bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional Characterization of Lpt3 and Lpt6, the Inner-Core Lipooligosaccharide Phosphoethanolamine Transferases from
Neisseria meningitidis
Authors: Wenzel Cory Q. , St. Michael Frank , Stupak Jacek , Li Jianjun , Cox Andrew D. , Richards James C.
Published: October 2009

The lipooligosaccharide (LOS) of
Neisseria meningitidis
contains heptose (Hep) residues that are modified with phosphoethanolamine (PEtn) at the 3 (3-PEtn) and/or 6 (6-PEtn) position. The
lpt3
(NMB2010) and
lpt6
(NMA0408) genes of
N. meningitidis
, which are proposed to encode the required HepII 3- and 6-PEtn transferases, respectively, were cloned and overexpressed as C-terminally polyhistidine-tagged fusion proteins in
Escherichia coli
and found to localize to the inner membrane, based on sucrose density gradient centrifugation. Lpt3-His
6
and Lpt6-His
6
were purified from Triton X-100-solubilized membranes by nickel chelation chromatography, and dot blot analysis of enzymatic reactions with 3-PEtn- and 6-PEtn-specific monoclonal antibodies demonstrated conclusively that Lpt3 and Lpt6 are phosphatidylethanolamine-dependent LOS HepII 3- and 6-PEtn transferases, respectively, and that both enzymes are capable of transferring PEtn to both fully acylated LOS and de-O-acylated (de-O-Ac) LOS. Further enzymatic studies using capillary electrophoresis-mass spectrometry (MS) demonstrated that both Lpt3 and Lpt6 are capable of transferring PEtn to de-O-Ac LOS molecules already containing PEtn at the 6 and 3 positions of HepII, respectively, demonstrating that there is no obligate order of PEtn addition in the generation of 3,6-di-PEtn LOS moieties in vitro.
Transcriptional and Functional Analysis of the
Neisseria gonorrhoeae
Fur Regulon
Authors: Jackson Lydgia A. , Ducey Thomas F. , Day Michael W. , Zaitshik Jeremy B. , Orvis Joshua , Dyer David W.
Published: October 2009

To ensure survival in the host, bacteria have evolved strategies to acquire the essential element iron. In
Neisseria gonorrhoeae
, the ferric uptake regulator Fur regulates metabolism through transcriptional control of iron-responsive genes by binding conserved Fur box (FB) sequences in promoters during iron-replete growth. Our previous studies showed that Fur also controls the transcription of secondary regulators that may, in turn, control pathways important to pathogenesis, indicating an indirect role for Fur in controlling these downstream genes. To better define the iron-regulated cascade of transcriptional control, we combined three global strategies—temporal transcriptome analysis, genomewide in silico FB prediction, and Fur titration assays (FURTA)—to detect genomic regions able to bind Fur in vivo. The majority of the 300 iron-repressed genes were predicted to be of unknown function, followed by genes involved in iron metabolism, cell communication, and intermediary metabolism. The 107 iron-induced genes encoded hypothetical proteins or energy metabolism functions. We found 28 predicted FBs in FURTA-positive clones in the promoters and within the open reading frames of iron-repressed genes. We found lower levels of conservation at critical thymidine residues involved in Fur binding in the FB sequence logos of FURTA-positive clones with intragenic FBs than in the sequence logos generated from FURTA-positive promoter regions. In electrophoretic mobility shift assay studies, intragenic FBs bound Fur with a lower affinity than intergenic FBs. Our findings further indicate that transcription under iron stress is indirectly controlled by Fur through 12 potential secondary regulators.
Molecular Basis for the Role of
Staphylococcus aureus
Penicillin Binding Protein 4 in Antimicrobial Resistance
Authors: Navratna Vikas , Nadig Savitha , Sood Varun , Prasad K. , Arakere Gayathri , Gopal B.
Published: October 2009

Penicillin binding proteins (PBPs) are membrane-associated proteins that catalyze the final step of murein biosynthesis. These proteins function as either transpeptidases or carboxypeptidases and in a few cases demonstrate transglycosylase activity. Both transpeptidase and carboxypeptidase activities of PBPs occur at the
d
-Ala-
d
-Ala terminus of a murein precursor containing a disaccharide pentapeptide comprising
N
-acetylglucosamine and
N
-acetyl-muramic acid-
l
-Ala-
d
-Glu-
l
-Lys-
d
-Ala-
d
-Ala. β-Lactam antibiotics inhibit these enzymes by competing with the pentapeptide precursor for binding to the active site of the enzyme. Here we describe the crystal structure, biochemical characteristics, and expression profile of PBP4, a low-molecular-mass PBP from
Staphylococcus aureus
strain COL. The crystal structures of PBP4-antibiotic complexes reported here were determined by molecular replacement, using the atomic coordinates deposited by the New York Structural Genomics Consortium. While the
pbp4
gene is not essential for the viability of
S. aureus
, the knockout phenotype of this gene is characterized by a marked reduction in cross-linked muropeptide and increased vancomycin resistance. Unlike other PBPs, we note that expression of PBP4 was not substantially altered under different experimental conditions, nor did it change across representative hospital- or community-associated strains of
S. aureus
that were examined.
In vitro
data on purified recombinant
S. aureus
PBP4 suggest that it is a β-lactamase and is not trapped as an acyl intermediate with β-lactam antibiotics. Put together, the expression analysis and biochemical features of PBP4 provide a framework for understanding the function of this protein in
S. aureus
and its role in antimicrobial resistance.
The AraC/XylS Family Activator RhaS Negatively Autoregulates
rhaSR
Expression by Preventing Cyclic AMP Receptor Protein Activation
Authors: Wickstrum Jason R. , Skredenske Jeff M. , Balasubramaniam Vinitha , Jones Kyle , Egan Susan M.
Published: October 2009

