bluuebunny/crossref_metadata_2025 huggingface dataset:
uvrD mutations enhance tandem repeat deletion in the Escherichia coli chromosome via SOS induction of the RecF recombination pathway
Authors: Bierne Hélène , Seigneur Marie , Ehrlich S. Dusko , Michel Bénédicte
Published: November 2003

It has previously been shown that recombination between tandem repeats is not significantly affected by a recA mutation in Escherichia coli. Here, we describe the activation of a RecA‐dependent recombination pathway in a hyper‐recombination mutant. In order to analyse how tandem repeat deletion may proceed, we searched for mutants that affect this process. Three hyper‐recombination clones were characterized and shown to be mutated in the uvrD gene. Two of the mutations were identified as opal mutations at codons 130 and 438. A uvrD ::Tn5 mutation was used to investigate the mechanism of deletion formation in these mutants. The uvrD‐mediated stimulation of deletion was abolished by a lexAind3 mutation or by inactivation of either the recA, recF, recQ or ruvA genes. We conclude that (i) this stimulation requires SOS induction and (ii) tandem repeat recombination in uvrD mutants occurs via the RecF pathway. In uvrD+ cells, constitutive expression of SOS genes is not sufficient to stimulate deletion formation. This suggests that the RecF recombination pathway activated by SOS induction is antagonized by the UvrD protein. Paradoxically, we observed that the overproduction of UvrD from a plasmid also stimulates tandem repeat deletion. However, this stimulation is RecA independent, as is deletion in a wild‐type strain. We propose that the presence of an excess of the UvrD helicase favours replication slippage. This work suggests that the UvrD helicase controls a balance between different routes of tandem repeat deletion.
Blocking rolling circle replication with a UV lesion creates a deletion hotspot
Authors: Seigneur Marie , Ehrlich S. Dusko , Michel Bénédicte
Published: November 2003

UV light irradiation increases genetic instability by causing mutations and deletions. The mechanism of UV‐induced rearrangements was investigated making use of deletion‐prone plasmids. Chimeric plasmids carrying pBR322 and M13 replication origins undergo deletions that join the M13 replication origin to a random nucleotide. A restriction fragment was UV irradiated, introduced into such a hybrid plasmid and deletions formed at the M13 origin were analysed. In most of the deletant molecules, the M13 replication nick site was linked to a nucleotide in the irradiated fragment, showing that UV lesions are deletion hotspots. These deletions were independent of the UvrABC excision repair proteins, suggesting that the deletogenic structure is the lesion itself and not a repair intermediate. They were not found in the absence of M13 replication, indicating that they result from the encounter of the M13 replication fork with the UV lesion. Furthermore, UV‐induced deletions occurred independently of pBR322 replication. We conclude that, in contrast to pBR322 replication forks, M13 replication forks blocked by UV lesions are deletion prone. We propose that the deletion‐prone properties of a UV‐arrested polymerase depend on the associated helicase.
Two distinct protein–protein interactions between the NIT2 and NMR regulatory proteins are required to establish nitrogen metabolite repression in Neurospora crassa
Authors: Pan Hongoo , Feng Bo , Marzluf George A.
Published: November 2003

Nitrogen metabolism is a highly regulated process in Neurospora crassa. The structural genes that encode nitrogen catabolic enzymes are subject to nitrogen metabolite repression, mediated by the positive‐acting NIT2 protein and by the negative‐acting NMR protein. NIT2, a globally acting factor, is a member of the GATA family of regulatory proteins and has a single Cys2/Cys2 zinc finger DNA‐binding domain. The negative‐acting NMR protein interacts via specific protein–protein binding with two distinct regions of the NIT2 protein, a short alpha‐helical motif within the NIT2 DNA‐binding domain and a second motif at its carboxy terminus. Deletions of segments of NIT2 throughout most of its length result in truncated proteins, which are still functional for activating gene expression; most of these mutant NIT2 proteins still allow proper nitrogen repression of nitrate reductase synthesis. In contrast, deletions or certain amino acid substitutions within the zinc finger and the carboxy‐terminal tail result in a loss of nitrogen metabolite repression. Those mutated forms of NIT2 that are insensitive to nitrogen repression have also lost one of the NIT2–NMR protein–protein interactions. These results provide compelling evidence that the specific NIT2–NMR interactions have a regulatory function and play a central role in establishing nitrogen metabolite repression.
Repair by recombination of DNA containing a palindromic sequence
Authors: Leach David R. F. , Okely Ewa A. , Pinder David J.
Published: November 2003

We report here that homologous recombination functions are required for the viability of Escherichia coli cells maintaining a 240 bp chromosomal inverted repeat (palindromic) sequence. Wild‐type cells can successfully replicate this palindrome but recA, recB or recC mutants carrying the palindrome are unviable. The dependence on homologous recombination for cell viability is overcome in sbcC mutants. Directly repeated copies of the DNA containing the palindrome are rapidly resolved to single copies in wild‐type cells but not in sbcC mutants. Our results suggest that double‐strand breaks introduced at the palindromic DNA sequence by the SbcCD nuclease are repaired by homologous recombination. The repair is conservative and the palindrome is retained in the repaired chromosome. We conclude that SbcCD can attack secondary structures but that repair conserves the DNA sequence with the potential to fold.
Structure and function of 59‐base element recombination sites associated with mobile gene cassettes
Authors: Stokes H. W. , O'Gorman D. B. , Recchia Gavin D. , Parsekhian Maryam , Hall Ruth M.
Published: November 2003

