bluuebunny/crossref_metadata_2025 huggingface dataset:
Mammalian urea transporters
Authors: Smith Craig P.
Published: November 2008

Urea transporters (UTs) encoded by the Slc14a1 (UT‐B) and Slc14a2 (UT‐A) genes mediate urea flux across cellular membranes. Considerable research has accrued detailing the function and distribution of members of both subfamilies. Much research effort has focused on the kidney, where UTs are highly expressed and function to promote urine concentration. Interestingly, UTs are also expressed in several other tissues that are historically not primarily associated with urea metabolism. In this review, I describe the phenotypes of UT knockout and transgenic mice and highlight the major advances made possible by use of these animal models. Where pertinent, I contrast these findings with known human phenotypes associated with UT mutations.
Chronic intermittent hypoxia and acetaminophen induce synergistic liver injury in mice
Authors: Savransky Vladimir , Reinke Christian , Jun Jonathan , Bevans‐Fonti Shannon , Nanayakkara Ashika , Li Jianguo , Myers Allen C. , Torbenson Michael S. , Polotsky Vsevolod Y.
Published: November 2008

Obstructive sleep apnoea (OSA) leads to chronic intermittent hypoxia (CIH) during sleep. Obstructive sleep apnoea has been associated with liver injury. Acetaminophen (APAP; known as paracetamol outside the USA) is one of the most commonly used drugs which has known hepatotoxicity. The goal of the present study was to examine whether CIH increases liver injury, hepatic oxidative stress and inflammation induced by chronic APAP treatment. Adult C57BL/6J mice were exposed to CIH or intermittent air (IA) for 4 weeks. Mice in both groups were treated with intraperitoneal injections of either APAP (200 mg kg−1) or normal saline daily. A combination of CIH and APAP caused liver injury, with marked increases in serum alanine aminotransferase, aspartate aminotransferase (AST), γ‐glutamyl transferase and total bilirubin levels, whereas CIH alone induced only elevation in serum AST levels. Acetaminophen alone did not affect serum levels of liver enzymes. Histopathology revealed hepatic necrosis and increased apoptosis in mice exposed to CIH and APAP, whereas the liver remained intact in all other groups. Mice exposed to CIH and APAP exhibited decreased hepatic glutathione in conjunction with a fivefold increase in nitrotyrosine levels, suggesting formation of toxic peroxynitrite in hepatocytes. Acetaminophen or CIH alone had no effect on either glutathione or nitrotyrosine. A combination of CIH and APAP caused marked increases in pro‐inflammatory chemokines, monocyte chemoattractant protein‐1 and macrophage inflammatory protein‐2, which were not observed in mice exposed to CIH or APAP alone. We conclude that CIH and chronic APAP treatment lead to synergistic liver injury, which may have clinical implications for patients with OSA.
Use‐dependent learning and memory of the Hering–Breuer inflation reflex in rats
Authors: MacDonald Shawna M. , Tin Chung , Song Gang , Poon Chi‐Sang
Published: November 2008

The classic Hering–Breuer inflation reflex (HBIR) is a widely held tenet for understanding the lung volume‐related vagal control of respiratory rhythm. Recent evidence, however, has revealed that the fictive HBIR elicited by electrical vagal stimulation in rats is not static but may be attenuated centrally by two forms of non‐associative learning (habituation and desensitization) that continually mitigate the reflex effects with exponential adaptations like a differentiator or high‐pass filter. Desensitization is analogous to habituation but exhibits an explicit short‐term memory (STM) in the form of a rebound response with exponential decay during recovery from stimulation. To investigate whether such learning and memory effects are lung volume related and use‐dependent (practice makes perfect), we compared the time‐dependent changes in inspiratory and expiratory durations (tI and tE) during and after 1 or 8 min unilateral lung inflation or high‐frequency, low‐intensity vagal stimulation in anaesthetized, uni‐ or bi‐vagotomized rats. Unilateral lung inflation and vagal stimulation both elicited abrupt shortening of tI and lengthening of tE (HBIR effects) and gradual habituation and desensitization throughout the 1 or 8 min test period, followed by rebound responses in tI and tE with exponential recovery (STM effects) in the post‐test period. In both cases, the STM time constants for tI and tE were significantly longer with the 8 min test than with the 1 min test (17–45 versus 4–11 s, P < 0.01). We conclude that the HBIR and its central habituation and desensitization are mediated peripherally by lung volume‐related vagal afferents, and that the STM of desensitization is use‐dependent. The translational implications of these findings are discussed.
Structural and Molecular Basis of the Role of Starch and Sucrose in
Streptococcus mutans
Biofilm Development
Authors: Klein M. I. , Duarte S. , Xiao J. , Mitra S. , Foster T. H. , Koo H.
Published: November 2008

The interaction of sucrose and starch with bacterial glucosyltransferases and human salivary amylase may enhance the pathogenic potential of
Streptococcus mutans
within biofilms by influencing the structural organization of the extracellular matrix and modulating the expression of genes involved in exopolysaccharide synthesis and specific sugar transport and two-component systems.
Differential Effects of
Bifidobacterium pseudolongum
Strain Patronus and Metronidazole in the Rat Gut
Authors: Vasquez Nadia , Suau Antonia , Magne Fabien , Pochart Philippe , Pélissier Marie-Agnès
Published: November 2008

