bluuebunny/crossref_metadata_2025 huggingface dataset:
The Genome Sequence of Avian PathogenicEscherichia coliStrain O1:K1:H7 Shares Strong Similarities with Human Extraintestinal PathogenicE. coliGenomes
Authors: Johnson Timothy J. , Kariyawasam Subhashinie , Wannemuehler Yvonne , Mangiamele Paul , Johnson Sara J. , Doetkott Curt , Skyberg Jerod A. , Lynne Aaron M. , Johnson James R. , Nolan Lisa K.
Published: February 2007

Escherichia colistrains that cause disease outside the intestine are known as extraintestinal pathogenicE. coli(ExPEC) and include human uropathogenicE. coli(UPEC) and avian pathogenicE. coli(APEC). Regardless of host of origin, ExPEC strains share many traits. It has been suggested that these commonalities may enable APEC to cause disease in humans. Here, we begin to test the hypothesis that certain APEC strains possess potential to cause human urinary tract infection through virulence genotyping of 1,000 APEC and UPEC strains, generation of the first complete genomic sequence of an APEC (APEC O1:K1:H7) strain, and comparison of this genome to all available human ExPEC genomic sequences. The genomes of APEC O1 and three human UPEC strains were found to be remarkably similar, with only 4.5% of APEC O1's genome not found in other sequenced ExPEC genomes. Also, use of multilocus sequence typing showed that some of the sequenced human ExPEC strains were more like APEC O1 than other human ExPEC strains. This work provides evidence that at least some human and avian ExPEC strains are highly similar to one another, and it supports the possibility that a food-borne link between some APEC and UPEC strains exists. Future studies are necessary to assess the ability of APEC to overcome the hurdles necessary for such a food-borne transmission, and epidemiological studies are required to confirm that such a phenomenon actually occurs.
Influence of Amino Acids on Low-Density
Escherichia coli
Responses to Nutrient Downshifts
Authors: Bodini Sergio , Nunziangeli Luisa , Santori Francesca
Published: February 2007

A vast bibliography on nutrient effects on high-density cultures exists, while it has been overlooked that low densities of starving cells are often the rule in natural environments. By means of a novel sensitive β-galactosidase assay, we examined
Escherichia coli
transitions to minimal media when the cell concentration was 100 to 10,000 cells per ml. As in high-density cultures, the enzyme activity depended on amino acid availability and was subject to catabolite repression and stringent control. In all conditions tested, despite the presence of other nutrient sources, the relationship between β-galactosidase activity and the
l
-amino acid pool was hyperbolic. The affinity constant when the amino acid pool was the only nutrient source averaged 14 μM after 90 min and increased up to 222 μM after 4.5 h. While investigating the transition from lag phase to exponential phase, we observed that the cells did not enter into starvation mode in the presence of amino acids, even when the nutrient amount was insufficient to support full survival. Based on these premises, the switch from starvation to hunger was investigated in relation to the amino acid pools. A critical range of concentrations at which
E. coli
linearly synthesized β-galactosidase despite, at the same time, suffering a large decrease in cell viability was then recognized. Since both β-galactosidase production and the dilution rate were reduced by more than half in the absence of leucine, we examined the contribution of leucine to cell recovery capabilities.
Mechanisms of Adaptation to Nitrosative Stress inBacillus subtilis
Authors: Rogstam Annika , Larsson Jonas T. , Kjelgaard Peter , von Wachenfeldt Claes
Published: February 2007

Bacteria use a number of mechanisms for coping with the toxic effects exerted by nitric oxide (NO) and its derivatives. Here we show that the flavohemoglobin encoded by thehmpgene has a vital role in an adaptive response to protect the soil bacteriumBacillus subtilisfrom nitrosative stress. We further show that nitrosative stress induced by the nitrosonium cation donor sodium nitroprusside (SNP) leads to deactivation of the transcriptional repressor NsrR, resulting in derepression ofhmp. Nitrosative stress induces the sigma B-controlled general stress regulon. However, asigBnull mutant did not show increased sensitivity to SNP, suggesting that the sigma B-dependent stress proteins are involved in a nonspecific protection against stress whereas the Hmp flavohemoglobin plays a central role in detoxification. Mutations in theyjbIHoperon, which encodes a truncated hemoglobin (YjbI) and a predicted 34-kDa cytosolic protein of unknown function (YjbH), renderedB. subtilishypersensitive to SNP, suggesting roles in nitrosative stress management.
A
pthA
Homolog from
Xanthomonas axonopodis
pv. citri Responsible for Host-Specific Suppression of Virulence
Authors: Shiotani Hiroshi , Fujikawa Takashi , Ishihara Hiromichi , Tsuyumu Sinji , Ozaki Katsumi
Published: February 2007

Strains of the plant-pathogenic bacterium
Xanthomonas axonopodis
pv. citri are differentiated into two groups with respect to aggressiveness (normal and weak) on
Citrus grandis
cultivars but not on other
Citrus
species such as
Citrus sinensis
. Random mutagenesis using the transposon Tn
5
in
X. axonopodis
pv. citri strain KC21, which showed weak aggressiveness on a
C. grandis
cultivar, was used to isolate mutant KC21T46, which regained a normal level of aggressiveness on the cultivar. The gene inactivated by the transposon,
hssB3.0
, was shown to be responsible for the suppression of virulence on
C. grandis
. Sequence analysis revealed it to be a new member of the
pthA
homologs, which was almost identical in sequence to the other homologs except for the number of tandem repeats in the central region of the gene.
hssB3.0
appears to be a chimera of other
pthA
homologs,
pB3.1
and
pB3.7
, and could have been generated by recombination between these two genes. Importantly, in
X. axonopodis
pv. citri,
hssB3.0
was found in all of the tested isolates belonging to the weakly aggressive group but not in the isolates of the normally aggressive group. Isolation of the virulence-deficient mutant KC21T14 from KC21, in which the pathogenicity gene
pthA-KC21
was disrupted, showed that
hssB3.0
induces a defense response on the host but partially interrupts canker development elicited by the pathogenicity gene in this bacterium.
YcfR (BhsA) InfluencesEscherichia coliBiofilm Formation through Stress Response and Surface Hydrophobicity
Authors: Zhang Xue-Song , García-Contreras Rodolfo , Wood Thomas K.
Published: February 2007

