bluuebunny/crossref_metadata_2025 huggingface dataset:
Consequences of reductive evolution for gene expression in an obligate endosymbiont
Authors: Wilcox Jennifer L. , Dunbar Helen E. , Wolfinger Russell D. , Moran Nancy A.
Published: June 2003

The smallest cellular genomes are found in obligate symbiotic and pathogenic bacteria living within eukaryotic hosts. In comparison with large genomes of free‐living relatives, these reduced genomes are rearranged and have lost most regulatory elements. To test whether reduced bacterial genomes incur reduced regulatory capacities, we used full‐genome microarrays to evaluate transcriptional response to environmental stress in Buchnera aphidicola, the obligate endosymbiont of aphids. The 580 genes of the B. aphidicola genome represent a subset of the 4500 genes known from the related organism, Escherichia coli. Although over 20 orthologues of E. coli heat stress (HS) genes are retained by B. aphidicola, only five were differentially expressed after near‐lethal heat stress treatments, and only modest shifts were observed. Analyses of upstream regulatory regions revealed loss or degradation of most HS (σ32) promoters. Genomic rearrangements downstream of an intact HS promoter yielded upregulation of a functionally unrelated and an inactivated gene. Reanalyses of comparable experimental array data for E. coli and Bacillus subtilis revealed that genome‐wide differential expression was significantly lower in B. aphidicola. Our demonstration of a diminished stress response validates reports of temperature sensitivity in B. aphidicola and suggests that this reduced bacterial genome exhibits transcriptional inflexibility.
The large linear plasmid pSLA2‐L of Streptomyces rochei has an unusually condensed gene organization for secondary metabolism
Authors: Mochizuki Susumu , Hiratsu Keiichiro , Suwa Masanori , Ishii Tatsuya , Sugino Fuminori , Yamada Kohei , Kinashi Haruyasu
Published: June 2003

The complete nucleotide sequence of the large linear plasmid pSLA2‐L in Streptomyces rochei strain 7434AN4 has been determined. pSLA2‐L was found to be 210 614 bp long with a GC content of 72.8% and carries 143 open reading frames. It is especially noteworthy that three‐quarters of the pSLA2‐L DNA is occupied by secondary metabolism‐related genes, namely two type I polyketide synthase (PKS) gene clusters for lankacidin and lankamycin, a mithramycin synthase‐like type II PKS gene cluster, a carotenoid biosynthetic gene cluster and many regulatory genes. In particular, the lankacidin PKS is unique, because it may be a mixture of modular‐ and iterative‐type PKSs and carries a fusion protein of non‐ribosomal peptide synthetase and PKS. It is also interesting that all the homologues of the afsA, arpA, adpA and strR genes in the A‐factor regulatory cascade in Streptomyces griseus were found on pSLA2‐L, and disruption of the afsA homologue caused non‐production of both lankacidin and lankamycin. These results, together with the finding of three possible replication origins at 50–63 kb from the right end, suggest that the present form of pSLA2‐L might have been generated by a series of insertions of the biosynthetic gene clusters into the left side of the original plasmid.
Alternative roles of ClpX and ClpP in Staphylococcus aureus stress tolerance and virulence
Authors: Frees Dorte , Qazi Saara N. A. , Hill Philip J. , Ingmer Hanne
Published: June 2003

Clp proteolytic complexes are essential for virulence and for survival under stress conditions in several pathogenic bacteria. Recently, a study using signature‐tagged mutagenesis identified the ClpX ATPase as also being required for virulence in Staphylococcus aureus. Presently, we have constructed deletion mutants removing either ClpX or the proteolytic subunit, ClpP, in S. aureus 8325‐4 in order to examine a putative link between stress tolerance and virulence. When exposed to stress, we found that, although clpP mutant cells were sensitive to conditions generating misfolded proteins, the absence of ClpX improved survival. In the presence of oxidative stress or at low temperature, both ClpP and ClpX were important for growth. Virulence was examined in a murine skin abscess model and was found to be severely attenuated for both mutants. S. aureus pathogenicity is largely dependent on a set of extracellular and cell wall‐associated proteins. In the mutant cells, the amount of α‐haemolysin (hla) and several other extracellular proteins was greatly decreased, and analysis of hla expression revealed that the reduction occurred at the transcriptional level. Essential for transcriptional regulation of hla is the quorum‐sensing agr locus. Interestingly, the absence of ClpX or ClpP reduced both transcription of the agr effector molecule, RNA III, and the activity of the autoinducing peptide (AIP). In addition, ClpX was required independently of ClpP for transcription of spa encoding Protein A. Thus, our results indicate that ClpX and ClpP contribute to virulence by controlling the activity of major virulence factors rather than by promoting stress tolerance.
Biofilm formation by Pseudomonas aeruginosa wild type, flagella and type IV pili mutants
Authors: Klausen Mikkel , Heydorn Arne , Ragas Paula , Lambertsen Lotte , Aaes‐Jørgensen Anders , Molin Søren , Tolker‐Nielsen Tim
Published: June 2003

Biofilm formation by Gfp‐tagged Pseudomonas aeruginosa PAO1 wild type, flagella and type IV pili mutants in flow chambers irrigated with citrate minimal medium was characterized by the use of confocal laser scanning microscopy and comstat image analysis. Flagella and type IV pili were not necessary for P. aeruginosa initial attachment or biofilm formation, but the cell appendages had roles in biofilm development, as wild type, flagella and type IV pili mutants formed biofilms with different structures. Dynamics and selection during biofilm formation were investigated by tagging the wild type and flagella/type IV mutants with Yfp and Cfp and performing time‐lapse confocal laser scanning microscopy in mixed colour biofilms. The initial microcolony formation occurred by clonal growth, after which wild‐type P. aeruginosa bacteria spread over the substratum by means of twitching motility. The wild‐type biofilms were dynamic compositions with extensive motility, competition and selection occurring during development. Bacterial migration prevented the formation of larger microcolonial structures in the wild‐type biofilms. The results are discussed in relation to the current model for P. aeruginosa biofilm development.
Autoinduction and signal transduction in the regulation of staphylococcal virulence
Authors: Novick Richard P.
Published: June 2003

