bluuebunny/crossref_metadata_2025_split huggingface dataset:
Control of DNA replication initiation by recruitment of an essential initiation protein to the membrane of Bacillus subtilis
Authors: Rokop Megan E. , Auchtung Jennifer M. , Grossman Alan D. 
Published: June 2004

SummaryThe Bacillus subtilis proteins DnaD and DnaB are essential for replication initiation and are conserved in low G+C content Gram‐positive bacteria. Previous work indicated that DnaD and DnaB are involved in helicase loading during the process of restarting stalled replication forks. We have investigated the roles of DnaD and DnaB in replication initiation at oriC in vivo. Using chromatin immunoprecipitation (ChIP), we found that DnaD and DnaB functions are needed to load the replicative helicase at oriC. To investigate further the functions of DnaD and DnaB in replication initiation, we isolated and characterized suppressors of the temperature sensitivity of dnaD and dnaB mutant cells. In both cases, we isolated the identical missense mutation in dnaB, dnaBS371P. Using yeast two‐hybrid analysis, we found that dnaBS371P uncovers a previously undetected physical interaction between DnaD and DnaB. We also found that DnaBS371P constitutively recruits DnaD to the membrane fraction of cells, where DnaB and oriC are enriched. Phenotypes of cells expressing DnaBS371P are consistent with aberrant replication control. We hypothesize that B. subtilis regulates replication initiation by regulating a physical interaction between two proteins essential for helicase loading at chromosomal origins.
Effects of RNA polymerase modifications on transcription‐induced negative supercoiling and associated R‐loop formation
Authors: Broccoli Sonia , Rallu Fabien , Sanscartier Patrick , Cerritelli Susana M. , Crouch Robert J. , Drolet Marc 
Published: June 2004

SummaryTranscription in the absence of topoisomerase I, but in the presence of DNA gyrase, can result in the formation of hypernegatively supercoiled DNA and associated R‐loops. In this paper, we have used several strategies to study the effects of elongation/termination properties of RNA polymerase on such transcription‐induced supercoiling. Effects on R‐loop formation were exacerbated when cells were exposed to translation inhibitors, a condition that stimulated the accumulation of R‐loop‐dependent hypernegative supercoiling. Translation inhibitors were not acting by decreasing (p)ppGpp levels as the absence of (p)ppGpp in spoT relA mutant strains had little effect on hypernegative supercoiling. However, an rpoB mutation leading to the accumulation of truncated RNAs considerably reduced R‐loop‐dependent hypernegative supercoiling. Transcription of an rrnB fragment preceded by a mutated and inactive boxA sequence to abolish the rrnB antitermination system also considerably reduced R‐loop‐dependent supercoiling. Taken together, our results indicate that RNA polymerase elongation/termination properties can have a major impact on R‐loop‐dependent supercoiling. We discuss different possibilities by which RNA polymerase directly or indirectly participates in R‐loop formation in Escherichia coli. Finally, our results also indicate that what determines the steady‐state level of hypernegatively supercoiled DNA in topA null mutants is likely to be complex and involves a multitude of factors, including the status of RNA polymerase, transcription–translation coupling, the cellular level of RNase HI, the status of DNA gyrase and the rate of relaxation of supercoiled DNA.
A pheromone receptor gene, pre‐1, is essential for mating type‐specific directional growth and fusion of trichogynes and female fertility in Neurospora crassa
Authors: Kim Hyojeong , Borkovich Katherine A. 
Published: June 2004

Summary Neurospora crassa is a heterothallic filamentous fungus with two mating types, mat a and mat A. Its mating involves differentiation of female reproductive structures (protoperithecia) and chemotropic growth of female‐specific hyphae (trichogynes) towards a cell of the opposite mating type in a pheromone‐mediated process. In this study, we characterize the pre‐1 gene, encoding a predicted G‐protein‐coupled receptor with sequence similarity to fungal pheromone receptors. pre‐1 is most highly expressed in mat A strains under mating conditions, but low levels can also be detected in mat a strains. Analysis of pre‐1 deletion mutants showed that loss of pre‐1 does not greatly affect vegetative growth, heterokaryon formation or male fertility in either mating type. Protoperithecia from Δpre‐1 mat A mutants do not undergo fertilization; this defect largely stems from an inability of their trichogynes to recognize and fuse with mat a cells. Previous work has demonstrated that the Gα subunit, GNA‐1, and the Gβ protein, GNB‐1, are essential for female fertility in N. crassa. Trichogynes of Δgna‐1 and Δgnb‐1 mutants displayed severe defects in growth towards and fusion with male cells, similar to that of Δpre‐1 mat A strains. However, the female sterility defect of the Δpre‐1 mat A mutant could not be complemented by constitutive activation of gna‐1, suggesting additional layers of regulation. We propose that PRE‐1 is a pheromone receptor coupled to GNA‐1 that is essential for the mating of mat A strains as females, consistent with a role in launching the pheromone response pathway in N. crassa.
Replication fork and SeqA focus distributions in Escherichia coli suggest a replication hyperstructure dependent on nucleotide metabolism
Authors: Molina Felipe , Skarstad Kirsten 
Published: June 2004

