bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphatidylinositol-4-Kinase Type II α Is a Component of Adaptor Protein-3-derived Vesicles
Authors: Salazar Gloria , Craige Branch , Wainer Bruce H. , Guo Jun , De Camilli Pietro , Faundez Victor
Published: June 2005

A membrane fraction enriched in vesicles containing the adaptor protein (AP) -3 cargo zinc transporter 3 was generated from PC12 cells and was used to identify new components of these organelles by mass spectrometry. Proteins prominently represented in the fraction included AP-3 subunits, synaptic vesicle proteins, and lysosomal proteins known to be sorted in an AP-3-dependent way or to interact genetically with AP-3. A protein enriched in this fraction was phosphatidylinositol-4-kinase type IIα (PI4KIIα). Biochemical, pharmacological, and morphological analyses supported the presence of PI4KIIα in AP-3-positive organelles. Furthermore, the subcellular localization of PI4KIIα was altered in cells from AP-3-deficient mocha mutant mice. The PI4KIIα normally present both in perinuclear and peripheral organelles was substantially decreased in the peripheral membranes of AP-3-deficient mocha fibroblasts. In addition, as is the case for other proteins sorted in an AP-3-dependent way, PI4KIIα content was strongly reduced in nerve terminals of mocha hippocampal mossy fibers. The functional relationship between AP-3 and PI4KIIα was further explored by PI4KIIα knockdown experiments. Reduction of the cellular content of PI4KIIα strongly decreased the punctate distribution of AP-3 observed in PC12 cells. These results indicate that PI4KIIα is present on AP-3 organelles where it regulates AP-3 function.
Adhesion-dependent and Contractile Ring-independent Equatorial Furrowing during Cytokinesis in Mammalian Cells
Authors: Kanada Masamitsu , Nagasaki Akira , Uyeda Taro Q.P.
Published: June 2005

Myosin II-dependent contraction of the contractile ring drives equatorial furrowing during cytokinesis in animal cells. Nonetheless, myosin II-null cells of the cellular slime mold Dictyostelium divide efficiently when adhering to substrates by making use of polar traction forces. Here, we show that in the presence of 30 μM blebbistatin, a potent myosin II inhibitor, normal rat kidney (NRK) cells adhering to fibronectin-coated surfaces formed equatorial furrows and divided in a manner strikingly similar to myosin II-null Dictyostelium cells. Such blebbistatin-resistant cytokinesis was absent in partially detached NRK cells and was disrupted in adherent cells if the advance of their polar lamellipodia was disturbed by neighboring cells. Y-27632 (40 μM), which inhibits Rho-kinase, was similar to 30 μM blebbistatin in that it inhibited cytokinesis of partially detached NRK cells but only prolonged furrow ingression in attached cells. In the presence of 100 μM blebbistatin, most NRK cells that initiated anaphase formed tight furrows, although scission never occurred. Adherent HT1080 fibrosarcoma cells also formed equatorial furrows efficiently in the presence of 100 μM blebbistatin. These results provide direct evidence for adhesion-dependent, contractile ring-independent equatorial furrowing in mammalian cells and demonstrate the importance of substrate adhesion for cytokinesis.
Genetic influences on variation in female orgasmic function: a twin study
Authors: Dunn Kate M , Cherkas Lynn F , Spector Tim D
Published: June 2005

Orgasmic dysfunction in females is commonly reported in the general population with little consensus on its aetiology. We performed a classical twin study to explore whether there were observable genetic influences on female orgasmic dysfunction. Adult females from the TwinsUK register were sent a confidential survey including questions on sexual problems. Complete responses to the questions on orgasmic dysfunction were obtained from 4037 women consisting of 683 monozygotic and 714 dizygotic pairs of female twins aged between 19 and 83 years. One in three women (32%) reported never or infrequently achieving orgasm during intercourse, with a corresponding figure of 21% during masturbation. A significant genetic influence was seen with an estimated heritability for difficulty reaching orgasm during intercourse of 34% (95% confidence interval 27–40%) and 45% (95% confidence interval 38–52%) for orgasm during masturbation. These results show that the wide variation in orgasmic dysfunction in females has a genetic basis and cannot be attributed solely to cultural influences. These results should stimulate further research into the biological and perhaps evolutionary processes governing female sexual function.
Do insect metabolic rates at rest and during flight scale with body mass?
Authors: Niven Jeremy E , Scharlemann Jörn P.W
Published: June 2005

Energetically costly behaviours, such as flight, push physiological systems to their limits requiring metabolic rates (MR) that are highly elevated above the resting MR (RMR). Both RMR and MR during exercise (e.g. flight or running) in birds and mammals scale allometrically, although there is little consensus about the underlying mechanisms or the scaling relationships themselves. Even less is known about the allometric scaling of RMR and MR during exercise in insects. We analysed data on the resting and flight MR (FMR) of over 50 insect species that fly to determine whether RMR and FMR scale allometrically. RMR scaled with body mass to the power of 0.66 (M0.66), whereas FMR scaled withM1.10. Further analysis suggested that FMR scaled with two separate relationships; insects weighing less than 10 mg had fourfold lower FMR than predicted from the scaling of FMR in insects weighing more than 10 mg, although both groups scaled withM0.86. The scaling exponents of RMR and FMR in insects were not significantly different from those of birds and mammals, suggesting that they might be determined by similar factors. We argue that low FMR in small insects suggests these insects may be making considerable energy savings during flight, which could be extremely important for the physiology and evolution of insect flight.
Novel male trait prolongs survival in suicidal mating
Authors: Andrade Maydianne C.B , Gu Lei , Stoltz Jeffrey A
Published: June 2005

