bluuebunny/crossref_metadata_2025 huggingface dataset:
Examination of Salmonella gene expression in an infected mammalian host using the green fluorescent protein and two‐colour flow cytometry
Authors: Bumann Dirk
Published: March 2003

Quantitative data on Salmonella gene expression in infected hosts are largely

lacking because of technical problems. One attractive reporter, the green fluorescent

protein (GFP), is widely used in vitro but is difficult to quantify in infected

tissues because of the preponderance of background particles with similar fluorescence.

Here, bacterial GFP emission was spectrally distinguished from host autofluorescence

by two‐colour flow cytometry. Using this technique, the in vivo activity of

three well‐characterized promoters (PsicA, PssaH

and PpagC) was determined. Their spatial and temporal activity

patterns are in close agreement with predictions based on previous data and the colonization

defects of corresponding deletion strains. To identify additional Salmonella

promoters that are induced in infected animals, a genomic library was sorted by flow

cytometry yielding four independent promoters. Genes expressed from PpibB

and PsifA contribute to virulence, and chorismate mutase expressed

from ParoQ might participate in aromatic acid biosynthesis, which

is also required for virulence. Promoter P3g appears to be part of a mobile genetic element that is lacking in the completely sequenced strain LT2.
Identification of genes that are associated with DNA repeats in prokaryotes
Authors: Jansen Ruud. , Embden Jan. D. A. van , Gaastra Wim. , Schouls Leo. M.
Published: March 2003

Using in silico analysis we studied a novel family of repetitive DNA sequences

that is present among both domains of the prokaryotes (Archaea and Bacteria), but

absent from eukaryotes or viruses. This family is characterized by direct repeats,

varying in size from 21 to 37 bp, interspaced by similarly sized non‐repetitive sequences.

To appreciate their characteri‐stic structure, we will refer to this family as the

clustered regularly interspaced short palindromic repeats (CRISPR). In most species

with two or more CRISPR loci, these loci were flanked on one side by a common leader

sequence of 300–500 b. The direct repeats and the leader sequences were conserved

within a species, but dissimilar between species. The presence of multiple chromosomal

CRISPR loci suggests that CRISPRs are mobile elements. Four CRISPR‐associated (cas)

genes were identified in CRISPR‐containing prokaryotes that were absent from CRISPR‐negative

prokaryotes. The cas genes were invariably located adjacent to a CRISPR locus,

indicating that the cas genes and CRISPR loci have a functional relationship.

The cas3 gene showed motifs characteristic for helicases of the superfamily

2, and the cas4 gene showed motifs of the RecB family of exonucleases, suggesting

that these genes are involved in DNA metabolism or gene expression. The spatial coherence

of CRISPR and cas genes may stimulate new research on the genesis and biological role of these repeats and genes.
NAD+‐glycohydrolase acts as an intracellular toxin to enhance the extracellular survival of group A streptococci
Authors: Bricker Angela L. , Cywes Colette , Ashbaugh Cameron D. , Wessels Michael R.
Published: March 2003

Group A streptococci (GAS) produce several secreted products that are thought to enhance pathogenicity by facilitating spread of the organisms through host tissues. Two such products, streptolysin O (SLO) and NAD+‐glycohydrolase,
appear to be functionally linked, in that SLO is required for transfer of NAD+‐glycohydrolase
into epithelial cells. However, the effects of NAD+‐glycohydrolase
on host cells are largely unexplored. We now report that SLO‐mediated delivery of
NAD+‐glycohydrolase to the cytoplasm of human keratinocytes results
in major changes in host cell biology that enhance GAS pathogenicity. We derived
isogenic mutant strains deficient in the expression of SLO, NAD+‐glycohydrolase
or both proteins in the background of a virulent, M‐type 3 strain of GAS. All three
mutant strains were internalized by human keratinocytes more rapidly and in higher
numbers than were organisms from the wild‐type strain. Association of the mutant
strains with keratinocytes also resulted in reduced cytotoxicity and reduced keratinocyte
apoptosis compared with wild‐type GAS. These results support a model in which NAD+‐glycohydrolase
contributes to GAS pathogenesis by modulating host cell signalling pathways to inhibit
GAS internalization, to augment SLO‐mediated cytotoxicity and to induce keratinocyte
apoptosis. We conclude that NAD+‐glycohydrolase is a novel type of bacterial toxin that acts intracellularly in the infected host to enhance the survival and proliferation of an extracellular pathogen.
Diversity in the serine recombinases
Authors: Smith Margaret C. M. , Thorpe Helena M.
Published: March 2003

Most site‐specific recombinases fall into one of two families, based on evolutionary and mechanistic relatedness. These are the tyrosine recombinases orλintegrase family and the serine recombinases or resolvase/invertase family. The tyrosine recombinases are structurally diverse and functionally versatile and include integrases, resolvases, invertases and transposases. Recent studies have revealed that the serine recombinase family is equally versatile and members have a variety of structural forms. The archetypal resolvase/invertases are highly regulated, only affect resolution or inversion and they have an N‐terminal catalytic domain and a C‐terminal DNA binding domain. Phage‐encoded serine recombinases (e.g.φC31 integrase) cause integration and excision with strictly controlled directionality, and have an N‐terminal catalytic domain but much longer C‐terminal domains compared with the resolvase/invertases. This high molecular weight group also contains transposases (e.g. TnpX from Tn4451). Other transposases, which belong to a third structurally different group, are similar in size to the resolvase/invertases but have the DNA binding domain N‐terminal to the catalytic domain (e.g. IS607transposase). These three structural groups represented by the resolvase/invertases, the large serine recombinases and relatives of IS607transposase correlate with three major groupings seen in a phylogeny of the catalytic domains. These observations indicate that the serine recombinases are modular and that fusion of the catalytic domain to unrelated sequences has generated structural and functional diversity.
ParE toxin encoded by the broad‐host‐range plasmid RK2 is an inhibitor of Escherichia coli gyrase
Authors: Jiang Yong , Pogliano Joe , Helinski Donald R. , Konieczny Igor
Published: March 2003

