bluuebunny/crossref_metadata_2025 huggingface dataset:
Mosquito larval consumption of toxic arborescent leaf‐litter, and its biocontrol potential
Authors: David J. P. , Tilquin M. , Rey D. , Ravanel P. , Meyran J. C.
Published: June 2003

Abstract.  Previously we described the mosquito larvicidal properties of decomposed leaf‐litter from deciduous trees, especially the alder Alnus glutinosa (L) Gaertn., due to toxic polyphenols and other secondary compounds. To further examine the biocontrol potential of toxic leaf‐litter for mosquito control, feeding rates of third‐instar mosquito larvae were assessed for examples of three genera: Anopheles stephensi Liston, Aedes aegypti (L) and Culex pipiens L. (Diptera: Culicidae).

When immersed in a suspension of non‐toxic leaf‐litter particles (∼0.4 mm), pre‐starved larvae of all three species ingested sufficient material in 30 min to fill the anterior gut lumen (thorax plus two to three abdominal segments). Gut filling peaked after 1–2 h ingestion time, filling the intestine up to six to seven abdominal segments for Ae. aegypti, but maxima of five abdominal segments for Cx. pipiens and An. stephensi.

Using three methods to quantify consumption of three materials by third‐instar larvae of Ae. aegypti, the average amount of leaf‐litter (non‐toxic 0.4 mm particles) ingested during 3 h was determined as ∼20 µg/larva (by dry weight and by lignin spectrophotometric assay). Consumption of humine (∼100 µm particles extracted from leaf‐litter) during 3 h was ∼80 µg/larva for Ae. aegypti, but only ∼30 µg/larva for Cx. pipiens and 15 µg/larva for An. stephensi, with good concordance of determinations by dry weight and by radiometric assay. Cellulose consumption by Ae. aegypti was intermediate: ∼40 µg/larva determined by radiometric assay. Apparent differences between the amounts of these materials ingested by Ae.aegypti larvae (humine four‐fold, cellulose two‐fold more than leaf‐litter) may be attributed to contrasts in palatability (perhaps related to particle size or form), rather than technical discrepancies, because there was good concordance between results of both methods used to determine the amounts of humine and leaf‐litter ingested.

Bioassays of toxic leaf‐litter (decomposed 10 months) with 4‐h exposure period (ingestion time) ranked the order of sensitivity: Ae. aegypti (LC50 < 0.03 g/L) > An. stephensi (LC50 = 0.35 g/L) > Cx. pipiens (LC20 > 0.4 g/L). When immersed in the high concentration of 0.5 g/L toxic leaf‐litter (0.4 mm particles), as little as 15–30 min ingestion time (exposure period) was sufficient to kill the majority of larvae of all three species, as soon as the gut lumen was filled for only the first few abdominal segments. Possibilities for mosquito larval control with toxic leaf‐litter products and the need for standardized ingestion bioassays of larvicidal particles are discussed.
Horizontal transfer of the insect growth regulator pyriproxyfen to larval microcosms by gravid Aedes albopictus and Ochlerotatus triseriatus mosquitoes in the laboratory
Authors: Dell Chism B. , Apperson C. S.
Published: June 2003

Abstract.  The insect growth regulator (IGR) pyriproxyfen is highly active against mosquitoes (Diptera: Culicidae). Through continuous emersion of large larvae (instars 3–4) the concentration causing 50% inhibition of adult emergence (EI50) was determined as 0.200 p.p.b. for Aedes albopictus (Skuse) and 3.5 to 7 times less for Ochlerotatus triseriatus (Say): IE50 0.0288 p.p.b.

As a possible method of application to larval microscosms of these species that oviposit in water containers and phytotelmata, the horizontal transfer of pyriproxyfen to larval microcosms by adult mosquitoes was evaluated under laboratory conditions. Gravid females were forced to walk on surfaces treated with pyriproxyfen (tarsal contact exposure) and then allowed to oviposit in larval microcosms. Using replicate bioassay cages, each with an oviposition container, and a factorial experimental design, we assessed Ae. albopictus for the effects of (i) pyriproxyfen concentration (0.2, 0.3 and 0.4 mg/cm2) contacted by gravid females, and (ii) the number of treated gravid females added to bioassay cages (one, three or five females/cage), on the mortality of larvae in oviposition containers. Only 0.2 mg/cm2 treatment rate was tested on Oc. triseriatus.

A significant (P < 0.05) curvilinear response in inhibition of emergence (IE) was achieved on both species. Densities of one or three treated Oc. triseriatus females/cage yielded IE rates of only 21–27%, whereas five treated females/cage resulted in 70% inhibition. With Ae. albopictus, densities of three or five treated females/cage yielded 48–67% and 59–73% IE, respectively, whereas one treated female/cage gave only 4–30% inhibition.

