bluuebunny/crossref_metadata_2025 huggingface dataset:
An operon encoding aspartokinase and purine phosphoribosyltransferase in Thermus flavus
Authors: Nishiyama Makoto , Kukimoto Mutsuko , Beppu Teruhiko , Horinouchi Sueharu
Published: June 2009

The nucleotide sequence of a 1:1 kb Xhol-HindIII fragment downstream of the malate dehydrogenase (mdh) gene of Thermus flavus revealed the presence of an ORF and an incomplete ORF lacking its NH2-terminal portion, in the opposite orientation to that of the mdh gene. These two genes overlapped with each other, sharing two base pairs, suggesting that these genes are co-transcribed in a single mRNA. One ORF (termed gpt) encoded a protein of 154 amino acids showing significant amino acid sequence similarity to purine phosphoribosyltransferases, such as xanthine-guanine phosphoribosyltransferase of Escherichia coli and human hypoxanthine phosphoribosyltransferase. Cloning and sequencing of the upstream region of the gpt gene, together with sequence comparison of the gene product encoded by the region upstream of gpt, suggested that the upstream ORF encoded two in-frame overlapping aspartokinase genes, askA, encoding the β-subunit of 405 amino acids, and askB, encoding the β-subunit of 161 amino acids, which was part of the 3′ portion of askA. Consistent with the sequence data, the askAB and the gpt genes conferred the heat-stable enzyme activities of aspartokinase and phosphoribosyltransferase, respectively, on E. coli. Preliminary characterization of these enzymes produced in E. coli is described.
Enzyme evolution in Rhodobacter sphaeroides: selection of a mutant expressing a new galactitol dehydrogenase and biochemical characterization of the enzyme
Authors: Schneider Karl-Heinz , Jakel Gregor , Hoffmann Ralf , Giffhorn Friedrich
Published: June 2009

A gain of function mutant of Rhodobacter sphaeroides Si4, capable of growing on galactitol, was isolated from a chemostat culture. Continuous cultivation was performed for 54 d with a limiting concentration (1 mM) of the substrate D-glucitol and an excess (20 mM) of the non-metabolizable galactitol. The mutant strain, R. sphaeroides D, grew in galactitol minimal medium with a growth rate of 0-11 h-1 (t
d = 6-3 h). In crude extracts of R. sphaeroides D, a specific galactitol dehydrogenase (GDH) activity of 380 mU mg-1 was found, while the wild-type strain exhibited GDH activities lower than 50 mU mg-1 when grown on different polyols. Unlike mannitol, sorbitol or ribitol dehydrogenase from the wild-type strain, the new GDH was expressed constitutively. To study whether it was a newly evolved enzyme or an improved side activity of one of the pre-existing polyol dehydrogenases, GDH was purified to apparent homogeneity by ammonium sulfate precipitation and chromatography on Phenyl-Sepharose, Q-Sepharose, Matrex Gel Red-A and Mono-Q. The relative molecular mass (M
r) of the native GDH was 110000. SDS-PAGE resulted in one single band that represented a polypeptide with a M
r of 28000, indicating that the native protein is a tetramer. The isoelectric point of GDH was determined to be pH 4-2. The enzyme was specific for NAD+ but catalysed the oxidation of different sugar alcohols as well as different diols and secondary alcohols. The apparent K
m values were: galactitol, 240 mM; D-threitol, 85 mM; 1,2-hexandiol, 0-2 mM; NAD+, 12μM; L-erythrulose, 144 mM; acetoin, 62 mM; dihydroxyacetone, 48 mM; and NADH, 4μM. GDH activity was strictly dependent on the presence of divalent cations. The properties of GDH are different to any of the three polyol dehydrogenases from R. sphaeroides Si4. In addition, comparison of the N-terminal amino acid sequence of the isolated GDH with the N-terminal sequence of the other three polyol dehydrogenases clearly demonstrates that GDH is an additional enzyme, so far unrecognized in the wild-type strain.
Hydrophobic properties of the cell surface of Candida albicans: a role in aggregation
Authors: Hobden Carole , Teevan Claire , Jones Lorraine , O’Shea Paul
Published: June 2009

The ability of Candida albicans to aggregate and adhere to biological surfaces is a topic of major biological and medical importance. One factor which has been implicated in such properties is the hydrophobic nature of the cell surface. Two simple spectroscopic techniques are described which permit the rapid determination of this property. The first involves the use of arylnaphthalenesulfonate, the fluorescence emission maximum of which was shown to be a sensitive indicator of dielectric polarity. This was used to identify the hydrophobic characteristics of the cell surface of C. albicans. The second technique involves the use of 90° Rayleigh-Debye light scattering as an indicator of the aggregation state of a fungal suspension. These techniques were then used to compare the surface properties of three different strains of C. albicans and the effects of culture conditions: the hydrophobicity of the strains varied, and galactose-based culture media promoted the greatest degree of cell surface hydrophobicity.
Identification of Pasteurella multocida tryptophan synthase β-subunit by antisera against strain P1059
Authors: Jablonski Peter E. , Jablonski Lynn M. , Pintado Oscar , Sriranganathan Namalamar , Hovde Carolyn J.
Published: June 2009