The accessory genes of Staphylococcus aureus, in‐cluding those involved in pathogenesis, are controlled by a complex regulatory network that includes at least four two‐component systems, one of which, agr, is a quorum sensor, an alternative sigma factor and a large set of transcription factors, including at least two of the superantigen genes, tst and seb. These regulatory genes are hypothesized to act in a time‐ and population density‐dependent manner to integrate signals received from the external environment with the internal metabolic machinery of the cell, in order to achieve the production of particular subsets of accessory/virulence factors at the time and in quantities that are appropriate to the needs of the organism at any given location. From the standpoint of pathogenesis, the regulatory agenda is presumably tuned to particular sites in the host organism. To address this hypothesis, it will be necessary to understand in considerable detail the regulatory interactions among the organism's numerous controlling systems. This review is an attempt to integrate a large body of data into the beginnings of a model that will hopefully help to guide research towards a full‐scale test.
Structural, genetic and functional characterization of the flagellin glycosylation process in Helicobacter pylori
Authors: Schirm M. , Soo E. C. , Aubry A. J. , Austin J. , Thibault P. , Logan S. M.
Published: June 2003

Mass spectrometry analyses of the complex polar flagella from Helicobacter pylori demonstrated that both FlaA and FlaB proteins are post‐translationally modified with pseudaminic acid (Pse5Ac7Ac, 5,7‐diacetamido‐3,5,7,9‐tetradeoxy‐l‐glycero‐l‐manno ‐n o n‐ulosonic acid). Unlike Campylobacter, flagellar glycosylation in Helicobacter displays little heterogeneity in isoform or glycoform distribution, although all glycosylation sites are located in the central core region of the protein monomer in a manner similar to that found in Campylobacter. Bioinformatic analysis revealed five genes (HP0840, HP0178, HP0326A, HP0326B, HP0114) homologous to other prokaryote genes previously reported to be involved in motility, flagellar glycosylation or polysaccharide biosynthesis. Insertional mutagenesis of four of these homologues in Helicobacter (HP0178, HP0326A, HP0326B, HP0114) resulted in a non‐motile phenotype, no structural flagella filament and only minor amounts of flagellin protein detectable by Western immunoblot. However, mRNA levels for the flagellin structural genes remained unaffected by each mutation. In view of the combined bioinformatic and structural evidence indicating a role for these gene products in glycan biosynthesis, subsequent investigations focused on the functional characterization of the respective gene products. A novel approach was devised to identify biosynthetic sugar nucleotide precursors from intracellular metabolic pools of parent and isogenic mutants using capillary electrophoresis‐electrospray mass spectrometry (CE‐ESMS) and precursor ion scanning. HP0326A, HP0326B and the HP0178 gene products are directly involved in the biosynthesis of the nucleotide‐activated form of Pse, CMP‐Pse. Mass spectral analyses of the cytosolic extract from the HP0326A and HP0326B isogenic mutants revealed the accumulation of a mono‐ and a diacetamido trideoxyhexose UDP sugar nucleotide precursor.
Identification of nuclear proteins that interact differentially with Plasmodium falciparum var gene promoters
Authors: Voss Till S. , Kaestli Mirjam , Vogel Denise , Bopp Selina , Beck Hans‐Peter
Published: June 2003

The Plasmodium falciparum virulence factor PfEMP1 is responsible for both antigenic variation and cytoadherence of infected erythrocytes in malaria. Approximately 50 var genes per parasite genome code for this highly polymorphic surface protein. We showed recently that chromosome‐central and subtelomeric var genes are controlled by different promoters. Here, we report that transcriptional repression of var genes located in different chromosomal regions occurs by different mechanisms. Subtelomeric var gene transcription is repressed 4–8 h before that of chromosome‐central var genes. Both repression events coincide with the shifted expression of two distinct nuclear proteins binding specifically to conserved sequence motifs, SPE1 and CPE, present in the respective promoter. Furthermore, a reiterated and highly conserved subtelomeric var promoter element (SPE2) interacts with a nuclear factor exclusively expressed during S‐phase. Promoter analysis by transient transfection suggested direct involvement of these interactions in var gene repression and silencing, and identified regions implicated in transcriptional activation of var genes.
Bile salts and fatty acids induce the expression of Escherichia coli AcrAB multidrug efflux pump through their interaction with Rob regulatory protein
Authors: Rosenberg Emiko Y. , Bertenthal Dan , Nilles Matthew L. , Bertrand Kevin P. , Nikaido Hiroshi
Published: June 2003

