Physiological interactions and development synchronisations between non-diapausing Ostrinia nubilalis larvae and the tachinid parasitoid Pseudoperichaeta nigrolineata

The physiological relationships between Ostrinia nubilalis Hübner and its tachinid parasitoid Pseudoperichaeta nigrolineata Walker are described under abiotic conditions which induce development of the host without diapause. The parasitoid lowers the larval growth of the host: the maximal weight attained by the parasitized larvae represented only 78% of that of healthy ones. The duration of the last larval host instar increased to 10.4 days in parasitized O. nubilalis compared to 8.0 days in unparasitized ones. The influence of the host on the parasitoid development was studied experimentally after parasitization of O. nubilalis larvae of instars 2 to 5. When the second larval instar of the host is parasitized, the overall duration of parasitoid larval development lasts twice as long as when the fifth instar is parasitized. The best yield of parasitoid pupariae (50%) is obtained when parasitization occurs in instar 3. We show that good synchronisation exists between the larval development of the host and its parasitoid. There are four phases of parasitoid development which would appear to require a signal from the host: the start of the growth of newly hatched parasitoid larvae and the 3rd to 4th instar ecdysis of the host; the first moulting of the parasitoid and the 4th to 5th instar ecdysis of the host; the growth resumption of the parasitoid instar II (weight about 1 mg) and the small rise of the ecdysteroid level in the middle of host instar 5; and in all probability, the second parasitoid moulting and the larval-pupal apolysis of the host.

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Résumé Les relations physiologiques entre O. nubilalis et le tachinidae P. nigrolineata ont été étudiées dans des conditions abiotiques telles que l'hôte présente un développement sans diapause. Le parasitoïde ralentit la prise de poids de l'hôte: le poids maximal des chenilles parasitées ne représente que 78% de celui des chenilles saines. Seule la durée du 5ème stade est significativement plus longue chez les chenilles parasitées que chez les saines (10,4 contre 8,0 jours). L'influence de l'hôte sur le développement du parasitoïde à été expérimentée en parasitant des stades 2 à 5 d'O. nubilalis. Lorsque l'infestation a lieu au stade 2, le développement larvaire complet du parasitoî de dure deux fois plus longtemps que lorsque l'infestation a lieu au stade 5. Le meilleur rendement en pupes (50%) est obtenu lorsque l'infestation a lieu au stade 3. Il a été montré qu'il existe une bonne synchronisation entre le développement de l'hôte et de son parasitoî de. Il y a 4 phases physiologiques du développement larvaire de P. nigrolineata qui semblent nécessiter un signal provenant de l'hôte pour être dépassées. Ainsi peuvent être mis en relation: — le début de la croissance de la larve néonate du parasitoî de et l'ecdysis 3/4 de l'hôte; — la première mue du parasitoïde et l'ecdysis 4/5 de l'hôte; — la reprise de la croissance du stade II du parasitoïde, vers un poids de 1 mg et la remontée des taux d'ecdystéroïdes au milieu du stade 5 de l'hôte. et probablement, — la seconde mue du parasitoïde et l'apolyse nymphale de l'hôte. Les expérimentations vont se poursuivre pour déterminer les facteurs en cause. Ces phénomènes de synchronisation seront aussi étudiés dans le cas de la diapause de l'hôte.

     

Dinucleoside tetraphosphate variations in cultured tumor cells during their cell cycle and growth

Asynchronous and synchronized cultures of A549 and HTC cells were used to detect possible, cell cycle or cell density specific variations in the intracellular pools of dinucleoside tetraphosphates (Ap4X). No important variations of the nucleotide pools were observed during cell growth. When HTC cells were released from mitotic arrest, a decrease by a factor of N3 Ap4X and ATP levels was observed when the cells entered the G1 phase. This decrease is essentially due to cell doubling. When A549 cells were released from an arrest at the G1/S boundary, the nucleotide pool size increased slightly during the G2 phase just before mitosis. This result is in agreement with both earlier data from our laboratory and the observed decrease in Ap4X pool after release from mitotic-arrested HTC cells. These results suggest that the Ap4X and ATP pools are only subjected to very small variations during the cell cycle, essentially in the G2 phase and after mitosis.     

