The Molecular Weight and Other Biophysical Properties of Bromegrass Mosaic Virus

Data are presented which show that bromegrass mosaic virus has a particularly low molecular weight and nucleic acid content. A molecular weight of 4.6 × 10⁶ was calculated from the sedimentation coefficient, S°_20,w = 86.2S, the diffusion coefficient, D_20,w = 1.55 × 10^-7 cm²/sec., and an assumed partial specific volume, [UNK] = 0.708 ml/gm. The virus has a ribonucleic acid content of 1.0 × 10⁶ atomic mass units. Electrophoresis experiments showed that the virus is stable in 0.10 ionic strength buffers in the pH range 3-6. Breakdown of the virus was observed outside this pH range. Some characteristics of the breakdown products are described.     

Basic trypsin-subtilisin inhibitor from marine turtle egg white: Hydrodynamic and inhibitory properties

A basic trypsin-subtilisin inhibitor has been isolated from the egg white of marine turtle (Caretta caretta Linn.) and purified to homogeneity by gel filtration followed by ion-exchange chromatography. It has a single polypeptide chain of 117 amino acid residues, having a molecular weight of 13,600. It lacks methionine and tryptophan. Its isoelectric point is atpH 10.0 and the sedimentation coefficient (s_20,w) value of 1.62 S is independent of protein concentration. It has a Stokes radius of 18.8 Å, an intrinsic viscosity of 0.048 dl g^–1 and a diffusion coefficient of 10.17×10^–7 cm² sec^–1. Its fluorescence emission spectrum is similar to that of free tyrosine and the bimolecular quencing rate constant of its tyrosine residues with acrylamide is 3.15×10⁹ M^–1 sec^–1. The inhibitor strongly inhibits both trypsin and subtilisin by forming enzyme-inhibitor complexes at a molar ratio of unity. The nature of inhibition toward both enzymes is not temporary. It has independent binding sites for inhibition of trypsin and subtilisin. Chemical modification with tetranitromethane suggests the presence of three tyrosine residues on the surface of the inhibitor molecule.     

Hydrodynamic properties of mushroom tyrosinase

The hydrodynamic properties of mushroom tyrosinase were determined at pH 6.5 using a Sephadex G-200 column. From the comparison of its gel-filtration behaviour with those of standard proteins, the following parameters were calculated: MW (122 500 ± 1%), Stokes' radius (42.75 × 10^?8 cm²/sec), diffusion coefficient (5.048 × 10^?7 cm²/sec) and frictional ratio (1.26). These values suggest a globular conformation of this enzyme.     

Frog striated muscle is permeable to hydroxide and buffer anions

Hydroxide, bicarbonate and buffer anion permeabilities in semitendinosus muscle fibers of Rana pipiens were measured. In all experiments, the fibers were initially equilibrated in isotonic, high K₂SO₄ solutions at pH _o =7.2 buffered with phosphate. Two different methods were used to estimate permeabilities: (i) membrane potential changes were recorded in response to changes in external ion concentrations, and (ii) intracellular pH changes were recorded in response to changes in external concentrations of ions that alter intracellular pH. Constant field equations were used to calculate relative or absolute permeabilities.In the first method, to increase the size of the membrane potential change produced by a sudden change in anion entry, external K⁺ was replaced by Cs⁺ prior to changes of the anion under study. At constant external Cs⁺ activity, a hyperpolarization results from increasing external pH from 7.2 to 10.0 or higher, using either CAPS (3-[cyclohexylamino]-1-propanesulfonic acid) or CHES (2-[N-cyclohexylamino]-ethanesulfonic acid) as buffer. For each buffer, the protonated form is a zwitterion of zero net charge and the nonprotonated form is an anion. Using reported values of H⁺ permeability, calculations show that the reduction in [H⁺] _o cannot account for the hyperpolarizations produced by alkaline solutions. Membrane hyperpolarization increases with increasing total external buffer concentration at constant external pH, and with increasing external pH at constant external buffer anion concentration. Taken together, these observations indicate that both OH^– and buffer anions permeate the surface membrane. The following relative permeabilities were obtained at pH_o, 10.0± 0.3: (P_OH/P_K) = 890 ± 150, (P_CAPS/P_K) = 12 ± 2 (P_CHIES/P_K) = 5.3 ± 0.9, and (P_NO3/P_K) = 4.7 ± 0.5 P_NO/P_K was independent of pH _o up to 10.75. At pH_o = 9.6, (P_HCO3/P_K) = 0.49 ± 0.03; at pH _o = 8.9, (P_Cl/P_K) = 18± 2 and at pH _o = 7.1, (P_HEPES/P_K) = 20 ± 2.In the second method, on increasing external pH from 7.2 to 10.0, using 2.5 mm CAPS (total buffer concentration), the internal pH increases linearly with time over the next 10 min. This alkalinization is due to the entry of OH^– and the absorption of internal H⁺ by entering CAPS^– anion. The rate of CAPS^– entry was determined in experiments in which the external CAPS concentration was increased at constant external pH. Such increases invariably produced an increase in the rate of internal alkalinization, which was reversed when the CAPS concentration was reduced to its initial value. From the internal buffer power, the diameter of the fiber under study and the rates of change of internal pH, the absolute permeability for both OH^– and CAPS^– were calculated. At external pH = 10.0, the average (±sem) permeabilities were: P_OH=1.68±0.19×10^–4 cm/sec and P_CAPS=2.10±0.74×10^–6cm/sec.We conclude that OH^– is about 50 times more permeable than Cl^– at alkaline pH and that the anionic forms of commonly used buffers have significant permeabilities.This research was supported by a grant from the National Institutes of Health (AR 31814). The authors wish to thank Dr. Peter G. Shrager and Dr. Bruce C. Spalding for reading an early draft of this report and for providing helpful suggestions.     

