Repetitive propagation of action potentials destabilizes the structure of the myelin sheath. A dynamic x-ray diffraction study

Time courses of myelin lattice swelling in toad sciatic nerves preexposed to different treatments were determined by x-ray diffraction using a one-dimensional position-sensitive detector. In the nerves supramaximally stimulated for 1 h at 200 Hz, the subsequent process of myelin swelling occurred 45.0 +/- 7.3 min (n = 24) sooner than in resting controls. Sciatic nerves incubated for 1 h in a Ringer's solution deprived of divalent cations (Ca++ and Mg++) exhibited a kinetics of swelling similar to that shown by the stimulated nerves, that is, 52.5 +/- 14.2 min (n = 6) sooner than controls preincubated for the same time in normal Ringer's solution (with divalent cations). The fact that both pretreatments supramaximal stimulation and removal of divalent cations from the perfusion solution produced a similar effect; namely, a decrease of the myelin lattice stability against swelling in distilled water, suggests that the repetitive propagation of action potentials could modify the ionic composition at either the intraperiod channel or the paranodal axoglial junction complexes.     

Loss and recovery of excitability by normal and by degenerating nerves deprived of sodium

A study has been made of the loss of excitability in a sodium-free medium and of the recovery of excitability in Ringer's solution by A fibers of normal frog nerves and of nerves in advanced stages of Wallerian degeneration. With normal nerves that are being kept in a sodium-free medium the number of conducting fibers does not undergo a readily detectable decrease in less than 1 to 2 hours; inexcitability of all the A fibers does not develop in less than 7 to 8 hours. During the development of inexcitability the speed of conduction of the still conducting fibers undergoes a progressive decrease; in advanced stages the speed of conduction is not more than one-fifth of the normal speed. The nerve fibers lose the ability to conduct rhythmic trains of impulses earlier than the ability to conduct single impulses. The recovery of excitability in Ringer's solution duplicates in a reverse order the sequence of changes that have been previously observed during the development of inexcitability. The rate of the recovery of excitability in Ringer's solution is higher than the rate of the loss of excitability in the sodium-free medium. With degenerating nerves the effect of the lack of sodium develops qualitatively in the same manner in which it develops with normal nerves. Degenerating nerve fibers, however, become inexcitable in a sodium-free medium earlier than normal fibers. The recovery of the excitability in Ringer's solution takes place in much the same manner in normal and in degenerating nerve fibers. The loss of excitability during Wallerian degeneration is a process that develops simultaneously, or practically so, throughout the entire length of the fibers. The nerve fibers retain a great deal of functional ability throughout the several days which precede the onset of inexcitability and then suddenly become inexcitable.     

Investigation of the conformational lability of serum albumin macromolecules in aqueous solution by the NMR spin-echo method.

The complex proton spin-echo decay curve was recorded for human serum albumin (SA) solutions with different concentrations in normal and heavy water. The curve included three fast-decaying components for SA, in addition to the slow-decaying component for the water. The total amplitude of these three components roughly corresponded to the number of protons in the SA (with isotopic exchange taken into account); the component ratio remained constant at different concentrations and different temperatures between 4 and 39 degrees. The relatively slow-decaying protein component, which accounted for similar to 10% of the SA protons, was produced by the side chains of the protein. The presence of two other faster-decaying SA components with approximately equal amplitudes indicated that only about half of the remaining protons in the SA macromolecule are incorporated into the comparatively rigid globule, the other half belonging to groups with high conformational lability in aqueous solution. The activation energy for the aqueous component was close to that for pure water, while the activation energies for the protein components were roughly twice as large.     