Broad‐host‐range plasmid RK2 encodes a post‐segregational killing system, parDE, which contributes to the stable maintenance of this plasmid in Escherichia coli and many distantly related bacteria. The ParE protein is a toxin that inhibits cell growth, causes cell filamentation and eventually cell death. The ParD protein is a specific ParE antitoxin. In this work, the in vitro activities of these two proteins were examined. The ParE protein was found to inhibit DNA synthesis using an E. coli oriC supercoiled template and a replication‐proficient E. coli extract. Moreover, ParE inhibited the early stages of both chromosomal and plasmid DNA replication, as measured by the DnaB helicase‐ and gyrase‐dependent formation of FI*, a highly unwound form of supercoiled DNA. The presence of ParD prevented these inhibitory activities of ParE. We also observed that the addition of ParE to supercoiled DNA plus gyrase alone resulted in the formation of a cleavable gyrase–DNA complex that was converted to a linear DNA form upon addition of sodium dodecyl sulphate (SDS). Adding ParD before or after the addition of ParE prevented the formation of this cleavable complex. These results demonstrate that the target of ParE toxin activity in vitro is E. coli gyrase.
An accessory sec locus of Streptococcus gordonii is required for export of the surface protein GspB and for normal levels of binding to human platelets
Authors: Bensing Barbara A. , Sullam Paul M.
Published: March 2003

The translocation of proteins across the bacterial cell membrane is carried out by highly conserved components of the Sec system. Most bacterial species have a single copy of the genes encoding SecA and SecY, which are essential for viability. However, Streptococcus gordonii strain M99 encodes SecA and SecY homologues that are
not required for viability or for the translocation of most exported proteins. The
genes (secA2 and secY2) reside in a region of the chromosome required
for the export of GspB, a 286 kDa cell wall‐anchored protein. Loss of GspB surface
expression is associated with a significant reduction in the binding of M99 to human
platelets, suggesting that it may be an adhesin. Genetic analyses indicate that M99
has a second, canonical SecA homologue that is essential for viability. At least
two other Gram‐positive species, Streptococcus pneumoniae and Staphylococcus
aureus, encode two sets of SecA and SecY homologues. One set is more similar
to SecA and SecY of Escherichia coli, whereas the other set is more similar
to SecA2 and SecY2 of strain M99. The conserved organization of genes in the secY2–secA2 loci suggests that, in each of these Gram‐positive species, SecA2 and SecY2 may constitute a specialized system for the transport of a very large serine‐rich repeat protein.
Deletion of the copper transporter CaCCC2 reveals two distinct pathways for iron acquisition in Candida albicans
Authors: Weissman Ziva , Shemer Revital , Kornitzer Daniel
Published: March 2003

Efficient iron acquisition is an essential requirement for growth of pathogenic organisms in the iron‐poor host environment. In Saccharomyces cerevisiae,

high‐affinity iron import depends on the multicopper ferroxidase ScFet3. ScFet3 biogenesis

in the trans‐Golgi compartment requires a copper‐transporting P‐type ATPase,

ScCcc2. Here, we describe the isolation by functional complementation of a Ccc2 homologue

from the pathogenic yeast Candida albicans. CaCcc2 is functionally distinct

from a previously described C. albicans copper‐transporting P‐type ATPase,

CaCrp1, which appears to be specifically involved in copper detoxification. Regulation

of CaCCC2 and the phenotype of the homozygous CaCCC2 deletion indicate

that it is required for high‐affinity iron import, making it the bona fide CCC2

homologue of C. albicans. Remarkably, in a mouse model of systemic infection,

the Caccc2Δ strain displayed robust proliferation and no significant

reduction in pathogenicity, suggesting the existence of alternative mechanisms of

iron uptake from host tissues. We identify haemin and haemoglobin as potential iron

sources that can be used by C. albicans in a CaCcc2‐independent manner.
Targeting and sequestration of truncated Pfs230 in an intraerythrocytic compartment during Plasmodium falciparum gametocytogenesis
Authors: Eksi Saliha , Stump Amy , Fanning Sarah L. , Shenouda Mary I. , Fujioka Hisashi , Williamson Kim C.
Published: March 2003

