bluuebunny/crossref_metadata_2025 huggingface dataset:
The effects of KT3‐671, a new angiotensin II (AT 1) receptor blocker in mild to moderate hypertension
Authors: Patterson D. , Webster J. , McInnes G. , Brady A. , MacDonald T.
Published: October 2003

Aims  To compare the antihypertensive effect, and tolerability and safety of once daily doses of KT3‐671 with that of placebo in patients with mild to moderate uncomplicated essential hypertension.

Methods  A randomised, multicentre, double blind, parallel‐group comparison of KT3‐671 with placebo. Hypertensive patients [Ambulatory Blood Pressure Monitoring (ABPM), mean daytime DBP > 90 mmHg, Office sitting mean DBP 95‐114 after a 7–28 day washout period] entered a 2‐week, single blind, run‐in phase. Patients eligible for the double‐blind phase were randomised to receive KT3‐671 40 mg, 80 mg, 160 mg or placebo once daily over 4 weeks. The primary end‐point was trough mean sitting office DBP. The study had 90% power to detect a 5 mmHg change between treatments and placebo at the 5% level of significance. The secondary end‐points were 24 hour, daytime and night time mean ABPM.

Results  Office DBP was significantly lower with KT3‐671 40 mg but not the other 2 dosage groups (−3.2; 95% CL −6.1 : −0.3 P < 0.03). Office SBP was significantly reduced with all dosage groups (40 mg −5.9, 95% CL −11 : −0.9; 80 mg −4.9, 95% CL −9.9 : 0.1 and 160 mg −5.7, 95% CL −10.8 : −0.7 P < 0.05). All doses of KT3‐671 reduced systolic and diastolic ABPM. The number of patients with treatment related adverse events were comparable to placebo (38.8% KT3‐671 vs 32.8% placebo). There was some evidence of a dose‐response relationship with fall in nocturnal ABPM.

Conclusions  Oral KT3‐671 was well tolerated. KT3‐671 reduced office systolic BP at all doses and diastolic BP at some of the doses. Due to greater precision and power, the falls in mean ambulatory systolic and diastolic pressure were all significantly lower than placebo.
Pathology of drug‐associated gastrointestinal disease
Authors: Price Ashley B.
Published: October 2003

A large number of drugs have gastrointestinal side‐effects of which diarrhoea or constipation, nausea and vomiting are amongst the commonest. In relatively few are there diagnostic pathological changes and this review draws attention to the most common. Incriminating a drug as a cause of specific pathological changes requires the drug to be associated with the changes, for the latter to resolve when the drug is withdrawn and for them to re‐appear when a patient is rechallenged with the drug. Individual histological features such as apoptosis, tissue infiltration by eosinophils and increased intra‐epithelial lymphocytes within the gut mucosa can be clues to an iatrogenic aetiology but these are by no means specific. Amongst the few pathognomonic patterns of drug reactions is pseudomembranous colitis and diaphragm disease. These, along with others such as reactive gastritis and the collagenous and lymphocytic forms of microscopic colitis, in which drugs have also been implicated, are described here.
Biopsy assessment of drug efficacy in the gastrointestinal tract
Authors: Walker Marjorie M.
Published: October 2003

When using biopsy pathology in clinical pharmacology to assess drug efficacy in the gastrointestinal tract, a number of questions must be answered: Is the biopsy necessary or more effective than macroscopic views by endoscopy? Can we extract maximal information from the specimen? Are there surrogate serum or other markers that give an overall measure of disease and/or improvement? Indeed, clinicopathological correlation is of paramount importance. If biopsy is to be used, it is important to utilize appropriate scoring systems. Many grading systems use continuous spectra, which are ordinal categorical variables and therefore a grading system of assigned ‘numbers’ which cannot be used in processes that require continuous variables such as linear regression. The use of grading vs a ‘true’ score with real numbers must be carefully considered, the site and number of biopsies must be precisely chosen and interobserver reproducibility of results evaluated before undertaking drug trials. Immunocytochemistry and in situ hybridization, however, can provide quantifiable molecular information related to mechanisms of drug action. The biopsy is of significant value as it is a true in vivo assessment if the above caveats are taken into account. However, further work is needed to determine sound histological criteria to assess the efficacy of drugs for use in gastrointestinal disease.
Root‐canal shaping with manual and rotary Ni‐Ti files performed by students
Authors: Sonntag D. , Delschen S. , Stachniss V.
Published: October 2003

Aim  To investigate root‐canal shaping with manual and rotary Ni‐Ti files performed by students.

Methodology  Thirty undergraduate dental students prepared 150 simulated curved root canals in resin blocks with manual Ni‐Ti files with a stepback technique and 450 simulated curved canals with rotary Ni‐Ti files with a crowndown technique. Incidence of fracture, preparation length, canal shape and preparation time were investigated. Questionnaires were then issued to the students for them to note their experience of the two preparation methods.

Results  Zips and elbows occurred significantly (P < 0.001) less frequently with rotary than with manual preparation. The correct preparation length was achieved significantly (P < 0.05) more often with rotary files than with manual files. Instrument fractures were recorded in only 1.3% of cases with both rotary and manual preparation. The mean time required for manual preparation was significantly (P < 0.001) longer than that required for rotary preparation. Prior experience with a hand preparation technique was not reflected in an improved quality of the subsequent rotary preparation. Approximately 83% of the students claimed to have a greater sense of security in rotary than in manual preparation. Overall 50% felt that manual and engine‐driven preparation should be given equal status in undergraduate dental education.