Male redback spiders (
Latrodectus hasselti
) maximize paternity if they copulate twice with their cannibalistic mate. Facilitating cannibalistic attack during their first copulation yields paternity benefits. However, females have paired sperm-storage organs inseminated during two separate copulations, so males that succumb to partial cannibalism during the first copulation lose at least 50% of their paternity to rivals. In this paper, we describe a novel male trait—an abdominal constriction that appears during courtship—that allows males to survive and mate with females for a second time, despite the substantial cannibalistic damage inflicted during the first copulation. Constricted males that were wounded to simulate early cannibalism had higher endurance, greater survivorship, longer subsequent courtship and higher mating success than wounded males that were not constricted. Constriction was not found in a non-sacrificial congener that rarely survived simulated cannibalism, and the protective effect of constriction in redbacks was specific to the type of damage inflicted by females during the first copulation. Thus, the abdominal constriction allows males to overcome the potential fitness limit imposed by their own suicidal strategy—paradoxically, by prolonging survival across two cannibalistic copulations.
Image content influences men's semen quality
Authors: Kilgallon Sarah J , Simmons Leigh W
Published: June 2005

There is increasing evidence from non-human animals that males adjust their ejaculate expenditure according to the risk of sperm competition. In this study we show that, after controlling for lifestyle factors known to influence semen quality, human males viewing images depicting sperm competition had a higher percentage of motile sperm in their ejaculates. Many lifestyle variables were confirmed to influence semen quality, including the recent suggestion that storage of mobile phones close to the testes can decrease semen quality.
Perspectives on the basic reproductive ratio
Authors: Heffernan J.M , Smith R.J , Wahl L.M
Published: June 2005

The basic reproductive ratio,R0, is defined as the expected number of secondary infections arising from a single individual during his or her entire infectious period, in a population of susceptibles. This concept is fundamental to the study of epidemiology and within-host pathogen dynamics. Most importantly,R0often serves as a threshold parameter that predicts whether an infection will spread. Related parameters which share this threshold behaviour, however, may or may not give the true value ofR0. In this paper we give a brief overview of common methods of formulatingR0and surrogate threshold parameters from deterministic, non-structured models. We also review common means of estimatingR0from epidemiological data. Finally, we survey the recent use ofR0in assessing emerging diseases, such as severe acute respiratory syndrome and avian influenza, a number of recent livestock diseases, and vector-borne diseases malaria, dengue and West Nile virus.
Convexity, Jensen's inequality and benefits of noisy mechanical ventilation
Authors: Brewster John F , Graham M. Ruth , Mutch W. Alan C
Published: June 2005

Mechanical ventilators breathe for you when you cannot or when your lungs are too sick to do their job. Most ventilators monotonously deliver the same-sized breaths, like clockwork; however, healthy people do not breathe this way. This has led to the development of a biologically variable ventilator—one that incorporates noise. There are indications that such a noisy ventilator may be beneficial for patients with very sick lungs. In this paper we use a probabilistic argument, based on Jensen's inequality, to identify the circumstances in which the addition of noise may be beneficial and, equally important, the circumstances in which it may not be beneficial. Using the local convexity of the relationship between airway pressure and tidal volume in the lung, we show that the addition of noise at low volume or low pressure results in higher mean volume (at the same mean pressure) or lower mean pressure (at the same mean volume). The consequence is enhanced gas exchange or less stress on the lungs, both clinically desirable. The argument has implications for other life support devices, such as cardiopulmonary bypass pumps. This paper illustrates the benefits of research that takes place at the interface between mathematics and medicine.
Improved understanding of biscuit checking using speckle interferometry and finite-element modelling techniques
Authors: Saleem Q , Wildman R.D , Huntley J.M , Whitworth M.B
Published: June 2005

For almost a century, manufacturers have tried to understand and predict the appearance of small hairline cracks in biscuits and crackers, an effect known as ‘checking’. Results are presented here from a combined experimental and numerical study of the phenomenon. Experimentally determined material properties of semi-sweet biscuits were used as inputs to an axisymmetric finite-element model, which predicts that, at low relative humidities (RHs), the rim of the biscuit expands and the centre contracts. The expansion is caused by uptake of moisture at the rim, which is initially dry after baking, and the contraction is attributable to loss of moisture from the more moist centre. The finite-element predictions were validated by strain measurements of freshly baked biscuits using speckle interferometry. These measurements are the most sensitive and the first whole-field measurements yet made of strains in biscuits. Experiments using a range of storage conditions enable us to confirm that it is the presence of moisture gradients, in combination with a low environmental RH, which increases the chance of checking. These results will aid biscuit manufacturers in the development of more effective strategies to avoid the damaging phenomenon of checking, potentially enabling consumers to benefit from fewer broken biscuits.
Prey survival by predator intimidation: an experimental study of peacock butterfly defence against blue tits
Authors: Vallin Adrian , Jakobsson Sven , Lind Johan , Wiklund Christer
Published: June 2005

