bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential Roles of the ChiB Chitinase in Autolysis and Cell Death of
Aspergillus nidulans
Authors: Shin Kwang-Soo , Kwon Nak-Jung , Kim Young Hwan , Park Hee-Soo , Kwon Gi-Seok , Yu Jae-Hyuk
Published: March 2009

Autolysis is a natural event that occurs in most filamentous fungi. Such self-degradation of fungal cells becomes a predominant phenomenon in the absence of the regulator of G protein signaling FlbA in
Aspergillus nidulans
. Among a number of potential hydrolytic enzymes in the
A. nidulans
genome, the secreted endochitinase ChiB was shown to play a major role in autolysis. In this report, we investigate the roles of ChiB in fungal autolysis and cell death processes through genetic, biochemical, and cellular analyses using a set of critical mutants. Determination of mycelial mass revealed that, while the
flbA
deletion (Δ
flbA
) mutant autolyzed completely after a 3-day incubation, the Δ
flbA
Δ
chiB
double mutant escaped from hyphal disintegration. These results indicate that ChiB is necessary for the Δ
flbA
-induced autolysis. However, importantly, both Δ
flbA
and Δ
flbA
Δ
chiB
strains displayed dramatically reduced cell viability compared to the wild type. These imply that ChiB is dispensable for cell death and that autolysis and cell death are separate processes. Liquid chromatography-tandem mass spectrometry analyses of the proteins that accumulate at high levels in the Δ
flbA
and Δ
flbA
Δ
chiB
mutants identify chitinase (ChiB), dipeptidyl peptidase V (DppV),
O
-glycosyl compound hydrolase, β-
N
-acetylhexosaminidase (NagA), and
myo
-inositol-1-phosphate synthase (InoB). Functional characterization of these four genes reveals that the deletion of
nagA
results in reduced cell death. A working model bridging G protein signaling and players in autolysis/cell death is proposed.
Type II NADH Dehydrogenase Inhibitor 1-Hydroxy-2-Dodecyl- 4(1H)Quinolone Leads to Collapse of Mitochondrial Inner- Membrane Potential and ATP Depletion inToxoplasma gondii
Authors: Lin San San , Groß Uwe , Bohne Wolfgang
Published: March 2009

The apicomplexan parasiteToxoplasma gondiiexpresses type II NADH dehydrogenases (NDH2s) instead of canonical complex I at the inner mitochondrial membrane. These non-proton-pumping enzymes are considered to be promising drug targets due to their absence in mammalian cells. We recently showed by inhibition kinetics thatT. gondiiNDH2-I is a target of the quinolone-like compound 1-hydroxy-2-dodecyl-4(1H)quinolone (HDQ), which inhibitsT. gondiireplication in the nanomolar range. In this study, the cationic fluorescent probes Mitotracker and DiOC6(3) (3,3′-dihexyloxacarbocyanine iodine) were used to monitor the influence of HDQ on the mitochondrial inner membrane potential (ΔΨm) inT. gondii. Real-time imaging revealed that nanomolar HDQ concentrations led to a ΔΨm collapse within minutes, which is followed by severe ATP depletions of 30% after 1 h and 70% after 24 h. ΔΨm depolarization was attenuated when substrates for other dehydrogenases that can donate electrons to ubiquinone were added to digitonin-permeabilized cells or when infected cultures were treated with the Fo-ATPase inhibitor oligomycin. A prolonged treatment with sublethal concentrations of HDQ induced differentiation into bradyzoites. This dormant stage is likely to be less dependent on the ΔΨm, since ΔΨm-positive parasites were found at a significantly lower frequency in alkaline-pH-induced bradyzoites than in tachyzoites. Together, our studies reveal that oxidative phosphorylation is essential for maintaining the ATP level in the fast-growing tachyzoite stage and that HDQ interferes with this pathway by inhibiting the electron transport chain at the level of ubiquinone reduction.
Nitrite Transport Activity of the ABC-Type Cyanate Transporter of the Cyanobacterium
Synechococcus elongatus
Authors: Maeda Shin-ichi , Omata Tatsuo
Published: March 2009

In addition to the ATP-binding cassette (ABC)-type nitrate/nitrite-bispecific transporter, which has a high affinity for both substrates (

K
m

, ∼1 μM),
Synechococcus elongatus
has an active nitrite transport system with an apparent

K
m

(NO
2
−
) value of 20 μM. We found that this activity depends on the
cynABD
genes, which encode a putative cyanate (NCO
−
) ABC-type transporter. Accordingly, nitrite transport by CynABD was competitively inhibited by NCO
−
with a

K
i

value of 0.025 μM. The transporter was induced under conditions of nitrogen deficiency, and the induced cells showed a
V
max
value of 11 to 13 μmol/mg of chlorophyll per h for cyanate or nitrite, which could supply ∼30% of the amount of nitrogen required for optimum growth. Its relative specificity for the substrates and regulation at transcriptional and posttranslational levels suggested that the physiological role of the bispecific cyanate/nitrite transporter in
S. elongatus
is to allow nitrogen-deficient cells to assimilate low concentrations of cyanate in the medium. Its contribution to nitrite assimilation was significant in a mutant lacking the ABC-type nitrate/nitrite transporter, suggesting a possible role for CynABD in nitrite assimilation by cyanobacterial species that lack another high-affinity mechanism(s) for nitrite transport.
A Novel Nitroreductase of
Staphylococcus aureus
with
S
-Nitrosoglutathione Reductase Activity
Authors: Tavares Ana Filipa N. , Nobre Lígia S. , Melo Ana M. P. , Saraiva Lígia M.
Published: March 2009

