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Determination of carbonic anhydrase activity by a pCO2 sensor.

A potentiometric method capable of determining carbonic anhydrase (CA) activity in vitro and based on the use of a pCO2 sensor is presented. By means of the procedure described here it is possible to follow the rate of CO2 diffusion that takes place in a buffered solution of NaHCO3 in either the presence or the absence of CA. All experimental parameters that affect the speed of HCO3- dehydration, as well as the speed of CO2 diffusion, can be fixed and kept constant for the duration of every assay. The advantage of this method is that the overall dehydration plus diffusion process can be followed as it actually takes place in open thermodynamic systems far from equilibrium. The results obtained strongly confirm the hypothesis of a facilitating role of CA toward the rate of CO2 diffusion.

Animals↗

Study of mechanisms of electric field-induced DNA transfection. I. DNA entry by surface binding and diffusion through membrane pores.

A study of mechanisms of electrotransfection using Escherichia coli (JM 105) and the plasmid DNA pBR322 as model system is reported. pBR322 DNA carries an ampicillin resistance gene: E. coli transformants are conveniently assayed by counting colonies in a selection medium containing 50 micrograms/ml ampicillin and 25 micrograms/ml streptomycin. Samples not exposed to the electric field showed no transfection. In the absence of added cations, the plasmid DNA remains in solution and the efficiency of the transfection was 2 x 10(6)/micrograms DNA for cells treated with a 8-kV/cm, 1-ms electric pulse (square wave). DNA binding to the cell membrane greatly enhanced the efficiency of the transfection and this binding was increased by milimolar concentrations of CaCl2, MgCl2, or NaCl (CaCl2 greater than MgCl2 greater than NaCl). For example, in the presence of 2.5 mM CaCl2, 55% of the DNA added bound to E. coli and the transfection efficiency was elevated by two orders of magnitude (2 x 10(8)/micrograms DNA). These ions did not cause cell aggregation. With a low ratio of DNA to cells (less than 1 copy/cell), transfection efficiency correlated with the amount of DNA bound to the cell surface irrespective of salts. When the DNA binding ratio approached zero, the transfection efficiency was reduced by two to three orders, indicating that DNA entry by diffusion through the bulk solution was less than 1%. Square pulses of up to 12 kV/cm and 1 ms were used in the electrotransfection experiments. When cell concentration was 1 x 1010 cell/ml and DNA was added before the pulse, a transfection efficiency of up to 5 x 108/ microg DNA was obtained under optimum conditions (a single pulse of 8 kV/cm, 1 ms, in the presence of 5 mM CaCl2). When DNA was added to E. coli after the electric pulse, the efficiency of the transfection was dramatically reduced owing to the resealing of pores. Transfection was reduced to zero when DNA was added 2 h after the electroporation. However, transfection as high as 5 x 104/microg DNA was still recorded when DNA was added 10 min after the electric field was turned off. Because DNA entry took place in the absence of an electric field it could not be driven by the electrophoretic forces. DNA entry was facilitated by surface binding followed by lateral diffusion of the bound DNA into the cells through the field-induced membrane pores.

Calcium Chloride↗

Kinetic assessment of apparent facilitation by albumin of cellular uptake of unbound ligands.

Previous studies of the effect of albumin on initial uptake of ligands by isolated cell suspensions or cultures found that the apparent uptake for unbound ligand appeared larger in the presence of binding to the albumin than when albumin was absent. Furthermore, when ligand and albumin were increased in a fixed molar ratio, uptake appeared to be competitively inhibited by the excess albumin. We examined the kinetics underlying this apparent facilitation phenomenon by incorporating unbound fraction of ligand in the medium (fu1) into the general model for diffusion between two compartments. The analysis showed that even in the absence of facilitation by albumin, the apparent rate constant for uptake of unbound ligand (k/fu1) increases as albumin concentration increases but the uptake clearance of unbound ligand remains constant. This theoretical analysis was verified experimentally by measuring the effect of albumin on uptake rates of 14C-taurocholate (12, 24, 48, 60, and 96 microM, with and without 0.87 mM albumin) in a nonphysiological system consisting of two solutions separated by a cellulose membrane. Moreover, when the taurocholate and albumin concentrations were increased in a fixed molar ratio of 0.06 (taurocholate 12-96 microM, albumin 0.2-1.6 mM), the initial uptake rate exhibited the same nonlinear pattern as the previous studies that used living cells. This pattern was due not to saturation of a putative albumin receptor but simply to the concomitant decrease in fu1 which tended to offset the increase in uptake rate due to the increasing total taurocholate concentration. The model was also used to evaluate published data describing the effect of albumin on the uptake of iopanoic acid by cultured hepatocytes. In accordance with the model, k1/fu1 increased as albumin concentration increased, but uptake clearance was independent of albumin concentration. Therefore, the kinetic pattern found in this and other studies with isolated cell suspensions or cultures argues against a special role for albumin in facilitating cellular ligand uptake.

