bluuebunny/crossref_metadata_2025 huggingface dataset:
The Bacillus subtilis cell division proteins FtsL and DivIC are intrinsically unstable and do not interact with one another in the absence of other septasomal components
Authors: Robson Scott A. , Michie Katharine A. , Mackay Joel P. , Harry Elizabeth , King Glenn F.
Published: March 2003

The bacterial septum appears to comprise a macromolecular assembly of essential cell division proteins (the ‘septasome’) that are responsible for physically dividing the cell during cytokinesis. FtsL and DivIC are essential components of this division machinery in Bacillus subtilis. We used yeast two‐hybrid analysis as well as a variety of biochemical and biophysical methods to examine the proposed interaction between Bacillus subtilis FtsL and DivIC. We show that FtsL and DivIC are thermodynamically unstable proteins that are likely to be unfolded and therefore targeted for degradation unless stabilized by interactions with other components of the septasome. However, we show that this stabilization does not result from a direct interaction between FtsL and DivIC. We propose that the observed interdepend‐ence of DivIC and FtsL stability is a result of indirect interactions that are mediated by other septasomal proteins.
Rho1p mutations specific for regulation of β(1→3)glucan synthesis and the order of assembly of the yeast cell wall
Authors: Roh Dong‐Hyun , Bowers Blair , Riezman Howard , Cabib Enrico
Published: March 2003

In the yeast Saccharomyces cerevisiae, the GTP‐binding protein Rho1 is required for β(1→3)glucan synthase activity, for activation of protein kinase C and the cell integrity pathway and for progression in G1, cell polarization and exocytosis. A genetic screen for cells that become permeabilized at non‐permissive temperature was used to isolate in vitro‐generated mutants of Rho1p. After undergoing a battery of tests, several of them appeared to be specifically defective in the β(1→3)glucan synthesis function of Rho1p. At the non‐permissive temperature (37°C), the mutants developed defects in the cell wall, especially at the tip of new buds. In the yeast cell wall, β(1→6)glucan is linked to both β(1→3)glucan and mannoprotein, as well as occasionally to chitin. We have used the rho1 mutants to study the order of assembly of the cell wall components. The incorporation of [14C]‐glucose into β(1→3)glucan at 37°C was decreased or abolished in the mutants. Concomitantly, a partial defect in the incorporation of label into cell wall mannoproteins and β(1→6)glucan was observed. In contrast, YW3458, an inhibitor of glycosylphosphatidylinositol anchor formation, prevented mannoprotein incorporation, whereas the β(1→3)–β(1→6)glucan complex was synthesized at almost normal levels. As β(1→3)glucan can be synthesized in vitro or in vivo independently, we conclude that the order of addition in vivo is β(1→3)glucan, β(1→6)glucan, mannoprotein. Previous observations indicate that chitin is the last component to be incorporated into the complex.
Multiple origins of hydrogenosomes: functional and phylogenetic evidence from the ADP/ATP carrier of the anaerobic chytridNeocallimastixsp.
Authors: Voncken Frank , Boxma Brigitte , Tjaden Joachim , Akhmanova Anna , Huynen Martijn , Verbeek Fons , Tielens Aloysius G. M. , Haferkamp Ilka , Neuhaus H. Ekkehard , Vogels Godfried , Veenhuis Marten , Hackstein Johannes H. P.
Published: March 2003

A mitochondrial‐type ADP/ATP carrier (AAC) has been identified in the hydrogenosomes of the anaerobic chytridiomycete fungusNeocallimastixsp. L2. Biochemical and immunocytochemical studies revealed that this ADP/ATP carrier is an integral component of hydrogenosomal membranes. Expression of the corresponding cDNA inEscherichia coliconfers the ability on the bacterial host to incorporate ADP at significantly higher rates than ATP – similar to isolated mitochondria of yeast and animals. Phylogenetic analysis of this AAC gene (hdgaac) confirmed with high statistical support that the hydrogenosomal ADP/ATP carrier ofNeocallimastixsp. L2 belongs to the family of veritable mitochondrial‐type AACs. Hydrogenosome‐bearing anaerobic ciliates possess clearly distinct mitochondrial‐type AACs, whereas the potential hydrogenosomal carrier Hmp31 of the anaerobic flagellateTrichomonas vaginalisand its homologue fromTrichomonas gallinaedo not belong to this family of proteins. Also, phylogenetic analysis of genes encoding mitochondrial‐type chaperonin 60 proteins (HSP 60) supports the conclusion that the hydrogenosomes of anaerobic chytrids and anaerobic ciliates had independent origins, although both of them arose from mitochondria.
Depression of Saccharomyces cerevisiae invasive growth on non‐glucose carbon sources requires the Snf1 kinase
Authors: Palecek Sean P. , Parikh Archita S. , Huh Joon H. , Kron Stephen J.
Published: March 2003

