bluuebunny/crossref_metadata_2025 huggingface dataset:
Microarray and Real-Time PCR Analyses of the Responses of High-Arctic Soil Bacteria to Hydrocarbon Pollution and Bioremediation Treatments
Authors: Yergeau Etienne , Arbour Mélanie , Brousseau Roland , Juck David , Lawrence John R. , Masson Luke , Whyte Lyle G. , Greer Charles W.
Published: August 2009

High-Arctic soils have low nutrient availability, low moisture content, and very low temperatures and, as such, they pose a particular problem in terms of hydrocarbon bioremediation. An in-depth knowledge of the microbiology involved in this process is likely to be crucial to understand and optimize the factors most influencing bioremediation. Here, we compared two distinct large-scale field bioremediation experiments, located at the Canadian high-Arctic stations of Alert (ex situ approach) and Eureka (in situ approach). Bacterial community structure and function were assessed using microarrays targeting the 16S rRNA genes of bacteria found in cold environments and hydrocarbon degradation genes as well as quantitative reverse transcriptase PCR targeting key functional genes. The results indicated a large difference between sampling sites in terms of both soil microbiology and decontamination rates. A rapid reorganization of the bacterial community structure and functional potential as well as rapid increases in the expression of alkane monooxygenases and polyaromatic hydrocarbon-ring-hydroxylating dioxygenases were observed 1 month after the bioremediation treatment commenced in the Alert soils. In contrast, no clear changes in community structure were observed in Eureka soils, while key gene expression increased after a relatively long lag period (1 year). Such discrepancies are likely caused by differences in bioremediation treatments (i.e., ex situ versus in situ), weathering of the hydrocarbons, indigenous microbial communities, and environmental factors such as soil humidity and temperature. In addition, this study demonstrates the value of molecular tools for the monitoring of polar bacteria and their associated functions during bioremediation.
Development and Validation of a Species-Independent Functional Gene Microarray That Targets Lactic Acid Bacteria
Authors: Weckx Stefan , Allemeersch Joke , Van der Meulen Roel , Vrancken Gino , Huys Geert , Vandamme Peter , Van Hummelen Paul , De Vuyst Luc
Published: August 2009

During the last few years, genome-related information has become available for many microorganisms, including important food-related bacteria. Lactic acid bacteria (LAB) are important industrially in the production of fermented foods such as dairy products, sausages, sourdoughs, and vegetables. Despite their limited metabolic capacity, LAB contribute considerably to important characteristics of fermented foods, such as flavor and texture. In the present study, a species-independent functional gene microarray was developed that targets 406 genes that play key roles in the production of sugar catabolites, bacteriocins, exopolysaccharides, and aromas, in probiotic and biosafety characteristics, and in the stress response. Also, genes linked to negative traits, such as antibiotic resistance and virulence, are represented. As LAB ecosystems contain a variety of species, there was a more global focus on these specific functional properties. Thus, an algorithm was used to design gene-specific oligonucleotides that preferably hybridize with multiple LAB species, thereby allowing controlled cross-hybridization. For proof of concept, the microarray composed of 2,269 30-mer oligonucleotides focused on LAB species that are prevalent in sourdough ecosystems. Validation hybridizations using DNA and RNA from 18 LAB strains, covering 86% of all the oligonucleotides, showed that there were wide ranges in intensity and high reproducibility between microarrays.
Identification of Particle Size Classes Inhibiting Protozoan Recovery from Surface Water Samples via U.S. Environmental Protection Agency Method 1623
Authors: Krometis Leigh-Anne H. , Characklis Gregory W. , Sobsey Mark D.
Published: August 2009

Giardia
species recovery by U.S. Environmental Protection Agency method 1623 appears significantly impacted by a wide size range (2 to 30 μm) of particles in water and organic matter.
Cryptospori
dium
species recovery seems negatively correlated only with smaller (2 to 10 μm), presumably inorganic particles. Results suggest constituents and mechanisms interfering with method performance may differ by protozoan type.
Acetolactate Synthase from
Bacillus subtilis
Serves as a 2-Ketoisovalerate Decarboxylase for Isobutanol Biosynthesis in
Escherichia coli
Authors: Atsumi Shota , Li Zhen , Liao James C.
Published: August 2009

A pathway toward isobutanol production previously constructed in
Escherichia coli
involves 2-ketoacid decarboxylase (Kdc) from
Lactococcus lactis
that decarboxylates 2-ketoisovalerate (KIV) to isobutyraldehyde. Here, we showed that a strain lacking Kdc is still capable of producing isobutanol. We found that acetolactate synthase from
Bacillus subtilis
(AlsS), which originally catalyzes the condensation of two molecules of pyruvate to form 2-acetolactate, is able to catalyze the decarboxylation of KIV like Kdc both in vivo and in vitro. Mutational studies revealed that the replacement of Q487 with amino acids with small side chains (Ala, Ser, and Gly) diminished only the decarboxylase activity but maintained the synthase activity.
Furfural Inhibits Growth by Limiting Sulfur Assimilation in EthanologenicEscherichia coliStrain LY180
Authors: Miller Elliot N. , Jarboe Laura R. , Turner Peter C. , Pharkya Priti , Yomano Lorraine P. , York Sean W. , Nunn David , Shanmugam K. T. , Ingram Lonnie O.
Published: August 2009

