bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of a preferential myosin loss on Ca2+ activation of force generation in single human skeletal muscle fibres
Authors: Ochala Julien , Larsson Lars
Published: February 2008

Preferential loss of the motor protein myosin, as observed in patients with acute quadriplegic myopathy (AQM) or cancer cachexia, causes generalized muscle wasting, muscle weakness and a decrease in muscle fibre force normalized to cross‐sectional area. It remains unclear, however, whether this myosin loss influences other important features of muscle fibre function, such as Ca2+ activation of the contractile proteins. To address this question, we have studied Ca2+ sensitivity of force generation using skinned muscle fibres from four patients with AQM or cancer cachexia and a preferential loss of myosin; and from seven healthy control individuals. Force and apparent rate constant of force redevelopment (ktr) were assessed in solutions with varying Ca2+ concentrations (pCa), allowing construction of relative force–pCa and ktr–pCa relationships. Results showed a rightward shift of the relative force–pCa relationship and a leftward shift of the relative ktr–pCa curve in muscle fibres with a preferential myosin loss. To improve the understanding of the mechanisms underlying these alterations, the relative stiffness–pCa relationship was evaluated. A rightward shift of this curve was observed, suggesting that the changes in the Ca2+ activation of force and ktr were predominantly due to a decrease in the relative number of attached cross‐bridges at different pCa values. Thus, a change in Ca2+ activation of the contractile apparatus in patients with preferential myosin loss is proposed as an additional factor contributing to the muscle function impairment in these patients.
Adaptive Evolution of a Lactose-Consuming
Saccharomyces cerevisiae
Recombinant
Authors: Guimarães Pedro M. R. , François Jean , Parrou Jean Luc , Teixeira José A. , Domingues Lucília
Published: February 2008

The construction of
Saccharomyces cerevisiae
strains that ferment lactose has biotechnological interest, particularly for cheese whey fermentation. A flocculent lactose-consuming
S. cerevisiae
recombinant expressing the
LAC12
(lactose permease) and
LAC4
(β-galactosidase) genes of
Kluyveromyces lactis
was constructed previously but showed poor efficiency in lactose fermentation. This strain was therefore subjected to an evolutionary engineering process (serial transfer and dilution in lactose medium), which yielded an evolved recombinant strain that consumed lactose twofold faster, producing 30% more ethanol than the original recombinant. We identified two molecular events that targeted the
LAC
construct in the evolved strain: a 1,593-bp deletion in the intergenic region (promoter) between
LAC4
and
LAC12
and a decrease of the plasmid copy number by about 10-fold compared to that in the original recombinant. The results suggest that the intact promoter was unable to mediate the induction of the transcription of
LAC4
and
LAC12
by lactose in the original recombinant and that the deletion established the transcriptional induction of both genes in the evolved strain. We propose that the tuning of the expression of the heterologous
LAC
genes in the evolved recombinant was accomplished by the interplay between the decreased copy number of both genes and the different levels of transcriptional induction for
LAC4
and
LAC12
resulting from the changed promoter structure. Nevertheless, our results do not exclude other possible mutations that may have contributed to the improved lactose fermentation phenotype. This study illustrates the usefulness of simple evolutionary engineering approaches in strain improvement. The evolved strain efficiently fermented threefold-concentrated cheese whey, providing an attractive alternative for the fermentation of lactose-based media.
Development of Inducible Systems To Engineer Conditional Mutants of Essential Genes of
Helicobacter pylori
Authors: Boneca Ivo G. , Ecobichon Chantal , Chaput Catherine , Mathieu Aurélie , Guadagnini Stéphanie , Prévost Marie-Christine , Colland Frédéric , Labigne Agnès , de Reuse Hilde
Published: February 2008

The
Escherichia coli
-
Helicobacter pylori
shuttle vector pHeL2 was modified to introduce the inducible LacI
q
-p
Tac
system of
E. coli
, in which the promoters were engineered to be under the control of
H. pylori
RNA polymerase. The
amiE
gene promoter of
H. pylori
was taken to constitutively express the LacI
q
repressor. Expression of the reporter gene
lacZ
was driven by either p
Tac
(pILL2150) or a modified version of the
ureI
gene promoter in which one or two LacI-binding sites and/or mutated nucleotides between the ribosomal binding site and the ATG start codon (pILL2153 and pILL2157) were introduced. Promoter activity was evaluated by measuring β-galactosidase activity. pILL2150 is a tightly regulated expression system suitable for the analysis of genes with low-level expression, while pILL2157 is well adapted for the controlled expression of genes encoding recombinant proteins in
H. pylori
. To exemplify the usefulness of these tools, we constructed conditional mutants of the putative essential
pbp1
and
ftsI
genes encoding penicillin-binding proteins 1 and 3 of
H. pylori
, respectively. Both genes were cloned into pILL2150 and introduced in the parental
H. pylori
strain N6. The chromosomally harbored
pbp1
and
ftsI
genes were then inactivated by replacing them with a nonpolar kanamycin cassette. Inactivation was strictly dependent upon addition of isopropyl-β-
d
-thiogalactopyranoside. Hence, we were able to construct the first conditional mutants of
H. pylori
. Finally, we demonstrated that following in vitro methylation of the recombinant plasmids, these could be introduced into a large variety of
H. pylori
isolates with different genetic backgrounds.
Quantitative Detection of Perchlorate-Reducing Bacteria by Real-Time PCR Targeting the Perchlorate Reductase Gene
Authors: Nozawa-Inoue Mamie , Jien Mercy , Hamilton Nicholas S. , Stewart Valley , Scow Kate M. , Hristova Krassimira R.
Published: February 2008

