bluuebunny/crossref_metadata_2025 huggingface dataset:
Involvement of multiple DNA elements in iron‐inducible transcription of the ap65‐1 gene in the protozoan parasite Trichomonas vaginalis
Authors: Ong Shiou‐Jeng , Huang Shen‐Chi , Liu Hsing‐Wei , Tai Jung‐Hsiang
Published: June 2004

A reputed iron‐responsive region, which contains multiple nuclear protein‐binding DNA sequences, was shown previously to regulate iron‐inducible transcription of the ap65‐1 gene in the protozoan pathogen, Trichomonas vaginalis. These DNA sequences include two overlapping MYB recognition elements (MRE‐1/MRE‐2r) and three abutted T‐tract elements. Additional nuclear protein‐binding DNA sequences flanking the 5′ (AGTGAAGTGA) and 3′ (MRE‐2f) of the iron‐responsive region were identified in the present study. A stable promoter assay and primer extension revealed that transcriptional activity of the ap65‐1 promoter is iron inducible as well as growth related, being lowest in the early logarithmic phase and highest in the mid‐logarithmic phase. Subsequent mutational analysis of individual DNA elements of the ap65‐1 promoter suggests that closely spaced T‐tract elements together with an intervening GAAGGAAG sequence within the iron‐responsive region are most critical for regulation of overall transcriptional activity, whereas an additional AGTGAAGTGA and MRE‐2f together with an upstream T‐rich region are required for optimal iron‐inducible activity, and the MRE‐1/MRE‐2r overlap is only involved in growth‐related activity. These observations suggest that expression of the ap65‐1 gene is dynamically regulated under various growth conditions via interactions among multiple DNA regulatory elements.
Role of the amino‐terminal GAF domain of the NifA activator in controlling the response to the antiactivator protein NifL
Authors: Martinez‐Argudo Isabel , Little Richard , Dixon Ray
Published: June 2004

The NifA protein from Azotobacter vinelandii belongs to a family of enhancer binding proteins (EBPs) that activate transcription by RNA polymerase containing the sigma factor σ54. These proteins have conserved AAA+ domains that catalyse ATP hydrolysis to drive conformational changes necessary for open complex formation by σ54‐RNA polymerase. The activity of the NifA protein is highly regulated in response to redox and fixed nitrogen through interaction with the antiactivator protein NifL. Binding of NifL to NifA inhibits the ATPase activity of NifA, and this interaction is controlled by the amino‐terminal GAF domain of NifA that binds 2‐oxoglutarate. Mutations conferring resistance to NifL are located in both the GAF and the AAA+ domains of NifA. To investigate the mechanism by which the GAF domain regulates the activity of the AAA+ domain, we screened for second‐site mutations that suppress the NifL‐resistant phenotype of mutations in the AAA+ domain. One suppressor mutation, F119S, in the GAF domain restores inhibition by NifL to an AAA+ domain mutation, E356K, in response to fixed nitrogen but not in response to oxygen. The biochemical properties of this mutant protein are consistent with the in vivo phenotype and demonstrate that interdomain suppression results in sensitivity to inhibition by NifL in the presence of the signal transduction protein GlnK, but not to the oxidized form of NifL. In the absence of an AAA+ domain mutation, the F119S mutation confers hypersensitivity to repression by NifL. Isothermal titration calorimetry demonstrates that this mutation prevents binding of 2‐oxoglutarate to the GAF domain. Our data support a model in which the GAF domain plays an essential role in preventing inhibition by NifL under conditions appropriate for nitrogen fixation. These observations are of general significance in considering how the activities of EBPs are controlled in response to environmental signals.
Biosynthesis of the angiogenesis inhibitor borrelidin by Streptomyces parvulus Tü4055: insights into nitrile formation†
Authors: Olano Carlos , Moss Steven J. , Braña Alfredo F. , Sheridan Rose M. , Math Vidya , Weston Alison J. , Méndez Carmen , Leadlay Peter F. , Wilkinson Barrie , Salas José A.
Published: June 2004

