Sulfated polysaccharides promote the assembly of amyloid beta(1-42) peptide into stable fibrils of reduced cytotoxicity

The histopathological hallmarks of Alzheimer disease are the self-aggregation of the amyloid beta peptide (Abeta) in extracellular amyloid fibrils and the formation of intraneuronal Tau filaments, but a convincing mechanism connecting both processes has yet to be provided. Here we show that the endogenous polysaccharide chondroitin sulfate B (CSB) promotes the formation of fibrillar structures of the 42-residue fragment, Abeta(1-42). Atomic force microscopy visualization, thioflavin T fluorescence, CD measurements, and cell viability assays indicate that CSB-induced fibrils are highly stable entities with abundant beta-sheet structure that have little toxicity for neuroblastoma cells. We propose a wedged cylinder model for Abeta(1-42) fibrils that is consistent with the majority of available data, it is an energetically favorable assembly that minimizes the exposure of hydrophobic areas, and it explains why fibrils do not grow in thickness. Fluorescence measurements of the effect of different Abeta(1-42) species on Ca(2+) homeostasis show that weakly structured nodular fibrils, but not CSB-induced smooth fibrils, trigger a rise in cytosolic Ca(2+) that depends on the presence of both extracellular and intracellular stocks. In vitro assays indicate that such transient, local Ca(2+) increases can have a direct effect in promoting the formation of Tau filaments similar to those isolated from Alzheimer disease brains.     

Collpas: Activity Hotspots for Frugivorous Bats (Phyllostomidae) in the Peruvian Amazon

In the SE Peruvian Amazon, large numbers of frugivorous bats regularly visit natural forest clearings known locally as collpas (which are also referred to as clay licks or mineral licks). Bats arrive at collpas to drink water that has accumulated in depressions created by larger geophagous mammals that consume exposed soil. Although collpa visitation is common, little is known about its causes and its ecological implications for the bat community. We compared patterns of use of collpas and non- collpa forest sites by bats in SE Peru. We mist netted bats at collpas and non- collpa sites during the dry season and compared abundance, species richness, species composition, sex ratio, and reproductive condition. More species were captured at collpas than at non- collpa sites, and collpas were visited almost exclusively by frugivores. Overall, bat-capture frequency and combined frugivorous bat-capture frequency were higher at collpas than at non- collpa sites, although some species of frugivorous bats were captured more frequently at non- collpa sites than at collpas ( e.g ., Carollia spp.). Irrespective of capture site, more female bats were pregnant or lactating than not, but there was a distinct female sex bias in bats that visited collpas : 70 percent of bats captured at collpas were female, whereas 44 percent of bats captured away from collpas were female. These patterns suggest that collpas may provide important resources for frugivorous bats in SE Peru, just as they are thought to provide important resources to the vertebrates that consume collpa soils. Accordingly, collpas are important conservation targets in the region.     

Binding of sulphonated indigo derivatives to RepA-WH1 inhibits DNA-induced protein amyloidogenesis

The quest for inducers and inhibitors of protein amyloidogenesis is of utmost interest, since they are key tools to understand the molecular bases of proteinopathies such as Alzheimer, Parkinson, Huntington and Creutzfeldt–Jakob diseases. It is also expected that such molecules could lead to valid therapeutic agents. In common with the mammalian prion protein (PrP), the N-terminal Winged-Helix (WH1) domain of the pPS10 plasmid replication protein (RepA) assembles in vitro into a variety of amyloid nanostructures upon binding to different specific dsDNA sequences. Here we show that di- (S2) and tetra-sulphonated (S4) derivatives of indigo stain dock at the DNA recognition interface in the RepA-WH1 dimer. They compete binding of RepA to its natural target dsDNA repeats, found at the repA operator and at the origin of replication of the plasmid. Calorimetry points to the existence of a major site, with micromolar affinity, for S4-indigo in RepA-WH1 dimers. As revealed by electron microscopy, in the presence of inducer dsDNA, both S2/S4 stains inhibit the assembly of RepA-WH1 into fibres. These results validate the concept that DNA can promote protein assembly into amyloids and reveal that the binding sites of effector molecules can be targeted to inhibit amyloidogenesis.     