The
Escherichia coli
RhaR protein activates expression of the
rhaSR
operon in the presence of its effector,
l
-rhamnose. The resulting RhaS protein (plus
l
-rhamnose) activates expression of the
l
-rhamnose catabolic and transport operons,
rhaBAD
and
rhaT
, respectively. Here, we further investigated our previous finding that
rhaS
deletion resulted in a threefold increase in
rhaSR
promoter activity, suggesting RhaS negative autoregulation of
rhaSR
. We found that RhaS autoregulation required the cyclic AMP receptor protein (CRP) binding site at
rhaSR
and that RhaS was able to bind to the RhaR binding site at
rhaSR
. In contrast to the expected repression, we found that in the absence of both RhaR and the CRP binding site at the
rhaSR
promoter, RhaS activated expression to a level comparable with RhaR activation of the same promoter. However, when the promoter included the RhaR and CRP binding sites, the level of activation by RhaS and CRP was much lower than that by RhaR and CRP, suggesting that CRP could not fully coactivate with RhaS. Taken together, our results indicate that RhaS negative autoregulation involves RhaS competition with RhaR for binding to the RhaR binding site at
rhaSR
. Although RhaS and RhaR activate
rhaSR
transcription to similar levels, CRP cannot effectively coactivate with RhaS. Therefore, once RhaS reaches a relatively high protein concentration, presumably sufficient to saturate the RhaS-activated promoters, there will be a decrease in
rhaSR
transcription. We propose a model in which differential DNA bending by RhaS and RhaR may be the basis for the difference in CRP coactivation.
Three-Dimensional Structure of Different Functional Forms of the
Vibrio cholerae
Hemolysin Oligomer: a Cryo-Electron Microscopic Study
Authors: Dutta Somnath , Mazumdar Budhaditya , Banerjee Kalyan K. , Ghosh Amar N.
Published: October 2009

Vibrio cholerae
hemolysin (HlyA) is a 65-kDa water-soluble pore-forming toxin that causes lysis of eukaryotic cells by destroying selective permeability of the plasma membrane bilayer. The HlyA monomer self-assembles on the target cell surface to the more stable β-barrel amphipathic heptamer, which inserts into the membrane bilayer to form a diffusion channel. Deletion of the 15-kDa β-prism lectin domain at the C terminus generates a 50-kDa hemolysin variant (HlyA50) with an ∼1,000-fold decrease in hemolytic activity. Because functional differences are eventually dictated by structural differences, we determined three-dimensional structures of 65- and 50-kDa HlyA oligomers, using cryo-electron microscopy and single-particle methods. Our study clearly shows that the HlyA oligomer has sevenfold symmetry but that the HlyA50 oligomer is an asymmetric molecule. The HlyA oligomer has bowl-like, arm-like, and ring-like domains. The bowl-like domain is coupled with the ring-like domain, and seven side openings are present just beneath the ring-like domain. Although a central channel is present in both HlyA and HlyA50 oligomers, they differ in pore size as well as in shape of the molecules and channel. These structural differences may be relevant to the striking difference in efficiencies of functional channel formation by the two toxin forms.
Crystal Structures of the Lumazine Protein fromPhotobacterium kishitaniiin Complexes with the Authentic Chromophore, 6,7-Dimethyl- 8-(1′-d-Ribityl) Lumazine, and Its Analogues, Riboflavin and Flavin Mononucleotide, at High Resolution
Authors: Sato Yuichi , Shimizu Satoshi , Ohtaki Akashi , Noguchi Keiichi , Miyatake Hideyuki , Dohmae Naoshi , Sasaki Satoshi , Odaka Masafumi , Yohda Masafumi
Published: October 2009

Lumazine protein (LumP) is a fluorescent accessory protein having 6,7-dimethyl-8-(1′-d-ribityl) lumazine (DMRL) as its authentic chromophore. It modulates the emission of bacterial luciferase to shorter wavelengths with increasing luminous strength. To obtain structural information on the native structure as well as the interaction with bacterial luciferase, we have determined the crystal structures of LumP fromPhotobacterium kishitaniiin complexes with DMRL and its analogues, riboflavin (RBF) and flavin mononucleotide (FMN), at resolutions of 2.00, 1.42, and 2.00 Å. LumP consists of two β barrels that have nearly identical folds, the N-terminal and C-terminal barrels. The structures of LumP in complex with all of the chromophores studied are all essentially identical, except around the chromophores. In all of the structures, the chromophore is tethered to the narrow cavity via many hydrogen bonds in the N-terminal domain. These are absent in the C-terminal domain. Hydrogen bonding in LumP-FMN is decreased in comparison with that in LumP-RBF because the phosphate moiety of FMN protrudes out of the narrow cavity. In LumP-DMRL, the side chain of Gln65 is close to the ring system, and a new water molecule that stabilizes the ligand is observed near Ser48. Therefore, DMRL packs more tightly in the ligand-binding site than RBF or FMN. A docking simulation of bacterial luciferase and LumP suggests that the chromophore is located close enough for direct energy transfer to occur. Moreover, the surface potentials around the ligand-binding sites of LumP and bacterial luciferase exhibit complementary charge distributions, which would have a significant effect on the interaction between LumP and luciferase.
Pantothenate Kinase from the Thermoacidophilic Archaeon
Picrophilus torridus
Authors: Takagi Masakazu , Tamaki Hideyuki , Miyamoto Yukiko , Leonardi Roberta , Hanada Satoshi , Jackowski Suzanne , Chohnan Shigeru
Published: October 2009

