bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular analysis of tyrosine‐ and phenylalanine‐mediated repression of the tyrB promoter by the TyrR protein of Escherichia coli
Authors: Yang Ji , Camakaris Helen , Pittard James
Published: March 2003

The mechanism of repression of the tyrB promoter by TyrR protein has been studied in vivo and in vitro. In tyrR+ strains, transcription of tyrB is repressed by either tyrosine or phenylalanine. Both of the TyrR binding sites (strong and weak TyrR boxes) lie downstream of the tyrB transcription start site and are required for tyrosine‐ or phenylalanine‐mediated repression. Our results establish that the binding of the TyrR protein to the weak box, induced by cofactor tyrosine or phenylalanine, is critical for repression to occur. Neither the binding of the TyrR protein dimer formed in the presence of phenylalanine, nor the binding of the hexamer formed in the presence of tyrosine, blocks the binding of RNA polymerase to the promoter. Instead, open complex formation is inhibited in the presence of tyrosine whereas a step(s) following open complex formation is inhibited in the presence of phenylalanine. Moving the TyrR boxes 3 bp or more further away from the promoter affects tyrosine‐mediated repression without affecting phenylalanine‐mediated repression which remains unaltered until 6 bp are inserted between the TyrR boxes and the promoter. Analysis of deletion and insertion mutants fails to reveal any face of the helix specificity for either tyrosine‐ or phenylalanine‐mediated ­repression.
A locus of group A streptococcus involved in invasive disease and DNA transfer
Authors: Hidalgo‐Grass Carlos , Ravins Miriam , Dan‐Goor Mary , Jaffe Joseph , Moses Allon E. , Hanski Emanuel
Published: March 2003

Group A streptococcus (GAS) causes diseases ranging from benign to severe infections such as necrotizing fasciitis (NF). The reasons for the differences in severity of streptococcal infections are unexplained. We developed the polymorphic‐tag‐lengths‐transposon‐mutagenesis (PTTM) method to identify virulence genes in vivo. We applied PTTM on an emm14 strain isolated from a patient with NF and screened for mutants of decreased virulence, using a mouse model of human soft‐tissue infection. A mutant that survived in the skin but was attenuated in its ability to reach the spleen and to cause a lethal infection was identified. The transposon was inserted into a small open reading frame (ORF) in a locus termed sil, streptococcal invasion locus. sil contains at least five genes (silA‐E) and is highly homologous to the quorum‐sensing competence regulons of Streptococcus pneumoniae. silA and silB encode a putative two‐component system whereas silD and silE encode two putative ABC transporters. silC is a small ORF of unknown function preceded by a combox promoter. Insertion and deletion mutants of sil had a diminished lethality in the animal model. Virulence of a deletion mutant of silC was restored when injected together with the avirulent emm14‐deletion mutant, but not when these mutants were injected into opposite flanks of a mouse. DNA transfer between these mutants occurred in vivo but could not account for the complementation of virulence. DNA exchange between the emm14‐deletion mutant and mutants of sil occurred also in vitro, at a frequency of ∼ 10‐8 for a single antibiotic marker. Whereas silC and silD mutants exchanged markers with the emm14 mutant, silB mutant did not. Thus, we identified a novel locus, which controls GAS spreading into deeper tissues and could be involved in DNA transfer.
Integration and excision of the Mycobacterium tuberculosis prophage‐like element, φRv1
Authors: Bibb Lori A. , Hatfull Graham F.
Published: March 2003

The genomes of Mycobacterium tuberculosis H37Rv and CDC1551 each contain two prophage‐like elements, φRv1 and φRv2. The φRv1 element is not only absent from Mycobacterium bovis BCG but is in different locations within the two sequenced M. tuberculosis genomes; in both cases φRv1 is inserted into a REP13E12 repeated sequence, which presumably contains the bacterial attachment site, attB, for φRv1. Although φRv1 is probably too small to encode infectious phage particles, it may nevertheless have an active integration/excision system and be capable of moving from one chromosomal position to another. We show here that the M. tuberculosis H37Rv φRv1 element does indeed encode an active site‐specific recombination system in which an integrase of the serine recombinase family (Rv1586c) catalyses integration and excision and a small, basic φRv1‐encoded protein (Rv1584c) controls the directionality of re‐combination. Integration‐proficient plasmid vectors derived from φRv1 efficiently transform BCG, can utilize four of the seven REP13E12 sites present in BCG as attachment sites, and can occupy more than one site simultaneously.
Point mutants of EHEC intimin that diminish Tir recognition and actin pedestal formation highlight a putative Tir binding pocket
Authors: Liu Hui , Radhakrishnan Padhma , Magoun Loranne , Prabu Moses , Campellone Kenneth G. , Savage Pamela , He Feng , Schiffer Celia A. , Leong John M.
Published: March 2003

Attachment to host cells by enterohaemorrhagic Escherichia coli (EHEC) is associated with the formation of a highly organized cytoskeletal structure containing filamentous actin, termed an attaching and effacing (AE) lesion. Intimin, an outer membrane protein of EHEC, is required for the formation of AE lesions, as is Tir, a bacterial protein that is translocated into the host cell to function as a receptor for intimin. We established a yeast two‐hybrid assay for intimin–Tir interaction and, after random mutagenesis, isolated 24 point mutants in intimin, which disrupted Tir recognition in this system. Analysis of 11 point mutants revealed a correlation between recognition of recombinant Tir and the ability to trigger AE lesions. Many of the mutations fell within a 50‐residue region near the C‐terminus of intimin. Alanine‐scanning mutagenesis of this region revealed four residues (Ser890, Thr909, Asn916 and Asn927) that are critical for Tir recognition. Mapping the sequences of EHEC intimin and Tir onto the crystal structure of the intimin–Tir complex of enteropathogenic E. coli predicts that each of these four intimin residues lies at the intimin–Tir interface and contributes to a pocket that interacts with Ile298 of EHEC Tir. Thus, this genetic approach to intimin function both identified residues critical for Tir binding and demonstrated a correlation between the ability to bind Tir and the ability to trigger actin focusing.
Compensatory adaptation to the deleterious effect of antibiotic resistance in Salmonella typhimurium
Authors: Maisnier‐Patin Sophie , Berg Otto G. , Liljas Lars , Andersson Dan I.
Published: March 2003

