Cloning, sequencing and heterologous expression of a new chitinase gene, chi92, from Streptomyces olivaceoviridis ATCC 11238

A new chitinase gene, chi92, encoding the largest known chitinase from Streptomyces olivaceoviridisATCC 11238 was sequenced by means of different PCR-methods. The cloned gene was expressed in E. coliand the recombinant protein could be detected by Western-blot analysis. The multiplicity of chitinolytic enzymes of this strain is discussed.     

Identification and heritability of fumonisin insensitivity in Zea mays

Landraces of maize (Zea mays ssp. mays) and its wild teosinte relatives (Zea mays spp. parviglumis and mexicana) were surveyed for sensitivity to fumonisin B(1), a phytotoxin produced by the maize pathogen Gibberella moniliformis. Only two of 42 Z. mays samples were highly insensitive to FB(1) (ED(50) = ca. 200 microM). The teosintes and 76% of the maize landraces were moderately or highly sensitive to FB(1) (ED(50) < or = 30 microM), which indicates that FB(1) sensitivity is likely to be an ancestral trait in Z. mays. F(1) generations derived from crosses between FB(1)-sensitive maize inbred B73 and insensitive landraces were significantly less sensitive than B73. Thus, our data indicate that FB(1)-insensitivity is a relatively rare but heritable trait in maize. We also report the sensitivity of maize to other Gibberella toxins - beauvericin, diacetoxyscirpenol, and moniliformin.     

Cumulation of mycotoxins in maize cobs infected with<Emphasis Type="Italic">Fusarium gramihearum</Emphasis>

Maize cobs withFusarium ear rot were collected at 1986 season and five infected byFusarium graminearum were analyzed for presence of triohothecenes and zearalenone. Collected material was subsampled forFusarium damaged kernels and corresponding axial stems and healthy looking kernels. All investigated cobs contained deoxynivalenol (DON) (range 18.0–131.5 mg/kg) and zearalenone (ZEA) (range 0.38–2.17 mg/kg), in four cobs 15-acetyl-deoxynivalenol (15-AcDON) (range 5.2–6.2 mg/kg) was present and two cobs besides three all metabolites contained 3-acetyl-deoxynivalenol (3-AcD0N) (range 0.5–0.8 mg/kg).The average of individual toxins amount in axial stems: in mg/kg was equal to: DON — 110.36, ZEA — 4.57, 15-AcD0N — 16.66, and 3-AcD0N — 1.32.Fusarium damaged kernels contained in average the following amount (mg/kg) of: DON 77.00, ZEA 0.98, 15-AcD0N 3.78 and 3-AcD0N 0.06. Healthy looking kernels contained DON 1.96 mg/kg and ZEA 0.07 mg/kg only. Cooccurrence of 3-AcDON and 15-AcDON in two samples was an interesting finding. The amount of DON in total cob was highly correlated (r = 0.94) with percentage ofFusarium damaged kernels in given ear.     

Quantitative calcium measurements in subcellular compartments of Plasmodium falciparum-infected erythrocytes

The acidic food vacuole exerts several important functions during intraerythrocytic development of the human malarial parasite Plasmodium falciparum. Hemoglobin taken up from the host erythrocyte is degraded in the food vacuole, and the heme liberated during this process is crystallized to inert hemozoin. Several anti-malarial drugs target food vacuolar pathways, such as hemoglobin degradation and heme crystallization. Resistance and sensitization to some antimalarials is associated with mutations in food vacuolar membrane proteins. Other studies suggest a role of the food vacuole in ion homeostasis, and release of Ca2+ from the food vacuole may mediate adopted physiological responses. To investigate whether the food vacuole is an intracellular Ca2+ store, which in turn may affect other physiological functions in which this organelle partakes, we have investigated total and exchangeable Ca2+ within the parasite's food vacuole using x-ray microanalysis and quantitative confocal live cell Ca2+ imaging. Apparent free Ca2+ concentrations of approximately 90, approximately 350, and approximately 400 nM were found in the host erythrocyte cytosol, the parasite cytoplasm, and the food vacuole, respectively. In our efforts to determine free intracellular Ca2+ concentrations, we evaluated several Ca2+-sensitive fluorochromes in a live cell confocal setting. We found that the ratiometric Ca2+ indicator Fura-Red provides reliable determinations, whereas measurements using the frequently used Fluo-4 are compromised due to problems arising from phototoxicity, photobleaching, and the strong pH dependence of the dye. Our data suggest that the food vacuole contains only moderate amounts of Ca2+, disfavoring a role as a major intracellular Ca2+ store.     