Conclusions  Inexperienced operators achieved better canal preparations with rotary instruments than with manual files. No difference in fracture rate was recorded between the two systems.
The effect of cleaning procedures on fracture properties and corrosion of NiTi files
Authors: O'Hoy P. Y. Z. , Messer H. H. , Palamara J. E. A.
Published: October 2003

Aim  To evaluate the effect of repeated cleaning procedures on fracture properties and corrosion of nickel‐titanium (NiTi) files.

Methodology  New NiTi instruments were subjected to 2, 5 and 10 cleaning cycles with the use of either diluted bleach (1% NaOCl) or Milton's solution (1% NaOCl plus 19% NaCl) as disinfectant. Each cleaning cycle consisted of scrubbing, rinsing and immersing in NaOCl for 10 min followed by 5 min of ultrasonication. Files were then tested for torsional failure and flexural fatigue, and observed for evidence of corrosion using scanning electron microscope (SEM). Four brands of NiTi files were immersed in either Milton's solution or diluted bleach overnight and evaluated for corrosion.

Results  Up to 10 cleaning cycles did not significantly reduce the torque at fracture or number of revolutions to flexural fatigue (P > 0.05, two‐way anova), although decreasing values were noted with increasing number of cleaning cycles using Milton's solution. No corrosion was detected on the surface of these files. Files immersed in 1% NaOCl overnight displayed a variety of corrosion patterns. The extent of corrosion was variable amongst different brands of files and amongst files in each brand. Overall, Milton's solution was much more corrosive than diluted bleach. Corrosion of file handles was often extreme.

Conclusions  Files can be cleaned up to 10 times without affecting fracture susceptibility or corrosion, but should not be immersed in NaOCl overnight. Milton's solution is much more corrosive than bleach with the same NaOCl concentration.
Effect of different irrigation solutions and calcium hydroxide on bacterial LPS
Authors: Tanomaru J. M. G. , Leonardo M. R. , Tanomaru Filho M. , Bonetti Filho I. , Silva L. A. B.
Published: October 2003

Aim  To evaluate the effect of biomechanical preparation with different irrigating solutions and calcium hydroxide dressing in dog root canals containing bacterial endotoxin (lipopolysaccharides; LPS).

Methodology  One hundred and forty premolar roots from seven dogs were filled with Escherichia coli LPS for 10 days (three roots were lost during histological processing). The following irrigating solutions were used for biomechanical preparation: 1% (group I, n = 20), 2.5% (group II, n = 19) and 5% sodium hypochlorite (group III, n = 19), 2% chlorhexidine digluconate (group IV, n = 20) and physiological saline solution (group V, n = 19). In group VI (n = 20), the LPS solution was maintained in the root canal during the entire experiment and in group VII (n = 20), after biomechanical preparation with saline solution, the root canals were filled with a calcium hydroxide dressing (Calen; control). After 60 days, the animals were sacrificed and the following parameters of periapical disease were evaluated: (a) inflammatory infiltrate, (b) periodontal ligament thickness, (c) cementum resorption and (d) bone resorption. Scores were given and data were analysed statistically with the Kruskal–Wallis and Dunn tests (P < 0.05).

Results  Histopathological evaluation showed that groups I–VI had more inflammatory infiltrate, greater periodontal ligament thickening and greater cementum and bone resorption (P < 0.05) compared to group VII, which received the calcium hydroxide intracanal dressing.

Conclusions  Biomechanical preparation with the irrigating solutions did not inactivate the effects of the endotoxin but the calcium hydroxide intracanal dressing did appear to inactivate the effects induced by the endotoxin in vivo.
Bacterial status in root‐filled teeth exposed to the oral environment by loss of restoration and fracture or caries – a histobacteriological study of treated cases
Authors: Ricucci D. , Bergenholtz G.
Published: October 2003

Aim  To describe histological and microbiological findings in teeth where root fillings had been exposed to caries and the oral environment for a prolonged period.

Methodology  For inclusion in the study, only teeth with a follow‐up period of 3 years or more and those that had been without proper restoration for at least a period of 3 months were considered. Some root fillings had been without restoration for several years. In all, 39 roots representing 32 teeth were examined by histology.

Results  The majority of the specimens were without a discernible periapical bone lesion as assessed by radiography. Osteolytic lesions were seen with five roots. Longitudinal tissue sections stained with a modified Brown/Brenn staining technique revealed presence of stainable bacteria in abundance at the canal entrance and in dentinal tubules but were absent mid‐root and apically in all but two specimens. Soft tissue attached to the root tip and in apical ramifications displayed distinct inflammatory cell infiltrates, suggesting microbial exposure in 7 of the 39 roots examined. In all other specimens, inflammatory cell infiltrates were either nonexistent or sparse and then associated with extruded sealer material.

Conclusions  Well‐prepared and filled root canals resist bacterial penetration even upon frank and long‐standing oral exposure by caries, fracture or loss of restoration.
Root‐canal preparation with FlexMaster: canal shapes analysed by micro‐computed tomography
Authors: Hübscher W. , Barbakow F. , Peters O. A.
Published: October 2003

Aim  To evaluate the relative performance of FlexMaster nickel–titanium instruments shaping maxillary molar root canals in vitro.