The 18‐membered polyketide macrolide borrelidin exhibits a number of important biological activities, including potent angiogenesis inhibition. This has prompted two recent total syntheses as well as the cloning of the biosynthetic gene cluster from Streptomyces parvulus Tü4055. Borrelidin possesses some unusual structural characteristics, including a cyclopentane carboxylic acid moiety at C17 and a nitrile moiety at C12 of the macrocyclic ring. Nitrile groups are relatively rare in nature, and little is known of their biosynthesis during secondary metabolism. The nitrile group of borrelidin is shown here to arise from the methyl group of a methylmalonyl‐CoA extender unit incorporated during polyketide chain extension. Insertional inactivation of two genes in the borrelidin gene cluster, borI (coding for a cytochrome P450 monooxygenase) and borJ (coding for an aminotransferase), generated borrelidin non‐producing mutants. These mutants accumulated different compounds lacking the C12 nitrile moiety, with the product of the borI‐minus mutant (12‐desnitrile‐12‐methyl‐borrelidin) possessing a methyl group and that of the borJ‐minus mutant (12‐desnitrile‐12‐carboxyl‐borrelidin) a carboxyl group at C12. The former but not the latter was converted into borrelidin when biotransformed by an S. parvulus mutant that is deficient in the biosynthesis of the borrelidin starter unit. This suggests that 12‐desnitrile‐12‐methyl‐borrelidin is a competent biosynthetic intermediate, whereas the carboxylated derivative is a shunt metabolite. Bioconversion of 12‐desnitrile‐12‐methyl‐borrelidin into borrelidin was also achieved in a heterologous system co‐expressing borI and borJ in Streptomyces albus J1074. This bioconversion was more efficient when borK, which is believed to encode a dehydrogenase, was simultaneously expressed with borI and borJ. On the basis of these findings, a pathway is proposed for the formation of the nitrile moiety during borrelidin biosynthesis.
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

The 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

Transcription 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

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

Replication 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

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

Endonucleolytic 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

Enteropathogenic 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

The 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

Numerous, 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.
Chemical Genetic Screening Identifies Sulfonamides That Raise Organellar pH and Interfere with Membrane Traffic
Authors: Nieland Thomas J. F. , Feng Yan , Brown Jing Xu , Chuang Tuan Daniel , Buckett Peter D. , Wang Jin , Xie Xiao‐Song , McGraw Timothy E. , Kirchhausen Tomas , Wessling‐Resnick Marianne
Published: June 2004

Chemical genetics seeks to identify small molecules that afford functional dissection of cell biological pathways. Previous screens for small molecule inhibitors of exocytic membrane traffic yielded the identification and characterization of several compounds that block traffic from the Golgi to the cell surface as well as transport from the endoplasmic reticulum to the Golgi network [Feng et al. Proc Natl Acad Sci USA 2003;100:6469–6474; Yarrow et al. Comb Chem High Throughput Screen 2003;6:279–286; Feng et al. EMBO Reports 2004: in press]. Here, we screened these inhibitors for potential effects on endocytic membrane traffic. Two structurally related sulfonamides were found to be potent and reversible inhibitors of transferrin‐mediated iron uptake. These inhibitors do not block endoplasmic reticulum‐to‐Golgi transport, but do disrupt Golgi‐to‐cell surface traffic. The compounds are members of a novel class of sulfonamides that elevate endosomal and lysosomal pH, down‐regulate cell surface receptors, and impair recycling of internalized transferrin receptors to the plasma membrane. In vitro experiments revealed that the sulfonamides directly inhibit adenosine triphosphate (ATP) hydrolysis by the V‐ATPase and that they also possess a potent proton ionophore activity. While maintenance of organellar pH is known to be a critical factor in both endocytosis and exocytosis, the precise role of acidification, beyond the uncoupling of ligands from their receptors, remains largely unknown. Identification of this novel class of sulfonamide inhibitors provides new chemical tools to better understand the function of organelle pH in membrane traffic and the activity of V‐ATPases in particular.
Fractionation and Characterization of Kinesin II Species in Vertebrate Brain
Authors: Berezuk Matthew A. , Schroer Trina A.
Published: June 2004

