bluuebunny/crossref_metadata_2025 huggingface dataset:
SulE, a Sulfonylurea Herbicide De-Esterification Esterase from Hansschlegelia zhihuaiae S113
Authors: Hang Bao-Jian , Hong Qing , Xie Xiang-Ting , Huang Xing , Wang Cheng-Hong , He Jian , Li Shun-Peng
Published: January 2012

De-esterification is an important degradation or detoxification mechanism of sulfonylurea herbicide in microbes and plants. However, the biochemical and molecular mechanisms of sulfonylurea herbicide de-esterification are still unknown. In this study, a novel esterase gene,
sulE
, responsible for sulfonylurea herbicide de-esterification, was cloned from
Hansschlegelia zhihuaiae
S113. The gene contained an open reading frame of 1,194 bp, and a putative signal peptide at the N terminal was identified with a predicted cleavage site between Ala37 and Glu38, resulting in a 361-residue mature protein. SulE minus the signal peptide was synthesized in
Escherichia coli
BL21 and purified to homogeneity. SulE catalyzed the de-esterification of a variety of sulfonylurea herbicides that gave rise to the corresponding herbicidally inactive parent acid and exhibited the highest catalytic efficiency toward thifensulfuron-methyl. SulE was a dimer without the requirement of a cofactor. The activity of the enzyme was completely inhibited by Ag
+
, Cd
2+
, Zn
2+
, methamidophos, and sodium dodecyl sulfate. A
sulE
-disrupted mutant strain, Δ
sulE
, was constructed by insertion mutation. Δ
sulE
lost the de-esterification ability and was more sensitive to the herbicides than the wild type of strain S113, suggesting that
sulE
played a vital role in the sulfonylurea herbicide resistance of the strain. The transfer of
sulE
into
Saccharomyces cerevisiae
BY4741 conferred on it the ability to de-esterify sulfonylurea herbicides and increased its resistance to the herbicides. This study has provided an excellent candidate for the mechanistic study of sulfonylurea herbicide metabolism and detoxification through de-esterification, construction of sulfonylurea herbicide-resistant transgenic crops, and bioremediation of sulfonylurea herbicide-contaminated environments.
Transcriptional Responses of Escherichia coli K-12 and O157:H7 Associated with Lettuce Leaves
Authors: Fink Ryan C. , Black Elaine P. , Hou Zhe , Sugawara Masayuki , Sadowsky Michael J. , Diez-Gonzalez Francisco
Published: January 2012

An increasing number of outbreaks of gastroenteritis recently caused by
Escherichia coli
O157:H7 have been linked to the consumption of leafy green vegetables. Although it is known that
E. coli
survives and grows in the phyllosphere of lettuce plants, the molecular mechanisms by which this bacterium associates with plants are largely unknown. The goal of this study was to identify
E. coli
genes relevant to its interaction, survival, or attachment to lettuce leaf surfaces, comparing
E. coli
K-12, a model system, and
E. coli
O157:H7, a pathogen associated with a large number of outbreaks. Using microarrays, we found that upon interaction with intact leaves, 10.1% and 8.7% of the 3,798 shared genes were differentially expressed in K-12 and O157:H7, respectively, whereas 3.1% changed transcript levels in both. The largest group of genes downregulated consisted of those involved in energy metabolism, including
tnaA
(33-fold change), encoding a tryptophanase that converts tryptophan into indole. Genes involved in biofilm modulation (
bhsA
and
ybiM
) and curli production (
csgA
and
csgB
) were significantly upregulated in
E. coli
K-12 and O157:H7. Both
csgA
and
bhsA
(
ycfR
) mutants were impaired in the long-term colonization of the leaf surface, but only
csgA
mutants had diminished ability in short-term attachment experiments. Our data suggested that the interaction of
E. coli
K-12 and O157:H7 with undamaged lettuce leaves likely is initiated via attachment to the leaf surface using curli fibers, a downward shift in their metabolism, and the suppression of biofilm formation.
Comparison of the Prevalences and Antimicrobial Resistances of Escherichia coli Isolates from Different Retail Meats in the United States, 2002 to 2008
Authors: Zhao S. , Blickenstaff K. , Bodeis-Jones S. , Gaines S. A. , Tong E. , McDermott P. F.
Published: January 2012

Escherichia coli
isolates were recovered from the National Antimicrobial Resistance Monitoring System retail meat program and examined for antimicrobial susceptibility. Retail meat samples (
n
= 11,921) from four U.S. states collected during 2002 to 2008, consisting of 2,988 chicken breast, 2,942 ground turkey, 2,991 ground beef, and 3,000 pork chop samples, were analyzed. A total of 8,286
E. coli
isolates were recovered. The greatest numbers of samples contaminated with the organism were chicken (83.5%) and turkey (82.0%), followed by beef (68.9%) and pork (44.0%). Resistance was most common to tetracycline (50.3%), followed by streptomycin (34.6%), sulfamethoxazole-sulfisoxazole (31.6%), ampicillin (22.5%), gentamicin (18.6%), kanamycin (8.4%), amoxicillin-clavulanic acid (6.4%), and cefoxitin (5.2%). Less than 5% of the isolates had resistance to trimethoprim, ceftriaxone, ceftiofur, nalidixic acid, chloramphenicol, and ciprofloxacin. All isolates were susceptible to amikacin. Compared to beef and pork isolates, the poultry meat isolates had a greater percentage of resistance to all tested drugs, with the exception of chloramphenicol, to which pork isolates had the most resistance. More than half of the turkey isolates (56%) were resistant to multidrugs (≥3 classes) compared to 38.9% of chicken, 17.3% of pork, and 9.3% of beef isolates. The
bla
CMY
gene was present in all ceftriaxone- and ceftiofur-resistant isolates. The
cmlA
,
flo
,
and catI
genes were present in 45%, 43%, and 40% of chloramphenicol-resistant isolates, respectively. Most nalidixic acid-resistant isolates (98.5%) had a
gyrA
mutation in S83 or D87 or both, whereas only 6.7% had a
parC
mutation in either S80 or E84. The results showed that
E. coli
was commonly present in the retail meats, and antimicrobial resistance profiles differed according to the animal origin of the isolates.
Relationship between Yield Components and Partial Resistance to Lecanicillium fungicola in the Button Mushroom, Agaricus bisporus, Assessed by Quantitative Trait Locus Mapping
Authors: Foulongne-Oriol Marie , Rodier Anne , Savoie Jean-Michel
Published: January 2012

