Stoichiometric binding of diacylglycerol to the phorbol ester receptor

The major phorbol ester receptor is the Ca++-activated, phospholipid-dependent protein kinase C. Diacylglycerol stimulates protein kinase C in a fashion similar to the phorbol esters. Likewise, it inhibits phorbol ester binding competitively. Both results suggest that diacylglycerol is the/an endogenous phorbol ester analogue. Alternatively, the diacylglycerol might simply be acting to modify the phospholipid environment of the protein. If diacylglycerol were indeed functioning as an analogue, it should interact with the receptor stoichiometrically. This interaction can be quantitated by measuring the perturbation in apparent diacylglycerol binding affinity as a function of the ratio of diacylglycerol to receptor. We report here that 1,2-dioleoylglycerol interacts with the receptor with the predicted stoichiometry.     

Inhibition of Bacillus spores by combinations of heat,potassium sorbate,NaCl and pH

Viability of spores of Bacillus cereus was totally inhibited at 85°C over 30 min by adding 0.4% (w/v) potassium sorbate with 6% (w/v) NaCl at pH 4.5. Viability of B. stearothermophilus spores was totally inhibited at 95°C for 45 min in a buffer at pH 4.2 containing 0.8% (w/v) potassium sorbate and 8% (w/v) NaCl. A synergistic inhibitory effect was demonstrated in some of the combinations. The inhibition may be due to interference with the heat-resistance apparatus of the spores.O.B. Oloyede was and J. Scholefield is with the Department of Bioscience & Biotechnology, Food Science Division, University of Strathclyde, Glasgow G1 1SD, UK. O.B. Oloyede is now with the Department of Biochemistry, University of Ilorin, Ilorin, Nigeria.     

Sound localisation in crickets

Intracellular recordings were made in the brain of the cricket Gryllus bimaculatus from an ascending auditory interneuron (AN1). Acoustic stimuli with calling song temporal pattern were delivered via earphones in a preparation with the acoustic trachea cut (attenuation of crossing sound > 30 dB). The input-output function of this cell was then determined by recording its responses to stimulation of the ipsilateral ear alone, of the contralateral ear alone and to stimulation of both ears simultaneously with the same or different carrier frequencies and intensities.This interneuron was excited by the ear ipsilateral to its axon and dendritic field and unresponsive to stimuli presented to the axon-contralateral ear alone. However, in binaural stimulation experiments, the response to a constant ipsilateral stimulus was progressively reduced as the intensity of a simultaneous contralateral stimulus was increased, above a threshold intensity.Tuning curves for threshold of this inhibition, determined in binaural stimulation experiments, indicated significant inhibition in the range 3–20 kHz with lowest threshold at 4–5 kHz. The inhibition was unaffected by sectioning of the contralateral circumoesophageal or neck connective, indicating that the inhibitory influence crosses the midline at the level of the prothoracic ganglion. Intracellular recordings from AN1 in the prothoracic ganglion confirmed that it was indeed neurally inhibited by inputs from the contralateral ear.Tuning curves for excitation of an omega neuron (ON1) by the ear ipsilateral to its soma and also the tuning of inhibition of ON1 by its contralateral ON1 partner, closely match the tuning of inhibition of AN1 and to a lesser extent, of AN2. This was taken as evidence that each AN1 is inhibited by the contralateral ON1. The significance of this interaction for directional hearing and phonotaxis is discussed.Abbreviations AP/CHP action potentials per chirp - AN1, AN2 ascending auditory interneurons 1, 2 - ON1 omega neuron 1 - ipsi ipsilateral contra contralateral - PTG prothoracic ganglion loc lateral ocellar nerve - On optic nerve an antennal nerve - coc circum-oesophageal connective so sound off     

