bluuebunny/crossref_metadata_2025 huggingface dataset:
Presidential Address
Authors: Duff Mountstuart E. Grant
Published: June 2009

Some fifteen years ago, before Mr. Lowe had developed into Viscount Sherbrooke, we were sitting next each other, one afternoon, on the front Opposition Bench. The House was in the possession of an orator of the kind to whom it pays ‘the genuine tribute of undissembled horror’ by reducing itself to a handful of members, but we, for some reason or other, could not escape, and fell accordingly to talking in self-defence. The conversation found its way to Thucydides, and I asked my companion whether it had ever struck him to write an article on the uses of that author to the modern statesman. He said, ‘No,’ but the idea pleased him; he dallied with it for some time, and had even, I believe, some correspondence on the subject with the editor of one of the monthly reviews. Nothing, however, came of his good intentions, and I am not aware that anyone else has done what he did not do. It seems to me, accordingly, that it might not be wholly un-interesting to this Society if I were to try to answer the question whether the study of Thucydides is useful at all to the modern statesman, and, if so, to what extent?
Snap! Recognising Implicit Actions in Static Point-Light Displays
Authors: Reid Russell , Brooks Anna , Blair Duncan , van der Zwan Rick
Published: June 2009

Johansson (1973 Perception & Psychophysics14 201–211) suggested that point-light displays that are static—so-called ‘snapshots’—contain little or no information about the actor or their action. Here we present data that suggest even naive observers can perceive such information from static point-light arrays. Observers were able, at rates better than chance, to discriminate the directions of facing of sagittally viewed static point-light walkers. The data show also that, without feedback, performances improved with experience. Our data have implications for assumptions made in designing experiments with point-light displays and for models of the neural mechanisms mediating biological motion perceptions.
Reintegrating young offenders into the community through discharge planning: a review of interventions and needs of youth in secure care
Authors: Wilson Anne , Tully Phillip
Published: June 2009

The health status of young offenders is often poorer than similar aged non-offenders. Addressing young offender needs is a key priority for their rehabilitation and reintegration into the community. The objectives were to identify programs and interventions that assist detained youth through focussed discharge planning by a systematic review of the literature. In addition, this review examined the identified health needs of young offenders in secure care. Our systematic review searched electronic databases using the keyword terms youth, secure care, health assessment and primary health care. Keyword browser terms were also entered as search strings, and only literature from 1997 onwards was retained. Twenty-two pieces of published literature were retained, and these documented the needs and service utilisation of young offenders. Mental health problems and trauma exposure were commonly identified in the literature, as was general physical health deficits and social and familial problems. Retrieved articles also made recommendations for health assessments and discharge planning, and some provided discharge planning models. Young offenders have diverse needs that can be ascertained from quality assessment measures. Effective discharge planning is important to attend to ongoing health issues and aid rehabilitation and reintegration into the community. By administering a comprehensive multidimensional screening measure upon admission to secure care, individualised care plans can be formulated with discharge planning measures. Recommendations for further study include the development and implementation of a needs assessment tool in youth secure-care facilities that complements the existing assessments and helps reintegrate young offenders to primary health care and community services.
Do older people perceive cancer prevention and early detection to be worthwhile? Implications for prevention
Authors: Ngune Irene , Howat Peter , Maycock Bruce , Slevin Terry
Published: June 2009

Despite evidence that suggests modifying risk factors at any age can reduce the risk of cancer, many older people fail to take relevant preventive actions. The objectives of the project were to assess whether older people perceived taking actions to prevent cancers as worthwhile after the age of 60 years. Fifty men (19) and women (31) 60 years and older (mean age 68 years) participated in seven focus groups to assess their perceptions of preventive health actions and barriers and motivators in relation to cancer prevention. Most participants reported not adhering to preventive actions for cancer. Many did not perceive the benefits if the actions are initiated after the age of 60 years, and some risk factors were not perceived to be related to cancer. There was strong agreement that screening for cancer was worthwhile after the age of 60 years. It appears that awareness programs specifically tailored to seniors may be desirable to promote cancer prevention within a context of chronic disease prevention in general.
Impaired cAMP and Rac 1 Signaling Contribute to TNF‐α‐induced Endothelial Barrier Breakdown in Microvascular Endothelium
Authors: SCHLEGEL NICOLAS , WASCHKE JENS
Published: June 2009

Objective: In sepsis, tumor necrosis factor‐alpha (TNF‐α) contributes to endothelial barrier breakdown. The involvement of Rho A/rho kinase signaling has recently been challenged. Here, we tested the role of cAMP and Rac 1 signaling. Materials and Methods: For this study, we took in vivo measurements of hydraulic conductivity in postcapillary mesenteric venules of adult rats. Measurements of transendothelial electrical resistence (TER), fluorescein isothiocyanate–dextran flux, Western blotting, immunostaining, and enzyme‐linked immunosorbent assay–based measurements of cAMP levels and Rho‐GTPase activity in human microvascular endothelial cells. Results: TNF‐α disrupted endothelial barrier functions in vivo and in vitro. Under these conditions, Rho A activity was significantly increased, whereas Rac 1 activity was decreased and Cdc42 was unaltered. Moreover, cAMP levels were reduced. Rho kinase inhibition, using Y27632, did not prevent TNF‐α‐induced barrier breakdown. In contrast, preincubation with forskolin and rolipram (F/R) to increase cAMP and cytotoxic necrotizing factor 1 to activate Rac 1 and Rho A abolished TNF‐α‐induced barrier breakdown in vivo and in vitro. Moreover, inactivation of Rac 1 was blocked by F/R‐mediated increase of cAMP, whereas Rho A activation was only partially inhibited. Conclusion: Our data indicate that decrease of cAMP and Rac 1 inactivation, rather than Rho A activation, contribute to TNF‐α‐induced endothelial barrier breakdown in vivo and in vitro.
SOAP2: an improved ultrafast tool for short read alignment
Authors: Li Ruiqiang , Yu Chang , Li Yingrui , Lam Tak-Wah , Yiu Siu-Ming , Kristiansen Karsten , Wang Jun
Published: June 2009

