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D Combes

Publications and source records attributed to D Combes.

At least 19 recordsLinked to original sources
PubMed1998

Long-term expression of two interacting motor pattern-generating networks in the stomatogastric system of freely behaving lobster.

Rhythmic movements of the gastric mill and pyloric regions of the crustacean foregut are controlled by two stomatogastric neuronal networks that have been intensively studied in vitro. By using electromyographic recordings from the European lobster, Homarus gammarus, we have monitored simultaneously the motor activity of pyloric and gastric mill muscles for </=3 mo in intact and freely behaving animals. Both pyloric and gastric mill networks are almost continuously active in vivo regardless of the presence of food. In unfed resting animals kept under "natural-like" conditions, the pyloric network expresses the typical triphasic pattern seen in vitro but at considerably slower cycle periods (2. 5-3.5 s instead of 1-1.5 s). Gastric mill activity occurs at mean cycle periods of 20-50 s compared with 5-10 s in vitro but may suddenly stop for up to tens of minutes, then restart without any apparent behavioral reason. When conjointly active, the two networks express a strict coupling that involves certain but not all motor neurons of the pyloric network. The posterior pyloric constrictor muscles, innervated by a total of 8 pyloric (PY) motor neurons, are influenced by the onset of each gastric mill medial gastric/lateral gastric(MG/LG) neuron powerstroke burst, and for one cycle, PY neuron bursts may attain >300% of their mean duration. However, the duration of activity in the lateral pyloric constrictor muscle, innervated by the unique lateral pyloric (LP) motor neuron, remains unaffected by this perturbation. During this period after gastric perturbation, LP neuron and PY neurons thus express opposite burst-to-period relationships in that LP neuron burst duration is independent of the ongoing cycle period, whereas PY neuron burst duration changes with period length. In vitro the same type of gastro-pyloric interaction is observed, indicating that it is not dependent on sensory inputs. Moreover, this interaction is intrinsic to the stomatogastric ganglion itself because the relationship between the two networks persists after suppression of descending inputs to the ganglion. Intracellular recordings reveal that this gastro-pyloric interaction originates from the gastric MG and LG neurons of the gastric network, which inhibit the pyloric pacemaker ensemble. As a consequence, the pyloric PY neurons, which are inhibited by the pyloric dilator (PD) neurons of the pyloric pacemaker group, extend their activity during the time that PD neuron is held silent. Moreover, there is evidence for a pyloro-gastric interaction, apparently rectifying, from the pyloric pacemakers back to the gastric MG/LG neuron group.

Animals↗
PubMed1997

Influence of surface hydrophilic/hydrophobic balance on enzyme properties.

Two different enzyme surface modifications were carried out in order to alter the protein hydrophilic/hydrophobic balance in opposite directions and to observe the effects induced on enzyme properties. First, a novel chemoenzymatic glycosylation method was applied, which resulted in a higher enzyme surface hydrophilic character. Then, an amphiphilic polymer, PEG, was bound to the enzymes by chemical means, and it brought about an increase in the global hydrophobic character. Two different enzymes, alpha-chymotrypsin and Candida rugosa lipase, were studied, and in all cases, several degrees of modification were obtained. Then, the modified biocatalysts were thoroughly investigated, and the influence of the variation of surface hydrophilic/hydrophobic balance on hydrolytic activity, hydrolysis kinetic parameters, synthetic activity and thermal stability was assessed.

Animals↗
PubMed1997

Conditional dendritic oscillators in a lobster mechanoreceptor neurone.

