bluuebunny/crossref_metadata_2025 huggingface dataset:
Polyphosphate kinase is a component of the Escherichia coli RNA degradosome
Authors: Blum Erwin , Py Beatrice , Carpousis Agamemnon J. , Higgins Christopher F.
Published: November 2003

The Escherichia coli degradosome is a multienzyme complex with four major protein components: the endoribonuclease RNase E, the exoribonuclease PNPase, the RNA helicase RhlB and enolase. The first three of these proteins are known to have important functions in mRNA processing and degradation. In this work, we identify an additional component of the degradosome, polyphosphate kinase (PPK), which catalyses the reversible polymerization of the γ‐phosphate of ATP into polyphosphate (poly(P)). An E. coli strain deleted for the ppk gene showed increased stability of the ompA mRNA. Purified His‐tagged PPK was shown to bind RNA, and RNA binding was prevented by hydrolysable ATP. Chemical modification of RNA by PPK, for example the addition or removal of 3′ or 5′ terminal phosphates, could not be detected. However, polyphosphate was found to inhibit RNA degradation by the degradosome in vitro. This inhibition was overcome by the addition of ADP, required for the degradation of polyphosphate and for the regeneration of ATP by PPK in the degradosome. Thus, PPK in the degradosome appears to maintain an appropriate microenvironment, removing inhibitory polyphosphate and NDPs and regenerating ATP.
Identification of Staphylococcus aureus virulence genes in a murine model of bacteraemia using signature‐tagged mutagenesis
Authors: Mei Ji‐Min , Nourbakhsh Fahimeh , Ford Charles W. , Holden David W.
Published: November 2003

Signature‐tagged mutagenesis with transposon Tn917 was used to identify genes of Staphylococcus aureus required for virulence in a murine model of bacteraemia. Screening 1248 mutant strains in pools of 96 resulted in the provisional identification of 50 mutants attenuated in virulence. Subsequent individual analysis of many of these mutants confirmed that they are attenuated in virulence. DNA sequence analysis of regions flanking their transposon insertion points revealed that approximately half of them represent genes with unknown function, while most of the remainder are involved in nutrient biosynthesis and cell surface metabolism. Three mutants were found with transposon insertions in different positions in femA, and one mutant had an insertion in femB. Both femA and femB are involved in the formation of cell wall peptidoglycan pentaglycine cross‐bridges. A further mutation occurred in a previously unknown gene that shares significant similarity to femB. Mutations were also obtained in recA and lsp (encoding the S. aureus prolipoprotein signal peptidase). On the basis of sequence similarities to proteins of known function, the products of other genes are probably involved in the synthesis of diaminopimelic acid (a component of peptidoglycan), maintenance of surface adhesins and cell surface membrane transport, showing that many components of the S. aureus cell surface are critical for the survival and replication of this pathogen in blood.
Outer membrane protein 100, a versatile virulence factor of Actinobacillus actinomycetemcomitans
Authors: Asakawa Ryuji , Komatsuzawa Hitoshi , Kawai Toshihisa , Yamada Sakuo , Goncalves Reginaldo Bruno , Izumi Shunsuke , Fujiwara Tamaki , Nakano Yoshio , Suzuki Nao , Uchida Yuushi , Ouhara Kazuhisa , Shiba Hideki , Taubman Martin A. , Kurihara Hidemi , Sugai Motoyuki
Published: November 2003

Actinobacillus actinomycetemcomitans (Aa) is one of the pathogenic bacteria involved in periodontal diseases. We have previously identified six major outer membrane proteins (Omps) of Aa Y4. Among them is an Omp with high molecular mass, designated Omp100, which has homology to a variety of virulence factors. Electron microscopic observation indicated that Omp100 is randomly localized on the cell surface of Aa. Aa Y4 has been shown to adhere and invade KB or normal human gingival keratinocytes. Anti‐Omp100 antibody inhibited 50% of adhesion and 70% of invasion of Aa Y4 to KB cells. An Omp100 knock‐out mutant had a decreased adhesion and invasion efficiency of 60%, compared with that of the wild type. Escherichia coli HB101 expressing Omp100 adhered twofold and invaded 10‐fold more than the wild‐type E. coli HB101. HB101 expressing Omp100 showed resistance to serum by trapping factor H, an inhibitor for C3b, with Omp100. Omp100 induced inflammatory cytokine responses of interleukin (IL)‐8, IL‐6 and tumour necrosis factor (TNF)α in epithelial cells, and induced IL‐1β and TNFα production in mouse macrophages. These results indicate that Omp100 is a versatile virulence factor that may demonstrate potential significance in the onset of periodontal diseases related to Aa.
A CLC‐type chloride channel gene is required for laccase activity and virulence in Cryptococcus neoformans
Authors: Zhu Xudong , Williamson Peter R.
Published: November 2003