Nuclear-magnetic-resonance imaging of leaves ofMesembryanthemum crystallinum L. plants grown at high salinity

Differences in water binding were measured in the leaf cells ofMesembryanthemum crystallinum L. plants grown under high-salinity conditions by using nuclear-magnetic-resonance (NMR) imaging. The 7-Tesla proton NMR imaging system yielded a spatial resolution of 20·20·100 m³. Images recorded with different spin-echo times (4.4 ms to 18 ms) showed that the water concentrations in the bladder cells (located on the upper and lower leaf surface), in the mesophyll cells and in the water-conducting vessels were nearly identical. All of the water in the bladder cells and in the water-conducting vessels was found to be mobile, whilst part of the water in the mesophyll cells was bound. Patches of mesophyll cells could be identified which bound water more strongly than the surrounding mesophyll cells. Optical investigations of leaf cross-sections revealed two types of mesophyll cells of different sizes and chloroplast contents. It is therefore likely that in the small-sized mesophyll cells water is strongly bound. A long-term asymmetric water exchange between the mesophyll cells and the bladder cells during Crassulacean acid metabolism has been described in the literature. The high density of these mesophyll cells in the lower epidermis is a possible cause of this asymmetry.Abbreviations CAM Crassulacean acid metabolism - NMR nuclear magnetic resonance - T_E spin-echo time     

The effect of regulatory peptides on water absorption in the large intestine of the frog]

Preparation of isolated large intestine of the frog was filled with Ringer's solution diluted with distilled water (1:5) and was placed into the glass with normal Ringer's solution. The preparation was weighed within every 30 min and the osmotic permeability was determined for water of the mucous and serous layers of the intestine. Then one of the peptides was added to Ringer's solution and the experiment continued. It is stated that bombesin, neurotensin, encephalins, substance P, somatostatin, pituitrin are able to change liquid absorption from the large intestine cavity when the concentration of Ringer's solution in the cavity and from its serous surface is the same. Bombesin and neurotensin inhibited while encephalins stimulated liquid absorption and these effects depended on the transport of ions. Liquid absorption by the osmotic gradient decreased using bombesin, substance P and increased using somatostatin. More complex peptide-peptide relations are observed if using pituitrin and other peptides. cAMP is shown to participate in bombesin effects.     

The loss of potassium from frog nerves in anoxia and other conditions

1. Frog nerves immersed in Ringer's solution lose on the average 23 per cent more potassium if the solution is equilibrated with pure nitrogen than if equilibrated with oxygen. 2. Tying off the ends of the nerves during immersion increased rather than diminished the loss of potassium. 3. There was some evidence that nerves tended to regain potassium if they were returned to oxygen after a period of anoxia. 4. Addition of acetylcholine to the solution increases the loss of potassium. 5. Equilibration of the solution and nerves with 20 per cent CO₂ in O₂ increases the loss of K from nerves in Ringer's solution but decreases it in frog blood.     

INFLUENCE OF A SLIGHT MODIFICATION OF THE COLLODION MEMBRANE ON THE SIGN OF THE ELECTRIFICATION OF WATER

1. It is shown that collodion membranes which have received one treatment with a 1 per cent gelatin solution show for a long time (if not permanently) afterwards a different osmotic behavior from collodion membranes not treated with gelatin. This difference shows itself only towards solutions of those electrolytes which have a tendency to induce a negative electrification of the water particles diffusing through the membrane, namely solutions of acids, acid salts, and of salts with trivalent and tetravalent cations; while the osmotic behavior of the two types of membranes towards solutions of salts and alkalies, which induce a positive electrification of the water particles diffusing through the membrane, is the same. 2. When we separate solutions of salts with trivalent cation, e.g. LaCl₃ or AlCl₃, from pure water by a collodion membrane treated with gelatin, water diffuses rapidly into the solution; while no water diffuses into the solution when the collodion membrane has received no gelatin treatment. 3. When we separate solutions of acid from pure water by a membrane previously treated with gelatin, negative osmosis occurs; i.e., practically no water can diffuse into the solution, while the molecules of solution and some water diffuse out. When we separate solutions of acid from pure water by collodion membranes not treated with gelatin, positive osmosis will occur; i.e., water will diffuse rapidly into the solution and the more rapidly the higher the valency of the anion. 4. These differences occur only in that range of concentrations of electrolytes inside of which the forces determining the rate of diffusion of water through the membrane are predominantly electrical; i.e., in concentrations from 0 to about M/16. For higher concentrations of the same electrolytes, where the forces determining the rate of diffusion are molecular, the osmotic behavior of the two types of membranes is essentially the same. 5. The differences in the osmotic behavior of the two types of membranes are not due to differences in the permeability of the membranes for solutes since it is shown that acids diffuse with the same rate through both kinds of membranes. 6. It is shown that the differences in the osmotic behavior of the two types of collodion membranes towards solutions of acids and of salts with trivalent cation are due to the fact that in the presence of these electrolytes water diffuses in the form of negatively charged particles through the membranes previously treated with gelatin, and in the form of positively charged particles through collodion membranes not treated with gelatin. 7. A treatment of the collodion membranes with casein, egg albumin, blood albumin, or edestin affects the behavior of the membrane towards salts with trivalent or tetravalent cations and towards acids in the same way as does a treatment with gelatin; while a treatment of the membranes with peptone prepared from egg albumin, with alanine, or with starch has no such effect.     