AcrAB of Escherichia coli, an archetype among bacterial multidrug efflux pumps, exports an extremely wide range of substrates including solvents, dyes, detergents and antimicrobial agents. Its expression is regulated by three XylS/AraC family regulators, MarA, SoxS and Rob. Although MarA and SoxS regulation works by the alteration of their own expression levels, it was not known how Rob, which is constitutively expressed, exerts its regulatory action. We show here that the induction of the AcrAB efflux pump by decanoate and the more lipophilic unconjugated bile salts is mediated by Rob, and that the low‐molecular‐weight inducers specifically bind to the C‐terminal, non‐DNA‐binding domain of Rob. Induction of Rob is not needed for induction of AcrAB, and we suggest that the inducers act by producing conformational alterations in pre‐existing Rob, as was suggested recently (Rosner, Dangi, Gronenborn and Martin, J Bacteriol 184: 1407–1416, 2002). Decanoate and unconjugated bile salts, which are present in the normal habitat of E. coli, were further shown to make the bacteria more resistant to lipophilic antibiotics, at least in part because of the induction of the AcrAB efflux pump. Thus, it is likely that E. coli is protecting itself by the Rob‐mediated upregulation of AcrAB against the harmful effects of bile salts and fatty acids in the intestinal tract.
Aromatic amino acids at the surface of InlB are essential for host cell invasion by Listeria monocytogenes
Authors: Machner Matthias P. , Frese Susanne , Schubert Wolf‐Dieter , Orian‐Rousseau Veronique , Gherardi Ermanno , Wehland Jürgen , Niemann Hartmut H. , Heinz Dirk W.
Published: June 2003

The surface protein InlB of the pathogen Listeria monocytogenes promotes invasion of this bacterium into host cells by binding to and activating the receptor tyrosine kinase Met. The curved leucine‐rich repeat (LRR) domain of InlB, which is essential for this process, contains a string of five surface‐exposed aromatic amino acid residues positioned along its concave face. Here, we show that the replacement of four of these residues (F104, W124, Y170 or Y214) by serine leads to a complete loss of uptake of latex beads coated with InlB′, a truncated functional variant of InlB. The mutants correspondingly display severely reduced binding to Met. To abrogate fully invasion of bacteria expressing full‐length InlB, exchange of at least four aromatic amino acids is required. We conclude that InlB binds to Met through its concave surface of the LRR domain, and that aromatic amino acids are critical for binding and signalling before invasion.
Escherichia coli SspA is a transcription activator for bacteriophage P1 late genes
Authors: Hansen Anne‐Marie , Lehnherr Hansjörg , Wang Xiandong , Mobley Victoria , Jin Ding Jun
Published: June 2003

The stringent starvation protein A (SspA), an Escherichia coli RNA polymerase (RNAP)‐associated protein, has been reported to be essential for lytic growth of bacteriophage P1. Unlike P1 early promoters, P1 late promoters are not recognized by RNAP alone. A phage‐encoded early protein, Lpa (late promoter activator protein, formerly called gp10), has been shown to be required for P1 late transcription in vivo. Here, we demonstrate that SspA is a transcription activator for P1 late genes. Our results indicated that Lpa is not limiting in an sspA mutant. However, the transcription of P1 late genes was deficient in an sspA mutant in vivo. We demonstrated that SspA/Lpa are required for transcription activation of the P1 late promoter Ps in vitro. In addition, SspA and Lpa were shown to facilitate the binding of RNAP to Ps late promoter DNA. Activation of late transcription by SspA/Lpa was dependent on holoenzyme containing σ70 but not σS, indicating that the two activators discriminate between the two forms of the holoenzyme. Furthermore, P1 early gene expression was downregulated in the wild‐type background, whereas it persisted in the sspA mutant background, indicating that SspA/Lpa mediate the transcriptional switch from the early to the late genes during P1 lytic growth. Thus, this work provides the first evidence for a function of the E. coli RNAP‐associated protein SspA.
Isolation of Listeria monocytogenes mutants with high‐level in vitro expression of host cytosol‐induced gene products
Authors: Shetron‐Rama Lynne M. , Mueller Kimberly , Bravo Juan M. , Bouwer H. G. Archie , Way Sing Sing , Freitag Nancy E.
Published: June 2003

The facultative intracellular bacterial pathogen Listeria monocytogenes dramatically increases the expression of several key virulence factors upon entry into the host cell cytosol. actA, the protein product of which is required for cell‐to‐cell spread of the bacterium, is expressed at low to undetectable levels in vitro and increases in expression more than 200‐fold after L. monocytogenes escape from the phagosome. To identify bacterial factors that participate in the intracellular induction of actA expression, L. monocytogenes mutants expressing high levels of actA during in vitro growth were selected after chemical mutagenesis. The resulting mutant isolates displayed a wide range of actA expression levels, and many were less sensitive to environmental signals that normally mediate repression of virulence gene expression. Several isolates contained mutations affecting actA gene expression that mapped at least 40 kb outside the PrfA regulon, supporting the existence of additional regulatory factors that contribute to virulence gene expression. Two actA in vitro expression mutants contained novel mutations within PrfA, a key regulator of L. monocytogenes virulence gene expression. PrfA E77K and PrfA G155S mutations resulted in high‐level expression of PrfA‐dependent genes, increased bacterial invasion of epithelial cells and increased virulence in mice. Both prfA mutant strains were significantly less motile than wild‐type L. monocytogenes. These results suggest that, although constitutive activation of PrfA and PrfA‐dependent gene expression may enhance L. monocytogenes virulence, it may conversely hamper the bacterium's ability to compete in environments outside host cells.
Global regulation by CsrA in Salmonella typhimurium
Authors: Lawhon Sara D. , Frye Jonathan G. , Suyemoto Mitsu , Porwollik Steffen , McClelland Michael , Altier Craig
Published: June 2003