In the luminal contents of metronidazole-treated rats, there was a dominant
Bifidobacterium
species. A strain has been isolated, its 16S rRNA gene has been sequenced, and the strain has been named
Bifidobacterium pseudolongum
strain Patronus. In this study, using an experimental model of healthy rats, the effects of metronidazole treatment and
B. pseudolongum
strain Patronus administration on the luminal and mucosa-associated microbiota and on gut oxidation processes were investigated. Metronidazole treatment and the daily gavage of rats with
B. pseudolongum
strain Patronus increased the numbers of bifidobacteria in cecal contents and in cecal mucosa-associated microbiota compared with those in control rats. Metronidazole reduced the colonic oxidative damage to proteins. This is the first evidence that
B. pseudolongum
strain Patronus exerts an effect on a biomarker of oxidative damage by reducing the susceptibility to oxidation of proteins in the colon and the small bowel. Antioxidant effects of metronidazole could be linked to the bifidobacterial increase but also to other bacterial modifications.
Comparative Proteomic Analysis of
Listeria monocytogenes
Strains F2365 and EGD
Authors: Donaldson Janet R. , Nanduri Bindu , Burgess Shane C. , Lawrence Mark L.
Published: November 2008

Listeria monocytogenes
is a gram-positive, food-borne pathogen that causes disease in both humans and animals. There are three major genetic lineages of
L. monocytogenes
and 13 serovars. To further our understanding of the differences that exist between different genetic lineages/serovars of
L. monocytogenes
, we analyzed the global protein expression of the serotype 1/2a strain EGD and the serotype 4b strain F2365 during early-stationary-phase growth at 37°C. Using multidimensional protein identification technology with electrospray ionization tandem mass spectrometry, we identified 1,754 proteins from EGD and 1,427 proteins from F2365, of which 1,077 were common to both. Analysis of proteins that had significantly altered expression between strains revealed potential biological differences between these two
L. monocytogenes
strains. In particular, the strains differed in expression of proteins involved in cell wall physiology and flagellar biosynthesis, as well as DNA repair proteins and stress response proteins.
Detection and Characterization of Waterborne Gastroenteritis Viruses in Urban Sewage and Sewage-Polluted River Waters in Caracas, Venezuela
Authors: Rodríguez-Díaz J. , Querales L. , Caraballo L. , Vizzi E. , Liprandi F. , Takiff H. , Betancourt W. Q.
Published: November 2008

The detection and molecular characterization of pathogenic human viruses in urban sewage have been used extensively to derive information on circulating viruses in given populations throughout the world. In this study, a similar approach was applied to provide an overview of the epidemiology of waterborne gastroenteritis viruses circulating in urban areas of Caracas, the capital city of Venezuela in South America. Dry season sampling was conducted in sewers and in a major river severely polluted with urban sewage discharges. Nested PCR was used for detection of human adenoviruses (HAds), while reverse transcription plus nested or seminested PCR was used for detection of enteroviruses (HuEVs), rotaviruses (HRVs), noroviruses (HuNoVs), and astroviruses (HAstVs). HRVs were fully characterized with genotype-specific primers for VP4 (genotype P), VP7 (genotype G), and the rotavirus nonstructural protein 4 (NSP4). HuNoVs and HAstVs were characterized by sequencing and phylogenetic analysis. The detection rates of all viruses were ≥50%, and all sampling events were positive for at least one of the pathogenic viruses studied. The predominant HRV types found were G1, P[8], P[4], and NSP4A and -B. Genogroup II of HuNoVs and HAstV type 8 were frequently detected in sewage and sewage-polluted river waters. This study reveals relevant epidemiological data on the distribution and persistence of human pathogenic viruses in sewage-polluted waters and addresses the potential health risks associated with transmission of these viruses through water-related environmental routes.
Production of Pyomelanin, a Second Type of Melanin, via the Tyrosine Degradation Pathway in
Aspergillus fumigatus
Authors: Schmaler-Ripcke Jeannette , Sugareva Venelina , Gebhardt Peter , Winkler Robert , Kniemeyer Olaf , Heinekamp Thorsten , Brakhage Axel A.
Published: November 2008

Aspergillus fumigatus
is the most important airborne fungal pathogen of immunosuppressed humans.
A. fumigatus
is able to produce dihydroxynaphthalene melanin, which is predominantly present in the conidia. Its biosynthesis is an important virulence determinant. Here, we show that
A. fumigatus
is able to produce an alternative melanin, i.e., pyomelanin, by a different pathway, starting from
l
-tyrosine. Proteome analysis indicated that the
l
-tyrosine degradation enzymes are synthesized when the fungus is grown with
l
-tyrosine in the medium. To investigate the pathway in detail, we deleted the genes encoding essential enzymes for pigment production, homogentisate dioxygenase (
hmgA
) and 4-hydroxyphenylpyruvate dioxygenase (
hppD
). Comparative Fourier transform infrared spectroscopy of synthetic pyomelanin and pigment extracted from
A. fumigatus
cultures confirmed the identity of the observed pigment as pyomelanin. In the
hmgA
deletion strain, HmgA activity was abolished and the accumulation of homogentisic acid provoked an increased pigment formation. In contrast, homogentisic acid and pyomelanin were not observed with an
hppD
deletion mutant. Germlings of the
hppD
deletion mutant showed an increased sensitivity to reactive oxygen intermediates. The transcription of both studied genes was induced by
l
-tyrosine. These results confirmed the function of the deleted genes and the predicted pathway in
A. fumigatus
. Homogentisic acid is the major intermediate, and the
l
-tyrosine degradation pathway leading to pyomelanin is similar to that in humans leading to alkaptomelanin.
Regulatory and DNA Repair Genes Contribute to the Desiccation Resistance of
Sinorhizobium meliloti
Rm1021
Authors: Humann Jodi L. , Ziemkiewicz Hope T. , Yurgel Svetlana N. , Kahn Michael L.
Published: November 2008

