bluuebunny/crossref_metadata_2025 huggingface dataset:
Unravelling the relationship between stem temperature and air temperature to correct for errors in sap-flow calculations using stem heat balance sensors
Authors: Steppe Kathy , Lemeur Raoul , Dierick Diego
Published: July 2005

Results from measurement of sap flow by heat balance sensors on the stem of a young oak tree (Quercus robur L.) revealed that thermal disequilibrium (i.e. heat storage) within the heated stem segment can introduce considerable errors in the measured sap-flow rates. The magnitude, sign and significance of these errors depend on the sap-flow rate and on the relationship between stem temperature and air temperature. Sap-flow rates were found to be more prone to errors caused by heat storage effects under low flow conditions than at higher rates of sap flow. Furthermore, daytime fluctuations of air temperature and stem temperature inside the heat balance sensor were either in phase when a low, or in opposite phase when a high sap-flow rate was passing through the stem of the young tree. To investigate this relationship, we developed an experimental set-up with cut stem segments through which tap water could be pressed. This set-up allowed the effects of air temperature and sap-flow rates on stem temperatures within heat balance sensors to be clearly separated. Good mathematical relationships were obtained and were successfully used to assess the relative importance of air temperature and sap-flow rate with respect to the fluctuations in stem temperature of the young oak tree. Based on the established relationships, a novel approach was put forward to correct for errors introduced into sap-flow calculations caused by heat storage effects if no measured data on stem temperature are available.
APOBEC3G hypermutates genomic DNA and inhibits Ty1 retrotransposition in yeast
Authors: Schumacher April J. , Nissley Dwight V. , Harris Reuben S.
Published: July 2005

Human cells harbor a variety of factors that function to block the proliferation of foreign nucleic acid. The APOBEC3G enzyme inhibits the replication of retroviruses by deaminating nascent retroviral cDNA cytosines to uracils, lesions that can result in lethal levels of hypermutation. Here, we demonstrate that APOBEC3G is capable of deaminating genomic cytosines in
Saccharomyces cerevisiae
. APOBEC3G expression caused a 20-fold increase in frequency of mutation to canavanine-resistance, which was further elevated in a uracil DNA glycosylase-deficient background. All APOBEC3G-induced base substitution mutations mapped to the nuclear
CAN1
gene and were exclusively C/G → T/A transition mutations within a 5′-C
C
consensus. The APOBEC3G preferred sites were found on both strands of the DNA duplex, but were otherwise located in hotspots nearly identical to those found previously in retroviral cDNA. This unique genetic system further enabled us to show that expression of APOBEC3G or its homolog APOBEC3F was able to inhibit the mobility of the retrotransposon Ty1 by a mechanism that involves the deamination of cDNA cytosines. Thus, these data expand the range of likely APOBEC3 targets to include nuclear DNA and endogenous retroelements, which have pathological and physiological implications, respectively. We postulate that the APOBEC3-dependent innate cellular defense constitutes a tightly regulated arm of a conserved mobile nucleic acid restriction mechanism that is poised to limit internal as well as external assaults.
Common pathways in circadian and cell cycle clocks: Light-dependent activation of Fos/AP-1 in zebrafish controls CRY-1a and WEE-1
Authors: Hirayama Jun , Cardone Luca , Doi Masao , Sassone-Corsi Paolo
Published: July 2005

The cell cycle and the circadian clock are endogenous pacemakers, which coexist in most eukaryotic cells and share a number of conceptual features. In the zebrafish, light directly regulates the timing of both clocks, although the signaling and transcriptional pathways that convey photic information to essential nuclear regulators have yet to be deciphered. We have previously established the Z3 cell line, which recapitulates the features of zebrafish circadian clock and represents an ideal system to study light-dependent signaling and gene regulation. We conducted a search for light-responsive transcription factors and found that AP-1 DNA binding is highly induced. Light induces the expression of
zWee1
, a cell cycle gene essential for G
2
/M transition, and
zCry1a
, a clock gene of the feedback regulatory loop. We have found consensus AP-1 sites in the regulatory regions of both
zWee1
and
zCry1a
genes, and we show that light inducibility of both genes is abrogated by inhibition of AP-1 function. Light also elicits chromatin remodeling by stimulating hyperacetylation at Lys-14 of histone H3 at both
zWee1
and
zCry1a
promoters, as assessed by chromatin immunoprecipitation assays by using anti-Fos antibody. These findings provide strong evidence that circadian and cell cycle clocks share unique light-responsive pathways in zebrafish.
An increase in telomere sister chromatid exchange in murine embryonic stem cells possessing critically shortened telomeres
Authors: Wang Yisong , Erdmann Natalie , Giannone Richard J. , Wu Jun , Gomez Marla , Liu Yie
Published: July 2005