Haploid Saccharomyces cerevisiae cells growing on media lacking glucose but containing high concentrations of carbon sources such as fructose, galactose, raffinose, and ethanol exhibit enhanced agar invasion. These carbon sources also promote diploid filamentous growth in response to nitrogen starvation. The enhanced invasive and filamentous growth phenotypes are suppressed by the addition of glucose to the media and require the Snf1 kinase. Mutations in the PGI1 and GND1 genes encoding carbon source utilization enzymes confer enhanced invasive growth that is unaffected by glucose but requires active Snf1. Carbon source does not modulate FLO11 flocculin expression, but enhanced polarized bud site selection is necessary for invasion on certain carbon sources. Interestingly, deletion of SNF1 blocks invasion without affecting bud site selection. Snf1 is also required for formation of spokes and hubs in multicellular mats. To examine glucose repression of invasive growth more broadly, we performed genome‐wide microarray expression analysis in wild‐type cells growing on glucose and galactose, and snf1Δ cells on galactose. SNF1 probably mediates glucose repression of multiple genes potentially involved in invasive and filamentous growth. FLO11‐independent cell‐cell attachment, cell wall integrity, and/or polarized growth are affected by carbon source metabolism. In addition, derepression of cell cycle genes and signalling via the cAMP–PKA pathway appears to depend upon SNF1 activity during growth on galactose.
The role of the G2 box, a conserved motif in the histidine kinase superfamily, in modulating the function of EnvZ
Authors: Zhu Yan , Inouye Masayori
Published: March 2003

Histidine kinase EnvZ, a transmembrane osmotic sensor for Escherichia coli, is a bifunctional enzyme having OmpR (its cognate response regulator) kinase and phosphorylated OmpR (OmpR‐P) phosphatase activities. Its cytoplasmic domain consists of domain A responsible for dimerization of EnvZ, histidine phosphotransfer and phosphatase activities, and domain B responsible for ATP binding. Here, we have constructed a number of substitution mutations at the G2 box, one of the conserved motifs in domain B, and demonstrated that they influence the phosphatase activity of EnvZ over a wide range. The effects of ADP, a cofactor for the phosphatase activity, were found to be substantially different depending upon the mutations. The effects of these mutations were also examined in vivo using a chimeric Tar–EnvZ construct (Taz1‐1), and the results agreed with the in vitro data for the phosphatase and kinase activities for all mutations. Using Taz1‐1 carrying the T402A mutation, three independent intragenic suppressor mutations (T235M, S269L and E276K) were isolated, and all were found in domain A. Together, the present results demonstrate for the first time that domain A and domain B are functionally co‐ordinated and topologi‐cally arranged in a specific manner. The G2 box may modulate the interaction between these two domains in response to extracellular osmolarity.
A third envelope stress signal transduction pathway in Escherichia coli
Authors: Raffa Robert G. , Raivio Tracy L.
Published: March 2003

Escherichia coli
uses overlapping envelope stress responses to adapt to insults to the bacterial envelope that cause protein misfolding. The σ
E
and Cpx envelope stress responses are activated by both common and distinct envelope stresses and respond by increasing the expression of the periplasmic protease DegP as well as target genes unique to each response. The σ
E
pathway is involved in outer membrane protein (OMP) folding quality control whereas the Cpx pathway plays an important role in the assembly of at least one pilus. Previously, we identified the
spy
gene as a new Cpx regulon member of unknown function. Interestingly, induction of
spy
expression by severe envelope stresses such as spheroplasting is only partially dependent on an intact Cpx signalling pathway, unlike other Cpx‐regulated genes. Here we show that the BaeS sensor kinase and BaeR response regulator also control expression of
spy
in response to envelope stress. BaeS and BaeR do not affect expression of other known Cpx‐regulated genes, however,
baeR cpxR
double mutants show increased sensitivity to envelope stresses relative to either single mutant alone. We propose that the Bae signal transduction pathway controls a third envelope stress response in
E. coli that induces expression of a distinct set of adaptive genes.
High resolution contact probing of the Lrp‐like DNA‐binding protein Ss‐Lrp from the hyperthermoacidophilic crenarchaeote Sulfolobus solfataricus P2
Authors: Enoru‐Eta Julius , Gigot Daniel , Glansdorff Nicolas , Charlier Daniel
Published: March 2003

Ss‐Lrp, from Sulfolobus solfataricus, is an archaeal homologue of the global bacterial regulator Lrp (Leucine‐responsive regulatory protein), which out of all genome‐encoded proteins is most similar to Escherichia coli Lrp (E‐value of 5.6 e−14). The recombinant protein has been purified as a 68 kDa homotetramer. The specific binding of Ss‐Lrp to its own control region is suggestive of negative autoregulation. A high resolution contact map of Ss‐Lrp binding was established by DNase I and hydroxyl radical footprinting, small non‐intercalating groove‐specific ligand‐binding interference, and various base‐specific premodification and base removal binding interference techniques. We show that Ss‐Lrp binds one face of the DNA helix and establishes the most salient contacts with two major groove segments and the intervening minor groove, in a region that overlaps the TATA‐box and BRE promoter elements. Therefore, Ss‐Lrp most likely exerts autoregulation by preventing promoter recognition by TBP and TFB. Moreover, the results demonstrate profound Ss‐Lrp induced structural alterations of sequence stretches flanking the core contact site, and reveal that the deformability of these regions significantly contributes to binding selectivity.
High‐frequency conjugative transfer of antibiotic resistance genes to Yersinia pestis in the flea midgut
Authors: Hinnebusch B. Joseph , Rosso Marie‐Laure , Schwan Tom G. , Carniel Elisabeth
Published: March 2003