Recent research on kinesin motors has outlined the diversity of the superfamily and defined specific cargoes moved by kinesin family (KIF) members. Owing to the difficulty of purifying large amounts of native motors, much of this work has relied on recombinant proteins expressed in vitro. This approach does not allow ready determination of the complement of kinesin motors present in a given tissue, the relative amounts of different motors, or comparison of their native activities. To address these questions, we isolated nucleotide‐dependent, microtubule‐binding proteins from 13‐day chick embryo brain. Proteins were enriched by microtubule affinity purification, then subjected to velocity sedimentation to separate the 20S dynein/dynactin pool from a slower sedimenting KIF containing pool. Analysis of the latter pool by anion exchange chromatography revealed three KIF species: kinesin I (KIF5), kinesin II (KIF3), and KIF1C (Unc104/KIF1). The most abundant species, kinesin I, exhibited the expected long range microtubule gliding activity. By contrast, KIF1C did not move microtubules. Kinesin II, the second most abundant KIF, could be fractionated into two pools, one containing predominantly A/B isoforms and the other containing A/C isoforms. The two motor species had similar activities, powering microtubule gliding at slower speeds and over shorter distances than kinesin I.
Amphiphysin is a Component of Clathrin Coats Formed During Synaptic Vesicle Recycling at the Lamprey Giant Synapse
Authors: Evergren Emma , Marcucci Melissa , Tomilin Nikolay , Löw Peter , Slepnev Vladimir , Andersson Fredrik , Gad Helge , Brodin Lennart , De Camilli Pietro , Shupliakov Oleg
Published: June 2004

Amphiphysin is a protein enriched at mammalian synapses thought to function as a clathrin accessory factor in synaptic vesicle endocytosis. Here we examine the involvement of amphiphysin in synaptic vesicle recycling at the giant synapse in the lamprey. We show that amphiphysin resides in the synaptic vesicle cluster at rest and relocates to sites of endocytosis during synaptic activity. It accumulates at coated pits where its SH3 domain, but not its central clathrin/AP‐2‐binding (CLAP) region, is accessible for antibody binding. Microinjection of antibodies specifically directed against the CLAP region inhibited recycling of synaptic vesicles and caused accumulation of clathrin‐coated intermediates with distorted morphology, including flat patches of coated presynaptic membrane. Our data provide evidence for an activity‐dependent redistribution of amphiphysin in intact nerve terminals and show that amphiphysin is a component of presynaptic clathrin‐coated intermediates formed during synaptic vesicle recycling.
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.
Recent advances in pathogenesis and management of hypereosinophilic syndromes
Authors: Roufosse F. , Cogan E. , Goldman M.
Published: June 2004

Idiopathic hypereosinophilic syndrome is a largely heterogeneous disorder defined until now as persistent marked hypereosinophilia of unknown origin generally complicated by end‐organ damage. Recent studies clearly indicate that many patients fulfilling the diagnostic criteria of this syndrome can now be classified as presenting one of two major disease variants: the myeloproliferative or the lymphocytic variant. Research in cellular and molecular biology has provided firm evidence for the existence of discrete hematological disorders underlying these variants, questioning the pertinence of continued reference to ‘idiopathic’ hypereosinophilic syndrome in such patients. Furthermore, identification of these variants has a number of prognostic and therapeutic implications that must be taken into consideration for adequate management of these patients.
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.
Rupatadine 10 mg and ebastine 10 mg in seasonal allergic rhinitis: a comparison study
Authors: Guadaño E. M. , Serra‐Batlles J. , Meseguer J. , Castillo J. A. , De Molina M. , Valero A. , Picado C. ,   Rupatadine Study Group
Published: June 2004

Background: The aim of this study is to establish the efficacy and safety of rupatadinevsebastine and placebo in the treatment of seasonal allergic rhinitis (SAR). Rupatadine is a new second generation H1‐antihistamine with once‐daily dosing that may provide better control of symptoms than the currently used H1‐receptor blockers because of its dual pharmacological profile (anti‐PAF and anti‐H1).

Methods: In a multicentre study, 250 patients with SAR were included in a double‐blind, randomized, parallel‐group and placebo‐controlled study. Patients received either rupatadine 10 mg, ebastine 10 mg or placebo once daily for 2 weeks. The main efficacy outcome was based on the patient's record of severity of nasal symptoms (sneezing, nasal itching, runny nose and nasal obstruction) and nonnasal symptoms (conjunctival itching, tearing and pharyngeal itching). The daily total symptom score (DTSS) was the mean of the DSS recorded for each of the seven symptoms assessed, and the mean DTSS (mDTSS) was the mean of the DTSS values for each study day.

