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.     

Delayed-type cutaneous hypersensitivity to melanoma antigens and its implication in active specific immunotherapy in cancer

Summary In this study, we explored whether soluble tumor-cell surface-associated antigens (TAA) might be derived from autochthonous as well as allogeneic sources as immunogens for active specific immunotherapy. Using two popular cell membrane-bound antigen extraction techniques (3 M KCl and isotonic-hypotonic NaCl), we examined the immunogenic potential of such TAA and the specificity of immunologic host reactivity through a delayed-type cutaneous hypersensitivity reaction (DTH) as a guideline for their immunogenic potential in a human malignant melanoma model system. We found that either extraction technique could provide soluble TAA from both autochthonous and allogeneic sources capable of eliciting DTH. While evidence of positive DTH with autochthonous TAA reaffirms the immunogenicity of such TAA, the specificity of host reactivity against TAA derived from allogeneic sources is extremely difficult to establish, even with TAA partially purified by column chromatography in Sephadex G-200. Patients exhibited reactivity to other TAA derived from tumors of different histologies and often to more than one component isolated by column chromatography. Furthermore, when a group of melanoma patients was tested against a panel of melanoma antigens in any random combination, DTH to allogeneic TAA was seen in an unpredictable order and with inconsistent frequency. We conclude, therefore, that while autochthonous antigen immunizations may be justified, more careful studies will be necessary to define the antigenic profile of a given tumor (individual specificity vs shared specificity), establish specificity of alloantigens, and devise suitable methods for testing immunologic specificity for alloantigens, before rational immunotherapy with allogeneic tumor antigens will be feasible.     

Gas chromatographic method for the simultaneous determination of hydralazine and its acetylated metabolite in serum using a nitrogen-selective detector

A relatively simple gas chromatographic method has been developed for the quantitative determination of hydralazine simultaneously with its acetylated metabolite, 3-methyl-s-triazolo[3,4-α]phthalazine (MTP). The proteins were removed by means of sulfosalicylic acid and Sure-Sep®. On treatment with formic acid, hydralazine and its internal standard were converted into their formylated derivatives. These derivatives, MTP and its internal standard were extracted with toluene and determined by gas chromatography with a nitrogen-selective detector. The lower limits of detection for hydralazine and MTP were 0.13 and 0.27 μmol/l, respectively.     

Amino acid transport in pig lymphocytes Enhanced activity of transport system asc following mitogenic stimulation

Changes in neutral amino acid transport activity caused by addition of phytohaemagglutinin-P to quiescent peripheral pig lymphocytes have been evaluated by measurements of ¹⁴C-labelled neutral and analogue amino acids under conditions approaching initial entry rates. Utilizing methylaminoisobutyric acid, the best model substrate of System A, we confirmed our previous report (Borghetti, A.F., Kay, J.E. and Wheeler, K.P. (1979) Biochem. J. 182, 27–32) on the absence of this transport system in quiescent cells and its emergence following stimulation. Furthermore, we demonstrated the presence in quiescent cells of an Na⁺-dependent transport system for neutral amino acids that has been characterized as System ASC by several criteria including intolerance to methylaminoisobutyric acid, strict Na⁺-dependence, the property of transtimulation and specificity for pertinent substrates such as alanine, serine, cysteine and threonine. Analysis of the relationship between influx and substrate concentration revealed that two independent saturable components contribute to entry of alanine in quiescent cells: a low affinity ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= ≈4}\text{mM}$) and a high affinity ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= ≈0.2}\text{mM}$) component. The high affinity component could be inhibited in a competitive way by serine, cysteine and threonine, but methylaminoisobutyric acid did not change appreciably its constants. The enhanced activity of alanine transport through the ASC system observed in activated cells resulted from a large increase in the capacity ($\text{V}$) of the high affinity component without any substantial change in the apparent affinity constant ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}$).     

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.