Biological Effects of Trichoderma harzianum Peptaibols on Mammalian Cells

Trichoderma species isolated from water-damaged buildings were screened for toxicity by using boar sperm cells as indicator cells. The crude methanolic cell extract from Trichoderma harzianum strain ES39 inhibited the boar sperm cell motility at a low exposure concentration (50% effective concentration, 1 to 5 μg [dry weight] ml of extended boar semen⁻¹). The same exposure concentration depleted the boar sperm cells of NADH₂. Inspection of the exposed boar sperm cells by transmission electron microscopy revealed damage to the plasma membrane. By using the black lipid membrane technique, it was shown that the semipurified metabolites (eluted from a SepPak C₁₈ cartridge) of T. harzianum strain ES39 induced voltage-dependent conductivity. The high-performance liquid chromatography-purified metabolites of T. harzianum strain ES39 dissipated the mitochondrial membrane potential (Δψ_m) of human lung epithelial carcinoma cells (cell line A549). The semipurified metabolites (eluted from a SepPak C₁₈ cartridge) of T. harzianum strain ES39 were analyzed by mass spectrometry (MS). Matrix-assisted laser desorption ionization and nanoflow electrospray ionization MS revealed five major peptaibols, each of which contained 18 residues and had a mass ranging from 1,719 to 1,775 Da. Their partial amino acid sequences were determined by collision-induced dissociation tandem MS.     

Priming-dependent phosphorylation and regulation of the tumor suppressor pVHL by glycogen synthase kinase 3

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is linked to the development of tumors of the eyes, kidneys, and central nervous system. VHL encodes two gene products, pVHL30 and pVHL19, of which one, pVHL30, associates functionally with microtubules (MTs) to regulate their stability. Here we report that pVHL30 is a novel substrate of glycogen synthase kinase 3 (GSK3) in vitro and in vivo. Phosphorylation of pVHL on serine 68 (S68) by GSK3 requires a priming phosphorylation event at serine 72 (S72) mediated in vitro by casein kinase I. Functional analysis of pVHL species carrying nonphosphorylatable or phosphomimicking mutations at S68 and/or S72 reveals a central role for these phosphorylation events in the regulation of pVHL's MT stabilization (but not binding) activity. Taken together, our results identify pVHL as a novel priming-dependent substrate of GSK3 and suggest a dual-kinase mechanism in the control of pVHL's MT stabilization function. Since GSK3 is a component of multiple signaling pathways that are altered in human cancer, our results further imply that normal operation of the GSK3-pVHL axis may be a critical aspect of pVHL's tumor suppressor mechanism through the regulation of MT dynamics.     

Crystal structures of interleukin-2 tyrosine kinase and their implications for the design of selective inhibitors

Interleukin-2 tyrosine kinase, Itk, is an important member of the Tec family of non-receptor tyrosine kinases that play a central role in signaling through antigen receptors such as the T-cell receptor, B-cell receptor, and Fcepsilon. Selective inhibition of Itk may be an important way of modulating many diseases involving heightened or inappropriate activation of the immune system. In addition to an unliganded nonphophorylated Itk catalytic kinase domain, we determined the crystal structures of the phosphorylated and nonphosphorylated kinase domain bound to staurosporine, a potent broad-spectrum kinase inhibitor. These structures are useful for the design of novel, highly potent and selective Itk inhibitors and provide insight into the influence of inhibitor binding and phosphorylation on the conformation of Itk.     

Impedance analysis of the organ of corti with magnetically actuated probes

An innovative method is presented to measure the mechanical driving point impedance of biological structures up to at least 40 kHz. The technique employs an atomic force cantilever with a ferromagnetic coating and an external magnetic field to apply a calibrated force to the cantilever. Measurement of the resulting cantilever velocity using a laser Doppler vibrometer yields the impedance. A key feature of the method is that it permits measurements for biological tissue in physiological solutions. The method was applied to measure the point impedance of the organ of Corti in situ, to elucidate the biophysical basis of cochlear amplification. The basilar membrane was mechanically clamped at its tympanic surface and the measurements conducted at different radial positions on the reticular lamina. The tectorial membrane was removed. The impedance was described by a generalized Voigt-Kelvin viscoelastic model, in which the stiffness was real-valued and independent of frequency, but the viscosity was complex-valued with positive real part, which was dependent on frequency and negative imaginary part, which was independent of frequency. There was no evidence for an inertial component. The magnitude of the impedance was greatest at the tunnel of Corti, and decreased monotonically in each of the radial directions. In the absence of inertia, the mechanical load on the outer hair cells causes their electromotile displacement responses to be reduced by only 10-fold over the entire range of auditory frequencies.     