The acquisition of foreign DNA by horizontal transfer from unrelated organisms is a major source of variation leading to new strains of bacterial pathogens. The extent to which this occurs varies widely, due in part to lifestyle factors that determine exposure to potential donors. Yersinia pestis, the plague bacillus, infects normally sterile sites in its mammalian host, but forms dense aggregates in the non‐sterile digestive tract of its flea vector to produce a transmissible infection. Here we show that unrelated co‐infecting bacteria in the flea midgut are readily incorporated into these aggregates, and that this close physical contact leads to high‐frequency conjugative genetic exchange. Transfer of an antibiotic resistance plasmid from an Escherichia coli donor to Y. pestis occurred in the flea midgut at a frequency of 10−3 after only 3 days of co‐infection, and after 4 weeks 95% of co‐infected fleas contained an average of 103 antibiotic‐resistant Y. pestis transconjugants. Thus, transit in its arthropod vector exposes Y. pestis to favourable conditions for efficient genetic exchange with microbial flora of the flea gut. Horizontal gene transfer in the flea may be the source of antibiotic‐resistant Y. pestis strains recently isolated from plague patients in Madagascar.
Cell cycle‐dependent expression of an essential SMC‐like protein and dynamic chromosome localization in the archaeon Halobacterium salinarum
Authors: Herrmann Ute , Soppa Jörg
Published: March 2003

The genome of Halobacterium salinarum encodes four proteins of the structural maintenance of chromosomes (SMC) protein superfamily. Two proteins form a novel subfamily and are named ‘SMC‐like proteins of H. salinarum’ (Sph1 and Sph2). Northern blot analyses revealed that sph1 and hp24, the adjacent gene, are solely transcribed in exponentially growing, but not in stationary phase, cells. A synchronization procedure was developed, which makes use of the DNA polymerase inhibitor aphidicolin and leads to highly synchronous cultures. It allowed us for the first time to study cell cycle‐dependent transcription in an archaeon. The sph1 transcript was found to be highly cell cycle regulated, with its maximal accumulation around the time of septum formation. The Sph1 protein level was also elevated at that time, but a basal protein level was found throughout the cell cycle. The hp24 transcript was sharply upregulated about 1 h before sph1 and had already declined at the time of sph1 induction. These and additional transcript patterns revealed that precisely controlled transcriptional regulation is involved in haloarchaeal cell cycle progression. A DNA staining protocol was developed, which opened the possibility of following the dynamic intracellular localization of haloarchaeal nucleoids using synchronized cultures. After an initial dispersed localization, the nucleoid is condensed at mid‐cell. Subsequently, DNA is rapidly transported to the 1/4 and 3/4 positions. All staining patterns were also observed in untreated exponentially growing cells, excluding synchronization artifacts. The Sph1 concentration is elevated when segregation of the new chromosomes is nearly complete; therefore, it is proposed to play a role in a late step of replication, e.g. DNA repair, similar to eukaryotic Rad18 proteins.
The segregation of the Escherichia coli origin and terminus of replication
Authors: Li Yongfang , Sergueev Kirill , Austin Stuart
Published: March 2003

Escherichia coli chromosome replication forks are tethered to the cell centre. Two opposing models describe how the chromosomes segregate. In the extrusion‐capture model, newly replicated DNA is fed bi‐directionally from the forks toward the cell poles, forming new chromosomes in each cell half. Starting with the origins, chromosomal regions segregate away from their sisters progressively as they are replicated. The termini segregate last. In the sister chromosome cohesion model, replication produces sister chromosomes that are paired along much of their length. The origins and most other chromosomal regions remain paired until late in the replication cycle, and all segregate together. We use a combination of microscopy and flow cytometry to determine the relationship of origin and terminus segregation to the cell cycle. Origin segregation frequently follows closely after initiation, in strong support of the extrusion‐capture model. The spatial disposition of the origin and terminus sequences also fits this model. Terminus segregation occurs extremely late in the cell cycle as the daughter cells separate. As the septum begins to invaginate, the termini of the completed sister chromosomes are transiently held apart at the cell centre, on opposite sides of the cell. This may facilitate the resolution of topological linkages between the chromosomes.
In vivo aggregation of a single enzyme limits growth of Escherichia coli at elevated temperatures
Authors: Gur Eyal , Biran Dvora , Gazit Ehud , Ron Eliora Z.
Published: March 2003

The formation of protein aggregates is associated with unfolding and denaturation of proteins. Recent studies have indicated that, in Escherichia coli, cellular proteins tend to aggregate when the bacteria are exposed to thermal stress. Here, we show that the aggregation of one single E. coli cytoplasmic protein limits growth at elevated temperatures in minimal media. Homoserine trans‐succinylase (HTS), the first enzyme in the methionine biosynthetic pathway, aggregates at temperatures higher than 44°C in vitro. Above this temperature, we can also observe in vivo aggregation that results in the complete disappearance of the enzyme from the soluble fraction. Moreover, reducing the in vivo level of HTS aggregation enables growth at non‐permissive temperatures. This is the first demonstration of the physiological role of aggregation of a specific protein in the growth of wild‐type bacteria.
Many of the genes required for mating in Saccharomyces cerevisiae are also required for mating in Candida albicans
Authors: Magee B. B. , Legrand Melanie , Alarco Anne‐Marie , Raymond Martine , Magee P. T.
Published: March 2003

