Fate and effects of the trehalase inhibitor trehazolin in the migratory locust (Locusta migratoria)

Trehalose is the main haemolymph sugar in many insect species. To be utilized trehalose must be hydrolysed into its glucose units by trehalase (EC 3.2.1.28). Inhibitors of trehalase have attracted interest as possible pesticides and tools for studying the regulation of trehalose metabolism in insects. To make full use of these inhibitors requires knowledge of their fate and effects in vivo. To this end we have measured trehazolin in locusts using a method based on the specific inhibition of a trehalase preparation. After injection of 20 μg, trehazolin decreased in haemolymph with a half-life of 2.6 days and after 10 days almost 95% had disappeared. Trehazolin did not reach the intracellular water space of locust tissues, but appeared with full inhibitory potency in locust faeces, suggesting that it was not metabolized, but quantitatively eliminated via the gut. Haemolymph trehalose increased transiently upon trehazolin injection, it was maximal after 3 days, then decreased and reached control level after 10 days. Inhibition of flight muscle trehalase by trehazolin was prolonged and still conspicuous 21 days post injection, suggesting that trehazolin inhibits trehalase activity irreversibly in vivo and that recovery requires de novo enzyme synthesis.     

Pathogenic mutations in the hydrophobic core of the human prion protein can promote structural instability and misfolding

Transmissible spongiform encephalopathies, or prion diseases, are caused by misfolding and aggregation of the prion protein PrP. These diseases can be hereditary in humans and four of the many disease-associated missense mutants of PrP are in the hydrophobic core: V180I, F198S, V203I and V210I. The T183A mutation is related to the hydrophobic core mutants as it is close to the hydrophobic core and known to cause instability. We used extensive molecular dynamics simulations of these five PrP mutants to compare their dynamics and conformations to those of the wild type PrP. The simulations highlight the changes that occur upon introduction of mutations and help to rationalize experimental findings. Changes can occur around the mutation site, but they can also be propagated over long distances. In particular, the F198S and T183A mutations lead to increased flexibility in parts of the structure that are normally stable, and the short β-sheet moves away from the rest of the protein. Mutations V180I, V210I and, to a lesser extent, V203I cause changes similar to those observed upon lowering the pH, which has been linked to misfolding. Early misfolding is observed in one V180I simulation. Overall, mutations in the hydrophobic core have a significant effect on the dynamics and stability of PrP, including the propensity to misfold, which helps to explain their role in the development of familial prion diseases.     

How fatty acids of different chain length enter and leave cells by free diffusion

Opposing views exist as to how unesterified fatty acids (FA) enter and leave cells. It is commonly believed that for short- and medium-chain FA free diffusion suffices whereas it is questioned whether proteins are required to facilitate transport of long-chain fatty acid (LCFA). Furthermore, it is unclear whether these proteins facilitate binding to the plasma membrane, trans-membrane movement, dissociation into the cytosol and/or transport in the cytosol. In this mini-review we approach the controversy from a different point of view by focusing on the membrane permeability constant (P) of FA with different chain length. We compare experimentally derived values of the P of short and medium-chain FA with values of apparent permeability coefficients for LCFA calculated from their dissociation rate constant (k(off)), flip-flop rate constant (k(flip)) and partition coefficient (Kp) in phospholipid bilayers. It was found that Overton's rule is valid as long as k(flip)相似文献   

Reconstructed human epidermis for skin absorption testing: results of the German prevalidation study

Exposure to chemicals absorbed by the skin can threaten human health. In order to standardise the predictive testing of percutaneous absorption for regulatory purposes, the OECD adopted guideline 428, which describes methods for assessing absorption by using human and animal skin. In this study, a protocol based on the OECD principles was developed and prevalidated by using reconstructed human epidermis (RHE). The permeation of the OECD standard compounds, caffeine and testosterone, through commercially available RHE models was compared to that of human epidermis and animal skin. In comparison to human epidermis, the permeation of the chemicals was overestimated when using RHE. The following ranking of the permeation coefficients for testosterone was obtained: SkinEthic > EpiDerm, EPISKIN > human epidermis, bovine udder skin, pig skin. The ranking for caffeine was: SkinEthic, EPISKIN > bovine udder skin, EpiDerm, pig skin, human epidermis. The inter-laboratory and intra-laboratory reproducibility was good. Long and variable lag times, which are a matter of concern when using human and pig skin, did not occur with RHE. Due to the successful transfer of the protocol, it is now in the validation process.     

Anaerobic Sulfide Oxidation with Nitrate by a Freshwater Beggiatoa Enrichment Culture

A lithotrophic freshwater Beggiatoa strain was enriched in O₂-H₂S gradient tubes to investigate its ability to oxidize sulfide with NO₃⁻ as an alternative electron acceptor. The gradient tubes contained different NO₃⁻ concentrations, and the chemotactic response of the Beggiatoa mats was observed. The effects of the Beggiatoa sp. on vertical gradients of O₂, H₂S, pH, and NO₃⁻ were determined with microsensors. The more NO₃⁻ that was added to the agar, the deeper the Beggiatoa filaments glided into anoxic agar layers, suggesting that the Beggiatoa sp. used NO₃⁻ to oxidize sulfide at depths below the depth that O₂ penetrated. In the presence of NO₃⁻ Beggiatoa formed thick mats (>8 mm), compared to the thin mats (ca. 0.4 mm) that were formed when no NO₃⁻ was added. These thick mats spatially separated O₂ and sulfide but not NO₃⁻ and sulfide, and therefore NO₃⁻ must have served as the electron acceptor for sulfide oxidation. This interpretation is consistent with a fourfold-lower O₂ flux and a twofold-higher sulfide flux into the NO₃⁻-exposed mats compared to the fluxes for controls without NO₃⁻. Additionally, a pronounced pH maximum was observed within the Beggiatoa mat; such a pH maximum is known to occur when sulfide is oxidized to S⁰ with NO₃⁻ as the electron acceptor.     