Pantothenate kinase (CoaA) catalyzes the first step of the coenzyme A (CoA) biosynthetic pathway and controls the intracellular concentrations of CoA through feedback inhibition in bacteria. An alternative enzyme found in archaea, pantoate kinase, is missing in the order
Thermoplasmatales
. The PTO0232 gene from
Picrophilus torridus
, a thermoacidophilic euryarchaeon, is shown to be a distant homologue of the prokaryotic type I CoaA. The cloned gene clearly complements the poor growth of the temperature-sensitive
Escherichia coli
CoaA mutant strain ts9, and the recombinant protein expressed in
E. coli
cells transfers phosphate to pantothenate at pH 5 and 55°C. In contrast to
E. coli
CoaA, the
P. torridus
enzyme is refractory to feedback regulation by CoA, indicating that in
P. torridus
cells the CoA levels are not regulated by the CoaA step. These data suggest the existence of two subtypes within the class of prokaryotic type I CoaAs.
Conservation of Structure and Protein-Protein Interactions Mediated by the Secreted Mycobacterial Proteins EsxA, EsxB, and EspA
Authors: Callahan Brian , Nguyen Kiet , Collins Alissa , Valdes Kayla , Caplow Michael , Crossman David K. , Steyn Adrie J. C. , Eisele Leslie , Derbyshire Keith M.
Published: October 2009

Mycobacterium tuberculosisEsxA and EsxB proteins are founding members of the WXG100 (WXG) protein family, characterized by their small size (∼100 amino acids) and conserved WXG amino acid motif.M. tuberculosiscontains 11 tandem pairs of WXG genes; each gene pair is thought to be coexpressed to form a heterodimer. The precise role of these proteins in the biology ofM. tuberculosisis unknown, but several of the heterodimers are secreted, which is important for virulence. However, WXG proteins are not simply virulence factors, since nonpathogenic mycobacteria also express and secrete these proteins. Here we show that three WXG heterodimers have structures and properties similar to those of theM. tuberculosisEsxBA (MtbEsxBA) heterodimer, regardless of their host species and apparent biological function. Biophysical studies indicate that the WXG proteins fromM. tuberculosis(EsxG and EsxH),Mycobacterium smegmatis(EsxA and EsxB), andCorynebacterium diphtheriae(EsxA and EsxB) are heterodimers and fold into a predominately α-helical structure. An in vivo protein-protein interaction assay was modified to identify proteins that interact specifically with the native WXG100 heterodimer. MtbEsxA and MtbEsxB were fused into a single polypeptide, MtbEsxBA, to create a biomimetic bait for the native heterodimer. The MtbEsxBA bait showed specific association with severalesx-1-encoded proteins and EspA, a virulence protein secreted by ESX-1. The MtbEsxBA fusion peptide was also utilized to identify residues in both EsxA and EsxB that are important for establishing protein interactions with Rv3871 and EspA. Together, the results are consistent with a model in which WXG proteins perform similar biological roles in virulent and nonvirulent species.
Structure and Regulation of thegabGene Cluster, Involved in the γ-Aminobutyric Acid Shunt, Are Controlled by a σ54Factor inBacillus thuringiensis
Authors: Zhu Li , Peng Qi , Song Fuping , Jiang Yanan , Sun Changpo , Zhang Jie , Huang Dafang
Published: October 2009

The structure and regulation of thegabgene cluster, involved in γ-aminobutyric acid (GABA) shunt, were studied by characterizinggabTandgabDgenes cloned fromBacillus thuringiensis. Deletions of thegabTandgabDgenes inB. thuringiensisstrain HD-73 did not affect the growth of mutant strains in rich culture media, but the growth of agabTdeletion mutant strain was reduced in basic media (containing 0.2% GABA). Genome analysis indicates that the structure of thegabgene cluster inB. thuringiensisHD-73 is different from that inEscherichia coliandBacillus subtilisbut is common in strains of theBacillus cereusgroup. This suggests that the gene cluster involved in GABA shunt is specific to theB. cereusgroup. Based on reverse transcription-PCR and transcriptional fusion analysis, we confirmed that thegabTandgabDgenes belong to different transcriptional units, while thegabDandgabRgenes form an operon. We also demonstrated that thegabRgene plays a positive regulatory role ingabDandgabTexpression. The GabR protein may be a σ54-dependent transcriptional activator, according to a conserved domain search in the NCBI database, and it is highly conserved in theB. cereusgroup. The −24/−12 consensus sequence of a promoter upstream fromgabTsuggests that the promoter can be recognized by a σ54factor. Further analysis of the genetic complementation studies also suggests that the expression of thegabTgene is controlled by a σ54factor. Thus, the expression of thegabcluster is regulated by a σ54factor by way of the transcription activator GabR.
Appendage-Mediated Surface Adherence of
Sulfolobus solfataricus
Authors: Zolghadr Behnam , Klingl Andreas , Koerdt Andrea , Driessen Arnold J. M. , Rachel Reinhard , Albers Sonja-Verena
Published: October 2009

Attachment of microorganisms to surfaces is a prerequisite for colonization and biofilm formation. The hyperthermophilic crenarchaeote
Sulfolobus solfataricus
was able to attach to a variety of surfaces, such as glass, mica, pyrite, and carbon-coated gold grids. Deletion mutant analysis showed that for initial attachment the presence of flagella and pili is essential. Attached cells produced extracellular polysaccharides containing mannose, galactose, and
N
-acetylglucosamine. Genes possibly involved in the production of the extracellular polysaccharides were identified.
Metabolic Control of Virulence Genes inBrucella abortus: HutC CoordinatesvirBExpression and the Histidine Utilization Pathway by Direct Binding to Both Promoters
Authors: Sieira Rodrigo , Arocena Gastón M. , Bukata Lucas , Comerci Diego J. , Ugalde Rodolfo A.
Published: October 2009

