bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of SufI (FtsP) in Cell Division of
Escherichia coli
: Evidence for Its Involvement in Stabilizing the Assembly of the Divisome
Authors: Samaluru Harish , SaiSree L. , Reddy Manjula
Published: September 2007

The function of SufI, a well-studied substrate of the TatABC translocase in
Escherichia coli
, is not known. It was earlier implicated in cell division, based on the finding that multiple copies of
sufI
suppressed the phenotypes of cells with mutations in
ftsI
(
ftsI23
), which encodes a divisomal transpeptidase. Recently,
sufI
was identified as both a multicopy suppressor gene and a synthetic lethal mutant of
ftsEX
, which codes for a division-specific putative ABC transporter. In this study, we show that
sufI
is essential for the viability of
E. coli
cells subjected to various forms of stress, including oxidative stress and DNA damage. The
sufI
mutant also exhibits
sulA
-independent filamentation, indicating a role in cell division. The phenotypes of the
sufI
mutant are suppressed by factors that stabilize FtsZ ring assembly, such as increased expression of cell division proteins FtsQAZ or FtsN or the presence of the gain-of-function
ftsA
* (FtsA R286W) mutation, suggesting that SufI is a divisomal protein required during stress conditions. In support of this, multicopy
sufI
suppressed the divisional defects of mutants carrying the
ftsA12
,
ftsQ1
, or
ftsK44
allele but not those of mutants carrying
ftsZ84
. Most of the division-defective mutants, in particular those carrying a Δ
ftsEX
or
ftsI23
allele, exhibited sensitivity to oxidative stress or DNA damage, and this sensitivity was also abolished by multiple copies of SufI. All of these data suggest that SufI is a division component involved in protecting or stabilizing the divisomal assembly under conditions of stress. Since
sufI
fulfils the requirements to be designated an
fts
gene, we propose that it be renamed
ftsP
.
Characterization of Phenylpyruvate Decarboxylase, Involved in Auxin Production of
Azospirillum brasilense
Authors: Spaepen Stijn , Versées Wim , Gocke Dörte , Pohl Martina , Steyaert Jan , Vanderleyden Jos
Published: September 2007

Azospirillum brasilense
belongs to the plant growth-promoting rhizobacteria with direct growth promotion through the production of the phytohormone indole-3-acetic acid (IAA). A key gene in the production of IAA, annotated as indole-3-pyruvate decarboxylase (
ipdC
), has been isolated from
A. brasilense
, and its regulation was reported previously (A. Vande Broek, P. Gysegom, O. Ona, N. Hendrickx, E. Prinsen, J. Van Impe, and J. Vanderleyden, Mol. Plant-Microbe Interact. 18:311-323, 2005). An
ipdC
-knockout mutant was found to produce only 10% (wt/vol) of the wild-type IAA production level. In this study, the encoded enzyme is characterized via a biochemical and phylogenetic analysis. Therefore, the recombinant enzyme was expressed and purified via heterologous overexpression in
Escherichia coli
and subsequent affinity chromatography. The molecular mass of the holoenzyme was determined by size-exclusion chromatography, suggesting a tetrameric structure, which is typical for 2-keto acid decarboxylases. The enzyme shows the highest
k
cat
value for phenylpyruvate. Comparing values for the specificity constant
k
cat
/

K
m

, indole-3-pyruvate is converted 10-fold less efficiently, while no activity could be detected with benzoylformate. The enzyme shows pronounced substrate activation with indole-3-pyruvate and some other aromatic substrates, while for phenylpyruvate it appears to obey classical Michaelis-Menten kinetics. Based on these data, we propose a reclassification of the
ipdC
gene product of
A. brasilense
as a phenylpyruvate decarboxylase (EC 4.1.1.43).
Genetic Relatedness of theStreptococcus pneumoniaeCapsular Biosynthetic Loci
Authors: Mavroidi Angeliki , Aanensen David M. , Godoy Daniel , Skovsted Ian C. , Kaltoft Margit S. , Reeves Peter R. , Bentley Stephen D. , Spratt Brian G.
Published: September 2007

Streptococcus pneumoniae(the pneumococcus) produces 1 of 91 capsular polysaccharides (CPS) that define the serotype. Thecpsloci of 88 pneumococcal serotypes whose CPS is synthesized by the Wzy-dependent pathway were compared with each other and with additional streptococcal polysaccharide biosynthetic loci and were clustered according to the proportion of shared homology groups (HGs), weighted for the sequence similarities between the genes encoding the shared HGs. Thecpsloci of the 88 pneumococcal serotypes were distributed into eight major clusters and 21 subclusters. All serotypes within the same serogroup fell into the same major cluster, but in six cases, serotypes within the same serogroup were in different subclusters and, conversely, nine subclusters included completely different serotypes. The closely relatedcpsloci within a subcluster were compared to the known CPS structures to relate gene content to structure. TheStreptococcus oralisandStreptococcus mitispolysaccharide biosynthetic loci clustered within the pneumococcalcpsloci and were in a subcluster that also included thecpslocus of pneumococcal serotype 21, whereas theStreptococcus agalactiae cpsloci formed a single cluster that was not closely related to any of the pneumococcalcpsclusters.
Predicted Functions and Linkage Specificities of the Products of theStreptococcus pneumoniaeCapsular Biosynthetic Loci
Authors: Aanensen David M. , Mavroidi Angeliki , Bentley Stephen D. , Reeves Peter R. , Spratt Brian G.
Published: September 2007

