Biological responses inCaenorhabditis elegans to high magnetic fields

Here we describe a device for testing possible influences of high magnetic fields on biological processes, by which alternating-current magnetic stimuli as high as 1.7 T can be administered. Experiments with a simple multicellular organism, the nematodeCaenorhabditis elegans, revealed that intermittent exposure to the magnetic fields modestly inhibited the animal's reproduction as well as its post-embryonic development, and caused a marked but transient derangement in its locomotory behavior. Available evidence indicates that alternating high magnetic fields can elicit both chronic and acute biological effects, but that the effects may be well tolerated or compensated for by the living organism.     

Resting potential of excitable neuroblastoma cells in weak magnetic fields

The mechanism by which static and low-frequency magnetic fields are transduced into biological signals responsible for reported effects on brain electrical activity is not yet ascertained. To test the hypothesis that fields can cause a subthreshold change in the resting membrane potential of excitable cells, we measured changes in transmembrane current under voltage clamp produced in SH-SY5Y neuroblastoma cells, using the patch-clamp method in the whole-cell configuration. In separate experiments, cells were exposed to static fields of 1, 5, and 75 G, to time-varying fields of 1 and 5 G, and to combined static and time-varying fields tuned for resonance of Na+, K+, Ca2+, or H+. To increase sensitivity, measurements were made on cells connected by gap junctions. For each cell, the effect of the field was evaluated on the basis of 100 trials consisting of a 5-s exposure immediately followed by a 5-s control period. In each experiment, the field had no discernible effect on the transmembrane current in the vicinity of zero current (- 50 mV voltage clamp). The sensitivity of the measuring system was such that we would have detected a current corresponding to a change in membrane potential as small as 38 microV. Consequently, if sensitivity of mammalian cells to magnetic fields is mediated by subthreshold changes in membrane potential, as in sensory transduction of sound, light, and other stimuli, then the ion channels responsible for the putative changes are probably present only in specialized sensory neurons or neuroepithelial cells. A change in transmembrane potential in response to magnetic fields is not a general property of excitable cells in culture.     

Der Hyaluronsäuregehalt und die «Hyaluronidase»-Aktivität der Leukozyten

Summary The hyaluronic acid content of leucocytes from the peritoneum of rats shows a significant concentration when measured either turbidimetrically or oxydimetrically, the average value calculated for wet weight being 0.7–0.8%. The hyaluronic acid break down capacity of the leucocytes is not a specific enzyme action but can be attributed to their high ascorbic acid content. The phenomenon has no well defined pH or temperature optimum and can be inhibited by oxine and by semicarbazide.     

Effect of benzimidazole on nicotinamide adenine dinucleotide phosphate phosphomonoesterase activity in wheat leaves.

Nicotinamide adenine dinucleotide phosphate phosphomonoesterase was isolated and partially purified from wheat (Triticum aestivum L. var. Selkirk) leaves. The enzyme had KNADP value of 1.4 X 10(-4) M and a pH optimum of 5.9. In vitro activity of this enzyme was unaffected by precursors of NAD (nicotinamide and nicotinic acid) or cytokinins (kinetin and benzimidazole). However, when detached wheat leaves were treated with solutions of these compounds, the precursors lowered the specific activity while the cytokinins enhanced the activity. It is suggested that spatial separation and compartmentation of the enzyme and its substrate NADP account for the similar effect of benzimidazole on both.     

Spontaneous phospholipase A activity of rat pancreatic homogenates]

Rat pancreas presents a spontaneous phospholipase A activity which appears before trypsin activation at optimal pH 6.5. The responsible enzyme is independent of pancreatic prophospholipase A, as can be seen through experiments done in the presence of trypsin inhibitors. On the other hand, this enzyme is distinct from excretory phospholipase which is more active and whose optimal pH is 8.8. Thermostability and insensibility of spontaneously active phospholipase A to DFP differentiate it from lipase, carboxyl-esterhydrolase and lysophospholipase, respectively.     

Immobilization of chicken liver fructose 1,6-bisphosphatase on CNBr-activated Sepharose

Chicken liver fructose 1,6-bisphosphatase is readily immobilized on CNBr-activated Sepharose. The immobilization alters some enzymatic properties. They include broader pH activity curve, loss of activation by K+ or NH+4, increased resistance to inactivation by trypsin, decreased sensitivity to AMP inhibition, and loss of cooperative interaction among AMP-binding sites. The immobilized enzyme retains about 38% or 19% of the specific activity of the native enzyme when the activity is measured in the absence or presence of K+, respectively.     