Venturia inaequalis: the causal agent of apple scab

The fungus Venturia inaequalis infects members of the Maloideae, and causes the disease apple scab, the most important disease of apple worldwide. The early elucidation of the gene-for-gene relationship between V. inaequalis and its host Malus has intrigued plant pathologists ever since, with the identification of 17 resistance (R)-avirulence (Avr) gene pairings. The Avr gene products are presumably a subset of the total effector arsenal of V. inaequalis (predominantly proteins secreted in planta assumed to facilitate infection). The supposition that effectors from V. inaequalis act as suppressors of plant defence is supported by the ability of the pathogen to penetrate the cuticle and differentiate into large pseudoparenchymatous structures, termed stromata, in the subcuticular space, without the initiation of an effective plant defence response. If effectors can be identified that are essential for pathogenicity, the corresponding R genes will be durable and would add significant value to breeding programmes. An R gene cluster in Malus has been cloned, but no V. inaequalis effectors have been characterized at the molecular level. However, the identification of effectors is likely to be facilitated by the resolution of the whole genome sequence of V. inaequalis. TAXONOMY: Teleomorph: Venturia inaequalis Cooke (Wint.); Kingdom Fungi; Phylum Ascomycota; Subphylum Euascomycota; Class Dothideomycetes; Family Venturiaceae; genus Venturia; species inaequalis. Anamorph: Fusicladium pomi (Fr.) Lind or Spilocaea pomi (Fr.). LIFE CYCLE: V. inaequalis is a hemibiotroph and overwinters as pseudothecia (sexual fruiting bodies) following a phase of saprobic growth in fallen leaf tissues. The primary inoculum consists of ascospores, which germinate and penetrate the cuticle. Stromata are formed above the epidermal cells but do not penetrate them. Cell wall-degrading enzymes are only produced late in the infection cycle, raising the as yet unanswered question as to how V. inaequalis gains nutrients from the host. Conidia (secondary inoculum) arise from the upper surface of the stromata, and are produced throughout the growing season, initiating multiple rounds of infection. VENTURIA INAEQUALIS AS A MODEL PATHOGEN OF A WOODY HOST: V. inaequalis can be cultured and is amenable to crossing in vitro, enabling map-based cloning strategies. It can be transformed readily, and functional analyses can be conducted by gene silencing. Expressed sequence tag collections are available to aid in gene identification. These will be complemented by the whole genome sequence, which, in turn, will contribute to the comparative analysis of different races of V. inaequalis and plant pathogens within the Dothideomycetes.     

Frequency and Rates of Outdoor Activities, and Perceptions of Places to Perform these Activities by Native Americans and Caucasians Interviewed in Tennessee

Activity patterns and perceptions play a key role in human health risk, management, and planning. A sample of 233 people attending a Native American festival in Cookeville, Tennessee was interviewed to determine the types, percent participation, and outdoor activities rates, and their perceptions of the importance of characteristics of nuclear sites. Results indicate that: (1) a high percentage of respondents used outdoor environments, (2) they used them for consumptive (hunting, fishing), non-consumptive (hiking, walking, bird-watching), and religious/sacred activities, (3) a higher percentage of respondents engaged in non-consumptive than consumptive activities, (4) praying or meditating, communing with nature, and bird-watching had the highest uses rates (5) the environmental characteristics rated the highest were lack of radionuclides that presented a health risk, no visible smog, clean air, and unpolluted water, (6) the presence of people, buildings and roads were rated the lowest, and (7) Native Americans had higher outdoor participation rates, participated more frequently, and evaluated environmental characteristics higher than did Caucasians. This information can be used by managers to create and maintain outdoor habitats that fit the needs of local people. Planning and management require information on public policy, human needs and requirements, and human perceptions and evaluations of environmental characteristics.     

Yersinia enterocolitica Infection and tcaA-Dependent Killing of Caenorhabditis elegans

