bluuebunny/crossref_metadata_2025 huggingface dataset:
Proposal to reclassify [Sphingomonas] xenophaga Stolz et al. 2000 and [Sphingomonas] taejonensis Lee et al. 2001 as Sphingobium xenophagum comb. nov. and Sphingopyxis taejonensis comb. nov., respectively
Authors: Pal Rinku , Bhasin V. K. , Lal Rup
Published: March 2006

The sphingomonad group contains bacterial isolates that are quite diverse in terms of their phylogenetic, ecological and physiological properties. Thus, the genus Sphingomonas was divided into four distinct genera, Sphingomonas sensu stricto, Sphingobium, Novosphingobium and Sphingopyxis on the basis of 16S rRNA gene sequence phylogenetic analysis, signature nucleotides, fatty acid profiles and polyamine patterns and this classification is currently widely accepted. In this study, a complete analysis of the 16S rRNA gene sequences of all the members of the group of sphingomonads encompassed in the genera Sphingomonas sensu stricto, Sphingobium, Novosphingobium and Sphingopyxis was inferred by using tree-making algorithms. [Sphingomonas] xenophaga DSM 6383T was found to form a distinct clade with the members of the genus Sphingobium, whereas [Sphingomonas] taejonensis DSM 15583T forms a clade with the members of the genus Sphingopyxis. The respective positions of these strains were also supported by the data for signature nucleotides, 2-hydroxy fatty acid profiles, polyamine patterns and the nitrate reduction properties of the strains. We therefore propose the reclassification of [Sphingomonas] xenophaga and [Sphingomonas] taejonensis as Sphingobium xenophagum comb. nov. (type strain DSM 6383T=CIP 107206T) and Sphingopyxis taejonensis comb. nov. (type strain DSM 15583T=KCTC 2884T=KCCM 41068T), respectively.
Enzymic systems proposed to be involved in the dissimilatory reduction of selenite in the purple non-sulfur bacteria Rhodospirillum rubrum and Rhodobacter capsulatus
Authors: Kessi Janine
Published: March 2006

Various enzymic systems, such as nitrite reductase, sulfite reductase and glutathione reductase, have been proposed for, or suspected to be involved in, the reduction of selenite in bacteria. As alphaproteobacteria have been shown to be highly tolerant to transition metal oxyanions, it seemed interesting to investigate the hypothetical involvement of these different enzymes in the reduction of selenite in the purple non-sulfur bacteriaRhodospirillum rubrumandRhodobacter capsulatus. The hypothetical involvement of nitrite reductase and sulfite reductase in the reduction of selenite in these bacteria was investigated by analysing the effects of nitrite and sulfite amendments on the growth and kinetics of selenite reduction. The reduction of selenite was not concomitant with that of either sulfite or nitrite inRs. rubrum, suggesting that the reduction pathways operate independently. InRb. capsulatus, strong interactions were observed between the nitrite reduction and selenite reduction pathways. However, in both organisms, selenite reduction took place during both the growth phase and the stationary phase, indicating that selenite metabolism is constitutively expressed. In contrast, neither nitrite nor sulfite was transformed during stationary phase, suggesting that the metabolism of both ions is induced, which implies that identical reduction pathways for selenite and nitrite or selenite and sulfite are excluded. Buthionine sulfoximine (BSO,S-n-butyl homocysteine sulfoximine), a specific inhibitor of glutathione synthesis, was used to depress the intracellular glutathione level. In stationary-phase cultures of bothRs. rubrumandRb. capsulatusamended with BSO, the rate of reduction of selenite was slowed, indicating that glutathione may be involved in the dissimilatory reduction of selenite in these organisms. The analysis of the headspace gases of the cultures indicated that the synthesis of methylated selenium compounds was prevented in the presence of 3·0 mM BSO in both organisms, implying that glutathione is also involved in the transformation of selenite to volatile selenium compounds.
Role of anionic phospholipids in the adaptation of Bacillus subtilis to high salinity
Authors: López Claudia S. , Alice Alejandro F. , Heras Horacio , Rivas Emilio A. , Sánchez-Rivas Carmen
Published: March 2006

The importance of the content of anionic phospholipids [cardiolipin (CL) and phosphatidylglycerol (PG)] in the osmotic adaptation and in the membrane structure ofBacillus subtiliscultures was investigated. Insertion mutations in the three putative cardiolipin synthase genes (ywiE,ywnEandywjE) were obtained. Only theywnEmutation resulted in a complete deficiency in cardiolipin and thus corresponds to a trueclsAgene. The osmotolerance of aclsAmutant was impaired: although at NaCl concentrations lower than 1·2 M the growth curves were similar to those of its wild-type control, at 1·5 M NaCl (LBN medium) the lag period increased and the maximal optical density reached was lower. The membrane of theclsAmutant strain showed an increased PG content, at both exponential and stationary phase, but no trace of CL in either LB or LBN medium. As well as the deficiency in CL synthesis, theclsA mutant showed other differences in lipid and fatty acids content compared to the wild-type, suggesting a cross-regulation in membrane lipid pathways, crucial for the maintenance of membrane functionality and integrity. The biophysical characteristics of membranes and large unilamellar vesicles from the wild-type andclsAmutant strains were studied by Laurdan's steady-state fluorescence spectroscopy. At physiological temperature, theclsAmutant showed a decreased lateral lipid packing in the protein-free vesicles and isolated membranes compared with the wild-type strain. Interestingly, the lateral lipid packing of the membranes of both the wild-type andclsAmutant strains increased when they were grown in LBN. In a conditional IPTG-controlledpgsAmutant, unable to synthesize PG and CL in the absence of IPTG, the osmoresistance of the cultures correlated with their content of anionic phospholipids. The transcriptional activity of theclsAandpgsAgenes was similar and increased twofold upon entry to stationary phase or under osmotic upshift. Overall, these results support the involvement of the anionic phospholipids in the growth ofB. subtilisin media containing elevated NaCl concentrations.
Identification of Fur-regulated genes in Actinobacillus actinomycetemcomitans
Authors: Haraszthy Violet I. , Jordan Shawn F. , Zambon Joseph J.
Published: March 2006

