The Salmonella wien virulence plasmid pZM3 carries Tn1935, a multiresistance transposon containing a composite IS1936-kanamycin resistance element

Tn1935, a 23.5-kb transposon mediating resistance to ampicillin, kanamycin, mercury, spectinomycin, and sulfonamide was isolated from pZM3, an IncFIme virulence plasmid from Salmonella wien. Tn1935 possesses the entire sequence of Tn21 and contains two additional DNA segments of 0.95 and 2.7 kb carrying the ampicillin and kanamycin resistance genes, respectively. The latter is part of a composite element since it is flanked by two IS15-like insertion sequences (IS1936) in direct orientation. IS1936 is about 800 bp long and is closely related to IS15 delta, IS26, IS46, IS140, and IS176. Functional analysis of IS1936-mediated cointegrates shows that both insertion sequences are active and able to form cointegrates at the same frequency. Resolution of the cointegrates requires the presence of the host Rec system. The presence of the composite IS1936-element within Tn1935 supports the hypothesis that multidrug resistance transposons evolved by insertion of antibiotic determinants which are themselves transposable.     

Thymocytes express a mRNA that is identical to hypothalamic luteinizing hormone-releasing hormone mRNA

1. A luteinizing hormone-releasing hormone (LHRH)-like molecule produced by thymocytes is similar to hypothalamic LHRH in both bioactivity and antigenicity. 2. We determined whether this thymic LHRH is identical to or only homologous with hypothalamic LHRH by synthesizing and sequencing the cDNA of rat thymus LHRH. 3. The thymocyte and hypothalamic LHRH cDNAs are identical, indicating, that the amino acid sequences of LHRH produced in the hypothalamus and the immune system are also identical. 4. This is the first report showing conclusively that cell of the immune system transcribe the authentic mRNA for a hypothalamic releasing factor, LHRH.     

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.     

The involvement of calpain in the activation of protein kinase C in neutrophils stimulated by phorbol myristic acid

The Ca2+/phospholipid-dependent protein kinase (protein kinase C) of human neutrophils is converted to a proteolytically modified Ca2+/phospholipid-independent form (Inoue, M., Kishimoto, A., Takai, Y.U., and Nishizuka, Y. (1977) J. Biol. Chem. 252, 7610-7616) on incubation with neutrophil membranes in the presence of micromolar concentrations of Ca2+ and an endogenous Ca2+-requiring proteinase (Melloni, E., Pontremoli, S., Michetti, M., Sacco, O., Sparatore, B., Salamino, F., and Horecker, B. L. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 6435-6439). We have now demonstrated the appearance of a similar Ca2+/phospholipid-independent kinase in intact human neutrophils stimulated by phorbol 12-myristate 13-acetate (PMA). The following evidence supports the conclusion that the Ca2+/phospholipid-independent protein kinase recovered from the PMA-treated cells is a proteolytically modified form of the "native" protein kinase C. 1) In cells exposed to PMA, the rate of disappearance of Ca2+/phospholipid-dependent protein kinase C activity is correlated with the rate of appearance of the Ca2+/phospholipid-independent kinase. 2) The chromatographic behavior of the new protein kinase and its molecular size (approximately 65 kDa) are identical to those previously reported for the proteolytically modified form of protein kinase C. 3) The modified protein kinase no longer binds to the cell membrane and is recovered almost entirely in the cytosol fraction. 4) In neutrophils preloaded with inhibitors of the Ca2+-requiring proteinase, stimulation with PMA results in translocation of protein kinase C from the cytosol fraction to the particulate fraction, but the appearance of the soluble, Ca2+/phospholipid-dependent form is prevented. We conclude that binding of protein kinase C to the plasma membrane and its proteolytic conversion are related, but independent, processes both elicited by exposure of neutrophils to the phorbol ester. Proteolytic cleavage of the membrane-bound protein kinase C provides an alternative mechanism for its activation and may account for certain of the cellular responses observed in PMA-stimulated neutrophils.     

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.     

Following association to the membrane, human erythrocyte procalpain is converted and released as fully active calpain

When exposed to inside-out human erythrocyte vesicles, in the presence of micromolar Ca2+, the 80 kDa catalytic subunit of procalpain is processed through three successive and sequential steps. These include binding to the cytosolic surface of the membrane, followed by a very rapid conversion into the 75 kDa active subunit, and ultimately by spontaneous and complete release of this active proteinase form. Binding to the membranes is competitively inhibited by the endogenous natural inhibitor through the formation of the proteinase-inhibitor complex, in which form the 80 kDa subunit can no longer be associated to the membranes. Calcium ions and the natural endogenous inhibitor appear to be crucially involved in the modulation of this novel membrane-bound mediated activation of human red cell procalpain.     

The reversible activation by Mn2+ ions of the Ca2+-requiring neutral proteinase of human erythrocytes

Mn2+ (50 microM) satisfies the requirement for activity of the purified Ca2+-dependent neutral proteinase from human erythrocytes. Unlike the activation by Ca2+ [E. Melloni et al. (1984) Biochem. Int. 8, 477-489], the effect of Mn2+ is fully reversible and does not involve autodigestion of the native 80-kDa catalytic subunit. However, the native dimeric proenzyme (procalpain), which contains both the 80-kDa subunit and a smaller 30-kDa subunit, is not activated by Mn2+ alone but also requires the presence of micromolar concentrations of Ca2+. Under these conditions, 40% of the maximum activity is expressed without dissociation of the 80- and 30-kDa subunits. Mn2+, but not micromolar Ca2+, can also partially satisfy the metal requirement of the native 80-kDa subunit isolated after dissociation of the heterodimer. This activity is further enhanced by the addition of 5 microM Ca2+, which is ineffective in the absence of Mn2+. After procalpain is converted to active calpain by incubation with Ca2+ and substrate [S. Pontremoli et al. (1984) Biochem. Biophys. Res. Commun. 123, 331-337] full activity is observed with 5 microM Mn2+, which now substitutes completely for Ca2+. Activation of procalpain by Mn2+ represents a new mechanism for modulation of the Ca2+-dependent proteinase activity.     

Binding to erythrocyte membrane is the physiological mechanism for activation of Ca2+-dependent neutral proteinase

In the presence of micromolar concentrations of Ca2+ the catalytic 80 kDa subunit of human erythrocyte procalpain binds to the cytosolic surface of the erythrocyte membrane. Binding is rapid, highly specific and is reversed by the removal of Ca2+. In the bound form the 80 kDa catalytic subunit undergoes a rapid conversion to calpain, the active 75 kDa Ca2+-requiring proteinase. The activated proteinase produces extensive degradation of membrane components, particularly of band 4.1 and 2.1 proteins. Binding to membranes may represent an obligatory physiological mechanism for the conversion of procalpain to calpain.     

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.