Animals↗

Developing methods for systematic reviewing in health services delivery and organization: an example from a review of access to health care for people with learning disabilities. Part 2. Evaluation of the literature--a practical guide.

OBJECTIVES: To develop methods to facilitate the 'systematic' review of evidence from a range of methodologies on diffuse or 'soft' topics, as exemplified by 'access to health care'. DATA SOURCES: Twenty-eight bibliographic databases, research registers, organizational websites or library catalogues. Reference lists from identified studies. Contact with experts and service users. Current awareness and contents alerting services in the area of learning disabilities. REVIEW METHODS: Inclusion criteria were English language literature from 1980 onwards, relating to people with learning disabilities of any age and all study designs. The main criteria for assessment was relevance to Guillifords' model of access to health care which was adapted to the circumstances of people with learning disabilities. Selected studies were evaluated for scientific rigour then data was extracted and the results synthesized. Quality assessment was by an initial set of 'generic' quality indicators. This enabled further evidence selection before evaluation of findings according to specific criteria for qualitative, quantitative or mixed-method studies. RESULTS: Eighty-two studies were fully evaluated. Five studies were rated 'highly rigorous', 22 'rigorous', 46 'less rigorous' and nine 'poor' papers were retained as the sole evidence covering aspects of the guiding model. The majority of studies were quantitative but used only descriptive statistics. Most evidence lacked methodological detail, which often lowered final quality ratings. CONCLUSIONS: The application of a consistent structure to quality evaluation can facilitate data appraisal, extraction and synthesis across a range of methodologies in diffuse or 'soft' topics. Synthesis can be facilitated further by using software, such as the microsoft 'access' database, for managing information.

Adult↗

Enhancement of the intestinal absorption of ergot peptide alkaloids in the rat by micellar solutions of polyoxyethylene-24-cholesteryl ether.

The incomplete intestinal absorption of hydrogenated ergot peptide alkaloids as measured in bile duct cannulated rats is much increased when the ergot compounds are administered as micellar solutions together with POE-24-cholesteryl ether. In vitro diffusion experiments with isolated intestinal mucus show that the ergot peptide alkaloids are strongly retained by the mucus layer. It is suggested that the diffusion of the ergot compounds across the mucus barrier is facilitated by micellar entrapment of the drug.

Animals↗

Mobility within the nucleus and neighboring cytosol is a key feature of prothymosin-alpha.

Prothymosin alpha is a small, unfolded, negatively charged, poorly antigenic mammalian protein with a potent nuclear localization signal. Although it is apparently essential for growth, its precise function is unknown. We examined the location and behavior of the protein bearing different epitope tags using in situ immunolocalization in COS-1 and NIH3T3 cells. Tagged prothymosin alpha appeared to be punctate and widely dispersed throughout the nucleus, with the exception of the nucleolus. A tiny cytoplasmic component, which persisted in the presence of cycloheximide and actinomycin D during interphase, became pronounced immediately before, during, and after mitosis. When nuclear uptake was abrogated, small tagged prothymosin alpha molecules, but not prothymosin alpha fused to beta-galactosidase, accumulated significantly in the cytoplasm. Tagged prothymosin alpha shared domains with mobile proteins such as Ran, transportin, and karyopherin beta, which also traverse the nuclear membrane, and co-localized with active RNA polymerase II. Mild digitonin treatment resulted in nuclei devoid of prothymosin alpha. The data do not support tight binding to any nuclear component. Therefore, we propose that prothymosin alpha is a highly diffusible bolus of salt and infer that it facilitates movement of charged molecules in highly charged environments within and near the nucleus.

