bluuebunny/crossref_metadata_2025 huggingface dataset:
Libraries and the R&D strategy: a way forward
Authors: HAINES MARGARET
Published: August 2003

As guest editor for the Decembrer 1994 issue of Health Libraries Review, I chose the theme of Evidence‐Based Practice. In my editorial I suggested that Evidence‐based Practice offered tremendous opportunities for NHS librarians to demonstrate their skills in supporting a knowledge‐based NHS, because many clinicians had complained that they did not have the time, retrieval skills or knowledge of relevant information resources to be effective at finding scientific evidence. Librarians, on the other hand have advanced online searching skills, rapid document retrieval and delivery services, and up‐to‐date knowledge of the world's medical information resources and networks. These skills mean that librarians are not only well‐placed to support clinicians in finding and sifting scientific evidence, but also in teaching clinicians how to search for and store information themselves.

NHS librarians have not been slow to recognize these opportunities and innovative professinal development programmes have appeared to help hone their skills, such as the Librarian of the 21st Century Programme in the Anglia and Oxford region. The success of NHS librarians in supporting evidence‐based health care has led to their formal involvement in evidence‐based medicine workshops for clinicians in a number of regions.

In March 1996, as NHS Library Adviser, I was asked to prepare a paper for the R&D Board of the NHS Executive about how this library support could be formally integrated into the R&D Strategy. My paper was unanimously endorsed by the Board and later by the Central R&D Committee of the NHS Executive. It suggests principles of library provision in support of R&D and is reprinted below.
Age‐related changes in cardiac autonomic control during sleep
Authors: Brandenberger Gabrielle , Viola Antoine U. , Ehrhart Jean , Charloux Anne , Geny Bernard , Piquard FranÇois , Simon Chantal
Published: August 2003

Aging is commonly associated with decreased sleep quality and increased periodic breathing (PB) that can influence heart rate variability (HRV). Cardiac autonomic control, as inferred from HRV analysis, was determined, taking into account the sleep quality and breathing patterns. Two groups of 12 young (21.1 ± 0.8 years) and 12 older (64.9 ± 1.9 years) volunteers underwent electroencephalographic, cardiac, and respiratory recordings during one experimental night. Time and frequency domain indices of HRV were calculated in 5‐min segments, together with electroencephalographic and respiratory power spectra. In the elderly, large R–R oscillations in the very‐low frequency (VLF) range emerged, that reflected the frequency of PB observed in 18% of the sleep time. PB occurred more frequently during rapid eye movement sleep (REM) sleep and caused a significant (P < 0.02) increase in the standard deviation of normal R–R intervals (SDNN) and absolute low‐frequency (LF) power. With normal respiratory patterns, SDNN, absolute VLF, LF, and high frequency (HF) power fell during each sleep stage (P < 0.01) compared with young subjects, with no significant sleep‐stage dependent variations. An overall decrease (P < 0.01) in normalized HF/(LF + HF) was observed in the elderly, suggesting a predominant loss of parasympathetic activity which may be related to decreased slow‐wave sleep duration. These results indicate that two distinct breathing features, implying different levels of autonomic drive to the heart, influence HRV in the elderly during sleep. The breathing pattern must be considered to correctly interpret HRV in the elderly.
Underlying sleep pathology may cause chronic high fatigue in shift‐workers
Authors: Hossain J. L. , Reinish L. W. , Kayumov L. , Bhuiya P. , Shapiro C. M.
Published: August 2003

About 20–25% of the population in primary healthcare settings complains of chronic fatigue but this symptom has been under‐emphasized compared with sleepiness in clinical practice. Shift‐workers are particularly vulnerable because of various fatigue‐related personal and public morbidity and mortality. The goal of this cross‐sectional study was to explore if fatigue severity could be used as an independent predictive tool to identify underlying sleep pathology. The 21 most‐fatigued (study group) and 23 least‐fatigued (control) miners were selected on the basis of the Fatigue Severity Scale (FSS), which was administered to 195 subjects in an underground mine in Timmins, a town in northern Ontario. The two groups were matched for age, gender, and body mass index (BMI). Mean FSS score for the most‐fatigued subjects was 4.9 ± 0.5 and the least‐fatigued was 2.2 ± 0.5 (P < 0.0001). The subjects from each group were studied polysomnographically to identify sleep disorders. The polysomnographic data in 15 of 21 (71.4%) of the most‐fatigued subjects displayed significant sleep pathology compared with only three of 23 (13.0%) in the least‐fatigued subjects. Based on Fisher's exact test, the difference between the two groups was highly significant (P < 0.0001). Also, in the total subject pool (n = 195), the correlation between subjective fatigue and sleepiness was not very strong (Pearson's r = 0.45), suggesting that these two symptoms can be independent phenomena. It is concluded that chronic high fatigue can be an independent manifestation of underlying sleep pathology, which warrants independent subjective and objective assessment.
Investigating the interaction between the homeostatic and circadian processes of sleep–wake regulation for the prediction of waking neurobehavioural performance
Authors: Van Dongen Hans P. A. , Dinges David F.
Published: August 2003

