Subcellular localization of the common shikimate-pathway enzymes in Pisum sativum L.

5-Enolpyruvylshikimate 3-phosphate (EPSP) synthase (3-phosphoshikimate 1-carboxyvinyltransferase; EC 2.5.1.19), 3-dehydroquinate dehydratase (EC 4.2.1.10) and shikimate: NADP⁺ oxidoreductase (EC 1.1.1.25) were present in intact chloroplasts and root plastids isolated from pea seedling extracts by sucrose and modified-silica density gradient centrifugation. In young (approx. 10-d-old) seedling shoots the enzymes were predominantly chloroplastic; high-performance anion-exchange chromatography resolved minor isoenzymic activities not observed in density-gradientpurified chloroplasts. The initial enzyme of the shikimate pathway, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (EC 4.1.2.15) was also associated with intact density-gradient-purified chloroplasts. 3-Dehydroquinate synthase (EC 4.6.1.3) and shikimate kinase (EC 2.7.1.71) were detected together with the other pathway enzymes in stromal preparations from washed chloroplasts. Plastidic EPSP synthase was inhibited by micromolar concentrations of the herbicide glyphosate.Abbreviations DAHP 3-deoxy-d-arabino-heptulosonate 7-phosphate - DEAE diethylaminoethyl - DHQase 3-dehydroquinate dehydratase - DTT dithiothreitol - EPSP 5-enolpyruvylshikimate 3-phosphate - SORase shikimate:NADP⁺ oxidoreductase     

Aspects of the numerical and functional respones of the aphid parasite, Aphidius sonchi, in the laboratory

The fecundity, reproductive rates, and adult survival of Aphidius sonchi Marshall (Hymenoptera: Aphidiidae) parasitizing second and third instar nymphs of the sowthistle aphid, Hyperomyzus lactucae (L.) (Homoptera: Aphididae) were measured at six different host densities under constant laboratory conditions. At host densities of less than 50 aphids per flowering shoot per female per day, oviposition constraints resulting from the lack of hosts reduced the number of eggs laid, enhanced the extent of superparasitization and, as a result, effectively lowered the fecundity and reproductive rates of the parasites. Above this host density the parasites laid on average 220–230 eggs, but the effective fecundity and reproductive rates continued to increase with the host density. By contrast, the survivorship of the parasites seemed unaffected by host density, with an average adult life span of 4–5 days at all densities. Analysis of the data showed that the intrinsic rate of increase (r_m) of the parasite varied with the host density and could reach values higher than that of the host under identical conditions. The response of r_m to changes in host density and parasite sex ratio is illustrated.Overall, A. sonchi showed a typical convex functional response, to host density. However, the response showed obvious changes through the parasite's adult life and, furthermore, the rates of changes were not consistent at all host densities. The frequency distributions of parasite eggs were generally indistinguishable from random, and the number of hosts parasitized were predicted satisfactorily by the random oviposition equation.

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Résumé L'étude a porté sure l'influence de 6 densités différentes d'Hyperomyzus lactucae (L.) (Homoptera: Aphididae), en conditions constantes de laboratoire, sur la fécondité, le taux de reproduction et la survie des adultes d'Aphidius sonchi Marshall (Hym. Aphidiidae, parasite des larves de 2e et 3e stades. A des densités inférieures à 50 pucerons par tige fleurie de Sonchus oleraceus L, par femelle et par jour, la limitation de la ponte due à l'absence d'hôtes a réduit le nombre d'oeufs émis, élevé le taux de superparasitisme et, en conséquence, diminué la fécondité et le taux de reproduction des parasites. Aux densités d'hôtes supérieures, les parasites ont pondu, en moyenne, 220 à 230 oeufs, mais la fécondité réelle et les taux de reproduction ont continué à augmenter avec la densité des pucerons. Par contre, la longévité des parasites n'a pas été affectée par la densité des hôtes, avec une durée moyenne de vie de 4 à 6 jours. L'analyse des données a montré que le taux d'accroissement intrinsèque (r_m) du parasite a changé avec la densité des hôtes, et pourrait atteindre des valeurs supérieures à celles de l'ôte sous des conditions identiques. Les réponses de r_m aux changements de densité des hôtes et au taux sexuel du parasite sont expliquées.Globalement, A. sonchi a présenté une réponse fonctionnelle convexe typique à la densité des hôtes. Cependant, cette réponse a changé au cours de la vie des images et, de plus, les taux de changement ne sont pas logiques à toutes les densités d'hôtes La fréquence de distribution des oeufs n'est généralement pas séparable d'une distribution au hasard, et le nombre d'hôtes parasites peut être prédit d'une façon satisfaisante en utilisant une équation de ponte au hasard.

