Nonspecific elicitation of defense reaction in suspension tobacco cells by elicitors from Armillaria.

Ergosterol and chitin oligomers were detected in water extracts from Armillaria gallica, A. cepistipes, A. tabescens, A. ostoyae and A. mellea containing as active components elicitors able to trigger early events of defense reaction in suspension tobacco cells. More virulent strains of A. ostoyae and A. mellea had the same ability of elicitation as weak pathogens A. gallica, A. cepistipes, A. tabescens. The elicitation of the defense reaction early events by chitin oligomers was markedly enhanced by ergosterol probably due to the activation of several signal pathways.

A lipid transfer protein binds to a receptor involved in the control of plant defence responses.

Lipid transfer proteins (LTPs) and elicitins are both able to load and transfer lipidic molecules and share some structural and functional properties. While elicitins are known as elicitors of plant defence mechanisms, the biological function of LTP is still an enigma. We show that a wheat LTP1 binds with high affinity sites. Binding and in vivo competition experiments point out that these binding sites are common to LTP1 and elicitins and confirm that they are the biological receptors of elicitins. A mathematical analysis suggests that these receptors could be represented by an allosteric model corresponding to an oligomeric structure with four identical subunits.

Mediation of elicitin activity on tobacco is assumed by elicitin-sterol complexes.

Elicitins secreted by phytopathogenic Phytophthora spp. are proteinaceous elicitors of plant defense mechanisms and were demonstrated to load, carry, and transfer sterols between membranes. The link between elicitor and sterol-loading properties was assessed with the use of site-directed mutagenesis of the 47 and 87 cryptogein tyrosine residues, postulated to be involved in sterol binding. Mutated cryptogeins were tested for their ability to load sterols, bind to plasma membrane putative receptors, and trigger biological responses. For each mutated elicitin, the chemical characterization of the corresponding complexes with stigmasterol (1:1 stoichiometry) demonstrated their full functionality. However, these proteins were strongly altered in their sterol-loading efficiency, specific binding to high-affinity sites, and activities on tobacco cells. Ligand replacement experiments strongly suggest that the formation of a sterol-elicitin complex is a requisite step before elicitins fasten to specific binding sites. This was confirmed with the use of two sterol-preloaded elicitins. Both more rapidly displaced labeled cryptogein from its specific binding sites than the unloaded proteins. Moreover, the binding kinetics of elicitins are related to their biological effects, which constitutes the first evidence that binding sites could be the biological receptors. The first event involved in elicitin-mediated cell responses is proposed to be the protein loading with a sterol molecule.

Fatty acids bind to the fungal elicitor cryptogein and compete with sterols.

Cryptogein is a proteinaceous elicitor of plant defense reactions which also exhibits sterol carrier properties. In this study, we report that this protein binds fatty acids. The stoichiometry of the fatty acid-cryptogein complex is 1:1. Linoleic acid and dehydroergosterol compete for the same site, but elicitin affinity is 27 times lower for fatty acid than for sterol. We show that C7 to C12 saturated and C16 to C22 unsaturated fatty acids are the best ligands. The presence of double bonds markedly increases the affinity of cryptogein for fatty acids. A comparison between elicitins and known lipid transfer proteins is discussed.

Elicitins trap and transfer sterols from micelles, liposomes and plant plasma membranes.

Using elicitins, proteins secreted by some phytopathogenic Oomycetes (Phytophthora) known to be able to transfer sterols between phospholipid vesicles, the transfer of sterols between micelles, liposomes and biological membranes was studied. Firstly, a simple fluorometric method to screen the sterol-carrier capacity of proteins, avoiding the preparation of sterol-containing phospholipidic vesicles, is proposed. The transfer of sterols between DHE micelles (donor) and stigmasterol or cholesterol micelles (acceptor) was directly measured, as the increase in DHE fluorescence signal. The results obtained with this rapid and easy method lead to the same conclusions as those previously reported, using fluorescence polarization of a mixture of donor and acceptor phospholipid vesicles, prepared in the presence of different sterols. Therefore, the micelles method can be useful to screen proteins for their sterol carrier activity. Secondly, elicitins are shown to trap sterols from purified plant plasma membranes and to transfer sterols from micelles to these biological membranes. This property should contribute to understand the molecular mechanism involved in sterol uptake by Phytophthora. It opens new perspectives concerning the role of such proteins in plant-microorganism interactions.