Actinobacillus actinomycetemcomitans is an oral pathogen that causes aggressive periodontitis as well as sometimes life-threatening, extra-oral infections. Iron regulation is thought to be important in the pathogenesis of A. actinomycetemcomitans infections and, consistent with this hypothesis, the fur gene has recently been identified and characterized in A. actinomycetemcomitans. In this study, 14 putatively Fur-regulated genes were identified by Fur titration assay (Furta) in A. actinomycetemcomitans, including afuA, dgt, eno, hemA, tbpA, recO and yfe – some of which are known to be Fur regulated in other species. A fur mutant A. actinomycetemcomitans strain was created by selecting for manganese resistance in order to study the Fur regulon. Comparisons between the fur gene sequences revealed that nucleotide 66 changed from C in the wild-type to T in the mutant strain, changing leucine to isoleucine. The fur mutant strain expressed a nonfunctional Fur protein as determined by Escherichia coli-based ferric uptake assays and Western blotting. It was also more sensitive to acid stress and expressed higher levels of minC than the wild-type strain. minC, which inhibits cell division in other bacterial species and whose regulation by iron has not been previously described, was found to be Fur regulated in A. actinomycetemcomitans by Furta, by gel shift assays, and by RT-qPCR assays for gene expression.
The impairment of superoxide dismutase coordinates the derepression of the PerR regulon in the response of Staphylococcus aureus to HOCl stress
Authors: Maalej Sami , Dammak Ines , Dukan Sam
Published: March 2006

The response ofStaphylococcus aureusto hypochlorous acid (HOCl) exposure was investigated. HOCl challenges were performed on cultures interrupted in the exponential phase. Pretreatment with HOCl conferred resistance to hydrogen peroxide in a PerR-dependent manner. Derepression of the PerR regulon was observed at low HOCl concentration (survival >50 %), using several fusions of different stress promoters tolacZreporter genes. At least four members of the PerR regulon (katA,mrgA,bcpandtrxA) encoding proteins with antioxidant properties were strongly induced following exposure to various HOCl concentrations. A striking result was the link between the derepression of the PerR regulon and the decreased superoxide dismutase (SOD) activity following exposure to increased HOCl concentrations. ThesodAmutant was more resistant than the wild-type and also had a higher level of 3-phosphoglycerate dehydrogenase (a measure of PerR regulon activity) without exposure to HOCl. Together, these results imply that derepression of PerR by HOCl is dependent on the level of SOD and protects exponentially arrested cells against HOCl stress.
Enterococcus faecalis strains show culture heterogeneity in cell surface charge
Authors: van Merode Annet E. J. , van der Mei Henny C. , Busscher Henk J. , Waar Karola , Krom Bastiaan P.
Published: March 2006

Adhesion of micro-organisms to biotic and abiotic surfaces is an important virulence factor and involves different types of interactions.Enterococcus faecalis, a human commensal and an important opportunistic pathogen, has the ability to adhere to surfaces. Biliary stents frequently become clogged with bacterial biofilms, withE. faecalisas one of the predominant species. SixE. faecalisstrains isolated from clogged biliary stents were investigated for the presence of specific biochemical factors involved in their adhesion: aggregation substances (Aggs) and the enterococcal surface protein (encoded by theespgene). In addition, physico-chemical factors involved in adhesion (zeta potential and cell surface hydrophobicity) were determined, as well as the influence of ox bile on these properties. Two-thirds of the biliary stent isolates displayed culture heterogeneity in the pH dependence of their zeta potentials. Moreover, 24 out of 46 clinical isolates ofE. faecalis, including 11 laboratory strains, also displayed such heterogeneity. The culture heterogeneity was demonstrated to be a stable trait, not caused by quorum sensing, not plasmid mediated, and independent of the presence ofespand Agg. Data presented show that culture heterogeneity in zeta potential enhances adhesion to an abiotic surface. A higher prevalence of culture heterogeneity in zeta potential in pathogenic as compared to non-pathogenic isolates could indicate that this phenomenon might play a role in virulence and putatively in pathogenesis.
The TraA relaxase autoregulates the putative type IV secretion-like system encoded by the broad-host-range Streptococcus agalactiae plasmid pIP501
Authors: Kurenbach Brigitta , Kopeć Jolanta , Mägdefrau Marion , Andreas Kristin , Keller Walter , Bohn Christine , Abajy Mouhammad Y. , Grohmann Elisabeth
Published: March 2006

