Isolation of methotrexate-resistant cell lines in Petunia hybrida upon stepwise selection procedure

Summary Cell suspensions of Petunia hybrida were subjected to a selection procedure in which the concentration of the selective agent, methotrexate (MTX), was gradually elevated. In mammalian cells, this procedure frequently results in MTX-resistant mutants due to amplification of the gene coding for dihydrofolate reductase (DHFR), the target protein of MTX.Five suspension lines were isolated, with degrees of resistance ranging from 10 to 500 M MTX (in wild type the LD_99.9 is 0.2 M). MTX^R phenotypes were unstable, as manifested by the loss of resistance upon prolonged growth in the absence of drug. All of the mutants also exhibited high values of MTX-binding protein (60- to 400-fold higher than that of the wild type), which declined to intermediate values upon MTX withdrawal. Finally, cellular extracts from all of the mutants also showed high specific staining of DHFR-activity in gels.The results suggest that the resistance of MTX in these plant cell-lines is mediated by the elevation of the amounts of DHFR, probably as a consequence of gene amplification.     

Innervation du muscle rétracteur antérieur du byssus (ABRM) de Mytilus edulis L. et de Mytilus galloprovincialis Lmk.

Summary Specific and non specific cholinesterase activities were demonstrated in the ABRM of Mytilus edulis L. and Mytilus galloprovincialis L. by means of different techniques. The results were found identical for both species: neuromuscular junctions en grappe-type scarcely distributed within the ABRM, contain AChE.According to the histochemical inhibition tests, (a) the eserine inhibits AChE activity of the ABRM with a level of 5·10^–5 M or higher, (b) the ChE non specific activities are inhibited by iso-OMPA level between 5·10^–5 to 10^–4 M.The histo- and cytochemical observations were completed by showing the existence of neuromuscular junctions containing small clear vesicles: they probably are the morphological support for ACh presence.Moreover, specific and non specific ChE activities were localized in the glio-interstitial cells. AChE precipitates were developped along the ABRM sarcolemma, some muscle mitochondria and in the intercellular spaces remain enigmatic.

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L'auteur dédicace cette contribution à son père, Mr. Anthime Gilloteaux à l'occasion de son 75ème anniversaire     

Action du cordo-mésoderme dorsal sur la régionalisation et la différenciation du massif endodermique chezRana dalmatina Bon. (Amphibien Anoure)

Résumé Du cordo-mésoderme dorsal greffé sur la région ventrale d'un massif endodermique de même âge provoque sa différenciation locale (st. 17/neurula). Des néo-formations nombreuses et variées sont obtenues en faisant varier les caractéristiques du greffon (cordo-mésoderme) et du porte-greffe (endoderme). L'existence, la nature et le volume de ces néo-formations dépendent: de l'étroitesse et de la durée du contact greffon/porte-greffe, de l'âge du cordo-mésoderme et de l'âge de l'endoderme, des niveaux de contact, plus ou moins antérieurs, du greffon et du porte-greffe.Les résultats des greffes (st. 17) sont interprétés grâce à la construction de modèles théoriques. Ces modèles sont bâtis sur les quatre paramètres suivants: 1) l'existence de 2 facteurs morphogénétiques, l'un de nature endodermique et l'autre de nature cordo-mésodermique. Ces 2 facteurs sont responsables de l'apparition de néo-formations; 2) la localisation de ces 2 facteurs actifs dans les deux tiers (antérieur et moyen) de l'endoderme et du cordo-mésoderme; 3) la décroissance de l'activité des 2 facteurs selon l'axe antéro-postérieur de l'embryon; 4) le rôle différent de chacun de ces 2 facteurs. Le facteur endodermique déterminerait la nature et la taille des néo-formations; le facteur cordo-mésodermique jouerait un rôle stimulant.Une discussion est engagée sur la méthode, la démarche et l'intérêt de ce type d'interprétation des résultats.

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Effect of dorsal chordo-mesoderm on regionalisation and differentiation of the endodermal mass inRana dalmatina bon (Amphibia Anura)Elaboration of a theoretical model

Summary Chordo-mesoderm grafted onto the ventral area of yolk endoderm of the same age causes its local differentiation (Rana dalmatina: st. 17/neurula). Numerous and various neo-formations are achieved by variation of the grafted tissue (chordo-mesoderm) and host (endoderm) characteristics. The existence, the constitution and the volume of the neo-formations are dependant on: the tightness and the duration of the graft/host contact, the age of the chordo-mesoderm and the age of the endoderm, and the antero-posterior level of contact with grafted tissue and host.The results of the grafts (st. 17) are explained by the elaboration of a theoretical model. These models are elaborated according to four parameters: (1) the existence of two morphogenetic factors, one endodermal and the other mesodermal. These two factors are responsible for the constitution of neo-formations; (2) the localization of these two factors, active in the anterior and median thirds of the endoderm and the chorda-mesoderm; (3) decreasing activity of these two factors along to the antero-posterior axis of the embryo; (4) the different notes of each of these two factors. The endodermal factor might determine the constitution and the size of the neo-formations; the chordo-mesodermal factor might play a stimulatory role.The method, the procedure and the interpretation of this kind of results are discussed.

