bluuebunny/crossref_metadata_2025 huggingface dataset:
Nitric Oxide Reductase Gene Expression and Nitrous Oxide Production in Nitrate-Grown
Pseudomonas mandelii
Authors: Saleh-Lakha Saleema , Shannon Kelly E. , Goyer Claudia , Trevors Jack T. , Zebarth Bernie J. , Burton David L.
Published: September 2008

Pure cultures of
Pseudomonas mandelii
were incubated with or without nitrate, which acts as a substrate and an electron acceptor for denitrification. Nitric oxide reductase (
cnorB
) gene expression was measured using a quantitative reverse transcription-PCR, and nitrous oxide emissions were measured by gas chromatography.
P. mandelii
cells in either the presence or absence of nitrate demonstrated an increase in
cnorB
gene expression during the first 3 h of growth. The level of expression of
cnorB
in nitrate-amended cells remained high (average, 2.06 × 10
8
transcripts/μg of RNA), while in untreated cells it decreased to an average of 3.63 × 10
6
transcripts/μg of RNA from 4 to 6 h. Nitrous oxide accumulation in the headspace was detected at 2 h, and cumulative emissions continued to increase over a 24-h period to 101 μmol in nitrate-amended cells.
P. mandelii cnorB
gene expression was not detected under aerobic conditions. These results demonstrate that
P. mandelii cnorB
gene expression was induced 203-fold at 4 h when nitrate was present in the medium. Accumulations of N
2
O indicated that the cNorB enzyme was synthesized and active.
csrA
Inhibits the Formation of Biofilms by
Vibrio vulnificus
Authors: Jones Melissa K. , Warner Elizabeth B. , Oliver James D.
Published: September 2008

PCR screening of the shellfish-borne pathogen
Vibrio vulnificus
revealed
csrA
-negative strains, and these strains formed increased biofilm compared to
csrA
-positive strains. Complementation in
trans
with
csrA
resulted in reduced biofilm formation, similar to that by
csrA
+
strains. Our results provide evidence that
csrA
inhibits biofilm formation in
V. vulnificus
.
UGE1
and
UGE2
Regulate the UDP-Glucose/UDP-Galactose Equilibrium in
Cryptococcus neoformans
Authors: Moyrand Frédérique , Lafontaine Ingrid , Fontaine Thierry , Janbon Guilhem
Published: September 2008

The genome of the basidiomycete pathogenic yeast
Cryptococcus neoformans
carries two UDP-glucose epimerase genes (
UGE1
and
UGE2
).
UGE2
maps within a galactose cluster composed of a galactokinase homologue gene and a galactose-1-phosphate uridylyltransferase. This clustered organization of the
GAL
genes is similar to that in most of the hemiascomycete yeast genomes and in
Schizosaccharomyces pombe
but is otherwise not generally conserved in the fungal kingdom.
UGE1
has been identified as necessary for galactoxylomannan biosynthesis and virulence. Here, we show that
UGE2
is necessary for
C. neoformans
cells to utilize galactose as a carbon source at 30°C but is not required for virulence. In contrast, deletion of
UGE1
does not affect cell growth on galactose at this temperature. At 37°C, a
uge2Δ
mutant grows on galactose in a
UGE1
-dependent manner. This compensation by
UGE1
of
UGE2
mutation for growth on galactose at 37°C was not associated with upregulation of
UGE1
transcription or with an increase of the affinity of the enzyme for UDP-galactose at this temperature. We studied the subcellular localization of the two enzymes. Whereas at 30°C, Uge1p is at least partially associated with intracellular vesicles and Uge2p is on the plasma membrane, in cells growing on galactose at 37°C, Uge1p colocalizes with Uge2p to the plasma membrane, suggesting that its activity is regulated through subcellular localization.
KMP-11, a Basal Body and Flagellar Protein, Is Required for Cell Division in
Trypanosoma brucei
Authors: Li Ziyin , Wang Ching C.
Published: September 2008

Kinetoplastid membrane protein 11 (KMP-11) has been identified as a flagellar protein and is conserved among kinetoplastid parasites, but its potential function remains unknown. In a recent study, we identified KMP-11 as a microtubule-bound protein localizing to the flagellum as well as the basal body in both procyclic and bloodstream forms of
Trypanosoma brucei
(Z. Li, J. H. Lee, F. Chu, A. L. Burlingame, A. Gunzl, and C. C. Wang, PLoS One 3:e2354, 2008). Silencing of KMP-11 by RNA interference inhibited basal body segregation and cytokinesis in both forms and resulted in multiple nuclei of various sizes, indicating a continuous, albeit somewhat defective, nuclear division while cell division was blocked. KMP-11 knockdown in the procyclic form led to severely compromised formation of the new flagellum attachment zone (FAZ) and detachment of the newly synthesized flagellum. However, a similar phenotype was not observed in the bloodstream form depleted of KMP-11. Thus, KMP-11 is a flagellar protein playing critical roles in regulating cytokinesis in both forms of the trypanosomes. Its distinct roles in regulating FAZ formation in the two forms may provide a clue to the different mechanisms of cytokinetic initiation in procyclic and bloodstream trypanosomes.
Changes in the
N
-Glycome, Glycoproteins with Asn-Linked Glycans, of
Giardia lamblia
with Differentiation from Trophozoites to Cysts
Authors: Ratner Daniel M. , Cui Jike , Steffen Martin , Moore Landon L. , Robbins Phillips W. , Samuelson John
Published: September 2008