Sinorhizobium meliloti
can form a nitrogen-fixing symbiotic relationship with alfalfa after bacteria in the soil infect emerging root hairs of the growing plant. To be successful at this, the bacteria must be able to survive in the soil between periods of active plant growth, including when conditions are dry. The ability of
S. meliloti
to withstand desiccation has been known for years, but genes that contribute to this phenotype have not been identified. Transposon mutagenesis was used in combination with novel screening techniques to identify four desiccation-sensitive mutants of
S. meliloti
Rm1021. DNA sequencing of the transposon insertion sites identified three genes with regulatory functions (
relA
,
rpoE2
, and
hpr
) and a DNA repair gene (
uvrC
). Various phenotypes of the mutants were determined, including their behavior on several indicator media and in symbiosis. All of the mutants formed an effective symbiosis with alfalfa. To test the hypothesis that UvrC-related excision repair was important in desiccation resistance,
uvrA
,
uvrB
, and
uvrC
deletion mutants were also constructed. These strains were sensitive to DNA damage induced by UV light and 4-NQO and were also desiccation sensitive. These data indicate that
uvr
gene-mediated DNA repair and the regulation of stress-induced pathways are important for desiccation resistance.
GzSNF1Is Required for Normal Sexual and Asexual Development in the AscomyceteGibberella zeae
Authors: Lee Seung-Ho , Lee Jungkwan , Lee Seunghoon , Park Eun-Hee , Kim Ki-Woo , Kim Myoung-Dong , Yun Sung-Hwan , Lee Yin-Won
Published: November 2008

The sucrose nonfermenting 1 (SNF1) protein kinase of yeast plays a central role in the transcription of glucose-repressible genes in response to glucose starvation. In this study, we deleted an ortholog ofSNF1fromGibberella zeaeto characterize its functions by using a gene replacement strategy. The mycelial growth of deletion mutants (ΔGzSNF1) was reduced by 21 to 74% on diverse carbon sources. The virulence of ΔGzSNF1mutants on barley decreased, and the expression of genes encoding cell-wall-degrading enzymes was reduced. The most distinct phenotypic changes were in sexual and asexual development. ΔGzSNF1mutants produced 30% fewer perithecia, which matured more slowly, and asci that contained one to eight abnormally shaped ascospores. Mutants in which only the GzSNF1catalytic domain was deleted had the same phenotype changes as the ΔGzSNF1strains, but the phenotype was less extreme in the mutants with the regulatory domain deleted. In outcrosses between the ΔGzSNF1mutants, each perithecium contained ∼70% of the abnormal ascospores, and ∼50% of the asci showed unexpected segregation patterns in a single locus tested. The asexual spores of the ΔGzSNF1mutants were shorter and had fewer septa than those of the wild-type strain. The germination and nucleation of both ascospores and conidia were delayed in ΔGzSNF1mutants in comparison with those of the wild-type strain. GzSNF1 expression and localization depended on the developmental stage of the fungus. These results suggest that GzSNF1is critical for normal sexual and asexual development in addition to virulence and the utilization of alternative carbon sources.
Trypanosome Spliced-Leader-Associated RNA (SLA1) Localization and Implications for Spliced-Leader RNA Biogenesis
Authors: Hury Avraham , Goldshmidt Hanoch , Tkacz Itai Dov , Michaeli Shulamit
Published: November 2008

Spliced-leader-associated RNA (SLA1) guides the pseudouridylation at position −12 (relative to the 5′ splice site) of the spliced-leader (SL) RNA in all trypanosomatid species. Nevertheless, the exact role of this RNA is currently unknown. Here, we demonstrate that the absence of pseudouridine onLeptomonas collosomaSL RNA has only a minor effect on the ability of this RNA to function intranssplicing in vivo. To investigate the possible role of SLA1 during SL RNA biogenesis, the structure of the SL RNA was examined in permeableTrypanosoma bruceicells depleted for CBF5, the H/ACA pseudouridine synthase, lacking SLA1. Our results suggest that in the absence of SLA1, the SL RNA secondary structure is changed, as was detected by differential sensitivity to oligonucleotide-directed RNase H cleavage, suggesting that the association of SLA1 maintains the SL RNA in a structural form which is distinct from the structure of the SL RNA in the steady state. InT. bruceicells depleted for the SL RNA core protein SmD1, SL RNA first accumulates in large amounts in the nucleus and then is expelled to the cytoplasm. Here, we demonstrate by in vivo aminomethyltrimethyl UV cross-linking studies that under SmD1 depletion, SLA1 remains bound to SL RNA and escorts the SL RNA to the cytoplasm. In situ hybridization with SLA1 and SL RNA demonstrates colocalization between SLA1 and the SL RNA transcription factor tSNAP42, as well as with Sm proteins, suggesting that SLA1 associates with SL RNA early in its biogenesis. These results demonstrate that SLA1 is a unique chaperonic RNA that functions during the early biogenesis of SL RNA to maintain a structure that is most probably suitable for cap 4 modification.
Growth Conditions Regulate the Requirements forCaulobacterChromosome Segregation
Authors: Shebelut Conrad W. , Jensen Rasmus B. , Gitai Zemer
Published: November 2008

Growth environments are important metabolic and developmental regulators. Here we demonstrate a growth environment-dependent effect onCaulobacterchromosome segregation of a small-molecule inhibitor of the MreB bacterial actin cytoskeleton. Our results also implicate ParAB as important segregation determinants, suggesting that multiple distinct mechanisms can mediateCaulobacterchromosome segregation and that their relative contributions can be environmentally regulated.
The Mycobacteriophage D29 Gene
65
Encodes an Early-Expressed Protein That Functions as a Structure-Specific Nuclease
Authors: Giri Nabanita , Bhowmik Priyanka , Bhattacharya Bidisha , Mitra Mahashweta , Das Gupta Sujoy K.
Published: November 2008

