bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the 101-Kilobase-Pair Megaplasmid pKB1, Isolated from the Rubber-Degrading BacteriumGordonia westfalicaKb1
Authors: Bröker Daniel , Arenskötter Matthias , Legatzki Antje , Nies Dietrich H. , Steinbüchel Alexander
Published: December 2003

The complete sequence of the circular 101,016-bp megaplasmid pKB1 from thecis-1,4-polyisoprene-degrading bacteriumGordonia westfalicaKb1, which represents the first described extrachromosomal DNA of a member of this genus, was determined. Plasmid pKB1 harbors 105 open reading frames. The predicted products of 46 of these are significantly related to proteins of known function. Plasmid pKB1 is organized into three functional regions that are flanked by insertion sequence (IS) elements: (i) a replication and putative partitioning region, (ii) a putative metabolic region, and (iii) a large putative conjugative transfer region, which is interrupted by an additional IS element. Southern hybridization experiments revealed the presence of another copy of this conjugational transfer region on the bacterial chromosome. The origin of replication (oriV) of pKB1 was identified and used for construction ofEscherichia coli-Gordoniashuttle vectors, which was also suitable for several otherGordoniaspecies and related genera. The metabolic region included the heavy-metal resistance genecadA, encoding a P-type ATPase. Expression ofcadAinE. colimediated resistance to cadmium, but not to zinc, and decreased the cellular content of cadmium in this host. WhenG. westfalicastrain Kb1 was cured of plasmid pKB1, the resulting derivative strains exhibited slightly decreased cadmium resistance. Furthermore, they had lost the ability to use isoprene rubber as a sole source of carbon and energy, suggesting that genes essential for rubber degradation are encoded by pKB1.
Multiple Formate Dehydrogenase Enzymes in the Facultative Methylotroph
Methylobacterium extorquens
AM1 Are Dispensable for Growth on Methanol
Authors: Chistoserdova Ludmila , Laukel Markus , Portais Jean-Charles , Vorholt Julia A. , Lidstrom Mary E.
Published: December 2003

Formate dehydrogenase has traditionally been assumed to play an essential role in energy generation during growth on C
1
compounds. However, this assumption has not yet been experimentally tested in methylotrophic bacteria. In this study, a whole-genome analysis approach was used to identify three different formate dehydrogenase systems in the facultative methylotroph
Methylobacterium extorquens
AM1 whose expression is affected by either molybdenum or tungsten. A complete set of single, double, and triple mutants was generated, and their phenotypes were analyzed. The growth phenotypes of the mutants suggest that any one of the three formate dehydrogenases is sufficient to sustain growth of
M. extorquens
AM1 on formate, while surprisingly, none is required for growth on methanol or methylamine. Nuclear magnetic resonance analysis of the fate of [
13
C]methanol revealed that while cells of wild-type
M. extorquens
AM1 as well as cells of all the single and the double mutants continuously produced [
13
C]bicarbonate and
13
CO
2
, cells of the triple mutant accumulated [
13
C]formate instead. Further studies of the triple mutant showed that formate was not produced quantitatively and was consumed later in growth. These results demonstrated that all three formate dehydrogenase systems must be inactivated in order to disrupt the formate-oxidizing capacity of the organism but that an alternative formate-consuming capacity exists in the triple mutant.
Null Mutation of HvrA Compensates for Loss of an Essential
relA/spoT
-Like Gene in
Rhodobacter capsulatus
Authors: Masuda Shinji , Bauer Carl E.
Published: December 2003

We report that a single
relA/spoT
-like gene exists on the
Rhodobacter capsulatus
chromosome, and its mutational loss is lethal. This gene could be mutated only under a mutational background of a null mutation in the nucleoid protein HvrA. This result suggests that there may be a direct link between HvrA-regulated promoters and the ppGpp-related stringent response.
Transcriptome Analysis of the Response of
Pseudomonas aeruginosa
to Hydrogen Peroxide
Authors: Palma Marco , DeLuca Darrow , Worgall Stefan , Quadri Luis E. N.
Published: December 2003

Pseudomonas aeruginosa
must often overcome a high concentration of oxidants to successfully infect the human host. We report here the results of a transcriptome profiling comparing cells treated with H
2
O
2
and untreated controls. The data indicate that the early response of
P. aeruginosa
to H
2
O
2
consists of an upregulation of protective mechanisms and a downregulation of primary metabolism.
Production of Heterologous and Chimeric Scaffoldins by
Clostridium acetobutylicum
ATCC 824
Authors: Perret S. , Casalot L. , Fierobe H.-P. , Tardif C. , Sabathe F. , Belaich J.-P. , Belaich A.
Published: December 2003

Clostridium acetobutylicum
ATCC 824 converts sugars and various polysaccharides into acids and solvents. This bacterium, however, is unable to utilize cellulosic substrates, since it is able to secrete very small amounts of cellulosomes. To promote the utilization of crystalline cellulose, the strategy we chose aims at producing heterologous minicellulosomes, containing two different cellulases bound to a miniscaffoldin, in
C. acetobutylicum
. A first step toward this goal describes the production of miniCipC1, a truncated form of CipC from
Clostridium cellulolyticum
, and the hybrid scaffoldin Scaf 3, which bears an additional cohesin domain derived from CipA from
Clostridium thermocellum
. Both proteins were correctly matured and secreted in the medium, and their various domains were found to be functional.
A Mother Cell-Specific Class B Penicillin-Binding Protein, PBP4b, in
Bacillus subtilis
Authors: Wei Yuping , McPherson Derrell C. , Popham David L.
Published: December 2003

