bluuebunny/crossref_metadata_2025 huggingface dataset:
Pyocin S2 (Sa) Kills
Pseudomonas aeruginosa
Strains via the FpvA Type I Ferripyoverdine Receptor
Authors: Denayer Sarah , Matthijs Sandra , Cornelis Pierre
Published: August 2007

Soluble (S-type) pyocins are
Pseudomonas aeruginosa
bacteriocins that kill nonimmune
P. aeruginosa
strains via a specific receptor. The genes coding for pyocin Sa (consisting of a killing protein and an immunity protein) were cloned and expressed in
Escherichia coli
. Sequence analysis revealed that Sa is identical to pyocin S2. Seventy-nine strains of
P. aeruginosa
were tested for their sensitivity to pyocins S1, S2, and S3, and their ferripyoverdine receptors were typed by multiplex PCR. No strain was found to be sensitive to both S2 and S3, suggesting that the receptors for these two pyocins cannot coexist in one strain. As expected, all S3-sensitive strains had the type II ferripyoverdine receptor
fpvA
gene, confirming our previous reports. S1 killed strains irrespective of the type of ferripyoverdine receptor they produced. All S2-sensitive strains had the type I
fpvA
gene, and the inactivation of type I
fpvA
in an S2-sensitive strain conferred resistance to the S2 pyocin. Accordingly, complementation with type I
fpvA
in
trans
restored sensitivity to S2. Some S2-resistant type I
fpvA
-positive strains were detected, the majority (all but five) of which had the S1-S2 immunity gene. Comparison of type I
fpvA
sequences from immunity gene-negative S2-sensitive and S2-resistant strains revealed only a valine-to-isoleucine substitution at position 46 of type I FpvA. However, both type I
fpvA
genes conferred the capacity for type I pyoverdine utilization and sensitivity to S2. When these two type I
fpvA
genes were introduced into strain 7NSK2 carrying mutations in type II
fpvA
(encoding the type II pyoverdine receptor) and
fpvB
(encoding the alternative type I receptor), growth in the presence of type I pyoverdine was observed and the strain became sensitive to S2. We also found that type I pyoverdine could signal type II pyoverdine production via the type I FpvA receptor in 7NSK2.
Genome-Wide Transcriptional Changes inStreptococcus gordoniiin Response to Competence Signaling Peptide
Authors: Vickerman M. M. , Iobst S. , Jesionowski A. M. , Gill S. R.
Published: August 2007

Streptococcus gordoniiis a primary colonizer of the multispecies biofilm on tooth surfaces forming dental plaque and a potential agent of endocarditis. The recent completion of the genome sequence of the naturally competent strain Challis allowed the design of a spotted oligonucleotide microarray to examine a genome-wide response of this organism to environmental stimuli such as signal peptides. Based on temporal responses to synthetic competence signaling peptide (CSP) as indicated by transformation frequencies, theS. gordoniitranscriptome was analyzed at various time points after CSP exposure. Microarray analysis identified 35 candidate early genes and 127 candidate late genes that were up-regulated at 5 and 15 min, respectively; these genes were often grouped in clusters. Results supported published findings onS. gordoniicompetence, showing up-regulation of 12 of 16 genes that have been reported to affect transformation frequencies in this species. Comparison of CSP-inducedS. gordoniitranscriptomes to results published forStreptococcus pneumoniaestrains identified both conserved and species-specific genes. Putative intergenic regulatory sites, such as the conserved combox sequence thought to be a binding site for competence sigma factor, were found precedingS. gordoniilate responsive genes. In contrast,S. gordoniiearly CSP-responsive genes were not preceded by the direct repeats found inS. pneumoniae. These studies provide the first insights into a genome-wide transcriptional response of an oral commensal organism. They offer an extensive analysis of transcriptional changes that accompany competence inS. gordoniiand form a basis for future intra- and interspecies comparative analyses of this ecologically important phenotype.
Role of Sigma Factors in Controlling Global Gene Expression in Light/Dark Transitions in the CyanobacteriumSynechocystissp. Strain PCC 6803
Authors: Summerfield Tina C. , Sherman Louis A.
Published: August 2007

We report on differential gene expression in the cyanobacteriumSynechocystissp. strain PCC 6803 after light-dark transitions in wild-type, ΔsigB, and ΔsigDstrains. We also studied the effect of day length in the presence of glucose on a ΔsigBΔsigEmutant. Our results indicated that the absence of SigB or SigD predominately altered gene expression in the dark or in the light, respectively. In the light, approximately 350 genes displayed transcript levels in the ΔsigDstrain that were different from those of the wild type, with over 200 of these up-regulated in the mutant. In the dark, removal of SigB altered more than 150 genes, and the levels of 136 of these were increased in the mutant compared to those in the wild type. The removal of both SigB and SigE had a major impact on gene expression under mixotrophic growth conditions and resulted in the inability of cells to grow in the presence of glucose with 8-h light and 16-h dark cycles. Our results indicated the importance of group II σ factors in the global regulation of transcription in this organism and are best explained by using the σ cycle paradigm with the stochastic release model described previously (R. A. Mooney, S. A. Darst, and R. Landick, Mol. Cell 20:335-345, 2005). We combined our results with the total protein levels of the σ factors in the light and dark as calculated previously (S. Imamura, S. Yoshihara, S. Nakano, N. Shiozaki, A. Yamada, K. Tanaka, H. Takahashi, M. Asayama, and M. Shirai, J. Mol. Biol.325:857-872, 2003; S. Imamura, M. Asayama, H. Takahashi, K. Tanaka, H. Takahashi, and M. Shirai, FEBS Lett.554:357-362, 2003). Thus, we concluded that the control of global transcription is based on the amount of the various σ factors present and able to bind RNA polymerase.
gerT
, a Newly Discovered Germination Gene under the Control of the Sporulation Transcription Factor σ
K
in
Bacillus subtilis
Authors: Ferguson Caitlin C. , Camp Amy H. , Losick Richard
Published: August 2007