Results: Significant differences in mDTSS were detected between rupatadine and placebo (33% lower for rupatadine group;P = 0.005) after 2 weeks of treatment. The TSS for rupatadine were 22% lower than for ebastine, although the differences were not statistically significant. No serious adverse events were reported during the study period.

Conclusions: Rupatadine 10 mg once daily was clearly superior to placebo in alleviating the symptoms of SAR over a 2‐week period. In comparison with ebastine, rupatadine shows a trend towards a better profile as regard several secondary efficacy variables.
C4d and/or immunoglobulins deposition in peritubular capillaries in perioperative graft biopsies in ABO‐incompatible renal transplantation
Authors: Kanetsuna Y , Yamaguchi Y , Horita S , Tanabe K , Toma H
Published: June 2004

Abstract:  We evaluated 0 h and/or 1 h graft biopsy specimens from 14 recipients in ABO‐incompatible renal transplantation using immunofluorescence for C4d, IgG, and IgM. All 0 h biopsy specimens revealed negative C4d, IgG, and IgM deposition in peritubular capillaries (PTC). In contrast, 8 of 14 1 h biopsy specimens revealed a positive C4d deposition in PTC. Eight specimens revealed positive IgM staining and seven of them had both C4d and IgM depositions. Three specimens had C4d, IgM, and IgG depositions in PTC. Three of eight patients with C4d deposition and two of six patients without C4d deposition in the 1 h biopsy group suffered from acute rejection within 1 month of transplantation. These findings suggest that complement fragments and immunoglobulin deposition in PTC in ABO‐incompatible renal grafts can start soon after reperfusion, although acute rejection may or may not develop.
Clinicopathological study of vesicoureteral reflux (VUR)‐associated pyelonephritis in renal transplantation
Authors: Ohba K , Matsuo M , Noguchi M , Nishikido M , Koga S , Kanetake H , Nazneen A , Liu D , Razzaque M S , Taguchi T
Published: June 2004

Abstract:  We retrospectively studied the occurrence of vesicoureteral reflux (VUR)‐associated pyelonephritis using renal biopsies obtained from the transplanted kidneys, and correlated the histological changes with clinical parameters. Out of a total of 131 renal biopsies performed between 1990 and 2001 on renal transplant patients at the department of Urology of Nagasaki University Graduate School of Biomedical Sciences, 12 patients showed pyuria more than twice in a single year. Seven of these 12 patients were available for determining VUR by voiding cystourethrography (VCUG). Cystoureterography demonstrated VUR in three of seven studied patients with pyuria. A histopathological examination revealed dilatation of both proximal and distal tubules in renal biopsies of transplant patients with VUR, compared to renal biopsies of transplant patients without VUR, or non‐transplanted patients with thin membrane disease. One of the patients with VUR showed advanced features of chronic pyelonephritis in four consecutive biopsies at different time points, suggesting a late stage of reflux nephropathy in the transplanted kidney. We conclude from our study that the occurrence of VUR‐related pyelonephritis may be one of the important long‐term complications in the survival of renal allografts.
Physico‐chemical characterization and synthesis of neuronally active α‐conotoxins
Authors: Loughnan Marion L. , Alewood Paul F.
Published: June 2004

The high specificity of α‐conotoxins for different neuronal nicotinic acetylcholine receptors makes them important probes for dissecting receptor subtype selectivity. New sequences continue to expand the diversity and utility of the pool of available α‐conotoxins. Their identification and characterization depend on a suite of techniques with increasing emphasis on mass spectrometry and microscale chromatography, which have benefited from recent advances in resolution and capability. Rigorous physico‐chemical analysis together with synthetic peptide chemistry is a prerequisite for detailed conformational analysis and to provide sufficient quantities of α‐conotoxins for activity assessment and structure–activity relationship studies.
Computational approaches to understand α‐conotoxin interactions at neuronal nicotinic receptors
Authors: Dutertre Sébastien , Lewis Richard J.
Published: June 2004