Pasteurella multocidastrain P1059 is a highly virulent bacterium which causes fowl cholera in turkeys and chickens. A genomic library ofP. multocidaP1059 DNA was constructed using pUC19, expressed inEscherichia coliDH5α, and screened with chicken antisera generated againstP. multocidaP1059. Twelve out of the 4100 clones screened were immunoreactive. Plasmids isolated from these twelve clones were transformed intoE. coliCSR603 for maxicell analysis. Five proteins, with molecular masses of 34, 37, 43, 46 and 55 kDa, were expressed. Further work focused on the 43 kDa protein because it was expressed at levels detectable by SDS-PAGE and immunoblot analysis. The nucleotide sequence of the 1.8 kbp insert containing the gene encoding this protein was determined. The sequence contained three open reading frames (ORFs). The first ORF (ORF1) did not appear to code for any known protein. The second ORF (ORF2) encoded a protein of 403 amino acids (43662 Da). The deduced amino acid sequence showed 77% identity (84% similarity) with the tryptophan synthase β subunit (TrpB) ofSalmonella typhimuriumandVibrio parahaemolyticus.The eight conserved regions of TrpB are observed in theP. multocidaenzyme, including the conserved lysine (Lys-88) and consensus sequence (GGGSNA) implicated in pyridoxal phosphate binding. The expression and identity of theP. multocidaTrpB were confirmed by complementation studies usingE. coliW3110tnaA2 trpB9578.The third ORF (ORF3) consisted of the first 77 nucleotides of the gene encoding the β-subunit of tryptophan synthase(trpA),and overlapped the 3'-end oftrpBby 14 nucleotides. The deduced amino acid sequence of the 77 nucleotides of theP. multocidaTrpA had 68% identity (92% similarity) with the analogous region of TrpA fromKlebsiella aerogenes (K. pneumoniae).
Correlation between carbon flux through the pentose phosphate pathway and production of the antibiotic methylenomycin in Streptomyces coelicolor A3(2)
Authors: Obanye A. I. C. , Hobbs G. , Gardner D. C. J. , Oliver S. G.
Published: June 2009

Radiorespirometry was employed to study carbon metabolism during the growth of Streptomyces coelicolor A3(2) in a minimal medium which permitted the production of methylenomycin as the sole detectable secondary metabolite. A switch in the pattern of carbon metabolism from the Embden-Myerhof-Parnas pathway to the pentose phosphate pathway occurred during the period of slower growth in batch culture which immediately preceded entry into the stationary phase. This coincided with the period of methylenomycin production. It is proposed that the biosynthesis of methylenomycin is supported by the generation of NADPH during the latter part of growth.
The 32 kDa major outer-membrane protein of Pasteurella multocida capsular serotype D
Authors: Vasfi Marandi M. , Dubreuil J. D. , Mittal K. R.
Published: June 2009

The major outer-membrane protein (MOMP) ofPasteurella multocidaserotype D strain P210, with an apparent molecular mass of 32 kDa, was purified and characterized. The purification method involved selective extraction of MOMP withN-lauroylsarcosine and SDS, followed by immunoaffinity chromatography using a murine monoclonal antibody (mAb). The N-terminal sequence and amino acid composition of the MOMP showed considerable similarity to other Gram-negative bacterial porins, notably to the 37 kDa MOMP (porin H) ofP. multocida.Immunoelectron microscopy and colony blotting assays were used to demonstrate the surface localization of the 32 kDa MOMP on bacterial cells. The colony blotting assay provided a simple, sensitive and rapid screening method for visualizing accessibility of the antibody on the cells. In a Western blot assay, murine polyclonal hyperimmune serum against the purified 32 kDa MOMP recognized both serotype B and D strains bearing either a 32 kDa or a 37 kDa MOMP, whereas the mAb recognized only serotype D strains bearing a 32 kDa but not a 37 kDa MOMP. The present data indicate that the 32 kDa MOMPs ofP. multocidaare antigenically heterogeneous and possess both specific and cross-reacting epitopes. Detection of type-specific epitopes on the 32 kDa MOMP using an mAb may have potential implications regarding the feasibility of developing a serotyping system forP. multocida.
Lactic acid excretion by Streptococcus mutans
Authors: Dashper Stuart G. , Reynolds Eric C.
Published: June 2009

Lactic acid is the major end-product of glycolysis by Streptococcus mutans under conditions of sugar excess or low environmental pH. However, the mechanism of lactic acid excretion by S. mutans is unknown. To characterize lactic acid efflux in S. mutans the transmembrane movement of radiolabelled lactate was monitored in de-energized cells. Lactate was found to equilibrate across the membrane in accordance with artificially imposed transmembrane pH gradient (Δψ). The imposition of a transmembrane electrical potential (Δψ) upon de-energized cells did not cause an accumulation of lactate within the cell. The efflux of lactate from lactate-loaded, deenergized cells created a ΔpH, but did not create a Δψ, indicating that lactate crosses the cell membrane in an electroneutral process, as lactic acid. ΔpH and Δψ were determined by the transmembrane equilibration of [14C]benzoic acid and [14C]tetraphenylphosphonium ion (TPP), respectively. The presence of a membrane carrier for lactic acid in S. mutans was suggested by counterflow. Enzymic determination of the intra- and extracellular lactate concentrations of S. mutans cells glycolysing at pHo 6.8 and 5.5 showed that lactate distributed across the cell membrane in accordance with the equation ΔpH = log[lact]i/[lact]o. The addition of high extracellular concentrations of lactate to glycolysing S. mutans at acidic pH resulted in a fall in ΔpH and a subsequent decrease in glycolysis. The fall in ΔpH was attributed to the F1F0 ATPase being unable to raise the pHi back to its initial level due to the build up of lactate anion within the cell creating a large Δψ. The increase in Δψ resulted in the overall proton motive force remaining constant at about −110 mV. The results demonstrate that lactate is transported across the cell membrane of S. mutans as lactic acid in an electroneutral process that is independent of metabolic energy and as such has important bioenergetic implications for the cell.
Development of a steady-state oral microbial biof ilrn community using the constant-depth film ferrnenter
Authors: Kinniment Sarah L. , Wimpenny Julian W. T. , Adams David , Marsh Philip D.
Published: June 2009