We report the identification of a gene, herein designated
gerT
(formerly
yozR
), that is involved in germination by spores of
Bacillus subtilis
. The
gerT
gene is induced late in sporulation under the positive control of the transcription factor σ
K
and under the negative control of the DNA-binding protein GerE. The
gerT
gene product (GerT) is a component of the spore coat, and its incorporation into the coat takes place in two stages. GerT initially assembles into foci, which then spread around the developing spore in a process that is dependent on the morphogenetic protein CotE. Mutant spores lacking GerT respond poorly to multiple germinants and are impaired at an early stage of germination.
Abh and AbrB Control of
Bacillus subtilis
Antimicrobial Gene Expression
Authors: Strauch Mark A. , Bobay Benjamin G. , Cavanagh John , Yao Fude , Wilson Angelo , Le Breton Yoann
Published: August 2007

The
Bacillus subtilis abh
gene encodes a protein whose N-terminal domain has 74% identity to the DNA-binding domain of the global regulatory protein AbrB. Strains with a mutation in
abh
showed alterations in the production of antimicrobial compounds directed against some other
Bacillus
species and gram-positive microbes. Relative to its wild-type parental strain, the
abh
mutant was found deficient, enhanced, or unaffected for the production of antimicrobial activity. Using
lacZ
fusions, we examined the effects of
abh
upon the expression of 10 promoters known to be regulated by AbrB, including five that transcribe well-characterized antimicrobial functions (SdpC, SkfA, TasA, sublancin, and subtilosin). For an otherwise wild-type background, the results show that Abh plays a negative regulatory role in the expression of four of the promoters, a positive role for the expression of three, and no apparent regulatory role in the expression of the other three promoters. Binding of AbrB and Abh to the promoter regions was examined using DNase I footprinting, and the results revealed significant differences. The transcription of
abh
is not autoregulated, but it is subject to a degree of AbrB-afforded negative regulation. The results indicate that Abh is part of the complex interconnected regulatory system that controls gene expression during the transition from active growth to stationary phase.
Complex Regulation of Urease Formation from the Two Promoters of the
ure
Operon of
Klebsiella pneumoniae
Authors: Liu Qiong , Bender Robert A.
Published: August 2007

Klebsiella pneumoniae
can use urea as the sole source of nitrogen, thanks to a urease encoded by the
ureDABCEFG
operon. Expression of this operon is independent of urea and is regulated by the supply of nitrogen in the growth medium. When cells were growth rate limited for nitrogen, the specific activity of urease was about 70 times higher than that in cells grown under conditions of excess nitrogen. Much of this nitrogen regulation of urease formation depended on the nitrogen regulatory system acting through the nitrogen assimilation control protein, NAC. In a strain deleted for the
nac
gene, nitrogen limitation resulted in only a 7-fold increase in the specific activity of urease, in contrast to the 70-fold increase seen in that of the wild type. The
ure
operon was transcribed from two promoters. The proximal promoter (P1) had an absolute requirement for NAC; little or no transcription was seen in the absence of NAC. The distal promoter (P2) was independent of NAC, but its activity increased about threefold when the growth rate of the cells was limited by the nitrogen source. Transcriptional regulation of P1 and P2 accounted for most of the changes in urease activity seen under various nitrogen conditions. However, when transcription of
ureDABCEFG
was less than 20% of its maximum, the amount of active urease formed per transcript of
ure
decreased almost linearly with decreasing transcription. This may reflect a defect in the assembly of active urease and accounted for as much as a threefold activity difference under the conditions tested here. Thus, the
ure
operon was transcribed from a NAC-independent promoter (P2) and the most strongly NAC-dependent promoter known (P1). Most of the regulation of urease formation was transcriptional, but when
ure
transcription was low, assembly of active urease also was defective.
Cloning of a Gene Cluster Involved in the Catabolism of
p
-Nitrophenol by
Arthrobacter
sp. Strain JS443 and Characterization of the
p
-Nitrophenol Monooxygenase
Authors: Perry Lynda L. , Zylstra Gerben J.
Published: August 2007

