bluuebunny/crossref_metadata_2025 huggingface dataset:
Mycobacterium tuberculosis
SigF Regulates Genes Encoding Cell Wall-Associated Proteins and Directly Regulates the Transcriptional Regulatory Gene
phoY1
Authors: Williams Ernest P. , Lee Jong-Hee , Bishai William R. , Colantuoni Carlo , Karakousis Petros C.
Published: March 2007

Mycobacterium tuberculosis
SigF is homologous to stress response and sporulation sigma factors in many bacteria. Consistent with a possible role in mycobacterial survival under stress conditions,
M. tuberculosis sigF
is strongly induced within cultured human macrophages and upon nutrient starvation, and SigF has been implicated in
M. tuberculosis
entry into stationary phase. On the other hand, SigF appears to contribute to the immune pathology of tuberculosis (TB), and a
sigF
-deficient mutant has altered cell membrane properties. Using an
M. tuberculosis sigF
deletion mutant, we show here that
sigF
is not required for bacillary survival under nutrient starvation conditions and within activated murine macrophages or for extracellular persistence in an in vivo granuloma model of latent TB infection. Using a chemically inducible recombinant strain to conditionally overexpress
sigF
, we did not observe arrest or retardation of growth in exponentially growing cultures or reduced susceptibility to rifampin and isoniazid. Consistent with our hypothesis that SigF may mediate TB immunopathogenesis by altering cell membrane properties, we found that overexpression of
sigF
resulted in the differential regulation of many cell wall-associated proteins, including members of the MmpL, PE, and PPE families, several of which have been shown to influence host-pathogen interactions. The most highly upregulated gene by quantitative reverse transcription-PCR at all time points following
sigF
induction was Rv3301c (
phoY1
), which encodes a probable transcriptional regulatory protein homologous to PhoU proteins involved in regulation of phosphate uptake. Using in vitro transcription analysis, we show that SigF directly regulates
phoY1
, whose proposed promoter sequence is GGATTG-N
16
-GGGTAT.
Multidrug-ResistantSalmonella entericaSerovar Paratyphi A Harbors IncHI1 Plasmids Similar to Those Found in Serovar Typhi
Authors: Holt Kathryn E. , Thomson Nicholas R. , Wain John , Phan Minh Duy , Nair Satheesh , Hasan Rumina , Bhutta Zulfiqar A. , Quail Michael A. , Norbertczak Halina , Walker Danielle , Dougan Gordon , Parkhill Julian
Published: March 2007

Salmonella entericaserovars Typhi and Paratyphi A cause systemic infections in humans which are referred to as enteric fever. Multidrug-resistant (MDR) serovar Typhi isolates emerged in the 1980s, and in recent years MDR serovar Paratyphi A infections have become established as a significant problem across Asia. MDR in serovar Typhi is almost invariably associated with IncHI1 plasmids, but the genetic basis of MDR in serovar Paratyphi A has remained predominantly undefined. The DNA sequence of an IncHI1 plasmid, pAKU_1, encoding MDR in a serovar Paratyphi A strain has been determined. Significantly, this plasmid shares a common IncHI1-associated DNA backbone with the serovar Typhi plasmid pHCM1 and anS. entericaserovar Typhimurium plasmid pR27. Plasmids pAKU_1 and pHCM1 share 14 antibiotic resistance genes encoded within similar mobile elements, which appear to form a 24-kb composite transposon that has transferred as a single unit into different positions into their IncHI1 backbones. Thus, these plasmids have acquired similar antibiotic resistance genes independently via the horizontal transfer of mobile DNA elements. Furthermore, two IncHI1 plasmids from a Vietnamese isolate of serovar Typhi were found to contain features of the backbone sequence of pAKU_1 rather than pHCM1, with the composite transposon inserted in the same location as in the pAKU_1 sequence. Our data show that these serovar Typhi and Paratyphi A IncHI1 plasmids share highly conserved core DNA and have acquired similar mobile elements encoding antibiotic resistance genes in past decades.
Coexistence of Two Distinct Versions of O-Antigen Polymerase, Wzy-Alpha and Wzy-Beta, in
Pseudomonas aeruginosa
Serogroup O2 and Their Contributions to Cell Surface Diversity
Authors: Kaluzny Katarina , Abeyrathne Priyanka D. , Lam Joseph S.
Published: March 2007

Assembly of B-band lipopolysaccharide (LPS) in
Pseudomonas aeruginosa
follows a Wzy-dependent pathway, requiring the O-antigen polymerase Wzy and other proteins. The peptide sequences of the
wzy
α
product from strains of serotypes O2, O5, and O16 are identical, but the O units in O5 are α-glycosidically linked, while those in O2 and O16 are β-linked. We hypothesized that a derivative of the D3 bacteriophage
wzy
β
is present in the chromosomes of O2 and O16 and that this gene is responsible for the β-linkage. By a combination of PCR and primer walking,
wzy
β
genes of both serotypes have been amplified and cloned. They are identical but share only 87.42% sequence identity with their xenolog in D3. A chromosomal knockout mutant of O16
wzy
β
was made, and it produces semirough LPS devoid of B-band O antigen. The cloned
wzy
β
is capable of complementing the O16
wzy
β
mutant, as well as cross-complementing a
wzy
α
knockout mutant. However, in the latter case, the restored O antigen was β-linked. Using reverse transcription-PCR, we showed that
wzy
α
was transcribed in O2 and O16 strains and was functional, since both of these genes could complement the
wzy
α
mutant of O5. With the coexistence of
wzy
α
and
wzy
β
in O2 and O16 and the B-band O polysaccharides in these being β-linked, we hypothesized that
iap
, an inhibitor of the alpha-polymerase gene, must be present in these serotypes. Indeed, through PCR, TOPO-cloning, and nucleotide-sequencing results, we verified the presence of
iap
in both O2 and O16 serotypes.
Roles of His-Rich Hpn and Hpn-Like Proteins in
Helicobacter pylori
Nickel Physiology
Authors: Seshadri Susmitha , Benoit Stéphane L. , Maier Robert J.
Published: March 2007

