bluuebunny/crossref_metadata_2025 huggingface dataset:
Extensive DNA Inversions in the
B. fragilis
Genome Control Variable Gene Expression
Authors: Cerdeño-Tárraga Ana M. , Patrick Sheila , Crossman Lisa C. , Blakely Garry , Abratt Val , Lennard Nicola , Poxton Ian , Duerden Brian , Harris Barbara , Quail Mike A. , Barron Andrew , Clark Louise , Corton Craig , Doggett Jonathan , Holden Matthew T. G. , Larke Natasha , Line Alexandra , Lord Angela , Norbertczak Halina , Ormond Doug , Price Claire , Rabbinowitsch Ester , Woodward John , Barrell Bart , Parkhill Julian
Published: March 2005

The obligately anaerobic bacterium
Bacteroides fragilis
, an opportunistic pathogen and inhabitant of the normal human colonic microbiota, exhibits considerable within-strain phase and antigenic variation of surface components. The complete genome sequence has revealed an unusual breadth (in number and in effect) of DNA inversion events that potentially control expression of many different components, including surface and secreted components, regulatory molecules, and restriction-modification proteins. Invertible promoters of two different types (12 group 1 and 11 group 2) were identified. One group has inversion crossover (
fix
) sites similar to the
hix
sites of
Salmonella typhimurium
. There are also four independent intergenic shufflons that potentially alter the expression and function of varied genes. The composition of the 10 different polysaccharide biosynthesis gene clusters identified (7 with associated invertible promoters) suggests a mechanism of synthesis similar to the O-antigen capsules of
Escherichia coli
.
How Visual Stimuli Activate Dopaminergic Neurons at Short Latency
Authors: Dommett Eleanor , Coizet Véronique , Blaha Charles D. , Martindale John , Lefebvre Véronique , Walton Natalie , Mayhew John E. W. , Overton Paul G. , Redgrave Peter
Published: March 2005

Unexpected, biologically salient stimuli elicit a short-latency, phasic response in midbrain dopaminergic (DA) neurons. Although this signal is important for reinforcement learning, the information it conveys to forebrain target structures remains uncertain. One way to decode the phasic DA signal would be to determine the perceptual properties of sensory inputs to DA neurons. After local disinhibition of the superior colliculus in anesthetized rats, DA neurons became visually responsive, whereas disinhibition of the visual cortex was ineffective. As the primary source of visual afferents, the limited processing capacities of the colliculus may constrain the visual information content of phasic DA responses.
OSBP Is a Cholesterol-Regulated Scaffolding Protein in Control of ERK1/2 Activation
Authors: Wang Ping-yuan , Weng Jian , Anderson Richard G. W.
Published: March 2005

Oxysterol-binding protein (OSBP) is the founding member of a family of sterol-binding proteins implicated in vesicle transport, lipid metabolism, and signal transduction. Here, OSBP was found to function as a cholesterol-binding scaffolding protein coordinating the activity of two phosphatases to control the extracellular signal–regulated kinase (ERK) signaling pathway. Cytosolic OSBP formed a ∼440-kilodalton oligomer with a member of the PTPPBS family of tyrosine phosphatases, the serine/threonine phosphatase PP2A, and cholesterol. This oligomer had dual specific phosphatase activity for phosphorylated ERK (pERK). When cell cholesterol was lowered, the oligomer disassembled and the level of pERK rose. The oligomer also disassembled when exposed to oxysterols. Increasing the amount of OSBP oligomer rendered cells resistant to the effects of cholesterol depletion and decreased the basal level of pERK. Thus, cholesterol functions through its interaction with OSBP outside of membranes to regulate the assembly of an oligomeric phosphatase that controls a key signaling pathway in the cell.
Nutritional Status and Diet Composition Affect the Value of Diatoms as Copepod Prey
Authors: Jones Ruth H. , Flynn Kevin J.
Published: March 2005

The role of diatoms as key food for copepods at the base of pelagic food chains has been questioned recently on the grounds of toxicity. We show, using unialgal versus mixed algal diets of different nutritional status (i.e., nitrogen:carbon ratio) fed to
Acartia tonsa
, that diatoms per se are not toxic but that single-diatom diets are inadequate. Additionally, the nutritional state of the phytoplankton has a profound effect on copepod growth and growth efficiency. The ecological significance of laboratory demonstrations of diatom toxicity needs to be reconsidered.
Deflection of the Interstellar Neutral Hydrogen Flow Across the Heliospheric Interface
Authors: Lallement R. , Quémerais E. , Bertaux J. L. , Ferron S. , Koutroumpa D. , Pellinen R.
Published: March 2005

Using an absorption cell, we measured the Doppler shifts of the interstellar hydrogen resonance glow to show the direction of the neutral hydrogen flow as it enters the inner heliosphere. The neutral hydrogen flow is found to be deflected relative to the helium flow by about 4°. The most likely explanation of this deflection is a distortion of the heliosphere under the action of an ambient interstellar magnetic field. In this case, the helium flow vector and the hydrogen flow vector constrain the direction of the magnetic field and act as an interstellar magnetic compass.
The Geometric Distance and Proper Motion of the Triangulum Galaxy (M33)
Authors: Brunthaler Andreas , Reid Mark J. , Falcke Heino , Greenhill Lincoln J. , Henkel Christian
Published: March 2005

