bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of dopaminergic neuron firing by heterogeneous dopamine autoreceptors in the substantia nigra pars compacta
Authors: Jang Jin Young , Jang Miae , Kim Shin Hye , Um Ki Bum , Kang Yun Kyung , Kim Hyun Jin , Chung Sungkwon , Park Myoung Kyu
Published: November 2010

J. Neurochem.(2011)116, 966–974.

Dopamine (DA) receptors generate many cellular signals and play various roles in locomotion, motivation, hormone production, and drug abuse. According to the location and expression types of the receptors in the brain, DA signals act in either stimulatory or inhibitory manners. Although DA autoreceptors in the substantia nigra pars compacta are known to regulate firing activity, the exact expression patterns and roles of DA autoreceptor types on the firing activity are highly debated. Therefore, we performed individual correlation studies between firing activity and receptor expression patterns using acutely isolated rat substantia nigra pars compacta DA neurons. When we performed single‐cell RT‐PCR experiments, D1, D2S, D2L, D3, and D5receptor mRNA were heterogeneously expressed in the order of D2L > D2S > D3 > D5 > D1. Stimulation of D2receptors with quinpirole suppressed spontaneous firing similarly among all neurons expressing mRNA solely for D2S, D2L, or D3receptors. However, quinpirole most strongly suppressed spontaneous firing in the neurons expressing mRNA for both D2and D3receptors. These data suggest that D2S, D2L, and D3receptors are able to equally suppress firing activity, but that D2and D3receptors synergistically suppress firing. This diversity in DA autoreceptors could explain the various actions of DA in the brain.
Pyrosequencing of the Genital Microbiotas of HIV-Seropositive and -Seronegative Women Reveals
Lactobacillus iners
as the Predominant
Lactobacillus
Species
Authors: Spear Gregory T. , Gilbert Douglas , Landay Alan L. , Zariffard Reza , French Audrey L. , Patel Pranjal , Gillevet Patrick M.
Published: November 2010

The species of vaginal lactobacilli in HIV-seropositive and -seronegative women were determined by 16S gene pyrosequencing.
Lactobacillus iners
sequences were the predominant lactobacillus sequences in 66% of HIV
+
women and 90% of HIV
−
women. This has implications for resistance of HIV
+
and HIV
−
women to genital colonization by pathogenic organisms.
Effects of Ionic Strength on Bacteriophage MS2 Behavior and Their Implications for the Assessment of Virus Retention by Ultrafiltration Membranes
Authors: Furiga Aurelie , Pierre Gwenaelle , Glories Marie , Aimar Pierre , Roques Christine , Causserand Christel , Berge Mathieu
Published: November 2010

Bacteriophage MS2 is widely used as a surrogate to estimate pathogenic virus elimination by membrane filtration processes used in water treatment. Given that this water technology may be conducted with different types of waters, we focused on investigating the effects of ionic strength on MS2 behavior. For this, MS2 was analyzed while suspended in solutions of various ionic strengths, first in a batch experiment and second during membrane ultrafiltration, and quantified using (i) quantitative reverse transcriptase PCR (qRT-PCR), which detects the total number of viral genomes, (ii) qRT-PCR without the RNA extraction step, which reflects only particles with a broken capsid (free RNA), and (iii) the PFU method, which detects only infectious viruses. At the beginning of the batch experiments using solutions containing small amounts of salts, losses of MS2 infectivity (90%) and broken particles (20%) were observed; these proportions did not change during filtration. In contrast, in high-ionic-strength solutions, bacteriophage kept its biological activity under static conditions, but it quickly lost its infectivity during the filtration process. Increasing the ionic strength decreased both the inactivation and the capsid breakup in the feed suspension and increased the loss of infectivity in the filtration retentate, while the numbers of MS2 genomes were identical in both experiments. In conclusion, the effects of ionic strength on MS2 behavior may significantly distort the results of membrane filtration processes, and therefore, the combination of classical and molecular methods used here is useful for an effective validation of the retention efficiency of ultrafiltration membranes.
Autoregulation of Lantibiotic Bovicin HJ50 Biosynthesis by the BovK-BovR Two-Component Signal Transduction System in
Streptococcus bovis
HJ50
Authors: Ni Jianqiang , Teng Kunling , Liu Gang , Qiao Caixia , Huan Liandong , Zhong Jin
Published: November 2010

Streptococcus bovis
HJ50 produces a lacticin 481-like 33-amino-acid-residue lantibiotic, designated bovicin HJ50.
bovK-bovR
in the bovicin HJ50 biosynthetic gene cluster is predicted to be a two-component signal transduction system involved in sensing signals and regulating gene expression. Disruption of
bovK
or
bovR
resulted in the abrogation of bovicin HJ50 production, suggesting both genes play important roles in bovicin HJ50 biosynthesis. Addition of exogenous bovicin HJ50 peptide to cultures of a
bovM
mutant that lost the capability for bovicin HJ50 production and structural gene
bovA
transcription in
S. bovis
HJ50 induced dose-dependent transcription of the
bovA
gene, demonstrating that bovicin HJ50 production was normally autoregulated. The transcription of
bovA
was no longer induced by bovicin HJ50 in
bovK
and
bovR
disruption mutants, suggesting that BovK-BovR plays an essential role in the signal transduction regulating bovicin HJ50 biosynthesis. A phosphorylation assay indicated that BovK has the ability to autophosphorylate and subsequently transfer the phosphoryl group to the downstream BovR protein to carry on signal transduction. Electromobility shift assays (EMSA) and green fluorescent protein (GFP) reporter gene expression assays showed the specific binding of BovR to the
bovA
promoter, indicating that BovR regulates
bovA
expression by direct binding between them. Taken together, bovicin HJ50 biosynthesis is induced by bovicin HJ50 itself and regulated via the two-component signal transduction system BovK-BovR.
Effect of Environmental Factors and Influence of Rumen and Hindgut Biogeography on Bacterial Communities in Steers
Authors: Romero-Pérez Gustavo A. , Ominski Kim H. , McAllister Tim A. , Krause Denis O.
Published: November 2010