Oxygen-17 and deuterium nuclear magnetic resonance studies of lysozyme hydration

Oxygen-17 and deuterium NMR studies of lysozyme hydration are reported for a wide range of lysozyme concentrations, and the relationship between water "activity" and water mobility in the lysozyme-water system as determined by high-field NMR is examined. In a first approximation, the effect of lysozyme activity on hydration is considered to be small because of the relatively low charge on lysozyme at pH 7 and the absence of salt in the lysozyme solutions. Correlation times are determined for tightly bound water, weakly bound water, and "multilayer" or trapped water in lysozyme at 20 degrees C. Hydration numbers are also determined for these three different water populations interacting with lysozyme. Good agreement is found between the hydration numbers determined by 17O NMR and the calculations based on the D'Arcy and Watt analysis of water sorption isotherms for proteins that considered three major water populations in hydrated lysozyme. A molecular interpretation for the three components in the D'Arcy and Watt theory of sorption isotherms is also proposed on the basis of our NMR results. Previous proton NMR spin-echo results are shown to be consistent with our findings by 17O NMR and support the view that there are at least four regions of distinct hydration behavior of lysozyme which span the whole range from solutions to solid powders.     

An analysis of physiological states responsible for antero-posterior disintegration in Planaria dorotocephala

Summary Planaria were treated with equi-molal solutions of ammonium, potassium, sodium, magnesium, and calcium chlorides, made up in distilled water and the rates of cytolysis compared with cytolysis in distilled water. Potassium and ammonium accelerate cytolysis; some protection is afforded by sodium; still more by magnesium, and complete protection by calcium in the concentrations employed.In distilled water solutions of calcium chloride no cytolysis occurs in concentrations from M/500 to M/40,000; cytolysis is distinctly delayed in M/100,000. The protective action of M/1,000,000 is detectable.Potassium oxalate accelerates disintegration in hypotonic solutions.One per cent ethyl alcohol in distilled water causes cytolysis more rapidly than does distilled water alone, but in M/500 molal calcium chloride the alcohol solution is much less effective.Ringer's solution minus calcium affords no protection against death due to absence of calcium and death due to potassium oxalate but completely protects against cytolysis. Death in Ringer's solution minus calcium and in Ringer's solution with potassium oxalate occurs first in the anterior region and describes an antero-posterior gradient.Cytolysis in distilled water, in potassium oxalate solutions, in alcohol solutions, and in hypotonic calcium solutions of extreme dilution is initiated in the anterior end and describes an antero-posterior gradient within a zooid.Earlier work of the writer on the disintegrative action of lipoid solvents, heat, KNC, hyper- and hypotonic solutions is discussed. It is concluded that inPlanaria dorotocephala the antero-posterior gradient in cytolytic disintegration represents an antero-posterior differential in sensitivity to disturbance of the calcium-lipoid-water relation in the organism.     