The complexity of biof ilm communities like dental plaque suggests that laboratory model biofilm growth systems may help to understand their structure and function. This study describes the use of a constant-depth film fermenter (CDFF) to investigate biofilm formation by a nine-membered community of oral bacteria. The community was grown to steady state in a chemostat incubated anaerobically. The chemostat output was fed into the CDFF incubated aerobically. Viable counts for each species from the chemostat and the CDFF at steady state showed major differences; however, all nine organisms were present under both conditions. There was evidence of succession during biofilm formation with obligately anaerobic species only establishing after several days. A steady-state biofilm community was achieved which remained stable over time. Electron microscopy showed evidence of spatial differentiation with what appeared to be Neisseria subflava dominant near the upper surface and Fusobacterium nudeatum largely confined to the middle portion.
Experimental verification of a mathematical model for pelleted growth of Streptomyces coelicolor A3(2) in submerged batch culture
Authors: Tough A. J. , Prosser J. I.
Published: June 2009

A published mathematical model for growth of pellets of filamentous microorganisms has been tested by comparison of model predictions with experimental data on growth of Streptomyces coelicolor in liquid batch culture. The original model considered the classification of pellets into a range of size classes. Growth resulted in movement of pellets to classes of increasing size, while shear forces produced mycelial fragments which entered the smallest size class, from which they grew to form further pellets. This model did not correctly describe changes in pellet size distributions during growth and was therefore modified in two ways. In the first, new pellets were assumed to be formed by the break-up, by shear forces, of existing pellets into two pellets of equal size, rather than removal of small hyphal fragments from the pellet surface. The second modification assumed that the outer shell of active mycelial biomass had a density less than 1 g cm–3 and that hyphal density within this shell decreased with distance from the pellet centre. The modified model generated predictions which agreed closely with experimental data on biomass concentration, pellet size distribution, pellet number and pellet radius during batch growth, thereby supporting the assumptions on which the model was based. The model did not accurately describe final biomass concentration, through lack of consideration of autolysis of mycelia at the centre of larger pellets in which growth was limited by diffusion of nutrients. Attempts to incorporate autolysis into the model improved prediction of biomass concentration but were not based on sound biological assumptions and increased the complexity of the model. Further experimental work is required for accurate description of the effects of autolysis on pellet growth.
Characterization of lethal factor binding and cell receptor binding domains of protective antigen of Bacillus anthracis using monoclonal anti bodies
Authors: Little Stephen F. , Novak Jeanne M. , Lowe John R. , Leppla Stephen H. , Singh Yogendra , Klimpel Kurt R. , Lidgerding Burton C. , Friedlander Arthur M.
Published: June 2009

Lethal toxin from Bacillus anthracisis composed of protective antigen (PA) and lethal factor (LF). Anti-PA mAbs that neutralized lethal toxin activity, either in vivo or in vitro, identified three non-overlapping antigenic regions on PA. Two distinct antigenic regions were recognized by the four mAbs that neutralized lethal toxin activity by inhibiting the binding of 125I-LF to cell-bound PA. Mapping showed that one mAb, 1G3PA63, recognized an epitope on a 17 IcDa fragment located between amino acid residues Ser-168 and Phe-314. The three other mAbs, 2D3PA, 2D5PA and 10D2PA, recognized an epitope between amino acids Ile-581 and Asn-601. A single antigenic region was recognized by the three mAbs, 3B6PA, 14B7PA and 10E10PA63, that inhibited binding of 125I-PA to cells. This region was located between amino acids Asp-671 and lle-721. These results confirm previously defined functional domains of PA and suggest that LF may interact with two different sites on PA to form lethal toxin.
Application of a suite of 16S rRNA-specific oligonucleotide probes designed to investigate bacteria of the phylum cytophaga-flavobacter-bacteroides in the natural environment
Authors: Manz Werner , Amann Rudolf , Ludwig Wolfgang , Vancanneyt Marc , Schleifer Karl-Heinz
Published: June 2009

We designed a panel of four 16S rRNA-targeted oligonucleotide probes specific for bacteria of the phylum cytophaga-flavobacter-bacteroides (CFB). Probes CF319a and CF319b are targeted to members of the flavobacteria-cytophaga group and the genus Porphyromonas, whereas probe BAC303 has a target region characteristic for the genera Prevotella and Bacteroides within the bacteroides group. The probe FFE8b was developed for species-specific hybridizations with Flavobacterium ferrugineum. All probes were designed by computer-assisted sequence analysis and compared to all currently accessible 16S and 23S rRNA sequences. The oligonucleotides were further evaluated by whole-cell and non-radioactive dot-blot hybridization against reference strains of the CFB phylum and other major lineages of Bacteria. The newly developed probes were used together with other higher-order probes to analyse the structure and community composition in complex environments. In activated sludge samples, members of the flavobacteria-cytophaga group were revealed by in situ hybridization as important constituents of sludge flocs and characteristic colonizers of filamentous bacteria. By application of fluorescent probe BAC303, members of the genera Bacteroides and Prevotella could be visualized without prior cultivation as an important part of the human faecal microflora.
Ether-bond scission in the biodegradation of alcohol ethoxylate nonionic surfactants by Pseudomonas sp. strain SC25A
Authors: Tidswell Edward C. , Russell Nicholas J. , White Graham F.
Published: June 2009