Feces from cattle production are considered important sources of bacterial contamination of food and the environment. Little is known about the combined effects of arctic temperatures and fodder tannins on rumen and hindgut bacterial populations. Individual rumen liquor and rectal fecal samples from donor steers fed either alfalfa silage or sainfoin (
Onobrychis viciifolia
Scop.) silage and water
ad libitum
were collected weekly on the first three sampling days and fortnightly afterwards. The daily ambient temperatures were registered and averaged to weekly mean temperatures. Steers fed sainfoin silage had lower (
P
< 0.05) concentrations of branched-chain volatile fatty acids (VFA) than those fed alfalfa silage. All VFA concentrations were higher (
P
< 0.001) in rumen liquor samples than in fecal samples. The interaction of sample type and diet showed a significant effect (
P
< 0.05) on the proportions of the bacterial community that were from the phyla
Proteobacteria
and
Verrucomicrobia.
Ambient temperature had an indirect effect (
P
< 0.05) on the phylum
Firmicutes
, as it affected its proportional balance. The bacterial population diversity in samples appeared to decrease concurrently with the ambient temperature. The phylum
Firmicutes
explained the first principal component at 64.83 and 42.58% of the total variance in rumen liquor and fecal samples, respectively. The sample type had a larger effect on bacterial communities than diet and temperature. Certain bacterial populations seemed to be better adapted than others to environmentally adverse conditions, such as less access time to nutrients due to higher motility and rate of passage of digesta caused by extreme temperatures, or antimicrobials such as tannins, possibly due to an influence of their biogeographical location within the gut.
Microbial Community Fingerprinting by Differential Display-Denaturing Gradient Gel Electrophoresis
Authors: Portillo M. C. , Villahermosa D. , Corzo A. , Gonzalez J. M.
Published: November 2010

Complex microbial communities exhibit a large diversity, hampering differentiation by DNA fingerprinting. Herein, differential display-denaturing gradient gel electrophoresis is proposed. By adding a nucleotide to the 3′ ends of PCR primers, 16 primer pairs and fingerprints were generated per community. Complexity reduction in each partial fingerprint facilitates sample comparison.
Influence of External Resistance on Electrogenesis, Methanogenesis, and Anode Prokaryotic Communities in Microbial Fuel Cells
Authors: Jung Sokhee , Regan John M.
Published: November 2010

The external resistance (
R
ext
) of microbial fuel cells (MFCs) regulates both the anode availability as an electron acceptor and the electron flux through the circuit. We evaluated the effects of
R
ext
on MFCs using acetate or glucose. The average current densities (
I
) ranged from 40.5 mA/m
2
(9,800 Ω) to 284.5 mA/m
2
(150 Ω) for acetate-fed MFCs (acetate-fed reactors [ARs]), with a corresponding anode potential (
E
an
) range of −188 to −4 mV (versus a standard hydrogen electrode [SHE]). For glucose-fed MFCs (glucose-fed reactors [GRs]),
I
ranged from 40.0 mA/m
2
(9,800 Ω) to 273.0 mA/m
2
(150 Ω), with a corresponding
E
an
range of −189 to −7 mV. ARs produced higher Coulombic efficiencies and energy efficiencies than GRs over all tested
R
ext
levels because of electron and potential losses from glucose fermentation. Biogas production accounted for 14 to 18% of electron flux in GRs but only 0 to 6% of that in ARs. GRs produced similar levels of methane, regardless of the
R
ext
. However, total methane production in ARs increased as
R
ext
increased, suggesting that
E
an
might influence the competition for substrates between exoelectrogens and methanogens in ARs. An increase of
R
ext
to 9,800 Ω significantly changed the anode bacterial communities for both ARs and GRs, while operating at 970 Ω and 150 Ω had little effect.
Deltaproteobacteria
and
Bacteroidetes
were the major groups found in anode communities in ARs and GRs.
Betaproteobacteria
and
Gammaproteobacteria
were found only in ARs.
Bacilli
were abundant only in GRs. The anode-methanogenic communities were dominated by
Methanosaetaceae
, with significantly lower numbers of
Methanomicrobiales
. These results show that
R
ext
affects not only the
E
an
and current generation but also the anode biofilm community and methanogenesis.
Different Contributions of HtrA Protease and Chaperone Activities to
Campylobacter jejuni
Stress Tolerance and Physiology
Authors: Bæk Kristoffer T. , Vegge Christina S. , Skórko-Glonek Joanna , Brøndsted Lone
Published: November 2010

The microaerophilic bacterium
Campylobacter jejuni
is the most common cause of bacterial food-borne infections in the developed world. Tolerance to environmental stress relies on proteases and chaperones in the cell envelope, such as HtrA and SurA. HtrA displays both chaperone and protease activities, but little is known about how each of these activities contributes to stress tolerance in bacteria.
In vitro
experiments showed temperature-dependent protease and chaperone activities of
C. jejuni
HtrA. A
C. jejuni
mutant lacking only the protease activity of HtrA was used to show that the HtrA chaperone activity is sufficient for growth at high temperature or under oxidative stress, whereas the HtrA protease activity is essential only under conditions close to the growth limit for
C. jejuni
. However, the protease activity was required to prevent induction of the cytoplasmic heat shock response even under optimal growth conditions. Interestingly, the requirement of HtrA at high temperatures was found to depend on the oxygen level, and our data suggest that HtrA may protect oxidatively damaged proteins. Finally, protease activity stimulates HtrA production and oligomer formation, suggesting that a regulatory role depends on the protease activity of HtrA. Studying a microaerophilic organism encoding only two known periplasmic chaperones (HtrA and SurA) revealed an efficient HtrA chaperone activity and proposed multiple roles of the protease activity, increasing our understanding of HtrA in bacterial physiology.
Environmental Patterns Are Imposed on the Population Structure of
Escherichia coli
after Fecal Deposition
Authors: Bergholz Peter W. , Noar Jesse D. , Buckley Daniel H.
Published: November 2010