Phenobarbital induces slow recovery from sodium inactivation at the nodal membrane

Voltage-clamp experiments were performed on single myelinated nerve fibres of Rana esculenta at 20 degrees C in Ringer's solution and in solutions containing phenobarbital-sodium ([PB] less than or equal to 5 mM). The reduction of the sodium current under phenobarbital could be explained by an increase in the resting sodium inactivation; h infinity (E) was shifted towards more negative membrane potentials. The recovery from sodium inactivation proceeded with two time constants. The fast process could be described with the same time constant as in Ringer's solution, whereas the slow process had a time constant approx. 40 times larger. The slow process was also potential-dependent and could be described by 1/(0.025 alpha h + beta h), where alpha h and beta h denoted the rate constants in Ringer's solution. With the measured blockage of sodium channels by phenobarbital, both the shift of h infinity (E) and the slow recovery from sodium inactivation could be explained.     

X-ray diffraction study of the kinetics of myelin lattice swelling. Effect of divalent cations.

The time-course of myelin lattice swelling and its reversal in dissected peripheral nerves was determined by small-angle x-ray diffraction using a position-sensitive proportional detector. The process of swelling can take place either in several hours or in less than 1 h depending on pretreatment of the nerves. The reversal of swelling was always completed within 1 h. The rapid structural transitions involved the disordering of membrane pairs as indicated by the transient appearance of a continuous intensity distribution similar to the membrane pair transform for myelin. The slow transitions involved the gradual replacement of the discrete reflections from the native structure by the reflections from the swollen lattice. Myelin membrane arrays reformed in normal Ringer's solution were much more stable to subsequent swelling than arrays reformed in Ca+2 and Mg+2-free Ringer's. These results suggest that these ions participate in stabilizing the interactions between the external surfaces of adjacent membrane pairs.     

Electron microprobe analysis of frog skin epithelium: Evidence for a syncytial sodium transport compartment

Summary For elucidation of the functional organization of frog skin epithelium with regard to transepithelial Na transport, electrolyte concentrations in individual epithelial cells were determined by electron microprobe analysis. The measurements were performed on 1-m thick freeze-dried cryosections by an energy-dispersive X-ray detecting system. Quantification of the electrolyte concentrations was achieved by comparing the X-ray intensities obtained in the cells with those of an internal albumin standard.The granular, spiny, and germinal cells, which constitute the various layers of the epithelium, showed an identical behavior of their Na and K concentrations under all experimental conditions. In the control, both sides of the skin bathed in frog Ringer's solution, the mean cellular concentrations (in mmole/kg wet wt) were 9 for Na and 118 for K. Almost no change in the cellular Na occurred when the inside bathing solution was replaced by a Na-free isotonic Ringer's solution, whereas replacing the outside solution by distilled water resulted in a decrease of Na to almost zero in all layers. Inhibition of the transepithelial Na transport by ouabain (10^–4 m) produced an increase in Na to 109 and a decrease in K to 16. The effect of ouabain on the cellular Na and K concentrations was completely cancelled when the Na influx from the outside was prevented, either by removing Na or adding amiloride (10^–4 m). When, after the action of ouabain, Na was removed from the outside bathing solution, the Na and K concentration in all layers returned to control values. The latter effect could be abolished by amiloride.The other cell types of the epithelium showed under some experimental conditions a different behavior. In the cornified cells and the light cells, which occurred occasionally in the stratum granulosum, the electrolyte concentrations approximated those of the outer bathing meium under all experimental conditions. In the mitochondria-rich cells, the Na influx after ouabain could not be, prevented by adding amiloride. In the gland cells, only a small change in the Na and K concentrations could be detected after ouabain.The results of the present study are consistent with a two-barrier concept of transepithelial Na transport. The Na transport compartment comprises all living epithelial layers. Therefore, with the exception of some epithelial cell types, the frog skin epithelium can be regarded as a functional syncytium for Na.     

Inhibition of packing of acetylcholine into quanta by ammonium

Soaking frog motor nerve terminals in a hypertonic solution produces an increase in the size of miniature end plate potentials (mepp's) and miniature end plate currents (mepc's) after the preparations are returned to normal Ringer's solution. There is substantial evidence that the size increase occurs because additional acetylcholine (ACh+) is incorporated into the quanta. It has been proposed that ACh+ loading into synaptic vesicles requires a proton gradient. As a step in testing this hypothesis the effects of millimolar concentrations of NH4+, methylamine+, or trimethylamine+ in the extracellular solution on the increase in quantal size were measured. These substances would be expected to accumulate in acid intracellular compartments, which would diminish the acidity. The increase in quantal size was blocked by these substances, in agreement with the idea that the proton gradient is involved in ACh+ accumulation. Tetanic stimulation in solutions containing 5 mM NH4Cl also produces a decrease in quantal size, not seen in controls in NH4+-free solution. The inhibition of transmitter packaging by ammonia may play a role in the neural sequelae of hepatic failure.     