The sequences of the capsular biosynthetic (cps) loci of 90 serotypes ofStreptococcus pneumoniaehave recently been determined. Bioinformatic procedures were used to predict the general functions of 1,973 of the 1,999 gene products and to identify proteins within the same homology group, Pfam family, and CAZy glycosyltransferase family. Correlatingcpsgene content with the 54 known capsular polysaccharide (CPS) structures provided tentative assignments of the specific functions of the different homology groups of each functional class (regulatory proteins, enzymes for synthesis of CPS constituents, polymerases, flippases, initial sugar transferases, glycosyltransferases [GTs], phosphotransferases, acetyltransferases, and pyruvyltransferases). Assignment of the glycosidic linkages catalyzed by the 342 GTs (92 homology groups) is problematic, but tentative assignments could be made by using this large set ofcpsloci and CPS structures to correlate the presence of particular GTs with specific glycosidic linkages, by correlating inverting or retaining linkages in CPS repeat units with the inverting or retaining mechanisms of the GTs predicted from their CAZy family membership, and by comparing the CPS structures of serotypes that have very similarcpsgene contents. These large-scale comparisons between structure and gene content assigned the linkages catalyzed by 72% of the GTs, and all linkages were assigned in 32 of the serotypes with known repeat unit structures. Clear examples where very similar initial sugar transferases or glycosyltransferases catalyze different linkages in different serotypes were also identified. These assignments should provide a stimulus for biochemical studies to evaluate the reactions that are proposed.
Identification of Novel Surface Proteins ofAnaplasma phagocytophilumby Affinity Purification and Proteomics
Authors: Ge Yan , Rikihisa Yasuko
Published: September 2007

Anaplasma phagocytophilumis the etiologic agent of human granulocytic anaplasmosis (HGA), one of the major tick-borne zoonoses in the United States. The surface ofA. phagocytophilumplays a crucial role in subverting the hostile host cell environment. However, except for the P44/Msp2 outer membrane protein family, the surface components ofA. phagocytophilumare largely unknown. To identify the major surface proteins ofA. phagocytophilum, a membrane-impermeable, cleavable biotin reagent, sulfosuccinimidyl-2-[biotinamido]ethyl-1,3-dithiopropionate (Sulfo-NHS-SS-Biotin), was used to label intact bacteria. The biotinylated bacterial surface proteins were isolated by streptavidin agarose affinity purification and then separated by electrophoresis, followed by capillary liquid chromatography-nanospray tandem mass spectrometry analysis. Among the major proteins captured by affinity purification were fiveA. phagocytophilumproteins, Omp85, hypothetical proteins APH_0404 (designated Asp62) and APH_0405 (designated Asp55), P44 family proteins, and Omp-1A. The surface exposure of Asp62 and Asp55 was verified by immunofluorescence microscopy. Recombinant Asp62 and Asp55 proteins were recognized by an HGA patient serum. Anti-Asp62 and anti-Asp55 peptide sera partially neutralizedA. phagocytophiluminfection of HL-60 cells in vitro. We found that the Asp62 and Asp55 genes were cotranscribed and conserved among members of the familyAnaplasmataceae. With the exception of P44-18, all of the proteins were newly revealed major surface-exposed proteins whose study should facilitate understanding the interaction betweenA. phagocytophilumand the host. These proteins may serve as targets for development of chemotherapy, diagnostics, and vaccines.
Analysis of Promoter Targets forEscherichia coliTranscription Elongation Factor GreA In Vivo and In Vitro
Authors: Stepanova Ekaterina , Lee Jookyung , Ozerova Maria , Semenova Ekaterina , Datsenko Kirill , Wanner Barry L. , Severinov Konstantin , Borukhov Sergei
Published: September 2007

Transcription elongation factor GreA induces nucleolytic activity of bacterial RNA polymerase (RNAP). In vitro, transcript cleavage by GreA contributes to transcription efficiency by (i) suppressing pauses and arrests, (ii) stimulating RNAP promoter escape, and (iii) enhancing transcription fidelity. However, it is unclear which of these functions is (are) most relevant in vivo. By comparing global gene expression profiles ofEscherichia colistrains lacking Gre factors and strains expressing either the wild type (wt) or a functionally inactive GreA mutant, we identified genes that are potential targets of GreA action. Data analysis revealed that in the presence of chromosomally expressed GreA, 19 genes are upregulated; an additional 105 genes are activated upon overexpression of the wt but not the mutant GreA. Primer extension reactions with selected transcription units confirmed the gene array data. The most prominent stimulatory effect (threefold to about sixfold) of GreA was observed for genes of ribosomal protein operons and thetnaoperon, suggesting that transcript cleavage by GreA contributes to optimal expression levels of these genes in vivo. In vitro transcription assays indicated that the stimulatory effect of GreA upon the transcription of these genes is mostly due to increased RNAP recycling due to facilitated promoter escape. We propose that transcript cleavage during early stages of initiation is thus the main in vivo function of GreA. Surprisingly, the presence of the wt GreA also led to the decreased transcription of many genes. The mechanism of this effect is unknown and may be indirect.
RelA-Dependent (p)ppGpp Production Controls Exoenzyme Synthesis inErwinia carotovorasubsp.atroseptica
Authors: Wang Jinhong , Gardiol Noemie , Burr Tom , Salmond George P. C. , Welch Martin
Published: September 2007

In this report, we investigate the link between nutrient limitation, RelA-mediated (p)ppGpp production, and virulence in the phytopathogenErwinia carotovorasubsp.atroseptica. ArelAnull mutant (JWC7) was constructed by allelic exchange, and we confirmed that, unlike the wild-type progenitor, this mutant did not produce elevated levels of (p)ppGpp upon nutrient downshift. However, (p)ppGpp production could be restored in strain JWC7 during nutrient limitation by supplyingrelAintrans. During growth on exoenzyme-inducing minimal medium, therelAmutant showed a diminution in secreted pectate lyase and protease activities and a severe defect in motility. TherelAmutant was also impaired in its ability to cause rot in potato tubers. In the presence of serine hydroxamate (a competitive inhibitor of seryl tRNA synthase and a potent inducer of the stringent response in wild-typeE. carotovorasubsp.atroseptica), exoenzyme production was essentially abolished in JWC7 but could be restored in the presence of plasmid-bornerelA. The inhibition of exoenzyme production in JWC7 caused by serine hydroxamate could not be overcome by addition of the quorum-sensing signal molecule,N-3-oxohexanoyl-l-homoserine lactone. Quantitative reverse transcription-PCR analysis of selected RNA species confirmed that the effects ofrelAon secreted pectate lyase activity and motility could be attributed to a reduction in transcription of the corresponding genes. We conclude that nutrient limitation is a potent environmental cue that triggers (p)ppGpp-dependent exoenzyme production inE. carotovorasubsp.atroseptica. Furthermore, our data suggest that nutrient limitation [or rather, (p)ppGpp accumulation] is a prerequisite for effective quorum-sensing-dependent activation of exoenzyme production.
Peptide Signals Encode Protein Localization
Authors: Russell Jay H. , Keiler Kenneth C.
Published: September 2007

