Effect of Lincocin Treatment on the Greening Process in Bean (Phaseolus vulgaris) Leaves

The effect of lincocin (a plastid protein synthesis inhibitor) treatment on the greening process of bean (Phaseolus vulgaris L.) leaves have been studied. In comparison with control leaves treated ones had a decreased rate of chloroplast development. They had a marked chlorophyll deficiency and a decreased chlorophyll a/b ratio. Some long and short wavelength forms of chlorophyll a were lacking as evidenced from the absorption spectra at 25°C and the fluorescence spectra at 77°K. The –¹⁴CO₂ fixation was inhibited by 80–90% in treated leaves. The fluorescence induced by the measuring light was greater in the treated leaves than in the control ones, and the kinetics of the decline of the relative fluorescence intensity were also different. Electron microscopic studies showed macrogranum-like structures and incomplete membrane vesicles in the treated plastids. After longer treatment a destruction of membranes was observed. The results indicate some structural and functional membrane deficiencies and instability of the membranes.     

Anionic micelles and vesicles induce tau fibrillization in vitro

Alzheimer's disease is defined in part by the intraneuronal accumulation of filaments comprised of the microtubule-associated protein tau. In vitro, fibrillization of recombinant tau can be induced by treatment with various agents, including phosphotransferases, polyanionic compounds, and fatty acids. Here we characterize the structural features required for the fatty acid class of tau fibrillization inducer using recombinant full-length tau protein, arachidonic acid, and a series of straight chain anionic, cationic, and nonionic detergents. Induction of measurable tau fibrillization required an alkyl chain length of at least 12 carbons and a negative charge consisting of carboxylate, sulfonate, or sulfate moieties. All detergents and fatty acids were micellar at active concentrations, due to a profound, taudependent depression of their critical micelle concentrations. Anionic surfaces larger than detergent micelles, such as those supplied by phosphatidylserine vesicles, also induced tau fibrillization with resultant filaments originating from their surface. These data suggest that anionic surfaces presented as micelles or vesicles can serve to nucleate tau fibrillization, that this mechanism underlies the activity of fatty acid inducers, and that anionic membranes may serve this function in vivo.     

Thioureides of 2-(phenoxymethyl)benzoic acid 4-R substituted: A novel class of anti-parasitic compounds

Fifty members of a novel class of antimicrobial compounds, 2-(4-R-phenoxymethyl)benzoic acid thioureides, were synthesized and characterized with respect to their activities against three parasites of human relevance, namely the protozoa Giardia lamblia and Toxoplasma gondii, and the larval (metacestode) stage of the tapeworm Echinococcus multilocularis. To determine the selective toxicity of these compounds, the human colon cancer cell line Caco2 and primary cultures of human foreskin fibroblasts (HFF) were also investigated. The new thioureides were obtained in a three-step-reaction process and subsequently characterized by their physical constants (melting point, solubility). The chemical structures were elucidated by ¹H NMR, ¹³C NMR, IR spectral methods and elemental analysis. The analyses confirmed the final and intermediate compound structures and the synthesis. The compounds were then tested on the parasites in vitro. All thioureides, except two compounds with a nitro group, were totally ineffective against Giardia lamblia. 23 compounds inhibited the proliferation of T. gondii, three of them with an IC₅₀ of approximately 1 μM. The structural integrity of E. multilocularis metacestodes was affected by 22 compounds. In contrast, HFF were not susceptible to any of these thioureides, while Caco2 cells were affected by 17 compounds, two of them inhibiting proliferation with an IC₅₀ in the micromolar range. Thioureides may thus present a promising class of anti-infective agents.     