CsrA is a regulator of invasion genes in Salmonella enterica serovar Typhimurium. To investigate the wider role of CsrA in gene regulation, we compared the expression of Salmonella genes in a csrA mutant with those in the wild type using a DNA microarray. As expected, we found that expression of Salmonella pathogenicity island 1 (SPI‐1) invasion genes was greatly reduced in the csrA mutant, as were genes outside the island that encode proteins translocated into eukaryotic cells by the SPI‐1 type III secretion apparatus. The flagellar synthesis operons, flg and fli, were also poorly expressed, and the csrA mutant was aflagellate and non‐motile. The genes of two metabolic pathways likely to be used by Salmonella in the intestinal milieu also showed reduced expression: the pdu operon for utilization of 1,2‐propanediol and the eut operon for ethanolamine catabolism. Reduced expression of reporter fusions in these two operons confirmed the microarray data. Moreover, csrA was found to regulate co‐ordinately the cob operon for synthesis of vitamin B12, required for the metabolism of either 1,2‐propanediol or ethanolamine. Additionally, the csrA mutant poorly expressed the genes of the mal operon, required for transport and use of maltose and maltodextrins, and had reduced amounts of maltoporin, normally a dominant protein of the outer membrane. These results show that csrA controls a number of gene classes in addition to those required for invasion, some of them unique to Salmonella, and suggests a co‐ordinated bacterial response to conditions that exist at the site of bacterial invasion, the intestinal tract of a host animal.
Regulation of Vibrio vulnificus virulence by the LuxS quorum‐sensing system
Authors: Kim Soo Young , Lee Shee Eun , Kim Young Ran , Kim Choon Mee , Ryu Phil Youl , Choy Hyon E. , Chung Sun Sik , Rhee Joon Haeng
Published: June 2003

Vibrio vulnificus is a halophilic estuarine bacterium that causes fatal septicaemia and necrotizing wound infections. We tested whether V. vulnificus produces signalling molecules (autoinducer 1 and/or 2) stimulating Vibrio harveyi quorum‐sensing system 1 and/or 2. Although there was no evidence for signalling system 1, we found that V. vulnificus produced a signalling activity in the culture supernatant that induced luminescence expression in V. harveyi through signalling system 2. Maximal autoinducer 2 (AI‐2) activity was observed during mid‐exponential to early stationary phase and disappeared in the late stationary phase when V. vulnificus was grown in heart infusion broth containing 2.5% NaCl. V. vulnificus showed increased signalling activity when it was cultured in the presence of glucose (0.5%) and at low pH (pH 6.0). From a cosmid library of V. vulnificus type strain ATCC 29307, we have identified the AI‐2 synthase gene (luxSVv) showing 80% identity with that of V. harveyi (luxSVh) at the amino acid level. To investigate the pathogenic role of luxSVv, a deletion mutant of the clinical isolate V. vulnificus MO6‐24/O was constructed. The luxSVv mutant showed a significant delay in protease production and an increase in haemolysin production. The decreased protease and increased haemolysin activities were restored to the isogenic wild‐type level by complementation with the wild‐type luxSVv allele. The change in phenotypes was also complemented by logarithmic phase spent media produced by the wild‐type bacteria. Transcriptional activities of the haemolysin gene (vvhA) and protease gene (vvpE) were also observed in the mutant using chromosomal PvvhA::lacZ and PvvpE::lacZ transcriptional reporter constructs: transcription of vvhA was increased and of vvpE decreased by the mutation. The mutation resulted in an attenuation of lethality to mice. Intraperitoneal LD50 of the luxSVv mutant increased by 10‐ and 750‐fold in ferric ammonium citrate‐non‐overloaded and ferric ammonium citrate‐overloaded mice respectively. The time required for the death of mice was also significantly delayed in the luxSVv mutant. Cytotoxic activity of the organism against HeLa cells, measured by lactate dehydrogenase (LDH) release assay, was also decreased significantly by the mutation. Taken together, the V. vulnificus LuxS quorum‐sensing system seems to play an important role in co‐ordinating the expression of virulence factors.
Transcriptional regulation of the ospAB and ospC promoters from Borrelia burgdorferi
Authors: Alverson Janet , Bundle Sharyl F. , Sohaskey Charles D. , Lybecker Meghan C. , Samuels D. Scott
Published: June 2003

OspA, OspB and OspC are the major outer surface proteins of Borrelia burgdorferi that are differentially synthesized in response to environmental conditions, including culture temperature. We found that DNA was more negatively supercoiled in B. burgdorferi cultures grown at 23°C compared with cultures grown at 35–37°C. We examined the regulation of ospAB and ospC transcription by temperature and DNA supercoiling. DNA supercoiling was relaxed by adding coumermycin A1, an antibiotic that inhibits DNA gyrase. Syntheses of the major outer surface proteins, expression of the ospA and ospC genes and the activities of the ospAB operon and ospC gene promoters were assayed. ospA product levels decreased, whereas ospC product levels increased after shifting from 23°C to 35°C or after adding coumermycin A1. In addition, OspC synthesis was higher in a gyrB mutant than in wild‐type B. burgdorferi. Promoter activity was quantified using cat reporter fusions. Increasing temperature or relaxing supercoiled DNA resulted in a decrease in ospAB promoter activity in B. burgdorferi, but not in Escherichia coli, as well as an increase in ospC promoter activity in both bacteria. ospC promoter activity was increased in an E. coli gyrB mutant with an attenuated DNA supercoiling phenotype. These results suggest that B. burgdorferi senses environmental changes in temperature by altering the level of DNA supercoiling, which then affects the expression of the ospAB operon and the ospC gene. This implies that DNA supercoiling acts as a signal transducer for environmental regulation of outer surface protein synthesis.
Ribosome–DnaK interactions in relation to protein folding
Authors: Ghosh Jaydip , Basu Arunima , Pal Saumen , Chowdhuri Saheli , Bhattacharya Arpita , Pal Debashis , Chattoraj Dhruba K. , DasGupta Chanchal
Published: June 2003