Dry bubble, caused byLecanicillium fungicola, is one of the most detrimental diseases affecting button mushroom cultivation. In a previous study, we demonstrated that breeding for resistance to this pathogen is quite challenging due to its quantitative inheritance. A second-generation hybrid progeny derived from an intervarietal cross between a wild strain and a commercial cultivar was characterized forL. fungicolaresistance under artificial inoculation in three independent experiments. Analysis of quantitative trait loci (QTL) was used to determine the locations, numbers, and effects of genomic regions associated with dry-bubble resistance. Four traits related to resistance were analyzed. Two to four QTL were detected per trait, depending on the experiment. Two genomic regions, on linkage group X (LGX) and LGVIII, were consistently detected in the three experiments. The genomic region on LGX was detected for three of the four variables studied. The total phenotypic variance accounted for by all QTL ranged from 19.3% to 42.1% over all traits in all experiments. For most of the QTL, the favorable allele for resistance came from the wild parent, but for some QTL, the allele that contributed to a higher level of resistance was carried by the cultivar. Comparative mapping with QTL for yield-related traits revealed five colocations between resistance and yield component loci, suggesting that the resistance results from both genetic factors and fitness expression. The consequences for mushroom breeding programs are discussed.
The Septin AspB in Aspergillus nidulans Forms Bars and Filaments and Plays Roles in Growth Emergence and Conidiation
Authors: Hernández-Rodríguez Yainitza , Hastings Susan , Momany Michelle
Published: January 2012

In yeast, septins form rings at the mother-bud neck and function as diffusion barriers. In animals, septins form filaments that can colocalize with other cytoskeletal elements. In the filamentous fungusAspergillus nidulansthere are five septin genes,aspA(an ortholog ofSaccharomyces cerevisiae CDC11),aspB(an ortholog ofS. cerevisiae CDC3),aspC(an ortholog ofS. cerevisiae CDC12),aspD(an ortholog ofS. cerevisiae CDC10), andaspE(found only in filamentous fungi). TheaspBgene was previously reported to be the most highly expressedAspergillus nidulansseptin and to be essential. Using improved gene targeting techniques, we found that deletion ofaspBis not lethal but results in delayed septation, increased emergence of germ tubes and branches, and greatly reduced conidiation. We also found that AspB-green fluorescent protein (GFP) localizes as rings and collars at septa, branches, and emerging layers of the conidiophore and as bars and filaments in conidia and hyphae. Bars are found in dormant and isotropically expanding conidia and in subapical nongrowing regions of hyphae and display fast movements. Filaments form as the germ tube emerges, localize to hyphal and branch tips, and display slower movements. All visible AspB-GFP structures are retained inΔaspDand lost inΔaspAandΔaspCstrains. Interestingly, in theΔaspEmutant, AspB-GFP rings, bars, and filaments are visible in early growth, but AspB-GFP rods and filaments disappear after septum formation. AspE orthologs are only found in filamentous fungi, suggesting that this class of septins might be required for stability of septin bars and filaments in highly polar cells.
The Giardia Median Body Protein Is a Ventral Disc Protein That Is Critical for Maintaining a Domed Disc Conformation during Attachment
Authors: Woessner David J. , Dawson Scott C.
Published: January 2012

Giardia
has unique microtubule structures, including the ventral disc, the primary organelle of attachment to the host, and the median body, a structure of undefined function. During attachment, the ventral disc has a domed conformation and enables
Giardia
to attach to the host intestinal epithelia within seconds. The mechanism of attachment via the ventral disc and the overall structure, function, and assembly of the ventral disc are not well understood. Our recent proteomic analysis of the ventral disc indicated that the median body protein (MBP), previously reported to localize exclusively to the median body, was primarily localized to the ventral disc. Using high-resolution light and electron microscopy, we confirm that the median body protein localizes primarily to the overlap zone of the ventral disc. The MBP also occasionally localized to the median body during prophase. To define the contribution of MBP to the ventral disc structure, we depleted MBP using an anti-MBP morpholino. We found that the ventral disc was no longer able to form properly and that the disc structure often had an aberrant nondomed or flattened horseshoe conformation. The ability of attached anti-MBP morpholino-treated trophozoites to withstand shear forces and normal forces was significantly decreased. Most notably, the plasma membrane contacts with the surface, including those of the bare area, were defective after the anti-MBP knockdown. To our knowledge, this is the first ventral disc protein whose depletion directly alters ventral disc structure, confirming that the domed ventral disc conformation is important for robust attachment.
Mutations at Several Loci Cause Increased Expression of Ribonucleotide Reductase in Escherichia coli
Authors: Feeney Morgan Anne , Ke Na , Beckwith Jon
Published: January 2012