Pseudomonas sp. strain SC25A, previously isolated for its ability to grow on alcohol ethoxylates (PEG dodecyl ethers) as sole source of carbon and energy, was shown to be capable of growth on the dodecyl ethers of mono-, di, tri- and octaethylene glycols. Comparative growth yields for this series of alcohol ethoxylate nonionic surfactants indicated that, whereas all of the carbon of monoethylene glycol dodecyl ether (MEGDE) was assimilable, only the alkyl chains were assimilated from the higher ethoxamers. These results are interpreted in terms of a primary biodegradation mechanism in which the scission of the dodecyl-ether bond is the first step. In the case of MEGDE this step separates the dodecyl chain from a C2 fragment, both of which are readily assimilable; for the higher ethoxamers, the assimilable dodecyl chain is accompanied by an ether-containing PEG derivative which would require further rounds of ether scission before assimilation. Whole cells and cell extracts converted [1-14C]MEGDE initially and very rapidly to radiolabelled dodecanol. Disappearance of [14C]dodecanol was accompanied by production of [14C]dodecanal. [14C]Dodecanoic acid was present at relatively low concentrations throughout the incubation periods. [14C]Dodecan-1, 12-dioic acid was produced in significant quantities (up to 25% of radiolabel), and the onset of its production coincided with the peak concentration of dodecanal, the disappearance of which mirrored the appearance of the dioic acid. Under anaerobic conditions in the presence of cell extracts, dodecanol (55% of radiolabel) and dodecanal (22%) accumulated rapidly from MEGDE, but there was little subsequent conversion to mono- or dicarboxylic acids. These results are interpreted in terms of a pathway initiated by dodecyl-ether cleavage to produce dodecanol, which is subsequently oxidized to dodecanal and dodecanoic acid. The formation of dodecan-1, 12-dioic acid, probably from dodecanal, may represent a means of harbouring carbon under non-growing conditions.
Carbon and electron flow in Clostridium butyricum grown in chemostat culture on glycerol and on glucose
Authors: Abbad-Andaloussi S. , Durr C. , Raval G. , Petitdemange H.
Published: June 2009

The metabolism of Clostridium butyricum DSM 5431 was studied in chemostat culture under carbon limitation using either glucose or glycerol. On glycerol, the enzymes glycerol dehydrogenase, diol dehydratase and 1,3-propanediol (1,3-PD) dehydrogenase constitute the branch point that partitions the carbon flux between the competing pathways, i.e. formation of either 1,3-PD or acetate and butyrate. The increasing levels of these enzyme activities with increasing dilution rates (D) explained the constant proportion of glycerol conversion into 1,3-PD. The production of acetate or butyrate constitutes another important branch point and when D increased (i) large amounts of intracellular acetyl-CoA accumulated, (ii) the carbon flux switched from butyric acid to acetic acid, (iii) the specific activity of thiolase was not affected, suggesting this enzyme may be the bottleneck for carbon flux to butyrate biosynthesis providing an explanation for the accumulation of large amounts of intracellular acetyl-CoA, and (iv) high levels of NADH were found in the cell. Oxidation of NADH by 1,3-PD dehydrogenase was linked to the production of 3-hydroxypropionaldehyde (3-HPA) by glycerol dehydratase. The fact that high intracellular concentrations of NADH were found means that diol dehydratase activity is the rate-limiting step in 1,3-PD formation, avoiding the accumulation of 3-HPA which is a very toxic compound. The specific rate of glucose catabolism (q
glucose = 11.1 mmol h-1 g-1) was around four times lower than the specific rate of glycerol catabolism (q
glucose = 57.4 mmol h-1 g-1). On glucose-grown cells, reducing equivalents which are released in the glycolytic pathway were reoxidized by the butyric pathway and the low specific formation rate of butyric acid led to an increase in the intracellular level of acetyl-CoA and NADH. Carbon flow was higher on glycerol due to the reoxidation of NADH by both butyric and PD pathways.
Demonstration of high-affinity Mn2+ uptake in Saccharomyces cerevisiae: specificity and kinetics
Authors: Gadd Geoffrey M. , Laurence Oliver S.
Published: June 2009

The existence of multiple transport systems for Mn2+inSaccharomyces cerevisiaehas been demonstrated in this study. Mn2+(supplied as MnCI2) was accumulated byS. cerevisiaeat all Mn2+concentrations examined (25 nM-1 mM) but a log-log plot of uptake rates and total amounts accumulated revealed the existence of at least two Mn2+concentration-dependent transport systems. Over a low Mn2+concentration range (25-1000 nM), high-affinity Mn2+uptake occurred with aKmvalue of 0.3 μM, while transformation of kinetic data obtained over the concentration range 5-200 μM revealed another system with aKmof 62 μM. Meaningful kinetic analyses were not possible at higher Mn2+concentrations because of toxicity: only about 30% of cells remained viable after 30 min incubation with 1000 μM MnCI2. Release of K+accompanied Mn2+accumulation and this increased with increasing Mn2+concentration. However, even in non-toxic Mn2+concentrations, the ratio of Mn2+uptake to K+release greatly exceeded electroneutral stoichiometric exchange. In 50 μM MnCI2, the ratio was 1: 123 and this increased to 1:2670 in 1000 μM MnCI2, a toxic concentration. External Mg2+was found to decrease Mn2+accumulation at all concentrations examined, but to differing extents. Over the low Mn2+concentration range (5-200 μM), Mg2+competitively inhibited Mn2+uptake with a half-maximal inhibitory concentration,Ki, of 5.5 μM Mg2+. However, even in the presence of a 50-fold excess of Mg2+, inhibition of Mn2+uptake was of the order of 72% and it appears that the cellular requirement for Mn2+could be maintained even in the presence of such a large excess of Mg2+. Over the high Mn2+concentration range (5-200 μM), theKifor Mg2+was 25.2 μM. At low Mn2+concentrations, Zn2+and Co2+, but not Cd2+, inhibited Mn2+uptake, which indicated that the high-affinity Mn2+uptake system was of low specificity, while at higher Mn2+concentrations, where the lower-affinity Mn2+transport system operated, inhibition was less marked. However, competition studies with potentially toxic metal cations were complicated due to toxic effects, particularly noticeable at 50 μM Co2+and Cd2+.
Genes responsible for nisin synthesis, regulation and immunity form a regulon of two operons and are induced by nisin in Lactoccocus lactis N8
Authors: Ra S. Runar , Qiao Mingqiang , Immonen Tiina , Pujana Idoia , Saris Per E. J.
Published: June 2009

