Effect of hydrophobic mutations in the H2-H3 subdomain of prion protein on stability and conversion in vitro and in vivo

Prion diseases are fatal neurodegenerative diseases, which can be acquired, sporadic or genetic, the latter being linked to mutations in the gene encoding prion protein. We have recently described the importance of subdomain separation in the conversion of prion protein (PrP). The goal of the present study was to investigate the effect of increasing the hydrophobic interactions within the H2-H3 subdomain on PrP conversion. Three hydrophobic mutations were introduced into PrP. The mutation V209I associated with human prion disease did not alter protein stability or in vitro fibrillization propensity of PrP. The designed mutations V175I and T187I on the other hand increased protein thermal stability. V175I mutant fibrillized faster than wild-type PrP. Conversion delay of T187I was slightly longer, but fluorescence intensity of amyloid specific dye thioflavin T was significantly higher. Surprisingly, cells expressing V209I variant exhibited inefficient proteinase K resistant PrP formation upon infection with 22L strain, which is in contrast to cell lines expressing wild-type, V175I and T187I mPrPs. In agreement with increased ThT fluorescence at the plateau T187I expressing cell lines accumulated an increased amount of the proteinase K-resistant prion protein. We showed that T187I induces formation of thin fibrils, which are absent from other samples. We propose that larger solvent accessibility of I187 in comparison to wild-type and other mutants may interfere with lateral annealing of filaments and may be the underlying reason for increased conversion efficiency.     

Natural images from the birthplace of the human eye

Here we introduce a database of calibrated natural images publicly available through an easy-to-use web interface. Using a Nikon D70 digital SLR camera, we acquired about six-megapixel images of Okavango Delta of Botswana, a tropical savanna habitat similar to where the human eye is thought to have evolved. Some sequences of images were captured unsystematically while following a baboon troop, while others were designed to vary a single parameter such as aperture, object distance, time of day or position on the horizon. Images are available in the raw RGB format and in grayscale. Images are also available in units relevant to the physiology of human cone photoreceptors, where pixel values represent the expected number of photoisomerizations per second for cones sensitive to long (L), medium (M) and short (S) wavelengths. This database is distributed under a Creative Commons Attribution-Noncommercial Unported license to facilitate research in computer vision, psychophysics of perception, and visual neuroscience.     

How Did the Spider Cross the River? Behavioral Adaptations for River-Bridging Webs in Caerostris darwini (Araneae: Araneidae)

Background

Interspecific coevolution is well described, but we know significantly less about how multiple traits coevolve within a species, particularly between behavioral traits and biomechanical properties of animals'' “extended phenotypes”. In orb weaving spiders, coevolution of spider behavior with ecological and physical traits of their webs is expected. Darwin''s bark spider (Caerostris darwini) bridges large water bodies, building the largest known orb webs utilizing the toughest known silk. Here, we examine C. darwini web building behaviors to establish how bridge lines are formed over water. We also test the prediction that this spider''s unique web ecology and architecture coevolved with new web building behaviors.

Methodology

We observed C. darwini in its natural habitat and filmed web building. We observed 90 web building events, and compared web building behaviors to other species of orb web spiders.

Conclusions

Caerostris darwini uses a unique set of behaviors, some unknown in other spiders, to construct its enormous webs. First, the spiders release unusually large amounts of bridging silk into the air, which is then carried downwind, across the water body, establishing bridge lines. Second, the spiders perform almost no web site exploration. Third, they construct the orb capture area below the initial bridge line. In contrast to all known orb-weavers, the web hub is therefore not part of the initial bridge line but is instead built de novo. Fourth, the orb contains two types of radial threads, with those in the upper half of the web doubled. These unique behaviors result in a giant, yet rather simplified web. Our results continue to build evidence for the coevolution of behavioral (web building), ecological (web microhabitat) and biomaterial (silk biomechanics) traits that combined allow C. darwini to occupy a unique niche among spiders.     

Predicting incursion of plant invaders into Kruger National Park, South Africa: the interplay of general drivers and species-specific factors

Background

Overcoming boundaries is crucial for incursion of alien plant species and their successful naturalization and invasion within protected areas. Previous work showed that in Kruger National Park, South Africa, this process can be quantified and that factors determining the incursion of invasive species can be identified and predicted confidently. Here we explore the similarity between determinants of incursions identified by the general model based on a multispecies assemblage, and those identified by species-specific models. We analyzed the presence and absence of six invasive plant species in 1.0×1.5 km segments along the border of the park as a function of environmental characteristics from outside and inside the KNP boundary, using two data-mining techniques: classification trees and random forests.