The conjugative multiple antibiotic resistance plasmid pIP501 can be transferred and stably maintained in a variety of Gram-positive genera, including multicellular Streptomyces lividans, as well as in Gram-negative Escherichia coli. The 15 putative pIP501 transfer (tra) genes are organized in an operon-like structure terminating in a strong transcriptional terminator. This paper reports co-transcription of the pIP501 tra genes in exponentially growing Enterococcus faecalis JH2-2 cells, as shown by RT-PCR. The tra genes are expressed throughout the life cycle of Ent. faecalis, and the expression level is independent of the growth phase. Electrophoretic mobility shift assays indicated that the TraA relaxase, the first gene of the tra operon, binds to the tra promoter P
tra
, which partially overlaps with the origin of transfer (oriT). DNase I footprinting experiments further delimited the TraA binding region and defined the nucleotides bound by TraA. β-Galactosidase assays with P
tra–lacZ fusions proved P
tra
promoter activity, which was strongly repressed when TraA was supplied in trans. Thus, it is concluded that the pIP501 tra operon is negatively autoregulated at the transcriptional level by the conjugative DNA relaxase TraA.
Hg(II) sequestration and protection by the MerR metal-binding domain (MBD)
Authors: Qin Jie , Song Lingyun , Brim Hassan , Daly Michael J. , Summers Anne O.
Published: March 2006

MerR, the metalloregulator of the bacterial mercury resistance (mer) operon, binds Hg(II) with high affinity. To study the mechanism of metal-induced activation, a small protein was previously engineered embodying in a single polypeptide the metal-binding domain (MBD) ordinarily formed between two monomers of MerR. Here the physiological and biochemical properties of MBD expressed on the cell surface or in the cytosol were examined, to better understand the environments in which specific metal binding can occur with this small derivative. Over 20 000 surface copies of MBD were expressed perEscherichia colicell, with metal stoichiometries of ∼1·0 Hg(II) per MBD monomer. Cells expressing MBD on their surface in rich medium bound 6·1-fold more Hg(II) than those not expressing MBD. Although in nature cells use the entiremeroperon to detoxify mercury, it was interesting to note that cells expressing only MBD survived Hg(II) challenge and recovered more quickly than cells without MBD. Cell-surface-expressed MBD bound Hg(II) preferentially even in the presence of a 22-fold molar excess of Zn(II) and when exposed to equimolar Cd(II) in addition. MBD expressed in the cystosol also afforded improved survival from Hg(II) exposure forE. coliand for the completely unrelated bacteriumDeinococcus radiodurans.
Plasmid-mediated genomic recombination at the pilin gene locus enhances the N-acetyl-d-galactosamine-specific haemagglutination activity and the growth rate of Eikenella corrodens
Authors: Azakami Hiroyuki , Akimichi Hiromi , Noiri Yuichiro , Ebisu Shigeyuki , Kato Akio
Published: March 2006

Eikenella corrodens belongs to a group of periodontopathogenic bacteria and forms unique corroding colonies on solid medium due to twitching motility. It is believed that an N-acetyl-d-galactosamine (GalNAc)-specific lectin on the cell surface contributes significantly to its pathogenicity and can be estimated by its haemagglutination (HA) activity. Recently, a plasmid, pMU1, from strain 1073 has been found; this plasmid affects pilus formation and colony morphology. To identify the gene involved in these phenomena, ORF 4 and ORFs 5–6 on pMU1 were separately subcloned into a shuttle vector, and the resultant plasmids were introduced into E. corrodens 23834. Transformants with the ORF 4 gene, which is identified to be a homologous gene of the type IV pilin gene-specific recombinase, lost their pilus structure and formed non-corroding colonies on a solid medium, whereas transformants with ORFs 5–6 exhibited the same phenotype as the host strain 23834. Southern analysis showed that the introduction of the ORF 4 gene into strain 23834 resulted in genomic recombination at the type IV pilin gene locus. The hybridization pattern of these transformants was similar to that of strain 1073. These results suggest that ORF 4 on pMU1 encodes a site-specific recombinase and causes genomic recombination of the type IV pilin gene locus. Furthermore, the introduction of ORF 4 into strain 23834 increased GalNAc-specific HA activity to a level equivalent to that of strain 1073. Although the morphological colony changes and loss of pilus structure are also observed in phase variation, genomic recombination of the type IV pilin gene locus did not occur in these variants. Moreover, an increase was not observed in the GalNAc-specific HA activity of these variants. These results suggested that the loss of pilus structure, the morphological change in colonies and the increase in HA activity due to plasmid pMU1 might be caused by a mechanism that differs from phase variation, such as a genomic recombination of the type IV pilin gene locus.
A putative sensor kinase, Hik31, is involved in the response of Synechocystis sp. strain PCC 6803 to the presence of glucose
Authors: Kahlon Shira , Beeri Karen , Ohkawa Hiroshi , Hihara Yukako , Murik Omer , Suzuki Iwane , Ogawa Teruo , Kaplan Aaron
Published: March 2006

The reason(s) for glucose sensitivity in certain cyanobacterial strains is poorly understood. Inactivation of genes encoding the putative sensor kinase Hik31 inSynechocystissp. strain PCC 6803 resulted in a mutant unable to grow in the presence ofd-glucose. Sensitivities tod-glucose, its analogue 2-deoxy-d-glucose, and fructose, were alleviated in mutants in whichglcP, encoding the glucose transporter, was inactivated. These data indicate that permeation of these substrates is required to inflict cell death. The mutant Δhik31, and the glucose-sensitive strain ofSynechocystis, do not possess glucokinase activity, although a transcript originating fromglk, encoding glucokinase, is present. Inactivation ofglkled to severe sensitivity to glucose, indicating that the presence of glucose itself, within the cells, inflicted this sensitivity. On the other hand, sensitivity to 2-deoxy-d-glucose was lower in Δglk, thus distinguishing between the effect of glucose itself and that of its analogue, which, in the absence of glucokinase activity, may not be phosphorylated. Addition of glucose led to a small rise in glucose-6-phosphate dehydrogenase activity in the wild type, but constitutive activity was observed in the Δhik31mutant regardless of the presence of glucose. Microarray analyses showed only small changes in the abundance of global transcripts inSynechocystisfollowing glucose addition, but the transcription levels of several genes, includingicfG, but notglk, were strongly affected by inactivation ofhik31. The mechanism(s) whereby Hik31 is involved in glucose sensing and response is discussed.
Adaptation of Bacillus subtilis to growth at low temperature: a combined transcriptomic and proteomic appraisal
Authors: Budde Ina , Steil Leif , Scharf Christian , Völker Uwe , Bremer Erhard
Published: March 2006