The genomes of mycobacteriophages of the L5 family, which includes the lytic phage D29, contain several genes putatively linked to DNA synthesis. One such gene is
65
, which encodes a protein belonging to the RecA/DnaB helicase superfamily. In this study a recombinant version of the mycobacteriophage D29 gp65 was functionally characterized. The results indicated that it is not a helicase as predicted but an exonuclease that removes 3′ arms from forked structures in an ATP-dependent manner. The gp65 exonuclease acts progressively from the 3′ end, until the fork junction is reached. As it goes past, its progress is stalled over a stretch of seven to eight nucleotides immediately downstream of the junction. It efficiently acts on forked structures with single stranded arms. It also acts upon 5′ and 3′ flaps, though with somewhat relaxed specificity, but not on double-stranded forks. Sequence comparison revealed the presence of a KNRXG motif in the C-terminal half of the protein. This is a conserved element found in the RadA/Sms family of DNA repair proteins. A mutation (R203G) in this motif led to complete loss of nuclease activity. This indicated that KNRXG plays an important role in the nuclease function of not only gp65, but possibly other RadA/Sms family proteins as well. This is the first characterization of a bacteriophage-derived RadA/Sms class protein. Given its mode of action, it is very likely that gp65 is involved in processing branched replication intermediates formed during the replication of phage DNA.
The
Pseudomonas aeruginosa pfpI
Gene Plays an Antimutator Role and Provides General Stress Protection
Authors: Rodríguez-Rojas Alexandro , Blázquez Jesús
Published: November 2008

Hypermutator
Pseudomonas aeruginosa
strains, characterized by an increased spontaneous-mutation rate, are found at high frequencies in chronic lung infections. Hypermutability is associated with the loss of antimutator genes related to DNA repair or damage avoidance systems. Only a few antimutator genes have been described in
P. aeruginosa
, although there is some evidence that additional genes may be involved in naturally occurring hypermutability. In order to find new
P. aeruginosa
antimutator genes, we constructed and screened a library of random insertions in the PA14 strain. Some previously described
P. aeruginosa
and/or
Escherichia coli
antimutator genes, such as
mutS
,
mutL
,
uvrD
,
mutT
,
ung
, and
mutY
, were detected, indicating a good coverage of our insertional library. One additional mutant contained an insertion in the
P. aeruginosa
PA14-04650 (
pfpI
) gene, putatively encoding a member of the DJ-1/ThiJ/PfpI superfamily, which includes chaperones, peptidases, and the Parkinson's disease protein DJ-1a. The
pfpI
-defective mutants in both PAO1 and PA14 showed higher spontaneous mutation rates than the wild-type strains, suggesting that PfpI plays a key role in DNA protection under nonstress conditions. Moreover, the inactivation of
pfpI
resulted in a dramatic increase in the H
2
O
2
-induced mutant frequency. Global transcription studies showed the induction of bacteriophage Pf1 genes and the repression of genes related to iron metabolism, suggesting that the increased spontaneous-mutant frequency may be due to reduced protection against the basal level of reactive oxygen species. Finally,
pfpI
mutants are more sensitive to different types of stress and are affected in biofilm formation.
Glycerol Metabolism Is Important for Cytotoxicity of
Mycoplasma pneumoniae
Authors: Hames Claudine , Halbedel Sven , Hoppert Michael , Frey Joachim , Stülke Jörg
Published: November 2008

Glycerol is one of the few carbon sources that can be utilized by
Mycoplasma pneumoniae
. Glycerol metabolism involves uptake by facilitated diffusion, phosphorylation, and the oxidation of glycerol 3-phosphate to dihydroxyacetone phosphate, a glycolytic intermediate. We have analyzed the expression of the genes involved in glycerol metabolism and observed constitutive expression irrespective of the presence of glycerol or preferred carbon sources. Similarly, the enzymatic activity of glycerol kinase is not modulated by HPr-dependent phosphorylation. This lack of regulation is unique among the bacteria for which glycerol metabolism has been studied so far. Two types of enzymes catalyze the oxidation of glycerol 3-phosphate: oxidases and dehydrogenases. Here, we demonstrate that the enzyme encoded by the
M. pneumoniae glpD
gene is a glycerol 3-phosphate oxidase that forms hydrogen peroxide rather than NADH
2
. The formation of hydrogen peroxide by GlpD is crucial for cytotoxic effects of
M. pneumoniae
. A
glpD
mutant exhibited a significantly reduced formation of hydrogen peroxide and a severely reduced cytotoxicity. Attempts to isolate mutants affected in the genes of glycerol metabolism revealed that only the
glpD
gene, encoding the glycerol 3-phosphate oxidase, is dispensable. In contrast, the
glpF
and
glpK
genes, encoding the glycerol facilitator and the glycerol kinase, respectively, are essential in
M. pneumoniae
. Thus, the enzymes of glycerol metabolism are crucial for the pathogenicity of
M. pneumoniae
but also for other essential, yet-to-be-identified functions in the
M. pneumoniae
cell.
Functional Analysis of Three Sulfide:Quinone Oxidoreductase Homologs in
Chlorobaculum tepidum
Authors: Chan Leong-Keat , Morgan-Kiss Rachael M. , Hanson Thomas E.
Published: November 2008