Methodology  Extracted human maxillary molars were scanned, before and after root‐canal shaping, with FlexMaster, employing micro‐computed tomography (µCT) at a resolution of 36 µm. Canals were three‐dimensionally reconstructed and evaluated for volume, surface area, ‘thickness’ (diameter), canal transportation and prepared surface. Based on median canal volume, the canals were divided into ‘wide’ and ‘constricted’ groups. Comparisons were made between mesiobuccal (mb), distobuccal (db) and palatal (p), as well as ‘wide’ and ‘constricted’ canals, using anova and Scheffé post‐hoc tests.

Results  Volume and surface area increased significantly and similarly in mb, db and p canals, and no gross preparation errors were found. Mean root‐canal diameters, 5 mm coronal to the apex, increased from 0.45 to 0.65 mm, from 0.41 to 0.56 mm and from 0.79 to 0.85 mm for mb, db and p canals, respectively. Apical canal transportation ranged from 0.01 to 0.29 mm and was independent of canal type; ‘wide’ canals had a significantly higher (P < 0.05) proportion of unprepared surfaces than ‘constricted’ canals had.

Conclusions  FlexMaster instruments shaped curved and narrow root canals in maxillary molars to sizes 40 and 45 without significant shaping errors. Canal anatomy had an insignificant impact on preparation, indicating that FlexMaster instruments were able to shape ‘constricted’ canals as well as ‘wide’ ones.
Induction of interleukin‐8 gene expression by black‐pigmented Bacteroides in human pulp fibroblasts and osteoblasts
Authors: Yang L.‐C. , Huang F.‐M. , Lin C.‐S. , Liu C.‐M. , Lai C.‐C. , Chang Y.‐C.
Published: October 2003

Aim  To investigate the effect of black‐pigmented Bacteroides on the expression of interleukin (IL)‐8 gene in human pulp fibroblasts and osteoblasts.

Methodology  The supernatants of Porphyromonas endodontalis, P. gingivalis and Prevotella intermedia were used to evaluate IL‐8 gene expression in human pulp fibroblasts and osteoblasts. The levels of mRNAs were measured by the quantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis.

Results  Investigations of the time‐dependence of IL‐8 mRNA expression in black‐pigmented Bacteroides‐treated pulp fibroblasts and osteoblasts revealed a rapid accumulation of the transcript after 2 h of exposure, and remained elevated throughout the 24‐h incubation period. In addition, IL‐8 mRNA gene expression was also found in human osteoblasts stimulated with black‐pigmented Bacteroides. However, black‐pigmented Bacteroides was found to be more effective in the induction of IL‐8 mRNA gene expression in osteoblasts than in pulp fibroblasts (P < 0.05).

Conclusions  Black‐pigmented Bacteroides are capable of amplifying the local immune response and promoting pulpal/periapical tissue inflammation by stimulating pulp fibroblasts and osteoblasts to express IL‐8.
Myosin heavy chain expression and atrophy in rat skeletal muscle during transition from cardiac hypertrophy to heart failure
Authors: Carvalho Robson Francisco , Cicogna Antonio Carlos , Campos Gerson Eduardo Rocha , De Assis Jeane Marlene FogaÇa , Padovani Carlos Roberto , Okoshi Marina Politi , Pai‐Silva Maeli Dal
Published: October 2003

Summary.  The purpose of this investigation was to determine whether changes in myosin heavy chain (MHC) expression and atrophy in rat skeletal muscle are observed during transition from cardiac hypertrophy to chronic heart failure (CHF) induced by aortic stenosis (AS). AS and control animals were studied 12 and 18 weeks after surgery and when overt CHF had developed in AS animals, 28 weeks after the surgery. The following parameters were studied in the soleus muscle: muscle atrophy index (soleus weight/body weight), muscle fibre diameter and frequency and MHC expression. AS animals presented decreases in both MHC1 and type I fibres and increases in both MHC2a and type IIa fibres during late cardiac hypertrophy and CHF. Type IIa fibre atrophy occurred during CHF. In conclusion, our data demonstrate that skeletal muscle phenotype changes occur in both late cardiac hypertrophy and heart failure; this suggests that attention should be given to the fact that skeletal muscle phenotype changes occur prior to overt heart failure symptoms.
Diverse range of fixed positional deformities and bone growth restraint provoked by flaccid paralysis in embryonic chicks
Authors: Lamb Katherine J. , Lewthwaite Jo C. , Lin Jean‐Pierre , Simon Dominic , Kavanagh Emma , Wheeler‐Jones Caroline P. D. , Pitsillides Andrew A.
Published: October 2003

Summary.  Pancuronium bromide (PB) is used in neonates and pregnant women to induce limp, flaccid paralysis in order to allow mechanical ventilation during intensive care. Such non‐depolarizing neuromuscular blocking drugs are administered to 0.1% of all human births in the UK. In this study, we examined PB effects on skeletal development in chick embryos. PB treatment produced skeletal deformities associated with significant reduction in longitudinal growth of all appendicular elements. This was associated with greater cartilage to bone ratios, indicating a preferential reduction in osteogenesis. PB also increased the incidence of knee joint flexion and tibiotarsal joint hyperextension. In addition to limb, spinal and craniofacial deformities, flaccid immobility appears to convert the normal geometric pattern of weight gain to a simple arithmetic accretion. This novel study highlights the potentially harmful effects of pharmacologically induced flaccid immobility on chick embryonic skeletal development. Whilst in ovo avian development clearly differs from human, our findings may have implications for the fetus, premature and term neonate receiving such non‐depolarizing neuromuscular blocking drugs.
Duchenne's muscular dystrophy: animal models used to investigate pathogenesis and develop therapeutic strategies
Authors: Collins C.A. , Morgan J.E.
Published: October 2003