     

Death of Cydia pomonella larvae and damage to apple fruit, after field application of codling moth granulosis virus

In field experiments, larvae of codling moth Cydia pomonella (L.) rarely acquired granulosis virus on hatching from the egg, but picked up most later, on the tree surface. Deposits of virus sprayed in 1.0% w/v skimmed milk did not affect neonate larval behaviour. Larvae died, usually in the first instar, after entering treated fruit, but they frequently entered via the calyx or near the base of the stalk or through cracks in the skin, where little feeding damage by first-and sometimes second-instar larvae was seen.

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Résumé En verger, la pulvérisation d'oeufs de carpocapse avec du virus de la granulose en suspension dans l'eau (additionnée de lait écrémé dilué à 1%) n'a pas modifié la survie des chenilles avant pénétration dans le fruit; par contre la pulvérisation des arbres a provoqué une forte mortalité. Bien que des chenilles consommant des poils et la surface des feuilles aient été observées avant leur pénétration dans le fruit, ce qui aurait pu provoquer leur contamination par le virus, il semble que la contamination létale provienne des fruits seuls.La présence de produit n'a modifié ni le comportement larvaire, ni le taux de pénétration dans les fruits; la mortalité y a lieu ensuite, généralement au premier stade. Dans 74 à 78% des cas, les chenilles ont pénétré dans le fruit par le calice ou près de la base du pédoncule — aucun dégât provenant de larves du premier stade n'y était visible, de même que dans le calice pour les larves du deuxième stade. Par contre, toute pénétration par la surface du fruit était repérable dès le premier stade. Il est possible que la répartition des lieux de pénétration dans le fruit influe sur la létalité due au virus et explique les variations d'efficacité observées en verger. Un système de classification des dégâts, provoqués lors de la pénétration dans le fruit, de chenilles du premier au troisième stade est proposé pour évaluer l'efficacité des essais en verger.

     

The endosomal compartment of rat hepatocytes. Its characterization in the course of [125I]insulin internalization

When freshly isolated hepatocytes are incubated with [125I]insulin in the presence of the microtubule-disrupting agent colchicine, internalization of the labelled hormone is not significantly altered. However, the drug limits the endocytosis of the labelled material to a peripheral band of cytoplasm extending 1 micron beyond the plasma membrane. Both in the presence and absence of colchicine, internalized [125I]insulin preferentially associates with clear vesicles (endosomes) and lysosome-like structures, but the relative amount of labelled material associated with clear vesicles is higher in the presence of the drug than in its absence. An inverse pattern is observed for the lysosome-like structures. As demonstrated by cytochemical methods, clear vesicles do not contain the lysosomal enzyme aryl sulfatase. Moreover, colchicine induces an increase of the clear vesicle diameter without affecting their frequency, while it perturbs multivesicular bodies and dense bodies in an opposite way by increasing their frequency without affecting their size. By reducing and/or delaying the fusion between internalized endocytotic vesicles and lysosomes, colchicine allows better characterization of the endosomal compartment of isolated rat hepatocytes and allows it to be distinguished from other compartments, such as multivesicular bodies and the Golgi apparatus.     