The
Bacillus subtilis
genome encodes 16 penicillin-binding proteins (PBPs), some of which are involved in synthesis of the spore peptidoglycan. The
pbpI
(
yrrR
) gene encodes a class B PBP, PBP4b, and is transcribed in the mother cell by RNA polymerase containing σ
E
. Loss of PBP4b, alone and in combination with other sporulation-specific PBPs, had no effect on spore peptidoglycan structure.
An H
+
-Coupled Multidrug Efflux Pump, PmpM, a Member of the MATE Family of Transporters, from
Pseudomonas aeruginosa
Authors: He Gui-Xin , Kuroda Teruo , Mima Takehiko , Morita Yuji , Mizushima Tohru , Tsuchiya Tomofusa
Published: December 2003

We cloned the gene PA1361 (we designated the gene
pmpM
), which seemed to encode a multidrug efflux pump belonging to the MATE family, of
Pseudomonas aeruginosa
by the PCR method using the drug-hypersensitive
Escherichia coli
KAM32 strain as a host. Cells of
E. coli
possessing the
pmpM
gene showed elevated resistance to several antimicrobial agents. We observed energy-dependent efflux of ethidium from cells possessing the
pmpM
gene. We found that PmpM is an H
+
-drug antiporter, and this finding is the first reported case of an H
+
-coupled efflux pump in the MATE family. Disruption and reintroduction of the
pmpM
gene in
P. aeruginosa
revealed that PmpM is functional and that benzalkonium chloride, fluoroquinolones, ethidium bromide, acriflavine, and tetraphenylphosphonium chloride are substrates for PmpM in this microorganism.
Influence of Growth Temperature on Lipid and Phosphate Contents of Surface Polysaccharides from the Antarctic Bacterium
Pseudoalteromonas haloplanktis
TAC 125
Authors: Corsaro M. Michela , Lanzetta Rosa , Parrilli Ermenegilda , Parrilli Michelangelo , Tutino M. Luisa , Ummarino Salvatore
Published: December 2003

The chemical structural variations induced by different growth temperatures in the lipooligosaccharide and exopolysaccharide components extracted from the Antarctic bacterium
Pseudoalteromonas haloplanktis
TAC 125 are described. The increase in phosphorylation with the increase in growth temperature seems to be general, because it happens not only for the lipooligosaccharide but also for the exopolysaccharide. Structural variations in the lipid components of lipid A also occur. In addition, free lipid A is found at both 25 and 4°C but not at 15°C, which is the optimal growth temperature, suggesting a incomplete biosynthesis of the lipooligosaccharide component under the first two temperature conditions.
Characterization of CmaA, an Adenylation-Thiolation Didomain Enzyme Involved in the Biosynthesis of Coronatine
Authors: Couch Robin , O'Connor Sarah E. , Seidle Heather , Walsh Christopher T. , Parry Ronald
Published: December 2003

Several pathovars of
Pseudomonas syringae
produce the phytotoxin coronatine (COR), which contains an unusual amino acid, the 1-amino-2-ethylcyclopropane carboxylic acid called coronamic acid (CMA), which is covalently linked to a polyketide-derived carboxylic acid, coronafacic acid, by an amide bond. The region of the COR biosynthetic gene cluster proposed to be responsible for CMA biosynthesis was resequenced, and errors in previously deposited
cmaA
sequences were corrected. These efforts allowed overproduction of
P. syringae
pv. glycinea PG4180 CmaA in
P. syringae
pv. syringae FF5 as a FLAG-tagged protein and overproduction of
P. syringae
pv. tomato CmaA in
Escherichia coli
as a His-tagged protein; both proteins were in an enzymatically active form. Sequence analysis of CmaA indicated that there were two domains, an adenylation domain (A domain) and a thiolation domain (T domain). ATP-
32
PP
i
exchange assays showed that the A domain of CmaA catalyzes the conversion of branched-chain
l
-amino acids and ATP into the corresponding aminoacyl-AMP derivatives, with a kinetic preference for
l
-
allo-
isoleucine. Additional experiments demonstrated that the T domain of CmaA, which is posttranslationally modified with a 4′-phosphopantetheinyl group, reacts with the AMP derivative of
l
-
allo-
isoleucine to produce an aminoacyl thiolester intermediate. This covalent species was detected by incubating CmaA with ATP and
l
-[G-
3
H]
allo
-isoleucine, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. It is postulated that the
l
-
allo-
isoleucine covalently tethered to CmaA serves as the substrate for additional enzymes in the CMA biosynthetic pathway that catalyze cyclopropane ring formation, which is followed by thiolester hydrolysis, yielding free CMA. The availability of catalytically active CmaA should facilitate elucidation of the details of the subsequent steps in the formation of this novel cyclopropyl amino acid.
Temporal Expression of Pertussis Toxin and Ptl Secretion Proteins byBordetella pertussis
Authors: Rambow-Larsen Amy A. , Weiss Alison A.
Published: December 2003

Pertussis toxin is an AB5toxin comprised of protein subunits S1 through S5. The individual subunits are secreted by a Sec-dependent mechanism into the periplasm, where the toxin is assembled. The Ptl type IV secretion system mediates secretion of assembled toxin past the outer membrane. In this study, we examined the time course of protein expression, toxin assembly, and secretion as a function of the bacterial growth cycle. Logarithmic growth was observed after a 1-h lag phase. Secreted toxin was first observed at 3 h. Secretion continued throughout the logarithmic growth phase and decreased as the culture entered the stationary phase after about 24 h. On a per cell basis, toxin secretion occurred at a constant rate of 3 molecules/min/cell from 2 to 18 h. More of toxin subunits S1, S2, and S3 were produced than were secreted, resulting in periplasmic accumulation. Periplasmic S1, S2, and S3 were found to be soluble in the periplasm, as well as membrane associated. About one-half of the periplasmic S1, S2 and S3 subunits were incorporated into holotoxin. Secretion component PtlF was present at a low level at time zero, and the level increased between 2 and 24 h from 30 to 1,000 molecules per cell; however, the initial level of PtlF, 30 molecules per cell, supported maximal secretion. The accumulation of both periplasmic toxin and secretion components suggests that translation rates exceed the rate of secretion and that secretion, not toxin and Ptl complex assembly, is rate limiting.
Analysis of Differences in the Functional Properties of the Substrate Binding Proteins of the
Borrelia burgdorferi
Oligopeptide Permease (
opp
) Operon
Authors: Wang Xing-Guo , Kidder J. Michael , Scagliotti Joanna P. , Klempner Mark S. , Noring Richard , Hu Linden T.
Published: December 2003