Candida albicans is the single, most frequently isolated human fungal pathogen. As with most fungal pathogens, the factors which contribute to pathogenesis in C. albicans are not known, despite more than a decade of molecular genetic analysis. Candida albicans was thought to be asexual until the discovery of the MTL loci homologous to the mating type (MAT) loci in Saccharomyces cerevisiae led to the demonstration that mating is possible. Using Candida albicans mutants in genes likely to be involved in mating, we analysed the process to determine its similarity to mating in Saccharomyces cerevisiae. We examined disruptions of three of the genes in the MAPK pathway which is involved in filamentous growth in both S. cerevisiae and C. albicans and is known to control pheromone response in the former fungus. Disruptions in HST7 and CPH1 blocked mating in both MTLa and MTLα strains, whereas disruptions in STE20 had no effect. A disruption in KEX2, a gene involved in processing the S. cerevisiae pheromone Mfα, prevented mating in MTLα but not MTLa cells, whereas a disruption in HST6, the orthologue of the STE6 gene which encodes an ABC transporter responsible for secretion of the Mfa pheromone, prevented mating in MTLa but not in MTLα cells. Disruption of two cell wall genes, ALS1 and INT1, had no effect on mating, even though ALS1 was identified by similarity to the S. cerevisiae sexual agglutinin, SAG1. The results reveal that these two diverged yeasts show a surprising similarity in their mating processes.
Lithium and valproate decrease the membrane phosphatidylinositol/phosphatidylcholine ratio
Authors: Ding Daobin , Greenberg Miriam L.
Published: March 2003

Lithium and valproate, two structurally different anti‐bipolar drugs, cause decreased intracellular inositol in the yeast Saccharomyces cerevisiae and an in‐crease in expression of a structural (INO1) and a regulatory (INO2) gene for phospholipid synthesis that responds to inositol depletion (Vaden, D., Ding, D., Peterson, B., and Greenberg, M.L., 2001, J Biol Chem 276: 15466–15471). We report here that both drugs decrease the relative rate of membrane phosphatidylinositol synthesis and, to a lesser but still significant degree, the steady state relative phosphatidylinositol composition. In addition, both drugs increase the rate of phosphatidylcholine (PC) synthesis. Finally, valproate, but not lithium, increases expression of phosphatidylcholine pathway genes CHO1 and OPI3. The overall effect on membrane phospholipid composition is a reduction in the phosphatidylinositol/phosphatidylcholine ratio by both drugs. Because maintenance of the appropriate phosphatidylinositol/phosphatidylcholine ratio is required for secretory vesicle formation, a decrease in this ratio may have far‐reaching implications for understanding the therapeutic mechanisms of action of these drugs.
Osmoregulation in Lactococcus lactis: BusR, a transcriptional repressor of the glycine betaine uptake system BusA
Authors: Romeo Yves , Obis David , Bouvier Jean , Guillot Alain , Fourçans Aude , Bouvier Isabelle , Gutierrez Claude , Mistou Michel‐Yves
Published: March 2003

The busA (opuA) locus of Lactococcus lactis encodes a glycine betaine uptake system. Transcription of busA is osmotically inducible and its induction after an osmotic stress is reduced in the presence of glycine betaine. Using a genetic screen in CLG802, an Escherichia coli strain carrying a lacZ transcriptional fusion expressed under the control of the busA promoter, we isolated a genomic fragment from the L. lactis subsp. cremoris strain MG1363, which represses transcription from busAp. The cloned locus responsible for this repression was identified as a gene present upstream from the busA operon, encoding a putative DNA binding protein. This gene was named busR. Electrophoretic mobility shift and footprinting experiments showed that BusR is able to bind a site that overlaps the busA promoter. Overexpression of busR in L. lactis reduced expression of busA. Its disruption led to increased and essentially constitutive transcription of busA at low osmolarity. Therefore, BusR is a major actor of the osmotic regulation of busA in L. lactis.
Expression of cnf1 by Escherichia coli J96 involves a large upstream DNA region including the hlyCABD operon, and is regulated by the RfaH protein
Authors: Landraud Luce , Gibert Maryse , Popoff Michel R. , Boquet Patrice , Gauthier Michel
Published: March 2003

Examination of 55 clinical isolates of uropathogenic Escherichia coli producing the CNF1 toxin demonstrated that the cnf1 gene is systematically associated with a hly operon via a highly conserved hlyD‐cnf1 intergenic region (igs, 943 bp) as shown in the J96 UPEC strain. We examined if this association could reflect a co‐regulation of the production of these toxins. Translation of cnf1 from an immediately upstream promoter has been shown to be controlled by means of an anti‐Shine–Dalgarno sequence present in the cnf1 coding sequence [fold‐back inhibition (cnf1 fbi)]. The cnf1 fbi was not regulated by elements present in the igs. An RNA covering the full hlyD sequence, the igs and extending on the cnf1 gene, was then detected in the J96 strain. This RNA could be part of a HlyCABD mRNA. Transcription of the haemolysin operon requires RfaH antitermination activity. Inactivation of rfaH in J96 resulted in a 100‐fold reduction of the CNF1 content of bacteria. The production of CNF1 from a plasmidic igscnf1 DNA was not sensitive to RfaH, indicating that this factor acted on cnf1 transcription via the hly promoter. This way the cnf1 fbi mechanism might be overcome by transcription of cnf1 from the haemolysin promoter and antitermination by RfaH. This constitutes a novel system of bacterial virulence factors co‐regulation.
Characterization of Cu,Zn superoxide dismutase (SOD1) gene knock‐out mutant of Cryptococcus neoformans var. gattii: role in biology and virulence
Authors: Narasipura Srinivas D. , Ault Jeffrey G. , Behr Melissa J. , Chaturvedi Vishnu , Chaturvedi Sudha
Published: March 2003