The localization of proteoglycans and glycoproteins in the hyaline cartilage

Summary The innervation of the cornea of newborn (two day old) and adult rats was investigated using glyoxylic-acid-induced fluorescence (GIF) for catecholamines and subsequent acetylcholinesterase reaction.Fluorescent nerves were observed around the limbal vessels and in the pericorneal nerve plexus, from which they branched towards the central parts of the cornea. The fluorescent corneal nerves were either nonvaricose or had varicosities at intervals of 10 micra. When the animals had been pretreated with nialamide, noradrenaline and propranolol, some fluorescent branching nerve terminals with numerous varicosities also appeared. All fluorescent nerves disappeared two days after ipsilateral superior cervical sympathectomy.When the acetylcholinesterase (AChE) reaction was performed subsequently to the GIF reaction the following nerve types could be identified: 1. nerves containing both catecholamine (CA) fluorescence and AChE, 2. nerves containing only AChE.     

Influence of paramagnetic ions bound to human serum albumin on water 1HNMR relaxation times

The extent to which various paramagnetic ions (Cu2+, Mn2+ and Gd3+) free and bound to human serum albumin alter the water proton relaxation times at two frequencies has been investigated. NMR relaxation parameters, T1 and T2, were measured at 5 and 10 MHz using a saturation recovery (90 degrees-tau-90 degrees) and a spin-echo (90 degrees-tau-180 degrees) sequence respectively. We found that all three ions enhance their effectiveness in inducing water proton magnetic relaxation when they are bound to human serum albumin and that Gd3+ is the most effective in pure water and Mn2+ in the presence of the protein. Cu2+ has a smaller effect, but it presents an interesting behaviour correlated with the existence of two different binding sites, which is also confirmed by electronic paramagnetic resonance spectra. The results indicate the potential usefulness of large molecular paramagnetic complexes as contrast agents in NMR Imaging.     

A Note on the Survival of some Bacteria in Different Diluents

The best diluent for four bacterial species was 0·1% (w/v) peptone solution. Tap water containing 0·1% (w/v) sodium thiosulphate was less satisfactory but tap water, tap water treated with charcoal, quarter-strength Ringer's solution, 0·85% (w/v) sodium chloride solution and glass distilled water were all bactericidal to one or more of the test species.     

2D 1H NMR studies of monomeric insulin

In order to establish the conditions required for the observation of monomeric insulin in solution, a series of proton nuclear magnetic resonance studies of insulin in a variety of solvents was undertaken. Optimal spectra were recorded in trifluoroethanol- water mixtures in a 1:2 ratio. Using the sequential assignment approach the proton nuclear magnetic resonance spectrum of insulin was then assigned. Aspects of the structure of monomeric insulin in solution have been determined using the observed NOE cross peaks and slow exchange protons.     

Red blood cells under mechanical stress

The effect of mechanical stress on erythrocytes suspended in various media was studied. The ability of the cells to increase their glucose consumption was found to be the major criterion allowing to divide the media into two groups. In plasma, serum or in Ringer's solution supplemented with albumin and glucose the energy consumption by mechanically stressed erythrocytes increased 20 to 50%; no morphological changes of the cells were observed either in suspension or on Giemsa smears. The cells behaved in the same way in Mg2(+)-free medium. The other group included protein-free medium (Ringer's solution supplemented with glucose) and Ca2(+)-free Ringer's solution supplemented with albumin and glucose; under these conditions erythrocytes were unable to raise their energy consumption in response to mechanical stress, and after some period structural impairment of the membrane could be observed on Giemsa smears. No differences in metabolism-associated nucleotide concentrations (ATP, ADP, NAD, NADP) were observed between the samples. Resealed red cell ghosts with high concentrations of intracellular components were prepared as a model of cells with damaged membrane. In these ghosts (with low ATP concentration) mechanical stress produced increased proportions of echinocytes, even in the "native" suspension. These results have confirmed the vital role of the energy-consuming contractile apparatus in the erythrocyte membrane, and supplied a clue to the role of Ca2+ in its activation and to the influence of extracellular proteins on the maintenance of in red cell shape.     

