Cyclic AMP inhibition of phosphoinositide turnover in human neutrophils

The effect of increased intracellular levels of cyclic AMP on phosphoinositide metabolism was studied in human neutrophils stimulated with fMet-Leu-Phe. Intracellular cyclic AMP was raised by preincubation either with dibutyryl cyclic AMP and theophylline or with prostaglandin E1. Concentrations of dibutyryl cyclic AMP and theophylline fully inhibitory for the metabolic responses inhibited phosphoinositide breakdown and phosphatidic acid formation to a large extent. The accumulation of the water-soluble inositol phosphates was also measured. In agreement with the data obtained on the phospholipids, inositol phosphate generation was found to be severely, though not completely, reduced. Treatment with dibutyryl cyclic AMP and theophylline also inhibited resynthesis of membrane inositol lipids. Treatment with prostaglandin E1 had a similar, though less, marked effect on inositol lipid turnover, which was parallel with a smaller inhibition of metabolic responses. We therefore suggest that the elevation of intracellular cyclic AMP mainly affects neutrophil responses by inhibiting the phosphoinositide cycle.     

Inhibition by verapamil of neutrophil responses to formylmethionylleucylphenylalanine and phorbol myristate acetate. Mechanisms involving Ca2+ changes, cyclic AMP and protein kinase C

Verapamil inhibits in human neutrophils the respiratory burst, the secretion and the change of transmembrane potential induced by formylmethionylleucylphenylalanine, a Ca2+-dependent stimulus, and by phorbol myristate acetate, a Ca2+-independent stimulus. Besides the blocking of Ca2+ channels, many mechanisms are responsible for the inhibition of neutrophil responses. In fact, verapamil (i) increases the intracellular cAMP concentration, potentiates the cAMP response induced by the chemotactic peptide and induces the appearance of a cAMP response also when the stimulant is phorbol myristate acetate; (ii) causes a decrease of Ca2+ association to cell membranes, so depleting the pools of exchangeable Ca2+ and depressing the 'Ca2+ response' in terms of rise in [Ca2+]i monitored with Quin 2 and of rapid mobilization from cell membranes monitored by chlorotetracycline fluorescence change; (iii) inhibits the Ca2+-activated phospholipid-dependent protein kinase C. The data, discussed in relation to the biochemical mechanisms of the stimulus-response coupling, are compatible with the hypothesis of an involvement of the activation of protein kinase C as key step in the sequence of transduction events for the induction of many neutrophil functions.     

HERSIM: a microcomputer program designed to compute the limits of conversion for real homogeneous isothermal enzymic reactors

HERSIM is a program written in BASIC designed to aid the investigatorinterested in determining the substrate conversion in a realhomogeneous isothermal enzymic reactor, for various kineticequations. The program runs after tracer data relative to aDirac impulse to the reactor have been entered, and computesthe two limits of real conversion: total segregation and maximummixedness. The kinetic constants of the reacting system areinput as data, and the variation of conversion with reactortemperature between given limits is computed as accurately asrequested. Received on November 6, 1986; accepted on March 4, 1987     

Studies on stimulus-response coupling in human neutrophils. II. Relationships between the effects of changes of external ionic composition on the properties of N-formylmethionylleucylphenylalanine receptors and on the respiratory and secretory responses

Studies were carried out on the mechanism responsible for the enhancement of the respiratory and secretory responses to N-formylmethionylleucylphenylalanine (fMet-Leu-Phe) exhibited by human neutrophils suspended in Na+-free, high-K+ buffered solution. The results demonstrate that: (a) the variation of Na+ concentration in the suspending solution induces in human neutrophils a marked modification of the recognition apparatus for the chemotactic peptide fMet-Leu-Phe, the lack of or low concentration of this ion increasing the number of the receptors and their specific affinity for the ligand; (b) the greater respiratory burst and secretion induced by fMet-Leu-Phe in human neutrophils suspended in Na+-free, high-K+ medium are due to the increased formation of receptor-ligand complexes at the cell membrane; (c) the greater respiratory response is partially due also to a higher efficiency of these receptor-ligand complexes. The molecular mechanism by which Na+ exerts a regulative role on the properties of the recognition apparatus for the chemotactic peptide and its possible significance are discussed.     