Adult spinal opioid receptor μ1 expression after incision is altered by early life repetitive tactile and noxious procedures in rats

Clinical and experimental data suggests that noxious stimulation at critical stages of development results in long‐term changes on nociceptive processing in later life. Here, we use an established, well‐documented rat model of repetitive noxious procedures closely mimicking the clinical situation in the NICU. In order to understand molecular changes underlying the long‐term consequences of repetitive stimulation of the developing nociceptive system the present study aims to analyze the presence of the µ‐opioid‐receptor‐1 (OPRM1). Neonatal rats received either four needle pricks per day in the left hind‐paw from postnatal day 0–7 as a model of procedural pain in infancy. Control pups were handled in the same way but were instead tactile stimulated, or were left undisturbed. At the age of 8 weeks, all animals received an ipsilateral hind‐paw incision as a model for post‐operative pain, and mechanical sensitivity was tested at multiple time‐points. Before, and 1 or 5 days post‐incision, spinal cord tissue was collected for immunostaining of opioid receptor OPRM1. Semi‐quantitative immunocytochemical analysis of superficial laminae in lumbar spinal dorsal horn revealed that: (1) early life repetitive tactile or noxious procedures do not alter baseline levels of OPRM1 staining intensity and (2) early life repetitive tactile or noxious procedures lead to a decrease in OPRM1 staining intensity 5 days after incision in adulthood compared to undisturbed controls. We conclude that early life repetitive tactile or noxious procedures affect the intensity of OPRM1‐immunoreactivity in the lumbar superficial spinal cord dorsal horn after adulthood injury, without affecting baseline intensity. © 2018 The Authors. Developmental Neurobiology Published by Wiley Periodicals, Inc. Develop Neurobiol 78: 417–426, 2018     

Evidence for an intermediate in tau filament formation

Alzheimer's disease is defined in part by the intraneuronal accumulation of filaments comprised of the microtubule-associated protein tau. In vitro, fibrillization of full-length, unphosphorylated recombinant tau can be induced under near-physiological conditions by treatment with various agents, including anionic surfactants. Here we examine the pathway through which anionic surfactants promote tau fibrillization using a combination of electron microscopy and fluorescence spectroscopy. Protein and surfactant first interacted in solution to form micelles, which then provided negatively charged surfaces that accumulated tau aggregates. Surface aggregation of tau protein was followed by the time-dependent appearance of a thioflavin S reactive intermediate that accumulated over a period of hours. The intermediate was unstable in the absence of anionic surfaces, suggesting it was not filamentous. Fibrillization proceeded after intermediate formation with classic nucleation-dependent kinetics, consisting of lag phase followed by the exponential increase in filament lengths, followed by an equilibrium phase reached in approximately 24 h. The pathway did not require protein insertion into the micelle hydrophobic core or conformational change arising from mixed micelle formation, because anionic microspheres constructed from impermeable polystyrene were capable of qualitatively reproducing all aspects of the fibrillization reaction. It is proposed that the progression from amorphous aggregation through intermediate formation and fibrillization may underlie the activity of other inducers such as hyperphosphorylation and may be operative in vivo.     

Ligand-dependent inhibition and reversal of tau filament formation

Alzheimer's disease is defined in part by the intraneuronal accumulation of filaments comprised of the microtubule associated protein tau. Because animal model studies suggest that a toxic gain of function accompanies tau aggregation in neurons, selective pharmacological inhibitors of the process may have utility in slowing neurodegeneration. Here, the properties of a candidate small molecule inhibitor of tau fibrillization, 3-(2-hydroxyethyl)-2-[2-[[3-(2-hydroxyethyl)-5-methoxy-2-benzothiazolylidene]methyl]-1-butenyl]-5-methoxybenzothiazolium (N744), were characterized in vitro using transmission electron microscopy. N744 inhibited arachidonic acid-induced aggregation of full-length, four-repeat tau protein at substoichiometric concentrations relative to total tau and with an IC(50) of approximately 300 nM. Inhibition was accompanied by a dose-dependent decrease in the number concentration of filaments, suggesting that N744 interfered with tau filament nucleation. Stoichiometric concentrations of N744 also promoted tau disaggregation when added to mature synthetic filaments. Disaggregation followed first-order kinetics and was accompanied by a steady decrease in filament number, suggesting that N744 promoted endwise loss of tau molecules with limited filament breakage. N744 at substoichiometric concentrations did not inhibit Abeta and alpha-synuclein aggregation, indicating it was tau selective under these conditions. Because of its activity in vitro, N744 may offer a pharmacological approach to the role of tau fibrillization in neurodegeneration.     