The soil bacterium Bacillus subtilis frequently encounters a reduction in temperature in its natural habitats. Here, a combined transcriptomic and proteomic approach has been used to analyse the adaptational responses of B. subtilis to low temperature. Propagation of B. subtilis in minimal medium at 15 °C triggered the induction of 279 genes and the repression of 301 genes in comparison to cells grown at 37 °C. The analysis thus revealed profound adjustments in the overall gene expression profile in chill-adapted cells. Important transcriptional changes in low-temperature-grown cells comprise the induction of the SigB-controlled general stress regulon, the induction of parts of the early sporulation regulons (SigF, SigE and SigG) and the induction of a regulatory circuit (RapA/PhrA and Opp) that is involved in the fine-tuning of the phosphorylation status of the Spo0A response regulator. The analysis of chill-stress-repressed genes revealed reductions in major catabolic (glycolysis, oxidative phosphorylation, ATP synthesis) and anabolic routes (biosynthesis of purines, pyrimidines, haem and fatty acids) that likely reflect the slower growth rates at low temperature. Low-temperature repression of part of the SigW regulon and of many genes with predicted functions in chemotaxis and motility was also noted. The proteome analysis of chill-adapted cells indicates a major contribution of post-transcriptional regulation phenomena in adaptation to low temperature. Comparative analysis of the previously reported transcriptional responses of cold-shocked B. subtilis cells with this data revealed that cold shock and growth in the cold constitute physiologically distinct phases of the adaptation of B. subtilis to low temperature.
Genetic diversity of the C protein β-antigen gene and its upstream regions within clonally related groups of type Ia and Ib group B streptococci
Authors: Nagano Noriyuki , Nagano Yukiko , Nakano Ryuichi , Okamoto Ryoichi , Inoue Matsuhisa
Published: March 2006

C protein β antigen (Bac), a surface protein of group B streptococci (GBS), is known to concurrently bind the Fc portion of IgA and factor H (FH). The authors' previous work has demonstrated that mRNA expression levels show diversity among clonally related strains containing genes (bac) encoding Bac, with high expression noted in invasive strains. In this study, the bac gene and upstream regions containing putative promoters, three ORFs and an IS1381 insertion sequence were characterized. Three invasive strains showed high bac expression levels and did not show any notable mutations except one strain producing Bac that was able to bind FH but not IgA. A deletion of 51 amino acid residues, including part of the Bac IgA-binding region, was identified and hypothesized to contribute to the loss of the IgA-binding ability of this strain. A vaginal strain that showed somewhat higher bac expression levels and produced Bac lacking immunoreactivity contained an 11 bp deletion, which generated a premature termination codon, in the region preceding the IgA-binding region. In another vaginal strain that did not express bac, disruption of the upstream ORFs of the sensor histidine kinase and DNA-binding response regulator, due to frameshift mutations, was noted although it is not known whether these proteins directly affect bac expression levels. An IS1381 insertion into the promoter region was found in another vaginal strain that showed low expression levels and produced Bac with a significantly larger proline-rich repeat region. These results demonstrate considerable genetic diversity of the bac and upstream regions of invasive and noninvasive GBS, which may contribute to the variability of bac expression levels among those strains.
The role of dsbA in colonization of the wheat rhizosphere by Pseudomonas fluorescens Q8r1-96
Authors: Mavrodi Olga V. , Mavrodi Dmitri V. , Park Amanda A. , Weller David M. , Thomashow Linda S.
Published: March 2006

Certain well-conserved genes in fluorescent Pseudomonas spp. are involved in pathogenic interactions between the bacteria and evolutionarily diverse hosts including plants, insects and vertebrate animals. One such gene, dsbA, encodes a periplasmic disulfide-bond-forming enzyme implicated in the biogenesis of exported proteins and cell surface structures. This study focused on the role of dsbA in Pseudomonas fluorescens Q8r1-96, a biological control strain that produces the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) and is known for its exceptional ability to colonize the roots of wheat and pea. The deduced DsbA protein from Q8r1-96 is similar to other predicted thiol : disulfide interchange proteins and contains a conserved DsbA catalytic site, a pattern associated with the thioredoxin family active site, and a signal peptide and cleavage site. A dsbA mutant of Q8r1-96 exhibited decreased motility and fluorescence, and altered colony morphology; however, it produced more 2,4-DAPG and total phloroglucinol-related compounds and was more inhibitory in vitro to the fungal root pathogen Gaeumannomyces graminis var. tritici than was the parental strain. When introduced separately into a natural soil, Q8r1-96 and the dsbA mutant did not differ in their ability to colonize the rhizosphere of wheat in greenhouse experiments lasting 12 weeks. However, when the two strains were co-inoculated, the parental strain consistently out-competed the dsbA mutant. It was concluded that dsbA does not contribute to the exceptional rhizosphere competence of Q8r1-96, although the dsbA mutation reduces competitiveness when the mutant competes with the parental strain in the same niche in the rhizosphere. The results also suggest that exoenzymes and multimeric cell surface structures are unlikely to have a critical role in root colonization by this strain.
Calcineurin, Mpk1 and Hog1 MAPK pathways independently control fludioxonil antifungal sensitivity in Cryptococcus neoformans
Authors: Kojima Kaihei , Bahn Yong-Sun , Heitman Joseph
Published: March 2006