The pathogenic yeast Cryptococcus neoformans (Cn) var. gattii causes meningoencephalitis in healthy individuals, unlike the better known Cn varieties grubii and neoformans, which are common in immunocompromised individuals. The virulence determinants and mechanisms of host predilection are poorly defined for var. gattii. The present study focused on the characterization of a Cu,Zn superoxide dismutase (SOD1) gene knock‐out mutant constructed by developing a DNA transformation system. The sod1 mutant was highly sensitive to the redox cycling agent menadione, and showed fragmentation of the large vacuole in the cytoplasm, but no other defects were seen in growth, capsule synthesis, mating, sporulation, stationary phase survival or auxotrophies for sulphur‐containing amino acids. The sod1 mutant was markedly attenuated in virulence in a mouse model, and it was significantly susceptible to in vitro killing by human neutrophils (PMNs). The deletion of SOD1 also resulted in defects in the expression of a number of virulence factors, i.e. laccase, urease and phospholipase. Complementation of the sod1 mutant with SOD1 resulted in recovery of virulence factor expression and menadione resistance, and in restoration of virulence. Overall, these results suggest that the antioxidant function of Cu,Zn SOD is critical for the pathogenesis of the fungus, but is dispensable in its saprobic life. This report constitutes the first instance in which superoxide dismutase has been directly implicated in the virulence of a fungal pathogen.
Association of mycothiol with protection of Mycobacterium tuberculosis from toxic oxidants and antibiotics
Authors: Buchmeier Nancy A. , Newton Gerald L. , Koledin Teresa , Fahey Robert C.
Published: March 2003

Mycothiol, MSH or 1d‐myo‐inosityl 2‐(N‐acetyl‐l‐cysteinyl)amido‐2‐deoxy‐α‐d‐glucopyranoside, is an unusual conjugate of N‐acetylcysteine (AcCys) with 1d‐myo‐inosityl 2‐acetamido‐2‐deoxy‐α‐d‐glucopyranoside (GlcN‐Ins), and is the major low‐molecular‐mass thiol in mycobacteria. Mycothiol has antioxidant activity as well as the ability to detoxify a variety of toxic compounds. Because of these activities, MSH is a candidate for protecting Mycobacterium tuberculosis from inactivation by the host during infections as well as for resisting antituberculosis drugs. In order to define the protective role of MSH for M. tuberculosis, we have constructed an M. tuberculosis mutant in Rv1170, one of the candidate MSH biosynthetic genes. During exponential growth, the Rv1170 mutant bacteria produced ≈ 20% of wild‐type levels of MSH. Levels of the Rv1170 substrate, GlcNAc‐Ins, were elevated, whereas those of the product, GlcN‐Ins, were reduced. This establishes that the Rv1170 gene encodes for the major GlcNAc‐Ins deacetylase activity (termed MshB) in the MSH biosynthetic pathway of M. tuberculosis. The Rv1170 mutant grew poorly on agar media lacking catalase and oleic acid, and had heightened sensitivities to the toxic oxidant cumene hydroperoxide and to the antibiotic rifampin. In addition, the mutant was more resistant to isoniazid, suggesting a role for MSH in activation of this prodrug. These data indicate that MSH contributes to the protection of M. tuberculosis from oxidants and influences resistance to two first‐line antituberculosis drugs.
Changes in pyloric resistance induced by erythromycin
Authors: MATHIS  , MALBERT
Published: March 2003

Erythromycin increases gastric emptying but the characteristics of transpyloric flow and the contribution of pyloric resistance to the mechanisms of increased flow are unknown. Transpyloric outflow and gastroduodenal pressures were studied in eight anaesthetized pigs during intravenous infusion of erythromycin (10 mg kg−1). Erythromycin increased emptying of saline through an increased stroke volume of individual flow pulses (13.7 ± 1.24 vs. 3.6 ± 1.30 mL) rather than by decreasing the interval between flow pulses (3.9 ± 0.55 vs. 3.2 ± 0.30 pulse.min−1). This flow pattern was associated with more frequent antral contractions of larger amplitude originating from the upper part of the gastric antrum, suppression of isolated pyloric pressure waves and significant reduction of the pyloric resistance to flow (2.3 ± 0.21 vs. 4.6 ± 0.46 mmHg mL−1 s−1). The decreased resistance originated from a different temporal relationship between antropyloric pressure event and flow pulses that occurred almost simultaneously during erythromycin.
Effect of transdermal application of nicotine on colonic transit in healthy nonsmoking volunteers
Authors: Rausch T , Beglinger C , Alam N , Gyr K , Meier R
Published: March 2003

The radio‐opaque marker technique (ROMT) is a safe and noninvasive method to determine total colonic (TCTT) and segmental colonic transit times (SCTT). Previous results have shown that smoking volunteers had significantly longer TCTT than nonsmokers, but the underlying mechanism was not clear. We investigated the effect of transdermal nicotine application in two different doses in a nonblind randomized experiment involving three distinct phases. In phase 1 baseline transit times were determined with an abdominal X‐ray after a 6‐day period of marker ingestion and again after the following bowel movement to study the influence of a bowel movement just before the X‐ray. TCTT was nearly twice as high before than after defaecation (42.6 h vs. 25.1 h, P < 0.05). The main acceleration was found in the rectosigmoid (RS) (18.6 h vs 7.1 h, P < 0.05) with no significant changes in right (RC) and left colon (LC). In phase 2 and 3 nicotine was applied in two doses of 17.5 mg day−1 and 35 mg day−1 in random order. Both doses resulted in a significant decrease of TCTT compared to the predefaecation baseline (42.6 h vs 32.2 h/28.2 h, respectively, P < 0.05). Again the main effect was located in the RS (18.6 h vs 9.9 h/7.6 h, P < 0.05). Short‐term application nicotine results in a decrease of TCTT which is due to an accelerated transit in the RS.
The patterns of small bowel motility: physiology and implications in organic disease and functional disorders
Authors: HUSEBYE
Published: March 2003