Inelastic light scattering studies of solutions of the chloroplast coupling factor (CF1) at high and low ionic strength

The translational diffusion coefficient of CF₁ at low and high protein concentration as well as at different ionic strength (0.05 – 1.65 M) wsa determined by means of quasi-elastic light scattering experiments. The diffusion coefficient changes from D_20,w^o = 3.12 × 10^?7 cm² · sec^?1 at 0.05 M, pH 7.8, 20°C, to D_20,w^o = 3.52 × 10^?7 cm² · sec^?1 at 1.6 M, pH 7.8, 20°C. At high enzyme concentration (20 mg/ml) and under crystallization conditions (Paradies, BBRC 91: 685, 1979) CF₁ behaves as a solution of “true” hard spheres, whereas at low salt concentration the ionic atmosphere has a larger spatial extent, resulting in a higher effective hydrodynamic radius (R_H = 65 Å).     

Molecular weight of cytochrome oxidase

The molecular weight of bovine heart mitochondrial cytochrome oxidase in 2% or 3% deoxycholate was determined by the sedimentation velocity method to be 228,000 daltons from an S_20,w=8.44×10^–13 sec, a D_20,w=3.21×10^–7 cm² sec^–1, and the reported by others. The S_20,w value was only slightly concentration-dependent. When the deoxycholate in our preparation was replaced with Tween 80 the S value increased to between 16 and 17. When one preparation in Tween 80 was allowed to stand at room temperature, the S value increased in successive determinations to reach 51.5 at the end of approximately 7 h. The minimum molecular weight of the enzyme as calculated from the heme content (determined from the absorbance at 603 nm) and total protein content (determined from total nitrogen) was 110,000. An amino acid analysis when related to the heme content yielded a minimum molecular weight of 85,000.Deceased.     