The two‐process model of sleep regulation has been applied successfully to describe, predict, and understand sleep–wake regulation in a variety of experimental protocols such as sleep deprivation and forced desynchrony. A non‐linear interaction between the homeostatic and circadian processes was reported when the model was applied to describe alertness and performance data obtained during forced desynchrony. This non‐linear interaction could also be due to intrinsic non‐linearity in the metrics used to measure alertness and performance, however. Distinguishing these possibilities would be of theoretical interest, but could also have important implications for the design and interpretation of experiments placing sleep at different circadian phases or varying the duration of sleep and/or wakefulness. Although to date no resolution to this controversy has been found, here we show that the issue can be addressed with existing data sets. The interaction between the homeostatic and circadian processes of sleep–wake regulation was investigated using neurobehavioural performance data from a laboratory experiment involving total sleep deprivation. The results provided evidence of an actual non‐linear interaction between the homeostatic and circadian processes of sleep–wake regulation for the prediction of waking neurobehavioural performance.
Cross‐resistance between strains of Bacillus sphaericus but not B. thuringiensisisraelensis in colonies of the mosquito Culex quinquefasciatus
Authors: Yuan Z. M. , Pei G. F. , Regis L. , Nielsen‐Leroux C. , Cai Q. X.
Published: August 2003

Abstract.  Two colonies of Culex quinquefasciatus Say (Diptera: Culicidae) were selected with Bacillus sphaericus strains C3‐41 and IAB59 in the laboratory for 13 and 18 generations; they attained 145 000‐ and 48.3‐fold resistance, respectively, in comparison with a susceptible laboratory colony (SLCq) and showed very high levels of cross‐resistance (8500‐ to 145 000‐fold) to B. sphaericus strains C3‐41, 1593, 2297 and 2362. They were relatively susceptible to B. sphaericus strains LP1‐G and 47‐6B (only 0.8‐ to 2.8‐fold tolerance), with 24.8‐ to 48.3‐fold cross‐resistance to strain IAB59. B. sphaericus‐resistant mosquito colonies remained highly susceptible to B. thuringiensisisraelensis, suggesting that B.t.i. would be of value in the management of B. sphaericus‐resistant Cx. quinquefasciatus colonies. The demonstration of low or no cross‐resistance of two selected resistant Cx. quinquefasciatus colonies to IAB59, LP1‐G and 47‐6B strains of B. sphaericus and the finding of a major 49 kDa protein in these strains suggest that there is likely to be another mosquitocidal factor in the three strains.
Effects of decomposition on carcass attendance in a guild of carrion‐breeding flies
Authors: Archer M. S. , Elgar M. A.
Published: August 2003

Abstract.  Many forensically important calliphorids, sarcophagids and muscids (Diptera) oviposit or larviposit on corpses only during the early stages of decomposition, yet individuals may attend bodies throughout decay. A field study was conducted to investigate how patterns of carcass use and attendance by some fly species are affected by decomposition. Five fly traps were placed in the forest and baited with whole, fresh piglet carcasses. Piglets decomposed in traps throughout the experiment, and all were skeletonized within 6 days. Flies were trapped at both early and late decomposition stages, and the species and population structures of trap catches were compared. More flies attended carcasses early rather than late in decay. For all species, flies attending early were mainly gravid females, but few gravid females attended late in decay. No females ovi‐ or larviposited late in decay, whereas females of all fly species deposited offspring early in decay. The number of males trapped of each species correlated positively with the number of females with eggs at early development stages. Observations were made of fly predation by European wasps Vespula germanica Fabricius (Hymenoptera, Vespidae) and jumper ants Myrmecia pilosula Smith (Hymenoptera, Formicidae) throughout the experiment. There was a higher risk for smaller fly species of being killed following predator attack. Ants and wasps attacked smaller fly species, whereas only wasps attacked larger fly species.
Growth characteristics of the veterinary vaccine candidate ChimeriVax™‐West Nile (WN) virus in Aedes and Culex mosquitoes
Authors: Johnson B. W. , Chambers T. V. , Crabtree M. B. , Arroyo J. , Monath T. P. , Miller B. R.
Published: August 2003

Abstract.  In 1999 West Nile (WN) virus was introduced to North America where this flavivirus has spread rapidly among wildlife (especially birds) transmitted by various species of mosquitoes (Diptera: Culicidae). Increasing numbers of cases and deaths among humans, horses and other domestic animals require development of effective vaccines. ‘ChimeriVax™‐West Nile(vet)’ is being developed for use as a veterinary vaccine to protect against WN infection. This chimeric virus contains the pre‐membrane (prM) and envelope (E) genes from the wild‐type WN NY99 virus (isolated from a flamingo in New York zoo during the 1999 WN epidemic) in the backbone of yellow fever (YF) 17D vaccine virus. Replication kinetics of ChimeriVax™‐WN(vet) virus were evaluated in mosquito cell culture (Aedes albopictus C6/36), in WN vector mosquitoes [Culex tritaeniorhynchus Giles, Cx. nigripalpus Theobald and Cx. quinquefasciatus Say (Diptera: Culicidae)] and in YF vectors [Aedes aegypti (L) and Ae. albopictus (Skuse)], to determine whether these mosquitoes become infected through feeding on a viraemic vaccine, and their potential infectivity to transmit the virus. Growth of ChimeriVax™‐WN(vet) virus was found to be restricted in mosquitoes, compared to WN virus in Ae. albopictus C6/36 cells. When inoculated intrathoracically, ChimeriVax™‐WN(vet) and YF 17D viruses did not replicate in Cx. tritaeniorhynchus or Cx. nigripalpus; replication was very restricted compared to the wild‐type WN virus in Cx. quinquefasciatus, Ae. aegypti and Ae. albopictus. When fed on hanging drops with ChimeriVax™‐WN(vet) virus (7.7 log10 PFU/mL), none of the Culex mosquitoes became infected; one Ae. albopictus and 10% of the Ae. aegypti became infected, but the titre was very low and virus did not disseminate to head tissue. ChimeriVax™‐WN(vet) virus had a replication profile similar to that of the attenuated vaccine virus YF 17D, which is not transmitted by mosquitoes. These results suggest that the natural mosquito vectors of WN and YF viruses, which may incidentally take a bloodmeal from a vaccinated host, will not become infected with ChimeriVax™‐WN(vet) virus.
Culicoides midges in Catalonia (Spain), with special reference to likely bluetongue virus vectors
Authors: Monteys V. Sarto I , Saiz‐Ardanaz M.
Published: August 2003