Cold resistance in Antarctic angiosperms

Deschampsia antarctica Desv. (Poaceae) and Colobanthus quitensis (Kunth) Bartl. (Cariophyllaceae) are the only two vascular plants that have colonized the Maritime Antarctic. The primary purpose of the present work was to determine cold resistance mechanisms in these two Antarctic plants. This was achieved by comparing thermal properties of leaves and the lethal freezing temperature to 50% of the tissue (LT50). The grass D. antarctica was able to tolerate freezing to a lower temperature than C. quitensis. The main freezing resistance mechanism for C. quitensis is supercooling. Thus, the grass is mainly a freezing‐tolerant species, while C. quitensis avoids freezing. D. antarctica cold acclimated; thus, reducing its LT50. C. quitensis showed little cold‐acclimation capacity. Because day length is highly variable in the Antarctic, the effect of day length on freezing tolerance, growth, various soluble carbohydrates, starch, and proline contents in leaves of D. antarctica growing in the laboratory under cold‐acclimation conditions was studied. During the cold‐acclimation treatment, the LT50 was lowered more effectively under long day (21/3 h light/dark) and medium day (16/8) light periods than under a short day period (8/16). The longer the day length treatment, the faster the growth rate for both acclimated and non‐acclimated plants. Similarly, the longer the day treatment during cold acclimation, the higher the sucrose content (up to 7‐fold with respect to non‐acclimated control values). Oligo and polyfructans accumulated significantly during cold acclimation only with the medium day length treatment. Oligofructans accounted for more than 80% of total fructans. The degrees of polymerization were mostly between 3 and 10. C. quitensis under cold acclimation accumulated a similar amount of sucrose than D. antarctica, but no fructans were detected. The suggestion that survival of Antarctic plants in the Antarctic could be at least partially explained by accumulation of these substances is discussed.     

Localization of growth hormone receptors in rat and human thyroid cells

Growth hormone (GH) exerts its multiple actions by binding to a specific receptor (GHR) widely distributed in the organism. It is well established that, in acromegaly, the thyroid gland is larger than normal and that GH increases triiodothyronin concentrations and decreases those of tetraiodothyronin (thyroxine). The aim of the present study was to analyze the presence of GHR and its mRNA in rat and human thyroid gland by Western blot, in situ hybridization techniques, and immunohistochemistry. A band of the expected size for GHR was shown in rat and human thyroid by Western blot. GHR immunoreactivity was found in virtually all follicles. The signal was mainly localized in the cytoplasm, although a nuclear positivity was also found. In situ hybridization techniques demonstrated the presence of GHR messenger RNA in the thyroid gland (cytoplasm of the follicular cells). These results provide direct morphological evidence that GHR is localized in the thyroid gland of mammals and opens up the possibility that GH regulates thyroid cell function directly or via local autocrine or paracrine production of insulin-like growth factor I.     

Substrate flow in catalases deduced from the crystal structures of active site variants of HPII from Escherichia coli.

The active site of heme catalases is buried deep inside a structurally highly conserved homotetramer. Channels leading to the active site have been identified as potential routes for substrate flow and product release, although evidence in support of this model is limited. To investigate further the role of protein structure and molecular channels in catalysis, the crystal structures of four active site variants of catalase HPII from Escherichia coli (His128Ala, His128Asn, Asn201Ala, and Asn201His) have been determined at approximately 2.0-A resolution. The solvent organization shows major rearrangements with respect to native HPII, not only in the vicinity of the replaced residues but also in the main molecular channel leading to the heme distal pocket. In the two inactive His128 variants, continuous chains of hydrogen bonded water molecules extend from the molecular surface to the heme distal pocket filling the main channel. The differences in continuity of solvent molecules between the native and variant structures illustrate how sensitive the solvent matrix is to subtle changes in structure. It is hypothesized that the slightly larger H(2)O(2) passing through the channel of the native enzyme will promote the formation of a continuous chain of solvent and peroxide. The structure of the His128Asn variant complexed with hydrogen peroxide has also been determined at 2.3-A resolution, revealing the existence of hydrogen peroxide binding sites both in the heme distal pocket and in the main channel. Unexpectedly, the largest changes in protein structure resulting from peroxide binding are clustered on the heme proximal side and mainly involve residues in only two subunits, leading to a departure from the 222-point group symmetry of the native enzyme. An active role for channels in the selective flow of substrates through the catalase molecule is proposed as an integral feature of the catalytic mechanism. The Asn201His variant of HPII was found to contain unoxidized heme b in combination with the proximal side His-Tyr bond suggesting that the mechanistic pathways of the two reactions can be uncoupled.     