Nisin is a small post-translationally modified lanthionine-containing peptide (lantibiotic) produced by certain Lactococcus lactis strains which has a high antimicrobial activity against several pathogenic Gram-positive bacteria. Northern blots and RT/PCR analyses of the nisin-producing strain N8 revealed that the nisZBTCIPRKFEG gene cluster, responsible for nisin biosynthesis, immunity and regulation, consists of two operons, nisZBTCIPRK and nisFEG. The promoter of the nisFEG operon was mapped. The −35 to −1 region upstream of the transcription start of the nisFEG promoter showed 73% identity with the corresponding region upstream of the nisA and nisZ gene. In contrast to earlier reports, nisin was found to be secreted during the early stages of growth as well as later in the growth cycle. The secreted nisin was adsorbed on the surface of the cells and was released to the medium during mid-exponential growth, when the pH in the medium fell below 5.5. In nisZB antisense and nisT deletion mutant strains constructed in this study the transcription of the nisin operons, nisin production and immunity were lost. Provision of external nisin restored the transcription of both operons in the mutant strains, showing that the operons are coordinately regulated by mature nisin. Nisin induction of the mutant strains also resulted in an increased amount of the Nisl protein and an increase in the level of immunity. Induction using higher concentrations of nisin yielded a higher level of immunity. These results showed that the nisin promoters are under positive control in an autoregulatory manner and that antimicrobial peptides can also function as signal molecules.
Identification of a 29 kDa protein in the envelope of Mycobacterium smegmatis as a putative ferri-exochelin receptor
Authors: Dover Lynn G. , Ratledge Colin
Published: June 2009

Evidence of a direct association between ferri-exochelin, the major extracellular siderophore ofMycobacterium smegmatis, and a 29 kDa protein has been obtained by three separate methods. (1) Direct binding of55Fe(III)-exochelin by the 29 kDa protein in an envelope preparation from iron-deficient cells was demonstrated by the extraction of a complex with the non-denaturing detergent CHAPS, and subsequent CHAPS-PAGE and autoradiography. (2) Affinity chromatography on a chemically synthesized ferri-exochelin-Sepharose 4B matrix led to the retention of the 29 kDa protein and a 25 kDa protein. The smaller protein was partially eluted with 1mM ferri-exochelin although it did not form a stable complex with ferri-exochelin. The 29 kDa protein could not be eluted from the affinity matrix with 1mM ferri-exochelin either alone or with 1 M NaCl. Only 2% (w/v) SDS could do this, but resulted in protein denaturation. (3) Incubation of55Fe-exochelin with CHAPS-solubilized envelope proteins in free solution followed by ion-exchange chromatography resolved three radioactive peaks; subsequent analysis by SDS-PAGE showed that the peak with the highest55Fe-binding activity per unit protein contained both the 29 and 25 kDa proteins. A direct association was demonstrated between the 29 kDa protein and55Fe-exochelin by gel filtration. The evidence suggests that the 29 kDa iron-regulated envelope protein ofM. smegmatisis a ferri-exochelin-binding protein and that the 25 kDa protein, which corresponds in size to a previously reported iron-regulated envelope protein in this bacterium, may have a role in the formation or maintenance of this complex. Proteins extracted from the cell envelope of iron-deficientM. smegmatiswith CHAPS were dialysed to remove the detergent, incorporated into liposome suspensions and then incubated with55Fe(III)-exochelin. This increased the retention of55Fe by 133-fold compared to proteins not placed in liposomes. Retention of55Fe was dependent upon the protein loading of the liposomes. Gel filtration confirmed that the iron was retained by these vesicles and even after dialysis the majority of55Fe was still retained by the vesicles. Re-solubilization of the labelled proteo-liposomes in various detergents gave limited recovery of a ferri-exochelin-protein complex. Attempts to resolve this complex by Triton X-100 PAGE led to separation of the two entities. The complex was stable, however, in a CHAPS-PAGE system.
Negotiating Informed Consent with Children in School‐Based Research: A Critical Review
Authors: Gallagher Michael , Haywood Sarah L. , Jones Manon W. , Milne Sue
Published: June 2009

The methods literature on research with children recognises the challenges of negotiating informed consent with this group. Special ‘child‐friendly’ techniques are advocated to overcome these challenges. We argue that, upon closer inspection, research with children foregrounds more fundamental problems with informed consent that are not easily resolved. Drawing from three ethical texts commonly consulted in our own research fields, we highlight problems of information, understanding, authority, capacity and voluntarity. We conclude that informed consent is more problematic than is generally admitted, and that researchers would benefit from more openly acknowledging its limitations.
Bacterial contamination of PCA and epidural infusion devices
Authors: Rothwell M. , Pearson D. , Wright K. , Barlow D.
Published: June 2009