DNA microarrays revealed that expression ofycfR, which encodes a putative outer membrane protein, is significantly induced inEscherichia colibiofilms and is also induced by several stress conditions. We show that deletion ofycfRincreased biofilm formation fivefold in the presence of glucose; the glucose effect was corroborated by showing binding of the cyclic AMP receptor protein to theycfRpromoter. It appears that YcfR is a multiple stress resistance protein, since deletingycfRalso rendered the cell more sensitive to acid, heat treatment, hydrogen peroxide, and cadmium. Increased biofilm formation through YcfR due to stress appears to be the result of decreasing indole synthesis, since a mutation in thetnaAgene encoding tryptophanase prevented enhanced biofilm formation upon stress and adding indole prevented enhanced biofilm formation upon stress. DeletingycfRalso affected outer membrane proteins and converted the cell from hydrophilic to hydrophobic, as well as increased cell aggregation fourfold. YcfR seems to be involved in the regulation ofE. coliK-12 biofilm formation by decreasing cell aggregation and cell surface adhesion, by influencing the concentration of signal molecules, and by interfering with stress responses. Based on our findings, we propose that this locus be namedbhsA, for influencingbiofilm throughhydrophobicity andstress response.
Ribosomal Features Essential for
tna
Operon Induction: Tryptophan Binding at the Peptidyl Transferase Center
Authors: Cruz-Vera Luis R. , New Aaron , Squires Catherine , Yanofsky Charles
Published: February 2007

Features of the amino acid sequence of the TnaC nascent peptide are recognized by the translating ribosome. Recognition leads to tryptophan binding within the translating ribosome, inhibiting the termination of
tnaC
translation and preventing Rho-dependent transcription termination in the
tna
operon leader region. It was previously shown that inserting an adenine residue at position 751 or introducing the U2609C change in 23S rRNA or introducing the K90W replacement in ribosomal protein L22 prevented tryptophan induction of
tna
operon expression. It was also observed that an adenine at position 752 of 23S rRNA was required for induction. In the current study, the explanation for the lack of induction by these altered ribosomes was investigated. Using isolated TnaC-ribosome complexes, it was shown that although tryptophan inhibits puromycin cleavage of TnaC-tRNA
Pro
with wild-type ribosome complexes, it does not inhibit cleavage with the four mutant ribosome complexes examined. Similarly, tryptophan prevents sparsomycin inhibition of TnaC-tRNA
Pro
cleavage with wild-type ribosome complexes but not with these mutant ribosome complexes. Additionally, a nucleotide located close to the peptidyl transferase center, A2572, which was protected from methylation by tryptophan with wild-type ribosome complexes, was not protected with mutant ribosome complexes. These findings identify specific ribosomal residues located in the ribosome exit tunnel that recognize features of the TnaC peptide. This recognition creates a free tryptophan-binding site in the peptidyl transferase center, where bound tryptophan inhibits peptidyl transferase activity.
Physiological Effects of Crl in
Salmonella
Are Modulated by σ
S
Level and Promoter Specificity
Authors: Robbe-Saule Véronique , Lopes Miguel Dias , Kolb Annie , Norel Françoise
Published: February 2007

The small regulatory protein Crl activates σ
S
(RpoS), the stationary-phase and general stress response sigma factor. Crl has been reported to bind σ
S
in vitro and to facilitate the formation of RNA polymerase holoenzyme. In
Salmonella enterica
serovar Typhimurium, Crl is required for the development of the rdar morphotype and transcription initiation of the σ
S
-dependent genes
csgD
and
adrA
, involved in curli and cellulose production. Here, we examined the expression of other σ
S
-dependent phenotypes and genes in a Δ
crl
mutant of
Salmonella
. Gene fusion analyses and in vitro transcription assays indicate that the magnitude of Crl activation differs between promoters and is highly dependent on σ
S
levels. We replaced the wild-type
rpoS
allele in
S. enterica
serovar Typhimurium strain ATCC 14028 with the
rpoS
LT2
allele that shows reduced expression of σ
S
; the result was an increased Crl activation ratio and larger physiological effects of Crl on oxidative, thermal, and acid stress resistance levels during stationary phase. We also found that
crl
,
rpoS
, and
crl rpoS
strains grew better on succinate than did the wild type and expressed the succinate dehydrogenase
sdhCDBA
operon more strongly. The
crl
and
rpoS
LT2
mutations also increased the competitive fitness of
Salmonella
in stationary phase. These results show that Crl contributes to negative regulation by σ
S
, a finding consistent with a role for Crl in sigma factor competition via the facilitation of σ
S
binding to core RNA polymerase.
In Vitro Analysis of the Two-Component System MtrB-MtrA from
Corynebacterium glutamicum
Authors: Möker Nina , Krämer Jens , Unden Gottfried , Krämer Reinhard , Morbach Susanne
Published: February 2007

The two-component system MtrBA is involved in the osmostress response of
Corynebacterium glutamicum
. MtrB was reconstituted in a functionally active form in liposomes and showed autophosphorylation and phosphatase activity. In proteoliposomes, MtrB activity was stimulated by monovalent cations used by many osmosensors for the detection of hypertonicity. Although MtrB was activated by monovalent cations, they lead in vitro to a general stabilization of histidine kinases and do not represent the stimulus for MtrB to sense hyperosmotic stress.
Psychological determinants of risk taking by children: an integrative model and implications for interventions: Figure 1
Authors: Morrongiello Barbara A , Lasenby-Lessard Jennifer
Published: February 2007

Objectives:To draw on empirical findings of the psychological factors that cause elementary-school children to engage in risky play behaviors that can lead to injury, with the aim of developing an integrative model that can support intervention-program planning.

