bluuebunny/crossref_metadata_2025 huggingface dataset:
An aminopeptidase nutritionally important to Fusobacterium nucleatum
Authors: Rogers A. H. , Gunadi A. , Gully N. J. , Zilm P. S.
Published: June 2009

The properties of an aminopeptidase (AP) from Fusobacterium nucleatum were studied in view of the fact that this organism, along with other Gram-negative anaerobes involved in periodontal diseases, survives in the subgingival environment by obtaining energy via the fermentation of a small number of peptide-derived amino acids. The AP was found to be cell-associated and was isolated from disrupted chemostat-grown cells. It was purified by (NH4)2SO4 fractionation, two column chromatographic steps and IEF. The enzyme was found to have a molecular mass of 54 kDa, a pl of 5.1, a pH optimum between 7.5 and 8.0 and, using Leu-Ala as substrate, it gave Km and Vmax values of 0.66 mM and 0.12 μmol min-1 mg-1, respectively. No complete homology was found between the N-terminal sequence of the first 20 amino acids (MDXKXYVDLKERFLRYVKFN.) and any other published sequence, but residues 8--20 gave a 62% match with residues 9--21 of an AP from Haemophilus influenzae. The enzyme was inactivated by chelating agents, bestatin, p-hydroxymercuribenzoate and some heavy metals. Cobalt ions restored EDTA-inactivated activity but did not reverse inhibition by 1,10-phenanthroline. In addition, bestatin and 1, 10-phenanthroline had an inhibitory effect on the batch growth of F. nucleatum in a complex medium in which peptidase activities would be nutritionally essential. No such inhibition was observed in a chemically defined medium in which growth was not dependent upon peptidase activities. The peptidase described in this paper therefore appears to be a cobalt-activated metallo-AP which, together with other peptidases, is considered to be important in the survival of F. nucleatum in the subgingival environment of the mouth.
Magnesium transport in Salmonella typhimurium: regulation of mgtA and mgtCB during invasion of epithelial and macrophage cells
Authors: Smith Ronald L. , Kaczmarek Michelle T. , Kucharski Lisa M. , Maguire Michael E.
Published: June 2009

Salmonella typhimurium contains two inducible Mg2+ transport systems, MgtA and MgtB, the latter encoded by a two-gene operon, mgtCB. Mg2+ deprivation of S. typhimurium increases transcription of both mgtA and mgtCB over a thousandfold and a similar increase occurs upon S. typhimurium invasion of epithelial cells. These increases are mediated by the phoPQ two-component signal transduction system, an essential system for S. typhimurium virulence. It was therefore hypothesized that expression of MgtA and MgtCB is increased upon invasion of eukaryotic cells because of a lack of intravacuolar Mg2+. However, when S. typhimurium was grown at pH 5.2, the capacity of the constitutive CorA transporter in mediating Mg2+ was greater than that at pH 7.4. Furthermore, induction of mgtA and mgtCB transcription was greater in the presence of a wild-type corA allele than in its absence. This implies that intravacuolar S. typhimurium could obtain sufficient Mg2+ via the CorA system. The effect of acid pH on mgtA and mgtCB transcription was also measured. Compared to induction at pH 7.4, exposure to pH 5.2 almost completely abolished induction of mgtA at low Mg2+ concentrations but diminished induction of mgtCB only twofold. Adaptation of cells to acid pH by overnight growth resulted in normal levels of induction of mgtA and mgtCB at low Mg2+ concentrations. These results imply an additional level of regulation for mgtA that is not present for mgtCB. Conversely, repression of mgtA and mgtCB expression by increased extracellular Mg2+ was relatively insensitive to acid. Transcription of both loci was strongly induced upon invasion of the Hep-2 or CMT-93 epithelial-like or J774 macrophage-like cell lines. However, the presence or absence of functional alleles of either or both mgtA or mgtCB had no effect on invasion efficiency or short-term survival of S. typhimurium within the eukaryotic cells. It was concluded that the strong Mg2+-dependent induction of mgtA and mgtCB upon invasion of eukaryotic cells is not required because S. typhimurium lacks sufficient Mg2+ during eukaryotic cell invasion and initial intravacuolar growth.
Membrane-bound and extracellular β-lactamase production with developmental regulation in Streptomyces griseus NRRL B-2682
Authors: Deák Eleonóra , SzabóA István , Kálmáczhelyi Attila , Gál Zsuzsanna , Barabás György , Penyige Andres
Published: June 2009

A new type ofβ-lactamase has been isolated and characterized in Streptomyces griseus NRRL B-2682. The enzyme has membrane-bound and extracellular forms. Biochemical characterization of some of the properties of the enzyme showed that it belongs to the class A group of penicillinases. Comparison of the membrane-bound and extracellular forms of theβ-lactamases suggests that they seem to be differently processed forms of the same enzyme. The N-terminal amino acid sequence of the extracellular form of the β-lactamase showed a high degree of similarity to a D-aminopeptidase of another Streptomyces griseus strain. Secretion of the β-lactamase was affected by the differentiation state of the strain since in spontaneous non-sporulating mutants only the membrane-bound form was present. In accordance with this when sporulation of the wild-type strain was inhibited it failed to secrete extracellular β-lactamase. Addition of globomycin to the non-sporulating cells liberated the enzyme from the membrane, indicating that the protein is processed normally by signal peptidase II and a glyceride-thioether group, together with a fatty acid amide-linkage, is responsible for the attachment of the enzyme to the cellular membrane. Under sporulation-repressed conditions addition of peptidoglycan fragments and analogues or inhibition of cell wall biosynthesis by penicillin-G induced β-lactamase secretion and also restored sporulation both in solid and submerged cultures. These results confirm that β-lactamase secretion is tightly coupled to the sporulation process in S. griseus.
Structural, functional and mutational analysis of the pfr gene encoding a ferritin from Helicobacter pylori
Authors: Bereswill Stefan , Waidner Uta , Odenbreit Stefan , Lichte Flavia , Fassbinder Frank , Bode G�nter , Kist Manfred
Published: June 2009