We prospectively audited the bacterial contamination of re‐useable analgesia infusion pumps. In a one‐month period, 112 samples from the handset and keypads of our analgesia infusion pumps were cultured for bacterial contamination. Forty‐five percent of handset swabs and 46% of keypad swabs grew bacteria; the commonest organism being coagulase‐negative staphylococcus. An additional cleaning step using 70% isopropyl alcohol wipes was introduced and the contamination rate was re‐audited in 100 samples. The contamination rate was reduced to 6% of handset swabs and 4% of keypad swabs. A high initial rate of bacterial contamination of re‐useable analgesia infusion pumps was significantly reduced by the implementation of a simple, additional cleaning procedure.
Optimal remifentanil dosage for providing excellent intubating conditions when co‐administered with a single standard dose of propofol
Authors: Bouvet L. , Stoian A. , Rimmelé T. , Allaouchiche B. , Chassard D. , Boselli E.
Published: June 2009

This dose–response study aimed to determine the dose of remifentanil combined with propofol 2.5 mg.kg−1 which provided excellent intubation conditions in 95% of patients. Ninety premedicated female ASA 1 and 2 patients were randomly allocated to five remifentanil dose groups (1, 2, 3, 4 or 5 μg.kg−1). Induction of anaesthesia was performed with a blinded dose of remifentanil infused over 60 s simultaneously co‐administered with propofol 2.5 mg.kg−1 infused over 45 s. Tracheal intubation was attempted 150 s after the beginning of induction. Intubating conditions were assessed with the Copenhagen score. A probit analysis was performed to calculate the intubating efficient doses (IED) of remifentanil in 95% of patients (IED95). Our data revealed that the IED95 of remifentanil was 4.0 (95% CI: 3.4–5.6) μg.kg−1, which was associated with a maximum decrease in heart rate and mean arterial pressure of < 30%, a finding which also applied to the other groups.
Agreement between children with cancer and their parents in reporting the child's health‐related quality of life during a stay at the hospital and at home
Authors: Speyer E. , Herbinet A. , Vuillemin A. , Chastagner P. , Briançon S.
Published: June 2009

Background  Although parent–child agreement is widely studied in the paediatric clinical sample, no study has compared parent–child agreement during home and hospital stays. We aimed to determine the level of agreement in reports of health‐related quality of life (HRQoL) between parents of children with cancer and the children themselves during a stay in the hospital and a stay at home.

Methods  The sample consisted of 28 children, aged 9–18 years old, hospitalized and treated for cancer in a Children's University Hospital in France. The child's HRQoL was assessed by the parent and child versions of the Child Health Questionnaire. Parent–child agreement was estimated by the intraclass correlation coefficient. Potential predictors influencing the parent–child agreement were investigated.

Results  For both locations of stay, parent scores from the HRQoL survey were lower than those for their children for all domains, except for the general behaviour during the home stay. Scores for both parents and children, for all domains, were lower during the hospital than the home stay. The parent–child agreement was better for mental health, self‐esteem, general health and family activities during the hospital stay (range 0.32–0.66), but was better for physical function, role/physical and general behaviour during the home stay (range 0.45–0.71). Few predictors of parent–child agreement were found.

Conclusions  Agreement between parents and their children who have cancer in reporting the child's HRQoL differed by location of stay. For the home stay, the agreement was better for domains related to the child's physical life, but for the hospital stay, it was better for domains related to the child's behaviour/psychological life.
A psychometric evaluation of the Chinese version of the Hospital Anxiety and Depression Scale in patients with coronary heart disease
Authors: Wang Wenru , Chair Sek Ying , Thompson David R , Twinn Sheila F
Published: June 2009

Aim.  To evaluate further the psychometric properties of the Chinese version of the Hospital Anxiety and Depression Scale (HADS) as a screening instrument for anxiety and depression in Chinese patients with coronary heart disease (CHD) in Xian, China.

Background.  There is considerable evidence that anxiety and depression are common in patients with CHD and are associated with increased morbidity and mortality. A valid, reliable and sensitive screening tool that can be used readily on this group of patients would be useful for assessment, intervention and outcome evaluation.

Design.  A single group, cross‐sectional study.

Method.  Measurement performance was tested on 314 Chinese patients with CHD and repeated on 173 of them two weeks later.

Results.  The Chinese version of HADS (C‐HADS) had acceptable internal consistency and test‐retest reliability, with a Cronbach's alpha of 0·85 and intraclass correlation coefficient of 0·90, respectively. There was acceptable concurrent validity with significant (p < 0·05) correlations between the anxiety and depression subscales of the C‐HADS and CHD patients’ perceived health status as measured by the Chinese‐Mandarin version of the Short Form‐36 health survey (CM:SF‐36). Principal components analysis revealed a three‐factor solution accounting for 53% of the total variance. The three underlying sub‐scale dimensions are depression, psychic anxiety and psychomotor anxiety. The responsiveness of the C‐HADS was also satisfactory with significant correlation between the changes in the C‐HADS score and the changes in the mental health domain of the CM:SF‐36 (p < 0·01). Finally, over one‐third of the patients demonstrated psychological distress.

Conclusion.  Empirical data support the C‐HADS as a reliable and valid screening instrument for the assessment of anxiety and depression in Chinese‐speaking patients with CHD. A tri‐dimensional scoring approach should be considered as potentially clinically useful for this group of patients.

