bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the Isophthalate Degradation Genes of
Comamonas
sp. Strain E6
Authors: Fukuhara Yuki , Inakazu Keisuke , Kodama Norimichi , Kamimura Naofumi , Kasai Daisuke , Katayama Yoshihiro , Fukuda Masao , Masai Eiji
Published: November 2009

The isophthalate (IPA) degradation gene cluster (
iphACBDR
) responsible for the conversion of IPA into protocatechuate (PCA) was isolated from
Comamonas
sp. strain E6, which utilizes phthalate isomers as sole carbon and energy sources via the PCA 4,5-cleavage pathway. Based on amino acid sequence similarity, the
iphA
,
iphC
,
iphB
,
iphD
, and
iphR
genes were predicted to code for an oxygenase component of IPA dioxygenase (IPADO), a periplasmic IPA binding receptor, a 1,2-dihydroxy-3,5-cyclohexadiene-1,5-dicarboxylate (1,5-DCD) dehydrogenase, a reductase component of IPADO, and an IclR-type transcriptional regulator, respectively. The
iphACBDR
genes constitute a single transcriptional unit, and transcription of the
iph
catabolic operon was induced during growth of E6 on IPA. The
iphA
,
iphD
, and
iphB
genes were expressed in
Escherichia coli
. Crude IphA and IphD converted IPA in the presence of NADPH into a product which was transformed to PCA by IphB. These results suggested that IPADO is a two-component dioxygenase that consists of a terminal oxygenase component (IphA) and a reductase component (IphD) and that
iphB
encodes the 1,5-DCD dehydrogenase. Disruption of
iphA
and
iphB
resulted in complete loss of growth of E6 on IPA. Inactivation of
iphD
significantly affected growth on IPA, and the
iphC
mutant did not grow on IPA at neutral pH. These results indicated that the
iphACBD
genes are essential for the catabolism of IPA in E6. Disruption of
iphR
resulted in faster growth of E6 on IPA, suggesting that
iphR
encodes a repressor for the
iph
catabolic operon. Promoter analysis of the operon supported this notion.
Multiplex PCR Method for Detection of Three
Aeromonas
Enterotoxin Genes
Authors: Bin Kingombe Cesar I. , D'Aoust Jean-Yves , Huys Geert , Hofmann Lisa , Rao Mary , Kwan Judy
Published: November 2009

A novel multiplex PCR method using three sets of specific primers was developed for the detection of the cytotoxic (
act
), heat-labile (
alt
), and heat-stable (
ast
) enterotoxin genes in
Aeromonas
spp. This assay was used to characterize 35 reference strains as well as 537 food-borne isolates. A total of seven gene pattern combinations were encountered, including
act
,
alt
,
act/alt
,
act/alt/ast
,
act/alt/
148-bp amplicon,
alt/ast
, and
alt
/148-bp amplicon. The
alt
gene was detected with 34 reference strains (97%) and occurred singly in 14% of these strains. The frequency of occurrence of the
act/alt
,
act/alt/ast
, and
alt/ast
gene patterns in reference strains was 14 (40%), 2 (6%), and 2 (6%), respectively. An unpredicted amplicon was detected in 11 reference strains (31%). Characterization of this amplicon showed that its size was 148 bp, as generated by the AHLF and AHLR primers, and that it uniquely aligned with the
Aeromonas salmonicida
A449 genome sequence (GenBank accession number CP000644). This amplicon was named
Aeromonas salmonicida
subsp.
salmonicida
hypothetical protein amplicon (AssHPA). In the 537 food-borne isolates, the
act
and
alt
genes were most dominant and were detected in 349 (65%) and 452 (84%) isolates, respectively, either alone or in combinations. The
act
and
alt
genes occurred singly in 30 (6%) and 128 (24%) of these strains, respectively. The
act/alt
gene pattern occurred in 315 isolates (59%), whereas the
ast
gene was always linked to strains exhibiting the
act/alt/ast
and
alt/ast
gene combinations in 4 (0.7%) and 5 (0.9%) isolates, respectively. The uniplex amplification of three enterotoxin genes separately confirms the specificity of the unique selected primers. This multiplex PCR is rapid and simple and can detect the presence of three
Aeromonas
enterotoxin genes in a single assay.
Association of Beta-Glucan Endogenous Production with Increased Stress Tolerance of Intestinal Lactobacilli
Authors: Stack Helena M. , Kearney Niamh , Stanton Catherine , Fitzgerald Gerald F. , Ross R. Paul
Published: November 2009

The exopolysaccharide beta-glucan has been reported to be associated with many health-promoting and prebiotic properties. The membrane-associated glycosyltransferase enzyme (encoded by the
gtf
gene), responsible for microbial beta-glucan production, catalyzes the conversion of sugar nucleotides into beta-glucan. In this study, the
gtf
gene from
Pediococcus parvulus
2.6 was heterologously expressed in
Lactobacillus paracasei
NFBC 338. When grown in the presence of glucose (7%, wt/vol), the recombinant strain (pNZ44-GTF
+
) displayed a “ropy” phenotype, while scanning electron microscopy (SEM) revealed strands of polysaccharide-linking neighboring cells. Beta-glucan biosynthesis was confirmed by agglutination tests carried out with
Streptococcus pneumoniae
type 37-specific antibodies, which specifically detect glucan-producing cells. Further analysis showed a ∼2-fold increase in viscosity in broth media for the beta-glucan-producing strain over 24 h compared to the control strain, which did not show any significant increase in viscosity. In addition, we analyzed the ability of beta-glucan-producing
Lactobacillus paracasei
NFBC 338 to survive both technological and gastrointestinal stresses. Heat stress assays revealed that production of the polysaccharide was associated with significantly increased protection during heat stress (60-fold), acid stress (20-fold), and simulated gastric juice stress (15-fold). Bile stress assays revealed a more modest but significant 5.5-fold increase in survival for the beta-glucan-producing strain compared to that of the control strain. These results suggest that production of a beta-glucan exopolysaccharide by strains destined for use as probiotics may afford them greater performance/protection during cultivation, processing, and ingestion. As such, expression of the
gtf
gene may prove to be a straightforward approach to improve strains that might otherwise prove sensitive in such applications.
Molecular and Genetic Characterization of a Novel Bacteriocin Locus in
Enterococcus avium
Isolates from Infants
Authors: Birri Dagim Jirata , Brede Dag A. , Forberg Torunn , Holo Helge , Nes Ingolf F.
Published: November 2009