The integration of gene cassettes into integrons is effected by site‐specific recombination catalysed by an integrase, IntI, encoded by the integron. The cassette‐associated recombination sites, 59‐base elements, are not highly conserved and vary in length from 57 to 141 bp. They can be identified by their location and the relationship of over 20 bp at their outer ends to consensus sequences that are imperfect inverted repeats of one another. The recombination cross‐over occurs close to one end of the 59‐base element, within a conserved core site with the consensus sequence GTTAGGC or GTTRRRY. By introducing single‐base changes at each of these positions in the aadB 59‐base element, bases that are critical for site activity were identified. The recombination cross‐over was also localized to a unique position between the adjacent G and T residues. Changes introduced in the conserved AAC of the inverse core site (GCCTAAC or RYYYAAC) located at the opposite end of the 59‐base element also reduced site activity but to a lesser extent. Sequences of rare recombinants revealed an alternative position for strand exchange and led to the conclusion that 59‐base elements comprise two simple sites, analogous to those recognized by other integrases, with each simple site made up of a pair of inversely oriented IntI binding domains separated by a spacer of 7 or 8 bp. Re‐examination of the sequences of all known 59‐base elements revealed that this simple site configuration was present at both the left and right ends in all 59‐base elements. The identity of bases in the spacer is not required for efficient recombination and the cross‐over is located at one end of the spacer, suggesting that during IntI1‐mediated recombination only one strand exchange occurs.
The Cryptococcus neoformans STE12α gene: a putative Saccharomyces cerevisiae STE12 homologue that is mating type specific
Authors: Wickes Brian L. , Edman Ursula , Edman Jeffrey C.
Published: November 2003

Cryptococcus neoformans possesses two mating types, MATα and MATa. α‐Cells are more virulent than a‐cells and are also, unlike a‐cells, capable of producing extensive hyphae in the haploid phase. The molecular analysis of hyphae production in C. neoformans has resulted in the identification of a gene which displays substantial similarity to other fungal STE12 genes, including the presence of a highly conserved homeodomain. Overexpression of the C. neoformans gene resulted in poor growth, altered morphology and the presence of hyphal projections, phenotypes reported in similar studies of the Saccharomyces cerevisiae STE12 gene. Overexpression was also found to induce MFα, a pheromone, and CNLAC1, a confirmed C. neoformans virulence gene. The C. neoformans STE12α gene, however, has one striking difference from other fungal STE12 genes; it is found only in α‐cells. The existence of STE12α in C. neoformans suggests that this fungus has elements of a conserved MAP kinase cascade, which may be organized in a novel manner.
Differential Opa specificities for CD66 receptors influence tissue interactions and cellular response to Neisseria gonorrhoeae
Authors: Gray‐Owen Scott D. , Lorenzen Dirk R. , Haude Anja , Meyer Thomas F. , Dehio Christoph
Published: November 2003

The ability of all 11 variable opacity (Opa) proteins encoded by Neisseria gonorrhoeae MS11 to interact directly with the five CD66 antigens was determined. Transfected HeLa cell lines expressing individual CD66 antigens were infected with recombinant N. gonorrhoeae and Escherichia coli strains expressing defined Opas. Based upon the ability of these bacteria to bind and invade and to isolate specifically CD66 antigens from detergent‐soluble extracts of the corresponding cell lines, distinct specificity groups of Opa interaction with CD66 were seen. Defining these specificity groups allowed us to assign a specific function for CD66a in the Opa‐mediated interaction of gonococci with two different target cell types, which are both known to co‐express multiple CD66 antigens. The competence of individual Opas to interact with CD66a was strictly correlated with their ability to induce an oxidative response by polymorphonuclear neutrophils. The same Opa specificity was observed for the level of gonococcal binding to primary endothelial cells after stimulation with TNFα, which was shown to increase the expression of CD66a rather than CD66e. As CD66e alone is expressed on other target tissues of gonococcal pathogenicity, Opa variation probably contributes to the cell tropism displayed by gonococci.
RfaH and the ops element, components of a novel system controlling bacterial transcription elongation
Authors: Bailey Marc J. A. , Hughes Colin , Koronakis Vassilis
Published: November 2003

The RfaH protein controls the transcription of a specialized group of Escherichia coli and Salmonella operons that direct the synthesis, assembly and export of the lipopolysaccharide core, exopolysaccharide, F conjugation pilus and haemolysin toxin. RfaH is a specific regulator of transcript elongation; its loss increases transcription polarity in these operons without affecting initiation from the operon promoters. The operons of the RfaH‐dependent regulon contain a short conserved 5′ sequence, the ops element, deletion of which increases operon polarity to an extent similar to that caused by loss of RfaH. The ops element is also present upstream of polysaccharide gene clusters of Shigella flexneri, Yersinia enterocolitica, Vibrio cholerae and Klebsiella pneumoniae and the RP4 fertility operon of Pseudomonas aeruginosa, suggesting that this is a widely spread control system. The mechanistic coupling of RfaH and the ops element has been demonstrated in vitro and in vivo, and we suggest that the ops element recruits RfaH and potentially other factors to the RNA polymerase complex, modifying the complex to increase its processivity and allowing transcription to proceed over long distances.
DspA, an essential pathogenicity factor of Erwinia amylovora showing homology with AvrE of Pseudomonas syringae, is secreted via the Hrp secretion pathway in a DspB‐dependent way
Authors: Gaudriault S. , Malandrin L. , Paulin J.‐P. , Barny M.‐A.
Published: November 2003