Long-lived butterflies that hibernate as adults are expected to have well-developed antipredation devices as a result of their long exposure to natural enemies. The peacock butterfly,Inachis io, for instance, is a cryptic leaf mimic when resting, but shifts to active defence when disturbed, performing a repeated sequence of movements exposing major eyespots on the wings accompanied by a hissing noise. We studied the effect of visual and auditory defence by staging experiments in which wild-caught blue tits,Parus caeruleus, were presented with one of six kinds of experimentally manipulated living peacock butterflies as follows: butterflies with eyespots painted over and their controls (painted on another part of the wing), butterflies with their sound production aborted (small part of wings removed) and their controls, and butterflies with eyespots painted over and sound production aborted and their controls. The results showed that eyespots alone, or in combination with sound, constituted an effective defence; only 1 out of 34 butterflies with intact eyespots was killed, whereas 13 out of 20 butterflies without eyespots were killed. The killed peacocks were eaten, indicating that they are not distasteful. Hence, intimidation by bluffing can be an efficient means of defence for an edible prey.
Long-wave theory for a new convective instability with exponential growth normal to the wall
Authors: Healey J.J
Published: June 2005

A linear stability theory is presented for the boundary-layer flow produced by an infinite disc rotating at constant angular velocity in otherwise undisturbed fluid. The theory is developed in the limit of long waves and when the effects of viscosity on the waves can be neglected. This is the parameter regime recently identified by the author in a numerical stability investigation where a curious new type of instability was found in which disturbances propagate and grow exponentially in the direction normal to the disc, (i.e. the growth takes place in a region of zero mean shear). The theory describes the mechanisms controlling the instability, the role and location of critical points, and presents a saddle-point analysis describing the large-time evolution of a wave packet in frames of reference moving normal to the disc. The theory also shows that the previously obtained numerical solutions for numerically large wavelengths do indeed lie in the asymptotic long-wave regime, and so the behaviour and mechanisms described here may apply to a number of cross-flow instability problems.
Stability of Bödewadt flow
Authors: MacKerrell Sharon O
Published: June 2005

The stability of the flow produced over an infinite stationary plane in a fluid rotating with uniform angular velocity at an infinite distance from the plane is considered. The basic flow is an exact solution of the Navier–Stokes equations making it amenable to theoretical study. An asymptotic investigation is presented in the limit of large Reynolds number. It is shown that the stationary spiral instabilities observed experimentally can be described by a linear inviscid stability analysis. The prediction obtained for the wave angle of the disturbances is found to agree well with the available experimental and numerical results.
The linear and nonlinear stability of thread-annular flow
Authors: Walton Andrew G
Published: June 2005

The surgical technique of thread injection of medical implants is modelled by the axial pressure-gradient-driven flow between concentric cylinders with a moving core. The linear stability of the flow to both axisymmetric and asymmetric perturbations is analysed asymptotically at large Reynolds number, and computationally at finite Reynolds number. The existence of multiple regions of instability is predicted and their dependence upon radius ratio and thread velocity is determined. A discrepancy in critical Reynolds numbers and cut-off velocity is found to exist between experimental results and the predictions of the linear theory. In order to account for this discrepancy, the high Reynolds number, nonlinear stability properties of the flow are analysed and a nonlinear, equilibrium critical layer structure is found, which leads to an enhanced correction to the basic flow. The predictions of the nonlinear theory are found to be in good agreement with the experimental data.
Determination of transmission patterns in multichannel data
Authors: Kamiński Maciej
Published: June 2005

The power of today's laboratory equipment allows more and more data channels to be easily recorded. However, some misunderstandings about processing such multivariate data may still be found in the literature. The typical mistake is to treat a multichannel system as comprising pairs of channels; this approach does not use all the collected information about the investigated system, and may lead to erroneous conclusions. In this paper, the differences between single- and multichannel approaches will be briefly summarized and some examples of problems will be described.
Multiscale brain modelling
Authors: Robinson P. A , Rennie C. J , Rowe D. L , O'Connor S. C , Gordon E
Published: June 2005

A central difficulty of brain modelling is to span the range of spatio-temporal scales from synapses to the whole brain. This paper overviews results from a recent model of the generation of brain electrical activity that incorporates both basic microscopic neurophysiology and large-scale brain anatomy to predict brain electrical activity at scales from a few tenths of a millimetre to the whole brain. This model incorporates synaptic and dendritic dynamics, nonlinearity of the firing response, axonal propagation and corticocortical and corticothalamic pathways. Its relatively few parameters measure quantities such as synaptic strengths, corticothalamic delays, synaptic and dendritic time constants, and axonal ranges, and are all constrained by independent physiological measurements. It reproduces quantitative forms of electroencephalograms seen in various states of arousal, evoked response potentials, coherence functions, seizure dynamics and other phenomena. Fitting model predictions to experimental data enables underlying physiological parameters to be inferred, giving a new non-invasive window into brain function that complements slower, but finer-resolution, techniques such as fMRI. Because the parameters measure physiological quantities relating to multiple scales, and probe deep structures such as the thalamus, this will permit the testing of a range of hypotheses about vigilance, cognition, drug action and brain function. In addition, referencing to a standardized database of subjects adds strength and specificity to characterizations obtained.
Relating connectional architecture to grey matter function using diffusion imaging
Authors: Behrens T.E.J , Johansen-Berg H
Published: June 2005

Understanding brain function in terms of connectional architecture is a major goal of neuroimaging. However, direct investigation of the influence of brain circuitry on function has been hindered by the lack of a technique for exploring anatomical connectivity in the
in vivo
brain. Recent advances in magnetic resonance diffusion imaging have given scientists access to data relating to local white matter architecture and, for the first time, have raised the possibility of
in vivo
investigations into brain circuitry. This review investigates whether diffusion imaging may be used to identify regions of grey matter that are distinct in their connectional architecture, and whether these connectional differences are reflected either in local cytoarchitecture or in local grey matter function.

