Effect of heat on frog sciatic nerve determined by X-ray diffraction

Low-angle X-ray diffraction patterns have been recorded from frog sciatic nerves in Ringer's solution after heat treatment from 20 to 80°C. The X-ray patterns were obtained from the heat treated specimens after cooling to room temperature. The normal X-ray pattern of frog sciatic nerve in Ringer's solution with $\text{d=171}\text{A}\text{?}$ was maintained from 20 to 58°C. Above 58°C, a new high temperature pattern based on a repeat period of $\text{d?435}\text{A}\text{?}$ was recorded from the nerve in Ringer's solution. The physical state of nerve myelin after heat teratment at a temperature ?58°C has been identified as the anomalous swollen state. Anomalous swelling takes place in units of four membranes.     

An X-ray study of the condensed and separated states of sciatic nerve myelin

Low-angle X-ray diffraction patterns of peripheral nerve myelin after modification by either rehydration in various solutions or by chemical treatment have been recorded. These X-ray patterns and the previously reported modified nerve myelin patterns demonstrate that nerve myelin has at least five different states: the normal state, condensed state I and II and separated state I and II. There are two membranes per unit cell in the normal state and in states II whereas there is one membrane per unit cell in states I. Under certain conditions normal nerve can go reversibly into either of states II. With continued treatment the nerve myelin structure moves irreversibly from state II to state I and, once in state I, the nerve myelin layers cannot return to the normal state. Our results demonstrate that there is a reversible transformation between condensed state I and separated state I. Fourier profiles of nerve myelin in the normal state, condensed state I and separated state I are presented.     

True Anomalous Osmosis in Multi-Solute Model Membrane Systems

The transport of liquid across charged porous membranes separating two electrolytic solutions of different composition consists of both a normal and an anomalous osmotic component. Anomalous osmosis does not occur with electroneutral membranes. Thus, with membranes which can be charged and discharged reversibly, normal osmosis can be measured with the membrane in the electroneutral state, and normal together with anomalous osmosis with the membrane in a charged state, the difference between these two effects being the true anomalous osmosis. Data are presented on the osmotic effects across an oxyhemoglobin membrane in the uncharged state at pH 6.75 and in two charged states, positive at pH 4.0 and negative at pH 10.0, in multi-solute systems with 0.2 and 0.4 osmolar solutions of a variety of electrolytes and of glucose against solutions of other solutes of the same, one-half, and twice these osmolarities. In the simpler systems the magnitude of the true anomalous osmosis can be predicted semiquantitatively by reference to appropriate single-solute systems. In isoosmolar systems with two electrolytic solutions the anomalous osmotic flow rates may reach 300 µl./cm.² hr. and more; systems with electrolytic solutions against solutions of glucose can produce twice this rate. These fluxes are of the same order of magnitude as the liquid transport rates across such living structures as the mucosa of dog gall bladder, ileum, and urinary bladder.     

Effect of pH of the external solution on posttetanic hyperpolarization of the denuded nerve

Changes in posttetanic hyperpolarization of the frog sciatic nerve during a change in pH of the external solution from 5.3 to 9.0 were studied. Reducing the pH of the external solution was shown to lead to an increase in the amplitude and time constant of posttetanic hyperpolarization of the nerve, and an increase in pH led to a decrease in the time constant and a considerable weaking or complete abolition of posttetanic hyperpolarization. It is concluded that these changes in posttetanic hyperpolarization of the nerve related to the pH of the external solution are probably the result of a change in the stoichiometry of the active transport of sodium and potassium ions and the consequent electrogenicity of the potassium — sodium pump.I. N. Ul'yanov Ul'yanovsk Pedagogic Institute. Translated from Neirofiziologiya, Vol. 8, No. 2, pp. 177–182, March–April, 1976.     