Sulfide:quinone oxidoreductase (SQR) catalyzes sulfide oxidation during sulfide-dependent chemo- and phototrophic growth in bacteria. The green sulfur bacterium
Chlorobaculum tepidum
(formerly
Chlorobium tepidum
) can grow on sulfide as the sole electron donor and sulfur source.
C. tepidum
contains genes encoding three SQR homologs: CT0117, CT0876, and CT1087. This study examined which, if any, of the SQR homologs possess sulfide-dependent ubiquinone reduction activity and are required for growth on sulfide. In contrast to CT0117 and CT0876, transcripts of CT1087 were detected only when cells actively oxidized sulfide. Mutation of CT0117 or CT1087 in
C. tepidum
decreased SQR activity in membrane fractions, and the CT1087 mutant could not grow with ≥6 mM sulfide. Mutation of both CT0117 and CT1087 in
C. tepidum
completely abolished SQR activity, and the double mutant failed to grow with ≥4 mM sulfide. A C-terminal His
6
-tagged CT1087 protein was membrane localized, as was SQR activity. Epitope-tagged CT1087 was detected only when sulfide was actively consumed by cells. Recombinantly produced CT1087 and CT0117 proteins had SQR activity, while CT0876 did not. In summary, we conclude that, under the conditions tested, both CT0117 and CT1087 function as SQR proteins in
C. tepidum
. CT0876 may support the growth of
C. tepidum
at low sulfide concentrations, but no evidence was found for SQR activity associated with this protein.
Identification and Characterization of a Bacterial Transport System for the Uptake of Pyruvate, Propionate, and Acetate in
Corynebacterium glutamicum
Authors: Jolkver Elena , Emer Denise , Ballan Stefan , Krämer Reinhard , Eikmanns Bernhard J. , Marin Kay
Published: November 2008

The metabolism of monocarboxylic acids is of central importance for bacteria in their natural habitat as well as during biotechnological production. Although biosynthesis and degradation are well understood, the transport of such compounds is still a matter of discussion. Here we present the identification and characterization of a new transport system in
Corynebacterium glutamicum
with high affinity for acetate and propionate and with lower affinity for pyruvate. Biochemical analysis of this monocarboxylic acid transporter (MctC) revealed for the first time a quantitative discrimination of passive diffusion and active transport of acetate by bacterial cells. MctC is a secondary transporter and belongs to the class of sodium solute symporters, but it is driven by the electrochemical proton potential. The
mctC
gene is preceded by and cotranscribed with cg0952, a locus encoding a small membrane protein, and the transcription of the cg0952-
mctC
operon is under the control of the transcriptional regulators RamA and RamB. Both of these proteins directly bind to the promoter region of the operon; RamA is essential for expression and RamB exerts a slightly negative control on expression of the cg0952-
mctC
operon.
mctC
expression is induced in the presence of pyruvate and beneficial under substrate-limiting conditions for
C. glutamicum
.
Influence of Operator Site Geometry on Transcriptional Control by the YefM-YoeB Toxin-Antitoxin Complex
Authors: Bailey Simon E. S. , Hayes Finbarr
Published: November 2008

YefM-YoeB is among the most prevalent and well-characterized toxin-antitoxin complexes. YoeB toxin is an endoribonuclease whose activity is inhibited by YefM antitoxin. The regions 5′ of
yefM-yoeB
in diverse bacteria possess conserved sequence motifs that mediate transcriptional autorepression. The
yefM-yoeB
operator site arrangement is exemplified in
Escherichia coli
: a pair of palindromes with core hexamer motifs and a center-to-center distance of 12 bp overlap the
yefM-yoeB
promoter. YefM is an autorepressor that initially recognizes a long palindrome containing the core hexamer, followed by binding to a short repeat. YoeB corepressor greatly enhances the YefM-operator interaction. Scanning mutagenesis demonstrated that the short repeat is crucial for correct interaction of YefM-YoeB with the operator site in vivo and in vitro. Moreover, altering the relative positions of the two palindromes on the DNA helix abrogated YefM-YoeB cooperative interactions with the repeats: complex binding to the long repeat was maintained but was perturbed to the short repeat. Although YefM lacks a canonical DNA binding motif, dual conserved arginine residues embedded in a basic patch of the protein are crucial for operator recognition. Deciphering the molecular basis of toxin-antitoxin transcriptional control will provide key insights into toxin-antitoxin activation and function.
Expression of the
cpdA
Gene, Encoding a 3′,5′-Cyclic AMP (cAMP) Phosphodiesterase, Is Positively Regulated by the cAMP-cAMP Receptor Protein Complex
Authors: Kim Han-Suk , Kim Sung-Min , Lee Hyun-Jung , Park Soon-Jung , Lee Kyu-Ho
Published: November 2008

The intracellular level of cyclic 3′,5′-AMP (cAMP), a signaling molecule that mediates a variety of cellular processes, is finely modulated by the regulation of its synthesis, excretion, and degradation. In this study, cAMP phosphodiesterase (CpdA), an enzyme that catalyzes the conversion of cAMP to AMP, was characterized in a pathogenic bacterium,
Vibrio vulnificus
. The
cpdA
gene exists in an operon composed of
mutT, yqiB, cpdA
, and
yqiA
, the transcription of which was initiated at position −22 upstream of
mutT
. A
cpdA
-null mutant of
V. vulnificus
contained significantly higher levels of cAMP than the wild type but showed no detectable cAMP when a multicopy plasmid of the
cpdA
gene was provided in
trans
, suggesting that CpdA is responsible for cAMP degradation. Cellular contents of the CpdA protein decreased dramatically in both
cya
and
crp
mutants. In addition, levels of expression of the
cpdA
::
luxAB
transcription fusion decreased in
cya
and
crp
mutants. The level of expression of
cpdA
::
luxAB
in the
cya
mutant increased in a concentration-dependent manner upon the exogenous addition of cAMP. The cAMP-cAMP receptor protein (CRP) complex bound directly to the upstream region of
mutT
, which includes a putative CRP-binding sequence centered at position −95.5 relative to the transcription start site. Site-directed mutagenesis or the deletion of this sequence in the
cpdA
::
luxAB
transcription fusion resulted in the loss of regulation by cAMP and CRP. Thus, this study demonstrates that CpdA plays a crucial role in determining the intracellular cAMP level and shows for the first time that the expression of
cpdA
is activated by the cAMP-CRP complex via direct binding to the regulatory region.
The Mevalonate Pathway ofStaphylococcus aureus
Authors: Balibar Carl J. , Shen Xiaoyu , Tao Jianshi
Published: November 2008