A wide variety of commercial products can be potentially made from monomeric sugars produced by the dilute acid hydrolysis of lignocellulosic biomass. However, this process is accompanied by side products such as furfural that hinder microbial growth and fermentation. To investigate the mechanism of furfural inhibition, mRNA microarrays of an ethanologenic strain ofEscherichia coli(LY180) were compared immediately prior to and 15 min after a moderate furfural challenge. Expression of genes and regulators associated with the biosynthesis of cysteine and methionine was increased by furfural, consistent with a limitation of these critical metabolites. This was in contrast to a general stringent response and decreased expression of many other biosynthetic genes. Of the 20 amino acids individually tested as supplements (100 μM each), cysteine and methionine were the most effective in increasing furfural tolerance with serine (precursor of cysteine), histidine, and arginine of lesser benefit. Supplementation with other reduced sulfur sources such asd-cysteine and thiosulfate also increased furfural tolerance. In contrast, supplementation with taurine, a sulfur source that requires 3 molecules of NADPH for sulfur assimilation, was of no benefit. Furfural tolerance was also increased by inserting a plasmid encodingpntAB, a cytoplasmic NADH/NADPH transhydrogenase. Based on these results, a model is proposed for the inhibition of growth in which the reduction of furfural by YqhD, an enzyme with a lowKmfor NADPH, depletes NADPH sufficiently to limit the assimilation of sulfur into amino acids (cysteine and methionine) by CysIJ (sulfite reductase).
Functional Assembly of Minicellulosomes on the
Saccharomyces cerevisiae
Cell Surface for Cellulose Hydrolysis and Ethanol Production
Authors: Tsai Shen-Long , Oh Jeongseok , Singh Shailendra , Chen Ruizhen , Chen Wilfred
Published: August 2009

We demonstrated the functional display of a miniscaffoldin on the
Saccharomyces cerevisiae
cell surface consisting of three divergent cohesin domains from
Clostridium thermocellum
(t),
Clostridium cellulolyticum
(c), and
Ruminococcus flavefaciens
(f). Incubation with
Escherichia coli
lysates containing an endoglucanase (CelA) fused with a dockerin domain from
C. thermocellum
(At), an exoglucanase (CelE) from
C. cellulolyticum
fused with a dockerin domain from the same species (Ec), and an endoglucanase (CelG) from
C. cellulolyticum
fused with a dockerin domain from
R. flavefaciens
(Gf) resulted in the assembly of a functional minicellulosome on the yeast cell surface. The displayed minicellulosome retained the synergistic effect for cellulose hydrolysis. When a β-glucosidase (BglA) from
C. thermocellum
tagged with the dockerin from
R. flavefaciens
was used in place of Gf, cells displaying the new minicellulosome exhibited significantly enhanced glucose liberation and produced ethanol directly from phosphoric acid-swollen cellulose. The final ethanol concentration of 3.5 g/liter was 2.6-fold higher than that obtained by using the same amounts of added purified cellulases. The overall yield was 0.49 g of ethanol produced per g of carbohydrate consumed, which corresponds to 95% of the theoretical value. This result confirms that simultaneous and synergistic saccharification and fermentation of cellulose to ethanol can be efficiently accomplished with a yeast strain displaying a functional minicellulosome containing all three required cellulolytic enzymes.
Inducible-Expression Plasmid for
Rhodobacter sphaeroides
and
Paracoccus denitrificans
Authors: Ind Alice C. , Porter Steven L. , Brown Mostyn T. , Byles Elaine D. , de Beyer Jennifer A. , Godfrey Scott A. , Armitage Judith P.
Published: August 2009

We have developed a stable isopropyl-β-
d
-thiogalactopyranoside (IPTG)-inducible-expression plasmid, pIND4, which allows graduated levels of protein expression in the alphaproteobacteria
Rhodobacter sphaeroides
and
Paracoccus denitrificans
. pIND4 confers kanamycin resistance and combines the stable replicon of pMG160 with the
lacI
q
gene from pYanni3 and the
lac
promoter, P
A1/04/03
, from pJBA24.
Independent Transport and Sorting of Functionally Distinct Protein Families in
Tetrahymena thermophila
Dense Core Secretory Granules
Authors: Rahaman Abdur , Miao Wei , Turkewitz Aaron P.
Published: August 2009

Dense core granules (DCGs) in
Tetrahymena thermophila
contain two protein classes. Proteins in the first class, called granule lattice (Grl), coassemble to form a crystalline lattice within the granule lumen. Lattice expansion acts as a propulsive mechanism during DCG release, and Grl proteins are essential for efficient exocytosis. The second protein class, defined by a C-terminal β/γ-crystallin domain, is poorly understood. Here, we have analyzed the function and sorting of Grt1p (
gr
anule
t
ip), which was previously identified as an abundant protein in this family. Cells lacking all copies of
GRT1
, together with the closely related
GRT2
, accumulate wild-type levels of docked DCGs. Unlike cells disrupted in any of the major
GRL
genes, Δ
GRT1
Δ
GRT2
cells show no defect in secretion, indicating that neither exocytic fusion nor core expansion depends on
GRT1
. These results suggest that Grl protein sorting to DCGs is independent of Grt proteins. Consistent with this, the granule core lattice in Δ
GRT1
Δ
GRT2
cells appears identical to that in wild-type cells by electron microscopy, and the only biochemical component visibly absent is Grt1p itself. Moreover, gel filtration showed that Grl and Grt proteins in cell homogenates exist in nonoverlapping complexes, and affinity-isolated Grt1p complexes do not contain Grl proteins. These data demonstrate that two major classes of proteins in
Tetrahymena
DCGs are likely to be independently transported during DCG biosynthesis and play distinct roles in granule function. The role of Grt1p may primarily be postexocytic; consistent with this idea, DCG contents from Δ
GRT1
Δ
GRT2
cells appear less adhesive than those from the wild type.
Identification of
Francisella tularensis
Live Vaccine Strain CuZn Superoxide Dismutase as Critical for Resistance to Extracellularly Generated Reactive Oxygen Species
Authors: Melillo Amanda A. , Mahawar Manish , Sellati Timothy J. , Malik Meenakshi , Metzger Dennis W. , Melendez J. Andres , Bakshi Chandra Shekhar
Published: August 2009