Establishing a direct relationship between regional boundaries based on diffusion imaging and borders between regions that perform different functions would not only be of great significance when interpreting functional results, but would also provide a first step towards the validation of diffusion-based anatomical connectivity studies.
Investigating the neural basis for functional and effective connectivity. Application to fMRI
Authors: Horwitz Barry , Warner Brent , Fitzer Julie , Tagamets M.-A , Husain Fatima T , Long Theresa W
Published: June 2005

Viewing cognitive functions as mediated by networks has begun to play a central role in interpreting neuroscientific data, and studies evaluating interregional functional and effective connectivity have become staples of the neuroimaging literature. The neurobiological substrates of functional and effective connectivity are, however, uncertain. We have constructed neurobiologically realistic models for visual and auditory object processing with multiple interconnected brain regions that perform delayed match-to-sample (DMS) tasks. We used these models to investigate how neurobiological parameters affect the interregional functional connectivity between functional magnetic resonance imaging (fMRI) time-series. Variability is included in the models as subject-to-subject differences in the strengths of anatomical connections, scan-to-scan changes in the level of attention, and trial-to-trial interactions with non-specific neurons processing noise stimuli. We find that time-series correlations between integrated synaptic activities between the anterior temporal and the prefrontal cortex were larger during the DMS task than during a control task. These results were less clear when the integrated synaptic activity was haemodynamically convolved to generate simulated fMRI activity. As the strength of the model anatomical connectivity between temporal and frontal cortex was weakened, so too was the strength of the corresponding functional connectivity. These results provide a partial validation for using fMRI functional connectivity to assess brain interregional relations.
Brain connectivity at different time-scales measured with EEG
Authors: Koenig T , Studer D , Hubl D , Melie L , Strik W.K
Published: June 2005

We present an overview of different methods for decomposing a multichannel spontaneous electroencephalogram (EEG) into sets of temporal patterns and topographic distributions. All of the methods presented here consider the scalp electric field as the basic analysis entity in space. In time, the resolution of the methods is between milliseconds (time-domain analysis), subseconds (time- and frequency-domain analysis) and seconds (frequency-domain analysis). For any of these methods, we show that large parts of the data can be explained by a small number of topographic distributions. Physically, this implies that the brain regions that generated one of those topographies must have been active with a common phase. If several brain regions are producing EEG signals at the same time and frequency, they have a strong tendency to do this in a synchronized mode. This view is illustrated by several examples (including combined EEG and functional magnetic resonance imaging (fMRI)) and a selective review of the literature. The findings are discussed in terms of short-lasting binding between different brain regions through synchronized oscillations, which could constitute a mechanism to form transient, functional neurocognitive networks.
The ferric iron uptake regulator (Fur) from the extreme acidophile Acidithiobacillus ferrooxidans
Authors: Quatrini R. , Lefimil C. , Holmes D. S. , Jedlicki E.
Published: June 2005

Acidithiobacillus ferrooxidansis a Gram-negative bacterium that lives at pH 2 in high concentrations of soluble ferrous and ferric iron, making it an interesting model for understanding the biological mechanisms of bacterial iron uptake and homeostasis in extremely acid conditions. A candidatefurAF(FerricUptakeRegulator) gene was identified in theA. ferrooxidansATCC 23270 genome. FurAFhas significant sequence similarity, including conservation of functional motifs, to known Fur orthologues and exhibits cross-reactivity toEscherichia coliFur antiserum. ThefurAFgene is able to complementfurdeficiency inE. coliin an iron-responsive manner. FurAFis also able to bind specifically toE. coliFur regulatory regions (Fur boxes) and to a candidate Fur box fromA. ferrooxidans, as judged by electrophoretic mobility shift assays. FurAFrepresses gene expression fromE. coliFur-responsive promotersfiuandfhuFwhen expressed at high protein levels. However, it increases gene expression from these promoters at low concentrations and possibly from other Fur-regulated promoters involved in iron-responsive oxidative stress responses.
The Bacillus subtilis YdfHI two-component system regulates the transcription of ydfJ, a member of the RND superfamily
Authors: Serizawa Masakuni , Sekiguchi Junichi
Published: June 2005

The ydfHI genes encode a sensor kinase and a response regulator forming a two-component system. ydfJ is located downstream of ydfHI, and belongs to the RND (resistance-nodulation-cell division) superfamily, which is present in most major organisms. Four genes (secDF, yerP, ydfJ and ydgH) in Bacillus subtilis belong to this family. This study revealed that the YdfHI two-component system regulates ydfJ transcription. A gel shift assay using histidine-tagged YdfI (h-YdfI) showed that it directly binds to the ydfJ promoter region. Moreover, DNase I footprinting analysis revealed a tandem repeat sequence consisting of two conserved 12-mer sequences (GCCCRAAYGTAC) within the h-YdfI-binding site.
Protein–protein interactions in the chemotaxis signalling pathway of Treponema denticola
Authors: Sim Jee-Hyun , Shi Wenyuan , Lux Renate
Published: June 2005