Telomerase deficiency leads to a progressive loss of telomeric DNA that eventually triggers cell apoptosis in human primary cells during prolonged growth in culture. Rare survivors can maintain telomere length through either activation of telomerase or recombination-based telomere lengthening, and thus proliferate indefinitely. We have explored the possibility that telomeres may be maintained through telomere sister chromatid exchange (T-SCE) in murine telomere reverse transcriptase-deficient (mTert-/-) splenocytes and ES cells. Because telomerase deficiency leads to gradual loss of telomeric DNA inmTert-/-splenocytes and ES cells and eventually to chromosomes with telomere signal-free ends (SFEs), we examined these cell types for evidence of sister chromatid exchange at telomeres, and observed an increase in T-SCEs only in a subset ofmTert-/-splenocytes or ES cells that possessed multiple SFEs. Furthermore, T-SCEs were more often detected in ES cells than in splenocytes that harbored a similar frequency of SFEs. InmTertheterozygous (mTert+/-) ES cells or splenocytes, which are known to exhibit a decrease in average telomere length but no SFEs, no increase in T-SCE was observed. In addition to T-SCE, other genomic rearrangements (i.e., SCE) were also significantly increased inmTert-/-ES cells possessing critically short telomeres, but not in splenocytes. Our results suggest that animals and cell culture differ in their ability to carry out genomic rearrangements as a means of maintaining telomere integrity when telomeres become critically shortened.
Cell Adhesion Strengthening: Contributions of Adhesive Area, Integrin Binding, and Focal Adhesion Assembly
Authors: Gallant Nathan D. , Michael Kristin E. , García Andrés J.
Published: July 2005

Mechanical interactions between a cell and its environment regulate migration, contractility, gene expression, and cell fate. We integrated micropatterned substrates to engineer adhesive area and a hydrodynamic assay to analyze fibroblast adhesion strengthening on fibronectin. Independently of cell spreading, integrin binding and focal adhesion assembly resulted in rapid sevenfold increases in adhesion strength to steady-state levels. Adhesive area strongly modulated adhesion strength, integrin binding, and vinculin and talin recruitment, exhibiting linear increases for small areas. However, above a threshold area, adhesion strength and focal adhesion assembly reached a saturation limit, whereas integrin binding transitioned from a uniform distribution to discrete complexes. Adhesion strength exhibited exponential increases with bound integrin numbers as well as vinculin and talin recruitment, and the relationship between adhesion strength and these biochemical events was accurately described by a simple mechanical model. Furthermore, adhesion strength was regulated by the position of an adhesive patch, comprised of bound integrins and cytoskeletal elements, which generated a constant 200-nN adhesive force. Unexpectedly, focal adhesion assembly, in particular vinculin recruitment, contributed only 30% of the adhesion strength. This work elucidates the roles of adhesive complex size and position in the generation of cell-extracellular matrix forces.
Conditional Expression ofToxoplasma gondiiApical Membrane Antigen-1 (TgAMA1) Demonstrates That TgAMA1 Plays a Critical Role in Host Cell Invasion
Authors: Mital Jeffrey , Meissner Markus , Soldati Dominique , Ward Gary E.
Published: July 2005

Toxoplasma gondii is an obligate intracellular parasite and an important human pathogen. Relatively little is known about the proteins that orchestrate host cell invasion by T. gondii or related apicomplexan parasites (including Plasmodium spp., which cause malaria), due to the difficulty of studying essential genes in these organisms. We have used a recently developed regulatable promoter to create a conditional knockout of T. gondii apical membrane antigen-1 (TgAMA1). TgAMA1 is a transmembrane protein that localizes to the parasite's micronemes, secretory organelles that discharge during invasion. AMA1 proteins are conserved among apicomplexan parasites and are of intense interest as malaria vaccine candidates. We show here that T. gondii tachyzoites depleted of TgAMA1 are severely compromised in their ability to invade host cells, providing direct genetic evidence that AMA1 functions during invasion. The TgAMA1 deficiency has no effect on microneme secretion or initial attachment of the parasite to the host cell, but it does inhibit secretion of the rhoptries, organelles whose discharge is coupled to active host cell penetration. The data suggest a model in which attachment of the parasite to the host cell occurs in two distinct stages, the second of which requires TgAMA1 and is involved in regulating rhoptry secretion.
The RNA-binding Protein Fragile X-related 1 Regulates Somite Formation inXenopus laevis
Authors: Huot Marc-Etienne , Bisson Nicolas , Davidovic Laetitia , Mazroui Rachid , Labelle Yves , Moss Tom , Khandjian Edouard W.
Published: July 2005

Fragile X-related 1 protein (FXR1P) is a member of a small family of RNA-binding proteins that includes the Fragile X mental retardation 1 protein (FMR1P) and the Fragile X-related 2 protein (FXR2P). These proteins are thought to transport mRNA and to control their translation. While FMR1P is highly expressed in neurons, substantial levels of FXR1P are found in striated muscles and heart, which are devoid of FMRP and FXR2P. However, little is known about the functions of FXR1P. We have isolated cDNAs for Xenopus Fxr1 and found that two specific splice variants are conserved in evolution. Knockdown of xFxr1p in Xenopus had highly muscle-specific effects, normal MyoD expression being disrupted, somitic myotomal cell rotation and segmentation being inhibited, and dermatome formation being abnormal. Consistent with the absence of the long muscle-specific xFxr1p isoform during early somite formation, these effects could be rescued by both the long and short mRNA variants. Microarray analyses showed that xFxr1p depletion affected the expression of 129 known genes of which 50% were implicated in muscle and nervous system formation. These studies shed significant new light on Fxr1p function(s).
Sprouty-2 Overexpression in C2C12 Cells Confers Myogenic Differentiation Properties in the Presence of FGF2
Authors: de Alvaro Cristina , Martinez Natalia , Rojas Jose M. , Lorenzo Margarita
Published: July 2005

