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Effects of bath-applied L-glutamate and related chemicals on the afferent synapse of the Plotosus electroreceptor.

Tonic electroreceptors of the marine catfish Plotosus were isolated, and effects of chemicals applied in the bath were examined in terms of firing rate (F) responses in single unit afferent nerve. L-Glutamate (L-Glu) and agonists caused marked F increase in the spontaneous discharge. Their potencies, estimated from concentrations for 50% of max F increase, were in the order of quisqualate (2 microM), kainate (7 microM), L-Glu (0.4 mM), L-homocysteate (0.4 mM), D-Glu (3 mM) and L-aspartate (L-Asp, greater than 10 mM). N-Methyl-D,L-aspartate (10 mM) had no effect. L-Glu induced F increase also in the receptors fully suppressed either by cathodal pulses or by high Mg (15 mM), which indicated the postsynaptic action. The synaptic responses were often affected differently in the fast and slow phases, here termed as the peak F and the adapted F, respectively. L-Asp potentiated only the adapted F. Kynurenic acid (Kyn) suppressed only the adapted F, but incompletely and rather dose-independently. Kyn, however, competitively antagonized the amino acid-induced responses. The present results suggest the presence of two distinct postsynaptic receptors, one a Kyn-sensitive Glu receptor that is responsible for part of the adapted F, and the other still undetermined that is responsible for most of the synaptic responses.

Action Potentials↗

Long-lasting physiological effects of bath applied N-methyl-D-aspartate.

The present experiments describe a long-lasting form of potentiation induced in field CA1 of rat hippocampal slices by bath application of N-methyl-D-aspartate (NMDA), in association with low magnesium concentrations, glycine and spermine. The potentiation effect consisted of a 50% increase in slope of field potentials and was stable for at least 80 min post treatment. It was not accompanied by detectable changes in antidromic responses and was completely blocked by an antagonist of NMDA receptor. The possible relationship of pharmacologically induced potentiation to long-term potentiation (LTP) is discussed.

2-Amino-5-phosphonovalerate↗

[Rapid gas chromatographic method for determination of acetate in human plasma and hemodialysis baths (author's transl)].

A rapid and sensitive method for acetate determination in human plasma and in hemodialysis baths (dialysates) is going to become necessary owing to the extensive using of sodium acetate solution in hemodialysis. The gas chromatographic method reported here allows, in about 35 minutes, the precise and reproducible measurement of acetate concentrations ranging from 0.2 to 20 mmol/1. This method can be used to investigate the kinetics of acetate concentration variations in the blood of patients undergoing hemodialysis with sodium acetate solution and to evaluate the amount of acetate absorbed during this treatment.

Acetates↗

The effects of bathing sodium ions upon the intracellular sodium activity in calcium-free media and the calcium paradox of isolated ferret ventricular muscle.

Exposure of fine trabeculae isolated from the ferret right ventricle to media depleted of Ca2+, with a rapid perfusion system, causes a depolarization of the membrane and a rise in aiNa. This rise in aiNa is sigmoidally dependent upon the bathing free [Ca2+], is antagonized by reduction of the [Na]o, raised [Mg]o and PN200-110 but augmented by removal of Mg2+ or inhibition of the Na-pump. On repletion of Ca2+, the strength of the resulting contracture depends upon the transmembrane. Na-gradient at the moment of Ca-repletion. The effects of reduced [Na]o, during Ca-depletion, depend upon the cation used with replacement by Li much greater than choline greater than tetramethylammonium. These are determined by the change in the transmembrane gradient for Na+ and any additional action the replacing ion has upon the Ca-channels, the Na-pump and the Na/Ca exchange. The effects described are consistent with the hypothesis that the intensity of the calcium paradox in mammalian ventricle is primarily determined by the entry of Na+ through L-type Ca-channels during the period of Ca-depletion and the Ca-loading on Ca-repletion due to the effect of the raised aiNa upon the Na/Ca exchange.

Acetylcholine↗

Crystallization and preliminary X-ray analysis of the methane monooxygenase hydroxylase protein from Methylococcus capsulatus (Bath).

