Mistletoe viscotoxins induce membrane permeabilization and spore death in phytopathogenic fungi

Viscotoxins (Vts) are basic peptides expressed in mistletoe leaves, seeds and stems which have been shown to be cytotoxic to mammalian cells. The aim of this study was to analyse whether Vts were able to control and/or inhibit the growth of phytopathogenic fungi to obtain a clue to their biological function. Incubation of two Vt isoforms, VtA₃ and VtB, at a final concentration of 10 µ M resulted in a complete blockage of the germination of spores from three different pathogenic fungi. It was also shown that lower concentrations than 10 µ M of VtA₃ and VtB inhibit their mycelial growth in a dose-dependent manner. The protein dose required to inhibit the growth of Fusarium solani and Sclerotinia sclerotiorum to a 50% was between 1.5 and 3.75 µ M , which represents a potent activity. No significant differences in the antifungal potency for each Vt isoform, either VtA₃ and VtB, were observed, although they have been shown to exert differential cytotoxicity on mammalian cells. It was also demonstrated that Vts act as fungicidal compounds. To explore the basis of the antifungal activity the ability of VtA₃ to induce changes in membrane permeability and on the oxidative status of F. solani spores was analysed. By using a specific fluorescent probe on intact spores, it was demonstrated that VtA₃ produces rapid changes in fungal membrane permeability. It also induces H₂O₂ production verified by a histochemical staining. The data presented in this study support a direct role of Vts in the plant defence determined by their lethal effect on fungal pathogens.     

Effect of angiotensin II and losartan on the phagocytic activity of peritoneal macrophages from Balb/C mice

Angiotensin II (AII), a product of rennin-angiotensin system, exerts an important role on the function of immune system cells. In this study, the effect of AII on the phagocytic activity of mouse peritoneal macrophages was assessed. Mice peritoneal macrophages were cultured for 48 h and the influence of different concentrations of AII (10(-14) to 10(-7) M) and/or losartan, 10(-16) to 10(-6) M), an AT1 angiotensin receptor antagonist, on phagocytic activity and superoxide anion production was determined. Dimethylthiazoldiphenyltetrazolium bromide reduction and the nucleic acid content were used to assess the cvtotoxicity of losartan. A stimulatory effect on phagocytic activity (P < 0.05) was observed with 10(-13) M and 10(-12 M) AII concentrations. The addition of losartan (up to10(-14) M) to the cell cultures blocked (P < 0.001) the phagocytosis indicating the involvement of AT1 receptors. In contrast, superoxide anion production was not affected by AII or losartan. The existence of AT1 and AT2 receptors in peritoneal macrophages was demonstrated by immunofluorescence microscopy. These results support the hypothesis that AII receptors can modulate murine macrophage activity and phagocytosis, and suggest that AII may have a therapeutic role as an immunomodulatory agent in modifying the host resistance to infection.     

Nitric oxide and iron: effect of iron overload on nitric oxide production in endotoxemia

The amount of iron within the cell is carefully regulated in order to provide an adequate level of micronutrient while preventing its accumulation and toxicity. Iron excess is believed to generate oxidative stress, understood as an increase in the steady-state concentration of oxygen radical intermediates. Nitric oxide (NO) is an inorganic free-radical gaseous molecule which has been shown over the last decade to play an unprecedented variety of roles in biological systems. The effect of nitrogen reactive species may explain the iron sequestration pattern that characterizes macrophages under inflammatory conditions. From a patho-physiological viewpoint, further studies are required to assess the usefulness of this mechanism to minimize formation and release of free radicals in diseased tissues. However, contrary to the deleterious effects of the reactive nitrogen oxide species formed from either NO/O(2) and NO/O(2)(-), it has been pointed out that NO shows antioxidant properties. A number of studies have described the complex relationships between iron and NO, but controversy remains as to the influence and significance of iron on inflammatory NO production. To explore the initial steps of the effects triggered by LPS administration in the presence of excess iron, male Wistar rats were treated with: lipopolysaccharide from Escherichia coli (serotype 0127:B8) (LPS); iron-dextran; or iron-dextran plus LPS and liver samples were taken after 6 h. EPR spectra of NO-Hb in the venous blood were determined at 77 K. Iron-dextran administered to rats intraperitoneally resulted predominantly in iron uptake by the liver Kupffer cells and led to an increased NO level in blood in the presence of LPS. Further studies will be required to assess the complex role of the Kupffer cells on iNOS induction and NO production.     