     

Isolated sheep heart hypothermic (5–13 °C) perfusion with fresh blood: Successful preservation for 24–72 hours with continuous strong ventricular activity

Isolated lamb hearts perfused with fresh whole blood at 10 and 13 °C in an ex vivo perfusion circuit continuously contracted at a rate of 15 to 20 times/min with a peak left ventricular systolic pressure (LVPSP) up to 70 mm Hg. These contractions persisted for the duration of the hypothermic study, up to three days with no change in vascular resistance. On rewarming to 38 °C, the hearts resumed regular and efficient contractions. Hearts perfused at 5 °C, however, exhibited no electrical or mechanical activity during hypothermic preservation and were uniformly poorly preserved.Quality of heart preservation was improved if, prior to final cooling, hearts were first rewarmed to 38 °C, followed by cooling. Change of the support animal, or interruption of flow of fresh blood into the perfusion circuit, resulted in cessation of ventricular contractions, ventricular fibrillation, and poor organ preservation.     

Histological and ultrastructural studies of Beauveria bassiana infection in Leptinotarsa decemlineta larvae during ecdysis

Studies of Leptinotarsa decemlineata larvae infected by Beauveria bassiana during ecdysis have enabled us to define the modes of fungal penetration employed to enter the ecdysial cuticle. We have observed the mechanically active passage of the penetrant hyphae and have followed the growth of the filaments and blastospore formation in the molting fluid. The attack of the new integument and its consequent alteration and the entry into the body cavity have also been studied. The infection develops rapidly in some of the larvae which die in premolt, while others are able to molt. Conditions rendered abnormal due to the presence of the fungus cause integumentary injuries which serve as an important factor in pathogenesis since they enhance the entry of fungal elements and bacteria thereby inducing septicemia. Contaminated larvae are able to molt, showing no signs of injury or disease, and survive for a long time, until the fungus finally invades the organism and causes death. This postponement of mortality shows that molting and hemocytic reactions are, to a certain extent, an effective defense mechanism. These last observations can be useful in the understanding of pathological processes associated with a hidden phase of fungal infection.     

Microspatial heterogeneity in the distribution of ciliates in a small pond

Five transects of contiguous samples from the surface of a small pond and one transect from its bottom were collected in order to quantify microspatial heterogeneity in the distribution of ciliated protozoa. Examination of the frequency-abundance relations for these transects suggests that they can be approximated by negative binomial distributions with a commonk of 1.87. Contagiousness or crowding increases with population density.Mean patch size and mean interpatch distance were measured for 4 transects as 1.5 to 2 cm and 3 to 4 cm, respectively. This heterogeneity is suggested to arise from behavioral aggregation about discrete food sources and be very ephemeral.Blocking of adjacent contiguous samples was used to investigate the effect of sample size on the apparent correlation between the numbers of pairs of taxa. In all cases examined, taxa were relatively independent in their distribution at small sample sizes and became more negatively or positively associated as samples were combined. This may reflect that the small scale patches are essentially monospecific.     

Contribution to the structural characterization of eucaryotic PSI reaction centre—I. Critical analysis of the polypeptidic composition of different P700 enriched fractions

In thylakoid membranes, several peptides of high MW are present which may interfere with the study of CP1's components. Modifying Cleveland's technique [7] for limited proteolysis, we have characterized the polypeptides found in the 60 kD region. Some may result from incomplete washing of the CF1 while others come from the CP1; indeed, this chlorophyll protein complex, which has a higher MW (above 100 kD), very often undergoes a dissociation into smaller components of about 60 KD MW.Analysis of the protein content of different preparations commonly used to obtain PSI reaction centre enriched fractions has been performed. The and subunits of CF1 are among the main contaminants of most of these preparations. A further purification step is described which can be applied to all these preparations, but numerous peptides are still present in the active fractions. It is most unlikely that all these polypeptides are required for the primary photochemical event, and this emphasizes the necessity to find a new simple method to purify PSI reaction centres.     

Contribution to the structural characterization of eucaryotic PSI reaction centre — II. Characterization of a highly purified photoactive SDS-CP1 complex

Under precise conditions, SDS PAGE allows purification of a photoactive P700-chla-protein complex from eucaryotic cells. The yield of P700 recovery is close to 100%. A total protein content equivalent to about 140 kD for one mole of P700 has been estimated by chemical analysis, and electrophoresis revealed the presence of two peptidic chains with MWs close to 65 kD. Photochemical and structural properties of this complex are given and compared with those of other complexes previously isolated.