Paracelsin; characterization by NMR spectroscopy and circular dichroism,and hemolytic properties of a peptaibol antibiotic from the cellulolytically active mnoldTrichoderma reesei. Part B

Summary Paracelsin, a hemolytic and membrane active polypeptide antibiotic of the peptaibol class which is excreted by the moldTrichderma reesei, was obtained by a simplified and isolation procedure utilziing hydrophobic adsorber resin. Investigation by¹³C nuclear magnetic resonance spectroscopy and circular dichroism revealed considerable helical portions in solution, and the very recently accomplished sequence determination of paracelsin allows the discussion of the results with regard to the closely related analogues, alamethicin and suzukacillin. A selective cleavage of the peptide was achieved by careful treatment with various acids, and a buffer of pH 8.25 and of high ionic strength made possible the quantitative determination of the C-terminal phenylalaninol released by means of ion-exchange chromatography. The significance of the production of paracelsin and related mycotoxins of the peptaibol class, exhibiting various kinds of biological activity, is discussed with respect to the extensive effort being made towards biotechnological applications of species, strains and cellulolytically highly active mutants of the fungusTrichoderma.Presented in part at the 5th European Symposium on Animal, Plant and Microbial Toxins, Hannover, August 29–September 2, 1983 and a lecture given at Ciby-Geigy/Basel in March 1984.Acknowledgment. We thank I. Ackermann for excellent and skilled technical assistance and gratefully acknowledge the help of R. Ratz for support in CD spectroscopy.     

Role of calcium in the histamine release induced by D-galactosamine from rat mast cells

Summary Rat peritoneal mast cells were isolated and purified by differential centrifugation in Ficoll. Cells pooled from three to four rats were suspended at approximately 10⁶ cells/ml in a buffered salt solution and incubated for 1 h at 37°C in 300 l volumes in the absence or presence (9×10^–4 M) of calcium chloride. Addition of D-galactosamine hydrochloride (DGM; 2.8×10^–4 M) caused (in addition to basal release) a mean ±SEM percent histamine release of 15.7±5.2 in the presence of Ca⁺⁺ and 19±4.9 in the absence of Ca⁺⁺ (p>0.05). It is suggested that D-galactosamine does not require extracellular Ca⁺⁺ for the release of histamine from the rat mast cell.A preliminary analysis of these results was presented at the International Symposium on calcium entry blockers and tissue protection, Rome, 15–16 March 1984.     

Recombinant synthesis of mouse Zn3-β and Zn4-α metallothionein 1 domains and characterization of their cadmium(II) binding capacity

Genetic engineering, coupled with spectro scopic analyses, has enabled the metal binding proper ties of the α and β subunits of mouse metallothionein 1 (MT) to be characterized. A heterologous expression system in E.coli has led to high yields of their pure zinc-complexed forms. The cadmium(II) binding properties of recombinant Zn₄-αMT and Zn₃-βMT have been studied by electronic absorption and circular dichroism. The former binds Cd(II) identically to α fragments obtained from mammalian organs, showing that the recombinant polypeptide behaves like the na tive protein. Titration of Zn₃-βMT with CdCl₂ results in the formation of Cd₃-βMT. The addition of excess Cd(II) leads to Cd₄-βMT which, with the extra loading of Cd(II), unravels to give rise isodichroically to Cd₉-βMT. The effect of cadmium-displaced Zn(II) ions and excess Cd(II) above the full metal occupancy of three has been studied using Chelex-100. The Cd₃-βMT species is stable in the presence of this strong metal-chelating agent. Received 20 May 1997; received after revision 7 July 1997; accepted 9 July 1997     

Cellular uptake of amelogenin,and its localization to CD63, and Lamp1-positive vesicles