Individual gene-targeted
hpn
and
hpn
-like mutants and a mutant with mutations in both
hpn
genes were more sensitive to nickel, cobalt, and cadmium toxicity than was the parent strain, with the
hpn
-like strain showing the most metal sensitivity of the two individual His-rich protein mutants. The mutant strains contained up to eightfold more urease activity than the parent under nickel-deficient conditions, and the parent strain was able to achieve mutant strain activity levels by nickel supplementation. The mutants contained 3- to 4-fold more and the double mutant about 10-fold more Ni associated with their total urease pools, even though all of the strains expressed similar levels of total urease protein. Hydrogenase activities in the mutants were like those in the parent strain; thus, hydrogenase is fully activated under nickel-deficient conditions. The histidine-rich proteins appear to compete with the Ni-dependent urease maturation machinery under low-nickel conditions. Upon lowering the pH of the growth medium from 7.3 to 5, the wild-type urease activity increased threefold, but the activity in the three mutant strains was relatively unaffected. This pH effect was attributed to a nickel storage role for the His-rich proteins. Under low-nickel conditions, the addition of a nickel chelator did not significantly affect the urease activity of the wild type but decreased the activity of all of the mutants, supporting a role for the His-rich proteins as Ni reservoirs. These nickel reservoirs significantly impact the active urease activities achieved. The His-rich proteins play dual roles, as Ni storage and as metal detoxification proteins, depending on the exogenous nickel levels.
Construction of Recombinant Hemagglutinin Derived from the Gingipain-Encoding Gene of
Porphyromonas gingivalis
, Identification of Its Target Protein on Erythrocytes, and Inhibition of Hemagglutination by an Interdomain Regional Peptide
Authors: Sakai Eiko , Naito Mariko , Sato Keiko , Hotokezaka Hitoshi , Kadowaki Tomoko , Kamaguchi Arihide , Yamamoto Kenji , Okamoto Kuniaki , Nakayama Koji
Published: March 2007

Porphyromonas gingivalis
, an anaerobic gram-negative bacterium associated with chronic periodontitis, can agglutinate human erythrocytes. In general, hemagglutination can be considered the ability to adhere to host cells; however,
P. gingivalis
-mediated hemagglutination has special significance because heme markedly accelerates growth of this bacterium. Although a number of studies have indicated that a major hemagglutinin of
P. gingivalis
is intragenically encoded by
rgpA
,
kgp
, and
hagA
, direct evidence has not been obtained. We demonstrated in this study that recombinant HGP44
720-1081
, a fully processed HGP44 domain protein, had hemagglutinating activity but that an unprocessed form, HGP44
720-1138
, did not. A peptide corresponding to residues 1083 to 1102, which was included in HGP44
720-1138
but not in HGP44
720-1081
, could bind HGP44
720-1081
in a dose-dependent manner and effectively inhibited HGP44
720-1081
-mediated hemagglutination, indicating that the interdomain regional amino acid sequence may function as an intramolecular suppressor of hemagglutinating activity. Analyses by solid-phase binding and chemical cross-linking suggested that HGP44 interacted with glycophorin A on the erythrocyte membrane. Glycophorin A and, more effectively, asialoglycophorin, which were added exogenously, inhibited HGP44
720-1081
-mediated hemagglutination. Treatment of erythrocytes with RgpB proteinase resulted in degradation of glycophorin A on the membrane and a decrease in HGP44
720-1081
-mediated hemagglutination. Surface plasmon resonance detection analysis revealed that HGP44
720-1081
could bind to asialoglycophorin with a dissociation constant of 3.0 × 10
−7
M. These results indicate that the target of HGP44 on the erythrocyte membrane appears to be glycophorin A.
Mutations in
rsmG
, Encoding a 16S rRNA Methyltransferase, Result in Low-Level Streptomycin Resistance and Antibiotic Overproduction in
Streptomyces coelicolor
A3(2)
Authors: Nishimura Kenji , Hosaka Takeshi , Tokuyama Shinji , Okamoto Susumu , Ochi Kozo
Published: March 2007

Certain
str
mutations that confer high- or low-level streptomycin resistance result in the overproduction of antibiotics by
Streptomyces
spp. The
str
mutations that confer the high-level resistance occur within
rpsL
, which encodes the ribosomal protein S12, while those that cause low-level resistance are not as well known. We have used comparative genome sequencing to determine that low-level resistance is caused by mutations of
rsmG
, which encodes an
S
-adenosylmethionine (SAM)-dependent 16S rRNA methyltransferase containing a SAM binding motif. Deletion of
rsmG
from wild-type
Streptomyces coelicolor
resulted in the acquisition of streptomycin resistance and the overproduction of the antibiotic actinorhodin. Introduction of wild-type
rsmG
into the deletion mutant completely abrogated the effects of the
rsmG
deletion, confirming that
rsmG
mutation underlies the observed phenotype. Consistent with earlier work using a spontaneous
rsmG
mutant, the strain carrying
ΔrsmG
exhibited increased SAM synthetase activity, which mediated the overproduction of antibiotic. Moreover, high-performance liquid chromatography analysis showed that the
ΔrsmG
mutant lacked a 7-methylguanosine modification in the 16S rRNA (possibly at position G518, which corresponds to G527 of
Escherichia coli
). Like certain
rpsL
mutants, the
ΔrsmG
mutant exhibited enhanced protein synthetic activity during the late growth phase. Unlike
rpsL
mutants, however, the
ΔrsmG
mutant showed neither greater stability of the 70S ribosomal complex nor increased expression of ribosome recycling factor, suggesting that the mechanism underlying increased protein synthesis differs in the
rsmG
and the
rpsL
mutants. Finally, spontaneous
rsmG
mutations arose at a 1,000-fold-higher frequency than
rpsL
mutations. These findings provide new insight into the role of rRNA modification in activating secondary metabolism in
Streptomyces
.
Transcriptomic Analysis Identifies Growth Rate Modulation as a Component of the Adaptation of Mycobacteria to Survival inside the Macrophage
Authors: Beste D. J. V. , Laing E. , Bonde B. , Avignone-Rossa C. , Bushell M. E. , McFadden J. J.
Published: March 2007

