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PubMed1992

Consequences of molecular engineering enhanced DNA binding in a DNA repair enzyme.

Facilitated one-dimensional diffusion is a general mechanism utilized by several DNA-interactive proteins as they search for their target sites within large domains of nontarget DNA. T4 endonuclease V is a protein which scans DNA in a nonspecifically bound state and processively incises DNA at ultraviolet (UV)-induced pyrimidine dimer sites. An electrostatic contribution to this mechanism of target location has been established. Previous studies indicate that a decrease in the affinity of endonuclease V for nontarget DNA results in a decreased ability to scan DNA and a concomitant decrease in the ability to enhance UV survival in repair-deficient Escherichia coli. This study was designed to question the contrasting effect of an increase in the affinity of endonuclease V for nontarget DNA. With this as a goal, a gradient of increasingly basic amino acid content was created along a proposed endonuclease V-nontarget DNA interface. This incremental increase in positive charge correlated with the stepwise enhancement of nontarget DNA binding, yet inversely correlated with enhanced UV survival in repair-deficient E. coli. Further analysis suggests that the observed reduction in UV survival is consistent with the hypothesis that enhanced nontarget DNA affinity results in reduced pyrimidine dimer-specific recognition and/or binding. The net effect is a reduction in the efficiency of pyrimidine dimer incision.

Base Sequence↗
PubMed1983

Two-dimensional crystallization technique for imaging macromolecules, with application to antigen--antibody--complement complexes.

Two-dimensional crystals are formed from macromolecules bound on the surface of a lipid monolayer. A ligand linked to the lipid orientates the binding, and lateral diffusion of the lipids facilitates crystallization. The crystals are suitable for structural analysis by image processing of electron micrographs. An example is the formation of ordered arrays of antibodies on a monolayer of a lipid hapten, and subsequent decoration of these arrays with the first component of complement. Image processing indicates the arrangement of antibodies and the site of complement binding. This approach should be widely applicable to molecular complexes, such as those in replication, protein synthesis, hormone-receptor interaction and metabolic processes.

Antigen-Antibody Complex↗
PubMed2001

Characterization of palmitoylethanolamide transport in mouse Neuro-2a neuroblastoma and rat RBL-2H3 basophilic leukaemia cells: comparison with anandamide.

The endogenous cannabinoid receptor agonist anandamide (AEA) and the related compound palmitoylethanolamide (PEA) are inactivated by transport into cells followed by metabolism by fatty acid amide hydrolase (FAAH). The cellular uptake of AEA has been characterized in detail, whereas less is known about the properties of the PEA uptake, in particular in neuronal cells. In the present study, the pharmacological and functional properties of PEA and AEA uptake have been investigated in mouse Neuro-2a neuroblastoma and, for comparison, in rat RBL-2H3 basophilic leukaemia cells. Saturable uptake of PEA and AEA into both cell lines were demonstrated with apparent K(M) values of 28 microM (PEA) and 10 microM (AEA) in Neuro-2a cells, and 30 microM (PEA) and 9.3 microM (AEA) in RBL-2H3 cells. Both PEA and AEA uptake showed temperature-dependence but only the AEA uptake was sensitive to treatment with Pronase and phenylmethylsulfonyl fluoride. The AEA uptake was inhibited by AM404, 2-arachidonoylglycerol (2-AG), R1- and S1-methanandamide, arachidonic acid and olvanil with similar potencies for the two cell types. PEA, up to a concentration of 100 microM, did not affect AEA uptake in either cell line. AEA, 2-AG, arachidonic acid, R1-methanandamide, (9)-THC, and cannabidiol inhibited PEA transport in both cell lines. The non-steroidal anti-inflammatory drug indomethacin inhibited the AEA uptake but had very weak effects on the uptake of PEA. From these data, it can be concluded that PEA is transported in to cells both by passive diffusion and by a facilitated transport that is pharmacologically distinguishable from AEA uptake.

Amides↗
PubMed2006

Molecular stenting with a crosslinked hyaluronan derivative inhibits collagen gel contraction.