Many bacterial proteins are localized to precise intracellular locations, but in most cases the mechanism for encoding localization information is not known. Screening libraries of peptides fused to green fluorescent protein identified sequences that directed the protein to helical structures or to midcell. These peptides indicate that protein localization can be encoded in 20-amino-acid peptides instead of complex protein-protein interactions and raise the possibility that the location of a protein within the cell could be predicted from bioinformatic data.
Proteins Associated with theMyxococcus xanthusExtracellular Matrix
Authors: Curtis Patrick D. , Atwood James , Orlando Ron , Shimkets Lawrence J.
Published: September 2007

Fruiting body formation ofMyxococcus xanthus, like biofilm formation of many other organisms, involves the production of an extracellular matrix (ECM). While the polysaccharide component has been studied, the protein component has been largely unexplored. Proteins associated with the ECM were solubilized from purified ECM by boiling with sodium dodecyl sulfate and were identified by liquid chromatography-tandem mass spectrometry of tryptic fragments. The ECM is enriched in proteins of novel function; putative functions were assigned for only 5 of the 21 proteins. Thirteen putative ECM proteins had lipoprotein secretion signals. The genes for many ECM proteins were disrupted in the wild-type (WT),fibA, andpilAbackgrounds. Disruption of the MXAN4860 gene had no effect in the WT orfibAbackground but in thepilAbackground resulted in a 24-h delay in aggregation and sporulation compared to its parent. The results of this study show that theM. xanthusECM proteome is diverse and novel.
Effect of a Small, Acid-Soluble Spore Protein from
Clostridium perfringens
on the Resistance Properties of
Bacillus subtilis
Spores
Authors: Leyva-Illades Juan Francisco , Setlow Barbara , Sarker Mahfuzur R. , Setlow Peter
Published: September 2007

α/β-type small, acid-soluble spore proteins (SASP) are essential for the resistance of DNA in spores of
Bacillus
species to damage. An α/β-type SASP, Ssp2, from
Clostridium perfringens
was expressed at significant levels in
B. subtilis
spores lacking one or both major α/β-type SASP (α
−
and α
−
β
−
strains, respectively). Ssp2 restored some of the resistance of α
−
β
−
spores to UV and nitrous acid and of α
−
spores to dry heat. Ssp2 also restored much of the resistance of α
−
spores to nitrous acid and restored full resistance of α
−
spores to UV and moist heat. These results further indicate the interchangeability of α/β-type SASP in DNA protection in spores.
Hexarelin suppresses cardiac fibroblast proliferation and collagen synthesis in rat
Authors: Xu Xiangbin , Pang Jinjiang , Yin Hongchao , Li Meixiu , Hao Wei , Chen Chen , Cao Ji-Min
Published: September 2007

Abnormal growth of cardiac fibroblasts is critically involved in the pathophysiology of cardiac hypertrophy/remodeling. Hexarelin is a synthetic growth hormone secretagogue (GHS), which possesses a variety of cardiovascular protective activities mediated via the GHS receptor (GHSR), including improving cardiac dysfunction and remodeling. The cellular and molecular mechanisms underlying the effect of GHS on cardiac fibrosis are, however, not clear. In this report, cultured cardiac fibroblasts from 8-day-old rats were stimulated with ANG II or FCS to induce proliferation. The fibroblast proliferation and DNA and collagen synthesis were evaluated utilizing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay,3H-thymidine incorporation, and3H-proline incorporation. The level of mRNA of transforming growth factor (TGF)-β was evaluated by RT-PCR, and the active TGF-β1 release from cardiac fibroblasts was evaluated by ELISA. The level of cellular cAMP was measured by radioimmunoassay. In addition, the effects of 3,7-dimethyl-l-propargylxanthine (DMPX; a specific adenosine receptor A2R antagonist) and 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; a specific A1R antagonist) were tested. It was found that incubation with 10−7mol/l hexarelin for 24 h 1) inhibited the ANG II-induced proliferation and collagen synthesis and the 5% FCS- and TGF-β-induced increase of DNA synthesis in cardiac fibroblast and 2) reduced ANG II-induced upregulation of TGF-β mRNA expression and active TGF-β1 release from fibroblasts. Hexarelin increased the cellular level of cAMP in cardiac fibroblasts. DMPX (10−8mol/l) but not DPCPX abolished the effect of hexarelin on cardiac fibroblast DNA synthesis. It is concluded that hexarelin inhibits DNA and collagen synthesis and proliferation of cardiac fibroblasts through activation of both GHSR and A2R and diminishment of ANG II-induced increase in TGF-β expression and release.
Effects of acute exercise, exercise training, and diabetes on the expression of lymphangiogenic growth factors and lymphatic vessels in skeletal muscle
Authors: Kivelä Riikka , Silvennoinen Mika , Lehti Maarit , Kainulainen Heikki , Vihko Veikko
Published: September 2007

