bluuebunny/crossref_metadata_2025 huggingface dataset:
Mammalian heterotrimeric G‐protein‐like proteins in mycobacteria: implications for cell signalling and survival in eukaryotic host cells
Authors: Shankar Sandeep , Kapatral Vinayak , Chakrabarty A. M.
Published: November 2003

Mammalian heterotrimeric GTP‐binding proteins (G proteins) are involved in transmembrane signalling that couples a number of receptors to effectors mediating various physiological processes in mammalian cells. We demonstrate that bacterial proteins such as a Ras‐like protein from Pseudomonas aeruginosa or a 65 kDa protein from Mycobacterium smegmatis can form complexes with human or yeast nucleoside diphosphate kinase (Ndk) to modulate their nucleoside triphosphate synthesizing specificity to GTP or UTP. In addition, we demonstrate that bacteria such as M. smegmatis or Mycobacterium tuberculosis harbour proteins that cross react with antibodies against the α‐, β‐ or the γ‐subunits of heterotrimeric G proteins. Such antibodies also alter the GTP synthesizing ability of specific membrane fractions isolated from glycerol gradients of such cells, suggesting that a membrane‐associated Ndk–G‐protein homologue complex is responsible for part of GTP synthesis in these bacteria. Indeed, purified Ndk from human erythrocytes and M. tuberculosis showed extensive complex formation with the purified mammalian α and β G‐protein subunits and allowed specific GTP synthesis, suggesting that such complexes may participate in transmembrane signalling in the eukaryotic host. We have purified the α‐, β‐ and γ‐subunit homologues from M. tuberculosis and we present their internal amino acid sequences as well as their putative homologies with mammalian subunits and the localization of their genes on the M. tuberculosis genome. Using oligonucleotide probes from the conserved regions of the α‐ and γ‐subunit of M. tuberculosis G‐protein homologue, we demonstrate hybridization of these probes with the genomic digest of M. tuberculosis H37Rv but not with that of M. smegmatis, suggesting that M. smegmatis might lack the genes present in M. tuberculosis H37Rv. Interestingly, the avirulent strain H37Ra showed weak hybridization with these two probes, suggesting that these genes might have been deleted in the avirulent strain or are present in limited copy numbers as opposed to those in the virulent strain H37Rv.
Secondary structures and starvation‐induced frameshifting
Authors: Atkinson Jenny , Dodge Matthew , Gallant Jonathan
Published: November 2003

We have examined the effect of a downstream secondary structure (the stem–loop sequence found downstream on the MMTV gag‐pro frameshift site) on frameshifting at a bacterial shifty site (U UUC AUA) that responds strongly to a isoleucine‐tRNA limitation. Our findings are as follows: (i) the downstream stem–loop has little effect on frameshifting in growing, unstarved cells; (ii) the stem–loop increases the frameshifting effect of isoleucine‐tRNA limitation about fourfold, and this synergism is maximal with a distance of 5–9 nucleotides between the ‘hungry’ AUA codon and the stem–loop; and (iii) a stem–loop of different sequence at the same position has the same effect.
In vivo cleavage of Escherichia coli BIME‐2 repeats by DNA gyrase: genetic characterization of the target and identification of the cut site
Authors: Espéli Olivier , Boccard Frédéric
Published: November 2003

The Escherichia coli chromosome contains about 300 bacterial interspersed mosaic elements (BIMEs). These elements, located at the 3′ end of genes, are composed of three types of alternating repetitive extragenic palindromes (REPs). Based on the type of REP they contain and on their ability to interact with the integration host factor (IHF), BIMEs are subdivided into two families: BIME‐1 elements contain an IHF binding site flanked by converging Y and Z1 REPs, whereas BIME‐2 elements contain a variable number of alternating Y and Z2 REPs without an IHF site. Although some BIMEs have been implicated in the protection of mRNA against 3′ exonucleolytic degradation, the main role of elements belonging to both families remains to be elucidated. In this paper, we used oxolinic acid, a drug that reveals potential sites of DNA gyrase action, to demonstrate that DNA gyrase interacts in vivo with BIME‐2 elements. The frequency of cleavage varied from one element to another, and the cleavage pattern observed in elements containing several REPs indicated that DNA gyrase cut DNA every two REPs. A single cleavage site has been identified in the Y REP in six out of seven instances, and the nucleotide sequence of a 44 bp fragment containing the scission point displayed conserved residues at six positions. The lack of one of the conserved residues accounted for the absence of cleavage in most of the Z2 REPs. Our results also showed that cleaved REPs were always associated with another REP, suggesting that a pair of diverging REPs constitutes the target of DNA gyrase. DNA gyrase cleavage at repetitive BIME‐2 elements may have consequences for DNA topology and genomic rearrangements.
Molecular mechanisms of Campylobacter fetus surface layer protein expression
Authors: Dworkin Joel , Blaser Martin J.
Published: November 2003

Cells of the Gram‐negative bacteria Campylobacter fetus are covered by monomolecular arrays of surface layer proteins (SLPs) critical for both persistence in their natural hosts and for virulence. For C. fetus cells, expression of SLPs essentially eliminates C3b binding and their antigenic variation thwarts host immunological defences. Each cell possesses multiple partially homologous and highly conserved SLP gene cassettes, tightly clustered in the genome, that encode SLPs of 97–149 kDa. These attach non‐covalently via a conserved N‐terminus to the cell wall lipopolysaccharide. Recent studies indicate that C. fetus reassorts a single promoter, controlling SLP expression, and one, or more, complete open reading frame strictly by DNA inversion, and that rearrangement is independent of the distance between sites of inversion. In contrast to previously reported programmed DNA inversion systems, inversion in C. fetus is recA‐ dependent. These rearrangements permit variation in protein expression from the family of SLP genes and suggest an expanding paradigm of programmed DNA rearrangements among microorganisms.
DNA sequestration and transcription in the oriC region of Escherichia coli
Authors: Bogan Joseph A. , Helmstetter Charles E.
Published: November 2003