Enterococci are among the most common human intestinal lactic acid bacteria, and they are known to produce bacteriocins. In this study, fecal enterococci were isolated from infants and screened for bacteriocin production. Bacteriocin-producing
Enterococcus avium
isolates were obtained, and a new pediocin-like bacteriocin was purified and characterized. This bacteriocin, termed avicin A, was found to be produced by isolates from two healthy infants. It was purified to homogeneity from culture supernatant by ion-exchange and reversed-phase chromatography, and part of its amino acid sequence was obtained. The sequence of a 7-kb DNA fragment of a bacteriocin locus was determined by PCR and DNA sequencing. The bacteriocin locus was organized into four operon-like structures consisting of (i) the structural genes encoding avicin A and its immunity protein, (ii) a divergicin-like bacteriocin (avicin B) gene, (iii) an ABC bacteriocin transporter gene and two regulatory genes (histamine protein kinase- and response regulator-encoding genes), and (iv) induction peptide pheromone- and transport accessory protein-encoding genes. It was shown that the production of avicin A was regulated by the peptide pheromone-inducible regulatory system. Avicin A shows very high levels of similarity to mundticin KS and enterocin CRL35. This bacteriocin showed strong antimicrobial activity against many species of Gram-positive bacteria, including the food-borne pathogen
Listeria monocytogenes
. The avicin A locus is the first bacteriocin locus identified in
E. avium
to be characterized at the molecular level.
Gene Clusters Involved in Isethionate Degradation by Terrestrial and Marine Bacteria
Authors: Weinitschke Sonja , Sharma Pia I. , Stingl Ulrich , Cook Alasdair M. , Smits Theo H. M.
Published: November 2009

Ubiquitous isethionate (2-hydroxyethanesulfonate) is dissimilated by diverse bacteria. Growth of
Cupriavidus necator
H16 with isethionate was observed, as was inducible membrane-bound isethionate dehydrogenase (IseJ) and inducible transcription of the genes predicted to encode IseJ and a transporter (IseU). Biodiversity in isethionate transport genes was observed and investigated by transcription experiments.
Production of Glycolic Acid by Chemolithotrophic Iron- and Sulfur-Oxidizing Bacteria and Its Role in Delineating and Sustaining Acidophilic Sulfide Mineral-Oxidizing Consortia
Authors: Ñancucheo Ivan , Johnson D. Barrie
Published: November 2009

Glycolic acid was detected as an exudate in actively growing cultures of three chemolithotrophic acidophiles that are important in biomining operations,
Leptospirillum ferriphilum
,
Acidithiobacillus
(
At
.)
ferrooxidans
, and
At. caldus
. Although similar concentrations of glycolic acid were found in all cases, the concentrations corresponded to ca. 24% of the total dissolved organic carbon (DOC) in cultures of
L. ferriphilum
but only ca. 5% of the total DOC in cultures of the two
Acidithiobacillus
spp. Rapid acidification (to pH 1.0) of the culture medium of
At. caldus
resulted in a large increase in the level of DOC, although the concentration of glycolic acid did not change in proportion. The archaeon
Ferroplasma acidiphilum
grew in the cell-free spent medium of
At. caldus
; glycolic acid was not metabolized, although other unidentified compounds in the DOC pool were metabolized. Glycolic acid exhibited levels of toxicity with 21 strains of acidophiles screened similar to those of acetic acid. The most sensitive species were chemolithotrophs (
L. ferriphilum
and
At. ferrivorans
), while the most tolerant species were chemoorganotrophs (
Acidocella
,
Acidobacterium
, and
Ferroplasma
species), and the ability to metabolize glycolic acid appeared to be restricted (among acidophiles) to
Firmicutes
(chiefly
Sulfobacillus
spp.). Results of this study help explain why
Sulfobacillus
spp. rather than other acidophiles are the main organic carbon-degrading bacteria in continuously fed stirred tanks used to bioprocess sulfide mineral concentrates and also why temporary cessation of pH control in these systems, resulting in rapid acidification, often results in a plume of the archaeon
Ferroplasma
.
Detection of Infective Poliovirus by a Simple, Rapid, and Sensitive Flow Cytometry Method Based on Fluorescence Resonance Energy Transfer Technology
Authors: Cantera Jason L. , Chen Wilfred , Yates Marylynn V.
Published: November 2009