Methane monooxygenase is a multicomponent enzyme system that catalyzes the conversion of methane to methanol in methanotrophic bacteria. Catalysis occurs at non-heme dinuclear iron centers contained in the hydroxylase component of the system, a dimer of composition alpha 2 beta 2 gamma 2. The hydroxylase protein from Methylococcus capsulatus (Bath) has been crystallized from aqueous solutions containing polyethylene glycol, lithium sulfate, and ammonium acetate. The crystals are orthorhombic, space group P2(1)2(1)2(1), with one dimer of relative molecular mass M(r) = 252,000 in the asymmetric unit. The unit cell dimensions are a = 62.6 A, b = 110.1 A, c = 333.5 A. The crystals diffract uniformly beyond 2.5 A resolution. Crystals of the related hydroxylase from Methylosinus trichosporium OB3b have also been obtained.

Crystallization↗

A comparison of the bond strengths of microwave- and water bath-cured denture material.

A comparison was made of the shear bond strengths between microwave-polymerized and conventional water bath-cured polymethyl methacrylate (PMMA) bonded to denture teeth. Four groups of 15 specimens were prepared and shear bond strengths obtained by use of standardized laboratory testing methods. The results showed that microwave-cured PMMA demonstrated significantly (p 0.0001) higher bond strengths than conventional PMMA, and priming of the denture tooth surface with monomer liquid yielded significantly higher bond strengths than other surface treatment (p 0.0001).

Analysis of Variance↗

Effects of bath-applied GABA on the firing pattern of cells in an in vitro preparation of mammalian cerebral cortex.

The patterns of spontaneous firing recorded from 25 cells in slice preparations of rat cerebral cortex were characterised using inter-spike interval plots (ISI). Histograms of the ISI showed two peaks having modal values of 20 and 82ms, corresponding to episodes of high frequency bursts of action potentials, and a lower frequency, steady pattern of activity. Bath-applied GABA was shown to reversibly block the bursting activity, but had little effect on the lower frequency component. This effect of GABA could be abolished by the simultaneous application of bicuculline.

Action Potentials↗

An attempt to localize the site of action of different agents within cholinergic motor neurones of the myenteric plexus-longitudinal muscle preparation of the guinea pig ileum by the triple bath method.

The site of action of cholinergic, adrenergic, peptidergic and opioid agents was studied in myenteric plexus-longitudinal muscle strips from the guinea pig ileum. A preparation in a special triple bath was drawn through two rubber membranes, dividing the strip into three segments. Neurogenic stimulation of the oral segment, set up nerve action potentials also in the neurones projecting axons up to the aboral segment. These axons, turning into varicose nerve terminals, conducted action potentials aborally across the middle segment, that was up to 10 mm wide. Finally, the nerve terminals, extending into the aboral segment, might be also invaded triggering twitches. Agents were added, either to the oral segment, to affect the genesis and spread of action potentials in the proximal parts of cholinergic neurones (cell bodies, axon hillocks, initial segments and axon preterminals) or they were added to the middle segment to affect propagation of action potentials in varicose nerve terminals. As a result, the amplitude of aboral twitches reflected their effects at each site, quantitatively. Noradrenaline and ethylketocyclazocine were more effective at the site of varicose nerve terminals, whereas substance P, acetylcholine and oxotremorine were more effective at the proximal parts; pilocarpine and nicotine were effective at both sites. Changes in membrane polarization might be the final common effect in the mechanism of action of all the stimulatory agents used.

Action Potentials↗

The role of copper in the pMMO of Methylococcus capsulatus bath: a structural vs. catalytic function.

