bluuebunny/crossref_metadata_2025 huggingface dataset:
Nac‐mediated repression of the serA promoter of Escherichia coli
Authors: Blauwkamp Timothy A. , Ninfa Alexander J.
Published: March 2003

Escherichia coli and related bacteria contain two paralogous PII‐like proteins involved in nitrogen regulation, the glnB product, PII, and the glnK product, GlnK. Previous studies have shown that cells lacking both PII and GlnK have a severe growth defect on minimal media, resulting from elevated expression of the Ntr regulon. Here, we show that this growth defect is caused by activity of the nac product, Nac, a LysR‐type transcription factor that is part of the Ntr regulon. Cells with elevated Ntr expression that also contain a null mutation in nac displayed growth rates on minimal medium similar to the wild type. When expressed from high‐copy plasmids, Nac imparts a growth defect to wild‐type cells in an expression level‐dependent manner. Neither expression of Nac nor lack thereof significantly affected Ntr gene expression, suggesting that the activity of Nac at one or more promoters outside the Ntr regulon was responsible for its effects. The growth defect of cells lacking both PII and GlnK was also eliminated upon supplementation of minimal medium with serine or glycine for solid medium or with serine or glycine and glutamine for liquid medium. These observations suggest that high Nac expression results in a reduction in serine biosynthesis. β‐Galactosidase activity expressed from a Mu d1 insertion in serA was reduced approximately 10‐fold in cells with high Nac expression. We hypothesize that one role of Nac is to limit serine biosynthesis as part of a cellular mechanism to reduce metabolism in a co‐ordinated manner when cells become starved for nitrogen.
Effect of var gene disruption on switching in Plasmodium falciparum
Authors: Horrocks Paul , Pinches Robert , Kyes Sue , Kriek Neline , Lee Sarah , Christodoulou Zóe , Newbold Chris I.
Published: March 2003

The molecular mechanisms underpinning switching of variant antigens on the surface of Plasmodium falciparum‐infected erythrocytes are poorly understood. We tested the hypothesis that insertional disruption of the A4var gene, one of two var genes located within the subtelomeric region of one end of chromosome 13, would result in a preferential switch in transcription to the adjacent R29var gene upon rosette selection. In this way, we aimed to mimic the preferential transcription of R29var in rosetting R29 parasites, a parasite line in which the A4var gene is deleted through a chromosome end truncation. Initial analysis of the knock‐out parasite lines shows that the insertional disruption of the A4var gene prevents A4 PfEMP1 expression, but that switching transcription to other var gene variants is unaffected. Furthermore, analysis of var transcription in the knock‐out parasite line during rosette selection shows that, rather than facilitating a switch to R29var gene transcription, this event was suppressed in the transfectants . These data, and the implications for epigenetic transcriptional control of var genes, are discussed.
Identification of Xenorhabdus nematophila genes required for mutualistic colonization of Steinernema carpocapsae nematodes
Authors: Heungens Kurt , Cowles Charles E. , Goodrich‐Blair Heidi
Published: March 2003

One stage in the symbiotic interaction between the bacterium Xenorhabdus nematophila and its nematode host, Steinernema carpocapsae, involves the species‐specific colonization of the nematode intestinal vesicle by the bacterium. To characterize the bacterial molecular determinants that are essential for vesicle colonization, we adapted and applied a signature‐tagged mutagenesis (STM) screen to this system. We identified 15 out of 3000 transposon mutants of X. nematophila with at least a 15‐fold reduction in average vesicle colonization. These 15 mutants harbour disruptions in nine separate loci. Three of these loci have predicted open reading frames (ORFs) with similarity to genes (rpoS, rpoE, lrp) encoding regulatory proteins; two have predicted ORFs with similarity to genes (aroA, serC) encoding amino acid biosynthetic enzymes; one, designated nilB (nematode intestine localization), has an ORF with similarity to a gene encoding a putative outer membrane protein (OmpU) in Neisseria; and three, nilA, nilC and nilD, have no apparent homologues in the public database. nilA, nilB and nilC are linked on a single 4 kb locus. nilB and nilC are > 104‐fold reduced in their ability to colonize the nematode vesicle and are predicted to encode membrane‐localized proteins. The nilD locus contains an extensive repeat region and several small putative ORFs. Other than reduced colonization, the nilB, nilC and nilD mutants did not display alterations in any other phenotype tested, suggesting a specific role for these genes in allowing X. nematophila to associate with the nematode host.
Mechanism of association of adenylate cyclase toxin with the surface of Bordetella pertussis: a role for toxin–filamentous haemagglutinin interaction
Authors: Zaretzky Franca R. , Gray Mary C. , Hewlett Erik L.
Published: March 2003

Adenylate cyclase (AC) toxin from Bordetella per‐tussis is unusual in that, unlike most other members of the repeats‐in‐toxin family that are released into the extracellular milieu, it remains associated with the bacterial surface. In this study, we investigated the nature of the association of this toxin with the surface of B. pertussis. AC toxin was extracted from crude outer membrane preparations of B. pertussis with 8 M urea, but only partially with alkaline sodium carbonate and not at all with octylglucoside, suggesting that denaturation of the toxin is necessary for its removal from the membrane. B. pertussis mutants lacking filamentous haemagglutinin (FHA) released significantly more AC toxin into the medium, and AC toxin association with the bacterial surface was partially restored by expression of FHA from a plasmid, suggesting a role for FHA in surface retention of AC toxin. AC toxin distribution was unaffected by the absence of pertactin, or full‐length lipopolysaccharide, or a defect in secretion of pertussis toxin. Using overlay and immunoprecipitation, we found that a direct physical association can occur between AC toxin and FHA. Combined, these findings suggest that FHA may play a role in AC toxin retention on the surface of B. pertussis and raise the possibility of an involvement of adherence mediated by FHA in delivery of AC toxin from the bacterium to the target cell.
The RHO1‐GAPs SAC7, BEM2 and BAG7 control distinct RHO1 functions in Saccharomyces cerevisiae
Authors: Schmidt Anja , Schmelzle Tobias , Hall Michael N.
Published: March 2003