A quantitative real-time PCR assay targeting thepcrAgene, encoding the catalytic subunit of perchlorate reductase, detectedpcrAgenes from perchlorate-reducing bacteria in three different genera and from soil microbial communities. PartialpcrAsequences indicated differences in the composition of perchlorate-reducing bacterial communities following exposure to different electron donors.
Bacterial Community Structure of Acid-Impacted Lakes: What Controls Diversity?
Authors: Percent Sascha F. , Frischer Marc E. , Vescio Paul A. , Duffy Ellen B. , Milano Vincenzo , McLellan Maggie , Stevens Brett M. , Boylen Charles W. , Nierzwicki-Bauer Sandra A.
Published: February 2008

Although it is recognized that acidification of freshwater systems results in decreased overall species richness of plants and animals, little is known about the response of aquatic microbial communities to acidification. In this study we examined bacterioplankton community diversity and structure in 18 lakes located in the Adirondack Park (in the state of New York in the United States) that were affected to various degrees by acidic deposition and assessed correlations with 31 physical and chemical parameters. The pH of these lakes ranged from 4.9 to 7.8. These studies were conducted as a component of the Adirondack Effects Assessment Program supported by the U.S. Environmental Protection Agency. Thirty-one independent 16S rRNA gene libraries consisting of 2,135 clones were constructed from epilimnion and hypolimnion water samples. Bacterioplankton community composition was determined by sequencing and amplified ribosomal DNA restriction analysis of the clone libraries. Nineteen bacterial classes representing 95 subclasses were observed, but clone libraries were dominated by representatives of the
Actinobacteria
and
Betaproteobacteria
classes. Although the diversity and richness of bacterioplankton communities were positively correlated with pH, the overall community composition assessed by principal component analysis was not. The strongest correlations were observed between bacterioplankton communities and lake depth, hydraulic retention time, dissolved inorganic carbon, and nonlabile monomeric aluminum concentrations. While there was not an overall correlation between bacterioplankton community structure and pH, several bacterial classes, including the
Alphaproteobacteria
, were directly correlated with acidity. These results indicate that unlike more identifiable correlations between acidity and species richness for higher trophic levels, controls on bacterioplankton community structure are likely more complex, involving both direct and indirect processes.
Physiological State, Growth Mode, and Oxidative Stress Play a Role in Cd(II)-Mediated Inhibition of
Nitrosomonas europaea
19718
Authors: Chandran Kartik , Love Nancy G.
Published: February 2008

The goal of this study was to determine the impact of physiological growth states (batch exponential and batch stationary growth) and growth modes (substrate-limited chemostat, substrate-sufficient exponential batch, and substrate-depleted stationary batch growth) on several measures of growth and responses to Cd(II)-mediated inhibition of
Nitrosomonas europaea
strain 19718. The specific oxygen uptake rate (sOUR) was the most sensitive indicator of inhibition among the different responses analyzed, including total cell abundance, membrane integrity, intracellular 16S rRNA/DNA ratio, and
amoA
expression. This observation remained true irrespective of the physiological state, the growth mode, or the mode of Cd(II) exposure. Based on the sOUR, a strong time-dependent exacerbation of inhibition (in terms of an inhibition coefficient [

K
i

]) in exponential batch cultures was observed. Long-term inhibition levels (based on

K
i

estimates) in metabolically active chemostat and exponential batch cultures were also especially severe and comparable. In contrast, the inhibition level in stationary-phase cultures was 10-fold lower and invariable with exposure time. Different strategies for surviving substrate limitation (a 10-fold increase in
amoA
expression) and starvation (the retention of 16S rRNA levels) in
N. europaea
cultures were observed.
amoA
expression was most negatively impacted by Cd(II) exposure in the chemostat cultures, was less impacted in exponential batch cultures, and was least impacted in stationary batch cultures. Although the
amoA
response was consistent with that of the sOUR, the
amoA
response was not as strong. The intracellular 16S rRNA/DNA ratio, as determined by fluorescence in situ hybridization, also did not uniformly correlate with the sOUR under conditions of inhibition or no inhibition. Finally, Cd(II)-mediated inhibition of
N. europaea
was attributed partially to oxidative stress.
On-Line Monitoring of Microbial Volatile Metabolites by Proton Transfer Reaction-Mass Spectrometry
Authors: Bunge Michael , Araghipour Nooshin , Mikoviny Tomas , Dunkl Jürgen , Schnitzhofer Ralf , Hansel Armin , Schinner Franz , Wisthaler Armin , Margesin Rosa , Märk Tilmann D.
Published: February 2008

A method for analysis of volatile organic compounds (VOCs) from microbial cultures was established using proton transfer reaction-mass spectrometry (PTR-MS). A newly developed sampling system was coupled to a PTR-MS instrument to allow on-line monitoring of VOCs in the dynamic headspaces of microbial cultures. The novel PTR-MS method was evaluated for four reference organisms:
Escherichia coli
,
Shigella flexneri
,
Salmonella enterica
, and
Candida tropicalis
. Headspace VOCs in sampling bottles containing actively growing cultures and uninoculated culture medium controls were sequentially analyzed by PTR-MS. Characteristic marker ions were found for certain microbial cultures:
C. tropicalis
could be identified by several unique markers compared with the other three organisms, and
E. coli
and
S. enterica
were distinguishable from each other and from
S. flexneri
by specific marker ions, demonstrating the potential of this method to differentiate between even closely related microorganisms. Although the temporal profiles of some VOCs were similar to the growth dynamics of the microbial cultures, most VOCs showed a different temporal profile, characterized by constant or decreasing VOC levels or by single or multiple peaks over 24 h of incubation. These findings strongly indicate that the temporal evolution of VOC emissions during growth must be considered if characterization or differentiation based on microbial VOC emissions is attempted. Our study may help to establish the analysis of VOCs by on-line PTR-MS as a routine method in microbiology and as a tool for monitoring environmental and biotechnological processes.
Capacity of Human Nisin- and Pediocin-Producing Lactic Acid Bacteria To Reduce Intestinal Colonization by Vancomycin-Resistant Enterococci
Authors: Millette Mathieu , Cornut Gilbert , Dupont Claude , Shareck François , Archambault Denis , Lacroix Monique
Published: February 2008