In this report we show that inactivation of the putative nitroreductase SA0UHSC_00833 (
ntrA
) increases the sensitivity of
Staphylococcus aureus
to
S
-nitrosoglutathione (GSNO) and augments its resistance to nitrofurans.
S. aureus
NtrA is a bifunctional enzyme that exhibits nitroreductase and GSNO reductase activity. A phylogenetic analysis suggests that NtrA is a member of a novel family of nitroreductases that seems to play a dual role in vivo, promoting nitrofuran activation and protecting the cell against transnitrosylation.
Structural Requirements of thePseudomonasQuinolone Signal for Membrane Vesicle Stimulation
Authors: Mashburn-Warren Lauren , Howe Jörg , Brandenburg Klaus , Whiteley Marvin
Published: March 2009

Pseudomonas aeruginosaproduces the quorum signal 2-heptyl-3-hydroxy-4-quinolone (Pseudomonasquinolone signal), which is important for stimulating outer membrane vesicle (MV) formation. Here we describe the importance of the 3-hydroxyl and 2-alkyl chain for MV production and the length of the 2-alkyl chain for association with MVs.
Identification and Characterization of a Novel Multidrug Resistance Operon,mdtRP(yusOP), ofBacillus subtilis
Authors: Kim Ji-Yun , Inaoka Takashi , Hirooka Kazutaka , Matsuoka Hiroshi , Murata Makiko , Ohki Reiko , Adachi Yoshikazu , Fujita Yasutaro , Ochi Kozo
Published: March 2009

Using comparative genome sequencing analysis, we identified a novel mutation inBacillus subtilisthat confers a low level of resistance to fusidic acid. This mutation was located in themdtR(formerlyyusO) gene, which encodes a MarR-type transcriptional regulator, and conferred a low level of resistance to several antibiotics, including novobiocin, streptomycin, and actinomycin D. Transformation experiments showed that thismdtRmutation was responsible for multidrug resistance. Northern blot analysis revealed that the downstream genemdtP(formerlyyusP), which encodes a multidrug efflux transporter, is cotranscribed withmdtRas an operon. Disruption of themdtPgene completely abolished the multidrug resistance phenotype observed in themdtRmutant. DNase I footprinting and primer extension analyses demonstrated that the MdtR protein binds directly to themdtRPpromoter, thus leading to repression of its transcription. Moreover, gel mobility shift analysis indicated that an Arg83 → Lys or Ala67 → Thr substitution in MdtR significantly reduces binding affinity to DNA, resulting in derepression ofmdtRPtranscription. Low concentrations of fusidic acid induced the expression ofmdtP, although the level ofmdtPexpression was much lower than that in themdtRdisruptant. These findings indicate that the MdtR protein is a repressor of themdtRPoperon and that the MdtP protein functions as a multidrug efflux transporter inB. subtilis.
Regulation of Superoxide Dismutase (sod) Genes by SarA inStaphylococcus aureus
Authors: Ballal Anand , Manna Adhar C.
Published: March 2009

The scavenging of reactive oxygen species (ROS) within cells is regulated by several interacting factors, including transcriptional regulators. Involvement ofsarAfamily genes in the regulation of proteins involved in the scavenging of ROS is largely unknown. In this report, we show that under aerobic conditions, the levels ofsodMand sodAtranscription, in particular thesodMtranscript, are markedly enhanced in thesarAmutant among the testedsarAfamily mutants. Increased levels ofsodexpression returned to near the parental level in a single-copysarAcomplemented strain. Under microaerophilc conditions, transcription of bothsodMandsodAwas considerably enhanced in thesarAmutant compared to the wild-type strain. Various genotypic, phenotypic, and DNA binding studies confirmed the involvement of SarA in the regulation ofsodtranscripts in different strains ofStaphylococcus aureus. ThesodAmutant was sensitive to an oxidative stress-inducing agent, methyl viologen, but thesarA sodAdouble mutant was more resistant to the same stressor than the singlesodAmutant. These results suggest that overexpression of SodM, which occurs in thesarAbackground, can rescue the methyl viologen-sensitive phenotype observed in the absence of thesodAgene. Analysis with various oxidative stress-inducing agents indicates that SarA may play a greater role in modulating oxidative stress resistance inS. aureus. This is the first report that demonstrates the direct involvement of a regulatory protein (SarA) in control ofsodexpression inS. aureus.
Crystal Structure of a Chimeric Receptor Binding Protein Constructed from Two Lactococcal Phages
Authors: Siponen Marina , Spinelli Silvia , Blangy Stéphanie , Moineau Sylvain , Cambillau Christian , Campanacci Valérie
Published: March 2009

Lactococcus lactis
, a gram-positive bacterium widely used by the dairy industry to manufacture cheeses, is subject to infection by a diverse population of virulent phages. We have previously determined the structures of three receptor binding proteins (RBPs) from lactococcal phages TP901-1, p2, and bIL170, each of them having a distinct host range. Virulent phages p2 and bIL170 are classified within the 936 group, while the temperate phage TP901-1 is a member of the genetically distinct P335 polythetic group. These RBPs comprise three domains: the N-terminal domain, binding to the virion particle; a β-helical linker domain; and the C-terminal domain, bearing the receptor binding site used for host recognition. Here, we have designed, expressed, and determined the structure of an RBP chimera in which the N-terminal and linker RBP domains of phage TP901-1 (P335) are fused to the C-terminal RBP domain of phage p2 (936). This chimera exhibits a stable structure that closely resembles the parental structures, while a slight displacement of the linker made RBP domain adaptation efficient. The receptor binding site is structurally indistinguishable from that of native p2 RBP and binds glycerol with excellent affinity.
Thioredoxins in Redox Maintenance and Survival during Oxidative Stress ofBacteroides fragilis
Authors: Reott Michael A. , Parker Anita C. , Rocha Edson R. , Smith C. Jeffrey
Published: March 2009