Interferon affects the formation of adhesion plaques in human monocyte cultures

When monocytes isolated from human blood adhere to glass substratum, actin- and vinculin-containing punctate plaques rapidly appear at the ventral surface of the cells. We show here that highly purified human leukocyte interferon (IFN) can inhibit formation of these adhesion plaques in a dose-dependent manner. Complete inhibition was obtained when 300 IU/ml IFN were added into the cell-seeding medium. Plaques already formed in the absence of IFN were only partially affected by subsequent addition of IFN into the culture medium. Prevention by IFN of the formation of the adhesion plaques was associated with loosened attachment of the cells to the substratum. Effect of IFN on cellular morphology was complex. At higher doses, IFN added to the cultures within 24 h of seeding almost completely inhibited the differentiation of monocytes to macrophages and most of the cells remained rounded. At lower doses, however, an enhancement of the bipolar spreading was seen and the end result was a culture with predominantly elongated fibroblastoid cells. The latter cells, unlike the fibroblastoid cells in untreated monocyte-macrophage cultures, were completely devoid of the actin plaques, while the reorganization of vimentin-type intermediate filaments took place in a normal manner. These results further support the view that the actin- and vinculin-containing plaques have a role in mediating firm adherence of human monocytes to growth substratum.     

Aedes aegypti: model for blood finding strategy and prediction of parasite manipulation

Aedes aegypti mosquitoes salivate during intradermal probing of vertebrate prey before ingesting blood (Griffiths and Gordon 1952). Nonsalivating mosquitoes locate blood more slowly; this difference was ascribed to an anti-platelet activity found in the mosquito's saliva (Ribeiro et al. 1984). Mosquitoes infected with Plasmodium gallinaceum suffer pathology that specifically impairs saliva anti-hemostatic activity but without reducing volume of output (Rossignol et al. 1984). The complexity of the feeding apparatus of mosquitoes provides opportunity for a variety of strategies in which pathogens may produce specific lesions that enhance their transmission, but the variables that affect the duration of probing by mosquitoes have not been defined. We sought to resolve this complexity by identifying and quantifying relevant parameters of probing behavior. Mosquitoes thrust their mouthparts repeatedly through their host's skin while searching for blood. Female A. aegypti thrust at 7-sec intervals. If this search results in success, feeding ensues. Alternatively, the mosquito "desists," the mouthparts stylets are withdrawn, and the mosquito attempts to feed at another site. Even after previous desistance, the probability of finding blood remains undiminished. Functions for the probability of feeding success and desistance over time were derived using data from observations on 300 mosquitoes. The probability of feeding success was interpreted as being a function of the density of vessels in the skin, their geometric distribution, and the conditions locally affecting hemostasis. During each probe, the probability of desisting increased linearly with time, and after desisting once, mosquitoes tended to desist more rapidly. A model was developed incorporating Monte Carlo simulation which closely fit observed data. By changing values for the several parameters of the probability functions, we predicted modes in which parasites may manipulate their hosts to enhance transmission, both to and from the vector. In particular, parasite strategies in the vector would include induced salivary pathology; increased duration of probing thrusts; decreased desistance time; and inhibited phagoreception. Predicted parasite strategies in the reservoir host would include increased skin vascular volume and impaired host hemostasis. Our model supports the hypothesis of a mutualistic interaction of malaria and mosquitoes.     

Inhibitory effect of molecular hydrogen on C2H2-reducing activity in Anabaena 7120 preilluminated in red or blue light

Molecular hydrogen inhibits nitrogenase activity in Anabaena pre-illuminated with red or blue light. The inhibitory effect of molecular hydrogen decreased in the presence of oxygen and several electron acceptors. When NH₄Cl and urea were added simultaneously with molecular hydrogen, marked synergistic inhibitory effects took place. The inhibitory effect of molecular hydrogen disappeared or was weakened after the suppression of hydrogenase activity. The addition of O₂ and electron acceptors to systems showed no enhancing effect on the C₂H₂-reducing activity.     