Relevance to clinical practice.  The C‐HADS can guide and evaluate the delivery of psychological care for Chinese patients with CHD.
An evaluation of strategies to improve the quality and content of hospital‐based postnatal care in a metropolitan Australian hospital
Authors: Schmied Virginia , Cooke Margaret , Gutwein Rosalind , Steinlein Elizabeth , Homer Caroline
Published: June 2009

Aim and objective.  This study aimed to design, implement and evaluate strategies to improve the quality and content of hospital‐based postnatal care.

Background.  Following birth, women report physical health problems, difficulties with breastfeeding, a lack of parenting self‐efficacy and there is high occurrence of postnatal distress and depression. Despite these significant needs, women are frequently dissatisfied with the advice and support they receive from hospital‐based postnatal care.

Design.  A pre/post test design compared the effect of multifaceted strategies on perceptions of quality and content of postnatal care, knowledge and experience of postnatal problems, parenting self‐efficacy and breastfeeding outcomes. The key strategy, ‘one‐to‐one time’, focused on providing women an uninterrupted period of time each day when a midwife would be available to discuss women’s concerns about their health and that of their baby.

Method.  A convenience sample of 146 women at baseline and 148 women postintervention completed a postal self‐report questionnaire between 2–4 weeks postpartum.

Results.  There were no significant differences between baseline and postintervention groups in perceived quality of care, breastfeeding outcomes and maternal self‐efficacy. Women experiencing health issues, including insufficient milk supply, backache, abnormal bleeding and urinary incontinence, were more likely to report that they received good or excellent care and advice in the postintervention group. Strategies to increase rest appeared effective with women less likely to report excessive tiredness postintervention. The key strategy, ‘one‐to‐one time’ was not consistently implemented, 57% of women reported they received 10 minutes or less of uninterrupted time with a midwife and only 11% reported that they were provided with 20 minutes or more.

Conclusion.  There is the potential for individualised care to impact on outcomes for women but established routines and institutional priorities are difficult to change.

Relevance to clinical practice.  Midwives require both skill development and time to be able to sensitively listen to women’s needs in the hospital postnatal setting.
Are older patients’ cardiac rehabilitation needs being met?
Authors: Tolmie Elizabeth P , Lindsay Grace M , Kelly Tim , Tolson Debbie , Baxter Susan , Belcher Philip R
Published: June 2009

Aims.  The primary aim of this study was to examine the needs of older people in relation to cardiac rehabilitation and to determine if these were currently being met. A secondary aim was to compare illness representations, quality of life and anxiety and depression in groups with different levels of attendance at a cardiac rehabilitation programme.

Background.  Coronary heart disease accounted for over seven million cardiovascular deaths globally in 2001. Associated deaths increase with age and are highest in those older than 65. Effective cardiac rehabilitation can assist independent function and maintain health but programme uptake rates are low. We have, therefore, focussed specifically on the older patient to determine reasons for the low uptake.

Design.  Mixed methods.

Methods.  A purposive sample of 31 older men and women (≥65 years) completed three questionnaires to determine illness representations, quality of life and anxiety and depression. They then underwent a brief clinical assessment and participated in a face‐to‐face audio‐taped interview.

Results.  Quantitative: Older adults, who did not attend a cardiac rehabilitation programme, had significantly poorer personal control and depression scores (p < 0·01) and lower quality of life scores than those who had attended. Few achieved recommended risk factor reduction targets. Qualitative: The three main themes identified as reflecting the views and experiences of and attendance at the cardiac rehabilitation programme were: ‘The sensible thing to do’, ‘Assessing the impact’ and ‘Nothing to gain’.

Conclusions.  Irrespective of level of attendance, cardiac rehabilitation programmes are not meeting the needs of many older people either in terms of risk factor reduction or programme uptake. More appropriate programmes are needed.

Relevance to clinical practice.  Cardiac rehabilitation nurses are ideally placed to identify the rehabilitation needs of older people. Identifying these from the older person’s perspective could help guide more appropriate intervention strategies.
Susceptibility of selected freshwater fish species to a UK Lactococcus garvieae isolate
Authors: Algöet M , Bayley A E , Roberts E G , Feist S W , Wheeler R W , Verner‐Jeffreys D W
Published: June 2009

Gram‐positive cocci recovered from diseased rainbow trout from a farm in England were characterized by different methods, including pulsed field gel electrophoresis, as virulent Lactococcus garvieae serogroup 2 (pulsotype A1). Groups of rainbow trout were kept at a range of temperatures and injected intraperitoneally (i.p.) with one of the UK isolates, L. garvieae 00021. The 18 °C and 16 °C groups showed 67% and 28% mortality, respectively, by day 27 post‐injection. Fish kept at 14 °C or lower were less susceptible (≤3% mortality). Raising the temperature of all groups to 18 °C at day 27 post‐injection did not result in recurrence of the disease, even though viable bacteria were recovered from all groups 42 days later. Grayling were highly susceptible, with 65% mortalities when challenged with 200 colony forming unit fish−1 by i.p. injection and 37% mortalities when exposed to effluent water from tanks containing affected rainbow trout. Other fish species tested, Atlantic salmon, brown trout and seven cyprinid species, were less susceptible. Viable L. garvieae was isolated from the internal organs of all species tested at the end of the trials, suggesting that they may pose a threat as possible carriers to susceptible farmed and wild fish.
Tetrahymena sp. infection in guppies, Poecilia reticulata Peters: parasite characterization and pathology of infected fish
Authors: Leibowitz M P , Zilberg D
Published: June 2009

