bluuebunny/crossref_metadata_2025 huggingface dataset:
GidA Posttranscriptionally Regulates
rhl
Quorum Sensing in
Pseudomonas aeruginosa
Authors: Gupta Rashmi , Gobble Timothy R. , Schuster Martin
Published: July 2009

The opportunistic pathogen
Pseudomonas aeruginosa
utilizes two interconnected acyl-homoserine lactone quorum-sensing (acyl-HSL QS) systems, LasRI and RhlRI, to regulate the expression of hundreds of genes. The QS circuitry itself is integrated into a complex network of regulation by other factors. However, our understanding of this network is still unlikely to be complete, as a comprehensive, saturating approach to identifying regulatory components has never been attempted. Here, we utilized a nonredundant
P. aeruginosa
PA14 transposon library to identify additional genes that regulate QS at the level of LasRI/RhlRI. We initially screened all 5,459 mutants for loss of function in one QS-controlled trait (skim milk proteolysis) and then rescreened attenuated candidates for defects in other QS phenotypes (LasA protease, rhamnolipid, and pyocyanin production) to exclude mutants defective in functions other than QS. We identified several known and novel genes, but only two novel genes,
gidA
and
pcnB
, affected all of the traits assayed. We characterized
gidA
, which exhibited the most striking QS phenotypes, further. This gene is predicted to encode a conserved flavin adenine dinucleotide-binding protein involved in tRNA modification. Inactivation of the gene primarily affected
rhlR
-dependent QS phenotypes such as LasA, pyocyanin, and rhamnolipid production. GidA affected RhlR protein but not transcript levels and also had no impact on LasR and acyl-HSL production. Overexpression of
rhlR
in a
gidA
mutant partially restored QS-dependent phenotypes. Taken together, these results indicate that GidA selectively controls QS gene expression posttranscriptionally via RhlR-dependent and -independent pathways.
Hopanoids Play a Role in Membrane Integrity and pH Homeostasis in
Rhodopseudomonas palustris
TIE-1
Authors: Welander Paula V. , Hunter Ryan C. , Zhang Lichun , Sessions Alex L. , Summons Roger E. , Newman Dianne K.
Published: July 2009

Sedimentary hopanes are pentacyclic triterpenoids that serve as biomarker proxies for bacteria and certain bacterial metabolisms, such as oxygenic photosynthesis and aerobic methanotrophy. Their parent molecules, the bacteriohopanepolyols (BHPs), have been hypothesized to be the bacterial equivalent of sterols. However, the actual function of BHPs in bacterial cells is poorly understood. Here, we report the physiological study of a mutant in
Rhodopseudomonas palustris
TIE-1 that is unable to produce any hopanoids. The deletion of the gene encoding the squalene-hopene cyclase protein (Shc), which cyclizes squalene to the basic hopene structure, resulted in a strain that no longer produced any polycyclic triterpenoids. This strain was able to grow chemoheterotrophically, photoheterotrophically, and photoautotrophically, demonstrating that hopanoids are not required for growth under normal conditions. A severe growth defect, as well as significant morphological damage, was observed when cells were grown under acidic and alkaline conditions. Although minimal changes in
shc
transcript expression were observed under certain conditions of pH shock, the total amount of hopanoid production was unaffected; however, the abundance of methylated hopanoids significantly increased. This suggests that hopanoids may play an indirect role in pH homeostasis, with certain hopanoid derivatives being of particular importance.
Comparisons between Geographically Diverse Samples of Carried
Staphylococcus aureus
Authors: Ruimy Raymond , Armand-Lefevre Laurence , Barbier Francois , Ruppé Etienne , Cocojaru Radu , Mesli Yasmine , Maiga Aminata , Benkalfat Mokhtar , Benchouk Samia , Hassaine Hafida , Dufourcq Jean-Baptiste , Nareth Chhor , Sarthou Jean-Louis , Andremont Antoine , Feil Edward J.
Published: July 2009

Approximately one-third of the human population is asymptomatically colonized by
Staphylococcus aureus
. However, much of the global diversity within the carriage populations remains uncharacterized, and it is unclear to what degree the variation is geographically partitioned. We isolated 300 carriage isolates from 1,531 adults contemporaneously in four countries: France, Algeria, Moldova, and Cambodia. All strains were characterized by multilocus sequence typing. Six clonal complexes (CCs) were present in all four samples (CC30, -45, -121, -15, -5, and -8). Analyses based on the genotype frequencies revealed the French and Algerian samples to be most similar and the Cambodian sample to be most distinct. While this pattern is consistent with likely rates of human migration and geographic distance, stochastic clonal expansion also contributes to regional differences. Phylogenetic analysis revealed a highly divergent and uncharacterized genotype (ST1223) within Cambodia. This lineage is related to CC75, which has previously been observed only in remote aboriginal populations in northern Australia.
Allelic Variation of Bile Salt Hydrolase Genes in
Lactobacillus salivarius
Does Not Determine Bile Resistance Levels
Authors: Fang Fang , Li Yin , Bumann Mario , Raftis Emma J. , Casey Pat G. , Cooney Jakki C. , Walsh Martin A. , O'Toole Paul W.
Published: July 2009