3T3 Cells↗

Changes in fluid secretion rate alter net transepithelial transport of MRP2 and P-glycoprotein substrates in Malpighian tubules of Drosophila melanogaster.

The effects of stimulants of fluid secretion on net transepithelial transport of the MRP2 substrate Texas Red and the p-glycoprotein substrate daunorubicin were examined in Malpighian tubules of Drosophila melanogaster. Fluid secretion rates were determined using the Ramsay assay and secreted fluid concentrations of Texas Red and daunorubicin were determined using a microfluorometric technique. Nanoliter droplets of secreted fluid were collected in optically flat glass capillaries and dye concentration was determined from fluorescence intensity measured by confocal laser scanning microscopy. Net transepithelial flux of each compound was then calculated as the product of its concentration in the secreted fluid and the fluid secretion rate. Net transepithelial flux of Texas Red increased when fluid secretion was stimulated by tyramine, cyclic AMP or hypoosmotic saline. Net flux decreased when fluid secretion rate of cAMP-stimulated tubules was reduced by elevating saline osmolality with sucrose. Net transepithelial flux of daunorubicin increased when fluid secretion was stimulated by cAMP. Significant increases in dye flux were seen only when the dyes were present at concentrations close to or greater than the concentration required for half maximal transport. Regression analyses showed that 57- 88% of the change in dye flux was attributable to the change in fluid secretion rate when tubules were stimulated with cAMP, cGMP, or tyramine. The results do not suggest that the effects of tyramine and cAMP are mediated through changes in transepithelial potential, nor do they indicate the direct effects of the stimulants on MRP2-like or p-glycoprotein-like transporters (e.g., via protein kinases). Instead, the results suggest that increases in fluid secretion rate minimize diffusive backflux of these dyes and, thus, facilitate higher rates of net transepithelial transport indirectly.

ATP Binding Cassette Transporter, Subfamily B↗

Physiologic models of hepatic drug clearance: influence of altered protein binding on the elimination of diclofenac in the isolated perfused rat liver.

The single-pass perfused rat liver preparation was used to assess the influence of binding to human serum albumin on the steady-state hepatic extraction of diclofenac (n = 8). In the absence of binding protein, the extraction ratio of diclofenac approached unity (range, 0.975-0.992), such that its clearance was perfusion-rate limited. As the binding of diclofenac to protein was increased by the addition of human serum albumin to the perfusion medium, its extraction ratio decreased dramatically, and clearance eventually became capacity limited. The relationship between diclofenac availability and fraction unbound was analyzed with various physiologic models of hepatic drug clearance. The dispersion model, which contains a parameter (the dispersion number) that quantifies the axial spreading of a substrate as it passes along the liver length, provided a significantly better description of the data (p < 0.05) than the undistributed parallel-tube model, which assumes that an eliminated substrate travels through the liver as an undispersed plug, and the well stirred (venous equilibrium) model, which assumes that substrate undergoes infinite mixing as soon as it enters the liver. The dispersion number estimated for diclofenac (mean, 3.03; range, 0.89-7.56) was significantly greater than that predicted from considerations of the transverse heterogeneity of blood flow within the hepatic sinusoidal bed, suggesting that additional factors influenced the relationship between availability and fraction unbound for this compound. Such factors may include transverse heterogeneity of the metabolizing enzyme system(s), axial flux of substrate created by diffusion within hepatic tissue, and protein-facilitated transfer of substrate across an unstirred fluid layer adjacent to the hepatocyte surface.

Animals↗

Bral1, a brain-specific link protein, colocalizing with the versican V2 isoform at the nodes of Ranvier in developing and adult mouse central nervous systems.

Bral1, a brain-specific hyaluronan-binding protein, has been cloned recently. To gain insight into the role of Bral1, we generated a specific antibody against this protein. We have examined the detailed localization pattern of Bral1 protein and compared it with that of other members of the lectican proteoglycan family, such as brevican and versican, with which Bral1 is predicted to interact. The immunoreactivity of Bral1 antibody was predominantly observed in myelinated fiber tracts in the adult brain and could be detected at P20 in the white matter of the developing cerebellum, suggesting that expression starts when axonal myelination takes place. Furthermore, immunostaining demonstrated that Bral1 colocalized with the versican V2 isoform at the nodes of Ranvier. The present data suggest that Bral1 may play a pivotal role in the formation of the hyaluronan-associated matrix in the CNS that facilitates neuronal conduction by forming an ion diffusion barrier at the nodes.

