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.     

Effects of heavy metals on immunocompetence of white-footed mice (Peromyscus leucopus)

Continued human population growth and industrialization result in increased contamination of wildlife habitats. Effects of such habitat deterioration on the well-being of natural populations are unclear. Exposure to contaminants may impair immunocompetence, thereby increasing disease susceptibility. The mammalian immune system is important in maintaining health and in its sensitivity to toxins. In our study conducted from May 1999 through May 2001, we examined assays of immnnoompetence in the white-footed mouse (Peromyscus leucopus) that inhabited reference sites and sites significantly contaminated with mixtures of heavy metals. We estimated potential exposure and uptake of heavy metals by measuring the level of each contaminant in representative soil and tissue samples. Intraindividual variation across mice, but not sex, explained a large portion of the overall variance in immune response, and spleen weight was significanltly afflicted by mouse age. We found no evidence that residence on contaminated sites had any effect on immunopathology and humoral immunity as measured in our study. We suggest that field and laboratory studies in ecotoxicology provide estimates of exposure to contaminants (i.e., tissue analyses) to establish a database suitable to clarify the dose-response relationship between contaminants and target systems.     

In vitro maturation, fertilization and early cleavage rates of bovine fetal oocytes

The in vitro developmental competence of oocytes harvested from 3 to 6 mm follicles from ovaries of 7.5 months to term fetuses and adult cows was compared. Cumulus oocyte complexes (COCs) were washed and placed in 200 microl droplets of maturation medium 199, supplemented with 10 microg/ml FSH, 10 microg/ml LH, 1.5 microg/ml estradiol, 75 microg/ml streptomycin, 100 IU/ml penicillin, 10 mM Hepes, and 10% fetal bovine serum (FBS) under oil and incubated for 24 h at 39 degrees C and 5% CO2. Matured oocytes were exposed to frozen-thawed TALP swim-up, heparin-capacitated sperm (20 h, 39 degrees C, 5% CO2). Presumptive zygotes were cultured in medium 199 containing 8 mg/ml BSA-V, 100 IU/ml penicillin G, 75 microg/ml streptomycin, and 10 mM Hepes (48 h, 39 degrees C, 5% CO2). Oocytes/embryos were fixed, stained with DAPI, and evaluated under fluorescent microscopy to assess maturation, fertilization, and subsequent embryonic development. There was a difference (P<0.05) between fetal and adult cow oocytes for in vitro maturation (IVM; 80.1% versus 92.0%), fertilization (69.3% versus 79.9%), and cleavage rates (36.7% versus 49.9%), respectively. Poor IVM, fertilization and embryonic development of fetal oocytes may be due to a higher incidence of blockage at germinal vesicle (GV) and metaphase-I (M-I) stage after IVM (12.0% versus 2.3% for fetal versus adult oocytes, respectively, P<0.05). Although the IVF results with fetal oocytes are poorer than with adult cow oocytes, they were still high enough to be considered for use in research and when death of the dam and/or fetus is pre-mature or sudden.     

Somatic embryogenesis and plant regeneration through leaf culture in <Emphasis Type="Italic">Arachis glabrata (Leguminosae)</Emphasis>

Plants of two accessions of Arachis glabrata were regenerated via somatic embryogenesis. Embryogenic calli were initiated from leaflet explants on Murashige and Skoog medium supplemented with picloram alone or picloram in combination with 6-benzylaminopurine. Leaflets of accession A6138 induced the highest percentage of somatic embryos in media composed of 10 mg dm⁻³ and 15 mg dm⁻³ picloram. In contrast, 5 mg dm⁻³ picloram with 0.1 mg dm⁻³ 6-benzylaminopurine was one of the most effective combinations in accession AF385. MS medium supplemented with 2 g dm⁻³ activated charcoal (AC) used for 30 days was the most effective for embryo maturation. After 20 days of culture on MS medium devoid of growth regulators, 6 % of embryos converted into plantlets in accession A6138.     