Summary: SOAP2 is a significantly improved version of the short oligonucleotide alignment program that both reduces computer memory usage and increases alignment speed at an unprecedented rate. We used a Burrows Wheeler Transformation (BWT) compression index to substitute the seed strategy for indexing the reference sequence in the main memory. We tested it on the whole human genome and found that this new algorithm reduced memory usage from 14.7 to 5.4 GB and improved alignment speed by 20–30 times. SOAP2 is compatible with both single- and paired-end reads. Additionally, this tool now supports multiple text and compressed file formats. A consensus builder has also been developed for consensus assembly and SNP detection from alignment of short reads on a reference genome.

Availability:  http://soap.genomics.org.cn

Contact:  soap@genomics.org.cn
Inhibition of membrane Ca2+-ATPase of Saccharomyces cerevisiae by mating pheromone α-factor in vitro
Authors: Hiraga Kazumi , Tahara Hidetaka , Taguchi Nobuhisa , Tsuchiya Eiko , Fukui Sakuzo , Miyakawa Tokichi
Published: June 2009

Plasma membrane Ca2+-ATPase of Saccharomyces cerevisiae was solubilized and partially purified by calmodulin-affinity chromatography. The activity of Ca2+-ATPase isolated from MATa cells was inhibited by physiological concentrations of the mating pheromone α-factor in a dose-dependent manner. The enzyme prepared from a receptor-deficient sterile mutant cells (Δste2) was similarly inhibited by α-factor, but the enzyme from MATα cells was resistant to the mating pheromone. We suggest that the inhibition may be involved in the α-factor-induced increase of Ca2+ uptake reaction of MATa cells.
Multilevel control of extracellular sucrose metabolism in Streptococcus salivarius by sucrose
Authors: Townsend-Lawman P. , Bleiweis A. S.
Published: June 2009

Standardized experimental conditions were established to test the role of sucrose in the regulation and control of its metabolism in Streptococcus salivarius. A fresh isolate of S. salivarius was used. The extracellular dextranase activity of cells grown on sucrose was 10-fold higher than that of cells grown on glucose, fructose or galactose. This activity increased in less than 5 min following the addition of sucrose to galactose-grown cells, a phenomenon which was affected by neither rifampicin nor chloramphenicol which inhibit transcription and translation, respectively. Extracellular fructanase activity was 2-fold higher when cells were grown on sucrose than when they were grown on the other sugars. This increase also occurred within 5 min, but was diminished by transcriptional and translational inhibitors. De novo synthesis was required for the production of extracellular glucosyltransferase (GTF) activity which, upon the addition of sucrose, became associated with the cell surface. Conversely, cell-associated fructosyltransferase (FTF) activity appeared to require genetic induction for its production and cell-surface association, but required sucrose for its release from the surface framework. Versatility in the control mechanisms of this complex set of enzymes allows their expression and function to be regulated at several widely separated stages in the life histories of these proteins.
The development of specific rRNA-derived oligonucleotide probes for Haemophilus ducreyi, the causative agent of chancroid
Authors: Rossau Rudi , Duhamel Maddy , Jannes Geert , Decourt Jean Luc , Van Heuverswyn Hugo
Published: June 2009

Part of a ribosomal ribonucleic acid (rRNA) cistron of Haemophilus ducreyi was enzymically amplified using conserved primers within the rRNA molecules, cloned in a plasmid vector, and sequenced. From the nucleotide sequence, eight oligonucleotides complementary to different regions in the 16S and 23S rRNA molecules were selected, chemically synthesized, and used as hybridization probes. Hybridization experiments with at least 41 H. ducreyi strains and 13 or 14 non-H. ducreyi strains revealed that all eight oligonucleotide probes were highly reliable and completely specific for H. ducreyi strains. Comparisons of 16S rRNA sequences confirm that H. ducreyi is a member of the Pasteurellaceae though not closely related to other species in this family.
Characterization of the temperate actinophage ϕA7 DNA and its deletion derivatives
Authors: Diaz Luis A. , Hardisson Carlos , Rodicio M. Rosario
Published: June 2009

A restriction map of ø A7 DNA (46·7 kb) was established for nine endonucleases (Bc/I,ClaI,EcoRI,EcoRV,HpaI,PvuI,SacII,SphI andXbaI) which cut the phage genome up to 11 times. There were no sites forBamHI,Bg/II,HindIII,PstI,PvuII,SacI orSa/I. ø A7 DNA, circularized through its cohesive ends, could integrate into the genome of severalStreptomyceshosts, to form stable lysogens. Integration occurred by recombination between unique attachment sites on the phage (attP) and the host (attB) genomes. TheattPsite has been located on the ø A7 restriction map. Deletion mutants of ø A7 DNA were obtained by selecting for pyrophosphate- or EDTA-resistant clones. The deletions occurred either near the left-hand end of the conventional restriction map, or about 18 kb from the right-hand end, close to, but not affecting the uniqueSacII site. Together, the deletions defined at least 7·9 of DNA (16·9% of the phage genome) non-essential for plaque formation. ø A7 DNA was introduced intoS. lividansprotoplasts by liposome-assisted transfection. Since the phage does not adsorb to intact cells of this strain, and therefore does not form plaques, an overlay ofS. antibioticusspores was used to detect the infectious progeny released by the protoplasts. Using this technique, ø A7 could be introduced intoS. antibioticuswith an efficiency of about 6 × 106p.f.u. per μg DNA (equivalent to 3 × 10-4p.f.u. per DNA molecule).
Trehalose-6-phosphate synthase/phosphatase complex from bakers’ yeast: purification of a proteolytically activated form
Authors: Londesborough John , Vuorio Outi
Published: June 2009