For malaria to be transmitted, the Plasmodium falciparum parasite must invade
an erythrocyte and undergo gametocytogenesis. When mature intraerythrocytic gametocytes
are taken up in a blood meal by a mosquito they emerge as gametes and, once fertilized,
continue to differentiate into infectious sporozoites. One of the major proteins
associated with the surface of the parasite during gamete differentiation is Pfs230,
a 360 kDa member of a family of P. falciparum proteins that contains a repeated
cysteine motif domain. To characterize the role of different regions of Pfs230, the
gene was disrupted by targeted integration and clones isolated that expressed distinct
sections of Pfs230. Independent clones D1.356 a and b express the first 452 amino
acids (aa) of Pfs230 and do not contain a cysteine motif domain, whereas clones D2.850
a and b express the first 950 aa, including the first cysteine motif domain. Although
both sets of clones undergo gametogenesis and produce morphologically normal gametes,
neither truncated Pfs230 is located on the surface of the gamete. In clones D1.356
a and b, the 452 aa Pfs230 is secreted into the parasitophorous vacuole and released
as a soluble protein when the parasite emerges from the erythrocyte as a gamete.
In marked contrast, the 950 aa form of Pfs230 expressed by clones D2.850 a and
b is sequestered in a novel tubular compartment in the erythrocyte cytoplasm. This
sexual‐stage tubular intraerythrocytic compartment (STIC) is not recognized by antibodies
specific for proteins associated with the parasitophorous vacuole membrane (Pfs16
or Exp‐1) or Maurer’s clefts (Pfsbp 1 or mAb LWL1) or intraerythrocytic asexual parasite proteins (PfEMP2 or HRP II).
Role of the extracytoplasmic‐function σ Factor σH in Mycobacterium tuberculosis global gene expression
Authors: Manganelli Riccardo , Voskuil Martin I. , Schoolnik Gary K. , Dubnau Eugenie , Gomez Manuel , Smith Issar
Published: March 2003

Like other bacterial species, Mycobacterium tuberculosis has multiple sigma (σ) factors encoded in its genome. In previously published work, we and others have shown that mutations in some of these transcriptional activators render M. tuberculosis sensitive to various environmental stresses and, in some cases, cause attenuated virulence phenotypes. In this paper, we characterize a M. tuberculosis mutant lacking the ECF σ factor σH. This mutant was more sensitive than the wild type to heat shock and to various oxidative stresses, but did not show de‐creased ability to grow inside macrophages. Using quantitative reverse transcription‐PCR and microarray technology, we have started to define the σH regulon and its involvement in the global regulation of the response to heat shock and the thiol‐specific oxidizing agent diamide. We identified 48 genes whose expression increased after exposure of M. tuberculosis to diamide; out of these, 39 were not induced in the sigH mutant, showing their direct or indirect dependence on σH. Some of these genes encode proteins whose predicted function is related to thiol metabolism, such as thioredoxin, thioredoxin reductase and enzymes involved in cysteine and molybdopterine biosynthesis. Other genes under σH control encode transcriptional regulators such as sigB, sigE, and sigH itself.
Histone deacetylases in Trypanosoma brucei: two are essential and another is required for normal cell cycle progression
Authors: Ingram Alexandra K. , Horn David
Published: March 2003

Reversible protein acetylation is established as a modification of major regulatory significance. In particular, histone acetylation regulates access to genetic information in eukaryotes. For example, class I and class II histone deacetylases are regulatory components of corepressor complexes involved in cell cycle progression and differentiation. Here, we have investigated the function of such enzymes in Trypanosoma brucei, mono‐flagellated parasitic protozoa that branched very early from the eukaryotic lineage. Four T. brucei genes encoding histone deacetylase orthologues have been identified, cloned and characterized. The predicted deacetylases, DAC1‐4 are approximately 43, 61, 75 and 64 kDa respectively. They share significant similarity with mammalian and yeast class I (DAC1 and DAC2) and class II (DAC3 and DAC4) histone deacetylases, and all except DAC2 have the critical residues predicted to be required for deacetylase activity. In gene targeting experiments, DAC1 and DAC3 appear to be essential whereas DAC2 and DAC4 are not required for viability. Of the two mutant cell types, the dac4 mutant displays a delay in the G2/M phase of the cell cycle. Our results provide genetic validation of DAC1 and DAC3 as potential chemotherapy targets and demonstrate that T. brucei expresses at least three probable histone deacetylases with distinct function.
Cladosporium fulvum overcomes Cf‐2‐mediated resistance by producing truncated AVR2 elicitor proteins
Authors: Luderer Rianne , Takken Frank L. W. , Wit Pierre J. G. M. de , Joosten Matthieu H. A. J.
Published: March 2003

The Cf‐2 gene of tomato confers resistance to strains of the biotrophic pathogenic fungus Cladosporium fulvum carrying avirulence gene Avr2. To allow dissection of the biochemical mechanism of perception of AVR2 by Cf‐2, we set out to clone the Avr2 gene. Here, we report the functional cloning of Avr2 cDNA, based on the induction of a hypersensitive response (HR) by the encoded AVR2 protein in Cf2 tomato plants. Analysis of strains of C. fulvum that are virulent on Cf2 tomato lines revealed various independent frameshift mutations in the Avr2 open reading frame (ORF) and a point mutation resulting in a premature stop codon. All modifications result in the production of truncated AVR2 proteins. Interestingly, an additional modification involves the insertion of a LINE‐like element, Cfl1, in the Avr2 ORF. Cfl1 is the first LINE‐like element identified in C. fulvum and provides the first example of loss of avirulence of a plant pathogen caused by insertion of a retrotransposable element in an Avr gene. Rcr3 represents an additional plant protein that is specifically required for Cf‐2‐mediated resistance. Analysis of two different rcr3 mutant Cf2 tomato plants revealed that their ability to respond to AVR2 with a HR correlates with their degree of resistance to AVR2‐producing strains of C. fulvum. These data support a role for Rcr3 in the perception of AVR2 by Cf‐2.
Listeria monocytogenes bile salt hydrolase is a PrfA‐regulated virulence factor involved in the intestinal and hepatic phases of listeriosis
Authors: Dussurget Olivier , Cabanes Didier , Dehoux Pierre , Lecuit Marc ,   the European Listeria Genome Consortium, Buchrieser Carmen , Glaser Philippe , Cossart Pascale
Published: March 2003

