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Biomedical subjects

F Ramón

Publications and source records attributed to F Ramón.

At least 19 recordsLinked to original sources
PubMed2000

Effect of hydrogen peroxide on d-amino acid oxidase from Rhodotorula gracilis.

D-amino acid oxidase from Rhodotorula gracilis is a FAD-containing enzyme that belongs to the oxidase class that is characterized by the ability of the reduced flavin to react quickly with oxygen, yielding hydrogen peroxide and the oxidized cofactor. Hydrogen peroxide, necessary for the production of glutaryl-7-ACA from cephalosporin C had a deleterious effect on the enzyme. H(2)O(2) induced the oxidation of tryptophan and cysteine residues of the protein that could be involved in the dimerization process, required for the attainment of a fully competent enzyme. H(2)O(2) had also a kinetic effect on the reaction catalyzed by D-amino acid oxidase. It was a pure noncompetitive inhibitor; the corresponding inhibition constants were K(is) = 0.52 mM and K(ii) = 0.70 mM.

Journal Article↗
PubMed1999

Enhanced production of penicillin V acylase from Streptomyces lavendulae.

A 28 degrees C, Streptomyces lavendulae produced high levels of penicillin V acylase (178 IU/l of culture) when grown on skim milk as the sole nutrient source for 275 h. The enzyme showed catabolite repression by glucose and was produced in the stationary phase of growth. Penicillin V was a good inducer of penicillin V acylase formation, while phenoxyacetic acid, the side-chain moiety of penicillin V, did not alter enzyme production significantly. The enzyme was stable between pH 6 and 11 and at temperatures from 20 degrees C to 55 degrees C. This extracellular enzyme was able to hydrolyse natural penicillins and unable to hydrolyse penicillin G.

Penicillin Amidase↗
PubMed1999

Evoked potentials elicited by natural stimuli in the brain of unanesthetized crayfish.

Experiments were conducted to test some characteristics of vision by crayfish underwater and in air, and determine possible motion reactions elicited in response to naturalistic or quasi-ethological visual stimuli. Chronically implanted electrodes on the brain were used to record visually evoked potentials in response to moving bars at different speeds or to fish of different sizes. Electroretinograms were also recorded to detect when an object or a shadow appeared in the crayfish visual field. Ongoing brain activity is mild under basal conditions, but increases in RMS by approximately 6% in response to bar passage and 12 to 53% in response to fish motionless or swimming in front of the crayfish. When crayfish are free to move, fish swimming in front of them elicit intense brain activity, together with displacement toward them and an attempt to grab them. Visual evoked potentials are elicited by moving objects as small as 1 degree at a distance of 30 cm in air as well as underwater. None of the stimuli used induced evident behavioral responses under our conditions. We conclude that vision-action activities can be divided into (a) vision of irrelevant objects with short lasting electrical activity and no motion in response to it; (b) vision of mildly interesting objects with long-lasting electrical effects, but no motion in response to it; and (c) vision of relevant objects with appropriate motion reaction.

Air↗
PubMed1998

Chemical mechanism of D-amino acid oxidase from Rhodotorula gracilis: pH dependence of kinetic parameters.

The variation of kinetic parameters of d-amino acid oxidase from Rhodotorula gracilis with pH was used to gain information about the chemical mechanism of the oxidation of D-amino acids catalysed by this flavoenzyme. d-Alanine was the substrate used. The pH dependence of Vmax and Vmax/Km for alanine as substrate showed that a group with a pK value of 6.26-7.95 (pK1) must be unprotonated and a group with a pK of 10.8-9.90 (pK2) must be protonated for activity. The lower pK value corresponded to a group on the enzyme involved in catalysis and whose protonation state was not important for binding. The higher pK value was assumed to be the amino group of the substrate. Profiles of pKi for D-aspartate as competitive inhibitor showed that binding is prevented when a group on the enzyme with a pK value of 8.4 becomes unprotonated; this basic group was not detected in Vmax/Km profiles suggesting its involvement in binding of the beta-carboxylic group of the inhibitor.

Aspartic Acid↗
PubMed1995

Chemical modification of histidyl residues in D-amino acid oxidase from Rhodotorula gracilis.