This study demonstrated the capacity of bacteriocin-producing lactic acid bacteria (LAB) to reduce intestinal colonization by vancomycin-resistant enterococci (VRE) in a mouse model.
Lactococcus lactis
MM19 and
Pediococcus acidilactici
MM33 are bacteriocin producers isolated from human feces. The bacteriocin secreted by
P. acidilactici
is identical to pediocin PA-1/AcH, while PCR analysis demonstrated that
L. lactis
harbors the nisin Z gene. LAB were acid and bile tolerant when assayed under simulated gastrointestinal conditions. A well diffusion assay using supernatants from LAB demonstrated strong activity against a clinical isolate of VRE. A first in vivo study was done using C57BL/6 mice that received daily intragastric doses of
L. lactis
MM19,
P. acidilactici
MM33,
P. acidilactici
MM33A (a pediocin mutant that had lost its ability to produce pediocin), or phosphate-buffered saline (PBS) for 18 days. This study showed that
L. lactis
and
P. acidilactici
MM33A increased the concentrations of total LAB and anaerobes while
P. acidilactici
MM33 decreased the
Enterobacteriaceae
populations. A second in vivo study was done using VRE-colonized mice that received the same inocula as those in the previous study for 16 days. In
L. lactis
-fed mice, fecal VRE levels 1.73 and 2.50 log
10
CFU/g lower than those in the PBS group were observed at 1 and 3 days postinfection. In the
P. acidilactici
MM33-fed mice, no reduction was observed at 1 day postinfection but a reduction of 1.85 log
10
CFU/g was measured at 3 days postinfection. Levels of VRE in both groups of mice treated with bacteriocin-producing LAB were undetectable at 6 days postinfection. No significant difference in mice fed the pediocin-negative strain compared to the control group was observed. This is the first demonstration that human
L. lactis
and
P. acidilactici
nisin- and pediocin-producing strains can reduce VRE intestinal colonization.
Antibiotic Resistance of Enterococci in American Bison (
Bison bison
) from a Nature Preserve Compared to That of Enterococci in Pastured Cattle
Authors: Anderson John F. , Parrish Torrey D. , Akhtar Mastura , Zurek Ludek , Hirt Helmut
Published: February 2008

Enterococci isolated from a bison population on a native tall-grass prairie preserve in Kansas were characterized and compared to enterococci isolated from pastured cattle. The species diversity was dominated by
Enterococcus casseliflavus
in bison (62.4%), while
Enterococcus hirae
was the most common isolate from cattle (39.7%).
Enterococcus faecalis
was the second most common species isolated from bison (16%). In cattle,
E. faecalis
and
Enterococcus faecium
were isolated at lower percentages (3.2% and 1.6%, respectively). No resistance to ampicillin, chloramphenicol, gentamicin, or high levels of vancomycin was detected from either source. Tetracycline and erythromycin resistance phenotypes, encoded by
tetO
and
ermB
, respectively, were common in cattle isolates (42.9% and 12.7%, respectively). A significant percentage of bison isolates (8% and 4%, respectively) were also resistant to these two antibiotics. The tetracycline resistance genes from both bison and cattle isolates resided on mobile genetic elements and showed a transfer frequency of 10
−6
per donor, whereas erythromycin resistance was not transferable. Resistance to ciprofloxacin was found to be higher in enterococci from bison (14.4%) than in enterococci isolated from cattle (9.5%). The bison population can serve as a sentinel population for studying the spread and origin of antibiotic resistance.
The
ygaVP
Genes of
Escherichia coli
Form a Tributyltin-Inducible Operon
Authors: Gueuné Hervé , Durand Marie-José , Thouand Gérald , DuBow Michael S.
Published: February 2008

A tributyltin (TBT)
luxAB
transcriptional fusion in
Escherichia coli
revealed that a TBT-activated promoter is located upstream of two cotranscribed orphan genes,
ygaV
and
ygaP
. We demonstrate that transcription from the promoter upstream of
ygaVP
is constitutive in a
ygaVP
mutant, suggesting that YgaV is an autoregulated, TBT-inducible repressor.
Fluorescence Correlation Spectroscopy To Study Diffusion and Reaction of Bacteriophages inside Biofilms
Authors: Briandet R. , Lacroix-Gueu P. , Renault M. , Lecart S. , Meylheuc T. , Bidnenko E. , Steenkeste K. , Bellon-Fontaine M.-N. , Fontaine-Aupart M.-P.
Published: February 2008

In the natural environment, most of the phages that target bacteria are thought to exist in biofilm ecosystems. The purpose of this study was to gain a clearer understanding of the reactivity of these viral particles when they come into contact with bacteria embedded in biofilms. Experimentally, we quantified lactococcal c2 phage diffusion and reaction through model biofilms using in situ fluorescence correlation spectroscopy with two-photon excitation. Correlation curves for fluorescently labeled c2 phage in nonreacting
Stenotrophomonas maltophilia
biofilms indicated that extracellular polymeric substances did not provide significant resistance to phage penetration and diffusion, even though penetration and diffusion were sometimes restricted because of the noncontractile tail of the viral particle. Fluctuations in the fluorescence intensity of the labeled phage were detected throughout the thickness of biofilms formed by c2-sensitive and c2-resistant strains of
Lactococcus lactis
but could never be correlated with time, revealing that the phage was immobile. This finding confirmed that recognition binding receptors for the viral particles were present on the resistant bacterial cell wall. Taken together, our results suggest that biofilms may act as “active” phage reservoirs that can entrap and amplify viral particles and protect them from harsh environments.
Microcyclamide Biosynthesis in Two Strains ofMicrocystis aeruginosa: from Structure to Genes and Vice Versa
Authors: Ziemert Nadine , Ishida Keishi , Quillardet Philippe , Bouchier Christiane , Hertweck Christian , de Marsac Nicole Tandeau , Dittmann Elke
Published: February 2008