The anaerobeBacteroides fragilisis a gram-negative, opportunistic pathogen that is highly aerotolerant and can persist in aerobic environments for extended periods. In this study, the sixB. fragilisthioredoxins (Trxs) were investigated to determine their role during oxidative stress. Phylogenetic analyses of Trx protein sequences indicated that four of the six Trxs (TrxA, TrxC, TrxD, and TrxF) belong to the M-type Trx class but were associated with two different M-type lineages. TrxE and TrxG were most closely associated to Y-type Trxs found primarily in cyanobacteria. Single and multipletrxgene deletions were generated to determine functional differences between the Trxs. ThetrxAgene was essential, but no anaerobic growth defects were observed for any other singletrxdeletion or for the ΔtrxCΔtrxD::cfxA ΔtrxE ΔtrxF ΔtrxGquintuple mutant. Regulation of thetrxgenes was linked to the oxidative stress response, and all were induced by aerobic conditions. The ΔtrxCΔtrxEΔtrxFΔtrxGand the ΔtrxCΔtrxD::cfxA ΔtrxE ΔtrxF ΔtrxGmultiple deletion strains were impaired during growth in oxidized media, but singletrxgene mutants did not have a phenotype in this assay. TrxD was protective during exposure to the thiol oxidant diamide, and expression oftrxDwas induced by diamide. Diamide-induced expression oftrxC,trxE, andtrxFincreased significantly in atrxDmutant strain, suggesting that there is some capacity for compensation in this complex Trx system. These data provide insight into the role of individual Trxs in theB. fragilisoxidative stress response.
Large Intergenic Cruciform-Like Supermotifs in the
Lactobacillus plantarum
Genome
Authors: Wels Michiel , Bongers Roger S. , Boekhorst Jos , Molenaar Douwe , Sturme Mark , de Vos Willem M. , Siezen Roland J. , Kleerebezem Michiel
Published: March 2009

Twenty-four
Lactobacillus plantarum
supermotifs (LPSMs) with lengths from ∼800 to 1,000 nucleotides were identified in the
L. plantarum
genome. LPSMs were conserved in other
L. plantarum
strains but not in other species. Secondary structure analysis predicted that LPSMs may fold into cruciform-like structures. Preliminary experiments indicate that the LPSMs are transcribed.
Oxidation of Cysteine 645 of Cobalamin-Independent Methionine Synthase Causes a Methionine Limitation in
Escherichia coli
Authors: Hondorp Elise R. , Matthews Rowena G.
Published: March 2009

Cobalamin-independent methionine synthase (MetE) catalyzes the final step in
Escherichia coli
methionine biosynthesis but is inactivated under oxidative conditions, triggering a methionine deficiency. This study demonstrates that the mutation of MetE cysteine 645 to alanine completely eliminates the methionine auxotrophy imposed by diamide treatment, suggesting that modulation of MetE activity via cysteine 645 oxidation has significant physiological consequences for oxidatively stressed cells.
Automation of the BD GeneOhm Methicillin-Resistant
Staphylococcus aureus
Assay for High-Throughput Screening of Nasal Swab Specimens
Authors: Wang Xue-Ping , Ginocchio Christine C.
Published: March 2009

This study demonstrated that an automated version of the BD-GeneOhm methicillin-resistant
Staphylococcus aureus
(MRSA) assay (BD-MRSA), using achromopeptidase sample lysis and PCR setup performed on the Hamilton MICROLAB STAR
let
(Auto-MRSA), gave results comparable to those obtained with BD-MRSA. The positive- and negative-result concordance rates and overall concordance of BD-MRSA and Auto-MRSA were 98.2, 97.7, and 97.6%, respectively. Auto-MRSA required 60% less technical time than BD-MRSA, and the time required to obtain results was 50% of that required for BD-MRSA. Auto-MRSA provides a reliable, robust system for high-volume clinical use.
Early and Strong Immune Responses Are Associated with Control of Viral Replication and Recovery in Lassa Virus-Infected Cynomolgus Monkeys
Authors: Baize Sylvain , Marianneau Philippe , Loth Philippe , Reynard Stéphanie , Journeaux Alexandra , Chevallier Michèle , Tordo Noël , Deubel Vincent , Contamin Hugues
Published: March 2009

Lassa virus causes a hemorrhagic fever endemic in West Africa. The pathogenesis and the immune responses associated with the disease are poorly understood, and no vaccine is available. We followed virological, pathological, and immunological markers associated with fatal and nonfatal Lassa virus infection of cynomolgus monkeys. The clinical picture was characterized by fever, weight loss, depression, and acute respiratory syndrome. Transient thrombocytopenia and lymphopenia, lymphadenopathy, splenomegaly, infiltration of mononuclear cells, and alterations of the liver, lungs, and endothelia were observed. Survivors exhibited fewer lesions and a lower viral load than nonsurvivors. Although all animals developed strong humoral responses, antibodies appeared more rapidly in survivors and were directed against GP
1
, GP
2
, and NP. Type I interferons were detected early after infection in survivors but only during the terminal stages in fatalities. The mRNAs for CXCL10 (IP-10) and CXCL11 (I-TAC) were abundant in peripheral blood mononuclear cells and lymph nodes from infected animals, but plasma interleukin-6 was detected only in fatalities. In survivors, high activated-monocyte counts were followed by a rise in the total number of circulating monocytes. Activated T lymphocytes circulated in survivors, whereas T-cell activation was low and delayed in fatalities. In vitro stimulation with inactivated Lassa virus induced activation of T lymphocytes from all infected monkeys, but only lymphocytes from survivors proliferated. Thus, early and strong immune responses and control of viral replication were associated with recovery, whereas fatal infection was characterized by major alterations of the blood formula and, in organs, weak immune responses and uncontrolled viral replication.
A case-crossover study on transient risk factors of work-related eye injuries
Authors: Chen S-Y , Fong P-C , Lin S-F , Chang C-H , Chan C-C
Published: March 2009

Objectives:

To investigate modifiable risk and preventive factors of work-related eye injuries.

