Relationship between Legionella pneumophila and Acanthamoeba polyphaga: physiological status and susceptibility to chemical inactivation.

Survival studies were conducted on Legionella pneumophila cells that had been grown intracellularly in Acanthamoeba polyphaga and then exposed to polyhexamethylene biguanide (PHMB), benzisothiazolone (BIT), and 5-chloro-N-methylisothiazolone (CMIT). Susceptibilities were also determined for L. pneumophila grown under iron-sufficient and iron-depleted conditions. BIT was relatively ineffective against cells grown under iron depletion; in contrast, iron-depleted conditions increased the susceptibilities of cells to PHMB and CMIT. The activities of all three biocides were greatly reduced against L. pneumophila grown in amoebae. PHMB (1 x MIC) gave 99.99% reductions in viability for cultures grown in broth within 6 h and no detectable survivors at 24 h but only 90 and 99.9% killing at 6 h and 24 h, respectively, for cells grown in amoebae. The antimicrobial properties of the three biocides against A. polyphaga were also determined. The majority of amoebae recovered from BIT treatment, but few, if any, survived CMIT treatment or exposure to PHMB. This study not only shows the profound effect that intra-amoebal growth has on the physiological status and antimicrobial susceptibility of L. pneumophila but also reveals PHMB to be a potential biocide for effective water treatment. In this respect, PHMB has significant activity, below its recommended use concentrations, against both the host amoeba and L. pneumophila.     

Inhibition of glutathione disulfide reductase by glutathione

Rat-liver glutathione disulfide reductase is significantly inhibited by physiological concentrations of the product, glutathione. GSH is a noncompetitive inhibitor against GSSG and an uncompetitive inhibitor against NADPH at saturating concentrations of the fixed substrate. In both cases, the inhibition by GSH is parabolic, consistent with the requirement for 2 eq. of GSH in the reverse reaction. The inhibition of GSSG reduction by physiological levels of the product, GSH, would result in a significantly more oxidizing intracellular environment than would be realized in the absence of inhibition. Considering inhibition by the high intracellular concentration of GSH, the steady-state concentration of GSSG required to maintain a basal glutathione peroxidase flux of 300 nmol/min/g in rat liver is estimated at 8-9 microM, about 1000-fold higher than the concentration of GSSG predicted from the equilibrium constant for glutathione reductase. The kinetic properties of glutathione reductase also provide a rationale for the increased glutathione (GSSG) efflux observed when cells are exposed to oxidative stress. The resulting decrease in intracellular GSH relieves the noncompetitive inhibition of glutathione reductase and results in an increased capacity (Vmax) and decreased Km for GSSG.     

Dimeric epoxy fatty acid methyl esters: formation,chromatography and mass spectrometry

The formation of dimers is reported from the thermal treatment of a series of epoxy fatty acid methyl esters. These compounds were isolated from the reaction mixture by steric exclusion chromatography and were subsequently characterised by their high resolution electron impact and ammonia chemical ionisation mass spectra. The spectra were consistent in each case with the presence of a mixture of four possible positional isomers each containing an ether bridge linking a pair of fatty acid methyl esters across the carbon chains, with a keto group on a carbon adjacent to the bridge on one of the esters.     

Correlations between epidermal cell structure and endogenous hormone titers during the fifth larval instar of the tobacco hornworm, Manduca sexta

Epidermal cell morphology and cuticle production in Manduca sexta are directly influenced by both ecdysterone and juvenile hormone. Up to day 6 of the last larval instar, post-molt endocuticle is continuously deposited even though cells undergo a partial and temporary separation from the overlying cuticle at the time when a small ecdysteroid peak is detected (approximately day 3.5). At about days 6--7 when another, larger ecdysteroid peak is present, apolysis occurs accompanied by the appearance of edcysial droplets. Following apolysis, layers of pupal cuticle are deposited. Increased quantities of rough endoplasmic reticulum characterize the epidermis at times of peak endocuticle deposition (day 3, larval cuticle; day 9, pupal cuticle). Dense pigment inclusions are found in epidermis from the day of ecdysis to the last larval instar until they are eliminated 5 days later. These dense bodies migrate from cell apex to base in the absence of juvenile hormone (or in the presence of a negligible amount of juvenile hormone) and probably contain insecticyanin.     

