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D Combes

Publications and source records attributed to D Combes.

At least 19 recordsLinked to original sources
PubMed2004

Developmental segregation of spinal networks driving axial- and hindlimb-based locomotion in metamorphosing Xenopus laevis.

Amphibian metamorphosis includes a complete reorganization of an organism's locomotory system from axial-based swimming in larvae to limbed propulsion in the young adult. At critical stages during this behavioural switch, larval and adult motor systems operate in the same animal, commensurate with a gradual and dynamic reconfiguration of spinal locomotor circuitry. To study this plasticity, we have developed isolated preparations of the spinal cord and brainstem from pre- to post-metamorphic stages of the amphibian Xenopus laevis, in which spinal motor output patterns expressed spontaneously or in the presence of NMDA correlate with locomotor behaviour in the freely swimming animal. Extracellular ventral root recordings along the spinal cord of pre-metamorphic tadpoles revealed motor output corresponding to larval axial swimming, whereas postmetamorphic animals expressed motor patterns appropriate for bilaterally synchronous hindlimb flexion-extension kicks. However, in vitro recordings from metamorphic climax stages, with the tail and the limbs both functional, revealed two distinct motor patterns that could occur either independently or simultaneously, albeit at very different frequencies. Activity at 0.5-1 Hz in lumbar ventral roots corresponded to bipedal extension-flexion cycles, while the second, faster pattern (2-5 Hz) recorded from tail ventral roots corresponded to larval-like swimming. These data indicate that at intermediate stages during metamorphosis separate networks, one responsible for segmentally organized axial locomotion and another for more localized appendicular rhythm generation, coexist in the spinal cord and remain functional after isolation in vitro. These preparations now afford the opportunity to explore the cellular basis of locomotor network plasticity and reconfiguration necessary for behavioural changes during development.

Animals↗
PubMed2002

Kinetic studies and mathematical model for sucrose conversion by Aspergillus niger fructosyl-transferase under high hydrostatic pressure.

The effect of pressure on the kinetically controlled synthesis reaction catalyzed from sucrose by Aspergillus niger fructosyl-transferase was investigated at pH 5.5 and 40 degrees C. The overall reaction was split up into five main reactions that were studied under pressure in initial rate conditions with various substrate concentrations in the absence or in the presence of glucose 50 g/l. A global reaction model was worked out according to the mathematical expression of the initial rates as the products of a polynomial rational function of substrate concentration and a corrective term introducing pressure. Experimental data from sugar concentrations were correctly described by the model during the course of the reaction under pressure. Raising the pressure induced a decrease in fructo-oligosaccharides yield by inhibiting the main transfer reaction without affecting sucrose hydrolysis.

Journal Article↗
PubMed2001

Acetylcholinesterase genes in the nematode Caenorhabditis elegans.

Acetylcholinesterase (AChE, EC 3.1.1.7) is responsible for the termination of cholinergic nerve transmission. It is the target of organophosphates and carbamates, two types of chemical pesticides being used extensively in agriculture and veterinary medicine against insects and nematodes. Whereas there is usually one single gene encoding AChE in insects, nematodes are one of the rare phyla where multiple ace genes have been unambiguously identified. We have taken advantage of the nematode Caenorhabditis elegans model to identify the four genes encoding AChE in this species. Two genes, ace-1 and ace-2, encode two major AChEs with different pharmacological properties and tissue repartition: ace-1 is expressed in muscle cells and a few neurons, whereas ace-2 is mainly expressed in motoneurons. ace-3 represents a minor proportion of the total AChE activity and is expressed only in a few cells, but it is able to sustain double null mutants ace-1; ace-2. It is resistant to usual cholinesterase inhibitors. ace-4 was transcribed but the corresponding enzyme was not detected in vivo.

Acetylcholinesterase↗
PubMed2000

Isolation of a type 2 metallothionein-like gene preferentially expressed in the tapetum in Zea mays.