Francisella tularensis
is an intracellular pathogen whose survival is in part dependent on its ability to resist the microbicidal activity of host-generated reactive oxygen species (ROS) and reactive nitrogen species (RNS). In numerous bacterial pathogens, CuZn-containing superoxide dismutases (SodC) are important virulence factors, localizing to the periplasm to offer protection from host-derived superoxide radicals (O
2
−
). In the present study, mutants of
F
.
tularensis
live vaccine strain (LVS) deficient in superoxide dismutases (SODs) were used to examine their role in defense against ROS/RNS-mediated microbicidal activity of infected macrophages. An in-frame deletion
F
.
tularensis
mutant of
sodC
(Δ
sodC
) and a
F
.
tularensis
Δ
sodC
mutant with attenuated Fe-superoxide dismutase (
sodB
) gene expression (
sodB
Δ
sodC
) were constructed and evaluated for susceptibility to ROS and RNS in gamma interferon (IFN-γ)-activated macrophages and a mouse model of respiratory tularemia. The
F
.
tularensis
Δ
sodC
and
sodB
Δ
sodC
mutants showed attenuated intramacrophage survival in IFN-γ-activated macrophages compared to the wild-type
F
.
tularensis
LVS. Transcomplementing the
sodC
gene in the Δ
sodC
mutant or inhibiting the IFN-γ-dependent production of O
2
−
or nitric oxide (NO) enhanced intramacrophage survival of the
sod
mutants. The Δ
sodC
and
sodB
Δ
sodC
mutants were also significantly attenuated for virulence in intranasally challenged C57BL/6 mice compared to the wild-type
F
.
tularensis
LVS. As observed for macrophages, the virulence of the Δ
sodC
mutant was restored in
ifn
-γ
−/−
,
inos
−
/
−
, and
phox
−
/
−
mice, indicating that SodC is required for resisting host-generated ROS. To conclude, this study demonstrates that SodB and SodC act to confer protection against host-derived oxidants and contribute to intramacrophage survival and virulence of
F
.
tularensis
in mice.
Structural and Mutational Analysis of Band 7 Proteins in the Cyanobacterium
Synechocystis
sp. Strain PCC 6803
Authors: Boehm Marko , Nield Jon , Zhang Pengpeng , Aro Eva-Mari , Komenda Josef , Nixon Peter J.
Published: August 2009

Band 7 proteins, which encompass members of the stomatin, prohibitin, flotillin, and HflK/C protein families, are integral membrane proteins that play important physiological roles in eukaryotes but are poorly characterized in bacteria. We have studied the band 7 proteins encoded by the cyanobacterium
Synechocystis
sp. strain PCC 6803, with emphasis on their structure and proposed role in the assembly and maintenance of the photosynthetic apparatus. Mutagenesis revealed that none of the five band 7 proteins (Slr1106, Slr1128, Slr1768, Sll0815, and Sll1021) was essential for growth under a range of conditions (including high light, salt, oxidative, and temperature stresses), although motility was compromised in an Slr1768 inactivation mutant. Accumulation of the major photosynthetic complexes in the thylakoid membrane and repair of the photosystem II complex following light damage were similar in the wild type and a quadruple mutant. Cellular fractionation experiments indicated that three of the band 7 proteins (Slr1106, Slr1768, and Slr1128) were associated with the cytoplasmic membrane, whereas Slr1106, a prohibitin homologue, was also found in the thylakoid membrane fraction. Blue native gel electrophoresis indicated that these three proteins, plus Sll0815, formed large (>669-kDa) independent complexes. Slr1128, a stomatin homologue, has a ring-like structure with an approximate diameter of 16 nm when visualized by negative stain electron microscopy. No evidence for band 7/FtsH supercomplexes was found. Overall, our results indicate that the band 7 proteins form large homo-oligomeric complexes but do not play a crucial role in the biogenesis of the photosynthetic apparatus in
Synechocystis
sp. strain PCC 6803.
Site-Specific Insertion of IS
492
in
Pseudoalteromonas atlantica
Authors: Higgins Brian P. , Popkowski Adam C. , Caruana Peter R. , Karls Anna C.
Published: August 2009

Reversible insertion of IS
492
at a site within
epsG
on the
Pseudoalteromonas atlantica
chromosome controls peripheral extracellular polysaccharide production and biofilm formation by
P. atlantica
. High-frequency precise excision of IS
492
from
epsG
requires 5 and 7 bp of flanking DNA, suggesting that IS
492
transposition involves a site-specific recombination mechanism. The site specificity of IS
492
insertion was examined in
P. atlantica
and shown to be specific for a 7-bp target, 5′-CTTGTTA-3′. Characterization of numerous insertion events at the target site in
epsG
indicated that insertion is also orientation specific. The frequency of IS
492
insertion at the
epsG
target site (2.7 × 10
−7
/cell/generation), determined by quantitative PCR, is 4 to 5 orders of magnitude lower than the frequency of IS
492
precise excision from the same site. Comparison of insertion sites for IS
492
and the highly related IS
Ptu2
from
Pseudoalteromonas tunicata
suggests DNA sequence and/or structural features that may contribute to site recognition and recombination by the transposase of IS
492
.
Inactivation of VicK Affects Acid Production and Acid Survival of
Streptococcus mutans
Authors: Senadheera D. , Krastel K. , Mair R. , Persadmehr A. , Abranches J. , Burne R. A. , Cvitkovitch Dennis G.
Published: August 2009

