Opposing modulatory effects of D1- and D2-like receptor activation on a spinal central pattern generator.

The role of dopamine in regulating spinal cord function is receiving increasing attention, but its actions on spinal motor networks responsible for rhythmic behaviors remain poorly understood. Here, we have explored the modulatory influence of dopamine on locomotory central pattern generator (CPG) circuitry in the spinal cord of premetamorphic Xenopus laevis tadpoles. Bath application of exogenous dopamine to isolated brain stem-spinal cords exerted divergent dose-dependent effects on spontaneous episodic patterns of locomotory-related activity recorded extracellularly from spinal ventral roots. At low concentration (2 µM), dopamine reduced the occurrence of bursts and fictive swim episodes and increased episode cycle periods. In contrast, at high concentration (50 µM) dopamine reversed its actions on fictive swimming, now increasing both burst and swim episode occurrences while reducing episode periods. The low-dopamine effects were mimicked by the D2-like receptor agonists bromocriptine and quinpirole, whereas the D1-like receptor agonist SKF 38393 reproduced the effects of high dopamine. Furthermore, the motor response to the D1-like antagonist SCH 23390 resembled that to the D2 agonists, whereas the D2-like antagonist raclopride mimicked the effects of the D1 agonist. Together, these findings indicate that dopamine plays an important role in modulating spinal locomotor activity. Moreover, the transmitter's opposing influences on the same target CPG are likely to be accomplished by a specific, concentration-dependent recruitment of independent D2- and D1-like receptor signaling pathways that differentially mediate inhibitory and excitatory actions.

Influence of subtilisin on the adhesion of a marine bacterium which produces mainly proteins as extracellular polymers.

AIMS: The nature of exopolymers involved in the adhesion of a marine biofilm-forming bacterium Pseudoalteromonas sp. D41 was investigated to evaluate and understand the antifouling potential of subtilisin. METHODS AND RESULTS: The exopolymers of D41 produced by fermentation were analysed by FTIR and SDS-PAGE showing the presence of polysaccharides, glycoproteins and proteins. A high content of proteins was detected both in soluble and capsular fractions. The microscopic observations of fluorescamine and calcofluor stained adhered D41 indicated mainly the presence of proteins in exopolymers produced during adhesion. Subtilisin, the broad spectrum protease, tested in natural sea water and in polystyrene microplates showed that antifouling activity was higher in the prevention of bacterial adhesion than in the detachment of adhered D41 cells. CONCLUSIONS: Overall, these results demonstrate the involvement of proteins in Pseudoalteromonas sp. D41 adhesion and confirm the high antifouling potential of subtilisin. SIGNIFICANCE AND IMPACT OF THE STUDY: This study emphasizes the major role of proteins instead of polysaccharides, thus extending our knowledge regarding the nature of extracellular polymers involved in bacterial adhesion. Furthermore, the high antifouling potential of subtilisin evaluated in the very first stages of fouling, bacterial adhesion, could lead to less toxic compounds than organometallic compounds in antifouling paint.

Effects of commercial enzymes on the adhesion of a marine biofilm-forming bacterium.

The antifouling potential of commercial hydrolases, four proteases, seven glycosidases and one lipase was evaluated on the adhesion of marine Pseudoalteromonas sp. D41. The experimental method, adapted to screen antifouling agents, was based on bacterial adhesion in natural sterile sea water in a microtiter plate and on total biomass quantification by the fluorescent dye DAPI (4[prime]6-diamidino-2-phenylindole). Savinase (subtilisin) was the most effective hydrolase in both the prevention of bacterial adhesion and the removal of adhered bacteria. However, some enzymatic preparations tested such as Amano protease were not only ineffective but also increased the number of adhered bacterial cells. Enumeration using epifluorescence microscopy of CTC (5-cyano-2,3-ditolyl tetrazolium chloride) and DAPI stained adhered D41 cells confirmed these observations. Overall, these results demonstrated that hydrolases could either prevent adhesion and remove adhered bacterial cells effectively, or conversely increase bacterial adhesion, depending on enzymatic concentrations and the type of enzymes tested.

A marine bacterial adhesion microplate test using the DAPI fluorescent dye: a new method to screen antifouling agents.

AIMS: To develop a method to screen antifouling agents against marine bacterial adhesion as a sensitive, rapid and quantitative microplate fluorescent test. METHODS AND RESULTS: Our experimental method is based on a natural biofilm formed by mono-incubation of the marine bacterium Pseudoalteromonas sp. D41 in sterile natural sea water in a 96-well polystyrene microplate. The 4'6-diamidino-2-phenylindole dye was used to quantify adhered bacteria in each well. The total measured fluorescence in the wells was correlated with the amount of bacteria showing a detection limit of one bacterium per 5 microm(2) and quantifying 2 x 10(7) to 2 x 10(8) bacteria adhered per cm(2). The antifouling properties of three commercial surface-active agents and chlorine were tested by this method in the prevention of adhesion and also in the detachment of already adhered bacteria. The marine bacterial adhesion inhibition rate depending on the agent concentration showed a sigmoid shaped dose-response curve. CONCLUSIONS: This test is well adapted for a rapid and quantitative first screening of antifouling agents directly in seawater in the early steps of marine biofilm formation. SIGNIFICANCE AND IMPACT OF THE STUDY: In contrast to the usual screenings of antifouling products which detect a bactericidal activity, this test is more appropriate to screen antifouling agents for bacterial adhesion removal or bacterial adhesion inhibition activities. This screening test focuses on the antifouling properties of the products, especially the initial steps of marine biofilm formation.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Four acetylcholinesterase genes in the nematode Caenorhabditis elegans.

Whereas a single gene encodes acetylcholinesterase (AChE) in vertebrates and most insect species, four distinct genes have been cloned and characterized in the nematode Caenorhabditis elegans. We found that ace-1 (mapped to chromosome X) is prominently expressed in muscle cells whereas ace-2 (located on chromosome I) is mainly expressed in neurons. Ace-x and ace-y genes are located in close proximity on chromosome II where they are separated by only a few hundred base pairs. The role of these two genes is still unknown.

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.

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.

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 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.

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.

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.