Giardia lamblia
is present in the intestinal lumen as a binucleate, flagellated trophozoite or a quadranucleate, immotile cyst. Here we used the plant lectin wheat germ agglutinin (WGA), which binds to the disaccharide di-
N
-acetyl-chitobiose (GlcNAc
2
), which is the truncated Asn-linked glycan (N-glycan) of
Giardia
, to affinity purify the
N
-glycomes (glycoproteins with N-glycans) of trophozoites and cysts. Fluorescent WGA bound to the perinuclear membranes, peripheral acidified vesicles, and plasma membranes of trophozoites. In contrast, WGA bound strongly to membranes adjacent to the wall of
Giardia
cysts and less strongly to the endoplasmic reticulum and acidified vesicles. WGA lectin-affinity chromatography dramatically enriched secreted and membrane proteins of
Giardia
, including proteases and acid phosphatases that retain their activities. With mass spectroscopy, we identified 91 glycopeptides with N-glycans and 194 trophozoite-secreted and membrane proteins, including 42 unique proteins. The
Giardia
oligosaccharyltransferase, which contains a single catalytic subunit, preferred N glycosylation sites with Thr to those with Ser in vivo but had no preference for flanking amino acids. The most-abundant glycoproteins in the
N
-glycome of trophozoites were lysosomal enzymes, folding-associated proteins, and unique transmembrane proteins with Cys-, Leu-, or Gly-rich repeats. We identified 157 secreted and membrane proteins in the
Giardia
cysts, including 20 unique proteins. Compared to trophozoites, cysts were enriched in Gly-rich repeat transmembrane proteins, cyst wall proteins, and unique membrane proteins but had relatively fewer Leu-rich repeat proteins, folding-associated proteins, and unique secreted proteins. In summary, there are major changes in the
Giardia N
-glycome with the differentiation from trophozoites to cysts.
Deletion of the
Plasmodium falciparum
Merozoite Surface Protein 7 Gene Impairs Parasite Invasion of Erythrocytes
Authors: Kadekoppala Madhusudan , O'Donnell Rebecca A. , Grainger Munira , Crabb Brendan S. , Holder Anthony A.
Published: September 2008

Merozoite surface proteins have been implicated in the initial attachment to the host red blood cell membrane that begins the process of invasion, an important step in the life cycle of the malaria parasite. In
Plasmodium falciparum
, merozoite surface proteins include several glycosylphosphatidyl inositol-anchored proteins and peripheral proteins attached to the membrane through protein-protein interactions. The most abundant of these proteins is the merozoite surface protein 1 (MSP1) complex, encoded by at least three genes:
msp1
,
msp6
, and
msp7
. The
msp7 g
ene is part of a six-member multigene family in
Plasmodium falciparum
. We have disrupted
msp7
in the
Plasmodium falciparum
D10 parasite, as confirmed by Southern hybridization. Immunoblot and indirect immunofluorescence analyses confirmed the MSP7 null phenotype of D10ΔMSP7 parasites. The synthesis, distribution, and processing of MSP1 were not affected in this parasite line. The level of expression and cellular distribution of the proteins MSP1, MSP3, MSP6, MSP9, and SERA5 remained comparable to those for the parental line. Furthermore, no significant change in the expression of MSP7-related proteins, except for that of MSRP5, was detected at the transcriptional level. The lack of MSP7 was not lethal at the asexual blood stage, but it did impair invasion of erythrocytes by merozoites to a significant degree. Despite this reduction in efficiency, D10ΔMSP7 parasites did not show any obvious preference for alternate pathways of invasion.
Maintenance Role of a Glutathionyl-Hydroquinone Lyase (PcpF) in Pentachlorophenol Degradation by
Sphingobium chlorophenolicum
ATCC 39723
Authors: Huang Yan , Xun Randy , Chen Guanjun , Xun Luying
Published: September 2008

Pentachlorophenol (PCP) is a toxic pollutant. Its biodegradation has been extensively studied in
Sphingobium chlorophenolicum
ATCC 39723. All enzymes required to convert PCP to a common metabolic intermediate before entering the tricarboxylic acid cycle have been characterized. One of the enzymes is tetrachloro-
p
-hydroquinone (TeCH) reductive dehalogenase (PcpC), which is a glutathione (GSH)
S
-transferase (GST). PcpC catalyzes the GSH-dependent conversion of TeCH to trichloro-
p
-hydroquinone (TriCH) and then to dichloro-
p
-hydroquinone (DiCH) in the PCP degradation pathway. PcpC is susceptible to oxidative damage, and the damaged PcpC produces glutathionyl (GS) conjugates, GS-TriCH and GS-DiCH, which cannot be further metabolized by PcpC. The fate and effect of GS-hydroquinone conjugates were unknown. A putative GST gene (
pcpF
) is located next to
pcpC
on the bacterial chromosome. The
pcpF
gene was cloned, and the recombinant PcpF was purified. The purified PcpF was able to convert GS-TriCH and GS-DiCH conjugates to TriCH and DiCH, respectively. The GS-hydroquinone lyase reactions catalyzed by PcpF are rather unusual for a GST. The disruption of
pcpF
in
S. chlorophenolicum
made the mutant lose the GS-hydroquinone lyase activities in the cell extracts. The mutant became more sensitive to PCP toxicity and had a significantly decreased PCP degradation rate, likely due to the accumulation of the GS-hydroquinone conjugates inside the cell. Thus, PcpF played a maintenance role in PCP degradation and converted the GS-hydroquinone conjugates back to the intermediates of the PCP degradation pathway.
Genome Sequence of a Nephritogenic and Highly Transformable M49 Strain ofStreptococcus pyogenes
Authors: McShan W. Michael , Ferretti Joseph J. , Karasawa Tadahiro , Suvorov Alexander N. , Lin Shaoping , Qin Biafang , Jia Honggui , Kenton Steve , Najar Fares , Wu Hongmin , Scott Julie , Roe Bruce A. , Savic Dragutin J.
Published: September 2008