Fludioxonil is employed as an agricultural fungicide to control plant-pathogenic fungi such as Botrytis cinerea. Cryptococcus neoformans is a basidiomycetous human fungal pathogen that causes fatal disease in immunocompromised hosts. This paper demonstrates that three different signalling cascades regulate sensitivity of C. neoformans to fludioxonil. Fludioxonil inhibited growth of the serotype A sequence reference strain H99 but not that of the sequenced serotype D strain JEC21. In the drug-sensitive wild-type strain, fludioxonil exposure activated the Hog1 osmosensing pathway, and hog1Δ mutations conferred fludioxonil resistance. Fludioxonil treatment caused cell growth inhibition following cell swelling and cytokinesis defects in the sensitive wild-type but not in a hog1Δ mutant strain, suggesting that Hog1 activation results in morphological cellular defects. Fludioxonil exerted a fungistatic effect on the wild-type strain H99, but exhibited fungicidal activity against calcineurin mutant strains, indicating that the calcineurin pathway contributes to drug resistance in this fungus. Combination of fludioxonil and the calcineurin inhibitor FK506 synergistically inhibited C. neoformans growth. mpk1Δ MAPK mutant strains exhibited fludioxonil hypersensitivity, indicating that this pathway also contributes to drug resistance. These studies provide evidence that the broad-spectrum antifungal drug fludioxonil exerts its action via activation of the Hog1 MAPK pathway and provide insight into novel targets for synergistic antifungal drug combinations.
The encapsulation of enterotoxigenic Escherichia coli colonization factor CS3 in biodegradable microspheres enhances the murine antibody response following intranasal administration
Authors: Byrd Wyatt , Cassels Frederick J.
Published: March 2006

The aim of this study was to measure serum and mucosal antibody responses following intranasal administration of biodegradable poly(dl-lactide-co-glycolide) (PLGA) microspheres loaded with the CS3 colonization factor isolated from enterotoxigenicEscherichia coli(ETEC). The response was compared against that measured in mice similarly administered the native CS3 antigen and in mice co-administered, along with the CS3 antigen, a known mucosal adjuvant, the R192G mutant heat-labile enterotoxin (mLT). The integrity of the CS3 antigen released from the microspheres was maintained as determined by SDS-PAGE and immunoblotting. Native CS3 induced serum and mucosal (bronchoalveolar, small intestinal and faecal) IgG and IgA responses. The co-administration of the mLT mucosal adjuvant significantly enhanced (P<0·001) serum and mucosal antibody responses to the CS3 protein. Likewise, the CS3-loaded PLGA microspheres induced significantly greater (P<0·001) serum and mucosal antibody responses than native CS3, as well as inducing antibody responses superior to those of the CS3 plus mLT formulation. Following administration of CS3 plus mLT, the mice became distressed (loss of activity, increased huddling, ruffled fur), a situation not seen following administration of the CS3-loaded PLGA microspheres. The results in this trial show that the CS3-loaded PLGA microspheres when administered intranasally to mice caused no observable distress to the mice and significantly (P<0·001) enhanced the immunogenicity of the CS3 protein.
Semi‐purification of the immunoglobulin E‐sweat antigen acting on mast cells and basophils in atopic dermatitis
Authors: Tanaka A. , Tanaka T. , Suzuki H. , Ishii K. , Kameyoshi Y. , Hide M.
Published: March 2006

Background:  Sweating aggravates the symptoms of atopic dermatitis (AD). We have recently reported positive skin reactions and histamine release from basophils in response to autologous sweat in patients with AD.

Objective:  To characterize the biochemical and immunological properties of the substance in sweat that evokes histamine release and to study the usability of the basophil‐histamine release test with the sweat antigen for AD.

Methods:  Sweat collected from healthy volunteers was purified using chromatographies. Serum immunoglobulin (Ig)E of four patients with AD were purified using an affinity‐chromatography column with anti‐IgE antibodies. The amount of semi‐purified sweat antigen (138 ng protein/ml) that induced a half‐maximum reaction of basophils of a patient with AD was utilized for the basophil histamine release test. The involvement of specific IgE and high‐affinity IgE receptor (FcɛRI) in the reactions was examined using basophils of healthy volunteers, a human mast cell line (LAD2), and a rat basophilic leukemia cell line transfected with human α‐subunit of FcɛRI (RBL‐48).

Results:  The semi‐purified sweat antigen induced histamine release from the basophils of 47 of 61 (74.6%) patients with AD and four of 46 (8.7%) healthy controls. Both basophils and mast cells sensitized with the patient‐derived IgE showed degranulation upon stimulation with the sweat antigen. However, no reaction was observed when cells were sensitized with myeloma IgE or the antigen was treated with proteases.