Isoprenoids are a class of ubiquitous organic molecules synthesized from the five-carbon starter unit isopentenyl pyrophosphate (IPP). Comprising more than 30,000 known natural products, isoprenoids serve various important biological functions in many organisms. In bacteria, undecaprenyl pyrophosphate is absolutely required for the formation of cell wall peptidoglycan and other cell surface structures, while ubiquinones and menaquinones, both containing an essential prenyl moiety, are key electron carriers in respiratory energy generation. There is scant knowledge on the nature and regulation of bacterial isoprenoid pathways. In order to explore the cellular responses to perturbations in the mevalonate pathway, responsible for producing the isoprenoid precursor IPP in many gram-positive bacteria and eukaryotes, we constructed three strains ofStaphylococcus aureusin which each of the mevalonate pathway genes is regulated by an IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible promoter. We used DNA microarrays to profile the transcriptional effects of downregulating the components of the mevalonate pathway inS. aureusand demonstrate that decreased expression of the mevalonate pathway leads to widespread downregulation of primary metabolism genes, an upregulation in virulence factors and cell wall biosynthetic determinants, and surprisingly little compensatory expression in other isoprenoid biosynthetic genes. We subsequently correlate these transcriptional changes with downstream metabolic consequences.
Switching Protein-DNA Recognition Specificity by Single-Amino-Acid Substitutions in the P1parFamily of Plasmid Partition Elements
Authors: Dabrazhynetskaya Alena , Brendler Therese , Ji Xinhua , Austin Stuart
Published: November 2008

The P1, P7, and pMT1parsystems are members of the P1parfamily of plasmid partition elements. Each has a ParA ATPase and a ParB protein that recognizes theparSpartition site of its own plasmid type to promote the active segregation of the plasmid DNA to daughter cells. ParB contacts twoparSmotifs known as BoxA and BoxB, the latter of which determines species specificity. We found that the substitution of a single orthologous amino acid in ParB for that of a different species has major effects on the specificity of recognition. A single change in ParB can cause a complete switch in recognition specificity to that of another species or can abolish specificity. Specificity changes do not necessarily correlate with changes in the gross DNA binding properties of the protein. Molecular modeling suggests that species specificity is determined by the capacity to form a hydrogen bond between ParB residue 288 and the second base in the BoxB sequence. As changes in just one ParB residue and one BoxB base can alter species specificity, plasmids may use such simple changes to evolve new species rapidly.
TheBacillus subtilisLate Competence OperoncomEIs Transcriptionally Regulated byyutBand under Post-Transcription Initiation Control bycomN(yrzD)
Authors: Ogura Mitsuo , Tanaka Teruo
Published: November 2008

TheBacillus subtilisgenome has been sequenced, and disruptants with disruptions in genes that were not characterized previously were systematically generated. We screened these gene disruptants for decreased transformation frequency and identified two genes,yrzDandyutB, whose disruption resulted in severely reduced transformation frequency and modestly reduced transformation frequency, respectively. In the regulation of competence development, various signals affect the expression ofcomK, which encodes a master regulator of genetic competence that drives late competence gene transcription. Epistatic analyses of both theyrzDandyutBgenes revealed no significant differences in the expression ofcomK. Further analysis of the expression of late competence genes in theyrzDdisruptant revealed thatyrzDis specifically required for regulation of thecomEoperon, which is one of the late competence operons, and thus was renamedcomN. An analysis of variouscomE-lacZfusions revealed that the targetciselement forcomNaction is in the large (approximately 1-kb) 5′ untranslated region ofcomE, while the activity of thecomEpromoter was not affected by disruption ofcomN. These results suggested that there is post-transcription initiation control ofcomEbycomN. A sequential deletion analysis of this region revealed the 35-bp region required forcomNaction. TheyutBgene encodes a putative lipoic acid synthetase and yet is specifically required for transcription ofcomE, based on the results oflacZfusion analyses. Therefore,yutBandcomNregulatecomEat the transcription and post-transcription initiation levels, respectively. These results demonstrate that acomE-specific regulatory mechanism is involved in development of genetic competence.
Analysis of Secretin-Induced Stress inPseudomonas aeruginosaSuggests Prevention Rather than Response and Identifies a Novel Protein Involved in Secretin Function
Authors: Seo Jin , Brencic Anja , Darwin Andrew J.
Published: November 2008