In Saccharomyces cerevisiae, the small GTPase RHO1 plays an essential role in the control of cell wall synthesis and organization of the actin cytoskeleton. Several regulators for RHO1 are known, including the GTPase‐activating proteins (GAPs) SAC7 and BEM2. Here we show that BAG7, identified as the closest homologue of SAC7, also acts as a GAP for RHO1 in vitro and in vivo. Furthermore, we find that BAG7, SAC7, and BEM2 are functionally different in vivo. Overexpression of BAG7 or SAC7, but not BEM2, suppresses the cold sensitivity of a sac7 mutation and the lethality of RHO1 hyperactivation in response to cell wall damage. In contrast, overexpression of BEM2 or SAC7, but not BAG7, downregulates the RHO1‐controlled PKC1‐MPK1 pathway, and disruption of BEM2 or SAC7, but not BAG7, results in increased MPK1 activation. We conclude that BEM2 and SAC7, but not BAG7, are involved in the control of the RHO1‐mediated activation of MPK1, whereas BAG7 and SAC7, but not BEM2, are involved in the regulation of other RHO1 functions. This suggests that different RHO1 GAPs control different RHO1 effector pathways, thus ensuring their individual regulation at the appropriate place and time.
Characterization of the promoter regions involved in galactose‐ and nisin‐mediated induction of the nisA gene in Lactococcus lactis ATCC 11454
Authors: Chandrapati Sailaja , O'Sullivan Daniel J.
Published: March 2003

The nisA promoter is positively regulated in Lactococcus lactis ATCC 11454 by autoinduction via a two‐component NisRK‐mediated system. However, induction of this promoter can also occur when introduced into the plasmid‐free L. lactis LM0230 during growth in galactose or lactose, independent of the NisRK system. In this study, we also characterized this galactose‐mediated induction by determining the nisA start site during growth in galactose, which was identical to the nisA start site upon nisin induction. The region involved in the galactose‐mediated induction of the nisA promoter was investigated by directed deletion analysis of a 200 bp region upstream of the nisA promoter in the transcription fusion pDOC99. The induction of the deletion derivatives by galactose and nisin was compared phenotypically using β‐galactosidase measurements, and the regions necessary for the induction were determined by sequence analysis. Analysis of these regions revealed two sets of a TCT direct repeat [TCT‐N8‐TCT] present at positions (−107 to −94) and (−39 to −26) relative to the transcription initiation site. Disruption of the upstream repeat abolished galactose induction and significantly reduced the nisin induction capacity, suggesting a potential pivotal role for these repeats in transcription induction of the nisA promoter. It was also observed that the galactose‐mediated induction was abolished when a plasmid containing the phosphotransferase system (PTS), phospho‐β‐galactosidase and tagatose pathway genes was introduced into this strain. As this effectively made the Leloir pathway redundant, it points to some component of this pathway as the specific inducer of the nisA promoter.
A novel assembly process of the multicomponent xenobiotic efflux pump in Pseudomonas aeruginosa
Authors: Maseda Hideaki , Kitao Masataka , Eda Shima , Yoshihara Eisaku , Nakae Taiji
Published: March 2003

The nfxC‐type cells of Pseudomonas aeruginosa show resistance to a wide range of structurally and functionally diverse antibiotics, which is a phenomenon that is mainly attributable to the expression of the MexEF‐OprN xenobiotic transporter. The MexF, MexE and OprN subunits of this transporter are located on the inner membrane, the periplasm and the outer membrane, respectively, and are assumed to function as an energy‐dependent transporter, a bridge connecting the inner and outer membranes and outer membrane channel respectively. The nfxC‐type cells showed a single protein band of MexF and OprN, whereas MexE appeared as three distinct bands in an SDS‐polyacrylamide gel electrophoretogram. The mutant cells lacking MexF produced undetectable OprN and only a full‐size of MexE even though the cells had unimpaired oprN and mexE. Expression of the plasmid‐borne MexF in this mutant fully restored OprN and three MexE bands. Another class of mutants producing a full amount of MexF yielded undetectable OprN and two MexE bands lacking the smallest protein species suggesting that the presence of the smallest MexE subunit is required for stabilization of OprN. To identify which part of MexE was needed for stabilization and assembly of OprN, the carboxyl‐terminal‐truncated MexE tagged with polyhistidine was constructed and protein bands were visualized in the presence of MexF with an antibody raised against polyhistidine or MexE. The results revealed that the proteolytic processing of MexE would occur at carboxyl terminal amino acids between 11 and 16, thereby suggesting that the presence of the C‐terminal truncated MexE is essential for stabilization and the proper assembly of OprN. Nucleotide sequencing of mutant mexFs, which produce a wild‐type level of MexF but are unable to support the production of the smallest MexE, thereby destabilizing OprN, revealed that all the mutations were located within two large periplasmic domains of MexF between transmembrane segments 1–2 and 7–8. Taking these findings together, we concluded that two large periplasmic domains of MexF interact with MexE thereby promoting programmed processing of MexE, and this complex eventually assists the correct assembly and sorting of OprN.
A unique, bifunctional site‐specific DNA recombinase from Mycoplasma pulmonis
Authors: Sitaraman Ramakrishnan , Denison Amy M. , Dybvig Kevin
Published: March 2003