In Erwinia amylovora, the dsp region, required for pathogenicity on the host plant but not for hypersensitive elicitation on tobacco, is separated from the hrp region by 4 kb. The genetic analysis reported in this paper showed that this 4 kb region is not required for pathogenicity on pear seedlings. The environmental conditions allowing expression of a dsp::lacZ fusion were examined: expression was barely detected in rich medium at 30°C, and the highest expression was observed in M9 galactose minimal medium at 25°C. A dsp::uidA fusion appeared to be expressed only in a HrpL‐proficient strain, indicating that the dsp region, like the hrp region, is positively controlled via the alternative σ factor HrpL. Sequence analysis revealed that the dsp cluster encodes two genes, dspA (5517 bp) and dspB (420 bp), and that the insertions leading to the dsp::lacZ and the dsp::uidA fusions were within dspA. A HrpL‐dependent promoter sequence (GGAACC‐ N15‐CAACA) was identified upstream of dspA, and primer extension analysis detected four transcriptional starts 7, 8, 9 and 10 bp downstream of this sequence. A σ70 promoter sequence (TTGCCC‐N16‐GATAAT) was observed upstream of dspB. The functionality of this second promoter was confirmed by complementation analysis. This promoter allowed constitutive expression of dspB, as measured by the expression of a dspB::uidA fusion in rich medium. In M9 galactose medium, however, HrpL was shown to activate dspB, as expression of the dspB::uidA fusion was twofold higher in a HrpL+ background than in a HrpL− background. Transposon insertions in either dspA or dspB led to a non‐pathogenic phenotype. Thus, both DspA and DspB were required for E. amylovora pathogenicity, as dspB could be expressed independently of dspA. DspA and DspB were visualized as polypeptides with apparent sizes of 190 kDa and 15.5 kDa, respectively, when encoded in the T7 polymerase/promoter system. DspA, which showed homology with the protein predicted from the partial sequence of Pseudomonas syringae pv. tomato avrE transcriptional unit III, was shown to be secreted into the external medium via the Hrp secretion pathway. DspB was predicted to be acidic, like the Syc chaperone of Yersinia. A chaperone role for DspB was suggested further by the fact that DspA secretion required a functional DspB protein.
Molecular analysis of the cos region of the Lactobacillus casei bacteriophage A2. Gene product 3, gp3, specifically binds to its downstream cos region
Authors: García Pilar , Alonso Juan Carlos , Suárez Juan Evaristo
Published: November 2003

The terminal nucleotide sequence of the Lactobacillus casei bacteriophage A2 DNA revealed a single‐stranded extension 13 bases in length (5′‐AACGGTCGGCCTC‐3′) at its 3′ termini that defines the packaging initiation nicking site (cosN). The cosN sequence is bisected by an axis of hyphenated twofold rotational symmetry. Directly and inverted repeated sequences located to the left (cosL) and the right (cosR) of the cosN site were observed. Analysis of the 3.4 kb EcoRI DNA sequence surrounding the cos region revealed four complete and one incomplete open reading frames (orfs). Northern blots indicated that all were cotranscribed in a single mRNA molecule in excess of 10 kb that appeared late during infection. Minicell studies indicated that the four orfs were translated into protein. From the ORF3 amino acid sequence DNA‐binding and NTP‐binding domains can be predicted. The purified ORF3 (predicted molecular mass 16.8 kDa) shows specific binding to the A2 cos region, so it was renamed gp3. Gp3 forms a specific complex with a 369 bp cos DNA segment in the presence of ATP. Gp3 interaction with the intrinsically bent cos DNA segment induces intramolecular ligation in the presence of T4 DNA ligase. The data presented here suggest that gp3 is the small subunit of the terminase enzyme.
Characterization of the ModE DNA‐binding sites in the control regions of modABCD and moaABCDE of Escherichia coli
Authors: McNicholas Paul M. , Rech Sabine A. , Gunsalus Robert P.
Published: November 2003

The Escherichia coli molybdate transporter, encoded by the modABCD operon, is negatively regulated by the modE gene product in response to the intracellular molybdate concentration. Utilizing an in vivo titration assay, we localized the ModE‐binding site to the start of modA transcription. This localization was further characterized using in vitro gel‐shift assays and DNase I footprinting. ModE bound the wild‐type modA promoter with an apparent dissociation constant (Kd) of 45 nM, and addition of molybdate, in physiologically relevant amounts, significantly increased DNA binding. Consistent with these data, modA promoter fragments containing mutations that reduced ModE repression in vivo displayed proportionately higher apparent Kd values in vitro. DNase I footprinting of the modA promoter revealed a single protected region that overlapped the start site of transcription and extended from position −18 to +10, relative to the transcript start site. Gel‐shifting assays, employing the promoter regions from the tor, nrf, moa and moe operons, revealed that ModE bound only the moa promoter region, with an apparent Kd of 24 nM. Footprint analysis of the moaA promoter revealed a single protected region located immediately upstream of the putative −35 consensus sequence and extending from position −202 to −174, relative to the start of translation. In vivo expression of a moaA–lacZ operon fusion was stimulated twofold by ModE. However, relative to modA, binding of ModE to the moaA promoter appeared to be largely molybdate independent both in vitro and in vivo. These findings demonstrate that ModE acts both as a repressor and activator of the mod and moa operons, respectively, depending on the properties of the binding site.
Transcriptional analysis of type 1 capsule genes in Staphylococcus aureus
Authors: Ouyang Shu , Lee Chia Y.
Published: November 2003