Bacterial ribosomes or their 50S subunit can refold many unfolded proteins. The folding activity resides in domain V of 23S RNA of the 50S subunit. Here we show that ribosomes can also refold a denatured chaperone, DnaK, in vitro, and the activity may apply in the folding of nascent DnaK polypeptides in vivo. The chaperone was unusual as the native protein associated with the 50S subunit stably with a 1:1 stoichiometry in vitro. The binding site of the native protein appears to be different from the domain V of 23S RNA, the region with which denatured proteins interact. The DnaK binding influenced the protein folding activity of domain V modestly. Conversely, denatured protein binding to domain V led to dissociation of the native chaperone from the 50S subunit. DnaK thus appears to depend on ribosomes for its own folding, and upon folding, can rebind to ribosome to modulate its general protein folding activity.
Different roles of the Mre11 complex in the DNA damage response in Aspergillus nidulans
Authors: Semighini Camile P. , Von Zeska Kress Fagundes Márcia Regina , Ferreira Joseane Cristina , Pascon Renata Castiglioni , De Souza Goldman Maria Helena , Goldman Gustavo Henrique
Published: June 2003

The Mre11‐Rad50‐Nbs1 protein complex has emerged as a central player in the cellular DNA damage response. Mutations in scaANBS1, which encodes the apparent homologue of human Nbs1 in Aspergillus nidulans, inhibit growth in the presence of the anti‐topoisomerase I drug camptothecin. We have used the scaANBS1 cDNA as a bait in a yeast two‐hybrid screening and report the identification of the A. nidulans Mre11 homologue (mreA). The inactivated mreA strain was more sensitive to several DNA damaging and oxidative stress agents. Septation in A. nidulans is dependent not only on the uvsBATR gene, but also on the mre11 complex. scaANBS1 and mreA genes are both involved in the DNA replication checkpoint whereas mreA is specifically involved in the intra‐S‐phase checkpoint. ScaANBS1 also participates in G2‐M checkpoint control upon DNA damage caused by MMS. In addition, the scaANBS1 gene is also important for ascospore viability, whereas mreA is required for successful meiosis in A. nidulans. Consistent with this view, the Mre11 complex and the uvsCRAD51 gene are highly expressed at the mRNA level during the sexual development.
RdgB acts to avoid chromosome fragmentation in Escherichia coli
Authors: Bradshaw Jill S. , Kuzminov Andrei
Published: June 2003

Bacterial RecA protein is required for repair of two‐strand DNA lesions that disable whole chromosomes. recA mutants are viable, suggesting a considerable cellular capacity to avoid these chromosome‐disabling lesions. recA‐dependent mutants reveal chromosomal lesion avoidance pathways. Here we characterize one such mutant, rdgB/yggV, deficient in a putative inosine/xanthosine triphosphatase, conserved throughout kingdoms of life. The rdgB recA lethality is suppressed by inactivation of endonuclease V (gpnfi) specific for DNA‐hypoxanthines/xanthines, suggesting that RdgB either intercepts improper DNA precursors dITP/dXTP or works downstream of EndoV in excision repair of incorporated hypoxathines/xanthines. We find that DNA isolated from rdgB mutants contains EndoV‐recognizable modifications, whereas DNA from nfi mutants does not, substantiating the dITP/dXTP interception by RdgB. rdgB recBC cells are inviable, whereas rdgB recF cells are healthy, suggesting that chromosomes in rdgB mutants suffer double‐strand breaks. Chromosomal fragmentation is indeed observed in rdgB recBC mutants and is suppressed in rdgB recBC nfi mutants. Thus, one way to avoid chromosomal lesions is to prevent hypoxanthine/xanthine incorporation into DNA via interception of dITP/dXTP.
Central tumour necrosis factor‐α mediates the early gastrointestinal motor disturbances induced by lipopolysaccharide in sheep
Authors: Guerrero‐Lindner E. , Castro M. , MuÑoz J. M. , Arruebo M. P. , Murillo M.D. , Buéno L. , Plaza M.A.
Published: June 2003

Cytokines are involved in fever and other symptoms of the acute phase response induced by endotoxins. The aim of this work was to study the involvement of central tumour necrosis factor‐α (TNF‐α) in the changes induced by lipopolysaccharide (LPS) on gastrointestinal (GI) motility in sheep. Body temperature and myoelectric activity of the antrum, duodenum and jejunum was recorded continuously. Intravenous (i.v.) administration of LPS (0.1 μg kg−1)‐induced hyperthermia, decreased gastrointestinal myoelectric activity and increased the frequency of the migrating motor complex (MMC). These effects started 40–50 min after LPS and lasted for 6–7 h. TNF‐α (50 and 100 ng kg−1) mimicked these effects when injected intracerebroventricularly (i.c.v.) but not i.v. Pretreatment with soluble recombinant TNF receptor (TNFR:Fc, 10 μg kg−1, i.c.v.) abolished the TNF‐induced actions and reduced those evoked by LPS. Furthermore, the effects induced by either LPS or TNF were suppressed by prior i.c.v. injection of indomethacin (100 μg kg−1). In contrast, the i.v. injections of TNFR:Fc or indomethacin were ineffective. Our data suggest that LPS disturbs GI motility in sheep through a central pathway that involves TNF‐α and prostaglandins sequentially.
Human submucosal neurones regulate intestinal epithelial cell proliferation: evidence from a novel co‐culture model
Authors: Toumi F. , Neunlist M. , Cassagnau E. , Parois S. , Laboisse C. L. , Galmiche J.‐P. , Jarry A.
Published: June 2003

