In Situ μGISAXS: I. Experimental Setup for Submicron Study of Protein Nucleation and Growth

In this study, we used microbeam grazing-incidence small-angle x-ray scattering (μGISAXS) to investigate in situ protein nucleation and crystal growth assisted by a protein nanotemplate, and introduced certain innovations to improve the method. Our aim was to understand the protein nanotemplate method in detail, as this method has been shown to be capable of accelerating and increasing crystal size and quality, as well as inducing crystallization of proteins that are not crystallizable by classical methods. The nanotemplate experimental setup was used for drops containing growing protein crystals at different stages of nucleation and growth. Two model proteins, lysozyme and thaumatin, were used under unique flow conditions to differentially probe protein crystal nucleation and growth.     

Cell-free propagation of prion strains

Prions are the infectious agents responsible for prion diseases, which appear to be composed exclusively by the misfolded prion protein (PrP(Sc)). Disease is transmitted by the autocatalytic propagation of PrP(Sc) misfolding at the expense of the normal prion protein. The biggest challenge of the prion hypothesis has been to explain the molecular mechanism by which prions can exist as different strains, producing diseases with distinguishable characteristics. Here, we show that PrP(Sc) generated in vitro by protein misfolding cyclic amplification from five different mouse prion strains maintains the strain-specific properties. Inoculation of wild-type mice with in vitro-generated PrP(Sc) caused a disease with indistinguishable incubation times as well as neuropathological and biochemical characteristics as the parental strains. Biochemical features were also maintained upon replication of four human prion strains. These results provide additional support for the prion hypothesis and indicate that strain characteristics can be faithfully propagated in the absence of living cells, suggesting that strain variation is dependent on PrP(Sc) properties.     

Chemotherapy and autophagy-mediated cell death in pancreatic cancer cells

Autophagy is an evolutionarily preserved degradation process of cytoplasmic cellular constituents and plays important physiological roles in human health and disease. It has been proposed that autophagy plays an important role both in tumor progression and in promotion of cancer cell death, although the molecular mechanisms responsible for this dual action of autophagy in cancer have not been elucidated. Pancreatic ductal adenocarcinoma is one of the most aggressive human malignancies with 2-3% five-year survival rate. Its poor prognosis has been attributed to the lack of specific symptoms and early detection tools, and its relatively refractory to traditional cytotoxic agents and radiotherapy. Experimental evidence pointed at autophagy as a pancreatic cancer cell mechanism to survive under adverse environmental conditions, or as a defective programmed cell death mechanism that favors pancreatic cancer cell resistance to treatment. Here, we consider several phenotypical alterations that have been related to increase or decrease the autophagic process in pancreatic tumor cells. We specially review autophagy as a cell death mechanism in response to chemotherapeutic drugs.     

Is loss of function of the prion protein the cause of prion disorders?

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases that involve misfolding of the prion protein. Recent studies have provided evidence that normal prion protein might have a physiological function in neuroprotective signaling, suggesting that loss of prion protein activity might contribute to the pathogenesis of prion disease. However, studies using knockout animals do not support the loss-of-function hypothesis and argue that prion neurodegeneration might be associated with a gain of a toxic activity by the misfolded prion protein. Thus, the mechanism of neurodegeneration in spongiform encephalopathies remains enigmatic.     

Toxic HypF-N Oligomers Selectively Bind the Plasma Membrane to Impair Cell Adhesion Capability

The deposition of fibrillar protein aggregates in human organs is the hallmark of several pathological states, including highly debilitating neurodegenerative disorders and systemic amyloidoses. It is widely accepted that small oligomers arising as intermediates in the aggregation process, released by fibrils, or growing in secondary nucleation steps are the cytotoxic entities in protein-misfolding diseases, notably neurodegenerative conditions. Increasing evidence indicates that cytotoxicity is triggered by the interaction between nanosized protein aggregates and cell membranes, even though little information on the molecular details of such interaction is presently available. In this work, we propose what is, to our knowledge, a new approach, based on the use of single-cell force spectroscopy applied to multifunctional substrates, to study the interaction between protein oligomers, cell membranes, and/or the extracellular matrix. We compared the interaction of single Chinese hamster ovary cells with two types of oligomers (toxic and nontoxic) grown from the N-terminal domain of the Escherichia coli protein HypF. We were able to quantify the affinity between both oligomer type and the cell membrane by measuring the mechanical work needed to detach the cells from the aggregates, and we could discriminate the contributions of the membrane lipid and protein fractions to such affinity. The fundamental role of the ganglioside GM1 in the membrane-oligomers interaction was also highlighted. Finally, we observed that the binding of toxic oligomers to the cell membrane significantly affects the functionality of adhesion molecules such as Arg-Gly-Asp binding integrins, and that this effect requires the presence of the negatively charged sialic acid moiety of GM1.     