Methods:

A case-crossover study conducted to explore the associations between transient risk factors and work-related eye injuries. Patients seen at seven medical centres in Taiwan with work-related eye injuries over a 4-year period were enrolled in the study. Clinical information was collected from medical charts and detailed information on exposure to eight potentially modifiable factors during the 60 minutes prior to the occurrence of each injury, as well as during the same time interval on the last work day prior to the injury, were obtained using questionnaire surveys. Matched-pair interval analysis was adopted to assess the odds ratios (ORs) for work-related eye injuries given exposure to the eight modifiable factors.

Results:

A total of 283 subjects were interviewed. Most of these injured workers were young, male, and self-employed or small enterprise workers. The most common injury type was photokeratitis (33.2%), mainly caused by welding (30.4%). The OR for a work-related eye injury was increased with the performance of an unfamiliar task (57.0), operation of a faulty tool or piece of equipment (48.5), distractions (24.0), being rushed (13.0), or fatigued (10.0), and a poor work environment (4.3). Wearing eye protection devices was found to have a significant protective effect on workers who might otherwise have been exposed to eye injuries (OR = 0.4; 95% CI 0.2 to 0.7).

Conclusion:

Potential modifiable risk and preventive factors for work-related eye injuries were identified using a case-crossover study. This information should be helpful in the development of preventive strategies.
Involvement of NO and MEK/ERK pathway in enhancement of endothelin-1-induced mesenteric artery contraction in later-stage type 2 diabetic Goto-Kakizaki rat
Authors: Matsumoto Takayuki , Ishida Keiko , Nakayama Naoaki , Kobayashi Tsuneo , Kamata Katsuo
Published: March 2009

Endothelin (ET)-1 is a likely candidate for a key role in diabetic vascular complications. However, no abnormalities in the vascular responsiveness to ET-1 have been identified in the chronic stage of type 2 diabetes. Our goal was to look for abnormalities in the roles played by ET receptors (ETAand ETB) in the mesenteric artery of the type 2 diabetic Goto-Kakizaki (GK) rat and to identify the molecular mechanisms involved. Using mesenteric arteries from later-stage (32–38 wk old) individuals, we compared the ET-1-induced contraction and the relaxation induced by the selective ETBreceptor agonist IRL1620 between GK rats and control Wistar rats. Mesenteric artery ERK activity and the protein expressions for ET receptors and MEK were also measured. In GK rats (vs. age-matched Wistar rats), we found as follows. 1) The ET-1-induced contraction was greater and was attenuated by BQ-123 (ETAantagonist) but not by BQ-788 (ETBantagonist). In the controls, BQ-788 augmented this contraction. 2) Both the relaxation and nitric oxide (NO) production induced by IRL1620 were reduced. 3) ET-1-induced contraction was enhanced by NG-nitro-l-arginine (l-NNA; NO synthase inhibitor) but suppressed by sodium nitroprusside (NO donor). 4) The enhanced ET-1-induced contraction was reduced by MEK/ERK pathway inhibitors (PD-98059 or U0126). 5) ET-1-stimulated ERK activation was increased, as were the ETAand MEK1/2 protein expressions. 6) Mesenteric ET-1 content was increased. These results suggest that upregulation of ETA, a defect in ETB-mediated NO signaling, and activation of the MEK/ERK pathway together represent a likely mechanism mediating the hyperreactivity to ET-1 examined in this study.
Lack of S100A1 in mice confers a gender-dependent hypertensive phenotype and increased mortality after myocardial infarction
Authors: Desjardins Jean-Francois , Pourdjabbar Ali , Quan Adrian , Leong-Poi Howard , Teichert-Kuliszewska Krystyna , Verma Subodh , Parker Thomas G.
Published: March 2009

S100A1 is a small Ca2+-binding protein expressed in the myocardium and blood vessels that is downregulated in the diseased heart and plays a role in the regulation of cardiac muscle Ca2+homeostasis and contractility. To understand its physiological role under basal conditions and after myocardial infarction (MI), we used a mouse strain with targeted deletion of the S100A1 gene [S100A1 knockout (KO) mice]. We compared 49 wild-type (WT) and 56 S100A1 KO mice (6–8 wk old) over 28 days after MI with sham-operated controls. We also examined the effect of S100A1 deficiency on vascular function of isolated blood vessels. S100A1 KO mice demonstrated worse survival compared with WT mice (21% vs. 69%, respectively, P < 0.001). Hemodynamic evaluation revealed a higher mean arterial pressure (MAP) in sham-operated KO animals compared with WT animals (99 ± 4 vs. 77 ± 3 mmHg, respectively, P < 0.001) that persisted in both groups after MI (86 ± 2 vs. 66 ± 4 mmHg, respectively, P < 0.001). Sham-operated male S100A1 KO mice had higher MAP than female KO mice (122 ± 5 vs. 93 ± 3 mmHg, respectively P < 0.05) and reduced survival after MI (4% vs. 27%, respectively, P < 0.05). In isolated aortas and mesenteric arteries, ACh-evoked vasodilatation in KO mice was significantly reduced compared with WT mice ( P < 0.05). Nitric oxide production was reduced in endothelial cells isolated from KO mice. Thus, absence of S100A1 results in acute functional impairment and high mortality after MI associated with a gender-specific hypertensive phenotype. S100A1 appears to play a role in the endothelium-dependent regulation of blood pressure.
ERK phosphorylation mediates sildenafil-induced myocardial protection against ischemia-reperfusion injury in mice
Authors: Das Anindita , Salloum Fadi N. , Xi Lei , Rao Yuan J. , Kukreja Rakesh C.
Published: March 2009