Secretins are bacterial outer membrane proteins that are important for protein export. However, they can also mislocalize and cause stress to the bacterial cell, which is dealt with by the well-conserved phage shock protein (Psp) system in a highly specific manner. Nevertheless, some bacteria have secretins but no Psp system. A notable example isPseudomonas aeruginosa, a prolific protein secretor with the potential to produce seven different secretins. We were interested in investigating howP. aeruginosamight deal with the potential for secretin-induced stress without a Psp system. Microarray analysis revealed the absence of any transcriptional response to XcpQ secretin overproduction. However, transposon insertions in eitherrpoN,truB, PA4068, PA4069, or PA0943 renderedP. aeruginosahypersensitive to XcpQ production. The PA0943 gene was studied further and found to encode a soluble periplasmic protein important for XcpQ localization to the outer membrane. Consistent with this, a PA0943 null mutation reduced the levels of type 2 secretion-dependent proteins in the culture supernatant. Therefore, this work has identified a novel protein required for normal secretin function inP. aeruginosa. Taken together, all of our data suggest thatP. aeruginosalacks a functional equivalent of the Psp stress response system. Rather, null mutations in genes such as PA0943 may cause increased secretin-induced stress to whichP. aeruginosacannot respond. Providing the PA0943 mutant with the ability to respond, in the form of critical Psp proteins from another species, alleviated its secretin sensitivity.
Definition and constituents of maltreatment in sport: establishing a conceptual framework for research practitioners
Authors: Stirling A E
Published: November 2008

There has recently been an increased emergence of research on the maltreatment of athletes in sport. It is suggested that research may play a particularly salient role with respect to athlete protection initiatives. However, as it stands, current research in this area is limited by a lack of consistency in definitions. The purpose of the paper, therefore, is to propose a conceptual framework of maltreatment in sport to be used among research practitioners. More specifically, a conceptual model of the different categories, constructs and constituents of maltreatment in sport is proposed. Sport-specific examples of the various maltreatments are outlined. Current literature is reviewed, and recommendations are made for future research.
A systematic review of four injection therapies for lateral epicondylosis: prolotherapy, polidocanol, whole blood and platelet-rich plasma
Authors: Rabago D , Best T M , Zgierska A E , Zeisig E , Ryan M , Crane D
Published: November 2008

Objective:

To appraise existing evidence for prolotherapy, polidocanol, autologous whole blood and platelet-rich plasma injection therapies for lateral epicondylosis (LE).

Design:

Systematic review.

Data sources:

Medline, Embase, CINAHL, Cochrane Central Register of Controlled Trials, Allied and Complementary Medicine. Search strategy: names and descriptors of the therapies and LE.

Study Selection:

All human studies assessing the four therapies for LE.

Main results:

Results of five prospective case series and four controlled trials (three prolotherapy, two polidocanol, three autologous whole blood and one platelet-rich plasma) suggest each of the four therapies is effective for LE. In follow-up periods ranging from 9 to 108 weeks, studies reported sustained, statistically significant (p<0.05) improvement in visual analogue scale primary outcome pain score measures and disease-specific questionnaires; relative effect sizes ranged from 51% to 94%; Cohen’s d ranged from 0.68 to 6.68. Secondary outcomes also improved, including biomechanical elbow function assessment (polidocanol and prolotherapy), presence of abnormalities and increased vascularity on ultrasound (autologous whole blood and polidocanol). Subjects reported satisfaction with therapies on single-item assessments. All studies were limited by small sample size.

Conclusions:

There is strong pilot-level evidence supporting the use of prolotherapy, polidocanol, autologous whole blood and platelet-rich plasma injections in the treatment of LE. Rigorous studies of sufficient sample size, assessing these injection therapies using validated clinical, radiological and biomechanical measures, and tissue injury/healing-responsive biomarkers, are needed to determine long-term effectiveness and safety, and whether these techniques can play a definitive role in the management of LE and other tendinopathies.
Prevalence of low back pain and concurrent psychological distress over a 16-year period: Table 1
Authors: Leijon O , Mulder M
Published: November 2008

Objectives:

To investigate time trends of low back pain (LBP) and concurrent psychological distress in the general population.

Methods:

Every 4 years between 1990 and 2006, a self-administered questionnaire including the General Health Questionnaire (GHQ-12) and a question on LBP were sent to a random sample of the population in the county of Stockholm (response rate 61–69%). All individuals aged 21–64 years in the five samples (n = 1976–26 611) were included in the study.

Results:

Among women, the prevalence of self-reported LBP rose rather moderately during the 16-year period, from 12.5% to 16.4% (prevalence rate ratio (PRR) 1.31; 95% CI 1.11 to 1.55). The prevalence did not change at all among men (PRR 1.02; 95% CI 0.85 to 1.23). In contrast, the prevalence of LBP with concurrent psychological distress rose more substantially, from 2.6% to 5.9% among women (PRR 2.23; 95% CI 1.53 to 3.24) and from 1.9% to 3.5% among men (PRR 1.82; 95% CI 1.14 to 2.90). The prevalence of both LBP and LBP with concurrent psychological distress seemed to fluctuate somewhat over the period.

Conclusions:

Between 1990 and 2006, the focus of discussion of the high societal costs for sickness absence and disability pensions gradually shifted from musculoskeletal disorders to psychological well-being. As a result, the general population’s awareness and perception of pain and psychological distress may have changed and, in turn, affected individuals’ willingness to report these symptoms. Further research is necessary to investigate the impact of cultural changes on subjective symptom reporting.
Effectiveness of a feedback signal in a computer mouse on upper extremity musculoskeletal symptoms: a randomised controlled trial with an 8-month follow-up
Authors: Meijer E M , Sluiter J K , Frings-Dresen M H W
Published: November 2008

Objectives:

To study the effectiveness of using a computer mouse with a feedback signal for upper extremity musculoskeletal symptoms in office workers.

Methods:

A randomised controlled trial with 8 months of follow-up was carried out. The intervention consisted of a computer mouse with a feedback signal. In total, 354 subjects were allocated to the intervention group or the control group. Measurements were performed with electronic questionnaires at baseline, after 4 months and after 8 months. Outcome variables were the prevalence and incidence of upper extremity musculoskeletal symptoms and disability in the upper extremities. The intervention process was evaluated by software registration.