A protein of about 800 kDa with trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) activity was purified from bakers’ yeast. This TPS/P complex contained 57, 86 and 93 kDa polypeptides. The 86 and 93 kDa polypeptides both appeared to be derived from a polypeptide of at least 115 kDa in the native enzyme. A TPS-activator (a dimer of 58 kDa subunits) was also purified. It decreased the Michaelis constants for both UDP-glucose (three-fold) and glucose 6-phosphate (G6P) (4·5-fold), and increased TPS activity at 5 mm-UDP-glucose/10 mm-G6P about three-fold. It did not affect TPP activity. The purification of TPS/P included an endogenous proteolytic step that increased TPS activity about three-fold and abolished its requirement for TPS-activator, but did not change TPP activity. This activation was accompanied by a decrease of some 20 kDa in the molecular mass of a cluster of SDS-PAGE bands at about 115 kDa recognized by antiserum to pure TPS/P, but by no change in the 57 kDa band. Phosphate inhibited TPS activity (K
i about 5 mm), but increased TPP activity about six-fold (K
a about 4 mm). Phosphate (6 mm) stimulated the synthesis of trehalose from G6P and UDP-glucose and decreased the accumulation of trehalose 6-phosphate.
The expression of the Acinetobacter calcoaceticus recA gene increases in response to DNA damage independently of RecA and of development of competence for natural transformation
Authors: Rauch Peter J. G. , Palmen Ronald , Burds Aurora A. , Gregg-Jolly Leslie A. , van der Zee J. Rob , Hellingwerf Klaas J.
Published: June 2009

Using the lacZ operon fusion technique, the transcriptional control of the Acinetobacter calcoaceticus recA gene was studied. A low (approximately twofold) inductive capacity was observed for compounds that damage DNA and/or inhibit DNA replication, e.g. methyl methanesulfonate, mitomycin C, UV light and nalidixic acid. Induction of the recA gene by DNA damage was independent of functional RecA. The presence of the recA promoter region on a multicopy plasmid had the same effect on recA transcription as the presence of DNA-damaging agents. Thus, recA expression in A. calcoaceticus appears to be regulated in a novel fashion, possibly involving a non-LexA-like repressor. Regulation of the recA gene in A. calcoaceticus appears not to be part of a regulon responsible for competence for natural transformation: in cells exhibiting extremely low transformation frequencies, the level of transcription of the recA gene was found to be comparable to the level found in cells in the state of maximal competence.
Characteristics of Sulfobacillus acidophilus sp. nov. and other moderately thermophilic mineral-sulphide-oxidizing bacteria
Authors: Norris Paul R. , Clark Darren A. , Owen Jonathan P. , Waterhouse Sara
Published: June 2009

Several isolates of Gram-positive, acidophilic, moderately thermophilic, ferrous-iron- and mineral-sulphide-oxidizing bacteria were examined to establish unequivocally the characteristics of Sulfobacillus-like bacteria. Two species were evident: Sulfobacillus thermosulfidooxidans with 48-50 mol% G + C and Sulfobacillus acidophilus sp. nov. with 55-57 mol% G + C. Both species grew autotrophically and mixotrophically on ferrous iron, on elemental sulphur in the presence of yeast extract, and heterotrophically on yeast extract. Autotrophic growth on sulphur was consistently obtained only with S. acidophilus.
Characterization of a prolinase gene and its product and an adjacent ABC transporter gene from Lactobacillus helveticus
Authors: Varmanen Pekka , Steele James , Palva Airi
Published: June 2009

A prolinase (pepR) gene was cloned from an industrial
Lactobacillus helveticus
strain (53/7). Three clones, hybridizing with a gene probe specific for a peptidase shown to have activity against di- and tripeptides, were detected from a L. helveticus genomic library constructed in
Escherichia coli
. None of the three clones, however, showed enzyme activity against the di- or tripeptide substrates tested. One of the clones, carrying a vector with a 5.5 kb insert, was further characterized by DNA sequencing. The sequence analysis revealed the presence of two ORFs, ORF1 and ORF2 of 912 and 1602 bp, respectively, ORF2, located upstream of and in the opposite orientation to ORF1, had a promoter region overlapping that of ORF1. ORF1 had the capacity to encode a 35083 Da protein. When amplified by PCR, ORF1 with its control regions specified a 35 kDa protein in E. coli that was able to hydrolyse dipeptides, with highest activity against Pro-Leu, whereas from the tripeptides tested, only Leu-Leu-Leu was slowly degraded. By the substrate-specificity profile and protein homologies, the 35 kDa protein was identified as a prolinase. The activity of the cloned prolinase was inhibited by p-hydroxymercuribenzoate. Northern and primer-extension analyses of ORF1 revealed a 1.25 kb transcript and two adjacent transcription start sites, respectively, thus confirming the DNA sequence data. ORF2 had encoding capacity for a 59.5 kDa protein that showed significant homology to several members of the family of ABC transporters. Determination of the mRNA levels at different growth phases revealed that the pep gene and ORF2 are transcribed in L. helveticus at the exponential and stationary phases of growth, respectively. Furthermore, two ORF2 deletion constructs, carrying the intact pepR gene, showed that this upstream operon adversely affected PepR activity in E. coli, which explains the enzymic inactivity of the original clones.
Polysaccharide lyases from gellan-producing Sphingomonas spp.
Authors: Kennedy Lynn , Sutherland Ian W.
Published: June 2009

