Characterization of Environmental Sources of the Human and Animal Pathogen Cryptococcus gattii in British Columbia, Canada, and the Pacific Northwest of the United States

Cryptococcus gattii has recently emerged as a primary pathogen of humans and wild and domesticated animals in British Columbia, particularly on Vancouver Island. C. gattii infections are typically infections of the pulmonary and/or the central nervous system, and the incidence of infection in British Columbia is currently the highest reported globally. Prior to this emergence, the environmental distribution of and the extent of colonization by C. gattii in British Columbia were unknown. We characterized the environmental sources and potential determinants of colonization in British Columbia. C. gattii was isolated from tree surfaces, soil, air, freshwater, and seawater, and no seasonal prevalence was observed. The C. gattii concentrations in air samples were significantly higher during the warm, dry summer months, although potentially infectious propagules (<3.3 μm in diameter) were present throughout the year. Positive samples were obtained from many different areas of British Columbia, and some locations were colonization “hot spots.” C. gattii was generally isolated from acidic soil, and geographic differences in soil pH may influence the extent of colonization. C. gattii soil colonization also was associated with low moisture and low organic carbon contents. Most of the C. gattii isolates recovered belonged to the VGIIa genetic subtype; however, sympatric colonization by the VGIIb strain was observed at most locations. At one sampling site, VGIIa, VGIIb, VGI, and the Cryptococcus neoformans serotype AD hybrid all were coisolated. Our findings indicate extensive colonization by C. gattii within British Columbia and highlight an expansion of the ecological niche of this pathogen.     

MalE of group A Streptococcus participates in the rapid transport of maltotriose and longer maltodextrins

Study of the maltose/maltodextrin binding protein MalE in Escherichia coli has resulted in fundamental insights into the molecular mechanisms of microbial transport. Whether gram-positive bacteria employ a similar pathway for maltodextrin transport is unclear. The maltodextrin binding protein MalE has previously been shown to be key to the ability of group A Streptococcus (GAS) to colonize the oropharynx, the major site of GAS infection in humans. Here we used a multifaceted approach to elucidate the function and binding characteristics of GAS MalE. We found that GAS MalE is a central part of a highly efficient maltodextrin transport system capable of transporting linear maltodextrins that are up to at least seven glucose molecules long. Of the carbohydrates tested, GAS MalE had the highest affinity for maltotriose, a major breakdown product of starch in the human oropharynx. The thermodynamics and fluorescence changes induced by GAS MalE-maltodextrin binding were essentially opposite those reported for E. coli MalE. Moreover, unlike E. coli MalE, GAS MalE exhibited no specific binding of maltose or cyclic maltodextrins. Our data show that GAS developed a transport system optimized for linear maltodextrins longer than two glucose molecules that has several key differences from its well-studied E. coli counterpart.     

Monitoring aminoglycoside-induced conformational changes in 16S rRNA through acrylamide quenching

Fluorescence of 2-aminopurine (2AP)-substituted A-site and acrylamide quenching were used to study the interactions of paromomycin and neamine with the decoding region of 16S rRNA. The results reveal that paromomycin binding to the A-site RNA leads to increased exposure of residue A1492. In contrast, neamine has little effect on the solvent accessibility of A1492. Electrospray ionization mass spectrometry was used to compare the affinity of paromomycin with the A-site and 2-AP-substituted A-site RNAs.     

Molecular and functional studies of tilapia (Oreochromis mossambicus) NMDA receptor NR1 subunits