D-Amino acid oxidase was inactivated by DEP at 30 degrees C and pH 7.5. The reaction followed pseudo-first-order kinetics with a second-order rate constant of 0.254 mM-1.min-1. The pH dependence of the inactivation showed the involvement of a group with a pK of 6.6. The presence of substrate or benzoate protected the enzyme against inactivation. Difference spectra at 242 nm and the reversal of the inactivation in the presence of 1 M hydroxylamine or 0.1 M NaOH pointed to the modification of histidine residues. The statistical analysis of the residual fractional activity versus the number of modified histidine residues led to the conclusion that one histidine residue is essential for the enzyme activity.

Animals↗
PubMed1994

Kinetic study of the aggregation and lipid mixing produced by alpha-sarcin on phosphatidylglycerol and phosphatidylserine vesicles: stopped-flow light scattering and fluorescence energy transfer measurements.

alpha-Sarcin is a fungal cytotoxic protein that inactivates the eukaryotic ribosomes. A kinetic study of the aggregation and lipid mixing promoted by this protein on phosphatidylglycerol (PG) and phosphatidylserine (PS) vesicles has been performed. Egg yolk PG, bovine brain PS, dimyristoyl-PG (DMPG) and dimyristoyl-PS (DMPS) vesicles have been considered. The initial rates of the vesicle aggregation induced by the protein have been measured by stopped-flow 90 degrees light scattering. The formation of a vesicle dimer as the initial step of this process was deduced from the second-order dependence of the initial rates on phospholipid concentration. The highest alpha-sarcin concentration studied did not inhibit the vesicle aggregation, indicating that many protein molecules are involved in the vesicle cross-linking. These are common characteristics of the initial steps of the aggregation produced by alpha-sarcin in the four types of phospholipid vesicles considered. However, the kinetics of the scattering values revealed that more complex changes occurred in the later steps of the aggregation process of egg PG and brain PS vesicles than in those of their synthetic counterparts. alpha-Sarcin produced lipid mixing in vesicles composed of DMPG or DMPS, which was measured by fluorescence resonance energy transfer assays. A delay in the onset of the process, dependent on the protein concentration, was observed. Measurement of the rates of lipid mixing revealed that the process is first order on phospholipid concentration. Egg PG and brain PS vesicles did not show lipid mixing, although they avidly aggregated. However, alpha-sarcin was able to promote lipid mixing in heterogeneous systems composed of egg PG+DMPG or brain PS+DMPS vesicles. The dilution of the fluorescence probes was faster when these were incorporated into the bilayers made of synthetic phospholipids than were present in those made of natural phospholipids. The bilayer destabilization produced by the protein in the vesices composed of the dimyristoyl-phospholipids should be transmitted to the more stable ones made of natural phospholipids. The obtained results are interpreted in terms of lipid mixing occurring within vesicle aggregates larger than dimer.

Animals↗
PubMed1993

Phenylalanine determined in plasma with use of phenylalanine dehydrogenase and a centrifugal analyzer.

Quantitative determination of plasma phenylalanine (Phe) is essential for the diagnosis of phenylketonuria (PKU) and the control of dietary therapy of PKU patients. We have adapted a spectrophotometric method (Clin Chim Acta 1991;201:95-8) based on phenylalanine dehydrogenase (EC 1.4.1.-) for rapid and accurate determinations of Phe with the Cobas Fara II centrifugal analyzer. The method is based on the NAD(H)-dependent reductive deamination of Phe with large amounts of phenylalanine dehydrogenase, which catalyzes the formation of phenylpyruvate at pH 10.8. Combining the specificity of the phenylalanine dehydrogenase with the precision, accuracy, and considerable time-saving of an automated system is useful for monitoring PKU patients in a children's hospital.

Amino Acid Oxidoreductases↗
PubMed1991

Humoral factors reduce gap junction sensitivity to cytoplasmic pH. I. Organ ablation studies.