The
Borrelia burgdorferi
genome encodes five orthologues of the substrate binding protein oligopeptide permease A (OppA). It was previously shown that these genes are under the control of separate promoters and are differentially expressed under various environmental conditions. We were interested in determining whether there are also differences in substrate specificities among the proteins. The substrate specificities of recombinant proteins were determined by screening for high-affinity peptides by use of a combinatorial phage display heptapeptide library. Different heptapeptides with high affinities for OppA-1, OppA-2, and OppA-3 were identified. No heptapeptide binding OppA-4 or OppA-5 could be identified. Competitive binding assays were performed under various conditions to determine the substrate preferences of the OppA proteins. OppA-1 retained maximal activity over a broad range of pHs (5.5 to 7.5), whereas OppA-2 and OppA-3 showed peak activities at pHs below 5.5. OppA-1 and OppA-2 showed preferences for tripeptides over dipeptides and longer-chain peptides. Although a wide variety of amino acyl side chains were tolerated by all three OppA proteins, OppA-1 showed the broadest substrate specificity and was able to accommodate peptides composed of bulky hydrophobic residues; OppA-2 and OppA-3 showed preferences for peptides composed of small nonpolar amino acids. All three OppA proteins showed preferences for peptides composed of
l
- rather than
d
-amino acids. OppA-3 showed the greatest tolerance for changes in stereochemistry. Substantial differences in the substrate specificities of the OppA proteins of
B. burgdorferi
suggest that they may have distinct functions in the organism.
Staphylococcus aureus
Mevalonate Kinase: Isolation and Characterization of an Enzyme of the Isoprenoid Biosynthetic Pathway
Authors: Voynova Natalya E. , Rios Sandra E. , Miziorko Henry M.
Published: December 2003

It has been proposed that isoprenoid biosynthesis in several gram-positive cocci depends on the mevalonate pathway for conversion of acetyl coenzyme A to isopentenyl diphosphate. Mevalonate kinase catalyzes a key reaction in this pathway. In this study the enzyme from
Staphylococcus aureus
was expressed in
Escherichia coli
, isolated in a highly purified form, and characterized. The overall amino acid sequence of this enzyme was very heterologous compared with the sequences of eukaryotic mevalonate kinases. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical gel filtration chromatography suggested that the native enzyme is a monomer with a molecular mass of approximately 33 kDa. The specific activity was 12 U/mg, and the pH optimum was 7.0 to 8.5. The apparent

K
m

values for
R,S
-mevalonate and ATP were 41 and 339 μM, respectively. There was substantial substrate inhibition at millimolar levels of mevalonate. The sensitivity to feedback inhibition by farnesyl diphosphate and its sulfur-containing analog, farnesyl thiodiphosphate, was characterized. These compounds were competitive inhibitors with respect to ATP; the

K
i

values were 46 and 45 μM for farnesyl diphosphate and its thio analog, respectively. Parallel measurements with heterologous eukaryotic mevalonate kinases indicated that
S. aureus
mevalonate kinase is much less sensitive to feedback inhibition (

K
i

difference, 3 orders of magnitude) than the human enzyme. In contrast, both enzymes tightly bound trinitrophenyl-ATP, a fluorescent substrate analog, suggesting that there are similarities in structural features that are important for catalytic function.
RtsA Coordinately Regulates DsbA and theSalmonellaPathogenicity Island 1 Type III Secretion System
Authors: Ellermeier Craig D. , Slauch James M.
Published: December 2003

Salmonellaserovars cause a wide variety of diseases ranging from mild gastroenteritis to life-threatening systemic infections. An important step inSalmonella entericaserovar Typhimurium infection is the invasion of nonphagocytic epithelial cells, mediated by a type III secretion system (TTSS) encoded onSalmonellapathogenicity island 1 (SPI1). The SPI1 TTSS forms a needle complex through which effector proteins are injected into the cytosol of host cells, where they promote actin rearrangement and engulfment of the bacteria. We previously identified theSalmonella-specific regulatory protein RtsA, which induces expression ofhilAand, thus, the SPI1 genes. Here we show that thehilAregulators RtsA, HilD, and HilC can each induce transcription ofdsbA, which encodes a periplasmic disulfide bond isomerase. RtsA induces expression ofdsbAindependent of either the SPI1 TTSS or the only known regulator ofdsbA, the CpxRA two-component system. We show that DsbA is required for both the SPI1 and SPI2 TTSS to translocate effector proteins into the cytosol of host cells. DsbA is also required for survival during the systemic stages of infection. We also present evidence that production of SPI1 effector proteins is coupled to assembly of the TTSS. This feedback regulation is mediated at either the transcriptional or posttranscriptional level, depending on the particular effector. Loss of DsbA leads to feedback inhibition, which is consistent with the hypothesis that disulfide bond formation plays a role in TTSS assembly or function.
Production of Muramic δ-Lactam in
Bacillus subtilis
Spore Peptidoglycan
Authors: Gilmore Meghan E. , Bandyopadhyay Dabanjan , Dean Amanda M. , Linnstaedt Sarah D. , Popham David L.
Published: December 2003