The function of the pfr gene encoding the ferritin from Helicobacter pylori was investigated using the Fur titration assay (FURTA) in Escherichia coli, and by characterization of a pfr-deficient mutant strain of H. pylori. Nucleotide sequence analysis revealed that the pfr region is conserved among strains (>95% nucleotide identity). Two transcriptional start sites, at least one of them preceded by a s70-dependent promoter, were identified. Provision of the H. pylori pfr gene on a multicopy plasmid resulted in reversal of the Fur-mediated repression of the fhuF gene in E. coli, thus enabling the use of the FURTA for cloning of the ferritin gene. Inactivation of the pfr gene, either by insertion of a resistance cassette or by deletion of the up- and downstream segments, abolished this function. Immunoblot analysis with a Pfr-specific antiserum detected the Pfr protein in H. pylori and in E. coli carrying the pfr gene on a plasmid. Pfr-deficient mutants of H. pylori were generated by marker-exchange mutagenesis. These were more susceptible than the parental strain to killing by various metal ions including iron, copper and manganese, whereas conditions of oxidative stress or iron deprivation were not discriminative. Analysis by element-specific electron microscopy revealed that growth of H. pylori in the presence of iron induces the formation of two kinds of cytoplasmic aggregates: large vacuole-like bodies and smaller granules containing iron in association with oxygen or phosphorus. Neither of these structures was detected in the pfr-deficient mutant strain. Furthermore, the ferritin accumulated under iron overload and the pfr-deficient mutant strains lacked expression of a 12 kDa protein which was negatively regulated by iron in the parental strain. The results indicate that the nonhaem-iron ferritin is involved in the formation of iron-containing subcellular structures and contributes to metal resistance of H. pylori. Further evidence for an interaction of ferritin with iron-dependent regulation mechanisms is provided.
Cellular fatty acyl and alkenyl residues in Megasphaera and Pectinatus species: contrasting profiles and detection of beer spoilage
Authors: Helander Ilkka M. , Haikara Auli
Published: June 2009

The strictly anaerobic Gram-negative beer spoilage bacteria Megasphaera cerevisiae, Pectinatus cerevisiiphilus and P. frisingensis were subjected to cellular fatty acid analysis, employing acid- and base-catalysed cleavage, gas chromatography and mass spectrometry. M. cerevisiae contained 12:0, 16:0, 16:1, 18:1, 17:cyc, 19:cyc, 12:0(3OH), 14:0(3OH) as the main fatty acids, and alk-1-enyl chains instead of acyl chains were detected to a considerable extent (14% of total fatty acids), indicating the presence of plasmalogens. The fatty acid pattern of M. cerevisiae was almost identical to that of M. elsdenii, the only species previously assigned to this genus. P. cerevisiiphilus and P. frisingensis yielded fatty acids that were heavily dominated by odd-numbered chains; 11:0, 15:0, 17:1, 18:cyc and 13:0(3OH) were the main fatty acids detected in both species. Alk-1-enyl chains with similar chain lengths were also found. Both Pectinatus species contained six different 3-hydroxy fatty acids with chain lengths between 11 and 15 carbons, 13:0(3OH) being dominant and the others accounting for generally less than 1% of total fatty acids. Among the minor components, an unsaturated 3-hydroxy fatty acid was detected which was shown to be 13:1(30H). In addition, fatty acid analysis was shown to be applicable to detection of bacterial contamination of beer.
Cloning of a Candida albicans peptide transport gene
Authors: Basrai Munira A. , Lubkowitz Mark A. , Perry Jack R. , Miller David , Krainer Eduardo , Naider Fred , Becker Jeffrey M.
Published: June 2009

A Candida albicans peptide transport gene, CaPTR2, was cloned from a C. albicans genomic library by functional complementation of a peptide transport deficient mutant (strain ptr2-2) of Saccharomyces cerevisiae. CaPTR2 restored peptide transport to transformants as determined by uptake of radiolabelled dileucine, growth on dipeptides as sources of required amino acids, and restoration of growth inhibition by toxic peptides. Plasmid curing experiments demonstrated that the peptide transport phenotype was plasmid borne. CaPTR2 was localized to chromosome R of C. albicans by contour-clamped homologous electric field gel chromosome blots. Deletion subclones and frameshift mutagenesis were used to narrow the peptide transport complementing region to a 5:1 kb DNA fragment. DNA sequencing of the complementing region identified an ORF of 1869 bp containing an 84 nucleotide intron. The deduced amino acid sequence predicts a protein of 70 kDa consisting of 623 amino acids with 12 hydrophobic segments. A high level of identity was found between the predicted protein and peptide transport proteins of S. cerevisiae and Arabidopsis thaliana. This study represents the first steps in the genetic characterization of peptide transport in C. albicans and initiates a molecular approach for the study of drug delivery against this pathogen.
Activity of mycobacterial promoters during intracellular and extracellular growth
Authors: Dellagostin Odir A. , Esposito Giovanna , Eales Lesley-Jane , Dale Jeremy W. , McFadden JohnJoe
Published: June 2009