Methanotrophs convert methane to methanol by the methane monooxygenase (MMO). It is well known that two forms of the MMO can be expressed: one form is found in the cytoplasm, or in the soluble fraction (sMMO); the other is associated with the membranes, or particulate fraction (pMMO). The sMMO has been extensively examined, and much is known about its structure and mechanism of dioxygen activation. The pMMO, however, is less well understood: the enzyme has proven difficult to purify as it loses activity once the membranes become solubilized. Furthermore, although copper is known to be important for the stability and activity of the pMMO, its role is still unclear. In a recent study, we reported the use of electron paramagnetic resonance (EPR) spectroscopy to probe the nature of the copper ions in these membranes. Two EPR signals were uncovered for the highly oxidized membranes: one set of signals arises from the type 2 Cu(II) centers, and the other has been assigned to trinuclear copper (Cu(II) clusters (H.-H. Nguyen, et al., J. Biol. Chem., 269, 14995 (1994). Here, we attempt to correlate the EPR spectra of the membrane fraction of Methylococcus capsulatus Bath with the amount of copper present in the membranes and to the activity of the pMMO as measured by the production of propylene oxide from propene by the pMMO. From these studies, we conclude that the primary role of copper is in the active site of the pMMO rather than simply a structural one.

Binding Sites↗

Presence of antigen sensitized leukocytes in carp (Cyprinus carpio L) following bath immunization against Flexibacter columnaris.

Bath immunization of carp (Cyprinus carpio L) resulted in protection of fish at natural challenge. Stimulation of leukocytes derived from thymus, spleen, anterior kidney and mid-kidney of fish immunized with Flexibacter columnaris bacterin revealed the presence of antigen sensitized cells in all lymphoid tissues except the anterior kidney. After 28 days a response was obtained in thymus and spleen leukocyte cultures.

Animals↗

Activation of the hydroxylase of sMMO from Methylococcus capsulatus (Bath) by hydrogen peroxide.

Hydrogen peroxide can activate the non-heme binuclear iron-containing hydroxylase of soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath) in the catalysis of oxidation of methane and other sMMO substrates. The reductase, protein B, O2 and NADH are normally required for catalytic activity, but can be replaced by H2O2 serving as the source of both oxygen and electrons for the reaction. Similar results have been observed in a different strain of MMO from Methylosinus trichosporium OB3b (Andersson, K.K., Froland, W.A., Lee, S.-K. and Lipscomb, J.D. (1991) New J. Chem. 15, 410-415). The Km,app for H2O2 was found to be 66 mM. Labelled oxygen experiments show that the oxygen atom in the product in the peroxide driven system is derived from H2O2 and not O2. Using C2-C5 alkanes and 2-butene as substrates it was shown that the product distribution differed in the complete sMMO and H2O2-driven systems, indicating that more than one pathway is available to the enzyme. Protein B, which is required for catalytic activity in the complete system, was found to be an inhibitor of the hydroxylase/H2O2 system. It was also observed that protein B not only affected the activity, but also the selectivity of carbon hydroxylation with 2-methylbutane as substrate.

Dose-Response Relationship, Drug↗

Acidification of the bathing fluid by the skin of Rana pipiens in metabolic acidosis.

The skin of Rana pipiens can be shown to excrete H+ in an in vitro preparation. This H+ excretion is increased by placing the frog in metabolic acidosis. In addition, H+ excretion is increased by the presence of HCO-3-CO2 on the serosal or inside surface of the skin. Removal of Na+ from the outside bathing solution of the skin has no apparent effect on H+ excretion. Ouabain inhibits H+ excretion by the skin of acidotic frogs almost completely, in the absence of exogenous CO2. In the presence of 5% CO2 ouabain inhibits H+ excretion by 50%. In the acidotic frog skin the H+ excretion was reduced by abolishing the spontaneous potential difference. While in the normal skin there was no effect. When the P.d. was clamped at -10 to -100 mV there was no effect on H+ excretion, while there was a slight depression of H+ excretion when the P.d. was clamped at +10 to +100 mV (outside to inside the skin). In the presence of 5% CO2 there was a marked depression of H+ excretion when clamped at -10 to -100 mV in the normal skin. In metabolic acidosis there was a marked stimulation when clamped at -10 to -100 mV.

Acidosis↗

Changes in the time course of miniature endplate currents induced by bath-applied acetylcholine.

Bath application of 0.5 and 2 microM acetylcholine (ACh) slowed the decay phase of miniature endplate currents (MEPC) recorded in isolated, voltage-clamped and prostigmine-treated frog sartorius muscle. Washout of ACh led to a decrease of the decay time constant of the MEPC to 72 +/- 5% (n = 5) and 51 +/- 3% (n = 6) of initial values, respectively, followed by very slow and incomplete recovery. MEPC amplitude changed slightly and recovered relatively fast. This discrepancy in the recovery rates is suggested to be due to a 'trapping' ability of desensitized receptors which can compete with the free receptors for ACh molecules and prevent repetitive binding. Thus the high affinity of desensitized receptors to ACh may partially compensate the absence of acetylcholinesterase activity.