Site‐specific DNA invertible elements often control the production of bacterial surface proteins that are subject to phase variation (ON/OFF switching). Inversion of the DNA element occurs as a result of the reciprocal exchange of DNA catalysed by a specialized enzyme (recombinase) that acts at specific sites. By continually switching the orientation of the invertible element in the chromosome, and consequently the production of the variable protein(s), the cell population remains continually responsive to environmental change such as immunological challenge. In addition to phase‐variable surface proteins, Mycoplasma pulmonis has a family of phase‐variable restriction‐modification enzymes. We report here that a single recombinase in M. pulmonis, HvsR, catalyses independent DNA inversions at non‐homologous loci, causing variations in surface lipoproteins and in the DNA recognition sequence specificity of restriction enzymes. Thus, HvsR is a site‐specific DNA recombinase with dual substrate specificity.
Ribosomal and non‐ribosomal resistance to oxazolidinones: species‐specific idiosyncrasy of ribosomal alterations
Authors: Sander P. , Belova L. , Kidan Y. G. , Pfister P. , Mankin A. S. , Böttger E. C.
Published: March 2003

A derivative of Mycobacterium smegmatis, which carries only one functional rRNA (rrn) operon, was used to isolate mutants resistant to the ribosome‐targeted antibiotic linezolid. Isolation and characterization of linezolid‐resistant clones revealed two classes of mutants. Ribosomes from class I mutants are resistant to oxazolidinones in an in vitro peptidyl transferase assay, indicating that resistance maps to the ribosome component. In contrast, ribosomes from class II mutants show wild‐type susceptibility to a linezolid derivative in vitro, pointing to a non‐ribosomal mechanism of resistance. Introduction of a wild‐type ribosomal RNA operon into linezolid‐resistant strains restored linezolid sensitivity in class I mutants, indicating that resistance (i) maps to the rRNA and (ii) is recessive. Sequencing of the entire rrn operon identified a single nucleotide alteration in 23S rRNA of class I mutant strains, 2447G → T (Escherichia coli numbering). Introduction of mutant rrl2447T into M. smegmatis rrn– resulted in a linezolid‐resistant phenotype, demonstrating a cause–effect relationship of the 2447G → T alteration. The 2447G → T mutation, which renders M. smegmatis linezolid resistant, confers lethality in E. coli. This finding is strong evidence of structural and pos‐sibly functional differences between the ribosomes of Gram‐positive and Gram‐negative bacteria. In agreement with the results of the in vitro assay, class II mutants show a wild‐type sequence of the complete rRNA operon. The lack of cross‐resistance of the class II mutants to other antibiotics suggests a resistance mechanism other than activation of a broad‐spectrum multidrug transporter.
An inhibitor of DNA binding and uptake events dictates the proficiency of genetic transformation in Neisseria gonorrhoeae: mechanism of action and links to Type IV pilus expression
Authors: Aas Finn Erik , Løvold Cecilia , Koomey Michael
Published: March 2003

Although natural genetic transformation is a widely disseminated form of genetic exchange in prokaryotic species, the proficiencies with which DNA recognition, uptake and processing occur in nature vary greatly. However, the molecular factors and interactions underlying intra‐ and interspecies diversity in levels of competence for natural genetic transformation are poorly understood. In Neisseria gonorrhoeae, the Gram‐negative aetiologic agent of gonorrhoea, DNA binding and uptake involve components required for Type IV pilus (Tfp) biogenesis as well as those which are structurally related to Tfp biogenesis components but dispensable for organelle expression. We demonstrate here that the gonococcal PilV protein, structurally related to Tfp pilin subunits, is an intrinsic inhibitor of natural genetic transformation which acts ultimately by reducing the levels of sequence‐specific DNA uptake into the cell. Specifically, we show that DNA uptake is enhanced in strains bearing pilV mutations and reduced in strains overexpressing PilV. Furthermore, we show that PilV exerts its effect by acting as an antagonist of ComP, a positive effector of sequence‐specific DNA binding. As it prevents the accumulation of ComP at a site where it can be purified by shear extraction of intact cells, the data are most consistent with PilV either obstructing ComP trafficking or altering ComP stability. In addition, we report that ComP and PilV play overlapping and partially redundant roles in Tfp biogenesis and document other genetic interactions between comP and pilV together with the pilE and pilT genes required for the expression of retractile Tfp. Together, the results reveal a novel mechanism by which the levels of competence are governed in prokaryotic species and suggest unique ways by which competence might be modulated.
The requirement of the LC8 dynein light chain for nuclear migration and septum positioning is temperature dependent in Aspergillus nidulans
Authors: Liu Bo , Xiang Xin , Lee Yuh‐Ru Julie
Published: March 2003