Methods:An extensive review of literature on this topic was conducted, determinants of risk taking for which there was empirical support were identified, and results were synthesized to create an integrative model of children’s risk taking.

Results:Research on risk taking in children is limited, but the findings support the importance of examining child, family and socio-environmental factors to understand children’s risk-taking behaviors.

Conclusions:Development of a model outlining the determinants of risk behaviors can provide a foundation for initiatives that aim to reduce such behaviors and prevent childhood injuries.
Geomatics in injury prevention: the science, the potential and the limitations: Figure 1
Authors: Cusimano M D , Chipman M , Glazier R H , Rinner C , Marshall S P
Published: February 2007

Background:Geomatics describes the activities involved in acquiring and managing geographical data and producing geographical information for scientific, administrative and technical endeavors. As an emerging science, geomatics has a great potential to support public health. Geomatics provides a conceptual foundation for the development of geographic information systems (GIS), computerized tools that manage and display geographical data for analytical applications. As descriptive epidemiology typically involves the examination of person, place and time in the occurrence of disease or injury, geomatics and GIS can play an important role in understanding and preventing injury.

Aim:This article provides a background to geomatics for those in the injury prevention field who are unfamiliar with spatial analysis. We hope to stimulate researchers and practitioners to begin to use geomatics to assist in the prevention of injury.

Methods:The authors illustrate the potential benefits and limitations of geomatics in injury prevention in a non-technical way through the use of maps and analysis.

Results:By analysing the location of patients treated for fall injuries in Central Toronto using GIS, some demographic and land use variables, such as household income, age, and the location of homeless shelters, were identified as explanatory factors for the spatial distribution.

Conclusion:By supporting novel approaches to injury prevention, geomatics has a great potential for efforts to combat the burden of injury. Despite some limitations, those with an interest in injury prevention could benefit from this science.
The economic cost of road traffic crashes in an urban setting
Authors: García-Altés A , Pérez K
Published: February 2007

The objective of this article is to assess the total economic costs of road traffic crashes in Barcelona, a metropolitan city located in Southern Europe. A cost-of-illness study was conducted using a prevalence approximation, a societal and healthcare system perspective, and a 1-year time horizon. Results were measured in terms of Euros in 2003. Total costs of road traffic crashes in Barcelona in 2003 were €367 million. Direct costs equalled €329 million (89.8% of total costs), including property damage costs, insurance administration costs and hospital costs. Police, emergency costs and transportation costs had a minimum effect on total direct costs. Indirect costs were €37 million, including lost productivity due to hospitalization and mortality. The results of the sensitivity analysis showed the upper limit of total economic cost of road traffic crashes in Barcelona to be €782 million. This is the first study to estimate the costs of road traffic crashes for a city in a developed country. The importance of the problem calls for further interventions to reduce road traffic crashes.
Attitudes associated with behavioral predictors of serious road traffic crashes: results from the GAZEL cohort
Authors: Nabi Hermann , Rachid Salmi Louis , Lafont Sylviane , Chiron Mireille , Zins Marie , Lagarde Emmanuel
Published: February 2007

Objectives:To test the hypothesis that behavioral predictors of serious road traffic crashes (RTC) are correlated with unfavorable attitudes towards traffic safety.

Design:Prospective and cross-sectional cohort study.

Setting:France

Participants:13 447 of the 19 894 living members of the GAZEL cohort, workers and recent retirees of a French national utility company followed up since 1989.

Main outcomes measures:Driving behavior and attitudes towards traffic safety in 2001 by questionnaire. Serious RTCs were recorded over the subsequent 3 years using the cohort annual questionnaire. Behavioral predictors of serious RTCs were assessed using generalized linear Poisson regression models with time-dependent covariates. Factor scores extracted from the first four attitudinal factors of principal component analysis were saved and then regressed on behavioral predictors as independent variables.

Results:After controlling for potential confounders, the best predictors of serious RTCs were: “exceeding speed limits on rural roads”, “risky use of cellular phone”, and “sleepy driving”. The adjusted rate ratio ranged from 1.47 to 2.16. Predictors of contravention of the highway code (the first two predictors) were found to be strongly associated with negative attitudes towards “enforcement” and “speed limitations” with an adjusted odds ratio ranging from 1.31 to 2.02.

Conclusion:Our study supports the view that individuals with a high propensity for driving behaviors associated with an increased risk of RTCs were more likely to have negative attitudes towards traffic safety. Changing drivers’ negative or distorted opinions of traffic “enforcement” as well as “speed limitations” and “alcohol prohibition on roads” could improve their compliance with road traffic rules.
Vancomycin administration: the impact of multidisciplinary interventions
Authors: Crowley R K , Fitzpatrick F , Solanki D , FitzGerald S , Humphreys H , Smyth E G
Published: February 2007

Background:

The clinical microbiology team observed that patients were not receiving all prescribed doses of vancomycin. Ward staff was confused about ordering and interpreting vancomycin therapeutic drug monitoring (TDM) levels.