The regulation of acid production in and the tolerance to low pH of the cariogenic bacterium
Streptococcus mutans
have garnered considerable attention since both of these properties contribute substantially to the virulence of this organism. Frequent or prolonged exposure to acid end products, mainly lactic acid, that are present following the consumption of dietary sugars erodes the dental enamel, thereby initiating dental caries. Here we report the involvement of the
S. mutans
VicK sensor kinase in both the acidogenicity and the aciduricity of this bacterium. When cultures were supplemented with glucose, the glycolytic rate of a VicK null mutant was significantly decreased compared to the glycolytic rate of the wild type (
P
< 0.05), suggesting that there was impaired acid production. Not surprisingly, the VicK deletion mutant produced less lactic acid, while an acid tolerance response assay revealed that loss of VicK significantly enhanced the survival of
S. mutans
(
P
< 0.05). Compared to the survival rates of the wild type, the survival rates of the VicK-deficient mutant were drastically increased when cultures were grown at pH 3.5 with or without preexposure to a signal pH (pH 5.5). Global transcriptional analysis using DNA microarrays and
S. mutans
wild-type UA159 and VicK deletion mutant strains grown at neutral and low pH values revealed that loss of VicK significantly affected expression of 89 transcripts more than twofold at pH 5.5 (
P
< 0.001). The affected transcripts included genes with putative functions in transport and maintenance of cell membrane integrity. While our results provide insight into the acid-inducible regulon of
S. mutans
, here we imply a novel role for VicK in regulating intracellular pH homeostasis in
S. mutans
.
Malonic Semialdehyde Reductase, Succinic Semialdehyde Reductase, and Succinyl-Coenzyme A Reductase from
Metallosphaera sedula
: Enzymes of the Autotrophic 3-Hydroxypropionate/4-Hydroxybutyrate Cycle in
Sulfolobales
Authors: Kockelkorn Daniel , Fuchs Georg
Published: August 2009

A 3-hydroxypropionate/4-hydroxybutyrate cycle operates during autotrophic CO
2
fixation in various members of the
Crenarchaea
. In this cycle, as determined using
Metallosphaera sedula
, malonyl-coenzyme A (malonyl-CoA) and succinyl-CoA are reductively converted via their semialdehydes to the corresponding alcohols 3-hydroxypropionate and 4-hydroxybutyrate. Here three missing oxidoreductases of this cycle were purified from
M. sedula
and studied. Malonic semialdehyde reductase, a member of the 3-hydroxyacyl-CoA dehydrogenase family, reduces malonic semialdehyde with NADPH to 3-hydroxypropionate. The latter compound is converted via propionyl-CoA to succinyl-CoA. Succinyl-CoA reduction to succinic semialdehyde is catalyzed by malonyl-CoA/succinyl-CoA reductase, a promiscuous NADPH-dependent enzyme that is a paralogue of aspartate semialdehyde dehydrogenase. Succinic semialdehyde is then reduced with NADPH to 4-hydroxybutyrate by succinic semialdehyde reductase, an enzyme belonging to the Zn-dependent alcohol dehydrogenase family. Genes highly similar to the
Metallosphaera
genes were found in other members of the
Sulfolobales
. Only distantly related genes were found in the genomes of autotrophic marine
Crenarchaeota
that may use a similar cycle in autotrophic carbon fixation.
Self-Enhanced Accumulation of FtsN at Division Sites and Roles for Other Proteins with a SPOR Domain (DamX, DedD, and RlpA) in
Escherichia coli
Cell Constriction
Authors: Gerding Matthew A. , Liu Bing , Bendezú Felipe O. , Hale Cynthia A. , Bernhardt Thomas G. , de Boer Piet A. J.
Published: August 2009

Of the known essential division proteins in
Escherichia coli
, FtsN is the last to join the septal ring organelle. FtsN is a bitopic membrane protein with a small cytoplasmic portion and a large periplasmic one. The latter is thought to form an α-helical juxtamembrane region, an unstructured linker, and a C-terminal, globular, murein-binding SPOR domain. We found that the essential function of FtsN is accomplished by a surprisingly small essential domain (
E
FtsN) of at most 35 residues that is centered about helix H2 in the periplasm.
E
FtsN contributed little, if any, to the accumulation of FtsN at constriction sites. However, the isolated SPOR domain (
S
FtsN) localized sharply to these sites, while SPOR-less FtsN derivatives localized poorly. Interestingly, localization of
S
FtsN depended on the ability of cells to constrict and, thus, on the activity of
E
FtsN. This and other results suggest that, compatible with a triggering function, FtsN joins the division apparatus in a self-enhancing fashion at the time of constriction initiation and that its SPOR domain specifically recognizes some form of septal murein that is only transiently available during the constriction process. SPOR domains are widely distributed in bacteria. The isolated SPOR domains of three additional
E. coli
proteins of unknown function, DamX, DedD, and RlpA, as well as that of
Bacillus subtilis
CwlC, also accumulated sharply at constriction sites in
E. coli
, suggesting that septal targeting is a common property of SPORs. Further analyses showed that DamX and, especially, DedD are genuine division proteins that contribute significantly to the cell constriction process.
A Genome-Scale Proteomic Screen Identifies a Role for DnaK in Chaperoning of Polar Autotransporters inShigella
Authors: Janakiraman Anuradha , Fixen Kathryn R. , Gray Andrew N. , Niki Hironori , Goldberg Marcia B.
Published: August 2009

Autotransporters are outer membrane proteins that are widely distributed among gram-negative bacteria. Like other autotransporters, theShigellaautotransporter IcsA, which is required for actin assembly during infection, is secreted at the bacterial pole. In the bacterial cytoplasm, IcsA localizes to poles and potential cell division sites independent of the cell division protein FtsZ. To identify bacterial proteins involved in the targeting of IcsA to the pole in the bacterial cytoplasm, we screened a genome-scale library ofEscherichia coliproteins tagged with green fluorescent protein (GFP) for those that displayed a localization pattern similar to that of IcsA-GFP in cells that lack functional FtsZ using a strain carrying a temperature-sensitiveftsZallele. For each protein that mimicked the localization of IcsA-GFP, we tested whether IcsA localization was dependent on the presence of the protein. Although these approaches did not identify a polar receptor for IcsA, the cytoplasmic chaperone DnaK both mimicked IcsA localization at elevated temperatures as a GFP fusion and was required for the localization of IcsA to the pole in the cytoplasm ofE. coli. DnaK was also required for IcsA secretion at the pole inShigella flexneri. The localization of DnaK-GFP to poles and potential cell division sites was dependent on elevated growth temperature and independent of the presence of IcsA or functional FtsZ; native DnaK was found to be enhanced at midcell and the poles. A secondShigellaautotransporter, SepA, also required DnaK for secretion, consistent with a role of DnaK more generally in the chaperoning of autotransporter proteins in the bacterial cytoplasm.
A New Member of the
Escherichia coli fad
Regulon: Transcriptional Regulation of
fadM
(
ybaW
)
Authors: Feng Youjun , Cronan John E.
Published: August 2009