Acetylcholine↗

Twitch responses dependent on external calcium ions in the mouse diaphragm in a potassium-free bathing solution.

We examined the effect of external Ca2+ on twitches of mouse diaphragm under the inhibition of Na(+)-K(+)-ATPase in vitro. The muscle was directly stimulated in presence of d-tubocurarine (dTc). K+ removal potentiated the amplitude of twitches with a transient prior reduction. This potentiation depended on external Ca2+. Membrane potentials decreased after removal of both external K+ and Ca2+ and were restored after an addition of Ca2+. The removal of both ions increased the content of Na+ in tissues and decreased K+. These changes were restored to the levels in the K(+)-free bathing solution by the addition of Ca2+. These results imply that Na+/Ca2+ exchange can support twitch contraction under the inhibition of Na(+)-K(+)-ATPase activity.

Animals↗

Cloning of nitrogenase structural genes from the obligate methanotroph Methylococcus capsulatus (Bath).

Southern hybridization techniques were used to examine the DNA homologies between the three nitrogenase structural genes nifH, nifD and nifK of Klebsiella pneumoniae and DNA from the obligate methane oxidizing bacterium Methylococcus capsulatus (Bath). The high degree of homology between methanotroph DNA sequences and the Klebsiella nifH and nifD genes was used to isolate and clone the corresponding Methylococcus nifH and nifD genes. Subsequent restriction analysis revealed that all three nif structural genes were contiguous in the Methylococcus genome in the order nifH, nifD and nifK, as found for the majority of other diazotrophs.

Cloning, Molecular↗

The methane monooxygenase gene cluster of Methylococcus capsulatus (Bath).

Methane is oxidised to methanol in methanotrophic bacteria by the enzyme methane monooxygenase (MMO). Methylococcus capsulatus (Bath) produces a soluble MMO which oxidises a range of aliphatic and aromatic compounds with potential for commercial exploitation. This multicomponent enzyme has been extensively characterised and biochemical data have been used to identify a 12-kb fragment of Methylococcus DNA carrying the structural genes mmoY and mmoZ, coding for the beta- and gamma-subunits of MMO component A, the methane-binding protein. We now report the complete nucleotide (nt) sequence of mmoX, the gene encoding the alpha-subunit of component A which is found to be 5' to mmoY and mmoZ. We also report the complete nt sequence of mmoC which encodes component C, the iron-sulfur flavoprotein of MMO, the N terminus of which is significantly homologous with spinach ferredoxin. The mmo structural genes are clustered within a 7-kb region and are closely linked to two small open reading frames of unknown function.

Amino Acid Sequence↗

Bathing-induced seizures.

Two unusual patients with sensory-induced seizures are presented. The clinical approach and evolution of seizures induced by bathing in patients with and without accompanying epilepsy are discussed.

Electroencephalography↗

Toluene diisocyanate-stimulated release of arachidonic acid metabolites in the organ bath from guinea-pig airways.

This study was designed to evaluate whether metabolites of arachidonic acid play a role in the contractile response to toluene diisocyanate in isolated guinea pig airways. In control experiments we collected the supernatant from an organ bath over a time period of 2 h, after the addition of toluene diisocyanate (100 and 300 microM), and after the addition of toluene diisocyanate (300 microM) in the presence of indomethacin (5 microM). We measured prostaglandin E2, 6-keto-prostaglandin F1 alpha, prostaglandin F2 alpha, thromboxane B2, leukotriene B4, leukotriene C4/D4/E4/F4 by radioimmunoassays. Levels of prostaglandin F2 alpha and 6-keto-prostaglandin F1 alpha increased significantly after addition of toluene diisocyanate in the absence of indomethacin. These results suggest that prostaglandins are involved in toluene diisocyanate-induced contractions in guinea-pig airways.

Animals↗