Aim:

To audit the incidence of vancomycin dose omission. To implement a series of interventions to improve vancomycin dose administration, and to repeat the audit process to assess these interventions.

Methods:

Three prospective audits were conducted to assess the impact of vancomycin TDM on administration of vancomycin. After the first audit, a number of changes in the TDM process were undertaken. After review of the second audit, a senior pharmacist coordinated ward-based pharmacists in assisting staff to interpret levels, and TDM interpretative charts were designed for drug charts. Following the third audit, feedback to hospital management and a plan for ongoing education were undertaken.

Results:

There was a significant reduction in the number of vancomycin doses held inappropriately in the third (10% (78/782) of prescribed doses) when compared to the first audit (16% (161/1007) of doses) (p<0.01). Of doses that were held inappropriately, there was a significant decrease in doses held for no apparent reason in audit 3 (16% (27/170) of prescribed doses) when compared to audit 1 (25% (69/282) of doses) (p<0.05).

Conclusions:

The interventions resulted in a 37.5% reduction in inappropriately held vancomycin doses over a one-year period; 10% of doses are still being held inappropriately. This study highlights the difficulties in identifying barriers to change and changing healthcare worker behaviour.
Frequency of epithelioid granulomas in colonoscopic biopsy specimens from paediatric and adult patients with Crohn’s colitis
Authors: Rubio C A , Orrego A , Nesi G , Finkel Y
Published: February 2007

Aims:

To test the assumption that epithelioid granulomas found in colonoscopic biopsy specimens in patients with Crohn’s colitis are markers of a different clinical behaviour.

Methods:

Sections from colonoscopic biopsy specimens from 352 consecutive patients (119 children and 233 adults) were investigated.

Results:

A total of 1117 colonoscopies were performed: 293 in children (mean 2.46 per patient) and 824 in adults (mean 3.53 per patient) (p<0.05). Granulomas at initial colonoscopy were recorded in 67.2% (43/64) of children and 65.9% (27/41) of adults (p>0.6), and at subsequent colonoscopies in 53.8% (64/119) of children and 17.6% (41/233) of adults (p<0.05). Surgical intervention was required in 6.3% (4/64) of the children having previous granuloma, but also in 14.5% (8/55) of those without previous granuloma, the rate for operated adults being 26.8% (11/41) and 24.5% (47/192), respectively (p>0.6).

Conclusions:

Granulomas in entry and/or in subsequent colonoscopic biopsy specimens in patients with Crohn’s colitis did not predict the need for subsequent surgical intervention. The fact that the frequency of granulomas was significantly higher in children than in adults with Crohn’s colitis (despite a higher mean number of colonoscopic biopsies in adults), and that granulomas were present in colonoscopic biopsy specimens but not in the subsequent surgical specimens from 50% of the paediatric and 36% of the adult patients strengthen the conviction that granulomas in Crohn’s colitis might evolve or regress at different time intervals during the course of the disease. This behaviour would reflect a particular immunological reaction, an epiphenomenon from immature tissues—as in children—when challenged by the so far elusive aetiological agent responsible for Crohn’s disease.
Losartan prevents stretch-induced electrical remodeling in cultured atrial neonatal myocytes
Authors: Saygili Erol , Rana Obaida R. , Saygili Esra , Reuter Hannes , Frank Konrad , Schwinger Robert H. G. , Müller-Ehmsen Jochen , Zobel Carsten
Published: February 2007

Atrial fibrillation (AF) is the most frequent arrhythmia found in clinical practice. In recent studies, a decrease in the development or recurrence of AF was found in hypertensive patients treated with angiotensin-converting enzyme inhibitors or angiotensin receptor-blocking agents. Hypertension is related to an increased wall tension in the atria, resulting in increased stretch of the individual myocyte, which is one of the major stimuli for the remodeling process. In the present study, we used a model of cultured atrial neonatal rat cardiomyocytes under conditions of stretch to provide insight into the mechanisms of the preventive effect of the angiotensin receptor-blocking agent losartan against AF on a molecular level. Stretch significantly increased protein-to-DNA ratio and atrial natriuretic factor mRNA expression, indicating hypertrophy. Expression of genes encoding for the inward rectifier K+current ( IK1), Kir2.1, and Kir2.3, as well as the gene encoding for the ultrarapid delayed rectifier K+current ( IKur), Kv1.5, was significantly increased. In contrast, mRNA expression of Kv4.2 was significantly reduced in stretched myocytes. Alterations of gene expression correlated with the corresponding current densities: IK1and IKurdensities were significantly increased in stretched myocytes, whereas transient outward K+current ( Ito) density was reduced. These alterations resulted in a significant abbreviation of the action potential duration. Losartan (1 μM) prevented stretch-induced increases in the protein-to-DNA ratio and atrial natriuretic peptide mRNA expression in stretched myocytes. Concomitantly, losartan attenuated stretch-induced alterations in IK1, IKur, and Itodensity and gene expression. This prevented the stretch-induced abbreviation of action potential duration. Prevention of stretch-induced electrical remodeling might contribute to the clinical effects of losartan against AF.
Endogenous interleukin-1α promotes a proliferative and proinflammatory phenotype in human vascular smooth muscle cells
Authors: Schultz Kelly , Murthy Vanishree , Tatro Jeffrey B. , Beasley Debbie
Published: February 2007