In the filamentous fungus Aspergillus nidulans, the multisubunit motor complex cytoplasmic dynein plays essential roles in nuclear migration and septum positioning. The 8 kDa light chain, LC8, the smallest subunit, is conserved among eukaryotic organisms. Besides being a component in the dynein complex, LC8 also interacts with a wide spectrum of mammalian and viral proteins. To date, the function of this small polypeptide is not well understood. To address this issue, we have created a deletion mutation (ΔnudG) at the nudG locus encoding LC8 in A. nidulans. At 42°C, the ΔnudG mutant forms minute colonies lacking asexual reproduction: this phenotype resembles the phenotype of the dynein heavy chain null mutant. The mutant nuclei largely clustered in the spore body after conidial germination, and the septum was often assembled distally toward the hyphal apex, whereas a control germling has its nuclei distributed along the hypha and the septum formed near the spore body. When the mutant was grown at 23°C, however, its colony resembled a control one, and so did the patterns of nuclear distribution and septum positioning. Elevation of the growth temperature gradually reduced colony size and abolished asexual sporulation. After a period of growth at 23°C that allowed the nuclei to move out of the spore end, a temperature shift to 42°C prevented newly divided nuclei from migrating apart, suggesting that LC8/NUDG was required for both initiating and maintaining dynein motor functions at elevated temperatures. A functional GFP‐NUDA fusion was used to test whether LC8/NUDG is required for DHC (dynein heavy chain)/NUDA localization. We found that at 23°C GFP‐NUDA localized to the hyphal apex and the septation site in ΔnudG cells as in control cells. Such localizations were absent at 42°C in mutant cells, but not in control cells. We conclude that LC8 plays a role in DHC localization/function, and the requirement for such a role in A. nidulans cells is temperature dependent.
Siderophore‐mediated cell signalling in Pseudomonas aeruginosa: divergent pathways regulate virulence factor production and siderophore receptor synthesis
Authors: Beare Paul A. , For Raewyn J. , Martin Lois W. , Lamont Iain L.
Published: March 2003

Under iron‐limiting conditions, Pseudomonas aeruginosa produces a siderophore called pyoverdine. Pyoverdine is secreted into the extracellular environment where it chelates iron, and the resulting ferri‐pyoverdine complexes are transported back into the bacteria by a cell surface receptor protein FpvA. Pyoverdine also acts as a signalling molecule inducing the production of three secreted virulence factors. Binding of ferri‐pyoverdine to FpvA transduces a signal to the periplasmic part of the membrane‐spanning antisigma factor FpvR. The signal is transmitted to the cytoplasmic part of FpvR, which controls the activity of an extracytoplasmic family (ECF) sigma factor protein PvdS. This results in the production of the virulence factors pyoverdine, exotoxin A and PrpL endoprotease. Here, we show that a second divergent branch of this signalling pathway regulates the production of the FpvA protein. FpvR negatively regulates the activity of a second ECF sigma factor, FpvI, which is required for the synthesis of FpvA, and the presence of ferri‐pyoverdine greatly increases the activity of FpvI so that production of FpvA is induced. To the best of our knowledge, this is the first example of a branched signalling system of this sort and the first example of an antisigma factor protein (FpvR) that directly regulates the activities of two different ECF sigma factor proteins (PvdS and FpvI).
EFG1 is a major regulator of cell wall dynamics in Candida albicans as revealed by DNA microarrays
Authors: Sohn K. , Urban C. , Brunner H. , Rupp S.
Published: March 2003

Cell wall dynamics in Candida albicans, the most common fungal pathogen in man, underlie regulatory processes during the yeast‐to‐hyphae transition. To analyse this regulation at the transcriptional level, we have established a DNA microarray representing genes implicated in cell wall biogenesis. Using these microarrays, we were able to identify YWP1 and HWP2 that are specifically transcribed in the yeast or hyphal growth form respectively. Cluster analysis revealed at least two major clusters of genes: cluster I comprised genes that were upregulated under at least one hyphae‐inducing condition. Three as yet not further characterized genes were attributed to cluster II. These genes were transcribed in the yeast form of C. albicans and were downregulated in an EFG1‐dependent manner under specific hyphae‐inducing conditions. We show further that, in contrast to CPH1, EFG1 plays a major role in the transcriptional regulation of cell wall proteins under the conditions investigated. EFG1 was essential for the transcription of both hyphae‐specific genes such as HWP1 and HWP2 as well as the yeast form‐specific gene YWP1. Moreover, we found that, under various conditions, EFG1 also can act as a strong repressor for the transcription of RBE1, another not yet characterized cell wall protein. Overall, our data show that EFG1 plays a major role in the induction and repression of cell wall genes, not only in the hyphal form but also in the yeast form of C. albicans.
Steroid biosynthesis in prokaryotes: identification of myxobacterial steroids and cloning of the first bacterial 2,3(S)‐oxidosqualene cyclase from the myxobacterium Stigmatella aurantiaca
Authors: Bode Helge Björn , Zeggel Bernd , Silakowski Barbara , Wenzel Silke C. , Reichenbach Hans , Müller Rolf
Published: March 2003

Steroids, such as cholesterol, are synthesized in almost all eukaryotic cells, which use these triterpenoid lipids to control the fluidity and flexibility of their cell membranes. Bacteria rarely synthesize such tetracyclic compounds but frequently replace them with a different class of triterpenoids, the pentacyclic hopanoids. The intriguing mechanisms involved in triterpene biosynthesis have attracted much attention, resulting in extensive studies of squalene‐hopene cyclase in bacteria and (S)‐2,3‐oxidosqualene cyclases in eukarya. Nevertheless, almost nothing is known about steroid biosynthesis in bacteria. Only three steroid‐synthesizing bacterial species have been identified before this study. Here, we report on a variety of sterol‐producing myxobacteria. Stigmatella aurantiaca is shown to produce cycloartenol, the well‐known first cyclization product of steroid biosynthesis in plants and algae. Additionally, we describe the cloning of the first bacterial steroid biosynthesis gene, cas, encoding the cycloartenol synthase (Cas) of S. aurantiaca. Mutants of cas generated via site‐directed mutagenesis do not produce the compound. They show neither growth retardation in comparison with wild type nor any increase in ethanol sensitivity. The protein encoded by cas is most similar to the Cas proteins from several plant species, indicating a close evolutionary relationship between myxobacterial and eukaryotic steroid biosynthesis.
Lack of SeqA focus formation, specific DNA binding and proper protein multimerization in the Escherichia coli sequestration mutant seqA2
Authors: Fossum Solveig , Søreide Sølvi , Skarstad Kirsten
Published: March 2003