Myoblast C2C12 cells cultured in the presence of FGF2 actively proliferate and showed a differentiation-defective phenotype compared with cells cultured in low serum or in the presence of insulin. These FGF2 effects are associated with sustained activation of p44/p42-MAPK and lack of activation of AKT. Here we demonstrate that Sprouty-2, a protein involved in the negative feedback of receptor tyrosine kinase signaling, when stably overexpressed in C2C12 cells and in the presence of FGF2 produces growth arrest (precluding the expression of PCNA and the phosphorylation of retinoblastoma and inducing the expression of p21CIP) and myogenesis (multinucleated myotubes formation, induction of creatine kinase and expression of myosin heavy chain protein). These events were accompanied by repression of p44/p42-MAPK and activation of AKT. When C2C12 cells were stably transfected with a Sprouty-2 (Y55F) mutant defective in inhibiting p44/p42-MAPK activation by FGF, myoblasts in the presence of FGF continue to grow and completely fail to form myotubes. This work is the first evidence of the contribution of sprouty genes to myogenic differentiation in the presence of FGF2.
FemaleBicyclus anynanabutterflies choose males on the basis of their dorsal UV-reflective eyespot pupils
Authors: Robertson Kendra A , Monteiro Antónia
Published: July 2005

Sexual and natural selection pressures are thought to shape the characteristic wing patterns of butterfly species. Here we test whether sexual selection by female choice plays a role in the maintenance of the male wing pattern in the butterflyBicyclus anynana. We perform one of the most extensive series of wing pattern manipulations in butterflies, dissecting every component of the ‘bulls-eye’ eyespot patterns in both ventral and dorsal wing surfaces of males to test the trait's appeal to females. We conclude that females select males on the basis of the size and brightness of the dorsal eyespot's ultraviolet reflecting pupils. Pupil absence is strongly selected against, as are artificially enlarged pupils. Small to intermediate (normal sized) pupils seem to function equally well. This work contradicts earlier experiments that suggest that the size of dorsal eyespots plays a role in female choice and explains why male dorsal eyespots are very variable in size and often have indistinct rings of coloration, as the only feature under selection by females seems to be the central white pupil. We propose that sexual selection by female choice, rather than predator avoidance, may have been an important selective factor in the early stages of eyespot evolution in ancestral Lepidopteran lineages.
Characterization and conservation of genetic diversity in subdivided populations
Authors: Toro M.A , Caballero A
Published: July 2005

We review the available tools for analysing genetic diversity in conservation programmes of subdivided populations. Ways for establishing conservation priorities have been developed in the context of livestock populations, both from the classical population genetic analysis and from the more recent Weitzman's approach. We discuss different reasons to emphasize either within or between-population variation in conservation decisions and the methodology to establish some compromise. The comparison between neutral and quantitative variation is reviewed from both theoretical and empirical points of view, and the different procedures for the dynamic management of conserved subdivided populations are discussed.
Genetic characterization of the β-glucuronidase enzyme from a human intestinal bacterium, Ruminococcus gnavus
Authors: Beaud Diane , Tailliez Patrick , Anba-Mondoloni Jamila
Published: July 2005

β-Glucuronidase activity (encoded by thegusgene) has been characterized for the first time fromRuminococcus gnavusE1, an anaerobic bacterium belonging to the dominant human gut microbiota.β-Glucuronidase activity plays a major role in the generation of toxic and carcinogenic metabolites in the large intestine, as well as in the absorption and enterohepatic circulation of many aglycone residues with protective effects, such as lignans, flavonoids, ceramide and glycyrrhetinic acid, that are liberated by the hydrolysis of the corresponding glucuronides. The complete nucleotide sequence of a 4537 bp DNA fragment containing theβ-glucuronidase locus fromR. gnavusE1 was determined. Five ORFs were detected on this fragment: three complete ORFs (ORF2,gusand ORF3) and two partial ORFs (ORF4 and ORF5). The products of ORF2 and ORF3 show strong similarities with manyβ-glucoside permeases of the phosphoenolpyruvate : β-glucoside phosphotransferase systems (PTSs), such asEscherichia coliBglC,Bacillus subtilisBglP andBacillus haloduransPTS Enzyme II. The product of ORF5 presents strong similarities with the amino-terminal domain ofClostridium acetobutylicumβ-glucosidase (bglA). Thegusgene product presents similarities with several knownβ-glucuronidase enzymes, including those ofLactobacillus gasseri(69 %),E. coli(61 %),Clostridium perfringens(59 %) andStaphylococcus aureus(58 %). By complementing anE. colistrain in which theuidAgene encoding the enzyme was deleted, it was confirmed that theR. gnavus gusgene encodes theβ-glucuronidase enzyme. Moreover, it was found that thegusgene was transcribed as part of an operon that includes ORF2, ORF3 and ORF5.
Prc protease promotes mucoidy in mucA mutants of Pseudomonas aeruginosa
Authors: Reiling S. A. , Jansen J. A. , Henley B. J. , Singh S. , Chattin C. , Chandler M. , Rowen D. W.
Published: July 2005