Adult burn wounds, which lack hyaluronan (HA), often undergo excessive tissue remodeling and contraction. In contrast fetal wounds, which contain large amounts of HA, undergo remodeling that culminates in a scarless repair or regeneration. Therefore, adding a HA derivative to burn wounds would better mimic the fetal extracellular matrix and could reduce contraction. To test this hypothesis, we determined the effects of HA and its two derivatives on fibroblast-mediated, collagen gel contraction, an assay widely used to mimic in vivo wound contraction. Interestingly, high molecular weight HA (HMW HA) facilitated collagen gel contraction, whereas a thiol-functionalized derivative HA-DTPH weakly inhibited contraction. In contrast, polyethylene glycol diacrylate (PEGDA)-crosslinked HA-DTPH (HA-DTPH-PEGDA) strongly inhibited contraction in a concentration-dependent manner. Immunofluorescence staining of cellular actin showed that this inhibition was not owing to reduced cell attachment or spreading. Furthermore, the supernatant of contracted collagen-HMW HA gels contained greater amounts of HA than those found in the supernatant of collagen-HA-DTPH-PEGDA gels, suggesting that HMW HA facilitates contraction by effectively diffusing out of the collagen gels. Therefore, the results suggest that the crosslinking of HA-DTPH enhances the structural mechanics of collagen/HA-DTPH composites, which resists the fibroblast contractile forces and may, therefore, be able to reduce excessive wound contraction observed in pathological conditions.

Adult↗
PubMed1980

Changes in the surface membrane during myoblast fusion.

1. During fusion of chick-embryo myoblasts in culture, the surface membrane is affected as follows. Uptake of 2-aminoisobutyrate and 2-deoxyglucose, each of which is concentrated 20-fold relative to its concentration in the medium, is unaltered; uptake of alpha-methyl glucoside and choline (15 mM), each of which equilibrates relative to its concentration in the medium, approximately doubles. An approximate doubling also occurs in iodinatable surface protein (and in total protein) and in cell surface area as judged by light-microscopy. Adenylate cyclase (in the absence or the presence of fluoride) increases by more than 2-fold. 2. It is concluded that, during myoblast fusion cells increase in size, and this is reflected in an increased rate of simple diffusion; the rate of facilitated processes such as the uptake of amino acids and sugars, on the other hand, remains unaltered, though the activity of certain enzymes is increased. These results indicate that specific changes in the function of surface membrane occur during myoblast fusion in vitro.

Aminoisobutyric Acids↗
PubMed2001

Allosteric phenomena in cytochrome P450-catalyzed monooxygenations.

Allosteric regulation of monooxygenase activity is shown to occur with diverse cytochrome P450 isoforms and is characterized by kinetic patterns deviating from the Michaelis-Menten model. Homotropic and heterotropic phenomena are encountered in both substrate activation and productive coupling of the electron donors NADPH-cytochrome P450 reductase and cytochrome b5, and the lipid environment of the system also appears to play a role as an effector. Circumstantial analysis reveals the components of the electron transfer chain to be mutually beneficial in interactions with each other depending on the substrate used and type of cytochrome P450 operative. It is noteworthy that association of diatomic gaseous ligands may be amenable to allosteric regulation as well. Thus, dioxygen binding to cytochrome P450 displays nonhyperbolic kinetic profiles in the presence of certain substrates; the latter, together with redox proteins such as cytochrome b5, can exert efficient control of the abortive breakdown of the oxyferrous intermediates formed. Similarly, substrates may modulate the structural features of the access channel for solutes such as carbon monoxide in specific cytochrome P450 isozymes to either facilitate or impair ligand diffusion to the heme iron. The in vivo importance of allosteric regulation of enzyme activity is discussed in detail.

Allosteric Regulation↗
PubMed1999

Absorption of hyaluronan applied to the surface of intact skin.

Hyaluronan has recently been introduced as a vehicle for topical application of drugs to the skin. We sought to determine whether hyaluronan acts solely as a hydrophilic reservoir on the surface of intact skin or might partly penetrate it. Drug-free hyaluronan gels were applied to the intact skin of hairless mice and human forearm in situ, with and without [3H] hyaluronan. [3H]hyaluronan was shown by autoradiography to disseminate through all layers of intact skin in mouse and human, reaching the dermis within 30 min of application in mice. Cellular uptake of [3H]hyaluronan was observed in the deeper layers of epidermis, dermis, and in lymphatic endothelium. Absorption through skin was confirmed in mice by chromatographic analysis of blood, urine, and extracts from skin and liver, which identified 3H as intact hyaluronan and its metabolites, free acetate and water. Hyaluronan absorption was similarly demonstrated without polyethylene glycol, which is usually included in the topical formulation. [3H]hyaluronan absorption was not restricted to its smaller polymers as demonstrated by the recovery of polymers of (360-400 kDa) from both blood and skin. This finding suggests that its passage through epidermis does not rely on passive diffusion but may be facilitated by active transport. This study establishes that hyaluronan is absorbed from the surface of the skin and passes rapidly through epidermis, which may allow associated drugs to be carried in relatively high concentration at least as far as the deeper layers of the dermis.