The physiology and pathophysiology of small bowel motility are reviewed with particular focus on the motility patterns and periods that are detected by intraluminal manometry. Motility patterns are groups of phasic pressure waves resulting from contractions of the circular muscle layer of the small bowel that are organized by the enteric nervous system. Phase III of the migrating motor complex, the hallmark of the fasting motility period, thus reflects enteric neuromuscular function. Response to meal challenge also involves the CNS, reflexes beyond the gut and endocrine responses. Although specific disease diagnosis cannot be made by motility studies of the small bowel, the functional integrity is revealed. The normal occurrence of the essential patterns and periods of motility and the absence of distinctly abnormal patterns evidence preserved function, whereas the opposite indicates clinically significant dysmotility. Certain motility patterns are occasionally seen both in health and disease, and increased prevalence indicates a moderate dysfunction of yet unclear significance. Bacterial overgrowth with Gram‐negative bacilli is the consequence of severe small bowel dysmotility, and a diagnosis that can be predicted by a motility study. Testing can be useful in the clinical management of paediatric and adult patients also by predicting the prognosis and response to enteral nutrition and medical therapy. Further studies are, however, needed to take full advantage of motility testing in clinical practise.
Development and plasticity of interstitial cells of Cajal
Authors: Sanders  , Ördög  , Koh  , Torihashi  , Ward
Published: March 2003

Interstitial cells of Cajal (ICC) are the pacemakers in gastrointestinal (GI) muscles, and these cells also mediate or transduce inputs from the enteric nervous system. Different classes of ICC are involved in pacemaking and neurotransmission. ICC express specific ionic conductances that make them unique in their ability to generate and propagate slow waves in GI muscles or transduce neural inputs. Much of what we know about the function of ICC comes from developmental studies that were made possible by the discoveries that ICC express c‐kit and proper development of ICC depends upon signalling via the Kit receptor pathway. Manipulating Kit signalling with reagents to block the receptor or downstream signalling pathways or by using mutant mice in which Kit or its ligand, stem cell factor, are defective has allowed novel studies into the specific functions of the different classes of ICC in several regions of the GI tract. Kit is also a surface antigen that can be used to conveniently label ICC in GI muscles. Immunohistochemical studies using Kit antibodies have expanded our knowledge about the ICC phenotype, the structure of ICC networks, the interactions of ICC with other cells in the gut wall, and the loss of ICC in some clinical disorders. Preparations made devoid of ICC have also allowed analysis of the consequences of losing specific classes of ICC on GI motility. This review describes recent advances in our knowledge about the development and plasticity of ICC and how developmental studies have contributed to our understanding of the functions of ICC. We have reviewed the clinical literature and discussed how loss or defects in ICC affect GI motor function.
Involvement of 5‐HT3 and 5‐HT4 receptors in the motor activity of isolated vascularly perfused rat duodenum
Authors: Yamamoto  , Kuwahara  , Fujimura  , Kadowaki  , Fujimiya
Published: March 2003

The involvement of serotonin (5‐HT) receptor subtypes in motor activity of the ex vivo vascularly perfused rat duodenum was investigated. Clusters of phasic contractions (CPCs), migrating in an oral to anal direction, were obtained without any stimulation. Drug effects were evaluated by changes in different components of the pressure waves, such as motor index (MI), frequency, amplitude and duration of the CPC. The effect of 5‐HT depletion on motor activity was examined in animals treated with p‐chlorophenylalanine (PCPA). The MI, frequency and duration of CPC were decreased by PCPA, but the amplitude was not affected, suggesting that endogenous 5‐HT may play an important role in regulation of the motor activity of the rat intestine. The importance of the 5‐HT receptor subtypes in the regulation of motor activity was examined. Neither the nonselective 5‐HT1 and 5‐HT2 receptor antagonist, methysergide, nor the 5‐HT2 receptor antagonist, ketanserin, affected motor activity. However, the 5‐HT3 receptor antagonists, granisetron and azasetron, decreased percentage MI, frequency, percentage amplitude and percentage duration of CPC. The 5‐HT4 receptor antagonist, SB204070, exerted both excitatory and inhibitory actions, with a higher dose (10 nM) stimulating percentage MI, frequency, percentage amplitude and percentage duration, and a lower dose (0.1 nMor 1 nM) decreasing percentage MI and percentage duration of CPC. These results suggest that endogenous 5‐HT regulates the motor activity of the rat duodenum through 5‐HT3 and 5‐HT4 receptors, with the former mediating the stimulatory influence and the latter mediating both stimulatory and inhibitory influences.
Acute intraduodenal bile salt depletion leads to strong gallbladder contraction, altered antroduodenal motility and high plasma motilin levels in humans
Authors: Portincasa  , Peeters  , Van Berge‐Henegouwen  , Van Solinge  , Palasciano  , Van Erpecum
Published: March 2003