Laccase is a major virulence factor required for infection caused by the human pathogenic yeast Cryptococcus neoformans. However, cellular processes involved in the regulation and expression of laccase remain largely unknown in C. neoformans. Here we report the identification of a chloride channel gene CLC‐A which is essential for laccase activity in C. neoformans. CLC‐A shares homology to CLC‐type voltage‐gated chloride channels from other organisms; for example, 63% homology to GEF1, a chloride channel gene from Saccharomyces cerevisiae. A clc‐a mutant, Mlac3, generated by insertional mutagenesis as well as a targeted Δclc‐a mutant produced undetectable laccase in a liquid assay and produced no melanin on asparagine agar containing norepinephrine. Mlac3 was complemented with wild‐type CLC‐A which restored laccase activity and melanin biosynthesis. The clc‐a mutants also showed reduced synthesis of another important virulence factor, capsule, and showed reduced growth at elevated pH. In addition, the clc‐a mutation resulted in attenuated virulence in a mouse cryptococcosis model that was restored by complementation with wild‐type CLC‐A, indicating that the chloride channel plays an important role in the virulence of the organism. Further analysis revealed that the basis for absent laccase expression in the clc‐a mutant was a laccase transcriptional defect that could be restored by adding exogenous copper. In conclusion, our findings show that CLC‐A plays a role in the expression of two important virulence factors, capsule and laccase expression, which are required for virulence of the fungal pathogen.
Identification of an anion‐specific channel in the cell wall of the Gram‐positive bacterium Corynebacterium glutamicum
Authors: Costa‐Riu Noelia , Maier Elke , Burkovski Andreas , Krämer Reinhard , Lottspeich Friedrich , Benz Roland
Published: November 2003

A cation‐selective channel (porin), designated PorA, facilitates the passage of hydrophilic solutes across the cell wall of the mycolic acid‐containing actinomycete Corynebacterium glutamicum. Biochemical and electrophysiological investigations of the cell wall of the mutant strain revealed the presence of an alternative channel‐forming protein. This porin was purified to homogeneity and studied in lipid bilayer membranes. It forms small anion‐selective channels with a diameter of about 1.4 nm and an average single‐channel conductance of about 700 pS in 1 M KCl. The PorBCglut channel could be blocked by citrate in a dose‐dependent manner. This result was in agreement with growth experiments in citrate as sole carbon source where growth in citrate was impaired as compared with growth in other carbon sources. The PorBCglut protein was partially sequenced and based on the resulting amino acid sequence of the corresponding gene, which was designated as porB, was identified as an unannotated 381 bp long open reading frame (ORF) in the published genome sequence of C. glutamicum ATCC13032. PorBCglut contains 126 amino acids with an N‐terminal extension of 27 amino acids. One hundred and thirty‐eight base pairs downstream of porB, we found an ORF that codes for a protein with about 30% identity to PorBCglut, which was named PorCCglut. The arrangement of porB and porC on the chromosome suggested that both genes belong to the same cluster. RT‐PCR from overlapping regions between genes from wild‐type C. glutamicum ATCC 13032 and its ATCC 13032ΔporA mutant demonstrated that this is the case and that porB and porC are cotranscribed. The gene products PorBCglut and PorCCglut represent obviously other permeability pathways for the transport of hydrophilic compounds through the cell wall of C. glutamicum.
Candida glabrata STE12 is required for wild‐type levels of virulence and nitrogen starvation induced filamentation
Authors: Calcagno Ana‐Maria , Bignell Elaine , Warn Peter , Jones Michael D. , Denning David W. , Mühlschlegel Fritz A. , Rogers Thomas R. , Haynes Ken
Published: November 2003

The highly conserved fungal Ste12 transcription factor family of proteins play critical roles in the regulation of many cellular processes including mating, cell wall biosynthesis, filamentation and invasive growth. They are also important mediators of fungal virulence. The Candida glabrata STE12 homologue was cloned. The encoded protein has a single DNA binding homeodomain but lacks both a C2H2 zinc finger DNA binding domain and an apparent Dig1/Dig2 regulatory motif. Candida glabrata STE12 can functionally complement the nitrogen starvation induced filamentation and mating defects of Saccharomyces cerevisiae ste12 mutants. We also show that C. glabrata STE12 is required for nitrogen starvation‐induced filamentation as ste12 mutants rarely produce pseudohyphae on nitrogen depeleted media. Finally we describe a novel murine model of C. glabrata systemic disease and use this to demonstrate that C. glabrata ste12 mutants, although still able to cause disease, are attenuated for virulence compared with STE12 reconstituted strains. Candida glabrata STE12 is therefore the first virulence factor encoding gene to be described in this increasingly important fungal pathogen.
An anti‐repression Fur operator upstream of the promoter is required for iron‐mediated transcriptional autoregulation in Helicobacter pylori
Authors: Delany Isabel , Spohn Gunther , Rappuoli Rino , Scarlato Vincenzo
Published: November 2003

The Fur protein acts as a regulator of iron‐dependent gene transcription in bacteria. In Helicobacter pylori, Fur regulates iron‐activated and iron‐repressed promoters. It also acts as an autoregulatory rheostat of transcription to fine‐tune its own expression in response to iron by binding to three operators at its own promoter Pfur . Using biochemical and genetic analyses, here we show that the distal upstream operator III (centred at −110) is essential for iron regulation of Pfur and functions as an anti‐repression site that is bound by the iron‐free form of Fur to induce transcription. Furthermore, operator I (centred at −50) may have a dual role both as a high‐affinity binding site for Fur and as an UP element. We propose that its role is ensuring that Fur expression is not repressed below a minimum threshold level. Our data supports a novel promoter architecture and mechanism of regulation by Fur.
The nuclear actin‐related protein Act3p/Arp4p of Saccharomyces cerevisiae is involved in transcription regulation of stress genes
Authors: Görzer Irene , Schüller Christoph , Heidenreich Erich , Krupanska Ludmila , Kuchler Karl , Wintersberger Ulrike
Published: November 2003