A number ofSphingomonasstrains capable of synthesizing the bacterial exopolysaccharide gellan and related polymers were shown to possess constitutive gellanase activity. In each case, the degradation of deacylated gellan was due to extracellular, eliminase-type enzymes (lyases) which cleave the sequence …ß-D-glucosyl 1,4-ß-D-glucuronosyl… in the tetrasaccharide repeat unit of the substrate polysaccharides. Deacetylated rhamsan was an alternative substrate but there was little or no action against most other polysaccharides with similar structures. Slight differences were found between the specificities of the lyases from different strains. Activities of gellan lyase preparations were generally low. As well as the extracellular ‘gellanase’ activity, all the bacteria possessed varying amounts of ß-D-glucosidase and ß-D-glucuronidase activities apparently located in the periplasm. The products from deacylated gellan and the chemically deacylated form of polysaccharide S194 (rhamsan gum), which is effectively a gentiobiosylated form of gellan, closely resembled those recently obtained by the authors from other, gellandegrading, non-gellan-producing bacteria. The enzymes had negligible activity against the natural, acylated gellan and rhamsan polysaccharides from bacteria now designated as strains ofSphingomonas.
A 28 kDa major immunogen of Chlamydia psittaci shares identity with Mip proteins of Legionella spp. and Chlamydia trachomatis - cloning and characterization of the C. psittaci mip-like gene
Authors: Rockey Daniel D. , Chesebro Brian B. , Heinzen Robert A. , Hackstadt Ted
Published: June 2009

Chlamydia psittaci strain guinea-pig inclusion conjunctivitis (GPIC) produces a self-limiting ocular infection of guinea-pigs, and this condition is a representative animal model of ocular chlamydial disease. Convalescent guinea-pigs, which are resistant to reinfection, produce antibodies to several elementary-body proteins, including an uncharacterized antigen of 28 kDa. Convalescent guinea-pig sera were used to identify, from a lambda expression library, two overlapping GPIC genomic clones that produced the 28 kDa antigenic protein. Nucleotide sequence analysis revealed that the gene coding for the 28 kDa protein was similar to the mip (macrophage infectivity potentiator) genes from Legionella pneumophila and Chlamydia trachomatis. The GPIC gene and its product were accordingly designated mip and Mip, respectively. Analysis of the regions flanking mip identified three tightly linked open reading frames coding for predicted products with sequence similarity to asparagine tRNA ligase (AspS), rRNA methylase (SpoU), and thioredoxin (TrxA). The arrangement of these genes in GPIC was aspS-mip-spoU-trxA. Sequence analysis of PCR products produced using genomic DNA from an ovine abortion strain of C. psittaci and from C. trachomatis strain LGV-434 demonstrated that the arrangement of mip, spoU and trxA is common among these chlamydiae.
A Mechanism for Production of Hydroxyl Radicals by the Brown-Rot Fungus Coniophora Puteana: Fe(III) Reduction by Cellobiose Dehydrogenase and Fe(II) Oxidation at a Distance from the Hyphae
Authors: Hyde Simon M. , Wood Paul M.
Published: June 2009

In timber infested by brown-rot fungi, a rapid loss of strength is attributed to production of hydroxyl radicals (HO.). The hydroxyl radicals are produced by the Fenton reaction [Fe(II)/H2O2], but the pathways leading to Fe(II) and H2O2have remained unclear. Cellobiose dehydrogenase, purified from cultures ofConiophora puteana, has been shown to couple oxidation of cellodextrins to conversion of Fe(III) to Fe(II). Two characteristics of brown rot are release of oxalic acid and lowering of the local pH, often to about pH 2. Modelling of Fe(II) speciation in the presence of oxalate has revealed that Fe(II)-oxalate complexes are important at pH 4-5, but at pH 2 almost all Fe(II) is in an uncomplexed state which reacts very slowly with dioxygen. Diffusion of Fe(II) away from the hyphae will promote conversion to Fe(II)-oxalate and autoxidation with H2O2as product. Thus the critical Fe(II)/H2O2combination will be generated at a distance, enabling hydroxyl radicals to be formed without damage to the hyphae.
Endotoxic Properties of Free Lipid a from Porphyromonas Gingivalis
Authors: Tanamoto Ken-ichi , Azumi Satoko , Haishima Yuji , Kumada Hidefumi , Umemoto Toshio
Published: June 2009