Summary.  Duchenne's muscular dystrophy (DMD) is a lethal childhood disease caused by mutations of the dystrophin gene, the protein product of which, dystrophin, has a vital role in maintaining muscle structure and function. Homologues of DMD have been identified in several animals including dogs, cats, mice, fish and invertebrates. The most notable of these are the extensively studied mdx mouse, a genetic and biochemical model of the human disease, and the muscular dystrophic Golden Retriever dog, which is the nearest pathological counterpart of DMD. These models have been used to explore potential therapeutic approaches along a number of avenues including gene replacement and cell transplantation strategies. High‐throughput screening of pharmacological and genetic therapies could potentially be carried out in recently available smaller models such as zebrafish and Caenorhabditis elegans. It is possible that a successful treatment will eventually be identified through the integration of studies in multiple species differentially suited to addressing particular questions.
End‐to‐end fusion of linear deleted chromosomes initiates a cycle of genome instability in Streptomyces ambofaciens
Authors: Wenner Thomas , Roth Virginie , Fischer Gilles , Fourrier Céline , Aigle Bertrand , Decaris Bernard , Leblond Pierre
Published: October 2003

Two mutant strains harbouring a linear chromosome whose size reached 13 Mb (versus approximately 8 Mb for the wild type) were characterized. This chromosomal structure resulted from the fusion in inverted orientation of two chromosomes partially deleted on the same arm. The fusion occurred by illegitimate recombination between 6 bp repeats. This chromosomal structure was inherited in strict association with a high level of genetic instability (30% of mutants in a single progeny, phenomenon also called hypervariability) and chromosomal instability. In contrast, derivatives, which did not retain the chromosome fusion, showed a wild‐type‐like instability frequency (c. 1%). Stabilization of the chromosomal structure occurred by chromosome arm replacement or circularization. A high variability of the terminal inverted repeat (TIR) length in the rescued chromosomes (from 5 kb to approximately 1.4 Mb for linear derivatives) was observed. Mutant lineages harbouring the chromosomal fusion are characterized by a highly heterogeneous distribution of DNA in the spores, by the presence of spores without DNA as well as aberrant sporulation figures, and by the production of spores with a low germination rate. The wild‐type characteristics were restored in the descendants, which lost the chromosomal fusion. Thus, the fusion of deleted chromosomes initiates a cycle of chromosome instability sharing several levels of analogy with the behaviour of dicentric chromosomes in eukaryotes. We propose that the high instability of the fused chromosomes results from the duplication of a region involved in partitioning of the chromosomes (parAB‐oriC ).
Vibrio cholerae H‐NS domain structure and function with respect to transcriptional repression of ToxR regulon genes reveals differences among H‐NS family members
Authors: Nye Melinda B. , Taylor Ronald K.
Published: October 2003

H‐NS is an abundant bacterial protein involved in transcriptional silencing of a variety of environmentally responsive genes during growth under non‐permissive conditions. We have previously demonstrated a direct role for H‐NS in the negative modulation of expression of several genes within the ToxR virulence regulon of Vibrio cholerae. Here we have undertaken extensive mutagenesis of the structural and functional domains of the H‐NS protein to determine the contribution of each to the regulation of gene expression. Insertions within, or truncations of, the C‐terminal conserved DNA‐binding domain prevent repression of toxT and ctx, as expected. Dominant negative experiments demonstrate that V. cholerae H‐NS represses gene expression as an oligomeric protein. Hydrophobic coiled–coil interactions have been shown to provide oligomerization capability in other H‐NS orthologues. We used site‐directed mutagenesis to construct altered V. cholerae H‐NS proteins, including an extensive internal deletion within the predicted coiled–coil domain. Remarkably, these proteins were competent to repress gene expression and to form oligomers. Chimeric H‐NS proteins, using sequences from both Escherichia coli and V. cholerae H‐NS orthologues, revealed that V. cholerae H‐NS possesses a second oligomerization domain in the N‐terminal 24 amino acids of the protein. Overall, our results suggest DNA binding and protein oligomerization, provided by either the central coiled–coil or N‐terminal domain, are required for repression of promoters responsive to H‐NS within the ToxR regulon.
Transformation of Streptococcus pneumoniae relies on DprA‐ and RecA‐dependent protection of incoming DNA single strands
Authors: Bergé Mathieu , Mortier‐Barrière Isabelle , Martin Bernard , Claverys Jean‐Pierre
Published: October 2003

Seventy‐five years after the discovery of transformation with Streptococcus pneumoniae, it is remarkable how little we know of the proteins that interact with incoming single strands in the early processing of transforming DNA. In this work, we used as donor DNA in transformation a radioactively labelled homologous fragment to examine the fate of the single‐stranded (ssDNA) products of uptake in cells mutant for DprA or RecA, two proteins essential for transformation. Fifteen minutes after uptake, the labelling of specific chromosomal restriction fragments that demonstrated homologous integration in the wild type was not detected in dprA or recA cells, indicating that in the mutants incoming ssDNA could not be processed into recombinants. Investigation of the fate of donor label 1 min after uptake revealed that incoming ssDNA was immediately degraded in the absence of DprA or RecA. Our results demonstrate that incoming ssDNA requires active protection prior to the RecA‐driven search for homology and that both DprA and RecA are needed for this protection.
Multiple mechanisms for choline transport and utilization in Haemophilus influenzae
Authors: Fan Xin , Pericone Christopher D. , Lysenko Elena , Goldfine Howard , Weiser Jeffrey N.
Published: October 2003