An efficient method for purification of cuprozinc superoxide dismutase from bovine erythrocytes

Cuprozinc superoxide dismutase (Cu,Zn-SOD) was isolated from bovine erythrocytes by pH-controlled ammonium sulfate-methanol extraction (ASME extraction). Adjustment of the pH of a suspension of the lysed red cells in the presence of ammonium sulfate (90% saturation) to pH 5.0, followed by partition with an equal amount of methanol, resulted in isolation of the enzyme with specific, activity of greater than 2000 units/mg of protein. Further purification using DEAE-cellulose column chromatography gave a highly purified Cu,Zn-SOD showing a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Using this procedure about 14 mg of pure Cu,Zn-SOD with a specific activity of 4728 units/mg of protein can be recovered from one liter of bovine blood. The enzyme was characterized and the results obtained were in agreement with earlier reports. This procedure appears, therefore, to be a convenient method for isolating the enzyme.     

Ambiquitous behavior of rabbit liver lactate dehydrogenase

Rabbit liver mitochondrial fraction shows lactate dehydrogenase activity. The enzyme can be released from particles by increasing the pH and the ionic strength of the medium. There is a narrow range of pH (6.8-7.4) and ionic strength (20-50 mM NaCl) in which the solubilization sharply increases. It has been shown that divalent anions (SO4(2-) and cations (Mg2+, Ca2+) are highly effective specific solubilizing agents. NADH (1.5 mM) and ATP (1.0 mM) were effective in solubilizing 50% of the enzyme bound, whereas the same concentrations of the analogs NAD+ and ADP had little effect. Cytosolic lactate dehydrogenase bound to the mitochondrial fraction and a saturation of particles by enzyme was observed in all experiments performed. The in vitro binding requires a short period of incubation between the enzyme and particles and the binding is independent of the temperature in the 0-37 degrees C range. Binding was prevented by 0.15 M NaCl. The bound enzyme is approximately 20% less active than the soluble one. The results described give support to the proposal that rabbit liver lactate dehydrogenase has an ambiquitous behavior, like other glycolytic enzymes, which have not a fixed intracellular localization.     

Inactivation of yeast glucose-6-P dehydrogenase by aspirin

Summary Glucose-6-P dehydrogenase is irreversibly inactivated by treatment with Na salts of aspirin. Kinetic data show that 1 molecule of aspirin reacts with each active unit when the enzyme is inactivated. The rate of inactivation is enhanced with increasing pH but is reduced in the presence of glucose-6-P or NADP⁺. Na salicylate fails to inactivate the enzyme.This work was supported by grant RR-8006 from the General Research Branch, Division of Research Resources, NIH (USA).     

The time course of plasma enzyme changes accompanying skeletal muscle stimulation

Summary 30 min electrical stimulation of hind limb skeletal muscle in nembutal anaesthetised dogs was accompanied by increases in arterial haematocrit, plasma GOT and plasma LDH, which were almost completed within the first 10 min of stimulation. This fast response indicated that a rapid change in either the entry and/or clearance of enzyme from the plasma must have occurred.     

Inactivation of human glutamate dehydrogenase by aluminum

Aluminum inactivated glutamate dehydrogenase (GDH) by a pseudo-first-order reaction at micromolar concentrations. A double-reciprocal plot gave a straight line with a k_inact of 2.7 min^-1 and indicated the presence of a binding step prior to inactivation. The inactivation was strictly pH dependent and a marked increase in sensitivity to aluminum was observed as the pH decreased. At a pH higher than 8.5, no inactivation was observed. The completely inactivated GDH contained 2 mol of aluminum per mole of enzyme subunit monomer. When preincubated with enzyme, several chelators such as citrate, NaF, N-(2-hydroxyethyl) ethylenediaminetriacetic acid or ethylenediaminetriacetic acid efficiently protected the enzyme against the aluminum inactivation. In a related experiment, only citrate and NaF released the aluminum from the completely inactivated aluminum-enzyme complex and fully recovered the enzyme activity. Ferritin, NADP⁺, or nerve growth factor did not show any effects on the recovery of the aluminum-inactivated GDH activity. The dissociation constant for the aluminum-enzyme complex was calculated to be 5.3 M. Although aluminum has been known to form a complex with nucleotides, no such effects were observed in the inactivation of GDH by aluminum as determined using GDHs mutated at the ADP-binding site, NAD⁺-binding site or GTP-binding site. Circular dichroism studies showed that the binding of aluminum to the enzyme induced a decrease in helices and sheets and an increase in random coil. Therefore, inactivation of GDH by aluminum is suggested to be due to the conformational change induced by aluminum binding. These results suggest a possibility that aluminum-induced alterations in enzymes of the glutamate system may be one of the causes of aluminum-induced neurotoxicity.Received 25 July 2003; received after revision 27 August 2003; accepted 15 September 2003     