Principal Findings

The occurrence of Ageratum houstonianum, Chromolaena odorata, Xanthium strumarium, Argemone ochroleuca, Opuntia stricta and Lantana camara can be reliably predicted based on landscape characteristics identified by the general multispecies model, namely water runoff from surrounding watersheds and road density in a 10 km radius. The presence of main rivers and species-specific combinations of vegetation types are reliable predictors from inside the park.

Conclusions

The predictors from the outside and inside of the park are complementary, and are approximately equally reliable for explaining the presence/absence of current invaders; those from the inside are, however, more reliable for predicting future invasions. Landscape characteristics determined as crucial predictors from outside the KNP serve as guidelines for management to enact proactive interventions to manipulate landscape features near the KNP to prevent further incursions. Predictors from the inside the KNP can be used reliably to identify high-risk areas to improve the cost-effectiveness of management, to locate invasive plants and target them for eradication.     

Strawberry polyphenols attenuate ethanol-induced gastric lesions in rats by activation of antioxidant enzymes and attenuation of MDA increase

Background and Aim

Free radicals are implicated in the aetiology of gastrointestinal disorders such as gastric ulcer, colorectal cancer and inflammatory bowel disease. Strawberries are common and important fruit due to their high content of essential nutrient and beneficial phytochemicals which seem to have relevant biological activity on human health. In the present study we investigated the antioxidant and protective effects of three strawberry extracts against ethanol-induced gastric mucosa damage in an experimental in vivo model and to test whether strawberry extracts affect antioxidant enzyme activities in gastric mucosa.

Methods/Principal Findings

Strawberry extracts were obtained from Adria, Sveva and Alba cultivars. Total antioxidant capacity and radical scavenging capacity were performed by TEAC, ORAC and electron paramagnetic resonance assays. Identification and quantification of anthocyanins was carried out by HPLC-DAD-MS analyses. Different groups of animals received 40 mg/day/kg body weight of strawberry crude extracts for 10 days. Gastric damage was induced by ethanol. The ulcer index was calculated together with the determination of catalase and SOD activities and MDA contents. Strawberry extracts are rich in anthocyanins and present important antioxidant capacity. Ethanol caused severe gastric damage and strawberry consumption protected against its deleterious role. Antioxidant enzyme activities increased significantly after strawberry extract intake and a concomitantly decrease in gastric lipid peroxidation was found. A significant correlation between total anthocyanin content and percent of inhibition of ulcer index was also found.

Conclusions

Strawberry extracts prevented exogenous ethanol-induced damage to rats'' gastric mucosa. These effects seem to be associated with the antioxidant activity and phenolic content in the extract as well as with the capacity of promoting the action of antioxidant enzymes. A diet rich in strawberries might exert a beneficial effect in the prevention of gastric diseases related to generation of reactive oxygen species.     

New noncovalent inhibitors of penicillin-binding proteins from penicillin-resistant bacteria

Background

Penicillin-binding proteins (PBPs) are well known and validated targets for antibacterial therapy. The most important clinically used inhibitors of PBPs β-lactams inhibit transpeptidase activity of PBPs by forming a covalent penicilloyl-enzyme complex that blocks the normal transpeptidation reaction; this finally results in bacterial death. In some resistant bacteria the resistance is acquired by active-site distortion of PBPs, which lowers their acylation efficiency for β-lactams. To address this problem we focused our attention to discovery of novel noncovalent inhibitors of PBPs.

Methodology/Principal Findings

Our in-house bank of compounds was screened for inhibition of three PBPs from resistant bacteria: PBP2a from Methicillin-resistant Staphylococcus aureus (MRSA), PBP2x from Streptococcus pneumoniae strain 5204, and PBP5fm from Enterococcus faecium strain D63r. Initial hit inhibitor obtained by screening was then used as a starting point for computational similarity searching for structurally related compounds and several new noncovalent inhibitors were discovered. Two compounds had promising inhibitory activities of both PBP2a and PBP2x 5204, and good in-vitro antibacterial activities against a panel of Gram-positive bacterial strains.

Conclusions

We found new noncovalent inhibitors of PBPs which represent important starting points for development of more potent inhibitors of PBPs that can target penicillin-resistant bacteria.     