The rapid and effective detection of virus infection is critical for clinical management and prevention of disease spread during an outbreak. Several methods have been developed for this purpose, of which classical serological and viral nucleic acid detection are the most common. We describe an alternative approach that utilizes engineered cells expressing fluorescent proteins undergoing fluorescence resonance energy transfer (FRET) upon cleavage by the viral 2A protease (2A
pro
) as an indication of infection. Quantification of the infectious-virus titers was resolved by using flow cytometry, and utility was demonstrated for the detection of poliovirus 1 (PV1) infection. Engineered buffalo green monkey kidney (BGMK) cells expressing the cyan fluorescent protein (CFP)-yellow fluorescent protein (YFP) substrate linked by a cleavage recognition site for PV1 2A
pro
were infected with different titers of PV1. After incubation at various time points, cells were harvested, washed, and subjected to flow cytometry analysis. The number of infected cells was determined by counting the number of cells with an increased CFP-to-YFP ratio. As early as 5 h postinfection, a significant number of infected cells (3%) was detected by flow cytometry, and cells infected with only 1 PFU were detected after 12 h postinfection. When applied to an environmental water sample spiked with PV1, the flow cytometry-based assay provided a level of sensitivity similar to that of the plaque assay for detecting and quantifying infectious virus particles. This approach, therefore, is more rapid than plaque assays and can be used to detect other viruses that frequently do not form clear plaques on cell cultures.
Spinacia oleracea
L. Leaf Stomata Harboring
Cryptosporidium parvum
Oocysts: a Potential Threat to Food Safety
Authors: Macarisin Dumitru , Bauchan Gary , Fayer Ronald
Published: November 2009

Cryptosporidium parvum
is a cosmopolitan microscopic protozoan parasite that causes severe diarrheal disease (cryptosporidiosis) in mammals, including humans and livestock. There is growing evidence of
Cryptosporidium
persistence in fresh produce that may result in food-borne infection, including sporadic cases as well as outbreaks. However, drinking and recreational waters are still considered the major sources of
Cryptosporidium
infection in humans, which has resulted in prioritization of studies of parasite etiology in aquatic environments, while the mechanisms of transmission and parasite persistence on edible plants remain poorly understood. Using laser scanning confocal microscopy together with fluorescein-labeled monoclonal antibodies,
C. parvum
oocysts were found to strongly adhere to spinach plants after contact with contaminated water, to infiltrate through the stomatal openings in spinach leaves, and to persist at the mesophyll level. These findings and the fact that this pathogenic parasite resists washing and disinfection raise concerns regarding food safety.
The Major Catalase Gene (
katA
) of
Pseudomonas aeruginosa
PA14 Is under both Positive and Negative Control of the Global Transactivator OxyR in Response to Hydrogen Peroxide
Authors: Heo Yun-Jeong , Chung In-Young , Cho Wan-Je , Lee Bo-Young , Kim Jung-Hoon , Choi Kyoung-Hee , Lee Jin-Won , Hassett Daniel J. , Cho You-Hee
Published: November 2009

The adaptive response to hydrogen peroxide (H
2
O
2
) in
Pseudomonas aeruginosa
involves the major catalase, KatA, and OxyR. However, neither the molecular basis nor the relationship between the aforementioned proteins has been established. Here, we demonstrate that the transcriptional activation of the
katA
promoter (
katAp
) in response to H
2
O
2
was abrogated in the
P. aeruginosa
PA14
oxyR
null mutant. Promoter deletion analyses revealed that H
2
O
2
-mediated induction was dependent on a region of DNA −76 to −36 upstream of the H
2
O
2
-responsive transcriptional start site. This region harbored the potential operator sites (OxyR-responsive element [ORE]) of the
Escherichia coli
OxyR binding consensus. Deletion of the entire ORE not only abolished H
2
O
2
-mediated induction but also elevated the basal transcription, suggesting the involvement of OxyR and the ORE in both transcriptional activation and repression. OxyR bound to the ORE both
in vivo
and
in vitro
, demonstrating that OxyR directly regulates the
katAp
. Three distinct mobility species of oxidized OxyR were observed in response to 1 mM H
2
O
2
, as assessed by free thiol trapping using 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid. These oxidized species were not observed for the double mutants with mutations in the conserved cysteine (Cys) residues (C199 and C208). The uninduced transcription of
katAp
was elevated in an
oxyR
mutant with a mutation of Cys to serine at 199 (C199S) and even higher in the
oxyR
mutant with a mutation of Cys to alanine at 199 (C199A) but not in
oxyR
mutants with mutations in C208 (C208S and C208A). In both the C199S and the C208S mutant, however,
katAp
transcription was still induced by H
2
O
2
treatment, unlike in the
oxyR
null mutant and the C199A mutant. The double mutants with mutations in both Cys residues (C199S C208S and C199A C208S) did not differ from the C199A mutant. Taken together, our results suggest that
P. aeruginosa
OxyR is a bona fide transcriptional regulator of the
katA
gene, sensing H
2
O
2
based on the conserved Cys residues, involving more than one oxidation as well as activation state
in vivo
.
Evidence that Ubiquinone Is a Required Intermediate for Rhodoquinone Biosynthesis in
Rhodospirillum rubrum
Authors: Brajcich Brian C. , Iarocci Andrew L. , Johnstone Lindsey A. G. , Morgan Rory K. , Lonjers Zachary T. , Hotchko Matthew J. , Muhs Jordan D. , Kieffer Amanda , Reynolds Bree J. , Mandel Sarah M. , Marbois Beth N. , Clarke Catherine F. , Shepherd Jennifer N.
Published: November 2009