Ionic currents in morphogenesis

Summary Morphogenetic fields must be generated by mechanisms based on known physical forces which include gravitational forces, mechanical forces, electrical forces, or some combination of these. While it is unrealistic to expect a single force, such as a voltage gradient, to be the sole cause of a morphogenetic event, spatial and temporal information about the electrical fields and ion concentration gradients in and around a cell or embryo undergoing morphogenesis can take us one step further toward understanding the entire morphogenetic mechanism. This is especially true because one of the handful of identified morphogens is Ca²⁺, an ion that will not only generate a current as it moves, but which is known to directly influence the plasma membrane's permeability to other ions, leading to other transcellular currents. It would be expected that movements of this morphogen across the plasma membrane might generate ionic currents and gradients of both electrical potential and intracellular concentration. Such ionic currents have been found to be integral components of the morphogenetic mechanism in some cases and only secondary components in other cases. My goal in this review is to discuss examples of both of these levels of involvement that have resulted from investigations conducted during the past several years, and to point to areas that are ripe for future investigation. This will include the history and theory of ionic current measurements, and a discussion of examples in both plant and animal systems in which ionic currents and intracellular concentration gradients are integral components of morphogenesis as well as cases in which they play only a secondary role. By far the strongest cases for a direct role of ionic currents in morphogenesis is the polarizing fucoid egg where the current is carried in part by Ca²⁺ and generates an intracellular concentration gradient of this ion that orients the outgrowth, and the insect follicle in which an intracellular voltage gradient is responsible for the polarized transport from nurse cell to oocyte. However, in most of the systems studied, the experiments to determine if the observed ionic currents are directly involved in the morphogenetic mechanism are yet to be done. Our experience with the fucoid egg and the fungal hypha ofAchlya suggest that it is the change in the intracellular ion concentration resulting from the ionic current that is critical for morphogenesis.     

Ionic currents in morphogenesis

Morphogenetic fields must be generated by mechanisms based on known physical forces which include gravitational forces, mechanical forces, electrical forces, or some combination of these. While it is unrealistic to expect a single force, such as a voltage gradient, to be the sole cause of a morphogenetic event, spatial and temporal information about the electrical fields and ion concentration gradients in and around a cell or embryo undergoing morphogenesis can take us one step further toward understanding the entire morphogenetic mechanism. This is especially true because one of the handful of identified morphogens is Ca2+, an ion that will not only generate a current as it moves, but which is known to directly influence the plasma membrane's permeability to other ions, leading to other transcellular currents. It would be expected that movements of this morphogen across the plasma membrane might generate ionic currents and gradients of both electrical potential and intracellular concentration. Such ionic currents have been found to be integral components of the morphogenetic mechanism in some cases and only secondary components in other cases. My goal in this review is to discuss examples of both of these levels of involvement that have resulted from investigations conducted during the past several years, and to point to areas that are ripe for future investigation. This will include the history and theory of ionic current measurements, and a discussion of examples in both plant and animal systems in which ionic currents and intracellular concentration gradients are integral components of morphogenesis as well as cases in which they play only a secondary role. By far the strongest cases for a direct role of ionic currents in morphogenesis is the polarizing fucoid egg where the current is carried in part by Ca2+ and generates an intracellular concentration gradient of this ion that orients the outgrowth, and the insect follicle in which an intracellular voltage gradient is responsible for the polarized transport from nurse cell to oocyte. However, in most of the systems studied, the experiments to determine if the observed ionic currents are directly involved in the morphogenetic mechanism are yet to be done. Our experience with the fucoid egg and the fungal hypha of Achlya suggest that it is the change in the intracellular ion concentration resulting from the ionic current that is critical for morphogenesis.     

Ambiquitous behavior of rabbit liver lactate dehydrogenase

Summary Rabbit liver mitochondrial fraction shows lactate dehydrogenase activity. The enzyme can be released from particles by increasing the pH and the ionic strength of the medium. There is a narrow range of pH (6.8–7.4) and ionic strength (20–50 mM NaCl) in which the solubilization sharply increases. It has been shown that divalent anions (SO ₄ ^2– ) and cations (Mg²⁺, Ca²⁺) are highly effective specific solubilizing agents. NADH (1.5 mM) and ATP (1.0 mM) were effective in solubilizing 50% of the enzyme bound, whereas the same concentrations of the analogs NAD⁺ and ADP had little effect. Cytosolic lactate dehydrogenase bound to the mitochondrial fraction and a saturation of particles by enzyme was observed in all experiments performed. The in vitro binding requires a short period of incubation between the enzyme and particles and the binding is independent of the temperature in the 0–37°C range. Binding was prevented by 0.15 M NaCl. The bound enzyme is approximately 20% less active than the soluble one. The results described give support to the proposal that rabbit liver lactate dehydrogenase has an ambiquitous behavior, like other glycolytic enzymes, which have not a fixed intracellular localization.     