The intestinal microbe
Escherichia coli
is subject to fecal deposition in secondary habitats, where it persists transiently, allowing for the opportunity to colonize new hosts. Selection in the secondary habitat can be postulated, but its impact on the genomic diversity of
E. coli
is unknown. Environmental selective pressure on extrahost
E. coli
can be revealed by landscape genetic analysis, which examines the influences of dispersal processes, landscape features, and the environment on the spatiotemporal distribution of genes in natural populations. We conducted multilocus sequence analysis of 353
E. coli
isolates from soil and fecal samples obtained in a recreational meadow to examine the ecological processes controlling their distributions. Soil isolates, as a group, were not genetically distinct from fecal isolates, with only 0.8% of genetic variation and no fixed mutations attributed to the isolate source. Analysis of the landscape genetic structure of
E. coli
populations showed a patchy spatial structure consistent with patterns of fecal deposition. Controlling for the spatial pattern made it possible to detect environmental gradients of pH, moisture, and organic matter corresponding to the genetic structure of
E. coli
in soil. Ecological distinctions among
E. coli
subpopulations (i.e.,
E. coli
reference collection [ECOR] groups) contributed to variation in subpopulation distributions. Therefore, while fecal deposition is the major predictor of
E. coli
distributions on the field scale, selection imposed by the soil environment has a significant impact on
E. coli
population structure and potentially amplifies the occasional introduction of stress-tolerant strains to new host individuals by transmission through water or food.
Army Ants Harbor a Host-Specific Clade of
Entomoplasmatales
Bacteria
Authors: Funaro Colin F. , Kronauer Daniel J. C. , Moreau Corrie S. , Goldman-Huertas Benjamin , Pierce Naomi E. , Russell Jacob A.
Published: November 2010

In this article, we describe the distributions of
Entomoplasmatales
bacteria across the ants, identifying a novel lineage of gut bacteria that is unique to the army ants. While our findings indicate that the
Entomoplasmatales
are not essential for growth or development, molecular analyses suggest that this relationship is host specific and potentially ancient. The documented trends add to a growing body of literature that hints at a diversity of undiscovered associations between ants and bacterial symbionts.
Extracellular Aldonolactonase from
Myceliophthora thermophila
Authors: Beeson William T. , Iavarone Anthony T. , Hausmann Corinne D. , Cate Jamie H. D. , Marletta Michael A.
Published: November 2010

Fungi secrete many different enzymes to deconstruct lignocellulosic biomass, including several families of hydrolases, oxidative enzymes, and many uncharacterized proteins. Here we describe the isolation, characterization, and primary sequence analysis of an extracellular aldonolactonase from the thermophilic fungus
Myceliophthora thermophila
(synonym
Sporotrichum thermophile
). The lactonase is a 48-kDa glycoprotein with a broad pH optimum. The enzyme catalyzes the hydrolysis of glucono-δ-lactone and cellobiono-δ-lactone with an apparent second-order rate constant,
k
cat
/

K
m

, of ∼1 × 10
6
M
−1
s
−1
at pH 5.0 and 25°C but is unable to hydrolyze xylono-γ-lactone or arabino-γ-lactone. Sequence analyses of the lactonase show that it has distant homology to
cis
-carboxy-muconate lactonizing enzymes (CMLE) as well as 6-phosphogluconolactonases present in some bacteria. The
M. thermophila
genome contains two predicted extracellular lactonase genes, and expression of both genes is induced by the presence of pure cellulose. Homologues of the
M. thermophila
lactonase, which are also predicted to be extracellular, are present in nearly all known cellulolytic ascomycetes.
Transformation of, and Heterologous Protein Expression in,
Lactobacillus agilis
and
Lactobacillus vaginalis
Isolates from the Chicken Gastrointestinal Tract
Authors: Stephenson David P. , Moore Robert J. , Allison Gwen E.
Published: November 2010

Lactobacilli are naturally found in the gastrointestinal tract of chickens, and there is interest in utilizing autochthonous strains for the delivery of therapeutic proteins. Previously we identified three chicken-derived
Lactobacillus
strains,
Lactobacillus agilis
La3,
Lactobacillus vaginalis
Lv5, and
Lactobacillus crispatus
Lc9, which persist in the gastrointestinal tract of chickens fed either a commercial or high-protein diet. In the current study, we investigated the ability to electrotransform these strains, determined plasmid vector stability, and compared reporter gene expression directed by several different promoters. The La3 and Lv5 strains were reproducibly transformed with efficiencies of 10
8
and 10
6
transformants per microgram of plasmid DNA, respectively. The third strain tested,
L. crispatus
Lc9, was recalcitrant to all transformation protocols examined. The plasmid vectors pTRK563 and pTRKH2 were maintained over 100 generations in La3 and Lv5, respectively. The ability of La3 and Lv5 to express the heterologous reporter gene
gfp
was analyzed using heterologous and homologous promoters. Transformants of both La3 and Lv5 containing the La3
ldhL
promoter were the most fluorescent. To our knowledge, this is the first report of successful transformation and heterologous protein expression in
L. agilis
and
L. vaginalis
. The ability of these strains to express heterologous proteins
in vitro
indicates their potential utility as
in vivo
delivery vectors for therapeutic peptides to the chicken gastrointestinal tract.
Recombination Shapes the Structure of an Environmental
Vibrio cholerae
Population
Authors: Keymer Daniel P. , Boehm Alexandria B.
Published: November 2010