Sildenafil, a selective inhibitor of phosphodiesterase type 5, induces powerful protection against myocardial ischemia-reperfusion injury through activation of cGMP-dependent protein kinase (PKG). We further hypothesized that PKG-dependent activation of survival kinase ERK may play a causative role in sildenafil-induced cardioprotection via induction of endothelial nitric oxide synthase (eNOS)/inducible nitric oxide synthase (iNOS) and Bcl-2. Our results show that acute intracoronary infusion of sildenafil in Langendorff isolated mouse hearts before global ischemia-reperfusion significantly reduced myocardial infarct size (from 29.4 ± 2.4% to 15.9 ± 3.0%; P < 0.05). Cotreatment with ERK inhibitor PD98059 abrogated sildenafil-induced protection (31.8 ± 4.4%). To further evaluate the role of ERK in delayed cardioprotection, mice were treated with sildenafil (ip) 24 h before global ischemia-reperfusion. PD98059 was administered (ip) 30 min before sildenafil treatment. Infarct size was reduced from 27.6 ± 3.3% in controls to 7.1 ± 1.5% in sildenafil-treated mice ( P < 0.05). The delayed protective effect of sildenafil was also abolished by PD98059 (22.5 ± 2.3%). Western blots revealed that sildenafil significantly increased phosphorylation of ERK1/2 and GSK-3β and induced iNOS, eNOS, Bcl-2, and PKG activity in the heart 24 h after treatment. PD98059 inhibited the enhanced expression of iNOS, eNOS, and Bcl-2 and the phosphorylation of GSK-3β. PD98059 had no effect on the sildenafil-induced activation of PKG. We conclude that these studies provide first direct evidence that PKG-dependent ERK phosphorylation is indispensable for the induction of eNOS/iNOS and Bcl-2 and the resulting cardioprotection by sildenafil.
Detrimental effect of combined exercise training and eNOS overexpression on cardiac function after myocardial infarction
Authors: de Waard Monique C. , van der Velden Jolanda , Boontje Nicky M. , Dekkers Dick H. W. , van Haperen Rien , Kuster Diederik W. D. , Lamers Jos M. J. , de Crom Rini , Duncker Dirk J.
Published: March 2009

It has been reported that exercise after myocardial infarction (MI) attenuates left ventricular (LV) pump dysfunction by normalization of myofilament function. This benefit could be due to an exercise-induced upregulation of endothelial nitric oxide synthase (eNOS) expression and activity. Consequently, we first tested the hypothesis that the effects of exercise after MI can be mimicked by elevated eNOS expression using transgenic mice with overexpression of human eNOS (eNOSTg). Both exercise and eNOSTg attenuated LV remodeling and dysfunction after MI in mice and improved cardiomyocyte maximal force development (Fmax). However, only exercise training restored myofilament Ca2+-sensitivity and sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a protein levels and improved the first derivative of LV pressure at 30 mmHg. Conversely, only eNOSTg improved survival. In view of these partly complementary actions, we subsequently tested the hypothesis that combining exercise and eNOSTg would provide additional protection against LV remodeling and dysfunction after MI. Unexpectedly, the combination of exercise and eNOSTg abolished the beneficial effects on LV remodeling and dysfunction of either treatment alone. The latter was likely due to perturbations in Ca2+homeostasis, as myofilament Fmaxactually increased despite marked reductions in the phosphorylation status of several myofilament proteins, whereas the exercise-induced increases in SERCA2a protein levels were lost in eNOSTg mice. Antioxidant treatment with N-acetylcysteine or supplementation of tetrahydrobiopterin and l-arginine prevented these detrimental effects on LV function while partly restoring the phosphorylation status of myofilament proteins and further enhancing myofilament Fmax. In conclusion, the combination of exercise and elevated eNOS expression abolished the cardioprotective effects of either treatment alone after MI, which appeared to be, at least in part, the result of increased oxidative stress secondary to eNOS “uncoupling.”
Role of superoxide, nitric oxide, and peroxynitrite in doxorubicin-induced cell death in vivo and in vitro
Authors: Mukhopadhyay Partha , Rajesh Mohanraj , Bátkai Sándor , Kashiwaya Yoshihiro , Haskó György , Liaudet Lucas , Szabó Csaba , Pacher Pál
Published: March 2009

Doxorubicin (DOX) is a potent available antitumor agent; however, its clinical use is limited because of its cardiotoxicity. Cell death is a key component in DOX-induced cardiotoxicity, but its mechanisms are elusive. Here, we explore the role of superoxide, nitric oxide (NO), and peroxynitrite in DOX-induced cell death using both in vivo and in vitro models of cardiotoxicity. Western blot analysis, real-time PCR, immunohistochemistry, flow cytometry, fluorescent microscopy, and biochemical assays were used to determine the markers of apoptosis/necrosis and sources of NO and superoxide and their production. Left ventricular function was measured by a pressure-volume system. We demonstrated increases in myocardial apoptosis (caspase-3 cleavage/activity, cytochrome c release, and TUNEL), inducible NO synthase (iNOS) expression, mitochondrial superoxide generation, 3-nitrotyrosine (NT) formation, matrix metalloproteinase (MMP)-2/MMP-9 gene expression, poly(ADP-ribose) polymerase activation [without major changes in NAD(P)H oxidase isoform 1, NAD(P)H oxidase isoform 2, p22phox, p40phox, p47phox, p67phox, xanthine oxidase, endothelial NOS, and neuronal NOS expression] and decreases in myocardial contractility, catalase, and glutathione peroxidase activities 5 days after DOX treatment to mice. All these effects of DOX were markedly attenuated by peroxynitrite scavengers. Doxorubicin dose dependently increased mitochondrial superoxide and NT generation and apoptosis/necrosis in cardiac-derived H9c2 cells. DOX- or peroxynitrite-induced apoptosis/necrosis positively correlated with intracellular NT formation and could be abolished by peroxynitrite scavengers. DOX-induced cell death and NT formation were also attenuated by selective iNOS inhibitors or in iNOS knockout mice. Various NO donors when coadministered with DOX but not alone dramatically enhanced DOX-induced cell death with concomitant increased NT formation. DOX-induced cell death was also attenuated by cell-permeable SOD but not by cell-permeable catalase, the xanthine oxidase inhibitor allopurinol, or the NADPH oxidase inhibitors apocynine or diphenylene iodonium. Thus, peroxynitrite is a major trigger of DOX-induced cell death both in vivo and in vivo, and the modulation of the pathways leading to its generation or its effective neutralization can be of significant therapeutic benefit.
Stretch-activated channel activation promotes early afterdepolarizations in rat ventricular myocytes under oxidative stress
Authors: Wang Yanggan , Joyner Ronald W. , Wagner Mary B. , Cheng Jun , Lai Dongwu , Crawford Brian H.
Published: March 2009