Protein toxicity to aphids: Anin vitro test onAcyrthosiphon pisum

Recent progress in plant transformation for insect resistance has increased the interest in the potential toxicity of proteins towards insect pests. While studies have been targeted to a large array of insect species, phloem-feeding Homoptera have not been investigated yet. The paper describes a routine test for screening toxicity and growth inhibition of purified proteins in artificial diets onAcyrthosiphon pisum (Harris). Twenty-five commercially available proteins of different classes were tested and compared to some non-protein chemicals (an insecticide, an antibiotic …).A. pisum proved to be very sensitive to all proteases tested and to some venoms with general cytolytic properties. A plant lectin, concanavalin A, displayed significant toxicity and growth inhibition, while various proteins such as a soybean proteinase inhibitor, a chitinase, and bovine serum albumin showed measurable impairments of growth only at higher dose (≥250 μg.ml⁻¹). Some proteins were without short-term effect onA. pisum physiology. The influence of these results on aphid-plant interactions are discussed.

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Résumé L'effet de protéines alimentaires sur les insectes phloémophages, dont les pucerons, n'a jamais été étudié. Nous proposons ici un test biologique standardisé sur milieu artificiel permettant d'analyser les effets de différentes classes de protéines sur la physiologie d'A. pisum. La validité de ce test est éprouvée (protocole, reproductibilité) et les différentes données récoltées (mortalité et inhibition de croissance) permettent de définir des paramètres toxicologiques tels que concentration létale 50 ou concentration inhibitrice 50. Cette caractérisation toxicologique a été réalisée sur 25 protéines appartenant à des classes différentes, ainsi que plusieurs substances non protéiques utilisées comme témoin de toxicité (insecticide, antibiotique, inhibiteur de synthèse protéique et glucoside phénolique). Les regroupements de protéines par proximités de profils toxicologiques ont été corrélés aux activités biochimiques des différentes protéines. Les implications de ces résultats sur les interactions plante-puceron sont discutées, ainsi que le potentiel d'une stratégie de création de variétés transgéniques résistantes aux pucerons.

     

A potent specific inhibitor of 6-phosphogluconate dehydrogenase ofCryptococcus neoformans and of certain other fungal enzymes

A particular lot of the zwitterionic buffer, 2(N-morpholino) ethane sulfonic acid (MES), contained a contaminant that inhibited a number of fungal NADP-dependent dehydrogenases. Enzymes that were particularly sensitive include 6-phosphogluconate dehydrogenases fromCryptococcus neoformans andSchizophyllum commune and glucose-6-phosphate dehydrogenase fromSchizophyllum commune. A number of NADP-dependent dehydrogenases of animal origin were tested and all were completely insensitive to inhibition except for rat liver 6-phosphogluconate dehydrogenase, which was 10-fold less sensitive than theCryptococcal enzyme. The pattern of inhibition in all cases was linear competitive versus NADP. The inhibitor has been purified and identified as an ethylenesulfonic acid oligomer. This inhibitor holds promise as a model compound for the development of a specific antifungal agent.     

Synthesis and evaluation of the antidepressant activity of the enantiomers of bupropion

The synthesis of the enantiomers of bupropion, (rac)-2-tert-butylamino-3′-chloropropiophenone 1 (Wellbutrin®) is described. The enantiomers were compared with the racemate in both the tetrabenazine-induced sedation model and the inhibition of uptake of biogenic amine assay. No significant differences were found in their potencies to reverse tetrabenazine-induced sedation in mice or in their IC₅₀ values as inhibitors of biogenic amine uptake into nerve endings obtained from mouse brain. © 1993 Wiley-Liss, Inc.     