1. Intra- and extracellular recordings were made from in vitro preparations of the lobster (Homarus gammarus) stomatogastric nervous system to study the nature and origin of pacemaker-like activity in a primary mechanoreceptor neurone, the anterior gastric receptor (AGR), whose two bilateral stretch-sensitive dendrites ramify in the tendon of powerstroke muscle GM1 of the gastric mill system. 2. Although the AGR is known to be autoactive, we report here that in 20% of our preparations, rather than autogenic tonic discharge, the receptor fired spontaneously in discrete bursts comprising three to ten action potentials and repeating at cycle frequencies of 0.5-2.5 Hz in the absence of mechanical stimulation. Intrasomatic recordings revealed that such rhythmic bursting was driven by slow oscillations in membrane potential, the frequency of which was voltage sensitive and dependent upon the level of stretch applied to the receptor terminals of the AGR. 3. Autoactive bursting of the AGR originated from an endogenous oscillatory mechanism in the sensory dendrites themselves, since (i) during both steady, repetitive firing and bursting, somatic and axonal impulses were always preceded 1:1 by dendritic action potentials, (ii) hyperpolarizing the AGR cell body to block triggering of axonal impulses revealed attenuated somatic spikes that continued to originate from the two peripheral dendrites, (iii) the timing of burst firing could be phase reset by brief electrical stimulation of either dendrite, and (iv) spontaneous bursting continued to be expressed by an AGR dendrite after physical isolation from the GM1 muscle and the stomatogastric nervous system. 4. Although a given AGR in vitro could switch spontaneously from dendritic bursting to tonic firing and vice versa, exogenous application of micromolar (or less) concentrations of the neuropeptide F1 (TNRNFLRFamide) to the dendritic membrane could rapidly and reversibly switch the receptor firing pattern from repetitive firing to the bursting mode. Exposure of the somatic and axonal membrane of the AGR to F1 was without effect, as were applications of other neuroactive substances such as serotonin, octopamine and proctolin. 5. We conclude that, as for many oscillatory neurones of the central nervous system, the intrinsic activity pattern of this peripheral sensory neurone may be dynamically conferred by extrinsic modulatory influences, presumably according to computational demands. Moreover, the ability of the AGR to behave as an endogenous burster imparts considerable integrative complexity since, in this activity mode, sensory coding not only occurs through the frequency modulation of on-going dendritic bursts but also via changes in the duration of individual bursts and their inherent spike frequencies.

Animals↗
PubMed1996

In vitro glycosylation of proteins: an enzymatic approach.

The glycosylation pathway is the most important post-translational modification of a protein and is moreover a highly specific process. The majority of proteins of pharmaceutical interest are glycoproteins. Therefore, it is necessary to identify the composition, the structure, the function and the biosynthesis of the glycoproteins. The present knowledge is described here. In addition, the performed studies about structure-function relationship of the glycoproteins have shown that the oligosaccharide part of a glycoprotein confers important and specific biological roles. Thus, the modification of the structure of the glycan chains can lead to a modification of the activity of the glycoprotein. This phenomenon is encountered at the time of the production of recombinant glycoprotein in a heterologous system. Indeed, the glycosylation profile of a protein is specific to both the host cell and the culture conditions of this cell. Thus, the advantages and the drawbacks of the different host cells used for the glycosylation engineering are presented. In this way, the identification of the different specific enzymes glycosyltransferases and glycosidases involved in the glycosylation pathway is now necessary to improve the production of recombinant glycoprotein. The structure and the characteristics of these enzymes, and more particularly the oligosaccharyltransferase and the galactosyltransferase, are also described.

Carbohydrate Conformation↗
PubMed1996

Is general practice audit alive and well? The view from Portsmouth.

A brief look at medical audit activity in the Portsmouth and South East Hampshire Health Authority area by use of a questionnaire mailed out to general practitioners, information from the medical audit advisory group database and the local health authority records. The details collected consisted of topics chosen, the stage of audit cycle reached, funding arrangements, practice personnel and the outside collaborators involved. Most general practices were found to be undertaking audit activity, and in some it had become integrated into the everyday routine.

England↗
PubMed1995

Effect of temperature and pressure on yeast invertase stability: a kinetic and conformational study.

Kinetics of the temperature- or pressure-induced denaturation of invertase from Saccharomyces cerevisiae were obtained in the temperature range 45-70 degrees C and in the pressure range 500-650 MPa. The investigation was done by measuring the residual activities after cooling or pressure release and the intrinsic fluorescence of aromatic amino-acids (tyrosine and tryptophan) upon excitation at 277 nm. The residual activity decreased exponentially as a function of time incubation according to a biphasic model either with pressure or temperature, whereas the fluorescence emission indicated a difference between these two parameters. When the enzyme was subjected to thermal treatment, the fluorescence of tyrosine and tryptophan decreased slowly, while after high-pressure treatment, these aromatic residues become more exposed to the aqueous solvent during unfolding, giving rise to a large decrease in fluorescence in the 330-340 nm region. Moreover, in the latter case, an enhancement of light scattering intensity showed changes in protein-protein interactions.

Biotechnology↗
PubMed1995

Structural and functional characterization of a muscle tendon proprioceptor in lobster.