Blood and lymphatic vessels together form the circulatory system, allowing the passage of fluids and molecules within the body. Recently we showed that lymphatic capillaries are also found in the capillary bed of skeletal muscle. Exercise is known to induce angiogenesis in skeletal muscle, but it is not known whether exercise has effects on lymphangiogenesis or lymphangiogenic growth factors. We studied lymphatic vessel density and expression of the main lymphangiogenic growth factors VEGF-C and VEGF-D and their receptor VEGFR-3 in response to acute running exercise and endurance exercise training in the skeletal muscle of healthy and diabetic mice. VEGF-C mRNA expression increased after the acute exercise bout ( P < 0.05) in healthy muscles, but there was no change in diabetic muscles. VEGF-C levels were not changed either in healthy or in diabetic muscle after the exercise training. Neither acute exercise nor exercise training had an effect on the mRNA expression of VEGF-D or VEGFR-3 in healthy or diabetic muscles. Lymphatic vessel density was similar in sedentary and trained mice and was >10-fold smaller than blood capillary density. Diabetes increased the mRNA expression of VEGF-D ( P < 0.01). Increased immunohistochemical staining of VEGF-D was found in degenerative muscle fibers in the diabetic mice. In conclusion, the results suggest that acute exercise or exercise training does not significantly affect lymphangiogenesis in skeletal muscle. Diabetes increased the expression of VEGF-D in skeletal muscle, and this increase may be related to muscle fiber damage.
Reactive oxygen species contribute to sleep apnea-induced hypertension in rats
Authors: Troncoso Brindeiro Carmen M. , da Silva Ana Q. , Allahdadi Kyan J. , Youngblood Victoria , Kanagy Nancy L.
Published: September 2007

In clinical studies, sleep apnea is associated with hypertension, oxidative stress, and increased circulating endothelin-1 (ET-1). We previously developed a model of sleep apnea by exposing rats to eucapnic intermittent hypoxia (IH-C) during sleep, which increases both blood pressure and plasma levels of ET-1. Because similar protocols in mice increase tissue and plasma markers of oxidative stress, we hypothesized that IH-C generation of reactive oxygen species (ROS) contributes to the development of ET-1-dependent hypertension in IH-C rats. To test this, male Sprague-Dawley rats were instrumented with indwelling blood pressure telemeters and drank either plain water or water containing the superoxide dismutase mimetic, Tempol (4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl, 1 mM). Mean arterial pressure (MAP) and heart rate (HR) were recorded for 3 control days and 14 treatment days with rats exposed 7 h/day to IH-C or air/air cycling (Sham). On day 14, MAP in IH-C rats treated with Tempol (107 ± 2.29 mmHg) was significantly lower than in untreated IH-C rats (118 ± 9 mmHg, P < 0.05). Tempol did not affect blood pressure in sham-operated rats (Tempol = 101 ± 3, water = 101 ± 2 mmHg). Immunoreactive ET-1 was greater in plasma from IH-C rats compared with plasma from sham-operated rats but was not different from Sham in Tempol-treated IH-C rats. Small mesenteric arteries from IH-C rats but not Tempol-treated IH-C rats had increased superoxide levels as measured by ferric cytochrome c reduction, lucigenin signaling, and dihydroethidium fluorescence. The data show that IH-C increases ET-1 production and vascular ROS levels and that scavenging superoxide prevents both. Thus oxidative stress appears to contribute to increases in ET-1 production and elevated arterial pressure in this rat model of sleep apnea-induced hypertension.
Hypertrophy signaling during peripartum cardiac remodeling
Authors: Gonzalez A. M. D. , Osorio J. C. , Manlhiot C. , Gruber D. , Homma S. , Mital S.
Published: September 2007

Molecular signaling pathways that regulate peripartum cardiac remodeling are not well understood. Our objectives were to study the role of mitogen-activated protein kinases (MAPKs), protein kinase B (Akt), and endothelial nitric oxide synthase (eNOS) in mediating pregnancy and postpartum (PP) cardiac remodeling. Methods: Adult female Sprague-Dawley rats were divided into nonpregnant ( n = 5), 18 days pregnant ( n = 5), 0 days PP ( n = 7), and 14 days PP ( n = 8). Rats underwent echocardiography under sedation to measure left ventricle (LV) size and function, and Western blots were performed to measure myocardial protein expression of MAPKs (p38, JNK, ERK), Akt, and eNOS. Results: 1) During pregnancy, there was an increase in LV mass (0.62 ± 0.03 to 1.1 ± 0.04 g, P < 0.001), mass/volume ratio (0.7 ± 0.02 to 1.28 ± 0.02 g/ml, P < 0.0001), and ejection fraction (EF) (64 ± 3 to 74 ± 2%). Whereas LV mass and mass/volume ratio returned to prepregnancy values in the PP period, EF remained below normal range (53 ± 3%, P < 0.05). 2) The expression of anti-hypertrophic factors (p38, JNK, Akt) decreased during pregnancy and normalized PP, except JNK, which increased to higher than normal levels. eNOS also increased to higher than baseline levels PP. 3) Activation of p38 and JNK was directly correlated with lower LV mass/volume ratio ( r = −0.81 and −0.71, respectively; P < 0.05). Conclusion: Pregnancy is associated with physiological cardiac hypertrophy. There is rapid reversal of hypertrophy in the PP period while recovery of cardiac function is delayed, possibly related to PP upregulation of JNK. A dysregulation of MAPK signaling may be an important determinant of PP cardiac dysfunction.
Proinflammatory and vasodilator effects of nociceptin/orphanin FQ in the rat mesenteric microcirculation are mediated by histamine
Authors: Brookes Zoë L. S. , Stedman Emily N. , Guerrini Remo , Lawton Bethan K. , Calo Girolamo , Lambert David G.
Published: September 2007