We measured the angular rotation and proper motion of the Triangulum Galaxy (M33) with the Very Long Baseline Array by observing two H
2
O masers on opposite sides of the galaxy. By comparing the angular rotation rate with the inclination and rotation speed, we obtained a distance of 730 ± 168 kiloparsecs. This distance is consistent with the most recent Cepheid distance measurement. M33 is moving with a velocity of 190 ± 59 kilometers per second relative to the Milky Way. These measurements promise a method to determine dynamical models for the Local Group and the mass and dark-matter halos of M31, M33, and the Milky Way.
Biosynthesis of Auxin by the Gram-Positive PhytopathogenRhodococcus fasciansIs Controlled by Compounds Specific to Infected Plant Tissues
Authors: Vandeputte Olivier , Öden Sevgi , Mol Adeline , Vereecke Danny , Goethals Koen , El Jaziri Mondher , Prinsen Els
Published: March 2005

The role and metabolism of indole-3-acetic acid in gram-negative bacteria is well documented, but little is known about indole-3-acetic acid biosynthesis and regulation in gram-positive bacteria. The phytopathogenRhodococcus fascians, a gram-positive organism, incites diverse developmental alterations, such as leafy galls, on a wide range of plants. Phenotypic analysis of a leafy gall suggests that auxin may play an important role in the development of the symptoms. We show here for the first time thatR. fasciansproduces and secretes the auxin indole-3-acetic acid. Interestingly, whereas noninfected-tobacco extracts have no effect, indole-3-acetic acid synthesis is highly induced in the presence of infected-tobacco extracts when tryptophan is not limiting. Indole-3-acetic acid production by a plasmid-free strain shows that the biosynthetic genes are located on the bacterial chromosome, although plasmid-encoded genes contribute to the kinetics and regulation of indole-3-acetic acid biosynthesis. The indole-3-acetic acid intermediates present in bacterial cells and secreted into the growth media show that the main biosynthetic route used byR. fasciansis the indole-3-pyruvic acid pathway with a possible rate-limiting role for indole-3-ethanol. The relationship between indole-3-acetic acid production and the symptoms induced byR. fasciansis discussed.
Acquisition of Resistance to Extended-Spectrum Cephalosporins by
Salmonella enterica
subsp.
enterica
Serovar Newport and
Escherichia coli
in the Turkey Poult Intestinal Tract
Authors: Poppe C. , Martin L. C. , Gyles C. L. , Reid-Smith R. , Boerlin P. , McEwen S. A. , Prescott J. F. , Forward K. R.
Published: March 2005

Salmonella enterica
subsp.
enterica
serovar Newport resistant to the extended-spectrum cephalosporins (ESCs) and other antimicrobials causes septicemic salmonellosis in humans and animals and is increasingly isolated from humans, animals, foods, and environmental sources. Mechanisms whereby serovar Newport bacteria become resistant to ESCs and other classes of antimicrobials while inhabiting the intestinal tract are not well understood. The present study shows that 25.3% of serovar Newport strains isolated from the turkey poult intestinal tract after the animals were dosed with
Escherichia coli
harboring a large conjugative plasmid encoding the CMY-2 β-lactamase and other drug resistance determinants acquired the plasmid and its associated drug resistance genes. The conjugative plasmid containing the
cmy-2
gene was transferred not only from the donor
E. coli
to
Salmonella
serovar Newport but also to another
E. coli
serotype present in the intestinal tract. Laboratory studies showed that the plasmid could be readily transferred between serovar Newport and
E. coli
intestinal isolates. Administration of a single dose of ceftiofur, used to prevent septicemic colibacillosis, to 1-day-old turkeys did not result in the isolation of ceftiofur-resistant
E. coli
or
Salmonella
serovar Newport. There was a remarkable association between serotype, drug resistance, and plasmid profile among the
E. coli
strains isolated from the poults. This study shows that
Salmonella
serovar Newport can become resistant to ESCs and other antibiotics by acquiring a conjugative drug resistance plasmid from
E. coli
in the intestines.
Influence of Starvation on Potential Ammonia-Oxidizing Activity and
amoA
mRNA Levels of
Nitrosospira briensis
Authors: Bollmann Annette , Schmidt Ingo , Saunders Aaron M. , Nicolaisen Mette H.
Published: March 2005

The effect of short-term ammonia starvation on
Nitrosospira briensis
was investigated. The ammonia-oxidizing activity was determined in a concentrated cell suspension with a NO
x
biosensor. The apparent half-saturation constant [

K
m(app)

] value of the NH
3
oxidation of
N. briensis
was 3 μM NH
3
for cultures grown both in continuous and batch cultures as determined by a NO
x
biosensor. Cells grown on the wall of the vessel had a lower

K
m(app)

value of 1.8 μM NH
3
. Nonstarving cultures of
N. briensis
showed potential ammonia-oxidizing activities of between 200 to 250 μM N h
−1
, and this activity decreased only slowly during starvation up to 10 days. Within 10 min after the addition of fresh NH
4
+
, 100% activity was regained. Parallel with activity measurements,
amoA
mRNA and 16S rRNA were investigated. No changes were observed in the 16S rRNA, but a relative decrease of
amoA
mRNA was observed during the starvation period. During resuscitation, an increase in
amoA
mRNA expression was detected simultaneously. The patterns of the soluble protein fraction of a 2-week-starved culture of
N. briensis
showed only small differences in comparison to a nonstarved control. From these results we conclude that
N. briensis
cells remain in a state allowing fast recovery of ammonia-oxidizing activity after addition of NH
4
+
to a starved culture. Maintaining cells in this kind of active state could be the survival strategy of ammonia-oxidizing bacteria in nature under fluctuating NH
4
+
availability.
Spatially Resolved Characterization of Biogenic Manganese Oxide Production within a Bacterial Biofilm
Authors: Toner Brandy , Fakra Sirine , Villalobos Mario , Warwick Tony , Sposito Garrison
Published: March 2005