Serotype 1 capsule of Staphylococcus aureus plays an important role in staphylococcal infections. A 14.6 kb chromosomal DNA fragment containing 13 cap1 genes responsible for the synthesis of type 1 capsule in S. aureus strain M has been previously cloned and sequenced in our laboratory. These genes are closely linked and are transcribed in one orientation. To study the transcription of these genes, we employed genetic complementation using a set of plasmids with various deletions in the 14.6 kb region to complement mutants mapped in each of the 13 genes. We found that there were six transcriptional units. By Northern hybridization using the entire gene cluster as a probe, we identified a large 14 kb band and several smaller bands ranging from 0.3 to 6 kb. The 14 kb band was also identified by various individual gene probes, suggesting that all 13 genes are transcribed into a large transcript. The 14 kb transcript and the smaller bands were not detected when a 533 bp fragment containing the potential promoter region upstream of the first gene (cap1A) was deleted from the chromosome, suggesting that the small transcripts are the degradation (processing) products of the 14 kb transcript. The activities of the promoters identified by genetic complementation tests were quantitatively measured by transcriptional fusions using xylE as the reporter gene. The result showed that the promoter preceding the first cap1 gene was 45–198‐fold higher than the downstream promoters. The result from gene‐fusion studies is in agreement with the Northern analysis in that only the transcript transcribed from the first promoter was detected. Taken together, these results suggest that the cap1 genes are organized as a large operon with at least five weak internal promoters, and the long polycistronic transcript is processed (degraded) into several smaller transcripts. The transcription initiation site of the primary transcript was mapped by S1 nuclease mapping and the major promoter was defined by deletion analysis of the promoter–xylE fusion. In addition, we showed that the internal promoters, despite their weak activities, were sufficiently effective at expressing their downstream genes required for producing capsule, compared to the wild‐type strain.
Antitermination of transcription of catabolic operons
Authors: Rutberg
Published: November 2003

Antitermination of transcription mediated by proteins interacting with mRNA sequences is described for nine operons/regulons. Eight of the systems are catabolic, while the ninth, the Klebsiella pneumoniae nas regulon, is involved in the assimilation of nitrate and nitrite. Six of the catabolic operons/regulons are found in Bacillus subtilis, one is found in Escherichia coli, and one in Pseudomonas aeruginosa. The antitermination system of five of the operons/regulons (E. coli blg, and sacPA, sacB, bgl, and lic from B. subtilis) are assigned to the bgl–sac family on the basis of extensive similarities with regard to antiterminator proteins and the sequences of the antiterminators. Other members of the bgl–sac family are the arb operon of Erwinia chrysanthemi and a presumed bgl operon of Lactococcus lactis. The antitermination systems of the other four operons/regulons (B. subtilis glp, B. subtilis hut, P. aeruginosa ami, and K. pneumoniae nas) seem to be unrelated both to the bgl–sac family and to each other. The antiterminator protein of the B. subtilis glp regulon has been found not only to cause antitermination but also to stabilize the resultant mRNA and to mediate glucose repression. If other antiterminator proteins, and antitermination factors, also prove to have additional functions, it will broaden the impact of antitermination as a means of controlling gene expression.
The S‐layer protein of Corynebacterium glutamicum is anchored to the cell wall by its C‐terminal hydrophobic domain
Authors: Chami Mohamed , Bayan Nicolas , Peyret Jean Louis , Gulik‐Krzywicki Thaddée , Leblon Gérard , Shechter Emanuel
Published: November 2003

PS2 is the S‐layer protein of Corynebacterium glutamicum. The S‐layer may be detached from the cell as organized sheets by detergents at room temperature. The solubilization of PS2 in the form of monomers requires detergent treatment at high temperature (70°C), conditions under which the protein is denatured. Treatment of the cells with proteinase K or trypsin results in the detachment of the organized S‐layer, which remains organized. Because we show that trypsin cleaves the C‐terminal part of the protein, we conclude that this domain is involved in the association of the S‐layer to the cell but is not essential in the interaction between individual PS2 proteins within the S‐layer. A modified form of PS2, deleted of its C‐terminal hydrophobic sequence, was constructed. The protein is almost unable to form an organized S‐layer and is mainly released into the medium. We suggest that PS2 is anchored via its C‐terminal hydrophobic sequence to a hydrophobic layer of the wall of the bacterium located some distance above the cytoplasmic membrane.
The iteron bases and spacers of the P1 replication origin contain information that specifies the formation of a complex structure involved in initiation
Authors: Brendler Therese G. , Abeles Ann L. , Reaves Lucretia D. , Austin Stuart J.
Published: November 2003