During vascular disease and following injury, vascular smooth muscle cells (VSMC) proliferate and produce inflammation-promoting cytokines and chemokines. Similar phenotypic changes can be elicited in vitro by activation of Toll-like receptors (TLR) within VSMC. TLR-activated VSMC also produce IL-1α, but it is unknown whether endogenous IL-1α stimulates VSMC in an autocrine manner. Here we tested the hypothesis that endogenous IL-1α contributes to TLR-induced proliferation and chemokine release in human VSMC by using RNA interference to knock down IL-1α expression. Knockdown of IL-1α abolished TLR-induced proliferation and suppressed TLR4-induced release of monocyte chemoattractant protein-1 (MCP-1) by VSMC, indicating that endogenous IL-1α plays a crucial role in both responses. Serum, PDGF, FGF-2, and EGF each increased cellular IL-1α concentrations, and IL-1α knockdown inhibited serum- and PDGF-induced DNA synthesis, further indicating that endogenous IL-1α also contributed to VSMC responses to growth factors. IL-1 receptor antagonist, a competitive inhibitor of IL-1 receptor I (IL-1RI), also attenuated TLR-induced proliferation and both basal and TLR-induced MCP-1 expression, indicating at least a partial role of the IL-1RI in mediating these responses. The results support the hypothesis that autocrine actions of endogenous IL-1α, mediated at least in part via IL-1RI signaling, contribute to a proproliferative and proinflammatory phenotypic shift in TLR-activated human VSMC, which might play a pathogenic role in vascular disorders.
Antithrombotic activity of kininogen is mediated by inhibitory effects of domain 3 during arterial injury in vivo
Authors: Hassan Sarmina , Sainz Irma M. , Khan Mohammad M. , Bradford Harlan N. , Isordia-Salas Irma , Kashem Sakeen W. , Sartor R. Balfour , Colman Robert W.
Published: February 2007

High-molecular-weight kininogen (HK) and its domain 3 (D3) exhibit anticoagulant properties and inhibit platelet activation at low thrombin concentration in vitro. We hypothesized that the rapid occlusive thrombosis in HK-deficient (HKd) rats following endothelial injury of the aorta results from enhanced platelet aggregation by thrombin. The effects of D3 (G235-M357) or D3-derived peptides on thrombosis in vivo were tested. D3 and its exon 7C terminal peptide (E7CP, K270-Q292), expressed as glutathione S-transferase (GST) fusion proteins (GST-D3, GST-E7CP), or GST alone, as well as cleaved HK (HKa) or synthetic peptide E7CP, were infused intravenously 10 min before endothelial injury. Blood flow was reduced down to 10% of baseline flow within 28 ± 5.2 min by a platelet-fibrin thrombus in GST-treated HKd rats compared with >240 min in GST-treated normal HK rats (wild type). GST-D3, GST-E7CP, HKa, or E7CP infusion prolonged the flow time to 233, >240, 223, and >240 min, respectively, in HKd rats. When GST-E7CP was infused 10 min after the injury, blood flow was maintained for >240 min. Thrombin-antithrombin concentrations were elevated by injury in HKd rats receiving GST from 35 to 55 μg/l and decreased with GST-E7CP, HKa, or E7CP reconstitution to 40, 15, and 9 μg/l, respectively. We conclude that HKd rats are prothrombotic and that HKa, kininogen D3, and its fragment E7CP modulate arterial thrombosis after endothelial injury.
Cardioprotective effects of novel tetrahydroisoquinoline analogs of nitrobenzylmercaptopurine riboside in an isolated perfused rat heart model of acute myocardial infarction
Authors: Zhu Z. , Hofmann P. A. , Buolamwini J. K.
Published: February 2007

We have investigated the cardioprotective effects of novel tetrahydroisoquinoline nitrobenzylmercaptopurine riboside (NBMPR) analog nucleoside transport (NT) inhibitors, compounds 2 and 4, in isolated perfused rat hearts. Langendorff-perfused heart preparations were subjected to 10 min of treatment with compound 2, compound 4, or vehicle (control) followed by 30 min of global ischemia and 120 min of reperfusion. For determination of infarct size, reperfusion time was 180 min. At 1 μM, compounds 2 and 4 provided excellent cardioprotection, with left ventricular developed pressure (LVDP) recovery and end-diastolic pressure (EDP) increase of 82.9 ± 4.0% ( P < 0.001) and 14.1 ± 2.0 mmHg ( P < 0.03) for compound 2-treated hearts and 79.2 ± 5.9% ( P < 0.002) and 7.5 ± 2.7 mmHg ( P < 0.01) for compound 4-treated hearts compared with 41.6 ± 5.2% and 42.5 ± 6.5 mmHg for control hearts. LVDP recovery and EDP increase were 64.1 ± 4.2% and 29.1 ± 2.5 mmHg for hearts treated with 1 μM NBMPR. Compound 4 was the best cardioprotective agent, affording significant cardioprotection, even at 0.1 μM, with LVDP recovery and EDP increase of 76.0 ± 4.9% ( P < 0.003) and 14.1 ± 1.0 mmHg ( P < 0.03). At 1 μM, compound 4 and NBMPR reduced infarct size, with infarct area-to-total risk area ratios of 29.13 ± 3.17 ( P < 0.001) for compound 4 and 37.5 ± 3.42 ( P < 0.01) for NBMPR vs. 51.08 ± 5.06% for control hearts. Infarct size was more effectively reduced by compound 4 than by NBMPR ( P < 0.02). These new tetrahydroisoquinoline NBMPR analogs are not only potent cardioprotective agents but are, also, more effective than NBMPR in this model.
Reduced baroreflex sensitivity in alcoholic cirrhosis: relations to hemodynamics and humoral systems
Authors: Møller Søren , Iversen Jens S. , Henriksen Jens H. , Bendtsen Flemming
Published: February 2007