Use of IGR‐treated oviposition containers to achieve horizontal transfer of pyriproxyfen to mosquito oviposition sites could be a field management technique based on mosquito biology and behaviour. In binary choice tests with Ae. albopictus, horizontal transfer of pyriproxyfen from a container with a treated ovistrip (0.3 or 0.4 mg/cm2) to an untreated microcosm resulted in 14–38% inhibition. In larval bioassays, pyriproxyfen activity declined markedly within 10 days. Forcibly exposing gravid female mosquitoes to pyriproxyfen‐treated paper surface did not affect their fecundity. However, from the 1st to 2nd gonotrophic cycles the egg hatch rate declined by 30% (P < 0.05). Some variation of results could be due to interactions between females at the oviposition site, possibly causing disproportionate transfer of pyriproxyfen to larval microcosms. Comparative studies of the oviposition behaviour of each mosquito are warranted and would potentially provide information needed to improve the technique.
Synergism between permethrin and propoxur against Culex quinquefasciatus mosquito larvae
Authors: Corbel V. , Chandre F. , Darriet F. , Lardeux F. , Hougard J‐M.
Published: June 2003

Abstract.  To see if synergism occurs between carbamate and pyrethroid insecticides, we tested permethrin and propoxur as representatives of these two classes of compounds used for mosquito control. Larvicidal activity of both insecticides was assessed separately and together on a susceptible strain of the mosquito Culex quinquefasciatus (Diptera: Culicidae) by two methods. When mixed at a constant ratio (permethrin : propoxur 1 : 60 based on LC50) and tested at serial concentrations to plot dose/mortality regression, significant synergy occurred between them (co‐toxicity coefficient = 2.2), not just an additive effect. For example, when the mixture gave 50% mortality, the same concentrations of permethrin and propoxur alone would have given merely 2 × 1% mortality. When a sublethal dose (LC0) of permethrin or propoxur was added to the other (range LC10–LC95), synergism occurred up to the LC80 level. Synergistic effects were attributed to the complementary modes of action by these two insecticide classes acting on different components of nerve impulse transmission. Apart from raising new possibilities for Culex control, it seems appropriate to consider using such mixtures or combinations for insecticide‐treated mosquito nets in situations with insecticide‐resistant Anopheles malaria vectors.
Potential vectors of loiasis and other tabanids on the island of Bioko, Equatorial Guinea
Authors: Cheke R. A. , Mas J. , Chainey J. E.
Published: June 2003

Abstract.  The biting flies Chrysops dimidiatus Wulp and C.silaceus Austen (Diptera: Tabanidae), vectors of Loa loa (Cobbold) (Nematoda: Onchocercidae) on the African mainland, were found to be widespread on the island of Bioko (Equatorial Guinea) during 1996–2001. These tabanids were particularly prevalent in the southern part of Bioko, indicating potential transmission of loiasis on the island. The only other tabanids previously recorded on Bioko, Tabanus argenteus Surcouf (from 1915) and Haematopota near heptagramma Speiser (from 1933), were also collected. The possibility of loiasis being endemic on Bioko contra‐indicates ivermectin treatment of onchocerciasis cases, due to risks of adverse side‐effects.
A mechanism by which nitric oxide accelerates the rate of oxidative DNA damage in Escherichia coli
Authors: Woodmansee Anh N. , Imlay James A.
Published: June 2003

The presence of nitric oxide (NO) greatly accelerates the rate at which hydrogen peroxide (H2O2) kills Escherichia coli. Workers have suggested that this effect may be important in the process of bacteriocide by phagocytes. The goal of this study was to determine the mechanism of this synergism. The filamentation of the dead cells, and their protection by cell‐permeable iron chelators, indicated that NO/H2O2 killed cells by damaging their DNA through the Fenton reaction. Indeed, the number of DNA lesions was far greater when NO was present during H2O2 exposure. In the Fenton reaction, free intracellular iron transfers electrons from adventitious donors to H2O2, producing hydroxyl radicals. Although NO damaged the [Fe‐S] clusters of dehydratases, this did not increase the amount of free iron and was therefore not the reason for acceleration of Fenton chemistry. However, NO also blocked respiration, an event that previous studies have shown can stimulate oxidative DNA damage. The resultant accumulation of NADH accelerates the reduction of free flavins by flavin reductase, and these reduced flavins drive Fenton chemistry by transferring electrons to free iron. Indeed, mutants lacking the respiratory quinol oxidases were sensitive to H2O2, and NO did not have any further effect. Further, mutants that lack flavin reductase were resistant to NO/H2O2, and overproducing strains were hypersensitive. We discuss the possibility that H2O2 and NO synergize when macrophages attack captive bacteria.
The interaction of Slt2 MAP kinase with Knr4 is necessary for signalling through the cell wall integrity pathway in Saccharomyces cerevisiae
Authors: Martin‐Yken Helene , Dagkessamanskaia Adilia , Basmaji Fadi , Lagorce Arnaud , Francois Jean
Published: June 2003

In budding yeast, PKC1 plays an essential role in cell integrity and proliferation through a linear MAP (Mitogen Activated Protein) kinase phosphorylation cascade, which ends up with the activation of the Slt2‐MAP kinase by dual phosphorylation on two conserved threonine and tyrosine residues. In this phosphorylated form, Slt2p kinase activates by phosphorylation at least two known downstream targets: Rlm1p, which is implicated in the expression of cell wall‐related genes, and SBF, required for transcription activation of cell cycle‐regulated genes at the G1 to S transition. In this paper, we demonstrate by two‐hybrid, in vitro immunoprecipitation and tandem affinity purification (TAP) methods that Knr4p physically interacts with Slt2p. Moreover, we show that the absence of Knr4p alters proper signalling of Slt2p to its two known downstream targets. In a knr4 null mutant, the SLT2‐dependent activation of Rlm1p is strongly reduced and the transcriptional activity of Rlm1p is decreased, although the phosphorylated form of Slt2p is more abundant than in wild‐type cells. On the contrary, SBF is abnormally activated in this mutant, as shown by a more abundant phosphorylated form of Swi6p, by higher β‐galactosidase levels from a SCB‐lacZ gene fusion, and by deregulation of the cyclic behaviour of several cell cycle‐regulated genes. These results, taken together with our recent finding that Bck2p requires Knr4p to activate additively with Cln3‐Cdc28p SBF target genes, lead to a model in which Knr4p is involved in co‐ordinating the Slt2p‐mediated cell wall integrity pathway with progression of the cell cycle.
Identification of a novel iron regulated staphylococcal surface protein with haptoglobin‐haemoglobin binding activity
Authors: Dryla Agnieszka , Gelbmann Dieter , Von Gabain Alexander , Nagy Eszter
Published: June 2003