Rhodoquinone (RQ) is an important cofactor used in the anaerobic energy metabolism of
Rhodospirillum rubrum
. RQ is structurally similar to ubiquinone (coenzyme Q or Q), a polyprenylated benzoquinone used in the aerobic respiratory chain. RQ is also found in several eukaryotic species that utilize a fumarate reductase pathway for anaerobic respiration, an important example being the parasitic helminths. RQ is not found in humans or other mammals, and therefore inhibition of its biosynthesis may provide a parasite-specific drug target. In this report, we describe several
in vivo
feeding experiments with
R. rubrum
used for the identification of RQ biosynthetic intermediates. Cultures of
R. rubrum
were grown in the presence of synthetic analogs of ubiquinone and the known Q biosynthetic precursors demethylubiquinone, demethoxyubiquinone, and demethyldemethoxyubiquinone, and assays were monitored for the formation of RQ
3
. Data from time course experiments and
S
-adenosyl-
l
-methionine-dependent
O
-methyltransferase inhibition studies are discussed. Based on the results presented, we have demonstrated that Q is a required intermediate for the biosynthesis of RQ in
R. rubrum
.
The Gene CBO0515 from
Clostridium botulinum
Strain Hall A Encodes the Rare Enzyme
N
5
-(Carboxyethyl) Ornithine Synthase, EC 1.5.1.24
Authors: Thompson John , Hill Karen K. , Smith Theresa J. , Pikis Andreas
Published: November 2009

Sequencing of the genome of
Clostridium botulinum
strain Hall A revealed a gene (CBO0515), whose putative amino acid sequence was suggestive of the rare enzyme
N
5
-(1-carboxyethyl) ornithine synthase. To test this hypothesis, CBO0515 has been cloned, and the encoded polypeptide was purified and characterized. This unusual gene appears to be confined to proteolytic strains assigned to group 1 of
C. botulinum
.
Localization of Proteins to Different Layers and Regions ofBacillus subtilisSpore Coats
Authors: Imamura Daisuke , Kuwana Ritsuko , Takamatsu Hiromu , Watabe Kazuhito
Published: November 2009

Bacterial spores are encased in a multilayered proteinaceous shell known as the coat. InBacillus subtilis, over 50 proteins are involved in spore coat assembly but the locations of these proteins in the spore coat are poorly understood. Here, we describe methods to estimate the positions of protein fusions to fluorescent proteins in the spore coat by using fluorescence microscopy. Our investigation suggested that CotD, CotF, CotT, GerQ, YaaH, YeeK, YmaG, YsnD, and YxeE are present in the inner coat and that CotA, CotB, CotC, and YtxO reside in the outer coat. In addition, CotZ and CgeA appeared in the outermost layer of the spore coat and were more abundant at the mother cell proximal pole of the forespore, whereas CotA and CotC were more abundant at the mother cell distal pole of the forespore. These polar localizations were observed both in sporangia prior to the release of the forespore from the mother cell and in mature spores after release. Moreover, CotB was observed at the middle of the spore as a ring- or spiral-like structure. Formation of this structure requiredcotGexpression. Thus, we conclude not only that the spore coat is a multilayered assembly but also that it exhibits uneven spatial distribution of particular proteins.
Characterization of the CtsR Stress Response Regulon in
Lactobacillus plantarum
Authors: Fiocco Daniela , Capozzi Vittorio , Collins Michael , Gallone Anna , Hols Pascal , Guzzo Jean , Weidmann Stephanie , Rieu Aurélie , Msadek Tarek , Spano Giuseppe
Published: November 2009

Lactobacillus plantarum ctsR
was characterized.
ctsR
was found to be cotranscribed with
clpC
and induced in response to various abiotic stresses.
ctsR
deletion conferred a heat-sensitive phenotype with peculiar cell morphological features. The transcriptional pattern of putative CtsR regulon genes was examined in the Δ
ctsR
mutant. Direct CtsR-dependent regulation was demonstrated by DNA-binding assays using recombinant CtsR and the promoters of the
ctsR
-
clpC
operon and
hsp1
.
The ArcBA Two-Component System of
Escherichia coli
Is Regulated by the Redox State of both the Ubiquinone and the Menaquinone Pool
Authors: Bekker Martijn , Alexeeva Svetlana , Laan Wouter , Sawers Gary , Teixeira de Mattos Joost , Hellingwerf Klaas
Published: November 2009

ArcBA is a two-component regulatory system of
Escherichia coli
involved in sensing oxygen availability and the concomitant transcriptional regulation of oxidative and fermentative catabolism. Based on
in vitro
data, it has been postulated that the redox state of the ubiquinone pool is the determinant for ArcB kinase activity. Here we report on the
in vivo
regulation of ArcB activation, as determined using a
lacZ
reporter specifically responsive to phosphorylated ArcA. Our results indicate that upon deletion of a ubiquinone biosynthetic enzyme, regulation of ArcB in the anaerobic-aerobic transition is not affected. In contrast, interference with menaquinone biosynthesis leads to inactivation of ArcB during anaerobic growth; this phenotype is fully rescued by addition of a menaquinone precursor. This clearly demonstrates that the menaquinones play a major role in ArcB activation. ArcB shows a complex pattern of regulation when
E. coli
is titrated through the entire aerobiosis range; ArcB is activated under anaerobic and subaerobic conditions and is much less active under fully aerobic and microaerobic conditions. Furthermore, there is no correlation between ArcB activation and the redox state of the ubiquinone pool, but there is a restricted correlation between the total cellular ubiquinone content and ArcB activity due to the considerable increase in the size of the ubiquinone pool with increasing degrees of aerobiosis. These results lead to the working hypothesis that the
in
vivo activity of ArcB in
E. coli
is modulated by the redox state of the menaquinone pool and that the ubiquinone/ubiquinol ratio
in vivo
surely is not the only determinant of ArcB activity.
SleC Is Essential for Germination of
Clostridium difficile
Spores in Nutrient-Rich Medium Supplemented with the Bile Salt Taurocholate
Authors: Burns David A. , Heap John T. , Minton Nigel P.
Published: November 2009

