A reassessment of the intervention of calmodulin in the regulation of stomatal movement

Commelina cammunis L., a monocotyledonous plant whose stomata are highly sensitive to calcium ions, was used to study calmodulin (CaM) involvement in stomatal movements. CaM was detected and quantified in guard cell and mesophyll cell protoplasts by western blot and by ⁴⁵Ca²⁺-overlays. CaM was found to be 3- to 7-fold more abundant on a per protein basis in guard cell than in mesophyll cell protoplasts. Numerous guard cell proteins that bind CaM in a Ca²⁺-dependent manner were detected by gold-labelled CaM overlays. Using bioassays with epidermal strips, different CaM-antagonists were found to induce a net stimulation of stomatal opening in darkness or under illumination (trifluoperazine > compound 48/80 ∼ fluphenazine > W7 > W5). As CaM is frequently involved in the regulation of phosphorylation processes, the effects of different inhibitors of protein kinases on stomatal movements were studied. In red plus blue light, a promotion of the stomatal aperture was observed in the nanomolar range with K252a and KT5926 and in the micromolar range with KT5720 ≫ ML7 ∼ ML9 ≫ H7 > KN62. Only the inhibitors with a high specificity for Ca²⁺-CaM dependent protein kinases (K252a, KT5926, ML7, ML9) triggered a stomatal opening in darkness and increased stomatal aperture in red plus blue light. Taken together, these data strongly suggest that a Ca²⁺- or a Ca²⁺-CaM-dependent protein kinase plays a central role in the calcium transduction pathway leading to the maintaining of stomatal closure.     

Some prerequisites for a study of the evolution of cognition in the animal kingdom

A distinction is made between two definitions of animal cognition: the one most frequently employed in cognitive sciences considers cognition as extracting and processing information; a more phenomenologically inspired model considers it as attributing to a form of the outside world a significance, linked to the state of the animal. The respective fields of validity of these two models are discussed along with the limitations they entail, and the questions they pose to evolutionary biologists are emphasized. This is followed by a presentation of a general overview of what might be the study of the evolution of knowledge in animals.     

Application of the multiple antigenic peptides (MAP) strategy to the production of prophormone convertases antibodies: Synthesis,characterization and use of 8-branched immunogenic peptides

Antiserum against an N-terminal sequence of murine prohormone convertase-1 (mPC1) incorporating the sequence immediatley following the junction between the putative pro-region and the active enzyme was obtained. This was accomplished using the multiple antigenic peptide (MAP) approach whereupon an 8-branched polylysine core to which are grafted multiple copies of a 16 amino acid peptide representing the N-terminal sequence of mPC1 (positions 84–99) was synthesized by solid-phase Fmoc chemistry. The ensuing peptide was purified and fully characterized by RP-HPLC, ¹H-NMR, amino acid composition, peptide sequencing and ion-spray mass spectrometry. The immunological properties of the resulting antibodies in detecting recombinant PC1 in both crude and purified preparations were compared with antibodies raised against a similar N-terminal segment of PC1 but using the conventioanl method of peptide–carrier protein conjugation and also developed against a C-terminal fusion protein of PC1. Our data indicate that the MAP antibody was as efficient as both the amino and carboxy-terminal antibodies in qualitative as well as quantitative analysis of PC1 encoded protein by radioimmunoassay. Following an identical approach, antibodies against other prohormone convertases like furin, PC5/6 and PACE4 were also developed and subsequently applied to a number of biochemical and immunological studies. In each case, the ease of preparation and high immunogenicity of the MAP approach were confirmed and reside in the simplicity and rapidity with which a potent and useful antiserum is obtained.     

Chemical synthesis of a 7 kDa insect gonadotropic neurohormone

An original insect neurohormone of 65 residues was synthesized by the solid-phase methodology using t-Boc strategy and Boc-Val-PAM–resin. The purification, conducted by several steps of liquid chromatography having mass, polarity or charge as separative criteria, yielded the product with the correct molecular weight of 6922 Da determined by mass spectrometry. The synthetic peptide had both the same affinity for the antinative neurohormone serum and the same biological activity as the native neurohormone.     