Secretion of HCO 3 − /OH− in cortical distal tubule of the rat

Secretion of bicarbonate has been described for distal nephron epithelium and attributed to apical Cl^–/HCO ₃ ^– exchange in beta-intercalated cells. We investigated the presence of this mechanism in cortical distal tubules by perfusing these segments with acid (pH 6) 10 mm phosphate Ringer. The kinetics of luminal alkalinization was studied in stationary microperfusion experiments by double-barreled pH (ion-exchange resin)/1 m KCl reference microelectrodes. Luminal alkalinization may be due to influx (into the lumen) of HCO ₃ ^– or OH^–, or efflux of H⁺. The magnitude of the Cl^–/ HCO ₃ ^– exchange component was measured by perfusing the lumen with solutions with or without chloride, which was substituted by gluconate. This component was not different from zero in control and alkalotic (chronic plus acute) Wistar rats. Homozygous Brattleboro rats (BRB), genetically devoid of antidiuretic hormone, were used since this hormone has been shown to stimulate H⁺ secretion, which could mask bicarbonate secretion. In these rats, no evidence for Cl^–/HCO ₃ ^– exchange was found in control BRB and in early distal segments of alkalotic animals, but in late distal tubule a significant component of 0.14±0.033 nmol/cm² · sec was observed, which, however, is small when compared to the reabsorptive flow found in control Wistar rats, of 0.95±0.10 nmol/cm² · sec. In addition, 5×10^–4 m SITS had no effect on distal bicarbonate reabsorption in controls as well as on secretion in alkalotic Wistar and Brattleboro rats, which is compatible with the absence of effect of this drug on the apical Cl^–/HCO ₃ ^– exchange in other tissues. It is concluded that most distal alkalinization is not Cl^– dependent, and that Cl^–/HCO ₃ ^– exchange may be found in cortical distal tubule, but its magnitude is, even in alkalosis, markedly smaller than the reabsorptive flux, which predominates in the rats studied in this paper, keeping luminal pH lower than that of blood.     

Conformation of the high molecular weight form ofDunaliella salina phosphofructokinase in solution by means of small-angle X-ray diffraction and viscosity measurements

Summary The intrinsic viscosity of phosphofructokinase fromDunaliella salina in different states of aggregation was determined. The instrinsic viscosity [], of the biologically active tetramer, with a molecular weight of 320,000, was found to be 6.5 ml·g^–1 at 4°C. Moreover, for the inactive dimer, with a molecular weight of 160,000, a value of []=8.0 ml·g^–1 was determined. The high molecular weight aggregate of phosphofructokinase fromDunaliella salina, that shows little activity, has an intrinsic viscosity of 23.2 ml·g^–1, which is significantly higher than that found for the active tetramer and the inactive dimer.Small angle X-ray scattering experiments in solution of this high molecular from of phosphofructokinase fromDunaliella salina reveal a radius of gyration of the cross section ofR _c=49.0 Å at an ionic strength of 0.15 M and_pH 7.2. Furthermore, a comparison of the values obtained for the tetramer and the radius of gyration (R _g=52.9 Å) with those of typical spherical proteins (3–4 ml·g^–1) shows that the values of [] andR _g are significantly larger for the high molecular weight form of phosphofructokinase than for the spherical proteins. The high intrinsic viscosity of the polymeric form of phosphofructokinase suggests an end-to-end aggregation consisting of monomeric units with heights,h=80–90 Å, and a cylindrical diameter of approximately 140.0 Å, resulting in a long rod of a total length of 1,800 Å and a molecular weight of two million. On the basis of the experimentally observedR _c and [] values, using a prolate ellipsoid of revolution as a model, the hydrodynamic volume and the hydration, the axial ratio could be determined to be 12. The native tetrameric form contains 0.4 g H₂O/g protein, whereas the higher aggregate structure corresponds to a hydration of 0.60 g H₂O/g protein.     

Influx of Ca ion in cultured excitable cells studied by fluorescence microscope imaging technique

The spatial distribution and temporal variation of intracellular Ca ion in differentiated Neuroblastoma-Glia Hybridoma 108–15 cells (NG108–15) were investigated using a fluorescence microscope imaging technique. Fura-2 was used as a probe. Electrical current pulses of 10–20 µA were applied to axons connecting to NG cells in order to elicit the influx of Ca ion. The concentration of intracellular Ca is usually 50–80 nM in NG cells in the resting state. Upon stimulation, the Ca level increases by a factor of 2–5. The entry of Ca⁺⁺ across cell membranes is followed by intracellular diffusion and the propagation of a wave front is clearly seen in digital images. The diffusion constant was calculated to be approximately 1.66×10^–6 cm²/sec. This value is about one-fifth of the free diffusion coefficient of Ca ion in aqueous solution (7.82 × 10^–6 cm²/sec). Cd ion, at the concentration of 1–2 mM, blocks the influx of Ca as expected whereas the influx is unaffected by TTX at the concentration of 0.1 – 0.2µM.     