In Escherichia coli wild‐type cells newly formed origins cannot be reinitiated. The prevention of reinitiation is termed sequestration and is dependent on the hemimethylated state of newly replicated DNA. Several mutants discovered in a screen for the inability to sequester hemimethylated origins have been mapped to the seqA gene. Here, one of these mutants, seqA2, harbouring a single amino acid change in the C‐terminal end of the SeqA protein, was found to also be unable to form foci in vivo. The SeqA foci seen in the wild‐type cells are believed to arise from multimerization of SeqA on hemimethylated DNA at the replication fork, presumably representing organization of newly formed DNA by SeqA. The result suggests that the process of origin sequestration is closely tied to the process of focus maintenance at the replication fork. In vitro, purified SeqA2 protein was found incapable of forming highly ordered multimers that bind hemimethylated oriC. The mutant protein was also incapable of restraining negative supercoils. Both in vivo and in vitro results support the idea that origin sequestration is an integral part of organization of newly formed DNA performed by SeqA.
A model for evaluation of gastric sensitivity in awake rats
Authors: Rouzade  , Fioramonti  , Bueno
Published: March 2003

We developed a model for the evaluation of gastric sensitivity to distension in awake rats. A balloon made from a latex condom was chronically placed in the stomach and three stainless steel electrodes were implanted in the neck muscles. Isobaric distensions were performed with a barostat by step of 5 mmHg with 10 min inflation and 2 min deflation. Gastric pressure, integrated neck electromyogram (EMG) and gastric volume were continuously monitored on a potentiometric recorder. Gastric distension at 15 or 20 mmHg induced a typical posture associated with contractions of the neck muscles. Pain threshold was defined as the pressure inducing an increase of integrated neck EMG greater than 100%.

The mean pain threshold was 18.5 ± 0.7 mmHg and was not modified 2, 4 and 7 days after the first experiment. However, gastric volumes were significantly higher on the 4th and the 7th days. Morphine at the doses of 0.4 and 4 mg kg−1 i.p. significantly increases the pain threshold. At the doses of 0.04 and 0.4 mg kg−1, morphine significantly increased gastric volume for the distending pressure of 10 mmHg. Naloxone (2.5 mg kg−1 i.p.) reversed the effects of morphine. In conclusion, our model permits simultaneous evaluation of pain threshold and gastric compliance associated with gastric distension in conscious rats.
Changes in the position and angulation of incisura during contractions of the isolated cat stomach
Authors: SCHULZE‐DELRIEU  , HERMAN
Published: March 2003

AbstractContractions change the configuration of the lesser curvature of the stomach while they indent the greater curvature. We studied these lesser curvature changes by measuring the position and angle of the gastric incisura on still frames captured from videotapes of isolated cat stomachs suspended in physiologic solution. In response to filling with 100 mL Krebs' solution stomachs generated a tonic contraction of the fundus/body segment and gave rise to a peristaltic contraction that spread from the body and through the antrum to the pylorus. In preparations where we left the duodenal cannula open we found that the incisura moves toward the gastro‐oesophageal (GO) junction and the angle of the incisura widens as the contraction passes through the stomach and empties its contents. Furthermore, the angle of the incisura is most acute when the full stomach starts contracting in its fundic segment and again when the contraction involves the gastric sinus (the wedge‐shaped segment adjacent to the incisura which forms the transition between the body and the antrum of the stomach). In preparations where the duodenal cannula was kept closed, the angle of the incisura becomes most acute when the contraction involves the gastric body and when the luminal pressure peaks. We conclude that changes in the position and angulation of the incisura are part of the mechanical response of the stomach to filling and emptying; unlike the peristaltic contraction along the greater curvature the net movement of the incisura goes in the orad direction. Movements of the incisura profoundly affect the configuration of the stomach and hence the distribution of luminal contents between various gastric segments. The gastric sling muscles are responsible for the formation of the gastric incisura but their role in any movements of the incisura remains to be defined.
Oesophagitis‐induced changes in capsaicin‐sensitive tachykininergic pathways in the ferret lower oesophageal sphincter
Authors: SMID  , PAGE  , O'DONNELL  , LANGMAN  , ROWLAND  , BLACKSHAW
Published: March 2003

Prolonged oesophageal acidification may impair lower oesophageal sphincter (LOS) function in reflux disease. The aim of this study was to investigate aspects of altered LOS innervation in a model of oeso‐phagitis. Oesophagitis was induced by acid (HCl, 0.15 M) and pepsin (0.1% w/v) infusions in anaesthetized ferrets. LOS muscle strip responses to the following stimuli were measured in vitro from control and acid/pepsin‐treated ferrets: electrical field stimulation (EFS; 1–50 Hz), potassium chloride KCl; 20 m
M), substance P, [β‐Ala8]‐neurokinin A 4‐10, [Sar9, Met (O2)11]‐substance P (all 10−10to 10−6 M) and capsaicin (10−8to 10−6 M). LOS relaxation occurred in response to all stimuli except [β‐Ala8]‐neurokinin A 4‐10, which evoked contraction. In muscle strips from acid/pepsin‐treated animals there were no differences in amplitude or sensitivity of relaxation following EFS, KCl or substance P vs controls. However, the inhibitory response to capsaicin was increased four‐fold (10−8 M; P < 0.05) and an increased sensitivity of the inhibitory response to [Sar9, Met(O2)11]‐substance P occurred (pD2 = 8.64 ± 0.12acid/pepsin‐treated vs 7.94 ± 0.24 control, P < 0.05). We conclude that in acute oesophagitis, increased sensitivity of capsaicin‐activated inhibitory pathways occurs in which activation of NK‐1 receptors plays an integral role in the ferret LOS.
Effect of somatostatin on human gallbladder motility: an in vitro study
Authors: MASELLI  , PIEPOLI  , PEZZOLLA  , CARUSO  , LORUSSO
Published: March 2003