In cirrhosis, arterial vasodilatation leads to central hypovolemia and activation of the sympathetic nervous and renin-angiotensin-aldosterone systems. As the liver disease and circulatory dysfunction may affect baroreflex sensitivity (BRS), we assessed BRS in a large group of patients with cirrhosis and in controls who were all supine and some after 60° passive head-up and 30° head-down tilting in relation to central hemodynamics and activity of the sympathetic nervous and renin-angiotensin-aldosterone systems. One-hundred and five patients (Child classes A/B/C: 21/55/29) and 25 ( n = 11 + 14) controls underwent a full hemodynamic investigation. BRS was assessed by cross-spectral analysis of variabilities between blood pressure and heart rate time series. The median BRS was significantly lower in the supine cirrhotic patients, 3.7 (range 0.3–30.7) ms/mmHg than in matched controls ( n = 11): 14.3 (6.1–23.6) ms/mmHg, P < 0.001. A stepwise multiple-regression analysis revealed that serum sodium ( P = 0.044), heart rate ( P = 0.027), and central circulation time ( P = 0.034) independently correlated with BRS. Head-down tilting had no effects on BRS, but, after head-up tilting, BRS was similar in the patients ( n = 23) and controls ( n = 14). In conclusion, BRS is reduced in cirrhosis in the supine position and relates to various aspects of cardiovascular dysfunction, but no further reduction was observed in parallel with the amelioration of the hyperdynamic circulation after head-up tilting. The results indicate that liver dysfunction and compensatory mechanisms to vasodilatation may be involved in the low BRS, which may contribute to poor cardiovascular adaptation in cirrhosis.
iNOS regulation by calcium/calmodulin-dependent protein kinase II in vascular smooth muscle
Authors: Jones Rachel J. , Jourd'heuil David , Salerno John C. , Smith Susan M. E. , Singer Harold A.
Published: February 2007

Nitric oxide synthase (NOS) expression is regulated transcriptionally in response to cytokine induction and posttranslationally by palmitoylation and trafficking into perinuclear aggresome-like structures. We investigated the effects of multifunctional calcium/calmodulin-dependent protein kinase II protein kinase (CaMKII) on inducible NOS (iNOS) trafficking in cultured rat aortic vascular smooth muscle cells (VSMCs). Immunofluorescence and confocal microscopy demonstrated colocalization of iNOS and CaMKIIδ2with a perinuclear distribution and concentration in aggresome-like structures identified by colocalization with γ-tubulin. Furthermore, CaMKIIδ2coimmunoprecipitated with iNOS in a CaMKII activity-dependent manner. Addition of Ca2+-mobilizing stimuli expected to activate CaMKII; a purinergic agonist (UTP) or calcium ionophore (ionomycin) caused a general redistribution of iNOS from cytosolic to membrane and nuclear fractions. Similarly, adenoviral expression of a constitutively active CaMKIIδ2mutant altered iNOS localization, shifting iNOS from the cytosolic fraction. Suppression of CaMKIIδ2using an adenovirus expressing a short hairpin, small interfering RNA increased nuclear iNOS localization in resting cells but inhibited ionomycin-induced translocation of iNOS to the nucleus. Following addition of these chronic and acute CaMKII modulators, there were fewer aggresome-like structures containing iNOS. All of the treatments that chronically affected CaMKII activity or expression significantly inhibited iNOS-specific activity following cytokine induction. The results suggest that CaMKIIδ2may be an important regulator of iNOS trafficking and activity in VSMCs.
Endothelium-derived nitric oxide mediates the antiadrenergic effect of human vasostatin-1 in rat ventricular myocardium
Authors: Gallo Maria Pia , Levi Renzo , Ramella Roberta , Brero Alessia , Boero Ombretta , Tota Bruno , Alloatti Giuseppe
Published: February 2007

Vasostatins (VSs) are vasoactive peptides derived from chromogranin A (CgA), a protein contained in secretory granules of chromaffin and other cells. The negative inotropic effect and the reduction of isoproterenol (Iso)-dependent inotropism induced by VSs in the heart suggest that they have an antiadrenergic function. However, further investigation of the mechanisms of action of VSs is needed. The aim of the present study was to define the signaling pathways activated by VS-1 in mammalian ventricular myocardium and cultured endothelial cells that lead to the modulation of cardiac contractility. Ca2+ and nitric oxide (NO) fluorometric confocal imaging was used to study the effects induced by recombinant human VS-1 [STA-CgA-(1-76)] on contractile force, L-type Ca2+ current, and Ca2+ transients under basal conditions and after β-adrenergic stimulation in rat papillary muscles and ventricular cells and the effects on intracellular Ca2+ concentration and NO production in cultured bovine aortic endothelial (BAE-1) cells. VS-1 had no effect on basal contractility of papillary muscle, but the effect of Iso stimulation was reduced by 27%. Removal of endocardial endothelium and inhibition of NO synthesis and phosphatidylinositol 3-kinase (PI3K) activity abolished the antiadrenergic effect of VS-1 on papillary muscle. In cardiomyocytes, 10 nM VS-1 was ineffective on basal and Iso (1 μM)-stimulated L-type Ca2+ current and Ca2+ transients. In BAE-1 cells, VS-1 induced a Ca2+-independent increase in NO production that was blocked by the PI3K inhibitor wortmannin. Our results suggest that the antiadrenergic effect of VS-1 is mainly due to a PI3K-dependent NO release by endothelial cells, rather than a direct action on cardiomyocytes.
Protein kinase C regulates vascular myogenic tone through activation of TRPM4
Authors: Earley Scott , Straub Stephen V. , Brayden Joseph E.
Published: February 2007