Listeria monocytogenes
is a bacterial pathogen causing severe food‐borne infections in humans and animals. It can sense and adapt to a variety of harsh microenvironments outside as well as inside the host. Once ingested by a mammalian host, the bacterial pathogen reaches the intestinal lumen, where it encounters bile salts which, in addition to their role in digestion, have antimicrobial activity. Comparison of the
L. monocytogenes
and
Listeria innocua
genomes has revealed the presence of an
L. monocytogenes
‐specific putative gene encoding a bile salt hydrolase (BSH). Here, we show that the
bsh
gene encodes a functional intracellular enzyme in all pathogenic
Listeria
species. The
bsh
gene is positively regulated by PrfA, the transcriptional activator of known
L. monocytogenes
virulence genes. Moreover, BSH activity increases at low oxygen concentration. Deletion of
bsh
results in decreased resistance to bile
in vitro
, reduced bacterial faecal carriage after oral infection of the guinea‐pigs, reduced virulence and liver colonization after intravenous inoculation of mice. Taken together, these results demonstrate that BSH is a novel PrfA‐regulated
L. monocytogenes virulence factor involved in the intestinal and hepatic phases of listeriosis.
Genome sequence of Streptococcus agalactiae, a pathogen causing invasive neonatal disease
Authors: Glaser Philippe , Rusniok Christophe , Buchrieser Carmen , Chevalier Fabien , Frangeul Lionel , Msadek Tarek , Zouine Mohamed , Couvé Elisabeth , Lalioui Lila , Poyart Claire , Trieu‐Cuot Patrick , Kunst Frank
Published: March 2003

Streptococcus agalactiae
is a commensal bacterium colonizing the intestinal tract of a significant proportion of the human population. However, it is also a pathogen which is the leading cause of invasive infections in neonates and causes septicaemia, meningitis and pneumonia. We sequenced the genome of the serogroup III strain NEM316, responsible for a fatal case of septicaemia. The genome is 2 211 485 base pairs long and contains 2118 protein coding genes. Fifty‐five per cent of the predicted genes have an ortholog in the
Streptococcus pyogenes
genome, representing a conserved backbone between these two streptococci. Among the genes in
S. agalactiae
that lack an ortholog in
S. pyogenes
, 50% are clustered within 14 islands. These islands contain known and putative virulence genes, mostly encoding surface proteins as well as a number of genes related to mobile elements. Some of these islands could therefore be considered as pathogenicity islands. Compared with other pathogenic streptococci,
S. agalactiae shows the unique feature that pathogenicity islands may have an important role in virulence acquisition and in genetic diversity.
An Inv/Mxi‐Spa‐like type III protein secretion system in Burkholderia pseudomallei modulates intracellular behaviour of the pathogen
Authors: Stevens Mark P. , Wood Michael W. , Taylor Lowrie A. , Monaghan Paul , Hawes Pippa , Jones Philip W. , Wallis Timothy S. , Galyov Edouard E.
Published: March 2003

Burkholderia pseudomallei is the causative agent of melioidosis, a serious infectious disease of humans and animals that is endemic in subtropical areas. B. pseudomallei is a facultative intracellular pathogen that may invade and survive within eukaryotic cells for prolonged periods. After internalization, the bacteria escape from endocytic vacuoles into the cytoplasm of infected cells and form membrane protrusions by inducing actin polymerization at one pole. It is believed that survival within phagocytic cells and cell‐to‐cell spread via actin protrusions is required for full virulence. We have studied the role of a putative type III protein secretion apparatus (Bsa) in the interaction between B. pseudomallei and host cells. The Bsa system is very similar to the Inv/Mxi‐Spa type III secretion systems of Salmonella and Shigella. Moreover, B. pseudomallei encodes proteins that are very similar to Salmonella and Shigella Inv/Mxi‐Spa secreted proteins required for invasion, escape from endocytic vacuoles, intercellular spread and pathogenesis. Antibodies to putative Bsa‐secreted proteins were detected in convalescent serum from a melioidosis patient, suggesting that the system is functionally expressed in vivo. B. pseudomallei mutant strains lacking components of the Bsa secretion and translocation apparatus were constructed. The mutant strains exhibited reduced replication in J774.2 murine macrophage‐like cells, an inability to escape from endocytic vacuoles and a complete absence of formation of membrane protrusions and actin tails. These findings indicate that the Bsa type III secretion system plays an essential role in modulating the intracellular behaviour of B. pseudomallei.
A site‐specific recombinase (RinQ) is required to exert incompatibility towards the symbiotic plasmid of Rhizobium etli
Authors: Quintero Verónica , Cevallos Miguel A. , Dávila Guillermo
Published: March 2003

The replication/partition region of the symbiotic plasmid p42d of Rhizobium etli CE3 is characterized by the presence of the repABC operon. A recombinant plasmid containing this region is able to replicate in a R. etli derivative cured from p42d, with the same stability and copy number shown by the parental plasmid. However, when this construct is introduced into the wild‐type strain, instead of exerting incompatibility against the p42d, it forms a stable cointegrate with it. In this paper, we show that a site‐specific resolvase, and its action sites are essential factors to displace the symbiotic p42d. We propose a model for this novel incompatibility mechanism.
Molecular diversity of the genetic loci responsible for lipopolysaccharide core oligosaccharide assembly within the genus Salmonella
Authors: Kaniuk Natalia A. , Monteiro Mario A. , Parker Craig T. , Whitfield Chris
Published: March 2003