Are elicitins cryptograms in plant-Oomycete communications?

Stimulation of plant natural defenses is an important challenge in phytoprotection prospects. In that context, elicitins, which are small proteins secreted by Phytophthora and Pythium species, have been shown to induce a hypersensitive-like reaction in tobacco plants. Moreover, these plants become resistant to their pathogens, and thus this interaction constitutes an excellent model to investigate the signaling pathways leading to plant resistance. However, most plants are not reactive to elicitins, although they possess the functional signaling pathways involved in tobacco responses to elicitin. The understanding of factors involved in this reactivity is needed to develop agronomic applications. In this review, it is proposed that elicitins could interact with regulating cell wall proteins before they reach the plasma membrane. Consequently, the plant reactivity or nonreactivity status could result from the equilibrium reached during this interaction. The possibility of overexpressing the elicitins directly from genomic DNA in Pichia pastoris allows site-directed mutagenesis experiments and structure/function studies. The recent discovery of the sterol carrier activity of elicitins brings a new insight on their molecular activity. This constitutes a crucial property, since the formation of a sterol-elicitin complex is required to trigger the biological responses of tobacco cells and plants. Only the elicitins loaded with a sterol are able to bind to their plasmalemma receptor, which is assumed to be an allosteric calcium channel. Moreover, Phytophthora and Pythium do not synthesize the sterols required for their growth and their fructification, and elicitins may act as shuttles trapping the sterols from the host plants. Sequence analysis of elicitin genes from several Phytophthora species sheds unexpected light on the phylogenetic relationships among the genus, and suggests that the expression of elicitins is under tight regulatory control. Finally, general involvement of these lipid transfer proteins in the biology of Pythiaceae, and in plant defense responses, is discussed. A possible scheme for the coevolution between Phytophthora and tobacco plants is approached.

Elicitins, proteinaceous elicitors of plant defense, are a new class of sterol carrier proteins.

Some phytopathogenic fungi within Phytophthora species are unable to synthesize sterols and therefore must pick them up from the membranes of their host-plant, using an unknown mechanism. These pseudo-fungi secrete elicitins which are small hydrophilic cystein-rich proteins. The results show that elicitins studied interact with dehydroergosterol in the same way, but with some time-dependent differences. Elicitins have one binding site with a similar strong affinity for dehydroergosterol. Using a non-steroid hydrophobic fluorescent probe, we showed that phytosterols are able to similarly bind to elicitins. Moreover, elicitins catalyze sterol transfer between phospholipidic artificial membranes. Our results afford the first evidence for a molecular activity of elicitins which appears to be extracellular sterol carrier proteins. This property should contribute to an understanding of the molecular mechanism involved in sterol uptake by Phytophthora. It opens new perspectives concerning the role of such proteins in plant-microorganism interactions, since elicitins trigger defence reactions in plants.

The fungal elicitor cryptogein is a sterol carrier protein.

Cryptogein is a protein secreted by the phytopathogenic pseudo-fungus, Phytophthora cryptogea. It is a basic 10 kDa hydrophilic protein having a hydrophobic pocket and three disulfide bridges. These common features with sterol carrier proteins led us to investigate its possible sterol transfer activity using the fluorescent sterol, dehydroergosterol. The results show that cryptogein has one binding site with strong affinity for dehydroergosterol. Moreover, this protein catalyzes the transfer of sterols between phospholipidic artificial membranes. This is the first evidence for the existence of an extracellular sterol carrier protein and for a molecular activity of cryptogein. This property should contribute to an understanding of the role of cryptogein in plant-microorganism interactions.