Comparative analysis of related biosynthetic gene clusters can provide new insights into the versatility of these pathways and allow the discovery of new natural products. The freshwater cyanobacteriumMicrocystis aeruginosaNIES298 produces the cytotoxic peptide microcyclamide. Here, we provide evidence that the cyclic hexapeptide is formed by a ribosomal pathway through the activity of a set of processing enzymes closely resembling those recently shown to be involved in patellamide biosynthesis in cyanobacterial symbionts of ascidians. Besides two subtilisin-type proteases and a heterocyclization enzyme, the gene cluster discovered in strain NIES298 encodes six further open reading frames, two of them without similarity to enzymes encoded by the patellamide gene cluster. Analyses of genomic data of a second cyanobacterial strain,M. aeruginosaPCC 7806, guided the discovery and structural elucidation of two novel peptides of the microcyclamide family. The identification of the microcyclamide biosynthetic genes provided an avenue by which to study the regulation of peptide synthesis at the transcriptional level. The precursor genes were strongly and constitutively expressed throughout the growth phase, excluding the autoinduction of these peptides, as has been observed for several peptide pheromone families in bacteria.
Molecular Variation of Satellite DNAβ Molecules Associated with
Malvastrum yellow vein virus
and Their Role in Pathogenicity
Authors: Guo Wei , Jiang Tong , Zhang Xian , Li Guixin , Zhou Xueping
Published: February 2008

Previous studies have found that the diversity of begomovirus-associated DNAβ satellites is related to host and geographical origin. In this study, we have cloned and sequenced 20 different isolates of DNAβ molecules associated with
Malvastrum yellow vein virus
(MYVV) isolated from
Malvastrum coromandelianum
plants in different geographical locations of Yunnan Province, China. Analyses of their molecular variation indicate that the satellites are clustered together according to their geographical location but that they have only limited sequence diversity. Infectivity tests using infectious clones of MYVV and its associated DNAβ molecule indicate that MYVV DNAβ is indispensable for symptom induction in
Nicotiana benthamiana
,
N. glutinosa
,
Petunia hybrida
, and
M. coromandelianum
plants. Furthermore, we showed that MYVV interacts functionally with heterologous DNAβ molecules in
N. benthamiana
plants.
Use of
Drosophila
S2 Cells as a Model for Studying
Ehrlichia chaffeensis
Infections
Authors: Luce-Fedrow Alison , Von Ohlen Tonia , Boyle Daniel , Ganta Roman R. , Chapes Stephen K.
Published: February 2008

Ehrlichia chaffeensis
is an obligate intracellular bacterium and the causative agent of human monocytic ehrlichiosis. Although this pathogen grows in several mammalian cell lines, no general model for eukaryotic cellular requirements for bacteria replication has yet been proposed. We found that
Drosophila
S2 cells are permissive for the growth of
E. chaffeensis
. We saw morulae (aggregates of bacteria) by microscopy, detected the
E. chaffeensis
16S rRNA gene by reverse transcriptase PCR, and used immunocytochemistry to detect
E. chaffeensis
in S2 and mammalian cells. Bacteria grown in S2 cells reinfected mammalian macrophages. S2 cells were made nonpermissive for
E. chaffeensis
through incubation with lipopolysaccharide. Our results demonstrate that S2 cells are an appropriate system for studying the pathogenesis of
E. chaffeensis
. The use of a
Drosophila
system has the potential to serve as a model system for studying
Ehrlichia
due to its completed genome, ease of genetic manipulation, and the availability of mutants.
Molecular Analysis as an Aid To Assess the Public Health Risk of Non-O157 Shiga Toxin-Producing
Escherichia coli
Strains
Authors: Coombes Brian K. , Wickham Mark E. , Mascarenhas Mariola , Gruenheid Samantha , Finlay B. Brett , Karmali Mohamed A.
Published: February 2008

Shiga toxin-producing
Escherichia coli
(STEC) strains are commensal bacteria in cattle with high potential for environmental and zoonotic transmission to humans. Although O157:H7 is the most common STEC serotype, there is growing concern over the emergence of more than 200 highly virulent non-O157 STEC serotypes that are globally distributed, several of which are associated with outbreaks and/or severe human illness such as hemolytic-uremic syndrome (HUS) and hemorrhagic colitis. At present, the underlying genetic basis of virulence potential in non-O157 STEC is unknown, although horizontal gene transfer and the acquisition of new pathogenicity islands are an expected origin. We used seropathotype classification as a framework to identify genetic elements that distinguish non-O157 STEC strains posing a serious risk to humans from STEC strains that are not associated with severe and epidemic disease. We report the identification of three genomic islands encoding non-LEE effector (
nle
) genes and 14 individual
nle
genes in non-O157 STEC strains that correlate independently with outbreak and HUS potential in humans. The implications for transmissible zoonotic spread and public health are discussed. These results and methods offer a molecular risk assessment strategy to rapidly recognize and respond to non-O157 STEC strains from environmental and animal sources that might pose serious public health risks to humans.
Biochemical and Physiological Characterization of the Pyruvate Formate-Lyase Pfl1 of
Chlamydomonas reinhardtii
, a Typically Bacterial Enzyme in a Eukaryotic Alga
Authors: Hemschemeier Anja , Jacobs Jessica , Happe Thomas
Published: February 2008

