Observation of sequence specificity in the seeding of protein amyloid fibrils

It is well established that the rate of formation of fibrils by amyloidogenic proteins is enhanced by the addition of preformed fibrils, a phenomenon known as seeding. We show that the efficiency of seeding fibril formation from solutions of hen lysozyme by a series of other proteins depends strongly on the similarity of their sequences. This observation is consistent with the importance of long-range interactions in stabilizing the core structure of amyloid fibrils and may be associated with the existence of a species barrier observed in the transmissible spongiform encephalopathies. In addition, it is consistent with the observation of a single dominant type of protein in the deposits associated with each form of amyloid disease.     

Functional identification of galactosyltransferases (SCGs) required for species-specific modifications of the lipophosphoglycan adhesin controlling Leishmania major-sand fly interactions

Lipophosphoglycan (LPG) is an abundant surface molecule that plays key roles in the infectious cycle of Leishmania major. The dominant feature of LPG is a polymer of phosphoglycan (PG) (6Galbeta1,4Manalpha1-PO(4)) repeating units. In L. major these are extensively substituted with Gal(beta1,3) side chains, which are required for binding to midgut lectins and survival. We utilized evolutionary polymorphisms in LPG structure and cross-species transfections to recover genes encoding the LPG side chain beta1,3-galactosyltransferases (betaGalTs). A dispersed family of six SCG genes was recovered, whose predicted proteins exhibited characteristics of eukaryotic GalTs. At least four of these proteins showed significant LPG side chain betaGalT activity; SCG3 exhibited initiating GalT activity whereas SCG2 showed both initiating and elongating GalT activity. However, the activity of SCG2 was context-dependent, being largely silent in its normal genomic milieu, and different strains show considerable variation in the extent of LPG galactosylation. Thus the L. major genome encodes a family of SCGs with varying specificity and activity, and we propose that strain-specific LPG galactosylation patterns reflect differences in their expression.     

Fluoranthene degradation in Pseudomonas alcaligenes PA-10

Pseudomonas alcaligenes strain PA-10 degrades thefour-ring polycyclic aromatic hydrocarbon fluoranthene, co-metabolically. HPLC analysisof the growth medium identified four intermediates, 9-fluorenone-1-carboxylicacid; 9-hydroxy-1-fluorene carboxylic acid; 9-fluorenone and 9-fluorenol, formedduring fluoranthene degradation. Pre-exposure of PA-10 to 9-fluorenone-1-carboxylic acidand 9-hydroxy-1-fluorene-carboxylic acid resulted inincreases in fluoranthene removal, while pre-exposure to9-fluorenone and 9-fluorenol resulted in a decrease influoranthene degradation. The rate of indole transformation was similarly affected by pre-exposureto these metabolic intermediates, indicating a link between fluoranthenedegradation and indigo formation in this strain.     

Characterization of Wolbachia host cell range via the in vitro establishment of infections

Maternally transmitted bacteria of the genus Wolbachia are obligate, intracellular symbionts that are frequently found in insects and cause a diverse array of reproductive manipulations, including cytoplasmic incompatibility, male killing, parthenogenesis, and feminization. Despite the existence of a broad range of scientific interest, many aspects of Wolbachia research have been limited to laboratories with insect-rearing facilities. The inability to culture these bacteria outside of the invertebrate host has also led to the existing bias of Wolbachia research toward infections that occur in host insects that are easily reared. Here, we demonstrate that Wolbachia infections can be simply established, stably maintained, and cryogenically stored in vitro using standard tissue culture techniques. We have examined Wolbachia host range by introducing different Wolbachia types into a single tissue culture. The results show that an Aedes albopictus (Diptera: Culicidae) cell line can support five different Wolbachia infection types derived from Drosophila simulans (Diptera: Drosophilidae), Culex pipiens (Culicidae), and Cadra cautella (Lepidoptera: Phycitidae). These bacterial types include infection types that have been assigned to two of the major Wolbachia clades. As an additional examination of Wolbachia host cell range, we demonstrated that a Wolbachia strain from D. simulans could be established in host insect cell lines derived from A. albopictus, Spodoptera frugiperda (Lepidoptera: Noctuidae), and Drosophila melanogaster. These results will facilitate the development of a Wolbachia stock center, permitting novel approaches for the study of Wolbachia infections and encouraging Wolbachia research in additional laboratories.     