Administration, Topical↗
PubMed2006

Effects of internal conductance on the temperature dependence of the photosynthetic rate in spinach leaves from contrasting growth temperatures.

The photosynthetic rate may be strongly limited by internal conductance from the intercellular airspace to the chloroplast stroma (g(i)). However, the effects of growth and leaf temperature on g(i) are still unclarified. In this work, we determined the temperature dependence of g(i) in spinach leaves grown at 30/25 degrees C (high temperature; HT) and 15/10 degrees C (low temperature; LT), using the concurrent measurements of the gas exchange rate and stable carbon isotope ratio. Moreover, we quantified the effects of g(i) on the temperature dependence of the photosynthetic rate. We measured g(i) and the photosynthetic rate at a CO(2) concentration of 360 microl l(-1) under saturating light (A(360)) at different leaf temperatures. The optimum temperature for A(360) was 28.5 degrees C in HT leaves and 22.9 degrees C in LT leaves. The optimum temperatures for g(i) were almost similar to those of A(360) in both HT and LT leaves. There was a strong linear relationship between A(360) and g(i). The photosynthetic rates predicted from the C(3) photosynthesis model taking account of g(i) agreed well with A(360) in both HT and LT leaves. The temperature coefficients (Q(10)) of g(i) between 10 and 20 degrees C were 2.0 and 1.8 in HT and LT leaves, respectively. This suggests that g(i) was determined not only by physical diffusion but by processes facilitated by protein(s). The limitation of the photosynthetic rate imposed by g(i) increased with leaf temperature and was greater than the limitation of the stomatal conductance at any temperature, in both HT and LT leaves. This study suggests that g(i) substantially limits the photosynthetic rate, especially at higher temperatures.

Carbon Dioxide↗
PubMed1985

Classification of the epilepsies.

We review the development of the classification of the epilepsies. Primary epilepsies are relatively benign, usually age-limited syndromes without clinical or radiologic evidence of brain lesions, and are related to a heritable constitutional predisposition to epilepsy. They usually respond well to antiepileptic drugs. The biochemical correlates of primary generalized epilepsy have been demonstrated in animals and man and have been related to diffuse cortical hyperexcitability, which has been linked to this disorder. The pathophysiology of the primary partial or focal epilepsies is poorly understood but does not appear to depend on focal brain lesions. We suggest that these are due to relatively localized areas of cortical hyperexcitability confined to isolated corticothalamic sectors and depend on interrelations between a constitutional cortical hyperexcitability and normal cortical maturation. The secondary epilepsies are associated with clinical and radiologic evidence of brain lesions and are often resistant to anticonvulsants. Secondary generalized epilepsies, with an associated diffuse encephalopathy, are typified by the Lennox-Gastaut syndrome. The presentation of the secondary partial epilepsies depends on the site of the lesion. The pathogenesis of epilepsy is multifactorial, and a preexisting constitutional predisposition can interact with an acquired diffuse or focal encephalopathy, facilitating the clinical expression of one or the other.

Adolescent↗
PubMed2005

Valproic acid binding to human serum and human placenta in vitro.

The binding characteristics of the antiepileptic agent and teratogen valproic acid for human serum and human placenta were investigated utilizing equilibrium dialysis of the drugs in serum and in homogenates of whole placenta so that the transplacental transfer of the drug could be better defined. A low-capacity, high-affinity binder and a high-capacity, low-affinity binder for valproic acid were found in serum. However, there was only minimal, nonspecific binding of the drug to placenta. It appears that transplacental transfer of valproic acid is not mediated by binding to placenta. It is likely that such transfer occurs by passive diffusion, which may be facilitated by the lipophilicity of the drug and by development of a pH gradient across the placenta. These findings indicate that the placenta is not a depot for valproic acid.