Myogenic vasoconstriction results from pressure-induced vascular smooth muscle cell depolarization and Ca2+influx via voltage-dependent Ca2+channels, a process that is significantly attenuated by inhibition of protein kinase C (PKC). It was recently reported that the melastatin transient receptor potential (TRP) channel TRPM4 is a critical mediator of pressure-induced smooth muscle depolarization and constriction in cerebral arteries. Interestingly, PKC activity enhances the activation of cloned TRPM4 channels expressed in cultured cells by increasing sensitivity of the channel to intracellular Ca2+. Thus we postulated that PKC-dependent activation of TRPM4 might be a critical mediator of vascular myogenic tone. We report here that PKC inhibition attenuated pressure-induced constriction of cerebral vessels and that stimulation of PKC activity with phorbol 12-myristate 13-acetate (PMA) enhanced the development of myogenic tone. In freshly isolated cerebral artery myocytes, we identified a Ca2+-dependent, rapidly inactivating, outwardly rectifying, iberiotoxin-insensitive cation current with properties similar to those of expressed TRPM4 channels. Stimulation of PKC activity with PMA increased the intracellular Ca2+sensitivity of this current in vascular smooth muscle cells. To validate TRPM4 as a target of PKC regulation, antisense technology was used to suppress TRPM4 expression in isolated cerebral arteries. Under these conditions, the magnitude of TRPM4-like currents was diminished in cells from arteries treated with antisense oligonucleotides compared with controls, identifying TRPM4 as the molecular entity responsible for the PKC-activated current. Furthermore, the extent of PKC-induced smooth muscle cell depolarization and vasoconstriction was significantly decreased in arteries treated with TRPM4 antisense oligonucleotides compared with controls. We conclude that PKC-dependent regulation of TRPM4 activity contributes to the control of cerebral artery myogenic tone.
Role of p38 mitogen-activated protein kinase pathway in estrogen-mediated cardioprotection following trauma-hemorrhage
Authors: Hsu Jun-Te , Hsieh Ya-Ching , Kan Wen Hong , Chen Jian Guo , Choudhry Mashkoor A. , Schwacha Martin G. , Bland Kirby I. , Chaudry Irshad H.
Published: February 2007

p38 mitogen-activated protein kinase (MAPK) activates a number of heat shock proteins (HSPs), including HSP27 and αB-crystallin, in response to stress. Activation of HSP27 or αB-crystallin is known to protect organs/cells by increasing the stability of actin microfilaments. Although our previous studies showed that 17β-estradiol (E2) improves cardiovascular function after trauma-hemorrhage, whether the salutary effects of E2under those conditions are mediated via p38 MAPK remains unknown. Male rats (275–325 g body wt) were subjected to soft tissue trauma and hemorrhage (35–40 mmHg mean blood pressure for ∼90 min) followed by fluid resuscitation. At the onset of resuscitation, rats were injected intravenously with vehicle, E2(1 mg/kg body wt), E2+ the p38 MAPK inhibitor SB-203580 (2 mg/kg body wt), or SB-203580 alone, and various parameters were measured 2 h thereafter. Cardiac functions that were depressed after trauma-hemorrhage were returned to normal levels by E2administration, and phosphorylation of cardiac p38 MAPK, HSP27, and αB-crystallin was increased. The E2-mediated improvement of cardiac function and increase in p38 MAPK, HSP27, and αB-crystallin phosphorylation were abolished with coadministration of SB-203580. These results suggest that the salutary effect of E2on cardiac function after trauma-hemorrhage is in part mediated via upregulation of p38 MAPK and subsequent phosphorylation of HSP27 and αB-crystallin.
Sphingosine 1-phosphate S1P2 and S1P3 receptor-mediated Akt activation protects against in vivo myocardial ischemia-reperfusion injury
Authors: Means Christopher K. , Xiao Chun-Yang , Li Zhuangjie , Zhang Tong , Omens Jeffrey H. , Ishii Isao , Chun Jerold , Brown Joan H.
Published: February 2007

Sphingosine 1-phosphate (S1P) is released at sites of tissue injury and effects cellular responses through activation of G protein-coupled receptors. The role of S1P in regulating cardiomyocyte survival following in vivo myocardial ischemia-reperfusion (I/R) injury was examined by using mice in which specific S1P receptor subtypes were deleted. Mice lacking either S1P2 or S1P3 receptors and subjected to 1-h coronary occlusion followed by 2 h of reperfusion developed infarcts equivalent to those of wild-type (WT) mice. However, in S1P2,3 receptor double-knockout mice, infarct size following I/R was increased by >50%. I/R leads to activation of ERK, JNK, and p38 MAP kinases; however, these responses were not diminished in S1P2,3 receptor knockout compared with WT mice. In contrast, activation of Akt in response to I/R was markedly attenuated in S1P2,3 receptor knockout mouse hearts. Neither S1P2 nor S1P3 receptor deletion alone impaired I/R-induced Akt activation, which suggests redundant signaling through these receptors and is consistent with the finding that deletion of either receptor alone did not increase I/R injury. The involvement of cardiomyocytes in S1P2 and S1P3 receptor mediated activation of Akt was tested by using cells from WT and S1P receptor knockout hearts. Akt was activated by S1P, and this was modestly diminished in cardiomyocytes from S1P2 or S1P3 receptor knockout mice and completely abolished in the S1P2,3 receptor double-knockout myocytes. Our data demonstrate that activation of S1P2 and S1P3 receptors plays a significant role in protecting cardiomyocytes from I/R damage in vivo and implicate the release of S1P and receptor-mediated Akt activation in this process.
Nonhematopoietic NADPH oxidase regulation of lung eosinophilia and airway hyperresponsiveness in experimentally induced asthma
Authors: Abdala-Valencia Hiam , Earwood Julie , Bansal Shelly , Jansen Michael , Babcock George , Garvy Beth , Wills-Karp Marsha , Cook-Mills Joan M.
Published: February 2007