Clostridium difficile
is the major cause of infectious diarrhea and a major burden to health care services. The ability of this organism to form endospores plays a pivotal role in infection and disease transmission. Spores are highly resistant to many forms of disinfection and thus are able to persist on hospital surfaces and disseminate infection. In order to cause disease, the spores must germinate and the organism must grow vegetatively. Spore germination in
Bacillus
is well understood, and genes important for this process have recently been identified in
Clostridium perfringens
; however, little is known about
C. difficile
. Apparent homologues of the spore cortex lytic enzyme genes
cwlJ
and
sleB
(
Bacillus subtilis
) and
sleC
(
C. perfringens
) are present in the
C. difficile
genome, and we describe inactivation of these homologues in
C. difficile
630Δ
erm
and a B1/NAP1/027 clinical isolate. Spores of a
sleC
mutant were unable to form colonies when germination was induced with taurocholate, although decoated
sleC
spores formed the same number of heat-resistant colonies as the parental control, even in the absence of germinants. This suggests that
sleC
is absolutely required for conversion of spores to vegetative cells, in contrast to CD3563 (a
cwlJ
/
sleB
homologue), inactivation of which had no effect on germination and outgrowth of
C. difficile
spores under the same conditions. The B1/NAP1/027 strain R20291 was found to sporulate more slowly and produce fewer spores than 630Δ
erm
. Furthermore, fewer R20291 spores germinated, indicating that there are differences in both sporulation and germination between these epidemic and nonepidemic
C. difficile
isolates.
Differential Target Gene Activation by the
Staphylococcus aureus
Two-Component System
saeRS
Authors: Mainiero Markus , Goerke Christiane , Geiger Tobias , Gonser Christoph , Herbert Silvia , Wolz Christiane
Published: November 2009

The
saePQRS
system of
Staphylococcus aureus
controls the expression of major virulence factors and encodes a histidine kinase (SaeS), a response regulator (SaeR), a membrane protein (SaeQ), and a lipoprotein (SaeP). The widely used strain Newman is characterized by a single amino acid change in the sensory domain of SaeS (Pro18 in strain Newman [SaeS
P
], compared with Leu18 in other strains [SaeS
L
]). SaeS
P
determines activation of the class I
sae
target genes (
coa
,
fnbA
,
eap
,
sib
,
efb
,
fib
,
sae
), which are highly expressed in strain Newman. In contrast, class II target genes (
hla
,
hlb
,
cap
) are not sensitive to the SaeS polymorphism. The SaeS
L
allele (

saeS
L

) is dominant over the SaeS
P
allele, as shown by single-copy integration of

saePQRS
L

in strain Newman, which results in severe repression of class I target genes. The differential effect on target gene expression is explained by different requirements for SaeR phosphorylation. From an analysis of
saeS
deletion strains and strains with mutated SaeR phosphorylation sites, we concluded that a high level of SaeR phosphorylation is required for activation of class I target genes. However, a low level of SaeR phosphorylation, which can occur independent of SaeS, is sufficient to activate class II target genes. Using inducible
saeRS
constructs, we showed that the expression of both types of target genes is independent of the
saeRS
dosage and that the typical growth phase-dependent gene expression pattern is not driven by SaeRS.
Bypassing Isophthalate Inhibition by Modulating Glutamate Dehydrogenase (GDH): Purification and Kinetic Characterization of NADP-GDHs from Isophthalate-Degrading
Pseudomonas aeruginosa
Strain PP4 and
Acinetobacter lwoffii
Strain ISP4
Authors: Vamsee-Krishna C. , Phale Prashant S.
Published: November 2009

Pseudomonas aeruginosa
strain PP4 and
Acinetobacter lwoffii
strain ISP4 metabolize isophthalate as a sole source of carbon and energy. Isophthalate is known to be a competitive inhibitor of glutamate dehydrogenase (GDH), which is involved in C and N metabolism. Strain PP4 showed carbon source-dependent modulation of NADP-GDH; GDH
I
was produced when cells were grown on isophthalate, while GDH
II
was produced when cells were grown on glucose. Strain ISP4 produced a single form of NADP-GDH, GDH
P
, when it was grown on either isophthalate or rich medium (2YT). All of the forms of GDH were purified to homogeneity and characterized. GDH
I
and GDH
II
were found to be homotetramers, while GDH
P
was found to be a homohexamer. GDH
II
was more sensitive to inhibition by isophthalate (2.5- and 5.5-fold more sensitive for amination and deamination reactions, respectively) than GDH
I
. Differences in the N-terminal sequences and electrophoretic mobilities in an activity-staining gel confirmed the presence of two forms of GDH, GDH
I
and GDH
II
, in strain PP4. In strain ISP4, irrespective of the carbon source, the GDH
P
produced showed similar levels of inhibition with isophthalate. However, the specific activity of GDH
P
from isophthalate-grown cells was 2.5- to 3-fold higher than that of GDH
P
from 2YT-grown cells. Identical N-terminal sequences and electrophoretic mobilities in the activity-staining gel suggested the presence of a single form of GDH
P
in strain ISP4. These results demonstrate the ability of organisms to modulate GDH either by producing an entirely different form or by increasing the level of the enzyme, thus enabling strains to utilize isophthalate more efficiently as a sole source of carbon and energy.
Application of OMICS technologies in occupational and environmental health research; current status and projections
Authors: Vlaanderen J , Moore L E , Smith M T , Lan Q , Zhang L , Skibola C F , Rothman N , Vermeulen R
Published: November 2009