Production of deoxyribonucleotides for DNA synthesis is an essential and tightly regulated process. The class Ia ribonucleotide reductase (RNR), the product of the
nrdAB
genes, is required for aerobic growth of
Escherichia coli
. In catalyzing the reduction of ribonucleotides, two of the cysteines of RNR become oxidized, forming a disulfide bond. To regenerate active RNR, the cell uses thioredoxins and glutaredoxins to reduce the disulfide bond. Strains that lack thioredoxins 1 and 2 and glutaredoxin 1 do not grow because RNR remains in its oxidized, inactive form. However, suppressor mutations that lead to RNR overproduction allow glutaredoxin 3 to reduce sufficient RNR for growth of these mutant strains. We previously described suppressor mutations in the
dnaA
and
dnaN
genes that had such effects. Here we report the isolation of new mutations that lead to increased levels of RNR. These include mutations that were not known to influence production of RNR previously, such as a mutation in the
hda
gene and insertions in the
nrdAB
promoter region of insertion elements IS
1
and IS
5
. Bioinformatic analysis raises the possibility that IS element insertion in this region represents an adaptive mechanism in
nrdAB
regulation in
E. coli
and closely related species. We also characterize mutations altering different amino acids in DnaA and DnaN from those isolated before.
Characterization of Escherichia coli
dinJ-yafQ
Toxin-Antitoxin System Using Insights from Mutagenesis Data
Authors: Armalytė Julija , Jurėnaitė Milda , Beinoravičiūtė Gina , Teišerskas Justinas , Sužiedėlienė Edita
Published: January 2012

Escherichia coli
dinJ-yafQ
operon codes for a functional toxin-antitoxin (TA) system. YafQ toxin is an RNase which, upon overproduction, specifically inhibits the translation process by cleaving cellular mRNA at specific sequences. DinJ is an antitoxin and counteracts YafQ-mediated toxicity by forming a strong protein complex. In the present study we used site-directed mutagenesis of YafQ to determine the amino acids important for its catalytic activity. His50Ala, His63Ala, Asp67Ala, Trp68Ala, Trp68Phe, Arg83Ala, His87Ala, and Phe91Ala substitutions of the predicted active-site residues of YafQ abolished mRNA cleavage
in vivo
, whereas Asp61Ala and Phe91Tyr mutations inhibited YafQ RNase activity only moderately. We show that YafQ, upon overexpression, cleaved mRNAs preferably 5′ to A between the second and third nucleotides in the codon
in vivo
. YafQ also showed RNase activity against mRNA, tRNA, and 5S rRNA molecules
in vitro
, albeit with no strong specificity. The endoribonuclease activity of YafQ was inhibited in the complex with DinJ antitoxin
in vitro
. DinJ-YafQ protein complex and DinJ antitoxin alone selectively bind to one of the two palindromic sequences present in the intergenic region upstream of the
dinJ-yafQ
operon, suggesting the autoregulation mode of this TA system.
AMP-Forming Acetyl Coenzyme A Synthetase in the Outermost Membrane of the Hyperthermophilic Crenarchaeon Ignicoccus hospitalis
Authors: Mayer Florian , Küper Ulf , Meyer Carolin , Daxer Stefanie , Müller Volker , Rachel Reinhard , Huber Harald
Published: January 2012

Ignicoccus hospitalis
, a hyperthermophilic, chemolithoautotrophic crenarchaeon was found to possess a new CO
2
fixation pathway, the dicarboxylate/4-hydroxybutyrate cycle. The primary acceptor molecule for this pathway is acetyl coenzyme A (acetyl-CoA), which is regenerated in the cycle via the characteristic intermediate 4-hydroxybutyrate. In the presence of acetate, acetyl-CoA can alternatively be formed in a one-step mechanism via an AMP-forming acetyl-CoA synthetase (ACS). This enzyme was identified after membrane preparation by two-dimensional native PAGE/SDS-PAGE, followed by matrix-assisted laser desorption ionization–time of flight tandem mass spectrometry and N-terminal sequencing. The ACS of
I. hospitalis
exhibits a molecular mass of ∼690 kDa with a monomeric molecular mass of 77 kDa. Activity tests on isolated membranes and bioinformatic analyses indicated that the ACS is a constitutive membrane-associated (but not an integral) protein complex. Unexpectedly, immunolabeling on cells of
I. hospitalis
and other described
Ignicoccus
species revealed that the ACS is localized at the outermost membrane. This perfectly coincides with recent results that the ATP synthase and the H
2
:sulfur oxidoreductase complexes are also located in the outermost membrane of
I. hospitalis
. These results imply that the intermembrane compartment of
I. hospitalis
is not only the site of ATP synthesis but may also be involved in the primary steps of CO
2
fixation.
Evolutionary Link between the Mycobacterial Plasmid pAL5000 Replication Protein RepB and the Extracytoplasmic Function Family of σ Factors
Authors: Basu Arnab , Chatterjee Sujoy , Chatterjee Soniya , Das Gupta Sujoy K.
Published: January 2012