The waa locus on the chromosome of Salmonella enterica encodes enzymes involved in the assembly of the core oligosaccharide region of the lipopolysaccharide (LPS) molecule. To date, there are two known core structures in Salmonella, represented by serovars Typhimurium (subspecies I) and Arizonae (subspecies IIIA). The waa locus for serovar Typhimurium has been characterized. Here, the corresponding locus from serovar Arizonae is described, and the molecular basis for the distinctive structures is established. Eleven of the 13 open reading frames (ORFs) are shared by the two loci and encode conserved proteins of known function. Two polymorphic regions distinguish the waa loci. One involves the waaK gene, the product of which adds a terminal α‐1,2‐linked N‐acetylglucosamine residue that characterizes the serovar Typhimurium core oligosaccharide. There is an extensive internal deletion within waaK of serovar Arizonae. The serovar Arizonae locus contains a novel ORF (waaH) between the waaB and waaP genes. Structural analyses and in vitro glycosyltransferase assays identified WaaH as the UDP‐glucose:(glucosyl) LPS α‐1,2‐glucosyltransferase responsible for the addition of the characteristic terminal glucose residue found in serovar Arizonae. Isolates comprising the Salmonella Reference Collections, SARC (representing the eight subspecies of S. enterica) and SARB (representing subspecies I), were examined to assess the distribution of the waa locus polymorphic regions in natural populations. These comparative studies identified additional waa locus polymorphisms, shedding light on the genetic basis for diversity in the LPS core oligosaccharides of Salmonella isolates and identifying potential sources of further novel LPS structures.
Divergence and transcriptional analysis of the division cell wall (dcw) gene cluster in Neisseria spp.
Authors: Snyder Lori A. S. , Shafer William M. , Saunders Nigel J.
Published: March 2003

Three of the 18 open reading frames in the division and cell wall synthesis cluster of the pathogenic Neisseria spp. are not present in the clusters of other bacterial species. The region containing two of these, dcaB and dcaC, displays interstrain and interspecies variability uncharacteristic of such clusters. 3′ of dcaB is a Correia repeat enclosed element (CREE), which is only present in some strains. It has been suggested that this CREE is a transcriptional terminator, although we demonstrate otherwise. A gearbox‐like promoter within this CREE is active in Escherichia coli but not in Neisseria meningitidis. There is an active promoter 5′ of dcaC, although its sequence is not conserved. The presence of similarly located promoters has not been demonstrated in other species. In Neisseria lactamica, this promoter involves another dcw‐associated CREE, the first demonstration of active promoter generation at the 5′ end of this common intergenic, apparently mobile, element. Upstream of this promoter is an inverted pair of neisserial uptake signal sequences, which are commonly considered to be transcriptional terminators. It has been proposed to terminate transcription in this location, although we have demonstrated transcript extending through this uptake signal sequence. dcaC contains a 108 bp tandem repeat, which is present in different copy numbers in the neisserial strains examined. This investigation reveals extensive sequence variation, disputes the presence of transcriptional terminators and identifies active internal promoters in this normally highly conserved cluster of essential genes, and addresses the transcriptional activity of two common neisserial intergenic components.
Regulation of Salmonella typhimurium lipopolysaccharide O antigen chain length is required for virulence; identification of FepE as a second Wzz
Authors: Murray Gerald L. , Attridge Stephen R. , Morona Renato
Published: March 2003

Wzz proteins regulate the degree of polymerization of the O antigen (Oag) subunits in lipopolysaccharide (LPS) biosynthesis. Although the pathogenic relevance of Oag is well recognized, the significance of Oag chain length regulation is not well defined. In this report, Salmonella typhimurium was shown to possess two functional wzz genes resulting in a bimodal Oag length distribution. In addition to the previously described wzzST that results in long (L) modal length LPS with 16–35 Oag repeat units (RUs), we now report that wzzfepE, a homologue of Escherichia coli fepE, is responsible for the production of very long (VL) modal length LPS Oag, estimated to contain> 100 Oag RUs. Analysis of a series of isogenic S. typhimurium C5 mutants found that the presence of either wzz gene (and hence either modal length) was sufficient for complement resistance and virulence in the mouse model of infection, suggesting a degree of redundancy in the role of these two wzz genes and their respective Oag modal lengths. In contrast, the wzzST/wzzfepE double mutant, with relatively short, random‐length Oag, displayed enhanced susceptibility to complement and was highly attenuated in the mouse. This clearly demonstrates the molecular genetic basis for the longer LPS Oag chains previously identified as the basis of complement resistance in Salmonella. The presence of wzzfepE homologues in the genomic sequences of strains of Escherichia coli, Shigella flexneri and multiple serovars of Salmonella suggests that bimodality of LPS Oag is a common phenomenon in the Enterobacteriaceae.
Stage‐specific gene expression of Candida albicans in human blood
Authors: Fradin Chantal , Kretschmar Marianne , Nichterlein Thomas , Gaillardin Claude , D’Enfert Christophe , Hube Bernhard
Published: March 2003

The pathogenic fungus Candida albicans commonly causes mucosal surface infections. In immunocompromised patients, C. albicans may penetrate into deeper tissue, enter the bloodstream and disseminate within the host causing life‐threatening systemic infections. In order to elucidate how C. albicans responds to the challenge of a blood environment, we analysed the transcription profile of C. albicans cells exposed to human blood using genomic arrays and a cDNA subtraction protocol. By combining data obtained with these two methods, we were able to identify unique sets of different fungal genes specifically expressed at different stages of this model that mimics bloodstream infections. By removing host cells and incubation in plasma, we were also able to identify several genes in which the expression level was significantly influenced by the presence of these cells. Differentially expressed genes included those that are involved in the general stress response, antioxidative response, glyoxylate cycle as well as putative virulence attributes. These data point to possible mechanisms by which C. albicans ensures survival in the hostile environment of the blood and how the fungus may escape the bloodstream as an essential step in its systemic dissemination.
Role of GABAA receptors in rat hindbrain nuclei controlling gastric motor function
Authors: SIVARAO  , KROWICKI  , HORNBY
Published: March 2003

