Small-molecule conversion of toxic oligomers to nontoxic β-sheet-rich amyloid fibrils

Several lines of evidence indicate that prefibrillar assemblies of amyloid-β (Aβ) polypeptides, such as soluble oligomers or protofibrils, rather than mature, end-stage amyloid fibrils cause neuronal dysfunction and memory impairment in Alzheimer's disease. These findings suggest that reducing the prevalence of transient intermediates by small molecule-mediated stimulation of amyloid polymerization might decrease toxicity. Here we demonstrate the acceleration of Aβ fibrillogenesis through the action of the orcein-related small molecule O4, which directly binds to hydrophobic amino acid residues in Aβ peptides and stabilizes the self-assembly of seeding-competent, β-sheet-rich protofibrils and fibrils. Notably, the O4-mediated acceleration of amyloid fibril formation efficiently decreases the concentration of small, toxic Aβ oligomers in complex, heterogeneous aggregation reactions. In addition, O4 treatment suppresses inhibition of long-term potentiation by Aβ oligomers in hippocampal brain slices. These results support the hypothesis that small, diffusible prefibrillar amyloid species rather than mature fibrillar aggregates are toxic for mammalian cells.     

Co-localized or randomly distributed? Pair cross correlation of in vivo grown subgingival biofilm bacteria quantified by digital image analysis

The polymicrobial nature of periodontal diseases is reflected by the diversity of phylotypes detected in subgingival plaque and the finding that consortia of suspected pathogens rather than single species are associated with disease development. A number of these microorganisms have been demonstrated in vitro to interact and enhance biofilm integration, survival or even pathogenic features. To examine the in vivo relevance of these proposed interactions, we extended the spatial arrangement analysis tool of the software daime (digital image analysis in microbial ecology). This modification enabled the quantitative analysis of microbial co-localization in images of subgingival biofilm species, where the biomass was confined to fractions of the whole-image area, a situation common for medical samples. Selected representatives of the disease-associated red and orange complexes that were previously suggested to interact with each other in vitro (Tannerella forsythia with Fusobacterium nucleatum and Porphyromonas gingivalis with Prevotella intermedia) were chosen for analysis and labeled with specific fluorescent probes via fluorescence in situ hybridization. Pair cross-correlation analysis of in vivo grown biofilms revealed tight clustering of F. nucleatum/periodonticum and T. forsythia at short distances (up to 6 μm) with a pronounced peak at 1.5 μm. While these results confirmed previous in vitro observations for F. nucleatum and T. forsythia, random spatial distribution was detected between P. gingivalis and P. intermedia in the in vivo samples. In conclusion, we successfully employed spatial arrangement analysis on the single cell level in clinically relevant medical samples and demonstrated the utility of this approach for the in vivo validation of in vitro observations by analyzing statistically relevant numbers of different patients. More importantly, the culture-independent nature of this approach enables similar quantitative analyses for "as-yet-uncultured" phylotypes which cannot be characterized in vitro.     

Yeast Ist2 recruits the endoplasmic reticulum to the plasma membrane and creates a ribosome-free membrane microcompartment

The endoplasmic reticulum (ER) forms contacts with the plasma membrane. These contacts are known to function in non-vesicular lipid transport and signaling. Ist2 resides in specific domains of the ER in Saccharomyces cerevisiae where it binds phosphoinositide lipids at the cytosolic face of the plasma membrane. Here, we report that Ist2 recruits domains of the yeast ER to the plasma membrane. Ist2 determines the amount of cortical ER present and the distance between the ER and the plasma membrane. Deletion of IST2 resulted in an increased distance between ER and plasma membrane and allowed access of ribosomes to the space between the two membranes. Cells that overexpress Ist2 showed an association of the nucleus with the plasma membrane. The morphology of the ER and yeast growth were sensitive to the abundance of Ist2. Moreover, Ist2-dependent effects on cytosolic pH and genetic interactions link Ist2 to the activity of the H(+) pump Pma1 in the plasma membrane during cellular adaptation to the growth phase of the culture. Consistently we found a partial colocalization of Ist2-containing cortical ER and Pma1-containing domains of the plasma membrane. Hence Ist2 may be critically positioned in domains that couple functions of the ER and the plasma membrane.     