Mucoid strains of Pseudomonas aeruginosa that overproduce the exopolysaccharide alginate are a frequent cause of chronic respiratory infections in cystic fibrosis (CF) patients. The overproduction of alginate by these strains is often caused by mutations within mucA of the algU mucABCD gene cluster. This gene cluster encodes an extreme stress response system composed of the ECF alternative sigma factor AlgU, the anti-sigma factor MucA located in the inner membrane and the negative regulator MucB located in the periplasm. Most of the mutations in mucA found in mucoid strains cause a truncation of the C-terminal, periplasmic domain of MucA. The most significant effect of these mutations appears to be to reduce the levels of MucA. PA3257 (prc) was identified as a regulator of alginate production in P. aeruginosa through the isolation and study of mutations that partially suppressed the mucoid phenotype of a mucA22 strain. The suppressor of mucoidy (som) mutants isolated produced very little alginate when grown on LB medium, but were still mucoid when grown on Pseudomonas isolation agar. These som mutations and another previously isolated suppressor mutation were complemented by cosmids or plasmids carrying PA3257. PA3257 is predicted to encode a periplasmic protease similar to Prc or Tsp of Escherichia coli. Sequencing of prc from three strains with som suppressor mutations confirmed that each had a mutation within the prc coding region. The authors propose that Prc acts to degrade mutant forms of MucA. Additional evidence in support of this hypothesis is: (1) transcription from the AlgU-regulated algD reporter was reduced in som mutants; (2) inactivation of prc affected alginate production in mucoid strains with other mucA mutations found in CF isolates; (3) inactivation or overexpression of prc did not affect alginate production in strains with wild-type MucA.
Multiple genotypes of Chlamydia pneumoniae identified in human carotid plaque
Authors: Cochrane Melanie , Walker Philip , Gibbs Harry , Timms Peter
Published: July 2005

Chlamydia pneumoniae is an obligate intracellular respiratory pathogen that causes 10 % of community-acquired pneumonia and has been associated with cardiovascular disease. Both whole-genome sequencing and specific gene typing suggest that there is relatively little genetic variation in human isolates of C. pneumoniae. To date, there has been little genomic analysis of strains from human cardiovascular sites. The genotypes of C. pneumoniae present in human atherosclerotic carotid plaque were analysed and several polymorphisms in the variable domain 4 (VD4) region of the outer-membrane protein-A (ompA) gene and the intergenic region between the ygeD and uridine kinase (ygeD–urk) genes were found. While one genotype was identified that was the same as one reported previously in humans (respiratory and cardiovascular), another genotype was found that was identical to a genotype from non-human sources (frog/koala).
Recovery from long-term stationary phase and stress survival in Escherichia coli require the l-isoaspartyl protein carboxyl methyltransferase at alkaline pH
Authors: Hicks Wade M. , Kotlajich Matthew V. , Visick Jonathan E.
Published: July 2005

Thel-isoaspartyl protein carboxyl methyltransferase (pcm) can stimulate repair of isoaspartyl residues arising spontaneously in proteins to normall-aspartyl residues. PCM is needed inEscherichia colifor maximal long-term survival when exposed to oxidative stress, osmotic stress, repeated heat stress or methanol. The effect of pH on apcmmutant during long-term stationary phase was examined. PCM was not required for long-term survival ofE. colisubjected to pH stress alone; however, PCM-deficient cells showed impaired resistance to paraquat and methanol only at elevated pH. The mutant also showed stress-survival phenotypes in minimal medium buffered to pH 9·0. Accumulation of isoaspartyl residues was accelerated at pH 8·0 or 9·0in vivo, though PCM-deficient cells did not show higher levels of damage. However, thepcmmutant displayed an extended lag phase in recovering from stationary phase at pH 9·0. Protein repair by PCM thus plays a key role in long-term stress survival only at alkaline pH inE. coli, and it may function primarily to repair damage in cells that are recovering from nutrient limitation and in those cells that are able to divide during long-term stationary phase.
Application of multilocus sequence analysis (MLSA) for rapid identification of Enterococcus species based on rpoA and pheS genes
Authors: Naser Sabri M. , Thompson Fabiano L. , Hoste Bart , Gevers Dirk , Dawyndt Peter , Vancanneyt Marc , Swings Jean
Published: July 2005

The aim of this study was to evaluate the use of RNA polymeraseαsubunit (rpoA) and phenylalanyl-tRNA synthase (pheS) gene sequences as species identification tools for enterococci. Ninety-six representative strains comprising all currently recognizedEnterococcusspecies were examined.rpoAgene sequences generated a robust classification into species groups similar to the one based on 16S rRNA gene sequence analysis. On the other hand, thepheSgene is a fast-evolving clock even better suited for species delineation than therpoAgene, but not for recognition of species groups withinEnterococcusas determined by bothrpoAand 16S rRNA genes. All enterococcal species were clearly differentiated on the basis of theirrpoAandpheSsequences. Evaluation of intraspecies variation showed that bothrpoAandpheSgenes have a high degree of homogeneity among strains of the same species. Strains of the same enterococcal species have at least 99 %rpoAand 97 %pheSgene sequence similarity, whereas, different enterococcal species have at maximum 97 %rpoAand 86 %pheSgene sequence similarity. It was concluded that both genes can be used as reliable tools for identification of clinical and environmental species ofEnterococcusand are efficient screening methods for the detection of novel species. The sequence data obtained in this study were compared to the availableatpAand 16S rRNA gene sequences. The MLSA approach toEnterococcustaxonomy provides portable, highly reproducible data with lower costs for rapid identification of all enterococcal species.
Phage therapy: the Escherichia coli experience
Authors: Brüssow Harald
Published: July 2005

Phages have been proposed as natural antimicrobial agents to fight bacterial infections in humans, in animals or in crops of agricultural importance. Phages have also been discussed as hygiene measures in food production facilities and hospitals. These proposals have a long history, but are currently going through a kind of renaissance as documented by a spate of recent reviews. This review discusses the potential of phage therapy with a specific example, namelyEscherichia coli.
Escherichia coli tol and rcs genes participate in the complex network affecting curli synthesis
Authors: Vianney Anne , Jubelin Grégory , Renault Sophie , Dorel Corine , Lejeune Philippe , Lazzaroni Jean Claude
Published: July 2005