Monogènes Dactylogyridae parasites de Cyprinidae du genreBarbus d'Afrique du Nord

Resumé Quinze nouvelles espèces de Monogènes Dactylogyridae sont décrites chez quinze espèces deBarbus (Teleostei, Cyprinidae) appartenant aux sous-genresB. (Barbus) etB. (Labeobarbus) en Afrique du Nord. Les barbeaux examinés proviennent des différents bassins hydrographiques du Maroc et d'une localité nommée Hamman Bourgiba en Tunisie. Dans cette dernière région, le genreBarbus n'est représenté que par une seule espèce:Barbus (B.) callensis. Au Maroc, on en dénombre actuellement quatorze dont quatre appartiennent au sous-genreLabeobarbus: il s'agit deBarbus (L.) fritschii; B. (L.) harteti; B. (L.) paytonii etB. (L.) reinii. Les dix espèces appartenant au sous-genreBarbus sont:Barbus (B.) figuiensis; B. (B.) ksibi; B. (B.) lepineyi; B. (B.) magniatlantis; B. (B.) massaensis; B. (B.) moulouyensis; B. (B.) nasus; B. (B.) pallaryi; B. (B.) setivemensis etB. (B.) issenensis.Chaque sous-genre possède son propre pool parasitaire, à l'exception deDactylogyrus marocanus n. sp., recontré sur des espèces appartenant aux deux sous-genres (B. (L.) fritschii, B. (L.) paytonii, B. (L.) harteti, B. (L.) reinii, B. (L.) nasus, B. (B.) setivimensis, B. (B.) ksibi). Sur les cinqDactylogyrus parasitant lesLabeobarbus, trois présentent une spécificité stricte vis à vis de leur hôte. Il s'agit deDactylogyrus reinii n. sp. surB. (L.) reinii; D. volutus n. sp. etD. zatensis n. sp. surB. (L.) fritschii. Les espècesD. oumiensis n. sp. etD. kulindrii n. sp. présentent une spécifité stenoxène et parasitent respectivementB. (L.) harteti, B. (L.) paytonii, B. (L.) reinii etB. (L.) fritschii, B. (L.) reinii.Nous avons recontré neufDactylogyrus chez les poisson-hôtes appartenent au sous-genreBarbus. Six d'entre eux ont une spécificité oïoxène; ce sont:D. guirensis n. sp.,D. atlasensis n. sp. etD. draaensis n. sp. surB. (B.) pallaryi; D. borjensis n. sp. surB. (B.) nasus etD. heteromorphus n. sp. etD. tunisiensis n. sp. surB. (B.) callensis. Les trois autres parasites ont un spectre d'hôtes plus large. Il s'agit deD. ksibii n. sp. recontré chezB. (B.) ksibi, B. (B.) setivimensis etB. (B.) magniatlantis; D. ksibioïdes n. sp. recontré chezB. (B.) setivimensis etB. (B.) moulouyensis. L'espèceD. fimbriphallus n. sp. stenoxène, se recontre chez les poisson-hôtes du versant Sud de l'Atlas et de la façade méditerranéenne à savoir:B. (B.) figuiensis, B. (B.) lepineyi, B. (B.) massaensis, B. (B.) moulouyensis, B. (B.) pallaryi etB. (B.) issenensis.Le rôle des Dactylogyridae en tant que marqueurs biogéographiques, phylogénétiques et taxonomiques est discuté à partir de la composition spécifique des communautés de Monogènes rencontrés et de leurs différents types morphologiques.

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Fifteen new species of the Dactylogyridae (Monogenea) parasitic on fifteen species of barbels (Barbus) from North Africa (Teleostei, Cyprinidae) are described. The fishes studied belong to two subgenera,B. (Labeobarbus) andB. (Barbus), collected from various hydrographical basins of Morocco and from the Hamman Bourgiba locality in Tunisia. In the latter area, the genusBarbus is represented by onlyBarbus (Barbus) callensis. In Morocco, fourteen species are listed, four of which belong to the subgenusLabeobarbus; these areBarbus (L.) fritschii; B. (L.) harteti; B. (L.) paytonii andB. (L.) reinii. The other ten species belong to the subgenusBarbus: these areBarbus (B.) figuiensis; B. (B.) ksibi; B. (B.) lepineyi; B. (B.) magniatlantis; B. (B.) massaensis; B. (B.) moulouyensis; B. (B.) nasus; B. (B.) pallaryi; B. (B.) setivimensis andB. (B.) issenensis. Each of the two subgenera has its unique parasitic fauna, except forDactylogyrus marocanus n. sp. collected on species belonging to both subgenera (B. (L.) fritschii, B. (L.) paytonii, B. (L.) harteti, B. (L.) reinii, B. (B.) nasus, B. (B.) setivimensis andB. (B.) ksibi). Of the five monogeneans found onLabeobarbus, three appear to be specific to one host: they areDactylogyrus reinii n. sp. onB. (L.) reinii, andD. volutus n. sp. andD. zatensis n. sp. onB. (L.) fritschii. D. kulindrii n. sp. parasitisedB. (L.) reinii andB. (L.) fritschii; andD. oumiensis n. sp. occurred onB. (L.) reinii, B. (L.) paytonii andB. (L.) harteti. NineDactylogyrus species were found in fishes belonging to the subgenusBarbus. Six of them have an oïoxenous specificity: these areD. guirensis n. sp.,D. atlasensis n. sp. andD. draaensis n. sp. onB. (B.) pallaryi; D. borjensis n. sp. onB. (B.) nasus andD. heteromorphus n. sp. andD. tunisiensis n. sp. on(B.) callensis. These other three have a wider range of hosts: they areD. ksibii n. sp. collected fromB. (B.) ksibi, B. (B.) setivimensis andB. (B.) magniatlantis, andD. ksibioïdes n. sp. found onB. (B.) setivimensis andB. (B.) moulouyensis. D. fimbriphallus n. sp. is a characteristic parasite of fishes from the southern side of the Atlas mountains and the Mediterranean coast (B. (B.) figuiensis, B. (B.) lepineyi, B. (B.) massaensis, B. (B.) moulouyensis, B. (B.) pallaryi andB. (B.) issenensis).The role of dactylogyrids as biogeographical phylogenetic and taxonomic indicators is discussed in relation to the specific structure of the monogenean communities and the different morphological types found.