Commensal lactobacilli frequently produce bile salt hydrolase (Bsh) enzymes whose roles in intestinal survival are unclear. Twenty-six
Lactobacillus salivarius
strains from different sources all harbored a
bsh1
allele on their respective megaplasmids. This allele was related to the plasmid-borne
bsh1
gene of the probiotic strain UCC118. A second locus (
bsh2
) was found in the chromosomes of two strains that had higher bile resistance levels. Four Bsh1-encoding allele groups were identified, defined by truncations or deletions involving a conserved residue. In vitro analyses showed that this allelic variation was correlated with widely varying bile deconjugation phenotypes. Despite very low activity of the UCC118 Bsh1 enzyme, a mutant lacking this protein had significantly lower bile resistance, both in vitro and during intestinal transit in mice. However, the overall bile resistance phenotype of this and other strains was independent of the
bsh1
allele type. Analysis of the
L. salivarius
transcriptome upon exposure to bile and cholate identified a multiplicity of stress response proteins and putative efflux proteins that appear to broadly compensate for, or mask, the effects of allelic variation of
bsh
genes. Bsh enzymes with different bile-degrading kinetics, though apparently not the primary determinants of bile resistance in
L. salivarius
, may have additional biological importance because of varying effects upon bile as a signaling molecule in the host.
TheMycobacterium tuberculosisSigma Factor σBIs Required for Full Response to Cell Envelope Stress and Hypoxia In Vitro, but It Is Dispensable for In Vivo Growth
Authors: Fontán P. A. , Voskuil M. I. , Gomez M. , Tan D. , Pardini M. , Manganelli R. , Fattorini L. , Schoolnik G. K. , Smith I.
Published: July 2009

The numerous sigma (σ) factors present inMycobacterium tuberculosisare indicative of the adaptability of this pathogen to different environmental conditions. In this report, we describe theM. tuberculosisσBregulon and the phenotypes of anM. tuberculosis sigBmutant strain exposed to cell envelope stress, oxidative stress, and hypoxia. ThesigBmutant was especially defective in survival under hypoxic conditions in vitro, but it was not attenuated for growth in THP-1 cells or during mouse and guinea pig infection.
Pilin Antigenic Variation Occurs Independently of the RecBCD Pathway in
Neisseria gonorrhoeae
Authors: Helm R. Allen , Seifert H. Steven
Published: July 2009

Type IV pilus expression has been strongly implicated in the virulence of
Neisseria gonorrhoeae
, the causative agent of gonorrhea. In
Neisseria
, these pili undergo frequent antigenic variation (Av), which is presumed to allow reinfection of high-risk groups. Pilin Av is the result of RecA-mediated recombination events between the gene encoding the major pilin subunit (
pilE
) and multiple silent pilin locus (
pilS
) copies, utilizing a RecF-like recombination pathway. The role of RecBCD in pilin Av has been controversial. Previous studies measuring pilin Av in
recB
and
recD
mutants in two independent strains of
N. gonorrhoeae
(MS11 and FA1090) by indirect methods yielded conflicting results. In addition, these two laboratory strains have been suggested to express very different DNA repair capabilities. We show that the FA1090 and MS11 parental strains have similar abilities to repair DNA damage via UV-induced DNA damage, nalidixic acid-induced double-strand breaks, and methyl methanesulfonate-induced alkylation and that RecB and RecD are involved in the repair of these lesions. To test the role of the RecBCD pathway in pilin Av, the rate and frequency of pilin Av were directly measured by sequencing the
pilE
locus in randomly selected piliated progeny of both MS11 and FA1090 in
recB
and
recD
mutants. Our results definitively show that
recB
and
recD
mutants undergo pilin Av at rates similar to those of the parents in both strain backgrounds, demonstrating that efficient pilin Av is neither enhanced nor inhibited by the RecBCD complex.
NtcA-Regulated Heterocyst Differentiation Genes
hetC
and
devB
from
Anabaena
sp. Strain PCC 7120 Exhibit a Similar Tandem Promoter Arrangement
Authors: Muro-Pastor Alicia M. , Flores Enrique , Herrero Antonia
Published: July 2009

Transcription of the
hetC
gene, whose product is required for heterocyst differentiation, takes place from a long promoter region that includes the previously described HetR-independent, NtcA-activated promoter producing transcripts with a 5′ end corresponding to position −571 with respect to the translational start site of
hetC
. Northern blot analysis indicated that the accumulation of
hetC
transcripts depends on HetR, and a second transcriptional start site located at position −293 that leads to NtcA-dependent, HetR-dependent inducible transcription of
hetC
was identified. Upon nitrogen stepdown, expression of a P

hetC

::
gfp
fusion was transiently induced in specific cells that were differentiating into heterocysts, both when the whole promoter region (containing transcription start points −571 and −293) or a short version (containing only the transcription start point −293) was used. Expression of
hetC
from the −293 position was delayed in a strain bearing a deleted promoter region lacking sequences upstream from position −570. Such a strain was still able to differentiate functional heterocysts and to grow diazotrophically, although diazotrophic growth was impaired under certain conditions. Similarly, a second, NtcA-dependent, HetR-dependent transcriptional start site was identified at position −454 in the promoter region upstream from the
devBCA
operon encoding an ABC transport system involved in heterocyst maturation, in which an NtcA-dependent promoter producing transcripts starting at position −704 had been previously noted. Thus, the
hetC
and
devBCA
promoter regions exhibit similar tandem promoter arrangements.
Acyl Enzyme Intermediates in Sortase-Catalyzed Pilus Morphogenesis in Gram-Positive Bacteria
Authors: Guttilla Irene K. , Gaspar Andrew H. , Swierczynski Arlene , Swaminathan Anu , Dwivedi Prabhat , Das Asis , Ton-That Hung
Published: July 2009