A mutational analysis of the essential nuclear actin‐related protein of Saccharomyces cerevisiae, Act3p/Arp4p, was performed. The five residues chosen for substitution were amino acids conserved between actin and Act3p/Arp4p, the tertiary structure of which most probably resembles that of actin. Two thermosensitive (ts) mutants, a single and a double point mutant, and one lethal double point mutant were obtained. Both ts mutants were formamide‐sensitive which supports a structural relatedness of Act3p/Arp4p to actin; they were also hypersensitive against hydroxyurea and ultraviolet irradiation pointing to a possible role of Act3p/Arp4p in DNA replication and repair. Their ‘suppressor of Ty’ (SPT) phenotype, ob‐served with another ts mutant of Act3p/Arp4p before, suggested involvement of Act3p/Arp4p in transcription regulation. Accordingly, genome‐wide expression profiling revealed misregulated transcription in a ts mutant of a number of genes, among which increased expression of various stress‐responsive genes (many of them requiring Msn2p/Msn4p for induction) was the most salient result. This provides an explanation for the mutant's enhanced resistance to severe thermal and oxidative stress. Thus, Act3p/Arp4p takes an important part in the repression of stress‐induced genes under non‐stress conditions.
From nose to lung: the regulation behind Streptococcus pneumoniae virulence factors
Authors: Hava David L. , LeMieux Julianna , Camilli Andrew
Published: November 2003

Streptococcus pneumoniae probably possesses a redundant set of factors required for colonization of the nasopharynx and invasive disease, because of its strict relationship with its human host and relatively small genome size (∼2.1 Mb). Nevertheless, transcriptional regulation of genes encoding factors required for in vivo growth is predicted to be important on two fronts: in the transition from carriage to invasive disease and within different microniches of the nasopharynx. The importance of both serotype‐specific and host tissue‐specific virulence factors during infection and disease has been highlighted by the recent identification of novel virulence factors in this organism coupled with the release of complete genome sequences from two strains. These studies add to the foundation of knowledge of classical S. pneumoniae virulence factors such as polysaccharide capsule and pneumolysin, which have well‐documented roles in pathogenesis
RsmY, a small regulatory RNA, is required in concert with RsmZ for GacA‐dependent expression of biocontrol traits in Pseudomonas fluorescens CHA0
Authors: Valverde Claudio , Heeb Stephan , Keel Christoph , Haas Dieter
Published: November 2003

In the plant‐beneficial soil bacterium and biocontrol model organism Pseudomonas fluorescens CHA0, the GacS/GacA two‐component system upregulates the production of biocontrol factors, i.e. antifungal secondary metabolites and extracellular enzymes, under conditions of slow, non‐exponential growth. When activated, the GacS/GacA system promotes the transcription of a small regulatory RNA (RsmZ), which sequesters the small RNA‐binding protein RsmA, a translational regulator of genes involved in biocontrol. The gene for a second GacA‐regulated small RNA (RsmY) was detected in silico in various pseudomonads, and was cloned from strain CHA0. RsmY, like RsmZ, contains several characteristic GGA motifs. The rsmY gene was expressed in strain CHA0 as a 118 nt transcript which was most abundant in stationary phase, as revealed by Northern blot and transcriptional fusion analysis. Transcription of rsmY was enhanced by the addition of the strain's own supernatant extract containing a quorum‐sensing signal and was abolished in gacS or gacA mutants. An rsmA mutation led to reduced rsmY expression, via a gacA‐independent mechanism. Overexpression of rsmY restored the expression of target genes (hcnA, aprA) to gacS or gacA mutants. Whereas mutants deleted for either the rsmY or the rsmZ structural gene were not significantly altered in the synthesis of extracellular products (hydrogen cyanide, 2,4‐diacetylphloroglucinol, exoprotease), an rsmY rsmZ double mutant was strongly impaired in this production and in its biocontrol properties in a cucumber‐Pythium ultimum microcosm. Mobility shift assays demonstrated that multiple molecules of RsmA bound specifically to RsmY and RsmZ RNAs. In conclusion, two small, untranslated RNAs, RsmY and RsmZ, are key factors that relieve RsmA‐mediated regulation of secondary metabolism and biocontrol traits in the GacS/GacA cascade of strain CHA0.
Response to culture aeration mediated by the nitrate and nitrite sensor NarQ of Escherichia coli K‐12
Authors: Stewart Valley , Chen Li‐Ling , Wu Hui‐chung
Published: November 2003

Respiratory enzyme synthesis in enterobacteria is controlled in response to electron acceptor availability. The iron–sulphur protein Fnr and the sensor–regulator proteins ArcB–ArcA control respiratory gene transcription in response to oxygen and quinone pool redox status respectively. The sensor–regulator proteins NarX–NarL and NarQ–NarP control anaerobic respiratory gene expression in response to nitrate and nitrite. Our laboratory recently engineered the lac operon to replace the primary operator O1‐lac with the NarL and NarP protein binding site from the nirB operon. Expression of the lacZ gene from this construct is repressed by nitrate in Nar+ strains. Here, we found that lacZ gene expression was repressed in aerated cultures of narQ+narX null strains. This repression was not observed in narX+narQ+ or narX+narQ null strains. Thus, the NarQ sensor responds to aeration as well as to nitrate and nitrite. The NarX and NarQ sensors are composed of three distinct modules: an amino‐terminal sensory module, a carboxyl‐terminal transmitter module and a central module of unknown function. Experiments with NarX–NarQ hybrid proteins suggest that the NarQ protein central module is necessary for response to aeration. The physiological significance of this additional sensory role for the NarQ sensor remains obscure.
Lsr‐mediated transport and processing of AI‐2 in Salmonella typhimurium
Authors: Taga Michiko E. , Miller Stephen T. , Bassler Bonnie L.
Published: November 2003

