Hypovirulence-associated traits induced by a mycovirus of Cryphonectria parasitica are mimicked by targeted inactivation of a host gene.

Expression of the Vir2 gene of Cryphonectria parasitica is down-regulated in strains of the fungus containing a double-stranded RNA genetic element that reduces fungal virulence (W. A. Powell and N. K. Van Alfen, Mol. Cell. Biol. 7:3688-3693, 1987). We have sequenced the Vir2 gene and characterized its structure; the mRNA contains a short open reading frame whose product has structural similarities to several fungal pheromones. A null mutant was constructed by homologous recombination to determine the function of the Vir2 gene and whether its disruption resulted in any of the altered phenotypes exhibited by many hypovirulent strains, such as reductions in virulence, pigmentation, and sporulation. The Vir2 null mutant (18dm) exhibited a wild-type phenotype with respect to gross colony morphology, growth rate, pigmentation, asexual spore viability, and virulence in apple fruit and chestnut trees. However, numbers of asexual fruiting bodies (pycnidia) and conidia were reduced significantly in comparison with the wild-type strain EP155/2. In sexual crosses of 18dm with a wild-type strain of the opposite mating type, perithecia (sexual fruiting bodies) developed but were barren. Deletion of the Vir2 gene results in a phenotype that mimics that of many double-stranded-RNA-containing hypovirulent strains; i.e., the null mutant exhibits significant reductions in asexual sporulation and pycinidum production as well as impaired sexual crossing ability. To our knowledge, this is the first report of the partial reproduction of a virus-induced phenotype by deletion of a virus-perturbed host gene.     

Intra-specific fusions in Solanum tuberosum

Summary Plants were regenerated from callus arising from protoplast fusion of two S. tuberosum diploids. Tetraploid progeny from the fusion of the two diploid partners had increased vigor. Isozyme analysis confirmed the presence of proteins from both partners in the fusion progeny. Pigmentation of tubers and anthers was heightened substantially in the fusion products. This fusion, the first intra-specific fusion within S. tuberosum, indicates that somatic fusion may be useful for transferring traits within this group.     

Susceptibility of the gray wolf (Canis lupus) to infection with the Lyme disease agent, Borrelia burgdorferi

Four juvenile gray wolves (Canis lupus) were inoculated with live Borrelia burgdorferi. One received an intravenous inoculum, a second was inoculated subcutaneously, and two more were fed Peromyscus maniculatus sucklings which had earlier been inoculated with B. burgdorferi. The intravenously inoculated wolf developed a generalized lymphadenopathy and a persistent serum antibody titer to the spirochete which peaked at 1:512. Borrelia burgdorferi was visualized in liver sections of this wolf using direct immunofluorescent staining. The subcutaneously inoculated wolf showed a low and transient antibody response which peaked at 1:64, and manifested no clinical or postmortem abnormalities. The wolves which were fed inoculated mice showed no detectable antibody response. They were clinically normal throughout the project, and there were no detectable lesions at necropsy. Two control wolves were inoculated intravenously with formalin killed B. burgdorferi. Serum antibody titers of these controls peaked at 1:64 and 1:32, respectively, and fell to 1:16 by day 48 postinoculation. A survey of serum samples from 78 wild-trapped wolves from Wisconsin and Minnesota revealed that one was positive and another was suspect for B. burgdorferi infection based on presence of antibody to the spirochete. We conclude that the wolf is susceptible to infection by B. burgdorferi and that wolves are being infected in the wild.     

The carbon monoxide releasing molecule CORM-2 attenuates Pseudomonas aeruginosa biofilm formation

Chronic infections resulting from biofilm formation are difficult to eradicate with current antimicrobial agents and consequently new therapies are needed. This work demonstrates that the carbon monoxide-releasing molecule CORM-2, previously shown to kill planktonic bacteria, also attenuates surface-associated growth of the gram-negative pathogen Pseudomonas aeruginosa by both preventing biofilm maturation and killing bacteria within the established biofilm. CORM-2 treatment has an additive effect when combined with tobramycin, a drug commonly used to treat P. aeruginosa lung infections. CORM-2 inhibited biofilm formation and planktonic growth of the majority of clinical P. aeruginosa isolates tested, for both mucoid and non-mucoid strains. While CORM-2 treatment increased the production of reactive oxygen species by P. aeruginosa biofilms, this increase did not correlate with bacterial death. These data demonstrate that CO-RMs possess potential novel therapeutic properties against a subset of P. aeruginosa biofilm related infections.     