It has been shown in cats that gastric motor control by the dorsal vagal complex and nucleus ambiguus is under a tonic GABAergic influence. Since much more work has been performed in rats to define vago‐vagal reflexes controlling gastrointestinal function, an understanding of the potential inhibition by candidate neurotransmitters such as GABA (gamma aminobutyric acid) in the rat dorsal vagal complex (DVC) is essential to assess. Multiple‐barrelled micropipettes were used to apply to the dorsal vagal complex the GABAA antagonist, bicuculline methiodide (0.1–1 nmol), and a GABAA agonist, muscimol (10 nmol) prior to micro‐injection of the GABAA antagonist. Micro‐injections of bicuculline (353 pmol and 1 nmol), which were localized primarily in the dorsal motor nucleus of the vagus, produced significant increases in intragastric pressure and pyloric motility. These responses were abolished by vagotomy and by a prior micro‐injection of muscimol. To determine whether GABAergic blockade in the dorsal vagal complex results in gastric motor excitation through excitatory amino acid receptors, kynurenic acid (5 nmol), a kainate/NMDA (N‐methyl
D‐aspartic acid) receptor antagonist, was micro‐injected prior to bicuculline. This abolished the increase in gastric motor function normally evoked by bicuculline. In the other two important hindbrain nuclei controlling gastric function, the nucleus raphe obscurus and nucleus ambiguus, bicuculline (353 pmol) significantly increased intragastric pressure via vagally mediated pathways. These data demonstrate that all three rat hindbrain nuclei known to influence gastric function via the vagus nerve are under tonic GABAergic control. In addition, in the dorsal vagal complex, relief from GABAergic inhibition results in increases in gastric motor function through kainate/NMDA receptor‐mediated excitation.
Distribution of calcitonin gene‐related peptide, somatostatin, substance P and vasoactive intestinal polypeptide in experimental colitis in rats
Authors: MIAMPAMBA  , SHARKEY
Published: March 2003

Immunohistochemistry was used to examine the distribution of calcitonin gene‐related peptide (CGRP), substance P, somatostatin and vasoactive intestinal polypeptide (VIP) in experimental colitis induced with trinitrobenzene sulphonic acid (TNBS) in rats. CGRP immunoreactivity was observed throughout the colonic wall. A significant reduction of CGRP‐immunoreactive (IR) nerve fibres was observed in the mucosa after the induction of colitis. After TNBS treatment substance P immunoreactivity was reduced throughout the colon; however, after 7 days there was a marked re‐innervation of the circular muscle. Somatostatin immunoreactivity was distributed sparsely within the colonic wall, and was comparatively less affected by colitis. VIP immuno‐ reactivity was abundantly distributed in the colonic wall and underwent an immediate reduction in the mucosa after TNBS treatment. After 2 days, there was a consistent and progressive increase in the number and density of VIP‐IR nerve fibres in the inflamed colon, particularly the circular muscle. This change was associated with a proliferation of nerve fibres within the muscle layers. It was concluded that the early decrease in these neuropeptides was consistent with release from peripheral nerve terminals or the loss of nerves during the initial stages of colonic inflammation, which may be an essential condition for the development of colitis in this model. The observation that the intensity and density of substance P and VIP‐IR nerve fibres increased in the circular muscle 7 days after the induction of colitis suggests their possible involvement in tissue repair.
Inhibition of stress‐stimulated colonic propulsion by α2‐adrenoceptor antagonists in rats
Authors: YAMAMOTO  , NIIDA  , TAJIMA  , SHIROUCHI  , MASUI  , UEDA  , KISE  , KIMURA
Published: March 2003

Alpha2‐adrenoceptor antagonists have been reported to stimulate colonic motor activity, but the effect on colonic motor dysfunction is unclear. We have investigated the effect of α2‐adrenoceptor antagonists on wrap‐restraint stress‐stimulated and normal colonic propulsion in rats. Colonic propulsion was evaluated by the transit of a charcoal marker along the colon. Faecal pellets output was also measured. A 30‐min exposure to wrap‐restraint stress starting 120 min after infusion of the charcoal marker significantly stimulated colonic transit with a concomitant increase in faecal pellets. Yohimbine and idazoxan, α2‐adrenoceptor antagonists, clonidine, an α2‐adrenoceptor agonist, and atropine suppressed wrap‐restraint stress‐stimulated colonic transit and faecal excretion in a dose‐dependent manner. Ondansetron and YM060, 5‐hydroxytryptamine3 (5‐HT3) receptor antagonists, potently inhibited wrap‐restraint stress‐stimulated colonic transit, but only weakly inhibited faecal excretion. Neither α2‐adrenoceptor antagonists nor atropine had any significant effect on normal colonic transit, whereas clonidine and the 5‐HT3 receptor antagonists inhibited it. α2‐Adrenoceptor antagonists as well as clonidine, atropine and 5‐HT3 receptor antagonists inhibit the stress‐induced colonic motor dysfunction in rats.
Cognitive evoked potentials to anticipated oesophageal stimulus in humans: quantitative assessment of the cognitive aspects of visceral perception
Authors: HOLLERBACH  , FITZPATRICK  , SHINE  , KAMATH  , UPTON  , TOUGAS
Published: March 2003