pUS933, a bifunctionalMycobacterium-Escherichia coli translationalfusion vector containing an amino-terminally truncatedE. coli lacZreporter gene, was constructed. Derivatives of pUS933, containing the promoter, RBS and start codon of theMycobacterium bovisBCGhsp60gene, theMycobacterium leprae28 kDa gene and theM. leprae18 kDa gene were constructed and introduced intoE. coli, Mycobacterium smegmatisandM. bovisBCG. -Galactosidase activity was measured for mycobacteria grown in liquid culture. Primerextension analysis was used to determine the transcriptional start point for the 18 kDa promoter inM. smegmatis.Murine macrophages were infected with recombinant BCG containing the pUS933 derivatives and expression levels were examined, by fluorescence microscopy and fluorometry, during intracellular growth of BCG. Both the BCGhsp60gene promoter and theM. leprae28 kDa gene promoter gave high levels of -galactosidase expression in all situations examined. In contrast, the M. leprae 18 kDa promoter fragment gave very low levels of expression inM. smegmatisand BCG grown in liquid culture, but in BCG growing within macrophages it was induced to levels almost as high as the other promoters. This indicated that the 18 kDa gene is specifically activated during intracellular growth and may therefore be involved in survival ofM. lepraewithin macrophages. This pattern of regulation may be useful for controlling expression of foreign genes in recombinant BCG strains.
Anti-Candida activity of a novel killer toxin from the yeast Williopsis mrakii
Authors: Hodgson Valerie J. , Button David , Walker Graeme M.
Published: June 2009

A screening of putative killer yeast strains showed that spore-forming ascomycetous yeasts of the generaPichiaandWilliopsisdisplayed the broadest range of activity against sensitive strains ofCandidaspp. andSaccharomyces cerevisiae. Williopsis mrakii(NCYC 500) showed extensive anti-Candidaactivity against strains isolated from clinical specimens.W. mrakiikiller factor was produced in minimal media as a function of growth and its activity reached constant levels as cells entered stationary phase. The proteinaceous killer toxin was found to be unstable outwith a specific range of temperature and pH (above 30 �C and pH 4�0), and further analysis showed that the active toxin molecule was an acidic polypeptide with a relative molecular mass between 1�8-5�0 kDa. At critical concentrations the killer factor exerted a greater effect on stationary phase cells ofCandidathan cells from an exponential phase of growth. At low concentrations, the killer toxin produced a fungistatic effect on sensitive yeasts but at higher concentrations there was evidence to suggest that membrane damage accounted for the zymocidal effects of the killer factor. The cidal nature of the toxin was reflected in a rapid decrease in sensitive cell viability. Findings presented suggest thatW. mrakiikiller toxin has potential as a novel antimycotic agent in combatting medically important strains ofCandida.
Nitrile hydrolysis by Rhodococcus erythropolis BL1, an acetonitrile-tolerant strain isolated from a marine sediment
Authors: Langdahl Bjarne R. , Bisp Peter , Ingvorsen Kjeld
Published: June 2009

A number of physiologically different nitrile-hydrolysing bacteria were isolated from coastal marine sediments in Denmark by enrichment culture. One strain, BL1, identified as Rhodococcus erythropolis, grew on acetonitrile as sole carbon and nitrogen source in a defined medium. Growth occurred between 0 and 8% NaCl with an optimum around 2%, thus reflecting the marine origin of the isolate. Intact cells of R. erythropolis BL1 could hydrolyse a large variety of saturated and unsaturated aliphatic nitriles to their corresponding acids. Benzonitrile and benzylcyanide were not hydrolysed, whereas some aromatic compounds containing a -CN group attached to a C3 or C4 aliphatic side chain were accepted as substrates. The substrate spectrum of R. erythropolis BL1 was thus markedly different from those of other Grampositive nitrile-hydrolysing bacteria isolated from non-marine environments. Nitrile hydrolysis during growth and in resting cell suspensions usually occurred without intermediate accumulation of amide outside the cells. Detailed studies, however, showed that nitrile hydrolysis by strain BL1 was due to a nitrile hydratase/amidase enzyme system. Nitrile hydratase activity was found to be inducible whereas amidase activity was constitutive. The amidase activity of cells could, however, be enhanced manyfold by growth in media containing acetamide or acetonitrile. In most cases amides were hydrolysed at a much higher rate than the corresponding nitriles, which explained why amides were rarely detected in the surrounding medium during nitrile hydrolysis. R. erythropolis BL1 exhibited the highest tolerance towards acetonitrile ever reported for a nitrile-hydrolysing bacterium, as demonstrated by its ability to grow exponentially in the presence of 900 mM acetonitrile.
High cAMP Levels Antagonize the Reprogramming of Gene Expression that Occurs at the Diauxic Shift in Saccharomyces Cerevisiae
Authors: Boy-Marcotte Emmanuelle , Tadi Djamila , Perrot Michel , Boucherie Helian , Jacquet Michel
Published: June 2009

In order to analyse the involvement of the cAMP pathway in the regulation of gene expression in Saccharomyces cerevisiae, we have examined the effect of cAMP on protein synthesis by using two-dimensional gel electrophoresis. cAMP had only a minor effect on the protein pattern of cells growing exponentially on glucose. However, it interfered with the changes in gene expression normally occurring upon glucose exhaustion in yeast cultures, maintaining a protein pattern typical of cells growing on glucose. This effect was accompanied by a delay before growth recovery on ethanol. We propose a model in which the cAMP-signalling pathway has a role in the maintenance of gene expression, rather than in the determination of a specific programme. A decrease of cAMP would then be required for metabolic transitions such as the diauxic phase.
Alteration in Membrane Fluidity and Lipid Composition, and Modulation of H+-ATPase Activity in Saccharomyces Cerevisiae caused by Decanoic Acid
Authors: Alexandre Herve , Mathieu Bruno , Charpentier Claudine
Published: June 2009