Bacterial spore heat resistance is primarily dependent upon dehydration of the spore cytoplasm, a state that is maintained by the spore peptidoglycan wall, the spore cortex. A peptidoglycan structural modification found uniquely in spores is the formation of muramic δ-lactam. Production of muramic δ-lactam in
Bacillus subtilis
requires removal of a peptide side chain from the
N
-acetylmuramic acid residue by a
cwlD
-encoded muramoyl-
l
-Alanine amidase. Expression of
cwlD
takes place in both the mother cell and forespore compartments of sporulating cells, though expression is expected to be required only in the mother cell, from which cortex synthesis derives. Expression of
cwlD
in the forespore is in a bicistronic message with the upstream gene
ybaK
. We show that
ybaK
plays no apparent role in spore peptidoglycan synthesis and that expression of
cwlD
in the forespore plays no significant role in spore peptidoglycan formation. Peptide cleavage by CwlD is apparently followed by deacetylation of muramic acid and lactam ring formation. The product of
pdaA
(
yfjS
), which encodes a putative deacetylase, has recently been shown to also be required for muramic δ-lactam formation. Expression of CwlD in
Escherichia coli
results in muramoyl
l
-Alanine amidase activity but no muramic δ-lactam formation. Expression of PdaA alone in
E. coli
had no effect on
E. coli
peptidoglycan structure, whereas expression of CwlD and PdaA together resulted in the formation of muramic δ-lactam. CwlD and PdaA are necessary and sufficient for muramic δ-lactam production, and no other
B. subtilis
gene product is required. PdaA probably carries out both deacetylation and lactam ring formation and requires the product of CwlD activity as a substrate.
NpdR, a Repressor Involved in 2,4,6-Trinitrophenol Degradation in
Rhodococcus opacus
HL PM-1
Authors: Nga Dang P. , Altenbuchner Josef , Heiss Gesche S.
Published: December 2003

Rhodococcus opacus
HL PM-1 utilizes 2,4,6-trinitrophenol (picric acid) as a sole nitrogen source. The initial attack on picric acid occurs through two hydrogenation reactions. Hydride transferase II (encoded by
npdI
) and hydride transferase I (encoded by
npdC
) are responsible for the hydride transfers. Database searches with the
npd
genes have indicated the presence of a putative transcriptional regulator,
npdR
. Here, the
npdR
gene was expressed in
Escherichia coli
, and the protein was purified and shown to form a complex with intergenic regions between open reading frames A and B and between
npdH
and
npdI
within the
npd
gene cluster. A change in DNA-NpdR complex formation occurred in the presence of 2,4-dinitrophenol, picric acid, 2-chloro-4,6-dinitrophenol, and 2-methyl-4,6-dinitrophenol. By constructing a promoter-probe vector, we demonstrated that both intergenic regions caused the expression of reporter gene
xylE
. Hence, both of these regions contain promoters. A deletion mutant of
R. opacus
HL PM-1 was constructed in which part of
npdR
was deleted. The expression of
npdI
and
npdC
was induced by 2,4-dinitrophenol in the wild-type strain, while in the mutant these genes were constitutively expressed. Hence, NpdR is a repressor involved in picric acid degradation.
CONFINING POTENTIAL IN A COLOR DIELECTRIC MEDIUM WITH PARALLEL DOMAIN WALLS
Authors: BAZEIA D. , BRITO F. A. , FREIRE W. , RIBEIRO R. F.
Published: December 2003

We study quark confinement in a system of two parallel domain walls interpolating different color dielectric media. We use the phenomenological approach in which the confinement of quarks appears considering the QCD vacuum as a color dielectric medium. We explore this phenomenon in QCD2, where the confinement of the color flux between the domain walls manifests, in a scenario where two 0-branes (representing external quark and antiquark) are connected by a QCD string. We obtain solutions of the equations of motion via first-order differential equations. We find a new color confining potential that increases monotonically with the distance between the domain walls.
UNIVERSAL CALABI–YAU ALGEBRA: TOWARDS AN UNIFICATION OF COMPLEX GEOMETRY
Authors: ANSELMO F. , ELLIS J. , NANOPOULOS D. V. , VOLKOV G.
Published: December 2003

We present a universal normal algebra suitable for constructing and classifying Calabi–Yau spaces in arbitrary dimensions. This algebraic approach includes natural extensions of reflexive weight vectors to higher dimensions, related to Batyrev's reflexive polyhedra, and their n-ary combinations. It also includes a "dual" construction based on the Diophantine decomposition of invariant monomials, which provides explicit recurrence formulae for the numbers of Calabi–Yau spaces in arbitrary dimensions with Weierstrass, K3, etc., fibrations. Our approach also yields simple algebraic relations between chains of Calabi–Yau spaces in different dimensions, and concrete visualizations of their singularities related to Cartan–Lie algebras. This Universal Calabi–Yau algebra is a powerful tool for deciphering the Calabi–Yau genome in all dimensions.
FINITE ORDER BFFT METHOD
Authors: MONEMZADEH M. , SHIRZAD A.
Published: December 2003

We propose a method in the context of BFFT approach that leads to truncation of the infinite series regarded as constraints in the extended phase space, as well as other physical quantities (such as Hamiltonian). This has been done for cases where the matrix of Poisson brackets among the constraints is symplectic or constant. The method is applied to the Proca model, single self dual chiral bosons and chiral Schwinger models as examples.
ON THE NATURE OF THE ANOMALIES IN THE SUPERSYMMETRIC KINK
Authors: FUJIKAWA KAZUO , REBHAN ANTON , VAN NIEUWENHUIZEN PETER
Published: December 2003

We discuss the possibility to absorb all anomalies in the supersymmetry algebra of the N=(1,1) Wess–Zumino model in d=1+1 by a local counterterm. This counterterm corresponds to the change of the vacuum parameter [Formula: see text] in the model and the transition to an unconventional but admissible renormalization scheme. It does not modify the physical consequences such as BPS saturation, and thus the situation is rather different from gauge theory where local counterterms are required to absorb spurious gauge anomalies.
High Glucose-Induced Upregulation of Osteopontin Is Mediated via Rho/Rho Kinase Pathway in Cultured Rat Aortic Smooth Muscle Cells
Authors: Kawamura Harukiyo , Yokote Koutaro , Asaumi Sunao , Kobayashi Kazuki , Fujimoto Masaki , Maezawa Yoshiro , Saito Yasushi , Mori Seijiro
Published: December 2003

Objective—

Osteopontin is upregulated in the diabetic vascular wall and in vascular smooth muscle cells cultured under high glucose concentration. In the present study, we analyzed the mechanism of high glucose-induced upregulation of osteopontin in cultured rat aortic smooth muscle cells.