A reversible abnormal form of myelin: an X-ray scattering study of human sural and rat sciatic nerves

A sural nerve dissected from a recently dead patient displayed an unusual X-ray diffraction pattern, suggesting that in situ and at the time of the patient's death the myelin sheaths were in a swollen state. Diffraction patterns of the swollen type were also recorded from: (1) a sural nerve from the corpse of a neurologically healthy person after soaking the nerve with Ringer solution at pH 5.5; (2) sciatic nerves dissected from rat cadavers at increasing time after death. In all the cases the swollen patterns reversed to the native type upon superfusion with Ringer solution at pH 7.3. The postmortem effect is to decrease the pH of the fluids surrounding the nerves in the cadavers. Our experiments show that the early postmortem processes have the effect of acidifying PNS nerves and that as a consequence of acidification the myelin sheaths swell.     

Kinetics of nerve impulse blocking by protein cross-linking aldehydes Apparent critical thermal points

The effect of formaldehyde, crotonaldehyde, butyraldehyde, glutaraldehyde and cinnamaldehyde on the compound action potential of frog sciatic nerve was studied in the temperature domain 20–35°C at various aldehyde concentrations. All these reagents gradually decrease the amplitude of nerve action potential, up to the complete block, the order of effectiveness being: crotonaldehyde > cinnamaldehyde > butyraldehyde > formaldehyde > glutaraldehyde. The effect of cinnamaldehyde is almost completely reversible, while all others have irreversible action. The dependence of the blocking time on temperature and concentration is well expressed in all cases by the same empirical equation. This dependence points to the existence of critical temperatures, specific for each aldehyde, at which impulse blocking would be instantaneous, regardless of concentration. These temperatures (obtained by extrapolation) lie between 43°C (for crotonaldehyde) and 57.5°C (for butyraldehyde). The existence of free amino groups within ionic channels, as main sites of aldehyde attack, is inferred.     

The contributions of normal and anomalous osmosis to the osmotic effects arising across charged membranes with solutions of electrolytes

The osmotic effect arising across a porous membrane separating the solution of an electrolyte from water (or a more dilute solution) is ordinarily due to both normal osmosis, as it occurs also with non-electrolytes, and to "anomalous" osmosis. It is shown that the normal osmotic component cannot be measured quantitatively by the conventional comparison with a non-electrolytic reference solute. Anomalous osmosis does not occur with electroneutral membranes. Accordingly, with membranes which can be charged and discharged reversibly (without changes in geometrical structure), such as many proteinized membranes, the osmotic effects caused by an electrolyte can be measured both when only normal osmosis arises (with the membrane in the electroneutral state) and when normal as well as anomalous osmosis occurs (with the membrane in a charged state). The difference between these two effects is the true anomalous osmosis. Data are presented on the osmotic effects across an oxyhemoglobin membrane in the uncharged state at pH 6.75 and in two charged states, positive at pH 4.0 and negative at pH 10.0, with solutions of a variety of electrolytes using a concentration ratio of 2:1 over a wide range of concentrations. The rates of the movement of liquid across the membrane against an inconsequentially small hydrostatic head are recorded instead of, as conventional, the physiologically less significant pressure rises after a standard time.     

Direct Determination of the Lamellar Structure of Peripheral Nerve Myelin at Low Resolution (17 Å)

New X-ray diffraction data from normal nerve and nerve swollen in glycerol solutions have been recorded. Direct methods of structure analysis have been used in the interpretation of the X-ray data, and the phases of the first five orders of diffraction of peripheral nerve myelin have been uniquely determined. The direct methods include deconvolution of the autocorrelation function, sampling theorem reconstructions, and Fourier synthesis comparisons. Electron density profiles of normal and swollen nerve myelin at a resolution of 17 Å together with an electron density scale in electrons per cubic angstrom are presented.     

An X-ray study of the effect of enzymes on frog sciatic nerve myelin

Low-angle X-ray diffraction patterns have been recorded from frog sciatic nerve after digestion with trypsin and Pronase. Reproducible X-ray patterns were obtained by swelling the nerves in distilled water before treatment with enzymes. The X-ray patterns of enzyme-treated nerves are distinctly different from the X-ray pattern of normal (live) nerve. It would appear that the normal asymmetric nerve myelin membrane becomes symmetric about its center after treatment with enzymes as a result of proteolytic cleavage and a subsequent redistribution of protein components.     

