Membrane lipid dynamics and enzymic activity in bovine adrenal cortex microsomes

The lipid dynamics of the adrenocortical microsomal membranes was studied by monitoring the fluorescence anisotropy and excited state lifetime of a set of anthroyloxy fatty acid probes (2-, 7-, 9- and 12-(9-anthroyloxy)-stearic acid (AP) and 16-(9-anthroyloxy)palmitic acid (AS). It was found that a decreasing polarity gradient from the aqueous membrane interface to the membrane interior, was present. This gradient was not modified by the proteins, as evidenced by comparison of complete membranes and derived liposomes, suggesting that the anthroyloxy probes were not in close contact with the proteins. An important change of the value of the mean rotational relaxation time as a function of the position of the anthroyl ring along the acyl chain was evidenced. In the complete membranes, a relatively more fluid medium was evidenced in the C₁₆ as compared to the C₂ region, while the rotational motion appeared to be the most hindered at the C₇–C₉ level. In the derived liposomes, a similar trend was observed but the mobility was higher at all levels. The decrease of the mean rotational relaxation time was more important for 12-AS and 16-AP. Temperature dependence of the mean rotational relaxation time of 2-AS, 12-AS and 16-AP in the complete membranes revealed the existence of a lipid reorganization occurring around 27°C and concerning mainly the C₁₆ region. The extent to which the acyl chain reacted to this perturbation at the C₁₂ level depended on pH. The presence of proteins increased the apparent magnitude of this reorganization and also modified the critical temperature from approx. 23°C in the derived liposomes to approx. 27°C in the complete membranes. Thermal dependence of the maximum velocity of the $\text{3-}\text{oxosteroid}\text{Δ}{}^5\text{?Δ}{}^4\text{-}\text{isomerase}$, the second enzyme in the enzymatic sequence, responsible for the biosynthesis of the $\text{3-}\text{oxo}\text{-Δ}{}^4\text{-}\text{steroids}$ in the adrenal cortex microsomes, was studied. The activation energy of the catalyzed reaction was found to be low and constant ($\text{2–5}\text{kcal}\text{·}\text{mol}{}^{\mathrm{?1}}$) in the temperature range 16–40°C at pH 7.5, 8.5 and 9, corresponding to the minimum, intermediate and maximum rate, respectively. A drastic increase of the activation energy ($\text{20}\text{kcal}\text{·}\text{mol}{}^{\mathrm{?1}}$) was observed at temperature below 16°C at pH 7.5. A correlated change of the $\text{p}\text{K}{}_{\mathrm{<mtext>ES</mtext>}}{}^{\mathrm{<mtext>app</mtext>}}$ as function of temperature was detected; at 36°C $\text{p}\text{K}{}_{\mathrm{<mtext>ES</mtext>}}{}^{\mathrm{<mtext>app</mtext>}}\text{= 8.3}$ while at 13°C the value shifted to 8.7. The pH range of the group ionization was narrower at 13°C. In contrast with the behaviour of the $\text{3β-}\text{hydroxy}\text{-Δ}{}^5\text{-}\text{steroid dehydrogenase}$, the $\text{3-}\text{oxosteroid}\text{Δ}{}^5\text{?Δ}{}^4\text{-}\text{isomerase}$ was apparently unaffected by the lipid reorganization at 27°C. It is suggested that this enzyme possesses a different and more fluid lipid environment than the bulk lipids.     

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.     

Rapid effects of nerve growth factor on the Na,K-pump in rat pheochromocytoma cells

Nerve growth factor (NGF) induces neuronal differentiation of rat pheochromocytoma cells (PC12). Here we show that NGF causes a stimulation of Na⁺,K⁺-pump mediated K⁺ influx, with a maximum at 30 min after addition of NGF. The stimulation of the Na⁺,K⁺-pump is completely blocked by the Na⁺-flux inhibitor amiloride (0.2 mM) and can be mimicked by the Na⁺ ionophore monensin. These results suggest that NGF causes a rapid enhancement of Na⁺ influx leading to an activation of the Na⁺,K⁺-pump, a mechanism similar to the action of other growth factors.     

Propidium iodide as a probe for the study of chromatin thermal denaturationin situ

Summary The possibility of using propidium iodide, a phenanthridinic fluorochrome specific for double-stranded nucleic acids, for the study of chromatin thermal denaturationin situ has been examined. Smears of lymphocytes and hepatocyte nuclei from 15-day-old rats were fixed in acetic acid-ethanol (13 v/v), treated with RNAse and submitted to different protein extraction procedures, namely, incubation with pepsin, trypsin and sodium chloride.Denaturation experiments were performed in Sörensen buffer at pH 7.4 containing 10% formamide at temperatures between 27 and 95°C. The samples were stained with propidium iodide and mounted in buffer or glycerol. Measurements were performed with a microfluorometer at a wavelength of 546 nm.The results indicate a higher thermostability of lymphocytes as compared to hepatocytes. The denaturation pattern suggests a certain organization complexity of chromatin, better emphasized by the derivative curves which show the presence of at least three fractions with different melting points. After protein extraction, the denaturation curves exhibit a somewhat simplified pattern, with the disappearance of the most stable peak in the derivative curves. The samples mounted in glycerine exhibit a better stability of staining with time, and an increased quantum efficiency of the fluorochrome with regard to those mounted in buffer.These data confirm the importance of protein-DNA interactions in the organization of chromatin and point to some differences, depending on the cell type and on functional activity.