Blood Proteins↗
PubMed2005

Real-time evolution of the distribution of nanoparticles in an ultrathin-polymer-film-based waveguide.

We report a novel application of an ultrathin-polymer-film-based, resonance-enhanced x-ray waveguide as a real-time nanoprobe for elucidating dilute, yet disordered, gold nanoparticles embedded in the polymer matrix. This nanoprobe promises a sensitivity enhancement of several orders of magnitude, hence revealing in real time the lateral nanoparticle distribution with subnanometer spatial resolution. We observed that the motion of the nanoparticles is strongly anisotropic, with in-plane coalescence taking place more rapidly than out-of-plane diffusion, which can ultimately facilitate the formation of two-dimensional structures.

Journal Article↗
PubMed1994

Various vectorial behaviours of a spatially structured substrate cycle.

The dynamic properties of the glucose/glucose-6-phosphate cycle are studied under conditions where the phosphatase and kinase interconverting enzymes are spatially distributed. A semi-artificial membrane made of compacted plant cell walls bearing active phosphatase in its natural state, separates two compartments, one of these compartments containing soluble hexokinase. Depending only upon the two enzyme activity levels and the initial distribution of the substrates, numerous asymmetrical and vectorial behaviours can be observed, such as facilitated glucose 6-phosphate diffusion, active transport of either glucose or glucose 6-phosphate and sequential (alternative) transport between glucose and glucose 6-phosphate. A diffusion-partition reaction coupling can account for these oriented mass transfers. The possibility of such a coupling in this model system is clearly dictated by the global analog of the Curie principle. These results may provide new insight on (a) the still obscure role played by the cell-wall phosphatase activities, particularly their involvement in the transport of exogenous phosphomonoesters, and (b) the actual in vivo operation of substrate and protein cycles in view of the heterogeneity and anisotropy of the cellular milieu.

Biological Transport↗
PubMed1985

Facilitated transport of glucose from blood into peripheral nerve.

D-Glucose is the major substrate for energy metabolism in peripheral nerve. The mechanism of transfer of glucose across the blood-nerve barrier is unclarified. In this study an in situ perfusion technique was utilized, in anesthetized rats, to examine monosaccharide transport from blood into peripheral nerve. Unidirectional influxes of D-[14C]glucose, L-[14C]glucose, and [14C]3-O-methyl-D-glucose across capillaries of the tibial nerve were measured at different perfusate concentrations of unlabelled D-glucose. The permeability-surface area product (PA) for D-[14C]glucose and [14C]3-O-methyl-D-glucose decreased, whereas the PA for L-[14C]glucose remained constant, as the perfusate concentration of D-glucose was increased. In the presence of no added unlabelled D-glucose in the perfusate, the PA for L-[14C]glucose equaled one-fifth the PA for D-[14C]glucose. These results demonstrate self-saturation, competitive inhibition, and stereospecificity of glucose transfer, and for the first time show a unidirectional facilitated transport mechanism for D-monosaccharides at capillaries of mammalian peripheral nerve. The data were fit to a model for facilitated transport and passive diffusion. The half-saturation constant and maximal rate of transport for the saturable component of D-glucose influx equaled 23 +/- 11 mumol X ml-1 and 6.6 +/- 3.2 X 10(-3) mumol X s-1 X g-1, respectively. The constant of nonsaturable glucose influx equaled 0.5 +/- 0.1 X 10(-4) s-1. At normal plasma glucose concentrations, the saturable component comprises about 80% of total D-glucose influx into nerve.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2005

Cultivating men's interest in family planning in rural El Salvador.

A pilot project in rural El Salvador tested the integration of family planning into a water and sanitation program as a strategy for increasing male involvement in family planning decison making and use. The organizations involved posited that integrating family planning into a resource management and community development project would facilitate male involvement by diffusing information, by referring men and women to services, and by expanding method choice to include the new Standard Days Method through networks established around issues men cared about and were already involved in. This article examines data from a community-based household survey to assess the impact of the intervention and finds significant changes in contraceptive knowledge, attitudes, and behavior from baseline to endline. Because the differences between baseline and endline are greater than the differences between participants and nonparticipants at endline, the study demonstrates the power of informal networks for spreading information.

Adult↗
PubMed1979

CO2 along the proximal tubules in the rat kidney.