In Escherichia coli, gidA and dnaA transcription are shut off transiently after initiation of chromosome replication, while mioC transcription is shut off before initiation. The possible involvements of seqA and dam in these transcriptional periodicities were evaluated by examining transcription of the genes in seqA and dam mutants of E. coli PC2 dnaC2(ts) aligned for initiation of chromosome replication. In both seqA− and dam− cells, gidA and dnaA continued to be transcribed after initiation, whereas the inhibition of mioC transcription before initiation was unaltered. After initiation, transcripts from mioC that traversed oriC reappeared more slowly in seqA+dam+ cells than in seqA− or dam− cells, but before the release of oriC from sequestration. Apparently, initiation of transcription at a promoter can be completely prevented by sequestration, but established transcription forks can traverse sequestered DNA. These findings, plus analyses of transcript levels in steady‐state cultures, support the idea that initiation capacity in seqA mutants is elevated because of the combined influences of increased durations of expression of both gidA and dnaA during the division cycle and defective sequestration at oriC. Accordingly, a proposal for the sequence of events during the interinitiation interval in E. coli is presented based on the evident coupling of transcription to replication.
The exoenzyme S regulon of Pseudomonas aeruginosa
Authors: Frank Dara W.
Published: November 2003

Pseudomonas aeruginosa can cause severe life‐threatening infections in which the bacterium disseminates rapidly from epithelial colonization sites to the bloodstream. In experimental models, the ability of P. aeruginosa to disseminate is linked to epithelial injury, in vitro cytotoxicity and expression of the exoenzyme S regulon. Using the expression of ExoS as a model, a series of genes that are important for regulation, secretion and, perhaps, intoxication of eukaryotic cells have been identified. Proteins encoded by the exoenzyme S regulon and the Yersinia Yop virulon show a high level of amino acid homology, suggesting that P. aeruginosa may use a contact‐mediated translocation mechanism to transfer anti‐host factors directly into eukaryotic cells. Potential anti‐host factors that may disrupt eukaryotic signal transduction through ADP‐ribosylation include ExoS and ExoT. Expression of ExoU, another candidate anti‐host factor, has been correlated with acute cytotoxicity and lung epithelial injury. Members of the exoenzyme S regulon represent only a portion of the virulence factor arsenal possessed by P. aeruginosa. It will be important to understand how the exoenzyme S regulon contributes to pathogenesis and whether these factors could serve as potential therapeutic targets.
YscM1 and YscM2, two Yersinia enterocolitica proteins causing downregulation of yop transcription
Authors: Stainier Isabelle , Iriarte Maite , Cornelis Guy R.
Published: November 2003

Synthesis of the Yop proteins by yersiniae is downregulated when secretion is prevented by closure or destruction of the contact (type III) secretion channel. In Yersinia pseudotuberculosis, a mutation in the lcrQ gene, encoding a secreted protein, reduces this feedback inhibition mechanism. Surprisingly, mutation in the yscM gene, the lcrQ homologue in Y. enterocolitica, does not lead to the same deregulated phenotype. In this paper, we addressed the question of this discrepancy. We found a new gene on the Y. enterocolitica pYV plasmid that encodes a protein with 57% identity to YscM (now called YscM1). Overexpression of this gene, called yscM2, like overexpression of lcrQ and yscM1, blocked Yop secretion. A double yscM1, yscM2 mutant had the same phenotype as that of the lcrQ mutant. The discrepancy can thus be explained by the existence of two functionally equivalent copies of yscM in Y. enterocolitica. Overexpression of yscM1 drastically reduced the expression of a yopH‐cat reporter gene when tested in a pYV+ background. However, no effect could be observed in the absence of a pYV plasmid, indicating that YscM1 does not act directly as a transcriptional repressor or as an anti‐VirF factor. We have also ruled out that YscM acts by obstructing the secretion channel.
The Rhodobacter capsulatus hupSLC promoter: identification of cis‐regulatory elements and of trans‐activating factors involved in H2 activation of hupSLC transcription
Authors: Toussaint Bertrand , De Sury d'Aspremont Régis , Delic‐Attree Ina , Berchet Véronique , Elsen Sylvie , Colbeau Annette , Dischert Wanda , Lazzaroni Yannick , Vignais Paulette M.
Published: November 2003

The [NiFe]hydrogenase of the photosynthetic bacterium Rhodobacter capsulatus is encoded by the structural hupSLC operon, the expression of which is induced by H2. H2 activation was no longer observable in chromosomal hupR mutants, an indication that HupR is implicated directly in the activation by H2 of hupS gene expression. The transcriptional start site of the hupS promoter, determined by primer extension mapping, was located 55 nucleotides upstream from the translational start codon of the hupS gene. Regulatory sequences were identified by serial 5′ deletions of the 300 bp hupS promoter‐regulatory region (phupS) and phupS–lacZ translational fusions. Cis‐regulatory sequences capable of interacting with two transcription factors, IHF and HupR, a response regulator of the NtrC subfamily, were studied by electrophoretic mobility shift assays (EMSAs). The R. capsulatus IHF and HupR proteins were overexpressed in Escherichia coli and purified by affinity chromatography. IHF binds to a site, 5′‐TCACACACCATTG, centred at −87 nt from the transcription start site. The HupR protein binds to one site within the −162 to −152 nt region, which contains the palindromic sequence 5′‐TTG‐R5‐CAA. By the use of 5′ deletions and site‐directed mutagenesis of the −162/−152 region, this palindrome was shown to be required for in vivo hupS transcriptional activation by H2.
Mechanism of bacteriophage SfII‐mediated serotype conversion in Shigella flexneri
Authors: Mavris Maria , Manning Paul A. , Morona Renato
Published: November 2003