Most chromosomal mutations that cause antibiotic resistance impose fitness costs on the bacteria. This biological cost can often be reduced by compensatory mutations. In Salmonella typhimurium, the nucleotide substitution AAA42 → AAC in the rpsL gene confers resistance to streptomycin. The resulting amino acid substitution (K42N) in ribosomal protein S12 causes an increased rate of ribosomal proofreading and, as a result, the rate of protein synthesis, bacterial growth and virulence are decreased. Eighty‐one independent lineages of the low‐fitness, K42N mutant were evolved in the absence of antibiotic to ameliorate the costs. From the rate of fixation of compensated mutants and their fitness, the rate of compensatory mutations was estimated to be ≥ 10−7 per cell per generation. The size of the population bottleneck during evolution affected fitness of the adapted mutants: a larger bottleneck resulted in higher average fitness. Only four of the evolved lineages contained streptomycin‐sensitive revertants. The remaining 77 lineages contained mutants that were still fully streptomycin resistant, had retained the original resistance mutation and also acquired compensatory mutations. Most of the compensatory mutations, resulting in at least 35 different amino acid substitutions, were novel single‐nucleotide substitutions in the rpsD, rpsE, rpsL or rplS genes encoding the ribosomal proteins S4, S5, S12 and L19 respectively. Our results show that the deleterious effects of a resistance mutation can be compensated by an unexpected variety of mutations.
Governor of the glnAp2 promoter of Escherichia coli
Authors: Atkinson Mariette R. , Pattaramanon Narinporn , Ninfa Alexander J.
Published: March 2003

Low‐affinity sites for the activator NRI∼P (NtrC∼P) that map between the enhancer and the glnAp2 promoter were responsible for limiting promoter activity at high concentrations of NRI∼P in intact cells and in an in vitro transcription system consisting of purified bacterial components. That is, the low‐affinity sites constitute a ‘governor’, limiting the maximum promoter activity. As the governor sites are themselves far from the promoter, they apparently act either by preventing the formation of the activation DNA loop that brings the enhancer‐bound activator and the promoter‐bound polymerase into proximity or by preventing a productive interaction between the enhancer‐bound activator and polymerase. The combination of potent enhancer and governor sites at the glnAp2 promoter provides for efficient activation of the promoter when the activator concentration is low, while limiting the maximum level of promoter activity when the activator concentration is high.
Thioredoxins are required for protection against a reductive stress in the yeast Saccharomyces cerevisiae
Authors: Trotter Eleanor W. , Grant Chris M.
Published: March 2003

Thioredoxins are small, highly conserved oxidoreductases that are required to maintain the redox homeostasis of the cell. They have been best characterized for their role as antioxidants in protection against reactive oxygen species. We show here that thioredoxins (TRX1, TRX2) and thioredoxin reductase (TRR1) are also required for protection against a reductive stress induced by exposure to dithiothreitol (DTT). This sensitivity to reducing conditions is not a general property of mutants affected in redox control, as mutants lacking components of the glutathione/glutaredoxin system are unaffected. Furthermore, TRX2 gene expression is induced in response to DTT treatment, indicating that thioredoxins form part of the cellular response to a reductive challenge. Our data indicate that the sensitivity of thioredoxin mutants to reducing stress appears to be a consequence of elevated glutathione levels, which is present predominantly in the reduced form (GSH). The elevated GSH levels also result in a constitutively high unfolded protein response (UPR), indicative of an accumulation of unfolded proteins in the endoplasmic reticulum (ER). However, there does not appear to be a general defect in ER function in thioredoxin mutants, as oxidative protein folding of the model protein carboxypeptidase Y occurs with similar kinetics to the wild‐type strain, and trx1 trx2 mutants are unaffected in sensitivity to the glycosylation inhibitor tunicamycin. Furthermore, trr1 mutants are resistant to tunicamycin, consistent with their high UPR. The high UPR seen in trr1 mutants can be abrogated by the GSH‐specific reagent 1‐chloro‐2,4‐dinitrobenzene. In summary, thioredoxins are required to maintain redox homeostasis in response to both oxidative and reductive stress conditions.
A conserved mitogen‐activated protein kinase pathway is required for mating in Candida albicans
Authors: Chen Jiangye , Chen Jing , Lane Shelley , Liu Haoping
Published: March 2003