Decanoic acid, a lipophilic agent, inhibitedin vitrothe plasma membrane H+-ATPase ofSaccharomyces cerevisiaegrown in YPD medium. Conversely, when decanoic acid (35 μM) was present in the growth medium, the measured H+-ATPase activity was four times higher than that of control cells.Km, and pH and orthovanadate sensitivity were the same for the two growth conditions, which indicated that H+-ATPase activation was not due to conformational changes in the enzyme. The activation process was not entirely reversible which showed that plasma membrane H+-ATPase activation is due to several mechanisms. 1,6-diphenyl-1,3,5-hexatriene anisotropy performed on protoplasts from cells grown in YPD revealed that as decanoic acid concentration was increased, anisotropy significantly decreased, i.e. membrance fluidity increased. Cells grown in media containing decanoic acid exhibited greater membrane fluidity compared with control cells. Furthermore, these cells did not show any fluidifying effect when increased concentrations of decanoic acid were added. Chemical analysis of cell membrane lipid composition revealed a modification in the distribution of the phospholipid fatty acids and sterols in cells grown in the presence of 35 μM decanoic acid compared with control cells. Our results support the view that the plasma membrane H+-ATPase activation induced by decanoic acid is correlated with an alteration in membrane lipid constituents.
Propionyl-CoA carboxylase from Streptomyces coelicolor A3(2) : cloning of the gene encoding the biotinlcontaining subunit
Authors: Bramwell Helena , Hunter Lain S. , Coggins John R. , Nimmo Hugh G.
Published: June 2009

In Streptomyces coelicolor A3(2), polyketides are made from malonyl-CoA, which is presumed to be derived from acetyl-CoA by the action of acetyl-CoA carboxylase (ACC). No ACC activity was found in cell-free extracts of S. coelicolor. However, propionyl-CoA carboxylase (PCC) activity was detected at substantial levels. Fixation of CO2 by ACC and PCC occurs by covalent bonding of CO2 to a biotin-containing protein. Most bacteria have a single small biotinylated protein of approximately 22 kDa, but S. coelicolor contains three larger biotin-containing proteins (approximately 145,88 and 70 kDa). To determine which biotinylated protein was associated with PCC activity, the enzyme was purified and shown to comprise an α subunit (biotin-containing) of 88 kDa and a β subunit of 66 kDa. The N-terminal sequences of these proteins were determined and, using an oligonucleotide probe, the gene for the α subunit (pccA) was cloned.
Exclusion of Long Heterologous Insertions and Deletions from the Pairing Synapsis in Pneumococcal Transformation
Authors: Pasta Franck , Sicard Michel A.
Published: June 2009

We have studied the mode of recombination of six insertions during genetic transformation of Streptococcus pneumoniae. The six heterologous insertions are located at the same site in the ami locus of the pneumococcal chromosome; insertion sizes range from 4 to 1374 bp. With respect to single-point markers we found that the number of transformants in one-point crosses is reduced, while the number of wild-type transformants in two-point crosses is drastically increased, what we call hyper-recombination. The magnitude of the shift is correlated with the size of the insert. This effect could result either from a special repair pathway of multibase heteroduplexes or from the exclusion of multibase heterologous insertions out of the pairing synapsis. To test these hypotheses we have used insertions in two kinds of three-point crosses. The repair model predicts that the excess of wild-type transformants remains in one set of crosses but is suppressed in the second set. The results we obtained are reversed, ruling out the hypothesis of a repair process, but in agreement with predictions based on the exclusion model. Moreover, we have re-examined the situation of deletions, our previous results suggesting that deletions were likely to be converted at the heteroduplex step. Genetic evidence we obtained in this work no longer supports this hypothesis. Thus, long heterologous insertions are partly excluded at the pairing step.
Transcription of the glnB and glnA genes in the photosynthetic bacterium Rhodospirillum rubrum
Authors: Johansson Magnus , Nordlund Stefan
Published: June 2009

The P11 protein, encoded by glnB, has a central role in the control of nitrogen metabolism in nitrogen-fixing prokaryotes. The glnB gene of Rhodospirillum rubrum was isolated and sequenced. The deduced amino acid sequence had very high sequence identity to other P11 proteins. The glnA gene, encoding glutamine synthetase, was located 135 bp downstream of glnB and was partially sequenced. glnB is cotranscribed with glnA from a promoter with high similarity to the s54-dependent promoter consensus sequence. A putative s70 promoter was also identified further upstream of glnB. Northern blotting analyses showed that in addition glnA is either transcribed from an unidentified promoter or, more likely, that the glnBA transcript is processed to give the glnA mRNA. The total level of the two transcripts was much higher in nitrogen-fixing cells than in ammonia-grown cells.
Molecular characterization of adiY, a regulatory gene which affects expression of the biodegradative acid-induced arginine decarboxylase gene (adiA) of Escherichia coli
Authors: Stim-Herndon Kathleen P. , Flores Theresa M. , Bennett George N.
Published: June 2009

A transcriptional regulator gene, designated adiY, was found downstream of the biodegradative arginine decarboxylase (adiA) gene (previously known as adi) of Escherichia coli. The arginine decarboxylase system is maximally induced under conditions of acidic pH, anaerobiosis and rich medium, and AdiY was found to increase the expression of adiA. The DNA sequence of adiY encodes a protein of 253 amino acids. Primer extension analysis defined the promoter. The amino acid sequence of AdiY showed homology to the XyIS/AraC family of transcriptional regulators, which includes EnvY and AppY. Studies suggested that sequences required for acid induction were also necessary to observe the stimulation by AdiY. An examination of the substitution of AdiY, AppY and EnvY showed that these three proteins can, to some extent, stimulate the other systems.
Molecular characterization of a chromosomal region involved in the oxidation of acetyl-CoA to glyoxylate in the isocitrate-lyase-negative methylotroph Methylobacterium extorquens AM1
Authors: Chistoserdova Ludmila V. , Lidstrom Mary E.
Published: June 2009

