β2-Microglobulin Amyloid Fibrils Are Nanoparticles That Disrupt Lysosomal Membrane Protein Trafficking and Inhibit Protein Degradation by Lysosomes

Fragmentation of amyloid fibrils produces fibrils that are reduced in length but have an otherwise unchanged molecular architecture. The resultant nanoscale fibril particles inhibit the cellular reduction of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), a substrate commonly used to measure cell viability, to a greater extent than unfragmented fibrils. Here we show that the internalization of β₂-microglobulin (β₂m) amyloid fibrils is dependent on fibril length, with fragmented fibrils being more efficiently internalized by cells. Correspondingly, inhibiting the internalization of fragmented β₂m fibrils rescued cellular MTT reduction. Incubation of cells with fragmented β₂m fibrils did not, however, cause cell death. Instead, fragmented β₂m fibrils accumulate in lysosomes, alter the trafficking of lysosomal membrane proteins, and inhibit the degradation of a model protein substrate by lysosomes. These findings suggest that nanoscale fibrils formed early during amyloid assembly reactions or by the fragmentation of longer fibrils could play a role in amyloid disease by disrupting protein degradation by lysosomes and trafficking in the endolysosomal pathway.     

Competition between Intramolecular and Intermolecular Interactions in an Amyloid-Forming Protein

Despite much progress in understanding the folding and the aggregation processes of proteins, the rules defining their interplay have yet to be fully defined. This problem is of particular importance since many diseases are initiated by protein unfolding and hence the propensity to aggregate competes with intramolecular collapse and other folding events. Here, we describe the roles of intramolecular and intermolecular interactions in defining the length of the lag time and the apparent rate of elongation of the 100-residue protein human β₂-microglobulin at pH 2.5, commencing from an acid-denatured state that lacks persistent structure but contains significant non-random hydrophobic interactions. Using a combination of site-directed mutagenesis, quantitative kinetic analysis and computational methods, we show that only a single region of about 10 residues in length, determines the rate of fibril formation, despite the fact that other regions exhibit a significant intrinsic propensity for aggregation. We rationalise these results by analysing the effect of incorporating the conformational properties of acid-unfolded β₂-microglobulin and its variants at pH 2.5 as measured by NMR spectroscopy into the Zyggregator aggregation prediction algorithm. These results demonstrate that residual structure in the precursor state modulates the intrinsic propensity of the polypeptide chain to aggregate and that the algorithm developed here allows the key regions for aggregation to be more clearly identified and the rates of their self-association to be predicted. Given the common propensity of unfolded chains to form non-random intramolecular interactions as monomers and to self-assemble subsequently into amyloid fibrils, the approach developed should find widespread utility for the prediction of regions important in amyloid formation and their rates of self-assembly.     

Invasiveness and comparative life-history traits of exotic and indigenous Senecio species in Australia

A comparative ecological study of closely related invasive and non-invasive species, Senecio madagascariensis and S. lautus (Asteraceae), investigated what traits might confer invasive ability in very similar species. Life-history attributes of the weed S. madagascariensis were compared to five habitat-specific subspecies of S. lautus: S. l. alpinus, S. l. dissectifolius, S. l. lanceolatus and two forms of S. l. maritimus. Field populations of each taxon were monitored to compare their population ecology. Relative rates of phenological development were compared at a single location. Seed germination was studied in a laboratory experiment. Transplant experiments were conducted in a range of S. madagascariensis and S. lautus habitats to compare performance in different environments. In monitored field populations S. madagascariensis produced seedlings and reproductive cohorts more frequently, flowered for longer periods, produced more seeds and had fewer germinable achenes in the soil compared to S. lautus taxa. S. madagascariensis achenes had higher rates of germination than S. lautus in both light and dark conditions. S. madagascariensis was found to have higher rates of survival than S. lautus taxa in a range of habitats and to be faster to flower in both transplant and standard glasshouse environments. Overall S. madagascariensis performed better than S. lautus ecotypes in terms of seedling, growth and fecundity measurements and second best for achenes. Despite relatively good performance in terms of life-history traits there is no evidence that S. madagascariensis is invading S. lautus habitats. We speculate that physiological and morphological adaptations to specialised environments are a better explanation for success of Senecio taxa/ecotypes than generalised life-history trait performance. We suggest that invasiveness is essentially unpredictable, due to habitat/plant specific interactions between invader and area of introduction. In the absence of predictive theory, quarantine authorities should use a combination of methods to assess invasive potential including a database of known weeds, performance comparisons between congeneric natives and exotics in a range of habitats at proposed point of introduction and monitoring of introduced species to determine if they spread.     