Motile bacteria employ sophisticated chemotaxis signal transduction systems to transform environmental cues into corresponding behavioural responses. The proteins involved in this signalling pathway have been extensively studied on a molecular level in various model organisms, including enterobacteria andBacillus subtilis, and specific protein–protein interactions have been identified. The chemotaxis operon of spirochaetes encodes a novel chemotaxis protein, CheX, in addition to homologues to the central components of established chemotaxis systems. Interestingly, the closest functionally characterized homologue of CheX is CheC of the complexB. subtilischemotaxis pathway. In this study, the yeast two-hybrid system was applied to investigate protein–protein interactions within the chemotaxis signalling pathway ofTreponema denticola, with special focus on CheX. CheX was found to interact with CheA and with itself. The other chemotaxis proteins exhibited interactions comparable to their homologues in known chemotaxis systems. Based on these findings, a model integrating CheX in the chemotaxis signal transduction pathway ofT. denticolais proposed.
Use of molecular diversity of Mycoplasma gallisepticum by gene-targeted sequencing (GTS) and random amplified polymorphic DNA (RAPD) analysis for epidemiological studies
Authors: Ferguson Naola M. , Hepp Diego , Sun Shulei , Ikuta Nilo , Levisohn Sharon , Kleven Stanley H. , García Maricarmen
Published: June 2005

A total of 67 Mycoplasma gallisepticum field isolates from the USA, Israel and Australia, and 10 reference strains, were characterized by gene-targeted sequencing (GTS) analysis of portions of the putative cytadhesin pvpA gene, the cytadhesin gapA gene, the cytadhesin mgc2 gene, and an uncharacterized hypothetical surface lipoprotein-encoding gene designated genome coding DNA sequence (CDS) MGA_0319. The regions of the surface-protein-encoding genes targeted in this analysis were found to be stable within a strain, after sequencing different in vitro passages of M. gallisepticum reference strains. Gene sequences were first analysed on the basis of gene size polymorphism. The pvpA and mgc2 genes are characterized by the presence of different nucleotide insertions/deletions. However, differentiation of isolates based solely on pvpA/mgc2 PCR size polymorphism was not found to be a reliable method to differentiate among M. gallisepticum isolates. On the other hand, GTS analysis based on the nucleotide sequence identities of individual and multiple genes correlated with epidemiologically linked isolates and with random amplified polymorphic DNA (RAPD) analysis. GTS analysis of individual genes, gapA, MGA_0319, mgc2 and pvpA, identified 17, 16, 20 and 22 sequence types, respectively. GTS analysis using multiple gene sequences mgc2/pvpa and gapA/MGA_0319/mgc2/pvpA identified 38 and 40 sequence types, respectively. GTS of multiple surface-protein-encoding genes showed better discriminatory power than RAPD analysis, which identified 36 pattern types from the same panel of M. gallisepticum strains. These results are believed to provide the first evidence that typing of M. gallisepticum isolates by GTS analysis of surface-protein genes is a sensitive and reproducible typing method and will allow rapid global comparisons between laboratories.
The leucyl aminopeptidase from Helicobacter pylori is an allosteric enzyme
Authors: Dong Lei , Cheng Ni , Wang Ming-Wei , Zhang Junfeng , Shu Chang , Zhu De-Xu
Published: June 2005

This study describes the cloning, genetic analysis and biochemical characterization of a leucyl aminopeptidase (LAP) fromHelicobacter pylori. A gene encoding LAP was cloned fromH. pyloriand the expressed 55 kDa protein displayed homology to aminopeptidases from Gram-negative bacteria, plants and mammals. This LAP demonstrated amidolytic activity againstl-leucine-p-nitroanilide. Optimal activity was observed at pH 8·0 and 45 °C, withVmaxof 232 μmol min−1(mg protein)−1andS0·5of 0·65 mM. The data suggest that LAP could be allosteric (nH=2·27), with regulatory homohexamers, and its activity was inhibited by ion chelators and enhanced by divalent manganese, cobalt and nickel cations. Bestatin inhibited both LAP activity (IC50=49·9 nM) andH. pylorigrowthin vitro. The results point to the potential use of LAP as a drug target to develop novel anti-H. pyloriagents.
Identification and regulation of cold-inducible factors of Bordetella bronchiseptica
Authors: Stübs Dorothee , Fuchs Thilo M. , Schneider Boris , Bosserhoff Armin , Gross Roy
Published: June 2005

