Template-directed polynucleotide synthesis on mineral surfaces

Ferric hydroxide, a plausible prebiotic material, strongly adsorbs polynucleotides. We show that adsorption on ferric hydroxide and on several other minerals has no effect, under the conditions studied, on the template-directed oligomerization of guanylic acid on polycytidylic acid.     

Template-directed polynucleotide synthesis on mineral surfaces

Summary Ferric hydroxide, a plausible prebiotic material, strongly adsorbs polynucleotides. We show that adsorption on ferric hydroxide and on several other minerals has no effect, under the conditions studies, on the template-directed oligomerization of guanylic acid on polycytidylic acid.     

Structural basis of M3 muscarinic receptor dimer/oligomer formation

Class A G protein-coupled receptors (GPCRs) are known to form dimers and/or oligomeric arrays in vitro and in vivo. These complexes are thought to play important roles in modulating class A GPCR function. Many studies suggest that residues located on the "outer" (lipid-facing) surface of the transmembrane (TM) receptor core are critically involved in the formation of class A receptor dimers (oligomers). However, no clear consensus has emerged regarding the identity of the TM helices or TM subsegments involved in this process. To shed light on this issue, we have used the M(3) muscarinic acetylcholine receptor (M3R), a prototypic class A GPCR, as a model system. Using a comprehensive and unbiased approach, we subjected all outward-facing residues (70 amino acids total) of the TM helical bundle (TM1-7) of the M3R to systematic alanine substitution mutagenesis. We then characterized the resulting mutant receptors in radioligand binding and functional studies and determined their ability to form dimers (oligomers) in bioluminescence resonance energy transfer saturation assays. We found that M3R/M3R interactions are not dependent on the presence of one specific structural motif but involve the outer surfaces of multiple TM subsegments (TM1-5 and -7) located within the central and endofacial portions of the TM receptor core. Moreover, we demonstrated that the outward-facing surfaces of most TM helices play critical roles in proper receptor folding and/or function. Guided by the bioluminescence resonance energy transfer data, molecular modeling studies suggested the existence of multiple dimeric/oligomeric M3R arrangements, which may exist in a dynamic equilibrium. Given the high structural homology found among all class A GPCRs, our results should be of considerable general relevance.     

Thermodynamics of peptide bond formation at clay mineral surfaces

Summary The possibility of surface catalysed condensation of unsubstituted amino acids on kaolinite in aqueous systems at elevated temperatures was investigated; no evidence of clay catalysed polycondensation has been found. The thermodynamic feasibility of the hypothetical lysine/dilysine condensation reaction in the temperature-range up to 90° C was evaluated for a range of experimental conditions by the combination of measured free energies of lysine/dilysine cation exchange on kaolinite and on montmorillonite, and free energies for the analogous condensation reaction in homogeneous solution. The results indicate that, in spite of the high selectivity of the clays for the adsorption of cation dimers from dilute solutions, the thermodynamic barrier to the surface condensation of unsubstituted amino acids on clay minerals in aqueous systems up to 90° C is not lower than it is in homogeneous solution.     

32S Tubulin oligomer. The resistance to factors suppressing the microtubule formation]

The resistance of brain 32S tubulin oligomer to factors suppressing the microtubules formation: colchicine, CaCl2, cooling and the absence of GTP is studied. The content of oligomer in the preparation and the polymerization degree were estimated by means of analytical centrifugation. Colchicine at 25 degrees and at a concentration of 10 muM does not change and at a concentration of 100 muM only slightly decreases the content of oligomer. The oligomer dissociated (but not completely) in 1 mM colchicine. Tubulin polymerization was partly suppressed by 10 muM and completely--by 100 muM colchicine. CaCl2 at 1 and 10 mM concentrations did not destroy the oligomer but inhibited its polymerization even in lesser of these concentrations. The cooling of the incubation medium to 14 degrees C or 4 degrees C completely inhibited the polymerization and did not affect the content of oligomer in the preparations. Tbulin preparations with low amount of exogenous GTP (less than or equal 3.10(-6) M) had a usual oligomer content, whereas GTP is necessary for polymerization at concentrations exceeding 10(-4) M. Thus, the reaction of tubulin oligomerization is relatively resistant to factors preventing the microtubules assembly. This probably means that there are at least two types of intereaction between tubulin molecules: 1) bonds in microtubules which are sensitive to colchicine, Ca2+ and cold, and which are formed only in the presence of nucleosidetriphosphates; 2) bonds in 32S tubulin oligomer which are more stable and do not need in exogenous nucleotides.     