Evoked potential studies provide an objective measure of the neural pathways involved with perception. The effects of cognitive factors, such as anticipation or awareness, on evoked potentials are not known. The aim was to compare the evoked potential response to oesophageal stimulation with the cortical activity associated with anticipation of the same stimulus. In 12 healthy men (23.5 ± 4 years), oesophageal electrical stimulation (15 mA, 0.2 Hz, 0.2 msec) was applied, and the evoked potentials recorded using scalp electrodes. A computerized model of randomly skipped stimuli (4:1 ratio) was used to separately record the evoked potentials associated with stimulation and those associated with an anticipated stimulus. The electrical stimulus represented the nontarget stimulus and the skipped impulse the target (anticipatory) stimulus. This anticipatory evoked potential was also compared to auditory P300 evoked potentials. Reproducible evoked potentials and auditory P300 responses were elicited in all subjects. Anticipatory evoked potentials (peak latency 282.1 ± 7.9 msec, amplitude 8.2 ± 0.7 μV, P < 0.05 vs auditory P300 evoked potential) were obtained with the skipped stimulus. This anticipatory evoked potential was located frontocentrally, while the auditory P300 potential was located in the centro‐parietal cortex. The anticipatory evoked potential associated with expectation of an oesophageal stimulus, although of similar latency to that of the auditory P300 evoked response, originates from a different cortical location. The recording of cognitive evoked potentials to an expected oesophageal stimulus depends on attention to, and awareness of, the actual stimulus. Anticipatory evoked potentials to GI stimuli may provide an objective electrophysiological tool for the assessment of the cognitive factors associated with visceral perception.
Preserved extrinsic neural connection between gallbladder and residual stomach is essential to prevent dysmotility of gallbladder after distal gastrectomy
Authors: Tsukamoto  , Enjoji  , Ura  , Kanematsu
Published: March 2003

Many reports have been made on gallbladder dysfunction after gastric surgery. We reported that the gallbladder contractions were abolished after antrectomy. Our hypothesis is that preservation of the neural connection along gastric vessels maintains normal gallbladder function during fasting after antrectomy. Six dogs underwent antrectomy with preservation of the extrinsic nerves and six other dogs underwent conventional antrectomy with dissection of the extrinsic nerves and vessels. Laparotomy alone was performed on another six control dogs. Motor activities of the gallbladder and upper gastrointestinal tract were recorded by strain gauges in conscious dogs. Motilin and CCK‐OP were used for pharmacological interventions. In conventional antrectomy, the amplitudes of the cyclic motor activity of the gallbladder and the stomach during fasting were significantly reduced, while this activity was maintained in dogs when extrinsic nerves were preserved. The coordination of the motor activity among the gallbladder, stomach and the duodenum was not impaired in the dogs when extrinsic nerves were preserved. The contractions of the gallbladder induced by exogenous motilin were reduced significantly in conventional antrectomy. Therefore, the preserved neural connection along the gastric vessels maintains normal gallbladder function in the fasting state even after distal gastrectomy.
Nissen‐type fundoplication and its effects on the emetic reflex and gastric motility in the ferret
Authors: Richards  , Carr  , Spitz  , Milla  , Andrews
Published: March 2003

Recurrent vomiting with failure to thrive is a common problem in neurologically impaired children. Many undergo fundoplication to control the underlying gastro‐oesophageal reflux. The results of surgery are not always satisfactory and post‐operative retching may be a major problem – a symptom indicative of activation of the emetic reflex. An animal model of antireflux surgery has been developed and used to investigate the effects of such surgery upon the emetic reflex and vagal influences on gastric motility. Following surgery, animals responded to a previously subemetic dose of a centrally acting opiate receptor agonist (loperamide), suggesting that fundoplication may sensitize the emetic reflex. A gastric vago‐vagal reflex (tonic inhibition of corpus tone) and responses to direct stimulation of vagal motor efferents (both cholinergic and nonadrenergic noncholinergic responses) were not significantly affected by antireflux surgery. Mechanisms by which neural damage may sensitize the emetic reflex are discussed, together with the possible clinical implications for the management of post‐operative symptoms in neurologically impaired children.
Abnormal visceral perception in patients with functional dyspepsia: use of cerebral potentials evoked by electrical stimulation of the oesophagus
Authors: Kanazawa  , Nomura  , Fukudo  , Hongo
Published: March 2003