Production of dextranase byLipomyces starkeyi

Summary The optimal growth rate ofLipomyces starkeyi, with dextran as sole carbon source, was found within the pH range 2.5–4.0, and temperature between 25–30°C. This yeast was unable to grow above 33°C. Dextranase production optima paralleled growth optima, except at pH 2.5. Decrease in enzyme yield at this pH could not be attributed to poor yeast growth or enzyme stability.     

Direct Determination of the Lamellar Structure of Peripheral Nerve Myelin at Moderate Resolution (7 Å)

Low-angle X-ray diffraction patterns have been recorded from normal nerve and nerve swollen in glycerol solutions. The new X-ray data have a resolution of 7 Å. Direct methods of structure analysis which include deconvolution of the auto-correlation function and sampling theorem reconstructions have been used in the interpretation of the X-ray data. Phases have been assigned to the first 12 orders of diffraction from normal nerve. Fourier syntheses at a resolution of 7 Å are described and an absolute electron density scale is derived. A possible molecular interpretation of the electron density profile is given.     

Determination of ketorolac in human plasma by reversed-phase high-performance liquid chromatography using solid-phase extraction and ultraviolet detection

An improved high-performance liquid chromatographic method has been developed to measure human plasma concentrations of the analgesic nonsteroidal anti-inflammatory drug ketorolac for use in pharmacokinetic studies. Samples were prepared for analysis by solid-phase extraction using Bond-Elut PH columns, with nearly complete recovery of both ketorolac and the internal standard tolmetin. The two compounds were separated on a Radial-Pak C₁₈ column using a mobile phase consisting of water–acetonitrile–1.0 mol/l dibutylamine phosphate (pH 2.5) (30:20:1) and detected at a UV wavelength of 313 nm. Using only 250 μl of plasma, the standard curve was linear from 0.05 to 10.0 μg/ml.     

Alkaline phosphatase activity in the brain of the American cockroach Periplaneta americana L

Summary The supra- and suboesophageal ganglia of the American cockroach contain material which catalyses the alkaline hydrolysis (pH 9.5) of 5-bromo-4-chloro-3-indolyl phosphate in the presence of Nitro blue tetrazolium. Histochemical studies on unfixed cryostat sections indicate that this type of alkaline phosphatase is restricted to discrete regions in the cockroach brain. Highest enzyme activity is encountered in the mushroom bodies, central body, antennal glomeruli and specific parts of some distinct neural connections including the optic nerve, antennal nerve, circumoesophageal connectives and nerves leaving the suboesophageal ganglion. Tissue fixation by use of formaldehyde-type fixatives, as well as routine paraffin-embedding, completely destroy all histochemically detectable enzyme activity.Native polyacrylamide gradient electrophoresis suggests that the alkaline phosphatase activity is present as multiple isozymic forms, which show up in the 120–130 kD range of standard proteins. Enzyme activity becomes undetectable after fixation (trichloroacetic acid, formaldehyde containing fixatives) of electrophoretically separated native proteins, as well as after electrophoresis in denaturing conditions (SDS and -mercapto-ethanol, boiling). However, the enzyme activity remains virtually unaffected after storage of the sample for prolonged periods at –20 to –80°C.     

X-ray diffraction of myelin membrane. I. Optimal conditions for obtaining unmodified small angle diffraction data from frog sciatic nerve

The X-ray diffraction pattern of myelin of frog sciatic nerve has been investigated, using a Kratky small angle slit camera to obtain the electron density distribution across the membrane. All major reflections observed were related to a fundamental repeat distance of 171 ± 2.8 A. There was no further increase in the number of reflections on varying the experimental conditions (varying pH, applying tension, immersion in various isotonic buffer solutions, etc.) or by varying the camera slit arrangement. The degree of disorder within the myelin sheath was examined by comparing the crystallite size to the half-width of the diffraction peak at half-height. The limiting of the diffraction spectra to five major reflections was determined not to be caused by disorder. It is concluded that the observed X-ray diffraction pattern is a consequence of the particular electron density distribution of the membrane. Therefore, the membrane cannot contain sharply distinct step-function regions of electron density, but approaches a modified cosine distribution.     