A region on the Methylobacterium extorquens AM1 chromosome previously shown to complement a chemically induced mutant (PCT48) unable to convert acetyl-CoA into glyoxylate was characterized in detail in order to identify the gene(s) involved in the unknown pathway for acetyl-CoA oxidation. Six complete and two partial ORFs were identified by sequencing. Sequence comparisons suggested these might code for, respectively, a dehydrogenase of unknown specificity, a polypeptide of at least 15 kDa with unknown function, a coenzyme-B12-linked mutase, a catalase, an alcohol dehydrogenase (ADH) of unknown function, a polypeptide of 28 kDa, a ketol-acid reductoisomerase and a propionyl-CoA carboxylase (PCC). Insertion mutations were introduced into each ORF in order to determine their involvement in C1 and C2 metabolism. Mutations in three genes, encoding the mutase, ADH and PCC, resulted in a phenotype characteristic of mutants unable to oxidize acetyl-CoA, i.e. they were C1- and C2-negative and their growth on these compounds was restored by the addition of glycolate or glyoxylate. Mutants in the genes thought to encode catalase and PCC were found to be deficient in the corresponding enzyme activity, confirming the identity of these genes, while physiological substrates for the mutase and ADH remain unidentified. This work, in which three new genes necessary for conversion of acetyl-CoA into glyoxylate were identified, is an intermediary step on the way to the solution of the unknown pathway for acetyl-CoA oxidation in isocitrate-lyase-negative methylotrophs.
Extracellular and surface-exposed polysaccharides of non-tuberculous mycobacteria
Authors: Lemassu Anne , Ortalo-Magné Annick , Bardou Fabienne , Silve Gaby , Lanéelle Marie-Antoinette , Daffé Mamadou
Published: June 2009

We studied the outermost constituents of the cell envelopes, which are involved in the interaction between the bacilli and the host cells, of five pathogenic and non-pathogenic mycobacterial species for comparison with those we have previously characterized fromM. tuberculosis. The extracellular materials (ECMs) were isolated by ethanol precipitation and compared to the surface-exposed materials (SXMs) extracted by mechanical means. The materials from both sources were composed almost exclusively of polysaccharides and proteins. Two groups of mycobacteria were clearly distinguishable. The first group comprised the pathogenic speciesM. kansasiiwhich produced large amounts of ECM, the glycosyl composition of which was similar to that of the SXM. The second group comprisedM. aviumand the non-pathogenic strains ofM. gastri, M. phleiandM. smegmatiswhich produced small amounts of ECM. This latter group could be subdivided into those which produced carbohydrate-rich ECM (M. aviumandM. gastri) and those forming protein-rich ECM (M. phleiandM. smegmatis), a classification that correlated with the difference in the growth rate of the two subgroups. The glycosyl composition of the ECM of a given species was qualitatively similar to that of the SXM, except forM. aviumandM. phleiwhose SXM were devoid of arabinose. In addition to glucose, mannose and arabinose, xylose was detected in the hydrolysis products of the ECM and SXM ofM. smegmatis, the SXM ofM. phleiand the ECM of some batches ofM. avium. The polysaccharide constituents of the ECM and SXM of the different mycobacteria were purified by anion-exchange and gel-filtration chromatography; all were found to be neutral compounds devoid of acyl substituents. The extracellular polysaccharides consisted of high-molecular-mass glycogen-like glucans, arabinomannans and mannans, structurally similar to the corresponding substances previously characterized from the capsule ofM. tuberculosis. The same types of polysaccharides were characterized from the SXM of all the strains, exceptM. aviumandM. phleiwhich were devoid of arabinomannans. This study questions the unique and universal representation of the mycobacterial cell envelope and the existence of the so-called acidic polysaccharide-rich outer layer.
MyCrystals– a simple visual data management program for laboratory-scale crystallization experiments
Authors: Løvgreen Monika Nøhr , Løvgreen Mikkel , Christensen Hans E. M. , Harris Pernille
Published: May 2009

MyCrystalsis designed as a user-friendly program to display crystal images and list crystallization conditions. The crystallization conditions entry fields can be customized to suit the experiments.MyCrystalsis also able to sort the images by the entered crystallization conditions, which presents a unique opportunity to easily assess the effect of, for example, changing pH or concentration and thus establish the best conditions to be used for optimization.
Integrated Fraud Risk Factors and Robust Methodology: A Review and Comment
Authors: Sitorus Tumpal , Scott Don
Published: June 2009

This paper introduces a range of literature from a broad area of social sciences. It identifies a need to gain a better understanding of the causation of fraud and the complexity of fraud risk factors. Its suggestions are under consideration by the Public Company Accounting Oversight Board (PCAOB) for possible inclusion in the year 2009 material. Later descriptive and robust fraud risk models, such as those proposed by Krambia‐Kapardis and Steane & Cockerell, have still not fully explained the aetiology of fraud and hence the causation of fraud, as well as the complexity of the problem. The paper concludes with a discussion of findings, resolutions and recommendations.
Improved therapeutic efficacy against murine carcinoma by combining honokiol with gene therapy of PNAS‐4, a novel pro‐apoptotic gene
Authors: Yuan Zhu , Liu Huanyi , Yan Fei , Wang Yongsheng , Gou Lantu , Nie Chunlai , Ding Zhenyu , Lai Songtao , Zhao Yuwei , Zhao Xinyu , Li Jiong , Deng Hongxin , Mao Yongqiu , Chen Lijuan , Wei Yuquan , Zhao Xia
Published: June 2009