A Zea mays cDNA, MZm3-4, was isolated by differential screening of a cDNA library obtained from meiotic stage anthers against a cDNA of 3-week-old seedlings. Northern blot analysis of RNA from different maize tissues and from male reproductive organs at various developmental stages demonstrated expression of a single transcript in anthers, from the pollen mother cell stage through the uninucleated microspore stage. In situ hybridization to anther sections resulted in a distinct signal only in the tapetum. The MZm3-4 cDNA is 743 nucleotides in length and has an open reading frame encoding a protein of 75 amino acids. Sequence comparisons with various databases revealed that MZm3-4 exhibits high similarities with type 2 plant metallothioneins at both the nucleotide and the amino-acid level. Primer extension analysis indicated that MZm3-4 cDNA is deleted of 13bp at the 5' end. Southern blot analysis showed that the MZm3-4 gene may be present in one or two copies in a Z. mays inbred line genome. This is the first report of the isolation of a type 2 metallothionein-like protein in maize. Moreover, the expression of this type 2 metallothionein-like gene is high in the male reproductive organs engaged in microsporogenesis.

Amino Acid Sequence↗
PubMed2000

Characterization of MZm3-3, a Zea mays tapetum-specific transcript.

A cDNA, MZm3-3, was isolated by differential screening of a cDNA library from Zea mays meiotic stage anthers against cDNA of 3-week-old seedlings. Characterization of this cDNA indicated that the MZm3-3 gene is expressed specifically during male gametogenesis. Its expression is highly and preferentially detected in the tapetum, from the pollen mother cell to uninucleated microspore stages. It encodes a short alkaline protein of 10.6 kDa, with a conserved pattern of eight cysteine residues. Sequence analysis showed that these features are shared with lipid transfer proteins and some male-flower-specific proteins. The presence of a putative signal peptide indicates that MZm3-3 enters into the secretory pathway to then be released into the anther loculus. Based on these features, the secretory activity of the tapetum and the temporal expression pattern of MZm3-3, a contribution to pollen coat formation is suggested. Southern blot analyses demonstrated the presence of closely related genes, indicating that MZm3-3 belongs to a multigene family.

Journal Article↗
PubMed2000

Four genes encode acetylcholinesterases in the nematodes Caenorhabditis elegans and Caenorhabditis briggsae. cDNA sequences, genomic structures, mutations and in vivo expression.

We report the full coding sequences and the genomic organization of the four genes encoding acetylcholinesterase (AChE) in Caenorhabditis elegans and Caenorhabditis briggsae, in relation to the properties of the encoded enzymes. ace-1 and ace-2, located on chromosome X and I, respectively, encode two AChEs (ACE-1 and ACE-2) that present 35% identity. The C-terminal end of ACE-1 is homologous to the C terminus of T subunits of vertebrate AChEs. ACE-1 oligomerizes into amphiphilic tetramers. ACE-2 has a hydrophobic C terminus of H type. It associates into glycolipid-anchored dimers. In C. elegans and C. briggsae, ace-3 and ace-4 are organized in tandem on chromosome II, with only 356 nt and 369 nt, respectively, between the stop codon of ace-4 (upstream gene) and the ATG of ace-3. ace-3 produces only 5 % of the total AChE activity. It encodes an H subunit that associates into dimers of glycolipid-anchored catalytic subunits, which are highly resistant to the usual AChE inhibitors, and which hydrolyze butyrylthiocholine faster than acetylthiocholine. ACE-4 is closer to ACE-3 (54 % identity) than to ACE-1 or ACE-2. The usual sequence FGESAG surrounding the active serine residue in cholinesterases is changed to FGQSAG in ace-4. ACE-4 was not detected by our current biochemical methods, although the gene is transcribed in vivo. However the level of ace-4 mRNAs is far lower than those of ace-1, ace-2 and ace-3. The ace-2, ace-3 and ace-4 transcripts were found to be trans-spliced by both SL1 and SL2, although these genes are not included in typical operons. The molecular bases of null mutations g72 (ace-2), p1304 and dc2 (ace-3) have been identified.