Ontogeny of the Na+−H+ exchanger in rat ileal brush-border membrane vesicles

Summary The developmental maturation of Na⁺–H⁺ antiporter was determined using a well-validated brush-border membrane vesicles (BBMV's) technique. Na⁺ uptake represented transport into an osmotically sensitive intravesicular space as evidenced by an osmolality study at equilibrium. An outwardly directed pH gradient (pH inside/pH outside=5.2/7.5) significantly stimulated Na⁺ uptake compared with no pH gradient conditions at all age groups; however, the magnitude of stimulation was significantly different between the age groups. Moreover, the imposition of greater pH gradient across the vesicles resulted in marked stimulation of Na⁺ uptake which increased with advancing age. Na⁺ uptake represented an electroneutral process.The amiloride sensitivity of the pH-stimulated Na⁺ uptake was investigated using [amiloride] 10^–2–10^–5 m. At 10^–3 m amiloride concentration, Na⁺ uptake under pH gradient conditions was inhibited 80, 45, and 20% in BBMV's of adolescent, weanling and suckling rats, respectively. Kinetic studies revealed aK _m for amiloride-sensitive Na⁺ uptake of 21.8±6.4, 24.9±10.9 and 11.8±4.17mm andV _max of 8.76±1.21, 5.38±1.16 and 1.99±0.28 nmol/mg protein/5 sec in adolescent, weanling and suckling rats, respectively. The rate of pH dissipation, as determined by the fluorescence quenching of acridine orange, was similar across membrane preparation of all age groups studied. These findings suggest for the first time the presence of an ileal brush-border membrane Na⁺–H⁺ antiporter system in all ages studied. This system exhibits changes in regard to amiloride sensitivity and kinetic parameters.     

Bounding fluid viscosity and translational diffusion in a fluid lipid bilayer

Fluorescence recovery after photobleaching was used to investigate the translational diffusion of a fluorescent derivative of a membrane-spanning lipid in L phase multibilayers of 1-palmitoyl-2-oleoylphosphatidylcholine prepared in water and in glycerol. The translational diffusion coefficient in hydrated bilayers (D _w) ranged between 2 and 5x10^–8 cm²/s and in glycerinated bilayers (D _g) the range was between 3 and 24×10^–10 cm²/s between 10° and 40°C. These results are discussed in terms of models for diffusion in membranes.     

Osmotic water permeabilities of human placental microvillous and basal membranes

Summary Literature data suggest that water accumulation by the human fetus is driven by osmotic gradients of small solutes. However, the existence of such gradients has not been supported by prior measurements. Attempts to estimate the size of the gradient necessary to drive net water movement have been seriously hampered by the lack of permeability data for the syncytiotrophoblast membranes. Stopped-flow light scattering techniques were employed to measure the osmotic water permeability (P _f )of microvillous (MVM) and basal membrane (BM) vesicles isolated from human term placenta. At 37°C, the P _f was determined to be 1.9±0.06 × 10^+–3 cm/sec for MVM and 3.1±0.20 × 10^+–3 cm/sec for BM (mean ±SD, n = 6). At 23°C, P _f was reduced to 0.7±0.04 × 10^+–3 cm/sec in MVM and 1.6±0.05 × 10^+–3 cm/sec in BM. These P _f values are comparable to those observed in membranes where water has been shown to permeate via a lipid diffusive mechanism. Arrhenius plots of P _f over the range 20–40°C were linear, with activation energies of 13.6 ± 0.6 kcal/mol for MVM and 12.9±1.0 kcal/mol for BM. Water permeation was not affected by mercurial sulfhydryl agents and glucose transport inhibitors. These data clearly suggest that water movement across human syncytiotrophoblast membranes occurs by a lipid diffusion pathway. As noted in several other epithelial tissues, the basal membrane has a higher water permeability than the microvillous membrane. It is speculated that water accumulation by the human fetus could be driven by a solute gradient small enough to be within the error of osmolarity measurements.We thank the staff of the labor and delivery ward at University of San Francisco Medical Center for help in obtaining placental tissue. This work was supported by NIH grant HD 26392. Dr. Jansson was supported by the Sweden-America Foundation, The Swedish Society of Medicine, The Swedish Society for Medical Research, and the Swedish Medical Research Council.     