The LuxS‐dependent autoinducer AI‐2 is proposed to function in interspecies cell–cell communication in bacteria. In Salmonella typhimurium, AI‐2 is produced and released during exponential growth and is subsequently imported into the bacteria via the Lsr (luxS regulated) ATP binding cassette (ABC) transporter. AI‐2 induces transcription of the lsrACDBFGE operon, the first four genes of which encode the Lsr transport apparatus. In this report, we identify and characterize LsrK, a new protein that is required for the regulation of the lsr operon and the AI‐2 uptake process. LsrK is a kinase that phosphorylates AI‐2 upon entry into the cell. Our data indicate that phosphorylation of AI‐2 results in its sequestration in the cytoplasm. We suggest that phospho‐AI‐2 is the inducer responsible for inactivation of LsrR, the repressor of the lsr operon. We also show that two previously uncharacterized members of the lsr operon, LsrF and LsrG, are necessary for the further processing of phospho‐AI‐2. Transport and processing of AI‐2 could be required for removing the quorum‐sensing signal, conveying the signal to an internal detector and/or scavenging boron.
Polar‐biased localization of the cold stress‐induced RNA helicase, CrhC, in the Cyanobacterium Anabaena sp. strain PCC 7120
Authors: El‐Fahmawi Bassam , Owttrim George W.
Published: November 2003

Shift of the filamentous cyanobacterium, Anabaena sp. strain PCC 7120, from 30°C to 20°C induces expression of a cold shock response gene encoding the RNA helicase CrhC. Subcellular localization using cellular fractionation and membrane purification indicated that CrhC is localized to the plasma membrane with no evidence of a soluble‐cytoplasmic form. Treatment of spheroplasts with trypsin and membrane fractions with various denaturing agents identified CrhC as an integral membrane protein associated with the cytoplasmic face of the plasma membrane. Immunoelectron microscopy confirmed the plasma membrane association of CrhC. Interestingly, a higher specific labelling was observed at the cell poles on the septa between adjacent cells within cell filaments. On a per cell area basis, CrhC localization to the cell pole was 3.5‐ and> 1000‐fold higher than to the lateral portion of the plasma membrane or cytoplasm respectively. In addition, CrhC also localizes to new cell poles forming within a dividing cell. Polar‐biased localization of the CrhC RNA helicase implies a role in RNA metabolism that is plasma membrane associated and preferentially occurs at the cell poles during cyanobacterial response to cold stress.
Characterization of the pathway for transport of the cytoadherence‐mediating protein, PfEMP1, to the host cell surface in malaria parasite‐infected erythrocytes
Authors: Kriek Neline , Tilley Leann , Horrocks Paul , Pinches Robert , Elford Barry C. , Ferguson David J. P. , Lingelbach Klaus , Newbold Chris I.
Published: November 2003

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family of antigenically diverse proteins is expressed on the surface of human erythrocytes infected with the malaria parasite P. falciparum, and mediates cytoadherence to the host vascular endothelium. In this report, we show that export of PfEMP1 is slow and inefficient as it takes several hours to traffic newly synthesized proteins to the erythrocyte membrane. Upon removal by trypsin treatment, the surface‐exposed population of PfEMP1 is not replenished during subsequent culture indicating that there is no cycling of PfEMP1 between the erythrocyte surface and an intracellular compartment. The role of Maurer's clefts as an intermediate sorting compartment in trafficking of PfEMP1 was investigated using immunoelectron microscopy and proteolytic digestion of streptolysin O‐permeabilized parasitized erythrocytes. We show that PfEMP1 is inserted into the Maurer's cleft membrane with the C‐terminal domain exposed to the erythrocyte cytoplasm, whereas the N‐terminal domain is buried inside the cleft. Transfer of PfEMP1 to the erythrocyte surface appears to involve electron‐lucent extensions of the Maurer's clefts. Thus, we have delineated some important aspects of the unusual trafficking mechanism for delivery of this critical parasite virulence factor to the erythrocyte surface.
Reactive oxygen species generated by microbial NADPH oxidase NoxA regulate sexual development in Aspergillus nidulans
Authors: Lara‐Ortíz Teresa , Riveros‐Rosas Héctor , Aguirre Jesús
Published: November 2003

NADPH oxidases (Nox) have been characterized as higher eukaryotic enzymes used deliberately to produce reactive oxygen species (ROS). The recent discovery of new functional members of the Nox family in plants and animals has led to the recognition of the increasing importance of ROS as signals involved in regulation of diverse cellular processes such as defence, growth and signalling. Here, we address the role of NADPH oxidase‐generated ROS in the biology of the filamentous fungus Aspergillus nidulans. We characterize the noxA gene and show that it encodes a member of a novel NADPH oxidase subfamily ubiquitous in lower eukaryotes. Deletion of noxA specifically blocks differentiation of sexual fruit bodies (cleistothecia), without affecting hyphal growth or asexual development. Accordingly, the noxA gene is induced during sexual development, peaking at the time of cleistothecia differentiation and in parallel with the hülle cell‐associated catalase peroxidase gene cpeA. This expression pattern is not dependent on transcription factors SteA and StuA, which are essential for cleistothecia formation. In contrast, noxA‐dependent premature sexual development correlates with noxA derepression in ΔsakA null mutants, connecting stress MAPK signalling to the regulated production of ROS. Using a nitroblue tetrazolium (NBT) assay to detect superoxide, we found that hülle cells and cleistothecia initials produce superoxide in a process inhibited by NADPH oxidase inhibitor DPI and markedly reduced in ΔnoxA mutants. Furthermore, using H2DCFDA, we detected that H2O2 and possibly other ROS are generated in a NoxA‐dependent fashion, mainly in the external walls from cleistothecia initials. The essential role of NoxA‐generated ROS in A. nidulans sexual differentiation and the presence of one or two noxA homologues in all analysed filamentous fungi suggest that NADPH oxidase‐generated ROS play important roles in fungal physiology and differentiation.
Partial characterization of a bipartite begomovirus infecting yellow passion flower in Brazil
Authors: Novaes Q. S. , Freitas‐Astua J. , Yuki V. A. , Kitajima E. W. , Camargo L. E. A. , Rezende J. A. M.
Published: November 2003