Amino Acid Sequence↗

Nontraumatic abdominal emergencies: acute abdominal pain: diagnostic strategies.

Common causes of acute abdominal pain include appendicitis, cholecystitis, bowel obstruction, urinary colic, perforated peptic ulcer, pancreatitis, diverticulitis, and nonspecific, nonsurgical abdominal pain. The topographic classification of acute abdominal pain (pain in one of the four abdominal quadrants, diffuse abdominal pain, flank or epigastric pain) facilitates the choice of the imaging technique. The initial radiological evaluation often consists of plain abdominal radiography, despite significant diagnostic limitations. The traditional indications for plain films--bowel obstruction, pneumoperitoneum, and the search of ureteral calculi--are questioned by helical computed tomography (CT). Although ultrasonography (US) is in many centers the modality of choice for imaging the gallbladder and the pelvis in children and women of reproductive age, CT is considered to be one of the most valued tools for triaging patients with acute abdominal pain. CT is particularly beneficial in patients with marked obesity, unclear US findings, bowel obstruction, and multiple lesions. The introduction of multidetector row CT (MDCT) has further enhanced the utility of CT in imaging patients with acute abdominal pain.

Abdominal Pain↗

Effects of hyaluronidase and hydrocortisone on myocardial necrosis after coronary occlusion.

In anesthetized open chest dogs, hydrocortisone (50 mg/kg body weight administered 30 minutes after occlusion and 25 mg/kg 12 hours later) substantially reduced the size of myocardial infarcts, as reflected by both myocardial creatine phosphokinase activity and histologic appearance 24 hours later. Similarly, hyaluronidase, which increases diffusion through the extracellular space and presumably facilitates delivery of substrate to ischemic cells, also reduced the extent of myocardial necrosis after coronary occlusion in the dog. In view of the salutary effects of hyaluronidase and the absence of serious side effects, this agent was administered clinically to two groups of patients, who were compared with two groups of untreated control subjects. Hyaluronidase (500 National Formulary units/kg X 8) was shown to result in a significantly more rapid reduction in the magnitude and the extent of precordial S-T segment elevations, and in patients treated within 4 hours a tendency to a lower incidence rate of Q waves and a smaller reduction of R waves.

Animals↗

Lactate transport and glycolytic activity in the freshly isolated rabbit cornea.

Studies on the intact avascular cornea reveal two types of lactate effluxes: exogenous glucose-elicited and spontaneous. The former type exhibits characteristics resembling the proton-lactate symport system previously found in tumor cells and erythrocytes, including an enhanced lactate efflux at a higher extracellular pH and in the presence of H+ and K+ ionophores, and an inhibition by mersalyl with subsequent lactate accumulation in the tissue and cessation of glycolytic activity. The latter type occurs immediately following the incubation of freshly isolated cornea in a medium containing no exogenous glucose, with a rate about 10 times that of exogenous glucose-elicited lactate efflux. It is insensitive to 10 mM iodoacetate and lacks the characteristics of the proton-lactate symport system. Findings reveal that about 50% of corneal glucose utilization occurs in the epithelium, with the stroma and endothelium sharing the other 50% approximately equally. Of the glucose utilized, the lactate formation to pyruvate oxidation rate ratios are approximately 1:1 in the epithelium, 2:1 in the stroma, and 1:2 in the endothelium. About 79% of total tissue lactate is formed in the epithelium and stroma, and in vivo, this is probably pumped into the stromal extracellular space (about 90% of total tissue volume) via the proton-lactate symport system, with spontaneous release into the aqueous humor via a simple diffusion process. The H+ and K+ ionophores facilitate lactate efflux at the expense of the cellular pyruvate pool, without significant effect on the glucose uptake and glycolytic activity. These findings suggest that the ionophore-mediated lactate efflux favors the reduction of low pyruvate concentration in the tissue, rather than parallel increases in glycolytic activity.

3-O-Methylglucose↗

Effect of HEMA on bonding to dentin.