PNAS‐4, a novel pro‐apoptotic gene activated during the early response to DNA damage, can inhibit proliferation via apoptosis when overexpressed in some tumor cells. Recent studies have indicated that honokiol can induce apoptosis, inhibit angiogenesis, and suppress tumor growth. In the present study, we investigated whether mouse PNAS‐4 (mPNAS‐4) could augment the apoptosis of tumor cells induced by honokiol in vitro, and whether the antiangiogenic activity of honokiol and induction of apoptosis by mPNAS‐4 could work cooperatively to improve the antitumor efficacy in vivo. In vitro, mPNAS‐4 inhibited proliferation of murine colorectal carcinoma CT26 and Lewis lung carcinoma LL2 cells through induction of apoptosis, and significantly augmented the apoptosis of CT26 and LL2 cells induced by honokiol. Compared with treatment with mPNAS‐4 or honokiol alone, in vivo systemic administration of an expression plasmid encoding mPNAS‐4 and low‐dose honokiol significantly suppressed tumor growth through the enhanced induction of apoptosis and the augmented inhibition of angiogenesis. Our data suggest that the combined treatment with mPNAS‐4 plus honokiol augments antitumor effects in vitro and in vivo, and that the improved antitumor activity in vivo may be associated with enhanced induction of apoptosis and augmented inhibition of angiogenesis. The present study may provide a novel and effective method for the treatment of cancer. (Cancer Sci 2009; 100: 1757–1766)
Spike‐monitoring of anaesthesia for corpus callosotomy using bilateral bispectral index
Authors: Ogawa S. , Okutani R. , Nakada K. , Suehiro K. , Shigemoto T.
Published: June 2009

During corpus callosotomy for intractable epilepsy, the electrocorticogram is commonly recorded from electrodes placed on the brain surface to monitor of epileptic activity and assess the synchronisation of epileptic signals between the left and the right hemispheres. We evaluated the usefulness of bilateral bispectral index monitoring using two monitors and two sensors placed above the frontal region. Spikes were readily detected on the electroencephalogram on the bispectral index monitor, and the frequency of their occurrence increased or decreased in response to adjustment of the sevoflurane concentration. The disappearance of synchronisation between the left and the right hemispheres was observed with use of the bispectral index – in concordance with the electrocorticogram. Thus, ‘spike‐monitoring anaesthesia’ using bilateral bispectral index was useful in assessing both the effect of anaesthetics on the electroencephalogram signals and the surgical therapeutic effect.
Impaired in vivo vasculogenic potential of endothelial progenitor cells in comparison to human umbilical vein endothelial cells in a spheroid‐based implantation model
Authors: Finkenzeller G. , Graner S. , Kirkpatrick C. J. , Fuchs S. , Stark G. B.
Published: May 2009

Objectives:  Neovascularization represents a major challenge in tissue engineering applications since implantation of voluminous grafts without sufficient vascularity results in hypoxic cell death of implanted cells. An attractive therapeutic approach to overcome this is based on co‐implantation of endothelial cells to create vascular networks. We have investigated the potential of human endothelial progenitor cells (EPC) to form functional blood vessels in vivo in direct comparison to vascular‐derived endothelial cells, represented by human umbilical vein endothelial cells (HUVEC).

Materials and methods:  EPCs were isolated from human peripheral blood, expanded in vitro and analysed in vitro for phenotypical and functional parameters. In vivo vasculogenic potential of EPCs and HUVECs was evaluated in a xenograft model where spheroidal endothelial aggregates were implanted subcutaneously into immunodeficient mice.

Results:  EPCs were indistinguishable from HUVECs in terms of expression of classical endothelial markers CD31, von Willebrand factor, VE‐cadherin and vascular endothelial growth factor‐R2, and in their ability to endocytose acetylated low‐density lipoprotein. Moreover, EPCs and HUVECs displayed almost identical angiogenic potential in vitro, as assessed by in vitro Matrigel sprouting assay. However in vivo, a striking and unexpected difference between EPCs and HUVECs was detected. Whereas implanted HUVEC spheroids gave rise to formation of a stable network of perfused microvessels, implanted EPC spheroids showed significantly impaired ability to form vascular structures under identical experimental conditions.

Conclusion:  Our results indicate that vascular‐derived endothelial cells, such as HUVECs are superior to EPCs in terms of promoting in vivo vascularization of engineered tissues.
Quality of life in young people with cystic fibrosis: effects of hospitalization, age and gender, and differences in parent/child perceptions
Authors: Hegarty M. , MacDonald J. , Watter P. , Wilson C.
Published: June 2009

Background  The Cystic Fibrosis Questionnaire‐Revised version (CFQ‐R) was used to evaluate age/gender effects on quality of life (QOL) in Australian young people with cystic fibrosis (CF) who were inpatients/outpatients aged 6–18 years. Parent/child agreement was also examined.

Method  The CFQ‐R was completed by 18 outpatients, and 15 inpatients at admission for an acute pulmonary exacerbation to a tertiary hospital, Brisbane, Australia, as well as by parents of those aged 6–13 years.

Results  Inpatients scored significantly lower than outpatients for the CFQ‐R domains ‘emotional state’, ‘social’, ‘body image’ and ‘respiratory symptoms’. Young people aged 6–13 years scored significantly better than those aged 14–18 years for ‘emotional state’, ‘body image’ and ‘treatment burden’. Women perceived less ‘treatment burden’ than did men. Young people aged 6–13 years perceived less ‘treatment burden’ than did their parents. A significant interaction occurred between child/parent report and gender for ‘emotional state’ and ‘eating disturbances’.