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the N/OFQ peptide receptor (NOP). N/OFQ causes hypotension and vasodilation, and we aimed to determine the role of histamine in inflammatory microvascular responses to N/OFQ. Male Wistar rats (220–300 g, n = 72) were anesthetized with thiopental (30 mg/kg bolus, 40–90 mg·kg−1·h−1 iv), and the mesentery was prepared for fluorescent intravital microscopy using fluorescein isothiocyanate-conjugated BSA (FITC-BSA, 0.25 ml/100 g iv) or 1 μm fluorescently labeled microspheres. N/OFQ (0.6–60 nmol/kg iv) caused hypotension (SAP, baseline: 154 ± 11 mmHg, 15 nmol/kg N/OFQ: 112 ± 10 mmHg, P = 0.009), vasodilation (venules: 23.9 ± 1.2 μm, 26.7 ± 1.2 μm, P = 0.006), macromolecular leak (interstitial gray level FITC-BSA: 103.7 ± 3.4, 123.5 ± 11.8, P = 0.009), and leukocyte adhesion (2.0 ± 0.9, 15.2 ± 0.9/100 μm, P = 0.036). Microsphere velocity also decreased (venules: 1,230 ± 370 μm/s, P = 0.037), but there were no significant changes in blood flow. Flow cytometry measured a concurrent increase in neutrophil expression of cd11b with N/OFQ vs. controls (Geo mean fluorescence: 4.19 ± 0.13 vs. 2.06 ± 0.38, P < 0.05). The NOP antagonist [Nphe1,Arg14,Lys15]N/OFQ-NH2 (UFP-101; 60 and 150 nmol/kg iv), H1 and H2antagonists pyrilamine (mepyramine, 1 mg/kg iv) and ranitidine (1 mg/kg iv), and mast cell stabilizer cromolyn (1 mg·kg−1·min−1) also abolished vasodilation and macromolecular leak to N/OFQ in vivo ( P < 0.05), but did not affect hypotension. Isolated mesenteric arteries (∼200 μm, n = 25) preconstricted with U-46619 were also mounted on a pressure myograph (60 mmHg), and both intraluminally and extraluminally administered N/OFQ (10−5 M) caused dilation, inhibited by pyrilamine in the extraluminal but not the intraluminal (control: −6.9 ± 3.8%; N/OFQ: 32.6 ± 8.4%; pyrilamine: 31.5 ± 6.8%, n = 18, P < 0.05) experiments. We conclude that, in vivo, mesenteric microvascular dilation and macromolecular leak occur via N/OFQ-NOP-mediated release of histamine from mast cells. Therefore, N/OFQ-NOP has an important role in microvascular inflammation, and this may be targeted during disease, particularly as we have proven that UFP-101 is an effective antagonist of microvascular responses in vivo.
Simulated ischemia enhances L-type calcium current in pacemaker cells isolated from the rabbit sinoatrial node
Authors: Du Yi-Mei , Nathan Richard D.
Published: September 2007

Ischemic-like conditions (a glucose-free, pH 6.6 Tyrode solution bubbled with 100% N2) enhance L-type Ca current ( ICa,L) in single pacemaker cells (PCs) isolated from the rabbit sinoatrial node (SAN). In contrast, studies of ventricular myocytes have shown that acidic extracellular pH, as employed in our “ischemic” Tyrode, reduces ICa,L. Therefore, our goal was to explain why ICa,Lis increased by “ischemia” in SAN PCs. The major findings were the following: 1) blockade of Ca-induced Ca release with ryanodine, exposure of PCs to BAPTA-AM, or replacement of extracellular Ca2+with Ba2+failed to prevent the ischemia-induced enhancement of ICa,L; 2) inhibition of protein kinase A with H-89, or calcium/calmodulin-dependent protein kinase II with KN-93, reduced ICa,Lbut did not prevent its augmentation by ischemia; 3) ischemic Tyrode or pH 6.6 Tyrode shifted the steady-state inactivation curve in the positive direction, thereby reducing inactivation; 4) ischemic Tyrode increased the maximum conductance but did not affect the activation curve; 5) in rabbit atrial myocytes isolated and studied with exactly the same techniques used for SAN PCs, ischemic Tyrode reduced the maximum conductance and shifted the activation curve in the positive direction; pH 6.6 Tyrode also shifted the steady-state inactivation curve in the positive direction. We conclude that the acidic pH of ischemic Tyrode enhances ICa,Lin SAN PCs, because it increases the maximum conductance and reduces inactivation. Furthermore, the opposite results obtained with rabbit atrial myocytes cannot be explained by differences in cell isolation or patch-clamp techniques.
Proteinase-activated receptor-2 activating peptides: distinct canine coronary artery receptor systems
Authors: Saifeddine Mahmoud , Seymour Michelle L. , Xiao Yu-Pei , Compton Steven J. , Houle Steeve , Ramachandran Rithwik , MacNaughton Wallace K. , Simonet Serge , Vayssettes-Courchay Christine , Verbeuren Tony J. , Hollenberg Morley D.
Published: September 2007

In canine coronary artery preparations, the proteinase-activated receptor-2 (PAR2) activating peptides (PAR2-APs) SLIGRL-NH2and 2-furoyl-LIGRLO-NH2caused both an endothelium-dependent relaxation and an endothelium-independent contraction. Relaxation was caused at peptide concentrations 10-fold lower than those causing a contractile response. Although trans-cinnamoyl-LIGRLO-NH2, like other PAR2-APs, caused relaxation, it was inactive as a contractile agonist and instead antagonized the contractile response to SLIGRL-NH2. RT-PCR-based sequencing of canine PAR2revealed a cleavage/activation (indicated by underlines) sequence (SKGR/SLIGKTDSSLQITGKG) that is very similar to the human PAR2sequence (R/SLIGKV). As a synthetic peptide, the canine PAR-AP (SLIGKT-NH2) was a much less potent agonist than either SLIGRL-NH2or 2-furoyl-LIGRLO-NH2, either in the coronary contractile assay or in a Madin-Darby canine kidney (MDCK) cell PAR2calcium signaling assay. In the MDCK signaling assay, the order of potencies was as follows: 2-furoyl-LIGRLO-NH2≫ SLIGRL-NH2= trans-cinnamoyl-LIGRLO-NH2≫ SLIGKT-NH2, as expected for PAR2responses. In the coronary contractile assay, however, the order of potencies was very different: SLIGRL-NH2≫ 2-furoyl-LIGRLO-NH2≫ SLIGKT-NH2, trans-cinnamoyl-LIGRLO-NH2= antagonist. Because of 1) the distinct agonist (relaxant) and antagonist (contractile) activity of trans-cinnamoyl-LIGRLO-NH2in the canine coronary contractile assays, 2) the different concentration ranges over which the peptides caused either relaxation or contraction in the same coronary preparation, and 3) the markedly distinct structure-activity profiles for the PAR-APs in the coronary contractile assay, compared with those for PAR2-mediated MDCK cell calcium signaling, we suggest that the canine coronary tissue possesses a receptor system for the PAR-APs that is distinct from PAR2itself.
Signals from type 1 sphingosine 1-phosphate receptors enhance adult mouse cardiac myocyte survival during hypoxia
Authors: Zhang Jianqing , Honbo Norman , Goetzl Edward J. , Chatterjee Kanu , Karliner Joel S. , Gray Mary O.
Published: September 2007