Abstract.  Midges of the genus Culicoides (Diptera: Ceratopogonidae) were trapped weekly from May 2001 through December 2002 on two farms in the province of Barcelona (Spain). Dosrius farm was stocked with sheep and goats whereas Bellaterra farm had only sheep. This trapping programme was carried out within the framework of an investigation into the occurrence/absence of possible vectors of bluetongue virus (BTV) in mainland Spain. A total of 30 079 Culicoides specimens were collected from 165 light trap collections, comprising nine species: C. obsoletus (Meigen), C. pulicaris (Linnaeus), C. circumscriptus Kieffer, C. newsteadi Austen, C. imicola Kieffer, C. scoticus Downes & Kettle, C. punctatus (Meigen), C. pallidicornis Kieffer and C. flavipulicaris Dzhafarov. The last five are new to Catalonia, C. pallidicornis is also new to Spain and C. flavipulicaris is new to Iberia. The seasonal occurrence of these species is described here. Of those species, C. imicola is the main BTV and African horse sickness virus (AHSV) vector in Europe, whereas species belonging to the so‐called Obsoletus group (C. obsoletus and C. scoticus in our study) are considered to be potential vectors for BTV.
Molecular characterization of the mitochondrial cytochrome oxidase I gene of Oestridae species causing obligate myiasis
Authors: Otranto D. , Traversa D. , Guida B. , Tarsitano E. , Fiorente P. , Stevens J. R.
Published: August 2003

Abstract.  A 688‐bp region of the mitochondrial cytochrome oxidase I gene was sequenced from larvae of 18 species of Oestridae causing obligate myiasis. Larvae belonged to the four Oestridae subfamilies (Cuterebrinae, Gasterophilinae, Hypodermatinae and Oestrinae), which are commonly found throughout the world. Analysis of both nucleotide and amino acid data was performed. Nucleotide sequences included 385 conserved sites and 303 variable sites; mean nucleotide variation between all species was 18.1% and variation within each subfamily ranged from 5.3% to 13.34%. Intraspecific pairwise divergences ranged from 0.14% to 1.59%, and interspecific variation ranged from 0.7% to 27%. Of the 229 amino acids, 76 were variable (60 of which were phylogenetically informative), with some highly conserved residues identified within each subfamily. Phylogenetic analysis showed a strong divergence among the four subfamilies, concordant with classical taxonomy based on morphological and biological features. This study provides the first molecular data set for myiasis‐causing Oestridae species, providing an essential database for the molecular identification of these parasites and the assessment of phylogenetic relationships within family Oestridae.
Fly larvicidal activity in the faeces of cattle and pigs treated with endectocide products
Authors: Farkas R. , Gyurcsó A. , Börzsönyi L.
Published: August 2003

Abstract.  Bioassays were conducted to study the effect of a single therapeutic dose of injectable ivermectin, doramectin or moxidectin given to cattle and pigs and excreted in their faeces, against larvae of the housefly, Musca domestica L. (Diptera: Muscidae). Five cattle were treated with each of the test products. Cattle faecal samples were collected before treatment and on days 1, 2, 3, 6, 10, 16, 20, 23 and 28 after treatment. Three groups of pigs, each comprising 12–14 pregnant sows and gilts, were used in the experiment. Pig faeces was collected from each group before treatment and on days 1, 3, 5, 7, 9, 11, 13, 15 and 20 after treatment. Thirty, first‐stage larvae were placed into 100 g of faeces. Five replicates were examined for each time‐point and for each endectocide group. Evaluation was based on the number of larvae surviving to adult emergence. Low numbers of adults emerged from samples taken from cattle 1 day after treatment, indicating that ivermectin and doramectin were rapidly excreted in the faeces and affected the development of the house fly. A larvicidal effect of both drugs in cattle faeces was present for a period of about 3–4 weeks and lasted a few days longer in cattle treated with doramectin than with ivermectin. In cattle, the larvicidal activity of moxidectin was first observed in faecal samples collected 2 days post‐treatment; however, it killed fewer larvae than the other two drugs. The larvicidal effect of moxidectin subsequently decreased. Ivermectin and doramectin exhibited a pronounced larvicidal effect against the house fly in the faeces of pigs. The effect of doramectin was of longer duration. Moxidectin gave the weakest larvicidal effect in pig faeces. The main difference between the results obtained for the two livestock species is that peak toxicity occurred relatively later and for a shorter duration in pig than in cattle faeces.
Comparison of the predation rate of freshwater cyclopoid copepod species on larvae of the mosquito Culex pipiens
Authors: Calliari D. , Sanz K. , Martínez M. , Cervetto G. , Gómez M. , Basso C.
Published: August 2003