AtPTR4 and AtPTR6 are differentially expressed,tonoplast-localized members of the peptide transporter/nitrate transporter 1 (PTR/NRT1) family

Members of the peptide transporter/nitrate transporter 1 (PTR/NRT1) family in plants transport a variety of substrates like nitrate, di- and tripepetides, auxin and carboxylates. We isolated two members of this family from Arabidopsis, AtPTR4 and AtPTR6, which are highly homologous to the characterized di- and tripeptide transporters AtPTR1, AtPTR2 and AtPTR5. All known substrates of members of the PTR/NRT1 family were tested using heterologous expression in Saccharomyces cerevisiae mutants and oocytes of Xenopus laevis, but none could be identified as substrate of AtPTR4 or AtPTR6. AtPTR4 and AtPTR6 show distinct expression patterns, while AtPTR4 is expressed in the vasculature of the plants, AtPTR6 is highly expressed in pollen and during senescence. Phylogenetic analyses revealed that AtPTR2, 4 and 6 belong to one clade of subgoup II, whereas AtPTR1 and 5 are found in a second clade. Like AtPTR2, AtPTR4-GFP and AtPTR6-GFP fusion proteins are localized at the tonoplast. Vacuolar localization was corroborated by co-localization of AtPTR2-YFP with the tonoplast marker protein GFP-AtTIP2;1 and AtTIP1;1-GFP. This indicates that the two clades reflect different intracellular localization at the tonoplast (AtPTR2, 4, 6) and plasma membrane (AtPTR1, 5), respectively.     

Optical imaging of plastic changes induced by fear conditioning in the auditory cortex

The plastic changes in the auditory cortex induced by a fear conditioning, through pairing a sound (CS) with an electric foot-shock (US), were investigated using an optical recording method with voltage sensitive dye, RH795. In order to investigate the effects of association learning, optical signals in the auditory cortex in response to CS (12 kHz pure tone) and non-CS (4, 8, 16 kHz pure tone) were recorded before and after normal and sham conditioning. As a result, the response area to CS enlarged only in the conditioning group after the conditioning. Additionally, the rise time constant of the auditory response to CS significantly decreased and the relative peak value and the decay time constant of the auditory response to CS significantly increased after the conditioning. This study introduces an optical approach to the investigation of fear conditioning, representational plasticity, and the cholinergic system. The findings are synthesized in a model of the synaptic mechanisms that underlie cortical plasticity.     

Laccase-induced cross-coupling of 4-aminobenzoic acid with para-dihydroxylated compounds 2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide and 2,5-dihydroxybenzoic acid methyl ester

A fungal laccase from Trametes villosa (EC 1.10.3.2 p-phenoloxidase) was used to mediate the oxidation and cross-coupling of two para-dihydroxylated benzoic acid derivatives with 4-aminobenzoic acid. The incubation of 2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide and 4-aminobenzoic acid with laccase under oxygen conditions resulted in the formation of 2-(4′-carboxy-anilino)-N-(2″-hydroxyethyl)-3,6-dioxo-1,4-cyclohexadien-1-carboxamide as the main product (yield > 85%). When 2,5-dihydroxybenzoic acid methyl ester was a co-substrate of 4-aminobenzoic acid, 2-(4′-carboxy-anilino)-N-(2″-hydroxyethyl)-3,6-dioxo-1,4-cyclohexadien-1-carboxy methyl ester was produced (yield > 75%). Both products were N–C coupling dimers consisting of para-quinone and benzoic acid moieties. The formation of quinone structures in the presence of T. villosa laccase may be useful in pharmaceutical synthesis. Because of high product yields and low amount of by-products laccase of T. villosa seems to be a suitable enzyme among laccases acting at pH 5 for the synthesis of heterologous dimers.     

Conversion of Daidzein and Genistein by an Anaerobic Bacterium Newly Isolated from the Mouse Intestine

The metabolism of isoflavones by gut bacteria plays a key role in the availability and bioactivation of these compounds in the intestine. Daidzein and genistein are the most common dietary soy isoflavones. While daidzein conversion yielding equol has been known for some time, the corresponding formation of 5-hydroxy-equol from genistein has not been reported previously. We isolated a strictly anaerobic bacterium (Mt1B8) from the mouse intestine which converted daidzein via dihydrodaidzein to equol as well as genistein via dihydrogenistein to 5-hydroxy-equol. Strain Mt1B8 was a gram-positive, rod-shaped bacterium identified as a member of the Coriobacteriaceae. Strain Mt1B8 also transformed dihydrodaidzein and dihydrogenistein to equol and 5-hydroxy-equol, respectively. The conversion of daidzein, genistein, dihydrodaidzein, and dihydrogenistein in the stationary growth phase depended on preincubation with the corresponding isoflavonoid, indicating enzyme induction. Moreover, dihydrogenistein was transformed even more rapidly in the stationary phase when strain Mt1B8 was grown on either genistein or daidzein. Growing the cells on daidzein also enabled conversion of genistein. This suggests that the same enzymes are involved in the conversion of the two isoflavones.     