The 1,815,783-bp genome of a serotype M49 strain ofStreptococcus pyogenes(group A streptococcus [GAS]), strain NZ131, has been determined. This GAS strain (FCT type 3;emmpattern E), originally isolated from a case of acute post-streptococcal glomerulonephritis, is unusually competent for electrotransformation and has been used extensively as a model organism for both basic genetic and pathogenesis investigations. As with the previously sequencedS. pyogenesgenomes, three unique prophages are a major source of genetic diversity. Two clustered regularly interspaced short palindromic repeat (CRISPR) regions were present in the genome, providing genetic information on previous prophage encounters. A unique cluster of genes was found in the pathogenicity island-likeemmregion that included a novel Nudix hydrolase, and, further, this cluster appears to be specific for serotype M49 and M82 strains. Nudix hydrolases eliminate potentially hazardous materials or prevent the unbalanced accumulation of normal metabolites; in bacteria, these enzymes may play a role in host cell invasion. Since M49S. pyogenesstrains have been known to be associated with skin infections, the Nudix hydrolase and its associated genes may have a role in facilitating survival in an environment that is more variable and unpredictable than the uniform warmth and moisture of the throat. The genome of NZ131 continues to shed light upon the evolutionary history of this human pathogen. Apparent horizontal transfer of genetic material has led to the existence of highly variable virulence-associated regions that are marked by multiple rearrangements and genetic diversification while other regions, even those associated with virulence, vary little between genomes. The genome regions that encode surface gene products that will interact with host targets or aid in immune avoidance are the ones that display the most sequence diversity. Thus, while natural selection favors stability in much of the genome, it favors diversity in these regions.
Processing of a Membrane Protein Required for Cell-to-Cell Signaling during Endospore Formation in
Bacillus subtilis
Authors: Serrano Mónica , Vieira Filipe , Moran Charles P. , Henriques Adriano O.
Published: September 2008

Activation of the late prespore-specific RNA polymerase sigma factor σ
G
during
Bacillus subtilis
sporulation coincides with completion of the engulfment process, when the prespore becomes a protoplast fully surrounded by the mother cell cytoplasm and separated from it by a double membrane system. Activation of σ
G
also requires expression of
spoIIIJ
, coding for a membrane protein translocase of the YidC/Oxa1p/Alb3 family, and of the mother cell-specific
spoIIIA
operon. Here we present genetic and biochemical evidence indicating that SpoIIIAE, the product of one of the
spoIIIA
cistrons, and SpoIIIJ interact in the membrane, thereby linking the function of the
spoIIIJ
and
spoIIIA
loci in the activation of σ
G
. We also show that SpoIIIAE has a functional Sec-type signal peptide, which is cleaved during sporulation. Furthermore, mutations that reduce or eliminate processing of the SpoIIIAE signal peptide arrest sporulation following engulfment completion and prevent activation of σ
G
. SpoIIIJ-type proteins can function in cooperation with or independently of the Sec system. In one model, SpoIIIJ interacts with SpoIIIAE in the context of the Sec translocon to promote its correct localization and/or topology in the membrane, so that it can signal the activation of σ
G
following engulfment completion.
Phenotypic and Transcriptomic Characterization of
Bacillus subtilis
Mutants with Grossly Altered Membrane Composition
Authors: Salzberg Letal I. , Helmann John D.
Published: September 2008

The
Bacillus subtilis
membrane contains diacylglycerol-based lipids with at least five distinct headgroups that together help to define the physical and chemical properties of the lipid bilayer. Here, we describe the phenotypic characterization of mutant strains lacking one or more of the following lipids: glycolipids (
ugtP
mutants), phosphatidylethanolamine (
pssA
and
psd
mutants), lysylphosphatidylglycerol (
mprF
), and cardiolipin (
ywnE
and
ywjE
). Alterations of membrane lipid headgroup composition are generally well-tolerated by the cell, and even severe alterations lead to only modest effects on growth proficiency. Mutants with decreased levels of positively charged lipids display an increased sensitivity to cationic antimicrobial compounds, and cells lacking glycolipids are more sensitive to the peptide antibiotic sublancin and are defective in swarming motility. A quadruple mutant strain (
ugtP pssA mprF ywnE
), with a membrane comprised predominantly of phosphatidylglycerol, is viable and grows at near-wild-type rates, although it forms long, coiled filaments. Transcriptome comparisons identified numerous regulons with altered expression in cells of the
ugtP
mutant, the
pssA mprF ywnE
triple mutant, and the
ugtP pssA mprF ywnE
quadruple mutant. These effects included a general decrease in expression of the SigD and FapR regulons and increased expression of cell envelope stress responses mediated by σ
M
and the YvrGHb two-component system.
Tricarboxylic Acid Cycle-Dependent Regulation of
Staphylococcus epidermidis
Polysaccharide Intercellular Adhesin Synthesis
Authors: Sadykov Marat R. , Olson Michael E. , Halouska Steven , Zhu Yefei , Fey Paul D. , Powers Robert , Somerville Greg A.
Published: September 2008

Staphylococcus epidermidis
is a major nosocomial pathogen primarily infecting immunocompromised individuals or those with implanted biomaterials (e.g., catheters). Biomaterial-associated infections often involve the formation of a biofilm on the surface of the medical device. In
S. epidermidis
, polysaccharide intercellular adhesin (PIA) is an important mediator of biofilm formation and pathogenesis. Synthesis of PIA is regulated by at least three DNA binding proteins (IcaR, SarA, and σ
B
) and several environmental and nutritional conditions. Previously, we observed the environmental conditions that increased PIA synthesis decreased tricarboxylic acid (TCA) cycle activity. In this study,
S. epidermidis
TCA cycle mutants were constructed, and the function of central metabolism in PIA biosynthesis was examined. TCA cycle inactivation altered the metabolic status of
S. epidermidis
, resulting in a massive derepression of PIA biosynthetic genes and a redirection of carbon from growth into PIA biosynthesis. These data demonstrate that the bacterial metabolic status is a critical regulatory determinant of PIA synthesis. In addition, these data lead us to propose that the TCA cycle acts as a signal transduction pathway to translate external environmental cues into intracellular metabolic signals that modulate the activity of transcriptional regulators.
Frequent Homologous Recombination Events in
Mycobacterium tuberculosis
PE/PPE Multigene Families: Potential Role in Antigenic Variability
Authors: Karboul Anis , Mazza Alberto , Gey van Pittius Nicolaas C. , Ho John L. , Brousseau Roland , Mardassi Helmi
Published: September 2008