Abstract.  The predation rates of six copepod species: Acanthocyclops robustus G.O. Sars, Eucyclops neumani Pesta, Macrocyclops albidus Jurine, Mesocyclops longisetus Thibaud, Metacyclops grandis Kiefer and Metacyclops mendocinus Wierzejski (Copepoda: Cyclopidae) on mosquito larvae, Culex pipiens Linnaeus (Diptera: Culicidae) were assessed. Experiments consisted of 24‐h incubations of one copepod and 10 larvae without extra food at 16°C and 26°C. Nine replicates were considered for each species and temperature. Predation rates (larvae per copepod per day) were: M. mendocinus (1.8), M. grandis (3.1), E. neumani (3.8), A. robustus (3.8), Ma. albidus (6.1) and Me. longisetus (7.0). There was a significant effect of both species and temperature on predation: all species experienced higher predation at 26°C than at 16°C, except for A. robustus whose predation rate was similar at both temperatures. These observations are consistent with previous results that point to Macrocyclops and Mesocyclops genera as important larval predators and suggest the need for field trials to evaluate the response of Ma. albidus and Me. longisetus under natural conditions in Uruguay.
First report of the oriental mosquito Aedes albopictus on the West African island of Bioko, Equatorial Guinea
Authors: Toto J.‐C. , Abaga S. , Carnevale P. , Simard F.
Published: August 2003

Abstract.  The invasive oriental mosquito Aedes (Stegomyia) albopictus (Skuse) (Diptera: Culicidae) was detected on Bioko Island for the first time in November 2001. It was found to be well established breeding in artificial containers at Planta, near Malabo, the capital of Equatorial Guinea. Associated species of mosquito larvae were Aedes aegypti (L.), Ae. africanus (Theobald), Culex near decens Theobald, Cx. duttoni Theobald, Cx. quinquefasciatus Say, Cx. tigripes De Grandpré & De Charmoy, Eretmapodites quinquevittatus Theobald and Mansonia africana (Theobald). This is the third tropical African country to be invaded by Ae. albopictus, which has recently spread to many parts of the Americas and Europe – with vector competence for dengue, yellow fever and other arboviruses. In the Afrotropical environment, it will be interesting to monitor the ecological balance and/or displacement between introduced Ae. albopictus and indigenous Ae. aegpyti (domestic, peri‐domestic and sylvatic populations).
Pyrethroid resistance/susceptibility and differential urban/rural distribution of Anopheles arabiensis and An. gambiae s.s. malaria vectors in Nigeria and Ghana
Authors: Kristan M. , Fleischmann H. , Della Torre A. , Stich A. , Curtis C. F.
Published: August 2003

Abstract.  Resistance to pyrethroid insecticides and DDT caused by the kdr gene in the malaria vector Anopheles gambiae Giles s.s. (Diptera: Culicidae) has been reported in several West African countries. To test for pyrethroid resistance in two more countries, we sampled populations of the An. gambiae complex from south‐western Ghana and from urban and rural localities in Ogun State, south‐west Nigeria. Adult mosquitoes, reared from field‐collected larvae, were exposed to the WHO‐recommended discriminating dosage of exposure for 1 h to DDT 4%, deltamethrin 0.05% or permethrin 0.75% and mortality was recorded 24 h post‐exposure. Susceptibility of An. gambiae s.l. to DDT was 94–100% in Ghana and 72–100% in Nigeria, indicating low levels of DDT resistance. Deltamethrin gave the highest mortality rates: 97–100% in Ghana, 95–100% in Nigeria. Ghanaian samples of An. gambiae s.l. were fully susceptible to permethrin, whereas some resistance to permethrin was detected at 4/5 Nigerian localities (percentage mortalities 75, 82, 88, 90 and 100%), with survivors including both An. arabiensis Patton and An. gambiae s.s. identified by PCR assay. Even so, the mean knockdown time was not significantly different from a susceptible reference strain, indicating absence or low frequency of kdr‐type resistance. Such low levels of pyrethroid resistance are unlikely to impair the effectiveness of pyrethroid‐impregnated bednets against malaria transmission. Among Nigerian samples of An. gambiae s.l., the majority from two urban localities were identified as An. arabiensis, whereas the majority from rural localities were An. gambiae s.s. These findings are consistent with those of M. Coluzzi et al. (1979). Differences of ecological distribution between molecular forms of An. gambiae s.s. were also found, with rural samples almost exclusively of the S‐form, whereas the M‐form predominated in urban samples. It is suggested that ‘urban island’ populations of An. arabiensis and of An. gambiae s.s. M‐form in the rainforest belt of West Africa might be appropriate targets for elimination of these malaria vectors by the sterile insect technique.
Comparative evaluation of carbosulfan‐ and permethrin‐impregnated curtains for preventing house‐entry by the malaria vector Anopheles gambiae in Burkina Faso
Authors: Fanello C. , Carneiro I. , Ilboudo‐Sanogo E. , Cuzin‐Ouattara N. , Badolo A. , Curtis C. F.
Published: August 2003