Pseudomonas putida
strain MnB1, a biofilm-forming bacterial culture, was used as a model for the study of bacterial Mn oxidation in freshwater and soil environments. The oxidation of aqueous Mn
+2
[Mn
+2
(aq)
] by
P. putida
was characterized by spatially and temporally resolving the oxidation state of Mn in the presence of a bacterial biofilm, using scanning transmission X-ray microscopy (STXM) combined with near-edge X-ray absorption fine structure (NEXAFS) spectroscopy at the Mn L
2,3
absorption edges. Subsamples were collected from growth flasks containing 0.1 and 1 mM total Mn at 16, 24, 36, and 48 h after inoculation. Immediately after collection, the unprocessed hydrated subsamples were imaged at a 40-nm resolution. Manganese NEXAFS spectra were extracted from X-ray energy sequences of STXM images (stacks) and fit with linear combinations of well-characterized reference spectra to obtain quantitative relative abundances of Mn(II), Mn(III), and Mn(IV). Careful consideration was given to uncertainty in the normalization of the reference spectra, choice of reference compounds, and chemical changes due to radiation damage. The STXM results confirm that Mn
+2
(aq)
was removed from solution by
P. putida
and was concentrated as Mn(III) and Mn(IV) immediately adjacent to the bacterial cells. The Mn precipitates were completely enveloped by bacterial biofilm material. The distribution of Mn oxidation states was spatially heterogeneous within and between the clusters of bacterial cells. Scanning transmission X-ray microscopy is a promising tool for advancing the study of hydrated interfaces between minerals and bacteria, particularly in cases where the structure of bacterial biofilms needs to be maintained.
Differential Activities of FourLactobacillus caseiPromoters during Bacterial Transit through the Gastrointestinal Tracts of Human-Microbiota-Associated Mice
Authors: Oozeer R. , Furet J. P. , Goupil-Feuillerat N. , Anba J. , Mengaud J. , Corthier G.
Published: March 2005

In a previous study using fusion of the deregulated lactose promoterlacTp*and reporter genes, we suggested thatLactobacillus caseicould initiate de novo protein synthesis during intestinal transit. In order to confirm this finding and extend it to other promoters, we adopted a reverse transcriptase quantitative PCR (RT-QPCR) approach combined with a transcriptional fusion system consisting of luciferase genes under the control of four promoters (ccpA,dlt,ldh, andlacT*) fromL. caseiDN-114 001. Promoter expression was monitored during cell growth, and variable luciferase activities were detected. In 3-day cultures, all the genetically modified strains survived but without exhibiting luciferase activity. Luciferase mRNA levels determined by RT-QPCR analysis (RNA/CFU) were not significant. The cultures were administered to human-microbiota-associated mice, and the feces were collected 6 h later.L. caseipromoterslacTp* andldhp initiated mRNA synthesis during gastrointestinal transit. The promoters,ccpAp anddltp, exhibited no luciferase activity, nor was de novo-synthesized luciferase mRNA detected in the feces.L. caseiseems to adapt its physiology to the gastrointestinal tract environment by modulating promoter activities. The approach (fecal transcriptional analysis) described herein may, moreover, be of value in studying gene expression of transiting bacteria in human fecal specimens.
Histamine-Producing Pathway Encoded on an Unstable Plasmid in
Lactobacillus hilgardii
0006
Authors: Lucas Patrick M. , Wolken Wout A. M. , Claisse Olivier , Lolkema Juke S. , Lonvaud-Funel Aline
Published: March 2005

Histamine production from histidine in fermented food products by lactic acid bacteria results in food spoilage and is harmful to consumers. We have isolated a histamine-producing lactic acid bacterium,
Lactobacillus hilgardii
strain IOEB 0006, which could retain or lose the ability to produce histamine depending on culture conditions. The
hdcA
gene, coding for the histidine decarboxylase of
L. hilgardii
IOEB 0006, was located on an 80-kb plasmid that proved to be unstable. Sequencing of the
hdcA
locus disclosed a four-gene cluster encoding the histidine decarboxylase, a protein of unknown function, a histidyl-tRNA synthetase, and a protein, which we named HdcP, showing similarities to integral membrane transporters driving substrate/product exchange. The gene coding for HdcP was cloned downstream of a sequence specifying a histidine tag and expressed in
Lactococcus lactis
. The recombinant HdcP could drive the uptake of histidine into the cell and the exchange of histidine and histamine. The combination of HdcP and the histidine decarboxylase forms a typical bacterial decarboxylation pathway that may generate metabolic energy or be involved in the acid stress response. Analyses of sequences present in databases suggest that the other two proteins have dispensable functions. These results describe for the first time the genes encoding a histamine-producing pathway and provide clues to the parsimonious distribution and the instability of histamine-producing lactic acid bacteria.
Posttranslational Modification of 6-Phosphofructo-1-Kinase in
Aspergillus niger
Authors: Mesojednik Suzana , Legiša Matic
Published: March 2005