Candida albicans had been thought to lack a mating process until the recent discovery of a mating type‐like locus and mating between MTLa and MTLα strains. To elucidate the molecular mechanisms that regulate mating in C. albicans, we examined the function of Cph1 and its upstream mitogen‐activated protein (MAP) kinase pathway in mating, as they are homologues of the pheromone‐responsive MAP kinase pathway in Saccharomyces cerevisiae. We found that overexpressing CPH1 in MTLa, but not in MTLa/α strains, induced the transcription of orthologues of S. cerevisiae pheromone‐induced genes and also increased mating efficiency. Furthermore, cph1 and hst7 mutants were completely defective in mating, and cst20 and cek1 mutants showed reduced mating efficiency, as in S. cerevisiae. The partial mating defect in cek1 results from the presence of a functionally redundant MAP kinase, Cek2. CEK2 complemented the mating defect of a fus3 kss1 mutant of S. cerevisiae and was expressed only in MTLa or MTLα, but not in MTLa/α cell types. Moreover, a cek1 cek2 double mutant was completely defective in mating. Our data suggest that the conserved MAP kinase pathway regulates mating in C. albicans. We also observed that C. albicans mating efficiency was greatly affected by medium composition, indicating the potential involvement of nutrient‐sensing pathways in mating in addition to the MAP kinase pathway.
Early targeting of Min proteins to the cell poles in germinated spores of Bacillus subtilis: evidence for division apparatus‐independent recruitment of Min proteins to the division site
Authors: Harry Elizabeth J. , Lewis Peter J.
Published: March 2003

The earliest event in bacterial cell division is the assembly of a tubulin‐like protein, FtsZ, at mid‐cell to form a ring. In rod‐shaped bacteria, the Min system plays an important role in division site placement by inhibiting FtsZ ring formation specifically at the polar regions of the cell. The Min system comprises MinD and MinC, which form an inhibitor complex and, in Bacillus subtilis, DivIVA, which ensures that division is inhibited only in the polar regions. All three proteins localize to the division site at mid‐cell and to cell poles. Their recruitment to the division site is dependent on localization of both ‘early’ and ‘late’ division proteins. We have examined the temporal and spatial localization of DivIVA relative to that of FtsZ during the first and second cell division after germination and outgrowth of B. subtilis spores. We show that, although the FtsZ ring assembles at mid‐cell about halfway through the cell cycle, DivIVA assembles at this site immediately before cell division and persists there during Z‐ring constriction and completion of division. We also show that both DivIVA and MinD localize to the cell poles immediately upon spore germination, well before a Z ring forms at mid‐cell. Furthermore, these proteins were found to be present in mature, dormant spores. These results suggest that targeting of Min proteins to division sites does not depend directly on the assembly of the division apparatus, as suggested previously, and that potential polar division sites are blocked at the earliest possible stage in the cell cycle in germinated spores as a mechanism to ensure that equal‐sized daughter cells are produced upon cell division.
Two different modes of transcription repression of the Escherichia coli acetate operon by IclR
Authors: Yamamoto Kaneyoshi , Ishihama Akira
Published: March 2003

IclR is a repressor for the Escherichia coli aceBAK operon, which encodes isocitrate lyase (aceB), malate synthase (aceA) and isocitrate dehydroge‐nase kinase/phosphorylase (aceK) in the glyoxylate bypass. IclR also represses the expression of iclR in an autogenous manner. DNase I footprinting and in vitro transcription assays indicated that IclR binds to an IclR box (−21 to +14), which overlaps the iclR promoter and thus competes with the RNA polymerase for DNA binding, leading to transcription repression. In the case of the aceBAK operon, IclR binds to IclR box II between −52 and −19 of the aceB promoter and interferes with binding of the RNA polymerase to this promoter. A secondary IclR binding site (IclR box I) was identified between −125 and −99 of the aceB promoter. IclR binds to this IclR box I even after formation of the aceB promoter open complex and, moreover, induces disassembly of the open complex, leading to repression of aceB transcription. In parallel, the location of the C‐terminal domain of the RNA polymerase α subunit (αCTD) on DNA is shifted close to the IclR box I, indicating that direct interaction between the αCTD and the IclR box I‐associated IclR caused the repression.
Control of the Salmonella ugd gene by three two‐component regulatory systems
Authors: Mouslim Chakib , Groisman Eduardo A.
Published: March 2003

The Salmonella ugd gene is required for the incorporation of 4‐aminoarabinose in the lipopolysaccharide and resistance to the antibiotic polymyxin B. Transcription of the ugd gene is induced by Fe3+ via the PmrA–PmrB two‐component system and by low Mg2+ in a process that requires the PhoP–PhoQ two‐component system, the PhoP‐activated PmrD protein and the PmrA–PmrB system. Here, we establish that mutation of the tolB gene promotes ugd transcription independently of both the PhoP–PhoQ and PmrA–PmrB systems. This activation is mediated by the RcsC–YojN–RcsB phosphorelay and the RcsA protein, suggesting a role for ugd in capsule synthesis. Binding sites for the RcsB, PmrA and PhoP proteins were identified in the ugd promoter. Although the PmrA–PmrB and RcsC–YojN–RcsB systems promoted ugd transcription independently of the PhoP–PhoQ system under different environmental conditions, ugd expression inside macrophages was strictly dependent on PhoP–PhoQ, suggesting that low Mg2+ is a cue for the intracellular environment.
Modified yeast cells to investigate the coupling of G protein‐coupled receptors to specific G proteins
Authors: Ladds Graham , Davis Kevin , Hillhouse Edward W. , Davey John
Published: March 2003