The relationship between chemical structure and biological activity of the lipid A fromPorphyromonas gingivalis, which we recently isolated and whose complete chemical structure was determined [Kumadaet al.(1995).J Bacteriol177, 2098--2106], was studied. The lipid A exhibited endotoxic activity in all the assay systems tested:Limulusgelation activity, lethal toxicity in galactosamine-sensitized mice, mitogenicity in mouse spleen cells and induction of nitric oxide (NO) and tumour necrosis factor alpha (TNF) release from both mouse peritoneal macrophages and the J774-1 mouse macrophage-like cell line. The activity was, however, about 100-fold less than that ofSalmonella minnesotaLPS used as a control. The moderate activity of the lipid A may be partially explained by its unique fatty acid composition and the lack of a phosphate group in position 4. In contrast, the lipid A as well as whole LPS ofP. gingivalisunexpectedly exhibited an even stronger induction of TNF from the human monocytic THP-1 cell line than control LPS when measured by the minimum stimulatory dose. The difference in sensitivity of human and mouse cells toP. gingivalislipid A suggests that the recognition mechanism, including that for the receptor for endotoxin, may be regulated in different ways in the two cells.
Plasmid-Encoded Genes Specifying Aniline Oxidation from Acinetobacter sp. Strain YAA
Authors: Fujii Toshiki , Takeo Masahiro , Maeda Yoshimichi
Published: June 2009

Acinetobacter sp. strain YAA is able to use aniline and o-toluidine as the sole carbon and energy source. This strain has several different plasmids and acridine orange curing suggested that aniline utilization in strain YAA was plasmid-encoded. The gene cluster involved in aniline oxidation was cloned in Escherichia coli JM109 from the total plasmid DNA of strain YAA. A recombinant E. coli containing an 18.5 kb insert fragment showed yellow colouration on aniline-containing plates, indicating the formation of 2-hydroxymuconic semialdehyde from aniline. In addition, subcloning of a 9.0 kb Sall fragment from the insert in E. coli resulted in the accumulation of catechol. Southern hybridization studies indicated that the aniline oxygenase gene (atdA) was present on one of the plasmids, pYA1. These results suggest that in strain YAA aniline is degraded via catechol through a pathway involving meta-cleavage of the benzene-ring by plasmid-encoded genes including atdA.
Low-resolution sequencing of Rhodobacter sphaeroides 2.A.1T: chromosome II is a true chromosome
Authors: Choudhary M. , Mackenzie C. , Nereng K. , Sodergren Erica , Weinstock G. M. , Kaplan S.
Published: June 2009

The photosynthetic bacterium Rhodobacter sphaeroides 2.4.1T has two chromosomes, CI (~3.0 Mb) and CII (~0.9 Mb). In this study a low-redundancy sequencing strategy was adopted to analyse 23 out of 47 cosmids from an ordered CII library. The sum of the lengths of these 23 cosmid inserts was ~495 kb, which comprised ~417 kb of unique DNA. A total of 1145 sequencing runs was carried out, with each run generating 559±268 bases of sequence to give ~640 kb of total sequence. After editing, ~2.8% bases per run were estimated to be ambiguous. After the removal of vector and Escherichia coli sequences, the remaining ~ 565 kb of R. sphaeroides sequences were assembled, generating ~291 kb of unique sequences. BLASTX analysis of these unique sequences suggested that ~131 kb (45% of the unique sequence) had matches to either known genes, or database ORFs of hypothetical or unknown function (dORFs). A total of 144 strong matches to the database was found; 101 of these matches represented genes encoding a wide variety of functions, e.g. amino acid biosynthesis, photosynthesis, nutrient transport, and various regulatory functions. Two rRNA operons (rrnB and rrnC) and five tRNAs were also identified. The remaining 160 kb of DNA sequence which did not yield database matches was then analysed using CODONPREFERENCE from the GCG package. This analysis suggested that 122 kb (42% of the total unique DNA sequence) could encode putative ORFs (ports), with the remaining 38 kb (13%) possibly representing non-coding intergenic DNA. From the data so far obtained, CII does not appear to be specialized for encoding any particular metabolic function, physiological state or growth condition. These data suggest that CII contains genes which are functionally as diverse as those found on any other bacterial chromosome and also contains sequences (pORFs) which may prove to be unique to this organism.
Escherichia coli is unable to produce pyrroloquinoline quinone (PQQ)
Authors: Matsushita K. , Arents J. C. , Bader R. , Yamada M. , Adachi O. , Postma P. W.
Published: June 2009

Many bacteria can synthesize the cofactor pyrroloquinoline quinone (PQQ), a cofactor of several dehydrogenases, including glucose dehydrogenase (GCD). Among the enteric bacteria, Klebsiella pneumoniae has been shown to contain the genes required for PQQ biosynthesis. Escherichia coli and Salmonella typhimurium were thought to be unable to synthesize PQQ but it has been reported that strain EF260, a derivative of E. coli FB8, can synthesize PQQ after mutation and can oxidize glucose to gluconate via the GCD/PQQ pathway (F. Biville, E. Turlin & F. Gasser, 1991, J Gen Microbiol 137, 1775-1782). We have reinvestigated this claim and conclude that it is most likely erroneous. (i) Strain EF260, isolated originally by Biville and coworkers, was unable to synthesize a holo-enzyme GCD unless PQQ was supplied to the growth medium. No GCD activity could be detected in membrane fractions. (ii) The amount of PQQ detected in the growth medium of EF260 was very low and not very different from that found in a medium with its parent strain or in a medium containing no cells. (iii) EF260 cells were unable to produce gluconate from glucose via the PQQ/GCD pathway. (iv) Introduction of a gcd::Cm deletion in EF260, eliminating GCD, did not affect glucose metabolism. This suggested a pathway for glucose metabolism other than the PQQ/GCD pathway, (v) Glucose uptake and metabolism in EF260 involved a low-affinity transport system of unknown identity, followed most likely by phosphorylation via glucokinase. It is concluded that E. coli cannot synthesize PQQ and that it lacks genes required for PQQ biosynthesis.
Acetate kinase from Clostridium acetobutylicum: a highly specific enzyme that is actively transcribed during acidogenesis and solventogenesis
Authors: Winzer Klaus , Lorenz Karin , DÜrre Peter
Published: June 2009