A model for the misfolded bis-His intermediate of cytochrome c: the 1-56 N-fragment

We have characterized the ferric and ferrous forms of the heme-containing (1-56 residues) N-fragment of horse heart cytochrome c (cyt c) at different pH values and low ionic strength by UV-visible absorption and resonance Raman (RR) scattering. The results are compared with native cyt c in the same experimental conditions as this may provide a deeper insight into the cyt c unfolding-folding process. Folding of cyt c leads to a state having the heme iron coordinated to a histidine (His18) and a methionine (Met80) as axial ligands. At neutral pH the N-fragment (which lacks Met80) shows absorption and RR spectra that are consistent with the presence of a bis-His low spin heme, like several non-native forms of the parental protein. In particular, the optical spectra are identical to those of cyt c in the presence of a high concentration of denaturants; this renders the N-fragment a suitable model to study the heme pocket microenvironment of the misfolded (His-His) intermediate formed during folding of cyt c. Acid pH affects the ligation state in both cyt c and the N-fragment. Data obtained as a function of pH allow a correlation between the structural properties in the heme pocket of the N-fragment and those of non-native forms of cyt c. The results underline that the (57-104 residues) segment under native-like conditions imparts structural stability to the protein by impeding solvent access into the heme pocket.     

Astrocyte Mitochondria in In Vitro Models of Ischemia

There is growing evidence that preservation of mitochondrial respiratory function during cerebral ischemia-reperfusion predicts the ultimate extent of tissue injury. Because neurons are selectively vulnerable to ischemic injury, many studies have focused on neuronal mitochondrial dysfunction in ischemia. However, positron emission tomography (PET) studies in animals and humans suggest that non-neuronal cells such as astrocytes may also experience mitochondrial metabolic compromise that contributes to ischemic necrosis. Astrocytes carry out a number of functions that are critical to normal nervous system function, including uptake of neurotransmitters, regulation of pH and ion concentrations, and metabolic support of neurons. Mitochondria are important for many of these actions. We have used a cell culture model of stroke, oxygen-glucose deprivation (OGD), to study the response of astrocyte mitochondria to ischemia, and to evaluate how changes in astrocyte mitochondrial function might affect neuronal survival and recovery after ischemia.     

Detection of Mycobacterium tuberculosis genotypic groups by a duplex real-time PCR targeting the katG and gyrA genes

A duplex real-time PCR assay was developed for the assignment of Mycobacterium tuberculosis isolates to the three genotypic groups based on the katG463/gyrA95 polymorphism. The assay was as sensitive and specific as nucleotide sequencing and proved also able to detect unambiguously the isolate genotype in clinical specimens.     

Heterologous expression of bovine and porcine beta-lactoglobulins in Pichia pastoris: towards a comparative functional characterisation

Bovine and porcine beta-lactoglobulins were cloned and expressed in host cells with the aim of developing the tools necessary for their structural, functional and conformational characterisation by NMR techniques. Both lipocalins were expressed in Pichia pastoris, where the use of a constitutive promoter turned out to allow the highest productivity. The yield of recombinant proteins was further improved through multiple integration of the encoding genes and by increasing aeration of the transformed cultures. Both proteins were obtained in the culture medium at the concentration of 200 microg/ml. Recombinant lipocalins were purified by ion-exchange chromatography from the culture medium. A preliminary NMR characterisation showed that both proteins were correctly folded.     

Circular dichroism spectra of human hemoglobin reveal a reversible structural transition at body temperature

Previously we have shown that human red blood cells (RBCs) undergo a sudden change from blocking to passing through a 1.3±0.2-µm micropipette when applying an aspiration pressure of 2.3 kPa at a critical transition temperature (T_c=36.4±0.3 °C). Low-shear viscosity measurements suggested that changes in the molecular properties of hemoglobin might be responsible for this effect. To evaluate structural changes in hemoglobin at the critical temperature, we have used circular dichroism (CD) spectroscopy. The thermal denaturation curves of human hemoglobin A (HbA) and hemoglobin S (HbS) upon heating between 25 and 60 °C were non-linear and showed accelerated denaturation between 35 and 39 °C with a midpoint at 37.2±0.6 °C. The transition was reversible below 39 °C and independent of solution pH (pH 6.8–7.8). It was also independent of the oxygenation state of hemoglobin, since a sample that was extensively deoxygenated with N₂ showed a similar transition by CD. These findings suggest that a structural change in hemoglobin may enable the cellular passage phenomenon as well as the temperature-dependent decrease in viscosity of RBC solutions.