Isolation and fractionation of CHO chromosomes in aqueous two phase systems using charged polymers and base specific macroligands

Summary Chromosomes were isolated in a preparative scale by synchronisation of CHO cells with a double Thymidine block followed by an arrest in the metaphase by addition of Colcemid. Under proper cultivation conditions a mitotic index of 77% total cells could be routinely achieved. Bulk chromosome preparations free of nuclei and other subcellular particles have been obtained by low speed centrifugation followed by a 60 transfer countercurrent distribution using aqueous two phase systems composed of polyethylenglycol and dextran. The partition of CHO chromosomes previously purified in aqueous two phase systems were studied further to develop a protocol for the separation and isolation of individual chromosomes. Partition experiments with chromosomes changing the electrostatic phase potential by addition of charged PEG-derivatives suggest the existence of relatively highly charged chromosome groups. Most promising results with regard to separation were obtained using two PEG-derivatives, which interact specifically with the bases in DNA. For this affinity partitioning a GC- and AT-specific macroligand were employed. Comparing CCD's using each of these ligands information on the GC and AT content of exposed DNA in the chromosomes groups could be derived, demonstrating that specific sequences of DNA are accessible at the surface of metaphase chromosomes.     

Synchrony as an Adaptive Mechanism for Large‐Scale Human Social Bonding

Humans have developed a number of specific mechanisms that allow us to maintain much larger social networks than would be expected given our brain size. For our primate cousins, social bonding is primarily supported using grooming, and the bonding effect this produces is primarily mechanistically underpinned by the release of endorphins (although other neurohormones are also likely to be involved). Given large group sizes and time budgeting constraints, grooming is not viable as the primary social bonding mechanism in humans. Instead, during our evolutionary history, we developed other behaviours that helped us to feel connected to our social communities. Here, we propose that synchrony might act as direct means to encourage group cohesion by causing the release of neurohormones that influence social bonding. By acting on ancient neurochemical bonding mechanisms, synchrony can act as a primal and direct social bonding agent, and this might explain its recurrence throughout diverse human cultures and contexts (e.g. dance, prayer, marching, music‐making). Recent evidence supports the theory that endorphins are released during synchronised human activities, including sport, but particularly during musical interaction. Thus, synchrony‐based activities are likely to have developed due to the fact that they allow the release of these hormones in large‐scale human communities, providing an alternative to social bonding mechanisms such as grooming.     

Eukaryotic cell locomotion depends on the propagation of self-organized reaction-diffusion waves and oscillations of actin filament assembly

Actin filament (F-actin) assembly kinetics determines the locomotion and shape of crawling eukaryotic cells, but the nature of these kinetics and their determining reactions are unclear. Live BHK21 fibroblasts, mouse melanoma cells, and Dictyostelium amoebae, locomoting on glass and expressing Green Fluorescent Protein-actin fusion proteins, were examined by confocal microscopy. The cells demonstrated three-dimensional bands of F-actin, which propagated throughout the cytoplasm at rates usually ranging between 2 and 5 microm/min in each cell type and produced lamellipodia or pseudopodia at the cell boundary. F-actin's dynamic behavior and supramolecular spatial patterns resembled in detail self-organized chemical waves in dissipative, physico-chemical systems. On this basis, the present observations provide the first evidence of self-organized, and probably autocatalytic, chemical reaction-diffusion waves of reversible actin filament assembly in vertebrate cells and a comprehensive record of wave and locomotory dynamics in vegetative-stage Dictyostelium cells. The intensity and frequency of F-actin wavefronts determine locomotory cell projections and the rotating oscillatory waves, which structure the cell surface. F-actin assembly waves thus provide a fundamental, deterministic, and nonlinear mechanism of cell locomotion and shape, which complements mechanisms based exclusively on stochastic molecular reaction kinetics.     

Modelling autocatalytic networks with artificial microbiology

Cells can usefully be equated to autocatalytic networks that increase in mass and then divide. To begin to model relationships between autocatalytic networks and cell division, we have written a program of artificial chemistry that simulates a cell fed by monomers. These monomers are symbols that can be assembled into linear (non-branched) polymers to give different lengths. A reaction is catalysed by a particular polymer or 'enzyme' that may itself be a reactant of that reaction (autocatalysis). These reactions are only studied within the confines of the 'cell' or 'reaction chamber'. There is a flux of material through the cell and eventually the mass of polymers reaches a threshold at which we analyse the cell. Our results indicate a similarity between the connectivity of the reaction network and that of real metabolic networks. Developing the model will entail attributing increased probabilities of reactions to polymers that are colocalised to evaluate the consequences of the dynamics of large assemblies of diverse molecules (hyperstructures) and of cell division.     