Staphylococcus aureus is an extremely adaptable pathogen causing a wide variety of infections. Staphylococcal surface proteins that directly interact with host extracellular proteins greatly contribute to virulence and are involved in adhesion, immune escape and nutrient acquisition. In our extensive search for highly immunogenic, in vivo‐expressed, staphylococcal proteins, previously, we identified a novel member of the family of Gram‐positive anchor motif proteins with a predicted 895 amino acid long sequence. In order to determine the ligand for this novel LPXTG cell wall protein, we employed affinity purification of human plasma using the recombinant form of the protein. Two‐dimensional electrophoresis of eluted plasma proteins identified haptoglobin as a specific binding partner. Importantly, we also observed this specific ligand binding when living S. aureus cells were exposed to biotin‐labelled haptoglobin (Hp) in a FACS‐based assay. Targeted deletion of the gene eliminated Hp‐binding, a function that has not been attributed to S. aureus before. Based on these data we specified the protein as the staphylococcal haptoglobin receptor A (HarA). Similarly to other haptoglobin receptors identified in Gram‐negative pathogens, HarA binds not only Hp, but also haptoglobin‐haemoglobin complexes with an even higher affinity, as demonstrated in in vitro binding assays. Employing specific deletion mutants, ligand binding was localized to two homologous regions with about 145 amino acid residues located within the N‐terminal part of the protein. In addition, we demonstrated that expression of HarA was strictly controlled by iron through the iron‐dependent transcriptional regulator Fur. Based on these data we propose that HarA can be added to the list of staphylococcal virulence factors with a most likely function related to iron acquisition.
Uropathogenic Escherichia coli use d‐serine deaminase to modulate infection of the murine urinary tract
Authors: Roesch Paula L. , Redford Peter , Batchelet Stephanie , Moritz Rebecca L. , Pellett Shahaireen , Haugen Brian J. , Blattner Frederick R. , Welch Rodney A.
Published: June 2003

Although once thought to be unique to bacteria, d‐amino acids are also produced by mammals. For example, d‐serine is excreted in human urine at concentrations ranging from 3.0 to 40 µg ml−1. An epidemiological survey demonstrated that urine isolates of E. coli are more likely to catabolise d‐serine via expression of d‐serine deaminase, DsdA than enteric disease isolates. The urosepsis strain, CFT073, and an isogenic dsdA mutant have similar growth kinetics in minimal or complex media. However, relative to the wild type, the dsdA mutant has a pleiomorphic cell shape and a prolonged, 4–6 h lag phase when grown in human urine. This suggests that d‐serine catabolism provides a growth advantage in the urinary tract. Unexpectedly, in a direct competition model of urinary tract infection, the dsdA mutant was recovered 300‐times more frequently than the wild type in the bladders of mice 48 h after infection. A new model of E. coli uropathogenesis is proposed where growth and gene expression are modulated in response to environmental d‐serine levels. In support of this, the CFT073 dsdA mutant is hyperflagellated and more motile than the wild type indicating that intracellular levels of d‐serine may directly or indirectly influence the expression of regulons associated with E. coli uropathogenesis.
Electron transport to periplasmic nitrate reductase (NapA) of Wolinella succinogenes is independent of a NapC protein
Authors: Simon Jörg , Sänger Monica , Schuster Stephan C. , Gross Roland
Published: June 2003

The rumen bacterium Wolinella succinogenes grows by respiratory nitrate ammonification with formate as electron donor. Whereas the enzymology and coupling mechanism of nitrite respiration is well known, nitrate reduction to nitrite has not yet been examined. We report here that intact cells and cell fractions catalyse nitrate and chlorate reduction by reduced viologen dyes with high specific activities. A gene cluster encoding components of a putative periplasmic nitrate reductase system (napA, G, H, B, F, L, D) was sequenced. The napA gene was inactivated by inserting a kanamycin resistance gene cassette. The resulting mutant did not grow by nitrate respiration and did not reduce nitrate during growth by fumarate respiration, in contrast to the wild type. An antigen was detected in wild‐type cells using an antiserum raised against the periplasmic nitrate reductase (NapA) from Paracoccus pantotrophus. This antigen was absent in the W. succinogenes napA mutant. It is concluded that the periplasmic nitrate reductase NapA is the only respiratory nitrate reductase in W. succinogenes, although a second nitrate‐reducing enzyme is apparently induced in the napA mutant. The nap cluster of W. succinogenes lacks a napC gene whose product is thought to function in quinol oxidation and electron transfer to NapA in other bacteria. The W. succinogenes genome encodes two members of the NapC/NirT family, NrfH and FccC. Characterization of corresponding deletion mutants indicates that neither of these two proteins is required for nitrate respiration. A mutant lacking the genes encoding respiratory nitrite reductase (nrfHA) had wild‐type properties with respect to nitrate respiration. A model of the electron transport chain of nitrate respiration is proposed in which one or more of the napF, G, H and L gene products mediate electron transport from menaquinol to the periplasmic NapAB complex. Inspection of the W. succinogenes genome sequence suggests that ammonia formation from nitrate is catalysed exclusively by periplasmic respiratory enzymes.
Secretion of a soluble colonization factor by the TCP type 4 pilus biogenesis pathway in Vibrio cholerae
Authors: Kirn Thomas J. , Bose Niranjan , Taylor Ronald K.
Published: June 2003