The Pin-tailed Manakin (Ilicura militaris) is a small, sexually dimorphic, frugivorous suboscine songbird (Pipridae; Passeriformes; Aves) endemic to the Atlantic Forest of Brazil. A variant individual of this species was recently described in which the red patches that characterise the male's Definitive plumage were replaced by orange-yellow ones. We show here that the pigments in the feathers of the colour variant are common dietary carotenoids (zeaxanthin, beta-cryptoxanthin), not carotenoids synthesised by birds, lending support to the suggestion that the individual is a colour mutant lacking the capability to transform yellow dietary pigments into the red pigments normally present in these feathers. By comparison, the yellow crown feathers of a close relative, the Golden-winged Manakin (Masius chrysopterus), contained predominantly endogenously produced epsilon-caroten-3'-ones. Surprisingly, the normal-coloured feathers of the male Pin-tailed Manakin owe their red hue to rhodoxanthin, an unusual carotenoid more commonly found in plants, rather than 4-keto-carotenoids typically found in red plumages and found lacking in previously characterised bird colour variants. The implication is that birds, like the tilapia fish, may be able to synthesise this unusual pigment endogenously from dietary precursors. A newly described carotenoid, 6-hydroxy-epsilon,epsilon-carotene-3,3'-dione, here named piprixanthin, present in the red feathers of the Pin-tailed Manakin, provides a plausible intermediate between epsilon,epsilon-carotene-3,3'-dione (canary-xanthophyll B), a bright yellow pigment found in this and other songbirds, and rhodoxanthin. It is apparent that pigeons (Columbidae, Columbiformes) also have the capability to produce rhodoxanthin, and a structurally related pigment, endogenously. The ability to synthesise rhodoxanthin might have arisen at least twice in birds.     

Induction of heat shock proteins in differentiated human and rodent neurons by celastrol

Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis have been termed protein misfolding disorders that are characterized by the neuronal accumulation of protein aggregates. Manipulation of the cellular stress-response involving induction of heat shock proteins (Hsps) in differentiated neurons offers a therapeutic strategy to counter conformational changes in neuronal proteins that trigger pathogenic cascades resulting in neurodegenerative diseases. Hsps are protein repair agents that provide a line of defense against misfolded, aggregation-prone proteins. These proteins are not induced in differentiated neurons by conventional heat shock. We have found that celastrol, a quinine methide triterpene, induced expression of a wider set of Hsps, including Hsp70B', in differentiated human neurons grown in tissue culture compared to cultured rodent neuronal cells. Hence the beneficial effect of celastrol against human neurodegenerative diseases may exceed its potential in rodent models of these diseases.     

Antibacterial activity of snake, scorpion and bee venoms: a comparison with purified venom phospholipase A2 enzymes

AIMS: Venoms of snakes, scorpions, bees and purified venom phospholipase A(2) (PLA(2)) enzymes were examined to evaluate the antibacterial activity of purified venom enzymes as compared with that of the crude venoms. METHODS AND RESULTS: Thirty-four crude venoms, nine purified PLA(2)s and two L-amino acid oxidases (LAAO) were studied for antibacterial activity by disc-diffusion assay (100 microg ml(-1)). Several snake venoms (Daboia russelli russelli, Crotalus adamanteus, Naja sumatrana, Pseudechis guttata, Agkistrodon halys, Acanthophis praelongus and Daboia russelli siamensis) showed activity against two to four different pathogenic bacteria. Daboia russelli russelli and Pseudechis australis venoms exhibited the most potent activity against Staphylococcus aureus, while the rest showed only a moderate activity against one or more bacteria. The order of susceptibility of the bacteria against viperidae venoms was -S. aureus > Proteus mirabilis > Proteus vulgaris > Enterobacter aerogenes > Pseudomonas aeruginosa and Escherichia coli. The minimum inhibitory concentrations (MIC) against S. aureus was studied by dilution method (160-1.25 microg ml(-1)). A stronger effect was noted with the viperidae venoms (20 microg ml(-11)) as compared with elapidae venoms (40 microg ml(-1)). The MIC were comparable with those of the standard drugs (chloramphenicol, streptomycin and penicillin). CONCLUSION: The present findings indicate that viperidae (D. russelli russelli) and elapidae (P. australis) venoms have significant antibacterial effects against gram (+) and gram (-) bacteria, which may be the result of the primary antibacterial components of laao, and in particular, the PLA(2) enzymes. The results would be useful for further purification and characterization of antibacterial agents from snake venoms. SIGNIFICANCE AND IMPACT OF THE STUDY: The activity of LAAO and PLA(2) enzymes may be associated with the antibacterial activity of snake venoms.     