The present study investigated the effectiveness of treating dentin with 2-hydroxyethyl methacrylate (HEMA) prior to application of an adhesive resin. The adhesive resin was 5% 4-methacryloxyethyl trimellitate anhydride (4-META) in methyl methacrylate (MMA) combined with poly-MMA powder. Polymerization of this resin was initiated by tri-n-butyl borane (TBB). Bovine dentin samples were ground with 600-grit Carbimet paper discs, and demineralized with either an aqueous solution of 10% citric acid/3% ferric chloride (10-3) or an aqueous solution of 10% citric acid (10-0). Improved bond strengths were achieved with HEMA treatment of bovine dentin samples, and improvement of bond strengths was dependent upon the time period of HEMA application. Scanning electron microscope (SEM) examination revealed the formation of a transitional zone of resin-reinforced dentin, termed the "hybrid" layer, in those specimens receiving 10-3 pre-treatment. The adhesive resin impregnated the exposed collagen bundles with which it entangled to create the "hybrid", essential in the attainment of high tensile bond strengths. Specimens pre-treated with 10-0 did not readily form "hybrid" layers. However, if HEMA application followed the 10-0 pre-treatment, "hybrids" were demonstrated on SEM, and bond strength increased to 13 MPa. The ferric ions in the 10-3 effectively improved the diffusivity of dentinal substrates, as did HEMA. This study indicates that HEMA applied to dentinal substrates enhances monomer diffusion and entanglement with dentinal components, and facilitates the formation of a "hybrid" layer.

Adhesiveness↗

Implantable cardioverter-defibrillator (ICD) indications in 1996: have they changed?

The recent progress in the technology of the implantable cardioverter-defibrillator (ICD) and the excellent clinical results achieved with ICD treatment in the prevention of sudden death have facilitated the wide-spread acceptance and diffusion of this therapeutic modality. However, ICD implantation is a costly therapy and its use is still associated with some important unresolved issues. In particular, owing to the absence of randomized controlled clinical trials, it is not yet known whether ICD really reduces overall mortality. Thus, at the present time, it appears logical to exercise restraint in expanding the use of ICDs as first-choice therapy in patients with life-threatening ventricular arrhythmias. ICD treatment should be restricted to those well-defined categories of high-risk patients who are most likely to benefit from device implantation in terms of life prolongation. Basically, this means patients with hemodynamically poorly tolerated ventricular tachycardia or ventricular fibrillation that are not inducible at electrophysiologic study and those who do not respond to, or do not tolerate, drug therapy with amiodarone, sotalol, or beta blockers. Patients with idiopathic ventricular fibrillation also seem to be suitable candidates for ICD implantation. Other indications for ICD therapy are, as yet, more controversial and should be carefully evaluated on a case-by-case basis.

Death, Sudden, Cardiac↗

Amplified electrocatalysis at DNA-modified nanowires.

Arrayed gold nanowires are a novel and useful platform for electrochemical DNA detection. Pilot studies testing the use of these templated structures with an electrocatalytic reporter system revealed that very low detection thresholds for target DNA sequences can be obtained. One factor contributing to the heightened sensitivity is the high signal-to-noise ratio achieved with the large electrocatalytic signals observed at DNA-modified nanowires. Here, we explain the improved sensitivity with evidence illustrating that electrocatalysis at DNA-modified nanostructures generates amplified signals that are significantly larger than those observed at bulk gold surfaces. The results presented strongly suggest that the three-dimensional architectures of the nanowires facilitate the electrocatalytic reaction because of enhanced diffusion occurring around these structures. Effects unique to the nanoscale are shown to underlie the utility of nanowires for DNA biosensing.

Biosensing Techniques↗

Ran at kinetochores.

The Ran GTPase controls many cellular functions, including nucleocytoplasmic trafficking, spindle assembly, nuclear assembly and cell-cycle progression. Considerable evidence suggests that diffusible Ran-GTP near mitotic chromatin facilitates the release of critical factors from nuclear transport receptors, thereby promoting organization of mitotic spindles with respect to chromosomes. In addition to this role of soluble Ran-GTP, Ran has two important but less understood roles at mitotic kinetochores. Namely, it is essential for regulation of the spindle assembly checkpoint and for assembly of microtubule fibres that attach kinetochores to spindle poles. Here, I will briefly summarize evidence for these kinetochore-associated functions and mention some of the issues that remain to be addressed regarding them.