Type IV secretion systems (T4SS) are multicomponent machineries involved in the translocation of effector molecules across the bacterial cell envelope. ThevirBoperon ofBrucella abortuscodes for a T4SS that is essential for virulence and intracellular multiplication of the bacterium in the host. Previous studies showed that thevirBoperon ofB. abortusis tightly regulated within the host cells. In order to identify factors implicated in the control ofvirBexpression, we searched for proteins ofBrucellathat directly bind to thevirBpromoter (PvirB). Using different procedures, we isolated a 27-kDa protein that binds specifically to PvirB. This protein was identified as HutC, the transcriptional repressor of the histidine utilization (hut) genes. Analyses ofvirBandhutpromoter activity revealed that HutC exerts two different roles: it acts as a coactivator of transcription of thevirBoperon, whereas it represses thehutgenes. Such activities were observed both intracellularly and in bacteria incubated under conditions that resemble the intracellular environment. Electrophoresis mobility shift assays (EMSA) and DNase I footprinting experiments revealed the structure, affinity, and localization of the HutC-binding sites and supported the regulatory role of HutC in bothhutandvirBpromoters. Taken together, these results indicate thatBrucellacoopted the function of HutC to coordinate the Hut pathway with transcriptional regulation of thevirBgenes, probably as a way to sense its own metabolic state and develop adaptive responses to overcome intracellular host defenses.
Accumulation of Intermediates of the Carbon-Phosphorus Lyase Pathway for Phosphonate Degradation in
phn
Mutants of
Escherichia coli
Authors: Hove-Jensen Bjarne , Rosenkrantz Tina J. , Zechel David L. , Willemoës Martin
Published: October 2009

The catabolism of phosphonic acids occurs in
Escherichia coli
by the carbon-phosphorus lyase pathway, which is governed by the 14-cistron
phn
operon. Here, several compounds are shown to accumulate in strains of
E. coli
with genetic blocks in various
phn
cistrons when the strains are fed with phosphonate.
Control of Thioredoxin Reductase Gene (
trxB
) Transcription by SarA in
Staphylococcus aureus
Authors: Ballal Anand , Manna Adhar C.
Published: October 2009

Thioredoxin reductase (encoded by
trxB
) protects
Staphylococcus aureus
against oxygen or disulfide stress and is indispensable for growth. Among the different
sarA
family mutants analyzed, transcription of
trxB
was markedly elevated in the
sarA
mutant under conditions of aerobic as well as microaerophilic growth, indicating that SarA acts as a negative regulator of
trxB
expression. Gel shift analysis showed that purified SarA protein binds directly to the
trxB
promoter region DNA
in vitro
. DNA binding of SarA was essential for repression of
trxB
transcription
in vivo
in
S. aureus
. Northern blot analysis and DNA binding studies of the purified wild-type SarA and the mutant SarAC9G with oxidizing agents indicated that oxidation of Cys-9 reduced the binding of SarA to the
trxB
promoter DNA. Oxidizing agents, in particular diamide, could further enhance transcription of the
trxB
gene in the
sarA
mutant, suggesting the presence of a SarA-independent mode of
trxB
induction. Analysis of two oxidative stress-responsive
sarA
regulatory target genes,
trxB
and
sodM
, with various mutant
sarA
constructs showed a differential ability of the SarA to regulate expression of the two above-mentioned genes
in vivo
. The overall data demonstrate the important role played by SarA in modulating expression of genes involved in oxidative stress resistance in
S. aureus
.
Identification and Characterization of Noncoding Small RNAs inStreptococcus pneumoniaeSerotype 2 Strain D39
Authors: Tsui Ho-Ching Tiffany , Mukherjee Dhriti , Ray Valerie A. , Sham Lok-To , Feig Andrew L. , Winkler Malcolm E.
Published: October 2009

We report a search for small RNAs (sRNAs) in the low-GC, Gram-positive human pathogenStreptococcus pneumoniae. Based on bioinformatic analyses by Livny et al. (J. Livny, A. Brencic, S. Lory, and M. K. Waldor, Nucleic Acids Res.34:3484-3493, 2006), we tested 40 candidates by Northern blotting and confirmed the expression of nine new and one previously reported (CcnA) sRNAs in strain D39. CcnA is one of five redundant sRNAs reported by Halfmann et al. (A. Halfmann, M. Kovacs, R. Hakenbeck, and R. Bruckner, Mol. Microbiol.66:110-126, 2007) that are positively controlled by the CiaR response regulator. We characterized 3 of these 14 sRNAs: Spd-sr17 (144 nucleotides [nt]; decreased in stationary phase), Spd-sr37 (80 nt; strongly expressed in all growth phases), and CcnA (93 nt; induced by competence stimulatory peptide). Spd-sr17 and CcnA likely fold into structures containing single-stranded regions between hairpin structures, whereas Spd-sr37 forms a base-paired structure. Primer extension mapping and ectopic expression in deletion/insertion mutants confirmed the independent expression of the three sRNAs. Microarray analyses indicated that insertion/deletion mutants inspd-sr37andccnAexerted strongcis-acting effects on the transcription of adjacent genes, indicating that these sRNA regions are also cotranscribed in operons. Deletion or overexpression of the three sRNAs did not cause changes in growth, certain stress responses, global transcription, or virulence. Constitutive ectopic expression of CcnA reversed some phenotypes of D39 ΔciaRmutants, but attempts to link CcnA to -E tocomCas a target were inconclusive inciaR+strains. These results show thatS. pneumoniae, which lacks known RNA chaperones, expresses numerous sRNAs, but three of these sRNAs do not strongly affect common phenotypes or transcription patterns.
Mismatch Correction Modulates Mutation Frequency and Pilus Phase and Antigenic Variation in
Neisseria gonorrhoeae
Authors: Criss Alison K. , Bonney Kevin M. , Chang Rhoda A. , Duffin Paul M. , LeCuyer Brian E. , Seifert H. Steven
Published: October 2009