Methods and Results—

We found that an inhibitor of Rho-associated protein kinase, Y-27632, suppressed osteopontin mRNA expression under high glucose concentration. Transfection of cells with a constitutive active Rho mutant, pSRα-myc-RhoDA, enhanced osteopontin mRNA expression. Furthermore, incubation of cells under high glucose concentration activated Rho, indicating that Rho/Rho kinase pathway mediates high-glucose–stimulated osteopontin expression. Treatment of cells with an inhibitor of protein kinase C, GF109203X, and azaserine, an inhibitor of the hexosamine pathway, suppressed high glucose-induced Rho activation. Glucosamine treatment was shown to activate Rho. Treatment of cells with an inhibitor of MEK1, PD98059, suppressed osteopontin mRNA expression under high glucose concentration. Incubation of cells under high glucose concentration activated ERK. Finally, transfection of cells with pSRα-myc-RhoDA also activated ERK.

Conclusions—

In conclusion, our present findings support a notion that Rho/Rho kinase pathway functions downstream of protein kinase C and the hexosamine pathways and upstream of ERK in mediating high-glucose–induced upregulation of osteopontin expression.
Hyperlipidemia Promotes Osteoclastic Potential of Bone Marrow Cells Ex Vivo
Authors: Tintut Yin , Morony Sean , Demer Linda L.
Published: December 2003

Objectives—

Osteoporosis is associated epidemiologically with atherosclerosis and hyperlipidemia. We previously found that atherogenic lipids regulate bone formation. To determine whether hyperlipidemia also affects bone resorption, we compared osteoclastogenesis in marrow preosteoclasts derived from hyperlipidemic versus control mice.

Methods—

Nonadherent marrow cells from low-density lipoprotein receptor−/− (LDLR−/−)and C57BL/6J mice were cultured with M-CSF and ligand for receptor activator of nuclear factor-kappaB (RANKL). Functional osteoclastic activity, measured as number of resorption pits, was significantly greater in 12-month-old LDLR−/−. Similar results were obtained in 5- and 10-month-old LDLR−/− versus C57BL/6J mice on a high-fat diet. Osteoclastic differentiation, indicated by tartrate resistant acid phosphatase (TRAP) activity, was significantly greater in the 12-month-old LDLR−/−, and there was a trend toward increased TRAP activity in LDLR−/− on a high-fat diet, at ages 5 and 10 months. Osteoclastic parameters correlated with total serum lipoproteins with a possible threshold effect. Osteoporotic human cortical bone stained positive for lipids in the perivascular space of Haversian canals by oil red O. The presence of lipid hydroperoxides was detected in bone marrow from hyperlipidemic mice.

Conclusions—

Hyperlipidemia may contribute to osteoporosis via increased osteoclastic bone resorption.
Distinct Subcellular Localizations of Nox1 and Nox4 in Vascular Smooth Muscle Cells
Authors: Hilenski Lula L. , Clempus Roza E. , Quinn Mark T. , Lambeth J. David , Griendling Kathy K.
Published: December 2003

Objective—

Reactive oxygen species (ROS) that act as signaling molecules in vascular smooth muscle cells (VSMC) and contribute to growth, hypertrophy, and migration in atherogenesis are produced by multi-subunit NAD(P)H oxidases. Nox1 and Nox4, two homologues to the phagocytic NAD(P)H subunit gp91
phox
, both generate ROS in VSMC but differ in their response to growth factors. We hypothesize that the opposing functions of Nox1 and Nox4 are reflected in their differential subcellular locations.

Methods and Results—

We used immunofluorescence to visualize the NAD(P)H subunits Nox1, Nox4, and p22
phox
in cultured rat and human VSMC. Optical sectioning using confocal microscopy showed that Nox1 is co-localized with caveolin in punctate patches on the surface and along the cellular margins, whereas Nox4 is co-localized with vinculin in focal adhesions. These immunocytochemical distributions are supported by membrane fractionation experiments. Interestingly, p22
phox
, a membrane subunit that interacts with the Nox proteins, is found in surface labeling and in focal adhesions in patterns similar to Nox1 and Nox4, respectively.

Conclusions—

The differential roles of Nox1 and Nox4 in VSMC may be correlated with their differential compartmentalization in specific signaling domains in the membrane and focal adhesions.
Apolipoprotein C-I Induces Apoptosis in Human Aortic Smooth Muscle Cells via Recruiting Neutral Sphingomyelinase
Authors: Kolmakova Antonina , Kwiterovich Peter , Virgil Donna , Alaupovic Petar , Knight-Gibson Carolyn , Martin Sergio F. , Chatterjee Subroto
Published: December 2003

Objectives—

Apolipoprotein C-I (apoC-I) influences lipoprotein metabolism, but little is known about its cellular effects in aortic smooth muscle cells (ASMC).

Methods and Results—

In cultured human ASMC, apoC-I and immunoaffinity purified apoC-I–enriched high-density lipoproteins (HDL) markedly induced apoptosis (5- to 25-fold), compared with control cells, apoC-I–poor HDL, and apolipoprotein C-III (apoC-III) as determined by 4′, 6-diamidino-2-phenylindole dihydrochloride staining and DNA ladder assay. Preincubation of cells with GW4869, an inhibitor of neutral sphingomyelinase (N-SMase), blocked apoC-I–induced apoptosis, an effect that was bypassed by C-2 ceramide. The activity of N-SMase was increased 2- to 3-fold in ASMC by apoC-I, apoC-I–enriched HDL, and tumor necrosis factor α (TNF-α) (positive control) after 10 minutes and then decreased over 60 minutes, which is a kinetic pattern not seen with controls, apoC-III, and apoC-I–poor HDL. ApoC-I and apoC-I–enriched HDL stimulated the generation of ceramide, the release of cytochrome c from mitochondria, and activation of caspase-3 greater than that found in controls, apoC-III, and apoC-I–poor HDL. GW4869 inhibited apoC-I–induced production of ceramide and cytochrome c release.