Two different enzymes exhibiting 6-phosphofructo-1-kinase (PFK1) activity were isolated from the mycelium of
Aspergillus niger
: the native enzyme with a molecular mass of 85 kDa, which corresponded to the calculated molecular mass of the deduced amino acid sequence of the
A. niger pfkA
gene, and a shorter protein of approximately 49 kDa. A fragment of identical size also was obtained in vitro by the proteolytic digestion of the partially purified native PFK1 with proteinase K. When PFK1 activity was measured during the proteolytic degradation of the native protein, it was found to be lost after 1 h of incubation, but it was reestablished after induction of phosphorylation by adding the catalytic subunit of cyclic AMP-dependent protein kinase to the system. By determining kinetic parameters, different ratios of activities measured at ATP concentrations of 0.1 and 1 mM were detected with fragmented PFK1, as with the native enzyme. Fructose-2,6-biphosphate significantly increased the
V
max
of the fragmented protein, while it had virtually no effect on the native protein. The native enzyme could be purified only from the early stages of growth on a minimal medium, while the 49-kDa fragment appeared later and was activated at the time of a sudden change in the growth rate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of sequential purifications of PFK1 enzymes by affinity chromatography during the early stages of the fungal development suggested spontaneous posttranslational modification of the native PFK1 in
A. niger
cells, while from the kinetic parameters determined for both isolated forms it could be concluded that the fragmented enzyme might be more efficient under physiological conditions.
Quantitative-PCR Assessment of
Cryptosporidium parvum
Cell Culture Infection
Authors: Di Giovanni George D. , LeChevallier Mark W.
Published: March 2005

A quantitative TaqMan PCR method was developed for assessing the
Cryptosporidium parvum
infection of in vitro cultivated human ileocecal adenocarcinoma (HCT-8) cell cultures. This method, termed cell culture quantitative sequence detection (CC-QSD), has numerous applications, several of which are presented. CC-QSD was used to investigate parasite infection in cell culture over time, the effects of oocyst treatment on infectivity and infectivity assessment of different
C. parvum
isolates. CC-QSD revealed that cell culture infection at 24 and 48 h postinoculation was approximately 20 and 60%, respectively, of the endpoint 72-h postinoculation infection. Evaluation of three different lots of
C. parvum
Iowa isolate oocysts revealed that the mean infection of 0.1 N HCl-treated oocysts was only 36% of the infection obtained with oocysts treated with acidified Hanks' balanced salt solution containing 1% trypsin. CC-QSD comparison of the
C. parvum
Iowa and TAMU isolates revealed significantly higher levels of infection for the TAMU isolate, which agrees with and supports previous human, animal, and cell culture studies. CC-QSD has the potential to aid in the optimization of
Cryptosporidium
cell culture methods and facilitate quantitative evaluation of cell culture infectivity experiments.
In Vivo Monitoring of Obligate Biotrophic Pathogen Growth by Kinetic PCR
Authors: Boyle Brian , Hamelin Richard C. , Séguin Armand
Published: March 2005

The plant kingdom is constantly challenged by a battery of evolving pathogens. New species or races of pathogens are discovered on crops that were initially bred for disease resistance, and globalization is facilitating the movement of exotic pests. Among these pests, obligate biotrophic parasites make up some of the most damaging groups and have been particularly challenging to study. Here we demonstrate the utility of kinetic PCR (kPCR) (real-time PCR, quantitative PCR) to assess the growth of poplar rust, caused by
Melampsora
species, by quantification of pathogen DNA. kPCR allowed the construction of reliable growth curves from inoculation through the final stages of uredinial maturation, as well as pathogen monitoring before symptoms become visible. Growth parameters, such as latency period, generation time in logarithmic growth, and the increase in DNA mass at saturation, were compared in compatible, incompatible, and nonhost interactions. Pathogen growth was monitored in different applications dealing with plant pathology, such as host and pathogen diversity and transgenic crop improvement. Finally, the capacity of kPCR to differentiate pathogens in the same sample has broad molecular ecology applications for dynamically monitoring the growth of fungi in their environments or in mixed populations or to measure the efficacy of pest control strategies.
Characterization of the Highly Efficient Sucrose Isomerase from
Pantoea dispersa
UQ68J and Cloning of the Sucrose Isomerase Gene
Authors: Wu Luguang , Birch Robert G.
Published: March 2005

Sucrose isomerase (SI) genes from
Pantoea dispersa
UQ68J,
Klebsiella planticola
UQ14S, and
Erwinia rhapontici
WAC2928 were cloned and expressed in
Escherichia coli
. The predicted products of the UQ14S and WAC2928 genes were similar to known SIs. The UQ68J SI differed substantially, and it showed the highest isomaltulose-producing efficiency in
E. coli
cells. The purified recombinant WAC2928 SI was unstable, whereas purified UQ68J and UQ14S SIs were very stable. UQ68J SI activity was optimal at pH 5 and 30 to 35°C, and it produced a high ratio of isomaltulose to trehalulose (>22:1) across its pH and temperature ranges for activity (pH 4 to 7 and 20 to 50°C). In contrast, UQ14S SI showed optimal activity at pH 6 and 35°C and produced a lower ratio of isomaltulose to trehalulose (<8:1) across its pH and temperature ranges for activity. UQ68J SI had much higher catalytic efficiency; the

K
m

was 39.9 mM, the
V
max
was 638 U mg
−1
, and the
K
cat
/

K
m

was 1.79 × 10
4
M
−1
s
−1
, compared to a

K
m

of 76.0 mM, a
V
max
of 423 U mg
−1
, and a
K
cat
/

K
m

of 0.62 × 10
4
M
−1
s
−1
for UQ14S SI. UQ68J SI also showed no apparent reverse reaction producing glucose, fructose, or trehalulose from isomaltulose. These properties of the
P. dispersa
UQ68J enzyme are exceptional among purified SIs, and they indicate likely differences in the mechanism at the enzyme active site. They may favor the production of isomaltulose as an inhibitor of competing microbes in high-sucrose environments, and they are likely to be highly beneficial for industrial production of isomaltulose.
Genomic Comparisons of
Salmonella enterica
Serovar Dublin, Agona, and Typhimurium Strains Recently Isolated from Milk Filters and Bovine Samples from Ireland, Using a
Salmonella
Microarray
Authors: Reen F. J. , Boyd E. F. , Porwollik S. , Murphy B. P. , Gilroy D. , Fanning S. , McClelland M.
Published: March 2005