Comparison of the orotidine 5'-monophosphate decarboxylase sequences of eight species

Predicted amino acid sequences of the enzyme orotidine 5'-phosphate decarboxylase (EC 4.1.1.23) from eight different organisms are compared. The comparisons are made on the basis of primary structural differences, primary amino acid sequence, hydropathy profiles, and secondary structure predictions. The organisms compared are Mus musculus, Aspergillus nidulans, Neurospora crassa, Kluyveromyces lactis, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Escherichia coli, and Salmonella typhimurium.     

Vocalisation Repertoire of Female Bluefin Gurnard (Chelidonichthys kumu) in Captivity: Sound Structure,Context and Vocal Activity

Fish vocalisation is often a major component of underwater soundscapes. Therefore, interpretation of these soundscapes requires an understanding of the vocalisation characteristics of common soniferous fish species. This study of captive female bluefin gurnard, Chelidonichthys kumu, aims to formally characterise their vocalisation sounds and daily pattern of sound production. Four types of sound were produced and characterised, twice as many as previously reported in this species. These sounds fit two aural categories; grunt and growl, the mean peak frequencies for which ranged between 129 to 215 Hz. This species vocalized throughout the 24 hour period at an average rate of (18.5 ± 2.0 sounds fish^-1 h^-1) with an increase in vocalization rate at dawn and dusk. Competitive feeding did not elevate vocalisation as has been found in other gurnard species. Bluefin gurnard are common in coastal waters of New Zealand, Australia and Japan and, given their vocalization rate, are likely to be significant contributors to ambient underwater soundscape in these areas.     

Climatic forcing and larval dispersal capabilities shape the replenishment of fishes and their habitat‐forming biota on a tropical coral reef

Fluctuations in marine populations often relate to the supply of recruits by oceanic currents. Variation in these currents is typically driven by large‐scale changes in climate, in particular ENSO (El Nino Southern Oscillation). The dependence on large‐scale climatic changes may, however, be modified by early life history traits of marine taxa. Based on eight years of annual surveys, along 150 km of coastline, we examined how ENSO influenced abundance of juvenile fish, coral spat, and canopy‐forming macroalgae. We then investigated what traits make populations of some fish families more reliant on the ENSO relationship than others. Abundance of juvenile fish and coral recruits was generally positively correlated with the Southern Oscillation Index (SOI), higher densities recorded during La Niña years, when the ENSO‐influenced Leeuwin Current is stronger and sea surface temperature higher. The relationship is typically positive and stronger among fish families with shorter pelagic larval durations and stronger swimming abilities. The relationship is also stronger at sites on the coral back reef, although the strongest of all relationships were among the lethrinids (r = .9), siganids (r = .9), and mullids (r = .8), which recruit to macroalgal meadows in the lagoon. ENSO effects on habitat seem to moderate SOI–juvenile abundance relationship. Macroalgal canopies are higher during La Niña years, providing more favorable habitat for juvenile fish and strengthening the SOI effect on juvenile abundance. Conversely, loss of coral following a La Niña‐related heat wave may have compromised postsettlement survival of coral dependent species, weakening the influence of SOI on their abundance. This assessment of ENSO effects on tropical fish and habitat‐forming biota and how it is mediated by functional ecology improves our ability to predict and manage changes in the replenishment of marine populations.     

Patterns of soluble cellular proteins during fetal rat hepatocyte development

Soluble cellular proteins from fetal rat hepatocytes, at 13-20 days of gestation were analyzed by 2-D gel electrophoresis and compared with protein patterns from adult hepatocytes and two hepatoma lines. The patterns of 13, 15 and 17 day fetal hepatocyte proteins were very similar. A number of proteins which were prominent in early fetal hepatocytes, were absent from the adult hepatocyte. Several proteins, which were also present in the adult pattern, appeared by day 17 of gestation. By day 20 of gestation, numerous proteins appeared and increased levels of a number of other proteins were observed. The adult hepatocyte protein pattern differed markedly from those of fetal hepatocytes. Many proteins present at low levels in fetal hepatocytes were increased in the adult, and at least thirty six new proteins appeared. The protein patterns of two hepatoma lines more closely resembled the early fetal rather than the adult hepatocyte pattern.     

Partial characterization of 5-fluoropyrimidine-resistant mutants of Neurospora crassa

Summary The primary lesion in a number of 5-fluoropyrimidine resistant mutants of Neurospora crassa has been identified. ud-1 mutants, previously designated fdu-2, are deficient in nucleoside uptake and show extensive intragenic complementation. uc-4 mutants lack uracil phosphoribosyl transferase with no complementation between 23 alleles. udk mutants lack uridine kinase activity. fdu-2 mutants affect the repression of the first two de novo pyrimidine biosynthetic enzymes, have no detectable uridine kinase activity and show decreased uridine uptake. Accordingly, fdu-2 may be involved in the regulation of pyrimidine uptake, salvage and de novo synthesis.Supported by S.R.C. grant GR/A/64655F. Buxton was supported during the period of this work by an S.R.C. Research Studentship