Regulation of the xylan-degrading apparatus of Cellvibrio japonicus by a novel two-component system

The microbial degradation of lignocellulose biomass is not only an important biological process but is of increasing industrial significance in the bioenergy sector. The mechanism by which the plant cell wall, an insoluble composite structure, activates the extensive repertoire of microbial hydrolytic enzymes required to catalyze its degradation is poorly understood. Here we have used a transposon mutagenesis strategy to identify a genetic locus, consisting of two genes that modulate the expression of xylan side chain-degrading enzymes in the saprophytic bacterium Cellvibrio japonicus. Significantly, the locus encodes a two-component signaling system, designated AbfS (sensor histidine kinase) and AbfR (response regulator). The AbfR/S two-component system is required to activate the expression of the suite of enzymes that remove the numerous side chains from xylan, but not the xylanases that hydrolyze the beta1,4-linked xylose polymeric backbone of this polysaccharide. Studies on the recombinant sensor domain of AbfS (AbfS(SD)) showed that it bound to decorated xylans and arabinoxylo-oligosaccharides, but not to undecorated xylo-oligosaccharides or other plant structural polysaccharides/oligosaccharides. The crystal structure of AbfS(SD) was determined to a resolution of 2.6A(.) The overall fold of AbfS(SD) is that of a classical Per Arndt Sim domain with a central antiparallel four-stranded beta-sheet flanked by alpha-helices. Our data expand the number of molecules known to bind to the sensor domain of two-component histidine kinases to include complex carbohydrates. The biological rationale for a regulatory system that induces enzymes that remove the side chains of xylan, but not the hydrolases that cleave the backbone of the polysaccharide, is discussed.     

Bovine papilloma virus plasmids replicate randomly in mouse fibroblasts throughout S phase of the cell cycle

Bovine papilloma virus (BPV) replicates as a multicopy nuclear plasmid in mouse fibroblasts. Using fluorescence activated cell sorting and mitotic selection procedures, we show that the replication of BPV occurs throughout S phase of the cell cycle and that replication is confined to S phase. After one round of chromosomal DNA replication, almost one quarter of BPV plasmids have replicated more than once, while a similar number of plasmids have not replicated at all. While multiple forms of BPV exist in the cell, all forms show the same pattern of replication. These results are consistent with a model in which BPV plasmids are chosen at random for replication throughout, and only during, S phase and support the view that the completion of S phase is a specifically activated event in the cell cycle rather than simply the end of one round of chromosomal DNA replication.     

Risks of Avian Influenza Transmission in Areas of Intensive Free-Ranging Duck Production with Wild Waterfowl

For decades, southern China has been considered to be an important source for emerging influenza viruses since key hosts live together in high densities in areas with intensive agriculture. However, the underlying conditions of emergence and spread of avian influenza viruses (AIV) have not been studied in detail, particularly the complex spatiotemporal interplay of viral transmission between wild and domestic ducks, two major actors of AIV epidemiology. In this synthesis, we examine the risks of avian influenza spread in Poyang Lake, an area of intensive free-ranging duck production and large numbers of wild waterfowl. Our synthesis shows that farming of free-grazing domestic ducks is intensive in this area and synchronized with wild duck migration. The presence of juvenile domestic ducks in harvested paddy fields prior to the arrival and departure of migrant ducks in the same fields may amplify the risk of AIV circulation and facilitate the transmission between wild and domestic populations. We provide evidence associating wild ducks migration with the spread of H5N1 in the spring of 2008 from southern China to South Korea, Russia, and Japan, supported by documented wild duck movements and phylogenetic analyses of highly pathogenic avian influenza H5N1 sequences. We suggest that prevention measures based on a modification of agricultural practices may be implemented in these areas to reduce the intensity of AIV transmission between wild and domestic ducks. This would require involving all local stakeholders to discuss feasible and acceptable solutions.     

Synthesis and degradation of phenylalanine ammonia-lyase of Rhodosporidium toruloides.

The regulation of the enzyme phenylalanine ammonia-lyase (PAL), which is of potential use in oral treatment of phenylketonuria, was investigated. Antiserum against PAL was prepared and was shown to be monospecific for the enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native enzyme and two inactive mutant forms of the enzyme were purified to homogeneity by immunoaffinity chromatography, using anti-PAL immunoglobulin G-Sepharose 4B. Both mutant enzymes contained intact prosthetic groups. The formation of PAL catalytic activity after phenylalanine was added to yeast cultures was paralleled by the appearance of enzyme antigen. During induction, uptake of [3H]leucine into the enzyme was higher than uptake into total protein. Our results are consistent with de novo synthesis of an enzyme induced by phenylalanine, rather than activation of a proenzyme. The half-lives of PAL and total protein were similar in both exponential and stationary phase cultures. No metabolite tested affected the rate of enzyme degradation. Glucose repressed enzyme synthesis, whereas ammonia reduced phenylalanine uptake and pool size and so may repress enzyme synthesis through inducer exclusion. The synthesis of enzyme antigen by a mutant unable to metabolize phenylalanine indicated that this amino acid is the physiological inducer of the enzyme.