Alternative diphtheria, tetanus and whooping cough immunization schedule to evoke a Th2 tetanus and a Th1 pertussis immune response

In a previous study, using BALB/c mice, we found that while diphtheria (D), tetanus (T) and whooping cough (Pw, whole-cell Bordetella pertussis) immunization induces a Th1/Th2 tetanus response and memory T cells able to proliferate in response to in vitro stimulation with B. pertussis, DTPa immunization induces a Th2 tetanus immune response and no memory T cells that recognize B. pertussis as stimulus. Considering that a pro-inflammatory cytokine production is not necessary for protection against tetanus and therefore should be avoided, an alternative DTP immunization schedule with minimal Pw exposure was assessed in order to obtain a Th2 tetanus response and a Th1 pertussis response. BALB/c mice were primed with DT vaccine at day 0, with Pw vaccine at day 14 and boosted with DTPa vaccine at days 21 and 28. A control group was inoculated with saline. Antibodies against B. pertussis surface antigens, tetanus and diphtheria toxoids were produced by mice. Spleen cells stimulated in vitro with B. pertussis produced IL-6 and IFNgamma. Only IL-5 was produced by cells in response to tetanus toxoid stimulation. These results are in line with the low IgG1/IgG2a ratio for pertussis antibodies compared with those corresponding to tetanus and diphtheria. The immunization protocol presented herein succeeded in producing tetanus and pertussis immune responses of Th2 and Th1 type, respectively. In contrast to previous results obtained with DTPw immunization, no IL-12 production was observed. Our findings provide direct evidence that an immunization protocol with an interval of 14 days between DT and Pw primings, followed by DTPa boosters, can induce appropriate immune responses against DTP vaccine antigens.     

In vitro interactions of thallium with components of the glutathione-dependent antioxidant defence system

We investigated the hypothesis that thallium (Tl) interactions with the glutathione-dependent antioxidant defence system could contribute to the oxidative stress associated with Tl toxicity. Working in vitro with reduced glutathione (GSH), glutathione reductase (GR) or glutathione peroxidase (GPx) in solution, we studied the effects of Tl⁺ and Tl³⁺ (1-25 μM) on: (a) the amount of free GSH, investigating whether the metal binds to GSH and/or oxidizes it; (b) the activity of the enzyme GR, that catalyzes GSH regeneration; and (c) the enzyme GPx, that reduces hydroperoxide at expense of GSH oxidation. We found that, while Tl⁺ had no effect on GSH concentration, Tl³⁺ oxidized it. Both cations inhibited the reduction of GSSG by GR and the diaphorase activity of this enzyme. In addition, Tl³⁺per se oxidized NADPH, the cofactor of GR. The effects of Tl on GPx activity depended on the metal charge: Tl⁺ inhibited GPx when cumene hydroperoxide (CuOOH) was the substrate, while Tl³⁺-mediated GPx inhibition occurred with both substrates. The present results show that Tl interacts with all the components of GSH/GSSG antioxidant defence system. Alterations of this protective pathway could be partially responsible for the oxidative stress associated with Tl toxicity.     

A germin-like protein of wheat leaf apoplast inhibits serine proteases

A protein resistant to heat and proteolysis that inhibits serine proteases was isolated from wheat leaf apoplasts. Based on trypsin inhibition, its more active form was a 66-69 kDa oligomer. It was dissociated in an 18-21 kDa monomer having an amino terminal sequence identical to the Box A of germins and germin-like proteins. Like these proteins, it was glycosylated and showed manganese superoxide dismutase activity. The monomer displayed three forms when examined by 2D western blot: two of 19 kDa, pI 5.8 and 6.2; and one of 21 kDa, pI 5.8. It was found that the protein controls serine protease activity in the apoplast of plants challenged with the fungus Septoria tritici.