The PE and PPE (PE/PPE) multigene families of
Mycobacterium tuberculosis
are particularly GC-rich and share extensive homologous repetitive sequences. We hypothesized that they may undergo homologous recombination events, a mechanism rarely described in the natural evolution of mycobacteria. To test our hypothesis, we developed a specific oligonucleotide-based microarray targeting nearly all of the PE/PPE genes, aimed at detecting signals for homologous recombination. Such a microarray has never before been reported due to the multiplicity and highly repetitive and homologous nature of these sequences. Application of the microarray to a collection of
M. tuberculosis
clinical isolates (
n
= 33) representing prevalent spoligotype strain families in Tunisia allowed successful detection of six deleted genomic regions involving a total of two PE and seven PPE genes. Some of these deleted genes are known to be immunodominant or involved in virulence. The four precisely determined deletions were flanked by 400- to 500-bp stretches of nearly identical sequences lying mainly at the conserved N-terminal region of the PE/PPE genes. These highly homologous sequences thus serve as substrates to mediate both intergenic and intragenic homologous recombination events, indicating an important function in generating strain variation. Importantly, all recombination events yielded a new in-frame fusion chimeric gene. Hence, homologous recombination within and between PE/PPE genes likely increased their antigenic variability, which may have profound implications in pathogenicity and/or host adaptation. The finding of high prevalence (∼45% and ∼58%) for at least two of the genomic deletions suggests that they likely confer advantageous biological attributes.
Suppression of ΔbipAPhenotypes inEscherichia coliby Abolishment of Pseudouridylation at Specific Sites on the 23S rRNA
Authors: Krishnan Karthik , Flower Ann M.
Published: September 2008

The BipA protein ofEscherichia colihas intriguing similarities to the elongation factor subfamily of GTPases, including EF-Tu, EF-G, and LepA. In addition, phenotypes of abipAdeletion mutant suggest that BipA is involved in regulation of a variety of pathways. These two points have led to speculation that BipA may be a novel regulatory protein that affects efficient translation of target genes through direct interaction with the ribosome. We isolated and characterized suppressors of the cold-sensitive growth phenotype exhibited by ΔbipAstrains and identified insertion mutations inrluC. TherluCgene encodes a pseudouridine synthase responsible for pseudouridine modification of 23S rRNA at three sites, all located near the peptidyl transferase center. Deletion ofrluCnot only suppressed cold sensitivity but also alleviated the decrease in capsule synthesis exhibited bybipAmutants, suggesting that the phenotypic effects of BipA are manifested through an effect on the ribosome. The suppressor effect is specific torluC, as deletion of otherrlugenes did not relieve cold sensitivity, and further, more than a single pseudouridine residue is involved, as alteration of single residues did not produce suppressors. These results are consistent with a role for BipA in either the structure or the function of the ribosome and imply that wild-type ribosomes are dependent on BipA for efficient expression of target mRNAs and that the lack of pseudouridylation at these three sites renders the ribosomes BipA independent.
A Two-Component Regulatory System Integrates Redox State and Population Density Sensing in
Pseudomonas putida
Authors: Fernández-Piñar Regina , Ramos Juan Luis , Rodríguez-Herva José Juan , Espinosa-Urgel Manuel
Published: September 2008

A two-component system formed by a sensor histidine kinase and a response regulator has been identified as an element participating in cell density signal transduction in
Pseudomonas putida
KT2440. It is a homolog of the
Pseudomonas aeruginosa
RoxS/RoxR system, which in turn belongs to the RegA/RegB family, described in photosynthetic bacteria as a key regulatory element. In KT2440, the two components are encoded by PP_0887 (
roxS
) and PP_0888 (
roxR
), which are transcribed in a single unit. Characterization of this two-component system has revealed its implication in redox signaling and cytochrome oxidase activity, as well as in expression of the cell density-dependent gene
ddcA
, involved in bacterial colonization of plant surfaces. Whole-genome transcriptional analysis has been performed to define the
P. putida
RoxS/RoxR regulon. It includes genes involved in sugar and amino acid metabolism and the sulfur starvation response and elements of the respiratory chain (a
cbb3
cytochrome oxidase, Fe-S clusters, and cytochrome
c
-related proteins) or genes participating in the maintenance of the redox balance. A putative RoxR recognition element containing a conserved hexamer (TGCCAG) has also been identified in promoters of genes regulated by this two-component system.
Characterization of the Oxygen-Responsive NreABC Regulon of
Staphylococcus aureus
Authors: Schlag Steffen , Fuchs Stephan , Nerz Christiane , Gaupp Rosmarie , Engelmann Susanne , Liebeke Manuel , Lalk Michael , Hecker Michael , Götz Friedrich
Published: September 2008