Caenorhabditis elegans is a validated model to study bacterial pathogenicity. We report that Yersinia enterocolitica strains {"type":"entrez-nucleotide","attrs":{"text":"W22703","term_id":"1299536","term_text":"W22703"}}W22703 (biovar 2, serovar O:9) and WA314 (biovar 1B, serovar O:8) kill C. elegans when feeding on the pathogens for at least 15 min before transfer to the feeding strain Escherichia coli OP50. The killing by Yersinia enterocolitica requires viable bacteria and, in contrast to that by Yersinia pestis and Yersinia pseudotuberculosis strains, is biofilm independent. The deletion of tcaA encoding an insecticidal toxin resulted in an OP50-like life span of C. elegans, indicating an essential role of TcaA in the nematocidal activity of Y. enterocolitica. TcaA alone is not sufficient for nematocidal activity because E. coli DH5α overexpressing TcaA did not result in a reduced C. elegans life span. Spatial-temporal analysis of C. elegans infected with green fluorescent protein-labeled Y. enterocolitica strains showed that Y. enterocolitica colonizes the nematode intestine, leading to an extreme expansion of the intestinal lumen. By low-dose infection with {"type":"entrez-nucleotide","attrs":{"text":"W22703","term_id":"1299536","term_text":"W22703"}}W22703 or DH5α followed by transfer to E. coli OP50, proliferation of Y. enterocolitica, but not E. coli, in the intestinal lumen of the nematode was observed. The titer of {"type":"entrez-nucleotide","attrs":{"text":"W22703","term_id":"1299536","term_text":"W22703"}}W22703 cells within the worm increased to over 10⁶ per worm 4 days after infection while a significantly lower number of a tcaA knockout mutant was recovered. A strong expression of tcaA was observed during the first 5 days of infection. Y. enterocolitica WA314 (biovar 1B, serovar O:8) mutant strains lacking the yadA, inv, yopE, and irp1 genes known to be important for virulence in mammals were not attenuated or only slightly attenuated in their toxicity toward the nematode, suggesting that these factors do not play a significant role in the colonization and persistence of this pathogen in nematodes. In summary, this study supports the hypothesis that C. elegans is a natural host and nutrient source of Y. enterocolitica.Yersinia enterocolitica belongs to the family of Enterobacteriaceae and is a psychrotolerant human pathogen that causes gastrointestinal syndromes ranging from acute enteritis to mesenteric lymphadenitis (5). It infects a number of mammals, and swine was identified as a major source for human infection (6). A multiphasic life cycle, which comprises a free-living phase and several host-associated phases, including cold-blooded and warm-blooded hosts, appears to be characteristic for biovars 1B and 2 to 5 of Y. enterocolitica (7, 24).Nonmammalian host organisms including Dictyostelium discoideum, Drosophila melanogaster, or Caenorhabditis elegans are increasingly used to study host-pathogen interactions (16, 26). Due to the obvious parallels between the mammalian and invertebrate defense mechanisms, it has been suggested that the bacteria-invertebrate interaction has shaped the evolution of microbial pathogenicity (53). Several human pathogens including Gram-positive and Gram-negative bacteria infect and kill the soil nematode C. elegans when they are supplied as a nutrient source (42). For example, Streptococcus pneumoniae (4), Listeria monocytogenes (50), extraintestinal Escherichia coli (15), and Staphylococcus aureus (43) but not Bacillus subtilis have been shown to kill the nematode. Upon infection of C. elegans with Enterococcus faecalis, Gram-positive virulence-related factors as well as putative antimicrobials have been identified (20, 35). The extensive conservation in virulence mechanisms directed against invertebrates as well as mammals was demonstrated using a screen with Pseudomonas aeruginosa (30). In this study, 10 of 13 genes whose knockout attenuated the nematode killing were also required for full virulence in a mouse model, confirming the suitability of the C. elegans model to study bacterial pathogenicity. C. elegans is also colonized by Salmonella enterica serovar Typhimurium (S. Typhimurium). This process requires Salmonella virulence factors and was used to study the innate immune response of the nematode (1, 2, 49).The effect of pathogenic Yersinia spp. on C. elegans has also been investigated. It could be demonstrated that both Yersinia pestis and Yersinia pseudotuberculosis block food intake by creating a biofilm around the worm''s mouth (13, 27). This biofilm formation requires the hemin storage locus (hms) and has been suggested to be responsible for the blockage of the digestive tract following uptake by fleas, thus acting as a bacterial defense against predation by invertebrates. In a study with 40 Y. pseudotuberculosis strains, one-quarter of them caused an infection of C. elegans by biofilm formation on the worm head (27). In contrast, a similar effect was not observed following nematode infection with 15 Y. enterocolitica strains. Using a Y. pestis strain lacking the hms genes, it could be demonstrated that this mutant can infect and kill the nematode by a biofilm-independent mechanism that includes the accumulation of Y. pestis in the intestine of the worm (47). This pathogenesis model was applied to show that putative virulence factors such as YapH, OmpT, or a metalloprotease, Y3857, but not the virulence plasmids pCD1 and pPCP1, are required for Y. pestis virulence in C. elegans. Six yet unknown genes required for full virulence in C. elegans were also identified, and one of them appeared to be a virulence factor in the mouse infection model.C. elegans has not been used to study the pathogenicity properties of Y. enterocolitica, mainly due to the fact that many of its virulence factors are upregulated at 37°C in comparison to growth at lower temperatures while C. elegans cannot be cultivated at temperatures above 25°C. In this study, we examined for the first time the infection of C. elegans by Y. enterocolitica strains, demonstrating that this pathogen colonizes and kills C. elegans and that the insecticidal toxin TcaA, which is expressed only at ambient temperature, is required for full nematocidal activity.