In vivo studies have demonstrated that somatostatin induces human gallbladder relaxation. To determine whether this polypeptide acts directly on the gallbladder muscle, its effect on strips of human gallbladder was studied in vitro. Strips of gallbladder were set up isometrically in an organ bath containing oxygenated Krebs' solution. Dose–response curves to cholecystokinin‐octapeptide and carbachol were first established. The ability of somatostatin to cause relaxation under basal conditions and during 50% maximal stimulation by cholecystokinin‐octapeptide (7.2 × 10−8 M) and carbachol (3.5 × 10−6 M) was assessed in 32 strips at 4.3 × 10−6 M concentration which mimics the plasma concentrations found in patients with somatostatinoma and in 12 additional strips at 4.3 × 10−8 M concentration. Somatostatin action on the intrinsic innervation by using electrical field stimulation (EFS) (200 mA 5 msec in duration, 30 Hz; 400 mA, 1 msec in duration, 10 Hz) was also evaluated in 39 strips. Somatostatin had no effect on the basal or carbachol‐generated tensions. On the contrary, somatostatin (4.3 × 10−6 M) reduced cholecystokinin‐octapeptide‐generated tensions by 8% (P < 0.001) and reduced EFS‐generated tensions at 30 Hz by 7.7% (P < 0.01) and those at 10 Hz by 41.2% (P < 0.01). All responses to cholecystokinin‐octapeptide and carbachol were abolished by dibutyryl‐guanosine 3′, 5′‐cyclic monophosphate (5 × 10−3 M) and atropine (10−5 M), respectively (P < 0.0002 and P < 0.0002). All responses to electrical field stimulation were reduced or abolished by tetrodotoxin (2 × 10−6 M) (P < 0.001 and P < 0.0001, respectively). Our findings show that somatostatin exerts its inhibitory action on the response to cholecystokinin‐octapeptide and on the intrinsic innervation of the gallbladder smooth muscle. The probable neurotransmitter is the acetylcholine.
Dysmotility of the small intestine in achalasia
Authors: SCHMIDT  , PFEIFFER  , HACKELSBERGER  , WIDMER  , PEHL  , KAESS
Published: March 2003

During recent years there has been increasing evidence for extraoesophageal dysfunction in achalasia. The aim was to investigate whether motility of the small intestine is abnormal in achalasia. Thirteen patients (eight men, five women) aged 52 (33–85) years were studied. They had all previously undergone treatment with pneumatic balloon dilatation and were free of dysphagia when examined. Ambulatory 24‐h motility was recorded in the upper jejunum under standardized caloric intake with a digital datalogger and catheter‐mounted pressure transducers located beyond the ligament of Treitz. Visual analysis was performed by two observers and data underwent quantitative analysis of phasic contractile events using a computer program. Normal values were obtained from 50 healthy controls. In the fasting state, a complete loss of cyclic MMC activity (n = 2), an abnormally prolonged phase II (n = 2) and disturbances in the aboral migration of phase III (n = 5) were observed. Postprandial motor response was absent (n = 2) or frequently showed a contraction frequency below the normal range (n = 5). Further abnormalities consisted in hypomotility during phase II (n = 3) and in a reduced frequency of migrating clustered contractions in the fasting (n = 2) or postprandial state (n = 2). In addition, motor events not present in any healthy subject, giant migrating contractions (n = 5), retrograde clustered contractions (n = 6) and repetitive retrograde contractions (n = 3) were identified. Each patient exhibited findings out of the range of normal. Dysmotility of the proximal small intestine is present in achalasia.
Influence of age on rectal tone and sensitivity to distension in healthy subjects
Authors: LAGIER  , DELVAUX  , VELLAS  , FIORAMONTI  , BUENO  , ALBAREDE  , FREXINOS
Published: March 2003

Hypersensitivity to rectal distension is frequently observed in patients with irritable bowel syndrome (IBS). However, few data are available about the influence of age on rectal sensory thresholds and tone. The aim of this study was to measure rectal sensory thresholds and tone with a barostat in 12 healthy subjects (aged 86 ± 4 years, eight females, four males) as compared with 12 young healthy male controls (26 ± 1 years). Isobaric phasic distensions were performed in the fasted state (increment of 4 mmHg, steps of 5 min, interval of 5 min). Rectal tone changes were then measured as changes in volume of the barostat bag, the pressure being kept constant. After a baseline recording of 1 h, a 1000‐kcal meal was served and the tone recorded until return to baseline.

Rectal sensory thresholds were significantly higher in aged subjects. First sensation, sensation of urge to defaecate and sensation of pain were triggered at 21.1 ± 3.2 mmHg, 30.4 ± 5.4 mmHg and 40.5 ± 5.0 mmHg, respectively, in aged subjects, vs 13.3 ± 4.6 mmHg (P < 0.05), 20.7 ± 1.0 mmHg (P < 0.001), 31.3 ± 1.7 mmHg (P < 0.001) in controls. Rectal compliance was not significantly different between the two groups. Mean barostat bag volume was 104 ± 13 mL in fasting aged subjects and 125 ± 23 mL in controls (NS). After the meal, the barostat bag volume decreased by 69 ± 11% during 85 ± 17 min in aged subjects and 75 ± 14% during 89 ± 15 min in young controls (NS). Rectal sensory thresholds triggered by distension are increased in aged healthy subjects while compliance and tone are not different. Age should be considered as a confounding factor when studying rectal sensitivity and further studies in aged patients with IBS should include a group of control subjects within the same range of age as studied patients.
Neurogenic pathways mediating ascending and descending reflexes at the porcine ileocolonic junction
Authors: Kajimoto  , Dinning  , Gibb  , De Carle  , Cook
Published: March 2003