The role of the human enteric nervous system (ENS) in the control of the intestinal epithelium organization and proliferation is unknown. To address this issue, we developed a novel co‐culture model, consisting of human submucosa containing the submucosal plexus and a human colonic epithelial monolayer. After 3 days in basal conditions (i.e. in absence of neuronal activation) epithelium disorganization and proliferation occurred. In contrast, electrical activation of submucosal neurones maintained monolayer organization and decreased cell proliferation. These effects were blocked by tetrodotoxin and a vasoactive intestinal peptide (VIP) receptor antagonist, and reproduced by VIP. In conclusion, our study suggests that the human ENS is involved in the control of epithelial cell proliferation.
Morphological characteristics and immunohistochemical detection of nicotinic acetylcholine receptors on intestinofugal afferent neurones in guinea‐pig colon
Authors: Ermilov L. G. , Miller S. M. , Schmalz P. F. , Hanani M. , Lennon V. A. , Szurszewski J. H.
Published: June 2003

Abstract Intestinofugal afferent neurones (IFANs) provide excitatory synaptic input to abdominal prevertebral ganglion neurones. Input is greatly reduced during blockade of nicotinic acetylcholine receptors (nAChRs) in the wall of the colon, suggesting two projection pathways: a direct pathway without synaptic interruption and an indirect pathway interrupted by at least one nicotinic cholinergic synapse. This study aimed to characterize the morphology of IFANs and examine the distribution of nAChRs on them. We identified IFANs in guinea‐pig colon by retrograde labelling with fluorescent tracer DiI placed either on the lumbar colonic nerves in vitro or inferior mesenteric ganglion in vivo. Confocal laser scanning microscopy and computerized image‐processing software were used for 3D image reconstruction. Approximately 70% of identified IFANs had Dogiel type I‐like morphology, the remainder were Dogiel type II‐like. In vivo labelled IFANs were injected with Lucifer Yellow and immunostained for nAChRs using monoclonal antibody MAb35. Approximately 3% of total plasma membrane surface of IFANs with Dogiel type I morphology had MAb35‐IR. In contrast, <1% of membrane surface of IFANs with Dogiel type II morphology had MAb35‐IR. The finding that IFANs displayed immunostaining for nAChRs suggests the presence of putative nicotinic synapses.
Motility in the isolated mouse colon: migrating motor complexes, myoelectric complexes and pressure waves
Authors: Powell A. K. , Fida R. , Bywater R. A. R.
Published: June 2003

This study has used mechanical, together with pressure/volume recordings or electrophysiological recordings, to investigate the spontaneous activity in isolated preparations of mouse colon. In the former preparations, when not distended with fluid, spontaneous colonic migrating motor complexes (CMMCs) were observed using isotonic transducers. When the colons were distended with fluid, CMMCs continued at an increased frequency and in addition were associated temporally, with rises in intraluminal pressure and pulses of distally ejected fluid. 5‐Hydroxytryptamine (1 μmol L−1) or NG‐nitro‐l‐arginine (100 μmol L−1) increased the frequency of propulsive activity and this activity was abolished by hexamethonium (500 μmol L−1). In a second preparation, myoelectric complexes recorded from circular muscle cells in colons using intracellular microelectrodes, were found to correlate in frequency and phase with CMMCs. The experiments indicate that CMMCs are intimately related to pressure waves in the fluid‐filled viscus and the muscle membrane potential changes that have been recorded during myoelectric complexes are likely to be analogous to those occurring during fluid‐filled propulsive activity.
Modulation of oesophago‐UOS contractile reflex: effect of proximal and distal esophageal distention and swallowing
Authors: Aslam M. , Kern M. , Shaker R.
Published: June 2003

Upper oesophageal sphincter (UOS) tone is influenced by intraoesophageal pressure events. Our aim was to test the hypothesis that UOS tone is responsive to simultaneous inhibitory and stimulatory signals originating from the oesophagus and compare effect of proximal and distal oesophageal air distention on oesophageal balloon‐stimulated UOS contraction. We studied 16 healthy volunteers, ages 19–80 years in two stages. We induced UOS contraction by distending various size balloons intraoesophageally and studied response of contracted UOS to oesophageal air distentions and swallowing. Intraoesophageal injections of 60‐ml room air resulted in UOS pressure augmentation (31%), relaxation (64%) and no effect in the remaining 5% of instances. The majority of air injections into the oesophageal segment proximal to the distended balloon were followed by relaxation of the contracted UOS, whereas, the majority of oesophageal air distentions distal to the balloon resulted in augmentation of UOS contraction (P < 0.01). Swallowing resulted in complete relaxation of the UOS. In conclusion, UOS contractile response to oesophageal balloon distention is overridden by further augmentation or relaxation as a result of oesophageal air distention and swallowing. Contractile and inhibitory responses of the contracted UOS to generalized oesophageal distention is region‐specific.
Role of abscisic acid in cadmium tolerance of rice (Oryza sativa L.) seedlings
Authors: HSU Y. T. , KAO C. H.
Published: June 2003