A morphological and electrophysiological study was made on a unique primary mechanosensory neuron, the anterior gastric receptor (AGR), previously shown to arise from power-stroke muscle gm1 of the gastric mill system in the lobster foregut. Ultrastructural analysis of horseradish peroxidase injected AGR demonstrated that its peripheral dendrites do not ramify in muscle but are confined strictly to the connective tissue/epidermal interface in the tendon of gm1. These terminals are rich in mitochondria and at their very endings are free of glial cell wrapping, suggesting that they are the site at which mechano-transduction occurs. Extracellular axonal recordings from an in vitro neuromuscular preparation consisting of the gm1 muscle still attached to the stomatogastric nervous system, revealed that AGR is activated by passive stretch of gm1. The response to ramp stimuli displays dynamic and static components, both of which increase with the amplitude of applied stretch, while the dynamic component is also velocity sensitive. AGR is also activated by muscle contraction here elicited either by application of exogenous acetylcholine, the excitatory neurotransmitter for gm1, or by electrical stimulation of the motoneurons (GM) themselves. Consistent with a receptor lying in-series with its muscle, therefore, the effective stimulus of AGR in vivo is probably an increase in tension exerted on the tendon during active muscle contraction. In neuromuscular preparations including the bilateral commissural ganglia, stretching gm1 reflexly activates GM motoneurons at low stimulus strengths but leads to an inactivation of GM motoneurons at high stimulus strengths. This is consistent with earlier findings that both responses can be elicited by direct electrical stimulation of AGR. The functional implications of AGR's anatomical relationship with muscle gm1, the receptor's response properties, and its central effects on motor output to gm1 are discussed. Comparison is also drawn between this first reported example of a true tendon receptor in invertebrates and muscle receptors of vertebrates.

Animals↗
PubMed1995

A novel chemoenzymatic glycosylation strategy: application to lysozyme modification.

Hen egg lysozyme has been non-specifically glycosylated using a novel two-step strategy. First, a number of sucrose molecules have been chemically bound to the protein surface lysines, then the glycosidic chains have been enzymically lengthened, using a glycosyltransferase. For this task, a fructosyltransferase and a levansucrase have been tested, the latter appearing as the most effective one. In all cases, reactions have been optimised and several degrees of modification have been obtained. Finally, the effects of the modifications on lysozyme hydrophobicity, hydrolytic activity, hydrolysis substrate affinity and thermostability have been assessed.

Animals↗
PubMed1995

Bitter peptide from hemoglobin hydrolysate: isolation and characterization.

Two separation methods, ultrafiltration and 2-butanol extraction, have shown that a peptide is the major agent responsible for bitterness in peptic hemoglobin hydrolysates. It was easily purified from these complex mixtures by specific hydrophobic adsorption on Superose 12, a gel-filtration column, which could constitute an original and interesting method for bitterness detection. The bitter peptide which corresponded to VV-hemorphin 7, the fragment 32-40 of the beta chain of bovine hemoglobin, is first generated during proteolysis, then hydrolysed by pepsin. It exhibited a strong bitterness at 0.25 mM equivalent to 0.073 mM quinine sulfate or 21 mM caffeine.

Amino Acid Sequence↗
PubMed1994

Effect of polyols on alpha-chymotrypsin thermostability: a mechanistic analysis of the enzyme stabilization.

The influence of the synthetic substrate (N-acetyl-L-tyrosine ethyl ester) and the different polyols (ethylene glycol, glycerol, erythritol, xylitol and sorbitol) on the thermostability of alpha-chymotrypsin at 60 degrees C have been studied. The results obtained showed an important stabilizing effect in the presence of both additives. In order to describe the kinetics of enzyme stabilization, the experimental results were analyzed by a four-parameters deactivation model with excellent agreement. In all cases, alpha-chymotrypsin exhibited non-first-order deactivation kinetics, corresponding to a two-step unimolecular mechanism, where the main protective effect of polyols was observed in the first-step of the deactivation profile. Thus, the presence of polyols increased the level of activity stabilization (alpha 1), and decreased the first-order deactivation rate constant (k1). Additionally, the experimental results were analyzed as a function of both, the change in the standard free energy of denaturation (delta(delta Gzero)), and a protective effect, defined as the ratio of alpha-chymotrypsin half-lives (with and without polyols), showing in both cases a clear stabilizing effect of these polyhydroxylic cosolvents for the enzyme. The overall protective effect of polyols was also simultaneously related to their concentration and their water-activity depressing power.

Chymotrypsin↗
PubMed1994

Use of supercritical CO2 for bone delipidation.