We have isolated the lysogenic bacteriophage SfII, which mediates glucosylation of Shigella flexneri O‐antigen, resulting in expression of the type II antigen. SfII belongs to group A of the Bradley classification and has a genome size of 42.3 kb. DNA sequencing of a 4 kb BamHI subclone identified four open reading frames (ORFs), of which only two were found to be necessary for serotype conversion. These genes were named bgt, which encodes a putative bactoprenol glucosyl transferase, and gtrII, encoding the putative type II antigen determining glucosyl transferase. These genes are adjacent to the integrase gene (int ) and attachment site (attP ), which are highly homologous to those of Salmonella bacteriophage P22. Another ORF encoded a highly hydrophobic protein of 120 amino acids with homologues in Escherichia coli, Salmonella bacteriophage P22 and S. flexneri. Previous studies identified gtrX, the glucosyl transferase gene, of bacteriophage SfX, which also glucosylates the O‐antigen specifically. We determined that gtrX‐mediated expression of the group 7,8 antigen also requires bgt. This allowed us to identify gtrII as being the serotype antigen II determining glucosyl transferase. Southern hybridization and polymerase chain reaction (PCR) analyses indicated that bgt homologues exist in the genomes of all S. flexneri serotypes and in E. coli K‐12, whereas gtrII was only detected in strains of serotype 2. Transposon TnphoA‐derived chromosomal mutations of bgt and gtrII in S. flexneri serotype 2a were isolated and characterized. [35S]‐methionine labelling and the use of a T7 RNA polymerase expression system identified a protein of 34 kDa corresponding to Bgt. However, GtrII, which has a predicted molecular weight of 55 kDa, was not detected. We propose that the function of Bgt is to transfer the glucose residues from the UDP‐glucose onto bactoprenol and GtrII then transfers the glucose onto the O‐antigen repeat unit at the rhamnose III position. The chromosomal organization of these serotype‐converting genes, when compared with their homologues in E. coli K‐12 chromosome and the P22 bacteriophage genome, were very similar. This suggests that the regions encode similar functions in these organisms and have a similar evolutionary origin.
Identification of novel intergenic repetitive units in a mycobacterial two‐component system operon
Authors: Supply Philip , Magdalena Juana , Himpens Sabine , Locht Camille
Published: November 2003

Mycobacterial interspersed repetitive units (MIRUs), a novel class of repeated sequences, were identified within the intercistronic region of an operon coding for a mycobacterial two‐component system, named senX3‐regX3. Southern blot analysis and homology searches revealed the presence of several homologous sequences in intergenic regions dispersed throughout the genomes of Mycobacterium bovis BCG, Mycobacterium tuberculosis and Mycobacterium leprae. These could be grouped into three major families, containing elements of 77–101 bp, 46–53 bp and 58–101 bp. Based on the available mycobacterial sequences, the total number of MIRUs is estimated to be about 40–50 per genome. Similar to previously identified small repetitive sequences, the MIRUs of the two‐component operon are transcribed on a polycistronic mRNA. Unlike previously identified small repetitive sequences, however, MIRUs do not contain dyad symmetries, comprise small open reading frames (ORFs) whose extremities overlap those of the contiguous ORFs and are oriented in the same translational direction as those of the adjacent genes. Analyses of the sequences at the insertion sites suggest that MIRUs disseminate by transposition into DTGA sites involved in translational coupling in polycistronic operons.
rpoB sequence analysis as a novel basis for bacterial identification
Authors: Mollet Christophe , Drancourt Michel , Raoult Didier
Published: November 2003

Comparison of the sequences of conserved genes, most commonly those encoding 16S rRNA, is used for bacterial genotypic identification. Among some taxa, such as the Enterobacteriaceae, variation within this gene does not allow confident species identification. We investigated the usefulness of RNA polymerase beta‐subunit encoding gene (rpoB ) sequences as an alternative tool for universal bacterial genotypic identification. We generated a database of partial rpoB for 14 Enterobacteriaceae species and then assessed the intra‐ and interspecies divergence between the rpoB and the 16S rRNA genes by pairwise comparisons. We found that levels of divergence between the rpoB sequences of different strains were markedly higher than those between their 16S rRNA genes. This higher discriminatory power was further confirmed by assigning 20 blindly selected clinical isolates to the correct enteric species on the basis of rpoB sequence comparison. Comparison of rpoB sequences from Enterobacteriaceae was also used as the basis for their phylogenetic analysis and demonstrated the genus Klebsiella to be polyphyletic. The trees obtained with rpoB were more compatible with the currently accepted classification of Enterobacteriaceae than those obtained with 16S rRNA. These data indicate that rpoB is a powerful identification tool, which may be useful for universal bacterial identification.
Antagonistic effect of CRP and KdgR in the transcription control of the Erwinia chrysanthemi pectinolysis genes
Authors: Nasser William , Robert‐Baudouy Janine , Reverchon Sylvie
Published: November 2003