The adaptation of the tubercle bacillus to the host environment is likely to involve a complex set of gene regulatory events and physiological switches in response to environmental signals. In order to deconstruct the physiological state of
Mycobacterium tuberculosis
in vivo, we used a chemostat model to study a single aspect of the organism's in vivo state, slow growth.
Mycobacterium bovis
BCG was cultivated at high and low growth rates in a carbon-limited chemostat, and transcriptomic analysis was performed to identify the gene regulation events associated with slow growth. The results demonstrated that slow growth was associated with the induction of expression of several genes of the dormancy survival regulon. There was also a striking overlap between the transcriptomic profile of BCG in the chemostat model and the response of
M. tuberculosis
to growth in the macrophage, implying that a significant component of the response of the pathogen to the macrophage environment is the response to slow growth in carbon-limited conditions. This demonstrated the importance of adaptation to a low growth rate to the virulence strategy of
M. tuberculosis
and also the value of the chemostat model for deconstructing components of the in vivo state of this important pathogen.
Transcription of All
amoC
Copies Is Associated with Recovery of
Nitrosomonas europaea
from Ammonia Starvation
Authors: Berube Paul M. , Samudrala Ram , Stahl David A.
Published: March 2007

The chemolithotrophic ammonia-oxidizing bacterium
Nitrosomonas europaea
is known to be highly resistant to starvation conditions. The transcriptional response of
N. europaea
to ammonia addition following short- and long-term starvation was examined by primer extension and S1 nuclease protection analyses of genes encoding enzymes for ammonia oxidation (
amoCAB
operons) and CO
2
fixation (
cbbLS
), a third, lone copy of
amoC
(
amoC

3

), and two representative housekeeping genes (
glyA
and
rpsJ
). Primer extension analysis of RNA isolated from growing, starved, and recovering cells revealed two differentially regulated promoters upstream of the two
amoCAB
operons. The distal σ
70
type
amoCAB
promoter was constitutively active in the presence of ammonia, but the proximal promoter was only active when cells were recovering from ammonia starvation. The lone, divergent copy of
amoC
(
amoC

3

) was expressed only during recovery. Both the proximal
amoC

1
,
2

promoter and the
amoC

3

promoter are similar to gram-negative σ
E
promoters, thus implicating σ
E
in the regulation of the recovery response. Although modeling of subunit interactions suggested that a nonconservative proline substitution in AmoC
3
may modify the activity of the holoenzyme, characterization of a Δ
amoC

3

strain showed no significant difference in starvation recovery under conditions evaluated. In contrast to the
amo
transcripts, a delayed appearance of transcripts for a gene required for CO
2
fixation (
cbbL
) suggested that its transcription is retarded until sufficient energy is available. Overall, these data revealed a programmed exit from starvation likely involving regulation by σ
E
and the coordinated regulation of catabolic and anabolic genes.
Exploring Knowledge Management and Knowledge Sharing in a Non-Profit Human Service Organisation
Authors: Stauss Kimberly
Published: March 2007

Non-profit, human service organisations are continuing to struggle for limited money and are being held accountable more than ever before to prove an increase in quality and efficacy of services. Agency administrators have to think creatively in order to help their agencies stand out. Efforts include tapping not only into the knowledge of their employees but also knowledge of their consumers. Utilising qualitative methods, with a highly successful non-profit agency, this document investigates the information sharing activities and the knowledge gained from consumers when knowledge transfer backflow was allowed. Shared are findings on how public service agencies, often dealing with those individuals in need, can use knowledge management processes to inform and improve future service delivery. The thick descriptions provided in this document give context to the multi-layered knowledge dimensions involved. Highlighted is the importance of the embedded knowledge their participants come in with and the need for agencies to consider the intricacies of this knowledge. Noticeably, this study also delineates how the historical and current oppression experienced contributes to this complexity. The data reported in this study is the first step in understanding the individual and organisation factors contributing to the collaboration efforts of non-profit agencies.
Acute myocardial infarction in mice: assessment of transmurality by strain rate imaging
Authors: Thibault Hélène , Gomez Ludovic , Donal Erwan , Pontier Gerard , Scherrer-Crosbie Marielle , Ovize Michel , Derumeaux Geneviève
Published: March 2007