OMICS technologies are relatively new biomarker discovery tools that can be applied to study large sets of biological molecules. Their application in human observational studies (HOS) has become feasible in recent years due to a spectacular increase in the sensitivity, resolution and throughput of OMICS-based assays. Although, the number of OMICS techniques is ever expanding, the five most developed OMICS technologies are genotyping, transcriptomics, epigenomics, proteomics and metabolomics. These techniques have been applied in HOS to various extents. However, their application in occupational environmental health (OEH) research has been limited. Here, we will discuss the opportunities these new techniques provide for OEH research. In addition we will address difficulties and limitations to the interpretation of the data that is generated by OMICS technologies. To illustrate the current status of the application of OMICS in OEH research, we will provide examples of studies that used OMICS technologies to investigate human health effects of two well-known toxicants, benzene and arsenic.
Short-axis epicardial volume change is a measure of cardiac left ventricular short-axis function, which is independent of myocardial wall thickness
Authors: Ugander Martin , Carlsson Marcus , Arheden Håkan
Published: November 2009

Fractional shortening (FS) by echocardiography is considered to represent the short-axis contribution to the stroke volume (SV), also called short-axis function. However, FS is mathematically coupled to the amount of myocardium, since it rearranges during atrioventricular plane displacement (AVPD). The SV is the sum of the volumes generated by 1) reduction in outer volume of the heart, and 2) inner AVPD. The long-axis contribution to the SV is generated by AVPD, and thus the short-axis contribution is the remaining outer volume change of the heart, which should be unrelated to myocardial wall thickness. We hypothesized that both endocardial and midwall shortening indexed to SV are dependent on myocardial wall thickness, whereas epicardial volume change (EVC) indexed to SV is not. Twelve healthy volunteers (normals), 12 athletes, and 12 patients with dilated cardiomyopathy (ejection fraction < 30%) underwent cine cardiac magnetic resonance imaging. Left ventricular long-axis function was measured as the portion of the SV, in milliliters, generated by AVPD. EVC was defined as SV minus long-axis function. Endocardial and midwall shortening were measured in a midventricular short-axis slice. Endocardial shortening/SV and midwall shortening/SV both varied in relation to end-diastolic myocardial wall thickness ( R2 = 0.16, P = 0.008 and R2 = 0.14, P = 0.012, respectively), whereas EVC/SV did not ( R2 = 0.00, P = 0.37). FS is dependent on myocardial wall thickness, whereas EVC is not and therefore represents true short-axis function. This is not surprising considering that FS is mainly caused by rearrangement of myocardium secondary to long-axis function. FS is therefore not synonymous with short-axis function.
Estrogen attenuates chronic volume overload induced structural and functional remodeling in male rat hearts
Authors: Gardner Jason D. , Murray David B. , Voloshenyuk Tetyana G. , Brower Gregory L. , Bradley Jessica M. , Janicki Joseph S.
Published: November 2009

We have previously reported gender differences in ventricular remodeling and development of heart failure using the aortocaval fistula model of chronic volume overload in rats. In contrast to males, female rats exhibited no adverse ventricular remodeling and less mortality in response to volume overload. This gender-specific cardioprotection was lost following ovariectomy and was partially restored using estrogen replacement. However, it is not known if estrogen treatment would be as effective in males. The purpose of this study was to evaluate the structural and functional effects of estrogen in male rats subjected to chronic volume overload. Four groups of male rats were studied at 3 days and 8 wk postsurgery as follows: fistula and sham-operated controls, with and without estrogen treatment. Biochemical and histological studies were performed at 3 days postsurgery, with chronic structural and functional effects studied at 8 wk. Measurement of systolic and diastolic pressure-volume relationships was obtained using a blood-perfused isolated heart preparation. Both fistula groups developed significant ventricular hypertrophy after 8 wk of volume overload. Untreated rats with fistula exhibited extensive ventricular dilatation, which was coupled with a loss of systolic function. Estrogen attenuated left ventricular dilatation and maintained function in treated rats. Estrogen treatment was also associated with a reduction in oxidative stress and circulating endothelin-1 levels, as well as prevention of matrix metalloproteinase-2 and -9 activation and breakdown of ventricular collagen in the early stage of remodeling. These data demonstrate that estrogen attenuates ventricular remodeling and disease progression in male rats subjected to chronic volume overload.
Inhibitory effects of angiotensin-(1–7) on the nerve stimulation-induced release of norepinephrine and neuropeptide Y from the mesenteric arterial bed
Authors: Byku Mirnela , Macarthur Heather , Westfall Thomas C.
Published: November 2009

Neuropeptide Y (NPY) is a cotransmitter with norepinephrine (NE) and ATP in sympathetic nerves. There is evidence for increased activity of the sympathetic nervous system and the renin-angiotensin system (RAS), as well as a role for NPY in the development of hypertension in experimental animal models and in humans. Angiotensin II (ANG II) is known to facilitate sympathetic neurotransmission, an effect greater in spontaneously hypertensive rats (SHR) than normotensive Wistar-Kyoto (WKY) rats. A newly discovered product of the RAS is angiotensin-(1–7) [ANG-(1–7)]. There is evidence suggesting that ANG-(1–7) opposes the actions of ANG II, resulting in hypotensive effects. The objective of this study was to investigate the role of ANG-(1–7) on the nerve-stimulated overflow of NE and NPY from the mesenteric arterial bed of SHR and the mechanisms involved in mediating any effects produced. ANG-(1–7) (0.001, 0.01, 0.1 μM) decreased nerve-stimulated NE and NPY overflow, as well as perfusion pressure in preparations obtained from SHR. This effect was greater in preparations of SHR than WKY controls. In addition, ANG-(1–7) decreased NE overflow to a greater extent than NPY overflow. Administration of the Mas receptor antagonist, d-Ala7ANG-(1–7), attenuated the decrease in both NE and NPY overflow due to ANG-(1–7) administration. However, the angiotensin type 2 receptor antagonist, PD-123391, attenuated the effect of ANG-(1–7) on NE overflow without affecting the decrease in NPY overflow. Moreover, in the presence of NG-nitro-l-arginine methyl ester, ANG-(1–7) decreased NPY overflow, but not NE overflow. ANG-(1–7) decreases the nerve-stimulated overflow of NE and NPY in preparations of SHR, whereas ANG II enhances it. Therefore, ANG-(1–7) may counteract the effects of ANG II by acting on ANG type 2 and Mas receptors.
Intimal cushions and endothelial nuclear elongation around mouse aortic branches and their spatial correspondence with patterns of lipid deposition
Authors: Bond Andrew R. , Ni Chih-Wen , Jo Hanjoong , Weinberg Peter D.
Published: November 2009