Mycobacterial plasmid pAL5000 represents a family of plasmids found mostly in the
Actinobacteria
. It replicates using two plasmid-encoded proteins, RepA and RepB. While BLAST searches indicate that RepA is a replicase family protein, the evolutionary connection of RepB cannot be established, as no significant homologous partner (E < 10
−3
) outside the RepB family can be identified. To obtain insight into the structure-function and evolutionary connections of RepB, an investigation was undertaken using homology modeling, phylogenetic, and mutational analysis methods. The results indicate that although they are synthesized from the same operon, the phylogenetic affinities of RepA and RepB differ. Thus, the operon may have evolved through random breaking and joining events. Homology modeling predicted the presence of a three-helical helix-turn-helix domain characteristic of region 4 of extracytoplasmic function (ECF) σ factors in the C-terminal region of RepB. At the N-terminal region, there is a helical stretch, which may be distantly related to region 3 of σ factors. Mutational analysis identified two arginines indispensable for RepB activity, one each located within the C- and N-terminal conserved regions. Apart from analyzing the domain organization of the protein, the significance of the presence of a highly conserved A/T-rich element within the RepB binding site was investigated. Mutational analysis revealed that although this motif does not bind RepB, its integrity is important for efficient DNA-protein interactions and replication to occur. The present investigation unravels the possibility that RepB-like proteins and their binding sites represent ancient DNA-protein interaction modules.
Outer Membrane Targeting, Ultrastructure, and Single Molecule Localization of the Enteropathogenic Escherichia coli Type IV Pilus Secretin BfpB
Authors: Lieberman Joshua A. , Frost Nicholas A. , Hoppert Michael , Fernandes Paula J. , Vogt Stefanie L. , Raivio Tracy L. , Blanpied Thomas A. , Donnenberg Michael S.
Published: January 2012

Type IV pili (T4P) are filamentous surface appendages required for tissue adherence, motility, aggregation, and transformation in a wide array of bacteria and archaea. The bundle-forming pilus (BFP) of enteropathogenic
Escherichia coli
(EPEC) is a prototypical T4P and confirmed virulence factor. T4P fibers are assembled by a complex biogenesis machine that extrudes pili through an outer membrane (OM) pore formed by the secretin protein. Secretins constitute a superfamily of proteins that assemble into multimers and support the transport of macromolecules by four evolutionarily ancient secretion systems: T4P, type II secretion, type III secretion, and phage assembly. Here, we determine that the lipoprotein transport pathway is not required for targeting the BfpB secretin protein of the EPEC T4P to the OM and describe the ultrastructure of the single particle averaged structures of the assembled complex by transmission electron microscopy. Furthermore, we use photoactivated localization microscopy to determine the distribution of single BfpB molecules fused to photoactivated mCherry. In contrast to findings in other T4P systems, we found that BFP components predominantly have an uneven distribution through the cell envelope and are only found at one or both poles in a minority of cells. In addition, we report that concurrent mutation of both the T4bP secretin and the retraction ATPase can result in viable cells and found that these cells display paradoxically low levels of cell envelope stress response activity. These results imply that secretins can direct their own targeting, have complex distributions and provide feedback information on the state of pilus biogenesis.
J-Western Forms of Helicobacter pylori cagA Constitute a Distinct Phylogenetic Group with a Widespread Geographic Distribution
Authors: Duncan Stacy S. , Valk Pieter L. , Shaffer Carrie L. , Bordenstein Seth R. , Cover Timothy L.
Published: January 2012

Chronic infection with
Helicobacter pylori
strains expressing the bacterial oncoprotein CagA confers an increased risk of gastric cancer. While much is known about the ancestry and molecular evolution of Western, East Asian, and Amerindian
cagA
sequences, relatively little is understood about a fourth group, known as “J-Western,” which has been detected mainly in strains from Okinawa, Japan. We show here that J-Western
cagA
sequences have a more widespread global distribution than previously recognized, occur in strains with multiple different ancestral origins (based on multilocus sequence typing [MLST] analysis), and did not arise recently. As shown by comparisons of Western and J-Western forms of CagA, there are 45 fixed or nearly fixed amino acid differences, and J-Western forms contain a unique 4-amino-acid insertion. The mean nucleotide diversity of synonymous sites (π
s
) is slightly lower in the J-Western group than in the Western and East Asian groups (0.066, 0.086, and 0.083, respectively), which suggests that the three groups have comparable, but not equivalent, effective population sizes. The reduced π
s
of the J-Western group is attributable to ancestral recombination events within the 5′ region of
cagA.
Population genetic analyses suggest that within the
cagA
region encoding EPIYA motifs, the East Asian group underwent a marked reduction in effective population size compared to the Western and J-Western groups, in association with positive selection. Finally, we show that J-Western
cagA
sequences are found mainly in strains producing m2 forms of the secreted VacA toxin and propose that these functionally interacting proteins coevolved to optimize the gastric colonization capacity of
H. pylori
.
Structure, Diversity, and Mobility of the Salmonella Pathogenicity Island 7 Family of Integrative and Conjugative Elements within Enterobacteriaceae
Authors: Seth-Smith Helena M. B. , Fookes Maria C. , Okoro Chinyere K. , Baker Stephen , Harris Simon R. , Scott Paul , Pickard Derek , Quail Michael A. , Churcher Carol , Sanders Mandy , Harmse Johan , Dougan Gordon , Parkhill Julian , Thomson Nicholas R.
Published: January 2012