Supercritical carbon dioxide was used for bone delipidation. It appeared that this technology is very efficient since supercritical CO2 is able to diffuse into microporous solids much better than liquids and that it has a good solvent capacity for lipids. This extraction is the ideal first step of any bone processing because microporosity of bone tissue becomes much more accessible, which may enhance osteoconduction once implanted. Moreover, it is safe since it involves no toxic chemical and is potentially usable with allografts as well as xenografts.

Animals↗
PubMed1993

Tetrodotoxin-sensitive dendritic spiking and control of axonal firing in a lobster mechanoreceptor neurone.

1. A primary mechanosensory neurone, the anterior gastric receptor (AGR) associated with gastric mill muscle in the lobster foregut was examined in vitro with extra- and intra-cellular recording techniques to understand processes of dendritic integration and dendro-axonal communication. 2. AGR has a 'T'-shaped geometry; its two long (> 3 mm) primary dendrites project distally to spatially separate, stretch sensitive terminals and converge centrally onto a common apical neurite that leads to a bipolar soma and single axon. 3. The receptor's bilateral dendrites are independently capable of generating action potentials. These appear to be Na+ dependent since they are blocked by tetrodotoxin, but not by Co2+ or a lack of Ca2+ in the bath saline. 4. Both dendrites are autogenically active, although impulses in the dendrite with the higher intrinsic excitability may cross over and activate the trigger zone on the contralateral side. Moreover, spikes arising on either dendrite do not actively invade the soma, but are conveyed as decremented potentials to a third trigger zone on the initial axon segment. 5. Focal applications of TTX (tetrodotoxin) demonstrated the existence and allowed precise definition of a central membrane compartment of AGR that appears to lack in functional Na+ channels. This inexcitable region includes the soma, the apical neurite and the central branch point of the two dendrites. A failure to observe collision block of bilateral dendritic potentials as they traverse the neurite supported this conclusion. 6. Horseradish peroxidase injections and staining revealed two morphological features of the apical neurite that differed markedly from other regions of the cell. In addition to a relatively large diameter, the neurite's plasma membrane is heavily convoluted and coiled to form a lamellar transverse profile. This latter feature may itself contribute to membrane inexcitability while the former is consistent with an elevated space constant for electrotonic conduction. 7. It is concluded that the inhomogeneous distribution of membrane excitability in AGR enhances the integrative capability of the receptor's dendrites, permitting mechanical input at diverse loci to be encoded and processed prior to transformation into axonal discharge.

Action Potentials↗
PubMed1991

alpha-Chymotrypsin in plastein synthesis: influence of substrate concentration on enzyme activity.

The role of alpha-chymotrypsin in the plastein reaction was studied using a peptic hydrolysate of albumin as substrate. Study of this reaction simultaneously by different methods showed that the plastein reaction is enzyme catalyzed and is highly dependent on environmental conditions. A gel permeation chromatography study of the plastein reaction showed simultaneous increases in the high- and low-molecular-weight oligopeptide fractions; a transpeptidation mechanism may be involved in the reaction. A study of the effect of substrate concentration on the plastein reaction catalyzed by alpha-chymotrypsin showed a profile with both hydrolytic and synthetic activities. This effect was also observed when the reaction course was followed by quantification of the free amino groups at different substrate concentrations, showing that a condensation mechanism is responsible for the synthetic activity when the substrate concentration is very high. These results have led us to conclude that the plastein reaction involves a transpeptidation and/or condensation mechanism, which is a function of the substrate concentration.

Albumins↗
PubMed1990

Effect of diffusional resistances on the action pattern of immobilized alpha-amylase.

Alpha-amylase from Aspergillus oryzae has been immobilized onto corn grits and porous silica (specific areas 180 and 440 m2 g-1). Kinetic parameters of immobilized enzyme have been determined. Immobilization of alpha-amylase results in the formation of less polymerized products resulting in an apparent decrease in the number of transglycosylation reactions, for both maltotetraose and starch as substrates, when compared with free enzyme. Diffusional limitations for substrate and products have been quantified in the case of the three supports used. External diffusional resistances were important in all cases for the reaction products, whilst they became negligible for the substrate in the case of silica supports. Moreover, internal transfer limitations were identified with silica 180 m2 g-1 support. It was demonstrated that diffusional resistances were in direct relation to the apparent modification of the enzyme action pattern after immobilization.

Aspergillus oryzae↗