Here, we investigate the functionality of the oxygen-responsive nitrogen regulation system NreABC in the human pathogen
Staphylococcus aureus
and evaluate its role in anaerobic gene regulation and virulence factor expression. Deletion of
nreABC
resulted in severe impairment of dissimilatory nitrate and nitrite reduction and led to a small-colony phenotype in the presence of nitrate during anaerobic growth. For characterization of the NreABC regulon, comparative DNA microarray and proteomic analyses between the wild type and
nreABC
mutant were performed under anoxic conditions in the absence and presence of nitrate. A reduced expression of virulence factors was not observed in the mutant. However, both the transcription of genes involved in nitrate and nitrite reduction and the accumulation of corresponding proteins were highly decreased in the
nreABC
mutant, which was unable to utilize nitrate as a respiratory oxidant and, hence, was forced to use fermentative pathways. These data were corroborated by the quantification of the extracellular metabolites lactate and acetate. Using an
Escherichia coli
-compatible two-plasmid system, the activation of the promoters of the nitrate and nitrite reductase operons and of the putative nitrate/nitrite transporter gene
narK
by NreBC was confirmed. Overall, our data indicate that NreABC is very likely a specific regulation system that is essential for the transcriptional activation of genes involved in dissimilatory reduction and transport of nitrate and nitrite. The study underscores the importance of NreABC as a fitness factor for
S. aureus
in anoxic environments.
The RNA-Binding Domain of Bacteriophage P22 N Protein Is Highly Mutable, and a Single Mutation Relaxes Specificity toward λ
Authors: Cocozaki Alexis I. , Ghattas Ingrid R. , Smith Colin A.
Published: September 2008

Antitermination in bacteriophage P22, a lambdoid phage, uses the arginine-rich domain of the N protein to recognize boxB RNAs in the nut site of two regulated transcripts. Using an antitermination reporter system, we screened libraries in which each nonconserved residue in the RNA-binding domain of P22 N was randomized. Mutants were assayed for the ability to complement N-deficient virus and for antitermination with P22 boxB
left
and boxB
right
reporters. Single amino acid substitutions complementing P22 N
−
virus were found at 12 of the 13 positions examined. We found evidence for defined structural roles for seven nonconserved residues, which was generally compatible with the nuclear magnetic resonance model. Interestingly, a histidine can be replaced by any other aromatic residue, although no planar partner is obvious. Few single substitutions showed bias between boxB
left
and boxB
right
, suggesting that the two RNAs impose similar constraints on genetic drift. A separate library comprising only hybrids of the RNA-binding domains of P22, λ, and φ21 N proteins produced mutants that displayed bias. P22 N
−
plaque size plotted against boxB
left
and boxB
right
reporter activities suggests that lytic viral fitness depends on balanced antitermination. A few N proteins were able to complement both λ N- and P22 N-deficient viruses, but no proteins were found to complement both P22 N- and φ21 N-deficient viruses. A single tryptophan substitution allowed P22 N to complement both P22 and λ N
−
. The existence of relaxed-specificity mutants suggests that conformational plasticity provides evolutionary transitions between distinct modes of RNA-protein recognition.
Identification of a Repressor of a Truncated Denitrification Pathway in
Moraxella catarrhalis
Authors: Wang Wei , Richardson Anthony R. , Martens-Habbena Willm , Stahl David A. , Fang Ferric C. , Hansen Eric J.
Published: September 2008

Growth of
Moraxella catarrhalis
in a biofilm resulted in marked upregulation of two open reading frames (ORFs),
aniA
and
norB
, predicted to encode a nitrite reductase and a nitric oxide reductase, respectively (W. Wang, L. Reitzer, D. A. Rasko, M. M. Pearson, R. J. Blick, C. Laurence, and E. J. Hansen, Infect. Immun. 75:4959-4971, 2007). An ORF designated
nsrR
, which was located between
aniA
and
norB
, was shown to encode a predicted transcriptional regulator. Inactivation of
nsrR
resulted in increased expression of
aniA
and
norB
in three different
M. catarrhalis
strains, as measured by both DNA microarray analysis and quantitative reverse transcriptase PCR. Provision of a wild-type
nsrR
gene in
trans
in an
nsrR
mutant resulted in decreased expression of the AniA protein. DNA microarray analysis revealed that two other ORFs (MC ORF 683 and MC ORF 1550) were also consistently upregulated in an
nsrR
mutant. Consumption of both nitrite and nitric oxide occurred more rapidly with cells of an
nsrR
mutant than with wild-type cells. However, growth of
nsrR
mutants was completely inhibited by a low level of sodium nitrite. This inhibition of growth by nitrite was significantly reversed by introduction of an
aniA
mutation into the
nsrR
mutant and was completely reversed by the presence of a wild-type
nsrR
gene in
trans
. NsrR regulation of the expression of
aniA
was sensitive to nitrite, whereas NsrR regulation of
norB
was sensitive to nitric oxide.
Nitric Oxide and Oxygen Regulate Truncated Hemoglobin Gene Expression inFrankiaStrain CcI3
Authors: Niemann James , Tisa Louis S.
Published: September 2008

TheFrankiagenome contains two truncated hemoglobin genes (hboNandhboO) whose functions remain to be determined. Nitric oxide (NO) generated by the addition of 400 μM SNAP (S-nitroso-N-acetylpenicillamine) caused a 10-fold increase inhboNgene expression but had no effect onhboOexpression. The addition of the NO scavenger, carboxy-PT10, reduced the effect of SNAP.hboOgene expression increased under low-oxygen conditions, whilehboNexpression was unaffected. These results suggest that HboN may function in protection from nitrosative stress and that HboO may act as an oxygen transport molecule for increased respiration in hypoxic environments.
Comparative Genomics of Regulation of Fatty Acid and Branched-Chain Amino Acid Utilization in Proteobacteria
Authors: Kazakov Alexey E. , Rodionov Dmitry A. , Alm Eric , Arkin Adam Paul , Dubchak Inna , Gelfand Mikhail S.
Published: September 2008