Proteins of the developing enamel matrix include amelogenin, ameloblastin and enamelin. Of these three proteins amelogenin predominates. Protein-protein interactions are likely to occur at the ameloblast Tomes’ processes between membrane-bound proteins and secreted enamel matrix proteins. Such protein-protein interactions could be associated with cell signaling or endocytosis. CD63 and Lamp1 are ubiquitously expressed, are lysosomal integral membrane proteins, and localize to the plasma membrane. CD63 and Lamp1 interact with amelogenin in vitro. In this study our objective was to study the molecular events of intercellular trafficking of an exogenous source of amelogenin, and related this movement to the spatiotemporal expression of CD63 and Lamp1 using various cell lineages. Exogenously added amelogenin moves rapidly into the cell into established Lamp1-positive vesicles that subsequently localize to the perinuclear region. These data indicate a possible mechanism by which amelogenin, or degraded amelogenin peptides, are removed from the extracellular matrix during enamel formation and maturation. Received 27 September 2006; received after revision 24 November 2006; accepted 5 December 2006     

Dual modes of 5-(N-ethyl-N-isopropyl)amiloride modulation of apical dipeptide uptake in the human small intestinal epithelial cell line Caco-2

Selective pharmacological Na⁺/H⁺ exchange (NHE) inhibitors were used to identify functional NHE isoforms in human small intestinal enterocytes (Caco-2) and to distinguish between direct and indirect effects on transport via the intestinal di/tripeptide transporter hPepT1. The relative potencies of these inhibitors to inhibit ²²Na⁺ influx identifies NHE3 and NHE1 as the apical and basolateral NHE isoforms. The Na⁺-dependent (NHE3-sensitive) component of apical dipeptide ([¹⁴C] Gly-Sar) uptake was inhibited by the selective NHE inhibitors with the same order of potency observed for inhibition of apical ²²Na⁺ uptake. However, 5-(N-ethyl-N-isopropyl) amiloride (EIPA) also reduced [¹⁴C]Gly-Sar uptake in the absence of Na⁺ and this inhibition was concentration and pH (maximal at pH 5.5) dependent. NHE3 inhibition by S1611 and S3226 modulates dipeptide uptake indirectly by reducing the transapical driving force (H⁺ electrochemical gradient). EIPA (at 100 μM) has similar effects, but at higher concentrations (>200 μM) also has direct inhibitory effects on hPepT1.Received 28 February 2005; received after revision 20 April 2005; accepted 20 May 2005     

Purification from human plasma of endogenous dodium transport inhibitor(s)

Summary Na⁺, K⁺-ATPase inhibitors extracted from plasma of healthy human subjects displaced³H-ouabain binding to human erythrocytes and inhibited the Na⁺ efflux catalyzed by the Na⁺, K⁺-pump and unexpectedly the Na⁺, K⁺-cotransport system without alteration of the Na⁺, Na⁺-exchange or the Na⁺ passive permeability. This suggests the presence in healthy human plasma of endogenous factors with ouabain-like and furosemide-like activities.Acknowledgments. We are indebted to Dr M. A. Devynck for her advice on chemical measurements and to Dr R. P. Garay for his help with flux measurements     

Distribution of (Na++K+)-ATPase in the hindgut ofGlossina morsitans morsitans Westwood

Summary (Na⁺+K⁺)-ATPase activity was higher in preparations from the ileum ofGlossina mortisans than in those from the rectum. This result suggests that the ileum as well as the rectum, may play a role in osmoregulation in the tsetse fly.     

Displacement activity of some natural cularine alkaloids at striatal3H-SCH 23 390 and3H-raclopride binding sites

Five natural cularines isolated from the aerial parts ofSarcocapnos crassifolia (Fumariaceae) and a cularioid isolated from the bark ofGuatteria ouregou (Annonaceae) were tested for their ability to displace³H-SCH 23 390 and³H-raclopride from their striatal binding sites. Celtisine, breoganine and cularidine were able to inhibit the binding at D-1 and D-2 dopaminergic sites at nanomolar concentrations. Other alkaloids were active at micromolar concentrations. These data suggest that the presence of an oxepine system in the isoquinoline skeleton could lead to compounds which would be very active and possibly selective at dopaminergic receptor sites.     