Haemophilus influenzae obtains choline from either its growth medium or host cell membrane lipids and expresses it on its lipopolysaccharide (LPS) in the form of phosphorylcholine (ChoP), which contributes to its pathogenesis by mimicry of host cell molecules. Two genes (licB and betT) revealed by whole genomic analysis as encoding potential choline transporters were tested for their role in LPS‐ChoP synthesis. The betT gene in H. influenzae is similar to betT in Escherichia coli, which functions in choline transport for the generation of betaine in osmoprotection. The licB gene has homology to bacterial permeases including betT and is encoded in the lic1 locus, which is essential for the expression of LPS‐ChoP. In the presence of high concentrations of choline, neither licB nor betT were necessary for expression of LPS‐ChoP raising the possibility that other unidentified choline uptake mechanisms may exist in this species. However, under choline limiting conditions, including growth in human nasal airway surface fluid, the licB, but not betT, gene was required for choline transport and synthesis of LPS‐ChoP suggesting that LicB functions as a high affinity choline permease. The betT, but not licB, gene was shown to function in osmoprotection in H. influenzae, similar to the role of betT in E. coli. Further analysis demonstrated growth condition dependent differences in the regulation of transcription of the licB and betT genes. We conclude that H. influenzae may have multiple mechanisms for choline uptake and distinct pathways for choline utilization in LPS‐ChoP biosynthesis and osmoregulation.
Error‐prone DNA polymerase IV is controlled by the stress‐response sigma factor, RpoS, in Escherichia coli
Authors: Layton Jill C. , Foster Patricia L.
Published: October 2003

An insertion in rpoS, which encodes the general stress response sigma factor σ38, was isolated as an antimutator for ‘stationary‐phase’ or ‘adaptive’ mutation. In the rpoS mutant strain the levels of error‐prone DNA polymerase Pol IV were reduced. Pol IV is encoded by the dinB gene, and the amount of its transcript was also reduced in rpoS mutant cells. In wild‐type cells, the levels of Pol IV increased in late stationary phase and stayed elevated for several days of continuous incubation, whereas in rpoS defective cells Pol IV was not induced and declined during prolonged incubation. Even in cells missing LexA, the repressor of dinB, maximum Pol IV expression required RpoS. These results suggest that induction of Pol IV is part of a cellular response to starvation and other stresses.
Legionella pneumophila CsrA is a pivotal repressor of transmission traits and activator of replication
Authors: Molofsky Ari B. , Swanson Michele S.
Published: October 2003

Legionella pneumophila can replicate inside amoebae and also alveolar macrophages to cause Legionnaires’ Disease in susceptible hosts. When nutrients become limiting, a stringent‐like response coordinates the differentiation of L. pneumophila to a transmissive form, a process mediated by the two‐component system LetA/S and the sigma factors RpoS and FliA. Here we demonstrate that the broadly conserved RNA binding protein CsrA is a global repressor of L. pneumophila transmission phenotypes and an essential activator of intracellular replication. By analysing csrA expression and the phenotypes of csrA single and double mutants and a strain that expresses csrA constitutively, we demonstrate that, during replication in broth, CsrA represses every post‐exponential phase phenotype examined, including cell shape shortening, motility, pigmentation, stress resistance, sodium sensitivity, cytotoxicity and efficient macrophage infection. At the transition to the post‐exponential phase, LetA/S relieves CsrA repression to induce transmission phenotypes by both FliA‐dependent and ‐independent pathways. For L. pneumophila to avoid lysosomal degradation in macrophages, CsrA repression must be relieved by LetA/S before phagocytosis; conversely, before intracellular bacteria can replicate, CsrA repression must be restored. The reciprocal regulation of replication and transmission exemplified by CsrA likely enhances the fitness of microbes faced with fluctuating environments.
Acidobacteria form a coherent but highly diverse group within the bacterial domain: evidence from environmental genomics
Authors: Quaiser Achim , Ochsenreiter Torsten , Lanz Christa , Schuster Stephan C. , Treusch Alexander H. , Eck Jürgen , Schleper Christa
Published: October 2003

Acidobacteria have been established as a novel phylum of Bacteria that is consistently detected in many different habitats around the globe by 16S rDNA‐based molecular surveys. The phylogenetic diversity, ubiquity and abundance of this group, particularly in soil habitats, suggest an important ecological role and extensive metabolic versatility. However, the genetic and physiological information about Acidobacteria is scarce. In order to gain insight into genome structure, evolution and diversity of these microorganisms we have initiated an environmental genomic approach by constructing large insert libraries directly from DNA of a calcerous grassland soil. Genomic fragments of Acidobacteria were identified with specific 16S rDNA probes and sequence analyses of six independently identified clones were performed, representing in total more than 210 000 bp. The 16S rRNA genes of the genomic fragments differed between 2.3% and 19.9% and were placed into two different subgroups of Acidobacteria (groups III and V). Although partial co‐linearity was found between genomic fragments, the gene content around the rRNA operons was generally not conserved. Phylogenetic reconstructions with orthologues that were encoded on two of the six genomic fragments (PurF, PurL, PurB and formamidopyrimidine‐DNA glycosylase) confirmed the coherence of the acidobacterial phylum. One genomic fragment harboured a cluster of eight genes which was syntenic and highly homologous to genomic regions in Rhodopseudomonas palustris and Bradyrhizobium japonicum, including a conserved two‐component system. Phylogenetic analysis of the putative response regulator confirmed that this similarity between Rhizobiales and Acidobacteria might be due to a horizontal gene transfer. In total, our data give first insight into the genome content and diversity of the ubiquitously distributed but poorly characterized phylum of Acidobacteria. Furthermore they support the phylogenetic inferences made from 16S rRNA gene libraries, suggesting that Acidobacteria form a broad group in the same sense and with a similar diversity as that of many well‐studied bacterial phyla.
σ28 RNA polymerase regulates hctB, a late developmental gene in Chlamydia
Authors: Hiu Yin Yu Hilda , Tan Ming
Published: October 2003