Acetate kinase (ATP:phosphotransferase, EC 2.7.2.1) has been purified 294-fold from acid-producing cells of Clostridium acetobutylicum DSM 1731 to a specific activity of 1087 U mg−1 (ADP-forming direction). The dimeric enzyme consisted of subunits with a molecular mass of 43 kDa. The molecular mass of the native acetate kinase was in the range 87-94 kDa as judged by gel filtration and native gel electrophoresis. The enzyme showed high specificity for the substrates acetate and ATP, and maximal activity was obtained with Mn2+ as divalent cation. The presence of mercury compounds such as HgCl2 and p-hydroxymercuribenzoate resulted in an essential loss of activity. The apparent K
m values for acetate, Mg-ATP, acetyl phosphate, and Mg-ADP were 73, 0.37, 0.58 and 0.71 mM. An activity-staining procedure for detection of acetate kinase in crude cell extracts after separation on native polyacrylamide gels was developed. A DNA fragment encoding 246 bp of the acetate kinase gene of C. acetobutylicum DSM 792 was cloned by a PCR-based approach. Northern blot analysis revealed transcription of the gene under acid- and solvent-producing conditions with no significant differences at the level of transcription.
Recombinant SINAP-25 is an effective substrate for Clostridium botulinum type A toxin endopeptidase activity in vitro
Authors: Ekong Theresa A. N , Feavers Ian M. , Sesardic Dorothea
Published: June 2009

Bacterial neurotoxins are now being used routinely for the treatment of neuromuscular conditions. Alternative assays to replace or to complementin vivobioassay methods for assessment of the safety and potency of these botulinum neurotoxin-based therapeutic products are urgently needed. Advances made in understanding the mode of action of clostridial neurotoxins have provided the basis for the development of alternative mechanism-based assay methods. Thus, the identification of SNAP-25 (synaptosomal-associated protein of molecular mass 25 kDa) as the intracellular protein target which is selectively cleaved during poisoning by botulinum neurotoxin type A (BoNT/A) has enabled the development of a functionalin vitroassay for this toxin. Using recombinant DNA methods, a segment of SNAP-25 (aa residues 134-206) spanning the toxin cleavage site was prepared as a fusion protein to the maltose-binding protein inEscherichia coli.The fusion protein was purified by affinity chromatography and the fragment isolated after cleavage with Factor Xa. Targeted antibodies specific for the N and C termini of SNAP-25, as well as the toxin cleavage site, were prepared and used in an immunoassay to demonstrate BoNT/A endopeptidase activity towards recombinant SNAP-25 substrates. The reaction required low concentrations of reducing agents which were inhibitory at higher concentrations as were metal chelators and some inhibitors of metallopeptidases. The endopeptidase assay has proved to be more sensitive than the mouse bioassay for detection of toxin in therapeutic preparations. A good correlation with results obtained in thein vivobioassay (r= 0·95,n= 23) was demonstrated. The endopeptidase assay described here may provide a suitable replacement assay for the estimation of the potency of type A toxin in therapeutic preparations.
Identification of staphylococcal and streptococcal causes of bovine mastitis using 16S-23S rRNA spacer regions
Authors: Forsman Pivi , Tilsaia-Timisjrvi Anu , Alatossava Tapani
Published: June 2009

Bovine mastitis is caused mainly by certain Staphylococcus and Streptococcus species. The sequences of the 16S-23S rRNA spacer regions were determined for the nine species which cause mastitis: Staphylococcus aureus, Staphylococcus chromogenes, Staphylococcus epidermidis. Staphylococcus hyicus, Staphylococcus simulans, Staphylococcus xylosus. Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis. Significant variation was found between the spacer sequences of different species with the lengths of the spacers varying from 240 to 461 bp. Between genera the spacers shared only short conserved regions (8-9 bp) and within genera the sequence identities varied from 53 to 85%. This variation made it possible to construct specific primer pairs for these species and genera. The specificities of these primers were tested with 25 bacterial species and 51 isolates from cattle with clinical mastitis. The DNA-based identification of the mastitis species was mostly successful.
Two transcripts, differing at their 3′ ends, are produced from the Candida albicans SEC14 gene
Authors: Riggle Perry J. , Slobodkin Igor V. , Brown Douglas H , Hanson Mark P. , Volkert Thomas L. , Kumamoto Carol A.
Published: June 2009

A search for Candida albicans mutants defective in filamentous growth led to the isolation of a mutant strain with an insertion mutation in the SEC14 gene. SEC14 encodes the phosphatidylinositol/phosphatidylcholine transfer protein, an essential protein in the yeast Saccharomyces cerevisiae. In the dimorphic yeast Yarrowia lipolytica, SEC14 is needed for growth only in the hyphal form and is not required for growth in the yeast form. However, unlike Y. lipolytica SEC14, C. albicans SEC14 is probably essential for growth. Northern blot analysis and PCR amplification of transcripts produced from the SEC14 gene demonstrated that two transcripts differing at their 3′ ends were produced. The two transcripts may regulate the activity of SEC14 so that Sec14p can perform two functions in C. albicans. One function may be an essential function analogous to the function of Sec14p in S. cerevisiae and the second function may be important during filamentous growth, analogous to the function of Sec14p in Y. lipolytica.
Thermotoga neapolitana bgIB gene, upstream of lamA, encodes a highly thermostable β-glucosidase that is a laminaribiase
Authors: Zverlov Vladimir V. , Volkov Ilia Y. , Velikodvorskaya Tatjana V. , Schwarz Wolfgang H.
Published: June 2009