Mechanical stretch and oxidative stress have been shown to prolong action potential duration (APD) and produce early afterdepolarizations (EADs). Here, we developed a simulation model to study the role of stretch-activated channel (SAC) currents in triggering EADs in ventricular myocytes under oxidative stress. We adapted our coupling clamp circuit so that a model ionic current representing the actual SAC current was injected into ventricular myocytes and added as a real-time current. This current was calculated as ISAC= GSAC* ( Vm− ESAC), where GSACis the stretch-activated conductance, Vmis the membrane potential, and ESACis the reversal potential. In rat ventricular myocytes, application of GSACdid not produce sustained automaticity or EADs, although turn-on of GSACdid produce some transient automaticity at high levels of GSAC. Exposure of myocytes to 100 μM H2O2induced significant APD prolongation and increase in intracellular Ca2+load and transient, but no EAD or sustained automaticity was generated in the absence of GSAC. However, the combination of GSACand H2O2consistently produced EADs at lower levels of GSAC(2.6 ± 0.4 nS, n = 14, P < 0.05). Pacing myocytes at a faster rate further prolonged APD and promoted the development of EADs. SAC activation plays an important role in facilitating the development of EADs in ventricular myocytes under acute oxidative stress. This mechanism may contribute to the increased propensity to lethal ventricular arrhythmias seen in cardiomyopathies, where the myocardium stretch and oxidative stress generally coexist.
The endothelial glycocalyx mediates shear-induced changes in hydraulic conductivity
Authors: Lopez-Quintero Sandra V. , Amaya Ronny , Pahakis Manolis , Tarbell John M.
Published: March 2009

Recent in vitro and in vivo studies have reported fluid shear stress-induced increases in endothelial layer hydraulic conductivity ( Lp) that are mediated by an increased production of nitric oxide (NO). Other recent studies have shown that NO induction by shear stress is mediated by the glycocalyx that decorates the surface of endothelial cells. Here we find that a selective depletion of the major components of the glycocalyx with enzymes can block the shear stress-induced response of Lp. Heparinase and hyaluronidase block shear-induced increases in Lp, which is consistent with their effects on NO production. But chondroitinase, which does not suppress shear-induced NO production, also inhibits shear-induced Lp. A further surprise is that treatment with the general proteolytic enzyme pronase does not suppress the shear Lp response. We also find that heparinase does not alter baseline Lp significantly, whereas chondroitinase, hyaluronidase, and pronase increase it significantly.
Is mean blood saturation a useful marker of tissue oxygenation?
Authors: Thorn Clare E. , Matcher Stephen J. , Meglinski Igor V. , Shore Angela C.
Published: March 2009

Increasingly we are monitoring the distribution of oxygen through the microcirculation using optical techniques such as optical reflectance spectroscopy (ORS) and near-infrared spectroscopy. Mean blood oxygen saturation (SmbO2) and tissue oxygenation index measured by these two techniques, respectively, evoke a concept of the measurement of oxygen delivery to tissue. This study aims to establish whether SmbO2 is an appropriate indicator of tissue oxygenation. Spontaneous fluctuations in SmbO2 observed as changes in concentration of oxyhemoglobin ([HbO2]) and deoxyhemoglobin ([Hb]) were measured by ORS in the skin microcirculation of 30 healthy subjects (15 men, age 21–42 yr). Fourier analysis identified two distinctly different spontaneous falls in SmbO2. The first type of swing, thought to be induced by fluctuations in arterial blood volume, resulted from the effects of respiration, endothelial, sympathetic, and myogenic activity. There was no apparent change in [Hb]. In contrast, a second type of swing resulted from a fall in [HbO2] accompanied by a rise in [Hb] and was only induced by endothelial and sympathetic activity. Thus the same fall in SmbO2 can be induced by two distinct responses. A “type I” swing does not suggest an inadequacy in oxygen delivery whereas a “type II” swing may indicate a change in oxygen delivery from blood to tissue. SmbO2 alone cannot therefore be accepted as a definitive marker of tissue oxygenation.
Myocardial oxygenation and functional recovery in infarct rat hearts transplanted with mesenchymal stem cells
Authors: Chacko Simi M. , Khan Mahmood , Kuppusamy M. Lakshmi , Pandian Ramasamy P. , Varadharaj Saradhadevi , Selvendiran Karuppaiyah , Bratasz Anna , Rivera Brian K. , Kuppusamy Periannan
Published: March 2009