Influence of IBA and aphidicolin on DNA synthesis and adventitious root regeneration from Malus ‘Jork 9’ stem discs

Adventitious root formation in Malus ‘Jork 9’ stem discs was studied through temporarily blocking DNA synthesis by application of aphidicolin (AD). Higher number of roots per disc (8.4) after 21 days of cultivation were formed after a 24-h pulse of 15 μM AD, compared to control without AD application (6.7), with significantly more roots (3.7) already appearing at day 7, compared to 1.5 roots on the control. The promotive effect of AD on rooting was lower at 5 μM, while a concentration of 30 μM was slightly inhibitory. Results show that DNA synthesis is effectively blocked by AD, and this blockage is overcome after AD withdrawal. The data indicate that AD treatment influences cell divisions, thereby, might synchronise root initiation. The effects of different treatments with and without AD were studied at the cellular level by visualising DNA replication through BrdU-labelling. BrdU labelling further revealed temporal changes in the competence of the explants to respond to applied IBA. Thus, it is shown that the proportion of replicating nuclei present during 28–32 h is significantly increased in the split IBA treatment (0–4 h and 28–32 h; treatment C3), compared with a single IBA application during 0–8 h (treatment C3.1). An erratum to this article can be found at     

A conserved threonine spring‐loads precursor for intein splicing

Protein splicing is an autocatalytic process where an “intein” self‐cleaves from a precursor and ligates the flanking N‐ and C‐“extein” polypeptides. Inteins occur in all domains of life and have myriad uses in biotechnology. Although the reaction steps of protein splicing are known, mechanistic details remain incomplete, particularly the initial peptide rearrangement at the N‐terminal extein/intein junction. Recently, we proposed that this transformation, an N‐S acyl shift, is accelerated by a localized conformational strain, between the intein's catalytic cysteine (Cys1) and the neighboring glycine (Gly‐1) in the N‐extein. That proposal was based on the crystal structure of a catalytically competent trapped precursor. Here, we define the structural origins and mechanistic relevance of the conformational strain using a combination of quantum mechanical simulations, mutational analysis, and X‐ray crystallography. Our results implicate a conserved, but largely unstudied, threonine residue of the Ssp DnaE intein (Thr69) as the mediator of conformational strain through hydrogen bonding. Further, the strain imposed by this residue is shown to position the splice junction in a manner that enhances the rate of the N‐S acyl shift substantially. Taken together, our results not only provide fundamental understanding of the control of the first step of protein splicing but also have important implications in various biotechnological applications that require precursor manipulation.     

Natural Synchronisation for the Study of Cell Division in the Green Unicellular Alga Ostreococcus tauri

Ostreococcus tauri (Prasinophyceae) is a marine unicellular green alga which diverged early in the green lineage. The interest of O. tauri as a potential model to study plant cell division is based on its key phylogenetic position, its simple binary division, a very simple cellular organisation and now the availability of the full genome sequence. In addition O. tauri has a minimal yet complete set of cell cycle control genes. Here we show that division can be naturally synchronised by light/dark cycles and that organelles divide before the nucleus. This natural synchronisation, although being only partial, enables the study of the expression of CDKs throughout the cell cycle. The expression patterns of OtCDKA and OtCDKB were determined both at the mRNA and protein levels. The single OtCDKA gene is constantly expressed throughout the cell cycle, whereas OtCDKB is highly regulated and expressed only in S/G2/M phases. More surprisingly, OtCDKA is not phosphorylated at the tyrosine residue, in contrast to OtCDKB which is strongly phosphorylated during cell division. OtCDKA kinase activity appears before the S phase, indicating a possible role of this protein in the G1/S transition. OtCDKB kinase activity occurs later than OtCDKA, and its tyrosine phosphorylation is correlated to G2/M, suggesting a possible control of the mitotic activity. To our knowledge this is the first organism in the green lineage which showed CDKB tyrosine phosphorylation during cell cycle progression.