Sphingosine 1-phosphate (S1P) is a biologically active lysophospholipid that serves as a key regulator of cellular differentiation and survival. Immune stimuli increase S1P synthesis and secretion by mast cells and platelets, implicating this molecule in tissue responses to injury and inflammation. Binding of S1P to Giprotein-coupled receptors activates phosphatidylinositol 3-kinase and Akt in a variety of tissues. To elucidate the mechanisms by which S1P enhances adult cardiac myocyte survival during hypoxia, we used a mouse cell culture system in which S1P1receptors were observed to transduce signals from exogenous S1P, an S1P1receptor antibody with agonist properties, and the pharmacological agents FTY720 and SEW2871. S1P1receptor mRNA and protein were abundantly expressed by adult mouse cardiac myocytes. S1P-S1P1receptor axis enhancement of myocyte survival during hypoxia was abolished by phosphatidylinositol 3-kinase inhibition. S1P1receptor function was closely associated with activation of Akt, inactivation of GSK-3β, and reduction of cytochrome c release from heart mitochondria. These observations highlight the importance of S1P1receptors on ventricular myocytes as mediators of inducible resistance against cellular injury during severe hypoxic stress.
WISE 2005: chronic bed rest impairs microcirculatory endothelium in women
Authors: Demiot Claire , Dignat-George Françoise , Fortrat Jacques-Olivier , Sabatier Florence , Gharib Claude , Larina Irina , Gauquelin-Koch Guillemette , Hughson Richard , Custaud Marc-Antoine
Published: September 2007

Sedentary behavior has deleterious effects on the cardiovascular system, including reduced endothelial functions. A 2-mo bed rest study in healthy women [women international space simulation for exploration (WISE) 2005 program] presented a unique opportunity to analyze the specific effects of prolonged inactivity without other vascular risk factors on the endothelium. We investigated endothelial properties before and after 56 days of bed rest in 8 subjects who performed no exercise (control group: No-EX) and in 8 subjects who regularly performed treadmill exercise in a lower body negative pressure chamber as well as resistance exercise (countermeasure group, EX). A functional evaluation of the microcirculation in the skin was assessed with laser Doppler. We studied endothelium-dependent and -independent vasodilation using iontophoresis of acetylcholine and sodium nitroprusside, respectively. We also measured circulating endothelial cells (CECs), an index of endothelial damage. In the No-EX group, endothelium-dependent vasodilation was significantly reduced (35.4 ± 4.8% vs. 24.1 ± 3.8%, P < 0.05) by bed rest with a significant increase in the number of CECs (3.6 ± 1.4 vs. 10.6 ± 2.7 ml−1, P < 0.05). In the EX group, endothelium-dependent vasodilation and number of CECs were preserved. Our study shows that in humans prolonged bed rest causes impairment of endothelium-dependent function at the microcirculatory level, along with an increase in circulating endothelial cells. Microcirculatory endothelial dysfunction might participate in cardiovascular deconditioning, as well as in several bed rest-induced pathologies. We therefore conclude that the endothelium should be a target for countermeasures during periods of prolonged deconditioning.
Roles of vasoconstrictor prostaglandins, COX-1 and -2, and AT1, AT2, and TP receptors in a rat model of early 2K,1C hypertension
Authors: Welch William J. , Patel Kinjal , Modlinger Paul , Mendonca Margarida , Kawada Noritaka , Dennehy Kathryn , Aslam Shakil , Wilcox Christopher S.
Published: September 2007

Angiotensin (ANG) II activating type 1 receptors (AT1Rs) enhances superoxide anion (O2•−) and arachidonate (AA) formation. AA is metabolized by cyclooxygenases (COXs) to PGH2, which is metabolized by thromboxane (Tx)A2synthase to TxA2or oxidized to 8-isoprostane PGF2α(8-Iso) by O2•−. PGH2, TxA2, and 8-Iso activate thromboxane-prostanoid receptors (TPRs). We investigated whether blood pressure in a rat model of early (3 wk) two-kidney, one-clip (2K,1C) Goldblatt hypertension is maintained by AT1Rs or AT2Rs, driving COX-1 or -2-dependent products that activate TPRs. Compared with sham-operated rats, 2K,1C Goldblatt rats had increased mean arterial pressure (MAP; 120 ± 4 vs. 155 ± 3 mmHg; P < 0.001), plasma renin activity (PRA; 22 ± 7 vs. 48 ± 5 ng·ml−1·h−1; P < 0.01), plasma malondialdehyde (1.07 ± 0.05 vs. 1.58 ± 0.16 nmol/l; P < 0.01), and TxB2excretion (26 ± 4 vs. 51 ± 7 ng/24 h; P < 0.01). Acute graded intravenous doses of benazeprilat (angiotensin-converting enzyme inhibitor) reduced MAP at 20 min (−36 ± 5 mmHg; P < 0.001) and excretion of TxA2metabolites. Indomethacin (nonselective COX antagonist) or SC-560 (COX-1 antagonist) reduced MAP at 20 min (−25 ± 5 and −28 ± 7 mmHg; P < 0.001), whereas valdecoxib (COX-2 antagonist) was ineffective (−9 ± 5 mmHg; not significant). Losartan (AT1R antagonist) or SQ-29548 (TPR antagonist) reduced MAP at 150 min (−24 ± 6 and −22 ± 3 mmHg; P < 0.001), whereas PD-123319 (AT2R antagonist) was ineffective. Acute blockade of TPRs, COX-1, or COX-2 did not change PRA, but TxB2generation by the clipped kidney was reduced by blockade of COX-1 and increased by blockade of COX-2. 2K,1C hypertension in rats activates renin, O2•−, and vasoconstrictor PGs. Hypertension is maintained by AT1Rs and by COX-1, but not COX-2, products that activate TPRs.
Hemoglobin oxygen fractional saturation regulates nitrite-dependent vasodilation of aortic ring bioassays
Authors: Isbell T. Scott , Gladwin Mark T. , Patel Rakesh P.
Published: September 2007