Measurement of short-term nutrient uptake rates in cranberry by aeroponics

Aeroponics, a soil-less plant culture system in which fresh nutrient solutions are intermittently or continuously misted on to plant roots, is capable of sustaining plant growth for extended periods of time while maintaining a constantly refreshed nutrient solution. Although used relatively extensively in commercial installations and in root physiology research, use of aeroponics in nutrient studies is rare. The object of this study was to examine whether nutrient uptake rates could be calculated for aeroponic systems by difference using measurements of concentrations and volumes of input and efflux solutions. Data were collected from an experiment with cranberry plants (Vaccinium macrocarpon Ait. cv. Stevens) cultured aeroponically with nutrient solutions containing various concentrations of ammonium-N and isotopically labelled nitrate-N. Validation of the calculated uptake rates was sought by: (1) evaluating charge balance of the solutions and total ion uptake (including proton efflux) and (2) comparison with N-isotope measurements. Charge balance and proton efflux calculations required use of chemical modelling of the solutions to determine speciation of dissolved phosphate and acid-neutralizing capacity (ANC). The results show that charge balance requirements were acceptably satisfied for individual solution analyses and for total ion uptake when proton efflux was included. Relative rates of nitrate/ammonium uptake determined by difference were in agreement with those determined by isotopic techniques. Additional information was easily obtained from this experimental technique, including evidence of diurnal variation in nutrient uptake, correlation between ammonium uptake and proton efflux, and the relationship between ion concentration and uptake. Use of aeroponic systems for non-destructive measurement of water and ion uptake rates for numerous other species and nutrients appears promising.     

Effects of acute hypobaric hypoxia on the appearance of ingested deuterium from a deuterium oxide-labelled carbohydrate beverage in body fluids of humans during prolonged cycling exercise

To determine whether or not acute hypobaric hypoxia alters the rate of water absorption from a carbohydrate beverage ingested during exercise, six men cycled for 80 min on three randomly assigned different occasions. In one trial, exercise was performed in hypoxia (barometric pressure, P(B) = 594 hPa, altitude 4,400 m) at an exercise intensity selected to elicit 75% of the individual's maximal oxygen uptake (VO2max) previously determined in such conditions. In the two other experiments, the subjects cycled in normoxia (P(B) = 992 hPa) at the same absolute and the same relative intensities as in hypoxia, which corresponded to 55% and 75%, respectively, of their VO2max determined in normoxia. The subjects consumed 400 ml of a 12.5% glucose beverage just prior to exercise, and 250 ml of the same drink at 20, 40 and 60 min from the beginning of exercise. The first drink contained 20 ml of deuterium oxide to serve as a tracer for the entry of water into body fluids. The heart rate (HR) during exercise was higher in hypoxia than in normoxia at the same absolute exercise intensity, whereas it was similar to HR measured in normoxia at the same relative exercise intensity. Both in normoxia and hypoxia, plasma noradrenaline concentrations were related to the relative exercise intensity up to 40 min of exercise. Beyond that duration, when exercise was performed at the highest absolute power in normoxia, the noradrenaline response was higher than in hypoxia at the same relative exercise intensity. No significant differences were observed among experimental conditions, either in temporal profiles of plasma D accumulation or in elimination of water ingested in sweat. Conversely, elimination in urine of the water ingested appeared to be related to the severity of exercise, either high absolute power or the same relative power combined with hypoxia. We concluded that water absorption into blood after drinking a 12.5% glucose beverage is not altered during cycling exercise in acute hypobaric hypoxia. It is suggested that the elimination of water ingested in sweat and urine may be dependent on local circulatory adjustments during exercise.