Interaction of caffeine-, IP3- and vanadate-sensitive Ca2+ pools in acinar cells of the exocrine pancreas

Summary Previous studies have shown the existence of functionally distinguishable inositol 1,4,5-trisphosphate- (IP₃) sensitive and IP₃-insensitive nonmitochondrial intracellular Ca²⁺ pools in acinar cells of the exocrine pancreas. For further characterization of Ca²⁺ pools, endoplasmic reticulum (ER) membrane vesicles were separated by Percoll gradient centrifugation which allowed us to distinguish five discrete fractions designatedP ₁ toP ₅ from the top to the bottom of the gradient. Measuring Ca²⁺ uptake and Ca²⁺ release with a Ca²⁺ electrode, we could differentiate three nonmitochondrial intracellular Ca²⁺ pools; (i) an IP₃-sensitive Ca²⁺ pool (IsCaP), vanadate- and caffeine-insensitive, (ii) a caffeine-sensitive Ca²⁺ pool (CasCaP), vanadate- and IP₃-insensitive, and (iii) a vanadate-sensitive Ca²⁺ pool (VasCaP), neither IP₃- nor caffeine-sensitive, into which Ca²⁺ uptake is mediated via a Ca²⁺ ATPase sensitive to vanadate at 10^–4 mol/liter. A fourth Ca²⁺ pool is neither IP₃- nor caffeine- or vanadate-sensitive. Percoll fractionP ₁ contained essentially the IsCaP, CasCaP and VasCaP and was mainly used for studies on Ca²⁺ uptake and Ca²⁺ release.When membrane vesicles were incubated in the presence of caffeine (2×10^–2 mol/liter), Ca²⁺ uptake up to the steady state [Ca²⁺] did not appear to be altered as compared to the control Ca²⁺ uptake. However, in control vesicles spontaneous Ca²⁺ release occurred after the steady state had been reached, whereas cfffeine-pretreated vesicles did not spontaneously release Ca²⁺. Addition of IP₃ at steady state [Ca²⁺] induced similar Ca²⁺ release followed by Ca²⁺ reuptake in both caffeine-pretreated and control vesicles. However, when caffeine was acutely added at steady state, Ca²⁺ was released from all Ca²⁺ pools including the IsCaP. Following Ca²⁺ reuptake after IP₃ had been added, a second addition of IP₃ to control vesicles induced further but smaller Ca²⁺ release, and a third addition resulted in a steady Ca²⁺ efflux by which all Ca²⁺ that had been taken up was released. This steady Ca²⁺ release started at a Ca²⁺ concentration between 5.5–8 ×10^–7 mol/liter and could also be induced by the IP₃ analogue inositol 1,4,5-trisphosphorothioate (IPS₃) or by addition of Ca²⁺ itself. Ruthenium red (10^–5 mol/liter) inhibited both caffeine-induced as well as Ca²⁺-induced but not IP₃-induced Ca²⁺ release. Heparin (100 g/m) inhibited IP₃-but not caffeine-induced Ca²⁺ release. The data indicate the presence of at least three separate Ca²⁺ pools in pancreatic acinar cells: the IsCaP, CasCaP and VasCaP. During Ca²⁺ uptake these Ca²⁺ pools appear to be separate. However, when steady state is reached, we assume that these Ca²⁺ pools come into contact and total Ca²⁺ release from all three pools can occur. The mechanism of this contact of Ca²⁺ pools is not clear but seems to be different from that induced by GTP in the presence of polyethylene glycol, which probably involves fusion of membranes.     

Mutations responsible for high light sensitivity in an atrazine-resistant mutant of Synechcystis 6714

The primary target of photoinhibition is the photosystem II reaction center. The process involves a reversible damage, followed by an irreversible inhibition of photosystem II activity. During cell exposition to high light intensity, the D₁ protein is specially degraded. An atrazine-resistant mutant of Synechocystis 6714, AzV, reaches the irreversible step of photoinhibition faster than wild-type cells. Two point mutations present in the psbA gene of AzV (coding for D₁) lead to the modification of Phe 211 to Ser and Ala 251 to Val in D₁. Transformation of wild-type cells with the AzV psbA gene shows that these two mutations are sufficient to induce a faster photodamage of PSII. Other DCMU-and/or atrazine-resistant mutants do not differ from the wild type when photoinhibited. We conclude that the Q_B pocket is involved in PSII photodamage and we propose that the mutation of Ala 251 might be related to a lower rate of proteolysis of the D₁ protein than in the wild type.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PSII photosystem II - RCII reaction center II     

