An improved method of measuring photosynthetic electron transport in Euglena under in vivo conditions

A method is described to measure photochemical activity in intact cells of Euglena under in vivo conditions. The method employs a cell wall digesting enzyme (cellulysin) to induce enough permeability in the cell walls and membranes in order to allow dyes, commonly used to investigate light-dependent electron transport reactions to enter, but without inducing a concomittant efflux of metabolites. Between 1 and 2 h of incubation in 5% (w/v) cellulysin provided conditions which allowed measurement of photosystem I-, II- and I+II-dependent electron transport with rates up to 600% higher than in control cells; whereas other cell wall degrading enzymes (cellulase and pectinase) still did not increase the entry of the dyes. Cellulysin up to 2 h of incubation had little or no effect on whole cell respiration, photosynthetic O₂ evolution, or the export of potassium and (¹⁴C) labeled compounds out of cells; therefore cellulysin obviously did not change the normal habit or physiology of Euglena. Cellulysin (4 h digestion), cellulase and pectinase (2–4 h of incubation) on the other hand led to a lowering of respiration and light-dependent O₂ evolution, and increased the efflux of K⁺, but apparently decreased that of (¹⁴C)labeled fixation products.Abbreviations DBMIB dibromothymoquinone - DCPIP 2,6-dichlorophenol-indophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMMIB 2,3-dimethyl-5,6-methylenedioxy-p-benzoquinone - MV methylviologen - PSI photosystem I - PS II photosystem II     

Presynaptic dense bars at neuromuscular synapses of the lobster,Homarus americanus

Summary The threedimensional ultrastructure of presynaptic dense bars was examined by serial section electron microscopy in the excitatory neuromuscular synapses of the accessory flexor muscle in the limbs of larval, juvenile, and adult lobsters. The cross-sectional profile of the dense bar resembles an asymmetric hourglass, the part contacting the presynaptic membrane being larger than that projecting into the terminal. The bar has a height of 55–65 nm and varies in length from 75–600 nm. In its dimensions it resembles the dense projections in the synapses of the CNS of insects and vertebrates. The usual location of these dense bars is at well defined synapses, though a few are found at extrasynaptic sites either in the axon or terminal. In the latter case the bars are close to synapse-bearing regions, particularly in the larval terminals, suggesting that the extrasynaptic bars denote early events in synapse formation. In all cases the bars are intimately associated with electron lucent, synaptic vesicles located on either side, in the indentation of its hourglass-shaped cross sectional profile. The vesicles occur along the length of the bar and contact the presynaptic membrane. Consequently the dense bar may serve to align the vesicles at the presynaptic membrane prior to exocytosis. A similar role has been suggested for the presynaptic dense bodies at the neuromuscular junction of the frog, where synaptic vesicles form a row on either side of this structure.Supported by Muscular Dystrophy Association of Canada and NSERCC. Generous use of laboratory facilities at Woods Hole was provided by the late Fred Lang     

Rat pancreas adenylate cyclase VII. Effect of extracellular calcium on pancreozymin-induced cyclic AMP formation

1. 1. The effect of stimulation of adenylate cyclase by pancreozymin-C-octapeptide on the cyclic AMP level of rat pancreatic fragments has been investigated.

2. 2. In normal Krebs-Ringer bicarbonate medium pancreozymin-C-octapeptide causes a slight increase in pancreatic cyclic AMP level; this increase can be considerably enhanced by incubation in a calcium-free incubation medium.

3. 3. The dose-responce curve for pancreazymin-C-octapeptide in calcium-free medium is shifted to lower peptide concentrations, compared to the curve in normal Krebs-Ringer bicarbonate medium.

4. 4. The maximal stimulatory effect of pancreozymin-C-octapeptide id obtained at a 1-methyl-3-isobutylxanthine concentration of 10 mM.

5. 5. It suffices to lower the Ca²⁺-concentration of the medium from 2.5 to 1.5 mM to get the maximal increase in cyclic AMP content under influence of pancreozymin-C-octapeptide.

6. 6. It is concluded that extracellular calcium antagonizes the stimulation of adenylate cyclase by pancreozymin-C-octapeptide. This suggest that a low cytoplasmic Ca²⁺-concentration is required for the maximal response of acinar cell adenylate cyclase to pancreozymin.