Hydrodynamic properties of mucins secreted by primary cultures of Guinea-pig tracheal epithelial cells: determination of diffusion coefficients by analytical ultracentrifugation and kinetic analysis of mucus gel hydration and dissolution

We have used two different approaches to determine hydrodynamic parameters for mucins secreted by guinea-pig tracheal epithelial cells in primary culture. Cells were cultured under conditions that promote mucous cell differentiation. Secreted mucins were isolated as the excluded fraction from a Sepharose CL-4B gel filtration column run under strongly dissociating conditions. Biochemical analysis confirmed the identity of the high molecular weight material as mucins. Analytical ultracentrifugation was used to study the physical properties of the purified mucins. The weight average molecular mass (M _w ) for three different preparations ranged from 3.3×10⁶ to 4.7×10⁶ g/mol (corresponding to an average structure of 1 – 2 subunits), and the sedimentation coefficient from 25.5 to 35 S. Diffusion coefficients ranging from 4.5×10^–8 to 6.4×10^–8 cm²/s were calculated using the Svedberg equation. A polydispersity index (M _z /M _w ) of ∼1.4 was obtained. Diffusivity values were also determined by image analysis of mucin granule exocytosis captured by videomicroscopy. The time course of hydration and dissolution of mucin was measured and a relationship is presented which models both phases, each with first order kinetics, in terms of a maximum radius and rate constants for hydration and dissolution. A median diffusivity value of 8.05×10^–8 cm²/s (inter-quartile range = 1.11×10^–7 to 6.08×10^–8 cm²/sec) was determined for the hydration phase. For the dissolution phase, a median diffusivity value of 6.98×10^–9 cm²/s (inter-quartile range = 1.47×10^–8 to 3.25×10^–9 cm²/sec) was determined. These values were compared with the macromolecular diffusion coefficients (D _20,w ) obtained by analytical ultracentrifugation. When differences in temperature and viscosity were taken into account, the resulting D _37,g was within the range of diffusivity values for dissolution. Our findings show that the physicochemical properties of mucins secreted by cultured guinea-pig tracheal epithelial cells are similar to those of mucins of the single or double subunit type purified from respiratory mucus or sputum. These data also suggest that measurement of the diffusivity of dissolution may be a useful means to estimate the diffusion coefficient of mucins in mucus gel at the time of exocytosis from a secretory cell. Received: 10 March 1998 / Accepted: 27 March 1998     

Volume flows and pressure changes during an action potential in cells ofChara australis

Summary Methods have been used for monitoring either volume flows or pressure changes, simultaneously with membrane potentials, in giant algal cells ofChara australis during an action potential. The volume flows were measured from the movement of a mercury bead in a capillary tube recorded by a photo-transducer. The pressure changes were measured by monitoring the deflection of a thin wedge, resting transversely across a cell, and using the same photo-transducer, the deflection of the wedge being directly related to the cell's turgor pressure. The average maximum rate of volume flow per unit area during an action potential was 0.88±0.11 nliter·sec^–1·cm^–2 in the direction of an outflow from the cell (total volume outflow being about 3 nliter·cm^–2 per action potential). Similarly, the maximum rate of change of pressure was 19.6±3.8×10^–3 atm·sec^–1 (peak change being 19.3±2.9×10^–3 atm equivalent to 14.7±2.2 mm Hg). The volume flow and pressure changes followed the vacuolar potential quite closely, the peak rate of volume flow lagging behind the peak of the action potential by 0.17±0.08 sec and the peak rate of pressure change leading it by 0.09±0.07 sec.     

Measurement of diffusion coefficient and electrophoretic mobility with a quasielastic light-scattering–band-electrophoresis apparatus