The
npd
gene cluster, which encodes the enzymes of a
p
-nitrophenol catabolic pathway from
Arthrobacter
sp. strain JS443, was cloned and sequenced. Three genes,
npdB
,
npdA1
, and
npdA2
, were independently expressed in
Escherichia coli
in order to confirm the identities of their gene products. NpdA2 is a
p
-nitrophenol monooxygenase belonging to the two-component flavin-diffusible monooxygenase family of reduced flavin-dependent monooxygenases. NpdA1 is an NADH-dependent flavin reductase, and NpdB is a hydroxyquinol 1,2-dioxygenase. The
npd
gene cluster also includes a putative maleylacetate reductase gene,
npdC
. In an in vitro assay containing NpdA2, an
E. coli
lysate transforms
p
-nitrophenol stoichiometrically to hydroquinone and hydroxyquinol. It was concluded that the
p
-nitrophenol catabolic pathway in JS443 most likely begins with a two-step transformation of
p
-nitrophenol to hydroxy-1,4-benzoquinone, catalyzed by NpdA2. Hydroxy-1,4-benzoquinone is reduced to hydroxyquinol, which is degraded through the hydroxyquinol
ortho
cleavage pathway. The hydroquinone detected in vitro is a dead-end product most likely resulting from chemical or enzymatic reduction of the hypothetical intermediate 1,4-benzoquinone. NpdA2 hydroxylates a broad range of chloro- and nitro-substituted phenols, resorcinols, and catechols. Only
p
-nitro- or
p
-chloro-substituted phenols are hydroxylated twice. Other substrates are hydroxylated once, always at a position
para
to a hydroxyl group.
Revision of the Dual-Substituent-Parameter Treatment; Reaction Series with a Donor Reaction Centre
Authors: Böhm Stanislav , Exner Otto
Published: August 2007

The dual-substituent-parameter (DSP) treatment was challenged previously as not generally valid. Just in the fundamental reaction, dissociation of 4-substituted benzoic acids and in similar reactions, DPS does not hold for acceptor substituents because the constant reaction centre is itself an acceptor. In this communication, the reverse case was examined, i.e., a reaction series with a donor reaction centre: basicity and acidity of 4-substituted anilines, and acidity of 4-substituted phenols. The reaction energies were calculated for 19 common substituents at the level B3LYP/6-311+G(d,p)//B3LYP/6-311+G(d,p); the substituents effects were also estimated separately in uncharged molecules and in the ions in terms of isodesmic reaction. DSP is valid and its resonance term is highly significant for molecules with acceptor substituents; donor substituents behave differently and cannot be described by simple resonance constants. Basicity of substituted anilines is more complex since the substituent effects are quite different in the free base and in the protonated form: basicity is controlled by a combination of various effects. It is recommended to use DSP only for acceptor substituents with a donor reaction centre or vice versa; otherwise the accuracy is decreased. All results were obtained with isolated molecules and with resonance constants derived on isolated molecules; however, they retain their validity even with the common constants σR determined from the reactions in solution.
Post-mortem evidence of idiopathic left ventricular hypertrophy and idiopathic interstitial myocardial fibrosis: is exercise the cause?
Authors: Whyte G , Sheppard M , George K , Shave R , Wilson M , Prasad S , O’Hanlon R , Sharma S
Published: August 2007

A growing body of evidence reporting altered cardiac function and myocardial damage after arduous exercise, together with the increased prevalence of arrhythmias observed in highly trained athletes, suggests that repetitive bouts of prolonged, arduous exercise may be deleterious to long-term cardiac health. We report the case of an experienced, highly trained marathon runner who died suddenly while running. On post-mortem examination, left ventricle hypertrophy and idiopathic interstitial myocardial fibrosis was found. We believe that life-long, repetitive bouts of arduous physical activity resulted in fibrous replacement of the myocardium, causing a pathological substrate for the propagation of fatal arrhythmias.
Physical examination tests of the shoulder: a systematic review with meta-analysis of individual tests
Authors: Hegedus E J , Goode A , Campbell S , Morin A , Tamaddoni M , Moorman C T , Cook C
Published: August 2007

Objective:

To compile and critique research on the diagnostic accuracy of individual orthopaedic physical examination tests in a manner that would allow clinicians to judge whether these tests are valuable to their practice.

Methods:

A computer-assisted literature search of MEDLINE, CINAHL, and SPORTDiscus databases (1966 to October 2006) using keywords related to diagnostic accuracy of physical examination tests of the shoulder. The Quality Assessment of Diagnostic Accuracy Studies (QUADAS) tool was used to critique the quality of each paper. Meta-analysis through meta-regression of the diagnostic odds ratio (DOR) was performed on the Neer test for impingement, the Hawkins−Kennedy test for impingement, and the Speed test for superior labral pathology.

Results:

Forty-five studies were critiqued with only half demonstrating acceptable high quality and only two having adequate sample size. For impingement, the meta-analysis revealed that the pooled sensitivity and specificity for the Neer test was 79% and 53%, respectively, and for the Hawkins−Kennedy test was 79% and 59%, respectively. For superior labral (SLAP) tears, the summary sensitivity and specificity of the Speed test was 32% and 61%, respectively. Regarding orthopaedic special tests (OSTs) where meta-analysis was not possible either due to lack of sufficient studies or heterogeneity between studies, the list that demonstrates both high sensitivity and high specificity is short: hornblowers’s sign and the external rotation lag sign for tears of the rotator cuff, biceps load II for superior labral anterior to posterior (SLAP) lesions, and apprehension, relocation and anterior release for anterior instability. Even these tests have been under-studied or are from lower quality studies or both. No tests for impingement or acromioclavicular (AC) joint pathology demonstrated significant diagnostic accuracy.