Conclusion:  The semi‐purified standardized sweat antigen consists of a protein that induces degranulation of basophils and mast cells via antigen‐specific IgE and FcɛRI in patients with AD.
Stat3 activation is required for cell proliferation and tumorigenesis but not for cell viability in cutaneous squamous cell carcinoma cell lines
Authors: Sumita Naoko , Bito Toshinori , Nakajima Koichi , Nishigori Chikako
Published: March 2006

Abstract:  Signal transducer and activator of transcription 3 (Stat3), a cytoplasmic transcription factor, is constitutively activated in various types of cancer. Previous investigations have demonstrated that Stat3 plays important roles in cell growth, survival, differentiation, and transformation. The constitutive activation of Stat3 in human malignancies is an important key to maintain the characteristics of a malignant tumor, such as the rate of proliferation and/or immortalization, and inhibition of Stat3 function could be a potent therapeutic approach. In order to elucidate the role of Stat3 in tumors, cutaneous squamous cell carcinoma (SCC) cells, which have constitutive activation of Stat3 in vivo and in vitro, were used for this study. To investigate the effect of specific inhibition of Stat3 in SCC cells, we developed small interfering RNAs (siRNAs) that target Stat3, and which effectively prevent its expression in vitro. Introduction of Stat3 siRNA into SCC cells led to inhibition of growth and changes in morphology but did not induce apoptosis. Stat3 siRNA‐transfected SCC cells had impaired tumor growth in nude mice. These findings demonstrate that Stat3 plays a critical role in the tumorigenesis, but not in the cell survival, of SCC cells and suggest that additional pro‐apoptotic signals are necessary for the induction of apoptosis.
Extracellular matrix protein 1 inhibits the activity of matrix metalloproteinase 9 through high‐affinity protein/protein interactions
Authors: Fujimoto Norihiro , Terlizzi Joseph , Aho Sirpa , Brittingham Raymond , Fertala Andrzej , Oyama Noritaka , McGrath John A. , Uitto Jouni
Published: March 2006

Abstract:  Extracellular matrix protein 1 (ECM1), an approximately 85‐kDa glycoprotein with broad tissue distribution, harbors mutations in lipoid proteinosis (LP), a heritable disease characterized by reduplication of basement membranes and hyalinization of dermis, associated with neurologic disorders. The mechanisms leading from ECM1 mutations to LP phenotype are unknown. In this study, we explored ECM1 protein‐protein interactions utilizing yeast two‐hybrid genetic screen of human placental library, which identified nine interacting proteins, including matrix metalloproteinase 9 (MMP9). The interactions were confirmed by β‐galactosidase assay with isolated clones and by co‐immunoprecipitation which narrowed the interacting segment in ECM1 to the C‐terminal tandem repeat 2 (amino acids 236–361). This peptide segment also inhibited MMP9 activity in a gelatin‐based ELISA assay. We propose that ECM1‐mediated reduction in MMP9 proteolytic activity may have relevance to pathogenesis of LP.
Pharmacokinetic modelling of the interaction between St John’s wort and ciclosporin A
Authors: Murakami Yuichi , Tanaka Tomoaki , Murakami Hideyasu , Tsujimoto Masayuki , Ohtani Hisakazu , Sawada Yasufumi
Published: March 2006

Aims

St John’s wort (SJW) decreases the blood concentration of ciclosporin A (CsA), which may result in allograft rejection. In addition, the time course of this interaction is not parallel with the administration of SJW. We aimed to develop a pharmacokinetic model to predict the time profile of blood CsA concentrations during and after the intake of SJW.

Methods

We developed a pharmacokinetic model incorporating turnover of detoxicating proteins, with the assumption that the amount of detoxicating proteins is in inverse proportion to the ratio of trough blood concentration to daily dose (C/D ratio) of CsA. First, we collected time profiles of blood CsA during and after the intake of SJW from the literature. Next, we analysed the relationship between D/C ratio and the daily dose of SJW at steady state. Subsequently, the developed model was simultaneously fitted to the time profiles of C/D ratios by using a nonlinear least‐squares method to obtain model parameters.

Results

The model analysis revealed that the induction of the detoxicating proteins by SJW was saturable with an elimination rate constant of the detoxicating proteins (ke) of 4.72 month−1. Elimination half‐life of the detoxicating proteins calculated from the ke value was 4.4 days, suggesting that the dose of CsA should be carefully monitored for up to 2 weeks after the cessation of SJW intake.

Conclusions

The present model may provide additional information for use in identifying optimal dosage regimens of CsA during and after the intake of SJW to prevent an adverse drug interaction between CsA and SJW.
Selectivity of NSAIDs for COX‐2 and cardiovascular outcome
Authors: Maxwell S. R. J. , Payne R. A. , Murray G. D. , Webb D. J.
Published: March 2006

Background

Nonsteroidal anti‐inflammatory drugs (NSAIDs) with increased selectivity for the cyclooxygenase‐2 (COX‐2) isoform reduce gastrotoxicity but may increase adverse cardiovascular events.

Methods

We searched the literature for studies that reported the odds ratio (OR) for such events following exposure to NSAIDs.

Results

For studies comparing NSAID use with no use, increased COX‐2 selectivity was significantly related to cardiovascular risk (log OR) amongst observational studies (R = −0.34, P < 0.001) and randomized controlled trials (RCTs) (R = −0.56, P < 0.001). For studies comparing NSAIDs, difference in selectivity was related to risk for observational studies (R = −0.28, P = 0.005) but not for RCTs (R = −0.23, P = 0.15).