Functional interactions between subunits of aspartate aminotransferase: Formation of monoliganded dimers during titration of the apoenzyme by pyridoxal 5′-phosphate

The interaction between apoaspartate aminotransferase and pyridoxal 5′-phosphate at either pH 8.3 (active form of holoenzyme) or pH 5.0 (inactive form) corresponds to a strong quenching of tryptophan fluorescence. The hybrid molecule containing one pyridoxal 5′-phosphate bound per dimer has been prepared both by electrofocusing and by ion exchange chromatography. At both pH values, the fluorescence of the hybrid is 80 to 85% of the arithmetic mean between the fluorescence of the symmetrical holoenzyme and apoenzyme. This is direct evidence of energy transfer from tryptophan residues of the subunit of apoenzyme to the coenzyme of the other subunit.Fluorescence intensity was used to determine the quantity of hybrid holoapoenzyme formed during titration of the apoenzyme by pyridoxal 5′-phosphate. At pH 8.3 a non-linear decrease in the fluorescence is observed, corresponding to 60% of hybrid for the point of half reactivation; this value corresponds to the percentage obtained by electrofocusing (Schlegel & Christen, 1974). At pH 5.0, the decrease in fluorescence is linear during pyridoxal binding; this indicates that at this pH the hybrid is never obtained at detectable concentrations. These results indicate strong interactions between subunits of aspartate aminotransferase corresponding to a weakly negative co-operativity at alkaline pH and a positive cooperativity at acidic pH for the binding of the coenzyme.     

Effects of the avermectin analogue MK-243 on vitellogenesis and reproduction in the ixodid tick,Amblyomma hebraeum

A dose-dependent reduction in weight of total egg mass and a slightly increased oviposition latency were observed following injection of the avermectin, analogue MK-243 (4-epi-methylamino-4-deoxyavermectin B₁) directly into the haemocoel of engorged femaleAmblyomma hebraeum Koch (Acari: Ixodidae). Egg laying was almost completely inhibited at 100 g/kg body weight. MK-243 markedly inhibited ovary development and vitellogenesis. Ticks treated with 100 g MK-243/kg also had one-tenth the haemolymph ecdysteroid concentration compared to controls 10 days post-engorgement. Thus, among its other effects on ticks, the avermectins also inhibit the process of vitellogenesis.     

Inter-relationships between pituitary-adrenal hormones and catecholamines during a 6-day Nordic ski race

The aim of the study was to investigate the inter-relationships between pituitary-adrenal hormones and catecholamines during a prolonged competition over 6 days. Plasma adrenocorticotropic hormone (ACTH), cortisol (C), beta-endorphin (beta EP), free and sulphated adrenaline (A) and noradrenaline (NA) were measured in 11 volunteer male subjects during a national Nordic-ski race (323 km). Blood samples were obtained before the competition in the evening as control (D0), and before and after each day's racing (D1-D6). The mean daily heart rate (fc) was calculated from fc values recorded every minute during the race. The results showed the following: changes in mean fc [from 147 (SEM 3) to 156 (SEM 3) beats.min-1 according to the day] were not significant during the race. Diurnal variations in ACTH, beta EP and C were no longer apparent after the race: evening levels were higher than their respective D0 values during the race, except on D3 when there was a lack of response to exercise in the three hormones. Unlike ACTH and beta EP, pre- and postexercise C values on D1 and D2 were higher than those on the subsequent days (P less than 0.001). In contrast, there was a progressive accumulation of A and NA in pre- and postrace concentrations which reached a plateau in about 4 days. Positive correlations between exercise responses in ACTH, C and beta EP were found especially on D3 and D6 (P less than 0.001) but there were no significant correlations between catecholamines and the other three hormones. Thus, prolonged competition over 6 days evoked different control mechanisms for hormones of the pituitary-adrenal axis and catecholamines.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of some growth regulators on in vitro tuberization in Dioscorea alata L. ‘Brazo fuerte’ and D. abyssinica Hoch.