Pulmonary eosinophilia is one of the most consistent hallmarks of asthma. Infiltration of eosinophils into the lung in experimental asthma is dependent on the adhesion molecule vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells. Ligation of VCAM-1 activates endothelial cell NADPH oxidase, which is required for VCAM-1-dependent leukocyte migration in vitro. To examine whether endothelial-derived NADPH oxidase modulates eosinophil recruitment in vivo, mice deficient in NADPH oxidase (CYBB mice) were irradiated and received wild-type hematopoietic cells to generate chimeric CYBB mice. In response to ovalbumin (OVA) challenge, the chimeric CYBB mice had increased numbers of eosinophils bound to the endothelium as well as reduced eosinophilia in the lung tissue and bronchoalveolar lavage. This occurred independent of changes in VCAM-1 expression, cytokine/chemokine levels (IL-5, IL-10, IL-13, IFNγ, or eotaxin), or numbers of T cells, neutrophils, or mononuclear cells in the lavage fluids or lung tissue of OVA-challenged mice. Importantly, the OVA-challenged chimeric CYBB mice had reduced airway hyperresponsiveness (AHR). The AHR in OVA-challenged chimeric CYBB mice was restored by bypassing the endothelium with intratracheal administration of eosinophils. These data suggest that VCAM-1 induction of NADPH oxidase in the endothelium is necessary for the eosinophil recruitment during allergic inflammation. Moreover, these studies provide a basis for targeting VCAM-1-dependent signaling pathways in asthma therapies.
Enhanced airway reactivity and inflammation in A2Aadenosine receptor-deficient allergic mice
Authors: Nadeem Ahmed , Fan Ming , Ansari Habib R. , Ledent Catherine , Jamal Mustafa S.
Published: February 2007

A2Aadenosine receptor (A2AAR) has potent anti-inflammatory properties, which may be important in the regulation of airway reactivity and inflammation. Inflammatory cells that possess A2AAR also produce nitrosative stress, which is associated with pathophysiology of asthma, so we hypothesized that A2AAR deficiency may lead to increased airway reactivity and inflammation through nitrosative stress. Thus the present study was carried out to investigate the role of A2AAR on airway reactivity, inflammation, NF-κB signaling, and nitrosative stress in A2AAR knockout (KO) and wild-type (WT) mice using our murine model of asthma. Animals were sensitized intraperitoneally on days 1 and 6 with 200 μg of ragweed, followed by aerosolized challenges with 0.5% ragweed on days 11, 12, and 13, twice a day. On day 14, airway reactivity to methacholine was assessed as enhanced pause (Penh) using whole body plethysmography followed by bronchoalveolar lavage (BAL) and lung collection for various analyses. Allergen challenge caused a significant decrease in expression of A2AAR in A2AWT sensitized mice, with A2AAR expression being undetected in A2AKO sensitized group leading to decreased lung cAMP levels in both groups. A2AAR deletion/downregulation led to an increase in Penh to methacholine and influx of total cells, eosinophils, lymphocytes, and neutrophils in BAL with highest values in A2AKO sensitized group. A2AKO sensitized group further had increased NF-κB expression and nitrosative stress compared with WT sensitized group. These data suggest that A2AAR deficiency leads to airway inflammation and airway hyperresponsiveness, possibly via involvement of nitrosative stress in this model of asthma.
Muc5b and Muc5ac are the major oligomeric mucins in equine airway mucus
Authors: Rousseau Karine , Kirkham Sara , McKane Shaun , Newton Richard , Clegg Peter , Thornton David J.
Published: February 2007

Horses frequently suffer from respiratory diseases, which, irrespective of etiology, are often associated with airway mucus accumulation. Studies on human airways have shown that the key structural components of the mucus layer are oligomeric mucins, which can undergo changes of expression and properties in disease. However, there is little information on these gel-forming glycoproteins in horse airways mucus. Therefore, the aims of this study were to isolate equine airways oligomeric mucins, characterize their macromolecular properties, and identify their gene products. To this end, pooled tracheal washes, collected from healthy horses and horses suffering from respiratory diseases, were solubilized with 6 M guanidinium chloride (GdmCl). The oligomeric mucins were purified by density gradient centrifugation followed by size exclusion chromatography. Biochemical and biophysical analyses showed the mucins were stiffened random coils in solution that were polydisperse in size ( Mr= 6–20 MDa, average Mr= 14 MDa) and comprised of disulfide-linked subunits (average Mr= 7 MDa). Agarose gel electrophoresis showed that the pooled mucus sample contained at least two populations of oligomeric mucins. Electrospray ionization tandem mass spectrometry of tryptic digests of the unfractionated mucin preparation showed that the oligomeric mucins Muc5b and Muc5ac were present. In summary, we have shown that equine airways mucus is a mixture of Muc5b and Muc5ac mucins that have a similar macromolecular organization to their human counterparts. This study will form the basis for future studies to analyze the contribution of these two mucins to equine airways pathology associated with mucus accumulation.