The mismatch correction (MMC) system repairs DNA mismatches and single nucleotide insertions or deletions postreplication. To test the functions of MMC in the obligate human pathogen
Neisseria gonorrhoeae
, homologues of the core MMC genes
mutS
and
mutL
were inactivated in strain FA1090. No
mutH
homologue was found in the FA1090 genome, suggesting that gonococcal MMC is not methyl directed. MMC mutants were compared to a mutant in
uvrD
, the helicase that functions with MMC in
Escherichia coli
. Inactivation of MMC or
uvrD
increased spontaneous resistance to rifampin and nalidixic acid, and MMC/
uvrD
double mutants exhibited higher mutation frequencies than any single mutant. Loss of MMC marginally enhanced the transformation efficiency of DNA carrying a single nucleotide mismatch but not that of DNA with a 1-kb insertion. Unlike the exquisite UV sensitivity of the
uvrD
mutant, inactivating MMC did not affect survival after UV irradiation. MMC and
uvrD
mutants exhibited increased PilC-dependent pilus phase variation.
mutS-
deficient gonococci underwent an increased frequency of pilin antigenic variation, whereas
uvrD
had no effect. Recombination tracts in the
mutS
pilin variants were longer than in parental gonococci but utilized the same donor
pilS
loci. These results show that gonococcal MMC repairs mismatches and small insertion/deletions in DNA and also affects the recombination events underlying pilin antigenic variation. The differential effects of MMC and
uvrD
in gonococci unexpectedly reveal that MMC can function independently of
uvrD
in this human-specific pathogen.
Response of the Oxygen Sensor NreB to Air
In Vivo
: Fe-S-Containing NreB and Apo-NreB in Aerobically and Anaerobically Growing
Staphylococcus carnosus
Authors: Reinhart F. , Huber A. , Thiele R. , Unden G.
Published: October 2009

The sensor kinase NreB from
Staphylococcus carnosus
contains an O
2
-sensitive [4Fe-4S]
2+
cluster which is converted by O
2
to a [2Fe-2S]
2+
cluster, followed by complete degradation and formation of Fe-S-less apo-NreB. NreB·[2Fe-2S]
2+
and apoNreB are devoid of kinase activity. NreB contains four Cys residues which ligate the Fe-S clusters. The accessibility of the Cys residues to alkylating agents was tested and used to differentiate Fe-S-containing and Fe-S-less NreB. In a two-step labeling procedure, accessible Cys residues in the native protein were first labeled by iodoacetate. In the second step, Cys residues not labeled in the first step were alkylated with the fluorescent monobromobimane (mBBr) after denaturing of the protein. In purified (aerobic) apoNreB, most (96%) of the Cys residues were alkylated in the first step, but in anaerobic (Fe-S-containing) NreB only a small portion (23%) were alkylated. In anaerobic bacteria, a very small portion of the Cys residues of NreB (9%) were accessible to alkylation in the native state, whereas most (89%) of the Cys residues from aerobic bacteria were accessible. The change in accessibility allowed determination of the half-time (6 min) for the conversion of NreB·[4Fe-4S]
2+
to apoNreB after the addition of air
in vitro
. Overall, in anaerobic bacteria most of the NreB exists as NreB·[4Fe-4S]
2+
, whereas in aerobic bacteria the (Fe-S-less) apoNreB is predominant and represents the physiological form. The number of accessible Cys residues was also determined by iodoacetate alkylation followed by mass spectrometry of Cys-containing peptides. The pattern of mass increases confirmed the results from the two-step labeling experiments.
Complete Genome Sequence of
Anaplasma marginale
subsp.
centrale
Authors: Herndon David R. , Palmer Guy H. , Shkap Varda , Knowles Donald P. , Brayton Kelly A.
Published: October 2009

Anaplasma marginale
subsp.
centrale
is a naturally attenuated subtype that has been used as a vaccine for a century. We sequenced the genome of this organism and compared it to those of virulent
senso stricto A. marginale
strains. The comparison markedly narrows the number of outer membrane protein candidates for development of a safer inactivated vaccine and provides insight into the diversity among strains of
senso lato A. marginale
.
Does physical activity reduce seniors' need for healthcare?: a study of 24 281 Canadians
Authors: Woolcott John C , Ashe Maureen C , Miller William C , Shi Peilin , Marra Carlo A ,   PACC Research Team
Published: October 2009

Objectives

Physical inactivity has been associated with significant increases in disease morbidity and mortality. This study assessed the association between physical activity and (1) health resource use and (2) health resource use costs.

Design and Participants

The responses from 24 281 respondents >65 years to the Canadian Community Health Survey Cycle 1.1 were used to find activity levels and determine health resource use and costs. Logistic regression models were used to assess risks of hospitalisation.

Results

Physical inactivity was associated with statistically significant increases to hospitalisations, lengths of stay and healthcare visits (p<0.01). Average healthcare costs (based on the 2007 value of the Canadian dollar) for the physically inactive were $C1214.15 higher than the healthcare costs of the physically active ($C2005.27 vs $C791.12, p<0.01).

Conclusion

Among those >65 years, physical activity is strongly associated with reduced health resource use and costs.
Biological markers of cardiac damage are not related to measures of cardiac systolic and diastolic function using cardiovascular magnetic resonance and echocardiography after an acute bout of prolonged endurance exercise
Authors: Wilson M , O'Hanlon R , Prasad S , Oxborough D , Godfrey R , Alpendurada F , Smith G , Wong J , Basavarajaiah S , Sharma S , Nevill A , Gaze David , George K , Whyte G
Published: October 2009

Objectives

Seventeen male participants (mean (SD) (range): age 33.5 (6.5) years (46–26 years), body mass 80 (9.2) kg (100–63 kg), height 1.81 (0.06) m (1.93– 1.70 m)) ran a marathon to investigate the relationship between systolic function (using cardiac magnetic resonance (CMR)) and diastolic function (using echocardiography) against biomarkers of cardiac damage.

Methods

Echocardiographic and cardiac troponin I (cTnI)/N-terminal pro-B-type natriuretic peptide (NTproBNP) data were collected 24 h premarathon, immediately postmarathon and 6 h postmarathon. CMR data were collected 24 h premarathon and at 6 h postmarathon.