The origin of replication of the P1 plasmid contains five direct, imperfect repeats (iterons) of a 19 bp sequence that binds the P1‐encoded RepA initiator protein. RepA binding to these iterons triggers origin initiation and represses transcription from the repA promoter that is nested within the iterons. The origin iterons were replaced with ligated oligonucleotides that insert five perfect 19 bp repeats with identical spacer sequences. This eliminates the natural variation in the iteron and spacer sequences and removes the repA promoter. The reconstructed origin is functional, showing that the repA promoter is not essential for origin function. The method used to make the reconstructed origin allows substitution of identical iterons with altered sequence or spacer length. Single changes of conserved iteron bases gave reduced or non‐existent origin activity, as did an increase in spacer length. Like the wild type, most of these mutant arrays retain avid primary binding activity for the RepA protein. However, although the wild‐type arrays readily form a mature complex in which all iterons are saturated, the most replication‐defective mutants were completely unable to do this, even at very high RepA concentrations. It appears that iteron spacing and contacts involving at least three of the conserved iteron bases play an important role in the assembly of the mature structure in which all sites are occupied. A model is presented in which an allosteric interaction between the DNA site and protein is needed for the saturated, mature complex required for initiation.
Replication terminus for DNA polymerase I during initiation of pAMβ1 replication: role of the plasmid‐encoded resolution system
Authors: Jannière L. , Bidnenko V. , McGovern S. , Ehrlich S. D. , Petit M. A.
Published: November 2003

Replication of plasmid pAMβ1 is initiated by DNA polymerase I (Pol I) and completed by DNA polymerase III holoenzyme contained in the replisome machinery. In this study we report that initiation of DNA replication generates D‐loop structures containing the nascent leading strand paired to its template, and that D‐loop extension is arrested ≈230 bp from the initiation site of DNA synthesis in the presence of the plasmid‐encoded resolvase. In vitro and in vivo data suggest that this arrest is caused by a collision between Pol I and the resolvase bound to its target. As the arrested D‐loop replication intermediates carry a single‐stranded primosome‐assembly site, we hypothesize that the biological role of the replication arrest is to limit the region replicated by Pol I and to promote the replacement of Pol I by the replisome in order to initiate concerted synthesis of the leading and lagging strands.
Substrate induction and glucose repression of maltose utilization by Streptomyces coelicolor A3(2) is controlled by malR, a member of the lacI–galR family of regulatory genes
Authors: van Wezel Gilles P. , White Janet , Young Peter , Postma Pieter W. , Bibb Mervyn J.
Published: November 2003

malR of Streptomyces coelicolor A3(2) encodes a homologue of the LacI/GalR family of repressor proteins, and is divergently transcribed from the malEFG gene cluster, which encodes components of an ATP‐dependent transport system that is required for maltose utilization. Transcription of malE was induced by maltose and repressed by glucose. Disruption or deletion of malR resulted in constitutive, glucose‐insensitive malE transcription at a level markedly above that observed in the parental malR+ strain, and overproduction of MalR prevented growth on maltose as carbon source. Consequently, MalR plays a crucial role in both substrate induction and glucose repression of maltose utilization. malR is expressed from a single promoter with transcription initiating at the first G of the predicted GTG translation start codon.
Characterization of Natronobacterium magadii phage ΦCh1, a unique archaeal phage containing DNA and RNA
Authors: Witte Angela , Baranyi Ulrike , Klein Reinhard , Sulzner Michael , Luo Cheng , Wanner Gerhard , Kru¨ger Detlev H. , Lubitz Werner
Published: November 2003

A novel archaeal bacteriophage, ΦCh1, was isolated from a haloalkalophilic archaeon Natronobacterium magadii upon spontaneous lysis. The phage‐cured strain N. magadii (L13) was used to demonstrate infectivity of phage ΦCh1. The turbid‐plaque morphology and the fact that N. magadii cells isolated from plaques were able to produce phage indicated that ΦCh1 is a temperate phage. The phage morphology resembles other members of Myoviridae‐infecting Halobacterium species. In solution below 2 M NaCl, the phage lost its morphological stability and infectivity. One‐ and two‐dimensional SDS–PAGE of phage particles revealed at least four major and five minor proteins with molecular masses ranging from 15 to 80 kDa and acidic isoelectric points. Southern blot analysis of chromosomal DNA of a lysogenic N. magadii strain showed that ΦCh1 exists as a chromosomally integrated prophage. The phage particles contain both double‐stranded, linear DNA (approx. 55 kbp) as well as several RNA species (80–700 nucleotides). Hybridization of labelled RNA fragments to total DNA from N. magadii and ΦCh1 showed that the virion‐associated RNA is host encoded. Part of the phage DNA population is modified and restriction analysis revealed evidence for adenine methylation. Phage ΦCh1 is the first virus described for the genus Natronobacterium, and the first phage containing DNA and RNA in mature phage particles.
Ambulatory long‐term jejunal manometry in diabetic patients with cardiac autonomic neuropathy
Authors: Hackelsberger N. , Schmidt T. , Renner R. , Widmer R. , Pfeiffer A. , Kaess H.
Published: November 2003

Concerning alteration of small bowel motility in diabetic patients with autonomic neuropathy controversial data were obtained with stationary manometry and over a limited period of time. The aim of our study was to examine ambulatory 24 h jejunal motility in 15 diabetic patients with cardiac autonomic neuropathy compared with data obtained in 50 healthy controls. Twenty‐four hour motility was recorded in the proximal jejunum with a portable datalogger and tube‐mounted miniature pressure sensors. Diurnal and nocturnal fasting motility and the motor response to a standardized evening meal of 600 kcal were evaluated by visual and computer‐aided analysis. The following abnormalities were found during fasting motility (n = number of patients): absence of phase III over 24 h (n = 2), retrograde migration or simultaneous occurrence of phase III (n = 5). During postprandial motility irregular bursts with tonic baseline elevation (n = 3) and contraction frequencies below the range of controls (n = 8) occurred. Furthermore patients exhibited an inversion of the normal relationship between phase I and phase II during nocturnal MMC – cycles, and discrete clustered contractions were diminished (P < 0.01) in the fasting and digestive state. All patients showed at least one abnormal manometric finding. We conclude that small bowel motility in diabetic autonomic neuropathy is characterized by disturbances in the generation and aboral migration of phase III, an altered circadian variability of the MMC cycle and by postprandial hypomotility.
Effect of truncal vagotomy and capsaicin on mast cells and IgA‐positive plasma cells in rat jejunal mucosa
Authors: Gottwald T. , LhotÁk Š. , Stead R. H.
Published: November 2003