G protein‐coupled receptors (GPCRs) help to regulate the physiology of all the major organ systems. They respond to a multitude of ligands and activate a range of effector proteins to bring about the appropriate cellular response. The choice of effector is largely determined by the interaction of individual GPCRs with different G proteins. Several factors influence this interaction, and a better understanding of the process may enable a more rational approach to identifying compounds that affect particular signalling pathways. A number of systems have been developed for the analysis of GPCRs. All provide useful information, but the genetic amenability and relative simplicity of yeast makes them a particularly attractive option for ligand identification and pharmaceutical screening. Many, but not all, GPCRs are functional in the budding yeast Saccharomyces cerevisiae, and we have developed reporter strains of the fission yeast Schizosaccharomyces pombe as an alternative host. To provide a more generic system for investigating GPCRs, we created a series of yeast–human Gα‐transplants, in which the last five residues at the C‐terminus of the yeast Gα‐subunit are replaced with the corresponding residues from different human G proteins. These enable GPCRs to be coupled to the Sz. pombe signalling machinery so that stimulation with an appropriate ligand induces the expression of a signal‐dependent lacZ reporter gene. We demonstrate the specificity of the system using corticotropin releasing factor (CRF) and CRF‐related peptides on two CRF receptors. We find that different combinations of ligand and receptor activate different Gα‐transplants, and the specificity of the coupling is similar to that in mammalian systems. Thus, CRF signalled through the Gs‐ and Gi‐transplants, consistent with its regulation of adenylate cyclase, and was more active against the CRF‐R1A receptor than against the CRF‐R2B receptor. In contrast, urocortin II and urocortin III were selective for the CRF‐R2B receptors. Furthermore, urocortin, but not CRF, induced signalling through the CRF‐R1A receptor and the Gq‐transplant. This is the first time that human GPCRs have been coupled to the signalling pathway in Sz. pombe, and the strains described in this study will complement the other systems available for studying this important family of receptors.
The transmembrane domains of the sensor kinase KdpD of Escherichia coli are not essential for sensing K+ limitation
Authors: Heermann Ralf , Fohrmann Andy , Altendorf Karlheinz , Jung Kirsten
Published: March 2003

The sensor kinase/response regulator system KdpD/KdpE of Escherichia coli regulates the expression of the kdpFABC operon, which encodes the high affinity K+ transport system KdpFABC. The membrane‐bound sensor kinase KdpD consists of four transmembrane domains, a large cytoplasmic N‐terminal domain and a cytoplasmic C‐terminal transmitter domain. To elucidate the role of the four transmembrane domains, various deletions were introduced in kdpD and the activities of the resulting truncated derivatives of KdpD were determined. A KdpD protein lacking all four transmembrane domains was able to sense low K+ concentrations, whereas at higher K+ concentrations kdpFABC expression was constitutive. These and further results with various truncated KdpD proteins lacking distinct parts of the transmembrane domains or derivatives in which a linker peptide or two transmembrane domains of PutP, the Na+/proline transporter of Escherichia coli, replaced the missing part indicated that the transmembrane domains are not essential for sensing of K+ limitation, but may be important for the correct positioning of the large N‐ and C‐terminal cytoplasmic domains to each other.
The Caulobacter crescentus polar organelle development protein PodJ is differentially localized and is required for polar targeting of the PleC development regulator
Authors: Hinz Aaron J. , Larson David E. , Smith Christopher S. , Brun Yves V.
Published: March 2003

Regulation of polar development and cell division in Caulobacter crescentus relies on the dynamic localization of several proteins to cell poles at specific stages of the cell cycle. The polar organelle development protein, PodJ, is required for the synthesis of the adhesive holdfast and pili. Here we show the cell cycle localization of PodJ and describe a novel role for this protein in controlling the dynamic localization of the developmental regulator PleC. In swarmer cells, a short form of PodJ is localized at the flagellated pole. Upon differentiation of the swarmer cell into a stalked cell, full length PodJ is synthesized and localizes to the pole opposite the stalk. In late predivisional cells, full length PodJ is processed into a short form which remains localized at the flagellar pole after cell division and is degraded during swarmer to stalked cell differentiation. Polar localization of the developmental regulator PleC requires the presence of PodJ. In contrast, the polar localization of PodJ is not dependent on the presence of PleC. These results indicate that PodJ is an important determinant for the localization of a major regulator of cell differentiation. Thus, PodJ acts directly or indirectly to target PleC to the incipient swarmer pole, to establish the cellular asymmetry that leads to the synthesis of holdfasts and pili at their proper subcellular location.
PriA supports two distinct pathways for replication restart in UV‐irradiated Escherichia coli cells
Authors: Jaktaji Razieh P. , Lloyd Robert G.
Published: March 2003

The Escherichia coli PriA protein loads the DnaB replicative helicase at branched DNA structures independently of the replication initiator protein, DnaA, and thereby facilitates assembly of functional replisomes at sites removed from oriC. It is therefore a critical factor in the rescue of replication forks stalled at DNA lesions. It is also a DNA helicase. We describe insertions near the 3′ end of priA that interfere with PriA activity. These insertions and the previously described priA300 encoding helicase‐defective PriA K230R are shown to be effective suppressors of the DNA repair defect in recG strains, but substantially reduce the ability of ruv mutants to survive DNA damage. The data presented suggest that PriA helicase in conjunction with RecG can promote direct rescue of stalled forks independently of the recombinational pathway promoted by the combined activities of the RuvABC, RecBCD and RecA proteins, which requires only the primosome assembly activity of PriA to load DnaB at D loops. In cells lacking the helicase activity of PriA, we propose that stalled forks can be redirected to the recombination pathway via a Holliday junction intermediate common to both pathways, thus explaining the resistance of these cells to DNA damage.
Molecular mechanisms of iron uptake in fungi
Authors: Kosman Daniel J.
Published: March 2003