Salmonella
-induced enterocolitis is the leading food-borne illness with a lethal outcome and causes millions of cases of gastroenteritis each year. We examined genomic variation among 12 environmental, veterinary, and clinical
Salmonella enterica
serovar Dublin, Agona, and Typhimurium strains isolated in Ireland between 2000 and 2003, as well as two clinical isolates from Canada and four archival isolates, which belonged to serovars Dublin and Agona. Using DNA-DNA hybridization to a microarray consisting of most of the predicted protein-encoding sequences of the
S. enterica
serovar Typhimurium LT2 genome, we identified a number of genomic regions that were absent in one or more serovars. The 34 genomic regions encoded proteins involved in sugar metabolism, transport, fimbrial and phage biogenesis, and transcriptional regulation, as well as inner and outer membrane-associated proteins. Two of the four prophages identified in strain LT2, prophages Gifsy-1 and Gifsy-2, were present in all six serovar Typhimurium strains examined. Prophage Fels-1 was absent from all 18 isolates examined, and Fels-2 was completely absent from the serovar Typhimurium isolates and the
Salmonella
Reference Collection B serovar Dublin strain Du2. All five
Salmonella
pathogenicity islands were present in all isolates. Plasmid pSLT was absent from all serovar Agona isolates, and only homologues of the
spv
genes were present in eight of the nine serovar Dublin strains. Only limited intraserovar diversity was found among the nine serovar Dublin, three serovar Agona, and six serovar Typhimurium isolates examined even though these isolates had extensive geographic, temporal, and source differences.
Dynamics of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase Gene Expression in the Coccolithophorid
Coccolithus pelagicus
during a Tracer Release Experiment in the Northeast Atlantic
Authors: Wyman Michael , Davies John T. , Hodgson Sylvia , Tarran Glen A. , Purdie Duncan A.
Published: March 2005

We report a pronounced diel rhythm in ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) gene expression in a natural population of the coccolithophorid
Coccolithus pelagicus
sampled during a Lagrangian experiment in the Northeast Atlantic. Our observations show that there is greater heterogeneity in the temporal regulation of RubisCO expression among planktonic chromophytes than has been reported hitherto.
Optical Brighteners Do Not Influence Covert Baculovirus Infection of
Spodoptera frugiperda
Authors: Martínez Ana-Mabel , Williams Trevor , López-Ferber Miguel , Caballero Primitivo
Published: March 2005

Covert infection with
Spodoptera frugiperda
multiple nucleopolyhedrovirus, detected by reverse transcription-PCR of virus gene transcripts (
ie-0
and
polh
), was not significantly affected by the presence of an optical brightener (Tinopal UNPA-GX), indicating no change in virus virulence. Detection of the covert infection was dependent on insect life stage and the viral mRNA used for diagnosis.
Structural characterization of the antigenic capsular polysaccharide and lipopolysaccharide O-chain produced byActinobacillus pleuropneumoniaeserotype 15
Authors: Perry Malcolm B , MacLean Leann L , Vinogradov Evgeny
Published: March 2005

The specific capsular polysaccharide produced by Actinobacillus pleuropneumoniae serotype 15 was determined to be a high-molecular-mass polymer having [α]D+ 69° (water) and composed of a linear backbone of phosphate diester linked disaccharide units of 2-acetamido-2-deoxy-D-glucose (D-GlcNAc) and 2-acetamido-2-deoxy-D-galactose (D-GalNAc) residues (1:1). Thirty percent of the D-GalNAc residues were substituted at O-4 by β-D-galactopyranose (β-D-Galp) residues. Through the application of chemical and NMR methods, the capsule, which defines the serotype specificity of the bacterium, was found to have the structure

                        [Formula: see text]

The O-polysaccharide (O-PS) component of the A. pleuro pneumoniae serotype 15 lipopolysaccharide (LPS) was characterized as a linear unbranched polymer of repeating pentasaccharide units composed of D-glucose (2 parts) and D-galactose (3 parts), shown to have the structure

[Formula: see text]

The O-PS was chemically identical with the O-antigen previously identified in the LPSs produced by A. pleuro pneumoniae serotypes 3 and 8. Key words: Capsule, lipopolysaccharide, Actinobacillus pleuropneumoniae serotype 15.
Ion Conduction and Selectivity in K+ Channels
Authors: Roux Benoît
Published: March 2005

▪ Abstract  Potassium (K+) channels are tetrameric membrane-spanning proteins that provide a selective pore for the conductance of K+ across the cell membranes. These channels are most remarkable in their ability to discriminate K+ from Na+ by more than a thousandfold and conduct at a throughput rate near diffusion limit. The recent progress in the structural characterization of K+ channel provides us with a unique opportunity to understand their function at the atomic level. With their ability to go beyond static structures, molecular dynamics simulations based on atomic models can play an important role in shaping our view of how ion channels carry out their function. The purpose of this review is to summarize the most important findings from experiments and computations and to highlight a number of fundamental mechanistic questions about ion conduction and selectivity that will require further work.
A role for Rho and Rac in secretagogue-induced amylase release by pancreatic acini
Authors: Bi Yan , Williams John A.
Published: March 2005