Chlamydia is predicted to encode two alternative sigma factors that could provide a mechanism for the regulation of gene expression via alternative forms of RNA polymerase. We have demonstrated that σ28, one of these alternative sigma factors, is transcriptionally active. Chlamydial σ28 RNA polymerase was reconstituted from recombinant σ28 protein and core enzyme that was biochemically isolated from chlamydiae. In an in vitro transcription assay, σ28 RNA polymerase transcribed the hctB promoter in a σ28‐dependent manner. Transcription by σ28 RNA polymerase was salt tolerant compared with transcription by σ66 RNA polymerase, the major form of chlamydial RNA polymerase. As hctB encodes a histone‐like protein that is only expressed late in the developmental cycle, our results suggest that σ28 RNA polymerase has a role in the regulation of late gene expression in Chlamydia.
Small heat shock proteins, ClpB and the DnaK system form a functional triade in reversing protein aggregation
Authors: Mogk Axel , Deuerling Elke , Vorderwülbecke Sonja , Vierling Elizabeth , Bukau Bernd
Published: October 2003

Small heat shock proteins (sHsps) can efficiently prevent the aggregation of unfolded proteins in vitro. However, how this in vitro activity translates to function in vivo is poorly understood. We demonstrate that sHsps of Escherichia coli, IbpA and IbpB, co‐operate with ClpB and the DnaK system in vitro and in vivo, forming a functional triade of chaperones. IbpA/IbpB and ClpB support independently and co‐operatively the DnaK system in reversing protein aggregation. A ΔibpABΔclpB double mutant exhibits strongly increased protein aggregation at 42°C compared with the single mutants. sHsp and ClpB function become essential for cell viability at 37°C if DnaK levels are reduced. The DnaK requirement for growth is increasingly higher for ΔibpAB, ΔclpB, and the double ΔibpABΔclpB mutant cells, establishing the positions of sHsps and ClpB in this chaperone triade.
Heat and DNA damage induction of the LexA‐like regulator HdiR from Lactococcus lactis is mediated by RecA and ClpP
Authors: Savijoki Kirsi , Ingmer Hanne , Frees Dorte , Vogensen Finn K. , Palva Airi , Varmanen Pekka
Published: October 2003

The SOS response is a paradigm for bacterial cells response to DNA damage. Yet some bacteria lack a homologue of the SOS regulator, LexA, including the Gram‐positive, Lactococcus lactis. In this organism we have identified a negative transcriptional regulator, HdiR that induces target gene expression both upon DNA damage and heat shock. Gel mobility shift assays revealed that the binding site for HdiR is located within an inverted repeat structure. HdiR is able to carry out a self‐cleavage reaction in vitro at high pHs, while in vivo it undergoes RecA‐dependent self‐cleavage in the presence of a DNA‐damaging agent. Intriguingly, the N‐terminal cleavage product of HdiR retains DNA binding activity, and only when degraded by the Clp protease, is gene expression induced. Thus, the activity of HdiR in response to DNA damage is controlled by sequential proteolysis, involving self‐cleavage and Clp‐dependent degradation of HdiR. During heat‐stress, limited self‐cleavage occurs; however, recA and clpP are still required for full induction of target gene expression. Thus, our data show that common elements are involved in both the DNA damage and the heat‐mediated induction of the HdiR regulon.
Two different mechanisms for ribosome/mRNA interaction in archaeal translation initiation
Authors: Benelli Dario , Maone Enzo , Londei Paola
Published: October 2003

In this study, we have analysed the features of mRNA/ribosome interaction in the thermophilic archeon Sulfolobus solfataricus. Leadered mRNAs endowed with Shine–Dalgarno (SD) motifs formed stable binary complexes with 30S subunits, optimally at high temperature (65–70°C) and without the aid of initiator tRNA (tRNAi) or any factor. ‘Toeprinting’ assays revealed that the SD motifs were necessary and sufficient to direct the 30S subunit to the translation initiation region. Leaderless mRNAs, i.e. mRNAs entirely lacking a 5′‐untranslated region (UTR), did not interact directly with 30S subunits but required the presence of tRNAi, indicating that codon–anticodon pairing was required for positioning the ribosome on the initiation codon. The data suggest that archaea such as Sulfolobus routinely use two distinct mechanisms for translational initiation. SD‐dependent initiation, resembling the pathway prevalent in present‐day bacteria, would operate on distal cistrons of polycistronic mRNAs, whereas ‘leaderless’ initiation, reminiscent of the eukaryotic pathway, would operate on monocistronic mRNAs and on opening cistrons of polycistronic mRNAs.
Genomic analysis in Escherichia coli demonstrates differential roles for polynucleotide phosphorylase and RNase II in mRNA abundance and decay
Authors: Mohanty Bijoy K. , Kushner Sidney R.
Published: October 2003