Curli are necessary for the adherence of Escherichia coli to surfaces, and to each other, during biofilm formation, and the csgBA and csgDEFG operons are both required for their synthesis. A recent survey of gene expression in Pseudomonas aeruginosa biofilms has identified tolA as a gene activated in biofilms. The tol genes play a fundamental role in maintaining the outer-membrane integrity of Gram-negative bacteria. RcsC, the sensor of the RcsBCD phosphorelay, is involved, together with RcsA, in colanic acid capsule synthesis, and also modulates the expression of tolQRA and csgDEFG. In addition, the RcsBCD phosphorelay is activated in tol mutants or when Tol proteins are overexpressed. These results led the authors to investigate the role of the tol genes in biofilm formation in laboratory and clinical isolates of E. coli. It was shown that the adherence of cells was lowered in the tol mutants. This could be the result of a drastic decrease in the expression of the csgBA operon, even though the expression of csgDEFG was slightly increased under such conditions. It was also shown that the Rcs system negatively controls the expression of the two csg operons in an RcsA-dependent manner. In the tol mutants, activation of csgDEFG occurred via OmpR and was dominant upon repression by RcsB and RcsA, while these two regulatory proteins repressed csgBA through a dominant effect on the activator protein CsgD, thus affecting curli synthesis. The results demonstrate that the Rcs system, previously known to control the synthesis of the capsule and the flagella, is an additional component involved in the regulation of curli. Furthermore, it is shown that the defect in cell motility observed in the tol mutants depends on RcsB and RcsA.
Clostridium pasteurianum W5 synthesizes two NifH-related polypeptides under nitrogen-fixing conditions
Authors: Kasap Murat , Chen Jiann-Shin
Published: July 2005

Previous studies identified fivenifH-like genes (nifH2throughnifH6) inClostridium pasteurianum(strain W5), where thenifH1gene encodes the nitrogenase iron protein. Transcripts of thesenifHgenes, with the exception ofnifH3, were detected in molybdenum-sufficient nitrogen-fixing cells. However, the size of the transcripts, the level of transcription and the presence of polypeptides encoded by thenifH-like genes were not reported. ThenifH2andnifH6genes were extremely similar, as they seemed to differ by only two bases in a span of 2481 bp, one in the coding region and another in the upstream region. Re-examination of the DNA sequences revealed that the coding region ofnifH2andnifH6was identical, whereas the difference in the upstream region was confirmed. Results from the authors' ongoing study of thenifgenes of single-colony isolates ofC. pasteurianumsuggest that thenifH6designation should be eliminated. Here the size of mRNA fromnifH2and the detection of the NifH2 polypeptide in nitrogen-fixing cells ofC. pasteurianumare reported. Northern blot analysis of periodically collected nitrogen-fixing cells showed that thenifH1andnifH2mRNAs were present throughout growth. Addition of ammonium acetate repressed the transcription of both these genes similarly. Using an antiserum raised against NifH ofAzotobacter vinelandii, two NifH-related bands were detected by Western blot analysis after electrophoretic separation of proteins in extracts of nitrogen-fixingC. pasteurianumcells. After separation of proteins by preparative SDS-PAGE, the NifH polypeptides were characterized by MALDI-TOF-MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) and by ES-MS/MS (electrospray tandem mass spectrometry) analyses. The results confirmed the presence of NifH2, in addition to NifH1, in nitrogen-fixingC. pasteurianumcells.
Functional analysis of the lysis genes of Staphylococcus aureus phage P68 in Escherichia coli
Authors: Takáč Marian , Witte Angela , Bläsi Udo
Published: July 2005

Double-stranded DNA phages of both Gram-positive and Gram-negative bacteria typically use a holin–endolysin system to achieve lysis of their host. In this study, the lysis genes of Staphylococcus aureus phage P68 were characterized. P68 gene lys16 was shown to encode a cell-wall-degrading enzyme, which causes cell lysis when externally added to clinical isolates of S. aureus. Another gene, hol15, was identified embedded in the −1 reading frame at the 3′ end of lys16. The deduced Hol15 protein has three putative transmembrane domains, and thus resembles class I holins. An additional candidate holin gene, hol12, was found downstream of the endolysin gene lys16 based on two predicted transmembrane domains of the encoded protein, which is a typical trait of class II holins. The synthesis of either Hol12 or Hol15 resulted in growth retardation of Escherichia coli, and both hol15 and hol12 were able to complement a phage λ
Sam mutation. The hol15 gene has a dual start motif beginning with the codons Met1-Lys2-Met3…. Evidence is presented that the hol15 gene encodes a lysis inhibitor (anti-holin) and a lysis effector (actual holin). As depolarization of the membrane converted the anti-holin to a functional holin, these studies suggested that hol15 functions as a typical dual start motif class I holin. The unusual arrangement of the P68 lysis genes is discussed.
Campylobacter jejuni inhibits the absorptive transport functions of Caco-2 cells and disrupts cellular tight junctions
Authors: MacCallum Amanda , Hardy Simon P. , Everest Paul H.
Published: July 2005