During the spring of 2001, approximately 10 000 yellow passion flower plants, from two orchards in the county of Livramento de Nossa Senhora, Bahia State, Brazil, exhibited intense yellow mosaic symptoms and drastic reduction of the leaf lamina and plant development. A large population of whiteflies (Bemisia tabaci) was also found colonizing the plants. All field samples collected tested positive for Passion fruit woodiness virus in DAS‐ELISA. Five out of 20 passion flower plants inoculated with adult whiteflies collected from diseased plants in the field developed symptoms 20–30 days after inoculation. Two of these plants gave a positive reaction in TAS‐ELISA using antiserum against a begomovirus. Degenerated PCR primers amplified viral DNA fragments from the DNA‐A and DNA‐B components of a begomovirus infecting these plants. The fragment corresponding to the core region of the coat protein (DNA‐A) was cloned and sequenced. A phylogenetic analysis placed this begomovirus isolated from passion flower in the same clade of the New World begomoviruses as several other species from Brazil. Based on the symptoms induced by this virus alone, the disease was tentatively named passion flower little leaf mosaic.
Conidial dispersal by Alternaria brassicicola on Chinese cabbage (Brassica pekinensis) in the field and under simulated conditions
Authors: Chen L. Y. , Price T. V. , Park‐Ng Z.
Published: November 2003

This study investigated conidial dispersal in the field, and effects of simulated wind and rain on the dispersal of A. brassicicola on Chinese cabbage (Brassica pekinensis). Spores were sampled using a Burkard volumetric spore sampler and rotorod samplers in a Chinese cabbage crop. Disease incidence in the field was well fitted by a Gompertz curve with an adjusted r2 of >0·99. Conidia of A. brassicicola were trapped in the field throughout the growing season. Peaks of high spore concentrations were usually associated with dry days, shortly after rain, high temperature or high wind speed. Diurnal periodicity of spore dispersal showed a peak of conidia trapped around 10·00 h. The number of conidia trapped at a height of 25 cm above ground level was greater than that at 50, 75 and 100 cm. Conidial dispersal was also studied under simulated conditions in a wind tunnel and a rain simulator. Generalized linear models were used to model these data. The number of conidia caught increased significantly at higher wind speeds and at higher rain intensities. Under simulated wind conditions, the number of conidia dispersed from source plants with wet leaves was only 22% of that for plants with dry leaves. Linear relationships were found between the number of conidia caught and the degree of infection of trap plants.
Predicting effective doses for the joint action of two fungicide applications
Authors: Paveley N. D. , Thomas J. M. , Vaughan T. B. , Havis N. D. , Jones D. R.
Published: November 2003

A function was derived to predict fungicide efficacy when more than one application of a single active ingredient is made to a crop, given parameters describing the dose–response curves of the component single‐spray applications. In the function, a second application is considered to act on that proportion of the total pathogen population which was uncontrollable at the time of the first application (represented by the lower asymptote of the dose–response curve for the first treatment), plus any additional part of the population which survived the first application as a result of a finite dose being applied. Data to estimate the single‐spray dose–response curve parameters and validate predictions of two‐spray programme efficacy were obtained from separate subsets of treatments in four field experiments. A systemic fungicide spray was applied to wheat at a range of doses, at one or both of two times (t1andt2), in all dose combinations. Observed values of the area under the disease progress curve (AUDPC) for septoria leaf blotch (Mycosphaerella graminicola) were used to construct response surfaces of dose att1by dose att2for each culm leaf layer. Parameters were estimated from single‐spray and zero‐dose treatment data only. The model predicted a high proportion (R2 = 71–95%) of the variation in efficacy of the two‐spray programmes. AUDPC isobols showed that the dose required att2was inversely related to the dose att1, but the slope of the relationship varied with the relative timings oft1andt2in relation to culm leaf emergence. Isobols were curved, so the effective dose – the total dose required to achieve a given level of disease suppression – was lower when administered as two applications.
SSR‐based genetic linkage analysis of resistance to crown rust (Puccinia coronata f. sp. lolii) in perennial ryegrass (Lolium perenne)
Authors: Dumsday J. L. , Smith K. F. , Forster J. W. , Jones E. S.
Published: November 2003