Partial purification and characterization of acid phosphatase from sporulated oocysts of Eimeria tenella

Acid phosphatase of Eimeria tenella oocysts (Peak II) was purified 77-fold with a recovery of 26% using protamine sulfate precipitation, DEAE-cellulose chromatography and Sephadex G-200 gel filtration. This enzyme occurs in multiple forms as indicated by two peaks which can be separated by DEAE-cellulose chromatography and polyacrylamide gel electrophoresis. The partially purified enzyme has optimal activity at pH 4.5. With p-nitrophenyl phosphate the Km and Vmax values for (Peak II) were 25 mM and 1.57 mumol/min/mg protein, respectively. The enzyme (Peak II) is strongly inhibited by Hg++, Cu++, iodoacetamide, fluoride and molybdate. Tartrate and other divalent metal ions have no effect on enzyme activity. The partially purified Peak II phosphatase is not a glycoprotein as it is not absorbed on concanavalin-A Sepharose and its treatment with bacterial neuraminidase does not alter its elution profile through DEAE cellulose.     

Action d'un inhibiteur de la carboanhydrase,l'acétazolamide,sur l'excitabilité du cortex,du thalamus et du rhinencéphale

Summary The electrical activity induced by stimulation of the cortex, diencephalon and rhinencephalon was studied in the rabbit under the influence of a carbonic anhydrase inhibitor, acetazolamide or Diamox. A significant decrease of the excitability was found in the diencephalon.The rapidity and the electivity of this action, as well as the blood pH variations simultaneously registered, suggest a specific inhibition of cerebral carbonic anhydrase by Diamox, although a participation of the metabolic acidosis cannot be fully excluded.     

On reversible changes in the steady state ion-conductance of the Ranvier node membrane under low frequency sound fields.

The influence of sound waves on the stationary membrane conductivity of the axon membrane in myelinated nerve was investigated. Sound fields with frequencies within the auditory limit shift the transition voltage Vtr and correspondingly the threshold voltage of the action potential in polarisation direction. The results indicate that these changes of the electrical membrane behavior are due to a direct mechanical interaction between low frequency sound fields and membrane structure.     

Immobilization of chicken liver fructose 1,6-bisphosphatase on CNBr-activated Sepharose

Summary Chicken liver fructose 1,6-bisphosphatase is readily immobilized on CNBr-activated Sepharose. The immobilization alters some enzymatic properties. They include broader pH activity curve, loss of activation by K⁺ or NH ₄ ⁺ , increased resistance to inactivation by trypsin, decreased sensitivity to AMP inhibition, and loss of cooperative interaction among AMP-binding sites. The immobilized enzyme retains about 38% or 19% of the specific activity of the native enzyme when the activity is measured in the absence or presence of K⁺, resepctively.This work was supported by grant RR-8006 from the General Research Branch, Division of Research Resources, NIH (USA).     

Effect of benzimidazole on nicotinamide adenine dinucleotide phosphate phosphomonosterase activity in wheat leaves

Summary Nicotinamide adenine dinucleotide phosphate phosphomonoesterase was isolated and partially purified from wheat (Triticum aestivum L. var. Selkirk) leaves. The enzyme hadK _NADP value of 1.4×10^–4 M and a pH optimum of 5.9.In vitro activity of this enzyme was unaffected by precursors of NAD (nicotinamide and nicotinic acid) or cytokinis (kinetin and benzimidazole). However, when detached wheat leaves were treated with solutions of these compounds, the precursors lowered the specific activity while the cytokinins enhanced the activity. It is suggested that spatial separation and compartmentation of the enzyme and its substrate NADP account for the similar effect of benzimidazole on both.This work was supported by a grant No. A2698 from the National Research Council, Canada.     

On reversible changes in the steady state ion-conductance of the Ranvier node membrane under low frequency sound fields

Summary The influence of sound waves on the stationary membrane conductivity of the axon membrane in myelinated nerve was investigated. Sound fields with frequencies within the auditory limit shift the transition voltage V_tr and correspondingly the threshold voltage of the action potential in polarisation direction. The results indicate that these changes of the electrical membrane behavior are due to a direct mechanical interaction between low frequency sound fields and membrane structure.