Vibrio cholerae
consists of pathogenic strains that cause sporadic gastrointestinal illness or epidemic cholera disease and nonpathogenic strains that grow and persist in coastal aquatic ecosystems. Previous studies of disease-causing strains have shown
V. cholerae
to be a primarily clonal bacterial species, but isolates analyzed have been strongly biased toward pathogenic genotypes, while representing only a small sample of the vast diversity in environmental strains. In this study, we characterized homologous recombination and structure among 152 environmental
V. cholerae
isolates and 13 other putative
Vibrio
isolates from coastal waters and sediments in central California, as well as four clinical
V. cholerae
isolates, using multilocus sequence analysis of seven housekeeping genes. Recombinant regions were identified by at least three detection methods in 72% of our
V. cholerae
isolates. Despite frequent recombination, significant linkage disequilibrium was still detected among the
V. cholerae
sequence types. Incongruent but nonrandom associations were observed for maximum likelihood topologies from the individual loci. Overall, our estimated recombination rate in
V. cholerae
of 6.5 times the mutation rate is similar to those of other sexual bacteria and appears frequently enough to restrict selection from purging much of the neutral intraspecies diversity. These data suggest that frequent recombination among
V. cholerae
may hinder the identification of ecotypes in this bacterioplankton population.
Type 2C Protein Phosphatases in Fungi
Authors: Ariño Joaquín , Casamayor Antonio , González Asier
Published: November 2010

Type 2C Ser/Thr phosphatases are a remarkable class of protein phosphatases, which are conserved in eukaryotes and involved in a large variety of functional processes. Unlike in other Ser/Thr phosphatases, the catalytic polypeptide is not usually associated with regulatory subunits, and functional specificity is achieved by encoding multiple isoforms. For fungi, most information comes from the study of type 2C protein phosphatase (PP2C) enzymes inSaccharomyces cerevisiae, where seven PP2C-encoding genes (PTC1to-7) with diverse functions can be found. More recently, data on severalCandida albicansPP2C proteins became available, suggesting that some of them can be involved in virulence. In this work we review the available literature on fungal PP2Cs and explore sequence databases to provide a comprehensive overview of these enzymes in fungi.
Genetic and Mass Spectrometry Analyses of the Unusual Type IV-Like Pili of the ArchaeonMethanococcus maripaludis
Authors: Ng Sandy Y. M. , Wu John , Nair Divya B. , Logan Susan M. , Robotham Anna , Tessier Luc , Kelly John F. , Uchida Kaoru , Aizawa Shin-Ichi , Jarrell Ken F.
Published: November 2010

The structure of pili from the archaeonMethanococcus maripaludisis unlike that of any bacterial pili. However, genetic analysis of the genes involved in the formation of these pili has been lacking until this study. Pili were isolated from a nonflagellated (ΔflaK) mutant and shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to consist primarily of subunits with an apparent molecular mass of 17 kDa. In-frame deletions were created in three genes,MMP0233,MMP0236, andMMP0237, which encode proteins with bacterial type IV pilin-like signal peptides previously identified byin silicomethodology as likely candidates for pilus structural proteins. Deletion ofMMP0236orMMP0237resulted in mutant cells completely devoid of pili on the cell surface, while deletion of the third pilin-like gene,MMP0233, resulted in cells greatly reduced in the number of pili on the surface. Complementation with the deleted gene in each case returned the cells to a piliated state. Surprisingly, mass spectrometry analysis of purified pili identified the major structural pilin as another type IV pilin-like protein, MMP1685, whose gene is located outside the first pilus locus. This protein was found to be glycosylated with an N-linked branched pentasaccharide glycan. Deletion and complementation analysis confirmed that MMP1685 is required for piliation.
Opposing Contributions of Polynucleotide Phosphorylase and the Membrane Protein NlpI to Biofilm Formation by
Salmonella enterica
Serovar Typhimurium
Authors: Rouf Syed Fazle , Ahmad Irfan , Anwar Naeem , Vodnala Suman Kumar , Kader Abdul , Römling Ute , Rhen Mikael
Published: November 2010

CsgD and cyclic-3′,5′-di-guanylate are key regulators of biofilm formation in
Salmonella enterica
serovar Typhimurium. Our results show that polynucleotide phosphorylase and NlpI oppositely altered expression of CsgD. Polynucleotide phosphorylase and NlpI also had opposite effects on the expression of
yjcC
, which codes for a cyclic-3′,5′-di-guanylate phosphodiesterase affecting CsgD expression.
Fur Negatively Regulates
hns
and Is Required for the Expression of HilA and Virulence in
Salmonella enterica
Serovar Typhimurium
Authors: Troxell Bryan , Sikes Michael L. , Fink Ryan C. , Vazquez-Torres Andres , Jones-Carson Jessica , Hassan Hosni M.
Published: November 2010

Iron is an essential element for the survival of living cells. However, excess iron is toxic, and its uptake is exquisitely regulated by the ferric uptake regulator, Fur. In
Salmonella
, the
Salmonella
pathogenicity island 1 (SPI-1) encodes a type three secretion system, which is required for invasion of host epithelial cells in the small intestine. A major activator of SPI-1 is HilA, which is encoded within SPI-1. One known regulator of
hilA
is Fur. The mechanism of
hilA
regulation by Fur is unknown. We report here that Fur is required for virulence in
Salmonella enterica
serovar Typhimurium and that Fur is required for the activation of
hilA
, as well as of other HilA-dependent genes,
invF
and
sipC
. The Fur-dependent regulation of
hilA
was independent of PhoP, a known repressor of
hilA
. Instead, the expression of the gene coding for the histone-like protein,
hns
, was significantly derepressed in the
fur
mutant. Indeed, the activation of
hilA
by Fur was dependent on 28 nucleotides located upstream of
hns
. Moreover, we used chromatin immunoprecipitation to show that Fur bound,
in vivo
, to the upstream region of
hns
in a metal-dependent fashion. Finally, deletion of
fur
in an
hns
mutant resulted in Fur-independent activation of
hilA
. In conclusion, Fur activates
hilA
by repressing the expression of
hns
.
XerCD-Mediated Site-Specific Recombination Leads to Loss of the 57-Kilobase Gonococcal Genetic Island
Authors: Domínguez Nadia M. , Hackett Kathleen T. , Dillard Joseph P.
Published: November 2010