Changes in abscisic acid (ABA) contents in Cd‐treated rice (Oryza sativa L.) seedlings of two cultivars were investigated. On treatment with CdCl2, the ABA content rapidly increased in the leaves and roots of Cd‐tolerant cultivar (cv. Tainung 67, TNG67) but not in the Cd‐sensitive cultivar (cv. Taichung Native 1, TN1). The reduction of transpiration rate of TN1 caused by Cd was less than that of TNG67. Exogenous application of ABA reduced transpiration rate, decreased Cd content, and enhanced Cd tolerance of TN1 seedlings. Exogenous application of the ABA biosynthesis inhibitor, fluridone, reduced ABA accumulation, increased transpiration rate and Cd content, and decreased Cd tolerance of TNG67 seedlings. Fluridone effect on Cd toxicity of TNG67 seedlings was reversed by the application of ABA. The roles of endogenous ABA in Cd tolerance of rice seedlings are discussed and suggested.
Three pathogen‐inducible genes encoding lipid transfer protein from pepper are differentially activated by pathogens, abiotic, and environmental stresses*
Authors: JUNG HO WON , KIM WOONBONG , HWANG BYUNG KOOK
Published: June 2003

The three cDNA clones, CALTPI, CALTPII, and CALTPIII, corresponding to pepper lipid transfer protein (LTP) genes were isolated from a pepper (Capsicum annuum) cDNA library from hypersensitive response (HR) lesions of leaves infected with Xanthomonas campestris pv. vesicatoria. The CALTP genes are well conserved in their coding region with 57–72% identity at the amino acid level, but display 72–83% identity at the nucleotide sequence level. The transcripts of the three CALTP genes differentially accumulated in pepper leaf, stem, and fruit tissues infected by X. campestris pv. vesicatoria, Phytophthora capsici and Colletotrichum gloeosporioides. The CALTP genes were also strongly induced in the systemic, upper leaves after immunization on lower leaves by either pathogenic or non‐pathogenic bacteria. In situ hybridization results showed that the CALTPI mRNA was localized in phloem cells of vascular tissues in pepper leaf, stem and fruit tissues after pathogen infection. CALTPI and CALTPIII genes were predominantly expressed in various pepper tissues infected by pathogens, while infection by P. capsici and C. gloeosporioides did not induce the transcription of the CALTPII gene. Ethylene, methyl jasmonate and abscisic acid induced CALTPI and III gene expression in pepper leaves. Drought, high salinity, low temperature and wounding stresses also induced the expression of the CALTPI and CALTPIII genes in a similar manner. In contrast, only high salinity induced the CALTPII expression that was not generally affected by abiotic and other environmental stimuli. When compared with each other and with LTPs from other plants, CALTPI is more distantly related than CALTPII and CALTPIII sequences, indicating that the three pepper CALTP genes represent two different classes. These results thus show that CALTPI and CALTPIII genes, although different in sequence structure, are transcriptionally activated in pepper tissues by pathogen infection as well as abiotic and environmental stresses.
Expression and Modulation of the Human Immunoglobulin A Fc Receptor (CD89) and the FcR γ Chain on Myeloid Cells in Blood and Tissue
Authors: Hamre R. , Farstad I. N. , Brandtzaeg P. , Morton H. C.
Published: June 2003

CD89, the human immunoglobulin A (IgA) Fc receptor (FcR), is a potential target for antibody‐based therapeutics, but little is known about its expression and modulation in vivo. In this study, we examined the expression pattern of CD89 and its signalling subunit, the FcR γ chain, on circulating myeloid cells and in various tissues. Our results showed a wide tissue distribution of CD89+ cells. Thus, CD89+ cells were evident as clusters in tonsils and appendix and scattered in varying numbers in lymph nodes, kidney, liver, intestinal mucosa, bronchoalveolar lavage and peritoneal fluid. Most CD89+ cells were identified as neutrophils with high levels of CD89. A few recently emigrated macrophages (CD14low), weakly positive for CD89, were occasionally found in the tissues and more often in the peritoneal fluid. The level of CD89 on neutrophils in tissues and peripheral blood was similar, whereas on monocytes it was much lower in the tissues than in blood, and it was absent on CD14–/CD68+ intestinal lamina propria macrophages. Conversely, we detected much higher levels of the FcR γ chain in monocytes than in neutrophils, but the FcR γ chain was also downregulated in tissue macrophages as well as in in vitro‐differentiated monocyte‐derived macrophages and dendritic cells. The implications of our current findings on the biological functioning of CD89 are discussed.
Increased Expression of Soluble Cytotoxic T‐Lymphocyte‐Associated Antigen‐4 Molecule in Patients with Systemic Lupus Erythematosus
Authors: Liu M.‐F. , Wang C.‐R. , Chen P.‐C. , Fung L.‐L.
Published: June 2003

A soluble form of cytotoxic T‐lymphocyte‐associated antigen‐4 (sCTLA‐4) was recently found and shown to possess a downregulatory function as a membrane‐bound CTLA‐4 molecule. The purpose of the study was to investigate the expression of sCTLA‐4 molecule in patients with systemic lupus erythematosus (SLE). One hundred patients with SLE and 40 age‐ and sex‐matched healthy individuals were enrolled in the study. The results showed that patients with SLE have significantly higher levels of sCTLA‐4 in sera than healthy controls (21.6 ± 12.3 ng/ml versus 5.9 ± 5.4 ng/ml, P < 0.001). Increased expression of sCTLA‐4 mRNA in peripheral blood mononuclear cells (PBMCs) was also found in SLE patients. However, we could not find a statistically significant correlation between the serum levels of sCTLA‐4 and lupus disease activities. The reported CTLA‐4 gene polymorphism in promoter region at position −318 did not affect the levels of sCTLA‐4. To the best of our knowledge, this is the first report showing that patients with SLE have increased sCTLA‐4 expression. However, the mechanism and role of increased sCTLA‐4 in the pathogenesis of SLE remains elucidated.
Egg Yolk Anti‐BfpA Antibodies as a Tool for Recognizing and Identifying Enteropathogenic Escherichia coli
Authors: De Almeida C. M. C. , Quintana‐Flores V. M. , Medina‐Acosta E. , Schriefer A. , Barral‐Netto M. , Dias da Silva W.
Published: June 2003