Conclusions—

ApoC-I and apoC-I–enriched HDL activate the N-SMase-ceramide signaling pathway, leading to apoptosis in human ASMC, which is an effect that may promote plaque rupture in vivo.
Impact of Type 2 Diabetes Mellitus on Sympathetic Neural Mechanisms in Hypertension
Authors: Huggett Robert J. , Scott Eleanor M. , Gilbey Stephen G. , Stoker John B. , Mackintosh Alan F. , Mary David A.S.G.
Published: December 2003

Background—

Essential hypertension (EHT) is a major cardiovascular risk factor, and the additional presence of type 2 diabetes mellitus (DM2) increases this risk. However, although the sympathetic nerve hyperactivity of EHT is known to play a role in cardiovascular risk, the level of sympathetic nerve activity is known neither in DM2 nor in hypertensive type 2 diabetic patients (EHT+DM2). Therefore, we planned to quantify the vasoconstrictor sympathetic nerve activity in patients with EHT+DM2 and with DM2 relative to that in matched groups with EHT and normal blood pressure (NT).

Methods and Results—

In 68 closely matched subjects with EHT+DM2 (n=17), DM2 (n=17), EHT (n=17), and NT (n=17), we measured resting muscle sympathetic nerve activity as the mean frequency of multiunit bursts (MSNA) and of single units (s-MSNA) with defined vasoconstrictor properties. The s-MSNA in EHT+DM2 (97±3.8 impulses/100 beats) was greater (at least
P
<0.001) than in EHT (69±3.4 impulses/100 beats) and DM2 (78±4.1 impulses/100 beats), and all these were significantly greater (at least
P
<0.01) than in NT (53±3.3 impulses/100 beats) despite similar age and body mass index. The MSNA followed a similar trend. In addition, the level of insulin was also raised in EHT+DM2 (20.4±3.6 μU/mL) and DM2 (18.1±3.1 μU/mL; at least
P
<0.05) compared with HT or NT.

Conclusions—

Patients with EHT+DM2, EHT, or DM2 had central sympathetic hyperactivity, although plasma insulin levels were raised only in EHT+DM2 and DM2. The combination of EHT and DM2 resulted in the greatest sympathetic hyperactivity and level of plasma insulin, and this hyperactivity could constitute a mechanism for the increased risks of this condition.
Targeting of Apoptotic Macrophages and Experimental Atheroma With Radiolabeled Annexin V
Authors: Kolodgie Frank D. , Petrov Artiom , Virmani Renu , Narula Navneet , Verjans Johan W. , Weber Deena K. , Hartung Dagmar , Steinmetz Neil , Vanderheyden Jean Luc , Vannan Mani A. , Gold Herman K. , Reutelingsperger Chris P.M. , Hofstra Leo , Narula Jagat
Published: December 2003

Background—

Apoptosis is common in advanced human atheroma and contributes to plaque instability. Because annexin V has a high affinity for exposed phosphatidylserine on apoptotic cells, radiolabeled annexin V may be used for noninvasive detection of apoptosis in atherosclerotic lesions.

Methods and Results—

Atherosclerotic plaques were produced in 5 rabbits by deendothelialization of the infradiaphragmatic aorta followed by 12 weeks of cholesterol diet; 5 controls were studied without manipulation. Animals were injected with human recombinant annexin V labeled with technetium-99m before imaging. Aortas were explanted for ex vivo imaging, macroautoradiography, and histological characterization of plaque. Radiolabeled annexin V cleared rapidly from the circulation (T
1/2
, α 9 and β 46 minutes). There was intense uptake of radiolabel within lesions by 2 hours; no uptake was seen in controls. The results were confirmed in the ex vivo imaging of the explanted aorta. Quantitative annexin uptake was 9.3-fold higher in lesion versus nonlesion areas; the lesion-to-blood ratio was 3.0±0.37. Annexin uptake paralleled lesion severity and macrophage burden; no correlation was observed with smooth muscle cells. DNA fragmentation staining of apoptotic nuclei was increased in advanced lesions with evolving necrotic cores, predominantly in macrophages; the uptake of radiolabel correlated with the apoptotic index.

Conclusions—

Because annexin V clears rapidly from blood and targets apoptotic macrophage population, it should constitute an attractive imaging agent for the noninvasive detection of unstable atherosclerotic plaques.
Estrogen-Mediated, Endothelial Nitric Oxide Synthase–Dependent Mobilization of Bone Marrow–Derived Endothelial Progenitor Cells Contributes to Reendothelialization After Arterial Injury
Authors: Iwakura Atsushi , Luedemann Corinne , Shastry Shubha , Hanley Allison , Kearney Marianne , Aikawa Ryuichi , Isner Jeffrey M. , Asahara Takayuki , Losordo Douglas W.
Published: December 2003

Background—

We hypothesized that estrogen-induced acceleration of reendothelialization might be mediated in part by effects involving mobilization and incorporation of bone marrow–derived endothelial progenitor cells (EPCs).