The actin cytoskeleton has long been implicated in protein secretion. We investigated whether Rho and Rac, known regulators of the cytoskeleton, are involved in amylase secretion by mouse pancreatic acini. Secretagogues, including cholecystokinin (CCK) and the acetylcholine analog carbachol, increased the amount of GTP-bound RhoA and Rac1 and induced translocation from cytosol to a membrane fraction. Immunocytochemistry revealed the translocation of Rho and Rac within the apical region of the cell. Expression by means of adenoviral vectors of dominant-negative Rho (RhoN19), dominant-negative Rac (RacN17), and Clostridium Botulinum C3 exotoxin, which ADP ribosylates and inactivates Rho, significantly inhibited amylase secretion by CCK and carbachol; inhibiting both Rho and Rac resulted in a greater reduction. This inhibitory effect of RhoN19 on CCK-induced amylase secretion was apparent in both the early and late phases of secretion, whereas RacN17 was more potent on the late phase of secretion. None of these three affected the basal Ca2+ or the peak intracellular Ca2+ concentration stimulated by CCK. Latrunculin, a marine toxin that sequesters actin monomers, time-dependently decreased the total amount of filamentous actin (F-actin) and dose-dependently decreased secretion by secretagogues without affecting Ca2+ signaling. These data suggest that Rho and Rac are both involved in CCK-induced amylase release in pancreatic acinar cell possibly through an effect on the actin cytoskeleton.
Effects of lactic acid and catecholamines on contractility in fast-twitch muscles exposed to hyperkalemia
Authors: Hansen Anders Krogh , Clausen Torben , Nielsen Ole Bækgaard
Published: March 2005

Intensive exercise is associated with a pronounced increase in extracellular K+ ([K+]o). Because of the ensuing depolarization and loss of excitability, this contributes to muscle fatigue. Intensive exercise also increases the level of circulating catecholamines and lactic acid, which both have been shown to alleviate the depressing effect of hyperkalemia in slow-twitch muscles. Because of their larger exercise-induced loss of K+, fast-twitch muscles are more prone to fatigue caused by increased [K+]o than slow-twitch muscles. Fast-twitch muscles also produce more lactic acid. We therefore compared the effects of catecholamines and lactic acid on the maintenance of contractility in rat fast-twitch [extensor digitorum longus (EDL)] and slow-twitch (soleus) muscles. Intact muscles were mounted on force transducers and stimulated electrically to evoke short isometric tetani. Elevated [K+]o (11 and 13 mM) was used to reduce force to ∼20% of control force at 4 mM K+. In EDL, the β2-agonist salbutamol (10−5 M) restored tetanic force to 83 ± 2% of control force, whereas in soleus salbutamol restored tetanic force to 93 ± 1%. In both muscles, salbutamol induced hyperpolarization (5–8 mV), reduced intracellular Na+ content and increased Na+-K+ pump activity, leading to an increased K+ tolerance. Lactic acid (24 mM) restored force from 22 ± 4% to 58 ± 2% of control force in EDL, an effect that was significantly lower than in soleus muscle. These results amplify and generalize the concept that the exercise-induced acidification and increase in plasma catecholamines counterbalance fatigue arising from rundown of Na+ and K+ gradients.
Frontoparietal Control of Spatial Attention and Motor Intention in Human EEG
Authors: Praamstra Peter , Boutsen Luc , Humphreys Glyn W.
Published: March 2005

Relations between spatial attention and motor intention were investigated by means of an EEG potential elicited by shifting attention to a location in space as well as by the selection of a hand for responding. High-density recordings traced this potential to a common frontoparietal network activated by attentional orienting and by response selection. Within this network, parietal and frontal cortex were activated sequentially, followed by an anterior-to-posterior migration of activity culminating in the lateral occipital cortex. Based on temporal and polarity information provided by EEG, we hypothesize that the frontoparietal activation, evoked by directional information, updates a task-defined preparatory state by deselecting or inhibiting the behavioral option competing with the cued response side or the cued direction of attention. These results from human EEG demonstrate a direct EEG manifestation of the frontoparietal attention network previously identified in functional imaging. EEG reveals the time-course of activation within this network and elucidates the generation and function of associated directing-attention EEG potentials. The results emphasize transient activation and a decision-related function of the frontoparietal attention network, contrasting with the sustained preparatory activation that is commonly inferred from neuroimaging.
Effects of Inhibitory Feedback in a Network Model of Avian Brain Stem
Authors: Dasika Vasant K. , White John A. , Carney Laurel H. , Colburn H. Steven
Published: March 2005

The avian auditory brain stem consists of a network of specialized nuclei, including nucleus laminaris (NL) and superior olivary nucleus (SON). NL cells show sensitivity to interaural time difference (ITD), a critical cue that underlies spatial hearing. SON cells provide inhibitory feedback to the rest of the network. Empirical data suggest that feedback inhibition from SON could increase the ITD sensitivity of NL across sound level. Using a bilateral network model, we assess the effects of SON feedback inhibition. Individual cells are specified as modified leaky-integrate-and-fire neurons with time constants and thresholds that vary with inhibitory input. Acoustic sound level is reflected in the discharge rates of the model auditory-nerve fibers, which innervate the network. Simulations show that with SON inhibitory feedback, ITD sensitivity is maintained in model NL cells over a threefold range in auditory-nerve discharge rate. In contrast, without SON feedback inhibition, ITD sensitivity is significantly reduced as input rates are increased. Feedback inhibition is most beneficial in maintaining ITD sensitivity at high-input rates (simulating high sound levels). With SON inhibition, ITD sensitivity is maintained for both interaurally balanced inputs (simulating an on-center sound source) and interaurally imbalanced inputs (simulating a lateralized source). Further, the empirically observed temporal build-up of SON inhibition and the presence of reciprocal inhibitory connections between the ipsi- and contralateral SON both improve ITD sensitivity. In sum, our network model shows that inhibitory feedback can substantially increase the sensitivity and dynamic range of ITD coding in the avian auditory brain stem.
Determination of Zinc‐Based Additives in Lubricating Oils by Flow‐Injection Analysis with Flame‐AAS Detection Exploiting Injection with a Computer‐Controlled Syringe
Authors: Pignalosa Gustavo , Knochen Moisés , Cabrera Noel
Published: March 2005