Spatial variation in hemodynamic stresses acting on the arterial wall may explain the nonuniform distribution of atherosclerosis. In thoracic aortas of LDL receptor/apolipoprotein E double knockout mice, lesions develop preferentially around the entire circumference of intercostal branch ostia, regardless of age, with the highest prevalence occurring upstream. Additional chevron-shaped lesions occur further upstream of the ostia. This pattern differs from the age-related ones occurring in people and rabbits. In the present study, patterns of near-wall blood flow around intercostal ostia in wild-type mice were estimated from the morphology of endothelial nuclei, which were shown in vitro to elongate in response to elevated shear stress and to align with the flow, and wall structure was assessed from confocal and scanning electron microscopy. A triangular intimal cushion surrounded the upstream part of most ostia. Nuclear length-to-width ratios were lowest over this cushion and highest at the sides of branches, regardless of age. Nuclear orientations were consistent with flow diverging around the branch. The pattern of nuclear morphology differed from the age-related ones observed in rabbits. The intimal cushion and the distribution of shear stress inferred from these observations can partly account for the pattern of lesions observed in knockout mice. Nuclear elongation in nonbranch regions was approximately constant across animals of different size, demonstrating the existence of a mechanism by which endothelial cells compensate for the dependence of mean aortic wall shear stress on body mass.
Tolerance to nitroglycerin-induced preconditioning of the endothelium: a human in vivo study
Authors: Gori Tommaso , Dragoni Saverio , Di Stolfo Giuseppe , Sicuro Silvia , Liuni Andrew , Luca Mary Clare , Thomas George , Oelze Matthias , Daiber Andreas , Parker John D.
Published: November 2009

Damage and dysfunction of the vascular endothelium critically influence clinical outcomes after ischemia and reperfusion (I/R). Brief exposure to organic nitrates can protect the vascular endothelium from I/R injury via a mechanism that is similar to ischemic preconditioning and is independent of hemodynamic changes. The clinical relevance of these protective effects clearly depends on whether they can be sustained over time. Twenty-four healthy (age 25–32) male volunteers were randomized to receive 1) transdermal nitroglycerin (GTN; 0.6 mg/h) administered for 2 h on 1 day only, 2) transdermal GTN for 2 h/day for 7 days, or 3) continuous therapy with transdermal GTN for 7 days. Eight volunteers underwent continuous GTN therapy followed by intra-arterial infusion of the antioxidant vitamin C. Finally, five additional subjects underwent no therapy and served as controls. Endothelial function measurements were performed before and after induction of I/R of the arm. I/R caused a significant blunting of the flow responses to acetylcholine in the control group ( P < 0.01 vs. before I/R). A single 2-h GTN dosage, given 24 h before I/R, prevented I/R-induced endothelial dysfunction [ P = not significant (NS) vs. before I/R], but this protective effect was completely lost after 1 wk of GTN administration 2 h/day ( P < 0.05 vs. before I/R; P = NS vs. control). In subjects who received continuous GTN, endothelial responses were blunted before I/R, and I/R did not cause further endothelial dysfunction. Finally, vitamin C normalized acetylcholine responses and prevented the loss of preconditioning associated with prolonged GTN. In a separate experimental model using isolated human endothelial cells, short-term incubation with GTN caused upregulation of heme oxygenase, an effect that was lost after prolonged GTN administration. Although a single administration of GTN is able to protect the endothelium from I/R-induced endothelial dysfunction, this protection is lost upon prolonged exposure, likely via an oxidative mechanism.
Impact of HIV Infection on Diastolic Function and Left Ventricular Mass
Authors: Hsue Priscilla Y. , Hunt Peter W. , Ho Jennifer E. , Farah Husam H. , Schnell Amanda , Hoh Rebecca , Martin Jeffrey N. , Deeks Steven G. , Bolger Ann F.
Published: November 2009

Background—

Patients with HIV have increased risk for cardiovascular disease, but the underlying mechanisms remain unknown. The purpose of this study was to determine the prevalence of echocardiographic abnormalities among asymptomatic HIV-infected individuals compared with HIV-uninfected individuals.

Methods/Results—

We performed echocardiography in 196 HIV-infected adults and 52 controls. Left ventricular ejection fraction, left ventricular mass indexed to the body surface area, and diastolic function were assessed according to American Society of Echocardiography standards. Left ventricular mass index was higher in HIV-infected patients (77.2 g/m
2
in patients with HIV versus 66.5 g/m
2
in controls,
P
<0.0001). Left ventricular ejection fraction was similar in both groups. Eight (4%) of the patients with HIV had evidence of left ventricular systolic dysfunction (defined as an EF <50%) versus none of the controls; 97 (50%) had mild diastolic dysfunction compared with 29% of the HIV-uninfected subjects (
P
=0.008). After adjustment for hypertension and race, HIV-infected participants had a mean 8 g/m
2
larger left ventricular mass index compared with controls (
P
=0.001). Higher left ventricular mass index was independently associated with lower nadir CD4 T-cell count, suggesting that immunodeficiency may play a role in this process. After adjustment for age and traditional risk factors, patients with HIV had a 2.4 greater odds of having diastolic dysfunction as compared with controls (
P
=0.019).