In gram-positive bacteria, covalently linked pilus polymers are assembled by a specific transpeptidase enzyme called pilus-specific sortase. This sortase is postulated to cleave the LPXTG motif of a pilin precursor between threonine and glycine and to form an acyl enzyme intermediate with the substrate. Pilus polymerization is believed to occur through the resolution of this intermediate upon specific nucleophilic attack by the conserved lysine located within the pilin motif of another pilin monomer, which joins two pilins with an isopeptide bond formed between threonine and lysine. Here, we present evidence for sortase reaction intermediates in
Corynebacterium diphtheriae
. We show that truncated SrtA mutants that are loosely bound to the cytoplasmic membrane form high-molecular-weight complexes with SpaA polymers secreted into the extracellular milieu. These complexes are not formed with SpaA pilin mutants that have alanine substitutions in place of threonine in the LPXTG motif or lysine in the pilin motif. The same phenotype is observed with alanine substitutions of either the conserved cysteine or histidine residue of SrtA known to be required for catalysis. Remarkably, the assembly of SpaA pili, or the formation of intermediates, is abolished with a SrtA mutant missing the membrane-anchoring domain. We infer that pilus polymerization involves the formation of covalent pilin-sortase intermediates, which occurs within a molecular platform on the exoplasmic face of the cytoplasmic membrane that brings together both sortase and its cognate substrates in close proximity to each other, likely surrounding a secretion apparatus. We present electron microscopic data in support of this picture.
Characterization of Two Outer Membrane Proteins, FlgO and FlgP, That Influence
Vibrio cholerae
Motility
Authors: Martinez Raquel M. , Dharmasena Madushini N. , Kirn Thomas J. , Taylor Ronald K.
Published: July 2009

Vibrio cholerae
is highly motile by the action of a single polar flagellum. The loss of motility reduces the infectivity of
V. cholerae
, demonstrating that motility is an important virulence factor. FlrC is the sigma-54-dependent positive regulator of flagellar genes. Recently, the genes VC2206 (
flgP
) and VC2207 (
flgO
) were identified as being regulated by FlrC via a microarray analysis of an
flrC
mutant (D. C. Morris, F. Peng, J. R. Barker, and K. E. Klose, J. Bacteriol. 190:231-239, 2008). FlgP is reported to be an outer membrane lipoprotein required for motility that functions as a colonization factor. The study reported here focuses on the characterization of
flgO
, the first gene in the
flgOP
operon. We show that FlgO and FlgP are important for motility, as strains with mutations in the
flgOP
genes have reduced motility phenotypes. The
flgO
and
flgP
mutant populations display fewer motile cells as well as reduced numbers of flagellated cells. The flagella produced by the
flgO
and
flgP
mutant strains are shorter in length than the wild-type flagella, which can be restored by inhibiting rotation of the flagellum. FlgO is an outer membrane protein that localizes throughout the membrane and not at the flagellar pole. Although FlgO and FlgP do not specifically localize to the flagellum, they are required for flagellar stability. Due to the nature of these motility defects, we established that the flagellum is not sufficient for adherence; rather, motility is the essential factor required for attachment and thus colonization by
V. cholerae
O1 of the classical biotype. This study reveals a novel mechanism for which the outer membrane proteins FlgO and FlgP function in motility to mediate flagellar stability and influence attachment and colonization.
Superdormant Spores of
Bacillus
Species Have Elevated Wet-Heat Resistance and Temperature Requirements for Heat Activation
Authors: Ghosh Sonali , Zhang Pengfei , Li Yong-qing , Setlow Peter
Published: July 2009

Purified superdormant spores of
Bacillus cereus
,
B. megaterium
, and
B. subtilis
isolated after optimal heat activation of dormant spores and subsequent germination with inosine,
d
-glucose, or
l
-valine, respectively, germinate very poorly with the original germinants used to remove dormant spores from spore populations, thus allowing isolation of the superdormant spores, and even with alternate germinants. However, these superdormant spores exhibited significant germination with the original or alternate germinants if the spores were heat activated at temperatures 8 to 15°C higher than the optimal temperatures for the original dormant spores, although the levels of superdormant spore germination were not as great as those of dormant spores. Use of mixtures of original and alternate germinants lowered the heat activation temperature optima for both dormant and superdormant spores. The superdormant spores had higher wet-heat resistance and lower core water content than the original dormant spore populations, and the environment of dipicolinic acid in the core of superdormant spores as determined by Raman spectroscopy of individual spores differed from that in dormant spores. These results provide new information about the germination, heat activation optima, and wet-heat resistance of superdormant spores and the heterogeneity in these properties between individual members of dormant spore populations.
Role of TonB1 in Pyoverdine-Mediated Signaling inPseudomonas aeruginosa
Authors: Shirley Matt , Lamont Iain L.
Published: July 2009

Pyoverdines are siderophores secreted byPseudomonas aeruginosa. Uptake of ferripyoverdine inP. aeruginosaPAO1 occurs via the FpvA receptor protein and requires the energy-transducing protein TonB1. Interaction of (ferri)pyoverdine with FpvA activates pyoverdine gene expression in a signaling process involving the cytoplasmic-membrane-spanning anti-sigma factor FpvR and the sigma factor PvdS. Here, we show that mutation of a region of FpvA that interacts with TonB1 (the TonB box) prevents this signaling process, as well as inhibiting bacterial growth in the presence of the iron-chelating compound ethylenediamine-di(o-hydroxy-phenylacetic acid). Signaling via wild-type FpvA was also eliminated in strains lacking TonB1 but was unaffected in strains lacking either (or both) of two other TonB proteins inP. aeruginosa, TonB2 and TonB3. An absence of pyoverdine-mediated signaling corresponded with proteolysis of PvdS. These data show that interactions between FpvA and TonB1 are required for (ferri)pyoverdine signal transduction, as well as for ferripyoverdine transport, consistent with a mechanistic link between the signaling and transport functions of FpvA.
The transport of LDL across the deformable arterial wall: the effect of endothelial cell turnover and intimal deformation under hypertension
Authors: Dabagh Mahsa , Jalali Payman , Tarbell John M.
Published: July 2009