Crown rust (caused by Puccinia coronata f. sp. lolii) is a serious foliar disease of the pasture and turfgrass perennial ryegrass (Lolium perenne). Previous genetic studies have detected both qualitative and quantitative resistance mechanisms, and interpretation of the genetic system is complicated by variation within the sexually reproducing pathogen. Resistant and susceptible parental genotypes of ryegrass were identified using a composite urediniospore population collected from three geographically distinct locations. A two‐way pseudo‐testcross mapping population was obtained as the F1 progeny of the pair‐cross between ryegrass parental genotypes Vedette6 and Victorian9. Both parents showed intermediate resistance against a pathogen population collected in a single geographical zone (Hamilton, Victoria), but in the F1 population, significant variation for a range of resistance‐associated characters was detected. Statistical analysis of phenotypic data suggested a major gene effect, hence bulked segregant analysis with map‐assigned simple sequence repeat (SSR) markers was used to scan the genome. A marker showing strong association with resistance was assigned to linkage group (LG) 2 of perennial ryegrass. Analysis of 11 LG2 SSR markers defined an interval between loci xlpssrh03f03 and xlpssrk02e02 as containing the gene or genes (LpPc1) conferring crown rust resistance. Resistance gene determinants were inherited from both parents, with up to 80% of the total phenotypic variation explained by markers segregating from Vedette6 and up to 26% of the variation explained by markers segregating from Victorian9. The two contributions together resulted in an additive increase in effect, with fully resistant individuals requiring determinants from both parents. A conserved syntenic relationship was observed with linkage group B of Avena strigosa, which is the location of a cluster of resistance genes to the oat form of crown rust. The implications of this study for marker‐assisted selection of disease resistance in perennial ryegrass are discussed.
Phoma–Didymella complex on hybrid arctic bramble with wilting symptoms
Authors: Lindqvist‐Kreuze H. , Hellqvist S. , Koponen H. , Valkonen J. P. T.
Published: November 2003

Pycnidia containing conidia characteristic of Phoma spp. and pseudothecia containing ascospores characteristic of Didymella applanata were isolated from edges of expanding stem lesions and dead stems of wilted cultivated hybrid arctic bramble plants (Rubus arcticus nothossp. stellarcticus) in Sweden in 1998 and 1999. The fungi were morphologically similar when grown on culture media, but some differences in growth rate were observed. They were also similar to the reference isolates of D. applanata (anamorph Phoma argillacea). However, they were different from an isolate of Phoma sp. (HPP 38) isolated from cultivated arctic bramble (R. arcticus ssp. arcticus) in Finland in 1980, and from reference isolates of Phoma glomerata isolated from other hosts. Multivariate analysis of growth rate data and conidial dimensions measured in vitro indicated that the fungi isolated from hybrid arctic bramble in Sweden were not distinguishable from D. applanata, but were clearly distinct from P. glomerata and P. exigua. Furthermore, they had identical ITS1 and ITS2 sequences, and were placed in a phylogenetic clade very closely related to the clade that contained isolates of D. applanata isolated from raspberry (Rubus idaeus). In contrast, isolate HPP 38 from Finland was placed in a clade with P. exigua. These data indicate that the Swedish isolates infecting arctic bramble belong to a strain of D. applanata that differs from the isolate infecting raspberry only by two common nucleotide substitutions in ITS2. Fungi of the Phoma–Didymella complex on wild and cultivated arctic bramble (a total of 291 plants showing symptoms sampled from 37 sites) were detected by a PCR‐based assay and found to be common in northern Sweden, but rare, albeit widely distributed, in Finland.
Ovary colonization by Claviceps africana is related to ergot resistance in male‐sterile sorghum lines
Authors: Komolong B. , Chakraborty S. , Ryley M. , Yates D.
Published: November 2003

Ergot, caused by Claviceps africana, has emerged as a serious threat to sorghum hybrid seed production worldwide. In the absence of gene‐for‐gene‐based qualitative resistance in commercial cultivars, varieties with high pollen production that can escape ergot infection are preferred. Recent demonstration of differences in ergot susceptibility among male‐sterile lines has indicated the presence of partial resistance. Using chitin‐specific fluorescin‐isothiocyanate‐conjugated wheat germ agglutin and callose‐specific aniline blue, this study investigated the process of sorghum ovary colonization by C. africana. Conidia germinated within 24 h after inoculation (a.i.); the pathogen was established in the ovary by 79 h a.i., and at least half of the ovary was converted into sphacelial tissue by 120 h a.i. Changes in fungal cell wall chitin content and strategic callose deposition in the host tissue were associated with penetration and invasion of the ovary. The rate of ovary colonization differed in three male‐sterile lines that also differed in ergot susceptibility. This work demonstrates a possible histological basis for partial resistance in male‐sterile sorghum lines that could lay the foundation for variety improvement through further breeding and selection.
Effects of temperature, relative humidity and duration of wetness period on germination and infection by conidia of the pear scab pathogen (Venturia nashicola)
Authors: Li B. , Zhao H. , Li B. , Xu X.‐M.
Published: November 2003

Experiments were conducted to determine the effects of temperature, relative humidity (RH) and duration of wetness period on in vitro germination of conidia and infection of detached pear leaves by Venturia nashicola, the causal agent of pear scab. Conidia germinated only in near‐saturation humidity (RH > 97%). The final percentage germination (24 h after inoculation) at 100% RH without free water was less than half that in free water. Conidia germinated over the range of temperatures tested (5–30°C); the optimum temperature for germination was ≈21°C. Changes in percentage germination of conidia over time were fitted by logistic models at each individual temperature. Polynomial models satisfactorily described the relationships between two (rate and time to 50% of maximum germination) of the three logistic model parameters and temperature. The minimum length of the wetness period for successful infection of detached pear leaves by conidia was observed at several temperatures. The shortest length of wetness period required for infection was 7 h at 22°C. Two polynomial models fitted well the relationship between the minimum wetness duration required for infection, and temperature.
Evaluating Verticillium dahliae for biological control of Ophiostoma novo‐ulmi in Ulmus minor
Authors: Solla A. , Gil L.
Published: November 2003