Acetylcholinesterase↗
PubMed1999

Structure and promoter activity of the 5' flanking region of ace-1, the gene encoding acetylcholinesterase of class A in Caenorhabditis elegans.

We report the structure and the functional activity of the promoter region of ace-1, the gene encoding acetylcholinesterase of class A in the nematode Caenorhabditis elegans. We found that ace-1 was trans -spliced to the SL1 spliced leader and that transcription was initiated at a cluster of multiple starts. There was neither a TATA nor a CAAT box at consensus distances from these starts. Interspecies sequence comparison of the 5' regions of ace-1 in C. elegans and in the related nematode Caenorhabditis briggsae identified four blocks of conserved sequences located within a sequence of 2.4 kilobases upstream from the initiator ATG. In vitro expression of CAT reporter genes in mammalian cells allowed the determination of a minimal promoter in the first 288 nucleotides. In phenotype rescue experiments in vivo, the ace-1 gene containing 2.4 kilobases of 5' flanking region of either C. elegans or C. briggsae was found to restore a coordinated mobility to the uncoordinated double mutants ace-1(-);ace-2(-)of C. elegans. This showed that the ace-1 promoter was contained in 2.4 kilobases of the 5' region, and indicated that cis -regulatory elements as well as coding sequences of ace-1 were functionally conserved between the two nematode species. The pattern of ace-1 expression was established through microinjection of Green Fluorescent Protein reporter gene constructs and showed a major mesodermal expression. Deletion analysis showed that two of the four blocks of conserved sequences act as tissue-specific activators. The distal block is a mesodermal enhancer responsible for the expression in body wall muscle cells, anal sphincter and vulval muscle cells. Another block of conserved sequence directs expression in pharyngeal muscle cells pm5 and three pairs of cephalic sensory neurons.

5' Untranslated Regions↗
PubMed1999

Motor pattern specification by dual descending pathways to a lobster rhythm-generating network.

In the European lobster Homarus gammarus, rhythmic masticatory movements of the three foregut gastric mill teeth are generated by antagonistic sets of striated muscles that are driven by a neural network in the stomatogastric ganglion. In vitro, this circuit can spontaneously generate a single (type I) motor program, unlike in vivo in which gastric mill patterns with different phase relationships are found. By using paired intrasomatic recordings, all elements of the gastric mill network, which consists mainly of motoneurons, have been identified and their synaptic relationships established. The gastric mill circuit of Homarus is similar to that of other decapod crustaceans, although some differences in neuron number and synaptic connectivity were found. Moreover, specific members of the lobster network receive input from two identified interneurons, one excitatory and one inhibitory, that project from each rostral commissural ganglion. Integration of input from these projection elements is mediated by synaptic interactions within the gastric mill network itself. In arrhythmic preparations, direct phasic stimulation of the previously identified commissural gastric (CG) interneuron evokes gastric mill output similar to the type I pattern spontaneously expressed in vitro and in vivo. The newly identified gastric inhibitor interneuron makes inhibitory synapses onto a different subset of gastric mill neurons and, when activated with the CG neuron, drives gastric mill output similar to the type II pattern that is only observed in the intact animal. Thus, two distinct phenotypes of gastric mill network activity can be specified by the concerted actions of parallel input pathways and synaptic connectivity within a target central pattern generator.

Animals↗
PubMed1999

Dynamic restructuring of a rhythmic motor program by a single mechanoreceptor neuron in lobster.