The sensitivity of gap junctions connecting crayfish lateral axons to uncoupling by axoplasmic acidification was studied after altering the hormonal balance of animals by 1) ablation of eyestalks or sinus glands or 2) inducing long-lasting defensive posturing behavior (stress). Internal pH (pHi) was measured with microelectrodes, and junctional resistance (Rj) was calculated from input and transfer resistances. In isolated nerve cords from intact animals, the maximal Rj (Rjmax) reached after acidification varied diurnally (Rjmax approximately 10 and 0.6 M omega at 0900 and 1800 h, respectively). Basal Rj (20-30 k omega) did not change during the 24-h period. Organ ablation (eyestalks or sinus glands) or stress rendered gap junctions less sensitive to uncoupling by low pHi within 1 h or 2 days; recovery toward control values had different time courses. The reduced pH sensitivity of crayfish junctions seen after eyestalk ablation is attributable to stress in its early phase (lasting 1-2 days) and to ablation of the endocrine organs in its late phase (2-7 days). No striking structural differences accompanied these changes, indicating that the altered properties are not due to major changes in gap junction expression.

Animals↗
PubMed1991

Humoral factors reduce gap junction sensitivity to cytoplasmic pH. II. In vitro manipulations.

Our previous studies demonstrated a diurnal rhythm in the response of gap junctions between crayfish giant axons to acidification and that the response was reduced after eyestalk ablation, sinus gland removal, or visual stress. In this paper we describe experiments to test whether compounds in the circulating hemolymph were responsible for modulation of the responsiveness gap junction channels to intracellular pH. In axons from destalked animals in which the hemolymph had been replaced with normal saline, the maximal junctional resistance after acidification (Rjmax) reached control values. In contrast, Rjmax reached only 30% of control after acidification in axons from animals that had been destalked but not perfused. Hemolymph drawn after eyestalk ablation was tested on axons from control animals. Treatment with hemolymph drawn 1 day after destalking resulted in control Rjmax values, while treatment with hemolymph drawn 7 days after destalking resulted in Rjmax values of only 5-40%. Similarly, pretreatment for 1 h with 100 microM ecdysterone resulted in low Rjmax values. These experimental results suggest that a circulating compound, most likely ecdysterone or a related molecule, regulates the physiological properties of gap junctions from crayfish lateral axons.

Animals↗
PubMed1990

Protein phosphorylation and hydrogen ions modulate calcium-induced closure of gap junction channels.

The regulation of the cell-to-cell pathway formed by gap junctions seems to involve the interaction of the junctional channels with either calcium or hydrogen ions, as well as protein phosphorylation and calmodulin. These mechanisms of junctional regulation have been considered to act independently on specific sites of the gap junction protein; however, the possibility that they may be interrelated has not been adequately explored mainly due to the difficulties involved in simultaneous measurement of intracellular cations and protein phosphorylation. To further understanding of mechanisms regulating gap junctions, we have internally perfused coupled lateral axons from crayfish with solutions containing different calcium and hydrogen concentrations under conditions favoring phosphorylation, while monitoring the junctional conductance. We found that calcium ions regulate cell communication probably through a direct interaction with the channel protein. Phosphorylation and low pH do not alter junctional conductance themselves, but appear only to modulate the effects of calcium, possibly by altering the affinity of the channel for calcium. We propose that a combination of free intracellular calcium and protein phosphorylation form an important physiological mechanism regulating intercellular communication.

Animals↗
PubMed1988

Calmodulin acts as an intermediary for the effects of calcium on gap junctions from crayfish lateral axons.

Lateral axons from the abdominal nerve cord of crayfish were internally perfused with the calcium receptor calmodulin (CaM) in solutions with low (pCa greater than 7.0) or high (pCa 5.5) calcium concentrations and studied electrophysiologically and morphologically. Results from these experiments show that when the internal solution contains calcium-activated calmodulin (Ca2+-CaM) the junctional resistance between the axons increases from control values of about 60 to 500-600 k omega in 60 min. In contrast, axons perfused with calmodulin in low calcium solutions maintain their junctional resistance at control levels during the 60-min perfusion. Similar results are obtained when only one or both coupled axons are perfused. The morphological study shows that in the perfused axons the axoplasmic organelles are replaced or grossly perturbed by the perfusion solution up to the region of the synapses. Additionally, in axons perfused with Ca2+-CaM there are regions where the synaptic gap between the membranes decreases from a control 4-6 to 2-3 nm. Both electrophysiological and morphological results can be interpreted as indicating that calcium-activated calmodulin acts directly on the junctional channels to induce their closure.