Cholecystokinin is the main hormone involved in postprandial gallbladder contraction. There is also considerable gallbladder contraction in the fasting state, associated with phase III of the gastrointestinal migrating motor complex and release of the hormone motilin. It has been proposed that intraduodenal bile salts exert a negative‐feedback control on postprandial cholecystokinin release and resulting gallbladder contraction. We wanted to elucidate whether a similar control mechanism on gallbladder contraction exists in the fasting state. We therefore performed gallbladder ultrasonography and 24‐h antroduodenal motility registrations and determined plasma cholecystokinin and motilin levels in six healthy subjects before and after acute (4 g) and chronic (8 days; 8 g day–1) oral cholestyramine. Acute cholestyramine strongly decreased gallbladder volumes and increased motilin without changed cholecystokinin levels. There was a negative relationship between gallbladder volumes and plasma motilin levels. Although there was a persistent fasting pattern of antroduodenal motility, its cycle length was increased (P < 0.03) with markedly longer phase II (P < 0.005). Fasting gallbladder volumes 24 h later were still strongly decreased but gradually increased to pretreatment levels. Before and after 8 days cholestyramine, interdigestive and postprandial gallbladder emptying, intestinal migrating motor complex and hormone levels did not differ. We conclude that acute (but not chronic) intraduodenal bile salt depletion with cholestyramine affects gallbladder and antroduodenal motility, possibly partly related to motilin release.
Comparison of cortical potentials evoked by mechanical and electrical stimulation of the rectum
Authors: Hobday D. I. , Hobson A. , Furlong P. L. , Thompson D. G. , Aziz Q.
Published: March 2003

Patients with irritable bowel syndrome have heightened perception of gut sensation. The mechanisms responsible for this remain unknown, due to current poor knowledge of the central processing of gut sensation. Cortical evoked potentials (CEPs) have been recorded following both electrical rectal stimulation (ERS) and mechanical rectal stimulation (MRS). Because of the lack of a direct comparison of these two methods, their robustness for future clinical use remains unknown. The aim of our study was to compare the characteristics of CEPs following ERS and MRS. CEPs were recorded from the vertex in 14 healthy volunteers following ERS with bipolar ring electrodes, and MRS by repeated rectal distension. CEPs were recorded in all subjects following electrical stimulation, but only in 11 subjects following mechanical stimulation. In comparison with electrical stimulation, mechanical stimulation produced CEPs with a smaller amplitude and longer latency. However, the morphology of CEPs following electrical and mechanical rectal stimulation was similar, with no difference in the interpeak latencies. In conclusion, we have demonstrated that electrical rectal stimulation is a more reliable stimulus for recording CEPs. The similarity of the morphology and interpeak latencies of the CEPs suggests that both stimuli are activating a similar network of cortical neurones.
Reproducibility of antroduodenal motility during prolonged ambulatory recording
Authors: Penning C. , Gielkens H. A. J. , Hemelaar M. , Lamers C. B. H. W. , Masclee A. A. M.
Published: March 2003

Ambulatory recording of antroduodenal manometry is a novel technique with several advantages over standard stationary manometry recording. Although the feasibility of this technique in clinical practice has been demonstrated, reproducibility of antroduodenal motility recorded by means of ambulatory manometry has not been investigated. To test whether antroduodenal motility recorded by ambulatory manometry is reproducible, we performed two 24‐h ambulatory antroduodenal manometry recordings in 18 healthy subjects according to an identical protocol with a 1‐week interval. Motility was recorded with a five‐channel solid‐state catheter. Postprandial motility was recorded after consumption of two test meals and interdigestive motility was recorded nocturnally. Postprandial antroduodenal motor characteristics were identical between the separate recordings. The number and duration of nocturnal cycles of the interdigestive migrating motor complex were also in the same range. Phase III characteristics in general were not different between the two recordings. Only minor alterations were observed in the duration of phase III motor fronts with duodenal onset and in the number of interdigestive cycles concluded by duodenal onset phase III. Parameters obtained by qualitative analysis were comparable between the two recordings. The antroduodenal motility pattern, when measured by ambulatory recording with solid state catheters under standardized conditions, is very reproducible.
Analysis of motor patterns in the isolated guinea‐pig large intestine by spatio‐temporal maps
Authors: D'Antona G. , Hennig G. W. , Costa M. , Humphreys C. M. , Brookes S. J. H.
Published: March 2003

We investigated and quantified the spontaneous patterns of motility in the isolated guinea‐pig proximal and distal colon taken from adult animals. During spontaneous emptying, profiles of proximal and distal colon were recorded with a video camera, and image analysis was used to construct spatio‐temporal maps of the motions of the intestinal wall. Four patterns of motility were recorded. In the proximal colon there were neurally mediated contractions that propagated in the aboral direction at 4.1 mm s–1, gently pushing the soft contents aborally; these are likely to represent spontaneous peristaltic behaviour. A second pattern, insensitive to tetrodotoxin (TTX; 0.6 μM), consisted, in both oral and aboral propagation, of shallow contractions of the circular muscle (ripples). These contractions propagated aborally at 2.8 ± 0.45 mm s–1 and orally at 2.03 ± 0.31 mm s–1 (n=10). Of these TTX‐resistant contractions, 22.5% propagated both orally and aborally from a common origin. The orally propagated component of these myogenic contractions is likely to correspond to the antiperistalsis widely described in the proximal colon. In the distal colon, two patterns of motor activity were observed. One, induced by natural or artificial pellets, consisted of peristaltic contractions that pushed the pellets aborally at 0.8 mm s–1 and expelled a pellet every 108 s. In the interval between pellet propulsion and after the distal colon had emptied all of its pellets a second, nerve‐mediated pattern of motor activity, consisting of clusters of annular circular muscle contractions separated by short dilated regions, slowly propagated aborally at 0.3 mm s–1. Both of these motor patterns were abolished by TTX (0.6 μM). A latex balloon, inserted at the oral end of the empty isolated distal colon and inflated to a size similar to faecal pellets, was propelled at 1.4 mm s–1. Epoxy resin‐covered natural pellets were propelled at a similar speed of 1.6 mm s−1.Our data revealed that myogenic and neurogenic patterns of propagated contractions in the colon occur in isolated preparations and are involved in emptying the colon.
Transient lower oesophageal sphincter relaxations play an insignificant role in gastro‐oesophageal reflux to the proximal oesophagus
Authors: Grossi L. , Ciccaglione A. F. , Marzio L.
Published: March 2003