Previous work has shown that simultaneous inactivation of polynucleotide phosphorylase (PNPase) and RNase II (both 3′→ 5′ exonucleases) in Escherichia coli leads to the loss of cell viability and the accumulation of partially degraded mRNA species. In order to help to distinguish how these two enzymes globally affect the abundance and decay of mRNAs, we have carried out a genome‐wide analysis of the steady‐state levels of E. coli transcripts using deletion mutations in either rnb or pnp. The data show that, in exponentially growing cells, inactivation of PNPase leads to an increase in the steady‐state level of more expressed mRNAs (17.3%) than inactivation of RNase II (7.3%). In contrast, the steady‐state levels of a large number of E. coli mRNAs (31%) are decreased in the absence of RNase II, including almost all the ribosomal protein genes, suggesting that a major function of this enzyme is to protect specific mRNAs from the activity of other ribonucleases. Array data were confirmed by Northern analysis of 12 individual mRNAs. A comparison between the steady‐state levels and the half‐lives of individual mRNAs indicates that there may be a direct interaction between transcription and mRNA decay for some of the transcripts. In addition, results are presented to show significant phenotypic differences between the pnp‐7 point mutant and the pnpΔ683 deletion allele.
amrA encodes a putative membrane protein necessary for maximal exponential phase expression of the Mga virulence regulon in Streptococcus pyogenes
Authors: Ribardo Deborah A. , McIver Kevin S.
Published: October 2003

The transcriptional regulator Mga activates a regulon of virulence genes important for colonization and immune evasion in GAS. Using transposon mutagenesis of a serotype M6 group A streptococcus (GAS) reporter strain KSM148, we have identified an open reading frame (ORF) designated amrA that is required for maximal activation of the Mga regulon during exponential phase. A deletion in amrA, but not in the downstream transcriptionally linked ORF Spy0798, was able to reproduce the phenotype seen in the transposon mutants. Northern analysis for mga and emm transcripts, as well as Western analysis of Mga, confirmed a reduction in mga expression leading to a decrease in transcription of the Mga‐regulated emm in the amrA deletion and transposon mutants. Furthermore, both the amrA deletion mutant and an original transposon mutant could be complemented using amrA expressed from a nisin‐inducible expression system. As amrA is strongly conserved across the sequenced streptococcal M types, and inactivation of amrA in an M3 serotype also resulted in reduction of emm transcripts, the role of amrA does not appear to be serotype specific. Although the specific function of AmrA is unknown, its putative membrane localization and homology to transporters involved in cell wall synthesis suggest a link between growth and virulence gene expression in GAS.
Xanthomonas type III effector XopD targets SUMO‐conjugated proteins in planta
Authors: Hotson Andrew , Chosed Renee , Shu Hongjun , Orth Kim , Mudgett Mary Beth
Published: October 2003

Xanthomonas campestris pathovar vesicatoria (Xcv) uses the type III secretion system (TTSS) to inject effector proteins into cells of Solanaceous plants during pathogenesis. A number of Xcv TTSS effectors have been identified; however, their function in planta remains elusive. Here, we provide direct evidence for a functional role for a phytopathogenic bacterial TTSS effector in planta by demonstrating that the Xcv effector XopD encodes an active cysteine protease with plant‐specific SUMO substrate specificity. XopD is injected into plant cells by the TTSS during Xcv pathogenesis, translocated to subnuclear foci and hydrolyses SUMO‐conjugated proteins in vivo. Our studies suggest that XopD mimics endogenous plant SUMO isopeptidases to interfere with the regulation of host proteins during Xcv infection.
Transcription of σ54‐dependent but not σ28‐dependent flagellar genes in Campylobacter jejuni is associated with formation of the flagellar secretory apparatus
Authors: Hendrixson David R. , DiRita Victor J.
Published: October 2003

We performed a genetic analysis of flagellar regulation in Campylobacter jejuni, from which we elucidated key portions of the flagellar transcriptional cascade in this bacterium. For this study, we developed a reporter gene system for C. jejuni involving astA, encoding arylsulphatase, and placed astA under control of the σ54‐regulated flgDE2 promoter in C. jejuni strain 81‐176. The astA reporter fusion combined with transposon mutagenesis allowed us to identify genes in which insertions abolished flgDE2 expression; genes identified were on both the chromosome and the plasmid pVir. Included among the chromosomal genes were genes encoding a putative sensor kinase and the σ54‐dependent transcriptional activator, FlgR. In addition, we identified specific flagellar genes, including flhA, flhB, fliP, fliR and flhF, that are also required for transcription of flgDE2 and are presumably at the beginning of the C. jejuni flagellar transcriptional cascade. Deletion of any of these genes reduced transcription of both flgDE2 and another σ54‐dependent flagellar gene, flaB, encoding a minor flagellin. Transcription of the σ28‐dependent gene flaA, encoding the major flagellin, was largely unaffected in the mutants. Further examination of flaA transcription revealed significant σ28‐independent transcription and only weak repressive activity of the putative anti‐σ28 factor FlgM. Our study suggests that σ54‐dependent transcription of flagellar genes in C. jejuni is linked to the formation of the flagellar secretory apparatus. A key difference in the C. jejuni flagellar transcriptional cascade compared with other bacteria that use σ28 for transcription of flagellar genes is that a mechanism to repress significantly σ28‐dependent transcription of flaA in flagellar assembly mutants is absent in C. jejuni.
Tag‐mediated fractionation of yeast ribosome populations proves the monomeric organization of the eukaryotic ribosomal stalk structure
Authors: Guarinos Esther , Santos Cruz , Sánchez Arancha , Qiu De‐Yi , Remacha Miguel , Ballesta Juan P. G.
Published: October 2003