Conclusion:

Based on pooled data, the diagnostic accuracy of the Neer test for impingement, the Hawkins−Kennedy test for impingement and the Speed test for labral pathology is limited. There is a great need for large, prospective, well-designed studies that examine the diagnostic accuracy of the numerous physical examination tests of the shoulder. Currently, almost without exception, there is a lack of clarity with regard to whether common OSTs used in clinical examination are useful in differentially diagnosing pathologies of the shoulder.
Expression of HDAC1 and CBP/p300 in human colorectal carcinomas
Authors: Ishihama Katsuyoshi , Yamakawa Mitsunori , Semba Shuho , Takeda Hiroaki , Kawata Sumio , Kimura Seishi , Kimura Wataru
Published: August 2007

Background:

The histone-modifying enzymes histone deacetylase (HDAC) and histone acetyltransferase (HAT) control gene transcriptional activation and repression in human malignancies.

Aims:

To analyse the expression of HDAC/HAT-associated molecules such as HDAC1, CREB-binding protein (CBP) and p300 in human colorectal carcinomas, and investigate the relationship between their expression levels and clinicopathological parameters.

Methods:

Expression levels of HDAC1, CBP, and p300 in human colorectal cancer were investigated by immunohistochemistry. In situ hybridisation (ISH) and reverse transcription (RT)-PCR analyses were also carried out to confirm mRNA expression levels of these genes. Immunoreactivity was evaluated semi-quantitatively using a staining index (SI). The relationships between the SIs and clinicopathological findings were analysed and survival curves were calculated using the Kaplan–Meier method and log-rank tests.

Results:

The mean SIs for HDAC1, CBP, and p300 in this series of tumours were much higher than those in normal colonic mucosa. The presence ofHDAC1andCBPmRNAs on colorectal carcinoma cells as well as normal epithelial cells was confirmed by ISH analysis. A marked increase inp300mRNA levels was detected in a majority of cases by RT-PCR. Among the patients with colorectal cancer, overexpression of p300 (SI>11.9) correlated with a poor prognosis, whereas high CBP expression levels (SI>16.6) indicated long-term survival.

Conclusion:

Results showed the up-regulation of these three histone-modifying molecules in this series of colorectal cancers and suggested that monitoring of CBP and p300 may assist prediction of the prognosis in patients with colorectal adenocarcinoma.
Expression of androgen receptor through androgen-converting enzymes is associated with biological aggressiveness in prostate cancer
Authors: Wako K , Kawasaki T , Yamana K , Suzuki K , Jiang S , Umezu H , Nishiyama T , Takahashi K , Hamakubo T , Kodama T , Naito M
Published: August 2007

Aims:

The association between the expression of androgen receptor (AR) or androgen-converting enzymes and malignant potential in prostate cancer (PCa) was examined.

Methods:

PCa specimens from 44 cases of stage II, 10 cases of stage III, four cases of stage IV and two recurrent cases were semi-quantitatively studied with immunohistochemistry for AR and androgen-converting enzymes.

Results:

The expression scores for AR, 5α-reductase type 1 (SRD5A1), 5α-reductase type 2 (SRD5A2), and aldo-keto reductase family 1 member C3 (AKR1C3) in the metastatic lesion of stage IV or recurrent cancer (n = 6) were 284.2 (30.1), 300 (0.0), 279.2 (51) and 254.2 (74.9), respectively; these scores were significantly higher than the respective scores of 121.8 (82.1), 135.1 (59.7), 167.0 (66.4) and 150.5 (62.8) for stage II and III cancer (n = 54) (p<0.001, p<0.001, p = 0.002 and p = 0.018, respectively). The expression scores for AR and SRD5A1 in stage II and III cancer with Gleason score 7 (n = 19) were 128.7 (72.3) and 150.5 (52.9); these were significantly higher than the scores of 78.8 (67.2) and 100.0 (39.6), respectively, for cancers with a Gleason score of ⩽6 (n = 20) (p = 0.032 and p = 0.002, respectively). The expression scores for AR, SRD5A1 and AKR1C3 in stage II and III cancer with primary Gleason pattern ⩾4 (n = 21) were 158.1 (84.3), 158.3 (61.1) and 173.8 (64.8); these were significantly higher than the scores of 98.6 (72.8), 120.3 (54.7) and 135.6 (57.6), respectively, for cancers with primary Gleason pattern ⩽3 (n = 33) (p = 0.011, p = 0.026 and p = 0.034, respectively). Within Gleason score 9 cancer, the expression scores for AR and SRD5A1 in the primary lesion of stage IV (n = 3) were 276.7 (5.8) and 283.3 (28.9); these scores were significantly higher than the scores of 182.1 (86.0) and 140.0 (56.6), respectively, for stage II and III cancer (n = 7) (p = 0.027 and p = 0.001, respectively).

Conclusions:

Both AR and androgen-converting enzymes were upregulated in high-grade or advanced PCa.
THE GENERATION AND ANALYSIS OF A NEW FOUR-DIMENSIONAL HYPERCHAOTIC SYSTEM
Authors: WANG JIEZHI , CHEN ZENGQIANG , YUAN ZHUZHI
Published: August 2007

A new four-dimensional continuous autonomous hyperchaotic system is considered. It possesses two parameters, and each equation of it has one quadratic cross product term. Some basic properties of it are studied. The dynamic behaviors of it are analyzed by the Lyapunov exponent (LE) spectrum, bifurcation diagrams, phase portraits, and Poincaré sections. The system has larger hyperchaotic region. When it is hyperchaotic, the two positive LE are both large and they are both larger than 1 if the system parameters are taken appropriately.
A FORMULA FOR THE SENSITIVITY TO sin2 2θ13 IN REACTOR EXPERIMENTS WITH A SPECTRAL ANALYSIS
Authors: SUGIYAMA H. , YASUDA O.
Published: August 2007