Results

Body mass was significantly reduced postmarathon (80 (9.2) vs 78.8 (8.6) kg; p<0.001). There was a significant E/A reduction postmarathon (1.11 (0.34) vs 1.72 (0.44); p<0.05) that remained depressed 6 h postmarathon (1.49 (0.43); p<0.05). CMR demonstrated left ventricular end-diastolic and end-systolic volumes were reduced postmarathon, with a preserved stroke volume. Left ventricular ejection fraction 6 h postmarathon significantly increased (64.4% (4.2%) vs 67.4% (5%); p<0.05). There were significant elevations in cTnI (0.00 vs 0.04 (0.03) μg/l; p<0.05) and NTproBNP (37.4 (24.15) ng/l vs 59.34 (43.3) ng/l; p<0.05) immediately postmarathon. Eight runners had cTnI elevations immediately postmarathon above acute myocardial infarction cutoff levels (≥0.03 μg/l). No correlations between cTnI/NTproBNP and measures of diastolic function (E, A, E/A, isovolumic relaxation time, E deceleration time and E/E′) or measures of systolic function (stroke volume or ejection fraction) were observed immediately postmarathon or 6 h postmarathon.

Conclusions

Biomarkers of cardiac damage after prolonged exercise are not associated with either systolic or diastolic functional measures.
Some sex hormone profiles are consistent over time in normal menstruating women: implications for sports injury epidemiology
Authors: Shultz S J , Wideman L , Montgomery M M , Levine B J
Published: October 2009

Purpose

It is unclear whether sex hormone profiles obtained in two consecutive months are consistent within women. Month-to-month consistency in daily, nadir, peak and mean hormone concentrations during the early follicular and luteal phases in recreationally active, young eumenorrheic women was prospectively examined.

Methods

60 healthy, non-smoking women who reported normal and consistent menstrual cycles lasting 26–32 days for the past 6 months were followed prospectively to obtain serum samples for the first 6 days of menses and for 8 days after a positive ovulation test over two consecutive months. Month-to-month consistency of daily concentrations of oestradiol (pg/ml), progesterone (ng/ml), testosterone (ng/dl), sex hormone-binding globulin (nmol/l) and free androgen index were determined using linear mixed models. Month-to-month consistency in nadir, peak and mean concentrations were then assessed using intraclass correlation coefficients and SEM to more precisely examine intraindividual consistency.

Results

Linear mixed models revealed stable hormone concentrations across cycles and cycles by day. Reliability estimates for nadir, peak, mean menses and mean postovulatory concentrations range from 0.56 to 0.86 for oestradiol, 0.44 to 0.91 for progesterone, 0.60 to 0.86 for testosterone, 0.88 to 0.97 for sex hormone-binding globulin and 0.78 to 0.91 for free androgen index.

Conclusions

Hormone profiles were reproducible over two consecutive months. To reduce month-to-month intraindividual variations and improve measurement consistency, it is recommended that multiple samples be taken over consecutive days as opposed to a single sample.
Neonatal giant pial arteriovenous malformation: genesis or rapid enlargement in the third trimester
Authors: Potter C A , Armstrong-Wells J , Fullerton H J , Young W L , Higashida R T , Dowd C F , Halbach V V , Hetts S W
Published: October 2009

A neonate with congestive heart failure at birth due to a nearly holohemispheric pial arteriovenous malformation is described. This occurred despite a normal second trimester prenatal sonogram. Successful treatment of heart failure was achieved by embolization alone. This case demonstrates that hemodynamically significant lesions may arise later or enlarge more rapidly in utero than previously thought.
Identifying pesticide use patterns among flower growers to assess occupational exposure to mixtures
Authors: Schilmann Astrid , Lacasaña Marina , Blanco-Muñoz Julia , Aguilar-Garduño Clemente , Salinas-Rodríguez Aarón , Flores-Aldana Mario , Cebrián Mariano E
Published: October 2009

Objectives

Exposure assessment to a single pesticide does not capture the complexity of the occupational exposure. Recently, pesticide use patterns analysis has emerged as an alternative to study these exposures. The aim of this study is to identify the pesticide use pattern among flower growers in Mexico participating in the study on the endocrine and reproductive effects associated with pesticide exposure.

Methods

A cross-sectional study was carried out to gather retrospective information on pesticide use applying a questionnaire to the person in charge of the participating flower growing farms. Information about seasonal frequency of pesticide use (rainy and dry) for the years 2004 and 2005 was obtained. Principal components analysis was performed.

Results

Complete information was obtained for 88 farms and 23 pesticides were included in the analysis. Six principal components were selected, which explained more than 70% of the data variability. The identified pesticide use patterns during both years were: 1. fungicides benomyl, carbendazim, thiophanate and metalaxyl (both seasons), including triadimephon during the rainy season, chlorotalonyl and insecticide permethrin during the dry season; 2. insecticides oxamyl, biphenthrin and fungicide iprodione (both seasons), including insecticide methomyl during the dry season; 3. fungicide mancozeb and herbicide glyphosate (only during the rainy season); 4. insecticides metamidophos and parathion (both seasons); 5. insecticides omethoate and methomyl (only rainy season); and 6. insecticides abamectin and carbofuran (only dry season). Some pesticides do not show a clear pattern of seasonal use during the studied years.

Conclusions

The principal component analysis is useful to summarise a large set of exposure variables into smaller groups of exposure patterns, identifying the mixtures of pesticides in the occupational environment that may have an interactive effect on a particular health effect.
Diagnostic strategies using physical examination are minimally useful in defining carpal tunnel syndrome in population-based research studies: Table 1
Authors: Descatha A , Dale A-M , Franzblau A , Coomes J , Evanoff B
Published: October 2009

Objective

We evaluated the utility of physical examination manoeuvres in the prediction of carpal tunnel syndrome (CTS) in a population-based research study.