Conclusion  The CFQ‐R found differences between inpatients and outpatients and between younger and older paediatric patients with CF, and between parent and child perceptions of QOL.
Skeletal linear extension rates of the foliose scleractinian coral Merulina ampliata (Ellis & Solander, 1786) in a turbid environment
Authors: Dikou Angela
Published: May 2009

Skeletal linear extension rates of a foliaceous, IndoPacific, skiophilous, heterotrophic, scleractinian Merulina ampliata (Ellis & Solander 1786) were obtained along a sediment/nutrient load gradient at the southern islands of Singapore. Measurements were made during November 1999– November 2000 using the alizarin red‐S staining technique. Suspended particulate matter concentration (r2adj = 0.76), turbidity (r2adj = 0.59), the organic content of suspended sediments (r2 = 0.50), and nitrite‐nitrate concentration (r2adj = 0.50) were significant predictors of the skeletal linear extension rate of M. ampliata. Maximum linear extension growth rates of M. ampliata (mean ± SD: 1.43 ± 0.67–3.26 ± 0.59 cm·year−1) were comparable to 15‐year‐old accounts at the same research sites, indicating adaptation to low‐light, high‐sediment waters.
Changes in abscisic acid, salicylic acid and phenylpropanoid concentrations during cold acclimation of androgenic forms of Festulolium (Festuca pratensis × Lolium multiflorum) in relation to resistance to pink snow mould (Microdochium nivale)
Authors: Pociecha E. , Płażek A. , Janowiak F. , Waligórski P. , Zwierzykowski Z.
Published: June 2009

We investigated changes in concentrations of abscisic (ABA) and salicylic acid (SA), phenolic compounds and phenylalanine ammonia‐lyase (PAL) activity in relation to cold‐induced tolerance of four androgenic genotypes of Festulolium (Festuca × Lolium hybrids) to frost and to the snow mould fungus Microdochium nivale. Cold acclimation increased frost tolerance and resistance to snow mould. Resistant genotypes were characterized by higher ABA concentrations during the first 54 h of cold acclimation and lower concentrations of SA than susceptible genotypes. After cold acclimation, the content of phenolics was significantly lower in genotypes tolerant to frost and M. nivale infection than in susceptible genotypes, while PAL activity was significantly higher. Signalling networks controlling cold acclimation to frost (abiotic) and mould infection (biotic) appears to involve increases in foliar concentrations of ABA and decreases in the SA level during successful cold acclimation. Higher PAL activity and lower concentrations of phenolic compounds also appear to be associated with enhanced tolerance to frost and fungal attack.
Decreased mass specific respiration under experimental warming is robust to the microbial biomass method employed
Authors: Bradford Mark A. , Wallenstein Matthew D. , Allison Steven D. , Treseder Kathleen K. , Frey Serita D. , Watts Brian W. , Davies Christian A. , Maddox Thomas R. , Melillo Jerry M. , Mohan Jacqueline E. , Reynolds James F.
Published: June 2009

Hartley et al. question whether reduction in Rmass, under experimental warming, arises because of the biomass method. We show the method they treat as independent yields the same result. We describe why the substrate‐depletion hypothesis may not solely explain observed responses, and urge caution in interpretation of the seasonal data.
Long‐term exendin‐4 treatment delays natural deterioration of glycaemic control in diabetic Goto–Kakizaki rats
Authors: Simonsen L. , Pilgaard S. , Orskov C. , Hartmann B. , Holst J. J. , Deacon C. F.
Published: June 2009

Aim: The glucagon‐like peptide‐1 (GLP‐1) receptor agonist, exendin‐4, has previously been shown to delay the onset of diabetes when administered to Goto–Kakizaki (GK) rats in the prediabetic period. The present study aimed to evaluate whether long‐term administration of exendin‐4 to GK rats in the diabetic period would improve their diabetes and how glycaemic control was affected following drug wash‐out.

Methods: Glycaemic control was assessed in diabetic GK rats during 12 weeks of exendin‐4 or vehicle treatment. Moreover, some animals were followed for an additional 9 weeks without treatment.

Results: Glycaemic control was seen to deteriorate in vehicle‐treated animals, as assessed by increased glycated haemoglobin A1c (HbA1c), whereas HbA1c improved in exendin‐4‐treated animals. Following an additional 9 weeks without treatment, glycaemic control in exendin‐4‐treated animals remained below baseline value and thus remained significantly lower than that of vehicle‐treated animals. Following exendin‐4 administration, oral glucose tolerance tests revealed greatly reduced glucose and insulin excursions compared with vehicle‐treated animals, whereas following overnight drug wash‐out, only little difference was seen, suggesting that the improvement in glycaemic control may have been obtained primarily by increased postprandial control. No significant differences were observed in pancreatic islet morphology or islet hormone content.

Conclusions: Exendin‐4 treatment improved glycaemic control in diabetic GK rats, independent of changes in β‐cell mass. Additionally, progression of the disease seemed to be delayed because the improvement in HbA1c was still apparent 9 weeks after cessation of treatment.
Material Space Texturing
Authors: Ray N. , Lévy B. , Wang H. , Turk G. , Vallet B.
Published: June 2009

Many objects have patterns that vary in appearance at different surface locations. We say that these are differences in materials, and we present a material‐space approach for interactively designing such textures. At the heart of our approach is a new method to pre‐calculate and use a 3D texture tile that is periodic in the spatial dimensions (s, t) and that also has a material axis along which the materials change smoothly. Given two textures and their feature masks, our algorithm produces such a tile in two steps. The first step resolves the features morphing by a level set advection approach, improved to ensure convergence. The second step performs the texture synthesis at each slice in material‐space, constrained by the morphed feature masks. With such tiles, our system lets a user interactively place and edit textures on a surface, and in particular, allows the user to specify which material appears at given positions on the object. Additional operations include changing the scale and orientation of the texture. We support these operations by using a global surface parameterization that is closely related to quad re‐meshing. Re‐parameterization is performed on‐the‐fly whenever the user's constraints are modified.
Commodified language in Chinatown: A contextualized approach to linguistic landscape1
Authors: Leeman Jennifer , Modan Gabriella
Published: June 2009