Using an analytical approach, the sensitivity to sin 2 2θ13 with infinite statistics in a spectral analysis is investigated in reactor neutrino oscillation experiments with one reactor and two identical detectors. We derive an useful formula for the sensitivity which depends only on two parameters [Formula: see text] (the uncorrelated bin-to-bin systematic error over the square-root of the number of bins) and σ dB (the bin-to-bin correlated detector specific systematic error) if they are smaller than other errors. It is pointed out that the spectrum analysis is advantageous over the rate analysis in search of neutrino oscillations for [Formula: see text].
A VARIATIONAL FORMULATION OF SYMPLECTIC NONCOMMUTATIVE MECHANICS
Authors: CORTESE IGNACIO , GARCÍA J. ANTONIO
Published: August 2007

The standard lore in noncommutative physics is the use of first order variational description of a dynamical system to probe the space noncommutativity and its consequences in the dynamics in phase space. As the ultimate goal is to understand the inherent space noncommutativity, we propose a variational principle for noncommutative dynamical systems in configuration space, based on results of our previous work [18]. We hope that this variational formulation in configuration space can be of help to elucidate the definition of some global and dynamical properties of classical and quantum noncommutative space.
ON STRATEGIES TOWARDS THE RIEMANN HYPOTHESIS: FRACTAL SUPERSYMMETRIC QM AND A TRACE FORMULA
Authors: CASTRO CARLOS
Published: August 2007

The Riemann hypothesis (RH) states that the non-trivial zeros of the Riemann zeta-function are of the form sn = 1/2+iλn. An improvement of our previous construction to prove the RH is presented by implementing the Hilbert–Polya proposal and furnishing the Fractal Supersymmetric Quantum Mechanical (SUSY-QM) model whose spectrum reproduces the imaginary parts of the zeta zeros. We model the fractal fluctuations of the smooth Wu–Sprung potential (that capture the average level density of zeros) by recurring to a weighted superposition of Weierstrass functions ∑p W(x, p, D) and where the summation has to be performed over all primes p in order to recapture the connection between the distribution of zeta zeros and prime numbers. We proceed next with the construction of a smooth version of the fractal QM wave equation by writing an ordinary Schroedinger equation whose fluctuating potential (relative to the smooth Wu–Sprung potential) has the same functional form as the fluctuating part of the level density of zeros. The second approach to prove the RH relies on the existence of a continuous family of scaling-like operators involving the Gauss–Jacobi theta series. An explicit completion relation ("trace formula") related to a superposition of eigenfunctions of these scaling-like operators is defined. If the completion relation is satisfied, this could be another test of the Riemann Hypothesis. In an appendix, we briefly describe our recent findings showing why the Riemann Hypothesis is a consequence of [Formula: see text]-invariant Quantum Mechanics, because [Formula: see text] where s are the complex eigenvalues of the scaling-like operators. We show why [Formula: see text] invariance requires that s(1 - s) = real , which implies that s is real and/or it lies in the critical Riemann line.
The energetic state within hibernating myocardium is normal during dobutamine despite inhibition of ATP-dependent potassium channel opening with glibenclamide
Authors: McFalls Edward O. , Kelly Rosemary F. , Hu Qingsong , Mansoor Abdul , Lee Joseph , Kuskowski Michael , Sikora Joseph , Ward Herbert B. , Zhang Jianyi
Published: August 2007

Within hibernating myocardium, it is uncertain whether a normal energetic state is present at baseline and whether maintaining that energy state during a catecholamine challenge is dependent on ATP-dependent potassium channel opening. In this study, 16 swine underwent a thoracotomy with placement of an external constrictor on the left anterior descending coronary artery (LAD) (hibernation model). Seven additional swine underwent a sham operation. At 10 wk, the myocardial energetic state in the LAD region was assessed by 31P-NMR spectroscopy, and the ratio of phosphocreatine to ATP (PCr/ATP) was determined at baseline, during glibenclamide treatment (0.5 mg/kg bolus with 50 μg/min iv), and during addition of dobutamine (40 μg·kg−1·min−1 iv). At baseline, transmural blood flow in the LAD and remote region was 0.75 ± 0.11 and 0.88 ± 0.09 ml·min−1·g−1, respectively ( P < 0.01), in hibernating hearts and 0.83 ± 0.12 and 0.88 ± 0.15 ml·min−1·g−1, respectively (not significant), in sham-operated hearts. Under basal conditions, PCr/ATP in the LAD region of hibernating and sham pigs was 2.15 ± 0.04 and 2.11 ± 0.05, respectively (not significant). In sham pigs, addition of dobutamine to glibenclamide increased the double product from 10.4 ± 0.8 to 23.9 ± 4.0 mmHg·beats·min−1 × 1,000 ( P < 0.05) and decreased transmural PCr/ATP from 2.06 ± 0.06 to 1.69 ± 0.06 ( P < 0.05). Dobutamine increased the double product in hibernating pigs in a similar fashion and, despite a 40% lower blood flow response, induced an equivalent decrease in PCr/ATP from 2.04 ± 0.04 to 1.73 ± 0.08 ( P < 0.05). In conclusion, we found that, in chronic hibernating swine myocardium with reduced basal blood flow and perfusion reserve, the transmural energetic state, defined by PCr/ATP, is normal during addition of dobutamine, despite inhibition of ATP-dependent potassium channel opening with glibenclamide. These data suggest that important adaptations other than the ATP-dependent potassium channel opening allow hibernating myocardium to operate over a lower range of the oxygen supply-demand relationship to protect against myocardial ischemia.
β2-Adrenergic receptor agonists stimulate L-type calcium current independent of PKA in newborn rabbit ventricular myocytes
Authors: Collis Leon P. , Srivastava Shekhar , Coetzee William A. , Artman Michael
Published: August 2007