Higher order multipole moments for molecular dynamics simulations

In conventional force fields, the electrostatic potential is represented by atom-centred point charges. This choice is in principle arbitrary, but technically convenient. Point charges can be understood as the first term of multipole expansions, which converge with an increasing number of terms towards the accurate representation of the molecular potential given by the electron density distribution. The use of multipole expansions can therefore improve the force field accuracy. Technically, the implementation of atomic multipoles is more involved than the use of point charges. Important points to consider are the orientation of the multipole moments during the trajectory, conformational dependence of the atomic moments and stability of the simulations which are discussed here.

Trichothecenes accumulated in liquid culture of a mutant of Fusarium sporotrichioides NRRL 3299.

A UV-generated mutant of Fusarium sporotrichioides NRRL 3299 was altered in its ability to biosynthesize T-2 toxin, as shown by a rapid screen with monoclonal antibodies to T-2. This stable mutant accumulated two trichothecenes that were not observed in liquid cultures of the parent strain. The two compounds were identified as 3,15-diol 12,13-epoxytrichothec-9-ene and 3,15-diol 12,13-epoxytrichothec-9-ene 3-acetate on the basis of their nuclear magnetic resonance and mass spectra. This is the first report of either of these two compounds as secondary metabolites of F. sporotrichioides and of a trichothecene acetylated at C-3 by this species.     

Production of Fumonisin B(inf1) and Moniliformin by Gibberella fujikuroi from Rice from Various Geographic Areas

Gibberella fujikuroi strains isolated from rice in the United States, Asia, and other geographic areas were tested for sexual fertility with members of mating population D and for production of fumonisin B(inf1) and moniliformin in culture. Of the 59 field strains tested, 32 (54%) were able to cross with tester strains of mating population D, but only a few ascospores were produced in most of these crosses. Thirty-four strains produced more than 10 (mu)g of fumonisin B(inf1) per g, but only three strains produced more than 1000 (mu)g/g. Twenty-five strains produced more than 100 (mu)g of moniliformin per g, and 15 produced more than 1,000 (mu)g/g. Seven field strains produced both fumonisin B(inf1) and moniliformin, but none of these strains produced a high level of fumonisin B(inf1) (>1,000 (mu)g/g). However, a genetic cross between a strain that produced fumonisin B(inf1) but no moniliformin and a strain that produced moniliformin but no fumonisin B(inf1) yielded progeny that produced high levels of both toxins. Strains of G. fujikuroi isolated from rice infected with bakanae disease are similar to strains of mating population D isolated from maize in their ability to produce both fumonisins and moniliformin. This finding suggests a potential for contamination of rice with both fumonisins and moniliformin.     

Membrane disruption, fusion and resealing in the course of exocytosis in Paramecium cells

Ca²⁺-ionophore-mediated trichocyst exocytosis was followed by scanning electron microscopy, freeze-cleaving and ultrathin sectioning after surface labelling in vivo with negatively charged hemepeptides. The apical trichocyst membrane and the superposed cell membrane portion (encircled by ˜300 nm large “rings” of membrane-intercalated particles) undergo fragmentation, while both membranes involved fuse with each other within the “rings”. Subsequently cell membrane materials spread centropetally to the region within the “rings” allowing the cell membrane to become resealed and the trichocyst membrane to become detached. Exocytosis does not result in any remarkable integration of trichocyst membrane materials into the cell membrane.