Abstract.  Pyrethroid‐impregnated bednets and curtains are widely employed to reduce the risk of malaria transmission, but pyrethroid‐resistance is becoming more prevalent among malaria vector Anopheles mosquitoes (Diptera: Culicidae). As an alternative treatment for curtains, we assessed carbosulfan (a carbamate insecticide) in comparison with permethrin as the standard pyrethroid, against endophilic female mosquitoes of the Anopheles gambiae Giles complex in a village near Ouagadougou, Burkina Faso. The main criterion evaluated was the impact of curtains (hung inside windows, eaves and doorways) on the number of An. gambiae s.l. females active indoors at night. Light‐traps were operated overnight (21.00–06.00 hours beside occupied untreated bednets) to sample mosquitoes in houses fitted with net curtains treated with carbosulfan 0.2 g ai/m2 or permethrin 1 g ai/m2 or untreated, compared with houses without curtains. The treated and untreated curtains significantly reduced the numbers of mosquitoes collected indoors, compared with houses without curtains. Carbosulfan‐treated curtains had a highly significantly greater effect than permethrin‐treated or untreated curtains, the scale of the difference being estimated as three‐fold. However, there was no significant difference between the impact of untreated and permethrin‐treated curtains on densities of An. gambiae s.l. trapped indoors. Samples of the An. gambiae complex comprised An. arabiensis Patton and both the S‐ and M‐forms of An. gambiae Giles s.s. Susceptibility tests revealed some resistance to DDT and low frequencies of permethrin‐resistance, insufficient to explain the poor performance of permethrin on curtains. Among survivors from the diagnostic dosage of permethrin were some specimens of all three members of the An. gambiae complex, but the kdr resistance mechanism was detected only in the S‐form of An. gambiae s.s. Questions arising for further investigation include clarification of resistance mechanisms in, and foraging behaviour of, each member of the An. gambiae complex in this situation and the need to decide whether carbosulfan‐treated curtains are acceptably safe for use to reduce risks of malaria transmission.
Analysis of the biosynthetic gene cluster for the polyether antibiotic monensin in Streptomyces cinnamonensis and evidence for the role of monB and monC genes in oxidative cyclization
Authors: Oliynyk Markiyan , Stark Christian B. W. , Bhatt Apoorva , Jones Michelle A. , Hughes‐Thomas Zoë A. , Wilkinson Christopher , Oliynyk Zoryana , Demydchuk Yuliya , Staunton James , Leadlay Peter F.
Published: August 2003

The analysis of a candidate biosynthetic gene cluster (97 kbp) for the polyether ionophore monensin from Streptomyces cinnamonensis has revealed a modular polyketide synthase composed of eight separate multienzyme subunits housing a total of 12 extension modules, and flanked by numerous other genes for which a plausible function in monensin biosynthesis can be ascribed. Deletion of essentially all these clustered genes specifically abolished monensin production, while overexpression in S. cinnamonensis of the putative pathway‐specific regulatory gene monR led to a fivefold increase in monensin production. Experimental support is presented for a recently‐proposed mechanism, for oxidative cyclization of a linear polyketide intermediate, involving four enzymes, the products of monBI, monBII, monCI and monCII. In frame deletion of either of the individual genes monCII (encoding a putative cyclase) or monBII (encoding a putative novel isomerase) specifically abolished monensin production. Also, heterologous expression of monCI, encoding a flavin‐linked epoxidase, in S. coelicolor was shown to significantly increase the ability of S. coelicolor to epoxidize linalool, a model substrate for the presumed linear polyketide intermediate in monensin biosynthesis.
Nuclear magnetic resonance spectroscopy reveals the functional state of the signalling protein CheY in vivo in Escherichia coli
Authors: Hubbard Julia A. , MacLachlan Lesley K. , King Gavin W. , Jones Joanna J. , Fosberry Andrew P.
Published: August 2003

Two‐component signal transduction (TCST) pathways are regulatory systems that are highly homologous throughout the bacterial kingdom. Their established role in virulence and absence in vertebrates has made TCST an attractive target for therapeutic intervention. However, such systems have yet to yield success in the development of novel antibiotics. CheY serves as a prototype for the analysis of response regulator function. The protein structure exhibits several conformations by both X‐ray and nuclear magnetic resonance (NMR) analyses. Knowledge of which structures are relevant in vivo would be valuable in a rational drug design project. Our aim was to probe the in vivo conformation and ligand binding of CheY in Escherichia coli under resting conditions by in‐cell NMR methods. CheY was selectively labelled with 15N by the control of growth and expression conditions. NMR spectra obtained in vivo demonstrated that the Mg2+ complex was the predominant form even though cells were resuspended in metal‐free buffers and the intracellular free Mg2+ was low. In‐cell NMR also confirmed the uptake and in vivo binding mode to CheY of small‐molecular‐weight compounds identified in vitro. This paper reports the first observation of the structure and interactions with a potential drug of a regulator protein in its native host in vivo using NMR spectroscopy.
Specific targeting of a DNA‐binding protein to the SPP1 procapsid by interaction with the portal oligomer
Authors: Stiege Asita C. , Isidro Anabela , Dröge Anja , Tavares Paulo
Published: August 2003

The icosahedral procapsid of tailed bacteriophages is composed of a large number of identical subunits and of minor proteins found in a few copies. Proteins present in a very low copy number are targeted to the viral procapsid by an unknown mechanism. Bacteriophage SPP1 procapsids and mature virions contain two copies of gp7 on average. Gp7 forms stable complexes with the SPP1 portal protein gp6. Deletion of the gp6 carboxyl‐terminus and the mutation Y467→C localized in the same region prevent gp6–gp7 complex formation. Gp7 binds double‐stranded and single‐stranded DNA. Gp6 competes for this interaction, and purified gp6–gp7 complexes do not bind DNA. Procapsid structures assembled in the absence of gp6 or carrying the mutant gp6 Y467→C lack gp7. The gp6–gp7 interaction thus targets gp7 to the procapsid where the portal protein is localized asymmetrically at a single vertex of the icosahedral structure. The interaction between the two proteins is disrupted during viral assembly. Proteins homologous to gp6 and gp7 are coded by contiguous genes in a variety of phage genomes from Gram‐positive bacteria, suggesting that the gp6–gp7 complex is widespread in this group of phages. Transient association with the portal protein, an essential component of tailed bacteriophages and herpes viruses, provides a novel strategy to target minor proteins to the virion structure that might be operative in a large number of viruses.
Elongator's toxin‐target (TOT) function is nuclear localization sequence dependent and suppressed by post‐translational modification
Authors: Fichtner Lars , Jablonowski Daniel , Schierhorn Angelika , Kitamoto Hiroko K. , Stark Michael J. R. , Schaffrath Raffael
Published: August 2003