The unicellular green alga
Chlamydomonas reinhardtii
has a special type of anaerobic metabolism that is quite unusual for eukaryotes. It has two oxygen-sensitive [Fe-Fe] hydrogenases (EC 1.12.7.2) that are coupled to photosynthesis and, in addition, a formate- and ethanol-producing fermentative metabolism, which was proposed to be initiated by pyruvate formate-lyase (Pfl; EC 2.3.1.54). Pfl enzymes are commonly found in prokaryotes but only rarely in eukaryotes. Both the hydrogen- and the formate/ethanol-producing pathways are involved in a sustained anaerobic metabolism of the alga, which can be induced by sulfur depletion in illuminated cultures. Before now, the presence of a Pfl protein in
C. reinhardtii
was predicted from formate secretion and the homology of the deduced protein of the
PFL1
gene model to known Pfl enzymes. In this study, we proved the formate-producing activity of the putative Pfl1 enzyme by heterologous expression of the
C. reinhardtii PFL1
cDNA in
Escherichia coli
and subsequent in vitro activity tests of the purified protein. Furthermore, a Pfl-deficient
E. coli
strain secretes formate when expressing the
PFL1
cDNA of
C. reinhardtii
. We also examined the Pfl1 fermentation pathway of
C. reinhardtii
under the physiological condition of sulfur depletion. Genetic and biochemical analyses show that sulfur-depleted algae express genes encoding enzymes acting downstream of Pfl1 and also potentially ethanol-producing enzymes, such as pyruvate decarboxylase (EC 4.1.1.1) or pyruvate ferredoxin oxidoreductase (EC 1.2.7.1). The latter enzymes might substitute for Pfl1 activity when Pfl1 is specifically inhibited by hypophosphite.
Mating Type Protein Mat1-2 from Asexual
Aspergillus fumigatus
Drives Sexual Reproduction in Fertile
Aspergillus nidulans
Authors: Pyrzak Wioletta , Miller Karen Y. , Miller Bruce L.
Published: February 2008

The lack of an experimentally amenable sexual genetic system in
Aspergillus fumigatus
is a major limitation in the study of the organism's pathogenesis. A recent comparative genome analysis revealed evidence for potential sexuality in
A. fumigatus
. Homologs of mating type genes as well as other genes of the “sexual machinery” have been identified in anamorphic
A. fumigatus
. The
mat1
-
2
gene encodes a homolog of MatA, an HMG box mating transcriptional factor (Mat
HMG
) that regulates sexual development in fertile
Aspergillus nidulans
. In this study, the functionalities of
A. fumigatus mat1
-
2
and the Mat1-2 protein were determined by interspecies gene exchange between sterile
A. fumigatus
and fertile
A. nidulans
. Ectopically integrated
A. fumigatus mat1
-
2
(driven by its own promoter) was not functional in a sterile
A. nidulans
Δ
matA
strain, and no sexual development was observed. In contrast, the
A. fumigatus mat1
-
2
open reading frame driven by the
A. nidulans matA
promoter and integrated by homologous gene replacement at the
matA
locus was functional and conferred full fertility. This is the first report showing that cross species mating type gene exchange between closely related
Ascomycetes
did not function in sexual development. This is also the first report demonstrating that a Mat
HMG
protein from an asexual species is fully functional, with viable ascospore differentiation, in a fertile homothallic species. The expression of
mat1
-
2
was assessed in
A. fumigatus
and
A. nidulans
. Our data suggest that
mat1
-
2
may not be properly regulated to allow sexuality in
A. fumigatus
. This study provides new insights about
A. fumigatus
asexuality and also suggests the possibility for the development of an experimentally amenable sexual cycle.
Coordinated Regulation of Expression ofSalmonellaPathogenicity Island 1 and Flagellar Type III Secretion Systems by ATP-Dependent ClpXP Protease
Authors: Kage Hirokazu , Takaya Akiko , Ohya Mai , Yamamoto Tomoko
Published: February 2008

Salmonella entericaserovar Typhimurium delivers a variety of proteins via theSalmonellapathogenicity island 1 (SPI1)-encoded type III secretion system into host cells, where they elicit several physiological changes, including bacterial invasion, macrophage apoptosis, and enteropathogenesis. OnceSalmonellahas established a systemic infection, excess macrophage apoptosis would be detrimental to the pathogen, as it utilizes macrophages as vectors for systemic dissemination throughout the host. Therefore, SPI1 expression must be restricted to one or a few specific locations in the host. In the present study, we have demonstrated that the expression of this complex of genes is repressed by the ATP-dependent ClpXP protease, which therefore suppresses macrophage apoptosis. Depletion of ClpXP caused significant increases in the amounts of two SPI1-encoded transcriptional regulators, HilC and HilD, leading to the stimulation ofhilAinduction and therefore activation of SPI1 expression. Our evidence shows that ClpXP regulates cellular levels of HilC and HilD via the control of flagellar gene expression. Subsequent experiments demonstrated that the flagellum-related gene product FliZ controls HilD posttranscriptionally, and this in turn activates HilC. These findings suggest that the ClpXP protease coregulates SPI1-related virulence phenotypes and motility. ClpXP is a member of the stress protein family induced in bacteria exposed to hostile environments such as macrophages.
Reduction of Turgor Is Not the Stimulus for the Sensor Kinase KdpD ofEscherichia coli
Authors: Hamann Knut , Zimmann Petra , Altendorf Karlheinz
Published: February 2008