Immunocompetent cells, including mast cells and plasma cells (PC), in the intestinal mucosa are closely apposed to nerve fibres. Recent work has shown that vagal afferent nerves penetrate the jejunal mucosa and contact intestinal mucosal mast cells (IMMC); and that electrical stimulation of the vagus results in increased IMMC histamine content. To determine if the vagus nerve exerts a trophic effect on immunocompetent cells in the gut mucosa, the effects of truncal vagotomy and neonatal capsaicin treatment on IMMC and IgA containing PC in the lamina propria of rat jejunum were investigated. Three weeks after vagotomy, microdensitometric assessment of Alcian blue stained sections revealed 25% fewer IMMC in vagotomized animals than in controls (P < 0.05). Three months after neonatal capsaicin administration 28% fewer IMMC were found in treated rat jejunum, compared with littermate controls (P < 0.05). Three weeks post‐surgery, IgA‐PC densities were increased in both vagotomized animals (that also underwent pyloroplasty) and pyloroplasty controls, compared to animals subjected to laparotomy only. The proportion of lamina propria areas remained stable, indicating that the observations reflected real reductions in the numbers of IMMC. We also determined the densities of B‐50 (a nerve growth‐associated protein, also called GAP‐43) immunoreactive nerve fibres in the lamina propria, as well as nerve profile areas, three weeks after vagotomy, and these parameters were unchanged. Taken together, these findings support the hypothesis that the vagus exerts a trophic effect on IMMC; and that capsaicin‐sensitive nerves also affect the IMMC population. These data add to the growing body of evidence for a functional connection between IMMC and the nervous system.
Neuropeptides in idiopathic chronic constipation (slow transit constipation)
Authors: Sjölund K. , Fasth S. , Ekman R. , Hultén L. , Jiborn H. , Nordgren S. , Sundler F.
Published: November 2003

Tissue specimens from the large bowel of 18 patients with long‐standing slow transit constipation were investigated to determine the distribution and density of several neuropeptides and amines in the enteric nerve system, and also of endocrine cells in comparison to normal individuals. CGRP (calcitonin gene‐related peptide), galanin, glucagon, GRP (gastrin‐releasing peptide), metenkephalin, motilin, neuropeptide Y (NPY), PACAP, peptide YY (PYY), serotonin, somatostatin, substance P and VIP were studied by immunohistochemistry. Tissue concentrations of VIP, substance P and galanin were also measured by radioimmunoassay. Significantly increased VIP, SP and galanin contents were found in specimens from the ascending colon. Levels of VIP and galanin were also increased in the transverse colon. Immunohistochemistry revealed only marginal changes with an increased density of PACAP nerve fibres in the smooth muscle and of VIP and PACAP nerves in the myenteric plexus of the transverse colon. In the descending colon substance P and NPY immunoreactivity were also increased in the myenteric plexus while the density of VIP nerve fibres was reduced in the mucosa/submucosa. The frequency of PYY‐containing cells and the 5‐HT‐containing cells in the ascending colon was significantly increased in the constipated patients.
Role of nitric oxide in norepinephrine release from myenteric plexus in vitro and in Trichinella spiralis‐infected rats
Authors: RÜHL A. , COLLINS S. M.
Published: November 2003

Nitric oxide (NO) has been implicated as modulator of neural function and inflammatory mediator. Previously, we have demonstrated suppression of norepinephrine (NE) release from myenteric nerves following Trichinella spiralis infection implicating interleukin‐1β (IL‐1β) as a mediator of these changes. In the present study, we have examined the role of NO in NE release from the myenteric plexus and in the suppression of NE release induced by IL‐1β in vitro, and we have determined whether NO is involved in the suppression of NE release from the myenteric plexus observed in T. spiralis‐infected rats. Electrically evoked NE release from jejunal longitudinal muscle–myenteric plexus preparations (LMMP) was measured following (a) in vitro exposure of the tissue to the NO donor 3‐morpholinosydnonimine (SIN‐1), L‐arginine, or IL‐1β, in the presence or absence of NOS inhibitors, and (b) in vivo treatment of control or T. spiralis‐infected rats with N6‐nitro‐L‐arginine‐methyl‐ester (L‐NAME), NG‐nitro‐D‐arginine‐methyl‐ester (D‐NAME) or vehicle for 6 days. In vitro inhibition of NO synthesis had no effect on NE release from the myenteric plexus. Treatment with SIN‐1 or L‐arginine suppressed NE release in a manner similar to that observed with IL‐1β. Moreover, the effect of IL‐1β was attenuated by L‐NAME. In contrast, treatment of T. spiralis‐infected rats with L‐NAME had no effect on the suppression of NE release. These results indicate that in the absence of inflammation, the myenteric plexus can generate sufficient NO to inhibit NE release and that NO mediates the action of IL‐1β on NE release in vitro. However, we have no evidence for the involvement of NO in the suppression of NE release in nematode‐infected rats.
Effects of gastrin‐releasing peptide (GRP) on the mechanical activity of the human ileocaecal region in vitro
Authors: Vadokas B. , Lüdtke F.‐E. , Lepsien G. , Golenhofen K. , Mandrek K.
Published: November 2003