The gene for thermostable 1,3-β-glucosidase BgIB was cloned from the chromosome of Thermotoga neapolitana and its primary sequence was determined. The purified recombinant β-glucosidase B had a monomer molecular mass of 81 kDa in accordance with the amino acid sequence predicted from the nucleotide sequence of clone pTT51. It was a member of glycosylhydrolase family 3 and belonged to enzyme class EC 3.2.1.21. β-Glucosidase B had a specific activity of 255 U mg-1on 4-nitrophenyl(PNP)-β-glucoside at the optima of pH (5.5) and temperature (90 °C), and K
m values of 0.1, 10 and 50 mM for PNP-β-glucoside, laminaribiose and cellobiose, respectively. The gene bgIB was located immediately upstream of the laminarinase gene IamA. Both genes were transcribed from the same DNA strand and were not separated by a palindromic transcription terminator. The two purified enzymes 1,3-β-glucosidase BgIB (laminaribiase) and 1,3-β-glucanase LamA (laminarinase) were together capable of completely degrading laminarin to glucose.
Characterization of a gene encoding dihydrolipoamide dehydrogenase of the cyanobacterium Synechocystis sp. strain PCC 6803
Authors: Engels Anke , Pistorius Elfriede K.
Published: June 2009

The authors previously reported the isolation and partial characterization of a periplasmically located dihydrolipoamide dehydrogenase (LPD) from the cyanobacteriumSynechocystissp. strain PCC 6803. In the present work the gene (IpdA;database accession number Z48564) encoding the apoprotein of this LPD inSynechocystisPCC 6803 has been identified, sequenced and analysed. TheIpdAgene codes for a protein starting with methionine, which is post-translationally removed. The mature protein contains an N-terminal serine and consists of 473 amino acids with a deduced molecular mass of 51421 Da (including one FAD). The LPD is an acidic protein with a calculated isoelectric point of 5.17. Comparison of the amino acid sequence of theSynechocystisLPD with protein sequences in the databases revealed that the enzyme shares identities of 31-35% with all 18 LPDs so far sequenced and published. As a first step in determining the role of this cyanobacterial LPD, attempts were made to generate an LPD-freeSynechocystismutant by insertionally inactivating theIpdAgene with a kanamycin-resistance cassette. However, the selected transformants appeared to be heteroallelic, containing both the intactIpdAgene and theIpdAgene inactivated by the drug-resistance cassette. The heteroallelic mutant studied, which had about 50% of the wild-type LPD activity, caused acidification of the growth medium. Growth over a prolonged time was only possible after an increased buffering of the medium. Since it is reported in the literature that inactivation of the pyruvate dehydrogenase complex (PDC) leads to acidosis, a function of the LPD in a cytoplasmic-membrane-associated PDC is conceivable.
Characterization and protective activity of a monoclonal antibody against a capsular epitope shared by Streptococcus suis serotypes 1, 2 and 1/2
Authors: Charland Nathalie , Jacques Mario , Lacouture Sonia , Gottschalk Marcelo
Published: June 2009

A monoclonal antibody (mAb Z3) was produced using BALB/c mice immunized with whole cells ofStreptococcus suisserotype 2 reference strain S735. Screening by dot-ELISA showed that mAb Z3, of isotype lgG2b, reacted only with reference strains and field isolates ofS. suisserotypes 1, 2 and 1/2. The recognized epitope was demonstrated to be polysaccharide in nature by periodate oxidation, and located in the capsule, since mAb Z3 reacted with purified capsular material by immunoblotting and was able to stabilize the capsule as shown by electron microscopy. Further characterization indicated that mAb Z3 may react specifically with the sialic acid moiety of the capsule, a common constituent of the polysaccharidic capsular material of the three capsular types, since sialidase-treated cells did not react with mAb Z3 in immunoblotting or indirect EILISA. Purified mAb Z3 was shown to significantly increase the rate of phagocytosis ofS. suiscells by porcine monocytes and to activate the clearance of bacteria from the circulation in experimentally infected mice. However, mAb Z3 only offered partial protection to mice challenged with a minimal lethal dose. Thus, even though the capsule ofS. suisseems to be an important virulence factor, the epitope recognized by mAb Z3 does not appear to be involved in complete protection against infection.
Expression of two alternative sigma factors of Synechococcus sp. strain PCC 7002 is modulated by carbon and nitrogen stress
Authors: Caslake Laurie F. , Gruber Tanja M. , Bryant Donald A.
Published: June 2009

The sigB and sigC genes, encoding two alternative sigma factors of the unicellular marine cyanobacterium Synechococcus sp. PCC 7002, were cloned and characterized. Strains in which the sigB and sigC genes were insertionally inactivated were viable under standard laboratory conditions, indicating that SigB and SigC are group 2 sigma factors. Starvation for either nitrogen or carbon caused an increase in sigB mRNA levels. Transcripts for the sigC gene initially increased but then decreased during nitrogen and carbon starvation. The SigC protein could not be identified in cyanobacterial extracts using antisera to Synechococcus sp. PCC 7002 SigA or RpoD from Bacillus subtilis. The ratio of the principal vegetative sigma factor, SigA, to SigB decreased during either nitrogen starvation or carbon starvation, and the levels of SigB also increased in the sigC mutant strain. These results imply that SigB and SigC play roles in modifying transcription in response to changes in carbon and nitrogen availability in this cyanobacterium.
The patB gene of Dictyostelium discoideum encodes a P-type H+-ATPase isoform essential for growth and development under acidic conditions
Authors: Coukell M. Barrie , Moniakis John , Cameron Anne M.
Published: June 2009