The proximal intratubular pH of the rat kidney was measured in vivo with an antimony electrode system. PCO2 and bicarbonate concentration of the proximal tubular fluid were determined with an ultramicro equilibration system. The tubular fluid to plasma inulin concentration ratio was evaluated by a microscope fluorometric method. The acid-base parameters and the inulin concentrations were determined under control conditions and during acetazolamide treatment. The intratubular PCO2 was higher than the PCO2 of the systemic arterial blood under control conditions and the difference in PCO2 was increased during acetazolamide treatment. In acetazolamide treated rats the rate of fractional bicarbonate reabsorption was decreased in the early part of the proximal tubule, while it was of about the same in the middle and late parts as compared with control rats. The total bicarbonate reabsorption in the proximal tubule was reduced by 50% due to the carbonic anhydrase inhibition. It seems possible that the bicarbonate is still reabsorbed as CO2 after carbonic anhydrase inhibition, as hydrogen ion secretion is not totally stopped by this treatment. The increase in intratubular PCO2 after acetazolamide treatment is assumed to be due to an inhibition of the carbonic anhydrase facilitating effect on outward diffusion of CO2 from the tubular lumen across the cell wall.

Acetazolamide↗
PubMed1967

Water uptake by Bufo melanostictus, as affected by osmotic gradients, vasopressin and temperature.

1. The rate of water uptake across the skin was studied in the live toad, Bufo melanostictus. When toads were kept in distilled water at 29 degrees C the uptake of water amounted to 16.9 +/- 1.3 mul./cm(2)/hr; when bathed in sucrose or urea solutions, the water uptake diminished with increasing osmotic pressure. There was no water uptake observed when toads were kept in 200 m-osmolar sucrose or urea.2. Intramuscular injections of vasopressin increased the rate of water uptake from distilled water. There was a good relation between doses and responses over various time intervals. A dose of 4 m-u. vasopressin/g body wt. doubled the rate of water uptake over a period of 1 hr. The same dose of vasopressin doubled the rate of water uptake when the toads were kept in solutions of sucrose or urea of different osmolarity.3. The rate of water uptake when the toads were bathed in sodium chloride solutions was consistently 8 mul./cm(2)/hr greater than when bathed in sucrose or urea solutions of equal osmolarity. There was no water uptake when the sodium chloride solution was 285 m-osmolar.4. Vasopressin (4 m-u./g) injected intramuscularly doubled the rate of water uptake from sodium chloride solutions of different osmolarity.5. With solutions of potassium chloride, sodium nitrate, and potassium nitrate, in concentrations up to 150 m-osmoles/l., the rate of water uptake was found to be the same as with solutions of sodium chloride of the same osmolarity. Similarly, it was doubled by injection of vasopressin (4m-u./g).6. The effect of temperature on the rate of water uptake before and after injection of vasopressin was investigated in toads kept in distilled water, sucrose, or sodium chloride solutions. For temperatures between 20 and 37 degrees C, vasopressin (4 m-u./g) reduced the activation energy involved in the process of water uptake by 4000 cal.7. The results agree with the view that water uptake follows a diffusion process which is facilitated by vasopressin, possibly as a result of increasing the size or number of available pores.

Animals↗
PubMed1981

Vesicle transport in arterial endothelium and the influence of mechanical factors on macromolecular permeability.

The time-dependent transport of labeled vesicles in arterial endothelium has been modeled, taking into account the space-varying electrodynamic and hydrodynamic forces and the steric hindrance of vesicle attachment by the already attached vesicles. With the aid of laboratory model experiments to assess the steric hindrance effect, theoretical computation has been made on time-dependent labeled vesicle concentration profiles, and the results agree reasonably well with the published experimental data. Oscillatory length variations (5-10 Hz for 15 min) and elevation of transmural pressure (from 0-100 and 200 mmHg) caused increases in 125I-albumin uptake by the canine common carotid artery. Theoretical computations based on ultrastructural determination of free vesicle density indicate that there was negligible enhancement of vesicle diffusion by these mechanical disturbances. The increases in albumin uptake following length oscillation and pressure elevation to 100 mmHg were accompanied by increases in luminal surface area, and the albumin permeability remained unchanged. The albumin permeability was elevated following pressure elevation to 200 mmHg, and this was attributable to (a) a decrease in transendothelial diffusion distance, and (b) facilitation of vesicle loading into the vesicles.

Animals↗