Stimulus perception by the KdpD/KdpE two-component system ofEscherichia coliis still controversial with respect to the nature of the stimulus that is perceived by the sensor kinase KdpD. Limiting potassium concentrations in the medium or high osmolality leads to KdpD/KdpE signal transduction, resulting inkdpFABCexpression. It has been hypothesized that changes in turgor are sensed by KdpD through alterations in the physical state of the cytoplasmic membrane. However, in this study the quantitative determination of expression levels of thekdpFABCoperon revealed that the system responds very effectively to K+-limiting conditions in the medium but barely and to various degrees to salt and sugar stress. Since the current view of stimulus perception calls for mainly intracellular parameters, which might be sensed by KdpD, we set out to test the cytoplasmic concentrations of ATP, K+, Na+, glutamate, proline, glycine, trehalose, putrescine, and spermidine under K+-limiting conditions. As a first result, the determination of the cytoplasmic volume, which is a prerequisite for such measurements, revealed that a transient shrinkage of the cytoplasmic volume, which is indicative of a reduction in turgor, occurred only under osmotic upshift but not under K+-limiting conditions. Furthermore, the intracellular ATP concentration significantly increased under osmotic upshift, whereas only a slight increase occurred after a potassium downshift. Finally, the cytoplasmic K+concentration rose severalfold only after an osmotic upshock. For the first time, these data indicate that stimulus perception by KdpD correlates neither with changes in the cytoplasmic volume nor with changes in the intracellular ATP or K+concentration or those of the other solutes tested. In conclusion, we propose that a reduction in turgor cannot be the stimulus for KdpD.
The Complete Genome Sequence ofEscherichia coliDH10B: Insights into the Biology of a Laboratory Workhorse
Authors: Durfee Tim , Nelson Richard , Baldwin Schuyler , Plunkett Guy , Burland Valerie , Mau Bob , Petrosino Joseph F. , Qin Xiang , Muzny Donna M. , Ayele Mulu , Gibbs Richard A. , Csörgő Bálint , Pósfai György , Weinstock George M. , Blattner Frederick R.
Published: February 2008

Escherichia coliDH10B was designed for the propagation of large insert DNA library clones. It is used extensively, taking advantage of properties such as high DNA transformation efficiency and maintenance of large plasmids. The strain was constructed by serial genetic recombination steps, but the underlying sequence changes remained unverified. We report the complete genomic sequence of DH10B by using reads accumulated from the bovine sequencing project at Baylor College of Medicine and assembled with DNAStar's SeqMan genome assembler. The DH10B genome is largely colinear with that of the wild-type K-12 strain MG1655, although it is substantially more complex than previously appreciated, allowing DH10B biology to be further explored. The 226 mutated genes in DH10B relative to MG1655 are mostly attributable to the extensive genetic manipulations the strain has undergone. However, we demonstrate that DH10B has a 13.5-fold higher mutation rate than MG1655, resulting from a dramatic increase in insertion sequence (IS) transposition, especially IS150. IS elements appear to have remodeled genome architecture, providing homologous recombination sites for a 113,260-bp tandem duplication and an inversion. DH10B requires leucine for growth on minimal medium due to the deletion ofleuLABCDand harbors both therelA1andspoT1alleles causing both sensitivity to nutritional downshifts and slightly lower growth rates relative to the wild type. Finally, while the sequence confirms most of the reported alleles, the sequence ofdeoRis wild type, necessitating reexamination of the assumed basis for the high transformability of DH10B.
Differential Substrate Specificity and Kinetic Behavior of
Escherichia coli
YfdW and
Oxalobacter formigenes
Formyl Coenzyme A Transferase
Authors: Toyota Cory G. , Berthold Catrine L. , Gruez Arnaud , Jónsson Stefán , Lindqvist Ylva , Cambillau Christian , Richards Nigel G. J.
Published: February 2008

The
yfdXWUVE
operon appears to encode proteins that enhance the ability of
Escherichia coli
MG1655 to survive under acidic conditions. Although the molecular mechanisms underlying this phenotypic behavior remain to be elucidated, findings from structural genomic studies have shown that the structure of YfdW, the protein encoded by the
yfdW
gene, is homologous to that of the enzyme that mediates oxalate catabolism in the obligate anaerobe
Oxalobacter formigenes
,
O. formigenes
formyl coenzyme A transferase (FRC). We now report the first detailed examination of the steady-state kinetic behavior and substrate specificity of recombinant, wild-type YfdW. Our studies confirm that YfdW is a formyl coenzyme A (formyl-CoA) transferase, and YfdW appears to be more stringent than the corresponding enzyme (FRC) in
Oxalobacter
in employing formyl-CoA and oxalate as substrates. We also report the effects of replacing Trp-48 in the FRC active site with the glutamine residue that occupies an equivalent position in the
E. coli
protein. The results of these experiments show that Trp-48 precludes oxalate binding to a site that mediates substrate inhibition for YfdW. In addition, the replacement of Trp-48 by Gln-48 yields an FRC variant for which oxalate-dependent substrate inhibition is modified to resemble that seen for YfdW. Our findings illustrate the utility of structural homology in assigning enzyme function and raise the question of whether oxalate catabolism takes place in
E. coli
upon the up-regulation of the
yfdXWUVE
operon under acidic conditions.
Flagellin and Outer Surface Proteins from
Borrelia burgdorferi
Are Not Glycosylated
Authors: Štěrba Ján , Vancová Marie , Rudenko Nataliia , Golovchenko Maryna , Tremblay Tammy-Lynn , Kelly John F. , MacKenzie C. Roger , Logan Susan M. , Grubhoffer Libor
Published: February 2008

We investigated the presence of glycoproteins in
Borrelia burgdorferi
. We did not find any evidence for glycosylation of the major outer membrane proteins OspA and OspB or the structural flagellar proteins FlaB and FlaA. We suggest that glycoproteins present on the surface of
B. burgdorferi
may be tightly bound culture medium glycoproteins.
Rhinovirus exposure impairs immune responses to bacterial products in human alveolar macrophages
Authors: Oliver B G G , Lim S , Wark P , Laza-Stanca V , King N , Black J L , Burgess J K , Roth M , Johnston S L
Published: February 2008

Background:

Rhinovirus infection is responsible for considerable morbidity and mortality as the major cause of exacerbations of asthma, and is also known to induce exacerbations of cystic fibrosis and chronic obstructive pulmonary disease. Exacerbations of these diseases are also frequently associated with bacterial and atypical bacterial infection. Alveolar macrophages are the major immune cells in the airways and are important in defence against bacterial infections.