Purification and Characterization of Glutamine Synthetase from the Basidiomycete Pleurotus ostreatus

The purification and some properties of glutamine synthetase (GS) from the mycelium of the basidiomycete Pleurotus ostreatus are described. The enzyme was purified to apparent homogeneity with ion exchange chromatography and a Dyematrex Green A column as the major purification steps. The GS has a molecular weight of 470 kDa and is composed of eight subunits with a molecular weight of 58 kDa. A tetrameric form of the enzyme may also be active. The apparent Km values for the biosynthetic reaction varied in different mycelial extracts from 2.5 to 3.5 mM and from 0.02 to 0.06 for glutamate and ammonium respectively. In the transferase reaction, Km values of 48 mM and 6.2 mM were found for L-glutamine and hydroxylamine, respectively. From the divalent cations tested, Mn2+ showed the strongest stimulatory effect both on the transferase and the biosynthetic reaction. ADP was the only nucleotide having an activating effect on the transferase reaction. The biosynthetic reaction was strongly inhibited by AMP and the transferase reaction by carbamoylphosphate. L-Alanine and glycine inhibited both reactions.

Cercospora beticola toxins. Part VI: preliminary studies of protonation and complexation equilibria.

The biological activity of Cercospora beticola toxins might be enhanced by the complex formation with magnesium. Therefore, protonation and complexation equilibria of beticolins were studied. Beticolins carry three dissociable functions (H3B) two of which dissociate at a physiological pH. In the presence of magnesium, the neutralisation and protonation curves provide evidence for the formation of complexes. At physiological pH, the uncharged complex, Mg2H2B2, is the predominant form. The nonionised forms of free beticolin-1 and -2 fluoresce in a 50% dioxan-water solution and their emission maxima shift to higher wavelengths in water. The dianion HB(2-) is non-fluorescent both in water and in less polar media. The formation of the Mg2H2B2 complex which strongly fluoresces in nonpolar media is confirmed by a marked increase in fluorescence at 520 nm and by a shift of the excitation maximum.

Cercospora beticola toxins. VII. Fluorometric study of their interactions with biological membranes.

The interactions of two beticolins, Cercospora beticola toxins, and of their magnesium complexes with liposomes or plasma membrane were studied. The fluorometric pH titration curves of beticolins in liposomes and in plasma membranes reveal the presence of the dissociated form of beticolins. The concentration of the magnesium complex in these membranes increases at high pH. The partition coefficient of beticolin-1 on liposomes is 3-fold higher than that of beticolin-2 and the fluorescence of both compounds on liposomes is similar. The addition of magnesium to liposomes causes a 40-fold and 20-fold increase in the partition coefficient of beticolin-1 and -2, respectively, as a result of the interactions between membrane, magnesium and beticolins. Beticolins react to a delta pH across the liposome membrane but the formation of the magnesium complex completely abolishes this effect.

Purification and some properties of glutamate dehydrogenase and glutamine synthetase from Paracoccus denitrificans.