Colonization of the human small intestine by Vibrio cholerae requires the type 4 toxin co‐regulated pilus (TCP). Genes encoding the structure and biogenesis functions of TCP are organized within an operon located on the Vibrio Pathogenicity Island (VPI). In an effort to elucidate the functions of proteins involved in TCP biogenesis, in frame deletions of all of the genes within the tcp operon coding for putative pilus biogenesis proteins have been constructed and the resulting mutants characterized with respect to the assembly and function of TCP. As a result of this analysis, we have identified the product of one of these genes, tcpF, as a novel secreted colonization factor. Chromosomal deletion of tcpF yields a mutant that retains in vitro phenotypes associated with the assembly of functional TCP yet is severely attenuated for colonization of the infant mouse intestine. Furthermore, we have determined that the mechanism by which TcpF is translocated across the bacterial outer membrane requires the TCP biogenesis machinery and is independent of the type II extracellular protein secretion (EPS) system. These results suggest a dual role for the TCP biogenesis apparatus in V. cholerae pathogenesis and a novel mechanism of intestinal colonization mediated by a soluble factor.
Control of rpoS transcription in Escherichia coli and Pseudomonas: why so different?
Authors: Venturi Vittorio
Published: June 2003

In Escherichia coli, the stationary phase alternative sigma factor σs controls the expression of genes involved cell survival in response to cessation of growth (stationary phase) and provides cross‐protection to various stresses. Levels of σs increase dramatically at the onset of stationary phase and are regulated at the transcriptional, post‐transcriptional and post‐translational level, making this one of the most complex regulatory systems in bacteria. The basic mechanisms for the control of translation and σs proteolysis have been understood. However, studies on the transcriptional control in E. coli lag behind and are controversial. The cAMP‐CRP complex and the two component BarA/UvrY system have been implicated and, ppGpp and polyphosphate appear to have a signalling role. σs has also been reported to be a general stress regulator in the fluorescent pseudomonads (Pseudomonas aeruginosa, P. fluorescens and P. putida) and recent studies on σs regulation highlight that transcriptional regulation in these bacteria apparently plays a major role. Global regulatory systems, the GacA/GacS two component system and quorum sensing all affect rpoS expression, as does the TetR family PsrA regulator that directly binds to‐ and activates the rpoS promoter in stationary phase. This striking difference in regulation between E. coli and Pseudomonas can be partly attributed to the differences in the functional role of σs in the two bacterial species. This report will review mainly recent studies on rpoS transcriptional regulation and will try to rationalize the current knowledge into a working model.
Characterization of two alternative promoters for integrase expression in the clc genomic island of Pseudomonas sp. strain B13
Authors: Sentchilo V. , Zehnder A. J. B. , Van Der Meer J. R.
Published: June 2003

The clc genomic island is a 105 kb integrative and conjugative element (ICE) in Pseudomonas sp. strain B13, which encodes metabolism of 3‐chlorocatechol. The clc island is integrated in a tRNAGly gene, but can excise and form a circular intermediate in which both ends are connected. The integrase gene (intB13) of the clc genomic island is located at the right end, 202 bp from the junction site facing inwards. Fragments upstream of intB13 in the circular form and in the integrated form were fused to a promoterless gfp gene for Green Fluorescent Protein and introduced in monocopy onto the chromosome of strain B13. Quantitative GFP fluorescence measurements in individual cells of the different B13‐derivatives revealed that the circular form fragment contained a strong constitutive promoter (Pcirc) driving intB13 expression in all cells. By using primer extension Pcirc could be mapped near the left end of the clc element and Pcirc can therefore only control intB13 expression when left and right ends are connected as in the circular form. Expression from intB13 upstream fragments from the integrated clc element was weaker than that from Pcirc and only occurred in maximally 15% of individual cells in a culture. A promoter (Pint) could be roughly mapped in this region by using reverse‐transcription PCR and by successively shortening the fragment from the 5′ end. Transposon mutants in cloned left end sequences of the clc element were selected which had lost the activation potential on the Pint promoter and those which resulted in overexpression of GFP from Pint. The DNA sequence of the region of the transposon insertions pointed to a relatively well conserved area among various other genomic islands. The activator mutants mapped in an open reading frame (ORF) encoding a 175 amino acid protein without any significant similarity to functionally characterized proteins in the databases.
A novel sheathed surface organelle of the Helicobacter pylori cag type IV secretion system
Authors: Rohde Manfred , Püls Jürgen , Buhrdorf Renate , Fischer Wolfgang , Haas Rainer
Published: June 2003

Type I strains of Helicobacter pylori (Hp) use a type IV secretion system (T4SS), encoded by the cag pathogenicity island (cag‐PAI), to deliver the bacterial protein CagA into eukaryotic cells and to induce interleukin‐8 secretion. Translocated CagA is activated by tyrosine phosphorylation involving Src‐family kinases. The mechanism and structural basis for type IV protein secretion is not well understood. We describe here, by confocal laser scanning microscopy and field emission scanning electron microscopy, a novel filamentous surface organelle which is part of the Hp T4SS. The organelle is often located at one bacterial pole but can be induced by cell contact also along the lateral side of the bacteria. It consists of a rigid needle, covered focally or completely by HP0527 (Cag7 or CagY), a VirB10‐homologous protein. HP0527 is also clustered in the outer membrane. The VirB7‐homologous protein HP0532 is found at the base of this organelle. These observations demonstrate for the first time by microscopic techniques a complex T4SS‐associated, sheathed surface organelle reminiscent to the needle structures of bacterial type III secretion systems.
Transgenic mice expressing the diphtheria toxin receptor are sensitive to the toxin
Authors: Cha Jeong‐Heon , Chang Mee Young , Richardson James A. , Eidels Leon
Published: June 2003