A multilayered model of the aortic wall is introduced to investigate the transport of low-density lipoprotein (LDL) under hypertension, taking into account the influences of increased endothelial cell turnover and deformation of the intima at higher pressure. Meanwhile, the thickness and properties of the endothelium, intima, internal elastic lamina (IEL), and media are affected by the transmural pressure. The LDL macromolecules enter the intima through leaky junctions over the endothelium, which are created by dying or dividing cells. Water molecules enter the intima via the paracellular pathway through breaks in tight junctions after passing the glycocalyx as well as through leaky junctions. The glycocalyx is modeled as a Brinkman porous medium to describe the fluid filtration associated with its structure. Combined Navier-Stokes and Brinkman equations are solved for the transmural flow, and the convective-diffusion equation is employed for LDL transport. The permeation of LDL over the surface of smooth muscle cells is modeled through a uniform reaction evenly distributed in the macroscopically homogeneous media layer. Simulations are performed in an axisymmetric plane centered at a leaky cell. The overriding issue addressed is that LDL fluxes across the leaky junction, the intima, fenestral pores in the IEL, and the media layer are highly affected by the transmural pressure, which affects the endothelial cell turnover rate and the compaction of intima. The present model, for the first time and with no adjustable parameters, is capable of making many realistic predictions including the proper magnitudes for the permeability of endothelium and intimal layers and the hydraulic conductivity of all layers as well as their trends with pressure. Results for the volume flux through the wall and the hydraulic conductivity of the entire arterial wall, the endothelium, and subendothelial layers at 70 and 180 mmHg are in good agreement with previous experimental studies.
A1adenosine receptor-mediated PKC and p42/p44 MAPK signaling in mouse coronary artery smooth muscle cells
Authors: Ansari Habib R. , Teng Bunyen , Nadeem Ahmed , Roush Kevin P. , Martin Karen H. , Schnermann J. , Mustafa S. Jamal
Published: July 2009

The A1adenosine receptor (A1AR) is coupled to Gi/Goproteins, but the downstream signaling pathways in smooth muscle cells are unclear. This study was performed in coronary artery smooth muscle cells (CASMCs) isolated from the mouse heart [A1AR wild type (A1WT) and A1AR knockout (A1KO)] to delineate A1AR signaling through the PKC pathway. In A1WT cells, treatment with (2 S)- N6-(2-endo-norbornyl)adenosine (ENBA; 10−5M) increased A1AR expression by 150%, which was inhibited significantly by the A1AR antagonist 1,3-dipropyl-8-cyclopentylxanthine (10−6M), but not in A1KO CASMCs. PKC isoforms were identified by Western blot analysis in the cytosolic and membrane fractions of cell homogenates of CASMCs. In A1WT and A1KO cells, significant levels of basal PKC-α were detected in the cytosolic fraction. Treatment with the A1AR agonist ENBA (10−5M) translocated PKC-α from the cytosolic to membrane fraction significantly in A1WT but not A1KO cells. Phospholipase C isoforms (βI, βIII, and γ1) were analyzed using specific antibodies where ENBA treatment led to the increased expression of PLC-βIII in A1WT CASMCs while having no effect in A1KO CASMCs. In A1WT cells, ENBA increased PKC-α expression and p42/p44 MAPK (ERK1/2) phospohorylation by 135% and 145%, respectively. These effects of ENBA were blocked by Gö-6976 (PKC-α inhibitor) and PD-98059 (p42/p44 MAPK inhibitor). We conclude that A1AR stimulation by ENBA activates the PKC-α signaling pathway, leading to p42/p44 MAPK phosphorylation in CASMCs.
Myocardial lysyl oxidase regulation of cardiac remodeling in a murine model of diet-induced metabolic syndrome
Authors: Zibadi Sherma , Vazquez Randy , Moore Derek , Larson Douglas F. , Watson Ronald R.
Published: July 2009

Metabolic syndrome (MetS) represents an increased risk of cardiovascular disease. Although its individual components adversely affect cardiac structure and function, the extent to which multiple components of MetS affect the cardiac extracellular matrix (ECM) has not been well characterized. Lysyl oxidase (LOX) is one of the cardiac ECM-modifying enzymes that catalyze the formation of collagen cross-linking. Our objective was to define the effect of diet-induced MetS on the LOX enzyme. MetS was induced in male C57BL/6 mice by administrating a high-fat, high-simple carbohydrate diet for 6 mo. Gene expression was determined by real-time PCR. The cardiac protein expression and enzymatic activity of LOX were measured. The severity of fibrosis was assessed by histology and hydroxylproline assay. Cardiac diastolic function was assessed by in vivo analysis of the pressure-volume relationship. LOX, matrix metalloproteinases, and their tissue inhibitors were analyzed, and of these three, LOX was most significantly changed in the MetS mice. Despite the blunted gene expression of LOX isoforms, MetS mice demonstrated a significant upregulation of bone morphogenetic protein-1. Correspondingly, there was an increase in the ratio of protein expression of mature to proenzyme LOX by 25.9%, enhanced LOX activity by 50.0%, and increased cardiac cross-linked collagen compared with the controls. This fibrotic response coincided with a marked increase in end-diastolic pressure, increased left ventricular stiffness, and impaired diastolic filling pattern. Our data signify that diet-induced MetS alters the remodeling enzymes, mainly LOX, thereby altering ECM structure by increasing the amount of cross-linking and inducing diastolic dysfunction.
Intermedin is a new angiogenic growth factor
Authors: Smith Robert S. , Gao Lin , Bledsoe Grant , Chao Lee , Chao Julie
Published: July 2009