Caco-2 cells are models of absorptive enterocytes. The net transport of fluid from apical to basolateral surfaces results in ‘domes' forming in differentiated monolayers. Here, the effect ofCampylobacter jejunion this process has been examined.C. jejunicaused no changes in short-circuit current upon infection of Caco-2 cell monolayers in Ussing chambers. Thus, no active secretory events could be demonstrated using this model. It was therefore hypothesized thatC. jejunicould inhibit the absorptive function of enterocytes and that this may contribute to diarrhoeal disease.C. jejuniinfection of fluid-transporting (‘doming’) Caco-2 cells resulted in a significant reduction in dome number, which correlated with a decrease in tight junction integrity in infected monolayers, when measured as transepithelial electrical resistance. Defined mutants ofC. jejunialso reduced dome numbers in infected monolayers.C. jejunialso altered the distribution of the tight junction protein occludin within cell monolayers. The addition to monolayers of extracellular gentamicin prevented these changes, indicating the contribution of extracellular bacteria to this process. Thus, tight junction integrity is required for fluid transport in Caco-2 cell monolayers as leaky tight junctions cannot maintain support of transported fluid at the basolateral surface of infected cell monolayers. Inhibition of absorptive cell function, changes in epithelial resistance and rearrangement of tight junctional proteins such as occludin represent a potential diarrhoeal mechanism ofC. jejuni.
Role of type III secretion in Edwardsiella tarda virulence
Authors: Tan Y. P. , Zheng J. , Tung S. L. , Rosenshine I. , Leung K. Y.
Published: July 2005

Edwardsiella tarda is a Gram-negative enteric bacterium affecting both animals and humans. Recently, a type III secretion system (TTSS) was found in Ed. tarda. Such systems are generally used by bacterial pathogens to deliver virulence factors into host cells to subvert normal cell functions. Genome-walking was performed from the eseB and esrB genes (homologues of Salmonella sseB and ssrB, respectively) identified in previous studies, to determine the sequences of the TTSS. Thirty-five ORFs were identified which encode the TTSS apparatus, chaperones, effectors and regulators. Mutants affected in genes representing each category were generated and found to have decreased survival and growth in fish phagocytes. LD50 values of the mutants were increased by at least 10-fold in comparison to those of the wild-type strain. The adherence and invasion rates of the esrA and esrB mutants were enhanced while those of the other mutants remained similar to the wild-type. The eseC and eseD mutants showed slight autoaggregation in Dulbecco's Modified Eagle Medium, whereas the rest of the mutants failed to autoaggregate. Regulation of the TTSS was found to involve the two-component regulatory system esrA–esrB. This study showed that the TTSS is important for Ed. tarda pathogenesis. An understanding of this system will provide greater insight into the virulence mechanisms of this bacterial pathogen.
Databases and software for the comparison of prokaryotic genomes
Authors: Field Dawn , Feil Edward J. , Wilson Gareth A.
Published: July 2005

The explosion in the number of complete genomes over the past decade has spawned a new and exciting discipline, that of comparative genomics. To exploit the full potential of this approach requires the development of novel algorithms, databases and software which are sophisticated enough to draw meaningful comparisons between complete genome sequences and are widely accessible to the scientific community at large. This article reviews progress towards the development of computational tools and databases for organizing and extracting biological meaning from the comparison of large collections of genomes.
High-level, inducible gene expression in Lactobacillus sakei and Lactobacillus plantarum using versatile expression vectors
Authors: Sørvig Elisabeth , Mathiesen Geir , Naterstad Kristine , Eijsink Vincent G. H. , Axelsson Lars
Published: July 2005

Vectors have been developed for inducible gene expression inLactobacillus sakeiandLactobacillus plantarumin which expression of the gene of interest is driven by strong, regulated promoters from bacteriocin operons found inL. sakeistrains. The activity of these promoters is controlled via a two-component signal transduction system, which responds to an externally added peptide pheromone. The vectors have a modular design, permitting easy exchange of all essential elements: the inducible promoter, the cognate regulatory system, the gene of interest, the antibiotic resistance marker and the replicon. Various variants of these so-called ‘pSIP’ vectors were constructed and tested, differing in terms of the bacteriocin regulon from which the regulatory elements were derived (sakacin A or sakacin P), the regulated promoter selected from these regulons, and the replicon (derived from p256 or pSH71). Usingβ-glucuronidase (GusA) and aminopeptidase N (PepN) as reporters, it was shown that the best vectors permitted inducible, pheromone-dose-dependent gene expression at very high levels, while displaying moderate basal activities when not induced. The most effective set-up was obtained using a vector containing the pSH71 replicon, theorfXpromoter from the sakacin P regulon, and the cognate regulatory genes, in aL. sakeihost. GusA levels obtained with this set-up were approximately ten times higher than the levels obtained with prototype pSIP versions, whereas PepN levels amounted to almost 50 % of total cellular protein.
The chemoresistance gene ABCG2 (MXR/BCRP1/ABCP1) is not expressed in melanomas but in single neuroendocrine carcinomas of the skin
Authors: Deichmann Martin , Thome Marianne , Egner Ursula , Hartschuh Wolfgang , Kurzen Hjalmar
Published: July 2005

Background:  As the molecular mechanisms in melanoma chemoresistance remain unknown to date, we hypothesized these tumors to express the ATP‐binding cassette (ABC) transporter G2 (ABCG2/MXR/BCRP1/ABCP1), a recently detected membrane transporter and putative stem‐cell marker. Besides melanoma, we addressed the neuroendocrine carcinoma of the skin (Merkel cell carcinoma), another cutaneous cancer supposed to originate from neuroectoderm and usually chemoresistant.