Whereas diphtheria and the mechanism of action of diphtheria toxin, the bacterial molecule that induces the disease, have been studied and understood for some time, the receptor that allows animal cells to bind the toxin escaped identification until recently. The receptor was identified by its ability to confer toxin‐sensitivity to mouse cells, which are normally toxin‐resistant. Although mice are also naturally resistant, we now demonstrate that transgenic mice expressing the diphtheria toxin receptor are as sensitive to the toxin as are humans and other toxin‐sensitive animals. These transgenic mice provide a suitable model for studying modern antidotes for diphtheria.
Interaction between the IncHI1 plasmid R27 coupling protein and type IV secretion system: TraG associates with the coiled‐coil mating pair formation protein TrhB
Authors: Gilmour Matthew W. , Gunton James E. , Lawley Trevor D. , Taylor Diane E.
Published: June 2003

Assemblies of plasmid‐encoded proteins direct the conjugative transfer of plasmid DNA molecules between bacteria. These include the membrane‐associated mating pair formation (Mpf) complex necessary for pilus production and the cytoplasmic relaxosome required for DNA processing. The proposed link between these distinct protein complexes is the coupling protein (the TraG family of proteins). Interactions between the coupling protein and relaxosome components have been previously characterized and we document here, for the first time, a direct interaction between the coupling protein and an Mpf protein. Using the adenylate cyclase bacterial two‐hybrid (BTH) system, we present in vivo evidence that the IncHI1 plasmid R27‐encoded proteins TraG and TrhB interact. This interaction was verified through a co‐immunoprecipitation reaction. We have also been able to delineate the interaction domain of TrhB to TraG by showing a positive interaction using the first 220 amino acids of TrhB (452 aa). TrhB has a proline‐rich domain from amino acids 135–173 which may serve to facilitate protein interactions and/or periplasmic extension. TrhB self association was detected using far‐Western, co‐immunoprecipitation, and also BTH analysis, which was used to define the homotypic interaction domain, comprising a predicted coiled‐coil region at residues 77–124 of TrhB. These data support a model in which the coupling protein interacts with an Mpf component to target the transferring DNA strand held by the relaxosome to the transmembrane Mpf complex.
A binuclear zinc transcription factor binds the host isoflavonoid‐responsive element in a fungal cytochrome p450 gene responsible for detoxification
Authors: Khan Rana , Tan Reynold , Mariscal Amanda Galvez , Straney David
Published: June 2003

The PDA1 gene of the filamentous fungus Nectria haematococca MPVI (anamorph: Fusarium solani) encodes pisatin demethylase, a cytochrome P450. Pisatin is a fungistatic isoflavonoid produced by garden pea (Pisum sativum), a host for this fungus. Pisatin demethylase detoxifies pisatin and functions as a virulence factor for this fungus. Pisatin induces PDA1 expression both in cultured mycelia as well as during pathogenesis on pea. The regulatory element within PDA1 that provides pisatin‐responsive expression was identified using a combination of in vivo functional analysis and in vitro binding analysis. The 40 bp pisatin‐responsive element is located 635 bp upstream of the PDA1 transcription start site. This element was sufficient to provide strong pisatin‐induced expression to a minimal promoter in vivo and was required for pisatin regulation of the PDA1 promoter. A gene encoding a DNA‐binding protein specific to this 40 bp element was isolated from a N. haematococca cDNA library using the yeast one‐hybrid screen. The cloned gene possesses sequence motifs found in the binuclear zinc (Cys 6‐Zn 2) family of transcription factors unique to fungi. The results suggest that it is a regulator of this fungal cytochrome P450 gene and may provide pisatin‐responsive regulation.
Glycerol dehydrogenase, encoded by gldB is essential for osmotolerance in Aspergillus nidulans
Authors: De Vries Ronald P. , Flitter Simon J. , Van De Vondervoort Peter J. I. , Chaveroche Marie‐Kim , Fontaine Thierry , Fillinger Sabine , Ruijter George J. G. , D’Enfert Christophe , Visser Jaap
Published: June 2003

We have characterized the Aspergillus nidulans gldB gene encoding a NADP+‐dependent glycerol dehydrogenase. A basal expression level was observed for gldB, which increased significantly under conditions of hyper‐osmotic shock (1 M NaCl). Growth of strains in which gldB was disrupted was severely reduced on plates containing 1% glucose and 1 M NaCl, but these strains were able to grow on plates containing 1 M NaCl and 1% glycerol, arabitol, mannitol or erythritol. Uptake of these polyols compensated for the inability of the gldB disruptants to produce glycerol. Presence of 1% glucose in these plates prevented growth restoration by all the polyols tested with the exemption of glycerol, indicating that uptake of mannitol, arabitol and erythritol is subject to glucose repression, whereas uptake of glycerol is significantly less or not repressed. No intracellular glycerol dehydrogenase activity could be detected in the gldB disruption strains. Intracellular glycerol levels in these strains were strongly decreased compared to wild type, whereas intracellular mannitol, erythritol and arabitol levels were increased. Conidia of the gldB disruption strain did not accumulate glycerol upon germination in glucose media with or without 1 M NaCl and germ tube emergence was significantly delayed in this strain in the presence of 1 M NaCl in comparison to the wild type. These data indicate that gldB is essential for osmotolerance in A. nidulans and that the pathways for glycerol biosynthesis under osmotic stress differ between yeast and filamentous fungi.
The extracellular proteome of Bacillus subtilis under secretion stress conditions
Authors: Antelmann Haike , Darmon Elise , Noone David , Veening Jan‐Willem , Westers Helga , Bron Sierd , Kuipers Oscar P. , Devine Kevin M. , Hecker Michael , Van Dijl Jan Maarten
Published: June 2003