Integrative and conjugative elements (ICEs) are self-mobile genetic elements found in the genomes of some bacteria. These elements may confer a fitness advantage upon their host bacteria through the cargo genes that they carry.
Salmonella
pathogenicity island 7 (SPI-7), found within some pathogenic strains of
Salmonella enterica
, possesses features indicative of an ICE and carries genes implicated in virulence. We aimed to identify and fully analyze ICEs related to SPI-7 within the genus
Salmonella
and other
Enterobacteriaceae
. We report the sequence of two novel SPI-7-like elements, found within strains of
Salmonella bongori
, which share 97% nucleotide identity over conserved regions with SPI-7 and with each other. Although SPI-7 within
Salmonella enterica
serovar Typhi appears to be fixed within the chromosome, we present evidence that these novel elements are capable of excision and self-mobility. Phylogenetic analyses show that these
Salmonella
mobile elements share an ancestor which existed approximately 3.6 to 15.8 million years ago. Additionally, we identified more distantly related ICEs, with distinct cargo regions, within other strains of
Salmonella
as well as within
Citrobacter
,
Erwinia
,
Escherichia
,
Photorhabdus
, and
Yersinia
species. In total, we report on a collection of 17 SPI-7 related ICEs within enterobacterial species, of which six are novel. Using comparative and mutational studies, we have defined a core of 27 genes essential for conjugation. We present a growing family of SPI-7-related ICEs whose mobility, abundance, and cargo variability indicate that these elements may have had a large impact on the evolution of the
Enterobacteriaceae
.
The
O
-Methyltransferase SrsB Catalyzes the Decarboxylative Methylation of Alkylresorcylic Acid during Phenolic Lipid Biosynthesis by Streptomyces griseus
Authors: Nakano Chiaki , Funa Nobutaka , Ohnishi Yasuo , Horinouchi Sueharu
Published: January 2012

Streptomyces griseus
contains the
srs
operon, which is required for phenolic lipid biosynthesis. The operon consists of
srsA
,
srsB
, and
srsC
, which encode a type III polyketide synthase, an
O
-methyltransferase, and a flavoprotein hydroxylase, respectively. We previously reported that the recombinant SrsA protein synthesized 3-(13′-methyltetradecyl)-4-methylresorcinol, using
iso
-C
16
fatty acyl-coenzyme A (CoA) as a starter substrate and malonyl-CoA and methylmalonyl-CoA as extender substrates. An
in vitro
SrsA reaction using [
13
C
3
]malonyl-CoA confirmed that the order of extender substrate condensation was methylmalonyl-CoA, followed by two extensions with malonyl-CoA. Furthermore, SrsA was revealed to produce an alkylresorcylic acid as its direct product rather than an alkylresorcinol. The functional SrsB protein was produced in the membrane fraction in
Streptomyces lividans
and used for the
in vitro
SrsB reaction. When the SrsA reaction was coupled, SrsB produced alkylresorcinol methyl ether in the presence of
S
-adenosyl-
l
-methionine (SAM). SrsB was incapable of catalyzing the
O
-methylation of alkylresorcinol, indicating that alkylresorcylic acid was the substrate of SrsB and that SrsB catalyzed the conversion of alkylresorcylic acid to alkylresorcinol methyl ether, namely, by both the
O
-methylation of the hydroxyl group (C-6) and the decarboxylation of the neighboring carboxyl group (C-1).
O
-methylated alkylresorcylic acid was not detected in the
in vitro
SrsAB reaction, although it was presumably stable, indicating that
O
-methylation did not precede decarboxylation. We therefore postulated that
O
-methylation was coupled with decarboxylation and proposed that SrsB catalyzed the feasible SAM-dependent decarboxylative methylation of alkylresorcylic acid. To the best of our knowledge, this is the first report of a methyltransferase that catalyzes decarboxylative methylation.
Further Unraveling the Regulatory Twist by Elucidating Metabolic Coinducer-Mediated CbbR-
cbb
I
Promoter Interactions in Rhodopseudomonas palustris CGA010
Authors: Joshi Gauri S. , Zianni Michael , Bobst Cedric E. , Tabita F. Robert
Published: January 2012

The
cbb
I
region of
Rhodopseudomonas palustris
(
Rp. palustris
) contains the
cbbLS
genes encoding form I ribulose-1,5-bisphosphate (RuBP) carboxylase oxygenase (RubisCO) along with a divergently transcribed regulator gene,
cbbR
. Juxtaposed between
cbbR
and
cbbLS
are the
cbbRRS
genes, encoding an unusual three-protein two-component (CbbRRS) system that modulates the ability of CbbR to influence
cbbLS
expression. The nature of the metabolic signals that
Rp. palustris
CbbR perceives to regulate
cbbLS
transcription is not known. Thus, in this study, the CbbR binding region was first mapped within the
cbbLS
promoter by the use of gel mobility shift assays and DNase I footprinting. In addition, potential metabolic coinducers (metabolites) were tested for their ability to alter the
cbbLS
promoter binding properties of CbbR. Gel mobility shift assays and surface plasmon resonance analyses together indicated that biosynthetic intermediates such as RuBP, ATP, fructose 1,6-bisphosphate, and NADPH enhanced DNA binding by CbbR. These coinducers did not yield identical CbbR-dependent DNase I footprints, indicating that the coinducers caused significant changes in DNA structure. These
in vitro
studies suggest that cellular signals such as fluctuating metabolite concentrations are perceived by and transduced to the
cbbLS
promoter via the master regulator CbbR.
Nitric Oxide Stress Resistance in Porphyromonas gingivalis Is Mediated by a Putative Hydroxylamine Reductase
Authors: Boutrin Marie-Claire , Wang Charles , Aruni Wilson , Li Xiaojin , Fletcher Hansel M.
Published: January 2012