In vivo evaluation of the transmural extension of myocardial infarction (TEI) is crucial to prediction of viability and prognosis. With the rise of transgenic technology, murine myocardial infarction (MI) models are increasingly used. Our study aimed to evaluate systolic strain rate (SR), a new parameter of regional function, to quantify TEI in a murine model of acute MI induced by various durations of ischemia followed by 24 h of reperfusion. Global and regional left ventricular (LV) function were assessed by echocardiography (13 MHz, Vivid 7, GE) in 4 groups of wild-type mice (C57BL/6, 2 mo old): a sham-treated group ( n = 10) and three MI groups [30 ( n = 11), 60 ( n = 10), and 90 ( n = 9) min of left coronary artery occlusion]. Conventional LV dimensions, anterior wall (AW) thickening, and peak systolic SR were measured before and 24 h after reperfusion. Area at risk (AR) was measured by blue dye and infarct size (area of necrosis, AN) and TEI by triphenyltetrazolium chloride staining. AN increased with ischemia duration (25 ± 2%, 56 ± 5%, 71 ± 6% of AR for 30, 60, and 90 min, respectively; P < 0.05). LV end-diastolic volume significantly increased with ischemia duration (30 ± 5, 34 ± 5, 43 ± 5 μl; P < 0.05), whereas LV ejection fraction decreased (63 ± 5%, 58 ± 6%, 46 ± 5%; P < 0.05). AW thickening decrease was not influenced by ischemia duration. Conversely, systolic SR decreased with ischemia duration (13 ± 5, 4 ± 3, −2 ± 6 s−1; P < 0.05) and was significantly correlated with TEI ( r = 0.89, P < 0.01). Receiver operating characteristic (ROC) curves identified systolic SR as the most accurate parameter to predict TEI. In conclusion, in a murine model of MI, SR imaging is superior to conventional echocardiography to predict TEI early after MI.
Myogenic contractility is more dependent on myofilament calcium sensitization in term fetal than adult ovine cerebral arteries
Authors: Sandoval Renan J. , Injeti Elisha R. , Williams James M. , Georthoffer William T. , Pearce William J.
Published: March 2007

Regulation of cytosolic calcium and myofilament calcium sensitivity varies considerably with postnatal age in cerebral arteries. Because these mechanisms also govern myogenic tone, the present study used graded stretch to examine the hypothesis that myogenic tone is less dependent on calcium influx and more dependent on myofilament calcium sensitization in term fetal compared with adult cerebral arteries. Term fetal and adult posterior communicating cerebral arteries exhibited similar myogenic responses, with peak tensions averaging 24 and 26% of maximum contractile force produced in any given tissue in response to an isotonic Krebs buffer containing 122 mM K+(Kmax) at optimum stretch ratios (working diameter/unstressed diameter) of 2.19 and 2.23, respectively. Graded stretch increased cytosolic Ca2+concentration at stretch ratios >2.0 in adult arteries, but increased Ca2+concentration only at stretch ratios >2.3 in fetal arteries. In permeabilized arteries, myogenic tone peaked at a stretch ratio of 2.1 in both fetal and adult arteries. The fetal %Kmaxvalues at peak myogenic tone were not significantly different at either pCa 7.0 (23%) or pCa 5.5 (25%) but were significantly less at pCa 8.0 (8.4 ± 2.3%). Conversely, adult %Kmaxvalues at peak myogenic tone were significantly less at both pCa 8.0 (10.4 ± 1.8%) and pCa 7.0 (16%) than at pCa 5.5 (27%). The maximal extents of stretch-induced increases in myosin light chain phosphorylation in intact fetal (20%) and adult (17%) arteries were similar. The data demonstrate that the cerebrovascular myogenic response is highly conserved during postnatal maturation but is mediated differently in fetal and adult cerebral arteries.
Darbepoetin alfa, a long-acting erythropoietin analog, offers novel and delayed cardioprotection for the ischemic heart
Authors: Gao Erhe , Boucher Matthieu , Chuprun J. Kurt , Zhou Rui-Hai , Eckhart Andrea D. , Koch Walter J.
Published: March 2007

Recent studies from our lab and others have shown that the hematopoietic cytokine erythropoietin (EPO) can protect the heart from ischemic damage in a red blood cell-independent manner. Here we examined any protective effects of the long-acting EPO analog darbepoetin alfa (DA) in a rat model of ischemia-reperfusion (I/R) injury. Rats were subjected to 30-min ischemia followed by 72-h reperfusion. In a dose-response study, DA (2, 7, 11, and 30 μg/kg) or vehicle was administered as a single bolus at the start of ischemia. To determine the time window of potential cardioprotection, a single high dose of DA (30 μg/kg) was given at either the initiation or the end of ischemia or at 1 or 24 h after reperfusion. After 3 days, cardiac function and infarct size were assessed. Acute myocyte apoptosis was quantified by TUNEL staining on myocardial sections and by caspase-3 activity assays. DA significantly reduced infarct size from 32.8 ± 3.5% (vehicle) to 11.0 ± 3.3% in a dose-dependent manner, while there was no difference in ischemic area between groups. Treatment with DA as late as 24 h after the beginning of reperfusion still demonstrated a significant reduction in infarct size (17.0 ± 1.6%). Consistent with infarction data, DA improved in vivo cardiac reserve compared with vehicle. Finally, DA significantly decreased myocyte apoptosis and caspase-3 activity after I/R. These data indicate that DA protects the heart against I/R injury and improves cardiac function, apparently through a reduction of myocyte apoptosis. Of clinical importance pointing toward a relevant therapeutic utility, we report that even if given 24 h after I/R injury, DA can significantly protect the myocardium.
Cannabidiol attenuates high glucose-induced endothelial cell inflammatory response and barrier disruption
Authors: Rajesh Mohanraj , Mukhopadhyay Partha , Bátkai Sándor , Haskó György , Liaudet Lucas , Drel Viktor R. , Obrosova Irina G. , Pacher Pál
Published: March 2007