Recently, Nie and coworkers (L. Nie, Y. Ren, A. Janakiraman, S. Smith, and H. Schulz, Biochemistry 47:9618-9626, 2008) reported a new
Escherichia coli
thioesterase encoded by the
ybaW
gene that cleaves the thioester bonds of inhibitory acyl-coenzyme A (CoA) by-products generated during β-oxidation of certain unsaturated fatty acids. These authors suggested that
ybaW
expression might be regulated by FadR, the repressor of the
fad
(fatty acid degradation) regulon. We report mapping of the
ybaW
promoter and show that
ybaW
transcription responded to FadR in vivo. Moreover, purified FadR bound to a DNA sequence similar to the canonical FadR binding site located upstream of the
ybaW
coding sequence and was released from the promoter upon the addition of long-chain acyl-CoA thioesters. We therefore propose the designation
fadM
in place of
ybaW
. Although FadR regulation of
fadM
expression had the pattern typical of
fad
regulon genes, its modulation by the cyclic AMP (cAMP) receptor protein-cAMP complex (CRP-cAMP) global regulator was the opposite of that normally observed. CRP-cAMP generally acts as an activator of
fad
gene expression, consistent with the low status of fatty acids as carbon sources. However, glucose growth stimulated
fadM
expression relative to acetate growth, as did inactivation of CRP-cAMP, indicating that the complex acts as a negative regulator of this gene. The stimulation of
fadM
expression seen upon deletion of the gene encoding adenylate cyclase (Δ
cya
) was reversed by supplementation of the growth medium with cAMP. Nie and coworkers also reported that growth on a conjugated linoleic acid isomer yields much higher levels of FadM thioesterase activity than does growth on oleic acid. In contrast, we found that the conjugated linoleic acid isomer was only a weak inducer of
fadM
expression. Although the gene is not essential for growth, the high basal level of
fadM
expression under diverse growth conditions suggests that the encoded thioesterase has functions in addition to β-oxidation.
The Iho670 Fibers of
Ignicoccus hospitalis
: a New Type of Archaeal Cell Surface Appendage
Authors: Müller Daniel W. , Meyer Carolin , Gürster Sonja , Küper Ulf , Huber Harald , Rachel Reinhard , Wanner Gerhard , Wirth Reinhard , Bellack Annett
Published: August 2009

Ignicoccus hospitalis
forms many cell surface appendages, the Iho670 fibers (width, 14 nm; length, up to 20 μm), which constitute up to 5% of cellular protein. They are composed mainly of protein Iho670, possessing no homology to archaeal flagellins or fimbrins. Their existence as structures different from archaeal flagella or fimbriae have gone unnoticed up to now because they are very brittle.
The Multifaceted Proteins MvaT and MvaU, Members of the H-NS Family, Control Arginine Metabolism, Pyocyanin Synthesis, and Prophage Activation in
Pseudomonas aeruginosa
PAO1
Authors: Li Congran , Wally Hassan , Miller Shearman J. , Lu Chung-Dar
Published: August 2009

The MvaT and MvaU proteins belonging to the H-NS family were identified as DNA-binding proteins that interact with the regulatory region of the
aotJQMOP-argR
operon for arginine uptake and regulation. Recombinant MvaT and MvaU proteins were purified, and binding of these purified proteins to the
aotJ
regulatory region was demonstrated using electromobility shift assays. Polyclonal antibodies against purified MvaT and MvaU were prepared and employed in supershift assays to support these observations. Knockout mutations resulting in a single lesion in
mvaT
or
mvaU
, as well as knockout mutations resulting in double lesions, were constructed using biparental conjugation, and the absence of MvaT and MvaU in the resulting mutants was confirmed by immunoblot analysis. Using measurements of the β-galactosidase activities from
aotJ
::
lacZ
fusions in the mutants and the parental strain, it was found that MvaT and MvaU serve as repressors in control of
aotJ
expression. The effects of MvaT and MvaU on pyocyanin synthesis and CupA fimbrial expression in these mutants were also analyzed. Pyocyanin synthesis was induced in the single mutants but was completely abolished in the double mutant, suggesting that there is a complicated regulatory scheme in which MvaT and MvaU are essential elements. In comparison, MvaT had a more profound role than MvaU as a repressor of
cupA
expression; however, a combination of MvaT depletion and MvaU depletion had a strong synergistic effect on
cupA
. Moreover, prophage Pf4 integrated into the chromosome of
Pseudomonas aeruginosa
PAO1 was activated in an
mvaT mvaU
double mutant but not in a single mutant. These results were supported by purification and nucleotide sequencing of replicative-form DNA and by the release of phage particles in plaque assays. In summary, the
mvaT mvaU
double mutant was viable, and depletion of MvaT and MvaU had serious effects on a variety of physiological functions in
P. aeruginosa
.
A systematic comparison of morphology and seed proteins of early- and late-flowering forms of Medicago scutellata
Authors: Small Ernest , Bauchan Gary R. , Salter Rosemary , Brookes Brenda , Auricht Geoff C.
Published: August 2009