Methods

We studied a cohort of 1108 newly employed workers in several industries. Each worker completed a symptom questionnaire, a structured physical examination and nerve conduction study. For each hand, our CTS case definition required both median nerve conduction abnormality and symptoms classified as “classic” or “probable” on a hand diagram. We calculated the positive predictive values and likelihood ratios for physical examination manoeuvres in subjects with and without symptoms.

Results

The prevalence of CTS in our cohort was 1.2% for the right hand and 1.0% for the left hand. The likelihood ratios of a positive test for physical provocative tests ranged from 2.0 to 3.3, and those of a negative test from 0.3 to 0.9. The post-test probability of positive testing was <50% for all strategies tested.

Conclusion

Our study found that physical examination, alone or in combination with symptoms, was not predictive of CTS in a working population. We suggest using specific symptoms as a first-level screening tool, and nerve conduction study as a confirmatory test, as a case definition strategy in research settings.
Airway inflammation in cement production workers
Authors: Fell Anne Kristin M , Sikkeland Liv Ingunn B , Svendsen Martin Veel , Kongerud Johny
Published: October 2009

Objective

Cement aerosol exposure is associated with increased morbidity of airway disease among exposed workers. Our aim was to compare levels of inflammatory cells and soluble inflammatory markers in induced sputum samples from cement production workers between exposed and unexposed periods, and to compare these variables between cement workers and references.

Methods

35 healthy, non-smoking aerosol-exposed cement production workers from Norway provided a blood sample and performed induced sputum and spirometry after 5 days without exposure and during a period of exposure. These values were compared with those from an internal low-exposed reference group of 15 office workers and an external reference group of 39 non-exposed workers. Differential cell counts and inflammatory markers were assessed.

Results

Median thoracic aerosol concentration over one work shift (8 h) was 0.6 mg/m3(range 0.2–8.1) in maintenance workers and 1.75 mg/m3(0.2–15.5) in furnace department workers. The median percentage of airway neutrophils in both groups combined was 51% (32–66) in the exposed period, which was significantly higher than in both the unexposed period (38%; 23–55) (p=0.04) and the external reference group (30%; 19–44) (p=0.001). Median interleukin-1β concentration was elevated compared with both office workers (p=0.05) and the external reference group (p=0.006).

Conclusions

A significantly higher percentage of neutrophils was observed in cement production workers during the exposed period compared with both the non-exposed period and the external reference group, and corresponded with elevated IL-1β concentration. These data indicate that cement aerosol exposure in concentrations below the Norwegian occupational limits (respirable dust 5 mg/m3; total dust 10 mg/m3) may cause airway inflammation.
Increased death receptor pathway of apoptotic signaling in septic mouse aorta: effect of systemic delivery of FADD siRNA
Authors: Matsuda Naoyuki , Teramae Hiroki , Yamamoto Seiji , Takano Ken-ichi , Takano Yasuo , Hattori Yuichi
Published: October 2009

Recent evidence suggests that apoptotic cell death plays an important role in the pathophysiology of sepsis. Because there is extensive apoptosis of vascular endothelial cells in sepsis, we examined whether the death receptor pathway of apoptotic signaling is altered in thoracic aortas from mice with polymicrobial sepsis, as produced by cecal ligation and puncture (CLP). In septic aorta, total and surface expression levels of the two death receptors tumor necrosis factor receptor 1 and Fas were highly upregulated. Furthermore, marked increases in the mRNA and protein levels of Fas-associated death domain (FADD), an adaptor molecule to recruit procaspase-8 into the death-inducing signal complex, were observed in septic aorta, which were strongly suppressed by systemic delivery of small interfering RNA (siRNA) against FADD. No increase in expression of death receptors and FADD was observed in endothelium-denuded aortic tissues from septic animals. Systemic administration of FADD siRNA also resulted in great attenuation of sepsis-induced increases in expression and activation of caspase-3, an effector protease in the apoptosis cascade. Terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL) revealed that the significant appearance of cell apoptosis in aortic endothelium after CLP-induced sepsis was eliminated when FADD siRNA was systemically applied. Light and electron microscopic examinations of septic aorta showed cell swelling, nuclear fragmentation, and partial detachment of endothelial cells from the basal membrane, which were prevented by systemic treatment with FADD siRNA. Finally, FADD siRNA administration dramatically improved survival of CLP mice, supporting the feasibility of this gene-based approach for treating septic shock.
Nonanticoagulant heparin reduces myocyte Na+and Ca2+loading during simulated ischemia and decreases reperfusion injury
Authors: Barry William H. , Zhang Xiu Q. , Halkos Michael E. , Vinten-Johansen Jakob , Saegusa Noriko , Spitzer Kenneth W. , Matsuoka Nobuhiro , Sheets Michael , Rao Narayanam V. , Kennedy Thomas P.
Published: October 2009