Effects of water deprivation on the water content of cattle skin

In cattle the water content of the skin was determined (1) in the normal animals; (2) after a 3-day period of water deprivation (dehydration); (3) one hour after the water deprived animals had resumed drinking (rehydration)and (4) one hour after the beginning of infusion of water into the rumen of normal animals (overhydration). Dehydration reduced the water content of the skin from 70.6 to 65.8% on average. Rehydration led to a partial restoration of the water content of the skin. Overhydration did not have a measurable effect on the water content of the skin. A rough estimation of the total amount of water lost during dehydration from the total skin of each animal indicated that on average the calves lost 315 ml,the oxen 1,336 ml of water from their skins.

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Zusammenfassung Beim Rind wurde der Wassergehalt der Haut bestimmt (1)in normalem Zustand der Tiere; (2) nach einer dreitägigen Wasserenthaltung der Tiere (Dehydration); (3) eine Stunde nachdem die dehydrierten Tiere wieder zu trinken begannen(Rehydration) und (4) eine Stunde nach begonnener Infusion von Wasser in den Pansen von normalen Tieren (Überhydration). Dehydration verursachte einen mittleren Abfall des Wassergehaltes der Haut von 70.6 auf 65.8%. Rehydration führte zu einer teilweisen Wiederherstellung des normalen Wassergehaltes der Haut, während Überhydration ohne messbaren Einfluss blieb. Der berechnete Wasserverlust von der gesamten Haut als Folge der Dehydration war 315 ml bei den Kälbern und 1.336 ml bei den Ochsen.

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Resume On a déterminé la teneur en eau de la peau des bovidés sous quatre conditions: (1) chez des bêtes à l'état normal; (2) par déshydratation (après 3 jours sans abreuvage); (3) une heure après que les bêtes déshydratées aient reçu à boire (réhydratation); (4) une heure après le début d'une infusion d'eau dans la panse de bêtes à l'état normal (surhydratation). L'état de déshydratation a provoqué une baisse moyenne du taux de l'eau cutanée de 70,6% à 65,8%. La réhydratation a eu pour conséquence la reconstitution partielle de la teneur en eau de la peau alors que la surhydratation ne semble pas avoir eu d'effets. La perte en eau calculée par suite de déshydratation fut de 315 ml pour les veaux, de 1,336 ml pour les boeufs.

     

Main Immunogenic Region of Torpedo Electroplax and Human Muscle Acetylcholine Receptor: Localization and Microheterogeneity Revealed by the Use of Synthetic Peptides

Most anti-nicotinic acetylcholine receptor (AChR) antibodies in myasthenia gravis are directed against an immunodominant epitope or epitopes [main immunogenic region (MIR)] on the AChR alpha-subunit. Thirty-two synthetic peptides, corresponding to the complete Torpedo alpha-subunit sequence and to a segment of human muscle alpha-subunit, were used to map the epitopes for 11 monoclonal antibodies (mAbs) directed against the Torpedo and/or the human MIR and for a panel of anti-AChR mAbs directed against epitopes on the alpha-subunit other than the MIR. A main constituent loop of the MIR was localized within residues alpha 67-76. Residues 70 and 75, which are different in the Torpedo and human alpha-subunits, seem to be crucial in determining the binding profile for several mAbs whose binding to the peptides correlated very well with their binding pattern to native Torpedo and human AChRs. This strongly supports the identification of the peptide loop alpha 67-76 as the actual location of the MIR on the intact AChR molecule. Residues 75 and 76 were necessary for binding of some mAbs and irrelevant for others, in agreement with earlier suggestions that the MIR comprises overlapping epitopes. Structural predictions for the sequence segment alpha 67-76 indicate that this segment has a relatively high segmental mobility and a very strong turning potential centered around residues 68-71. The most stable structure predicted for this segment, in both the Torpedo and human alpha-subunits, is a hairpin loop, whose apex is a type I beta-turn and whose arms are beta-strands. This loop is highly hydrophilic, and its apex is negatively charged. All these structural properties have been proposed as characteristic of antibody binding sites. We also localized the epitopes for mAbs against non-MIR regions. Among these, the epitope for a monoclonal antibody (mAb 13) that noncompetitively inhibits channel function was localized within residues alpha 331-351.     