Results:

The use of the mouse with a feedback signal resulted in a significant decrease in duration of mouse usage over time. No differences were found in the number of mouse usage rest breaks. No differences were found in the prevalence (p = 0.29) or incidence (p = 0.832) of upper extremity musculoskeletal symptoms between the groups 8 months after baseline. The prevalence decreased from 49% at baseline to 44% after 8 months in the control group, while it remained at 36% in the intervention group. The incidence was 21% in the control group and 22% in the intervention group. Among the population with upper extremity musculoskeletal symptoms at baseline, the risk of experiencing symptoms after 8 months did not differ between the groups (p = 0.49). Minor disability was found in both groups. In the intervention group, a lower level of physical disability over time was reported than in the control group (p = 0.02).

Conclusions:

A feedback signal computer mouse does not affect the prevalence and incidence of upper extremity musculoskeletal symptoms, but it does lower disability scores. Given the high prevalence, studying preventive interventions for upper extremity musculoskeletal symptoms is of high importance.

Trial registration number:

ISRCTN13222474.
PDGF-DD, a novel mediator of smooth muscle cell phenotypic modulation, is upregulated in endothelial cells exposed to atherosclerosis-prone flow patterns
Authors: Thomas James A. , Deaton Rebecca A. , Hastings Nicole E. , Shang Yueting , Moehle Christopher W. , Eriksson Ulf , Topouzis Stavros , Wamhoff Brian R. , Blackman Brett R. , Owens Gary K.
Published: November 2008

Platelet-derived growth factor (PDGF)-BB is a well-known smooth muscle (SM) cell (SMC) phenotypic modulator that signals by binding to PDGF αα-, αβ-, and ββ-membrane receptors. PDGF-DD is a recently identified PDGF family member, and its role in SMC phenotypic modulation is unknown. Here we demonstrate that PDGF-DD inhibited expression of multiple SMC genes, including SM α-actin and SM myosin heavy chain, and upregulated expression of the potent SMC differentiation repressor gene Kruppel-like factor-4 at the mRNA and protein levels. On the basis of the results of promoter-reporter assays, changes in SMC gene expression were mediated, at least in part, at the level of transcription. Attenuation of the SMC phenotypic modulatory activity of PDGF-DD by pharmacological inhibitors of ERK phosphorylation and by a small interfering RNA to Kruppel-like factor-4 highlight the role of these two pathways in this process. PDGF-DD failed to repress SM α-actin and SM myosin heavy chain in mouse SMCs lacking a functional PDGF β-receptor. Importantly, PDGF-DD expression was increased in neointimal lesions in the aortic arch region of apolipoprotein C-deficient (ApoE−/−) mice. Furthermore, human endothelial cells exposed to an atherosclerosis-prone flow pattern, as in vascular regions susceptible to the development of atherosclerosis, exhibited a significant increase in PDGF-DD expression. These findings demonstrate a novel activity for PDGF-DD in SMC biology and highlight the potential contribution of this molecule to SMC phenotypic modulation in the setting of disturbed blood flow.
Regulation of coronary vascular tone via redox modulation in the α1-adrenergic-angiotensin-endothelin axis of the myocardium
Authors: Yamaguchi Osamu , Kaneshiro Takashi , Saitoh Shu-ichi , Ishibashi Toshiyuki , Maruyama Yukio , Takeishi Yasuchika
Published: November 2008

We hypothesized that α1-adrenoceptor stimulation of cardiac myocytes results in the production of an endothelin (ET)-releasing factor that stimulates the coronary vasculature to release ET and, by manipulating the redox state of cardiac and vascular cells, may influence the extent of α1-adrenergic-ET-1 vasoconstriction. Dihydroethidium (DHE) and dichlorodihydrofluorescein (DCF) intensities were increased by phenylephrine stimulation in isolated rat cardiac myocytes, which were enhanced by the mitochondrial electron transport chain complex I inhibitor rotenone (DHE: 20.4 ± 1.2-fold and DCF: 25.2 ± 0.9-fold, n = 8, P < 0.01, respectively) but not by the NADPH oxidase inhibitor apocynin. Olmesartan, an angiotensin II type 1 receptor antagonist, and enalaprilate did not change DHE and DCF intensities by phenylephrine. Next, we measured the vasoconstriction of isolated, pressurized rat coronary arterioles (diameter: 74 ± 8 μm) in response to supernatant collected from isolated cardiac myocytes. The addition of supernatant from phenylephrine-stimulated myocytes to a 2-ml vessel bath ( n = 8 each) caused volume-dependent vasoconstriction (500 μl: −14.8 ± 2.2%). Olmesartan and TA0201, an ET type A receptor antagonist, converted vasoconstriction into vasodilation (8.5 ± 1.2% and 10.5 ± 0.5%, P < 0.01, respectively) in response to supernatant from phenylephrine-stimulated myocytes, which was eliminated with catalase. Vasoconstriction was weakened using supernatant from phenylephrine with rotenone-treated myocytes. Treatment of arterioles with apocynin to myocyte supernatant converted vasoconstriction into vasodilation (7.8 ± 0.8%, P < 0.01). These results suggest that α1-adrenergic stimulation in cardiac myocytes produces angiotensin I and H2O2 and that angiotensin releases ET-1 through NADPH oxidase in coronary arterioles. Thus, coronary vasoconstriction via the α-adrenergic-angiotensin-ET axis appears to require redox-mediated signaling in cardiac and vascular cells.