Hydrolysis of isoprenyl diphosphates with the acid phosphatase fromCinnamomum camphora

Direct observations of the enzymatic hydrolysis of C₁₀ acyclic allylic isoprenyl diphosphates by an acid phosphatase from the leaves ofCinnamomum camphora (camphor tree) were made using¹H and³¹P NMR spectrometers. The measurements indicated that the allylic primary diphosphates, geranyl diphosphate and neryl diphosphate, were hydrolyzed to their corresponding alcohols in a sequential manner via their corresponding monophosphates, whereas the allylic tertiary diphosphate, linalyl diphosphate, was hydrolyzed only to its corresponding monophosphate.     

The effect of cyclic AMP on Na+ and K+ transport systems in mouse macrophages

Summary Exogenous cyclic AMP (cAMP) inhibits the Na⁺, K⁺-cotransport system and stimulates the Na⁺, K⁺-pump and Na⁺, Ca²⁺ exchange in mouse macrophages. These effects are enhanced by inhibition of phosphodiesterase with methylisobutylxanthine (MIX). MIX alone showed little or no effect. A similar response was observed after stimulation of endogenous production of cAMP by isoproterenol.     

Inhibition of the sodium pump does not cause a stoichiometric decrease of ATP-production in energy limited fish hepatocytes

In isolated goldfish hepatocytes underaerobic conditions the energy requirement for the sodium pump (calculated from Rb⁺ flux) is closely matched by the ouabain-sensitive fraction of oxygen consumption, whereas during in vitroanoxia (cyanide inhibition of the electron transport chain) the measured ATP demand of the sodium pump clearly exceeds ouabain-sensitive ATP production by anaerobic glycolysis. We conclude that when the energy status of cells is low, part or all of the ATP spared by the inhibition of a particular function may be used for fuelling other ATP-consuming functions.     

Cationic and secretory effects of BPDZ 44 and diazoxide in rat pancreatic islets

The present study aimed at comparing the effects of low concentrations of BPDZ 44, a new pyridothiadiazine derivative, and diazoxide on⁸⁶Rb outflow,⁴⁵Ca outflow,⁴⁵Ca uptake and insulin release from rat pancreatic islets. Both drugs caused similar modifications, but the effects of BPDZ 44 on the cationic and secretory events were much more marked than those of diazoxide. It is suggested that BPDZ 44 could be valuable tool for further studies of the K_ATP channels.     

Self and non-self discrimination is needed for the existence rather than deletion of autoimmunity: the role of regulatory T cells in protective autoimmunity

Autoimmune T cells have been viewed for decades as an outcome of immune system malfunction, and specifically as a failure to distinguish between components of self and non-self. The need for discrimination between self and non-self as a way to avoid autoimmunity has been repeatedly debated over the years. Recent studies suggest that autoimmunity, at least in the nervous system, is the bodys defense mechanism against deviations from the normal. The ability to harness neuroprotective autoimmunity upon need is evidently allowed by naturally occurring CD4+CD25+ regulatory T cells, which are themselves controlled by brain-derived compounds. These findings challenge widely accepted concepts of the need for discrimination between self and non-self, as they suggest that while such discrimination is indeed required, it is needed not as a way to avoid an anti-self response but to ensure its proper regulation. Whereas a response to non-self can be self-limited by a decreased presence of the relevant antigen, the response to self needs a mechanism for strict control, such as that provided by the naturally occurring regulatory T cells.Received 8 June 2004; accepted 6 July 2004     

Studies of total and protein-bound plasma Mg++ in wild and hatchery-reared coho salmon smolts in freshwater and in seawater

Summary Total plasma Mg⁺⁺ and Ca⁺⁺, Mg⁺⁺ in erythrocytes as well as protein-bound plasma Mg⁺⁺ were investigated in wild and hatchery-reared smolts. The proportion of plasma Mg⁺⁺ which was bound to plasma protein did not change significantly during entry into seawater, even though the in vitro addition of exogenous Mg⁺⁺ to the plasma showed that additional binding was possible.     