Redox intermediates of Desulfovibrio gigas [NiFe] hydrogenase generated under hydrogen. M?ssbauer and EPR characterization of the metal centers

The hydrogenase (EC 1.2.2.1) of Desulfovibrio gigas is a complex enzyme containing one nickel center, one [3Fe-4S] and two [4Fe-4S] clusters. Redox intermediates of this enzyme were generated under hydrogen (the natural substrate) using a redox-titration technique and were studied by EPR and M?ssbauer spectroscopy. In the oxidized states, the two [4Fe-4S]2+ clusters exhibit a broad quadrupole doublet with parameters (apparent delta EQ = 1.10 mm/s and delta = 0.35 mm/s) typical for this type of cluster. Upon reduction, the two [4Fe-4S]1+ clusters are spectroscopically distinguishable, allowing the determination of their midpoint redox potentials. The cluster with higher midpoint potential (-290 +/- 20 mV) was labeled Fe-S center I and the other with lower potential (-340 +/- 20 mV), Fe-S center II. Both reduced clusters show atypical magnetic hyperfine coupling constants, suggesting structural differences from the clusters of bacterial ferredoxins. Also, an unusually broad EPR signal, labeled Fe-S signal B', extending from approximately 150 to approximately 450 mT was observed concomitantly with the reduction of the [4Fe-4S] clusters. The following two EPR signals observed at the weak-field region were tentatively attributed to the reduced [3Fe-4S] cluster: (i) a signal with crossover point at g approximately 12, labeled the g = 12 signal, and (ii) a broad signal at the very weak-field region (approximately 3 mT), labeled the Fe-S signal B. The midpoint redox potential associated with the appearance of the g = 12 signal was determined to be -70 +/- 10 mV. At potentials below -250 mV, the g = 12 signal began to decrease in intensity, and simultaneously, the Fe-S signal B appeared. The transformation of the g = 12 signal into the Fe-S signal B was found to parallel the reduction of the two [4Fe-4S] clusters indicating that the [3Fe-4S]o cluster is sensitive to the redox state of the [4Fe-4S] clusters. Detailed redox profiles for the previously reported Ni-signal C and the g = 2.21 signal were obtained in this study, and evidence was found to indicate that these two signals represent two different oxidation states of the enzyme. Finally, the mechanistic implications of our results are discussed.     

GROWTH PATTERN,REPRODUCTION, AND SELF-THINNING IN SEAWEEDS1

In unispecific plant stands, the logarithm of mean individual weight (w) depends on the logarithm of density (d) by the -3/4 power law (a slope of -1.5 and an intercept ranging from 2.3 to 5.0). The analysis of the w and d relationships in whole cohorts of two seaweed species from the Strait of Gibraltar shows deviations from the canonical equation. The kelp Phyllariopsis purpurascens (C. Agardh) Henry et South (Phaeophyta) growing at a 30-m depth has the lowest intercept value (0.6) recorded for any plant species and a slope not significantly different from -1.5. The slope value is in accordance with those found in species whose growth is not stopped by reproduction. Irradiance under a single layer of blades was lower than the photosynthetic light compensation point, and this could be due to overdispersion of the shoots and, in consequence, to the low intercept value of the self-thinning equation. The w to d relationships in Asparagopsis armata Harvey (Rhodophyta) show two different components: no dependence between these two variables (slope not significantly different from 0) at densities < 500 shoots·m^?2, and a slope more negative (-2.1) than proposed by the -3/2 power law at densities > 500 shoots·m^?2. The pattern at high densities could be due to intraspecific competition for light, whereas the slope ～0 at low densities could be related to inhibition of growth by reproduction (cystocarp and carpospore production). Therefore, rather than being considered exceptional, we suggest that a gradient of variability could be expected in the dependence of w on d when specific growth patterns and reproduction are considered.     

Small-scale association measures in epibenthic communities as a clue for allelochemical interactions

The small-scale associations in a rocky subtidal community in the northwestern Mediterranean were studied by a development of the continuous line transect method. This method allowed the overall measurement of non-randomness in interspecific contacts and the assignment of an association index to each species-pair, whose, significance was tested by Monte Carlo procedures. At the same time, the continuous recording allowed the study of the weakening of the interactions with increasing distances. Our purpose was to uncover evidence for allelochemical mechanisms of space occupation and maintenance. A strong non-randomness was found in the interspecific associations. This was mostly due to the interactions of the poecilosclerid sponge Crambe crambe (Schmidt) with its neighbours, especially its negative associations with other sponge species. The strength of the relationships fell drastically over the first few centimeters from the contact borders of the different species. The results pointed strongly to an allelochemical mechanism. The extracts of this sponge featured high bioactivity in laboratory assays, and field experiments demonstrated that the sponge can inhibit the growth of species in the community studied. Standard sampling techniques would have overlooked the spatial structure present in the data. The study emphasizes the need for both contact data and distance data in order to identify the underlying processes reliably. The line transect method provides both types of information easily and allows testing of models and identification of organisms likely to use chemical defenses in space competition. Its use as a preliminary step in studies of chemical ecology might help to detect presumptive allelochemical processes prior to experimental work on the potentially active species.