Porphyromonas gingivalis
, the causative agent of adult periodontitis, must maintain nitric oxide (NO) homeostasis and surmount nitric oxide stress from host immune responses or other oral bacteria to survive in the periodontal pocket. To determine the involvement of a putative hydroxylamine reductase (PG0893) and a putative nitrite reductase-related protein (PG2213) in
P. gingivalis
W83 NO stress resistance, genes encoding those proteins were inactivated by allelic exchange mutagenesis. The isogenic mutants
P. gingivalis
FLL455 (
PG0893
∷
ermF
) and FLL456 (
PG2213
∷
ermF
) were black pigmented and showed growth rates and gingipain and hemolytic activities similar to those of the wild-type strain.
P. gingivalis
FLL455 was more sensitive to NO than the wild type. Complementation of
P. gingivalis
FLL455 with the wild-type gene restored the level of NO sensitivity to a level similar to that of the parent strain.
P. gingivalis
FLL455 and FLL456 showed sensitivity to oxidative stress similar to that of the wild-type strain. DNA microarray analysis showed that
PG0893
and
PG2213
were upregulated 1.4- and 2-fold, respectively, in cells exposed to NO. In addition, 178 genes were upregulated and 201 genes downregulated more than 2-fold. The majority of these modulated genes were hypothetical or of unknown function.
PG1181
, predicted to encode a transcriptional regulator, was upregulated 76-fold. Transcriptome
in silico
analysis of the microarray data showed major metabolomic variations in key pathways. Collectively, these findings indicate that
PG0893
and several other genes may play an important role in
P. gingivalis
NO stress resistance.
CtsR Regulation in
mcsAB
-Deficient Gram-Positive Bacteria
Authors: Tao Liang , Chattoraj Partho , Biswas Indranil
Published: January 2012

CtsR is an important repressor that modulates the transcription of class III stress genes in Gram-positive bacteria. In
Bacillus subtilis
, a model Gram-positive organism, the DNA binding activity of CtsR is regulated by McsAB-mediated phosphorylation of the protein where phosphorylated CtsR is a substrate for degradation by the ClpCP complex. Surprisingly, the
mcsAB
genes are absent from many Gram-positive bacteria, including streptococci; therefore, how CtsR activity is modulated in those bacteria remains unknown. Here we show that the posttranslational modulation of CtsR activity is different in
Streptococcus mutans
, a dental pathogen. We observed that of all of the Clp-related proteins, only ClpL is involved in the degradation of CtsR. Neither ClpP nor ClpC had any effect on the degradation of CtsR. We also found that phosphorylation of CtsR on a conserved arginine residue within the winged helix-turn-helix domain is necessary for modulation of the repressor activity of CtsR, as demonstrated by both
in vitro
and
in vivo
assays. We speculate that CtsR is regulated posttranslationally by a different mechanism in
S. mutans
and possibly in other streptococci.
Airway injury during high-level exercise
Authors: Kippelen Pascale , Anderson Sandra D
Published: January 2012

Airway epithelial cells act as a physical barrier against environmental toxins and injury, and modulate inflammation and the immune response. As such, maintenance of their integrity is critical. Evidence is accumulating to suggest that exercise can cause injury to the airway epithelium. This seems the case particularly for competitive athletes performing high-level exercise, or when exercise takes place in extreme environmental conditions such as in cold dry air or in polluted air. Dehydration of the small airways and increased forces exerted on to the airway surface during severe hyperpnoea are thought to be key factors in determining the occurrence of injury of the airway epithelium. The injury-repair process of the airway epithelium may contribute to the development of the bronchial hyper-responsiveness that is documented in many elite athletes.
Improvement of knowledge, attitude and perception of healthcare workers about ADR, a pre- and post-clinical pharmacists' interventional study
Authors: Khalili Hossein , Mohebbi Niayesh , Hendoiee Narjes , Keshtkar Abbas-Ali , Dashti-Khavidaki Simin
Published: January 2012

Purpose

Healthcare workers have a main role in detection, assessment and spontaneous reporting of adverse drug reactions (ADRs), and improvement of their related knowledge, attitude and perception is essential. The goal of this study was evaluation of clinical pharmacists' interventions in improvement of knowledge, attitude and perception of healthcare workers about ADRs in a teaching referral hospital, Tehran, Iran.

Method

Changes in knowledge, attitude and perception of healthcare workers of Imam teaching hospital about ADRs were evaluated before and after clinical pharmacists' interventions including workshops, meetings and presentations.

Results

From the 100 participated subjects, 82 of them completed the study. 51% of the health workers have been aware of the Iranian Pharmacovigilance Center at the ministry of health before intervention and after that all the participants knew this centre. About awareness and detection of ADRs in patients, 69 (84.1%) healthcare workers recognised at least one, and following interventions, it was improved to 73 (89%). Only seven (8.5%) subjects have reported ADRs in before intervention phase that were increased significantly to 18 (22%) after intervention.

Conclusion

Clinical pharmacists' interventions were successful in improvement of healthcare workers' knowledge, attitude and perception about ADRs and spontaneous reporting in our hospital.
T/NK cell type chronic active Epstein–Barr virus disease in adults: an underlying condition for Epstein–Barr virus-associated T/NK-cell lymphoma
Authors: Isobe Yasushi , Aritaka Nanae , Setoguchi Yasuhiro , Ito Yoshinori , Kimura Hiroshi , Hamano Yasuharu , Sugimoto Koichi , Komatsu Norio
Published: January 2012