Heparin desulfated at the 2- O and 3- O positions (ODSH) decreases canine myocardial reperfusion injury. We hypothesized that this occurs from effects on ion channels rather than solely from anti-inflammatory activities, as previously proposed. We studied closed-chest pigs with balloon left anterior descending coronary artery occlusion (75-min) and reperfusion (3-h). ODSH effects on [Na+]i(Na Green) and [Ca2+]i(Fluo-3) were measured by flow cytometry in rabbit ventricular myocytes after 45-min of simulated ischemia [metabolic inhibition with 2 mM cyanide, 0 glucose, 37°C, pacing at 0.5 Hz; i.e., pacing-metabolic inhibition (PMI)]. Na+/Ca2+exchange (NCX) activity and Na+channel function were assessed by voltage clamping. ODSH (15 mg/kg) 5 min before reperfusion significantly decreased myocardial necrosis, but neutrophil influx into reperfused myocardium was not consistently reduced. ODSH (100 μg/ml) reduced [Na+]iand [Ca2+]iduring PMI. The NCX inhibitor KB-R7943 (10 μM) or the late Na+current ( INa-L) inhibitor ranolazine (10 μM) reduced [Ca2+]iduring PMI and prevented effects of ODSH on Ca2+loading. ODSH also reduced the increase in Na+loading in paced myocytes caused by 10 nM sea anemone toxin II, a selective activator of INa-L. ODSH directly stimulated NCX and reduced INa-L. These results suggest that in the intact heart ODSH reduces Na+influx during early reperfusion, when INa-Lis activated by a burst of reactive oxygen production. This reduces Na+overload and thus Ca2+influx via NCX. Stimulation of Ca2+extrusion via NCX later after reperfusion may also reduce myocyte Ca2+loading and decrease infarct size.
Cardioprotection leads to novel changes in the mitochondrial proteome
Authors: Wong Renee , Aponte Angel M. , Steenbergen Charles , Murphy Elizabeth
Published: October 2009

It is proposed that ischemic preconditioning (PC) initiates signaling that converges on mitochondria and results in cardioprotection. The outcome of this signaling on mitochondrial enzyme complexes is yet to be understood. We therefore used proteomic methods to test the hypothesis that PC and pharmacological preconditioning similarly alter mitochondrial signaling complexes. Langendorff-perfused murine hearts were treated with the specific GSK-3 inhibitor AR-A014418 (GSK Inhib VIII) for 10 min or subjected to four cycles of 5-min ischemia-reperfusion (PC) before 20-min global ischemia and 120-min reperfusion. PC and GSK Inhib VIII both improved recovery of postischemic left ventricular developed pressure, decreased infarct size, and reduced lactate production during ischemia compared with their time-matched controls. We used proteomics to examine mitochondrial protein levels/posttranslational modifications that were common between PC and GSK Inhib VIII. Levels of cytochrome- c oxidase subunits Va and VIb, ATP synthase-coupling factor 6, and cytochrome b- c1 complex subunit 6 were increased while cytochrome c was decreased with PC and GSK Inhib VIII. Furthermore, the amount of cytochrome- c oxidase subunit VIb was found to be increased in PC and GSK Inhib VIII mitochondrial supercomplexes, which are comprised of complexes I, III, and IV. This result would suggest that changes in complex subunits associated with cardioprotection may affect supercomplex composition. Thus the ability of PC and GSK inhibition to alter the expression levels of electron transport complexes will have important implications for mitochondrial function.
A mathematical model of vasoreactivity in rat mesenteric arterioles. II. Conducted vasoreactivity
Authors: Kapela Adam , Nagaraja Sridevi , Tsoukias Nikolaos M.
Published: October 2009

This study presents a multicellular computational model of a rat mesenteric arteriole to investigate the signal transduction mechanisms involved in the generation of conducted vasoreactivity. The model comprises detailed descriptions of endothelial (ECs) and smooth muscle (SM) cells (SMCs), coupled by nonselective gap junctions. With strong myoendothelial coupling, local agonist stimulation of the EC or SM layer causes local changes in membrane potential ( Vm) that are conducted electrotonically, primarily through the endothelium. When myoendothelial coupling is weak, signals initiated in the SM conduct poorly, but the sensitivity of the SMCs to current injection and agonist stimulation increases. Thus physiological transmembrane currents can induce different levels of local Vmchange, depending on cell's gap junction connectivity. The physiological relevance of current and voltage clamp stimulations in intact vessels is discussed. Focal agonist stimulation of the endothelium reduces cytosolic calcium (intracellular Ca2+concentration) in the prestimulated SM layer. This SMC Ca2+reduction is attributed to a spread of EC hyperpolarization via gap junctions. Inositol (1,4,5)-trisphosphate, but not Ca2+, diffusion through homocellular gap junctions can increase intracellular Ca2+concentration in neighboring ECs. The small endothelial Ca2+spread can amplify the total current generated at the local site by the ECs and through the nitric oxide pathway, by the SMCs, and thus reduces the number of stimulated cells required to induce distant responses. The distance of the electrotonic and Ca2+spread depends on the magnitude of SM prestimulation and the number of SM layers. Model results are consistent with experimental data for vasoreactivity in rat mesenteric resistance arteries.
A transatrial pericardial access: lead placement as proof of concept
Authors: Kassab G. S. , Svendsen M. , Combs W. , Choy J. S. , Berbari E. J. , Navia J. A.
Published: October 2009

A safe, easy, and quick access into the pericardial space may provide a window for diagnostics and therapeutics to the heart. The objective of this study was to provide proof of concept for an engagement and access catheter that allows access to the pericardial space percutaneously. A multilumen catheter was developed to allow navigation and suction fixation to the right atrial appendage/wall in a normal swine model. Advancement through the multilumen catheter using a second catheter with a distal needle tip allows access to the pericardial space without pericardial puncture and advancement of a standard guide wire into the space. Navigation into the pericardial space was undertaken by fluoroscopy alone and was accomplished in 10 swine (5 acute and 5 chronic). As a specific application of this pericardial access method, a pacing lead was implanted on the epicardial surface. Five chronic swine experiments were conducted with successful pacing engagement verified by lead impedance and pacing threshold and sensing. Lead impedance exceeded 1,000 Ω preengagement and dropped by an average of 200 Ω upon implant (769 ± 498 Ω). Pacing thresholds at 0.4 ms ranged from ∼0.5 to 2.1 V acutely (1.03 ± 0.92 V). No cardiac effusion or tamponade was observed in any of the acute or chronic studies. The ability to engage, maintain, and retract the right atrial appendage/wall and to engage an epicardial lead was successfully demonstrated. These findings support the feasibility of safe access into the pericardial space in a normal swine model and warrant further investigations for clinical translation.