The ability of isolate Vd‐48 of Verticillium dahliae to induce resistance against subsequent challenge with Ophiostoma novo‐ulmi was examined in Ulmus minor. In the first experiment, conditioning inoculation of 5‐year‐old elm trees (2–3 m in height) with Vd‐48 15 days prior to challenge inoculation with O. novo‐ulmi significantly reduced wilting (P ≤ 0·05) compared with trees not conditioned with Vd‐48. However, in another experiment on 6‐year‐old trees (2–3·5 m in height), no protection was achieved when the length of time between conditioning and challenge inoculations was 45 or 60 days. In a further experiment, inoculations with Vd‐48 alone resulted in severe wilting in 22 out of 118 trees (6–10 years old and 4–7 m in height). Across the 2 years of this last trial, nine trees showed massive feeding wounds made by Scolytus sp., while O. novo‐ulmi strains were isolated from six trees. Vd‐48 provided a variable prophylactic effect against O. novo‐ulmi in U. minor. Major difficulties with this approach to control Dutch elm disease are discussed.
Relative significance of nursery infections and orchard inoculum in the development and spread of apple canker (Nectria galligena) in young orchards
Authors: McCracken A. R. , Berrie A. , Barbara D. J. , Locke T. , Cooke L. R. , Phelps K. , Swinburne T. R. , Brown A. E. , Ellerker B. , Langrell S. R. H.
Published: November 2003

Three nurseries produced apple rootstocks (M9) and budwood (cv. Royal Gala), which they exchanged at the end of the first year. Each nursery then budded its own budwood onto the rootstocks it had produced and that from the other two nurseries. Budded trees were grown on for a further year before being planted at HRI, East Malling in southern England; NIHPBS, Loughgall in Northern Ireland; and ADAS, Rosemaund in the West Midlands of England. Canker development was monitored twice a year. The position of the infected trees within the orchard was recorded, as was the position of the canker on each tree (main‐stem or peripheral). Nectria galligena was isolated from representative cankers and analysed using molecular techniques. At the sites in Northern Ireland and HRI there was a strong positional effect, especially of peripheral cankers, indicating that most of the inoculum was external and had been spread from neighbouring orchards. There was little or no positional effect on main‐stem cankers at any of the three sites. The proportions of different isolates taken from peripheral cankers was different in Northern Ireland from that in England, suggesting different populations associated with the geographic areas. In contrast, the populations of N. galligena obtained from main‐stem cankers were very similar in England and Northern Ireland. It was concluded that a small proportion of trees developing canker were infected during propagation, with no symptom development until after planting. In a second trial it was demonstrated that trees infected during the propagation phase, and particularly at budding and heading back, could develop canker up to 3 years later. While it is clear that some canker developing in the orchard can be associated with the nursery of production, in climatic conditions conducive to the formation and dissemination of conidia, inoculum from surrounding infected orchards is the primary source of the pathogen. Aerial spread is therefore an essential element of the epidemiology of N. galligena, and its control is a crucial part of any canker‐control programme.
Impact of the Cropping Systems of a Minor Dry Season on the Growth, Yields and Nitrogen Uptake of Maize (Zea mays L) Grown in the Humid Tropics during the Major Rainy Season
Authors: Sangakkara U. R. , Richner W. , Steinebrunner F. , Stamp P
Published: November 2003

A field study evaluated the residual effect of a cropping system in two minor (dry) seasons on the productivity pattern and nitrogen utilization of a maize crop grown in the subsequent major (wet) seasons. The cropping systems established in the minor seasons, where evaporation exceeds rainfall, were either monocultures of maize (Zea mays L), or maize intercropped with either common bean (Phaseolus vulgaris L) or sunhemp (Crotolaria juncea L). In addition, monocultures of two green manures, namely sunhemp (Crotolaria juncea) or Tithonia (Tithonia diversifolia), were established. The residues of maize and beans and the green manures were incorporated at the end of the minor season; at the onset of rains in the major season, maize crops were established on the same plots. Germination of maize was not affected by the previous cropping system. In contrast, crop growth and yields of maize and nitrogen utilization were affected by the previous cropping system. Optimum growth and highest yields were procured in maize that was grown after a green manure crop. Similarly, although the yields were high, the planting of a green manure crop reduced nitrogen utilization by maize in the major season, thereby indicating its potential contribution to sustainability, due to its lower mining of soil nitrogen. On the basis of the results of this two‐year study, the impact of cropping systems in minor seasons on the productivity of maize, a very important highland cereal in the tropics, grown under rain‐fed conditions in a major season, is presented.
Suprasellar chordoid glioma combined with Rathke's cleft cyst
Authors: Suh Yeon‐Lim , Kim Na Rae , Kim Jong‐Hyun , Park Sung‐Hye
Published: November 2003

Chordoid glioma has been recently described as a slow‐growing neoplasm with chordoid appearance, occurring exclusively in the regions of the third ventricle and hypothalamus of middle‐aged women. We experienced a case of a  48‐year‐old  woman  with  a  suprasellar  tumor  composed of chordoid glioma and Rathke's cleft cyst, which was confirmed by histopathological, immunohistochemical and electron microscopic examinations. Histologically, chordoid glioma comprised the major part of the tumor, and the prominent Rathke's cleft cysts were distributed focally in the same tumor tissue without any transitions. Chordoid glioma was immunoreactive for glial fibrillary acidic protein, S‐100 protein and vimentin, and focally positive for epithelial membrane antigen and CD34, while cytokeratin highlighted epithelial cells lining Rathke's cleft cysts. Ultrastructural examination of the chordoid glioma revealed short cytoplasmic processes, intermediate filaments, intercellular junctions of zonular adherens type, basal lamina, secretory granules and pinocytic vesicles. The ultrastructural observations of the current case are similar to those of the subcommisural organ, although cell body zonation or microvilli were not evident. The coexistence of chordoid glioma and Rathke's cleft cyst has not been reported previously and may represent a collision tumor.
Correlation between the expression of telomerase reverse transcriptase and proliferative activity in breast cancer cells using an immunocytochemical restaining method
Authors: Ikeda Satoshi , Shibata Toshikatsu , Eishi Yoshinobu , Takizawa Touichiro , Koike Morio
Published: November 2003