SummaryReplication from the origin of Escherichia coli has traditionally been visualized as two replisomes moving away from each other, each containing a leading and a lagging strand polymerase. Fluorescence microscopy studies of tagged polymerases or forks have, however, indicated that the polymerases may be confined to a single location (or a few locations in cells with overlapping replication cycles). Here, we have analysed the exact replication patterns of cells growing with four different growth and replication rates, and compared these with the distributions of SeqA foci. The SeqA foci represent replication forks because the SeqA protein binds to the newly formed hemimethylated DNA immediately following the forks. The results show that pairs of forks originating from the same origin stay coupled for most of the cell cycle and thus support the replication factory model. They also suggest that the factories consisting of four polymerases are, at the time immediately after initiation, organized into higher order structures consisting of eight or 12 polymerases. The organization into replication factories was lost when replication forks experienced a limitation in the supply of nucleotides or when the thymidylate synthetase gene was mutated. These results support the idea that the nucleotide synthesis apparatus co‐localizes with the replisomes forming a ‘hyperstructure’ and further suggest that the integrity of the replication factories and hyperstructures is dependent on nucleotide metabolism.
The Che4 pathway of Myxococcus xanthus regulates type IV pilus‐mediated motility
Authors: Vlamakis Hera C. , Kirby John R. , Zusman David R. 
Published: June 2004

Summary Myxococcus xanthus co‐ordinates cell movement during its complex life cycle using multiple chemotaxis‐like signal transduction pathways. These pathways regulate both type IV pilus‐mediated social (S) motility and adventurous (A) motility. During a search for new chemoreceptors, we identified the che4 operon, which encodes homologues to a MCP (methyl‐accepting chemotaxis protein), two CheWs, a hybrid CheA–CheY, a response regulator and a CheR. Deletion of the che4 operon did not cause swarming or developmental defects in either the wild‐type (A+S+) strain or in a strain sustaining only A motility (A+S–). However, in a strain displaying only S motility (A–S+), deletion of the che4 operon or the gene encoding the response regulator, cheY4, caused enhanced vegetative swarming and prevented aggregation and sporulation. In contrast, deletion of mcp4 caused reduced vegetative swarming and enhanced development compared with the parent strain. Single‐cell analysis of the motility of the A–S+ parent strain revealed a previously unknown inverse correlation between velocity and reversal frequency. Thus, cells that moved at higher velocities showed a reduced reversal frequency. This co‐ordination of reversal frequency and velocity was lost in the mcp4 and cheY4 mutants. The structural components of the S motility apparatus were unaffected in the che4 mutants, suggesting that the Che4 system affects reversal frequency of cells by modulating the function of the type IV pilus.
HrpA, a DEAH‐box RNA helicase, is involved in mRNA processing of a fimbrial operon in Escherichia coli
Authors: Koo Jovanka T. , Choe Juno , Moseley Steve L. 
Published: June 2004

SummaryEndonucleolytic cleavage of mRNA in the daa operon of Escherichia coli is responsible for co‐ordinate regulation of genes involved in F1845 fimbrial biogenesis. Cleavage occurs by an unidentified endoribonuclease, is translation dependent and involves a unique recognition mechanism. Here, we present the results of a genetic strategy used to identify factors involved in daa mRNA processing. We used a reporter construct consisting of the daa mRNA processing region fused to the gene encoding green fluorescent protein (GFP). A mutant defective in daa mRNA processing and expressing high levels of GFP was isolated by flow cytometry. To determine the location of mutations, two different genetic approaches, Hfr crosses and P1 transductions, were used. The mutation responsible for the processing defect was subsequently mapped to the 32 min region of the E. coli chromosome. A putative DEAH‐box RNA helicase‐encoding gene at this position, hrpA, was able to restore the ability of the mutant to cleave daa mRNA. Site‐directed mutagenesis of the hrpA regions predicted to encode nucleotide triphosphate binding and hydrolysis functions abolished the ability of the gene to restore the processing defect in the mutant. We propose that HrpA is a novel enzyme involved in mRNA processing in E. coli.
SepL, a protein required for enteropathogenic Escherichia coli type III translocation, interacts with secretion component SepD
Authors: O'Connell Colin B. , Creasey Elizabeth A. , Knutton Stuart , Elliott Simon , Crowther Lynette J. , Luo Wensheng , Albert M. John , Kaper James B. , Frankel Gad , Donnenberg Michael S. 
Published: June 2004