We have constructed an apparatus for the simultaneous measurement of electrophoretic mobility, μ, and diffusion coefficient, D, of macromolecules and cells. It combines band electrophoresis in a vertical, sucrose-gradient stabilized column, with quasielastic laser light-scattering determination of the diffusion coefficient of the species within the band. The entire electrophoresis cell is scanned through the laser beam of the quasielastic laser light-scattering apparatus by a vertical translation stage. Total intensity light-scattering measurement at each point in the cell gives the macromolecular concentration at that point. Solvent viscosity and electrical potential are measured at each point in the cell. Application of this apparatus to resealed red blood cell ghosts and to bovine hemoglobin indicates that measurements of field, viscosity, and migration distance are reliable, and that electroosmosis is insignificant. Application to T4D bacteriophage gives μ_20,w = (?1.05 ± 0.05) × 10^?4 cm²/V sec and D_20,w = (3.35 ± 0.10) × 10^?8 cm²/sec for fiberless particles, and μ_20,w = ?(0.59 ± 0.03) × 10^?4 cm²/V sec and D_20,w = (2.86 ± 0.09) × 10^?8 cm²/sec for whole phage with 6 fibers. Approximate analysis of these results with the Henry electrophoresis theory for spheres in dicates that each fiber contributes about 193 positive charges to the phage particle, compared with 327 from amino-acid analysis. The advantages and disadvantages of this apparatus, relative to conventional electrophoresis and to electrophoretic light scattering, are discussed.     

Na+/H+ exchanger and reperfusion-induced ventricular arrhythmias in isolated perfused heart: possible role of amiloride

The roles of the Na⁺/H⁺ exchange system in the development and cessation of reperfusion induced ventricular arrhythmias were studied in the isolated perfused rat heart. The hearts were perfused in the working heart mode with modified Krebs Henseleit bicarbonate (KHB) buffer and whole heart ischemia was induced by a one-way ball valve with 330 beat/min pacing. Ischemia was continued for 15 min followed by 20 min of aerobic reperfusion (control). Amiloride (1.0mM), an inhibitor of the Na⁺/H⁺ exchange system, was added to the KHB buffer only during reperfusion (group B) or only during ischemic periods (group C). Electrocardiographic and hemodynamic parameters were monitored throughout the perfusion. Coronary effluent was collected through pulmonary artery cannulation and PO₂, PCO₂, HCO ₃ ^– and pH were measured by blood-gas analyzer.The incidence of reperfusion induced ventricular arrhythmias was 100%, 100% and 0% in control, group B and group C, respectively. The mean onset time of termination of reperfusion arrhythmias was significantly shorter in group B than in control. PCO₂ increased from 39.0±0.9 to 89.3±6.0 mmHg at the end of ischemia in control and from 40.6±0.4 to 60.5±5.8 in group C, the difference between groups was statistically significant. HCO ₃ ^– level decreased from 21.8±0.1 to 18.3±0.5 mmol/l in control, however, this decrease was significantly inhibited in group C (from 22.0±0.5 to 20.3±0.2). The increase in PCO₂ and the decrease in HCO ₃ ^– in group B were similar over time to those observed in control. The decrease in pH produced by ischemia was marked in control (from 7.35±0.01 to 6.92±0.04) and group B (from 7.34±0.01 to 6.94±0.02), whereas a decrease in pH was significantly prevented in group C (from 7.34±0.01 to 7.15±0.04). There were no significant differences in PCO₂, HCO ₃ ^– or pH among the three groups during reperfusion.These experiments provide evidence that amiloride significantly prevented the incidence of reperfusion arrhythmias when added only during ischemia and significantly terminated reperfusion arrhythmias when added only during reperfusion. Amiloride may prevent a decrease in pH, due to alterations in PCO₂ and/or HCO ₃ ^– . These changes in PCO₂ and HCO ₃ ^– might be indirectly influenced by inhibition of the Na⁺/H⁺ exchange system via Cl^–/HCO ₃ ^– exchange. The mechanism by which amiloride terminates reperfusion arrhythmias seems to involve electrophysiological effects which were not directly addressed in this experiment.     