Two hundred and thirty-nine germ plasm accessions of Medicago scutellata grown under greenhouse conditions exhibited a strongly bimodal distribution of flowering time. Numerical taxonomic analysis showed that the early- and late-flowering forms are substantially different morphologically. The early-flowering form is smaller and less vigorous, has fewer flowers in the inflorescence, and fewer serrations on the leaf blades and stipules than the late-flowering form. Analysis of seed proteins using polyacrylamide gel electrophoresis identified 11 seed proteins that can be used to distinguish the two forms and putative hybrids. Although both forms appear widespread in the circum-Mediterranean indigenous distribution area, only about one-third of the accessions represented the early-flowering form, which seemed to predominate only on isolated Mediterranean islands. The two forms may merit infraspecific taxonomic recognition. Key words: Medicago scutellata, Fabaceae, taxonomy, classification, systematics, seed proteins, electrophoresis, flowering time.
HANDWRITTEN SHORTHAND AND ITS FUTURE POTENTIAL FOR FAST MOBILE TEXT ENTRY
Authors: LEEDHAM GRAHAM , MA YANG , BLUMENSTEIN MICHAEL
Published: August 2009

Handwritten shorthand systems were devised to enable writers to record information on paper at fast speeds, ideally at the speed of speech. While they have been in existence for many years it is only since the 17th Century that widespread usage appeared. Several shorthand systems flourished in the first half of the 20th century until the introduction and widespread use of electronic recording and dictation machines in the 1970's. Since then, shorthand usage has been in rapid decline, but has not yet become a lost skill. Pitman shorthand has been shown to possess unique advantages as a means of fast text entry which is particularly applicable to hand-held devices in mobile environments. This paper presents progress and critical research issues for a Pitman/Renqun Shorthand Online Recognition System. Recognition and transcription experiments are reported which indicate that a correct recognition and transcription rate of around 90% is currently possible.
Postinfarction gene therapy with adenoviral vector expressing decorin mitigates cardiac remodeling and dysfunction
Authors: Li Longhu , Okada Hideshi , Takemura Genzou , Kosai Ken-ichiro , Kanamori Hiromitsu , Esaki Masayasu , Takahashi Tomoyuki , Goto Kazuko , Tsujimoto Akiko , Maruyama Rumi , Kawamura Itta , Kawaguchi Tomonori , Takeyama Toshiaki , Fujiwara Takako , Fujiwara Hisayoshi , Minatoguchi Shinya
Published: August 2009

The small leucine-rich proteoglycan decorin is a natural inhibitor of transforming growth factor-β (TGF-β) and exerts antifibrotic effects in heart and to stimulate skeletal muscle regeneration. We investigated decorin's chronic effects on postinfarction cardiac remodeling and dysfunction. Myocardial infarction (MI) was induced in mice by left coronary artery ligation. An adenoviral vector encoding human decorin (Ad. CAG-decorin) was then injected into the hindlimbs on day 3 post-MI (control, Ad.CAG-LacZ). Four weeks post-MI, the decorin-treated mice showed significant mitigation of the left ventricular dilatation and dysfunction seen in control mice. Although infarct size did not differ between the two groups, the infarcted wall thickness was greater and the segmental length of the infarct was smaller in decorin-treated mice. In addition, cellular components, including myofibroblasts and blood vessels, were more abundant within the infarcted area in decorin-treated mice, and fibrosis was significantly reduced in both the infarcted and noninfarcted areas of the left ventricular wall. Ten days post-MI, there was greater cell proliferation and less apoptosis among granulation tissue cells in the infarcted areas of decorin-treated mice. The treatment, however, did not affect proliferation and apoptosis of salvaged cardiomyocytes. Although decorin gene therapy did not affect TGF-β1 expression in the infarcted heart, it inhibited Smad2/3 activation (downstream mediators of TGF-β signaling). In summary, postinfarction decorin gene therapy mitigated cardiac remodeling and dysfunction by altering infarct tissue noncardiomyocyte dynamics and preventing cardiac fibrosis, accompanying inhibition of Smad2/3 activation.
Role of sarcoplasmic reticulum in mitochondrial permeability transition and cardiomyocyte death during reperfusion
Authors: Ruiz-Meana Marisol , Abellán Arancha , Miró-Casas Elisabet , Agulló Esperanza , Garcia-Dorado David
Published: August 2009

There is solid evidence that a sudden change in mitochondrial membrane permeability (mitochondrial permeability transition, MPT) plays a critical role in reperfusion-induced myocardial necrosis. We hypothesized that sarcoplasmic reticulum (SR) Ca2+cycling may induce partial MPT in microdomains of close anatomic proximity between mitochondria and SR, resulting in hypercontracture and cell death. MPT (mitochondrial calcein release), cell length, and sarcolemmal rupture (Trypan blue and lactate dehydrogenase release) were measured in adult rat cardiomyocytes submitted to simulated ischemia (NaCN/2-deoxyglucose, pH 6.4) and reperfusion. On simulated reperfusion, 83 ± 2% of myocytes developed hypercontracture. In 22 ± 6% of cases, hypercontracture was associated with sarcolemmal disruption [Trypan blue(+)]. During simulated reperfusion there was a 25% release of cyclosporin A-sensitive mitochondrial calcein (with respect to total mitochondrial calcein content). Simultaneous blockade of SR Ca2+uptake and release with thapsigargin and ryanodine, respectively, significantly reduced mitochondrial calcein release, hypercontracture, and cell death during simulated reperfusion. SR Ca2+blockers delayed mitochondrial Ca2+uptake in digitonin-permeabilized cardiomyocytes but did not have any effect on isolated mitochondria. Pretreatment with colchicine to disrupt microtubule network reduced the degree of fluorescent overlap between SR and mitochondria and abolished the protective effect of SR Ca2+blockers on MPT, hypercontracture, and cell death during reperfusion. We conclude that SR Ca2+cycling during reperfusion facilitates partial mitochondrial permeabilization due to the close anatomic proximity between both organelles, favoring hypercontracture and cell death.
Pharmacological stimulation of soluble guanylate cyclase modulates hypoxia-inducible factor-1α in rat heart
Authors: Tsuruda Toshihiro , Hatakeyama Kinta , Masuyama Hiroyuki , Sekita Yoko , Imamura Takuroh , Asada Yujiro , Kitamura Kazuo
Published: August 2009