A nonpsychoactive cannabinoid cannabidiol (CBD) has been shown to exert potent anti-inflammatory and antioxidant effects and has recently been reported to lower the incidence of diabetes in nonobese diabetic mice and to preserve the blood-retinal barrier in experimental diabetes. In this study we have investigated the effects of CBD on high glucose (HG)-induced, mitochondrial superoxide generation, NF-κB activation, nitrotyrosine formation, inducible nitric oxide synthase (iNOS) and adhesion molecules ICAM-1 and VCAM-1 expression, monocyte-endothelial adhesion, transendothelial migration of monocytes, and disruption of endothelial barrier function in human coronary artery endothelial cells (HCAECs). HG markedly increased mitochondrial superoxide generation (measured by flow cytometry using MitoSOX), NF-κB activation, nitrotyrosine formation, upregulation of iNOS and adhesion molecules ICAM-1 and VCAM-1, transendothelial migration of monocytes, and monocyte-endothelial adhesion in HCAECs. HG also decreased endothelial barrier function measured by increased permeability and diminished expression of vascular endothelial cadherin in HCAECs. Remarkably, all the above mentioned effects of HG were attenuated by CBD pretreatment. Since a disruption of the endothelial function and integrity by HG is a crucial early event underlying the development of various diabetic complications, our results suggest that CBD, which has recently been approved for the treatment of inflammation, pain, and spasticity associated with multiple sclerosis in humans, may have significant therapeutic benefits against diabetic complications and atherosclerosis.
Blockade of both α1A- and α1B-adrenergic receptor subtype signaling is required to inhibit neointimal formation in the mouse femoral artery
Authors: Hosoda Chihiro , Hiroyama Masami , Sanbe Atsushi , Birumachi Jun-ichi , Kitamura Tadaichi , Cotecchia Susanna , Simpson Paul C. , Tsujimoto Gozoh , Tanoue Akito
Published: March 2007

Attenuation of early restenosis after percutaneous coronary intervention (PCI) is important for the successful treatment of coronary artery disease. Some clinical studies have shown that hypertension is a risk factor for early restenosis after PCI. These findings suggest that α1-adrenergic receptors (α1-ARs) may facilitate restenosis after PCI because of α1-AR's remarkable contribution to the onset of hypertension. In this study, we examined the neointimal formation after vascular injury in the femoral artery of α1A-knockout (α1A-KO), α1B-KO, α1D-KO, α1A-/α1B-AR double-KO (α1AB-KO), and wild-type mice to investigate the functional role of each α1-AR subtype in neointimal formation, which is known to promote restenosis. Neointimal formation 4 wk after wire injury was significantly ( P < 0.05) smaller in α1AB-KO mice than in any other group of mice, while blood pressures were not altered in any of the groups of mice after wire injury compared with those before it. These results suggest that lack of both α1A- and α1B-ARs could be necessary to inhibit neointimal formation in the mouse femoral artery.
Rho kinase contributes to basal vascular tone in humans: role of endothelium-derived nitric oxide
Authors: Büssemaker E. , Pistrosch Frank , Förster Sarah , Herbrig Kay , Gross Peter , Passauer Jens , Brandes Ralf P.
Published: March 2007

Our objective was to determine the role of the Rho-associated kinase (ROK) for the regulation of FBF (FBF) and to unmask a potential role of ROK for the regulation of endothelium-derived nitric oxide (NO). Moreover, the effect of fasudil on the constrictor response to endothelin-1 was recorded. Regarding background, phosphorylation of the myosin light chain (MLC) determines the calcium sensitivity of the contractile apparatus. MLC phosphorylation depends on the activity of the MLC kinase and the MLC phosphatase. The latter enzyme is inhibited through phosphorylation by ROK. ROK has been suggested to inhibit NO generation, possibly via the inhibition of the Akt pathway. In this study, the effect of intra-arterial infusion of the ROK inhibitor fasudil on FBF in 12 healthy volunteers was examined by venous occlusion plethysmography. To unmask the role of NO, fasudil was infused during NO clamp. As a result, fasudil markedly increased FBF in a dose-dependent manner from 2.34 ± 0.21 to 6.96 ± 0.93 ml/100 ml forearm volume at 80 μg/min ( P < 0.001). At 1,600 μg/min, fasudil reduced systolic, diastolic, and mean arterial pressure while increasing heart rate. Fasudil abolished the vasoconstrictor effect of endothelin-1. The vascular response to fasudil (80 μmol/min) was blunted during NO clamp (104 ± 18% vs. 244 ± 48% for NO clamp + fasudil vs. fasudil alone; data as ratio between infused and noninfused arm with baseline = 0%, P < 0.05). In conclusion, 1) basal peripheral and systemic vascular tone depends on ROK; 2) a significant portion of fasudil-induced vasodilation is mediated by NO, suggesting that vascular bioavailable NO is negatively regulated by ROK; and 3) the constrictor response to endothelin involves the activation of ROK.
Interaction between spiral and paced waves in cardiac tissue
Authors: Agladze Konstantin , Kay Matthew W. , Krinsky Valentin , Sarvazyan Narine
Published: March 2007

For prevention of lethal arrhythmias, patients at risk receive implantable cardioverter-defibrillators, which use high-frequency antitachycardia pacing (ATP) to convert tachycardias to a normal rhythm. One of the suggested ATP mechanisms involves paced-induced drift of rotating waves followed by their collision with the boundary of excitable tissue. This study provides direct experimental evidence of this mechanism. In monolayers of neonatal rat cardiomyocytes in which rotating waves of activity were initiated by premature stimuli, we used the Ca2+-sensitive indicator fluo 4 to observe propagating wave patterns. The interaction of the spiral tip with a paced wave was then monitored at a high spatial resolution. In the course of the experiments, we observed spiral wave pinning to local heterogeneities within the myocyte layer. High-frequency pacing led, in a majority of cases, to successful termination of spiral activity. Our data show that 1) stable spiral waves in cardiac monolayers tend to be pinned to local heterogeneities or areas of altered conduction, 2) overdrive pacing can shift a rotating wave from its original site, and 3) the wave break, formed as a result of interaction between the spiral tip and a paced wave front, moves by a paced-induced drift mechanism to an area where it may become unstable or collide with a boundary. The data were complemented by numerical simulations, which was used to further analyze experimentally observed behavior.
Chronic intermittent hypoxia impairs baroreflex control of heart rate but enhances heart rate responses to vagal efferent stimulation in anesthetized mice
Authors: Lin Min , Liu Rugao , Gozal David , Wead William B. , Chapleau Mark W. , Wurster Robert , Cheng Zixi (Jack)
Published: March 2007