Intermedin (IMD) is a newly discovered peptide closely related to adrenomedullin. We recently reported that IMD gene delivery prevented kidney damage and capillary loss in a rat model of chronic renal injury. In this study, we evaluated the role of IMD in angiogenesis in the ischemic hindlimb. Adenovirus containing human IMD or control adenovirus (Ad.Null) was injected into the adductor muscles of rats immediately after femoral artery ligation. The expression of human IMD was detected in the skeletal muscle 5 days after the viral injection. Blood perfusion in the ischemic hindlimb was monitored by laser-Doppler imaging from 1 to 3 wk after gene delivery. When compared with animals receiving Ad.Null, those with IMD gene transfer resulted in a time-dependent increase in blood perfusion. IMD gene delivery also increased capillary and arteriole density in ischemic hindlimb, identified by anti-CD-31 and α-smooth muscle actin immunostaining. Angiogenesis promoted by IMD was confirmed by increased capillary formation and hemoglobin content in Matrigel implants containing IMD peptide in mice. In cultured endothelial cells, IMD induced cell migration and tube formation, and these effects were blocked by the inhibition of extracellular signal-regulated kinase (ERK), Akt, nitric oxide (NO) synthase (NOS), vascular endothelial growth factor receptor-2 (VEGFR-2), and anti-IMD-neutralizing antibody. IMD was found to increase the phosphorylation of ERK, Akt, and endothelial NOS, as well as to augment NO formation, VEGF, and VEGFR-2 synthesis. Taken together, these results indicate that IMD enhances angiogenesis through ERK, Akt/NOS/NO, and VEGF/VEGFR-2 signaling pathways and raises the potential of IMD gene or peptide administration in the modulation of endothelial dysfunction.
Absence of gp130 in dopamine β-hydroxylase-expressing neurons leads to autonomic imbalance and increased reperfusion arrhythmias
Authors: Parrish Diana C. , Alston Eric N. , Rohrer Hermann , Hermes Sam M. , Aicher Sue A. , Nkadi Paul , Woodward William R. , Stubbusch Jutta , Gardner Ryan T. , Habecker Beth A.
Published: July 2009

Inflammatory cytokines that act through glycoprotein (gp)130 are elevated in the heart after myocardial infarction and in heart failure. These cytokines are potent regulators of neurotransmitter and neuropeptide production in sympathetic neurons but are also important for the survival of cardiac myocytes after damage to the heart. To examine the effect of gp130 cytokines on cardiac nerves, we used gp130DBH-Cre/loxmice, which have a selective deletion of the gp130 cytokine receptor in neurons expressing dopamine β-hydroxylase (DBH). Basal sympathetic parameters, including norepinephrine (NE) content, tyrosine hydroxylase expression, NE transporter expression, and sympathetic innervation density, appeared normal in gp130DBH-Cre/loxcompared with wild-type mice. Likewise, basal cardiovascular parameters measured under isoflurane anesthesia were similar in both genotypes, including mean arterial pressure, left ventricular peak systolic pressure, dP/d tmax, and dP/d tmin. However, pharmacological interventions revealed an autonomic imbalance in gp130DBH-Cre/loxmice that was correlated with an increased incidence of premature ventricular complexes after reperfusion. Stimulation of NE release with tyramine and infusion of the β-agonist dobutamine revealed blunted adrenergic transmission that correlated with decreased β-receptor expression in gp130DBH-Cre/loxhearts. Due to the developmental expression of the DBH-Cre transgene in parasympathetic ganglia, gp130 was eliminated. Cholinergic transmission was impaired in gp130DBH-Cre/loxhearts due to decreased parasympathetic drive, but tyrosine hydroxylase immunohistochemistry in the brain stem revealed that catecholaminergic nuclei appeared grossly normal. Thus, the apparently normal basal parameters in gp130DBH-Cre/loxmice mask an autonomic imbalance that includes alterations in sympathetic and parasympathetic transmission.
Senescence, apoptosis, and stem cell biology: the rationale for an expanded view of intracrine action
Authors: Re Richard N. , Cook Julia L.
Published: July 2009

Some extracellular-signaling peptides also at times function within the intracellular space. We have termed these peptides intracrines and have argued that intracrine function is associated with a wide variety of peptides/proteins including hormones, growth factors, cytokines, enzymes, and DNA-binding proteins among others. Here we consider the possibility that intracrines participate in the related phenomena of senescence, apoptosis, and stem cell regulation of tissue biology. Based on this analysis, we also suggest that the concept of intracrine action be expanded to include possible regulatory peptide transfer via exosomes/microvesicles and possibly by nanotubes. Moreover, the process of microvesicular and nanotube transfer of peptides and other biologically relevant molecules, which we inclusively term laterality, is explored. These notions have potentially important therapeutic implications, including implications for the therapy of cardiovascular disease.
Reduction of repolarization reserve unmasks the proarrhythmic role of endogenous late Na+current in the heart
Authors: Wu Lin , Rajamani Sridharan , Li Hong , January Craig T. , Shryock John C. , Belardinelli Luiz
Published: July 2009