The main virulence factors of the phytopathogenic bacteria Erwinia chrysanthemi are pectinases that cleave pectin, a major constituent of the plant cell wall. The cyclic AMP receptor protein (CRP) was identified as the main activator of the pectinolysis genes. Gel shift and DNase I footprinting experiments showed that the purified E. chrysanthemi CRP protein binds specifically to the promoter regions of seven pectinolysis genes (pelB, pelC, pelD, pelE, ogl, kduI and kdgT ) whose expression is positively regulated in vivo by CRP. In contrast, no interaction was observed between CRP and the promoter–operator region of pelA, whose expression is negatively regulated in vivo by CRP. Primer extension experiments demonstrated that each of the pelB, pelC, pelE and kduI genes is expressed from a unique σ70 promoter, whereas ogl and kdgT possess three and two functional promoters respectively. The position of the CRP binding site relative to the transcription start site suggests that CRP acts as a primary activator at the pelB (via the CRP binding site 1), pelC, pelE, pelD, kdgT P1 and oglP2 promoters. In contrast, transcription at the kduI, oglP1 promoters seems to require another transcriptional activator in synergy with CRP. Investigation of the simultaneous binding of CRP and KdgR, the main repressor of pectinolysis genes, to the regulatory regions of pelB, pelC, pelD, pelE, ogl, kduI and kdgT genes showed that binding of KdgR is preferential and exclusive in the case of ogl and kdgT, whereas the binding of these two regulators is independent in the case of pelB, pelC, pelD, pelE and kduI. Taken together, our data suggest that the antagonistic effects of CRP and KdgR on the expression of the pectinolysis genes occur by different mechanisms, including direct competition between the two regulators or between the repressor and RNA polymerase for the occupation of a common DNA region on the target genes.
KapB is a lipoprotein required for KinB signal transduction and activation of the phosphorelay to sporulation in Bacillus subtilis
Authors: Dartois Véronique , Djavakhishvili Tsotne , Hoch James A.
Published: November 2003

KinB is one of the two major histidine kinases that provide phosphate input in the phosphorelay to produce Spo0A∼P, the key transcription factor controlling the initiation of sporulation. A search for insertion mutants affected in activation of KinB‐dependent sporulation led to the identification of the lgt locus encoding the lipoprotein glyceryltransferase required for the lipid modification of prolipoproteins before their cleavage and translocation across the cytoplasmic membrane. In parallel, a putative lipoprotein signal peptide cleavage site was detected in KapB, known to be strictly required for KinB‐mediated sporulation and located downstream of KinB in a single transcription unit. Using PhoA peptide fusions, we have shown that KapB signal‐peptide can direct active alkaline phosphatase to the outer surface of the cytoplasmic membrane in an LGT‐dependent manner, strongly suggesting that KapB is a lipoprotein tethered to the outer face of the cytoplasmic membrane via a lipid anchor. As KapB proved to be dispensable for expression of the kinBkapB operon, a chimeric kinase was built consisting of KinA sensor domain fused to KinB kinase domain (KinA′‐′B) to assess (i) the involvement of KapB in catalysis of the kinase reaction, and (ii) the ability of KinB to phosphorylate Spo0F in vitro. It was shown that KapB is dispensable for both in vivo and in vitro activation of the phosphorelay by the KinA′‐′B chimera and that KinA′‐′B phosphorylates Spo0F directly in vitro. Models for the role of KapB in regulating KinB activity are discussed.
Escherichia coli RNase III (rnc) autoregulation occurs independently of rnc gene translation
Authors: Matsunaga James , Simons Elizabeth L. , Simons Robert W.
Published: November 2003

Control of mRNA stability is an established means of regulating gene expression. However, the detailed mechanisms by which such control is achieved are only now emerging. In particular, there remains a question about the involvement of translation. Escherichia coli ribonuclease III (RNase III) negatively autoregulates expression of its own gene (rnc) approximately 10‐fold, by cleaving the untranslated leader and initiating approximately 10‐fold more rapid decay of the rnc mRNA, after which RNase III plays no further role. Here, we define the mechanism of this control further. Mutations that increase rnc gene translation abolish autoregulation by increasing the stability of the RNase III‐cleaved transcript RNA approximately 10‐fold, with no effect on the uncleaved species. Mutations that decrease translation destabilize the rnc mRNA in the presence or absence of RNase III. In so doing, they reveal a pathway of rnc transcript decay distinct from the RNase III‐dependent pathway. Stability of a ‘mini‐rnc’ transcript containing the rnc leader and only the first two codons of the rnc gene is unaffected by decreased translation, presumably because sequences required for this pathway were removed. Importantly, this mini‐rnc transcript is regulated normally by RNase III. Moreover, rnc transcripts synthesized in vitro do not decay in cell‐free extracts lacking ribosomes, unless they are first cleaved by RNase III. These two results show that RNase III cleavage can initiate rnc transcript decay independently of rnc gene translation, unambiguously establishing that control of mRNA decay need not involve changes in translation. How rnc gene translation is optimized for efficient autoregulation will also be discussed.
Characterization of SKM1, a Saccharomyces cerevisiae gene encoding a novel Ste20/PAK‐like protein kinase
Authors: Martín Humberto , Mendoza Alfonso , Rodríguez‐Pachón Jose M. , Molina María , Nombela César
Published: November 2003