Mechanical activity was recorded in muscle preparations isolated from the human ileocaecal region. Gastrin‐releasing peptide (GRP, 10−9− 10−7 mol L−1) produced two types of response in the different muscle layers. Longitudinally cut strips showed a concentration‐dependent increase in the rhythmic activity, whereas the circularly orientated layers generally reacted with a small decrease in tone. These effects could not be influenced by blockade of adrenergic or cholinergic receptors or nerve blockade with tetrodotoxin (TTX). Application of pentagastrin did not mimic the action of GRP. These findings suggest a direct action of GRP on smooth muscle via distinct receptors which have already been demonstrated to exist in human gastrointestinal tract. The opposite effects on circular and longitudinal strips might indicate a modulatory role of GRP in the control of ileocolonic transit.
A new nausea model in humans produces mild nausea without electrogastrogram and vasopressin changes
Authors: Kiernan B. D. , Soykan I. , Lin Z. , Dale A. , Mccallum R. W.
Published: November 2003

The standard human vection model utilized for nausea has been an optokinetic drum. This model may be difficult to extrapolate to the usual clinical setting. Our goals were to develop an experimental model which could induce low grade nausea in humans and to determine the relationship between mild nausea and changes in the electrogastrogram and plasma vasopressin concentrations. Methods: twenty‐one volunteers (11 males, mean age: 37 years) participated. At baseline and throughout the study the electro­gastrogram was monitored, blood was drawn for vasopressin assay and symptoms of nausea, dizziness and headache were rated on a 0–10‐point scale. Subjects were semireclined in a darkened room while viewing moving bars of light rotating at a rate of 85° s−1. Subjects were asked to rate their proneness to motion sickness and their current level of anxiety at baseline. Results: eight subjects developed mild to moderate (mean: 3.6) nausea during vection. Symptoms of nausea were correlated with a reported history of motion sickness (r = 0.49, P < 0.05) but not with anxiety (r = 0.14, P = 0.54). Degrees of nausea correlated with degrees of dizziness (r = 0.47, P < 0.05) but not with headache (r = 0.29, P = 0.14). Subjects who developed mild nausea were not significantly more likely to exhibit altered electrogastrogram or vasopressin than subjects who did not report nausea. Vasopressin levels during baseline and experimental conditions were highly correlated (r = 0.66, P < 0.005 and r = 0.55 P < 0.005, respectively) with reported baseline anxiety. Conclusions: (1) this new model in humans induced mild nausea that was unrelated to electrogastrogram and vasopressin abnormalities, (2) high correlation between anxiety and vasopressin suggests that vasopressin may not be directly related to nausea, and (3) these data indicate that onset of nausea mediated centrally can occur without associated electrogastrogram changes.
5‐HT receptor types in the rat ileum longitudinal muscle: focus on 5‐HT2 receptors mediating contraction
Authors: Briejer M. R. , Mathis C. , Schuurkes J. A. J.
Published: November 2003

The 5‐hydroxytryptamine (5‐HT) receptor(s) that mediate(s) contraction of the rat ileum longitudinal muscle was studied.

5‐HT and α‐methyl‐5‐HT equipotently induced contractions, whereas 5‐methoxytryptamine and 2‐methyl‐5‐HT (partial agonist) were less potent; this rank order of potency suggests involvement of a 5‐HT2 receptor. Neither tetrodotoxin nor atropine affected the contraction to 5‐HT, suggesting a smooth muscle localization of these 5‐HT2 receptors. The presence of either a selective 5‐HT2B (SB 204741), 5‐HT3 (granisetron) or 5‐HT4 (SB 204070) antagonist, slightly affected the contractions to 5‐HT. Thus, they were also included in the organ bath solution in all subsequent experiments in order to pharmacologically isolate the main contractile component. Using (if possible) 5‐HT2A receptor‐selective concentrations, ketanserin, ritanserin, metergoline, spiperone, mianserin, methiothepin, mesulergine, methysergide and cisapride all inhibited the contractions to 5‐HT, causing a depression of the curve to 5‐HT (i.e. surmountable antagonism was not observed with any of the above agents). Comparison of the affinities of these compounds for the various 5‐HT2 receptor subtypes revealed that the receptor involved in the contractions to 5‐HT most closely resembles the 5‐HT2A receptor. However, cinanserin at a concentration expected to inhibit 5‐HT2A receptor‐mediated effects, failed to affect the contractions to 5‐HT.

It is thus concluded that on the longitudinal smooth muscle of the rat ileum, at least a part of the contraction to 5‐HT is mediated by 5‐HT receptors resembling the 5‐HT2A receptor subtype.
Trade in pegasid fishes (sea moths), primarily for traditional Chinese medicine
Authors: Vincent Amanda C. J.
Published: November 2003

Pegasid fishes (sea moths) have only entered the arsenal of traditional Chinese medicine within the past few decades, but are now used in southern China and Hong Kong to treat respiratory ailments and cancers. Brief trade surveys suggest that millions of individuals of two pegasid species are used each year, and that they cost relatively little compared with other ingredients. Most pegasids are apparently a bycatch of trawl fishing, which has recently intensified near China. Trade and use of these fishes is expanding – they have been sought in the Philippines, Indonesia and Vietnam – and should be monitored, particularly because the biology of pegasids makes these fishes vulnerable to exploitation.
Planning conservation areas in Uganda's natural forests
Authors: Howard Peter , Davenport Tim , Kigenyi Fred
Published: November 2003