Bacteria can use branched-chain amino acids (ILV, i.e., isoleucine, leucine, valine) and fatty acids (FAs) as sole carbon and energy sources converting ILV into acetyl-coenzyme A (CoA), propanoyl-CoA, and propionyl-CoA, respectively. In this work, we used the comparative genomic approach to identify candidate transcriptional factors and DNA motifs that control ILV and FA utilization pathways in proteobacteria. The metabolic regulons were characterized based on the identification and comparison of candidate transcription factor binding sites in groups of phylogenetically related genomes. The reconstructed ILV/FA regulatory network demonstrates considerable variability and involves six transcriptional factors from the MerR, TetR, and GntR families binding to 11 distinct DNA motifs. The ILV degradation genes in gamma- and betaproteobacteria are regulated mainly by a novel regulator from the MerR family (e.g., LiuR in
Pseudomonas aeruginosa
) (40 species); in addition, the TetR-type regulator LiuQ was identified in some betaproteobacteria (eight species). Besides the core set of ILV utilization genes, the LiuR regulon in some lineages is expanded to include genes from other metabolic pathways, such as the glyoxylate shunt and glutamate synthase in
Shewanella
species. The FA degradation genes are controlled by four regulators including FadR in gammaproteobacteria (34 species), PsrA in gamma- and betaproteobacteria (45 species), FadP in betaproteobacteria (14 species), and LiuR orthologs in alphaproteobacteria (22 species). The remarkable variability of the regulatory systems associated with the FA degradation pathway is discussed from functional and evolutionary points of view.
Dipeptidyl Aminopeptidase IV fromStenotrophomonas maltophiliaExhibits Activity against a Substrate Containing a 4-Hydroxyproline Residue
Authors: Nakajima Yoshitaka , Ito Kiyoshi , Toshima Tsubasa , Egawa Takashi , Zheng Heng , Oyama Hiroshi , Wu Yu-Fan , Takahashi Eiji , Kyono Kiyoshi , Yoshimoto Tadashi
Published: September 2008

The crystal structure of dipeptidyl aminopeptidase IV fromStenotrophomonas maltophiliawas determined at 2.8-Å resolution by the multiple isomorphous replacement method, using platinum and selenomethionine derivatives. The crystals belong to space groupP43212, with unit cell parametersa=b= 105.9 Å andc= 161.9 Å. Dipeptidyl aminopeptidase IV is a homodimer, and the subunit structure is composed of two domains, namely, N-terminal β-propeller and C-terminal catalytic domains. At the active site, a hydrophobic pocket to accommodate a proline residue of the substrate is conserved as well as those of mammalian enzymes.Stenotrophomonasdipeptidyl aminopeptidase IV exhibited activity toward a substrate containing a 4-hydroxyproline residue at the second position from the N terminus. In theStenotrophomonasenzyme, one of the residues composing the hydrophobic pocket at the active site is changed to Asn611 from the corresponding residue of Tyr631 in the porcine enzyme, which showed very low activity against the substrate containing 4-hydroxyproline. The N611Y mutant enzyme was generated by site-directed mutagenesis. The activity of this mutant enzyme toward a substrate containing 4-hydroxyproline decreased to 30.6% of that of the wild-type enzyme. Accordingly, it was considered that Asn611 would be one of the major factors involved in the recognition of substrates containing 4-hydroxyproline.
The impact of urinary stress incontinence in young and middle-age women practising recreational sports activity: an epidemiological study
Authors: Salvatore S , Serati M , Laterza R , Uccella S , Torella M , Bolis P-F
Published: September 2008

Objectives:

To evaluate the prevalence of urinary stress incontinence (USI) in menstruating women practising recreational sports activity, to detect specific sports with a stronger association with urinary incontinence (UI) and to evaluate risk factors possibly related to this condition.

Design:

Epidemiological study.

Setting:

Non-competitive sports organisations in the province of Varese, Italy.

Participants:

679 women of fertile age, practising recreational sports activity.

Intervention:

Anonymous questionnaire on UI.

Main outcome measurements:

The questionnaire included questions about patients’ general characteristics, occurrence of UI in relation to sport or daily general activities, time of onset of this condition, frequency of leakage episodes, correlation of incontinence with types of movements or sports, subjective impression of being limited on such occasions and/or necessity to modify the type of sport.

Results:

UI was reported by 101 women (14.9%). Of these, 32 (31.7%) complained of UI only during sports activity, 48 (47.5%) only during daily life and 21 (20.8%) in both circumstances. Body mass index and parity were significantly associated with the risk of UI. Looking at the different sports activities, a higher rate of incontinence was found in women participating in basketball (16.6%), athletics (15%), and tennis or squash (11%). 10.4% of women abandoned their favourite sport, because of USI, and a further 20% limited the way they practised their favourite sport to reduce leakage episodes.

Conclusions:

Female UI affects a significant proportion of young women practising non-competitive sports activity; it can cause abandonment of the sport or limitation of its practice.
A clinical prediction rule for identifying patients with patellofemoral pain who are likely to benefit from foot orthoses: a preliminary determination
Authors: Vicenzino Bill , Collins Natalie , Cleland Joshua , McPoil Thomas
Published: September 2008

Objective

To develop a clinical prediction rule to identify patients with patellofemoral pain (PFP) who are more likely to benefit from foot orthoses.

Design

Posthoc analysis of one treatment arm of a randomised clinical trial.

Setting

Single-centre trial in a community setting in Brisbane, Australia.

Participants

42 participants (mean age 27.9 years) with a clinical diagnosis of PFP (median duration 36 months).

Interventions

Foot orthoses fitted by a physiotherapist.

Main Outcome Measures

Five-point global improvement scale at 12-week follow-up, dichotomised with marked improvement equalling success.

Results

Potential predictor variables identified by univariate analyses were age, height, pain severity, anterior knee pain scale score, functional index questionnaire score, foot morphometry (arch height ratio, mid-foot width difference from non-weight bearing to weight bearing) and overall orthoses comfort. Parsimonious fitting of these variables to a model that explained success with orthoses identified the following: age (>25 years), height (<165 cm), worst pain visual analogue scale (<53.25 mm) and a difference in mid-foot width from non-weight bearing to weight bearing (>10.96 mm). The pretest success rate of 40% increased to 86% if the patient exhibited three of these variables (positive likelihood ratio 8.8; 95% CI 1.2 to 66.9).