Methods and Results:  Upon semiquantitative reverse transcription polymerase chain reaction, ABCG2 mRNA expression was not upregulated in 18 melanoma resection specimens when compared with 19 acquired melanocytic nevi from which melanomas are known to often arise (Mantel–Haenszel test, p = 0.3). At protein level, immunohistochemistry was negative in all 66 investigated melanoma resection specimens (50 primary melanomas and 16 cutaneous/subcutaneous metastases) and in 19 acquired melanocytic nevi. Among 29 neuroendocrine carcinomas of the skin, ABCG2 protein was detected in single clusters of cells in three tumors. As a positive control, three dermatofibrosarcomas were also stained and showed ABCG2 protein expression of the endothelial cells of the blood vessels.

Conclusion:  Altogether, chemoresistance of melanomas and neuroendocrine carcinomas of the skin cannot be explained by expression of the ABCG2‐chemoresistance gene. Most of these tumors do not exhibit this potential stem‐cell feature.
Histopathologic comparison of nephrogenic fibrosing dermopathy and scleromyxedema
Authors: Kucher Cynthia , Xu Xiaowei , Pasha Theresa , Elenitsas Rosalie
Published: July 2005

Background:  Nephrogenic fibrosing dermopathy (NFD) clinically presents as indurated plaques and papules in patients with renal dysfunction. The differential diagnosis generally includes scleromyxedema (SMX), an idiopathic systemic disorder with cutaneous manifestations, in which patients also develop indurated papules and plaques. The two entities can be extremely difficult to distinguish microscopically. Histopathologic differences with immunophenotypic comparison, to our knowledge, have not been thoroughly studied. We compared these two entities with an emphasis on immunohistochemistry.

Design:  Nine biopsies diagnosed as NFD and seven biopsies diagnosed as SMX were retrospectively collected from the University of Pennsylvania Medical Center's surgical pathology and dermatopathology archives. Immunohistochemical staining for CD34, factor XIIIa, CD31, smooth muscle actin, CD68, and procollagen‐I, as well as colloidal iron, were performed on each biopsy. Amount of expression for each of these markers, as well as degree of inflammation, for each biopsy was evaluated using a grading system of 0–3.

Results:  Overall, NFD and SMX showed similar expression for all markers except procollagen‐I, which showed increased expression in SMX.

Discussion:  Although some immunophenotypic differences were found, our study did not demonstrate microscopic characteristics that can be easily used diagnostically to distinguish NFD from SMX. Clinical pathologic correlation is paramount in distinguishing these two entities.
Anti‐cytokeratin 20 staining of Merkel cells helps differentiate basaloid proliferations overlying dermatofibromas from basal cell carcinoma
Authors: Mahmoodi Mandana , Asad Haider , Salim Sadia , Kantor Gary , Minimo Corrado
Published: July 2005

Background:  Basaloid epidermal proliferations (BEP), morphologically resembling basal cell carcinoma (BCC), have been described overlying dermatofibromas. Distinguishing the two is important because of non‐aggressiveness of BEP and local aggressiveness of BCC. The aim of this study is to determine whether CK20 antibody staining for Merkel cells can be used as an adjunct method to differentiate BEP from BCC.

Methods:  Ten cases of BEP overlying dermatofibromas were selected. Ten cases of BCC were used as control. The two groups were stained with CK20 antibody. Numerical density of CK20 stained Merkel cells in peri‐lesional epidermis, BEP and BCC was determined by examining 300 cells at 400X in two separate areas by three independent pathologists. To determine statistical significance, the results were compared using t‐test method.

Results:  Density of Merkel cells in peri‐lesional epidermis was 0.2–0.3%. No merkel cells were detected in the BCC. BEP overlying dermatofibromas showed an obvious increase in CK 20 stained Merkel cells. The difference was statistically significant (P < 0.02)

Conclusions:  We report a significant increase in CK20 stained Merkel cells in BEP overlying dermatofibromas as compared to BCC. CK20 antibody staining for Merkel cells can be used as an adjunct method to differentiate BEP overlying dermatofibromas from BCC.
Autologous serum skin test reactivity in patients with non‐allergic asthma
Authors: Tedeschi A. , Comi A. L. , Lorini M. , Tosini C. , Miadonna A.
Published: July 2005

Background
Inflammatory alterations of respiratory airways have been found in patients with non‐allergic asthma, but the triggering event has not been defined. An autoimmune activation of inflammatory cells has been hypothesized.

Objective
To evaluate whether histamine‐releasing factors are present in sera from non‐allergic asthmatics.

Methods
Twenty‐four patients with non‐allergic asthma underwent in vivo autologous serum skin test (ASST) and in vitro basophil histamine release assay using autologous basophils as well as basophils from normal donors. Twenty‐seven subjects with respiratory allergy and three normal subjects were chosen as control.

Results
ASST was positive in 14/24 non‐allergic asthmatics (58%) whereas it was negative in all 30 control subjects (P<0.001). The serum of only one ASST‐positive patient out of 12 (8.4%) induced in vitro histamine release from autologous basophils. The serum from another ASST‐positive patient induced histamine release from membrane IgE‐stripped autologous basophils. Sera from either non‐allergic asthmatics or from control subjects did not provoke significant histamine release from basophils from three normal donors.