Ste20/PAK serine/threonine protein kinases have been suggested as playing essential roles in cell signalling and morphogenesis as potential targets of Cdc42 and Rac GTPases. We have isolated and characterized the Saccharomyces cerevisiaeSKM1 gene, which codes for a novel member of this family of protein kinases. The amino acid sequence analysis of Skm1p revealed the presence of a PH domain and a putative p21‐binding domain near its amino terminus, suggesting its involvement in cellular signalling or cytoskeletal functions. However, deletion of SKM1 produced no detectable phenotype under standard laboratory conditions. Moreover, disruption of each of the two other S. cerevisiae Ste20/PAK‐like kinase‐encoding genes, STE20 and CLA4, in skm1 backgrounds, showed that Skm1p is not redundant with Ste20p or Cla4p. Interestingly, overexpression of SKM1 led to morphological alterations, indicating a possible role for this protein in morphogenetic control. Furthermore, overproduction of Skm1p lacking its N‐terminus caused growth arrest. This effect was also seen when similarly truncated versions of Ste20p or Cla4p were overexpressed. We further observed that overproduction of this C‐terminal fragment of Skm1p complements the mating defect of a ste20 mutant strain. These results suggest that the N‐terminal domains of S. cerevisiae Ste20/PAK‐like protein kinases share a negative regulatory function and play a role in substrate specificity.
Pkn9, a Ser/Thr protein kinase involved in the development of Myxococcus xanthus
Authors: Hanlon William A. , Inouye Masayori , Inouye Sumiko
Published: November 2003

The Myxococcus xanthus gene, pkn9, encodes a protein that contains significant homology with eukaryotic Ser/Thr protein kinases. The pkn9 gene was singled out of a previously identified family of kinase genes by amplification techniques that displayed differences in kinase gene expression during selected periods of the M. xanthus life cycle. Pkn9 was constitutively expressed during vegetative growth and upregulated during the aggregation stage of early development. It consists of 589 amino acids, and its N‐terminal 394 residues show 38% identity with both Pkn1 and Pkn2 of M. xanthus. This region also shows 29, 25 and 29% identity with myosin light‐chain kinase, protein kinase C, and cAMP‐dependent protein kinase, respectively. A 22‐residue hydrophobic transmembrane domain separates the kinase domain from the 173‐residue C‐terminal domain that resides on the outside of the inner membrane. The C‐terminal domain contains two sets of tandem repeats of 13 and 10 residues which have no known function. When expressed in Escherichia coli under the T7 promoter, Pkn9 was found to be phosphorylated on serine and threonine residues. Disruption of the pkn9 kinase catalytic subdomains I–III by the insertion of a kanamycin‐resistance gene resulted in slightly delayed, smaller and more‐crowded fruiting bodies, while spore formation was normal. Total deletion of the pkn9 gene caused severely reduced progression through development resulting in light loose mounds that become slightly more compact over time. Development progressed further at the centre than at the edge of the spot, and spore formation was significantly reduced. Two‐dimensional gel analysis revealed that both the disruption and the deletion of pkn9 prevented the expression of five membrane proteins (KREP9‐1‐4). These results suggest that the loss of Pkn9 kinase activity caused altered fruiting‐body formation, the absence of the KREP9 proteins in the membrane, and reduced spore production.
Generation of a membrane‐bound, oligomerized pre‐pore complex is necessary for pore formation by Clostridium septicum alpha toxin
Authors: Sellman Bret R. , Kagan Bruce L. , Tweten Rodney K.
Published: November 2003

Low‐temperature inhibition of the cytolytic activity of alpha toxin has facilitated the identification of an important step in the cytolytic mechanism of this toxin. When alpha toxin‐dependent haemolysis was measured on erythrocytes at various temperatures it was clear that at temperatures ≤15°C the haemolysis rate was significantly inhibited with little or no haemolysis occurring at 4°C. Alpha toxin appeared to bind to and oligomerize on erythrocyte membranes with similar kinetics at 4°C and 37°C. The slight differences in these two processes at 4°C and 37°C could not account for the loss of cytolytic activity at low temperature. At 4°C alpha toxin neither stimulated potassium release from erythrocytes nor formed pores in planar membranes. In contrast, at temperatures ≥25°C both processes proceeded rapidly. Pores that were opened in osmotically stabilized erythrocytes could not be closed by low temperature. Therefore, low temperature appeared to prevent the oligomerized complex from forming a pore in the membrane. These data support the hypothesis that alpha toxin oligomerizes into a membrane‐bound, pre‐pore complex prior to formation of a pore in a lipid bilayer.
Characterization of monoclonal antibodies recognizing three distinct, phosphorylated carbohydrate epitopes in the lipopolysaccharide of the deep rough mutant I‐69 Rd−/b+ of Haemophilus influenzae
Authors: Rozalski Antoni , Brade Lore , Kosma Paul , Moxon Richard , Kusumoto Shoichi , Brade Helmut
Published: November 2003