Nitrite reacts with deoxyhemoglobin to generate nitric oxide (NO). This reaction has been proposed to contribute to nitrite-dependent vasodilation in vivo and potentially regulate physiological hypoxic vasodilation. Paradoxically, while deoxyhemoglobin can generate NO via nitrite reduction, both oxyhemoglobin and deoxyhemoglobin potently scavenge NO. Furthermore, at the very low O2tensions required to deoxygenate cell-free hemoglobin solutions in aortic ring bioassays, surprisingly low doses of nitrite can be reduced to NO directly by the blood vessel, independent of the presence of hemoglobin; this makes assessments of the role of hemoglobin in the bioactivation of nitrite difficult to characterize in these systems. Therefore, to study the O2dependence and ability of deoxhemoglobin to generate vasodilatory NO from nitrite, we performed full factorial experiments of oxyhemoglobin, deoxyhemoglobin, and nitrite and found a highly significant interaction between hemoglobin deoxygenation and nitrite-dependent vasodilation ( P ≤ 0.0002). Furthermore, we compared the effect of hemoglobin oxygenation on authentic NO-dependent vasodilation using a NONOate NO donor and found that there was no such interaction, i.e., both oxyhemoglobin and deoxyhemoglobin inhibited NO-mediated vasodilation. Finally, we showed that another NO scavenger, 2-carboxyphenyl-4,4–5,5-tetramethylimidazoline-1-oxyl-3-oxide, inhibits nitrite-dependent vasodilation under normoxia and hypoxia, illustrating the uniqueness of the interaction of nitrite with deoxyhemoglobin. While both oxyhemoglobin and deoxyhemoglobin potently inhibit NO, deoxyhemoglobin exhibits unique functional duality as an NO scavenger and nitrite-dependent NO generator, suggesting a model in which intravascular NO homeostasis is regulated by a balance between NO scavenging and NO generation that is dynamically regulated by hemoglobin's O2fractional saturation and allosteric nitrite reductase activity.
Profound biopterin oxidation and protein tyrosine nitration in tissues of ApoE-null mice on an atherogenic diet: contribution of inducible nitric oxide synthase
Authors: Upmacis Rita K. , Crabtree Mark J. , Deeb Ruba S. , Shen Hao , Lane Paul B. , Benguigui Lea Esther S. , Maeda Nobuyo , Hajjar David P. , Gross Steven S.
Published: September 2007

Diminished nitric oxide (NO) bioactivity and enhanced peroxynitrite formation have been implicated as major contributors to atherosclerotic vascular dysfunctions. Hallmark reactions of peroxynitrite include the accumulation of 3-nitrotyrosine (3-NT) in proteins and oxidation of the NO synthase (NOS) cofactor, tetrahydrobiopterin (BH4). The present study sought to 1) quantify the extent to which 3-NT accumulates and BH4becomes oxidized in organs of apolipoprotein E-deficient (ApoE−/−) atherosclerotic mice and 2) determine the specific contribution of inducible NOS (iNOS) to these processes. Whereas protein 3-NT and oxidized BH4were undetected or near the detection limit in heart, lung, and kidney of 3-wk-old ApoE−/−mice or ApoE−/−mice fed a regular chow diet for 24 wk, robust accumulation was evident after 24 wk on a Western (atherogenic) diet. Since 3-NT accumulation was diminished 3- to 20-fold in heart, lung, and liver in ApoE−/−mice missing iNOS, iNOS-derived species are involved in this reaction. In contrast, iNOS-derived species did not contribute to elevated protein 3-NT formation in kidney or brain. iNOS deletion also afforded marked protection against BH4oxidation in heart, lung, and kidney of atherogenic ApoE−/−mice but not in brain or liver. These findings demonstrate that iNOS-derived species are increased during atherogenesis in ApoE−/−mice and that these species differentially contribute to protein 3-NT accumulation and BH4oxidation in a tissue-selective manner. Since BH4oxidation can switch the predominant NOS product from NO to superoxide, we predict that progressive NOS uncoupling is likely to drive atherogenic vascular dysfunctions.
Atrial myocardium is the predominant inotropic target of adrenomedullin in the human heart
Authors: Bisping Egbert , Tenderich Gero , Barckhausen Paul , Stumme Burkhard , Bruns Sebastian , von Lewinski Dirk , Pieske Burkert
Published: September 2007

Adrenomedullin (ADM) is an endogenous peptide with favorable hemodynamic effects in vivo. In this study, we characterized the direct functional effects of ADM in isolated preparations from human atria and ventricles. In electrically stimulated human nonfailing right atrial trabeculae, ADM (0.0001–1 μmol/l) increased force of contraction in a concentration-dependent manner, with a maximal increase by 35 ± 8% (at 1 μmol/l; P < 0.05). The positive inotropic effect was accompanied by a disproportionate increase in calcium transients assessed by aequorin light emission [by 76 ± 20%; force/light ratio (ΔF/ΔL) 0.58 ± 0.15]. In contrast, elevation of extracellular calcium (from 2.5 to 3.2 mmol/l) proportionally increased force and aequorin light emission (ΔF/ΔL 1.0 ± 0.1; P < 0.05 vs. ADM). Consistent with a cAMP-dependent mechanism, ADM (1 μmol/l) increased atrial cAMP levels by 90 ± 12%, and its inotropic effects could be blocked by the protein kinase A (PKA) inhibitor H-89. ADM also exerted positive inotropic effects in failing atrial myocardium and in nonfailing and failing ventricular myocardium. The inotropic response was significantly weaker in ventricular vs. atrial myocardium and in failing vs. nonfailing myocardium. In conclusion, ADM exerts Ca2+-dependent positive inotropic effects in human atrial and less-pronounced effects in ventricular myocardium. The inotropic effects are related to increased cAMP levels and stimulation of PKA. In heart failure, the responsiveness to ADM is reduced in atria and ventricles.
Tumor necrosis factor-α causes barrier dysfunction mediated by tyrosine198and tyrosine218in β-actin
Authors: Gertzberg Nancy , Gurnani Tina , Neumann Paul , Forbes Anne-Kay , Jean-Louis Natacha , Johnson Arnold
Published: September 2007