A chronic infectious mononucleosis-like illness caused by Epstein–Barr virus (EBV) is called ‘chronic active EBV disease’, which is defined as an EBV-associated lymphoproliferative disease. This lymphoproliferative disease is rare and predominantly occurs in Japanese children. Between 1998 and 2010, seven adult-onset cases (aged 20–45 years, median 39 years) were identified, which initially presented with inflammatory diseases, including hepatitis, interstitial pneumonitis, uveitis, nephritis and hypersensitivity to mosquito bites. They showed an EBV viral load in the peripheral blood and evidence of EBV infection of T or natural killer (NK) cells. Five cases (71.4%) developed EBV-positive T/NK-cell lymphoma/leukaemia at a median of 5 years (range 1–7 years) after the diagnosis. Althoughl-asparaginase-containing chemotherapy was effective for the lymphomas, only allogeneic haematopoietic cell transplantation eradicated EBV-infected cells. This observation indicates that persistent EBV infection of T or NK cells defines a distinct disease entity, which provides an underlying condition for EBV-positive T/NK-cell lymphoma/leukaemia.
Hospital consultants and workplace based assessments: how foundation doctors view these educational interactions?
Authors: McKavanagh P , Smyth A , Carragher A
Published: January 2012

Aim

To determine how foundation year 2 (F2) doctors view the input of hospital consultants into their workplace based assessments (WPBAs).

Study design

F2 doctors in Northern Ireland participated in an electronic survey to evaluate their experiences of foundation programme WPBAs. The opportunity to participate was available to all F2 doctors. The survey was performed using questions displayed electronically and the responses were collated using Turning Point technology. Two weeks later a focus group was convened to assess the issues raised by the electronic survey.

Results

Consultant input into foundation doctor's WPBAs was an infrequent occurrence. The F2 doctors expressed a clear view that they valued consultant input, when this occurred. The WPBAs gave the foundation doctors an opportunity to have a one to one learning opportunity with their supervising consultants. However, many of the WPBAs were completed by other doctors in training, in the grades immediately above the foundation doctors. It was suggested that friendship could influence these assessments.

Conclusions

Completion of foundation doctors' assessments by hospital consultants is viewed as a low priority. These assessments are being completed to a large extent by fellow doctors in training. The learning opportunities are consequently less educationally productive. F2 doctors want more opportunities for valued consultant interaction with timely feedback. Suggestions are proposed to improve WPBA implementation. The present WPBA process lacks integrity and a change in approach is urgently required.
Junior doctors' reflections on patient safety
Authors: Ahmed Maria , Arora Sonal , Carley Simon , Sevdalis Nick , Neale Graham
Published: January 2012

Aim

To determine whether foundation year 1 (FY1) doctors reflect upon patient safety incidents (PSIs) within their portfolios and the potential value of such reflections for quality of care.

Methods

A cross-sectional retrospective review of every ‘reflective practice’ portfolio entry made by all FY1 doctors within an Acute Teaching Hospital Trust was conducted in February 2010. Entries were reviewed by two independent blinded researchers to determine whether they related to a PSI that is, any unintended or unexpected incident that could have or did lead to patient harm. For all entries rated positive by both reviewers, a content analysis approach was used to code PSI into incident type, contributing factors and patient outcome according to validated frameworks developed by the National Patient Safety Agency.

Results

139 reflective practice entries were completed by 30 trainees (15 men, 15 women, mean age 24 years). Of the 139 entries, 49% reflected on a PSI. Of these, 22% were due to errors in clinical assessment; 22% were due to delayed access to care; 18% were due to infrastructure/staffing deficiencies; and 16% were due to medication errors. The most common contributing factors were team/social factors (23%), patient factors (22%), communication and task factors (both 17%). The majority of PSIs led to no harm. Six entries described PSIs resulting in patient death, the majority of which were attributable to diagnostic errors.

Conclusions

FY1 doctors commonly reflect on PSIs within their professional portfolios. Such critical reflection can encourage learning but may also promote patient safety and the quality of healthcare across all medical specialties.
Age at first anal sex and HIV/STI vulnerability among gay men in Australia
Authors: Lyons Anthony , Pitts Marian , Grierson Jeffrey , Smith Anthony , McNally Stephen , Couch Murray
Published: January 2012

Objectives

To determine whether there is a link between age at first anal intercourse (AFAI) and gay men's HIV/sexually transmissible infection (STI) vulnerability, including tendencies to engage in higher risk sexual behaviour.

Methods

A nationwide cross-sectional survey was conducted online involving 845 Australian gay men born between 1944 and 1993.

Results

Median AFAI fell from 35 years for men born between 1944 and 1953 to 18 years for men born between 1984 and 1993. Of those who reported having had anal intercourse (N=822), HIV-positive men were found to be significantly younger on average when they first had anal intercourse compared with HIV-negative men (18.5 vs 21.3 years, p<0.001). Men with a history of other STIs were also significantly younger. Engaging in higher risk sexual behaviour is a likely factor, with AFAI generally younger among men who reported >10 sexual partners in the past year (p<0.001) and who engaged in group sex (p<0.001), receptive anal intercourse (p=0.008) or were drug or alcohol affected (p=0.06) during their most recent sexual encounter.

Conclusions

There appears to be a strong link between AFAI and infection with HIV/STIs, as well as tendencies to engage in higher risk sexual behaviour. While further research is needed to understand this link, these findings highlight a need for sexuality education aimed at gay-identified youth to ensure their sexual debut does not lead to poorer sexual health outcomes.
Protective effect of 20-HETE inhibition in a model of oxygen-glucose deprivation in hippocampal slice cultures
Authors: Renic Marija , Kumar Suresh N. , Gebremedhin Debebe , Florence Matthew A. , Gerges Nashaat Z. , Falck John R. , Harder David R. , Roman Richard J.
Published: January 2012