Monoclonal antibodies against the lipopolysaccharide (LPS) of the deep rough mutant I‐69 Rd−/b+ of Haemophilus influenzae were obtained after immunization of mice with sheep erythrocytes which had been coated with de‐O‐acylated LPS. Characterization of antibodies was performed by enzyme immuno assay (EIA) using LPS or neoglycoconjugates containing partial structures of LPS as solid‐phase antigens and by haemagglutination with sheep erythrocytes coated with de‐O‐acylated LPS. Binding data were confirmed by EIA inhibition experiments using deacylated LPS or synthetic partial structures thereof. Three antibodies were specific for 3‐deoxy‐d‐manno‐octulopyranosonic acid‐ (Kdo) 5‐phosphate, one for Kdo‐4‐phosphate, and one required, in addition to a Kdo‐phosphate, parts of the phosphorylated glucosamine backbone of lipid A. All antibodies also bound in (i) Western blots to bacterial whole‐cell lysates or isolated LPS separated by SDS–PAGE, (ii) bacterial colony blots, and (iii) immunofluorescence with live bacteria. The latter result indicated that Kdo‐4‐ and Kdo‐5‐phosphate are synthesized by the bacteria and are not the result of phosphate migration.
Structure of TolC, the outer membrane component of the bacterial type I efflux system, derived from two‐dimensional crystals
Authors: Koronakis Vassilis , Li Jade , Koronakis Eva , Stauffer Kathrin
Published: November 2003

TolC is an outer membrane protein required for the export of virulence proteins and toxic compounds without a periplasmic intermediate. We show that TolC is an integral part of the translocator, interacting with inner membrane components, by demonstrating a need for TolC in protein export not only from intact cells but also from sphaeroplasts. To establish the structure of TolC, and thus gain information on how this might be achieved, the protein was purified from the Escherichia coli outer membrane, as a trimer, and crystallized in two‐dimensional lattices by reconstitution in phospholipid bilayers. The projection structure at 12 Å resolution showed a threefold symmetric molecule of 58 Å outer diameter, and a single pool of stain filling its centre. Side views parallel to the membrane plane revealed an additional domain outside the membrane. Eighteen membrane‐spanning β‐strands were predicted for the 51.5 kDa monomer, excluding a 7 kDa C‐terminal segment, and this segment was shown to contain a proteinase K‐sensitive site that was exposed in reconstituted membranes and sphaeroplasts, but which was protected in intact cells. The combined data suggest that TolC is a trimeric outer membrane protein with each monomer comprising a membrane domain, predicted to be β‐barrel, and a C‐terminal periplasmic domain. The latter could form part of the bridge to the energized inner membrane component of the translocation complex.
Gastric emptying patterns of a liquid meal in newborn infants measured by epigastric impedance
Authors: Lange A. , Funch‐Jensen P. , Thommesen P. , Schiøtz P. O.
Published: November 2003

Epigastric impedance was used to measure the gastric emptying patterns of a liquid non‐caloric meal (5 mL water kg−1) in 30 healthy newborn infants. Twenty‐six mature infants were examined in the first eight days of life, and four preterm infants were examined within 6 weeks after birth. The recordings consisted of two components: the emptying signal (the DC component), and a phasic 3 cycles per minutes (CPM) signal (the AC component). In some of the infants the phasic 3 CPM signal was also seen during the fasting state.

For mature infants the median half emptying time (T50) was 6.9 min. For a second meal given within one hour after the first meal the half emptying time was 5.5 min (P < 0.01). In preterm infants the emptying times were not significantly different from mature infants.

Day‐to‐day variation was low with a coefficient of variation of 17% in nine infants.

A periodic change of the impedance signal, the phasic 3 CPM signal, was observed after a meal in 24 of the infants. The median frequency was 3.0 CPM in 20 mature and 2.9 CPM in four preterm infants. In nine infants a phasic 3 CPM signal was also observed during the fasting state, with a median frequency of 2.9 CPM.

Measurement of gastric emptying pattern with epigastric impedance is a simple investigation for the evaluation of gastric emptying time and phasic activity in mature and preterm infants. However, the method is sensitive to spontaneous movements of the children, resulting in non‐valid measurements in around one fourth of the infants.
Responses of the rat lower oesophageal sphincter (LOS) to vagal efferent activation
Authors: Kawahara H. , Blackshaw L. A. , Lehmann A. , Dent J.
Published: November 2003

Nitric oxide (NO) is a major candidate in vagal‐induced LOS relaxation. Vagal adrenergic fibres also innervate the gastrointestinal tract including the LOS. This study investigates the role of these two and other mechanisms in LOS responses to vagal activation in the rat, and provides functional and anatomical evidence for a smooth muscle LOS in this species. LOS, gastric and oesophageal pressures were measured in urethane anaesthetized rats during vagal stimulation. The LOS pressure (LOSP) response to vagal stimulation (5 mA, 10 Hz, 0.5 msec pulses, 5 sec) comprised three consecutive stages: (1) brief reduction of LOSP, (2) transient increase of LOSP and (3) prolonged reduction of LOSP. The influences of additive treatment with several antagonist drugs on the LOS response to vagal stimulation were investigated. L‐NAME (100 mg kg−1) reduced stage 1 and increased stage 2. Subsequent treatment with either phentolamine (1 mg kg−1) or prazosin (200 μg kg−1) abolished stage 1. After phentolamine, atropine treatment (400 μg kg−1) abolished stage 2. Stage 3 was evident throughout experiments. In five additional studies, treatment with hexamethonium (30 mg kg−1) abolished stages 2 and 3 leaving stage 1, which was later abolished by phentolamine or atropine. In the LOS response to vagal stimulation, the following major mechanisms are therefore evident: nicotinic transmission in both excitation and inhibition, alpha‐adrenergic and NO‐mediated inhibition, muscarinic excitation, and non‐adrenergic, non‐NO inhibition (not characterized further). Characteristics of these different neurotransmitter influences may be important in LOS relaxation associated with swallowing and gastro‐oesophageal reflux.
PYY and NPY: control of gastric motility via action on Y1 and Y2 receptors in the DVC
Authors: Chen C.‐H. , Stephens Jr R. L. , Rogers R. C.
Published: November 2003