We studied the pharmacology of the neural pathways mediating the responses of ileo‐ and coloileo‐colonic junction (ICJ) to regional distension in ten anaesthetized pigs. Using manometric pullthroughs and a sleeve sensor, we found the ICJ demonstrated sustained tone that was resistant to tetrodotoxin. Ileal distension decreased ICJ pressure by 22.2 ± 10.1% (11.9 ± 2.7–10.1 ± 2.6 mmHg; P=0.002) and colonic distension augmented ICJ pressure by 23.5 ± 8.6% (12.8 ± 1.5–15.6 ± 2.1 mmHg; P=0.02). Bethanecol and Nω‐nitro‐ L‐arginine methyl ester (
L‐NAME) increased ICJ pressure (P=0.002, P=0.01, respectively). Sodium nitroprusside and isoproterenol reduced ICJ pressure (P=0.004, P=0.02, respectively). In the presence of
L‐NAME, the early inhibitory ileo‐ICJ response was abolished, while early and late inhibitory responses were abolished by further addition of propranolol but not by the addition of hexamethonium, atropine, prazosin or yohimbine. The excitatory colo‐ICJ response was replaced by inhibition in the presence of
L‐NAME. We concluded that:

1 the porcine ICJ displays myogenic tone which is influenced by excitatory muscarinic and inhibitory nitrergic and beta adrenergic pathways

2 an inhibitory ileo‐sphincteric reflex mediated by nitrergic and beta adrenergic postganglionic neural pathways

3 both excitatory and inhibitory neurogenic colo‐sphincteric reflexes exist, and the excitatory pathway involves nitrergic neurotransmission.
Schistosoma mansoni infection causing diffuse enteric inflammation and damage of the enteric nervous system in the mouse small intestine
Authors: Bogers  , Moreels  , De Man  , Vrolix  , Jacobs  , Pelckmans  , Van Marck
Published: March 2003

Schistosomiasis mansoni is a major health problem, mainly occurring in developing countries. A large proportion of infected individuals suffers from motility‐related gastrointestinal problems. In the present study, the diffuse inflammatory response in the small bowel wall, as compared to the egg‐induced granulomatous inflammation, was investigated. For this purpose, OF1 mice infected with Schistosoma mansoni 8–16 weeks prior to the experiment, and uninfected control mice were studied. The ileum showed both a diffuse mucosal inflammation as well as a granulomatous reaction. The diffuse mucosal inflammation caused an increase in the thickness of the mucosa, with blunting of the villi. A significant, transient increase of thickness of the muscularis propria after 12 weeks of infection was noted. There was an infection‐related mast cell infiltrate in the muscularis propria, consisting of formalin fixation‐insensitive connective tissue mast cells. Ganglionitis of the myenteric plexus was noted. Rarely, ganglia of the myenteric plexus contained apoptotic cells. A general pharmacological set of experiments showed a significant increase in intestinal contractility, both to exogenously administered, as well as to endogenously released neurotransmitters. Our results demonstrate that S. mansoni infection in the mouse ileum leads to diffuse specific enteric inflammation that is associated with an enhanced response to contractile agents.
IL‐1β and IL‐6 excite neurones and suppress cholinergic neurotransmission in the myenteric plexus of the guinea pig
Authors: Kelles A. , Janssens J. , Tack J.
Published: March 2003

The effects of the inflammatory mediators interleukin‐1β (IL‐1β) and interleukin‐6 (IL‐6) on myenteric neurones were investigated by intracellular recordings in a conventional myenteric plexus preparation of guinea pig ileum. Micropressure ejection of IL‐1β and IL‐6 (10−7 mol L−1) both caused an excitatory effect in, respectively, 19% (13/70) and 7% (5/70) of the myenteric neurones. The IL‐1β‐induced depolarizations were inhibited by superfusion of the IL‐1β receptor antagonist. The responses seen were tetrodotoxin‐resistant, indicating a direct neuronal effect. Responses to both cytokines were seen in nitric oxide synthase‐immunoreactive as well as choline acetyltransferase‐immunoreactive neurones. In addition, both IL‐1β and IL‐6 reversibly caused a presynaptic inhibition of acetylcholine release from cholinergic nerve terminals. Both cytokines had no effect on the slow excitatory postsynaptic potentials. Therefore, we can conclude that the inflammatory mediators IL‐1β and IL‐6 can act as excitatory neuromodulators of gastrointestinal motility through direct excitatory actions on a subset of myenteric neurones and through the presynaptic inhibition of acetylcholine release.
Colonic mechanoreceptor inputs to rat lumbo‐sacral dorsal horn neurones: distribution, thresholds and chemosensory modulation
Authors: Andrew L. K. , Blackshaw L. A.
Published: March 2003