Reduction of repolarization reserve increases the risk of arrhythmia. We hypothesized that inhibition of K+current ( IK) to decrease repolarization reserve would unmask the proarrhythmic role of endogenous, physiological late Na+current (late INa). Monophasic action potentials (MAP) and 12-lead electrocardiogram were recorded from female rabbit isolated hearts. To block IKand reduce repolarization reserve, E-4031, 4-aminopyridine, and BaCl2were used; to block endogenous late INa, tetrodotoxin (TTX) and ranolazine were used. E-4031 (1–60 nM) concentration-dependently prolonged MAP duration (MAPD90) and increased duration of the T wave from Tpeakto Tend(Tpeak-Tend), transmural dispersion of repolarization (TDR), and beat-to-beat variability (BVR) of MAPD90. E-4031 caused spontaneous and pause-triggered polymorphic ventricular tachycardia [ torsade de pointes (TdP)]. In the presence of 60 nM E-4031, TTX (0.6–3 μM) and ranolazine (5–10 μM) shortened MAPD90, decreased TDR, BVR, and Tpeak-Tend( n = 9–20, P < 0.01), and abolished episodes of TdP. In hearts treated with BaCl2or 4-aminopyridine plus E-4031, TTX (0.6–3 μM) shortened MAPD90and decreased Tpeak-Tend. Ranolazine could not reverse the effect of E-4031 to inhibit human ether-a-go-go-related gene (HERG) K+current; thus, the reversal by ranolazine of effects of E-4031 was likely due to inhibition of late INaand not to antagonism of the HERG-blocking action of E-4031. We conclude that endogenous, physiological late INacontributes to arrhythmogenesis in hearts with reduced repolarization reserve. Inhibition of this current partially reverses MAPD prolongation and abolishes arrhythmic activity caused by IKinhibitors.
Determinants of left ventricular shear strain
Authors: Bovendeerd Peter H. M. , Kroon Wilco , Delhaas Tammo
Published: July 2009

Mathematical models of cardiac mechanics can potentially be used to relate abnormal cardiac deformation, as measured noninvasively by ultrasound strain rate imaging or magnetic resonance tagging (MRT), to the underlying pathology. However, with current models, the correct prediction of wall shear strain has proven to be difficult, even for the normal healthy heart. Discrepancies between simulated and measured strains have been attributed to 1) inadequate modeling of passive tissue behavior, 2) neglecting active stress development perpendicular to the myofiber direction, or 3) neglecting crossover of myofibers in between subendocardial and subepicardial layers. In this study, we used a finite-element model of left ventricular (LV) mechanics to investigate the sensitivity of midwall circumferential-radial shear strain ( Ecr) to settings of parameters determining passive shear stiffness, cross-fiber active stress development, and transmural crossover of myofibers. Simulated time courses of midwall LV Ecrwere compared with time courses obtained in three healthy volunteers using MRT. Ecras measured in the volunteers during the cardiac cycle was characterized by an amplitude of ∼0.1. In the simulations, a realistic amplitude of the Ecrsignal could be obtained by tuning either of the three model components mentioned above. However, a realistic time course of Ecr, with virtually no change of Ecrduring isovolumic contraction and a correct base-to-apex gradient of Ecrduring ejection, could only be obtained by including transmural crossover of myofibers. Thus, accounting for this crossover seems to be essential for a realistic model of LV wall mechanics.
Regulation of soluble guanylyl cyclase-α1 expression in chronic hypoxia-induced pulmonary hypertension: role of NFATc3 and HuR
Authors: de Frutos Sergio , Nitta Carlos H. , Caldwell Elizabeth , Friedman Jessica , González Bosc Laura V.
Published: July 2009

The nitric oxide/soluble guanylyl cyclase (sGC) signal transduction pathway plays an important role in smooth muscle relaxation and phenotypic regulation. However, the transcriptional regulation of sGC gene expression is largely unknown. It has been shown that sGC expression increases in pulmonary arteries from chronic hypoxia-induced pulmonary hypertensive animals. Since the transcription factor NFATc3 is required for the upregulation of the smooth muscle hypertrophic/differentiation marker α-actin in pulmonary artery smooth muscle cells from chronically hypoxic mice, we hypothesized that NFATc3 is required for the regulation of sGC-α1 expression during chronic hypoxia. Exposure to chronic hypoxia for 2 days induced a decrease in sGC-α1 expression in mouse pulmonary arteries. This reduction was independent of NFATc3 but mediated by nuclear accumulation of the mRNA-stabilizing protein human antigen R (HuR). Consistent with our hypothesis, chronic hypoxia (21 days) upregulated pulmonary artery sGC-α1 expression, bringing it back to the level of the normoxic controls. This response was prevented in NFATc3 knockout and cyclosporin (calcineurin/NFATc inhibitor)-treated mice. Furthermore, we identified effective binding sites for NFATc in the mouse sGC-α1 promoter. Activation of NFATc3 increased sGC-α1 promoter activity in human embryonic derived kidney cells, rat aortic-derived smooth muscle cells, and human pulmonary artery smooth muscle cells. Our results suggest that NFATc3 and HuR are important regulators of sGC-α1 expression in pulmonary vascular smooth muscle cells during chronic hypoxia-induced pulmonary hypertension.
Role of HIF-1α in the regulation ACE and ACE2 expression in hypoxic human pulmonary artery smooth muscle cells
Authors: Zhang Ruifeng , Wu Yingli , Zhao Meng , Liu Chuanxu , Zhou Lin , Shen Shaoming , Liao Shihua , Yang Kun , Li Qingyun , Wan Huanying
Published: July 2009