The toxin target (TOT) function of the Saccharomyces cerevisiae Elongator complex enables Kluyveromyces lactis zymocin to induce a G1 cell cycle arrest. Loss of a ubiquitin‐related system (URM1–UBA4 ) and KTI11 enhances post‐translational modification/proteolysis of Elongator subunit Tot1p (Elp1p) and abrogates its TOT function. Using TAP tagging, Kti11p contacts Elongator and translational proteins (Rps7Ap, Rps19Ap Eft2p, Yil103wp, Dph2p). Loss of YIL103w and DPH2 (involved in diphtheria toxicity) suppresses zymocicity implying that both toxins overlap in a manner mediated by Kti11p. Among the pool that co‐fractionates with RNA polymerase II (pol II) and nucleolin, Nop1p, unmodified Tot1p dominates. Thus, modification/proteolysis may affect association of Elongator with pol II or its localization. Consistently, an Elongator‐nuclear localization sequence (NLS) targets green fluorescent protein (GFP) to the nucleus, and its truncation yields TOT deficiency. Similarly, KAP120 deletion rescues cells from zymocin, suggesting that Elongator's TOT function requires NLS‐ and karyopherin‐dependent nuclear import.
GadE (YhiE) activates glutamate decarboxylase‐dependent acid resistance in Escherichia coli K‐12
Authors: Ma Zhuo , Gong Shimei , Richard Hope , Tucker Don L. , Conway Tyrrell , Foster John W.
Published: August 2003

Commensal and pathogenic strains of Escherichia coli possess three inducible acid resistance systems that collaboratively protect cells against acid stress to pH 2 or below. The most effective system requires glutamate in the acid challenge media and relies on two glutamate decarboxylases (GadA and B) combined with a putative glutamate:γ‐aminobutyric acid antiporter (GadC). A complex network of regulators mediates induction of this system in response to various media, pH and growth phase signals. We report that the LuxR‐like regulator GadE (formerly YhiE) is required for expression of gadA and gadBC regardless of media or growth conditions. This protein binds directly to the 20 bp GAD box sequence found in the control regions of both loci. Two previously identified AraC‐like regulators, GadX and GadW, are only needed for gadA/BC expression under some circumstances. Overexpression of GadX or GadW will not overcome a need for GadE. However, overexpression of GadE can supplant a requirement for GadX and W. Data provided also indicate that GadX and GadE can simultaneously bind the area around the GAD box region and probably form a complex. The gadA, gadBC and gadE genes are all induced by low pH in exponential phase cells grown in minimal glucose media. The acid induction of gadA/BC results primarily from the acid induction of gadE. Constitutive expression of GadE removes most pH control over the glutamate decarboxylase and antiporter genes. The small amount of remaining pH control is governed by GadX and W. The finding that gadE mutations also diminish the effectiveness of the other two acid resistance systems suggests that GadE influences the expression of additional acid resistance components. The number of regulatory proteins (five), sigma factors (two) and regulatory feedback loops focused on gadA/BC expression make this one of the most intensively regulated systems in E. coli.
The type III secretion chaperone FlgN regulates flagellar assembly via a negative feedback loop containing its chaperone substrates FlgK and FlgL
Authors: Aldridge Phillip , Karlinsey Joyce , Hughes Kelly T.
Published: August 2003

The type III secretion (TTS) chaperones are small proteins that act either as cytoplasmic bodyguards, protecting their secretion substrates from degradation and aggregation, facilitators of their cognate substrate secretion or both. FlgN has been previously shown to be a TTS chaperone for the hook‐associated proteins FlgK and FlgL (FlgKL), and a translational regulator of the anti‐σ28 factor FlgM. Protein stability assays indicate that a flgN mutation leads to a dramatic decrease in the half‐life of intracellular FlgK. However, using gene reporter fusions to flgK we show that a flgN mutation does not affect the translation of a flgK–lacZ fusion. Quantification of FlgM protein levels showed that FlgKL inhibit the positive regulation on flgM translation by FlgN when secretion of FlgKL is inhibited. Suppressors of the motility‐defective phenotype of a flgN mutant were isolated and mapped to the clpXP and fliDST loci. Overexpression of flgKL on a plasmid also suppressed the motility defect of a flgN null mutant. These results suggest that FlgN is not required for secretion of FlgKL and that FlgN typifies a class of TTS chaperones that allows for the minimal amount of their substrates expression required in the assembly process by protecting the substrate from proteolysis. Our data leads us to propose a model in which the interaction between FlgN and FlgK or FlgL is a sensing mechanism to determine the stage of flagellar assembly. Furthermore, the interaction between FlgN and FlgK or FlgL inhibits the translational regulation of flgM via FlgN in response to the stage of flagellar assembly.
Regulation of tryptophan synthase gene expression in Chlamydia trachomatis
Authors: Wood Heidi , Fehlner‐Gardner Christine , Berry Jody , Fischer Elizabeth , Graham Bonnie , Hackstadt Ted , Roshick Christine , McClarty Grant
Published: August 2003