Selective stimulation of β2-adrenergic receptors (ARs) in newborn rabbit ventricular myocardium invokes a positive inotropic effect that is lost during postnatal maturation. The underlying mechanisms for this age-related stimulatory response remain unresolved. We examined the effects of β2-AR stimulation on L-type Ca2+current ( ICa,L) during postnatal development. ICa,Lwas measured (37°C; either Ca2+or Ba2+as the charge carrier) using the whole-cell patch-clamp technique in newborn (1 to 5 days old) and adult rabbit ventricular myocytes. Ca2+transients were measured concomitantly by dialyzing the cell with indo-1. Activation of β2-ARs (with either 100 nM zinterol or 1 μM isoproterenol in the presence of the β1-AR antagonist, CGP20712A) stimulated ICa,Ltwofold in newborns but not in adults. The β2-AR-mediated increase in Ca2+transient amplitude in newborns was due exclusively to the augmentation of ICa,L. Zinterol increased the rate of inactivation of ICa,Land increased the Ca2+flux integral. The β2-AR inverse agonist, ICI-118551 (500 nM), but not the β1-AR antagonist, CGP20712A (500 nM), blocked the response to zinterol. Unexpectedly, the PKA blockers, H-89 (10 μM), PKI 6-22 amide (10 μM), and Rp-cAMP (100 μM), all failed to prevent the response to zinterol but completely blocked responses to selective β1-AR stimulation of ICa,Lin newborns. Our results demonstrate that in addition to the conventional β1-AR/cAMP/PKA pathway, newborn rabbit myocardium exhibits a novel β2-AR-mediated, PKA-insensitive pathway that stimulates ICa,L. This striking developmental difference plays a major role in the age-related differences in inotropic responses to β2-AR agonists.
PAR-2 activation at the time of reperfusion salvages myocardium via an ERK1/2 pathway in in vivo rat hearts
Authors: Jiang Rong , Zatta Amanda , Kin Hajime , Wang Ningping , Reeves James G. , Mykytenko James , Deneve Jeremiah , Zhao Zhi-Qing , Guyton Robert A. , Vinten-Johansen Jakob
Published: August 2007

Protease-activated receptor-2 (PAR-2) may have proinflammatory effects in some tissues and protective effects in other tissues. The role of PAR-2 in in vivo myocardial ischemia-reperfusion has not yet been determined. This study tested the hypothesis that PAR-2 activation with the PAR-2 agonist peptide SLIGRL (PAR-2 AP) reduces myocardial infarct size when given at reperfusion in vivo, and this cardioprotection involves the ERK1/2 pathway. Anesthetized rats were randomly assigned to the following groups with 30 min of regional ischemia and 3 h reperfusion: 1) control with saline; 2) vehicle (DMSO); 3) PAR-2 AP, 1 mg/kg given intravenously 5 min before reperfusion; 4) scrambled peptide (SP), 1 mg/kg; 5) the ERK1/2 inhibitor PD-98059 (PD), 0.3 mg/kg given 10 min before reperfusion; 6) the phosphatidylinositol 3-kinase inhibitor LY-294002 (LY), 0.3 mg/kg given 10 min before reperfusion; 7) PD + PAR-2 AP, 0.3 mg/kg PD given 5 min before PAR-2 AP; 8) LY + PAR-2 AP, 0.3 mg/kg LY given 5 min before PAR-2 AP; 9) chelerythrine (Chel) alone, 5 mg/kg given 10 min before reperfusion; and 10) Chel + PAR-2 AP, Chel was given 5 min before PAR-2 AP (10 min before reperfusion). Activation of ERK1/2, ERK5, Akt, and the downstream targets of ERK1/2 [P90 RSK and bcl-xl/bcl-2-associated death promoter (BAD)] was determined by Western blot analysis in separate experiments. PAR-2 AP significantly reduced infarct size compared with control (36 ± 2% vs. 53 ± 1%, P < 0.05), and SP had no effect on infarct size (53 ± 3%). PAR-2 AP significantly increased phosphorylation of ERK1/2, p90RSK, and BAD but not Akt or ERK5. Accordingly, the infarct-size sparing effect of PAR-2 AP was abolished by PD (PAR-2 AP, 36 ± 2% vs. PD + PAR-2 AP, 50 ± 1%; P < 0.05) and by Chel (Chel + PAR-2 AP, 58 ± 2%) but not by LY (PAR-2 AP, 36 ± 2% vs. LY + PAR-2 AP, 38 ± 3%; P > 0.05). Therefore, PAR-2 activation is cardioprotective in the in vivo rat heart ischemia-reperfusion model, and this protection involves the ERK1/2 pathway and PKC.
ROK contribution to endothelin-mediated contraction in aorta and mesenteric arteries following intermittent hypoxia/hypercapnia in rats
Authors: Allahdadi Kyan J. , Walker Benjimen R. , Kanagy Nancy L.
Published: August 2007