Conclusions—

HIV-infected patients had a higher prevalence of diastolic dysfunction and higher left ventricular mass index compared with controls. These differences were not readily explained by differences in traditional risk factors and were independently associated with HIV infection. These results suggest that contemporary asymptomatic patients with HIV manifest mild functional and morphological cardiac abnormalities, which are independently associated with HIV infection.
β-Blocker Therapy and Mortality of Patients With Chagas Cardiomyopathy
Authors: Issa Victor S. , Amaral Alexandre F. , Cruz Fátima D. , Ferreira Silvia M.A. , Guimarães Guilherme V. , Chizzola Paulo R. , Souza Germano E.C. , Bacal Fernando , Bocchi Edimar A.
Published: November 2009

Background—

Peculiar aspects of Chagas cardiomyopathy raise concerns about efficacy and safety of sympathetic blockade. We studied the influence of β-blockers in patients with Chagas cardiomyopathy.

Methods and Results—

We examined REMADHE trial and grouped patients according to etiology (Chagas versus non-Chagas) and β-blocker therapy. Primary end point was all-cause mortality or heart transplantation. Altogether 456 patients were studied; 27 (5.9%) were submitted to heart transplantation and 202 (44.3%) died. Chagas etiology was present in 68 (14.9%) patients; they had lower body mass index (24.1±4.1 versus 26.3±5.1,
P
=0.001), smaller end-diastolic left ventricle diameter (6.7±1.0 mm versus 7.0±0.9 mm,
P
=0.001), smaller proportion of β-blocker therapy (35.8% versus 68%,
P
<0.001), and higher proportion of spironolactone therapy (74.6% versus 57.8%,
P
=0.003). Twenty-four (35.8%) patients with Chagas disease were under β-blocker therapy and had lower serum sodium (136.6±3.1 versus 138.4±3.1 mEqs,
P
=0.05) and lower body mass index (22.5±3.3 versus 24.9±4.3,
P
=0.03) compared with those who received β-blockers. Survival was lower in patients with Chagas heart disease as compared with other etiologies. When only patients under β-blockers were considered, the survival of patients with Chagas disease was similar to that of other etiologies. The survival of patients with β-blockers was higher than that of patients without β-blockers. In Cox regression model, left ventricle end-diastolic diameter (hazard ratio, 1.78; CI, 1.15 to 2.76;
P
=0.009) and β-blockers (hazard ratio, 0.37; CI, 0.14 to 0.97;
P
=0.044) were associated with better survival.

Conclusions—

Our study suggests that β-blockers may have beneficial effects on survival of patients with heart failure and Chagas heart disease and warrants further investigation in a prospective, randomized trial.

Clinical Trial Registration—

clinicaltrials.gov. Identifier: NCT00505050.
Quantification of Regional Myocardial Oxygenation by Magnetic Resonance Imaging
Authors: McCommis Kyle S. , Goldstein Thomas A. , Abendschein Dana R. , Herrero Pilar , Misselwitz Bernd , Gropler Robert J. , Zheng Jie
Published: November 2009

Background—

A comprehensive evaluation of myocardial ischemia requires measures of both oxygen supply and demand. Positron emission tomography (PET) is currently the gold standard for such evaluations, but its use is limited because of its ionizing radiation, limited availability, and high cost. A cardiac MRI method was developed for assessing myocardial oxygenation. The purpose of this study was to evaluate and validate this technique compared with PET during pharmacological stress in a canine model of coronary artery stenosis.

Methods and Results—

Twenty-one beagles and small mongrel dogs without coronary artery stenosis (controls) or with moderate to severe acute coronary artery stenosis underwent MRI and PET imaging at rest and during dipyridamole vasodilation or dobutamine stress to induce a wide range of changes in cardiac perfusion and oxygenation. MRI first-pass perfusion imaging was performed to quantify myocardial blood flow and volume. The MRI blood oxygen level-dependent technique was used to determine the myocardial oxygen extraction fraction during pharmacological hyperemia. Myocardial oxygen consumption was determined by the Fick law. In the same dogs,
15
O-water and
11
C-acetate were used to measure myocardial blood flow and myocardial oxygen consumption, respectively, by PET. Regional assessments were performed for both MR and PET. MRI data correlated nicely with PET values for myocardial blood flow (
R
2
=0.79,
P
<0.001), myocardial oxygen consumption (
R
2
=0.74,
P
<0.001), and oxygen extraction fraction (
R
2
=0.66,
P
<0.01).

Conclusions—

Cardiac MRI methods may provide an alternative to radionuclide imaging in settings of myocardial ischemia. Our newly developed quantitative MRI oxygenation imaging technique may be a valuable noninvasive tool to directly evaluate myocardial energetics and efficiency.
On the Kubelka—Munk Single-Constant/Two-Constant Theories
Authors: Hongying Yang  , Sukang Zhu  , Ning Pan
Published: November 2009

As the basic theory for color mixing in intensely light-scattering materials, the Kubelka— Munk single-constant/two-constant theories have played an important role in color science and technology. However, in practice there are some shortcomings. This paper checked the theories with a series of systematical experiments, data processing and analyzing, and has revealed several disagreements with the experimental evidence in the case of the additivity color-mixing law for turbid materials. Explanations on some of these problems are given, which are different from the conventional views.