We have explored the synaptic and cellular mechanisms by which a single primary mechanosensory neuron, the anterior gastric receptor (AGR), reconfigures motor output of the gastric mill central pattern generator (CPG) in the stomatogastric nervous system (STNS) of the lobster Homarus gammarus. AGR is activated in vivo by contraction of the medial tooth protractor muscle gm1 and accesses the gastric CPG via excitation of two in-parallel interneurons, the excitatory commissural gastric (CG) and the inhibitory gastric inhibitor (GI). In the spontaneously active STNS in vitro, weak firing of AGR in time with gastric mill motoneurons (GM) reinforces an ongoing type I gastric mill rhythm in which all gastric teeth power-stroke motoneurons are synchronously active. With strong AGR firing, these phase relationships switch abruptly to a type II pattern in which lateral and medial teeth power-stroke motoneurons fire in antiphase. Our results suggest that these bimodal actions on the gastric mill rhythm depend on the balance of firing of the CG and GI interneurons and that selection of the pathway resides in their different postsynaptic sensitivities to AGR. Whereas high intrinsic firing rates of the CG neuron ensure that the excitatory pathway predominates during low levels of sensory input, strong synaptic facilitation in the GI neuron favors the inhibitory pathway during high levels of receptor activity. Feedback from a single mechanosensory neuron is thus able, in an activity-dependent manner, to specify different motor programs from a single central pattern-generating network.

Animals↗
PubMed1998

Influence of polyols on the structural properties of Kluyveromyces lactis beta-galactosidase under high hydrostatic pressure.

The conformational changes in dimeric Kluyveromyces lactis beta-galactosidase induced by hydrostatic pressure were investigated by means of its intrinsic tryptophan fluorescence. At high pressure, the fluorescence emission spectrum was shifted to the red, indicating the exposure of buried Trp residues to the aqueous solvent. This spectral change was paralleled by a loss of enzyme activity. The shift of the emission spectrum was quantified by evaluating the centre of spectral mass ((nu(g))), which is an intensity-weighted mean wavenumber. The experimental data could be fitted to a two-state transition (native<-->denatured), corrected for a linear pressure dependence of (nu(g)), and allowed the determination of thermodynamic parameters deltaG0(app), V(app) and P(1/2). The results were consistent with a partial unfolding of the protein and not simply with dissociation of this dimeric enzyme. In the presence of polyols, the native conformation of beta-galactosidase was considerably more resistant to pressure. This protective effect of polyols is probably due to a reduced accessibility of water inside the protein structure, through the direct or indirect action of these additives on the enzyme.

Animals↗
PubMed1998

Irreversible high pressure inactivation of beta-galactosidase from Kluyveromyces lactis: comparison with thermal inactivation.

High hydrostatic pressure and high temperature are both shown to induce inactivation of Kluyveromyces lactis beta-galactosidase in deionised water and their respective effects are compared. These two physical parameters lead to similar inactivation kinetics which can be suitably represented by series-type models. The plot of half-lives as a function of pressure is close to the same plot towards temperature. Thus, the same inactivation rate constant can be obtained in two different ways: an increase in pressure at room temperature or an increase in temperature at atmospheric pressure (e.g. 125 MPa at 25 degrees C or 45 degrees C at 0.1 MPa for a kappa 1 value about 28 x 10(-2) min -1). When beta-galactosidase was prepared in 0.1 M potassium phosphate buffer pH 7.3, its stability in extreme conditions of pressure as at high temperature was strongly enhanced. This stabilizing effect of the buffer was essentially attributed to a pH-effect by comparison with the behaviour of the enzyme in a similar buffer but with a 10-fold lower ionic strength.

Biotechnology↗
PubMed1998

Existence of four acetylcholinesterase genes in the nematodes Caenorhabditis elegans and Caenorhabditis briggsae.

Three genes, ace-1, ace-2 and ace-3, respectively located on chromosomes X, I and II, were reported to encode acetylcholinesterases (AChEs) of classes A, B and C in the nematode Caenorhabditis elegans. We have previously cloned and sequenced ace-1 in the two related species C. elegans and C. briggsae. We report here partial sequences of ace-2 (encoding class B) and of two other ace sequences located in close proximity on chromosome II in C. elegans and C. briggsae. These two sequences are provisionally named ace-x and ace-y, because it is not possible at the moment to establish which of these two genes corresponds to ace-3. Ace-x and ace-y are transcribed in vivo as shown by RT-PCR and they are likely to be included in a single operon.

Acetylcholinesterase↗
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↗