Rapid anionic micelle-mediated alpha-synuclein fibrillization in vitro

Parkinson's disease is characterized by the aggregation of alpha-synuclein into filamentous forms within affected neurons of the basal ganglia. Fibrillization of purified recombinant alpha-synuclein is inefficient in vitro but can be enhanced by the addition of various agents including glycosaminoglycans and polycations. Here we report that fatty acids and structurally related anionic detergents greatly accelerate fibrillization of recombinant alpha-synuclein at low micromolar concentrations with lag times as short as 11 min and apparent first order growth rate constants as fast as 10.4 h-1. All detergents and fatty acids were micellar at active concentrations because of an alpha-synuclein-dependent depression of their critical micelle concentrations. Other anionic surfaces, such as those supplied by anionic phospholipid vesicles, also induced alpha-synuclein fibrillization, with resultant filaments originating from their surface. These data suggest that anionic surfaces presented as micelles or vesicles can serve to nucleate alpha-synuclein fibrillization, that this mechanism underlies the inducer activity of anionic surfactants, and that anionic membranes may serve this function in vivo.     

Triggers of full-length tau aggregation: a role for partially folded intermediates

Alzheimer's disease is characterized in part by the accumulation of full-length tau proteins into intracellular filamentous inclusions. To clarify the events that trigger lesion formation, the aggregation of recombinant full-length four-repeat tau (htau40) was examined in vitro under near-physiological conditions using transmission electron microscopy and spectroscopy methods. In the absence of exogenous inducers, tau protein behaved as an assembly-incompetent monomer with little tertiary structure. The addition of anionic inducers led to fibrillization with nucleation-dependent kinetics. On the basis of circular dichroism spectroscopy and reactivity with thioflavin S and 8-anilino-1-naphthalenesulfonic acid fluorescent probes, the inducer stabilized a monomeric species with the folding characteristics of a premolten globule state. Planar aromatic dyes capable of binding the intermediate state with high affinity were also capable of triggering fibrillization in the absence of other inducers. Dye-mediated aggregation was characterized by concentration-dependent decreases in lag time, indicating increased nucleation rates, and submicromolar critical concentrations, indicating a final equilibrium that favored the filamentous state. The data suggest that the rate-limiting barrier for filament formation from full-length tau is conformational and that the aggregation reaction is triggered by environmental conditions that stabilize assembly-competent conformations.     

Egg size and composition in an ageing capital breeder – consequences for offspring performance

1. Egg size is often used as a proxy of egg quality although size and composition may vary, e.g. in insects egg size usually decreases as female ages. Whether this decrease in size reflects reduced concentrations of essential nutrients such as lipids and proteins of eggs laid by ageing females, or does reduced size per se explain often observed lower fitness of later laid eggs is poorly explored. 2. Egg properties were compared with fitness parameters of offspring laid on the first and fourth night during the oviposition period of a capital breeding moth, Cleorodes lichenaria (Hufnagel). The study aim was to explore whether decreased egg size is caused by decreased provisioning into later laid eggs measured as egg protein and lipid concentration, and whether it results in lower fitness of later laid offspring. 3. The fresh and dry weight of eggs decreased over the oviposition period, but the protein and lipid concentration remained constant. Survival of larvae was lower among the fourth night laid offspring on a low quality host Parmelia sulcata Taylor compared to a high quality host Ramalina fraxinea (L.) Ach. No differences were observed in egg fertility or hatchability, neonate survival without food and pupal mass between the offspring produced on different nights. 4. Decreased survival of offspring produced later was rather attributable to absolute provisioning (i.e. lower weight of eggs) than relative provisioning (i.e. decreased concentrations of nutrients in eggs). It is argued that lower survival of later laid smaller eggs on low quality diet is likely attributable to physical and chemical characteristics of host lichens and/or physical properties of tiny neonate larvae.