Conclusion

Post-hoc analysis identified age, height, pain severity and mid-foot morphometry as possible predictors of successful treatment of PFP with foot orthoses, thereby providing practitioners with information for prescribing foot orthoses in PFP and stimulating further research.
Homing of Atlantic Herring (Clupea harengus harengus) in Newfoundland Waters as Indicated by Tagging Data
Authors: Wheeler J. P. , Winters G. H.
Published: September 2008

An extensive tagging program was carried out on the herring populations along the east coast of Newfoundland during the 1970s as a basis for defining stock management units. The resulting tag recapture data have been examined in relation to the hypothesis that the majority of Atlantic herring (Clupea harengus harengus) return to the same area to spawn in successive years. Our analyses support this hypothesis and indicate that homing rates for Atlantic herring are high (approximately 90%). We concluded that herring fisheries restricted to spawning fish would allow a more rational distribution of fishing effort such that individual spawning stocks could be exploited in varying fashion according to the desired management objectives.
Recycling of Elements Transported Upstream by Runs of Pacific Salmon: II. δ15N and δ13C Evidence in the Kvichak River Watershed, Bristol Bay, Southwestern Alaska
Authors: Kline Jr. Thomas C. , Goering John J. , Mathisen Ole A. , Poe Patrick H. , Parker Patrick L. , Scalan Richard S.
Published: September 2008

Biota δ15N and δ13C values (deviations from recognized isotope standards) from lliamna Lake (a major anadromous sockeye salmon (Oncorhynchus nerka) nursery lake supporting peak-year runs > 10 million) and several other anadromous-salmon-free lakes in the Kvichak River watershed, Bristol Bay, southwestern Alaska, were compared to determine the significance of marine-derived nitrogen (MDN) delivered by returning adult salmon. Biota in lliamna Lake had higher δ15N compared with control lakes, verifying a mixing model correlating δ15N with MDN. Periphyton δ15N values reflected localized input from populations of spawning salmon. Juvenile sockeye MDN varied in response to escapement size, suggesting the importance of large escapements (> 10 million) for maintaining a predominantly MDN lacustrine N pool. Other resident fishes showed shifts in δ15N between years of high and low escapement. The dual-isotope approach, using δ15N and δ13C together, suggested that fish production is primarily dependent on limnetic primary and secondary production. The dual-isotope approach indicated that the coast range sculpin (Cottus aleuticus) was the only fish with an appreciable dietary component consisting of salmon eggs or emergent fry.
Geographic Variation in the Spawning of Atlantic Cod, Gadus morhua, in the Northwest Atlantic
Authors: Hutchings Jeffrey A. , Myers Ransom A. , Lilly George R.
Published: September 2008

We analyzed 47 yr (1946–92) of research trawl data and 5 yr (1964–68) of research gillnet data to identify spawning locations of Atlantic cod, Gadus morhua, in the Newfoundland–Labrador region. Offshore, spawners are common on the continental shelf but generally rare on the slope. Relative abundance of spawning individuals on the shelf is comparably high off northeast Newfoundland, within 100 km of the Newfoundland coast from Cape Freels to Cape Race, on central Grand Bank, and on St. Pierre Bank. Slope spawning is largely restricted to the eastern slope of Hamilton Bank, a small section of northern Grand Bank, and Flemish Cap. Inshore spawning is evident in southeastern Labrador and southeastern Newfoundland, particularly in St. Mary's, Placentia, Trinity, and Bonavista bays. Trajectories of satellite-tracked drifter buoys indicate that it is highly improbable that eggs spawned on the slope of Grand Bank and much of the northeastern Newfoundland slope will be transported into shelf and coastal waters. We conclude that cod spawn in areas in which their eggs and larvae are likely to be retained and that inshore spawning populations may provide a considerably larger contribution to cod recruitment in coastal Newfoundland than has previously been believed.
Nitrite consumption in ischemic rat heart catalyzed by distinct blood-borne and tissue factors
Authors: McNulty Patrick H. , Scott Sophia , Kehoe Valerie , Kozak Mark , Sinoway Lawrence I. , Li Jinhua
Published: September 2008

Nitric oxide (NO) may limit myocardial ischemia-reperfusion injury by slowing the mitochondrial metabolism. We examined whether rat heart contains catalysts potentially capable of reducing nitrite to NO during an episode of regional myocardial ischemia produced by temporary coronary artery occlusion. In intact Sprague-Dawley rats, a 15-min coronary occlusion lowered the nitrite concentration of the myocardial regions exhibiting ischemic glucose metabolism to ∼50% that of nonischemic regions (185 ± 223 vs. 420 ± 203 nmol/l). Nitrite was rapidly repleted during subsequent reperfusion. The heart tissue tested in vitro acquired a substantial ability to consume nitrite when made hypoxic at neutral pH, and this ability was slightly enhanced by simultaneously lowering the pH to 5.5. More than 70% of this activity could be abolished by flushing the coronary circulation with crystalloid to remove trapped erythrocytes. Correspondingly, erythrocytes demonstrated the ability to reduce exogenous nitrite to NO under hypoxic conditions in vitro. In erythrocyte-free heart tissue, the nitrite consumption increased fivefold when the pH was lowered to 5.5. Approximately 40% of this pH-sensitive increase in nitrite consumption could be blocked by the xanthine oxidoreductase inhibitor allopurinol, whereas lowering the Po2sufficiently to desaturate myoglobin accelerated it further. We conclude that rat heart contains several factors capable of catalyzing ischemic nitrite reduction; the most potent is contained within erythrocytes and activated by hypoxia, whereas the remainder includes xanthine oxidoreductase and other pH-sensitive factors endogenous to heart tissue, including deoxymyoglobin.
Phasic hemodynamics and reverse blood flows in the aortic isthmus and pulmonary arteries of preterm lambs with pulmonary vascular dysfunction
Authors: Rolland Pierre-Henri , de Lagausie Pascal , Stathopoulos Euletheris , Leprètre Olivier , Viudes Gilles , Gorincour Guillaume , Hery Géraldine , de Magnée Catherine , Paut Olivier , Guys Jean-Michel
Published: September 2008