In Washington DC's newly gentrified Chinatown, recent commercial establishments, primarily non‐Chinese owned chains, use Chinese‐language signs as design features targeted towards people who neither read nor have ethnic ties to Chinese. Using this neighborhood as a case study, we advocate a contextualized, historicized and spatialized perspective on linguistic landscape which highlights that landscapes are not simply physical spaces but are instead ideologically charged constructions. Drawing from cultural geography and urban studies, we analyze how written language interacts with other features of the built environment to construct commodified urban places. Taking a contextually informed, qualitative approach, we link micro‐level analysis of individual Chinese‐language signs to the specific local socio‐geographic processes of spatial commodification. Such a qualitative approach to linguistic landscape, which emphasizes the importance of sociohistorical context, and which includes analysis of signage use, function, and history, leads to a greater understanding of the larger sociopolitical meanings of linguistic landscapes.
Relationship between alpha synuclein phosphorylation, proteasomal inhibition and cell death: relevance to Parkinson’s disease pathogenesis
Authors: Chau Kai‐Yin , Ching Hey Long , Schapira Anthony H. V. , Cooper J. Mark
Published: May 2009

Alpha synuclein can be phosphorylated at serine129 (P‐S129), and the presence of highly phosphorylated α‐synuclein in Lewy bodies suggests changes to its phosphorylation status has an important pathological role. We demonstrate that the kinase(s) responsible for α‐synuclein S129 phosphorylation is constitutively active in SH‐SY5Y cells and involves casein kinase 2 activity. Increased oxidative stress or proteasomal inhibition caused significant elevation of P‐S129 α‐synuclein levels. Under these conditions, similar increases in P‐S129 α‐synuclein were found in both sodium dodecyl sulphate lysates and Triton extracts indicating the phosphorylated protein was soluble and did not lead to aggregation. The rate of S129 phosphorylation was increased in response to proteasomal inhibition indicating a higher activity of the relevant kinase. Cells expressing the phosphorylation mimic, S129D α‐synuclein increased cell death and enhanced sensitivity to epoxomycin exposure. Proteasomal inhibition markedly decreased S129D α‐synuclein turnover suggesting proteasomal inhibition leads to the accumulation of P‐S129 α‐synuclein through an increase in the kinase activity and a decrease in protein turnover resulting in increased cell death. We conclude that S129 phosphorylation is toxic to dopaminergic cells and both the levels of S129 phosphorylated protein and its toxicity are increased with proteasomal inhibition emphasising the interdependence of these pathways in Parkinson’s disease pathogenesis.
Functional characterization of muscarinic autoreceptors in rat and human neocortex
Authors: Stoll Christoph , Eltze Manfrid , Lambrecht Günter , Zentner Josef , Feuerstein Thomas J. , Jackisch Rolf
Published: May 2009

Electrically evoked overflow of [3H]acetylcholine in slices of rat neocortex and of human neocortex (freshly obtained during neurosurgical treatment of epilepsy or deep‐seated tumors) was used tofunctionallycharacterize the muscarinic receptor subtype, which mediates autoinhibition of acetylcholine release in these tissues. In the rat neocortex, the following pKBvalues [CI95] were calculated from the shifts to the right of the concentration–response curves of the full agonist oxotremorine in presence of subtype preferring muscarinic receptor antagonists: tripitramine: 9.1 [8.8, 9.4], tripinamide: 8.6 [8.5, 8.7], AQ‐RA 741 (11‐[[4‐[4‐(diethylamino)butyl]‐1‐piperidinyl]acetyl]‐5,11‐dihydro‐6H‐pyrido(2,3‐b)[1,4]benzodiazepine‐6‐one): 8.2 [8.0, 8.4], himbacine: 8.0 [7.9, 8.1], 4‐diphenylacetoxy‐N‐methylpiperidine methobromide: 8.0 [7.8, 8.1], methoctramine: 7.5 [7.4, 7.6], AF‐DX 116 (11[[2‐[(diethyl‐amino)methyl]‐1‐piperidinyl] acetyl] 5,11‐dihydro‐6H‐pyrido(2,3‐b)[1,4]benzodiazepine‐6‐one): 7.1 [7.0, 7.3], hexahydro‐sila‐difenidol: 6.8 [6.7, 6.9], pirenzepine: 6.6 [6.4, 6.7], and 3,6a,11,14‐tetrahydro‐9‐methoxy‐2‐methyl‐12H‐isoquino[1,2‐b]pyrrolo[3,2‐f] [1,3]benzoxazine‐1‐carboxylic acid ethyl ester (PD 102807): 6.0 [5.8, 6.2]. In the human neocortex the following values were found: tripitramine: 9.4 [9.3, 9.6], tripinamide: 9.0 [8.9, 9.2], AF‐DX 116: 6.7 [6.4, 6.9], hexahydro‐sila‐difenidol: 6.6 [6.2, 6.9], and PD 102807: 6.5 [6.3, 6.6]. In correlation plots, these pKBvalues correspond best to published binding data on native or recombinant M2receptors but not to those on M1, M3, M4, and M5receptors, suggesting that muscarinic autoreceptors of both the rat and human neocortex belong to the M2subtype. This observation lends further support to the development of M2receptor selective brain penetrating antagonists for application in Alzheimer’s disease.