The pancreatic polypeptide family of hormones and neurotransmitters including pancreatic polypeptide (PP), peptide YY (PYY) and neuropeptide Y (NPY) are emerging as potent central regulators of gastric function. There is, however, considerable debate concerning the mechanisms and even the direction of effects mediated by these peptides. Good evidence exists showing that PYY is the ‘enterogastrone’ released by the ileum after feeding which acts on vagal reflex control circuits to reduce gastric motility (i.e. the ‘ileal brake’). However, equally convincing evidence is available to suggest that PYY and its close structural relative NPY may act in the same region of the brain to increase gastric motility through vagal mechanisms. We hypothesize that the confounding observations are due to agonist effects on two different receptor types – Y1 and Y2, which are both present in the dorsal vagal complex (DVC) but may be accessed differentially by peripheral humoral (PYY) vs central (NPY) pathways. In our initial approach to this problem, we studied the effects of NPY, PYY, Y1 and Y2 agonists microinjected into the DVC on gastric motility in the stimulated (by central thyrotropin‐releasing hormone (TRH)) and basal conditions. Our results show that Y2 agonist applied to the DVC during conditions of TRH‐stimulated gastric motility mimicked the suppressive effects of PYY applied under the same conditions. Under basal conditions, Y2 agonist has no effect on motility. The DVC effect of the Y1 agonist is the opposite; Y1 agonist has no further effect to stimulate gastric motility in the TRH stimulated condition while Y1 agonist strongly stimulates motility from the basal condition. The effects of NPY depend upon the condition of study. Under TRH stimulation (maximal motility), NPY in the DVC reduces (but does not completely suppress) gastric motility while in the basal state, NPY is a strong activator of motility. These results are discussed in terms of the possible differential localization of Y1 vs Y2 receptors within the DVC and in terms of recent findings suggesting that PYY is rapidly converted to a Y2 agonist by a ubiquitous dipeptidyl aminopeptidase (DAP‐IV).
Central regulation of gastric emptying of solid nutrient meals by corticotropin releasing factor
Authors: Lee C. , Sarna S. K.
Published: November 2003

Central regulation of gastric emptying of a solid nutrient meal and spatial and temporal parameters of gastro‐pyloro‐duodenal contractions by corticotropin‐releasing factor (CRF) were investigated in conscious dogs. Intracerebroventricular (ICV) infusion of CRF at 0.033 nmol kg−1 min−1 for 15 min in a volume of 0.2 mL significantly delayed the total gastric emptying time of the meal. ICV infusion of CRF also increased the mean frequency of proximal duodenal contractions and decreased the percentage of distally propagating contractions in the whole duodenum. The remaining parameters of pyloric and duodenal contractions were not affected. A prior ICV infusion of I‐helical CRF9–41 (0.166 nmol kg−1 min−1 for 15 min in a volume of 0.2 mL) blocked the central effects of CRF on gastric emptying time and the duodenal contractions. Central infusion of CRF had no significant effect on the lag phase of gastric emptying. Bilateral truncal vagotomy significantly delayed the gastric emptying time of the solid nutrient meal. However, after vagotomy, ICV infusion of CRF had no effect on gastric emptying time or the spatial and temporal parameters of gastro‐pyloro‐duodenal contractions. In conclusion, CRF, the mediator of stress response, delays the total gastric emptying time of solid nutrient meals. The delay in gastric emptying may not be due to a change in the spatial and temporal parameters of gastric or pyloric contractions, but mainly due to changes in the parameters of duodenal contractions. The central effects of CRF on gastric emptying and duodenal contractions may be mediated by the vagus nerves.
Use of echo planar imaging to demonstrate the effect of posture on the intragastric distribution and emptying of an oil/water meal
Authors: Boulby P. , Gowland P. , Adams V. , Spiller R. C.
Published: November 2003

Intragastric distribution of solids and liquids is far from uniform but until recently technical limitations have prevented detailed study. Echo planar imaging (EPI), which can provide high‐quality images of intragastric contents, has been used in this study to assess the intragastric distribution of oil and water and gastric emptying in subjects lying on either their left or right side. Eight healthy volunteers underwent four gastric emptying studies after consuming either an aqueous meal (400 mL beef consomme soup + 100 mL water) or a fat/aqueous meal (400 mL soup + 100 mL olive oil) lying on either their left or right side.

Using a water suppressed imaging mode the lipid phase was clearly seen layering above the aqueous phase with little evidence of mixing. Gastric emptying of the aqueous meal (A) was not significantly different when lying on the left compared to the right side. However, gastric emptying of the aqueous phase of the fat/aqueous meal (B) was considerably slower when lying on the left compared to the right side, gastric volumes actually increasing over the 90 min of the study P ≤ 0.05. When lying on the right side the aqueous phase of meal B emptied significantly slower than meal A. Oil was clearly observed to layer above the water and fill the duodenal cap when subjects lay on their left and the fundus when they lay on their right side.