Time-domain representations of the fetal aortopulmonary circulation were carried out in lamb fetuses to study hemodynamic consequences of congenital diaphragmatic hernia (CDH) and the effects of endothelin-receptor antagonist tezosentan (3 mg/45 min). From the isthmic aortic and left pulmonary artery (PA) flows (Q) and isthmic aortic, PA, and left auricle pressures (P) on day 135 in 10 controls and 7 CDH fetuses (28 ewes), discrete-triggered P and Q waveforms were modelized as Ptand Qtfunctions to obtain basic hemodynamic profiles, pulsatile waves [P, Q, and entry impedance ( Ze)], and P and Q hysteresis loops. In the controls, blood propelling energy was accounted for by biventricular ejection flow waves (kinetic energy) with low Zeand by flow-driven pressure waves (potential energy) with low Ze. Weak fetal pulmonary perfusion was ensured by reflux (reverse flows) from PA branches to the ductus anteriosus and aortic isthmus as reverse flows. Endothelin-receptor antagonist blockade using tezosentan slightly increased the forward flow but largely increased diastolic backward flow with a diminished left auricle pre- and postloading. In CHD fetuses, the static component overrode phasic flows that were detrimental to reverse flows and the direction of the diastolic isthmic flow changed to forward during the diastole period. Decreased cardiac output, flattened pressure waves, and increased forward Zepromoted backward flow to the detriment of forward flow (especially during diastole). Additionally, the intrapulmonary arteriovenous shunting was ineffective. The slowing of cardiac output, the dampening of energetic pressure waves and pulsatility, and the heightening of phasic impedances contributed to the lowering of aortopulmonary blood flows. We speculate that reverse pulmonary flow is a physiological requirement to protect the fetal pulmonary circulation from the prominent right ventricular stream and to enhance blood flow to the fetal heart and brain.
Aging and prostacyclin responses in aorta and platelets from WKY and SHR rats
Authors: Gomez Elodie , Schwendemann Cédric , Roger Séverine , Simonet Serge , Paysant Jerôme , Courchay Christine , Verbeuren Tony J. , Félétou Michel
Published: September 2008

In spontaneously hypertensive rat (SHR) aorta, prostacyclin is an endothelium-derived contracting factor contributing to the endothelial dysfunction. This study was designed to determine whether the impairment of the prostacyclin response is influenced by aging and whether such a dysfunction is observed in platelets. Isometric tension was measured in aortic rings, and aggregation was studied in platelet-rich plasma taken from 3-, 6-, and 15-mo-old Wistar-Kyoto rats (WKY) and SHR. In aorta from 3- and 6-mo-old WKY, prostacyclin and beraprost [prostacyclin receptor (IP) agonists] produced relaxations that were enhanced by Triplion (thromboxane-prostanoid receptor antagonist). In 15-mo-old WKY, the relaxations to beraprost were maintained, but not those to prostacyclin. In SHR aorta, prostacyclin or beraprost produced no or minor relaxations, which, in younger SHR, were enhanced by Triplion. In both strains, the relaxations were inhibited by CAY-10441 (IP receptor antagonist). The relaxations to forskolin and isoproterenol were reduced with aging. When compared with those of WKY, the relaxations to isoproterenol were reduced in 3- but not in 6- or 15-mo-old SHR, whereas those to forskolin were consistently diminished at any given age. Whatever the age, prostacyclin and beraprost produced CAY-10441-sensitive inhibitions of ADP-induced platelet aggregation. Both agonists were more potent in SHR than in WKY. Therefore, in platelets from WKY and SHR, the IP receptor-dependent antiaggregant response is functional and maintained during aging. In aorta from WKY those responses are reduced by aging and, in SHR, are already compromised at 3 mo. This dysfunction of the IP receptor is only partially explained by a general dysfunction of the adenylate cyclase pathway.
LXR-induced reverse cholesterol transport in human airway smooth muscle is mediated exclusively by ABCA1
Authors: Delvecchio Christopher J. , Bilan Patricia , Nair Parameswaran , Capone John P.
Published: September 2008

The association of hypercholesterolemia and obesity with airway hyperresponsiveness has drawn increasing attention to the potential role of cholesterol and lipid homeostasis in lung physiology and in chronic pulmonary diseases such as asthma. We have recently shown that activation of the nuclear hormone receptor liver X receptor (LXR) stimulates cholesterol efflux in human airway smooth muscle (hASM) cells and induces expression of the ATP-binding cassette (ABC) transporters ABCA1 and ABCG1, members of a family of proteins that mediate reverse cholesterol and phospholipid transport. We show here that ABCA1 is responsible for all LXR-mediated cholesterol and phospholipid efflux to both apolipoprotein AI and high-density lipoprotein acceptors. In contrast, ABCG1 does not appear to be required for this process. Moreover, we show that hASM cells respond to increased levels of cholesterol by inducing expression of ABCA1 and ABCG1 transporters, a process that is dependent on LXR expression. These findings establish a critical role for ABCA1 in reverse cholesterol and phospholipid transport in airway smooth muscle cells and suggest that dysregulation of cholesterol homeostasis in these cells may be important in the pathogenesis of diseases such as asthma.