Animals↗

Identification of detergent-resistant plasma membrane microdomains in dictyostelium: enrichment of signal transduction proteins.

Unlike most other cellular proteins, the chemoattractant receptor, cAR1, of Dictyostelium is resistant to extraction by the zwitterionic detergent, CHAPS. We exploited this property to isolate a subcellular fraction highly enriched in cAR1 by flotation of CHAPS lysates of cells in sucrose density gradients. Immunogold electron microscopy studies revealed a homogeneous preparation of membrane bilayer sheets. This preparation, designated CHAPS-insoluble floating fraction (CHIEF), also contained a defined set of 20 other proteins and a single uncharged lipid. Cell surface biotinylation and preembedding immunoelectron microscopy both confirmed the plasma membrane origin of this preparation. The cell surface phosphodiesterase (PDE) and a downstream effector of cAR1, adenylate cyclase (ACA), were specifically localized in these structures, whereas the cell adhesion molecule gp80, most of the major cell surface membrane proteins, cytoskeletal components, the actin-binding integral membrane protein ponticulin, and G-protein alpha- and beta-subunits were absent. Overall, CHIFF represents about 3-5% of cell externally exposed membrane proteins. All of these results indicate that CHIFF is derived from specialized microdomains of the plasma membrane. The method of isolation is analogous to that of caveolae. However, we were unable to detect distinct caveolae-like structures on the cell surface associated with cAR1, which showed a diffuse staining profile. The discovery of CHIFF facilitates the purification of cAR1 and related signaling proteins and the biochemical characterization of receptor-mediated processes such as G-protein activation and desensitization. It also has important implications for the "fluid mosaic" model of the plasma membrane structures.

Animals↗

Contrasting oxygen dynamics in the freshwater isoetid Lobelia dortmanna and the marine seagrass Zostera marina.

BACKGROUND: and Aims Submerged plants possess well-developed aerenchyma facilitating intra-plant gas-phase diffusion of O2 to below-ground tissues, which are usually buried in anoxic sediments. However, aquatic habitats differ in terms of O2 fluctuations in the water column and in O2 consumption of the sediment, and aquatic plants differ in aerenchymal volume and resistance to O2 diffusion through the plant and across leaf and root surfaces. The hypothesis that the freshwater isoetid Lobelia dortmanna and the marine seagrass Zostera marina should display pronounced contrasts in intra-plant O2 dynamics because of differences in morphology/anatomy, physiology and growth habitat was tested. METHODS: In order to determine the O2 dynamics and relate this to the anatomy and morphology of the two species, O2 microelectrodes were inserted in the aerenchyma of leaves and roots, the sediment pore-water, and the water column in the field. Manipulation of water column O2 in the laboratory was also carried out. KEY RESULTS: It was found that intra-plant transport of O2 between leaf and root tips takes place more readily in L. dortmanna than in Z. marina due to shorter distances and greater cross-sections of the aerenchyma. The major exchange of O2 across roots of L. dortmanna can be accounted for by small intra-plant resistances to diffusion, larger root than leaf surfaces, and greater radial diffusive resistance of leaves than roots. In contrast, the major O2 exchange across leaves than roots of Z. marina can be accounted for by the opposite anatomical-morphological features. The larger aerenchymal volume and the smaller metabolic rates of L. dortmanna compared to Z. marina imply that turnover of O2 is slower in the aerenchyma of L. dortmanna and O2 fluctuations are more dampened following changes in irradiance. Also, O2 accumulated in the aerenchyma can theoretically support dark respiration for a few hours in L. dortmanna but for only a few minutes in Z. marina. CONCLUSIONS: The build-up of O2 in the pore-water of L. dortmanna sediments during the day as a result of high release of photosynthetic O2 from roots and low O2 consumption of sediments means that sediment, aerenchyma and water are important O2 sources for respiration during the following night, while Z. marina relies on the water column as the sole source of O2 because its sediments are anoxic. These differences between L. dortmanna and Z. marina appear to represent a general difference between the isoetid species mainly inhabiting sediments of low reducing capacity of oligotrophic lakes and the elodeid freshwater species and marine seagrasses mainly inhabiting sediments of higher reducing capacity in more nutrient-rich habitats.

Circadian Rhythm↗