SummaryEnteropathogenic Escherichia coli (EPEC), an important cause of infantile diarrhoea in the developing world, disrupts host cell microvilli, causes actin rearrangements and attaches intimately to the host cell surface. This characteristic phenotype, referred to as the attaching and effacing (A/E) effect, is encoded on a 36 kb pathogenicity island called the locus of enterocyte effacement (LEE). The LEE includes genes involved in type III secretion and translocation, the eae gene encoding an outer membrane adhesin known as intimin, the tir gene for the translocated intimin receptor, a regulator and various genes of unknown function. Among this last group is sepL. To determine the role of SepL in EPEC pathogenesis, we constructed and tested a non‐polar sepL mutant. We found that this sepL mutant is deficient for A/E and that it secretes markedly reduced quantities of those proteins involved in translocation (EspA, EspB and EspD), but normal levels of those proteins presumed to be effectors (Tir, EspF and EspG). Despite normal levels of secretion, the mutant strain was unable to translocate EspF and Tir into host cells and formed no EspA filaments. Fractionation studies revealed that SepL is a soluble cytoplasmic protein. Yeast two‐hybrid and affinity purification studies indicated that SepL interacts with the LEE‐encoded protein SepD. In contrast to SepL, we found that SepD is required for type III secretion of both translocation and effector proteins. Together, these results demonstrate that SepL has a unique role in type III secretion as a functional component of the translocation system that interacts with an essential element of the secretion machinery.
Quorum‐sensing antiactivator TraM forms a dimer that dissociates to inhibit TraR
Authors: Chen Guozhou , Malenkos James W. , Cha Mee‐Rye , Fuqua Clay , Chen Lingling 
Published: June 2004

SummaryThe quorum‐sensing transcriptional activator TraR of Agrobacterium tumefaciens, which controls the replication and conjugal transfer of the tumour‐inducing (Ti) virulence plasmid, is inhibited by the TraM antiactivator. The crystal structure of TraM reveals this protein to form a homodimer in which the monomer primarily consists of two long coiled α‐helices, and one of the helices from each monomer also bundles to form the dimeric interface. The importance of dimerization is addressed by mutational studies in which disruption of the hydrophobic dimer interface leads to aggregation of TraM. Biochemical studies confirm that TraM exists as a homodimer in solution in equilibrium with the monomeric form, and also establish that the TraM–TraR complex is a heterodimer. Thus, the TraM homodimer undergoes dissociation in forming the antiactivation complex. Combined with the structure of TraR (Zhang et al., 2002, Nature 417: 971–974; Vannini et al., 2002, EMBO J 21: 4393–4401), our structural analysis suggests overlapping interactive surfaces in homodimeric TraM with those in the TraM–TraR complex and a mechanism for TraM inhibition on TraR.
The bacterial signal molecule, ppGpp, regulates Salmonella virulence gene expression
Authors: Pizarro‐Cerdá Javier , Tedin Karsten 
Published: June 2004

SummaryNumerous, overlapping global regulatory systems mediate the environmental signalling controlling the virulence of Salmonella typhimurium. With both extra‐ and intracellular lifestyles, unravelling the mechanisms involved in regulating Salmonella pathogenesis has been complex. Here, we report a factor co‐ordinating environmental signals with global regulators involved in pathogenesis. An S. typhimuriumΔrelAΔspoT strain deficient in guanosine tetraphosphate (ppGpp) synthesis was found to be highly attenuated in vivo and non‐invasive in vitro. The ΔrelAΔspoT strain exhibited severely reduced expression of hilA and invF, encoding major transcriptional activators required for Salmonella pathogenicity island 1 (SPI‐1) gene expression and at least two other pathogenicity islands. None of the growth conditions intended to mimic the intestinal milieu was capable of inducing hilA expression in the absence of ppGpp. However, the expression of global regulators of Salmonella virulence, RpoS and PhoP/Q, and RpoS‐ and PhoP/Q‐dependent, non‐virulence‐related genes was not significantly different from the wild‐type strain. The results indicate that ppGpp plays a central role as a regulator of virulence gene expression in S. typhimurium and implicates ppGpp as a major factor in the environmental and host‐dependent regulation of Salmonella pathogenesis.
Does my partner cause my allergy?
Authors: Schäfer T. , Merkl J. , Klemm E. , Wichmann H.‐E. , Ring J. ,   KORA Study Group
Published: June 2004