PcF protein from <Emphasis Type="Italic">Phytophthora cactorum</Emphasis> and its recombinant homologue elicit phenylalanine ammonia lyase activation in tomato

The phytotoxic protein PcF (Phytophthora cactorum-Fragaria) is a 5.6-kDa cysteine-rich, hydroxyproline- containing protein that is secreted in limited amounts by P. cactorum, an oomycete pathogen of tomato, strawberry and other relevant crop plants. Although we have shown that pure PcF triggers plant reactivity, its mechanism of action is not yet understood. Here we show that PcF, like other known fungal protein elicitors involved in pathogen-plant interaction, stimulates the activity of the defense enzyme phenylalanine ammonia a key step in understanding the mechanism of action of PcF at a molecular level is knowledge of its three-dimensional structure, we overexpressed this protein extracellularly in Pichia pastoris. The preliminary structural and functional characterization of a recombinant PcF homologue, N4-rPcF, is reported. Interestingly, although N4-rPcF is devoid of proline hydroxylation and has four additional amino acid residues attached to its N terminus, its secondary structure and biological activity are indistinguishable from wild-type PcF.Received 22 February 2003; received after revision 25 March 2003; accepted 14 April 2003     

Oviposition-deterring pheromone inRhagoletis cerasi L.: Purification and determination of the chemical constitution

Summary An oviposition-deterring pheromone (ODP) of the European cherry fruit flyRhagoletis cerasi L. was isolated from faeces using cellulose and several reverse phase TLC and HPLC procedures. The biological activity was evaluated by means of behavior tests and by electrophysiological recordings from tarsal contact chemoreceptors. The compound was structurally characterized as a N[15(-glucopyranosyl)oxy-8-hydroxypalmitoyl]-taurine by spectroscopic means. The configurations of C-8 and C-15 of the fatty acid constituent remain to be established by synthetic work.     

The effect of a phorbol ester upon the cholinergic regulation of potassium permeability in the rat submandibular gland

Acetylcholine releases calcium from cytoplasmic stores and permits an influx of calcium in salivary acinar cells. The resultant rise in [Ca²⁺]_i causes an increase in potassium permeability which is an important part of the secretory response. We have investigated the effects of 12-0-tetradecanoyl phorbol-13-acetate, a potent activator of protein kinase C, upon this regulation of potassium permeability in superfused pieces of rat submandibular salivary gland. This compound inhibited the initial [Ca²⁺]_o-independent component of the response of acetylcholine but had no effect upon the subsequent [Ca²⁺]_o-dependent phase. This compound does not, therefore, appear to inhibit receptor-regulated calcium influx.     

42K spaces in submandibular gland of early postnatal rats: effects of carbachol and transport inhibitors

Summary Equivalent spaces of⁴²K were measured in fragments of the submandibular gland of 1-, 7-, 14- and 21-day-old and adult rats in the absence and presence of carbachol and the transport inhibitors ouabain and furosemide. The results indicate that the⁴²K space was increased by carbachol in an ouabain-sensitive manner at all ages studied and that part of the secretagogue-stimulated K uptake occurred by way of a furosemide-sensitive transport system in the latter part of the postnatal period and in the adult.     

Decreased rates of Ca2+-dependent heat production in slow- and fast-twitch muscles from the dystrophic (mdx) mouse

Using a newly developed microcalorimetric approach to assess the rate of energy expenditure for intracellular [Ca²⁺] homeostasis in isolated muscles at rest, we found this was lower inmdx than in control mouse muscles, by 62% and 29% in soleus and extensor digitorum longus, respectively. Differences in total and Ca²⁺-dependent rates of specific heat production betweenmdx and control were enhanced during sustained, KCl-induced stimulation of energy dissipation. These results suggest that the low sacroplasmic energy status of dystrophic muscles is not due to any excessive energy expenditure for intracellular [Ca²⁺] homeostasis.