Role of protein phosphatase 2A in calcium-dependent chloride secretion by human colonic epithelial cells

EGF inhibits carbachol-induced chloride secretion by regulating a basolateral potassium channel via phosphatidylinositol 3-kinase (PI 3-kinase) and PKC activation. Although both EGF and carbachol cause tyrosine phosphorylation of p85 of PI 3-kinase, only EGF activates the enzyme. Serine phosphorylation of p85 is thought to suppress the lipid kinase of PI 3-kinase. Our present study examined whether the differential effects of carbachol and EGF on PI 3-kinase activity correspond to varying phosphorylation of p85, and the mechanisms and consequences. T₈₄ colonic epithelial cells were treated with either EGF or carbachol. Cell lysates were immunoprecipitated with p85 antibody and blotted with either phosphotyrosine or phosphoserine antibodies. Protein phosphatase (PP) 1 and 2A activities were also measured. Both tyrosine and serine residues of p85 were phosphorylated by carbachol, whereas EGF induced only tyrosine phosphorylation. Moreover, EGF abolished carbachol-induced serine phosphorylation of p85 and activated PP2A without affecting PP1. Carbachol did not affect either phosphatase. Calyculin A or okadaic acid pretreatment reversed the inhibitory action of EGF on carbachol-induced chloride secretion and restored serine phosphorylation of p85. Although carbachol recruits p85, it phosphorylates both serine and tyrosine residues so that the lipid kinase of PI 3-kinase is inhibited. EGF results in p85 tyrosine phosphorylation as well as dephosphorylation of serine residues via the activation of PP2A. This explains the differential induction of PI 3-kinase enzyme activity in response to EGF and/or carbachol and has functional implications. Our data provide further insights into negative signals that regulate chloride secretion and into the molecular basis of signaling diversification in the intestinal epithelium. epithelial secretion; PI 3-kinase; EGF     

The truncated ghrelin receptor polypeptide (GHS-R1b) acts as a dominant-negative mutant of the ghrelin receptor

The dimerization properties of the ghrelin receptor (GRLN-R) and its non-signalling, naturally occurring, truncated splice variant (GHS-R1b) have been investigated in human embryonic kidney 293 cells heterologously expressing these proteins. Using the techniques of bioluminescence resonance energy transfer and co-immunoprecipitation, we detected the formation of GRLN-R homodimers and GRLN-R/GHS-R1b heterodimers, but ghrelin-induced conformational changes were only detected in the GRLN-R homodimers. When the expression of GHS-R1b exceeded that of GRLN-R, there was a decrease in the cell surface expression of GRLN-R with a consequent decrease in constitutive activation of phosphatidylinositol-specific phospholipase C (PI-PLC). Furthermore, there was no change in ghrelin affinity, and the efficacy of cell signalling as measured by stimulation of PI-PLC and extracellular signal-regulated kinase 1/2 was unchanged. Cellular localization studies suggest that GRLN-R is normally distributed between the plasma membrane and cytosolic fractions, but in the presence of GHS-R1b, GRLN-R is localized to the nucleus. Therefore, we propose that the decrease in GRLN-R constitutive signalling was due to translocation of GRLN-R to the nucleus due to the formation of GRLN-R/GHS-R1b heterodimers. Therefore, GHS-R1b appears to act as a dominant-negative mutant of the full-length GRLN-R.     

Growth rate and carrageenan analyses in four strains of <Emphasis Type="Italic">Kappaphycus alvarezii</Emphasis> (Rhodophyta,Gigartinales) farmed in the subtropical waters of São Paulo State,Brazil

Growth rate, semi-refined and refined carrageenan yields, 3,6-anhydrogalactose and sulphate contents, and gel strength were investigated in four strains of Kappaphycus alvarezii (brown, green and red tetrasporophytic strains, and one strain derived from tetraspores progeny, called G11) farmed in subtropical waters of São Paulo State, Brazil. All studied strains showed higher growth rates from February to May, decreasing from July to December. The G11 strain exhibited lower growth rates, but had semi-refined and refined carrageenan yields significantly higher than the others and similar to a commercial sample. A negative correlation between growth rate and semi-refined carrageenan yield was verified for all strains. The brown strain had the lowest content of 3,6-anhydrogalactose, while G11 and green strains showed higher values. No clear pattern of variation and no relationship were observed for sulphate and gel strength. However, all strains presented gel strength values near the one obtained from commercial sample. These results show that the carrageenan extracted from brown, green, red, and G11 strains of K. alvarezii cultured in subtropical waters of São Paulo State, Brazil with commercial potentials.