Background:  The study of partners can help to understand the impact of environmental influences on the development of allergies. We aimed to test the hypothesis that subjects whose partners have hay fever are at increased risk for the same disease and that the risk increases with the time subjects live together with an affected partner.Methods:  A nested unmatched case–control study was performed in a random sample of 4261 inhabitants, aged 25–74 years, of the City of Augsburg, Germany, and two adjacent counties. Using standardized computer‐assisted face‐to‐face interviews, we determined the risk of doctor‐diagnosed hay fever in subjects who lived together with a partner having the same disease as opposed to subjects living with an unaffected partner. Furthermore, the risk of doctor‐diagnosed hay fever depending on the time the subjects had lived together with an affected partner was calculated.Results:  After adjustment for age, sex, parental predisposition and social status, the risk of hay fever was more than double in subjects who lived together with a partner having the same disease (odds ratio, ORadj., 2.41; 95% confidence interval, CI, 1.48–3.92). If subjects lived together with an affected partner, the risk of developing the disease increased with the time the partners lived together (1–11 years, OR 1; 12–23 years, OR 1.8; 24–35 years, OR 7.4; 36–54 years, OR 13.7).Conclusion:  The risk of developing hay fever is significantly elevated in subjects who live together with a partner having the same disease. The risk further increases with the time the partners live together. This points to important shared environmental influences or behaviours and raises speculations on a transmissible cause.
Alternaria spores at different heights from the ground
Authors: Bergamini B. M. , Grillenzoni S. , Andreoni A. D. , Natali P. , Ranzi A. , Bertolani M. F. 
Published: June 2004

Background:  Alternaria tenuis (Alt) is one of the main allergens in pediatric age. In temperate climates, airborne Alt spores are detectable from May to November with peaks in late summer and autumn. Sensitized children display symptoms even in the absence of airborne Alt spores. Alt spore concentration, as well as pollen, is usually detected by fixed devices located on the roof of a building at a height of 10–20 m. The aim of the current study is to find out whether ground‐level (50 cm) Alt spore concentrations are different from those at roof‐top level, even during low‐concentration periods.Methods:  Alt samples were taken simultaneously using a Hirst fixed volumetric collector (FVC) placed on a 15 m‐high roof and by a portable volumetric collector (PVC). Firstly, the results of FVC and PVC, both placed on the roof‐top, were compared to verify the correlation coefficient of the two samplers. Subsequently, the PVC was placed 50 cm above the ground in a courtyard (30 samplings) and in private green areas (50 samplings). The results were compared by statistical analysis (Student's t‐test or K–S test).Results:  The values of the 20 samples taken jointly in summer time (FVC 195 ± 134 spores/m3; PVC = 134 ± 131 spores/m3) showed a good correlation between the two samplers (r = 0.850; P < 0.01), with a correction factor equal to 1.177.

Thirty samples obtained in summer and winter when the PVC was positioned in an enclosed courtyard directly below the FVC showed no significant difference (PVC, 181 ± 194 spores/m3; FVC, 152 ± 145 spores/m3; P = 0.221).
Fifty samples taken by PVC placed in private green areas in a low‐concentration period, showed significantly higher concentrations than by FVC: PVC, 531 ± 925 spores/m3; FVC, 25 ± 51 spores/m3 (K–S test: P < 0.0001). In particular, 33 samples taken in winter when Alt counts by FVC were <10 spores/m3 still demonstrated highly significant differences: PVC, 398 ± 961 spores/m3; FVC, 2.0 ± 2 spores/m3 (K–S test: P < 0.0001).