Methods:

The authors investigated whether rhinovirus modifies cytokine release, the pattern recognition receptor expression and phagocytosis by human alveolar macrophages in response to bacterial products.

Results:

Viable rhinovirus was detected in macrophages up to 3 days after exposure and viral RNA expression persisted for 10 days. Infectious but not UV inactivated rhinovirus increased tumour necrosis factor α (TNFα) and interleukin (IL)8 release by macrophages. In contrast, infectious rhinovirus impaired lipopolysaccharide and lipoteichoic acid induced TNFα and IL8 secretion by macrophages. Rhinovirus induced impairment of macrophage antibacterial immune responses did not involve IL10, prostaglandin E2 or downregulation of Toll-like receptor 2. Furthermore, the macrophage phagocytic response to labelled bacterial particles, but not to latex beads, was impaired.

Conclusion:

The authors have identified impairment of cytokine responses to bacterial lipopolysaccharide and lipoteichoic acid by alveolar macrophages in response to infectious rhinovirus. Virus induced impairment of antibacterial host defence has important implications in the pathogenesis of exacerbations of respiratory diseases.
Short term improvement in exercise capacity and symptoms following exercise training in interstitial lung disease
Authors: Holland A E , Hill C J , Conron M , Munro P , McDonald C F
Published: February 2008

Background:

Interstitial lung disease (ILD) is characterised by exertional dyspnoea, exercise limitation and reduced quality of life. The role of exercise training in this diverse patient group is unclear. The aims of this study were to establish the safety of exercise training in ILD; its effects on exercise capacity, dyspnoea and quality of life; and whether patients with idiopathic pulmonary fibrosis (IPF) had similar responses to those with other types of ILD.

Methods:

57 subjects with ILD (34 IPF) were randomised to receive 8 weeks of supervised exercise training or weekly telephone support. The 6 min walk distance (6MWD), incremental exercise test, modified Medical Research Council (MRC) dyspnoea score and Chronic Respiratory Disease Questionnaire (CRDQ) were performed at baseline, following intervention and at 6 months.

Results:

80% of subjects completed the exercise programme and no adverse events were recorded. The 6MWD increased following training (mean difference to control 35 m, 95% CI 6 to 64 m). A significant reduction in MRC score was observed (0.7 points, 95% CI 0.1 to 1.3) along with improvements in dyspnoea (p = 0.04) and fatigue (p<0.01) on the CRDQ. There was no change in peak oxygen uptake; however, exercise training reduced heart rate at maximum isoworkload (p = 0.01). There were no significant differences in response between those with and without IPF. After 6 months there were no differences between the training and control group for any outcome variable.

Conclusions:

Exercise training improves exercise capacity and symptoms in patients with ILD, but these benefits are not sustained 6 months following intervention.

Trial registration number:

NCT00168285
Effects of posture on shear rates in human brachial and superficial femoral arteries
Authors: Newcomer S. C. , Sauder C. L. , Kuipers N. T. , Laughlin M. H. , Ray C. A.
Published: February 2008

Shear rate is significantly lower in the superficial femoral compared with the brachial artery in the supine posture. The relative shear rates in these arteries of subjects in the upright posture (seated and/or standing) are unknown. The purpose of this investigation was to test the hypothesis that upright posture (seated and/or standing) would produce greater shear rates in the superficial femoral compared with the brachial artery. To test this hypothesis, Doppler ultrasound was used to measure mean blood velocity (MBV) and diameter in the brachial and superficial femoral arteries of 21 healthy subjects after being in the supine, seated, and standing postures for 10 min. MBV was significantly higher in the brachial compared with the superficial femoral artery during upright postures. Superficial femoral artery diameter was significantly larger than brachial artery diameter. However, posture had no significant effect on either brachial or superficial femoral artery diameter. The calculated shear rate was significantly greater in the brachial (73 ± 5, 91 ± 11, and 97 ± 13 s−1) compared with the superficial femoral (53 ± 4, 39 ± 77, and 44 ± 5 s−1) artery in the supine, seated, and standing postures, respectively. Contrary to our hypothesis, our current findings indicate that mean shear rate is lower in the superficial femoral compared with the brachial artery in the supine, seated, and standing postures. These findings of lower shear rates in the superficial femoral artery may be one mechanism for the higher propensity for atherosclerosis in the arteries of the leg than of the arm.
RAGE modulates myocardial injury consequent to LAD infarction via impact on JNK and STAT signaling in a murine model
Authors: Aleshin Alexey , Ananthakrishnan Radha , Li Qing , Rosario Rosa , Lu Yan , Qu Wu , Song Fei , Bakr Soliman , Szabolcs Matthias , D'Agati Vivette , Liu Rui , Homma Shunichi , Schmidt Ann Marie , Yan Shi Fang , Ramasamy Ravichandran
Published: February 2008