Fungi, like all free‐living organisms, are in competition for limiting nutrients. In accumulating iron, fungi are faced also with a trace metal whose aqueous and redox chemistry make it both relatively bio‐unavailable and strongly cytotoxic. Successful adaptation to this environmental context has provided fungi with an iron uptake strategy that has three features: it relies on redox cycling to enhance iron bio‐availability and reduce iron cytotoxicity; it includes both high‐ and low‐affinity pathways that are mechanistically distinct; and it is autoregulating so as to maintain intracellular iron homeostasis. Using Saccharomyces cerevisiae as a paradigm, this review summarizes current knowledge about the four pathways by which this yeast accumulates iron. These four pathways include: siderophore iron accumulation; high affinity iron uptake via an iron permease; and two lower affinity uptake pathways through relatively non‐specific divalent metal ion transporters. All of these four pathways are directly or indirectly dependent on the activity of metalloreductase activity expressed extracellularly on the plasma membrane. A variety of experimental and genomics data indicate that this resourcefulness is shared by many, if not most, fungi. On the other hand, while the autoregulation of iron metabolism in Baker's yeast is well‐understood, little is known about the apparent homeostatic mechanisms in these other yeasts and fungi. The integration of these multiple uptake mechanisms and their regulation into over‐all iron homeostasis in yeast concludes this brief review.
F1‐catalysed ATP hydrolysis is required for mitochondrial biogenesis in Saccharomyces cerevisiae growing under conditions where it cannot respire
Authors: Lefebvre‐Legendre Linnka , Balguerie Axelle , Duvezin‐Caubet Stéphane , Giraud Marie‐France , Slonimski Piotr P. , Di Rago Jean‐Paul
Published: March 2003

Mutant strains of yeast Saccharomyces cerevisiae lacking a functional F1‐ATPase were found to grow very poorly under anaerobic conditions. A single amino acid replacement (K222 > E222) that locally disrupts the adenine nucleotide catalytic site in the β‐F1 subunit was sufficient to compromise anaerobic growth. This mutation also affected growth in aerated conditions when ethidium bromide (an intercalating agent impairing mtDNA propagation) or antimycin (an inhibitor of respiration) was included in the medium. F1‐deficient cells forced to grow in oxygen‐limited conditions were shown to lose their mtDNA completely and to accumulate Hsp60p mainly under its precursor form. Fluorescence microscopy analyses with a modified GFP containing a mitochondrial targeting presequence revealed that aerobically growing F1‐deficient cells stopped importing the GFP when antimycin was added to the medium. Finally, after total inactivation of the catalytic α3β3 subcomplex of F1, mitochondria could no longer be energized by externally added ATP because of either a block in assembly or local disruption of the adenine nucleotide processing site. Altogether these data strengthen the notion that in the absence of respiration, and whether the proton translocating domain (F0) of complex V is present or not, F1‐catalysed hydrolysis of ATP is essential for the occurrence of vital cellular processes depending on the maintenance of an electrochemical potential across the mitochondrial inner membrane.
Cell shape, division and development: the 2002 American Society for Microbiology (ASM) conference on prokaryotic development
Authors: Figge Rainer M. , Gober James W.
Published: March 2003

In the last decade, the use of cytological techniques, together with the analysis of complete genomes, has dramatically advanced our understanding of bacterial development. Work on several well‐developed model systems such as Bacillus subtilis, Caulobacter crescentus, Myxococcus xanthus and Streptomyces spp., has provided us with an in‐depth understanding of processes such as sporulation, multicellular behaviour and the bacterial cell cycle. At the same time, these studies have revolutionized our view of the bacterial cell and shown it to be a highly complex entity with spatial and temporal organization. The recent American Society for Microbiology (ASM) conference on prokaryotic development demonstrated that several laboratories have now started to connect data obtained through functional genomic analysis with subcellular organization, thereby generating three‐dimensional regulatory networks. This meeting report highlights new findings in the field, such as regulation of protein localization during sporulation and the cell cycle, control of cell–cell interaction and the initiation of cell division.
Activation of non‐adrenergic non‐cholinergic inhibitory pathways by endogenous and exogenous tachykinins in the ferret lower oesophageal sphincter
Authors: SMID  , LYNN  , TEMPLEMAN  , BLACKSHAW
Published: March 2003

Repeated oesophageal acidification causes lower oesophageal sphincter (LOS) relaxation in the anaesthetized ferret which is mediated by a peripheral neurokinin (NK‐1) receptor mechanism. Our aim in this study was to characterize neural pathways in the LOS activated by capsaicin and tachykinin receptor agonists in vitro. Circular muscle strips of LOS (two per animal) from a total of 24 ferrets were maintained in organ baths. Electrical field stimulation (EFS, 50 V, 5–50 Hz) caused frequency‐dependent LOS relaxation which was abolished by tetrodotoxin (TTX; 10−6 M: P < 0.001) and reduced by NG‐nitro‐L‐arginine (L‐NNA; 10−4 M: P < 0.01). Substance P and [Sar9, Met (O2)11]‐substance P (selective NK‐1 agonist) caused dose‐dependent relaxation, while the NK‐2 receptor agonist [β‐Ala8]‐NKA 4‐10 evoked excitation. Capsaicin (10−6 M) caused relaxation and desensitization that was overcome by long recovery periods and substance P dosing (10−8 M). After pretreatment with the NK‐1 receptor antagonist CP 99994 (10−7 M), substance P (10−8 M; P < 0.001) and capsaicin (10−6 M: P < 0.01)‐induced relaxations were reduced. In the presence of TTX (10−6 M), excitation resulted in response to substance P (10−8 M; P < 0.05) and [Sar9, Met (O2)11]‐substance P (10−8 M; P < 0.001), while the response to [β‐Ala8]‐NKA 4‐10 (10−7 M) was unaffected. In the presence of L‐NNA (10−4 M), substance P and [Sar9, Met (O2)11]‐substance P‐induced relaxations were reduced (10−8 M; P < 0.01), while the response to [β‐Ala8]‐NKA 4‐10 (10−7 M) was unaffected. These results show that functional coupling between capsaicin‐sensitive sensory neurones and NANC inhibitory neural pathways occurs via NK‐1 receptors in the ferret LOS. NK‐2 (and some NK‐1) receptors activate non‐neural excitatory mechanisms. Substance P and NK‐1 receptors coupli ng sensory and NANC inhibitory neurones may be important in the reflex control of LOS motility.
In vitro and in vivo effects of nitric oxide synthase inhibitors and nitric oxide inactivators on the South American opossum ileocolonic junction*
Authors: Matsuda N.M. , Feitosa Jr R.L. , Lemos M.C. , Oliveira R.B. , Ballejo G.
Published: March 2003