The expression of bacterial cold-shock proteins (CSPs) is highly induced in response to cold shock, and some CSPs are essential for cells to resume growth at low temperature.Bordetella bronchisepticaencodes five CSPs (named CspA to CspE) with significant amino acid homology to CspA ofEscherichia coli. In contrast toE. coli, the insertional knock-out of a singlecspgene (cspB) strongly affected growth ofB. bronchisepticaindependent of temperature. In the case of three of thecspgenes (cspA,cspB,cspC) more than one specific transcript could be detected. The net amount ofcspA,cspBandcspCtranscripts increased strongly after cold shock, while no such effect could be observed forcspDandcspE. The exposure to other stress conditions, including translation inhibitors, heat shock, osmotic stress and nutrient deprivation in the stationary phase, indicated that thecspgenes are also responsive to these conditions. The coding regions of all of the cold-shock genes are preceded by a long non-translated upstream region (5′-UTR). In the case of thecspBgene, a deletion of parts of this region led to a significant reduction of translation of the resulting truncated transcript, indicating a role of the 5′-UTR in translational control. The cold-shock stimulon was investigated by 2D-PAGE and mass spectrometric characterization, leading to the identification of additional cold-inducible proteins (CIPs). Interestingly, two cold-shock genes (cspCandcspD) were found to be under the negative control of the BvgAS system, the main transcriptional regulator ofBordetellavirulence genes. Moreover, a negative effect of slight overexpression of CspB, but not of the other CSPs, on the transcription of the adenylate cyclase toxin CyaA ofBordetella pertussiswas observed, suggesting cross-talk between the CSP-mediated stress response stimulon and theBordetellavirulence regulon.
Diverse humoral immune responses and changes in IgG antibody levels against mycobacterial lipid antigens in active tuberculosis
Authors: Fujita Yukiko , Doi Takeshi , Sato Koji , Yano Ikuya
Published: June 2005

Humoral immune responses of active TB patients against six mycobacterial lipid antigens [trehalose 6,6′-dimycolate (TDM) from Mycobacterium bovis BCG (TDM-T) and Mycobacterium avium complex (TDM-M), trehalose 6-monomycolate (TMM) from M. bovis BCG (TMM-T) and M. avium complex (TMM-M), triacyl (PL-2) and tetraacyl (PL-1) phosphatidylinositol dimannosides] were examined by ELISA. IgG antibodies of TB patients with active disease reacted against the six lipid antigens distinctively, but heterogeneously. If tests were combined and an overall positive was scored cumulatively when any one of the six tests was positive, a good discrimination between patient and normal subject was obtained. A positive result in any one of the six tests was obtained in 91·5 % of all 924 hospitalized patients and 93·3 % of 210 patients at their first visit to the outpatient clinic. The IgG antibody response differed considerably from patient to patient, and the response patterns were grouped into several types. IgG antibody levels paralleled the bacterial burden; however, the smear-negative (culture-positive) patient group also showed high positive rates and mean ELISA ΔA values against the six lipid antigens. There were also marked differences in positive rate and mean ΔA values between cavity-positive and -negative groups, the former being higher than the latter. After anti-TB chemotherapy was initiated, IgG antibody levels decreased dramatically, paralleling the decrease in the amount of excretion of bacteria. Since multiple-antigen ELISA using particular lipid antigens was highly sensitive, and IgG antibody levels vary greatly at different stages of the disease, this technique is applicable for early diagnosis of smear-negative (and -positive) active TB and the prognosis for completion of anti-TB chemotherapy.
The DNA-binding specificity of the Bacillus anthracis AbrB protein
Authors: Strauch Mark A. , Ballar Petek , Rowshan Austin J. , Zoller Katherine L.
Published: June 2005

TheBacillus subtilisAbrB protein is a DNA-binding global regulator of a plethora of functions that are expressed during the transition from exponential growth to stationary phase and under suboptimal growth conditions. AbrB orthologues have been identified in a variety of prokaryotic organisms, notably in all species ofBacillus,ClostridiumandListeriathat have been examined. Based on amino acid sequence identity in the N-terminal domains of the orthologues fromB. subtilisandBacillus anthracis, it was predicted that the proteins might display identical DNA-binding specificities. The binding of purifiedB. anthracisAbrB (AbrBBA) and purifiedB. subtilisAbrB (AbrBBS) at DNA targets ofB. subtilis,B. anthracisand a synthetic origin was compared. In all cases examined, DNA-binding specificity was identical as judged by DNase I footprinting. InB. subtiliscells, theB. anthracispromoters from theatxAandabrBgenes were regulated by AbrBBS, and theB. subtilispromoter from theyxbBoperon was regulated by AbrBBA.
Mutations affecting predation ability of the soil bacterium Myxococcus xanthus
Authors: Pham Vinh D. , Shebelut Conrad W. , Diodati Michelle E. , Bull Carolee T. , Singer Mitchell
Published: June 2005

Myxococcus xanthusgenetic mutants with characterized phenotypes were analysed for the ability to prey on susceptible bacteria. Quantification of predatory ability was scored by a newly developed method under conditions in which prey bacteria provided the only source of nutrients. These results were corroborated by data derived using a previously published protocol that measures predation in the presence of limited external nutrients. First, early developmental regulatory mutants were examined, because their likely functions in assessing the local nutrient status were predicted to be also important for predation. The results showed that predation efficiency is reduced by 64–80 % for mutants of three A-signalling components, AsgA, AsgC and AsgE, but not for AsgB. This suggests that an Asg regulon function that is separate from A-signal production is needed for predation. Besides the Asg components, mutations in the early developmental genessdeKandcsgAwere also consistently observed to reduce predatory efficacy by 36 and 33 %, respectively. In contrast, later developmental components, such as DevRS, 4406 and PhoP4, did not appear to play significant roles in predation. The predatory abilities of mutants defective for motility were also tested. The data showed that adventurous, but not social, motility is required for predation in the assay. Also, mutants for components in the chemotaxis-like Frz system were found to be reduced in predation efficiency by between 62 and 85 %. In sum, it was demonstrated here that defects in development and development-related processes affect the ability ofM. xanthusto prey on other bacteria.
Population structure of group B streptococcus from a low-incidence region for invasive neonatal disease
Authors: Bisharat Naiel , Jones Nicola , Marchaim Dror , Block Colin , Harding Rosalind M. , Yagupsky Pablo , Peto Tim , Crook Derrick W.
Published: June 2005