Mechanical load and ischemia induce a series of adaptive physiological responses by activating the expression of O2-regulated genes, such as hypoxia inducible factor-1α (HIF-1α). The aim of this study was to explore the interaction between HIF-1α and soluble guanylate cyclase (sGC) and its second messenger cGMP in cultured cardiomyocytes exposed to hypoxia and in pressure-overloaded heart. In cultured cardiomyocytes of neonatal rats, either sGC stimulator BAY 41-2272 or cGMP analog 8-bromo-cGMP decreased the hypoxia (1% O2/5% CO2)-induced HIF-1α expression, whereas the inhibition of protein kinase G by KT-5823 reversed the effect of BAY 41-2272 on the expression under hypoxic conditions. In pressure-overloaded heart induced by suprarenal aortic constriction (AC) in 7-wk-old male Wistar rats, the administration of BAY 41-2272 (2 mg·kg−1·day−1) for 14 days significantly suppressed the protein expression of HIF-1α ( P < 0.05), vascular endothelial growth factor ( P < 0.01), and the number of capillary vessels ( P < 0.01) induced by pressure overload. This study suggests that the pharmacological sGC-cGMP stimulation modulates the HIF-1α expression in response to hypoxia or mechanical load in the heart.
Cardiac autonomic neural remodeling and susceptibility to sudden cardiac death: effect of endurance exercise training
Authors: Billman George E.
Published: August 2009

Sudden cardiac death resulting from ventricular tachyarrhythmias remains the leading cause of death in industrially developed countries, accounting for between 300,000 and 500,000 deaths each year in the United States. Yet, despite the enormity of this problem, both the identification of factors contributing to ventricular fibrillation as well as the development of safe and effective antiarrhythmic agents remain elusive. Subnormal cardiac parasympathetic regulation coupled with an elevated cardiac sympathetic activation may allow for the formation of malignant ventricular arrhythmias. In particular, myocardial infarction can reduce cardiac parasympathetic regulation and alter β-adrenoceptor subtype expression enhancing β2-adrenoceptor sensitivity that can lead to intracellular calcium dysregulation and arrhythmias. As such, myocardial infarction can induce a remodeling of cardiac autonomic regulation that may be required to maintain cardiac pump function. If alterations in cardiac autonomic regulation play an important role in the genesis of life-threatening arrhythmias, then one would predict that interventions designed to either augment parasympathetic activity and/or reduce cardiac adrenergic activity would also protect against ventricular fibrillation. Recently, studies using a canine model of sudden death demonstrate that endurance exercise training (treadmill running) enhanced cardiac parasympathetic regulation (increased heart rate variability), restored a more normal β-adrenoceptor balance (i.e., reduced β2-adrenoceptor sensitivity and expression), and protected against ventricular fibrillation induced by acute myocardial ischemia. Thus exercise training may reverse the autonomic neural remodeling induced by myocardial infarction and thereby enhance the electrical stability of the heart in individuals shown to be at an increased risk for sudden cardiac death.
Hypophosphatemia-mediated hypotension in transgenic mice overexpressing human FGF-23
Authors: Liu Peidang , Bai Xiuying , Wang Heming , Karaplis Andrew , Goltzman David , Miao Dengshun
Published: August 2009

Fibroblast growth factor-23 (FGF-23) is a potent circulating phosphaturic factor associated with renal phosphate wasting. The effects of FGF-23 on skeletal and phosphate homeostasis have been investigated widely; however, the effect of FGF-23 on the cardiovascular system (CVS) is unknown. To assess whether FGF-23 influences the function and structure of the CVS and whether the effect of FGF-23 on the CVS is mediated by FGF receptors directly or indirectly by hypophosphatemia, FGF-23 transgenic mice and their wild-type littermates were fed a normal diet or a high-phosphate diet comprising a normal diet plus 1.25% phosphate in drinking water from weaning for 5 wk, and the phenotypes of the CVS were compared between FGF-23 transgenic mice and their wild-type littermates on the same diet. At the end of this time period, transgenic animals on the normal diet developed hypotension. The left ventricle was appropriately hypertrophic, and plasma catecholamine and renin-angiotensin system components were upregulated, indicating compensatory mechanisms in response to the hypotension. Transgenic mice also exhibited an impaired vascular reactivity and a downregulation of vasoconstrictor receptor gene expression, possibly as pathogenetic factors contributing to the hypotension. The high-phosphate diet improved the hypophosphatemia, resulting in a rescue of the cardiovascular phenotype. This study demonstrates that FGF-23 overexpression can result in abnormalities in the CVS and that the effect of FGF-23 overexpression on the CVS is mediated by the secondary severe hypophosphatemia.
Angiotensin II maintains cerebral vascular relaxation via EGF receptor transactivation and ERK1/2
Authors: McEwen Scott T. , Balus Sarah F. , Durand Matthew J. , Lombard Julian H.
Published: August 2009