Effects of aluminum,calcium and low pH on egg hatching and nymphal survival of Cloeon triangulifer McDunnough (Ephemeroptera: Baetidae)

Laboratory experiments were conducted to assess the effects of aluminum, calcium and low pH on egg hatching and nymphal survival of the mayfly Cloeon triangulifer. Percent successful hatch (living nymphs breaking free of the chorion) decreased and percent partial hatch (nymphs dying attached to the chorion) increased with increasing acidity (pH 7.5–3.0). Most hatches occurring below pH 5.0 were partial hatches. Decreased time of exposure to acidic waters increased percent successful hatch and decreased percent partial hatch. Time to first hatch was not affected by pH. Eggs were incubated in acidic waters (pH 4.0 and 5.5) with additions of calcium (10 and 100 mg l^–1) and aluminum (100 and 500 g l^–1). Aluminum decreased percent successful hatch and increased percent partial hatch and calcium increased both percent successful hatch and percent partial hatch. Time to first hatch was increased by both aluminum and calcium. The 96 h LC50 for small nymphs was pH 4.75. Addition of aluminum (100 and 500 µg l^–1) to acidic waters (pH 4.0 and 5.0) reduced nymphal mortality by 8–22%. Toxic effects of low pH on egg hatching and early nymphs may contribute to the absence of mayflies from acidified habitats.Contribution No. 1469 of the Maine Agricultural Experiment Station, University of Maine, Orono, Maine 04469 USA.Contribution No. 1469 of the Maine Agricultural Experiment Station, University of Maine, Orono, Maine 04469 USA.     

Enzymatic preparation of wheat bran xylooligosaccharides and their stability during pasteurization and autoclave sterilization at low pH

Xylooligosaccharides (XOS) were prepared from wheat bran insoluble dietary fiber (WBIDF) by treatment with commercial xylanase preparation Sunzymes. XOS, with a purity of 95% (w/w) and degree of polymerization of 2-7 and the ratio of arabinose to xylose of 0.27, was obtained with a yield of approximately 31.2% of WBIDF. Their stability was evaluated by comparing with that of commercial fructooligosaccharides (FOS) during pasteurization (60–100 °C, 30 min) and autoclave sterilization (121 °C, 1 kg/cm², 10–50 min) at pH 2.0–4.0. XOS was characterized by a high thermal stability during pasteurization at pH 2.5–4.0 and sterilization at pH 3.0–4.0. Even at pH 2.0, the remaining XOS reached 97.2% (w/w) and 84.2% (w/w) during pasteurization (100 °C, 30 min) and sterilization (50 min), respectively. Compared with FOS, XOS was strongly resistant to lower acidic conditions. The results revealed that XOS was considered to be more suitable for use as functional food ingredients.     

Effect of pH changes on after potentials of single frog Ranvier nodes

In experiments on single nodes of Ranvier of isolated frog nerve fibers a decrease in pH of the medium to 5 caused a small increase in after-depolarization but an increase in pH to 9 caused virtually no change in it. The after-hyperpolarization arising in potassium-free Ringer's solution was abolished by a decrease in pH but remained substantially unchanged as a result of an increase in pH; posttetanic hyperpolarization was reduced in amplitude and slightly increased in duration by a decrease in pH, but not appreciably changed by an increase in pH. It is concluded that the character of changes in after-potentials of single nodes of Ranvier of isolated nerve fibers in medium of different pH is entirely determined by the influence of this factor on the kinetics of the potassium permeability of the excitable membrane.I. N. Ul'yanov Pedagogic Institute, Ul'yanovsk. Translated from Neirofiziologiya, Vol. 8, No. 1, pp. 62–66, January–February, 1976.     

Pullulanases of alkaline and broad pH range from a newly isolated alkalophilicBacillus sp. S-1 and aMicrococcus sp. Y-1