EPI clearly demonstrates the intragastric layering of oil which causes posture to strongly influence gastric emptying. EPI is a tool with great potential to describe the intragastric events following ingestion of complex multiphase meals.
Fictive oesophageal peristalsis evoked by activation of muscarinic acetylcholine receptors in rat nucleus tractus solitarii
Authors: Lu W. , Zhang M. , Neuman R. S. , Bieger D.
Published: November 2003

The aim of the present study was to determine if muscarinic acetylcholine receptor‐mediated peristaltic rhythmogenesis in the rat oesophagus is a central motor program that can be generated without peripheral sensory support. In anaesthetized male Sprague‐Dawley rats, pressure‐ejection of glutamate (10–20 pmol) and muscarine (5–10 pmol) in the subnucleus centralis of the nucleus tractus solitarii (NTSC) evoked monophasic pressure waves and rhythmic oesophageal peristalsis, respectively, but did not change mean arterial blood pressure or respiration. Application of muscarine (50–100 pmol) to the NTS extraventricular surface evoked rhythmic multi‐unit burst discharges in the compact formation of the nucleus ambiguus (AMBC) that led to oesophageal peristalsis in a phase‐locked manner. Evoked rhythmic AMBC activity persisted during neuromuscular blockade with curare, although the peak frequency of individual bursts was decreased. In a brainstem slice preparation, intracellular and whole cell patch recordings from AMBC neurones during focal stimulation of the NTSC region with muscarine revealed rhythmic depolarizing waves that showed a pattern similar to that of rhythmic oesophageal peristalsis. The present findings support the concept that medullary circuits comprising premotor neurones of the NTSC are intrinsically capable of generating rhythmic oesophagomotor output, but are subject to a powerful modulation by peripheral sensory feedback.
Stress‐induced visceral hypersensitivity to rectal distension in rats: role of CRF and mast cells
Authors: GUÉ M. , DEL RIO‐LACHEZE C. , EUTAMENE H. , THÉODOROU V. , FIORAMONTI J. , BUÉNO L.
Published: November 2003

Background: psychological factors have long been implicated in the aetiology of irritable bowel syndrome often associated with abdominal pain. This work was designed to study, in rats, the influence of partial restraint stress on the abdominal cramps induced by rectal distension and to determine the role of corticotropin releasing factor (CRF) and mast cells degranulation in this response. Methods: abdominal contractions were electromyographically recorded. Thirty minutes after stress or intracerebroventricular CRF, rectal distension was performed by inflation of a balloon (0.4–1.2 mL). α‐helical CRF9–41 or doxantrazole were administered centrally (15 min) and intraperitoneally (30 min), respectively, before stress. Histamine release and the number of mast cells were determined in colonic pieces from stressed and control rats. Results: stress and CRF enhanced the number of abdominal cramps evoked by rectal distension without affecting rectal compliance. α‐helical CRF9–41 and doxantrazole antagonized the stress and CRF‐induced enhancement of abdominal cramps. Stress increased the colonic histamine content whereas the number of colonic mast cells was unchanged. Conclusions: stress enhances abdominal contractions in response to rectal distension in rats via pathways involving central CRF and intestinal mast cells.
Mortality of olive ridley turtles Lepidochelys olivacea due to incidental capture in fishing nets along the Orissa coast, India
Authors: Pandav B. , Choudhury B. C. , Kar C. S.
Published: November 2003

The coastal state of Orissa, India, harbours three important mass-nesting beaches of the olive ridley sea turtle Lepidochelys olivacea. During a survey of sea turtle nesting beaches from December 1993 to May 1994, 5282 dead olive ridleys were counted along the 480 km coastline of Orissa. Almost all the deaths were due to incidental capture in offshore fishing nets. Increased fishing activities in the coastal waters near important sea-turtle nesting beaches pose a serious threat to the survival of this endangered sea turtle. It is therefore suggested that coastal waters adjacent to major sea-turtle nesting beaches be declared as closed areas for commercial fishing activities in order to ensure the sea turtle's long-term survival.
Challenges to nature conservation with community development in central African forests
Authors: Noss Andrew J.
Published: November 2003

Based on field research in the Central African Republic, this article discusses several social and economic challenges to conservation programmes that include community development components. These interrelated challenges include immigration as people elsewhere are attracted to economic opportunities, the lack of tenure of land and natural resources, diversification of economic and subsistence strategies, ethnic diversity and the lack of a conservation ethic. Addressing these challenges requires fundamental socio-economic change.
A new park in the Bolivian Gran Chaco – an advance in tropical dry forest conservation and community-based management
Authors: Taber Andrew , Navarro Gonzalo , Arribas Miguel Angel
Published: November 2003

The Kaa-Iya del Gran Chaco National Park and Integrated Management Area was established in September 1995. At 3.44 million hectares it is one of South America's largest protected areas. The tropical dry forest of the Chaco, which this reserve protects, is Bolivia's most threatened major lowland habitat type. With the creation of this reserve the protected-area coverage of the Gran Chaco increased to 4.7 per cent. With at least 69 species of mammals (the Chiroptera have not yet been surveyed), it is one of the richest Neotropical sites for this taxonomic group. The Kaa-Iya park is being administered by the Izoceño-Guaraní Indian organization, the Capitanía del Alto y Bajo Izozog, and puts community-based conservation into practice. Threats to the park include encroachment by colonists, ranchers and farmers; the Bolivia-Brazil gas pipeline; and hunting.
The progress and problems of the ‘Endemic Section’ of St Helena Island
Authors: Smith Doug
Published: November 2003

St Helena is a small Atlantic island with an unusual and remarkable flora and fauna but with a legacy of ecological destruction typical of oceanic islands. The efforts of the island's governmental Endemic Section in the conservation of the flora and fauna is examined alongside the objectives of the section as listed in its current business plan. The worthy level of progress that is evident (including the rediscovery of species, habitat restoration and a high level of environmental awareness) is attributed to local ownership, an emphasis on education, and pragmatism and flexibility. The problems that limit the section's long-term effectiveness are funding, limited baseline ecological information and limited technical understanding.