Structural aspects of aldehyde dehydrogenase that influence dimer-tetramer formation

Aldehyde dehydrogenases are isolated as dimers or tetramers but have essentially identical structures. The homotetramer (ALDH1 or ALDH2) is a dimer of dimers (A-B + C-D). In the tetrameric enzyme, Ser500 from subunit "D" interacts with Arg84, a conserved residue, from subunit "A". In the dimeric ALDH3 form, the interaction cannot exist. It has been proposed that the formation of the tetramer is prevented by the presence of a C-terminal tail in ALDH3 that is not present in ALDH1 or 2. To understand the forces that maintain the tetramer, deletion of the tail in ALDH3, addition of different tails in ALDH1, and mutations of different residues located in the dimer-dimer interface were made. Gel filtration of the recombinantly expressed enzymes demonstrated that no change in their oligomerization occurred. Urea denaturation showed a diminution to the stability of the ALDH1 mutants. The K(m) for propionaldehyde was similar to that of the wild-type enzyme, but the K(m) for NAD was altered. A double mutant of D80G and S82A produced an enzyme with the ability to form dimers and tetramers in a protein-concentration-dependent manner. Though stable, this dimeric form was inactive. The tetramer exhibited 10% activity compared with the wild type. Sequence alignment demonstrated that the hydrophobic surface area is increased in the tetrameric enzymes. The hydrophobic surface seems to be the main force that drives the formation of tetramers. The results indicated that residues 80 and 82 are involved in maintaining the tetramer after its assembly but the C-terminal extension contributes to the overall stability of the assembled protein.     

Soluble age-related factors from skeletal muscle which influence muscle development

Successful regeneration of damaged striated muscle in adult mice is dependent on the regeneration of newly differentiated myofibers from proliferating satellite cells and inhibition of scar tissue formation by fibroblasts. As with most tissues, the ability of skeletal muscle to regenerate decreases in older animals. In this study, we have analysed soluble extracts from intact and regenerating skeletal muscle from mice of different ages for their ability to affect avian myogenesis in tissue culture. We were interested in determining whether an age-dependent difference could be detected with this tissue culture bioassay system. Total cell proliferation in the cultures, measured by [3H]thymidine incorporation was increased equally by muscle extracts from both young and older mice but the resulting cell populations differed in proportion of cell types. The ratio of myoblasts to fibroblasts was significantly greater in cultures exposed to extracts from younger mouse muscle as compared with cultures exposed to extracts from older animals. This age-related activity was found to reside in a low molecular weight (MW) (greater than 12 kD) component of the extract. This fraction had dissimilar effects on myoblasts and fibroblasts. Relative to saline controls, myoblast proliferation was increased and fibroblast proliferation decreased. The low MW fraction from younger mouse muscle extracts stimulated myogenic cell proliferation and myotube formation to a greater extent than the similar fraction prepared from older mouse muscle. Conversely, younger mouse muscle fractions had significantly greater inhibitory activity against fibroblast proliferation than did older mouse muscle fractions.     

Model system studies of the influence of bacterial biofilm formation on mineral surface reactivity

Biofilm development on mineral surfaces and related changes in surface reactivity were studied using batch and flow through experiments. An artificial groundwater was used as the primary nutrient medium, Pseudomonas aeruginosa (PAO1) was the model microbial organism and ‘mineral’ surfaces were kept as simple as possible by using glass or a polished quartz tile. Experiments were also completed with very low concentrations (100 mg l^?1) of iron, Fe²⁺ , in the solution. In situ confocal laser scanning microscopy of developing colonies during the live growth phase, and of thick, mature biofilms, revealed only sporadic coverage of biofilm cells and associated polymers at the ‘mineral–microbe interface’. Imaging and analysis of biofilm-conditioned surfaces doped with Fe²⁺ -rich solutions allowed the locus and form of Fe-rich mineral precipitation to be determined and show that biological surface components can cause mineral precipitation from dilute dissolved species which might otherwise remain in solution.     

Microbial selectivity on mineral surfaces: possible implications for weathering processes

In order to understand how microorganisms influence mineral surface processes, a better assessment of how microorganisms colonise mineral surfaces in situ is necessary. A crucial question in understanding mineral–microbial processes is whether colonization by microbial cells on mineral surfaces is a random process or whether it follows a selective pattern related primarily to the chemical composition of the mineral.     

Synergistic influence of phosphorylation and metal ions on tau oligomer formation and coaggregation with alpha-synuclein at the single molecule level

ABSTRACT: BACKGROUND: Fibrillar amyloid-like deposits and co-deposits of tau and alpha-synuclein are found in several common neurodegenerative diseases. Recent evidence indicates that small oligomers are the most relevant toxic aggregate species. While tau fibril formation is well-characterized, factors influencing tau oligomerization and molecular interactions of tau and alpha-synuclein are not well understood. RESULTS: We used a novel approach applying confocal single-particle fluorescence to investigate the influence of tau phosphorylation and metal ions on tau oligomer formation and its coaggregation with alpha-synuclein at the level of individual oligomers. We show that Al3+ at physiologically relevant concentrations and tau phosphorylation by GSK-3beta exert synergistic effects on the formation of a distinct SDS-resistant tau oligomer species even at nanomolar protein concentration. Moreover, tau phosphorylation and Al3+ as well as Fe3+ enhanced both formation of mixed oligomers and recruitment of alpha-synuclein in pre-formed tau oligomers. CONCLUSIONS: Our findings provide a new perspective on interactions of tau phosphorylation, metal ions, and the formation of potentially toxic oligomer species, and elucidate molecular crosstalks between different aggregation pathways involved in neurodegeneration.