Telomerase activity is thought to contribute to the immortality of cancers. Recently, some investigators described a correlation between the activity of telomerase and the proliferative activity of cancer cells. The aim of this study was to evaluate the correlation between the expression of telomerase‐associated protein and proliferative activity. Telomerase reverse transcriptase (TERT) is one of the proteins that correlates with telomerase activity. We investigated TERT protein and its mRNA, and examined the correlation between the TERT protein and Ki‐67, which reflects proliferative activity with immunostaining, and its mRNA, which correlates with telomerase activity, using in situ hybridization. Imprint smears from 17 invasive ductal adenocarcinomas were investigated. In most cases positive for TERT mRNA, the percentage of TERT protein‐positive cells was also high and was closely related to mRNA (P = 0.024). The positive rates of TERT for the cases with lymph node metastasis were significantly higher than those for the cases without metastasis (P = 0.046). The positivity of TERT protein also correlated significantly with the Ki‐67‐positive rate (r = 0.82). As the proliferation activity increased, the number of cells positive for both proteins also increased (r = 0.89). In conclusion, it was suggested that the expression of TERT  protein is associated with the expression of Ki‐67, and is concerned with maintenance of the high proliferative activity in cancer cells with aggressive proliferation.
Hepatobiliary cystadenocarcinoma with cystadenoma elements of the gall bladder in an old man
Authors: Terada Tadashi , Takeuchi Tetsu , Taniguchi Masami
Published: November 2003

Hepatobiliary cystadenoma and cystadenocarcinoma of the gall bladder have rarely been reported. An 88‐year‐old Japanese man was admitted to our clinic because of hypochondralgia and jaundice. Imaging techniques revealed hemobilia and a multilocular cystic tumor in the fundus of the gall bladder, and cholecystectomy was performed. Grossly, the tumor (3.5 × 3 × 3 cm) was multicystic, containing seromucous fluid. The tumor was located in the fibromuscular layer and subserosa of the gall bladder fundus, and protruded into the serosal surface, not into gall bladder lumen. The mucosa appeared free of tumor involvement, and no gall stones were recognized. Microscopically, the tumor was located in the fibromuscular layer, subserosa and tiny focus of the mucosal surface. The tumor consisted of mucin‐rich benign columnar cells, dysplastic mucous cells, malignant papillotubular cells and invasive carcinoma cells. Malignant and atypical tumor cells were located in the center of the tumor and in the tiny area of the mucosal surface, while benign tumor cells were located in the peripheral portions of the tumor and in the serosal side. Neither ovarian stroma‐like mesenchymal stroma nor an oncocytic change in tumor cells was recognized. Non‐tumorous gall bladder showed chronic cholecystitis. Immunohistochemically, benign and carcinoma cells were positive for cytokeratins, epithelial membrane antigen, CA19‐9, MUC1, MUC5AC and MUC6, and carcinoma cells were also positive for carcinoembryonic antigen and p53 protein. The present case indicates that hepatobiliary cystadenocarcinoma without mesenchymal stroma may occur in the gall bladder of old men, and suggests that hepatobiliary cystadenoma without mesenchymal stroma may transform into hepatobiliary cystadenocarcinoma in the gall bladder.
Increased expression of 11β‐hydroxysteroid dehydrogenase type 2 in the lungs of patients with acute respiratory distress syndrome
Authors: Suzuki Satoshi , Tsubochi Hiroyoshi , Ishibashi Hironori , Suzuki Takashi , Kondo Takashi , Sasano Hironobu
Published: November 2003

We  examined  the  immunohistochemical  distribution  of 11β‐hydroxysteroid dehydrogenase type 2 (11β‐HSD2), the enzyme responsible for the conversion of bioactive glucocorticoids to their receptor‐inactive forms, in lung tissue obtained at autopsy from 14 patients who had died due to acute respiratory distress syndrome (ARDS). We found positive immunoreactivity for 11β‐HSD2 in 13 cases. The cells expressing 11β‐HSD2 in the alveolar wall were positive for surfactant apoprotein‐A as well as cytokeratin. Immunoreactivity for 11β‐HSD2 was also detected in the CD68+ cells, which were found in the alveolar spaces. All patients had been treated with glucocorticoids for ARDS and/or the underlying diseases. There was no statistically significant correlation between the use of glucocorticoids and 11β‐HSD2 immunoreactivity in the alveolar wall (P = 0.0729). However, expression of grade + + was found in three out of five patients who received dexamethasone pulse therapy at relatively large doses, as well as in three other patients treated with prednisolone for a long period of time for the underlying disease. An increase in the expression of 11β‐HSD2 may result in faster glucocorticoid breakdown in lung cells in patients with ARDS. Impaired glucocorticoid availability in the lungs of such patients may explain, in part, the fact that glucocorticoid therapy does not always rescue patients with ARDS.