Animals↗
PubMed1988

Structural characteristics of gap junctions. I. Channel number in coupled and uncoupled conditions.

Gap junctions between crayfish lateral axons were studied by combining anatomical and electrophysiological measurements to determine structural changes associated during uncoupling by axoplasmic acidification. In basal conditions, the junctional resistance, Rj, was approximately 60-80 k omega and the synapses appeared as two adhering membranes; 18-20-nm overall thickness, containing transverse densities (channels) spanning both membranes and the narrow extracellular gap (4-6 nm). In freeze-fracture replicas, the synapses contained greater than 3 X 10(3) gap junction plaques having a total of approximately 3.5 X 10(5) intramembrane particles. "Single" gap junction particles represented approximately 10% of the total number of gap junction particles present in the synapse. Therefore, in basal conditions, most of the gap junction particles were organized in plaques. Moreover, correlations of the total number of gap junction particles with Rj suggested that most of the junctional particles in plaques corresponded to conducting channels. Upon acidification of the axoplasm to pH 6.7-6.8, the junctional resistance increased to approximately 300 k omega and action potentials failed to propagate across the septum. Morphological measurements showed that the total number of gap junction particles in plaques decreased approximately 11-fold to 3.1 X 10(4) whereas the number of single particles dispersed in the axolemmae increased significantly. Thin sections of these synapses showed that the width of the extracellular gap increased from 4-6 nm in basal conditions to 10-20 nm under conditions where axoplasmic pH was 6.7-6.8. These observations suggest that single gap junction particles dispersed in the synapse most likely represent hemi-channels produced by the dissasembly of channels previously arranged in plaques.

Animals↗
PubMed1986

Lowering of pH does not directly affect the junctional resistance of crayfish lateral axons.

The effect of pH was tested on the junction between crayfish lateral axons. By means of a glass capillary inserted into one of the axons, one side of the junction was perfused with solutions of known pH while the junctional resistance, Rj, was monitored. Integrity of the gap junction was checked electron microscopically. Rj remained unchanged when the pH of the perfusate was lowered from 7.1 to 6.0. However, when the pH of the unperfused side of the junction was lowered by substituting acetate for chloride in the external solution, Rj rose, attesting to the integrity of the junction and its capacity to uncouple in the perfused state. We suggest that H+ does not affect the junctional channels directly, but acts through an intermediary which is inactivated or removed by the perfusion.

Animals↗
PubMed1981

Electrotonic coupling in internally perfused crayfish segmented axons.

We have developed a technique for cannulation and internal perfusion of crayfish segmented lateral axons. Experiments on perfused and non-perfused axons lead to the following conclusions: 1. Internally perfused segmented axons behave very similarly to non-perfused axons. 2. The axial electrical resistance of the junctional region is almost as low as a comparable segment of axon. 3. Neither intracellular Ca2+ nor H+ is effective in disrupting the intercellular communication pathway in perfused axons. On the basis of these findings we have formulated a hypothesis for cellular control of intercellular coupling based on the existence of a soluble intermediate for Ca2+ or H+-induced uncoupling. This hypothesis is consistent with data from both internally perfused and non-perfused axons.

Animals↗
PubMed1980

Interaction of anaesthetics with electrical synapses.

Studies of the interaction of anaesthetics with various preparations, from whole animals to organic solvents, have been continuing since Overton and Meyer found a correlation between anaesthetic potency and solubility in olive oil. Although the physiological basis of anaesthesia is far from clear, one popular hypothesis is that anaesthetics act primarily by interfering with the normal functioning of chemical synapses. This hypothesis is supported by experiments showing that these synapses are more sensitive to both local and general anaesthetics than are axons. The effects of anaesthetics on electrical synapses (gap-junctions or nexus) have not previously been studied. These ubiquitous structures, presumably responsible for cell-to-cell communication, are found in most vertebrate and invertebrate tissues. We report here the effects of several anaesthetics on electronic coupling between nerve cells, and show that electrical synapses are less sensitive to most anaesthetics than are chemical synapses and axonal membranes.

Alcohols↗