Diffusion von45Ca,85Sr und32P in Hydroxylapatit

Zusammenfassung Es wurde der Transport von⁴⁵Ca,⁸⁵Sr und³²P in polykristallinen Sinterkörpern von synthetischem Hydroxylapatit im Temperaturbereich 1000 bis 1400 °C untersucht. Nach sorgfältiger Berücksichtigung von Korngrenzen-Diffusionseffekten ergaben sich für die Diffusion von⁴⁵Ca und⁸⁵Sr gleiche Werte für die Aktivierungsenthalpien und Frequenzfaktoren, und zwar beipH₂O<30 Torr:Q=3,50 ± 0,02 eV;D ₀=41 ± 5 cm²s^–1 und beipH₂O=230 Torr:Q=3,55 ± 0,02 eV;D ₀=20 ± 3 cm²s^–1 Die Abhängigkeit des Kationen-Diffusionskoeffizienten vom Wasserdampfpartialdruck ist vermutlich dadurch bedingt, daß im untersuchten Temperaturbereich feste Lösungen von Hydroxylapatit und Oxyapatit entstehen und Leerstellen im OH^–-Teilgitter den Kationentransport beschleunigen. Der³²P-Transport wurde nur bei 1360 °C undpH₂O < 30 Torr gemessen. Der Diffusionskoeffizient ist um einen Faktor 400 ± 50 kleiner als der entsprechende Diffusionskoeffizient der Kationen.Die Ergebnisse der Diffusionsuntersuchungen werden in Verbindung mit einer einfachen Modellvorstellung zum Retentionsmechanismus der Erdalkalien im Skelett diskutiert.

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Diffusion of⁴⁵Ca,⁸⁵Sr, and³²P in hydroxyapatite

Summary The transport of⁴⁵Ca,⁸⁵Sr, and³²P in polycrystalline sinter pellets of synthetic hydroxyapatite has been investigated in the temperature range 1000 to 1400 °C. After subtraction of activity transports by grain boundary diffusion processes, equal values of activation enthalpy and frequency factor were found for the lattice diffusion of⁴⁵Ca and⁸⁵Sr: atpH₂O<30 Torr:Q=3,50 ± 0,02 eV,D ₀=41 ± 5 cm²s^–1 and atpH₂O=230 Torr:Q=3,55 ± 0,02 eV,D ₀=20 ± 3 cm²s^–.The dependence of the cation diffusion coefficient on the partial vapour pressure is probably caused by formation of solid solutions of hydroxyapatite and oxyapatite where vacancies of the OH^– sublattice accelerate the cation transport. The diffusion of³²P was investigated only atT=1360 °C andpH₂O<30 Torr. The value obtained is smaller by a factor of 400 ± 50 then the cation diffusion coefficient.The results of the diffusion experiments are discussed in the light of a simple model for the retention mechanism of the alkaline earth metals in the skeleton.

     

Electrical effects of potassium and bicarbonate on proximal tubule cells ofNecturus

Summary The effects of stepwise concentration changes of K⁺ and HCO ₃ ^– in the basolateral solution on the basolateral membrane potential (V _bl) of proximal tubule cells of the doubly-perfusedNecturus kidney were examined using conventional microelectrodes. Apparent transference numbers were calculated from changes inV _bl after alterations in external K⁺ concentration from 1.0 to 2.5mm (t _{K, 1.0–2.5}), 2.5 to 10, and in external HCO ₃ ^– concentration (at constant pH) from 5 to 10mm (t _{HCO3, 5–10}), 10 to 20, or 10 to 50.t _{K, 2.5–10} was 0.38±0.02 under control conditions but was sharply reduced to 0.08±0.03 (P>0.001) by 4mm Ba⁺⁺. This concentration of Ba⁺⁺ reducedV _bl by 9±1 mV (at 2.5 external K⁺). Perfusion with SITS (5×10^–4 m) for 1 hr hyperpolarizedV _bl by 10±3 mV and increasedt _{K, 2.5–10} significantly to 0.52±0.01 (P<0.001). Ba⁺⁺ application in the presence of SITS depolarizedV _bl by 22±3 mV. In control conditionst _{HCO3, 10–50} was 0.63±0.05 and was increased to 0.89±0.07 (P<0.01) by Ba⁺⁺ but was decreased to 0.14±0.02 (P<0.001) by SITS. In the absence of apical and basolateral chloride, the response ofV _bl to bicarbonate was diminished but still present (t _{HCO3, 10–20} was 0.35±0.03). Intracellular pH, measured with liquid ion-exchange microelectrodes, increased from 7.42±0.19 to 7.57±0.17 (P<0.02) when basolateral bicarbonate was increased from 10 to 20mm at constant pH. These data show that the effects of bicarbonate onV _bl are largely independent of effects on the K⁺ conductance and that there is a significant current-carrying bicarbonate pathway in the basolateral membrane. Hence, both K⁺ and HCO ₃ ^– gradients are important in the generation ofV _bl, and their relative effects vary reciprocally.     