Polyphenolic compounds are novel protective agents against lipid membrane damage by α-synuclein aggregates in vitro

Cumulative evidence now suggests that the abnormal aggregation of the protein α-synuclein (αS) is a critical factor in triggering neurodegeneration in Parkinson's disease (PD). In particular, a fundamental pathogenetic mechanism appears to involve targeting of neuronal membranes by soluble oligomeric intermediates of αS, leading to their disruption or permeabilisation. Therefore, a model assay was developed in which fluorophore-loaded unilamellar vesicles were permeabilised by soluble oligomers, the latter formed by aggregation of human recombinant αS protein. The αS oligomers induced an impairment of membrane integrity similar to that of the pore-forming bacterial peptide gramicidin. The lipid vesicle permeabilisation assay was then utilised to screen 11 natural polyphenolic compounds, 8 synthetic N'-benzylidene-benzohydrazide compounds and black tea extract for protection against membrane damage by wild-type and mutant (A30P, A53T) synuclein aggregates. A select group of potent inhibitory compounds included apigenin, baicalein, morin, nordihydroguaiaretic acid, and black tea extract. Structure-activity analysis further suggests that a 5,7-dihydroxy-chromen-4-one moiety appears to be favourable for the inhibition reaction. In conclusion, we have identified a group of polyphenols that can effectively hinder membrane damage by αS aggregates. These may serve as a viable source of lead compounds for the development and design of novel therapeutic agents in PD.     

Two different binding modes of α-synuclein to lipid vesicles depending on its aggregation state

Aggregation of α-synuclein is involved in the pathogenesis of Parkinson's disease (PD). Studies of in vitro aggregation of α-synuclein are rendered complex because of the formation of a heterogeneous population of oligomers. With the use of confocal single-molecule fluorescence techniques, we demonstrate that small aggregates (oligomers) of α-synuclein formed from unbound monomeric species in the presence of organic solvent (DMSO) and iron (Fe³⁺) ions have a high affinity to bind to model membranes, regardless of the lipid-composition or membrane curvature. This binding mode contrasts with the well-established membrane binding of α-synuclein monomers, which is accompanied with α-helix formation and requires membranes with high curvature, defects in the lipid packing, and/or negatively charged lipids. Additionally, we demonstrate that membrane-bound α-synuclein monomers are protected from aggregation. Finally, we identified compounds that potently dissolved vesicle-bound α-synuclein oligomers into monomers, leaving the lipid vesicles intact. As it is commonly believed that formation of oligomers is related PD progression, such compounds may provide a promising strategy for the design of novel therapeutic drugs in Parkinson's disease.     

Endogenous fluorescence signatures in living pluripotent stem cells change with loss of potency

The therapeutic potential of stem cells is limited by the non-uniformity of their phenotypic state. Thus it would be advantageous to noninvasively monitor stem cell status. Driven by this challenge, we employed multidimensional multiphoton microscopy to quantify changes in endogenous fluorescence occurring with pluripotent stem cell differentiation. We found that global and cellular-scale fluorescence lifetime of human embryonic stem cells (hESC) and murine embryonic stem cells (mESC) consistently decreased with differentiation. Less consistent were trends in endogenous fluorescence intensity with differentiation, suggesting intensity is more readily impacted by nuances of species and scale of analysis. What emerges is a practical and accessible approach to evaluate, and ultimately enrich, living stem cell populations based on changes in metabolism that could be exploited for both research and clinical applications.     

Contrast-enhanced versus non-enhanced three-dimensional echocardiography of left ventricular volumes

Background In three-dimensional echocardiography (3DE), individual endocardial trabeculae are not clearly visible necessitating left ventricular (LV) volumes to be measured by tracing the innermost endocardial contour. Ultrasound contrast agents aim to improve endocardial definition, but may delineate the outermost endocardial contour by filling up intertrabecular space. Although measurement reproducibility may benefit, there may be a significant influence on absolute LV volume measurements. Methods Twenty patients with a recent myocardial infarction and good ultrasound image quality underwent 3DE using the TomTec Freehand method before and during continuous intravenous contrast infusion. LV volumes were measured offline using TomTec Echo-Scan software. Results The use of contrast enhancement increased end-diastolic (110±35 vs. 144±53 ml; p<0.01) and end-systolic volume measurements (68±31 vs. 87±45 ml; p<0.01) significantly compared with non-contrast; the ejection fraction remained unchanged (40±13 vs. 41±14%, p=NS). Measurement reproducibility did not improve significantly, however. Conclusion Volumes measured by 3DE are significantly larger when ultrasound contrast is used. Possibly, intertrabecular space comprises a substantial part of the LV cavity. In the presence of an adequate apical acoustic window, ultrasound contrast does not improve LV volume measurement reproducibility. (Neth Heart J 2008;16:47-52.)