We reported previously that intermittent hypoxia with CO2to maintain eucapnia (IH-C) elevates plasma endothelin-1 (ET-1) and arterial pressure. In small mesenteric arteries (sMA; inner diameter = 150 μm), IH-C augments ET-1 constrictor sensitivity but diminishes ET-1-induced increases in intracellular Ca2+concentration, suggesting IH-C exposure increases both ET-1 levels and ET-1-stimulated Ca2+sensitization. Because Rho-associated kinase (ROK) can mediate Ca2+sensitization, we hypothesized that augmented vasoconstrictor sensitivity to ET-1 in arteries from IH-C-exposed rats is dependent on ROK activation. In thoracic aortic rings, ET-1 contraction was not different between groups, but ROK inhibition (Y-27632, 3 and 10 μM) attenuated ET-1 contraction more in IH-C than in sham arteries (50 ± 11 and 78 ± 7% vs. 41 ± 12 and 48 ± 9% inhibition, respectively). Therefore, ROK appears to contribute more to ET-1 contraction in IH-C than in sham aorta. In sMA, ROK inhibitors did not affect ET-1-mediated constriction in sham arteries and only modestly inhibited it in IH-C arteries. In ionomycin-permeabilized sMA with intracellular Ca2+concentration held at basal levels, Y-27632 did not affect ET-1-mediated constriction in either IH-C or sham sMA and ET-1 did not stimulate ROK translocation. In contrast, inhibition of myosin light-chain kinase (ML-9, 100 μM) prevented ET-1-mediated constriction in sMA from both groups. Therefore, IH-C exposure increases ET-1 vasoconstrictor sensitivity in sMA but not in aorta. Furthermore, ET-1 constriction is myosin light-chain kinase dependent and mediated by Ca2+sensitization that is independent of ROK activation in sMA but not aorta. Thus ET-1-mediated signaling in aorta and sMA is altered by IH-C but is dependent on different second messenger systems in small vs. large arteries.
Cardiac proinflammatory pathways are altered with different dietary n-6 linoleic to n-3 α-linolenic acid ratios in normal, fat-fed pigs
Authors: Ghosh Sanjoy , Novak Elizabeth M. , Innis Sheila M.
Published: August 2007

Although dietary fat has been associated with inflammation and cardiovascular diseases (CVD), most studies have focused on individuals with preexisting diseases. However, the role of dietary fatty acids on inflammatory pathways before the onset of any abnormality may be more relevant for identifying initiating factors and interventions for CVD prevention. We fed young male pigs one of three diets differing in n-6 and n-3 polyunsaturated fatty acids (PUFA) linoleic acid (LA, 18:2n-6) and α-linolenic acid (ALA, 18:3n-3) for 30 days. Cardiac membrane phospholipid fatty acids, phospholipase A2(PLA2) isoform activities, and cyclooxygenase (COX)-1 and -2 and 5-lipoxygenase (5-LO) expression were measured. The low PUFA diet (% energy, 1.2% LA+0.06% ALA) increased arachidonic acid (AA) and decreased eicosapentaenoic acid (EPA) in heart membranes and increased Ca2+-independent iPLA2activity, COX-2 expression, and activation of 5-LO. Increasing dietary ALA while keeping LA constant (1.4% LA+1.2% ALA) decreased the heart membrane AA, increased EPA, and prevented proinflammatory enzyme activation. However, regardless of high ALA, high dietary LA (11.6% LA and 1.2% ALA) decreased EPA and led to a high heart membrane AA, and Ca2+-dependent cPLA2with a marked increase in nitrosative stress. Our results suggest that the potential cardiovascular benefit of ALA is achieved only when dietary LA is reduced concomitantly rather than fed with high LA diet. The increased nitrosative stress in the unstressed heart with high dietary LA suggests that biomarkers of nitrosative stress may offer a useful early marker of the effects of dietary fat on oxidative tissue stress.
Exercise training normalizes β-adrenoceptor expression in dogs susceptible to ventricular fibrillation
Authors: Holycross Bethany J. , Kukielka Monica , Nishijima Yoshinori , Altschuld Ruth A. , Carnes Cynthia A. , Billman George E.
Published: August 2007

Previous studies demonstrated an enhanced β2-adrenoceptor (AR) responsiveness in animals susceptible to ventricular fibrillation (VF) that was eliminated by exercise training. The present study investigated the effects of endurance exercise training on β1-AR and β2-AR expression in dogs susceptible to VF. Myocardial ischemia was induced by a 2-min occlusion of the left circumflex artery during the last minute of exercise in dogs with healed infarctions: 20 had VF [susceptible (S)] and 13 did not [resistant (R)]. These dogs were randomly assigned to either 10-wk exercise training [treadmill running; n = 9 (S) or 8 (R)] or an equivalent sedentary period [ n = 11 (S) or 5 (R)]. Left ventricular tissue β-AR protein and mRNA were quantified by Western blot analysis and RT-PCR, respectively. Because β2-ARs are located in caveolae, caveolin-3 was also quantified. β1-AR gene expression decreased (∼5-fold), β2-AR gene expression was not changed, and the ratio of β2-AR to β1-AR gene expression was significantly increased in susceptible compared with resistant dogs. β1-AR protein decreased (∼50%) and β2-AR protein increased (400%) in noncaveolar fractions of the cell membrane in susceptible dogs. Exercise training returned β1-AR gene expression to levels seen in resistant animals but did not alter β2-AR protein levels in susceptible dogs. These data suggest that β1-AR gene expression was decreased in susceptible dogs compared with resistant dogs and, further, that exercise training improves β1-AR gene expression, thereby restoring a more normal β-AR balance.
Transendothelial flow inhibits neutrophil transmigration through a nitric oxide-dependent mechanism: potential role for cleft shear stress
Authors: Burns Alan R. , Zheng Zhilan , Soubra Said H. , Chen Jie , Rumbaut Rolando E.
Published: August 2007