Summary Nodal cuttings of Dioscorea alata L. Brazo fuerte and D. abyssinica Hoch. were cultured in vitro to assess the influence of NAA on the production of microtubers. In D. alata, high concentrations of NAA (27 and 54 M) favored the production of large microtubers, whereas the highest number of microtubers was obtained with 2.7 M. D. abyssinica was found to be more sensitive to NAA since concentrations higher than 0.27 M promoted the growth of callus on the root system. In this species, the production of the largest microtubers was obtained at 2.7 M whereas the number of microtubers was not affected by any concentration tested. In D. alata, the effects of ABA and BAP were also evaluated. The weight of the microtubers increased with increasing concentrations of ABA. This effect, however, was observed only on expiants cultured under 8 h photoperiod, but not on those cultured under 16 h. Finally the presence of BAP at concentrations as low as 0.22 M adversely affected the survival of the explants.Abbreviations ABA abscissic acid - BAP benzyl aminopurine - NAA -naphthaleneacetic acid - MS Murashige and Skoog     

Unequal Day-Night Terbutaline I.V. Dosing in Acute Severe Asthma: Effect on Nocturnal Bronchial Patency, Heart Rate, and Arterial Pressure

Our study investigated the differential effects of continuous or unequal day-night terbutaline dosing on circadian bronchial patency, heart rate, and arterial pressure in severe acute asthma. Forty-five hospitalized asthmatic patients (19 women and 26 men, mean age 45.4 years, mean weight 63.5 kg) were included in this multicenter study. Three groups of patients (corresponding to three dosing schedules) were randomized; the three groups were comparable, since no statistically significant difference was detected in the age, weight, or peak expiratory flow values at the beginning of the study. In order to reach immediately the concentrations of terbutaline corresponding to the desired unequal day-night concentrations, a theoretical pharmacokinetic simulation was done to predict the outcome in terms of the plasma concentrations after the three dosing regimens; the results of this simulation allowed us to calculate the initial bolus dose to be given over 5 min to groups A, B, and C, i.e., 1.47, 2.94, and 4.41 Mg/kg, respectively. This bolus was given to all patients at 0700 h, the beginning of the study. The patients were randomly divided into three groups (A, B, C) receiving one of these treatments: 0.0111 mg/kg of terbutaline i.v. from 0700 to 1900 h at a constant rate delivered by an electrical pump and 0.0222 mg/kg of terbutaline i.v. from 1900 to 0700 h at a constant rate (A) (one third the total daily dose during the day and the remaining two thirds at night), 0.0166 mg/kg of terbutaline i.v. from 0700 to 1900 h at a constant rate and 0.0166 mg/kg of terbutaline i.v. from 1900 to 0700 h at a constant rate (B) (one half the total daily dose during the day and the remaining one half at night), or 0.0222 mg/kg of terbutaline i.v. from 0700 to 1900 h at a constant rate and 0.0111 mg/kg of terbutaline i.v. from 1900 to 0700 h at a constant rate (C) (two thirds the total daily dose during the day and the remaining one third at night). Since acute severe asthma could not be treated without steroids, a 40 mg dose of SoluMedrol was injected into all patients at 0700. Peak expiratory flow rate, heart rate, systolic arterial pressure, and possible side effects were recorded at different times during the 24-h scale: 0700, 1000, 1300, 1600, 1900, 2300, 0300, and 0700 h. Our results have shown a significant therapeutic effect of terbutaline i.v. dosing in severe acute asthma whatever the unequal daynight dosing, but did not demonstrate the efficacy of one of the three dosing schedules over the others.     

Protein kinase activities during maturation in xenopus laevis oocytes

Protein kinase activities have been compared in ovarian oocytes and in ovulated eggs of $\text{Xenopus laeyis}$.In ovaries and ovarian oocytes, we have detected, in addition to an already known (1) cyclic AMP stimulated phosphoprotein kinase, a second very active phosphoprotein kinase which is cAMP-independent.Besides these two activities, a third protein kinase activity becomes detectable after maturation and ovulation: it is a cAMP and cGMP-dependent histone kinase.