Stem cell therapy for myocardial tissue repair is limited by the poor survival of transplanted cells, possibly because of inadequate supply of oxygen and nutrients. The purpose of this study was to assess the oxygenation level and functional recovery after allogenic transplantation of mesenchymal stem cells (MSC) in a rat model of myocardial infarction (MI). Myocardial oxygen tension (Po2) was measured by electron paramagnetic resonance oximetry using an implantable oxygen-sensing spin probe (OxySpin). MSCs incubated with OxySpins showed substantial uptake of the probe without affecting its oxygen sensitivity or calibration. The cells internalized with OxySpins were able to differentiate into osteogenic, adipogenic, cardiomyocyte, and endothelial cell lineages. The labeled cells tested positive for CD44 and CD29 markers and negative for the hematopoietic markers CD14 and CD45. For the in vivo studies, MI was induced in rats by permanently ligating the left anterior descending coronary artery. MSCs with OxySpins were transplanted in the infarct region of hearts. A significant increase in Po2 was observed in the MSC group compared with the untreated MI group (18.1 ± 2.6 vs. 13.0 ± 1.8 mmHg, n = 4, P < 0.05) at 4 wk after transplantation. Echocardiography showed a significant improvement in ejection fraction and fraction shortening, which inversely correlated with the magnitude of fibrosis in the treated hearts. The cell-transplanted hearts also showed an increase in vascular endothelial growth factor level and capillary density in the infarct region. The study established our ability to measure and correlate changes in myocardial tissue oxygenation with cardiac function in infarcted rat hearts treated with MSCs.
Functionally important role for arginase 1 in the airway hyperresponsiveness of asthma
Authors: North Michelle L. , Khanna Nivedita , Marsden Philip A. , Grasemann Hartmut , Scott Jeremy A.
Published: March 2009

l-Arginine metabolism by the arginase and nitric oxide (NO) synthase (NOS) families of enzymes is important in NO production, and imbalances between these pathways contribute to airway hyperresponsiveness (AHR) in asthma. To investigate the role of arginase isozymes (ARG1 and ARG2) in AHR, we determined the protein expression of ARG1, ARG2, the NOS isozymes, and other proteins involved in l-arginine metabolism in lung tissues from asthma patients and in acute (3-wk) and chronic (12-wk) murine models of ovalbumin-induced airway inflammation. Expression of ARG1 was increased in human asthma, whereas ARG2, NOS isoforms, and the other l-arginine-related proteins (i.e., cationic amino acid transporters 1 and 2, agmatinase, and ornithine decarboxylase) were unchanged. In the acute murine model of allergic airway inflammation, augmentation of ARG1 expression was similarly the most dramatic change in protein expression. However, ARG2, NOS1, NOS2, and agmatinase were also increased, whereas NOS3 expression was decreased. Arginase inhibition in vivo with nebulized S-(2-boronoethyl)-l-cysteine attenuated the methacholine responsiveness of the central airways in mice from the acute model. Further investigations in the chronic murine model revealed an expression profile that more closely paralleled the human asthma samples: only ARG1 expression was significantly increased. Interestingly, in the chronic mouse model, which generates a remodeling phenotype, arginase inhibition attenuated methacholine responsiveness of the central and peripheral airways. The similarity in arginase expression between human asthma and the chronic model and the attenuation of AHR after in vivo treatment with an arginase inhibitor suggest the potential for therapeutic modification of arginase activity in asthma.
Identification of putative endothelial progenitor cells (CD34+CD133+Flk-1+) in endarterectomized tissue of patients with chronic thromboembolic pulmonary hypertension
Authors: Yao Weijuan , Firth Amy L. , Sacks Richard S. , Ogawa Aiko , Auger William R. , Fedullo Peter F. , Madani Michael M. , Lin Grace Y. , Sakakibara Naohide , Thistlethwaite Patricia A. , Jamieson Stuart W. , Rubin Lewis J. , Yuan Jason X.-J.
Published: March 2009

Chronic thromboembolic pulmonary hypertension (CTEPH) is characterized by a fibrotic thrombus persisting and obliterating the lumen of pulmonary arteries; its pathogenesis remains poorly defined. This study investigates a potential contribution for progenitor cell types in the development of vascular obliteration and remodeling in CTEPH patients. Endarterectomized tissue from patients undergoing pulmonary thromboendarterectomy was collected and examined for the structure and cellular composition. Our data show an organized fibrin network structure in unresolved thromboemboli and intimal remodeling in vascular wall tissues, characterized by smooth muscle α-actin (SM-αA)-positive cell proliferation in proximal regions (adjacent to thromboemboli) and neoangiogenesis/recanalization in distal regions (downstream from thromboemboli). Cells that are positively stained with CD34 and fetal liver kinase-1 (Flk-1) (CD34+Flk-1+) were identified in both the proximal and distal vascular tissues; a subpopulation of CD34+Flk-1+CD133+cells were further identified by immunostaining. Triple-positive cells are indicative of a population of putative endothelial progenitor cells or potential colony-forming units of endothelial cells. In addition, inflammatory cells (CD45+) and collagen-secreting cells (procollagen-1+) were detected in the proximal vascular wall. Some of the CD34+cells in CTEPH cells isolated from proximal regions were also positive for SM-αA. Our data indicate that putative progenitor cell types are present in the neointima of occluded vessels of CTEPH patients. It is possible that the microenvironment provided by thromboemboli may promote these putative progenitor cells to differentiate and enhance intimal remodeling.
Ceramide-dependent PP2A regulation of TNFα-induced IL-8 production in respiratory epithelial cells
Authors: Cornell Timothy T. , Hinkovska-Galcheva Vania , Sun Lei , Cai Qing , Hershenson Marc B. , Vanway Susan , Shanley Thomas P.
Published: March 2009