Methods and Results—

Carotid injury was induced in ovariectomized wild-type mice receiving either 17β-estradiol or placebo. Estradiol treatment significantly accelerated reendothelialization of injured arterial segments within 7 days and resulted in a significant reduction of medial thickness 14 and 21 days after the injury. Significant increases in circulating EPCs 3 days after the injury were observed in the estradiol group compared with placebo-treated mice. These data were further supported by fluorescence-activated cell sorting analysis, which disclosed a significant increase in Sca-1/Flk-1–positive cells in estradiol versus control mice. To evaluate the effects of estradiol on bone marrow–derived EPC incorporation at sites of reendothelialization, carotid injury was established in ovariectomized wild-type mice transplanted with bone marrow from transgenic donors expressing β-galactosidase transcriptionally regulated by the Tie-2 promoter. Significantly greater numbers of X-gal–positive cells were observed at reendothelialized areas in the estradiol group 3 days after injury as compared with placebo. Fluorescent immunohistochemistry 14 days after the injury documented a marked increase in cells expressing both β-gal, indicating bone marrow origin and Tie-2 expression, and isolectin B4, also indicating endothelial lineage, in the estradiol group compared with control. In contrast, estradiol did not accelerate reendothelialization or augment EPC mobilization into the peripheral circulation after injury in endothelial nitric oxide synthase–deficient mice (eNOS
−/−
). Furthermore, estradiol exhibited direct stimulatory effects on EPC mitogenic and migration activity and inhibited EPC apoptosis.

Conclusions—

Estradiol accelerates reendothelialization and attenuates medial thickening after carotid injury in part by augmenting mobilization and proliferation of bone marrow–derived EPCs and their incorporation into the recovering endothelium at the site of injury.
Expression of Cholesterol Sulfotransferase (SULT2B1b) in Human Platelets
Authors: Yanai Hidekatsu , Javitt Norman B. , Higashi Yuko , Fuda Hirotoshi , Strott Charles A.
Published: December 2003

Background—

Cholesterol sulfate, the most important sterol sulfate in the human circulation, has emerged as a multifaceted molecule. Among its many demonstrated regulatory actions is its ability to influence blood clotting and fibrinolysis. Additionally, cholesterol sulfate is a constituent of human platelets, where it has been shown to support platelet aggregation.

Methods and Results—

We have documented the presence of the enzyme (SULT2B1b) that sulfonates cholesterol in human platelets and examined the influence of plasma lipoproteins on the expression and activity of this enzyme. SULT2B1b mRNA was detected by reverse transcription-polymerase chain reaction and found to be the only steroid/sterol sulfotransferase expressed in these discoid anucleate particles. Using real-time polymerase chain reaction for quantification, we found that the level of SULT2B1b mRNA in platelets was maintained at 4°C but substantially diminished over a period of 4 hours at 37°C. The loss of SULT2B1b mRNA, however, was markedly reduced in the presence of HDL but not LDL. The stabilizing influence of HDL was attributable specifically to its apolipoprotein (apo) A-I component, whereas apoA-II and apoE were without effect. Importantly, there was a direct correlation between platelet SULT2B1b mRNA and protein levels in the presence or absence of lipoprotein that was reflected in enzymatic activity and cholesterol sulfate production.

Conclusions—

Human platelets selectively express SULT2B1b, the physiological cholesterol sulfotransferase. Furthermore, the stability of SULT2B1b mRNA and protein in platelets maintained at 37°C is subject to regulation by the apoA-I component of HDL.
Association of the Metabolic Syndrome With History of Myocardial Infarction and Stroke in the Third National Health and Nutrition Examination Survey
Authors: Ninomiya John K. , L’Italien Gilbert , Criqui Michael H. , Whyte Joanna L. , Gamst Anthony , Chen Roland S.
Published: December 2003

Background—

The combination of cardiovascular risk factors known as the metabolic syndrome is receiving increased attention from physicians, but data on the syndrome’s association with morbidity are limited.

Methods and Results—

Applying National Cholesterol Education Program Adult Treatment Panel III (NCEP-ATP III) criteria, we evaluated 10 357 NHANES III subjects for the 5 component conditions of the metabolic syndrome: insulin resistance, abdominal obesity based on waist circumference, hypertriglyceridemia, low HDL cholesterol (HDL-C), and hypertension, as well as the full syndrome, defined as at least 3 of the 5 conditions. Logistic regression was used to estimate the cross-sectional association of the syndrome and each of its 5 component conditions separately with history of myocardial infarction (MI), stroke, and either MI or stroke (MI/stroke). Models were adjusted for age, sex, race, and cigarette smoking. The metabolic syndrome was significantly related in multivariate analysis to MI (OR, 2.01; 95% CI, 1.53 to 2.64), stroke (OR, 2.16; 95% CI, 1.48 to 3.16), and MI/stroke (OR, 2.05; 95% CI, 1.64 to 2.57). The syndrome was significantly associated with MI/stroke in both women and men. Among the component conditions, insulin resistance (OR, 1.30; 95% CI, 1.03 to 1.66), low HDL-C (OR, 1.35; 95% CI, 1.05 to 1.74), hypertension (OR, 1.44; 95% CI, 1.00 to 2.08), and hypertriglyceridemia (OR, 1.66; 95% CI=1.20 to 2.30) were independently and significantly related to MI/stroke.

Conclusions—

These results indicate a strong, consistent relationship of the metabolic syndrome with prevalent MI and stroke.
Cardiovascular Mortality and Long-Term Exposure to Particulate Air Pollution
Authors: Pope C. Arden , Burnett Richard T. , Thurston George D. , Thun Michael J. , Calle Eugenia E. , Krewski Daniel , Godleski John J.
Published: December 2003

Background—

Epidemiologic studies have linked long-term exposure to fine particulate matter air pollution (PM) to broad cause-of-death mortality. Associations with specific cardiopulmonary diseases might be useful in exploring potential mechanistic pathways linking exposure and mortality.

Methods and Results—

General pathophysiological pathways linking long-term PM exposure with mortality and expected patterns of PM mortality with specific causes of death were proposed a priori. Vital status, risk factor, and cause-of-death data, collected by the American Cancer Society as part of the Cancer Prevention II study, were linked with air pollution data from United States metropolitan areas. Cox Proportional Hazard regression models were used to estimate PM-mortality associations with specific causes of death. Long-term PM exposures were most strongly associated with mortality attributable to ischemic heart disease, dysrhythmias, heart failure, and cardiac arrest. For these cardiovascular causes of death, a 10-μg/m
3
elevation in fine PM was associated with 8% to 18% increases in mortality risk, with comparable or larger risks being observed for smokers relative to nonsmokers. Mortality attributable to respiratory disease had relatively weak associations.