Conserved folding pathways of alpha-lactalbumin and lysozyme revealed by kinetic CD, fluorescence, NMR, and interrupted refolding experiments

In this report, it is shown by a combination of stopped-flow CD, fluorescence, and time-resolved NMR studies that the Ca^2 +-induced refolding of bovine α-lactalbumin (BLA) at constant denaturant concentration (4 M urea) exhibits triple-exponential kinetics. In order to distinguish between parallel folding pathways and a strictly sequential formation of the native state, interrupted refolding experiments were conducted. We show here that the Ca^2 +-induced refolding of BLA involves parallel pathways and the transient formation of a folding intermediate on the millisecond timescale. Our data furthermore suggest that the two structurally homologous proteins BLA and hen egg white lysozyme share a common folding mechanism. We provide evidence that the guiding role of long-range interactions in the unfolded state of lysozyme in mediating intersubdomain interactions during folding is replaced in the case of BLA by the Ca^2 +-binding site. Time-resolved NMR spectroscopy, in combination with fast ion release from caged compounds, enables the measurement of complex protein folding kinetics at protein concentrations as low as 100 μM and the concomitant detection of conformational transitions with rate constants of up to 8 s^− 1.     

"Candidatus Cloacamonas acidaminovorans": genome sequence reconstruction provides a first glimpse of a new bacterial division

Many microorganisms live in anaerobic environments. Most of these microorganisms have not yet been cultivated. Here, we present, from a metagenomic analysis of an anaerobic digester of a municipal wastewater treatment plant, a reconstruction of the complete genome of a bacterium belonging to the WWE1 candidate division. In silico proteome analysis indicated that this bacterium might derive most of its carbon and energy from the fermentation of amino acids, and hence, it was provisionally classified as "Candidatus Cloacamonas acidaminovorans." "Candidatus Cloacamonas acidaminovorans" is probably a syntrophic bacterium that is present in many anaerobic digesters. This report highlights how environmental sequence data might provide genomic and functional information about a new bacterial clade whose members are involved in anaerobic digestion.     

Uracil nucleotides stimulate human neural precursor cell proliferation and dopaminergic differentiation: involvement of MEK/ERK signalling

Isolation and propagation of neural stem cells derived from human brain tissue uniquely enables the study of human neurogenesis in vitro. In addition, ex vivo-expanded human neural stem/precursor cells (NPCs) may offer novel therapeutic strategies. We investigated the effects of extracellular nucleotides on the proliferation and differentiation of human mesencephalic neural stem/precursor cells (hmNPCs). When combined with the mitogens epidermal growth factor and fibroblast growth factor 2, UTP (1 microm) boosted proliferation of hmNPCs as shown by increased expression of the proliferation marker proliferating cell nuclear antigen (330%). UTP-induced proliferation was abrogated by the preferential P2Y receptor blocker pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). UTP also stimulated dopaminergic differentiation. Treatment with UTP (100 microm) increased the number of tyrosine hydroxylase (TH)-positive cells and TH protein by 267 and 319% respectively. UTP-stimulated dopaminergic differentiation of hmNPCs was blocked by the P2 receptor antagonists suramin (10 microm) and PPADS (100 microm). In addition, UDP (1 microm) enhanced TH protein expression by 194%. During differentiation, treatment with UTP stimulated the extracellular signal-regulated kinase (ERK) pathway. Both ERK1/2 phosphorylation and dopaminergic differentiation were inhibited by U0126, a selective ERK kinase inhibitor, as well as by suramin. When other P2 receptor agonists (ATP, ADP and adenosine 5'-O-(2-thiophosphate) (ADPbetaS); all 100 microm) were applied, both proliferation and dopaminergic differentiation of NPCs were compromised. We conclude that uracil nucleotides exert specific P2 receptor-mediated effects on midbrain-derived human NPCs, and may be used to enhance both proliferation and dopaminergic differentiation.