The population structure of group B streptococcus (GBS) from a low-incidence region for invasive neonatal disease (Israel) was investigated using multilocus genotype data. The strain collection consisted of isolates from maternal carriage (n=104) and invasive neonatal disease (n=50), resolving into 46 sequence types. The most prevalent sequence types were ST-1 (17·5 %), ST-19 (10·4 %), ST-17 (9·7 %), ST-22 (8·4 %) and ST-23 (6·5 %). Serotype III was the most common, accounting for 29·2 % of the isolates. None of the serotypes was significantly associated with invasive neonatal disease. burst analysis resolved the 46 sequence types into seven lineages (clonal complexes), from which only lineage ST-17, expressing serotype III only, was significantly associated with invasive neonatal disease. Lineage ST-22 expressed mainly serotype II, and was significantly associated with carriage. The distribution of the various sequence types and lineages, and the association of lineage ST-17 with invasive disease, are consistent with the results of analyses from a global GBS isolate collection. These findings could imply that the global variation in disease incidence is independent of the circulating GBS populations, and may be more affected by other risk factors for invasive GBS disease, or by different prevention strategies.
Up-regulation of competence- but not stress-responsive proteins accompanies an altered metabolic phenotype in Streptococcus mutans biofilms
Authors: Rathsam Catherine , Eaton Ruth E. , Simpson Christine L. , Browne Gina V. , Berg Tracey , Harty Derek W. S. , Jacques N. A.
Published: June 2005

Mature biofilm and planktonic cells ofStreptococcus mutanscultured in a neutral pH environment were subjected to comparative proteome analysis. Of the 242 protein spots identified, 48 were significantly altered in their level of expression (P<0·050) or were unique to planktonic or biofilm-grown cells. Among these were four hypothetical proteins as well as proteins known to be associated with the maintenance of competence or found to possess acin-box-like element upstream of their coding gene. Most notable among the non-responsive genes were those encoding the molecular chaperones DnaK, GroEL and GroES, which are considered to be up-regulated by sessile growth. Analysis of the rest of the proteome indicated that a number of cellular functions associated with carbon uptake and cell division were down-regulated. The data obtained were consistent with the hypothesis that a reduction in the general growth rate of mature biofilms ofS. mutansin a neutral pH environment is associated with the maintenance of transformation without the concomitant stress response observed during the transient state of competence in bacterial batch cultures.
An acyl-CoA dehydrogenase is involved in the formation of the Δcis3 double bond in the acyl residue of the lipopeptide antibiotic friulimicin in Actinoplanes friuliensis
Authors: Heinzelmann Eva , Berger Susanne , Müller Claudia , Härtner Thomas , Poralla Karl , Wohlleben Wolfgang , Schwartz Dirk
Published: June 2005

The lipopeptide antibiotic friulimicin, produced by Actinoplanes friuliensis, is an effective drug against Gram-positive bacteria, such as methicillin-resistant Staphylococcus epidermidis and Staphylococcus aureus strains. Friulimicin consists of a cyclic peptide core of ten amino acids and an acyl residue linked to an exocyclic amino acid. The acyl residue is essential for antibiotic activity, varies in length from C13 to C15, and carries a characteristic double bond at position Δcis3. Sequencing of a DNA fragment adjacent to a previously described fragment encoding some of the friulimicin biosynthetic genes revealed several genes whose gene products resemble enzymes of lipid metabolism. One of these genes, lipB, encodes an acyl-CoA dehydrogenase homologue. To elucidate the function of the LipB protein, a lipB insertion mutant was generated and the friulimicin derivative (FR242) produced by the mutant was purified. FR242 had antibiotic activity lower than friulimicin in a bioassay. Gas chromatography showed that the acyl residue of wild-type friulimicin contains a double bond, whereas a saturated bond was present in FR242. These results were confirmed by the heterologous expression of lipB in Streptomyces lividans T7, which led to the production of unsaturated fatty acids not found in the S. lividans T7 parent strain. These results indicate that the acyl-CoA dehydrogenase LipB is involved in the introduction of the unusual Δcis3 double bond into the acyl residue of friulimicin.
Comparative analysis of the exopolysaccharide biosynthesis gene clusters from four strains of Lactobacillus rhamnosus
Authors: Péant B. , LaPointe G. , Gilbert C. , Atlan D. , Ward P. , Roy D.
Published: June 2005