The purification and some properties of NADP-dependent glutamate dehydrogenase (GDH) and glutamine synthetase (GS) from the facultatively anaerobic Gram-negative bacterium Paracoccus denitrificans were investigated. The enzymes were purified to homogeneity using a procedure which involved affinity chromatography on Blue Sepharose CL-6B as the major purification step. The recoveries in the purification of GDH and GS were 28% and 64%, respectively. The specific activity of purified GDH was 183 nkat (mg protein)-1 (deaminating reaction). GDH was composed of subunits of molecular mass 47 kDa and the native enzyme was either a tetramer or hexamer. The apparent Km values for L-glutamate, NADP, 2-oxoglutarate, NADPH and ammonia were 1.5 mM, 5.9 microM, 0.47 microM, 12.5 microM and 14 mM, respectively. The specific activity of purified GS was 1125 nkat (mg protein)-1 (transferase reaction). The molecular mass of native GS was 570 kDa; it was composed of 12 subunits of molecular mass 50.1 kDa. The apparent Km values for L-glutamine and hydroxylamine in the transferase reaction were 2.1 and 2.4 mM, respectively; those of ammonia, L-glutamate and ATP in the biosynthetic reaction were 0.03, 1 and 0.17 mM, respectively. After the adenylylation of GS, the Km for L-glutamine and L-glutamate increased and reached the values of 8.0 and 27 mM, respectively. The effects of the changes in GS activity on the ammonia metabolism of Paracoccus denitrificans are discussed.

Assimilation of ammonia in Paracoccus denitrificans.

Two pathways serve for assimilation of ammonia in Paracoccus denitrificans. Glutamate dehydrogenase (NADP+) catalyzes the assimilation at a high NH4+ concentration. If nitrate serves as the nitrogen source, glutamate is synthesized by glutamate-ammonia ligase and glutamate synthase (NADPH). At a very low NH4+ concentration, all three enzymes are synthesized simultaneously. No direct relationship exists between glutamate dehydrogenase (NADP+) and glutamate-ammonia ligase in P. denitrificans, while the glutamate synthase (NADPH) activity changes in parallel with that of the latter enzyme. Ammonia does not influence the induction or repression of glutamate dehydrogenase (NADP+). The inner concentration of metabolites indicates a possible repression of glutamate dehydrogenase (NADP+) by the high concentration of glutamine or its metabolic products as in the case when NH4+ is formed by assimilative nitrate reduction. No direct effect of the intermediates of nitrate assimilation on the synthesis of glutamate dehydrogenase (NADP+) was observed.

Interaction of fluorescent berberine alkyl derivatives with respiratory chain of rat liver mitochondria.

The cationic fluorescent dyes, berberines, have been observed to inhibit NAD-linked respiration in rat liver mitochondria. Low concentrations inhibit electron transport in the NAD-ubiquinone span after penetration into mitochondria. More hydrophobic alkyl derivatives proved to be stronger inhibitors showing more rapid onset of inhibition. The inhibition was totally dependent on the energization of the membrane; however, the addition of a hydrophobic anion stimulated the inhibition effects in uncoupled mitochondria. Substantially higher concentrations of berberines are needed for the inhibition of the oxidation of succinate. The excess of dye interacting with surface dipoles in the energized state can inhibit the energy transduction through the complex bc1. On the basis of the difference in the rate of fluorescence response when berberines are added to coupled mitochondria and the corresponding inhibition effects, the presence minimally of two binding sites was suggested. The dye bound on the outer surface is highly fluorescent and inhibits the energy transduction if added in excess. The remaining dye interacting with NADH dehydrogenase does not fluoresce. The accumulation of alkylberberine in mitochondria results in additional effects in the region of cytochrome b the nature of which is not fully understood.

Elevated levels of substances immunologically cross-reactive with insulin in blood of patients with malignant and nonmalignant pulmonary tissue proliferation.

Results of a survey of concentrations of substances immunologically cross-reactive with insulin (SICRI), glucose and growth hormone in preprandially drawn blood of 84 patients suffering from bronchial epidermoid, microcellular and adenocarcinomas and 22 patients from sarcoidosis, tuberculosis and obstructive bronchitis are presented. In all these diseases, tissue proliferation takes place. Supranormal SICRI concentrations were frequently associated with these diseases, whereas concentrations of glucose and growth hormone remained unaffected; this shows that physiological effects of SICRI in these diseases differ from the effects in patients suffering from some lympho-proliferative and solid tumors. These results indicate that elevated levels of circulating substances detectable by insulin-specific radioimmunoassay can accompany both malignant and nonmalignant proliferation in lungs.