Most strains of
Neisseria gonorrhoeae
carry the 57-kb gonococcal genetic island (GGI), as do a few strains of
Neisseria meningitidis
. The GGI is inserted into the chromosome at the
dif
site (
difA
) and is flanked by a partial repeat of the
dif
site (
difB
). Since
dif
is a sequence recognized by the site-specific recombinases XerC and XerD and the GGI shows evidence of horizontal acquisition, we hypothesized that the GGI may be acquired or lost by XerCD-mediated site-specific recombination. We show that while the GGI flanked by wild-type
dif
sites,
difA
and
difB
, is not readily lost from the gonococcal chromosome, the substitution of
difB
with another copy of
difA
allows the frequent excision and loss of the GGI. In mutants carrying two
difA
sites (
difA

+

difA

+

), the GGI can be detected as an extrachromosomal circle that exists transiently. A mutation of
xerD
diminished GGI excision from the chromosome of a
difA

+

difA

+

strain, while mutations in
recA
or type IV secretion genes had no effect on the loss of the GGI. These data indicate that the GGI is maintained by the replication of the chromosome and that GGI excision and loss are dependent upon the
dif
sequence and
xerD
. The detection of a circular form of the GGI in a wild-type strain suggests that GGI excision may occur naturally and could function to facilitate GGI transfer. These data suggest a model of GGI excision and loss explaining the absence of the GGI from some gonococcal strains and the maintenance of variant GGIs in some gonococcal and meningococcal isolates.
MexT Regulates the Type III Secretion System through MexS and PtrC in
Pseudomonas aeruginosa
Authors: Jin Yongxin , Yang Hongjiang , Qiao Mingqiang , Jin Shouguang
Published: November 2010

The type III secretion system (T3SS) is the most important virulence factor in
Pseudomonas aeruginosa
, and its expression level varies in different isolates. We studied the molecular basis for such differences in two laboratory strains, PAK and PAO1. A chromosomal clone library from the high-T3SS-producer strain PAK was introduced into the low-producer strain PAO1, and we found that a
mexS
gene from PAK confers high T3SS expression in the PAO1 background. Further tests demonstrated that both
mexS
and its neighboring
mexT
gene are required for the repression of the T3SS in PAO1, while the PAK genome encodes a defective MexS, accounting for the derepression of the T3SS in PAK and the dominant negative effect when it is introduced into PAO1. MexS is a probable oxidoreductase whose expression is dependent on MexT, a LysR-type transcriptional regulator. Various genetic data support the idea that MexS modulates the transcriptional regulator function of MexT. In searching for the MexT-dependent repressor of the T3SS, a small gene product of PA2486 (
ptrC
) was found effective in suppressing the T3SS upon overexpression. However, deletion of
ptrC
in the PAO1 background did not result in derepression of the T3SS, indicating the presence of another repressor for the T3SS. Interestingly, overexpression of functional
mexS
alone was sufficient to repress T3SS even in the absence of MexT, suggesting that MexS is another mediator of MexT-dependent T3SS repression. Overexpression of
mexS
alone had no effect on the well-known MexT-dependent genes, including those encoding MexEF efflux pump, elastase, and pyocyanin, indicating alternative regulatory mechanisms. A model has been proposed for the MexS/MexT-mediated regulation of the T3SS, the MexEF efflux pump, and the production of elastase and pyocyanin.
Genome Sequence of the Marine
Janibacter
Sp. Strain HTCC2649
Authors: Thrash J. Cameron , Cho Jang-Cheon , Bertagnolli Anthony D. , Ferriera Steve , Johnson Justin , Vergin Kevin L. , Giovannoni Stephen J.
Published: November 2010

Janibacter
sp. strain HTCC2649 is a novel marine member of the
Actinobacteria
, family
Intrasporangiaceae
, and is closely related to
Janibacter melonis
CM2104
T
and
Knoellia sinensis
HKI 0119
T
. The organism was isolated from a sample collected at Hydrostation S south of Bermuda by using high-throughput culturing techniques. Here we present the genome sequence of
Janibacter
sp. strain HTCC2649.
Understanding the effect of compensation on recovery from severe motor vehicle crash injuries: a qualitative study
Authors: Murgatroyd Darnel F , Cameron Ian D , Harris Ian A
Published: November 2010

Objective

To explore the factors that influence recovery from serious injuries sustained in motor vehicle crashes, particularly differences between those with compensable and non-compensable injuries.

Design and setting

Qualitative study using grounded theory and focus group methods within the trauma service of a university teaching hospital.

Participants

34 subjects (27 male, 7 female), of whom 21 were participants with a compensation claim and 13 were not. Each had sustained injuries in motor vehicle crashes between two and seven years previously.

Main outcome measure

Themes identified from transcripts of the focus groups.

Results

The themes identified from participants claiming compensation were a strong sense of entitlement and injustice, a difficult claims and settlement process, an inability to move on with life during the claims process, an extreme dislike of medico-legal assessments, the necessity of legal representation to assist with the claims process, and a perceived lack of trust about having to prove an injury or disability. The themes common to all participants were the significance of the trauma experience, the importance of family and social support, and, if self-employed, financial hardship and difficult experiences in returning to work.