We previously reported that Chlamydia trachomatis expresses the genes encoding tryptophan synthase (trpA and trpB). The results presented here indicate that C. trachomatis also expresses the tryptophan repressor gene (trpR). The complement of genes regulated by tryptophan levels in C. trachomatis is limited to trpBA and trpR. trp gene expression was repressed if chlamydiae‐infected HeLa cells were cultured the presence of tryptophan and induced if grown in tryptophan‐depleted medium or in the presence of IFN‐γ. Furthermore, expression of the trp genes in strains which encode a functional tryptophan synthase is repressed when infected cells are cultured in the presence of the tryptophan precursor indole. Results from experiments with cycloheximide, an inhibitor of eukaryotic protein synthesis, indicate that in addition to the absolute size of the intracellular tryptophan pool, host competition for available tryptophan plays a key role in regulating expression of the trp genes. The tryptophan analogue, 5‐fluorotryptophan, repressed trp gene expression and induced the formation of aberrant organisms of C. trachomatis. The growth‐inhibitory properties of 5‐fluorotryptophan could be reversed with exogenous tryptophan but not indole. In total, our results indicate that the ability to regulate trp gene expression in response to tryptophan availability is advantageous for the intracellular survival of this organism. Furthermore, the fact that C. trachomatis has retained the capacity to respond to tryptophan limitation supports the view that the in vivo antichlamydial effect of IFN‐γ is via the induction of the tryptophan‐degrading enzyme, indoleamine 2,3‐dioxygenase.
Ena/VASP proteins contribute to Listeria monocytogenes pathogenesis by controlling temporal and spatial persistence of bacterial actin‐based motility
Authors: Auerbuch Victoria , Loureiro Joseph J. , Gertler Frank B. , Theriot Julie A. , Portnoy Daniel A.
Published: August 2003

The Listeria monocytogenes surface protein ActA mediates actin‐based motility by interacting with a number of host cytoskeletal components, including Ena/VASP family proteins, which in turn interact with actin and the actin‐binding protein profilin. We employed a bidirectional genetic approach to study Ena/VASP′s contribution to L. monocytogenes movement and pathogenesis. We generated an ActA allelic series within the defined Ena/VASP‐binding sites and introduced the resulting mutant L. monocytogenes into cell lines expressing different Ena/VASP derivatives. Our findings indicate that Ena/VASP proteins contribute to the persistence of both speed and directionality of L. monocytogenes movement. In the absence of the Ena/VASP proline‐rich central domain, speed consistency decreased by sixfold. In addition, the   Ena/VASP   F‐actin‐binding   region   increased directionality     of     bacterial     movement     by     fourfold. We further show that both regions of Ena/VASP enhanced L. monocytogenes cell‐to‐cell spread to a similar degree, although the Ena/VASP F‐actin‐binding region did so in an ActA‐independent manner. Surprisingly, our ActA allelic series enabled us to uncouple L. monocytogenes speed from directionality although both were controlled by Ena/VASP proteins. Lastly, we showed the pathogenic relevance of these findings by the observation that L. monocytogenes lacking   ActA   Ena/VASP‐binding   sites   were   up   to 400‐fold less virulent during an adaptive immune response.
A group II intron has invaded the genus Azotobacter and is inserted within the termination codon of the essential groEL gene
Authors: Ferat Jean‐Luc , Le Gouar Martine , Michel François
Published: August 2003

A group II intron that was previously identified within Azotobacter  vinelandii by  polymerase  chain  reac‐tion with consensus primers has been completely sequenced, together with its flanking exons. In contrast to other bacterial members of group II, which are associated with mobile or other presumably non‐essential DNA, the A. vinelandii intron is inserted within the termination codon of the groEL coding sequence, which it changes from UAA to UAG. Both the host gene and the intron appear to be functional as (i) the ribozyme component of the intron self‐splices in vitro and (ii) both intron‐carrying and intronless versions of the single‐copy groEL gene from A. vinelandii complement groEL mutations in Escherichia coli. Moreover, analysis of nucleotide substitutions within and around a closely related intron sequence that is present at the same site in Azotobacter chroococcum provides indirect evidence of intron transposition posterior to the divergence of the two Azotobacter taxa. Somewhat surprisingly, however, analyses of RNA extracted from cells that had or had not undergone a heat shock show that the bulk of groEL transcripts end within the first 140 nucleotides of the intron. These findings are discussed in the light of our current knowledge of the biochemistry of group II introns.
A bacterial conjugation machinery recruited for pathogenesis
Authors: Seubert Anja , Hiestand Rosemarie , De La Cruz Fernando , Dehio Christoph
Published: August 2003

Type IV secretion systems (T4SS) are multicomponent transporters of Gram‐negative bacteria adapted to functions as diverse as DNA transfer in bacterial conjugation or the delivery of effector proteins into eukaryotic target cells in pathogenesis. The generally modest sequence conservation between T4SS may reflect their evolutionary distance and/or functional divergence. Here, we show that the establishment of intraerythrocytic parasitism by Bartonella tribocorum requires a putative T4SS, which shares an unprecedented level of sequence identity with the Trw conjugation machinery of the broad‐host‐range antibiotic resistance plasmid R388 (up to 80% amino acid identity for individual T4SS components). The highly conserved T4SS loci are collinear except for the presence of numerous tandem gene duplications in B. tribocorum, which mostly encode variant forms of presumed surface‐exposed pilus subunits. Conservation is not only structural, but also functional: R388 mutated in either trwD or trwH encoding essential T4SS components could be trans‐complemented for conjugation by the homologues of the B. tribocorum system. Conservation also includes the transcription regulatory circuit: both T4SS loci encode a highly homologous and interchangeable KorA/KorB repressor system that negatively regulates the expression of all T4SS components. This striking example of adaptive evolution reveals the capacity of T4SS to assume dedicated functions in either DNA transfer or pathogenesis over rather short evolutionary distance and implies a novel role for the conjugation systems of widespread broad‐host‐range plasmids in the evolution of bacterial pathogens.
Mycobacterial porins – new channel proteins in unique outer membranes
Authors: Niederweis Michael
Published: August 2003