Chronic intermittent hypoxia (CIH) leads to increased sympathetic nerve activity and arterial hypertension. In this study, we tested the hypothesis that CIH impairs baroreflex (BR) control of heart rate (HR) in mice, and that decreased cardiac chronotropic responsiveness to vagal efferent activity contributes to such impairment. C57BL/6J mice were exposed to either room air (RA) or CIH (6-min alternations of 21% O2and 5.7% O2, 12 h/day) for 90 days. After the treatment period, mice were anesthetized (Avertin) and arterial blood pressure (ABP) was measured from the femoral artery. Mean ABP (MABP) was significantly increased in mice exposed to CIH (98.7 ± 2.5 vs. RA: 78.9 ± 1.4 mmHg, P < 0.001). CIH increased HR significantly (584.7 ± 8.9 beats/min; RA: 518.2 ± 17.9 beats/min, P < 0.05). Sustained infusion of phenylephrine (PE) at different doses (0.1–0.4 μg/min) significantly increased MABP in both CIH and RA mice, but the ABP-mediated decreases in HR were significantly attenuated in mice exposed to CIH ( P < 0.001). In contrast, decreases in HR in response to electrical stimulation of the left vagus nerve (30 μA, 2-ms pulses) were significantly enhanced in mice exposed to CIH compared with RA mice at low frequencies. We conclude that CIH elicits a sustained impairment of baroreflex control of HR in mice. The blunted BR-mediated bradycardia occurs despite enhanced cardiac chronotropic responsiveness to vagal efferent stimulation. This suggests that an afferent and/or a central defect is responsible for the baroreflex impairment following CIH.
CCSP regulates cross talk between secretory cells and both ciliated cells and macrophages of the conducting airway
Authors: Reynolds Susan D. , Reynolds Paul R. , Snyder Joshua C. , Whyte Fadra , Paavola Kevin J. , Stripp Barry R.
Published: March 2007

Pulmonary host defense employs a combination of biochemical and biophysical activities to recognize, inactivate, and mediate clearance of environmental agents as well as modulate the overall response to such challenge. Dysregulation of the inflammatory arm of this response is associated with chronic lung diseases (CLD) including cystic fibrosis and chronic obstructive lung disease. Although mechanisms mediating immunoregulation are incompletely characterized, decrements in levels of the nonciliated secretory cell product Clara cell secretory protein (CCSP) in numerous CLD and identification of proinflammatory state in mice homozygous for a null allele of the CCSP gene (CCSP−/−) suggest a central role for the nonciliated secretory cell in this process. In an effort to determine the molecular basis for immunoregulatory defects associated with CCSP deficiency, we utilized difference gel electrophoresis in combination with matrix-assisted laser desorption ionization time-of-flight to compare the proteomes of wild-type and CCSP−/− mice. We demonstrate a shift in the isoelectric point of the immunomodulatory protein annexin A1 (ANXA1) to more acidic isoforms in CCSP−/− mice. Similar ANXA1 mRNA and protein abundance in wild-type and CCSP−/− tissue and identical localization of ANXA1 protein to alveolar macrophages and the ciliary bed of ciliated cells demonstrated that CCSP deficiency was associated exclusively with altered posttranslational modification of ANXA1. These results suggest that both long- and short-range paracrine signaling between nonciliated secretory cells and cells of the immune system and epithelium impact modification of cell type-specific proteins and implicate nonciliated secretory cells in a regulatory axis that might integrate critical aspects of host defense.
CD14 is an essential mediator of LPS-induced airway disease
Authors: Brass David M. , Hollingsworth John W. , McElvania-Tekippe Erin , Garantziotis Stavros , Hossain Imtaz , Schwartz David A.
Published: March 2007

Chronic lipopolysaccharide (LPS) inhalation in rodents recapitulates many classic features of chronic obstructive pulmonary disease seen in humans, including airways hyperresponsiveness, neutrophilic inflammation, cytokine production in the lung, and small airways remodeling. CD14-deficient mice (C57BL/6CD14−/−) have an altered response to systemic LPS, and yet the role of CD14 in the response to inhaled LPS has not been defined. We observed that C57BL/6CD14−/−mice demonstrate no discernable physiological or inflammatory response to a single LPS inhalation challenge. However, the physiological (airways hyperresponsiveness) and inflammatory (presence of neutrophils and TNF-α in whole lung lavage fluid) responsiveness to inhaled LPS in C57BL/6CD14−/−mice was restored by instilling soluble CD14 intratracheally. Intratracheal instillation of wild-type macrophages into C57BL/6CD14−/−mice restored neutrophilic inflammation only and failed to restore airways hyperresponsiveness or TNF-α protein in whole lung lavage. These findings demonstrate that CD14 is critical to LPS-induced airway disease and that macrophage CD14 is sufficient to initiate neutrophil recruitment into the airways but that CD14 may need to interact with other cell types as well for the development of airways hyperresponsiveness and for cytokine production.
Early growth response gene-1 promotes airway allograft rejection
Authors: Harada Hiroaki , Lama Vibha N. , Badri Linda N. , Ohtsuka Takashi , Petrovic-Djergovic Danica , Liao Hui , Yoshikawa Yasushi , Iwanaga Koichiro , Lau Chris L. , Pinsky David J.
Published: March 2007