Colchicine Treatment Differently Affects Releasable Thyrotropin-Releasing Hormone (TRH) Pools in the Hypothalamic Paraventricular Nucleus (PVN) and the Median Eminence (ME)

1. Hypophysiotropic thyrotropin-releasing hormone (TRH) is synthesized in the hypothalamic paraventricular nucleus (PVN) and transported to the median eminence (ME) where it enters the hypophyseal portal blood. TRH in the ME is situated exclusively in nerve terminals, whereas TRH in the PVN and septum is of extrinsic (nerve terminals) as well as intrinsic (perikarya) origin. 2. To determine the source and possible differential regulation of TRH release from these structures, we blocked TRH axonal delivery by i.c.v. administration of colchicine into the lateral cerebral ventricle of euthyroid or hypothyroid rats in doses of 7.5 μg or 7.5, 75 and 100 μg, respectively, two days prior to the evaluation of the TRH secretion from the PVN, ME and the septum in vitro. 3. In euthyroid rats a low dose of colchicine did not significantly affect plasma TSH. The secretory response to both ethanol in an isosmolar medium and a high K⁺ in the ME as well as the PVN explants was well preserved. However, colchicine treatment resulted in the significant increase of basal secretion of TRH from the PVN. 4. Hypothyroidism induced by 200 mg/l methimazole in drinking water for two weeks resulted in growth arrest, elevated plasma thyrotropin and decreased TRH content in the PVN and the ME. Colchicine partially decreased elevated plasma thyrotropin and increased the TRH content in the PVN and its basal release in vitro which was independent of extracellular Ca²⁺. Interestingly, a TRH release from the PVN could not be further stimulated either by K⁺ membrane depolarization or by ethanol. TRH responsiveness to the stimulation remained unaffected in the ME. The effect of colchicine on the septal TRH secretion was intermediate between the effect observed in the PVN and the ME. 5. In conclusion, the absence of a TRH secretory response to stimuli in the PVN after colchicine disruption of the microtubules and Golgi system suggests that stimulated TRH release observed from the PVN explants in vitro occurs from nerve terminals projecting to the PVN from other brain regions. The independence from extracellular calcium implies that TRH released under the non-stimulating conditions occurs most likely via the constitutive secretory pathway from dendrites and/or perikarya. Regulation of septal TRH is markedly different from the hypophysiotropic one. An erratum to this article is available at .     

Cholesterol conjugated oligonucleotide and LNA: A comparison of cellular and nuclear uptake by Hep2 cells enhanced by Streptolysin-O

Antisense and antigene oligonucleotides (ONs) are attractive drugs for gene therapy, but major limiting factors for their routine use are inefficient cellular uptake and low accessibility to the target sites. Adding various lipophilic conjugates to the ON improves intracellular delivery as has been previously reported.We studied the cellular delivery of various ON modifications, as well as their cytosolic and nuclear distribution in mammalian Hep2-EGFP-NLS cell line. We compared uptake efficacy of ON and LNA, both conjugated with cholesterol at the 5′ end. All ONs were 3′ labeled with fluorescent Cy5 dye. We made a comparison of the ONs uptake efficacy and the kinetics, both adding ONs to the culture medium, and using streptolysin-O (SL-O) permeabilization.The cellular uptake of each ON used in this study was visualized by fluorescent microscopy. We confirmed the results by FACS analysis. We determined the ratio between initial ON-chol concentration (0.4 μM) and the final amount in nucleus.SL-O can highly improve kinetics of ON delivery; not only into the cytoplasm but also to the nucleus, the presumed site of antigene ON action. The most effective nuclear uptake was observed when ON conjugated with cholesterol (ON-chol) and SL-O was used. Nuclear distribution of ON was reached within few minutes. In contrast, ON simply added to the medium reached cytoplasm only and the process of delivery took several hours. (Mol Cell Biochem 276: 61–69, 2005)     

Photosynthetic performance of vegetative and reproductive structures of green hellebore (<Emphasis Type="Italic">Helleborus viridis</Emphasis> L. agg.)

Photosynthetic irradiance response of vegetative and reproductive structures of the green-flowered deciduous perennial green hellebore was studied by the comparative use of chlorophyll (Chl) fluorescence techniques and gas exchange measurements. All the Chl-containing organs (leaves, sepals, stalks, and fruits) examined were photosynthetically active showing high intrinsic efficiencies of photosystem 2 (F_v/F_m: 0.75–0.79) after dark adaptation. Even in the smaller fertile and sterile parts of the flower (nectaries and anthers) a remarkable photosynthetic competence was detected. With increasing photon flux densities (PFD) electron transport rates, actual quantum yields, and photochemical quenching coefficients of the main photosynthetic organs decreased in the order: leaf>sepal>fruit>stalk. At moderate to high PFDs the sepals achieved maximum electron transport rates corresponding to about 80 % of concomitant mature leaves. In contrast, maximum net photosynthetic rate of the sepals [2.3 mol(CO₂) m^–2 s^–1] were less than one fourth of the leaves [10.6 mol(CO₂) m^–2 s^–1]. This difference is explained by a 70–80 % lower stomatal density of sepals in comparison to leaves. As the basal leaves emerge late during fruit development, the photosynthetically active sepals are a major source of assimilates, contributing more than 60 % of whole-plant CO₂ gain in early spring. The ripening dehiscent fruits are characterized by an effective internal re-fixation of the respirational carbon loss and thus additionally improve the overall carbon budget.