A flow‐injection system is proposed for the determination of metal‐based additives in lubricating oils. The system, operating under computer control uses a motorised syringe for measuring and injecting the oil sample (200 μL) in a kerosene stream, where it is dispersed by means of a packed mixing reactor and carried to an atomic absorption spectrometer which is used as detector. Zinc was used as model analyte. Two different systems were evaluated, one for low concentrations (range 0–10 ppm) and the second capable of providing higher dilution rates for high concentrations (range 0.02%–0.2% w/w). The sampling frequency was about 30 samples/h. Calibration curves fitted a second‐degree regression model (r2 = 0.996). Commercial samples with high and low zinc levels were analysed by the proposed method and the results were compared with those obtained with the standard ASTM method. The t test for mean values showed no significant differences at the 95% confidence level. Precision (RSD%) was better than 5% (2% typical) for the high concentrations system. The carryover between successive injections was found to be
negligible.
Organization and evolution of highly repeated satellite DNA sequences in plant chromosomes
Authors: Sharma S. , Raina S.N.
Published: March 2005

A major component of the plant nuclear genome is constituted by different classes of repetitive DNA sequences. The structural, functional and evolutionary aspects of the satellite repetitive DNA families, and their organization in the chromosomes is reviewed. The tandem satellite DNA sequences exhibit characteristic chromosomal locations, usually at subtelomeric and centromeric regions. The repetitive DNA family(ies) may be widely distributed in a taxonomic family or a genus, or may be specific for a species, genome or even a chromosome. They may acquire large-scale variations in their sequence and copy number over an evolutionary time-scale. These features have formed the basis of extensive utilization of repetitive sequences for taxonomic and phylogenetic studies. Hybrid polyploids have especially proven to be excellent models for studying the evolution of repetitive DNA sequences. Recent studies explicitly show that some repetitive DNA families localized at the telomeres and centromeres have acquired important structural and functional significance. The repetitive elements are under different evolutionary constraints as compared to the genes. Satellite DNA families are thought to arise de novo as a consequence of molecular mechanisms such as unequal crossing over, rolling circle amplification, replication slippage and mutation that constitute “molecular drive”.
Unraveling the genome structure of polyploids using FISH and GISH; examples of sugarcane and banana
Authors: D’Hont A.
Published: March 2005

We review here the progress that has been achieved using molecular cytogenetics to analyze the genome structure of sugarcane (Saccharum spp) and banana (Musa spp), two crops that are polyploid, of interspecific origin and with chromosomes not distinguishable by their gross morphology. In Saccharum, molecular cytogenetics enabled us to determine the basic chromosome number of two species, Saccharum officinarum and S. spontaneum, involved in the origin of modern cultivars, to quantify the proportion of chromosomes of these species in the genome of modern cultivars, to assess the extent of interspecific chromosome recombination and to clarify the origin of the related species S. barberi. These techniques are also used to monitor introgression with related genera. In Musa, GISH enabled us to differentiate the four genomes involved in banana cultivars and allowed us to determine the genome constitution of several cultivars. FISH was used to analyze the distribution of repeated sequences along the genome.
Variability of the chromosomal distribution of Ty3-gypsy retrotransposons in the populations of two wild Triticeae species
Authors: Belyayev A. , Raskina O. , Nevo E.
Published: March 2005

Here, we report data on the population variability of Ty3-gypsy retrotransposons in genomes of Aegilops speltoides (2n = 2x = 14) and Hordeum spontaneum (2n = 2x = 14). Based on the sequence analysis or reverse transcriptase (RT) gene conserved domains, two groups of elements were recognized. Elements of Group I show relatedness to such a known element as RIRE2, and elements of Group II show relatedness to Fatima and Cereba. Cloned and sequenced fragments of Ty3-gypsy RT that show the closest relatedness to known elements (Fatima and RIRE2) were used as probes for fluorescent in situ hybridization (FISH). FISH experiments revealed mini-cluster organization of the Ty3-gypsy element chromosomal distribution in wild Triticeae species. Mini-clusters can be divided into three categories according to their intraspecific stability: (i) stable species-specific clusters that are mainly adjusted to the regions of rRNA genes; (ii) variable clusters that represent 68% of clusters in the genome of Ae. speltoides and 20% in the genome of H. spontaneum; and (iii) population-specific clusters that are mainly insertions into centromeric central domains of different chromosomes and the majority of these insertions were detected in populations with hot, dry environments. Significant interpopulation variability of Ty3-gypsy element chromosomal distribution in the Ae. speltoides genome contrasts with the uniform genome of H. spontaneum and may reflect differences in adaptive strategies between investigated species.
Nuclear genome size and genomic distribution of ribosomal DNA in Musa and Ensete (Musaceae): taxonomic implications
Authors: Bartoš J. , Alkhimova O. , Doleželová M. , De Langhe E. , Doležel J.
Published: March 2005