In order to better understand the central processing of visceral sensory information, we studied the responses of lumbo‐sacral dorsal horn (L4‐S1) neurones to colonic stimuli in anaesthetized rats. Twenty‐four neurones responded to distal colonic distension with a 2.5‐cm balloon; six of these were tested with proximal colonic distension, to which none responded. All neurones tested responded to somatic non‐noxious inputs (tail movement). Responses to colonic distension were excitatory (n=22) or inhibitory (n=2). Sixteen neurones responded at a threshold of 20 mmHg or less, five at 20–40 mmHg, and three at 40–80 mmHg. Three of 10 neurones tested showed increased responses to colonic distension after intraluminal perfusion with bile. Bile itself did not evoke a response. We conclude that lumbo‐sacral spinal neurones selectively receive mechanosensory inputs from the distal colon. Neurones respond at thresholds within and above the physiological range. Dorsal horn neurones receiving colonic mechanosensory inputs are not directly modulated by chemosensory inputs, but their responsiveness to distension may be augmented.
The relationship between propagated contractions and pseudoaffective changes in blood pressure in response to intestinal distension
Authors: Timar‐Peregrin A. , Kumano K. , Khalil Z. , Sanger G. J. , Furness J. B.
Published: March 2003

We investigated the relationship between changes in small intestinal motility and changes in blood pressure and heart rate in response to intestinal distension. Rats were maintained under stable anaesthesia with α‐chloralose, and jejunal motility, blood pressure and heart rate were recorded. Pressure changes during propagated contractions of the circular muscle were recorded in the jejunum when the intraluminal pressure was maintained at 10 mmHg. Raising the pressure in 10 mmHg increments from 10 mmHg to 40 mmHg increased the frequency of propagated contractions from 0.30 ± 0.06 min–1 (mean ± SEM) to 1.29 ± 0.09 per min. In contrast, amplitudes of contractions above baseline pressure decreased from 19.5 ± 0.6 mmHg to 7.8 ± 0.5 mmHg. Simultaneously, blood pressure and heart rate were both increased. Pretreatment of rats with capsaicin, or severing the mesenteric nerves acutely, prevented these cardiovascular responses, but did not influence the changes in propagated activity caused by distension. Propagated contractions were blocked by hexamethonium (10 mg kg–1, intravenously [i.v.]) and by local application of 2% lidocaine, but propulsion was unchanged by hyoscine (1 mg kg–1, i.v.). Phentolamine (1 mg kg–1, i.v.) increased the frequency of propagated contractions. The methods described in this work allow the effects of drugs on intrinsic intestinal reflexes to be distinguished from their effects on extra‐intestinal, pseudoaffective reflexes. In addition, unlike other experiments using anaesthetized rats, blood pressure increased in response to distension, as it does in mammals that are not anaesthetized. The experiments demonstrate that the neural pathways for propagated contractions that rely on intrinsic nerve circuits, including intrinsic primary afferent neurones, and the neural pathways for extrinsic reflexes that signal pain or discomfort in the intestine, which involve capsaicin‐sensitive spinal afferent neurones, are independent.
Altered periodic rectal motor activity: a mechanism for slow transit constipation
Authors: Rao S. S. C. , Sadeghi P. , Batterson K. , Beaty J.
Published: March 2003

The pathophysiology of slow transit constipation is poorly understood. Both decreased and increased distal colonic motility have been reported. In healthy humans, a 3 cycles per minute (cpm), periodic rectal motor activity (PRMA) has been described. Our aim was to investigate the characteristics of PRMA and to assess its role in the pathogenesis of constipation. A six‐sensor solid‐state probe was placed with the tip sensor in the mid‐transverse colon, without sedation, and prolonged colonic motility was recorded in nine patients with slow transit constipation (1M, 8F) and in 11 healthy subjects (3M, 8F). Subjects were free to ambulate. We examined the frequency, nocturnal vs. diurnal variation, and characteristics of PRMA, and its relationship to proximal colonic motility. All subjects showed PRMA. The rhythm was similar (2.5–4 cpm) in both groups. However, constipated patients exhibited a greater (P < 0.001) number of PRMA cycles than controls. The duration of each cycle and amplitude of pressure waves during PRMA were also greater (P < 0.05) at night in patients compared with controls. In patients, 40% of PRMA cycles were associated with a proximal colonic motor event compared with 81% in controls (P < 0.02). The area under the curve of all colonic pressure waves and incidence of specialized propagating pressure waves was lower (P < 0.05) in patients during daytime. When compared with controls, constipated patients exhibited reduced daytime colonic pressure waves and a higher frequency of PRMA. Most of the PRMA was unrelated to proximal colonic activity in constipated patients in contrast with findings in control patients. In addition to decreased colonic motility, this excessive and unco‐ordinated phasic rectal activity may further impede stool transport and contribute to the pathogenesis of slow transit constipation.
Bile flow to the duodenum is reduced in hypothyreosis and enhanced in hyperthyreosis
Authors: LAUKKARINEN J. , KÖÖBI P. , KALLIOVALKAMA J. , SAND J. , MATTILA J. , TURJANMAA V. , PÖRSTI I. , NORDBACK I.
Published: March 2003

Disturbances in sphincter of Oddi (SO) function may prevent normal bile flow and thus enhance probability of common bile duct stone (CBDS) formation. We have previously shown increased prevalence of diagnosed hypothyroidism in CBDS patients, which may be explained by thyroxine‐induced inhibition of SO contractility, in addition to previously suggested changes in bile composition and hepatocytic excretion. The aim of this study was to investigate biliary dynamics in relation to altered thyroid gland function in rat, a rodent without a gallbladder. Euthyroid, hypothyroid or hyperthyroid Spraque–Dawley rats were anaesthetized with i.p. urethane, and exsanguinated at 15, 45, or 60 min after intravenous 99mTc HIDA injection. At these timepoints, the bile flow to intestine was determined by measuring the relative intestine vs. liver radioactivity. At 45 min this was 44% lower in hypothyroid rats and at 60 min 73% higher in hyperthyroid rats compared to euthyroid rats, while hepatic radioactivity at 15 min and blood pressure at injection were similar in the groups. We conclude that the bile flow to duodenum is reduced in hypothyreosis and enhanced in hyperthyreosis.