This study identified, on the integrative level, two components of the ANG II signaling pathway that lay downstream from the ANG II type 1 (AT1) receptor and are critically involved in maintaining vascular relaxation in cerebral resistance arteries. In these experiments, the relaxation of isolated middle cerebral arteries (MCA) in response to ACh (10−9-10−5M), iloprost (10−16-10−11g/ml), and reduced PO2was lost and the ratio of phospho-ERK/ERK1/2 was significantly reduced in aortas of male Sprague-Dawley rats fed a high-salt (HS; 4% NaCl) diet to suppress plasma ANG II levels. In salt-fed rats, relaxation of MCA in response to these vasodilator stimuli was restored by chronic (3 days) intravenous infusion of either ANG II (5 ng·kg−1·min−1) or epidermal growth factor (EGF; 2 μg/h). The protective effect of ANG II infusion to restore vascular relaxation was eliminated by coinfusion of either the EGF receptor kinase inhibitor AG-1478 (20 μg/h), the ERK1/2 inhibitor PD-98059 (10 μg/h), or the protein synthesis inhibitor cycloheximide (5 μg/h). In rats fed a low-salt (0.4% NaCl) diet, MCA relaxation in response to ACh, reduced PO2, and iloprost was eliminated by intravenous infusion of AG-1478, PD-98059, or cycloheximide. In ANG II-infused rats fed HS diet, and in rats fed LS diet, vasodilator responses to reduced PO2and iloprost were unaffected by the p38 MAP kinase inhibitor SB-203580 and the phosphatidylinositol 3-kinase inhibitor wortmannin. These findings indicate that maintenance of normal vascular relaxation mechanisms by ANG II in rat MCA requires activation of the EGF receptor kinase and ERK1/2.
Effect of ventilation pressure on alveolar fluid clearance and β-agonist responses in mice
Authors: Yu Erin N. Z. , Traylor Zachary P. , Davis Ian C.
Published: August 2009

High tidal volume ventilation is detrimental to alveolar fluid clearance (AFC), but effects of ventilation pressure (P) on AFC are unknown. In anesthetized BALB/c mice ventilated at constant tidal volume (8 ml/kg), mean AFC rate was 12.8% at 6 cmH2O P, but increased to 37.3% at 18 cmH2O P. AFC rate declined at 22 cmH2O P, which also induced lung damage. Increased AFC at 18 cmH2O P did not result from elevated plasma catecholamines, hypercapnia, or hypocapnia, but was due to augmented Na+ and Cl− absorption. PKA agonists and β-agonists stimulated AFC at 10 cmH2O P by upregulating amiloride-sensitive Na+ transport. However, at 18 cmH2O P, PKA agonists and β-agonists reduced AFC. At 15 cmH2O P, the AFC rate was intermediate (mean 26.6%), and forskolin and β-agonists had no effect. Comparable P dependency of AFC and β-agonist responsiveness was found in C57BL/6 mice. The effect on AFC of increasing P to 18 cmH2O was blocked by adenosine deaminase or an A2b-adenosine receptor antagonist, and could be mimicked by adenosine in mice ventilated at 10 cmH2O P. Modulation of adenosine signaling also resulted in altered responsiveness to β-agonists. These findings indicate that, in the normal mouse lung, basal AFC rates and responses to β-agonists are impacted by ventilation pressure in an adenosine-dependent manner.
EphA2 receptor mediates increased vascular permeability in lung injury due to viral infection and hypoxia
Authors: Cercone Melissa A. , Schroeder William , Schomberg Stacey , Carpenter Todd C.
Published: August 2009

Ephrin family receptor tyrosine kinases are mediators of angiogenesis that may also regulate endothelial barrier function in the lung. Previous work has demonstrated that stimulation of EphA ephrin receptors causes increased vascular leak in the intact lung and increased permeability in cultured endothelial cells. Whether EphA receptors are involved in the permeability changes associated with lung injury is unknown. We studied this question in young rats exposed to viral respiratory infection combined with exposure to moderate hypoxia, a previously described lung injury model. We found that the EphA2 receptor is expressed in normal lung and that EphA2 expression is markedly upregulated in the lungs of hypoxic infected (HV) rats compared with normal control animals. Immunohistochemistry showed increased EphA2 expression principally in areas of edematous alveolar septae. In HV rats, EphA2 antagonism with either the soluble decoy receptor EphA2/Fc or with monoclonal anti-EphA2 antibody reduced albumin extravasation and histological evidence of edema formation ( P < 0.01). Vascular leak in HV rats is mediated in large part by increased lung endothelin (ET) levels. In HV rats, ET receptor antagonism with bosentan resulted in reduced EphA2 mRNA and protein expression ( P < 0.01). Experiments with cultured rat lung microvascular endothelial cells demonstrated that ET increases endothelial EphA2 expression. These results suggest that EphA2 expression is increased in lung injury, contributes to vascular leak in the injured lung, and is regulated in endothelial cells by ET. EphA2 may be a previously unrecognized contributor to the pathophysiology of lung injury.
Modulation of reactive oxygen species by Rac1 or catalase prevents asbestos-induced pulmonary fibrosis
Authors: Murthy Shubha , Adamcakova-Dodd Andrea , Perry Sarah S. , Tephly Linda A. , Keller Richard M. , Metwali Nervana , Meyerholz David K. , Wang Yongqiang , Glogauer Michael , Thorne Peter S. , Carter A. Brent
Published: August 2009

The release of reactive oxygen species (ROS) and cytokines by alveolar macrophages has been demonstrated in asbestos-induced pulmonary fibrosis, but the mechanism linking alveolar macrophages to the pathogenesis is not known. The GTPase Rac1 is a second messenger that plays an important role in host defense. In this study, we demonstrate that Rac1 null mice are protected from asbestos-induced pulmonary fibrosis, as determined by histological and biochemical analysis. We hypothesized that Rac1 induced pulmonary fibrosis via generation of ROS. Asbestos increased TNF-α and ROS in a Rac1-dependent manner. TNF-α was elevated only 1 day after exposure, whereas ROS generation progressively increased in bronchoalveolar lavage cells obtained from wild-type (WT) mice. To determine whether ROS generation contributed to pulmonary fibrosis, we overexpressed catalase in WT monocytes and observed a decrease in ROS generation in vitro . More importantly, administration of catalase to WT mice attenuated the development of fibrosis in vivo. For the first time, these results demonstrate that Rac1 plays a crucial role in asbestos-induced pulmonary fibrosis. Moreover, it suggests that a simple intervention may be useful to prevent progression of the disease.