The exopolysaccharide (EPS) biosynthesis gene clusters of four Lactobacillus rhamnosus strains consist of chromosomal DNA regions of 18·5 kb encoding 17 ORFs that are highly similar among the strains. However, under identical conditions, EPS production varies considerably among these strains, from 61 to 1611 mg l−1. Fifteen genes are co-transcribed starting from the first promoter upstream of wzd. Nevertheless, five transcription start sites were identified by 5′-RACE PCR analysis, and these were associated with promoter sequences upstream of wzd, rmlA, welE, wzr and wzb. Six potential glycosyltransferase genes were identified that account for the assembly of the heptasaccharide repeat unit containing an unusually high proportion of rhamnose. Four genes involved in the biosynthesis of the sugar nucleotide precursor dTDP-l-rhamnose were identified in the EPS biosynthesis locus, which is unusual for lactic acid bacteria. These four genes are expressed from their own promoter (P2), as well as co-transcribed with the upstream EPS genes, resulting in coordinated production of the rhamnose precursor with the enzymes involved in EPS biosynthesis. This is believed to be the first report demonstrating that the sequence, original organization and transcription of genes encoding EPS production are highly similar among four strains of Lb. rhamnosus, and do not vary with the amount of EPS produced.
Immobilization-based isolation of capsule-negative mutants of Streptococcus pneumoniae
Authors: Llull Daniel , Veiga Patrick , Tremblay Josselyne , Kulakauskas Saulius
Published: June 2005

The capsular polysaccharide (CPS) is the most important identified virulence factor of Streptococcus pneumoniae, a human pathogen of the upper respiratory tract. One limitation in studies of S. pneumoniae surface virulence factors is the lack of a reliable procedure for isolation of capsule-negative mutants of clinical strains. This paper presents an approach, based on the immobilization of pneumococci in semi-liquid (0·04 % agar) medium, to easily distinguish and select for non-capsulated mutants. A clinical S. pneumoniae type 37 strain was used as a model to show that CPS production results in bacterial immobilization in semi-liquid agar medium and restricts cell sedimentation. Descendants of CPS− mutants sedimented faster under these conditions and therefore could be separated from immobilized parental cells. The CPS− phenotype of the obtained mutants was confirmed by both immunoagglutination and immunostaining experiments using specific type 37 capsular antibodies. Complementation of immobilization with the cloned tts gene, encoding type 37 CPS synthase, confirmed that faster sedimentation of mutants was specifically due to loss of the capsule. DNA sequence determination of three independent mutants revealed a point mutation, a 46 nt deletion and a heptanucleotide duplication in the tts gene. Immobilization of strains producing other CPSs (type 2, 3 and 6) also resulted in the appearance of CPS− mutants, thus showing that immobilization-based isolation is not restricted to type 37 pneumococci. Bacterial growth in semi-liquid medium proved to be a useful model system to identify the genetic consequences of immobilization. The results indicate that immobilization due to CPS may impose selective pressure against capsule production and thus contribute to capsule plasticity.
The cell envelope structure and properties of Mycobacterium smegmatis mc2155: is there a clue for the unique transformability of the strain?
Authors: Etienne Gilles , Laval Françoise , Villeneuve Christelle , Dinadayala Premkumar , Abouwarda Ahmed , Zerbib Didier , Galamba Alexandra , Daffé Mamadou
Published: June 2005

Mycobacterium smegmatisis often used as a surrogate host for pathogenic mycobacteria, especially since the isolation of the transformable smooth morphotype strain mc2155 from the isogenic rough wild-type strain ATCC 607. Biochemical analysis of the cell envelope components revealed a lack of polar glycolipids, namely the lipooligosaccharides and the polar subfamilies of glycopeptidolipids, in the mc2155 strain. In addition, the latter strain differs from its parent by the distribution of various species of glycolipids and phospholipids between the outermost and deeper layers of the cell envelope. The presence of filamentous and rope-like structures at the cell surface of mc2155 cells grown in complex media further supported an ultrastructural change in the cell envelope of the mutant. Importantly, a significantly more rapid uptake of the hydrophobic chenodeoxycholate was observed for the mutant compared to wild-type cells. Taken together, these data indicate that the nature of the surface-exposed and envelope constituents is crucial for the surface properties, cell wall permeability and bacterial phenotype, and suggest that the transformable character of the mc2155 strain may be in part explained by these profound modifications of its cell envelope.
Pectin utilization by the methylotrophic yeast Pichia methanolica
Authors: Nakagawa Tomoyuki , Yamada Kaichiro , Fujimura Shuki , Ito Takashi , Miyaji Tatsuro , Tomizuka Noboru
Published: June 2005

The methylotrophic yeastPichia methanolicawas able to grow on pectic compounds, pectin and polygalacturonate, as sole carbon sources. Under the growth conditions used,P. methanolicaexhibited increased levels of pectin methylesterase, and pectin-depolymerizing and methanol-metabolizing enzyme activities. On the other hand,P. methanolicahas two alcohol oxidase (AOD) genes,MOD1andMOD2. On growth on pectin, theP. methanolica mod1Δ andmod1Δmod2Δ strains showed a severe defect in the growth yield, although themod2Δ strain could grow on polygalacturonate to the same extent as the wild-type strain. The expression ofMOD1was detected in pectin-grown cells, but theMOD2-gene expression detected by pectin was much lower than that ofMOD1. Moreover, pectin could induce peroxisome proliferation inP. methanolica, like methanol and oleic acid. These findings showed thatP. methanolicawas able to utilize the methylester moiety of pectin by means of methanol-metabolic enzymes in peroxisomes, and that the functional AOD subunit for pectin utilization was Mod1p inP. methanolica.