IL-8 is a key mediator in the pathophysiology of acute lung injury. TNFα stimulates IL-8 production in respiratory epithelial cells by activating both the NF-κB and MAP kinase pathways. The precise mechanism by which these pathways are downregulated to terminate IL-8 production remains unclear. We studied the regulatory role of the serine/threonine phosphatase, PP2A, on the signaling pathways involved in IL-8 production from respiratory epithelial cells. Inhibition of PP2A using okadaic acid or gene knockdown using siRNA resulted in an augmentation of TNFα-induced IL-8 production. We also found that PP2A inhibition resulted in prolonged activation of JNK, p38, and ERK resulting in both increased transcriptional activation of the IL-8 promoter and posttranscriptional stabilization of IL-8 mRNA. Because TNFα had been shown to activate ceramide accumulation, and separate studies had linked ceramide with activation of PP2A, we hypothesized the pathway of TNFα-inducing ceramide to activate PP2A comprised an endogenous regulatory pathway. Inhibition of the immediate sphingomyelinase-dependent pathway as well as the de novo synthesis pathway of ceramide production reduced serine/threonine phosphatase activity and augmented IL-8 production. These data suggest that ceramide plays a role in activating PP2A to terminate ongoing IL-8 production. In summary, our data suggest that in respiratory epithelium, TNFα induces ceramide accumulation, resulting in subsequent activation of PP2A, which targets those kinases responsible for transcriptional activation of IL-8.
α,β-Unsaturated aldehydes contained in cigarette smoke elicit IL-8 release in pulmonary cells through mitogen-activated protein kinases
Authors: Moretto Nadia , Facchinetti Fabrizio , Southworth Thomas , Civelli Maurizio , Singh Dave , Patacchini Riccardo
Published: March 2009

Cigarette smoking is the major risk factor for chronic obstructive pulmonary disease (COPD), a syndrome characterized by pulmonary neutrophil infiltration, chronic inflammation, and progressive tissue destruction. We examined here the acute effect of aqueous cigarette smoke extract (CSE) and of two α,β-unsaturated aldehydes (acrolein and crotonaldehyde) contained in CSE in cultured normal human lung fibroblasts and small airway epithelial cells. By examining a panel of 19 cytokines and chemokines, we found that IL-8 release was elevated by CSE as well as by acrolein, whereas other inflammatory mediators were mostly unaffected. CSE-evoked IL-8 release was mimicked by acrolein and crotonaldehyde at concentrations (3–60 μM each) found in CSE and fully prevented by 1 mM α,β-unsaturated aldehydes scavengers N-acetylcysteine (NAC) or sodium 2-mercaptoethanesulfonate. Neither the saturated aldehyde acetaldehyde nor H2O2evoked IL-8 release. In addition, CSE or crotonaldehyde upregulated the release of IL-8 from alveolar macrophages from both COPD patients and healthy nonsmokers, indicating that this is a response common to cells involved in lung inflammation. CSE-evoked IL-8 release was accompanied by increased phosphorylation of p38 MAPK and ERK1/2. CSE-evoked p38 and ERK1/2 phosphorylation was mimicked by acrolein and inhibited by NAC. IL-8 release elicited by both acrolein and CSE was blocked by pharmacological inhibition of p38 and ERK1/2 phosphorylation. In summary, our data show that α,β-unsaturated aldehydes-evoked phosphorylation of p38 and ERK1/2 underlies IL-8 release elicited by CSE, thus shedding light on the mechanisms through which cigarette smoke can initiate inflammation in the lung.
Secretory group V phospholipase A2regulates acute lung injury and neutrophilic inflammation caused by LPS in mice
Authors: Muñoz Nilda M. , Meliton Angelo Y. , Meliton Lucille N. , Dudek Steven M. , Leff Alan R.
Published: March 2009

We investigated the regulatory role of 14-kDa secretory group V phospholipase A2(gVPLA2) in the development of acute lung injury (ALI) and neutrophilic inflammation (NI) caused by intratracheal administration of LPS. Experiments were conducted in gVPLA2knockout ( pla2g5−/−) mice, which lack the gene, and gVPLA2wild-type littermate control ( pla2g5+/+) mice. Indices of pulmonary injury were evaluated 24 h after intratracheal administration of LPS. Expression of gVPLA2in microsections of airways and mRNA content in lung homogenates were increased substantially in pla2g5+/+mice after LPS-administered compared with saline-treated pla2g5+/+mice. By contrast, expression of gVPLA2was neither localized in LPS- nor saline-treated pla2g5−/−mice. LPS also caused 1) reduced transthoracic static compliance, 2) lung edema, 3) neutrophilic infiltration, and 4) increased neutrophil myeloperoxidase activity in pla2g5+/+mice. These events were attenuated in pla2g5−/−mice exposed to LPS or in pla2g5+/+mice receiving MCL-3G1, a neutralizing MAb directed against gVPLA2, before LPS administration. Our data demonstrate that gVPLA2is an inducible protein in pla2g5+/+mice but not in pla2g5−/−mice within 24 h after LPS treatment. Specific inhibition of gVPLA2with MCL-3G1 or gene-targeted mice lacking gVPLA2blocks ALI and attenuates NI caused by LPS.
A high-fat diet attenuates the central response to within-meal satiation signals and modifies the receptor expression of vagal afferents in mice
Authors: Nefti Wahiba , Chaumontet Catherine , Fromentin Gilles , Tomé Daniel , Darcel Nicolas
Published: March 2009

During digestion, macronutrients are sensed within the small intestine. This sensory process is dependent upon the action of gut mediators, such as cholecystokinin (CCK) or serotonin (5-HT), on vagal afferents that, in turn, convey peripheral information to the brain to influence the control of food intake. Recent studies have suggested that dietary conditions alter vagal sensitivity to CCK and 5-HT. This phenomenon may be of importance to the onset of eating disorders. The aim of the present study was thus to investigate the effects of subjecting mice to 15 days of either an HF diet (30% fat, 54% carbohydrate) or an NF diet (10% fat, 74% carbohydrate) on 1) daily and short-term food intake, 2) vagal sensitivity to peripheral anorectic factors and macronutrient loads, and 3) vagal afferent neuron receptor expression. The results indicated that compared with an NF diet, and while increasing food intake and body weight gain, an HF diet altered the short-term response to CCK-8 and intragastric macronutrient loads, while decreasing vagal activation by CCK-8 and modifying the receptor expression of vagal neurons. These findings, therefore, suggest that dietary intervention effect on food intake could be linked to changes in vagal afferent receptor profiles.