Synaptosomal phospholipid pool in rabbit brain and its effect on GABA uptake

Rabbit synaptosomes have been used to study the effect of the base-exchange reaction in membrane phospholipids on -aminobutyric acid (GABA) transport in vitro. The uptake of GABA was measured after a base-exchange reaction with ethanolamine, choline, orl-serine and after subsequent displacement of these exchanged moieties from lipid by bases of similar or different structures which were added to the synaptosomal medium. Serine incorporation stimulated GABA transport, but its displacement from membrane lipid by choline or ethanolamine induced an inhibition of GABA transport. Ethanolamine incorporation inhibited GABA transport, but its displacement by serine or choline resulted in stimulation of GABA uptake. Choline incorporation also inhibited GABA transport, although less than ethanolamine. The pool size of synaptosomal phospholipids, presumably involved in GABA uptake, accounted for 0.2 to 10% of the total content of membrane phospholipid. Thus, alteration of phospholipid compositior by exchange of the lipid hydrophilic head-groups influences the extent GABA uptake into rabbit synaptosomes.     

Effect of psoralen-induced photodermatitis on tryptophan metabolism in rats

Tryptophan metabolism ‘via kynurenine’ has been studied in rats before and after induction of experimental light-conditioned dermatitis with psoralen. Tryptophan load in animals during the acute phase of dermatitis (one day after induction) causes a markedly increased urinary excretion of total metabolites in comparison with that obtained before dermatitis. During this phase of the skin disease tryptophan pyrrolase activity is significantly increased and kynureninase activity significantly decreased in liver in respect to the control animals. Kynurenine aminotransferase activity shows no significant variations in both liver and kidneys. After 6 days of dermatitis, when the skin damage is in repair, both the excretory values of the urinary metabolites after L-tryptophan load and the enzymic activities are similar to those before dermatitis.     

Longevity and fecundity in the colorado potato beetle, Leptinotarsa decemlineata

Longevity, egg production and sensitivity towards induction of diapause of two laboratory cultures of Leptinotarsa decemlineata Say, the Colorado potato beetle, were compared. One culture was kept ab ovo under long-day conditions (LD: 16 hr photophase; 25°=LD culture); the other was raised ab ovo in short-day condition (SD: 10 hr photophase; 25°), kept in diapause during 9 months at 25° in dry sand and after termination of diapause brought to LD (=DP-LD culture). During 97 days, there were no significant differences in the longevities of the cultures (50% mortality: day 52 in DP-LD; day 58 in LD). The survival curve of each was almost linear, which is rather unusual in animals. If the regression line, representing each survival curve was linearly extrapolated beyond day 97 (end of experiment), a theoretical maximal life span of 121 days was found for the LD culture, and of 110 days for the DP-LD culture. Beetles did not enter a second diapause as easily as the first. The haemolymph protein pattern of old animals that had ceased reproduction was almost similar to that of young reproducing animals. In both cultures, the last egg was laid at day 86. Mated females produced 30–60 eggs per day as compared with virgin females which produced less than ten per day. Application of JH 1, 20-hydroxyecdysone, prostaglandin E₂, haemolymph or brain extracts of females did not increase egg production in virgins.

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Résumé La longévité, la fécondité et la sensibilité à l'induction de la diapause ont été comparées chez deux souches de laboratoire du doryphore, Leptinotarsa decemlineata. Une souche a été conservée ab ovo en jours longs (LD: 16 h de photophase; 25°=LD culture). L'autre souche a été élevée ab ovo en jours courts (SD: 10 h de photophase; 25°); elle est restée en diapause pendant 9 mois à 25° dans du sable sec et a été placée en LD après la fin de diapause (= DP-LD culture). Jusqu'au 97ième jour de l'expérience, il n'y avait pas de différence significative entre les longévités des deux souches. Les courbes de survie des deux souches étaient presque rectilignes, ce qui est plutôt rare chez des animaux.En extrapolant linéairement après le 97ième jour (fin de l'expérience) les lignes de régression linéaire correspondant aux deux courbes, une durée de vie maximale théorique de 121 jours a pu être déduite dans la souche LD et de 110 jours pour la souche DP-LD. Aucune différence significative n'a été observée entre les fécondités des deux souches. Le dernier uf a été pondu le 86ième jour. Les doryphores qui avaient déjà été en diapause, n'y retournaient pas aussi aisément que ceux qui n'avaient jamais été en diapause avant leur vie reproductive. Le protéinogramme de l'hémolymphe des animaux âgés ne différait pas de celui des jeunes animaux reproducteurs. Tandis que les femelles accouplées ont pondu 30 à 60 ufs par jour, les femelles vierges n'en ont pas émis généralement plus de 10. L'application d'hormone juvénile 1, de 20-hydroxyecdysone, de prostaglatine E², d'hémolymphe ou d'extraits céphaliques de femelles n'a pas accru la production d'ufs.

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This research was supported by the National Foundation of Scientific Research (N.F.W.O.) of Belgium.