The analysis of the not well understood composition of the stalk, a key ribosomal structure, in eukaryotes having multiple 12 kDa P1/P2 acidic protein components has been approached using these proteins tagged with a histidine tail at the C‐terminus. Tagged Saccharomyces cerevisiae ribosomes, which contain two P1 proteins (P1α and P1β) and two P2 proteins (P2α and P2β), were fractionated by affinity chromatography and their stalk composition was determined. Different yeast strains expressing one or two tagged proteins and containing either a complete or a defective stalk were used. No indication of protein dimers was found in the tested strains. The results are only compatible with a stalk structure containing a single copy of each one of the four 12 kDa proteins per ribosome. Ribosomes having an incomplete stalk are found in wild‐type cells. When one of the four proteins is missing, the ribosomes do not carry the three remaining proteins simultaneously, containing only two of them distributed in pairs made of one P1 and one P2. Ribosomes can carry two, one or no acidic protein pairs. The P1α/P2β and P1β/P2α pairs are preferentially found in the ribosome, but they are not essential either for stalk assembly or function.
Cell cycle regulator Cdc14 is expressed during sporulation but not hyphal growth in the fungus‐like oomycete Phytophthora infestans
Authors: Ah Fong Audrey M. V. , Judelson Howard S.
Published: October 2003

Cdc14 proteins are important regulators of mitosis and the cell cycle. These phosphatases have been studied previously only in yeasts and metazoans, which grow by fission or budding. Here we describe a homologue (piCdc14) from the oomycete Phytophthora infestans, a primitive eukaryote lacking a classical cell cycle. PiCdc14 complements a cdc14ts mutant of Saccharomyces cerevisiae and may function like other Cdc14 proteins, but displays a strikingly different pattern of expression. Whereas previously studied Cdc14 genes are constitutively transcribed, piCdc14 is not expressed during normal growth but instead only during asexual sporulation. In transformants of P. infestans expressing a fusion between the piCdc14 promoter and the β‐glucuronidase reporter, expression was first detected in sporangiophore initials, persisted in sporangiophores bearing immature sporangia, and later became restricted to mature sporangia. After germination, expression ended a few hours before the resumption of mitosis in hyphae emerged from the spores. Homology‐dependent silencing experiments supported an essential role of piCdc14 in sporulation. It is proposed that the function of piCdc14 may be to synchronise nuclear behaviour during sporulation and maintain dormancy in spores until germination. These results help illuminate the process of sporulation in oomycetes and the evolution of the cell cycle in eukaryotes.
SetB: an integral membrane protein that affects chromosome segregation in Escherichia coli
Authors: Espeli Olivier , Nurse Pearl , Levine Cindy , Lee Chong , Marians Kenneth J.
Published: October 2003

setB was identified as a high‐copy suppressor of the partition defect of a mutation in parC, encoding one of the subunits of topoisomerase IV. Deletion of this integral inner membrane protein causes a delay in chromosome segregation, whereas its overproduction causes nucleoid disintegration and stretching, leading to a cell division defect. setB deletion mutants also exhibit a synthetic phenotype when combined with mutations that delete the C‐terminal motor domain of the septal ring protein FtsK. SetB localizes in the cell as a helix and interacts with MreB, the bacterial actin homologue, which also forms a helix. These observations suggest that there may be a link between chromosome segregation and cellular infrastructure.
Partition operon expression in the linear plasmid prophage N15 is controlled by both Sop proteins and protelomerase
Authors: Dorokhov Boris D. , Lane David , Ravin Nikolai V.
Published: October 2003

The temperate coliphage N15, unlike most low copy‐number prokaryotic replicons, is maintained as a linear DNA molecule with covalently closed ends. Accurate partitioning of the plasmid prophage is assured by a close homologue of the sop locus of the F plasmid. However, the region upstream of the N15 sopAB genes contains multiple putative promoters, in contrast to F sop whose expression is driven by one negatively autoregulated promoter. In addition, the centromere of N15 is represented by four inverted repeats located at widely separated sites within the region essential for replication and control of lytic functions. We have analysed expression of N15 sop genes. We find that transcription of N15 sop is driven by two major promoters. The first, P1, is similar in sequence and function to the F sop promoter; it is repressed by Sop proteins. The second promoter, P2, is upstream of P1 and is several times stronger. It is insensitive to regulation by Sop proteins but is tightly repressed by protelomerase, the N15 enzyme that completes prophage replication by generating hairpin telomeres. These results establish a regulatory link between the partition system and other processes of N15 maintenance.
New effects of type III effectors
Authors: Innes Roger
Published: October 2003

The enzymatic activities and/or targets of four type III effector proteins from plant pathogens have been reported in a flurry of new papers. In this issue, XopD is shown to remove SUMO groups from host cell proteins, while in previous issues of Molecular Microbiology, HopPtoD2 was shown to function as a tyrosine phosphatase and AvrRpt2 as probably a cysteine protease that targets the host RIN4 protein. Finally, AvrPphB is revealed in a recent Science paper to function as a cysteine protease that targets the host PBS1 kinase. This work is providing some of the first insights into how plant pathogens subvert host cell signalling machinery to cause disease.