The receptor for advanced glycation end-products (RAGE) has been implicated in the pathogenesis of ischemia-reperfusion (I/R) injury in the isolated perfused heart. To test the hypothesis that RAGE-dependent mechanisms modulated responses to I/R in a murine model of transient occlusion and reperfusion of the left anterior descending coronary artery (LAD), we subjected male homozygous RAGE−/− mice and their wild-type age-matched littermates to 30 min of occlusion of the LAD followed by reperfusion. At 48 h of reperfusion, hematoxylin and eosin staining revealed significantly larger infarct size in wild-type versus RAGE−/− mice. Contractile function, as evaluated by echocardiography 48 h after reperfusion, revealed that fractional shortening was significantly higher in RAGE−/− versus wild-type mice. Plasma levels of creatine kinase were markedly decreased in RAGE−/− versus wild-type animals. Integral to the impact of RAGE deletion on diminished myocardial damage after infarction was significantly decreased apoptosis in the heart, as assessed by TUNEL staining, release of cytochrome c, and caspase-3 activity. Experiments investigating the impact of RAGE on early signaling pathways influencing myocardial ischemic injury revealed attenuation of JNK and STAT5 phosphorylation in RAGE−/− mouse hearts versus robust activation observed in wild-type mice upon ischemia and reperfusion. Solidifying the link to RAGE, these experiments revealed that infarction stimulated the rapid production of advanced glycation end-products in the heart. Thus, we tested the effect of ligand decoy soluble RAGE (sRAGE). Administration of sRAGE protected the myocardium from ischemic damage, similar to the effects observed in RAGE−/− mouse hearts. Taken together, these data implicate RAGE and its ligands in the pathogenesis of I/R injury and identify JNK and STAT signal transduction as central downstream effector pathways of the ligand-RAGE axis in the heart subjected to I/R injury.
The intrinsic resistance of female hearts to an ischemic insult is abrogated in primary cardiac hypertrophy
Authors: Bell James R. , Porrello Enzo R. , Huggins Catherine E. , Harrap Stephen B. , Delbridge Lea M. D.
Published: February 2008

Important sex differences in cardiovascular disease outcomes exist, including conditions of hypertrophic cardiomyopathy and cardiac ischemia. Studies of sex differences in the extent to which load-independent (primary) hypertrophy modulates the response to ischemia-reperfusion (I/R) damage have not been characterized. We have previously described a model of primary genetic cardiac hypertrophy, the hypertrophic heart rat (HHR). In this study the sex differences in HHR cardiac function and responses to I/R [compared to control normal heart rat (NHR)] were investigated ex vivo. The ventricular weight index was markedly increased in HHR female (7.82 ± 0.49 vs. 4.80 ± 0.10 mg/g; P < 0.05) and male (5.76 ± 0.22 vs. 4.62 ± 0.07 mg/g; P < 0.05) hearts. Female hearts of both strains exhibited a reduced basal contractility compared with strain-matched males [maximum first derivative of pressure (dP/d tmax): NHR, 4,036 ± 171 vs. 4,258 ± 152 mmHg/s; and HHR, 3,974 ± 160 vs. 4,540 ± 259 mmHg/s; P < 0.05]. HHR hearts were more susceptible to I/R (I = 25 min, and R = 30 min) injury than NHR hearts (decreased functional recovery, and increased lactate dehydrogenase efflux). Female NHR hearts exhibited a significantly greater recovery (dP/d tmax) post-I/R relative to male NHR (95.0 ± 12.2% vs. 60.5 ± 9.4%), a resistance to postischemic dysfunction not evident in female HHR (29.0 ± 5.6% vs. 25.9 ± 6.3%). Ventricular fibrillation was suppressed, and expression levels of Akt and ERK1/2 were selectively elevated in female NHR hearts. Thus the occurrence of load-independent primary cardiac hypertrophy undermines the intrinsic resistance of female hearts to I/R insult, with the observed abrogation of endogenous cardioprotective signaling pathways consistent with a potential mechanistic role in this loss of protection.
Effect of dietary sodium on estrogen regulation of blood pressure in Dahl salt-sensitive rats
Authors: Zheng Wei , Ji Hong , Maric Christine , Wu Xie , Sandberg Kathryn
Published: February 2008

The effects of high-sodium (HS) and normal-sodium (NS) diets on ovarian hormone modulation of mean arterial pressure (MAP) were examined in Dahl salt-resistant (DR) and salt-sensitive (DS) rats. Ovariectomy increased MAP (OVX-Sham) to a greater extent in DS rats maintained for 2 wk on a HS (22 mmHg) compared with a NS (6 mmHg) diet. Ovariectomy had no effect on MAP in DR rats on NS but did increase MAP in rats on HS (10 mmHg) diets. On HS diets, glomerular filtration rate (GFR) was 36% less in the DS-Sham than DR-Sham animals; ovariectomy increased GFR in both strains by 1.4–1.5-fold; glomerular angiotensin II type 1 receptor (AT1R) densities were 1.6-fold higher in the DS-Sham than in the DR-Sham group; ovariectomy increased glomerular AT1R densities by 1.3-fold in DR rats but had no effect in DS rats; 17β-estradiol (E2) downregulated adrenal AT1R densities in both strains on either diet; ovariectomy reduced estrogen receptor-α (ER-α) protein expression in the renal cortex by 40–50% although renal ER-α expression was 34% lower in DS than in DR rats. These observed effects of gonadectomy were prevented by E2treatment, suggesting that E2deficiency mediates the effects of ovariectomy on MAP, GFR, AT1R densities, and renal ER-α protein expression. In conclusion, ovariectomy-induced increases in MAP are augmented by HS diet in both strains, and this effect is not mediated by a reduction in GFR. Aberrant renal AT1R regulation and reduced renal ER-α expression are potential contributors to the hypertensive effects of E2deficiency in DS rats. These findings have implications for women with salt-sensitive hypertension and women who are E2deficient, such as postmenopausal women.