Conclusions

The injury recovery experience was difficult for all subjects, but it was particularly stressful for those claiming compensation. Based on this study, the claims process, particularly medico-legal examinations, and other factors that could impact on injury recovery, are targets for further research, possible policy review, or legislative change.
Impact of common mental disorders on sickness absence in an occupational cohort study
Authors: Stansfeld Stephen A , Fuhrer Rebecca , Head Jenny
Published: November 2010

Objectives

Common mental disorders are associated with impaired functioning and sickness absence. We examine whether sub-clinical as well as clinical psychiatric morbidity predict long spells of sickness absence for both psychiatric and non-psychiatric illness. We also examine whether recent common mental disorders and those present on two occasions have a stronger association with sickness absence than less recent and single episodes of disorder.

Methods

Common mental disorders measured by the General Health Questionnaire were linked with long spells of sickness absence in 5104 civil servants from the longitudinal Whitehall II Study. Negative binomial models were used to estimate rate ratios for long spells of sickness absence with and without a psychiatric diagnosis (mean follow-up 5.3 years).

Results

Clinical but not sub-threshold common mental disorders were associated with increased risk of long spells of psychiatric sickness absence for men, but not for women, after adjusting for covariates (rate ratios (RR) 1.67, 95% CI 1.13 to 2.46). Risk of psychiatric sickness absence was associated with recent common mental disorders (RR 2.08, 95% CI 1.29 to 3.35) and disorder present on two occasions (RR 1.65, 95% CI 0.98 to 2.71) for men only. Common mental disorders were not associated with increased risk of non-psychiatric sickness absence after adjustment for covariates.

Conclusions

Identification and treatment of common mental disorders may reduce the economic burden of long term psychiatric sickness absence. Our results suggest that public health and clinical services should focus on the identification of workers with elevated mental health symptoms. Studies are needed of the efficacy of early identification and management of mental health symptoms for the prevention of long spells of sickness absence.
Sprouty2 downregulates angiogenesis during mouse skin wound healing
Authors: Wietecha Mateusz S. , Chen Lin , Ranzer Matthew J. , Anderson Kimberly , Ying Chunyi , Patel Tarun B. , DiPietro Luisa A.
Published: November 2010

Angiogenesis is regulated by signals received by receptor tyrosine kinases such as vascular endothelial growth factor receptors. Mammalian Sprouty (Spry) proteins are known to function by specifically antagonizing the activation of the mitogen-activated protein kinase signaling pathway by receptor tyrosine kinases, a pathway known to promote angiogenesis. To examine the role of Spry2 in the regulation of angiogenesis during wound repair, we used a model of murine dermal wound healing. Full-thickness excisional wounds (3 mm) were made on the dorsum of anesthetized adult female FVB mice. Samples were harvested at multiple time points postwounding and analyzed using real-time RT-PCR, Western blot analysis, and immunofluorescent histochemistry. Spry2 mRNA and protein levels in the wound bed increased significantly during the resolving phases of healing, coincident with the onset of vascular regression in this wound model. In another experiment, intracellular levels of Spry2 or its dominant-negative mutant (Y55F) were elevated by a topical application to the wounds of controlled-release gel containing cell permeable, transactivator of transcription-tagged Spry2, Spry2Y55F, or green fluorescent protein (as control). Wound samples were analyzed for vascularity using CD31 immunofluorescent histochemistry as well as for total and phospho-Erk1/2 protein content. The treatment of wounds with Spry2 resulted in a significant decrease in vascularity and a reduced abundance of phospho-Erk1/2 compared with wounds treated with the green fluorescent protein control. In contrast, the wounds treated with the dominant-negative Spry2Y55Fexhibited a moderate increase in vascularity and elevated phospho-Erk1/2 content. These results indicate that endogenous Spry2 functions to downregulate angiogenesis in the healing murine skin wound, potentially by inhibiting the mitogen-activated protein kinase signaling pathway.
Cardiovascular responses elicited by a new endogenous angiotensin in the nucleus tractus solitarius of the rat
Authors: Chitravanshi Vineet C. , Sapru Hreday N.
Published: November 2010

Cardiovascular effects of angiotensin-(1–12) [ANG-(1–12)] were studied in the medial nucleus of the tractus solitarius (mNTS) in anesthetized, artificially ventilated, adult male Wistar rats. Microinjections (100 nl) of ANG-(1–12) (0.06 mM) into the mNTS elicited maximum decreases in mean arterial pressure (MAP; 34 ± 5.8 mmHg) and heart rate (HR; 39 ± 3.7 beats/min). Bilateral vagotomy abolished ANG-(1–12)-induced bradycardia. Efferent greater splanchnic nerve activity was decreased by microinjections of ANG-(1–12) into the mNTS. Blockade of ANG type 1 receptors (AT1Rs; using ZD-7155 or L-158,809), but not ANG type 2 receptors (AT2Rs; using PD-123319), significantly attenuated ANG-(1–12)-induced cardiovascular responses. Simultaneous inhibition of both angiotensin-converting enzyme (ACE; using captopril) and chymase (using chymostatin) completely blocked the effects of ANG-(1–12). Microinjections of A-779 [ANG-(1–7) antagonist] did not attenuate ANG-(1–12)-induced responses. Pressure ejection of ANG-(1–12) (0.06 mM, 2 nl) caused excitation of barosensitive mNTS neurons, which was blocked by prior application of the AT1R antagonist. ANG-(1–12)-induced excitation of mNTS neurons was also blocked by prior sequential applications of captopril and chymostatin. These results indicate that 1) microinjections of ANG-(1–12) into the mNTS elicited depressor and bradycardic responses by exciting barosensitive mNTS neurons; 2) the decreases in MAP and HR were mediated via sympathetic and vagus nerves, respectively; 3) AT1Rs, but not AT2Rs, mediated these actions of ANG-(1–12); 4) the responses were mediated via the conversion of ANG-(1–12) to ANG II and both ACE and chymase were involved in this conversion; and 5) ANG-(1–7) was not one of the metabolites of ANG-(1–12) in the mNTS.
Calcium and zinc dyshomeostasis during isoproterenol-induced acute stressor state
Authors: Shahbaz Atta U. , Zhao Tieqiang , Zhao Wenyuan , Johnson Patti L. , Ahokas Robert A. , Bhattacharya Syamal K. , Sun Yao , Gerling Ivan C. , Weber Karl T.
Published: November 2010