Recent and increasing use of computational tools in the field of nicotinic receptors has led to the publication of several models of ligand–receptor interactions. These models are all based on the crystal structure at 2.7 Å resolution of a protein related to the extracellular N‐terminus of nicotinic acetylcholine receptors (nAChRs), the acetylcholine binding protein. In the absence of any X‐ray or NMR information on nAChRs, this new structure has provided a reliable alternative to study the nAChR structure. We are now able to build homology models of the binding domain of any nAChR subtype and fit in different ligands using docking programs. This strategy has already been performed successfully for the docking of several nAChR agonists and antagonists. This minireview focuses on the interaction of α‐conotoxins with neuronal nicotinic receptors in light of our new understanding of the receptor structure. Computational tools are expected to reveal the molecular recognition mechanisms that govern the interaction between α‐conotoxins and neuronal nAChRs at the molecular level. An accurate determination of their binding modes on the neuronal nAChR may allow the rational design of α‐conotoxin‐based ligands with novel nAChR selectivity.
Structure‐activity relationships of α‐conotoxins targeting neuronal nicotinic acetylcholine receptors
Authors: Millard Emma L. , Daly Norelle L. , Craik David J.
Published: June 2004

α‐Conotoxins that target the neuronal nicotinic acetylcholine receptor have a range of potential therapeutic applications and are valuable probes for examining receptor subtype selectivity. The three‐dimensional structures of about half of the known neuronal specific α‐conotoxins have now been determined and have a consensus fold containing a helical region braced by two conserved disulfide bonds. These disulfide bonds define the two‐loop framework characteristic for α‐conotoxins, CCXmCXnC, where loop 1 comprises four residues (m = 4) and loop 2 between three and seven residues (n = 3, 6 or 7). Structural studies, particularly using NMR spectroscopy have provided an insight into the role and spatial location of residues implicated in receptor binding and biological activity.
Cloning and characterization of the genes encoding toxic lectins in mistletoe (Viscum album L)
Authors: Kourmanova Alma G. , Soudarkina Olga J. , Olsnes Sjur , Kozlov Jurij V.
Published: June 2004

Leaves of mistletoe (Viscum album L) contain three toxic lectins (type 2 ribosome‐inactivating proteins) MLI, MLII, and MLIII, differing in molecular mass and carbohydrate specificity. Clones, containing sequences of three gene variants designated ml1p, ml2p, and ml3p, were obtained using PCR amplification from cDNA and from mistletoe genomic DNA. The quantitative ratio of the ml1p, ml2p, and ml3p genes in genomic DNA was found to be 1.5 : 1 : 4, respectively, whereas the ratio of their mRNA was 50 : 10 : 1. The quantitative prevalence of the ml1p transcript correlates well with the observation that MLI is quantitatively dominant over MLII and MLIII in the mistletoe extract. The sequences of the proteins encoded by the ml1p, ml2p, and ml3p genes are identical to MLI by 98, 88, and 77%, respectively. The similarity to MLI of the amino acid sequence encoded by the gene ml1p, the quantitative prevalent of its mRNA, as well as structural properties of the B‐chain indicate that the gene, ml1p, corresponds to MLI. Western blot analysis of recombinant A‐chains encoded by the three variants of mlp genes with the monoclonal antibody MNA4 having differential affinity to MLI, MLII and MLIII A‐chains suggests that the ml2p and ml3p genes correspond to MLII and MLIII, respectively. Structural differences in the carbohydrate‐binding sites of the B‐subunits of ML1p, ML2p, and ML3p probably explain the difference in sugar specificity of MLI, MLII and MLIII.
Copper‐containing nitrite reductase from Pseudomonas chlororaphis DSM 50135
Authors: Pinho Dora , Besson Stéphane , Brondino Carlos D. , de Castro Baltazar , Moura Isabel
Published: June 2004

The nitrite reductase (Nir) isolated from Pseudomonas chlororaphis DSM 50135 is a blue enzyme, with type 1 and type 2 copper centers, as in all copper‐containing Nirs described so far. For the first time, a direct determination of the reduction potentials of both copper centers in a Cu‐Nir was performed: type 2 copper (T2Cu), 172 mV and type 1 copper (T1Cu), 298 mV at pH 7.6. Although the obtained values seem to be inconsistent with the established electron‐transfer mechanism, EPR data indicate that the binding of nitrite to the T2Cu center increases its potential, favoring the electron‐transfer process. Analysis of the EPR spectrum of the turnover form of the enzyme also suggests that the electron‐transfer process between T1Cu and T2Cu is the fastest of the three redox processes involved in the catalysis: (a) reduction of T1Cu; (b) oxidation of T1Cu by T2Cu; and (c) reoxidation of T2Cu by NO2–. Electrochemical experiments show that azurin from the same organism can donate electrons to this enzyme.