Angiotensin-converting enzyme (ACE) enhances the proliferation and migration of pulmonary artery smooth muscle cells (PASMCs), which contribute to the pathogenesis of hypoxic pulmonary hypertension (HPH). Previous reports have demonstrated that hypoxia upregulates ACE expression, but the underlying mechanism is unknown. Here, we found that ACE is persistently upregulated in PASMCs on the transcriptional level during hypoxia. Hypoxia-inducible factor 1α (HIF-1α), a key transcription factor activated during hypoxia, was able to upregulate ACE protein expression under normoxia, whereas knockdown of HIF-1α expression in PASMCs inhibited hypoxia-induced ACE upregulation. Furthermore, HIF-1α can bind and transactivate the ACE promoter directly. Therefore, we report that ACE is a novel target of HIF-1α. Recently, a homolog of ACE, ACE2, was reported to counterbalance the function of ACE. In contrast to ACE, we found that ACE2 mRNA and protein levels increased during the early stages of hypoxia and decreased to near-baseline levels at the later stages after HIF-1α accumulation. Thus HIF-1α inhibited ACE2 expression, and the accumulated ANG II catalyzed by ACE is a key mediator in the downregulation of ACE2 by HIF-1α. Moreover, a reduction of ACE2 expression in PASMCs by RNA interference was accompanied by significantly enhanced proliferation and migration during hypoxia. We conclude that ACE is directly regulated by HIF-1α, whereas ACE2 is regulated in a bidirectional way during hypoxia and may play a protective role during the development of HPH. In sum, these findings contribute to the understanding of the pathogenesis of HPH.
Mutation of protein kinase C phosphorylation site S1076 on α-subunits affects BKCachannel activity in HEK-293 cells
Authors: Zhu Shu , Browning Darren D. , White Richard E. , Fulton David , Barman Scott A.
Published: July 2009

Large conductance, calcium- and voltage-activated potassium (BKCa) channels are important modulators of pulmonary vascular smooth muscle membrane potential, and phosphorylation of BKCachannels by protein kinases regulates pulmonary arterial smooth muscle function. However, little is known about the effect of phosphorylating specific channel subunits on BKCachannel activity. The present study was done to determine the effect of mutating protein kinase C (PKC) phosphorylation site serine 1076 (S1076) on transfected human BKCachannel α-subunits in human embryonic kidney (HEK-293) cells, a heterologous expression system devoid of endogenous BKCachannels. Results showed that mutating S1076 altered the effect of PKC activation on BKCachannels in HEK-293 cells. Specifically, the phospho-deficient mutation BKCa-α(S1076A)/β1attenuated the excitatory effect of the PKC activator phorbol myristate acetate (PMA) on BKCachannels, whereas the phospho-mimetic mutation BKCa-α(S1076E)/β1increased the excitatory effect of PMA on BKCachannels. In addition, the phospho-null mutation S1076A blocked the activating effect of cGMP-dependent protein kinase G (PKG) on BKCachannels. Collectively, these results suggest that specific putative PKC phosphorylation site(s) on human BKCachannel α-subunits influences BKCachannel activity, which may subsequently alter pulmonary vascular smooth muscle function and tone.
NADPH oxidases and reactive oxygen species at different stages of chronic hypoxia-induced pulmonary hypertension in newborn piglets
Authors: Dennis Kathleen E. , Aschner J. L. , Milatovic D. , Schmidt J. W. , Aschner M. , Kaplowitz M. R. , Zhang Y. , Fike Candice D.
Published: July 2009

Recently, we reported that reactive oxygen species (ROS) generated by NADPH oxidase (NOX) contribute to aberrant responses in pulmonary resistance arteries (PRAs) of piglets exposed to 3 days of hypoxia ( Am J Physiol Lung Cell Mol Physiol 295: L881–L888, 2008). An objective of the present study was to determine whether NOX-derived ROS also contribute to altered PRA responses at a more advanced stage of pulmonary hypertension, after 10 days of hypoxia. We further wished to advance knowledge about the specific NOX and antioxidant enzymes that are altered at early and later stages of pulmonary hypertension. Piglets were raised in room air (control) or hypoxia for 3 or 10 days. Using a cannulated artery technique, we found that treatments with agents that inhibit NOX (apocynin) or remove ROS [an SOD mimetic (M40403) + polyethylene glycol-catalase] diminished responses to ACh in PRAs from piglets exposed to 10 days of hypoxia. Western blot analysis showed an increase in expression of NOX1 and the membrane fraction of p67phox. Expression of NOX4, SOD2, and catalase were unchanged, whereas expression of SOD1 was reduced, in arteries from piglets raised in hypoxia for 3 or 10 days. Markers of oxidant stress, F2-isoprostanes, measured by gas chromatography-mass spectrometry, were increased in PRAs from piglets raised in hypoxia for 3 days, but not 10 days. We conclude that ROS derived from some, but not all, NOX family members, as well as alterations in the antioxidant enzyme SOD1, contribute to aberrant PRA responses at an early and a more progressive stage of chronic hypoxia-induced pulmonary hypertension in newborn piglets.
β-Adrenoceptor stimulation of alveolar fluid clearance is increased in rats with heart failure
Authors: Maron Michael B. , Luther Daniel J. , Pilati Charles F. , Ohanyan Vahagn , Li Tianbo , Koshy Shyny , Horne Walter I. , Meszaros J. Gary , Walro Jon M. , Folkesson Hans G.
Published: July 2009