Acute hyperadrenergic stressor states are accompanied by cation dyshomeostasis, together with the release of cardiac troponins predictive of necrosis. The signal-transducer-effector pathway accounting for this pathophysiological scenario remains unclear. We hypothesized that a dyshomeostasis of extra- and intracellular Ca2+ and Zn2+ occurs in rats in response to isoproterenol (Isop) including excessive intracellular Ca2+ accumulation (EICA) and mitochondrial [Ca2+]m-induced oxidative stress. Contemporaneously, the selective translocation of Ca2+ and Zn2+ to tissues contributes to their fallen plasma levels. Rats received a single subcutaneous injection of Isop (1 mg/kg body wt). Other groups of rats received pretreatment for 10 days with either carvedilol (C), a β-adrenergic receptor antagonist with mitochondrial Ca2+ uniporter-inhibiting properties, or quercetin (Q), a flavonoid with mitochondrial-targeted antioxidant properties, before Isop. We monitored temporal responses in the following: [Ca2+] and [Zn2+] in plasma, left ventricular (LV) apex, equator and base, skeletal muscle, liver, spleen, and peripheral blood mononuclear cells (PBMC), indices of oxidative stress and antioxidant defenses, mitochondrial permeability transition pore (mPTP) opening, and myocardial fibrosis. We found ionized hypocalcemia and hypozincemia attributable to their tissue translocation and also a heterogeneous distribution of these cations among tissues with a preferential Ca2+ accumulation in the LV apex, muscle, and PBMC, whereas Zn2+ declined except in liver, where it increased corresponding with upregulation of metallothionein, a Zn2+-binding protein. EICA was associated with a simultaneous increase in tissue 8-isoprostane and increased [Ca2+]m accompanied by a rise in H2O2 generation, mPTP opening, and scarring, each of which were prevented by either C or Q. Thus excessive [Ca2+]m, coupled with the induction of oxidative stress and increased mPTP opening, suggests that this signal-transducer-effector pathway is responsible for Isop-induced cardiomyocyte necrosis at the LV apex.
Titin-based mechanosensing and signaling: role in diaphragm atrophy during unloading?
Authors: Ottenheijm Coen A. C. , van Hees Hieronymus W. H. , Heunks Leo M. A. , Granzier Henk
Published: November 2010

The diaphragm, the main muscle of inspiration, is constantly subjected to mechanical loading. One of the very few occasions during which diaphragm loading is arrested is during controlled mechanical ventilation in the intensive care unit. Recent animal studies indicate that the diaphragm is extremely sensitive to unloading, causing rapid muscle fiber atrophy: unloading-induced diaphragm atrophy and the concomitant diaphragm weakness has been suggested to contribute to the difficulties in weaning patients from ventilatory support. Little is known about the molecular triggers that initiate the rapid unloading atrophy of the diaphragm, although proteolytic pathways and oxidative signaling have been shown to be involved. Mechanical stress is known to play an important role in the maintenance of muscle mass. Within the muscle's sarcomere titin is considered to play an important role in the stress-response machinery. Titin is the largest protein known to date and acts as a mechanosensor that regulates muscle protein expression in a sarcomere strain-dependent fashion. Thus, titin is an attractive candidate for sensing the sudden mechanical arrest of the diaphragm when patients are mechanically ventilated, leading to changes in muscle protein expression. Here, we provide a novel perspective on how titin, and its biomechanical sensing and signaling, might be involved in the development of mechanical unloading-induced diaphragm weakness.
A Novel Fusion Toxin Derived from an EpCAM-Specific Designed Ankyrin Repeat Protein Has Potent Antitumor Activity
Authors: Martin-Killias Patricia , Stefan Nikolas , Rothschild Sacha , Plückthun Andreas , Zangemeister-Wittke Uwe
Published: November 2010

Purpose: Designed ankyrin repeat proteins (DARPins) hold great promise as a new class of binding molecules to overcome the limitations of antibodies for biomedical applications. Here, we assessed the potential of an epithelial cell adhesion molecule (EpCAM)–specific DARPin (Ec4) for tumor targeting as a fusion toxin with Pseudomonas aeruginosa exotoxin A.

Experimental design: DARPin Ec4 was genetically fused to a truncated form of Pseudomonas aeruginosa exotoxin A (ETA″) and expressed in Escherichia coli. The cytotoxicity of Ec4-ETA″ was measured against tumor cell lines of various histotypes in vitro. Tumor localization and antitumor activity were determined in mice bearing 2 different EpCAM-positive tumor xenografts.

Results: Ec4-ETA″ expressed very well in soluble form in the cytoplasm of E. coli and yielded up to 40 mg after purification per liter of culture. The protein was monomeric and the disulfides of ETA″ formed spontaneously. Ec4-ETA″ bound to EpCAM with low nanomolar affinity, similar to free Ec4. Furthermore, it was highly cytotoxic against various EpCAM-positive tumor cell lines in vitro with IC50 values less than 0.005 pmol/L. This effect was competed by free Ec4, but not by unspecific DARPins. Upon systemic administration in athymic mice, Ec4-ETA″ efficiently localized to EpCAM-positive tumors to achieve maximum accumulation 48 to 72 hours after injection, whereas an irrelevant control fusion toxin did not accumulate. Tumor targeting with Ec4-ETA″ resulted in a strong antitumor response including complete regressions in some animals.