Mycobacteria protect themselves with an outer lipid bilayer, which is the thickest biological membrane hitherto known and has an exceptionally low permeability rendering mycobacteria intrinsically resistant to many antibiotics. Pore proteins spanning the outer membrane mediate the diffusion of hydrophilic nutrients. Mycobacterium tuberculosis possesses at least two porins in addition to the low activity channel protein OmpATb. OmpATb is essential for adaptation of M. tuberculosis to low pH and survival in macrophages and mice. The channel activity of OmpATb is likely to play a major role in the defence of M. tuberculosis against acidification within the phagosome of macrophages. MspA is the main porin of Mycobacterium smegmatis. It forms a tetrameric complex with a single central pore of 10 nm length and a cone‐like structure. This structure differs clearly from that of the trimeric porins of Gram‐negative bacteria, which form one 4 nm long pore per monomer. The 45‐fold lower number of porins compared to Gram‐negative bacteria and the exceptional length of the pores are two major determinants of the low permeability of the outer membrane of M. smegmatis for hydrophilic solutes. The importance of the synergism between slow transport through the porins and drug efflux or inactivation for the development of drugs against M. tuberculosis is discussed.
Cytokine response and outcome of infection depends on the infective dose of parasites in experimental infection by Echinococcus granulosus
Authors: Dematteis Sylvia , Rottenberg Martin , Baz Adriana
Published: August 2003

We here analysed whether the cytokine responses in early and late experimental infection with Echinococcus granulosus depend on the dose of parasites to which the host is exposed. To this purpose Balb/c mice were inoculated intraperitoneally (ip) with either 500 or 2000 protoscoleces. Splenocytes of mice were obtained at days 3, 7, 14 and 21 and also on week 37 post‐infection and cultured in vitro with protoscolex antigens. Type‐1 and type‐2 cytokines were analysed in supernatants by ELISA. Results showed that the inoculation of 500 protoscoleces induced an early type‐0 and a late type‐2 cytokine response, whereas the inoculation of 2000 protoscoleces induced an early type‐2 and a late type‐0 cytokine response. Parasite growth was lower in the group inoculated with the low infective dose. These results indicate that the cytokine response during the infection by the helminth E. granulosus depends on the dose of parasites to which the host has been exposed.
The innate immune response in calves to Boophilus microplus tick transmitted Babesia bovis involves type‐1 cytokine induction and NK‐like cells in the spleen
Authors: Goff W. L. , Johnson W. C. , Horn R. H. , Barrington G. M. , Knowles D. P.
Published: August 2003

The innate immune response to Babesia bovis infection in cattle is age‐related, spleen‐dependent and, in stabilate inoculated calves, has type‐1 characteristics, including the early induction of IL‐12 and IFN‐γ. In this study with three calves, parameters of innate immunity were followed for 2 weeks after tick transmission of B. bovis. Each calf survived the acute disease episode without drug intervention, and responded with increased levels of plasma interferon‐γ and type‐1 cytokine expression, monocyte/macrophage activation, and CD8+cellular proliferation in the spleen. The proliferating CD8+population consisted primarily of NK‐like cells, and the expansion occurred in parallel with an increase in IL‐15 mRNA expression in the spleen.
Characterizing cellular immune response to kinetoplastid membrane protein‐11 (KMP‐11) during Leishmania (Viannia) panamensis infection using dendritic cells (DCs) as antigen presenting cells (APCs)
Authors: Delgado Gabriela , Parra‐López Carlos A. , Vargas Luis Eduardo , Hoya Rubén , Estupiñán Mónica , Guzmán Fanny , Torres Angela , Alonso Carlos , Velez Iván Dario , Spinel Clara , Patarroyo Manuel E.
Published: August 2003

In vitro peptide binding assays and DCs pulsed with recombinant KMP‐11 (rKMP‐11) plus six 20‐mer overlapping peptides covering the entire protein of Leishmania (Viannia) panamensis (L(V)p) promastigotes were used to identify T‐cell epitopes in this protein. Such in vitro binding assays, using HLA DRB1* 0101, ‐0401, ‐0701 and ‐1101 alleles, demonstrated that two peptide sequences (

DEEFNKKMQEQNAKFFADKP and FKHKFAELLEQQKAAQYPSK) exhibited high HLA DRB1* 0401 allele binding capacity. rKMP‐11 specific T‐cell proliferation and cytokine production, derived from 13 volunteers exposed to the parasite, suggested that using autologous DCs as APCs becomes advantageous in uncovering T‐cell epitopes promoting proliferation and differences in IFN‐γ and IL‐4 production in T‐cells from volunteers with ACTIVE and CURED undetectable disease when other APCs were used. The two peptides which bound in vitro to the HLA DRB1* 0401 allele were immunogenic in HLA DRB1* 04 volunteers, thus validating the use of in vitro binding assays for predicting epitopes in this protein. The experimental approach used here may prove useful for characterizing T‐cell epitopes in a protein useful in designing peptide‐based vaccine candidates for Leishmania and other intracellular pathogens.