Conclusions

Although increased COX‐2 selectivity may reduce gastrotoxicity, this may be at the cost of increasing cardiovascular risk.
An update and appraisal of the cilomilast Phase III clinical development programme for chronic obstructive pulmonary disease
Authors: Giembycz Mark A.
Published: March 2006

Cilomilast (ArifloTM, SB 207499) is an orally active, second‐generation phosphodiesterase (PDE) 4 inhibitor that is being developed by GlaxoSmithkline for the treatment of chronic obstructive pulmonary disease (COPD). The results of Phase I and Phase II studies have demonstrated that cilomilast significantly improves lung function and quality of life to a clinically meaningful extent, which has led to a comprehensive Phase III programme of research evaluating efficacy, safety and mechanism of action. However, the results of those Phase III studies are unremarkable and disappointing, raising doubt over the future of cilomilast as a novel therapy for COPD. This review summarizes data obtained from the Phase III clinical development programme, highlights some of the potential concerns both specific to cilomilast and to PDE4 inhibitors in general and assesses the likelihood that cilomilast will reach the market.
Mucin genes have different expression patterns in healthy and diseased upper airway mucosa
Authors: Martínez‐Antón A. , DeBolós C. , Garrido M. , Roca‐Ferrer J. , Barranco C. , Alobid I. , Xaubet A. , Picado C. , Mullol J.
Published: March 2006

Background
Mucus hyper‐secretion is a feature of several airways diseases such as chronic rhinosinusitis, asthma, and cystic fibrosis (CF). Since mucins are major components of mucus, the knowledge of their distribution and regulation in nasal tissues is likely to improve mucus hyper‐secretion therapy.

Objective
The aim of this study was to evaluate and compare mucin gene expression at epithelial and glandular levels, and to identify potential mucin expression patterns for specific upper airways pathologies.

Methods
Immunohistochemistry for MUC1, MUC2, and MUC4–MUC8 mucins was performed on healthy nasal mucosa (NM; n=12), bilateral nasal polyps (NP; n=38), NP from CF patients (n=10), and antrochoanal (AC) polyps (n=11). MUC2, MUC4, MUC5AC, and MUC6 mRNA expression were also analysed by in situ hybridization.

Results
MUC1, MUC4, and MUC5AC mucins were highly expressed in the epithelium and their expression pattern was similar in all NP types, MUC1 and MUC4 being increased and MUC5AC decreased compared with NM. MUC8 was highly detected at both epithelial and glandular levels with marked variability between groups. MUC5B was mainly detected in glands and the expression in all polyp types was higher than in NM. Moreover, MUC5B expression was higher in NP epithelia from CF patients than in bilateral NP and healthy NM. Although MUC2 expression was low, especially in AC polyps, it was detected in most samples. In NM, MUC6 and MUC7 were scarcely detected and MUC7 expression was restricted to glands.

Conclusions
These results suggest that NP have a different pattern of mucin expression than healthy NM and that CF polyps (increased MUC5B) and AC polyps (decreased MUC2) have a different mucin expression pattern than bilateral NP.
Cysteinyl leukotriene receptor 1 promoter polymorphism is associated with aspirin‐intolerant asthma in males
Authors: Kim S.‐H. , Oh J.‐M. , Kim Y.‐S. , Palmer L. J. , Suh C.‐H. , Nahm D.‐H. , Park H.‐S.
Published: March 2006

Background
Cysteinyl leukotrienes (CysLTs) play important roles in the pathogenesis of eosinophilic airway inflammation characterized by bronchoconstriction, mucus secretion and airway hyper‐responsiveness via cysteinyl leukotriene receptor 1 (CysLTR1)‐mediated mechanism. CysLTR1‐selective antagonists have anti‐bronchoconstrictive and anti‐inflammatory effects in asthma, particularly aspirin‐intolerant asthma (AIA).

Methods
To investigate the association of CysLTR1 with AIA development, we identified three single nucleotide polymorphisms (SNPs), −634C>T, −475A>C, −336A>G, in the 5′ upstream region of CysLTR1 gene using a direct sequencing method in 105 AIA patients, 110 ASA‐tolerant asthma (ATA) patients and 125 normal healthy controls (NC).

Results
Significant differences were observed in allele frequencies of the three SNPs within male subjects; Male AIA patients had higher frequencies of the minor alleles of these three SNPs than male control groups (P=0.03 for AIA vs. NC; P=0.02 for AIA vs. ATA). Moreover, three‐SNP haplotype, ht2 [T–C–G], was associated with increased disease risk (odds ratio (OR)=2.71, P=0.03 for AIA vs. NC; OR=2.89, P=0.02 for AIA vs. ATA) in males. CysLTR1 haplotypes were also associated with altered gene expression; luciferase activity was significantly enhanced with the ht2 [T–C–G] construct in comparison with the ht1 [C–A–A] construct in human Jurkat cells (P=0.04).

Conclusion
These results suggest that genetic variants of CysLTR1 are associated with AIA in a Korean population, and may modulate CysLTR1 expression.
Short-term simvastatin treatment has no effect on plasma cytokine response in a human in vivo model of low-grade inflammation
Authors: Erikstrup C , Ullum H , Pedersen B K
Published: March 2006