Recent studies have indicated that inhibitors of the synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE) may have direct neuroprotective actions since they reduce infarct volume after ischemia reperfusion in the brain without altering blood flow. To explore this possibility, the present study used organotypic hippocampal slice cultures subjected to oxygen-glucose deprivation (OGD) and reoxygenation to examine whether 20-HETE is released by organotypic hippocampal slices after OGD and whether it contributes to neuronal death through the generation of ROS and activation of caspase-3. The production of 20-HETE increased twofold after OGD and reoxygenation. Blockade of the synthesis of 20-HETE with N-hydroxy- N′-(4-butyl-2-methylphenol)formamidine (HET0016) or its actions with a 20-HETE antagonist, 20-hydroxyeicosa-6( Z),15( Z)-dienoic acid, reduced cell death, as measured by the release of lactate dehydrogenase and propidium iodide uptake. Administration of a 20-HETE mimetic, 20-hydroxyeicosa-5( Z),14( Z)-dienoic acid (5,14-20-HEDE), had the opposite effect and increased injury after OGD. The death of neurons after OGD was associated with an increase in the production of ROS and activation of caspase-3. These effects were attenuated by HET0016 and potentiated after the administration of 5,14-20-HEDE. These findings indicate that the production of 20-HETE by hippocampal slices is increased after OGD and that inhibitors of the synthesis or actions of 20-HETE protect neurons from ischemic cell death. The protective effect of 20-HETE inhibitors is associated with a decrease in superoxide production and activation of caspase-3.
Normal pregnancy: mechanisms underlying the paradox of a ouabain-resistant state with elevated endogenous ouabain, suppressed arterial sodium calcium exchange, and low blood pressure
Authors: Jacobs Brandiese E. , Liu Yong , Pulina Maria V. , Golovina Vera A. , Hamlyn John M.
Published: January 2012

Endogenous cardiotonic steroids (CTS) raise blood pressure (BP) via vascular sodium calcium exchange (NCX1.3) and transient receptor-operated channels (TRPCs). Circulating CTS are superelevated in pregnancy-induced hypertension and preeclampsia. However, their significance in normal pregnancy, where BP is low, is paradoxical. Here we test the hypothesis that vascular resistance to endogenous ouabain (EO) develops in normal pregnancy and is mediated by reduced expression of NCX1.3 and TRPCs. We determined plasma and adrenal levels of EO and the impact of exogenous ouabain in pregnancy on arterial expression of Na+pumps, NCX1.3, TRPC3, and TRPC6 and BP. Pregnant (embryonic day 4) and nonpregnant rats received infusions of ouabain or vehicle. At 14–16 days, tissues and plasma were collected for blotting and EO assay by radioimmunoassay (RIA), liquid chromatography (LC)-RIA, and LC-multidimensional mass spectrometry (MS3). BP (−8 mmHg; P < 0.05) and NCX1.3 expression fell (aorta −60% and mesenteric artery −30%; P < 0.001) in pregnancy while TRPC expression was unchanged. Circulating EO increased (1.14 ± 0.13 nM) vs. nonpregnant (0.6 ± 0.08 nM; P < 0.05) and was confirmed by LC-MS3 and LC-RIA. LC-MS3 revealed two previously unknown isomers of EO; one increased ∼90-fold in pregnancy. Adrenal EO but not isomers were increased in pregnancy. In nonpregnant rats, similar infusions of ouabain raised BP (+24 ± 3 mmHg; P < 0.001). In ouabain-infused rats, impaired fetal and placental growth occurred with no BP increase. In summary, normal pregnancy is an ouabain-resistant state associated with low BP, elevated circulating levels of EO, two novel steroidal EO isomers, and increased adrenal mass and EO content. Ouabain raises BP only in nonpregnant animals. Vascular resistance to the chronic pressor activity of endogenous and exogenous ouabain is mediated by suppressed NCX1.3 and reduced sensitivity of events downstream of Ca2+entry. The mechanisms of EO resistance and the impaired fetal and placental growth due to elevated ouabain may be important in pregnancy-induced hypertension (PIH) and preeclampsia (PE).
Hemin causes mitochondrial dysfunction in endothelial cells through promoting lipid peroxidation: the protective role of autophagy
Authors: Higdon Ashlee N. , Benavides Gloria A. , Chacko Balu K. , Ouyang Xiaosen , Johnson Michelle S. , Landar Aimee , Zhang Jianhua , Darley-Usmar Victor M.
Published: January 2012

The hemolysis of red blood cells and muscle damage results in the release of the heme proteins myoglobin, hemoglobin, and free heme into the vasculature. The mechanisms of heme toxicity are not clear but may involve lipid peroxidation, which we hypothesized would result in mitochondrial damage in endothelial cells. To test this, we used bovine aortic endothelial cells (BAEC) in culture and exposed them to hemin. Hemin led to mitochondrial dysfunction, activation of autophagy, mitophagy, and, at high concentrations, apoptosis. To detect whether hemin induced lipid peroxidation and damaged proteins, we used derivatives of arachidonic acid tagged with biotin or Bodipy (Bt-AA, BD-AA). We found that in cells treated with hemin, Bt-AA was oxidized and formed adducts with proteins, which were inhibited by α-tocopherol. Hemin-dependent mitochondrial dysfunction was also attenuated by α-tocopherol. Protein thiol modification and carbonyl formation occurred on exposure and was not inhibited by α-tocopherol. Supporting a protective role of autophagy, the inhibitor 3-methyladenine potentiated cell death. These data demonstrate that hemin mediates cytotoxicity through a mechanism which involves protein modification by oxidized lipids and other oxidants, decreased respiratory capacity, and a protective role for the autophagic process. Attenuation of lipid peroxidation may be able to preserve mitochondrial function in the endothelium and protect cells from heme-dependent toxicity.