Enteropathogenic Escherichia coli (EPEC) is a major aetiological agent of childhood diarrhoea in developing countries. The structural repeating protein A subunit, BfpA, found in the bundle‐forming pilus, is one of the virulent factors for EPEC pathogenesis. Recombinant BfpA in laying hens elicited sustained and vigorous antibody production. Immunoglobulin Y (IgY) anti‐BfpA antibodies were recovered from egg yolk, purified and characterized. Immunoadsorption with whole extracts of the isogenic E. coli EPEC adherence factor (EAF) strain that lacks BfpA rendered the resulting IgY preparations capable of: (a) recognizing purified or recombinant BfpA proteins in a dose‐dependent fashion; (b) blocking the colonization of HeLa cells by EPEC EAF+, in vitro; (c) specifically identifying E. coli bearing EAF+; and (d) inhibiting the growth of E. coli EAF+ but not the EAF strain. IgY anti‐BfpA is potentially useful as a specific, low‐cost immunobiological reagent to screen human faecal specimens for the presence of EPEC.
Was It There All the Time?
Authors: Bennett M. , Leanderson T.
Published: June 2003

The complement system is old, yet it may still have something new to teach us. For many years, research has existed which shows that C3d, in addition to its established role as an adjuvant, could have an immunosuppressive activity. Being true, it suggests that a common mechanism may be used both by organisms and by their pathogens to prevent unwanted immune responses.
Probing the Specificity of Human Myeloma Proteins with a Random Peptide Phage Library
Authors: Dybwad A. , Lambin P. , Sioud M. , Zouali M.
Published: June 2003

Human myeloma proteins (HMPs) from 10 patients with multiple myeloma (MM) were used to affinity‐select peptides from a random phage‐display peptide library. Binding peptides were identified for the 10 analysed antibodies (eight, immunoglobulin G (IgG), and two, immunoglobulin A (IgA)). The specificity of the binding was confirmed by competitive experiments using phages and chemically synthesized peptides. Interestingly, some phage‐displayed peptides were immuno‐selected with HMPs isolated from different patients. Sequence alignments and homology searches revealed a significant homology with human proteins (e.g. neural cell adhesion proteins) and pathogen‐derived proteins (e.g. herpes simplex virus capsid proteins). The selected peptides could be useful as targeting agents for myeloma cells expressing surface immunoglobulins.
The Use of Fluorometric Assays to Assess the Immune Response to DNA in Murine Systemic Lupus Erythematosus
Authors: Björkman L. , Reich C. F. , Pisetsky D. S.
Published: June 2003

Antibodies to DNA (anti‐DNA) play an important role in the pathogenesis of systemic lupus erythematosus (SLE). In blood, these antibodies may exist in a free, unbound state or as part of complexes with DNA. Furthermore, circulating DNA may be either complexed or free. Because of the central role of these immunoreactants (anti‐DNA and DNA) in the disease, monitoring of their levels could provide valuable information for both clinical and investigative purposes. In these studies, we have explored the use of a DNA‐binding dye, PicoGreen, for the detection of circulating DNA, either total or immune complex bound. In addition, we have used this dye for Farr‐type antibody assays. Using autoimmune MRL/lpr mice as a model, we have shown that, while the levels of free DNA in the plasma of these mice were comparable with those of normal BALB/c mice, the amounts in complexes precipitable by ammonium sulfate were significantly greater. Furthermore, we showed that Farr assays using PicoGreen reliably detect levels of free anti‐DNA, with values correlated with anti‐DNA levels by an enzyme‐linked immunosorbent assay. Together, our results suggest that a fluorometric dye can accurately monitor DNA and anti‐DNA antibody levels in SLE and may provide important information on immunopathogenesis.
Liver Metastasis of Cancer Facilitated by Chemokine Receptor CCR6
Authors: Dellacasagrande J. , Schreurs O. J. F. , Hofgaard P. O. , Omholt H. , Steinsvoll S. , Schenck K. , Bogen B. , Dembic Z.
Published: June 2003

When injected subcutaneously, mouse plasmacytoma (MOPC315) grew rapidly in situ, and metastatic cells became detectable first in the lymph nodes (LNs) and bone marrow, and later in the liver and lungs. We studied MOPC315 cell migration by tracking metastatic cells labelled with green fluorescent protein (GFP). We measured the levels of their chemokine receptor mRNA (by semiquantitative and real‐time quantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR), because chemokines can regulate organ predilection of metastasis. Freshly sorted metastatic cells and tumour cell lines derived from the liver of BALB/c mice overexpressed functional CCR6 and CCR7 molecules compared with primary tumour. Preincubation with the CCR6 ligand (CCL20) induced liver‐sorted tumour cells to preferentially colonize the liver, demonstrating an association between liver metastasis and CCR6 expression in the mouse. Because the liver is a common site for metastasis, second only to draining LNs, we wished to ascertain whether this finding could be generalized, i.e. whether other cancers can use the similar mechanism of metastasis to the liver, and whether it holds true for humans. We found that CCR6 is overexpressed in small liver metastases of colon, thyroid and ovarian carcinomas compared with normal liver. Because human liver constitutively expresses CCL20, it could attract and select CCR6+ cancer cells. We suggest that chemotaxis via CCR6 might be a common mechanism by which malignant cancers metastasize to the liver. As metastasis in patients with cancer poses the biggest peril for survival, inhibition of CCR6 signalling, either during or after medical or surgical treatment, might be useful in preventing liver metastasis.