Conclusions: Our data show for the first time the potential of DARPins for the generation of protein therapeutics for tumor targeting, and that Ec4-ETA″ deserves attention for clinical development. Clin Cancer Res; 17(1); 100–10. ©2010 AACR.
A Small-Molecule Inhibitor of MDMX Activates p53 and Induces Apoptosis
Authors: Wang Hongbo , Ma Xujun , Ren Shumei , Buolamwini John K. , Yan Chunhong
Published: November 2010

The p53 inactivation caused by aberrant expression of its major regulators (e.g., MDM2 and MDMX) contributes to the genesis of a large number of human cancers. Recent studies have shown that restoration of p53 activity by counteracting p53 repressors is a promising anticancer strategy. Although agents (e.g., nutlin-3a) that disrupt MDM2–p53 interaction can inhibit tumor growth, they are less effective in cancer cells that express high levels of MDMX. MDMX binds to p53 and can repress the tumor suppressor function of p53 through inhibiting its trans-activation activity and/or destabilizing the protein. Here we report the identification of a benzofuroxan derivative [7-(4-methylpiperazin-1-yl)-4-nitro-1-oxido-2,1,3-benzoxadiazol-1-ium, NSC207895] that could inhibit MDMX expression in cancer cells through a reporter-based drug screening. Treatments of MCF-7 cells with this small-molecule MDMX inhibitor activated p53, resulting in elevated expression of proapoptotic genes (e.g., PUMA, BAX, and PIG3). Importantly, this novel small-molecule p53 activator caused MCF-7 cells to undergo apoptosis and acted additively with nutlin-3a to activate p53 and decrease the viability of cancer cells. These results thus show that small molecules targeting MDMX expression would be of therapeutic benefits. Mol Cancer Ther; 10(1); 69–79. ©2010 AACR.
B1, a Novel Amonafide Analogue, Overcomes the Resistance Conferred by Bcl-2 in Human Promyelocytic Leukemia HL60 Cells
Authors: Liang Xin , Xu Yufang , Xu Ke , Liu Jianwen , Qian Xuhong
Published: November 2010

In the course of screening for novel anticancer compounds, B1 [N-(2-(dimethylamino)ethyl)-2-aminothiazonaphthalimide], a novel amonafide analogue, was generated as a new anticancer candidate. In the present study, B1 displayed stronger antitumor effects than amonafide in HL60 cells. We further examined whether B1 overcomes the resistance conferred by Bcl-2, as overcoming the resistance conferred by Bcl-2 represents an attractive therapeutic strategy against cancer. Our viability assay showed that B1 overcomes the resistance conferred by Bcl-2 in human promyelocytic leukemia HL60 cells. Various apoptosis assessment assays showed that B1 overcomes the resistance conferred by Bcl-2 in HL60 cells by inducing apoptosis. Noticeably, we elucidated the marked downregulation of 14-3-3σ protein by B1, indicating that B1 overcomes the resistance conferred by Bcl-2 in HL60 cells via 14-3-3σ. The analysis of chromatin immunoprecipitation assay indicated that MBD2 was associated with the methylated 14-3-3σ promoter-associated CpG island and thus interfered with transcriptional activity of the methylated promoter. Furthermore, knockdown of MBD2, using siRNA transfection, inhibited B1-induced apoptosis and overcame the resistance conferred by Bcl-2. Accordingly, these data showed the involvement of MBD2 in B1-induced apoptosis and overcoming the resistance conferred by Bcl-2, which suggested that MBD2 might guide the development of future anticancer agents. Mol Cancer Res; 8(12);1619–32. ©2010 AACR.
Screening for Obstructive Sleep Apnea by Cyclic Variation of Heart Rate
Authors: Hayano Junichiro , Watanabe Eiichi , Saito Yuji , Sasaki Fumihiko , Fujimoto Keisaku , Nomiyama Tetsuo , Kawai Kiyohiro , Kodama Itsuo , Sakakibara Hiroki
Published: November 2010

Background—

Despite the adverse cardiovascular consequences of obstructive sleep apnea, the majority of patients remain undiagnosed. To explore an efficient ECG-based screening tool for obstructive sleep apnea, we examined the usefulness of automated detection of cyclic variation of heart rate (CVHR) in a large-scale controlled clinical setting.

Methods and Results—

We developed an algorithm of autocorrelated wave detection with adaptive threshold (ACAT). The algorithm was optimized with 63 sleep studies in a training cohort, and its performance was confirmed with 70 sleep studies of the Physionet Apnea-ECG database. We then applied the algorithm to ECGs extracted from all-night polysomnograms in 862 consecutive subjects referred for diagnostic sleep study. The number of CVHR per hour (the CVHR index) closely correlated (
r
=0.84) with the apnea-hypopnea index, although the absolute agreement with the apnea-hypopnea index was modest (the upper and lower limits of agreement, 21 per hour and −19 per hour) with periodic leg movement causing most of the disagreement (
P
<0.001). The CVHR index showed a good performance in identifying the patients with an apnea-hypopnea index ≥15 per hour (area under the receiver-operating characteristic curve, 0.913; 83% sensitivity and 88% specificity, with the predetermined cutoff threshold of CVHR index ≥15 per hour). The classification performance was unaffected by older age (≥65 years) or cardiac autonomic dysfunction (SD of normal-to-normal R-R intervals over the entire length of recording <65 ms; area under the receiver-operating characteristic curve, 0.915 and 0.911, respectively).

Conclusions—

The automated detection of CVHR with the ACAT algorithm provides a powerful ECG-based screening tool for moderate-to-severe obstructive sleep apnea, even in older subjects and in those with cardiac autonomic dysfunction.