The accumulation of malfolded proteins in the cell envelope of the Gram‐positive eubacterium Bacillus subtilis was previously shown to provoke a so‐called secretion stress response. In the present studies, proteomic approaches were employed to identify changes in the extracellular proteome of B. subtilis in response to secretion stress. The data shows that, irrespective of the way in which secretion stress is imposed on the cells, the levels of only two extracellular proteins, HtrA and YqxI, display major variations in a parallel manner. Whereas the extracellular level of the HtrA protease is determined through transcriptional regulation, the level of YqxI in the growth medium is determined post‐transcriptionally in an HtrA‐dependent manner. In the absence of secretion stress, the extracellular levels of HtrA and YqxI are low because of extracytoplasmic proteolysis. Finally, the protease active site of HtrA is dispensable for post‐transcriptional YqxI regulation. It is known that Escherichia coli HtrA has combined protease and chaperone‐like activities. As this protein shares a high degree of similarity with B. subtilis HtrA, it can be hypothesized that both activities are conserved in B. subtilis HtrA. Thus, a chaperone‐like activity of B. subtilis HtrA could be involved in the appearance of YqxI on the extracellular proteome.
Identification of additional TnrA‐regulated genes of Bacillus subtilis associated with a TnrA box
Authors: Yoshida Ken‐ichi , Yamaguchi Hirotake , Kinehara Masaki , Ohki Yo‐hei , Nakaura Yoshiko , Fujita Yasutaro
Published: June 2003

Bacillus subtilis TnrA is a global regulator that responds to the availability of nitrogen sources and both activates and represses many genes during nitrogen‐limited growth. In order to obtain a holistic view of the gene regulation depending on TnrA, we performed a genome‐wide screening for TnrA‐regulated genes associated with a TnrA box. A combination of DNA microarray hybridization and a genome‐wide search for TnrA boxes allowed us to find 36 TnrA‐regulated transcription units associated with a putative TnrA box. Gel retardation assaying, using probes carrying at least one putative TnrA box and the deletion derivatives of each box, indicated that 17 out of 36 transcription units were likely TnrA targets associated with the TnrA boxes, two of which (nasA and nasBCDEF) possessed a common TnrA box. The sequences of these TnrA boxes contained a consensus one, TGTNANAWWWTMTNACA. The TnrA targets detected in this study were nrgAB, pucJKLM, glnQHMP, nasDEF, oppABCDF, nasA, nasBCDEF and ywrD for positive regulation, and gltAB, pel, ywdIJK, yycCB, yttA, yxkC, ywlFG, yodF and alsT for negative regulation, nrgAB and gltAB being well‐studied TnrA targets. It was unexpected that the negatively regulated TnrA targets were as many as the positively regulated targets. The physiological role of the TnrA regulon is discussed.
A mutation in the membrane subunit of an ABC transporter LolCDE complex causing outer membrane localization of lipoproteins against their inner membrane‐specific signals
Authors: Narita Shin‐ichiro , Kanamaru Kyoko , Matsuyama Shin‐ichi , Tokuda Hajime
Published: June 2003

Lipoproteins in Gram‐negative bacteria are anchored to the inner or outer membrane via fatty acids attached to the N‐terminal cysteine. The residue at position 2 determines the membrane specificity. An ATP binding cassette transporter LolCDE complex releases lipoproteins with residues other than aspartate at position 2 from the inner membrane, whereas those with aspartate at position 2 are rejected by LolCDE and therefore remain in the inner membrane. For further understanding of this rejection mechanism, a novel strategy was developed to select mutants in which lipoproteins with aspartate at position 2 are released. The isolated mutants carried an alanine to proline mutation at position 40 of LolC, a membrane subunit of the LolCDE complex. A significant portion of an inner membrane lipoprotein, L10P(DQ), was localized to the outer membrane when the LolC mutant was expressed. Periplasmic chaperone LolA formed a complex with the released L10P(DQ), which was subsequently incorporated into the outer membrane in a LolB‐dependent manner, indicating that neither LolA nor LolB rejects lipoproteins with aspartate at position 2. The amount of the LolC mutant co‐purified with LolD and LolE after membrane solubilization was reduced significantly. Taken together, these results indicate that the mutation causes destabilization of the LolCDE complex and concomitantly prevents the accurate recognition of lipoprotein‐sorting signals.
Selective upregulation of a single distinctly structured var gene in chondroitin sulphate A‐adhering Plasmodium falciparum involved in pregnancy‐associated malaria
Authors: Salanti Ali , Staalsoe Trine , Lavstsen Thomas , Jensen Anja T. R. , Sowa M. P. Kordai , Arnot David E. , Hviid Lars , Theander Thor G.
Published: June 2003