Mutual support and selection between branches of damaged plants

Snow (1931) showed that the fate of a branch of a plant that had suffered local herbivory could be determined by correlative effects of other branches. This neglected work was continued in the current study, herbivory being simulated by the removal of leaves of different ages from pea plants with two branches. A damaged branch was suppressed when an undamaged alternative branch was present; otherwise the damage never prevented continued development. The removal of mature leaves had a smaller effect than the removal of immature, expanding leaves. When leaves were removed from both branches it was the branches that suffered less damage to their immature leaves that continued shoot development. Branches from which all photosynthetic leaves were repeatedly removed developed only when they retained their immature leaves and remained dominant, inhibiting the development — but not the photosynthesis — of the other branch on the same plant. Accounting for these results requires mechanisms that compare the different branches of the plant and select for development the ones that have the greatest potential for future, rather than present, photosynthesis. It is concluded that a compartmentalized or sectorial distribution of essential substrates can be modified by correlative relations that are probably mediated by hormones.     

A dual fluorescence technique for visualization ofStaphylococcus epidermidis biofilm using scanning confocal laser microscopy

A new dual fluorescence technique is described which, when combined with scanning confocal laser microscopy (SCLM), can be used to visualize the components of biofilm produced byStaphylococcus epidermidis. Chemostat cultures of RP62A (a well-characterized slime-producing strain ofS. epidermidis) were used to produce mature biofilm on polyvinylcholoride (PVC) disks immobilized in a modified Robbins device using a seed and feed model system. Serial horizontal and vertical optical thin sections, as well as three-dimensional computer reconstructions, were obtained onin situ biofilm using the dual fluorescence procedure. Bacteria were visualized by green autofluorescence excited at 488 nm with an Argon laser. Cell-associated and exocellular matrix material (slime) was visualized by red fluorescence excited at 568 nm with a Krypton laser after interaction of the biofilm with Texas Red-labeled wheat germ agglutinin which is a slime-specific lectin marker. Structural analysis revealed that the cocci grew in slime-embedded cell clusters forming distinct conical-shaped microcolonies. Interspersed open channels served to connect the bulk liquid with the deepest layers of the mature, hydrated biofilm which increased overall surface area and likely facilitated the exchange of nutrients and waste products throughout the biofilm. The combined dual fluorescence technique and SCLM is potentially useful as a specific noninvasive tool for studying the effect of antimicrobial agents on the process of biofilm formation and for the characterization of the architecture ofS. epidermidis biofilm formedin vivo andin vitro on medical grade virgin or modified inert polymer surfaces.     

Steady-state kinetics of the overall oxidative phosphorylation reaction in heart mitochondria. Determination of the coupling relationships between the respiratory reactions and miscellaneous observations concerning rate-limiting steps

The linear sequence of steps involved in the oxidation of extramitochondrial succinate by O₂ in bovine heart mitochondria was examined by a steady-state kinetic method to determine whether or not freely diffusible intermediates occur between the various inhibitor-sensitive steps. The kinetic method is based on the facts (1) that if two inhibitor-sensitive steps within a sequence are linked by a freely diffusible intermediate, inhibition of one will make the other less rate limiting in the overall reaction and thus will increase the amount of inhibitor of the other step required for half-maximal inhibition of the overall reaction, and (2) that if the two steps are not linked in this manner, inhibition of one will make the other more rate limiting and thus will decrease the amount of inhibitor of the other required for half-maximal inhibition. These two types of coupling relationships between steps were designated as sequential and fixed, respectively. The results indicate the existence of freely diffusible intermediates (sequential coupling relationships) between the succinate transport and succinate dehydrogenase reactions, between the succinate dehydrogenase and cytochromebc ₁ reactions, and between the cytochromesbc ₁ andaa ₃ reactions. Uncoupling respiration from phosphorylation results in the coupling relationship between thebc ₁ andaa ₃ reactions becoming partially fixed. This change is accompanied by marked decreases in the degrees to which thebc ₁ andaa ₃ reactions limit the overall reaction and appears to account for the large uncoupler-induced releases of inhibition at the levels of thebc ₁ andaa ₃ reactions observed previously by others. It is suggested that cytochromec is the freely diffusible intermediate between thebc ₁ andaa ₃ reactions and that the uncoupler-induced changes occur as a result of formation of functional and highly efficient supercomplexes between cytochromec and the cytochromesbc ₁ andaa ₃ complexes.