     

Effect of ischemia/reperfusion sequence on cytosolic iron status and its release in the coronary effluent in isolated rat hearts

The hypothesis that oxygen-derived free radicals play an important role in myocardial ischemic and reperfusion injury has received a lot of support. In the presence of catalytic amounts of transition metals such as iron, superoxide anions, and hydrogen peroxide can be transformed into a highly reactive hydroxyl radical °OH (Haber-Weiss reaction). In view of this, we have undertaken this study to investigate whether iron is involved in the reperfusion syndrome and therefore could aggravate free radicals injury. Coronary effluent iron concentrations and cardiac cytosolic iron levels were evaluated in rat hearts subjected to an ischemia/reperfusion sequences. In the case of total ischemia, iron concentration in coronary effluents peaked immediately in the first sample collected upon reperfusion. However, in the case of partial ischemia, iron concentration in coronary effluents peaked rather exclusively during ischemia period. Cardiac cytosolic iron level augmented significantly after 30 min of total ischemia and non significantly in the other ischemia protocols compared to perfused control hearts. It also appears that the iron released is not protein-bound, and could therefore have a marked catalytic activity. The results of the present study suggest that in the oxygen paradox, iron plays an important role in inducing alterations during reoxygenation.     

Uptake,accumulation and metabolism of auxins in tobacco leaf protoplasts

Uptake and metabolism of exogenous naphthalene-1-acetic acid (NAA) and indole-3-acetic acid (IAA) have been studied in tobacco (Nicotiana tabacum L. cv. Xanthi) mesophyll protoplasts. Both auxins entered protoplasts by diffusion under the action of the transmembrane pH gradient without any detectable participation of an influx carrier. Molecules were accumulated by an anion-trapping mechanism and most of them were metabolized within hours, essentially as glucose-ester and amino-acid conjugates. Protoplasts were equipped with a functional auxin-efflux carrier as evidenced by the inhibitory effect of naphthylphtalamic acid on IAA efflux. Basically, similar mechanisms of NAA and IAA uptake occurred in protoplasts. However, the two auxins differed in their levels of accumulation, due to different membrane-transport characteristics, and the nature of the metabolites produced. This shows the need to estimate the accumulation and the metabolism of auxins when analyzing their effects in a given cell system. The internal auxin concentration could be modulated by changing the transmembrane pH gradient, giving an interesting perspective for discriminating between the effects of intra- and extracellular auxin on physiological processes.Abbreviations BA benzoic acid - C_i/C_e accumulation ratio of auxin - IAAasp N-[3-indolylacetyl]-dl-aspartic acid - NAA naphthalene-1-acetic acid - NAAasp N-[1-naphthylacetyl]-l-aspartic acid - NPA N-1-naphthylphthalamic acid The authors thank Dr. M. Caboche (I.N.R.A, Versailles, France) for his generous gifts of some amide derivatives of 1-NAA, Mr. P. Varennes and Dr. B. Das (I.C.S.N., C.N.R.S., Gif-sur-Yvette, France) for recording and interpreting the mass spectra of NAA glucose ester, and Prof. P. Manigault (Institut des Sciences Végétales, Gif-sur-Yvette) for microscopy measurements of protoplast dimensions. This work was supported by funds from the C.N.R.S, I.N.R.A, and E.E.C.