF-4272 enantiomers by hepatocytes was found to be temperature and dose dependent. The temperature-dependent uptake of the S(-) and R(+) enantiomers showed saturation kinetics. The Km values for the S(-) and R(+) enantiomers were 59.3 and 25.7 microM, respectively, which was a significant difference (p < 0.05). However, the maximal uptake rate was comparable for both enantiomers. Metabolic inhibitors such as antimycin, oligomycin, rotenone, carbonylcyanide m-chlorophenyl hydrazone, and carbonyl cyanide-p-(trifluromethoxy)-phenylhydrazone significantly inhibited uptake of the R(+) enantiomer, but had little effect on uptake of the S(-) enantiomer. Ouabain (an inhibitor of Na+/K(+)-ATPase) and p-nitrobenzylthioinosine (NBMPR, a nucleoside transporter inhibitor) showed no significant effects on the uptake of either enantiomer. Organic anions such as taurocholate and cholate reduced the uptake of both enantiomers. These results suggest that the hepatic uptake of both BOF-4272 enantiomers is not due to simple diffusion but also involves carrier-mediated uptake. We suggest that the carrier-mediated uptake of BOF-4272 enantiomers includes both NBMPR-insensitive facilitated diffusion and an active transport system in liver plasma membrane, and that the enantioselective uptake of BOF-4272 is due to differences in affinity for the active transporter.

Animals↗

Strategies of nutrient transport by ruminal bacteria.

The survival of bacteria in natural environments like the rumen depends on the ability of the bacteria to scavenge nutrients. It is now evident that ruminal bacteria use a variety of transport mechanisms. Hydrophobic substances, such as ammonia and acetate, are permeable to the lipid bilayers of cell membranes and can be taken up by passive diffusion. Hydrophilic compounds (e.g., sugars, amino acids, peptides) do not easily pass through lipid bilayers and must be transported across cell membranes on carrier proteins. Facilitated diffusion can display saturable kinetics but does not result in accumulation of solute. Active transport can establish extremely high concentration gradients, and this work may be driven by the hydrolysis of chemical bonds (e.g., ATP) or ion gradients, which are coupled to solute symport. Many solute symports involve protons, but sodium systems also are common in ruminal bacteria. The phosphotransferase system chemically modifies sugars as they pass across the cell membrane, and several ruminal bacteria have this method of group translocation. Many feed additives have either a direct or indirect effect on rumen bacterial transport. For instance, ionophores can inhibit transport by destroying (sometimes even reversing) ion gradients, lowering intracellular pH, or causing excessive ATP hydrolysis.

Animal Feed↗

Interaction of blood flow, diffusive transport and cell metabolism in isovolemic anemia.

1) High blood flow can compensate for half-normal hematocrit, leaving the rate at which O2 is offered to the capillaries unchanged. Nevertheless, intracellular PO2 is lower in anemia, indicating impaired diffusive transport. 2) Anemia increases O2 flux per red cell and decreases functional capillary surface area. These changes increase flux density and the extracellular component of resistance to diffusive O2 transport, in accord with current theory (Federspiel and Popel, 1986; Groebe, 1990; Hellums, 1977). 3) Maintenance of diffusive flux in presence of anemia required a larger delta PO2 between Hb and Mb, and higher intracellular O2 conductance brought about by greater Mb-facilitated diffusion. Both compensations depend on lower PmbO2. 4) PmbO2 and creatine charge fall with increasing VO2 and ATP demand. These responses, as well as adaptive changes in redox help maintain VO2 in the presence of a lower O2 drive on electron transport. 5) Greater engagement of reserves of both transport and metabolism limits the range of aerobic performance in anemia. 6) The match between the transcapillary and mitochondrial O2 fluxes depends on interaction of transport and metabolism as a system.

Anemia↗

[Glucose transporter].

All mammalian cells contain trans-membrane transport systems for the stereo specific uptake of glucose. These systems are divided into two groups, a facilitated diffusion system driven by the concentration differences across the membrane and an energy-dependent, active transport system. Both are mediated by specific membrane proteins, termed glucose transporter. The facilitated glucose transporters comprise a structurally related family of proteins having 12 membrane-spanning domains, with the amino terminus, a relatively large middle loop and the carboxy-terminus all oriented towards the cytoplasm. The active glucose transport system is also designated Na+/glucose cotransporter, SGLT, the predicted secondary structure of which is still controversial.

Animals↗

Urea transport in the dogfish kidney.

There is increasing evidence that urea movement across many epithelia involves more than passive diffusion. Of particular interest is the observation that urea transport across the erythrocyte membrane and across the vasopressin-stimulated urinary bladder of the toad occurs by facilitated diffusion, and can be selectively inhibited by phloretin and chromate. These inhibitory agents have been employed in studies of renal urea reabsorption by the spiny dogfish Squalus acanthias. Both agents inhibit urea reabsorption; the effect of chromate is of particular interest, since it blocks urea reabsorption to a proportionately greater extent than sodium reabsorption, and does so irreversibly.

Animals↗

Amino acid transport in Mycobacterium smegmatis.

The transport of d-alanine, d-glutamic acid, and d-valine in Mycobacterium smegmatis was compared quantitatively with that of their l-isomers. It appeared that the uptake of d-alanine was mediated by an active process displaying saturation kinetics characteristic of enzyme function, whereas the uptake of d-glutamic acid was accomplished by a passive process showing diffusion kinetics. Both processes were involved in the uptake of l-alanine, l-glutamic acid, d-valine, and l-valine. d-Valine competed with l-valine for entry into the cell through a single active process. d-Alanine and l-alanine also utilized the same active process, but the d-isomer could not enter the cell through the passive process. The passive process exhibited characteristics of diffusion, but was sensitive to sulfhydryl-blocking reagents and showed competition among structurally related amino acids. These last findings suggested that the passive process is a facilitated diffusion.

Alanine↗