Conclusion:  Our results lead to the conclusion that Alt spore concentration is significantly higher at ground level in the presence of vegetation, even when the spore concentration is very low (<10 spores/m3). These results further suggest that the individual's exposure to Alt, especially in the case of children, is underestimated by samples taken at roof‐top level by FVC.
Importance of including Blomia tropicalis in the routine diagnosis of Venezuelan patients with persistent allergic symptoms
Authors: Puccio F. A. , Lynch N. R. , Noga O. , Noda A. , Hagel I. , López E. , López R. , Caraballo L. , Mercado D. , DiPrisco M.C. 
Published: June 2004

Background:  Blomia tropicalis is a common mite found in the house dust of many tropical countries including Venezuela. The prevalence of skin test and specific serum immunoglobulin (Ig)E reactivity to B. tropicalis in Venezuela has not been previously evaluated.Methods:  In the present study we evaluated the skin reactivity by skin prick test and specific IgE by a multiple antigen blot assay, against B. tropicalis and Dermatophagoides pteronyssinus, in a group of 115 subjects who attended the Allergy Clinic of the Institute of Biomedicine, Caracas, Venezuela, and we studied possible cross reactions between similar proteins of these two mites.Results:  One hundred and six patients with persistent allergic respiratory symptoms showed a positive skin prick test to at least one of the mite extracts, with the frequency of positive reactions to B. tropicalis being as high as to D. pteronyssinus. Twelve patients reacted only to D. pteronyssinus and 13 different patients only to B. tropicalis. Specific IgE to each of the mite extracts was found with similar frequency, and the results coincided with the skin test reactivity.Conclusions:  The study indicated the importance of including B. tropicalis in routine diagnostic testing in tropical and sub‐tropical situations.
Antigen characterization of major cork moulds in Suberosis (cork worker's pneumonitis) by immunoblotting
Authors: Winck J. C. , Delgado L. , Murta R. , Lopez M. , Marques J. A. 
Published: June 2004

Background:  We characterized by immunoblotting the antigenicity of the most frequent fungi colonizing cork during its industrial processing, Penicillium glabrum and Chrysonilia sitophila. Penicillium glabrum is the main causative agent of Suberosis, a hypersensitivity pneumonitis of cork workers. Chrysonilia sitophila induces both IgE sensitization and occupational asthma in the wood processing industry.Methods:  Serum‐specific IgG, IgG4 and IgE to P. glabrum and C. sitophila from nine cork workers with hypersensitivity pneumonitis (HP) and seven with asthma (four with occupational asthma) were analysed by immunoblotting.Results:  Both HP and asthmatic patients’ sera showed immunoreactivity to several proteins resolved in the specific immunoblot strips. The frequency of specific IgG recognition to 12–13.5 and 33 kDa proteins of P. glabrum was significantly higher in HP patients. The sera of HP patients had significantly higher specific IgG recognition to 16 and 51–55 kDa proteins of C. sitophila. There was no specific IgE recognition in the sera of HP or asthmatic patients to both fungi.Conclusions:  Different patterns of antibody reactivity to P. glabrum and C. sitophila are seen in cork workers with hypersensitivity pneumonitis or asthma. The 12–13.5 and 33 kDa proteins of P. glabrum and the 16 and 51–55 kDa proteins of C. sitophila may be major antigens in Suberosis.
Efficacy and tolerability of anti‐immunoglobulin E therapy with omalizumab in patients with poorly controlled (moderate‐to‐severe) allergic asthma
Authors: Ayres J. G. , Higgins B. , Chilvers E. R. , Ayre G. , Blogg M. , Fox H. 
Published: June 2004

Background:  Patients with poorly controlled asthma have greater morbidity and mortality. This study evaluated the efficacy and tolerability of omalizumab in patients with poorly controlled, moderate‐to‐severe allergic asthma.Methods:  This was a randomized, open‐label, multicentre, parallel‐group study. A total of 312 patients (12–73 years) receiving ≥400 μg/day (adolescent) or ≥800 μg/day (adult) inhaled beclomethasone dipropionate, or equivalent were included. Patients received best standard care (BSC) with or without omalizumab [at least 0.016 mg/kg/IgE (IU/ml) every 4 weeks] for 12 months.Results:  The annualized mean number of asthma deterioration‐related incidents was reduced from 9.76 with BSC alone (n = 106) to 4.92 per patient‐year with omalizumab (n = 206) (P < 0.001). Mean clinically significant asthma exacerbation rates were 2.86 and 1.12 per patient‐year, respectively (P < 0.001). Omalizumab‐treated patients (41.4%) required rescue medication <1 day/week compared with 20.7% for BSC alone (P < 0.001). Omalizumab improved absolute forced expiratory volume in 1 s (FEV1) compared with BSC alone (2.48 and 2.28l, respectively; P < 0.05) and reduced symptom scores relative to BSC alone (decrease of 6.5 and 0.7 respectively; P < 0.001). Omalizumab was well‐tolerated.Conclusions:  Omalizumab administered as add‐on therapy to BSC benefits patients with poorly controlled, moderate‐to‐severe allergic asthma.