Berberine derivatives as cationic fluorescent probes for the investigation of the energized state of mitochondria.

The interaction of rat liver and bovine heart mitochondria with a series of fluorescent, cationic berberine derivatives varying in the length of alkyl chain has been investigated. An increase in the hydrophobicity of the derivative was accompanied by a larger value of the partition coefficient and by binding to a more hydrophobic region of the inner mitochondrial membrane. It was found that berberines could be used as sensitive indicators of processes which take place on the outer surface of the mitochondrial membrane; the greatest (15-fold) increase in fluorescence was obtained with 13-methylberberine in the energized state of mitochondria. The fluorescence increase was due to the increase in fluorescence quantum yield although a small increase in the amount of bound derivative could also be detected upon energization. The fluorescence was linearly dependent on the magnitude of the membrane potential. In parallel with an observed fluorescence enhancement a considerable decrease in rotational mobility was found. We suggest that berberines move in the inner membrane according to the polarity of the membrane potential; consequently, deeper immersion in the less polar region in the energized state brings about a larger fluorescence increase. More hydrophobic derivatives inhibited NAD-linked respiration in rat liver mitochondria but exerted no effect on succinate oxidation up to 10 microM concentration.

[Positive feedback mechanisms by which immunoproliferative tumors stimulate their own growth]. / O mehanizmu pozitivne povratne sprege kojom imunoproliferativni tumori podsticu vlastiti rast.

Based on clinical findings of patients with some proliferative tumors and experimental data, a hypothesis of positive feedback mechanism by which tumor stimulates its own growth has been formulated. In some patients with Hodgkin's disease and non-Hodgkin's lymphomas, i.e. leukemias, the high levels of substances immunologically cross reactive with insulin (SICRI), low glycemic values and increased values of growth hormone were found in the blood. These findings were in correlation with the status and stages of the disease. In a more advanced stage of the disease, the concentration of insulin-like substances was higher and glucose levels were lower in patients in remission. The high correlation was found between the increased SICRI levels of insulin-like substances showed faster growth. It is certain that some tumor cells excrete these substances. In mice with melanomas, high concentrations of these substances, growth hormone and low glucose levels were found in the blood. On the basis of these findings a hypothesis was formulated about positive feedback mechanism by which tumors stimulates their own growth. Tumor excretes SICRI which decreases glucose concentration in the blood. Hypoglycemia is a stimulation for the pituitary to release growth hormone into the blood. This hormone probably stimulates protein synthesis and replication of tumor cells contributing to increased SICRI excretion, etc. The final results of this positive feedback mechanism is faster growth of tumor and death of host.

Hormonal changes in patients with haematological malignancies.

Levels of immunologically reactive insulin (IRI), growth hormone (GH), thyroxine, cortisol and ACTH in sera of patients with various haematological malignancies were determined. IRI and GH levels were increased in 80% of the patients regardless of the type of disease. IRI was more elevated in relapse than in remission. Cytostatic treatment returned IRI serum levels to normal. Thyroxine, ACTH and cortisol in serum were higher in only two percent of the patient with haematological malignancies.

Interaction of liposomes with homologous series of fluorescent berberine derivatives. New cationic probes for measuring membrane potential.

The interaction of liposomes with a series of fluorescent berberine derivatives having different alkyl chain lengths has been investigated. The hydrophobicity of the binding site on the phospholipid membrane increases and mobility decreases with the length of the alkyl chain. If lauryl sulphate micelles are used to bind berberines, the hydrophobicity of the binding site is the same for all derivatives. The dye series represents a model with constant charge and growing lipophilicity. Both electrostatic forces and lipophilicity play an important role in binding. By virtue of the excellent sensitivity of the dyes to medium polarity, berberines prove to be suitable probes for measuring membrane potential, but only in cases when a negative charge is generated in the liposomal interior. The fluorescent response is a linear function of the potential magnitude.