Gastro‐oesophageal reflux to the proximal oesophagus may cause atypical symptoms of gastro‐oesophageal reflux disease (GORD). The motor abnormalities underlying reflux into the proximal oesophagus are still unclear. The aim of this study was to analyse the oesophageal motility during reflux into the proximal oesophagus in a group of healthy subjects and in patients with atypical symptoms of GORD. We concentrated particularly on lower oesophageal sphincter (LOS) activity and transient lower oesophageal sphincter relaxations (TLOSRs). Ten patients (7M, 3F, age 25–51 years) with mild oesophagitis (Savary–Miller grade I–II) and 10 healthy subjects (6M, 4F, age 23–54 years) underwent a 24‐h dual pH‐metric and manometric recording, using an electronic portable device. This recorded distal and proximal oesophageal pH values, oesophageal body and LOS motility. GORD patients had more distal and proximal reflux (DR and PR) compared with healthy controls (DR P < 0.001; PR P < 0.05). TLOSRs were the most frequent event during reflux into the distal oesophagus, whereas TLOSR frequency was much lower during reflux to the proximal oesophagus in GORD patients and in healthy controls (P < 0.05 and P < 0.01 vs. distal reflux, respectively). A significant relationship between TLOSRs and distal refluxes was present but no relationship with proximal reflux was detected. We conclude that TLOSRs are much less frequent during reflux to the proximal oesophagus than distal oesophageal reflux in patients with mild GORD suffering from atypical manifestations. The mechanism of acid reflux to the proximal oesophagus is unclear.
Diurnal variation of abdominal motor responses to colorectal distension and plasma cortisol levels in rats
Authors: Gschossmann J. M. , Buenger L. , Adam B. , Liebregts T. , Saller B. , Mann K. , Gerken G. , Holtmann G.
Published: March 2003

Most patients with functional bowel disorders complain of daytime symptoms while they remain asymptomatic at night. As symptoms are associated with heightened visceral sensitivity, we hypothesized that circadian fluctuations of the visceral sensory function occur. At four different timepoints (06.00, 12.00, 18.00 and 24.00 h), colorectal distensions (CRD) were performed in fasting conscious male Lewis rats using a balloon catheter and a barostat device. The abdominal wall contractions (behavioural pain response) were assessed during colorectal distension by abdominal wall electromyography (EMG). Plasma levels for endogenous cortisol were determined simultaneously at these timepoints. EMG responses to CRD were significantly (P < 0.05) higher at midnight and in the early morning. Plasma cortisol levels peaked in the evening. In night‐active Lewis rats, the behavioural pain response to noxious visceral stimulation is augmented at night and fluctuations of visceral sensitivity are accompanied by circadian changes of plasma concentrations of endogenous cortisol. We conclude that there are marked circadian fluctuations in visceral sensory functions. Thresholds are low during time periods of normal behavioural activity. These findings suggest that fluctuation of the sensory functions may be linked to the circadian variability of symptoms in patients with functional GI disorders.
Acute and chronic stress differently affect visceral sensitivity to rectal distension in female rats
Authors: BRADESI S. , EUTAMENE H. , GARCIA‐VILLAR R. , FIORAMONTI J. , BUÉNO L.
Published: March 2003

Stressful life events are frequently associated with outward signs of irritable bowel syndrome (IBS). Increasing evidence suggests that acute and chronic stress stimuli implicate different physiological mechanisms and neuroendocrine responses. Therefore, we investigated the influence of acute and chronic stress on visceral nociception in female rats and the involvement of colonic mast cells in this effect. The effect of acute and chronic partial restraint stress (PRS) on visceral sensitivity to rectal distension (RD) was assessed by abdominal muscle electromyography. Colonic mast cell activation was determined by measuring histamine release after in vitro stimulation with substance P (SP) in colonic samples from rats experiencing RD vs. controls. Acute PRS significantly enhanced abdominal response to RD compared with sham PRS for all volumes of distension. In contrast, chronic PRS induced a hyperalgesic response for the highest volumes of distension (0.8 and 1.2 mL), but did not affect the number of abdominal contractions for the lowest volume (0.4 mL) compared with controls. Both acute and chronic PRS increased in vitro SP‐induced histamine release without affecting mast cell numbers. RD induced similar in vitro histamine release from colonic samples from both acute and chronic PRS rats; this release, however, was significantly higher than that measured in sham‐PRS rats. Acute and chronic PRS differently influence visceral sensitivity in response to RD in female rats. This difference, however, cannot be attributed to a different effect of either stress paradigm on mast cell histamine release.