In the late 1980s the Ugandan Government decided to dedicate a fifth (3000 sq km) of the country's 15,000-sq-km forest estate to management as Strict Nature Reserves (SNRs)for the protection of biodiversity. The Forest Department subsequently undertook a 5-year programme of biological inventory and socioeconomic evaluation to select appropriate areas for designation. Sixty-five of the country's principal forests (including five now designated as National Parks) were systematically evaluated for biodiversity, focusing on five ‘indicator’ taxa (woody plants, birds, small mammals, butterflies and large moths). A scoring system was developed to compare and rank sites according to their suitability for nature reserve establishment and 11 key sites were identified, which, when combined with the country's 10 national parks, account for more than 95 per cent of Uganda's species. In order to satisfy multiple-use management objectives, the Man and the Biosphere model of reserve design is being applied at each forest, by designating a centrally located core area as SNR, with increasingly intensive resource use permitted towards the periphery of each reserve and adjacent rural communities.
Population status and conservation of bats (Chiroptera) in Kenting National Park, Taiwan
Authors: Hsu Minna J.
Published: November 2003

This paper presents the results of field work conducted between 1994 and 1996 to establish the occurrence, and population, breeding and conservation status of bats in and around Kenting National Park, southern Taiwan. Of the 20 bat species that occur in Taiwan, Kenting National Park and its vicinity harbour eight species; three are endemic to Taiwan and two are endemic subspecies. Six of the eight species roost in natural caves and man-made tunnels. Two species of forest bats were captured during mist-netting. The most predominant species were the horseshoe bat Rhinolophus monoceros and the common bent-winged bat Miniopterus schreibersii. The major threat for the cave-dwelling bats around Kenting is the closure of cave entrances by farmers. Recommendations are given for conserving the bats and their habitats in Taiwan.
The wild Bactrian camel Camelus bactrianus ferus in China: the need for urgent action
Authors: Hare John
Published: November 2003

Between 31 March and 28 May 1995 and 5 April and 2 May 1996 two expeditions carried out surveys of the status and distribution of the wild Bactrian camel Camelus bactrianus ferus in the Xinjiang Uygur Autonomous Region and Gansu Province in the People's Republic of China, the last known habitat of the subspecies in the country. The surveys revealed that the wild camel is highly endangered in China, due to illegal mining and hunting. An estimated total of 380–500 individuals remain and immediate action is needed to ensure their survival.
Status and distribution of the Pemba flying fox Pteropus voeltzkowi
Authors: Entwistle Abigail , Corp Nadia
Published: November 2003

The Pemba flying fox Pteropus voeltzkowi is a fruit bat endemic to the island of Pemba, off Tanzania. A total of 41 reported roosting areas were visited in June and July 1995, and 19 occupied roosts were located, most of them in the west of the island, and on small islets off the west and south of Pemba. Roosts were situated in primary forest, secondary forest (overgrown clove plantations), traditional graveyards and mangrove areas. A range of sizes and species of trees were used as roosts. The minimum population of P. voeltzkowi was estimated to be between 4600 and 5500 individuals. In total 94 per cent of the population was located at 10 roost sites. Larger colonies were associated with roosts located in forests, which together supported 75 per cent of the total bat population. Colonies were of mixed sex, but no young or obviously pregnant females were observed. Major threats to this species appeared to be hunting and deforestation (both logging and clearing for agriculture) and P. voeltzkowi is considered to be endangered.
Distribution, abundance and conservation status of Grauer's gorilla
Authors: Hall Jefferson S. , Saltonstall Kristin , Inogwabini Bila-Lsia , Omari Ilambu
Published: November 2003

This report updates the distribution and provides abundance estimates for Grauer's gorilla Gorilla gorilla graueri across its 90,000-sq-km range. The authors divide the range of Grauer's gorilla into four regions within which they identify 11 populations and estimate a total of c. 16,900 individuals. Gorillas found in the Kahuzi-Biega lowland-Kasese region represent 86 per cent of the subspecies's total population. Further, approximately 67 per cent of known Grauer's gorillas inhabit Kahuzi-Biega, Maiko and Virunga National Parks. The eastern region of the Democratic Republic of Congo (formerly Zaire) has an extremely high human population growth rate indicating that deforestation and wildlife use trends will continue to increase. Thus, in spite of the encouraging results of surveys to identify populations and characterize abundance, no Grauer's gorilla population should be considered safe from extirpation.
Not peripheral parasitaemia but the level of soluble parasite antigen in plasma correlates with vaccine efficacy against Babesia canis
Authors: SCHETTERS Th.P.M. , SCHOLTES N.C. , KLEUSKENS J.A.G.M. , BOS H.J.
Published: November 2003

Groups of five dogs were vaccinated against Babesia canis using soluble parasite (SPA) antigens from in vitro cultures. Although vaccination did not significantly alter peripheral parasitaemia upon challenge, protected animals had lower levels of SPA in the plasma after a challenge infection. The severity of anaemia correlated with the SPA‐load during the post‐challenge period in that high levels of SPA were associated with low haematocrit values. In addition, it was found that recovery was associated with the production of antibodies against SPA. The results suggest that SPA induce anaemia during B. canis infection, and that vaccination with SPA results in antibody production that can neutralize the effects of SPA after a challenge infection.