Nuclear DNA content and genomic distributions of 5S and 45S rDNA were examined in nineteen diploid accessions of the genus Musa representing its four sections Eumusa, Rhodochlamys, Callimusa and Australimusa, and in Ensete gilletii, which was the outgroup in this study. In the Eumusa (x = 11), 2C DNA content ranged from 1.130 to 1.377 pg, M. balbisiana having the lowest DNA content of all sections. M. beccarii (x = 9), a representative of Callimusa, had the highest 2C nuclear DNA content (1.561 pg). Species belonging to Rhodochlamys (x = 11) and Australimusa (x = 10) had 2C DNA contents ranging from 1.191 to 1.299 pg and from 1.435 to 1.547 pg, respectively. E. gilletii (x = 9) had 2C DNA content of 1.210 pg. The number of 5S rDNA loci in Musa varied from 4 to 8 per diploid cell. While different numbers of 5S rDNA loci were observed within Eumusa and Rhodochlamys, four 5S rDNA loci were observed in all accessions of Australimusa. M. beccarii (Callimusa) and E. gilletii contained 5S rRNA gene clusters on five and six chromosomes, respectively. The number of 45S rDNA loci was conserved within individual sections. Hierarchical cluster analysis of genome size, number of chromosomes and 45S rDNA sites suggested a close relationship between Rhodochlamys and Eumusa; Australimusa was clearly separated as were M. beccarii and E. gilletii. Within the Eumusa-Rhodochlamys group, M. balbisiana, M. schizocarpa and M. ornata formed distinct subgroups, clearly separated from the accessions of M. acuminata, M. mannii, M. laterita and M. velutina, which formed a tight subgroup. The results expand the knowledge of genome size and genomic distribution of ribosomal DNA in Musa and Ensete. They aid in clarification of the taxonomical classification of Musa and show a need to supplement the analyses on the DNA sequence level with cytogenetic studies.
Long-range organization of plant satellite repeats investigated using strand-specific FISH
Authors: Navrátilová A. , Neumann P. , Macas J.
Published: March 2005

The technique of chromosomal orientation and direction fluorescence in situ hybridization (COD-FISH) was adapted for plant chromosomes in order to study long-range organization of two families of satellite repeats, VicTR-B of Vicia sativa and PisTR-B of Pisum sativum. The technique allowed FISH to be performed on mitotic chromosomes in a strand-specific manner, resulting in visualization of the repeat orientation along the chromosomes and with respect to the direction of telomeric repeats. The VicTR-B probe applied to V. sativa chromosomes produced signals on a single chromatid at most regions containing corresponding sequences, thus confirming a presence of long arrays of head-to-tail arranged repeat monomers which is typical for satellite DNA. However, hybridization signals of different or equal intensities on both chromatids were also detected at some loci, suggesting a more complex arrangement of the repeats. Similar observations were made for PisTR-B repeats on P. sativum chromosomes, although the proportion of loci displaying signals on both chromatids was lower. In contrast to VicTR-B, orientation of the PisTR-B clusters with respect to telomeric sequences appeared to be conserved among subtelomeric regions of metacentric chromosomes and of the short arms of acrocentric chromosomes.
McClintock’s controlling elements: the full story
Authors: Jones R.N.
Published: March 2005

The chapter describes the early part of Barbara McClintock’s work on DNA transposons in maize, in which she uncovered the Ac-Ds family of mobile “controlling elements”. As a basis to understanding the work there is a preamble on the reproductive biology of maize, which explains the particular qualities of the maize plant as an experimental organism for genetics. An account is then given of the cytology of the system that was used to generate intact chromosomes having “sticky” (broken) ends, which is the starting point of the story. Cytogenetic aspects of the chromatid and chromosome breakage-fusion-bridge cycles, deriving from breakage, are then described; this leads on to a discussion about the way in which variegation phenotypes of the maize kernels can be “read” in terms of chromosome breakage. The “genetic earthquake” event of 1944, triggered by introducing broken chromosomes into a zygote from both parents, is then described; and the sequence of events leading to the discovery of Ds and Ac is traced. Finding mobility of Ds from one chromosomal location to another was pure serendipity, and the transposition showed itself while experiments were being undertaken to accurately map Ds. A similar chance observation revealed transposition of Ac as well, and then the relationship between the two elements was elucidated in terms of their autonomous and non-autonomous nature. The chapter concludes with a brief reference to the molecular cloning of Ac and Ds.
Molecular analysis of holocentric centromeres of Luzula species
Authors: Haizel T. , Lim Y.K. , Leitch A.R. , Moore G.
Published: March 2005

Luzula spp, like the rest of the members of the Juncaceae family, have holocentric chromosomes. Using the rice 155-bp centromeric tandem repeat sequence (RCS2) as a probe, we have isolated and characterized a 178-bp tandem sequence repeat (LCS1) from Luzula nivea. The LCS1 sequence is present in all Luzula species tested so far (except L. pilosa) and like other satellite repeats found in heterochromatin, the cytosine residues are methylated within the LCS1 repeats. Using fluorescent in situ hybridization (FISH) experiments we have shown that there are at least 5 large clusters of LCS1 sequences distributed at heterochromatin regions along each of the 12 chromosomes of L. nivea. We have shown that a centromeric antibody Skp1 co-localizes with these heterochromatin regions and with the LCS1 sequences. This suggests that the LCS1 sequences are part of regions which function as centromeres on these holocentric chromosomes. Furthermore, using the BrdU assay to identify replication sites, we have shown that these heterochromatin sites containing LCS1 associate when being replicated in root interphase nuclei. Our results also show premeiotic chromosome association during anther development as indicated by single-copy BAC in situ and the presence of fewer LCS1 containing heterochromatin sites in these cells.