Conclusion
Skin reactivity to autologous serum is common among non‐allergic asthmatics, indicating the presence of circulating histamine‐releasing factors. However, only in a minority of patients in vitro functional evidence of histamine‐releasing autoantibodies (anti‐FcɛRI or anti‐IgE) was obtained. The presence of circulating histamine‐releasing factors might contribute to initiation/maintenance of inflammation in respiratory airways of non‐allergic asthmatics.
High levels and gender difference of exhaled nitric oxide in Chinese schoolchildren
Authors: Wong G. W. K. , Liu E. K. H. , Leung T. F. , Yung E. , Ko F. W. S. , Hui D. S. C. , Fok T. F. , Lai C. K. W.
Published: July 2005

Background
Exhaled nitric oxide (eNO) may represent a useful noninvasive marker of airway inflammation, but data on the reference population values in schoolchildren are limited. No reference eNO study in Asian children has been published.

Methods
Levels of eNO in a sample of 531 schoolchildren aged 11–18 years recruited from five schools (three international schools) in Hong Kong were measured online by a chemiluminescence analyser according to ERS/ATS standard. Each student also completed an International Study of Asthma and Allergic disease in Childhood questionnaire.

Results
Among the children without a physician's diagnosis of asthma or symptoms of wheeze, rhinitis and eczema, there were 258 Chinese and 33 Caucasians. In control Chinese children, the eNO level (median: interquantile range) was significantly higher (P<0.001) in males (17.0 parts per billion (p.p.b.); 10.7–36.6) than in females (10.8 p.p.b.; 7.8–17.6). When compared with Caucasian control males (11.6 p.p.b.; 8.2–19.3) and females (9.1 p.p.b.; 7.5–11.9), the Chinese children had significantly higher eNO levels for both males (P=0.011) and females (P=0.037). For Chinese asthmatic males, the median eNO (interquartile range) was 39.8 p.p.b. (12.5–73.8), and for asthmatic females, 18.0 (9.6–56.3). After controlling for sex in Chinese controls, eNO did not have any significant correlation with height, weight and body mass index or body surface area.

Conclusions
This study demonstrates a gender difference of eNO level in healthy Chinese schoolchildren. When compared with Caucasians, Chinese children have significantly higher eNO levels.
Association of CARD15 polymorphisms with atopy‐related traits in a population‐based cohort of Caucasian adults
Authors: Weidinger S. , Klopp N. , Rümmler L. , Wagenpfeil S. , Baurecht H. J. , Gauger A. , Darsow U. , Jakob T. , Novak N. , Schäfer T. , Heinrich J. , Behrendt H. , Wichmann H. E. , Ring J. , Illig T. ,   for the KORA study group
Published: July 2005

Background Influences of microbial pathogens are crucial for the maturation of the immune system. Caspase‐recruitment domain containing protein 15 (CARD15) is a cytosolic receptor involved in bacterial recognition by antigen‐presenting cells. CARD15 polymorphisms have been associated with Crohn's disease. Recently, associations with atopic phenotypes have been reported in children.

Objective Within a large population of German adults (n=1875), we evaluated eight CARD15 polymorphisms for associations with atopic phenotypes.

Methods Subjects were phenotyped by standardized questionnaires and interviews as well as total and allergen‐specific IgE measurements. Genotyping was performed using matrix‐assisted laser desorption ionization – time of flight mass spectrometry. Haplotypes were estimated using the SAS/Genetics module.

Results Subjects with a T allele at rs1077861 had a decreased risk of developing asthma (odds ratio OR=0.648, P=0.013), whereas the presence of an A allele at rs3135500 was significantly associated with an increased risk (OR=1.374, P=0.023). In addition, a CARD15 haplotype revealed to be protective against the development of asthma (OR=0.326, P=0.003). Subjects with an A allele at position rs5743266 or a T allele at rs2066842 had a significantly decreased risk of developing allergic rhinoconjunctivitis with ORs of 0.820 (P=0.049) and 0.801 (P=0.025). Polymorphism rs2066845 showed a significant association with increased total serum IgE (OR=2.155, P=0.006).

Conclusion Genetic variants of CARD15 that might result in inappropriate immunomodulation are not only associated with autoimmune diseases but also with atopic disorders.
Changes in indoor allergen and fungal levels predict changes in asthma activity among young adults
Authors: Matheson M. C. , Abramson M. J. , Dharmage S. C. , Forbes A. B. , Raven J. M. , Thien F. C. K. , Walters E. H.
Published: July 2005

Background
Exposures to allergens are thought to be important risk factors for asthma. We conducted a longitudinal study of indoor allergen and fungal levels in Melbourne homes between 1996 and 1998 to examine the effect of changes in allergen exposure upon asthma and associated outcomes.

Methods
Participants were visited at home in 1996 (n=485) and 1998 (n=360), when dust and air samples were collected from their bedrooms and assayed for Der p 1, Fel d 1, ergosterol and fungal propagules. Subjects then attended the lung function laboratory to complete a questionnaire, spirometry, methacholine challenge and skin prick testing. The associations between the change in allergen levels and change in clinical outcomes were examined using multiple logistic and linear regression.

Results
Participants whose Cladosporium fungal exposure doubled had 52% greater odds of having had an attack of asthma in the last 12 months. A doubling of fungal exposure was also associated with 53% greater odds of developing atopy. A doubling of Fel d 1 floor levels was associated with 73% increased odds of doctor‐diagnosed asthma. A doubling of Der p 1 levels in bed dust was associated with a 64% greater odds of persistent bronchial hyper‐reactivity.

Conclusions
These findings provide evidence that changes in indoor levels of fungi and house dust mites can affect the risk of development and persistence of asthma and atopy in adults. Further studies are required to establish any benefit of sustained reductions in indoor allergen exposures, and to determine whether these effects are truly ‘allergic’ or because of immune stimulation in the airway through other less specific mechanisms.