Cytoadhesion of infected red blood cells (iRBC) is mediated through parasite‐encoded, clonally variant surface antigens (VSA) and is a central process in the pathogenesis of Plasmodium falciparum malaria. Pregnancy‐associated malaria (PAM) has been linked to VSA‐mediated adhesion of iRBC to the glycosaminoglycan chondroitin sulphate A (CSA) in the placental intervillous space. Several studies have pointed to members of the PfEMP1 VSA family as mediators of CSA‐specific iRBC sequestration in the placenta. Here, we report marked upregulation of a single var gene in several P. falciparum parasite isolates after selection for adhesion to CSA in vitro. The gene belongs to a highly conserved and common var gene subfamily (var2csa). The var2csa genes are structurally distinct from all other var genes in the parasite genome in lacking both CIDR and DBL‐γ domains. These domains have previously been implicated in PfEMP1‐mediated adhesion to CD36 and CSA. We also show that var2csa was transcribed at higher levels in three placental parasite isolates compared with transcription in parasites from peripheral blood of two children with P. falciparum malaria. This var gene thus has the properties expected of a gene encoding the parasite adhesion molecule that initiates the pathology associated with PAM.
Species specificity in the activation of Xer recombination at dif by FtsK
Authors: Yates James , Aroyo Mira , Sherratt David J. , Barre François‐Xavier
Published: June 2003

In Escherichia coli, chromosome dimers are resolved to monomers by the addition of a single cross‐over at a specific locus on the chromosome, dif. Recombination is performed by two tyrosine recombinases, XerC and XerD, and requires the action of an additional protein, FtsK. We show that Haemophilus influenzae FtsK activates recombination by H. influenzae XerCD at H. influenzae dif. However, it cannot activate recombination by E. coli XerCD. Reciprocally, E. coli FtsK cannot activate recombination by the H. influenzae recombinases at H. influenzae dif. We took advantage of this species specificity to gain further insight into the mechanism of activation of Xer recombination at dif by FtsK. We mapped the region of FtsK implicated in species specificity to the extreme 140‐amino‐acid C‐terminal residues of the protein. Our results suggest that FtsK interacts directly with XerCD in order to activate recombination at dif.
Differentiation of Trypanosoma brucei may be stage non‐specific and does not require progression of cell cycle
Authors: Li Yan , Li Ziyin , Wang Ching C.
Published: June 2003

Ubiquitination and proteasomal degradation of cell cycle regulatory proteins are known to play a pivotal role in controlling the progression of the eukaryotic cell cycle. Using the technique of RNA interference (RNAi) on the bloodstream form of Trypanosoma brucei, we were able to knock down expression of each of the 11 non‐ATPase regulatory subunit proteins (Rpns) in the 19S regulatory complex of the 26S proteasome. In each case, the knock‐down led to arrest of cells within the G1 and G2 phases, suggesting blockage of cell cycle progression at both G1/S and G2/M boundaries. This finding differs from that observed previously in the procyclic form of T. brucei, in which loss of individual Rpns blocks only passage across the G2/M boundary. Thus, proteasomal degradation of additional regulatory protein(s) may be required for exiting from G1 phase in the bloodstream form. In vitro differentiation of each of the 11 Rpn‐depleted bloodstream form cell lines into the procyclic form was monitored. Each cell line proceeded to completion of the differentiation process like the wild‐type cells with the total percentage of differentiated cells about equivalent to the sum of G1 and G2 cells. Thus, cells trapped in either G1 or G2 phase can apparently still enter and complete the process of differentiation, which is probably neither stage specific nor dependent on the progression of the T. brucei cell cycle. The process is probably a simple pattern change of gene expression in the trypanosome induced by a temperature decrease from 37°C to 26°C in the presence of citrate and cis‐aconitate.
Sip, an integrase protein with excision, circularization and integration activities, defines a new family of mobile Staphylococcus aureus pathogenicity islands
Authors: Úbeda Carles , Tormo Ma. Ángeles , Cucarella Carme , Trotonda Pilar , Foster Timothy J. , Lasa Íñigo , Penadés José R.
Published: June 2003

We report the complete sequence of Staphylococcal pathogenicity island bovine 2 (SaPIbov2), encoding the biofilm‐associated protein Bap. SaPIbov2 contains 24 open reading frames, including sip, which encodes a functional staphylococcal integrase protein. SaPIbov2 is bordered by 18 bp direct repeats. The integration site into the chromosome lies at the 3′ end of a gene encoding GMP synthase. SaPIbov2 has extensive similarity to previously described pathogenicity islands of Staphylococcus aureus. The principal difference is that toxin genes present in the other pathogenicity islands are exchanged for a transposon‐like element that carries the bap gene and genes encoding an ABC transporter and a transposase. Also, SaPIbov2 can be excised to form a circular element and can integrate site‐specifically and RecA‐independently at a chromosomal att site in a Sip‐dependent manner. This was demonstrated both in S. aureus and with plasmid substrates ectopically in Escherichia coli. Thus, SaPIbov2 encodes a functional recombinase of the integrase family that promotes element excision and insertion/integration. In addition, we demonstrated that the presence of SaPIbov2 facilitated the persistence of S. aureus in an intramammary gland infection model. Finally, different bovine isolates of S. aureus were found to carry islands related to SaPIbov2, suggesting the existence of a family of related pathogenicity islands.
In vivo evidence of TonB shuttling between the cytoplasmic and outer membrane in Escherichia coli
Authors: Larsen Ray A. , Letain Tracy E. , Postle Kathleen
Published: June 2003