Innate immune responses to Pseudomonas aeruginosa infection

Innate immune responses play a critical role in controlling acute infections due to Pseudomonas aeruginosa in both mice and in humans. In this review we focus on innate immune recognition and clearance mechanisms that are important for controlling P. aeruginosa in the mammalian lung, with particular attention to those that influence the outcome of in vivo infection in murine models.     

Regulation of kinase activity by diffusion and feedback

In living cells proteins motilities regulate the spatiotemporal dynamics of molecular pathways. We consider here a reaction-diffusion model of mutual kinase-receptor activation showing that the strength of positive feedback is controlled by the kinase diffusion coefficient. For high diffusion, the activated kinase molecules quickly leave the vicinity of the cell membrane and cannot efficiently activate the receptors. As a result, in a broad range of parameters, the cell can be activated only if the kinase diffusion coefficient is sufficiently small. Our simple model shows that change in the motility of substrates may dramatically influence the cell responses.     

Borreliosis in free-ranging black bears from Wisconsin

Blood, kidney and tick samples were obtained from 18 hunter-killed black bears (Ursus americanus) from three sites in northern Wisconsin. A Borrelia sp., morphologically and antigenically similar to Borrelia burgdorferi, was isolated from the blood of two of the animals, and from the kidney of a third. Ixodes dammini and Dermacentor variabilis were found on the bears. This is the first report of borreliosis in the Ursidae, and of the primary vector of Lyme disease, I. dammini, from this host.     

Calcium carbonate precipitation in cyanobacterial mats from sandy tidal flats of the North Sea

Precipitated calcium carbonate was found in annual cyanobacterial mats developing on the beaches of the North Sea barrier island Schiermonnikoog (the Netherlands). A variety of different calcium carbonate morphs were found in the cyanobacterial mucous secretions and identified by light- and scanning electron microscopy and X-ray diffraction. Most of the calcium carbonate seemed to be associated with degrading extracellular polymer. It is conceived that supersaturation of calcium carbonate resulted from the periodic evaporation of the mats and from the release of calcium from the cyanobacterial mucous as a result of its degradation. The analysis of the carbon stable isotopic composition of the calcium carbonate showed only a slight depletion of ¹³C, indicating that it did not in major part originated from the decomposition of organic matter. The δ¹⁸O values of the calcium carbonate confirmed the temperature differences between spring and summer but excluded the possibility that excessive evaporative events controlled precipitation. The precipitation of calcium carbonate could represent a potential factor enhancing the stabilization of intertidal siliciclastic sediments through cementing the sand. The discovery of massive calcium carbonate precipitation in these cyanobacterial mats was unexpected and hitherto unknown.     

alpha-Synuclein enhances secretion and toxicity of amyloid beta peptides in PC12 cells

α-Synuclein is the fundamental component of Lewy bodies which occur in the brain of 60% of sporadic and familial Alzheimer’s disease patients. Moreover, a proteolytic fragment of α-synuclein, the so-called non-amyloid component of Alzheimer’s disease amyloid, was found to be an integral part of Alzheimer’s dementia related plaques. However, the role of α-synuclein in pathomechanism of Alzheimer’s disease remains elusive. In particular, the relationship between α-synuclein and amyloid beta is unknown. In the present study we showed the involvement of α-synuclein in amyloid beta secretion and in the mechanism of amyloid beta evoked mitochondria dysfunction and cell death. Rat pheochromocytoma PC12 cells transfected with amyloid beta precursor protein bearing Swedish double mutation (APPsw) and control PC12 cells transfected with empty vector were used in this study. α-Synuclein (10 μM) was found to increase by twofold amyloid beta secretion from control and APPsw PC12 cells. Moreover, α-synuclein decreased the viability of PC12 cells by about 50% and potentiated amyloid beta toxicity leading to mitochondrial dysfunction and caspase-dependent programmed cell death. Inhibitor of caspase-3 (Z-DEVD-FMK, 100 μM), and a mitochondrial permeability transition pore blocker, cyclosporine A (2 μM) protected PC12 cells against α-synuclein or amyloid beta evoked cell death. In contrast Z-DEVD-FMK and cyclosporine A were ineffective in APPsw cells containing elevated amount of amyloid beta treated with α-synuclein. It was found that the inhibition of neuronal and inducible nitric oxide synthase reversed the toxic effect of α-synuclein in control but not in APPsw cells. Our results indicate that α-synuclein enhances the release and toxicity of amyloid beta leading to nitric oxide mediated irreversible mitochondria dysfunction and caspase-dependent programmed cell death.