The potential role of nitrergic nerves in the regulation of the South American (SA) opossum ileocolonic junction (ICJ) function was investigated. In vitro, the effects of nitric oxide (NO) synthase inhibitors and NO inactivators on the non‐adrenergic non‐cholinergic (NANC) nerve‐mediated relaxations of the circular muscle of the SA opossum ICJ were determined by employing isolated strips. Electrical field stimulation (0.2–8.0 Hz) caused frequency‐dependent NANC relaxations. Nicotine and ATP also induced concentration dependent NANC relaxations that were abolished by tetrodotoxin (TTX). The relaxation response induced by NANC nerve activation was reduced in a dose dependent manner by NO synthase inhibitors while vasoactive intestinal peptide (VIP) and sodium nitroprusside (SNP) induced relaxations were uninfluenced by these drugs. In vivo, the NO synthase inhibitor, L‐NAME, administered into the local artery caused a raise in intraluminal pressure of the ICJ in anaesthetized SA opossums in a L‐arginine‐preventable manner. Hydroquinone and pyrogallol, while being able to reduce, in a superoxide dimutase (SOD) reversible manner, the relaxations induced by exogenous NO failed to affect the NANC nerve‐induced relaxations. Finally, neurones and nerve fibres in the myenteric plexus as well as varicose nerve fibres on the circular smooth layer were positive for NADPH‐diaphorase activity. These findings indicate that nitrergic nerves inhibit ICJ circular smooth muscle in vitro and in vivo but cast doubts on the neuromediator being the NO radical.
Discharge patterns of intramural mechanoreceptive afferents during selective distension of the cat's rectum
Authors: Rühl A. , Thewißen M. , Ross H.‐G. , Cleveland S. , Frieling T. , Enck P.
Published: March 2003

The afferent input from the rectum to the central nervous system (CNS) has yet to be thoroughly characterized. The characteristics of mechanoreceptive rectal afferents have been studied in unanaesthetized decerebrate cats. Following lumbo‐sacral laminectomy, single‐unit activity (occasionally multi‐unit activity) was recorded from centrally cut filaments of the sacral dorsal roots (predominantly S2), while a balloon was inflated in the rectum. Starting from their background activities (mean 15.1 imp sec−1, SD 7.6 imp sec−1), afferent discharge rate increased with increasing balloon pressure (mean threshold 6.3 mmHg, SD 3.6 mmHg). The dependence of firing rate on intrarectal pressure began to flatten out at 25 mmHg (mean; SD 10 mmHg). For 22 out of 29 units (76%) complete saturation occurred at 35 mmHg (mean; SD 15 mmHg) with a maximum discharge rate of 31 imp sec−1 (mean; SD 12.6 imp sec−1). In a number of recording sessions, cyclical rectal contractions were observed. In these cases, changes in firing of the units were closely related to changes in intrarectal pressure. Pressure‐related afferent activity could be enhanced by parasympathomimetic drugs which augmented rectal contractions. We conclude that sacral dorsal roots contain afferents from low‐threshold mechanoreceptors located in the rectal wall, and that these afferents monitor the filling state and contraction level of the rectum.
Motility in the Roux‐Y limb after distal gastrectomy: relation to the length of the limb and the afferent duodenojejunal segment – an experimental study
Authors: Blanc‐Louvry I. Le , Ducrotté  , Lemeland  , Metayer  , Denis  , TéniÈre
Published: March 2003

Following gastrectomy, the longer is a Roux‐Y limb constructed to restore digestive continuity the higher the frequency of postoperative symptoms. The aim of this experimental study was to test how the level of the jejunal transection and the length of the Roux limb affect the motility of the constructed limb and in particular the onset and the propagation of activity fronts (AFs).

Three months after a distal Roux‐en‐Y gastrectomy, electromyographic tracings were recorded in six groups of rats grouped according to the level of the transection (20 or 40 cm from the pylorus) and the length of the limb (10, 20 or 30 cm). Animals in which a simple laparotomy or laparotomy + jejunal transection was performed, served as controls.

During the interdigestive period, all animals had AFs in the limb which were independent from those recorded in the duodenum. In the limb, the mean time interval between two AFs was shorter (P < 0.01) and more irregular than in controls. An increase in limb length was associated with a lower incidence of completely propagated AFs (P < 0.05) and a higher incidence of irregularly propagated AFs (P < 0.01). When propagation of the AFs was analysed both in the limb and in the jejunum distal to the anastomosis, propagation abnormalities were more frequent. Below the gastrojejunal anastomosis, for an intestinal length of either 20 or 30 cm, the frequency of abnormal AFs was not different when this length was either only a limb or a limb with the 10 cm of distal jejunum below the jejuno‐jejunal anastomosis. Interruption of AFs by a meal was irregular in the limb and more rarely observed in the 30‐cm than in 10‐cm limbs (P < 0.05). Interruption of AFs was shorter than in controls (P < 0.01). In the duodenum and the jejunum proximal to the limb, the interval between AFs was higher than in controls and in the Roux‐Y limbs (P < 0.001). Intraluminal concentrations of bacterial strains were not different in the different types of limb while lactobacillus concentrations and pooled concentrations of bacteria were higher than in controls (P < 0.05). No relationship was found between the incidence of myoelectric abnormalities and intraluminal bacterial concentrations.