Thermodynamics of the pyruvate kinase reaction and the reversal of glycolysis in heart and skeletal muscle

The effect of temperature, pH, and free [Mg(2+)] on the apparent equilibrium constant of pyruvate kinase (phosphoenol transphosphorylase) (EC ) was investigated. The apparent equilibrium constant, K', for the biochemical reaction P-enolpyruvate + ADP = ATP + Pyr was defined as K' = [ATP][Pyr]/[ADP][P-enolpyruvate], where each reactant represents the sum of all the ionic and metal complexed species in M. The K' at pH 7.0, 1.0 mm free Mg(2+) and I of 0.25 m was 3.89 x 10(4) (n = 8) at 25 degrees C. The standard apparent enthalpy (DeltaH' degrees ) for the biochemical reaction was -4.31 kJmol(-1) in the direction of ATP formation. The corresponding standard apparent entropy (DeltaS' degrees ) was +73.4 J K(-1) mol(-1). The DeltaH degrees and DeltaS degrees values for the reference reaction, P-enolpyruvate(3-) + ADP(3-) + H(+) = ATP(4-) + Pyr(1-), were -6.43 kJmol(-1) and +180 J K(-1) mol(-1), respectively (5 to 38 degrees C). We examined further the mass action ratio in rat heart and skeletal muscle at rest and found that the pyruvate kinase reaction in vivo was close to equilibrium i.e. within a factor of about 3 to 6 of K' in the direction of ATP at the same pH, free [Mg(2+)], and T. We conclude that the pyruvate kinase reaction may be reversed under some conditions in vivo, a finding that challenges the long held dogma that the reaction is displaced far from equilibrium.     

Enhanced mtDNA repair capacity protects pulmonary artery endothelial cells from oxidant-mediated death

In rat cultured pulmonary arterial (PA), microvascular, and venous endothelial cells (ECs), the rate of mitochondrial (mt) DNA repair is predictive of the severity of xanthine oxidase (XO)-induced mtDNA damage and the sensitivity to XO-mediated cell death. To examine the importance of mtDNA damage and repair more directly, we determined the impact of mitochondrial overexpression of the DNA repair enzyme, Ogg1, on XO-induced mtDNA damage and cell death in PAECs. PAECs were transiently transfected with an Ogg1-mitochondrial targeting sequence construct. Mitochondria-selective overexpression of the transgene product was confirmed microscopically by the observation that immunoreactive Ogg1 colocalized with a mitochondria-specific tracer and, with an oligonucleotide cleavage assay, by a selective enhancement of mitochondrial Ogg1 activity. Overexpression of Ogg1 protected against both XO-induced mtDNA damage, determined by quantitative Southern analysis, and cell death as assessed by trypan blue exclusion and MTS assays. These findings show that mtDNA damage is a direct cause of cell death in XO-treated PAECs.     

Density and diversity of protozoa in some arid Australian soils

This is the first extensive study of soil protozoa of arid lands. Twenty-six samples from litters, soils, termitaria, and a cyanobacterial crust, collected from central and south Australian arid lands, were analyzed for numbers and species of gymnamoebae, ciliates, and testacea. Amoebae ranged from 1,000-5,000/g of material, and were two orders of magnitude more abundant than ciliates. Both groups increased in abundance and species richness from bare soils through spinifex to mulga to chenopod vegetations. Testacea ranged 900-5,000/g with similar species richness throughout vegetations, but reached 11,900/g with a doubling of species in a refugium in Kings Canyon. The most prevalent species of amoebae, ciliates, and testacea were taxa associated with ephemeral and disturbed habitats (r-selection). The cyanobacterial crust might be considered a micro-refugium because it contained a number of non-encysting protozoa, including Thecamoeba sp. and Nassula picta, feeding on cyanobacterial filaments. The numbers and species richness of protozoa under shrubs were greater than in bare soils, supporting the resource island hypothesis that desert plants create soil heterogeneity by localizing soil fertility under their canopies.     

Biochemistry,genetics and physiology of microbial styrene degradation

The last few decades have seen a steady increase in the global production and utilisation of the alkenylbenzene, styrene. The compound is of major importance in the petrochemical and polymer-processing industries, which can contribute to the pollution of natural resources via the release of styrene-contaminated effluents and off-gases. This is a cause for some concern as human over-exposure to styrene, and/or its early catabolic intermediates, can have a range of destructive health effects. These features have prompted researchers to investigate routes of styrene degradation in microorganisms, given the potential application of these organisms in bioremediation/biodegradation strategies. This review aims to examine the recent advances which have been made in elucidating the underlying biochemistry, genetics and physiology of microbial styrene catabolism, identifying areas of interest for the future and highlighting the potential industrial importance of individual catabolic pathway enzymes.