Characteristics of Tyrosine Hydroxylase Activation by K+-Induced Depolarization and/or Forskolin in Rat Striatal Slices

The mechanisms of tyrosine hydroxylase (TH) activation by depolarization or exposure of dopaminergic terminals to cyclic AMP have been compared using rat striatal slices. Tissues were incubated with veratridine or 60 mM K+ (depolarizing conditions), on the one hand, and forskolin or dibutyryl cyclic AMP, on the other. K+-(or veratridine-)induced depolarization triggered an activation of TH (+75%) that persisted in soluble extracts of incubated tissues. This effect disappeared when drugs (EGTA, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, Gallopamil) preventing Ca2+- and calmodulin-dependent processes were included in the incubating medium. In contrast, prior in vivo reserpine treatment or in vitro addition of benztropine did not affect the depolarization-induced activation of TH. In vitro studies of soluble TH extracted from depolarized tissues indicated that activation was associated with a marked increase in the enzyme Vmax but with no change in its apparent affinity for the pteridin cofactor 6-methyl-5,6,7,8-tetrahydropterin (6-MPH4) or tyrosine. Furthermore, the activated enzyme from depolarized tissues exhibited the same optimal pH (5.8) as native TH extracted from control striatal slices. In contrast, TH activation resulting from tissue incubation in the presence of forskolin or dibutyryl cyclic AMP was associated with a selective increase in the apparent affinity for 6-MPH4 and a shift in the optimal pH from 5.8 to 7.0-7.2. Clear distinction between the two activating processes was further confirmed by the facts that heparin- and cyclic AMP-dependent phosphorylation stimulated TH activity from K+-exposed (and control) tissues but not that from striatal slices incubated with forskolin (or dibutyryl cyclic AMP). In contrast, the latter enzyme but not that from depolarized tissues could be activated by Ca2+-dependent phosphorylation. These data strongly support the concept that Ca2+- but not cyclic AMP-dependent phosphorylation is responsible for TH activation in depolarized dopaminergic terminals.     

Drug Potencies on Partially Purified Brain D2 Dopamine Receptors

To examine the sensitivities of partially purified dopamine receptors to various dopaminergic agonists and antagonists, canine brain striatum dopamine receptors were enriched by isoelectric focusing. The digitonin-solubilized receptors were prelabelled with [3H]spiperone and focused for two time periods. After 5 h (incomplete focusing), radioactive peaks were detected at pH 6 and 9-11. Only the pH 6 peak revealed drug sensitivities expected of D2 receptors. Receptor recovery of the pH 6 peak was 79% with purification being sevenfold. After focusing overnight to equilibrium, the pH 6 peak further separated into peaks at pH 4.6 and 6.8. The receptor was identified only in the pH 4.6 fraction. The recovery of receptors in the pH 4.6 peak was low (10%), indicating little enrichment of the receptor. The rank order of binding of neuroleptics and dopamine agonists to the purified material was similar to that of the original preparation of soluble receptors. Dopamine did not bind to the purified pH 4.6 fraction unless the phosphate buffer (used during focusing) was replaced with Tris buffer. The absence of receptors in the pH 6.8 and pH 10 fractions, although both contained prelabeled [3H]spiperone, indicates the importance of testing agonists and antagonists on each fraction at each step in purification.     

Deprenyl Protects Dopamine Neurons from the Neurotoxic Effect of 1-Methyl-4-Phenylpyridinium Ion

1-Methyl-4-phenylpyridinium ion (MPP+) is the product of the metabolic oxidation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) by monoamine oxidase (MAO). MPP+ is toxic to 3,4-dihydroxyphenylethylamine (dopamine, DA) neurons in explant cultures of rat embryonic midbrain. Addition of 2.5 microM MPP+ to the feeding medium for 6 days results in significant reduction of the DA levels in the cultures (to 19% of control) as well as in the uptake of [3H]DA (to 32% of control). When the cultures are treated with the MAO inhibitor deprenyl (10 microM) 24 h prior to and during exposure to MPP+, the DA neurons are protected from the toxicity of the drug. In the combined deprenyl plus MPP+ treatment, the levels of DA in the cultures remain at the control range and the [3H]DA uptake is reduced to only 73% of control. These results indicate that MAO is involved in the toxicity of MPP+ on DA neurons.