The alveolar epithelium plays a critical role in resolving pulmonary edema. We thus hypothesized that its function might be upregulated in rats with heart failure, a condition that severely challenges the lung's ability to maintain fluid balance. Heart failure was induced by left coronary artery ligation. Echocardiographic and cardiovascular hemodynamics confirmed its development at 16 wk postligation. At that time, alveolar fluid clearance was measured by an increase in protein concentration over 1 h of a 5% albumin solution instilled into the lungs. Baseline alveolar fluid clearance was similar in heart failure and age-matched control rats. Terbutaline was added to the instillate to determine whether heart failure rats responded to β-adrenoceptor stimulation. Alveolar fluid clearance in heart failure rats was increased by 194% after terbutaline stimulation compared with a 153% increase by terbutaline in control rats. To determine the mechanisms responsible for this accelerated alveolar fluid clearance, we measured ion transporter expression (ENaC, Na-K- ATPase, CFTR). No significant upregulation was observed for these ion transporters in the heart failure rats. Lung morphology showed significant alveolar epithelial type II cell hyperplasia in heart failure rats. Thus, alveolar epithelial type II cell hyperplasia is the likely explanation for the increased terbutaline-stimulated alveolar fluid clearance in heart failure rats. These data provide evidence for previously unrecognized mechanisms that can protect against or hasten resolution of alveolar edema in heart failure.
Sphingosine 1-Phosphate
Authors: Daum G. , Grabski A. , Reidy M.A.
Published: July 2009

Sphingosine-1 phosphate (S1P) is a bioactive sphingolipid that is critical in the development of blood vessels, and in the adult regulates vascular functions including vascular tone, endothelial integrity, and angiogenesis. Further, S1P may regulate arterial lesions in disease and after injury by controlling leukocyte recruitment and smooth muscle cell functions.
Tissue Factor in Coagulation
Authors: Butenas Saulius , Orfeo Thomas , Mann Kenneth G.
Published: July 2009

Tissue factor (TF) is an integral membrane protein, normally separated from the blood by the vascular endothelium, which plays a key role in the initiation of blood coagulation. With a perforating vascular injury, TF becomes exposed to blood and binds plasma factor VIIa. The resulting complex initiates a series of enzymatic reactions leading to clot formation and vascular sealing. In some pathological states, circulating blood cells express TF as a result of exposure to an inflammatory stimulus leading to intravascular clotting, vessel occlusion, and thrombotic pathology. Numerous controversies have arisen related to the influence of structural features of TF, its presentation, and its function. There are contradictory reports about the synthesis and presentation of TF on blood cells and the presence (or absence) of functionally active TF circulating in normal blood either on microparticles or as a soluble protein. In this review we discuss TF structure-function relationships and the role of TF during various phases of the blood coagulation process. We also highlight controversies concerning the expression/presence of TF on various cells and in blood in normal and pathological states.
Osteoprotegerin Promotes Fibrous Cap Formation in Atherosclerotic Lesions of ApoE-Deficient Mice—Brief Report
Authors: Ovchinnikova Olga , Gylfe Åsa , Bailey Leslie , Nordström Anna , Rudling Mats , Jung Christian , Bergström Sven , Waldenström Anders , Hansson Göran K , Nordström Peter
Published: July 2009

Objective—

Osteoprotegerin (OPG) is a tumor necrosis factor receptor–related cytokine, initially found to inhibit osteoclastogenesis. In the present study we investigated the effect of OPG treatment on atherosclerosis.

Methods and Results—

Hypercholesterolemic
apoe
−/−
mice were treated with recombinant 15 mg/kg OPG or vehicle injections twice a week for 10 consecutive weeks. Mice treated with OPG showed increased amounts of smooth muscle cells and collagen within the atherosclerotic lesions. OPG treatment did not affect atherosclerotic lesion size (8.2% versus 7.6%) or total vessel area but led to a 250% increase in lesion collagen, formation of mature collagen fibers in subendothelial fibrous caps, and upregulated mRNA for lysyl oxidase that promotes collagen crosslinking. In cell culture studies, OPG promoted cell proliferation in rat aortic smooth muscle cells. In contrast, OPG treatment did not affect markers of vascular or systemic inflammation.

Conclusion—

OPG treatment promotes smooth muscle accumulation, collagen fiber formation, and development of fibrous caps but does not affect inflammatory properties of atherosclerotic lesions. Its effects may contribute to plaque stabilization.
Nrf2 Protects Against Maladaptive Cardiac Responses to Hemodynamic Stress
Authors: Li Jinqing , Ichikawa Tomonaga , Villacorta Luis , Janicki Joseph S. , Brower Gregory L. , Yamamoto Masayuki , Cui Taixing
Published: July 2009

Background—

Reactive oxygen species (ROS) play an important role in the maintenance of cardiovascular homeostasis. The present study sought to determine whether nuclear factor erythroid-2 related factor 2 (Nrf2), a master gene of the endogenous antioxidant defense system, is a critical regulator of the cardiac hypertrophic response to pathological stress.

Methods and Results—

Cardiac hypertrophy and dysfunction were established in mice by transverse aortic constriction (TAC). Nrf2 expression was transiently increased and then declined to the basal level while impairment of cardiac function proceeded. The knockout of Nrf2 (Nrf2
−/−
) did not cause any apparent structural and functional abnormalities in the unstressed heart. However, Nrf2
−/−
mice after TAC developed pathological cardiac hypertrophy, significant myocardial fibrosis and apoptosis, overt heart failure, and increased mortality, which were associated with elevated myocardial levels of 4-hydroxy-2-nonenal and 8-hydroxydeoxyguanosine and a complete blockade of the myocardial expression of several antioxidant genes. Overexpression of Nrf2 dramatically inhibited hypertrophic factor–induced ROS production and growth in both cardiomyocytes and cardiac fibroblasts, whereas knockdown of Nrf2 exerted opposite effects in both cells.

Conclusions—

These findings demonstrate that activation of Nrf2 provides a novel mechanism to protect the murine heart against pathological cardiac hypertrophy and heart failure via suppressing oxidative stress.