Permeability parameters of the toad isolatedstratum corneum

Summary A technique for isolating thestratum corneum from the subjacent layers of the epithelium was developed which permits studying thestratum corneum as an isolated membrane mounted between half-chambers. The method basically consists of an osmotic shock induced by immersing a piece of skin in distilled water at 50°C for 2 min. When the membrane is bathed on each surface by NaCl-Ringer's solution, its electrical resistance is 14.1±1.3 cm² (n=10). This value is about 1/100 of the whole skin resistance in the presence of the same solution. The hydraulic filtration coefficient (L _p ) measured by a hydrostatic pressure method, with identical solutions on each side of the membrane, is 8.8×10^–5±1.5×10^–5 cm sec^–1 atm^–1 (n=10) in distilled water and 9.2×10^–5±1.4×10^–5 cm sec^–1 atm^–1 (n=10) in NaCl-Ringer's solution. These values are not statistically different and are within the range of 1/80 to 1/120 of the whole skinL _p . Thestratum corneum shows an amphoteric character when studied by KCl diffusion potentials at different pH's. The membrane presents an isoelectric pH of 4.6±0.3 (n=10). Above the isoelectric pH the potassium transport number is higher than the chloride transport number; below it, the reverse situation is valid. Divalent cations (Ca⁺⁺ or Cu⁺⁺) reduce membrane ionic discrimination when the membrane is negatively charged and are ineffective when the membrane fixed charges are protonated at low pH.     

UVB radiation affects the mobility of epidermal growth factor receptors in human keratinocytes and fibroblasts

Growth factor receptors transmit biological signals for the stimulation of cell growth in vitro and in vivo and their autocrine stimulation may be involved in tumorigenesis. It is therefore, of great value to understand receptor reactions in response to ultraviolet (UV) light which certain normal human cells are invaribly exposed to during their growth cycle. UV irradiation has recently been shown to deplete antioxidant enzymes in human skin. The aims of the present study were a) to compare the lateral mobility of epidermal growth factor receptors (EGF-R) in cultured human keratinocytes and human foreskin fibroblasts, b) to investigate effects of ultraviolet B radiation on the mobility of EGF-R in these cells, and c) study the response of EGF-R on addition of antioxidant enzymes. The epidermal growth factor receptors were labeled with rhodaminated EGF, the lateral diffusion was determined and the fraction of mobile EGF-R assessed with the fluorescence recovery after photobleaching (FRAP). We found that human keratinocytes display a higher basal level of EGF-R mobility than human skin fibroblasts, viz. with diffusion coefficients (D ± standard error of the mean, SEM) of 4.2±0.2 × 10^–10 cm²/s, and 1.8±0.2 × 10^–10 cm²/s, respectively. UVB-irradiated fibroblasts showed an almost four-fold increase in the diffusion coefficient; D was 6.3±0.3 × 10^–10 cm²/s. The keratinocytes, however, displayed no significant increase in receptor diffusion after irradiation; D was 5.1±0.8 × 10^–10 cm²/s. In both cell types the percentage of EGF-R fluorescence recovery after photobleaching, i.e. the fraction of mobile receptors, was significantly increased after irradiation. In keratinocytes it increased from 69% before irradiation to 78% after irradiation. Analogous figures for fibroblasts were 61% and 73%. The effect of UVB on fibroblast receptors was abolished by prior addition of superoxide dismutase (SOD) and catalase (CAT). It is concluded that UVB radiation of fibroblasts and keratinocytes can affect their biophysical properties of EGF-R. The finding that addition of antioxidant enzymes prevented the UVB effect in fibroblasts may indicate the involvement of reactive oxygen metabolites.Abbreviations CAT Catalase - D Lateral diffusion coefficient - EDTA Ethylenediaminetetraacetic acid - EGF Epidermal growth factor - E-MEM Eagle's minimum essential medium - FCS Fetal calf serum - FRAP Fluorescence recovery after photobleaching - KRG Krebs-Ringer phosphate buffer - PBS Phosphate-buffered saline - R Mobile fraction - ROS Reactive oxygen species - SEM Standard error of the mean - SOD Superoxide dismutase - UVA Ultraviolet light-A (315-400 nm) - UVB Ultraviolet light-B (280-315 nm)