Tetrahymena sp. infection was diagnosed in guppies imported from Singapore. The parasite was isolated (Tet‐NI) and optimally cultured in vitro in RM‐9 medium. Cytological analyses [silver‐staining and scanning electron microscopy (SEM)] revealed a pyriform‐shaped, 64 × 41‐μm holotrich ciliate without caudal cilium, containing a macro‐nucleus (18.25 × 16.83 μm) and micro‐nucleus (5.73 × 5.40 μm). Wet‐mount examination and histological analyses of fish exposed to the parasite by co‐habitation, immersion and infection by i.p. (intra‐peritoneal) and i.m. (intra‐muscular) injection revealed numerous ciliates on the skin, and in the gill and caudal fin blood vessels. Ciliates surrounded internal organs, the peri‐orbital region of the eye, and were observed inside developing guppy embryos. Some muscle necrosis was associated with infection, but little or no inflammatory response. Immersion, co‐habitation and i.m. injection caused relatively high infection rates and levels in the skin and tail, and lower infection in the gill blood vessels and internal organs; i.p. injection caused higher infection in the gill blood vessels and internal organs. Co‐habited fish had relatively high infection levels in the hind‐gut sub‐mucosa. This is the first report of controlled systemic infection by Tetrahymena sp.
Foliar and tuber late blight resistance in a Solanum tuberosum breeding population
Authors: Mayton H. , Griffiths H. , Simko I. , Cheng S. , Lorenzen J. , De Jong W. , Fry W. E.
Published: June 2009

With 2 figures and 2 tables

The purpose of this research was to identify the genetic basis of foliar and tuber resistance to Phytophthora infestans in a potato breeding population developed from a cross between two tetraploid Solanum tuberosum lines, NY121 and NY115. The parent with high foliar resistance, NY121, was highly susceptible to tuber blight. Foliar resistance was assessed in field trials, while tuber blight was assessed both in the field and in the laboratory. A quantitative trait locus (QTL) explaining ca. 50% of the phenotypic variance for foliar resistance was located on the lower arm of linkage group V, and was loosely associated with tuber blight resistance (significantly in one of two trials). This QTL was confirmed in a separate sample from the same population. Tuber blight detected via laboratory assays was not correlated with tuber blight incidence in the field. Most markers associated with tuber blight resistance were not associated with foliar resistance and most markers associated with foliar resistance were not associated with tuber blight resistance.
No evidence for compensatory thermal adaptation of soil microbial respiration in the study of Bradford et al. (2008)
Authors: Hartley Iain P. , Hopkins David W. , Garnett Mark H. , Sommerkorn Martin , Wookey Philip A.
Published: June 2009

Bradford et al. (2008) conclude that thermal adaptation will reduce the response of soil microbial respiration to rising global temperatures. However, we question both the methods used to calculate mass‐specific respiration rates and the interpretation of the results. No clear evidence of thermal adaptation reducing soil microbial activity was produced.
New nitrogen uptake strategy: specialized snow roots
Authors: Onipchenko Vladimir G. , Makarov Mikhail I. , Van Logtestijn Richard S. P. , Ivanov Viktor B. , Akhmetzhanova Assem A. , Tekeev Dzhamal K. , Ermak Anton A. , Salpagarova Fatima S. , Kozhevnikova Anna D. , Cornelissen Johannes H. C.
Published: June 2009

The evolution of plants has yielded a wealth of adaptations for the acquisition of key mineral nutrients. These include the structure, physiology and positioning of root systems. We report the discovery of specialized snow roots as a plant strategy to cope with the very short season for nutrient uptake and growth in alpine snow‐beds, i.e. patches in the landscape that remain snow‐covered well into the summer. We provide anatomical, chemical and experimental 15N isotope tracking evidence that the Caucasian snow‐bed plant Corydalis conorhiza forms extensive networks of specialized above‐ground roots, which grow against gravity to acquire nitrogen directly from within snow packs. Snow roots capture nitrogen that would otherwise partly run off down‐slope over a frozen surface, thereby helping to nourish these alpine ecosystems. Climate warming is changing and will change mountain snow regimes, while large‐scale anthropogenic N deposition has increased snow N contents. These global changes are likely to impact on the distribution, abundance and functional significance of snow roots.
Palmitoylation of the synaptic vesicle fusion machinery
Authors: Prescott Gerald R. , Gorleku Oforiwa A. , Greaves Jennifer , Chamberlain Luke H.
Published: June 2009

The fusion of synaptic vesicles with the pre‐synaptic plasma membrane mediates the secretion of neurotransmitters at nerve terminals. This pathway is regulated by an array of protein–protein interactions. Of central importance are the soluble NSF (N‐ethylmaleimide‐sensitive factor) attachment protein receptor (SNARE) proteins syntaxin 1 and SNAP25, which are associated with the pre‐synaptic plasma membrane and vesicle‐associated membrane protein (VAMP2), a synaptic vesicle SNARE. Syntaxin 1, SNAP25 and VAMP2 interact to form a tight complex bridging the vesicle and plasma membranes, which has been suggested to represent the minimal membrane fusion machinery. Synaptic vesicle fusion is stimulated by a rise in intraterminal Ca2+ levels, and a major Ca2+ sensor for vesicle fusion is synaptotagmin I. Synaptotagmin is likely to couple Ca2+ entry to vesicle fusion via Ca2+‐dependent and independent interactions with membrane phospholipids and the SNARE proteins. Intriguingly, syntaxin 1, SNAP25, VAMP2 and synaptotagmin I have all been reported to be modified by palmitoylation in neurons. In this review, we discuss the mechanisms and dynamics of palmitoylation of these proteins and speculate on how palmitoylation might contribute to the regulation of synaptic vesicle fusion.