Chronic airway rejection, characterized by lymphocytic bronchitis, epithelial cell damage, and obliterative bronchiolitis (OB), limits long-term survival after lung transplantation. The transcription factor early growth response gene-1 (Egr-1) induces diverse inflammatory mediators, some involved in OB pathogenesis. An orthotopic mouse tracheal transplant model was used to determine whether Egr-1 promotes development of airway allograft rejection. Significantly higher Egr-1 mRNA levels were seen in allografts (3.2-fold increase vs. isografts, P = 0.012). Allografts revealed thickening of epithelial and subepithelial airway layers (51 ± 4% luminal encroachment for allografts vs. 20 ± 3% for isografts, P < 0.0001) marked by significant lymphocytic infiltration. Absence of the Egr-1 gene in donor (but not recipient) tissue resulted in significant reduction in luminal narrowing (34 ± 4%, P = 0.0001) with corresponding diminution of T cell infiltration. Egr-1 null allografts exhibited a striking reduction in inducible nitric oxide synthase (iNOS) expression. Effector cytokines previously implicated in OB pathogenesis with known Egr-1 promoter motifs (IL-1β and JE/monocyte chemoattractant protein-1) were reduced in Egr-1 null allografts. These data suggest a paradigm wherein local induction of Egr-1 in tracheal allografts drives expression of inflammatory mediators responsible for lymphocyte recruitment and tissue destruction characteristic of airway rejection.
Bronchial epithelial cell growth regulation in fibroblast cocultures: the role of hepatocyte growth factor
Authors: Skibinski Grzegorz , Elborn J. Stuart , Ennis Madeleine
Published: March 2007

Proliferation of bronchial epithelial cells is an important biological process in physiological conditions and various lung diseases. The objective of this study was to determine how bronchial fibroblasts influence bronchial epithelial cell proliferation. The proliferative activity in cocultures was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and direct cells counts. Concentration of cytokines was measured in cell culture supernatants by means of ELISA. In primary cell cocultures, fibroblasts or fibroblast-conditioned medium enhanced 1.85-fold the proliferation of primary bronchial epithelial cells ( P < 0.02) compared with bronchial epithelial cells cultured alone. The proliferative activity in cocultures and in fibroblast-conditioned medium was reduced by neutralizing antibody to hepatocyte growth factor (HGF) and HGF receptor c-met. Neutralizing antibodies to FGF-7 and IGF-1 had no effect. Treatment of fibroblast-epithelial cocultures with anti-IL-6 and anti-TNF-α neutralizing antibodies and with indomethacin decreased production of HGF. These results indicate that cytokines and PGE2may indirectly mediate epithelial cell proliferation via the regulation of HGF in bronchial stromal cells and that HGF plays a crucial role in proinflammatory cytokine-induced proliferation in the experimental system studied.
Reduction in soluble guanylyl cyclase-specific activity following prolonged treatment of porcine pulmonary artery with nitric oxide
Authors: Perkins William J. , Taniguchi Miwa , Warner David O. , Chini Eduardo N. , Jones Keith A.
Published: March 2007

In a newly characterized cultured porcine pulmonary artery (PA) preparation, 24-h treatment with the nitric oxide (NO) donor ( Z)-1-[ N-(2-aminoethyl)- N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO) decreased the response to acutely applied DETA-NO compared with 24-h control (−log EC506.55 ± 0.12 and 5.02 ± 0.21, respectively). Treatment of PA with the cell-permeable superoxide dismutase mimetic, Mn(III) tetra(4-benzoic acid) porphyrin chloride, did not change NO responsiveness in either freshly prepared or 24-h DETA-NO-treated PA. cGMP and cAMP phosphodiesterase activities were approximately equal in PA. Twenty-four-hour DETA-NO treatment did not change either cGMP or cAMP phosphodiesterase activities. Twenty-four hours in culture had no significant effect on soluble guanylyl cyclase (sGC) subunit mRNA expression, but 24-h DETA-NO treatment significantly decreased the expression of both sGCα1and sGCβ1. sGCβ1protein expression was 42 ± 4 ng/mg soluble protein. Twenty-four hours in culture without and with DETA-NO reduced sGCβ1protein expression (36 ± 3 and 31 ± 3 ng/mg soluble protein, respectively, P < 0.025). Basal tissue cGMP [(cGMP)i] was significantly increased, and NO-induced (cGMP)iwas significantly decreased by 24-h DETA-NO treatment. (cGMP)inormalized to the amount of sGC protein expressed in PA was significantly lower in PA treated for 24 h with DETA-NO compared with both freshly isolated and 24-h cultured PA. We conclude that prolonged NO treatment induces decreased acute NO responsiveness in part by decreasing both sGC expression and sGC-specific activity.
Perspectives on endothelial-to-mesenchymal transition: potential contribution to vascular remodeling in chronic pulmonary hypertension
Authors: Arciniegas Enrique , Frid Maria G. , Douglas Ivor S. , Stenmark Kurt R.
Published: March 2007