Glucocorticoid-regulated and constitutive trafficking of proteolytically processed cell surface-associated glycoproteins in wild type and variant rat hepatoma cells

Glucocorticoids regulate the trafficking of mouse mammary tumor virus (MMTV) glycoproteins to the cell surface in the rat hepatoma cell line M1.54, but not in the immunoselected sorting variant CR4. To compare the localization of MMTV glycoproteins to another proteolytically processed glycoprotein, both wild type M1.54 cells and variant CR4 cells were transfected with a human insulin receptor (hIR) expression vector, pRSVhIR. The production of cell surface hIR was monitored in dexamethasone-treated and -untreated wild type M1.54 and variant CR4 cells by indirect immunofluorescence, direct plasma membrane immunoprecipitation, and by [125I] insulin binding. In both wild type and variant rat hepatoma cells, hIR were localized at the cell surface in the presence or in the absence of 1 microM dexamethasone. In contrast, the glucocorticoid-regulated trafficking of cell surface MMTV glycoproteins occurred only in wild type M1.54 cells. We conclude that the hIR, which undergoes posttranslational processing reactions similar to MMTV glycoproteins, does not require glucocorticoids to be transported to the plasma membrane and is representative of a subset of cell surface glycoproteins whose trafficking is constitutive in rat hepatoma cells. Thus, MMTV glycoproteins and hIR provide specific cell surface markers to characterize the glucocorticoid-regulated and constitutive sorting pathways.     

Photosynthetic Light Responses May Explain Vertical Distribution of Hymenophyllaceae Species in a Temperate Rainforest of Southern Chile

Some epiphytic Hymenophyllaceae are restricted to lower parts of the host (<60 cm; 10–100 μmol photons m^-2 s^-1) in a secondary forest of Southern Chile; other species occupy the whole host height (≥10 m; max PPFD >1000 μmol photons m^-2 s^-1). Our aim was to study the photosynthetic light responses of two Hymenophyllaceae species in relation to their contrasting distribution. We determined light tolerance of Hymenoglossum cruentum and Hymenophyllum dentatum by measuring gas exchange, PSI and PSII light energy partitioning, NPQ components, and pigment contents. H. dentatum showed lower maximum photosynthesis rates (A_max) than H. cruentum, but the former species kept its net rates (A_n) near A_max across a wide light range. In contrast, in the latter one, A_n declined at PPFDs >60 μmol photons m^-2 s^-1. H. cruentum, the shadiest plant, showed higher chlorophyll contents than H. dentatum. Differences in energy partitioning at PSI and PSII were consistent with gas exchange results. H. dentatum exhibited a higher light compensation point of the partitioning of absorbed energy between photochemical Y(PSII) and non-photochemical Y(NPQ) processes. Hence, both species allocated energy mainly toward photochemistry instead of heat dissipation at their light saturation points. Above saturation, H. cruentum had higher heat dissipation than H. dentatum. PSI yield (YPSI) remained higher in H. dentatum than H. cruentum in a wider light range. In both species, the main cause of heat dissipation at PSI was a donor side limitation. An early dynamic photo-inhibition of PSII may have caused an over reduction of the Qa⁺ pool decreasing the efficiency of electron donation to PSI. In H. dentatum, a slight increase in heat dissipation due to acceptor side limitation of PSI was observed above 300 μmol photons m^-2s^-1. Differences in photosynthetic responses to light suggest that light tolerance and species plasticity could explain their contrasting vertical distribution.