Increasing the length of a Roux‐Y limb resulted in more frequent disturbances in AFs in the limb but had no significant consequence on the overall rate of abnormal AFs in the jejunum distal to the transection. Motor response to food intake was also reduced. Motor changes were not related to intraluminal bacterial concentrations.
Inhibitory effects of NPY on ganglionic transmission in myenteric neurones of the guinea‐pig descending colon
Authors: Browning  , Lees
Published: March 2003

Intracellular recordings were made from myenteric neurones of the guinea‐pig descending colon. Neuropeptide Y (NPY) and related pancreatic polypeptides were applied by superfusion and the effects upon the amplitude of fast excitatory synaptic potentials (ESPs) and the ratio of paired fast ESPs evoked by stimulation of internodal fibre tracts were noted. NPY produced a concentration‐dependent inhibition in fast ESP amplitude in the majority of neurones (17/21) with a calculated IC50 value of 7 nM; in some neurones this inhibition was mediated via the local release of noradrenaline. Peptide YY (PYY) (eight out of 11 neurones; IC50 = 1 nM), NPY(3–36) (three out of three neurones) and [Leu31, Pro34]NPY (four out of five neurones) also decreased the amplitude of fast ESPs. The effects of two or more pancreatic polypeptides or analogues on fast synaptic transmission were compared directly in six neurones; the apparent relative potency of agonists suggested the involvement of Y2‐receptors and at least one other Y‐receptor type. In the absence of any direct postsynaptic effects of pancreatic polypeptides on the active or passive properties of myenteric neurones, or on their sensitivity to ionophoretically applied acetylcholine, inhibition of fast ganglionic transmission was presumed to be presynaptic in origin. It is concluded that, in addition to their previously described depressant actions on neuro‐effector transmission to colonic smooth muscle, pancreatic polypeptides can exert powerful inhibitory effects on myenteric neurones of the descending colon.
The effect of fentanyl, DNQX and MK‐801 on dorsal horn neurones responsive to colorectal distension in the anaesthetized rat
Authors: Kozlowski C. M. , Bountra C. , Grundy D.
Published: March 2003

Certain dorsal horn neurones respond in a graded manner to noxious colorectal distension (CRD). Morphine inhibits these responses in the spinalized rat, but the role of excitatory amino acids in baseline visceral nociceptive transmission is less clear. This study examines the effect of the μ‐opiate receptor agonist fentanyl, and the non‐NMDA and NMDA antagonists DNQX and MK‐801, respectively, on such responses to CRD in the sodium pentobarbitone‐anaesthetized rat.

Male rats were prepared for extracellular recording from the lumbosacral spinal cord. 90 neurones responsive to CRD, located throughout the dorsal horn, were classified according to their response duration and latency to 60 mmHg distension, as SL‐A (short latency‐abrupt; 59%), SL‐S (short latency‐sustained; 23%), L‐L (long‐latency; 10%) and Inhib (inhibited; 8%). Convergent cutaneous receptive fields were mapped for 79/90 neurones and classified as LT (low threshold), WDR (wide dynamic range) or HT (high threshold). CRD (20–100 mm Hg) elicited graded responses in most neurones.

In 6/6 SL‐S neurones, fentanyl (1–8 μg kg–1) dose‐dependently inhibited the response to 60 mm Hg CRD, in a naloxone‐sensitive manner, with an ID50 value (±95% confidence limits) of 2.48 (1.7–3.7) μg kg–1. In 6/6 SL‐A neurones, fentanyl had no significant effect on the response to CRD. DNQX (0.03–3 mg kg–1) produced a dose‐dependent inhibition of the response to CRD in 5/5 SL‐A neurones, with an ID50 value of 0.32 (0.01–41.1) mg kg–1. MK‐801 (0.03–0.3 mg kg–1) had no significant effect on responses to CRD in 6/6 SL‐A neurones.

The differential inhibitory effects of fentanyl on two neuronal subtypes may indicate functional differences. In SL‐A neurones AMPA/kainate, but not NMDA receptors are involved in mediating baseline nociceptive neurotransmission.
Contractile effects and intracellular Ca2+ signalling induced by motilin and erythromycin in the circular smooth muscle of human colon
Authors: Van Assche G. , Depoortere I. , Thijs T. , Missiaen L. , Penninckx F. , Takanashi H. , Geboes K. , Janssens J. , Peeters T. L.
Published: March 2003