During growth and early development ofDictyostelium discoideum,the amoebae exhibit transient pH changes in their cytosol (pHi) and external medium which correlate with the extrusion of H+from the cell by a plasma membrane pump. Moreover, the changes in pHihave been postulated to influence early prestalk/prespore differentiation during development. To learn more about the role of H+fluxes inDictyostelium.we cloned and analysed cDNAs of the genepatB,which appears to encode a P-type H+-ATPase. ThepatBORF encodes a protein (termed PAT2) of 1058 amino acids with a calculated molecular mass of 117460 Da. When aligned with other P-type ion-transport ATPases, PAT2 showed the greatest amino acid sequence identity with plasma membrane H+-ATPases of plants and fungi and considerably lower identity with other monovalent cation pumps and with Ca2+pumps. Northern and Western analyses revealed that patB is expressed at very low levels in cells growing at neutral pH, but it is up-regulated rapidly and dramatically when the cells are shifted to an acidic medium. Immunofluorescence analysis indicated that PAT2 resides on the plasma membrane. WhenpatBwas disrupted by homologous recombination, the cells grew and developed normally at neutral and slightly alkaline pHs but they were unable to grow or develop at pH 5.0, and they slowly died. In growth medium at pH 6.8,patB+andpatBcells exhibited similar levels of vanadate-sensitive ATPase activity. However, when the cells were shifted to pH 5.0, this activity rapidly increased about twofold in the control cells but not in the mutant cells. Despite the lower ATPase activity inpatBcells, they showed relatively normal H+fluxes and only a slight decrease in pHiwhen incubated in acidic medium. Together, these results suggest thatpatBencodes an acid-inducible P-type H+-ATPase which is indispensable for the survival ofDictyosteliumcells in moderately acidic external environments.
Yeast-enhanced green fluorescent protein (yEGFP): a reporter of gene expression in Candida albicans
Authors: Cormack Brendan P. , Bertram Gwyneth , Egerton Mark , Gow Neil A. R. , Falkow Stanley , Brown Alistair J. P.
Published: June 2009

The green fluorescent protein (GFP) of Aequorea victoria has been developed here as a reporter for gene expression and protein localization in Candida albicans. When wild-type (wt) GFP was expressed in C. albicans, it was not possible to detect fluorescence or a translation product for the wt protein. Since this was probably due in part to the presence of the non-canonical CTG serine codon in the Aequorea sequence, this codon was changed to the leucine codon TTG. C. albicans cells expressing this construct contained GFP mRNA but were non-fluorescent and contained no detectable translation product. Hence a codon-optimized GFP gene was constructed in which all of the 239 amino acids are encoded by optimal codons for C albicans. In this gene were also incorporated two previously identified mutations in the chromophore that increase GFP fluorescence. C. albicans cells expressing this yeast-enhanced GFP gene (yEGFP3) are fluorescent and contain GFP protein. yEGFP3 can be used as a versatile reporter of gene expression in C. albicans and Saccharomyces cerevisiae and the optimized GFP described here should have broad applications in these and other fungal species.
Cloning and characterization of a gene (LIP1) which encodes a lipase from the pathogenic yeast Candida albicans
Authors: Fu Yue , Ibrahim Ashraf S. , Fonzi William , Zhou Xiang , Ramos Clarisa F. , Ghannoum Mahmoud A.
Published: June 2009

Extracellular phospholipases are demonstrated virulence factors for a number of pathogenic microbes. The opportunistic pathogen Candida albicans is known to secrete phospholipases and these have been correlated with strain virulence. In an attempt to clone C. albicans genes encoding secreted phospholipases, Saccharomyces cerevisiae was transformed with a C. albicans genomic library and screened for lipolytic activity on egg-yolk agar plates, a traditional screen for phospholipase activity. Two identical clones were obtained which exhibited lipolytic activity. Nucleotide sequence analysis identified an ORF encoding a protein of 351 amino acid residues. Although no extensive homologies were identified, the sequence contained the Gly-X-Ser-X-Gly motif found in prokaryotic and eukaryotic lipases, suggesting a similar activity for the encoded protein. Indeed, culture supernatants from complemented yeast cells contained abundant hydrolytic activity against a triglyceride substrate and had no phospholipase activity. The data suggest that C. albicans, in addition to phospholipases, also has lipases. Southern blot analyses revealed that C. albicans may contain a lipase gene (LIP) family, and that a lipase gene(s) may be present in Candida parapsilosis, Candida tropicalis and Candida krusei, but not in Candida pseudotropicalis, Candida glabrata or S. cerevisiae. Northern blot analyses showed that expression of the LIP1 transcript, the cloned gene which encodes a lipase, was detected only when C. albicans was grown in media containing Tween 80, other Tweens or triglycerides as the sole carbon source, and not in Sabouraud Dextrose Broth or yeast/peptone/dextrose media. Additionally, carbohydrate supplementation inhibited LIP1 expression. Cloning this gene will allow the construction of LIP1-deficient null mutants which will be critical in determining the role of this gene in candidal virulence.