Sgt1, a co‐chaperone of Hsp90 stabilizes Polo and is required for centrosome organization

Sgt1 was described previously in yeast and humans to be a Hsp90 co‐chaperone and required for kinetochore assembly. We have identified a mutant allele of Sgt1 in Drosophila and characterized its function. Mutations in sgt1 do not affect overall kinetochore assembly or spindle assembly checkpoint. sgt1 mutant cells enter less frequently into mitosis and arrest in a prometaphase‐like state. Mutations in sgt1 severely compromise the organization and function of the mitotic apparatus. In these cells, centrioles replicate but centrosomes fail to mature, and pericentriolar material components do not localize normally resulting in highly abnormal spindles. Interestingly, a similar phenotype was described previously in Hsp90 mutant cells and correlated with a decrease in Polo protein levels. In sgt1 mutant neuroblasts, we also observe a decrease in overall levels of Polo. Overexpression of the kinase results in a substantial rescue of the centrosome defects; most cells form normal bipolar spindles and progress through mitosis normally. Taken together, these findings suggest that Sgt1 is involved in the stabilization of Polo allowing normal centrosome maturation, entry and progression though mitosis.     

Pyrolysis-gas chromatography of the fungus Metarhizium anisopliae: An aid to strain identification

Six strains of the entomopathogenic fungus Matarhizium anisopliae var. anisopliae from North and South America and one strain of M. anisopliae var. major from Samoa were compared by pyrolysis-gas chromatography of conidia. Two strains established to be very similar by other methods proved 98% similar by pyrolysis-gas chromatography. Similarities of the other strains ranged from 62 to 88%. The method is proposed as a simple technique for routine identification of M. anisopliae strains.     

Drought‐stress and plant resistance affect herbivore performance and proteome: the case of the green peach aphid Myzus persicae (Hemiptera: Aphididae)

Little is known about the simultaneous effects of drought stress and plant resistance on herbivorous insects. By subjecting the green peach aphid Myzus persicae Sulzer to well‐watered and drought‐stressed plants of both susceptible and resistant peach (Prunus persica), the effects of both stressors on aphid performance and proteomics are tested. Overall, the influence of the water treatment on aphid performance is less pronounced than the effect of host plant genetic resistance. On the susceptible cultivar, aphid survival, host acceptance and ability to colonize the plant do not depend on water treatment. On the resistant cultivar, aphid survival and ability to colonize are higher on drought‐stressed than on well‐watered plants. A study examining the pattern of protein expression aiming to explain the variation in aphid performance finds higher protein expression in aphids on the drought‐stressed susceptible cultivars compared with the well‐watered ones. In the susceptible cultivar, the regulated proteins are related to energy metabolism and exoskeleton functionality, whereas, in the resistant cultivar, the proteins are involved with the cytoskeleton. Comparison of the protein expression ratios for resistant versus susceptible plants reveals that four proteins are down‐regulated in well‐watered plants and 15 proteins are down‐regulated in drought‐stressed plants. Drought stress applied to the susceptible cultivar induces the regulation of proteins in M. persicae that enable physiological adaptation to maintain an almost unaltered aphid performance. By contrast, for aphids on the resistant cultivar subjected to drought stress, the down‐regulation of proteins responds to an induced host susceptibility effect.     

Two new Dematiaceous Hyphomycetes from humid tropic forest litter

Abstract

Two Dematiaceous Hyphomycetes, Beltraniella lyrata and Beltraniopsis fabularis, found on tropical forest litter in the Tai National Park in Ivory Coast are proposed as new species and described.     

The mode of action of Aescin and the release of prostaglandins

The horse-chestnut saponin Aescin, an anti-exudative compound, induces contraction of isolated portal vein of rat and rabbit. This effect appears to be mediated by Prostaglandins of F_α type.The ability of Aescin to stimulate generation and release of Prostaglandins has been demonstrated in isolated lung of the rat. Mass-fragmentographic analysis of the lung effluent indicate that when Aescin is perfused through this organ the release of PGF_2α is increased. The capability of Aescin to generate Prostaglandins is discussed in connection with its anti-exudative activity.