Motilin has excitatory effects on the colon of the rabbit and the dog, but little is known of its effect on the human colon. The aim of this study was to investigate the effects induced by motilin and erythromycin A (EMA) on muscle strips and on single cells from primary cultures from human colon. Isotonic contraction was recorded in circular muscle strips from macroscopically normal resection specimens of patients operated on for colonic neoplasm. Agonist‐induced intracellular Ca2+ ([Ca2+]i) signalling was studied in primary cultures of colonic smooth‐muscle cells using the ratiometric Ca2+ indicator Indo 1, on a laser‐scanning confocal epifluorescence microscope. In circular muscle strips, norleucine13‐porcine motilin ([Nle13]‐pm)and EMA induced tonic contractions with an EC50 of 92 ± 21 nmol L−1 and 31 ± 16 μmol L−1, respectively. The maximal contraction was 21 ± 4% (motilin) and 33 ± 12% (EMA) of the response to 10−4 mol L−1 acetylcholine (ACh). The motilin antagonist OHM‐11526 (10−5.5 mol L−1) abolished the effects of both [Nle13]‐pm and EMA. Neither tetrodotoxin (10−5.5 mol L−1), L‐nitro‐D‐arginine methyl ester (L‐NAME) (10−3.5 mol L−1) nor guanethidine (10−5 mol L−1) interfered with the effects of [Nle13]‐pm or EMA. [Nle13]‐pm (10−11−10−6 mol L−1) induced rises of [Ca2+]i in cultured colonic myocytes. At 10−6 mol L−1, 94% of the cells responded, and half of the cells responded at 1.4 nmol L−1 [Nle13]‐pm. 81% (35/43) and 95% (75/79) responded to EMA (10−6 mol L−1) and acetylcholine (ACh, 10−4 mol L−1), respectively. The motilin antagonist GM‐109 inhibited motilin‐ and EMA‐induced [Ca2+]i rises. In the absence of extracellular Ca2+, only 13% (7/52) of the cells responded to [Nle13]‐pm (10−6 mol L−1) vs. 90% (47/52) to ACh (10−4 mol L−1). Motilin and EMA have direct excitatory effects on circular smooth muscle from the human colon and these effects are mediated via a smooth‐muscle motilin receptor. These findings suggest that motilin may regulate colonic motility and that motilides may have therapeutic potential for the treatment of colonic hypomotility.
Low antroduodenal pressure gradients are responsible for gastric emptying of a low‐caloric liquid meal in humans
Authors: HAUSKEN T. , MUNDT M. , SAMSOM M.
Published: March 2003

The motor mechanisms responsible for transpyloric flow of gastric contents are still poorly understood. The aim of our study was to investigate the relationship between luminal pressures and gastric wall motion and between gastroduodenal pressure gradients and pressure waves, and ante‐ and retro‐grade transpyloric flow. In eight healthy volunteers, intraluminal pressures were recorded from the antrum and proximal duodenum. Transpyloric flow was monitored simultaneously using duplex ultrasonography, before, during and after ingestion of 300 mL meat soup. Transpyloric emptying occurred as sequences of alternating periods of emptying–reflux–emptying. Approximately one‐third of the sequences were not associated with peristalsis. The antroduodenal pressure gradients were significantly lower during nonperistaltic‐related emptying than during peristaltic‐related emptying (0.15 (0–0.3) kPa, and 1.7 (0.2–2.0) kPa, respectively [mean ± (range)], P < 0.005). The duration of emptying episodes not associated with peristalsis were significantly longer than those associated with peristalsis at (6.5 (3–8.7) s and 4.4 (2–6) s, respectively, P=0.059). Manometry detected only 56% of the antral contractions seen on ultrasound. We concluded that gastric emptying of a low‐calorie liquid meal occurs both during peristaltic and nonperistaltic antral activity. In spite of lower antroduodenal pressure gradients, the emptying episodes were longer for nonperistaltic emptying, which is likely to be caused by low pyloric resistance. Considerable flow seems to occur without peristalsis during gastric emptying of a low‐calorie, liquid meal in humans.
Physiological study of interstitial cells of Cajal identified by vital staining
Authors: RICH A. , HANANI M. , ERMILOV L. G. , MALYSZ J. , BELZER V. , SZURSZEWSKI J. H. , FARRUGIA G
Published: March 2003

Interstitial cells of Cajal (ICC) form networks that intercalate between the enteric nervous system and smooth muscle cells and play a fundamental role in the control of gastrointestinal motility by initiating rhythmic electrical activity. In this report, we used a method to examine the physiological and morphological properties of ICC in living, intact tissues. ACK2, an anti‐Kit antibody, was conjugated to a fluorescent probe and used to identify individual ICC for intracellular electrical recordings, to record changes in intracellular calcium concentration using fluorescent dyes and for confocal microscopy. Cyclic changes in intracellular calcium concentration were recorded in ICC with a frequency similar to the electrical slow wave. In addition, injection of a fluorescent dye into single ICC enabled the three‐dimensional reconstruction of single myenteric plexus ICC within the intact network. The data show that ICC in intact networks from the myenteric plexus region in living tissues in the guinea‐pig antrum exhibit an electrical slow wave, and that intracellular calcium oscillates at a frequency similar to the slow wave.
Periodic colonic motor activity identified by 24‐h pancolonic ambulatory manometry in humans
Authors: Hagger R. , Kumar D. , Benson M. , Grundy A.
Published: March 2003

Abstract
 
The pattern of colonic motor activity in healthy humans has not been fully elucidated to date. The aim of this study was to evaluate colorectal motor activity employing 24‐h ambulant pancolonic manometry. Ten healthy volunteers (6F, 4M), aged 19–31 years were studied. Motor activity was measured using two custom‐made silicone coated catheters, each with five solid‐state pressure transducers. No bowel preparation or sedation was used. The study period was 24 h. A total of 232 h of recording was obtained. Sixty‐three high amplitude propagated contractions were observed, median six per 24‐h period. Low‐amplitude colonic contractile activity showed regional and diurnal variations. Frequency of contraction was highest in the right colon [median 5.26 cpm (cycles per minute)], and transverse colon and splenic flexure (median 5.15 cpm). The interval between colonic motor complexes was shortest in the transverse colon and splenic flexure. This study introduces a new technique for the evaluation of colorectal motor activity. Subjects were studied in an ambulant setting in their own environment ensuring that this method of study is as physiological as possible. This study demonstrates that colonic motor activity has two main components: high amplitude propagated contractions and low amplitude colonic contractile activity.