Altered visceral perception is thought to be included in the pathogenesis of functional dyspepsia. However, in previous studies, the assessment of visceral perception has been based solely on patients’ self‐reported symptoms. Cerebral evoked potential (EP), either by mechanical or electrical stimulation (ES) of the visceral organ, is used to evaluate visceral perception via afferent neural pathways. In this study, we investigated the visceral perception in patients with functional dyspepsia by EP to eliminate the possibility of self‐reported bias. EP responses were recorded by oesophageal ES at 37 cm from the nostril in 14 patients with functional dyspepsia and 14 normal healthy control subjects. Threshold levels of perception, peak latencies and peak‐to‐peak amplitudes of EP were evaluated. There was no difference in the sensory threshold between the dyspeptic patients and the control subjects (median 6 mA, range 2–12 mA, vs. 8 mA, range 6–14 mA; Pthinsp;= 0.09). There was a strong trend towards a decreased discomfort threshold in the patients when compared to the control subjects (median 14 mA, range 6–24 mA vs. 20 mA, range 14–26 mA; Pthinsp;= 0.05). The latency of the later EP peak (N2) among the patients (154 ± 4 ms) was significantly shorter than that of the control subjects (171 ± 3 ms, P < 0.01) although there was no difference between the earlier peaks (N1 and P1). There was also no difference in the amplitudes (N1/P1 and P1/N2) of EP between the patients and the control subjects. Half of the patients (seven out of 14) complained of nausea during ES but the control subjects were unaffected. The latency of the first EP peak (N1) in the patients with nausea was significantly shorter (66 ± 3 ms) than that of the patients without nausea (79 ± 4 ms, P < 0.05) or among the control subjects (80 ± 3 ms, P < 0.05). These results suggest that dyspeptic patients may recruit a greater number of fast conducting myelinated nerve fibres that convey visceral afferent impulses to the brain and/or that dyspeptic patients may have an altered central processing of the visceral perception. We conclude that EP recording by oesophageal ES provides an objective measurement of altered visceral perception in patients with functional dyspepsia.
Opioid‐receptor‐mediated excitation of rat mesenteric afferent fibres supplying the rat jejunum
Authors: Eastwood C. , Grundy D.
Published: March 2003

The aim of the present study was to examine the sensitivity to opioid‐receptor agonists of mesenteric afferents supplying the small intestine and to characterize the subpopulations of any responsive fibres. Mesenteric afferent discharge was recorded electrophysiologically in response to cumulative doses (1–400 μg kg–1) of the μ‐receptor agonist [
D‐ala,2 N‐ me‐Phe4,Gly5‐ol]‐enkephalin (DAMGO), the δ‐receptor agonist [
D‐ala,2D‐leu5]‐enkephalin (DADLE) and the κ‐receptor agonist U‐50488. DAMGO and DADLE, but not U‐50488, markedly stimulated whole nerve mesenteric afferent discharge (P < 0.05) that was unrelated to intestinal motor events. Subpopulations of afferent fibres responding to DAMGO were examined using waveform analysis to identify single units from within the whole mesenteric nerve bundles. One population was CCK‐sensitive (15/15 fibres) and the other was a subpopulation of mechanosensitive afferents that responded to distension (17/28). 5‐HT‐sensitive afferents did not respond to DAMGO (0/11). We conclude that specific subpopulations of mesenteric afferents respond to μ‐ and possibly δ‐ but not κ‐receptor agonists. This sensitivity to opioids may contribute to the antinociceptive property of vagal afferents.
Prostaglandin E2 stimulates motilin release via a cholinergic muscarinic pathway in the dog
Authors: Mochiki E. , Nakabayashi T. , Suzuki H. , Haga N. , Asao T. , Kuwano H. , Itoh Z.
Published: March 2003

Prostaglandins are well known to be widely distributed in mammalian gastrointestinal tissues and to play a role in the regulation of gastrointestinal hormones and contractions. The present study was undertaken to determine whether prostaglandins have an effect on the endogenous release of motilin in the dog. In six conscious dogs, gastrointestinal contractions were monitored by means of chronically implanted force transducers. Prostaglandin E2 (PGE2; 3, 10, 30 μg kg−1) was given intravenously during the interdigestive phase I period with or without a muscarinic or nicotinic receptor antagonist. Blood samples were collected from 10 min before, to 30 min after, prostaglandin injection. Indomethacin (5 mg kg−1) was given intravenously to investigate the effect of endogenous prostaglandins on motilin release. PGE2 significantly stimulated motilin release but not gastric contractions. Atropine, but not hexamethonium, blocked PGE2‐induced motilin release. Motilin release in response to PGE2 was significantly increased by pretreatment with hexamethonium. Indomethacin inhibited the cyclic release of motilin and gastric phase III contractions. We conclude that PGE2 appears to stimulate motilin release via cholinergic muscarinic pathways, and nicotinic receptors modulate this reaction. PGE2 may be involved in part in the regulation of the cyclic release of motilin and the occurrence of gastric phase III.
Differential gene expression in the small intestines of wildtype and W/WV mice
Authors: Takayama I. , Daigo Y. , Ward S. M. , Sanders K. M. , Yamanaka T. , Fujino M. A.
Published: March 2003

Much of the evidence demonstrating the role of interstitial cells of Cajal (ICC) in pacemaking and neurotransmission in the gastrointestinal tract comes from studies of W/WV mice. These animals have few pacemaker ICC in the small bowel due to reduced functional Kit protein. We examined gene expression in the small intestines of wildtype and W/WV mice. RNA expression in the jejunums of wildtype and W/WV mutants was studied using a differential gene expression method. Seven known genes were differentially expressed in wildtype and W/WV mice. COX7B (cytochrome c oxidase, subunit VIIb) and SORCIN (encoding multidrug‐resistance complex, class 4) were suppressed in both fed and fasted W/WV mice. Expression of another five genes was increased in W/WV mice: ADA (adenosine deaminase), MDH1 (malate dehydrogenase), RPL‐8 (ribosomal protein L8), SPTB2 (spectrin, nonerythroid, beta subunit), and p6–5 (encoding phosphorylcholine [PC] T‐cell suppressor factor [TsF]). Differential expression was the same in fasted and fed animals, suggesting that the differences were independent of the dietetic state. We conclude that several genes are differentially expressed in the small intestines of W/WV mice where the major lesion is loss of pacemaker ICC. Differential gene display may help develop a molecular profile of motility disorders in which ICC are lost.