Gram‐negative bacteria are able to convert potential energy inherent in the proton gradient of the cytoplasmic membrane into active nutrient transport across the outer membrane. The transduction of energy is mediated by TonB protein. Previous studies suggest a model in which TonB makes sequential and cyclic contact with proteins in each membrane, a process called shuttling. A key feature of shuttling is that the amino‐terminal signal anchor must quit its association with the cytoplasmic membrane, and TonB becomes associated solely with the outer membrane. However, the initial studies did not exclude the possibility that TonB was artifactually pulled from the cytoplasmic membrane by the fractionation process. To resolve this ambiguity, we devised a method to test whether the extreme TonB amino‐terminus, located in the cytoplasm, ever became accessible to the cys‐specific, cytoplasmic membrane‐impermeant molecule, Oregon Green® 488 maleimide (OGM) in vivo. A full‐length TonB and a truncated TonB were modified to carry a sole cysteine at position 3. Both full‐length TonB and truncated TonB (consisting of the amino‐terminal two‐thirds) achieved identical conformations in the cytoplasmic membrane, as determined by their abilities to cross‐link to the cytoplasmic membrane protein ExbB and their abilities to respond conformationally to the presence or absence of proton motive force. Full‐length TonB could be amino‐terminally labelled in vivo, suggesting that it was periplasmically exposed. In contrast, truncated TonB, which did not associate with the outer membrane, was not specifically labelled in vivo. The truncated TonB also acted as a control for leakage of OGM across the cytoplasmic membrane. Further, the extent of labelling for full‐length TonB correlated roughly with the proportion of TonB found at the outer membrane. These findings suggest that TonB does indeed disengage from the cytoplasmic membrane during energy transduction and shuttle to the outer membrane.
VFK1, a Vicia faba K+ channel involved in phloem unloading
Authors: Ache Peter , Becker Dirk , Deeken Rosalia , Dreyer Ingo , Weber Hans , Fromm Jörg , Hedrich Rainer
Published: June 2003

In search of a K+ channel involved in phloem transport we screened a Vicia faba cotyledon cDNA library taking advantage of a set of degenerated primers, flanking regions conserved among K+ uptake channels. We cloned VFK1 (for Vicia faba K+ channel 1) characterised by a structure known from the Shaker family of plant K+ channels. When co‐expressed with a KAT1 mutant in Xenopus oocytes, heteromers revealed the biophysical properties of a K+ selective, proton‐blocked channel. Northern blot analyses showed high levels of expression in cotyledons, flowers, stem and leaves. Using in situ PCR techniques we could localise the K+ channel mRNA in the phloem. In the stem VFK1 expression levels were higher in the lower internodes. There channel transcripts increased in the light and thus under conditions of increased photosynthate allocation. VFK1 transcripts are elevated in sink leaves, and rise in source leaves during the experimental transition into sinks. Fructose‐ rather than sucrose‐ or glucose‐feeding via the petiole induced VFK1 gene activity. We therefore monitored the fructose sensitivity of the sieve tube potential through cut aphid stylets. In response to an 1 h fructose treatment the sieve tube potential shift increased from 19 mV to 53 mV per 10‐fold change in K+ concentration. Under these conditions K+ channels dominated the electrical properties of the plasma membrane. Based on the phloem localisation and expression patterns of VFK1 we conclude that this K+ channel is involved in sugar unloading and K+ retrieval.
Prognostic factors in neonatal tetanus
Authors: Davies‐Adetugbo Anita A. , Davies‐Adetugbo Anita A. , Torimiro S. E. A. , Ako‐Nai K. A.
Published: June 2003

objective
 
 Neonatal tetanus (NNT) is the leading cause of neonatal deaths in developing countries. The objective of this study was to determine prognostic indicators in NNT.methods  We reviewed the clinical records of all neonates (n= 174) admitted to Ife State Hospital with the diagnosis of NNT from 1991 through 1995.results  Delivery had occurred at home in 73.3% of cases. Only 37/164 of the mothers had had adequate immunization with tetanus toxoid. The umbilical cord appeared to be the portal of entry in 58.6% of cases. Mean age of infants at presentation was 7.2 days. Mortality was 57.5%; non‐survivors succumbed after mean stay in the hospital of 5.0 days. Mortality was significantly associated with an incubation period of 6 days or less (P= 0.0026), infant's weight of less than 2.5 kg (P= 0.0113), lack of antenatal care in a health facility (P= 0.0279), birth at home (P= 0.0455), but not with lack of adequate maternal immunization (P = 0.2081; not significant). Multivariable analysis showed that a short (6 d) incubation period was the strongest predictor of mortality (OR = 3.11, P= 0.0030) while low infant weight (< 2.5 kg) was also a significant predictor (OR = 2.46, P= 0.0408).conclusions  Hygienic deliveries and adequate cord care are very important for the prevention of neonatal tetanus deaths, and universal prenatal care, including education programmes on appropriate perinatal and cord care, can significantly reduce NNT incidence and mortality in developing countries.
Intramuscular artemether vs intravenous quinine: an open, randomized trial in Malawian children with cerebral malaria
Authors: Taylor T. E. , Taylor T. E. , Wills B. A. , Courval J. M. , Molyneux M. E. , Molyneux M. E.
Published: June 2003

objectives
 
 To compare artemether (by intramuscular injection) and quinine (by intravenous infusion) as treatments for cerebral malaria in African children.methods  An open, randomized trial conducted at the Queen Elizabeth Central Hospital in Blantyre, Malawi. This trial was part of a multicentre study designed to determine if treatment with artemether would significantly lower mortality rates compared with quinine. Data from 83 artemether recipients and 81 quinine recipients are reported here.results  Overall mortality rates and coma resolution times were not significantly different in the two treatment groups. Parasite and fever clearance times were significantly more rapid in the artemether recipients. Analyses which took into account the possible confounding variables did not significantly alter the findings of these unadjusted analyses.conclusion  These results do not suggest that treatment with artemether would confer a survival advantage in children with life‐threatening malaria. The power and precision of the estimated treatment effects of artemether would be enhanced by a meta‐analysis of all relevant clinical trials.