Statins reduce plasma cholesterol, but clinical trials and in vitro studies indicate that they might also possess anti-inflammatory properties. The effect of simvastatin on circulating cytokines and leucocytes was evaluated in a human in vivo model of low-grade inflammation. Thirty young healthy male participants received an injection of the bacterial cell wall product endotoxin (0·06 ng/kg) to induce systemic inflammation. Participants were then randomized into a control and a simvastatin group. The simvastatin group received simvastatin 20 mg daily for 14 days. All participants returned after 14 days to receive a second endotoxin injection. Plasma concentrations of tumour necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1 receptor antagonist were measured by enzyme-linked immunosorbent assay (ELISA) before and hourly for 6 hours after endotoxin administration. Plasma cytokines as well as total leucocyte counts increased in all participants upon endotoxin challenge but were not affected by simvastatin treatment. Tolerance to endotoxin was observed in both groups after 14 days. Short-term treatment with simvastatin (20 mg/day) did not influence circulating cytokine levels during endotoxaemia in this human in vivo study.
Interferon-γ independent formation of pulmonary granuloma in mice by injections with trehalose dimycolate (cord factor), lipoarabinomannan and phosphatidylinositol mannosides isolated fromMycobacterium tuberculosis
Authors: Takimoto H , Maruyama H , Shimada K-I , Yakabe R , Yano I , Kumazawa Y
Published: March 2006

The mechanisms by which pulmonary granuloma formation is caused by administration of mycobacterial glycolipids such as trehalose dimycolate (TDM), lipoarabinomannan (LAM) and phosphatidylinositol mannosides (PIM) were investigated. When peritoneal and alveolar macrophages were stimulated with TDM, LAM and PIM in vitro, TDM exhibited the strongest tumour necrosis factor (TNF)-inducing activity. Responsiveness of macrophages from mice defected Toll-like receptor 4 (TLR4) was much higher than that of the wild-type mice. Although PIM and LAM also had a significant activity, LAM rather than PIM stimulated higher TNF-α production by alveolar macrophage. When mycobacterial glycolipids were injected as water-in-oil-in-water emulsion into mice via the tail vein, development of pulmonary granuloma in response to glycolipids were related closely to their TNF-inducing activity and TDM exhibited the strongest activity. Granuloma formation was observed not only in mice lacking interleukin (IL)-12 signalling but also interferon (IFN)-γ knock-out mice. Granuloma formation caused by glycolipids correlated with TNF-α levels in lungs. Administration of anti-TNF-α monoclonal antibody into TDM-injected IFN-γ knock-out mice decreased in granuloma formation, suggesting that development of pulmonary granuloma by mycobacterial glycolipids such as TDM is due to IFN-γ-independent and TNF-α-dependent pathway.
Development of an immunocapture method for measuring IgA antibodies to tissue transglutaminase in the sera of patients with coeliac disease
Authors: El Alaoui S , Gresti C
Published: March 2006

One of the most reliable sero-diagnostic tests for coeliac disease (CD) is the measurement, by ELISA, of serum IgA antibodies to tissue transglutaminase (tTG) adsorbed to the wells of microtitre plates. In spite of its reliability, however, some discrepancies exist with the results obtained by the antiendomysium histological assay (EMA) and by biopsy the accepted gold standard. Among the reasons for these differences in titres between the ELISA and the last 2 mentioned assays are the conformational changes that proteins undergo on adsorption and the importance of conformational epitopes on tTG for diagnosing CD. To address this problem, a novel procedure was developed using guinea-pig tTG (gptTG) free in solution to interact with IgA antibodies in the sera of CD patients. Any immune complexes so formed are then captured by anti-tTG antibodies preadsorbed to the wells of microtitre plates. This immunocapture method was optimized for the amount of soluble gptTG needed to interact with all the IgA’s anti-tTG present in fixed dilutions of serum samples, the amount of rabbit IgG anti-gptTG used to coat the wells of microtitre plates and the order of addition of the reaction components. Comparison of the IgA titres obtained by immunocapture with those by EMA and ELISA (adsorbed tTG) on 9 highly positive and 6 weakly positive sera from clinically characterized CD patients and 5 negative sera from non-CD control subjects revealed that the IgA titres by the immunocapture procedure were well correlated with those obtained by EMA, whereas the titres on ELISA showed discrepancies with both immunocapture and EMA.
Interleukin 18 and human immunodeficiency virus type I infection in adolescents and adults
Authors: Song W , Wilson C M , Allen S , Wang C , Li Y , Kaslow R A , Tang J
Published: March 2006

Interleukin (IL)-18, a proinflammatory cytokine, has been recognized recently as an important factor in both treated and untreated patients with human immunodeficiency virus type 1 (HIV-1) infection. Consistent with all earlier reports, our quantification of serum IL-18 concentrations in 88 HIV-1 seropositive, North American adolescents (14–18 years old) revealed a positive correlation with cell-free HIV-1 viral load at two separate visits (Spearman’s r = 0·31 and 0·50, respectively, P < 0·01 for both), along with a negative correlation with CD4+ T cell counts (r = –0·31 and −0·35, P < 0·01 for both). In additional analyses of 66 adults (21–58 years old) from Zambia, HIV-1 seroconversion was associated uniformly with elevated IL-18 production (P < 0·0001). These epidemiological relationships were independent of other population-related characteristics, including age, gender and ethnicity. In neither study population could serum IL-18 concentrations be associated with the IL-18 gene (IL18) promoter genotypes defined by five major single nucleotide polymorphisms. Collectively, these findings suggest that circulating IL-18 rather than the IL18 genotype may provide a useful biomarker for HIV-1-related events or outcomes.
Delivering customer services online: identifying best practices of medium‐sized enterprises
Authors: Levenburg Nancy M. , Klein Helen A.
Published: March 2006

Abstract. The importance of using the internet to achieve competitive advantage has been well documented. Many companies have benefited from capturing customers’ interest in buying via the web. Additionally, an ever‐expanding of technologies exist that enable firms to provide additional customer services online. Yet for many firms, determining which customer service applications to use can be perplexing. This study examines the practices of small‐ and medium‐sized enterprises and analyses performance results of adopting selected customer service applications on the internet.