Conclusions—

Fine particulate air pollution is a risk factor for cause-specific cardiovascular disease mortality via mechanisms that likely include pulmonary and systemic inflammation, accelerated atherosclerosis, and altered cardiac autonomic function. Although smoking is a much larger risk factor for cardiovascular disease mortality, exposure to fine PM imposes additional effects that seem to be at least additive to if not synergistic with smoking.
Nuclear Factor-κB Protects the Adult Cardiac Myocyte Against Ischemia-Induced Apoptosis in a Murine Model of Acute Myocardial Infarction
Authors: Misra Arunima , Haudek Sandra B. , Knuefermann Pascal , Vallejo Jesus G. , Chen Zhijian J. , Michael Lloyd H. , Sivasubramanian Natarajan , Olson Eric N. , Entman Mark L. , Mann Douglas L.
Published: December 2003

Background—

Previous studies have shown that tumor necrosis factor (TNF) confers cytoprotective responses in cardiac myocytes. However, the mechanisms for the cytoprotective effects of TNF remain unknown. Given that TNF signals through nuclear factor κB (NF-κB) and given that NF-κB mediates cytoprotective responses, we asked whether NF-κB activation conferred cytoprotective responses in acute myocardial ischemia/infarction.

Methods and Results—

We examined infarct size and the prevalence of apoptosis in transgenic mice harboring cardiac-restricted expression of a mutated IκBα protein (IκBαΔN) that prevents nuclear translocation of NF-κB in cardiac myocytes. Triphenyltetrazolium chloride staining showed that infarct size was ≈50% greater (
P
<0.02) in the IκBαΔN mice compared with littermate controls at 24 hours. The prevalence of cardiac myocyte apoptosis was significantly greater (
P
<0.008) in the IκBαΔN mice compared with the littermate control mice 3 and 6 hours after left anterior descending occlusion. To explore the mechanism for these findings, we examined protein levels of c-IAP1, c-IAP2, and Bcl-2 as well as manganese superoxide dismutase and c-Jun NH2-terminal kinase activity. These studies showed that protein levels of c-IAP1 and Bcl-2 were significantly lower in the IκBαΔN mice, whereas there was no change in c-IAP2 levels, manganese superoxide dismutase, or c-Jun NH2-terminal kinase activity.

Conclusions—

Transgenic mice with a defect in activation of NF-κB have increased susceptibility to tissue injury after acute left anterior descending occlusion. These studies suggest that the cytoprotective effects of NF-κB are mediated, at least in part, by Bcl-2 or c-IAP1.
Cardiac Systolic and Diastolic Dysfunction After a Cholesterol-Rich Diet
Authors: Huang Y. , Walker K.E. , Hanley F. , Narula J. , Houser S.R. , Tulenko T.N.
Published: December 2003

Background—

Although hypercholesterolemia is a well-established risk factor for coronary artery disease, little is known regarding its direct effects on cardiac function.

Methods and Results—

We examined the effects of cholesterol feeding (0.5%) on cardiac function in rabbits. After 10 weeks, both systolic shortening and diastolic relaxation rates were impaired without any change in aortic pressure or ventricular hypertrophy. However, sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA)-2 mRNA levels were reduced within 4 days after initiation of cholesterol feeding. After this effect, SERCA-2 protein and SERCA-mediated Ca uptake into sarcoplasmic reticulum vesicles were impaired, and the ratio of MHC-β to MHC-α mRNA increased 5-fold. Suppression of the SERCA-2 message correlated temporally with enrichment of the cardiac sarcolemma with cholesterol.

Conclusions—

These data demonstrate that dietary hypercholesterolemia induces a “cholesterol cardiomyopathy” characterized by systolic and diastolic dysfunction. These alterations were independent of vascular disease and demonstrate a dietary link to cardiac dysfunction.
Sudden Death Associated With Short-QT Syndrome Linked to Mutations in HERG
Authors: Brugada Ramon , Hong Kui , Dumaine Robert , Cordeiro Jonathan , Gaita Fiorenzo , Borggrefe Martin , Menendez Teresa M. , Brugada Josep , Pollevick Guido D. , Wolpert Christian , Burashnikov Elena , Matsuo Kiyotaka , Sheng Wu Yue , Guerchicoff Alejandra , Bianchi Francesca , Giustetto Carla , Schimpf Rainer , Brugada Pedro , Antzelevitch Charles
Published: December 2003

Background—

Sudden cardiac death takes the lives of more than 300 000 Americans annually. Malignant ventricular arrhythmias occurring in individuals with structurally normal hearts account for a subgroup of these sudden deaths. The present study describes the genetic basis for a new clinical entity characterized by sudden death and short-QT intervals in the ECG.

Methods and Results—

Three families with hereditary short-QT syndrome and a high incidence of ventricular arrhythmias and sudden cardiac death were studied. In 2 of them, we identified 2 different missense mutations resulting in the same amino acid change (N588K) in the S5-P loop region of the cardiac
I
Kr
channel HERG (KCNH2). The mutations dramatically increase
I
Kr
, leading to heterogeneous abbreviation of action potential duration and refractoriness, and reduce the affinity of the channels to
I
Kr
blockers.

Conclusions—

We demonstrate a novel genetic and biophysical mechanism responsible for sudden death in infants, children, and young adults caused by mutations in KCNH2. The occurrence of sudden cardiac death in the first 12 months of life in 2 patients suggests the possibility of a link between KCNH2 gain of function mutations and sudden infant death syndrome. KCNH2 is the binding target for a wide spectrum of cardiac and noncardiac pharmacological compounds. Our findings may provide better understanding of drug interaction with KCNH2 and have implications for diagnosis and therapy of this and other arrhythmogenic diseases.