Summary Two highly alkalophilic bacteria, and potent producers of alkaline pullulanase, were isolated from Korean soils. The two isolates, identified asBacillus sp. S-1 andMicrococcus sp. Y-1, grow on starch under alkaline conditions and effectively secrete extracellular pullulanases. The two isolates were extremely alkalophilic since bacterial growth and enzyme production occurred at pH values ranging from pH 6.0 to 12.0 forMicrococcus sp. Y-1 and pH 6.0 to 10.0 forBacillus sp. S-1. Both strains secrete enzymes that possess amylolytic and pullulanolytic acitivities. Extracellular crude enzymes of both isolates gave maltotriose as the major product formed from soluble starch and pullulan hydrolysis. Compared to other alkalophilic microbes such asMicrococcus sp. (0.57 units ml^–1),Bacillus sp. KSM-1876 (0.56 units ml^–1) andBacillus No. 202-1 (1.89 units ml^–1) these isolates secreted extremely high concentrations (7.0 units ml^–1 forBacillus sp. S-1 and 7.6 units ml^–1 forMicrococcus sp. Y-1) of pullulanases in batch culture. The pullulanase activities from both strains were mostly found in the culture medium (85–90%). The extracellular enzymes of both bacteria were alkalophilic and moderately thermoactive; optimal activity was detected at pH 8.0–10.0 and between 50 and 60°C. Even at pH 12.0, 65% of original Y-1 pullulanase activity and 10% of S-1 pullulanase activity remained. The two newly isolated strains had broad pH ranges and moderate thermostability for their enzyme activities. These result strongly indicate that these new bacterial isolates have potential as producers of pullulanases for use in the starch industry.     

pH-induced domain interaction and conformational transitions of lipoxygenase-1

The multidomain structure of soybean LOX1 was examined over the pH range 1-12. Lipoxygenase-1 activity was reversible over broad pH range of 4-10 due to the reversibility of conformational states of the molecule. Below pH 4.0, due to collapse in hydrophobic interactions, the enzyme unfolded to an irreversible conformation with the properties of molten globule state with a mid point of transition at pH 2.4. This intermediate state lost iron irreversibly. In alkaline pH at 11.5, LOX1 underwent partial unfolding with the exposure of cysteine residues with subsequent oxidation of a pair of cysteine residues in the C-terminal domain and this intermediate showed some properties of molten globule state and retained 35% of activity. Beyond pH 12.0, the enzyme was completely inactivated irreversibly due to irreversible conformational changes. The pH-dependent urea-induced unfolding of LOX1 suggested that LOX1 was more stable at pH 7.0 and least stable at pH 9.0. Furthermore, the urea-induced unfolding of LOX1 indicated that the unfolding was biphasic due to pH-dependent domain interactions and involved sequential unfolding of domains. The loss of enzyme activity at pH 4. 0 and 7.0 occurred much earlier to unfolding of the C-domain at all pHs studied. The combination of urea-induced unfolding measurements and limited proteolysis experiments suggested that at pH 4.0, the domains in LOX1 were less interactive and existed as tightly folded units. Furthermore, these results confirmed the contribution of ionic interactions in the interdomain contacts.     

Paired pulse facilitation of summated intracellular synaptic potentials in single retinotectal fibers in the frog

Facilitation of the second of two consecutive test EEG quanta (the summated monosynaptic potentials of the synapses of one axon arborizing in layer F of the frog tectum) was investigated in the normal and under conditions of raised extracellular Ca²⁺ and Mg²⁺ concentration. Intensification of paired-pulse facilitation (×1.4–2.4) was observed at the shortest interstimulus intervals (of 2.5–5 msec). The distribution of maximum levels for facilitation of EEG quanta was bimodal at levels 1.95 and 1.65, on the basis of which two groups were identified, one potentiating EEG quanta more than the other. The time course of paired-pulse facilitation of both groups of EEG quanta can be broken down into two exponential components with time constants of 5–6, 140–150 and 6–8, 60–70 msec respectively. Bimodal distribution of maximum paired-pulse levels in the normal, together with findings from experiments involving raised Ca²⁺ and Mg²⁺ concentrations would indicate that facilitation of frog retinotectal synapses is dependent on the quantal release of transmitter; it may thus be postulated that this release reaches near-saturation point in the normal. It is suggested that two types of axonal terminal arborizations whose synapses differ in the quantal content of transmitter release are present in layer F of the frog tectum. These axonal arbors could well originate from different class 3 and 5 retinal detectors.Z. Yanushkevichyus Institute of Physiology and Pathology of the Cardiovascular System of the Kaunas Medical Institute. Translated from Neirofiziologiya, Vol. 18, No. 1, pp. 45–55, January–February, 1986.