We tested the hypothesis that tumor necrosis factor-α (TNF) induces barrier dysfunction of pulmonary microvessel endothelial monolayers (PMEM) mediated by specific tyrosine residues in β-actin. PMEM were transfected with a wild-type, mutant [tyrosine198to phenylalanine198(Y198F)], mutant Y218F, or mutant Y306F β-actin construct tagged with enhanced yellow fluorescent protein (EYFP-β-actin). The cellular compartmentalization of wild-type and mutant EYFP-β-actin was displayed using EYFP fluorescence of the tagged β-actin. β-Actin was quantified for the EYFP-tagged and native β-actin using Western blot assay. The effect of the EYFP-β-actin on a cell junction protein was assessed by association of EYFP-β-actin with β-catenin using confocal microscopy and coimmunoprecipitation. The permeability of PMEM was assessed by the clearance rate of Evans blue-labeled albumin. The cellular compartmentalization of wild-type and mutant EYFP-β-actin was similar to the native β-actin. Incubation of PMEM with TNF (100 ng/ml) for 0.5 h resulted in increases in permeability to albumin and a decrease in association of the EYFP-β-actin with β-catenin. However, the expression of the EYFP-Y198F β-actin and EYFP-Y218F β-actin prevented the effect of TNF on β-catenin and barrier function. The vehicle, wild-type EYFP-β-actin, and mutant Y306F β-actin had no affect on the response to TNF. The data indicate that TNF induces an increase in endothelial permeability that is dependent on tyrosine198and tyrosine218in β-actin.
PAR-2 activation and LPS synergistically enhance inflammatory signaling in airway epithelial cells by raising PAR expression level and interleukin-8 release
Authors: Ostrowska Ewa , Sokolova Elena , Reiser Georg
Published: September 2007

Protease-activated receptors (PARs) are involved in the contribution of airway epithelial cells to the development of inflammation by release of pro- and anti-inflammatory mediators. Here, we evaluated in epithelial cells the influence of LPS and continuous PAR activation on PAR expression level and the release of the proinflammatory chemokine IL-8. We studied primary human small airway epithelial cells and two airway epithelial cell lines, A549 and HBE cells. LPS specifically upregulated expression of PAR-2 but not of PAR-1. Exposure of epithelial cells to PAR-1 or PAR-2 agonists increased the PAR-1 expression level. The PAR-2 agonist exhibited higher potency than PAR-1 activators. However, the combined exposure of epithelial cells to LPS and PAR agonists abrogated the PAR-1 upregulation. The PAR-2 expression level was also upregulated after exposure to PAR-1 or PAR-2 agonists. This elevation was higher than the effect of PAR agonists on the PAR-1 level. In contrast to the PAR-1 level, the PAR-2 level remained elevated under concomitant stimulation with LPS and PAR-2 agonist. Furthermore, activation of PAR-2, but not of PAR-1, caused production of IL-8 from the epithelial cells. Interestingly, both in the epithelial cell line and in primary epithelial cells, there was a potentiation of the stimulation of the IL-8 synthesis and release by PAR-2 agonist together with LPS. In summary, these results underline the important role of PAR-2 in human lung epithelial cells. Moreover, our study shows an intricate interplay between LPS and PAR agonists in affecting PAR regulation and IL-8 production.
Rho kinase and Ca2+entry mediate increased pulmonary and systemic vascular resistance inl-NAME-treated rats
Authors: Dhaliwal Jasdeep S. , Casey David B. , Greco Anthony J. , Badejo Adeleke M. , Gallen Thomas B. , Murthy Subramanyam N. , Nossaman Bobby D. , Hyman Albert L. , Kadowitz Philip J.
Published: September 2007

The small GTP-binding protein and its downstream effector Rho kinase play an important role in the regulation of vasoconstrictor tone. Rho kinase activation maintains increased pulmonary vascular tone and mediates the vasoconstrictor response to nitric oxide (NO) synthesis inhibition in chronically hypoxic rats and in the ovine fetal lung. However, the role of Rho kinase in mediating pulmonary vasoconstriction after NO synthesis inhibition has not been examined in the intact rat. To address this question, cardiovascular responses to the Rho kinase inhibitor fasudil were studied at baseline and after administration of an NO synthesis inhibitor. In the intact rat, intravenous injections of fasudil cause dose-dependent decreases in systemic arterial pressure, small decreases in pulmonary arterial pressure, and increases in cardiac output. l-NAME caused a significant increase in pulmonary and systemic arterial pressures and a decrease in cardiac output. The intravenous injections of fasudil after l-NAME caused dose-dependent decreases in pulmonary and systemic arterial pressure and increases in cardiac output, and the percent decreases in pulmonary arterial pressure in response to the lower doses of fasudil were greater than decreases in systemic arterial pressure. The Ca++entry blocker isradipine also decreased pulmonary and systemic arterial pressure in l-NAME-treated rats. Infusion of sodium nitroprusside restored pulmonary arterial pressure to baseline values after administration of l-NAME. These data provide evidence in support of the hypothesis that increases in pulmonary and systemic vascular resistance following l-NAME treatment are mediated by Rho kinase and Ca++entry through L-type channels, and that responses to l-NAME can be reversed by an NO donor.