All forms of pulmonary hypertension are characterized by structural changes in pulmonary arteries. Increased numbers of cells expressing α-smooth muscle (α-SM) actin is a nearly universal finding in the remodeled artery. Traditionally, it was assumed that resident smooth muscle cells were the exclusive source of these newly appearing α-SM actin-expressing cells. However, rapidly emerging experimental evidence suggests other, alternative cellular sources of these cells. One possibility is that endothelial cells can transition into mesenchymal cells expressing α-SM actin and that this process contributes to the accumulation of SM-like cells in vascular pathologies. We review the evidence that endothelial-mesenchymal transition is an important contributor to cardiac and vascular development as well as to pathophysiological vascular remodeling. Recent work has provided evidence for the role of transforming growth factor-β, Wnt, and Notch signaling in this process. The potential roles of matrix metalloproteinases and serine proteases are also discussed. Importantly, endothelial-mesenchymal transition may be reversible. Thus insights into the mechanisms controlling endothelial-mesenchymal transition are relevant to vascular remodeling and are important as we consider new therapies aimed at reversing pulmonary vascular remodeling.
Inhaled nitric oxide attenuates pulmonary inflammation and fibrin deposition and prolongs survival in neonatal hyperoxic lung injury
Authors: ter Horst Simone A. J. , Walther Frans J. , Poorthuis Ben J. H. M. , Hiemstra Pieter S. , Wagenaar Gerry T. M.
Published: March 2007

Administration of inhaled nitric oxide (iNO) is a potential therapeutic strategy to prevent bronchopulmonary dysplasia (BPD) in premature newborns with respiratory distress syndrome. We evaluated this approach in a rat model, in which premature pups were exposed to room air, hyperoxia, or a combination of hyperoxia and NO (8.5 and 17 ppm). We investigated the anti-inflammatory effects of prolonged iNO therapy by studying survival, histopathology, fibrin deposition, and differential mRNA expression (real-time RT-PCR) of key genes involved in the development of BPD. iNO therapy prolonged median survival 1.5 days ( P = 0.0003), reduced fibrin deposition in a dosage-dependent way up to 4.3-fold ( P < 0.001), improved alveolar development by reducing septal thickness, and reduced the influx of leukocytes. Analysis of mRNA expression revealed an iNO-induced downregulation of genes involved in inflammation (IL-6, cytokine-induced neutrophilic chemoattractant-1, and amphiregulin), coagulation, fibrinolysis (plasminogen activator inhibitor 1 and urokinase-type plasminogen activator receptor), cell cycle regulation (p21), and an upregulation of fibroblast growth factor receptor-4 (alveolar formation). We conclude that iNO therapy improves lung pathology and prolongs survival by reducing septum thickness, inhibiting inflammation, and reducing alveolar fibrin deposition in premature rat pups with neonatal hyperoxic lung injury.
Comparison of Physicomechanical Properties of NBR—PVC Blend Cured by Sulfur and Electron Beam
Authors: Hafezi M. , Khorasani S.Nouri , Ziaei F. , Azim H.R.
Published: March 2007

In this research, physicomechanical properties including tensile strength, elongation at break, hardness, resilience, and aging of acrylonitrile butadiene rubber—poly vinyl chloride (NBR—PVC) blend cured by sulfur and electron beam are investigated. The Taguchi method is employed in order to formulate the sulfur curing system. Electron beams in dose rates from 0 to 150 kGy are applied to cure NBR—PVC blends without sulfur curing system. Results show that the physicomechanical properties, except resilience, of NBR— PVC blend cured by the electron beam improved by 15% compared with blends cured by the sulfur system.
Mining maximal frequent itemsets from data streams
Authors: Mao Guojun , Wu Xindong , Zhu Xingquan , Chen Gong , Liu Chunnian
Published: March 2007

Frequent pattern mining from data streams is an active research topic in data mining. Existing research efforts often rely on a two-phase framework to discover frequent patterns: (1) using internal data structures to store meta-patterns obtained by scanning the stream data; and (2) re-mining the meta-patterns to finalize and output frequent patterns. The defectiveness of such a two-phase framework lies in the fact that the two stages provide barriers to dynamically and immediately finding frequent patterns with online functionalities. It is expected that a single-phase algorithm can fulfil frequent pattern mining from data streams in such a way that the users can see patterns in an immediate and dynamic manner, as soon as the patterns have become frequent. In this paper, we propose INSTANT, a single-phase algorithm for discovering frequent itemsets from data streams. The theoretical foundation of INSTANT is based on a framework theory on a set of itemsets, which is also presented in the paper. The novel design of INSTANT ensures that it employs compact data structures to mine frequent patterns from data streams in a single phase. Our experimental results demonstrate the time and space efficiency of the proposed algorithm.
How much is information worth? Willingness to pay for expert and non-expert informational goods compared to material goods in lay economic thinking
Authors: Sakalaki Maria , Kazi Smaragda
Published: March 2007

Economists consider information a paradoxical good of uncertain value. This experimental study compares the valuation of economic information to the valuation of material goods when subjects possess perfect information so that uncertainty about the outcome of the transaction is eliminated. It also aims to study the participants' justifications of their valuations, in order to examine the underlying cognitive processes. The main hypotheses were that: (a) even if subjects deal with perfect information they will underestimate its value compared to the value of material goods; (b) underestimation of the value of information will be greater in conditions of high involvement, that is when expected payoff and therefore investment and risk are high; and (c) expert information will be valued more highly than non-expert information, since it should be regarded as more reliable. In the main study, two groups of students in the same university were asked to value and justify their valuation of material goods, under conditions of certain low (1000) and high (100,000) payoffs, respectively. Two other groups were asked to value and to justify their valuation of non-expert information under the same conditions of profit. Finally, two more groups of students were asked to value and justify their valuation of expert information, again under conditions of low or high expected profit. The findings of the study show that under all conditions subjects underestimate the value of information both absolutely and relatively in comparison to material goods, especially under conditions of high involvement. The participants' justifications of their own valuations are heuristics which seem to take into account the uncertain character of informational goods.