Endothelial cells in vivo are well known to respond to parallel shear stress induced by luminal blood flow. In addition, fluid filtration across endothelium (transendothelial flow) may trigger nitric oxide (NO) production, presumably via shear stress within intercellular clefts. Since NO regulates neutrophil-endothelial interactions, we determined whether transendothelial flow regulates neutrophil transmigration. Interleukin-1β-treated human umbilical vein endothelial cell (HUVEC) monolayers cultured on a polycarbonate filter were placed in a custom chamber with or without a modest hydrostatic pressure gradient (ΔP, 10 cmH2O) to induce transendothelial flow. In other experiments, cells were studied in a parallel plate flow chamber at various transendothelial flows (ΔP = 0, 5, and 10 cmH2O) and luminal flows (shear stress of 0, 1, and 2 dyn/cm2). In the absence of luminal flow, transendothelial flow reduced transmigration of freshly isolated human neutrophils from 57% to 14% ( P < 0.05) and induced an increase in NO detected with a fluorescent assay (DAF-2DA). The NO synthase inhibitor l-NAME prevented the effects of transendothelial flow on neutrophil transmigration, while a NO donor (DETA/NO, 1 mM) inhibited neutrophil transmigration. Finally, in the presence of luminal flow (1 and 2 dyn/cm2), transendothelial flow also inhibited transmigration. On the basis of HUVEC morphometry and measured transendothelial volume flow, we estimated cleft shear stress to range from 49 to 198 dyn/cm2. These shear stress estimates, while substantial, are of similar magnitude to those reported by others with similar analyses. These data are consistent with the hypothesis that endothelial cleft shear stress inhibits neutrophil transmigration via a NO-dependent mechanism.
Tranilast attenuates diastolic dysfunction and structural injury in experimental diabetic cardiomyopathy
Authors: Kelly Darren J. , Zhang Yuan , Connelly Kim , Cox Alison J. , Martin Jennifer , Krum Henry , Gilbert Richard E.
Published: August 2007

Diastolic dysfunction is an increasingly recognized complication of diabetes that develops in relatively young patients as a result of diabetic cardiomyopathy (DCM). With recent advances in echocardiographic technology now permitting the reliable assessment of diastolic function in the rat, we examined cardiac function and structure in diabetic rodents and assessed the effects of intervening with tranilast, an antifibrotic compound that has been shown to attenuate the actions of transforming growth factor-β (TGF-β) in cardiac fibroblasts. We also sought to examine the mechanism whereby tranilast inhibits the actions of TGF-β. Six-week-old heterozygous (mRen-2)27 rats were randomized to receive either streptozotocin or citrate buffer and then further randomized to receive either tranilast (400 mg·kg−1·day−1by twice daily gavage) or vehicle for another 8 wk. Cell signaling was examined in neonatal cardiac fibroblasts. After 8 wk, diabetic rats showed evidence of impaired diastolic function with reduced early-to-late atrial wave ratio and prolonged deceleration time in association with fibrosis, apoptosis, and hypertrophy (all P < 0.05). Treatment with tranilast prevented the development of diastolic dysfunction and the histopathological features of DCM. While tranilast did not affect Smad phosphorylation, it significantly attenuated TGF-β-induced p44/42 mitogen-activated protein kinase phosphorylation.
Diminished NO release in chronic hypoxic human endothelial cells
Authors: Østergaard Louise , Stankevicius Edgaras , Andersen Malene R. , Eskildsen-Helmond Yvonne , Ledet Thomas , Mulvany Michael J. , Simonsen Ulf
Published: August 2007

The present study addressed whether chronic hypoxia is associated with reduced nitric oxide (NO) release due to decreased activation of endothelial NO synthase (eNOS). Primary cultures of endothelial cells from human umbilical veins (HUVECs) were used and exposed to different oxygen levels for 24 h, after which NO release, intracellular calcium, and eNOS activity and phosphorylation were measured after 24 h. Direct measurements using a NO microsensor showed that in contrast to 1-h exposure to 5% and 1% oxygen (acute hypoxia), histamine-evoked (10 μM) NO release from endothelial cells exposed to 5% and 1% oxygen for 24 h (chronic hypoxia) was reduced by, respectively, 58% and 40%. Furthermore, chronic hypoxia also lowered the amount and activity of eNOS enzyme. The decrease in activity could be accounted for by reduced intracellular calcium and altered eNOS phosphorylation. eNOS Ser1177and eNOS Thr495phosphorylations were reduced and increased, respectively, consistent with lowered enzyme activity. Akt kinase, which can phosphorylate eNOS Ser1177, was also decreased by hypoxia, regarding both total protein content and the phosphorylated (active) form. Moreover, the protein content of β- actin, which is known to influence the activity of eNOS, was almost halved by hypoxia, further supporting the fall in eNOS activity. In conclusion, chronic hypoxia in HUVECs reduces histamine-induced NO release as well as eNOS expression and activity. The decreased activity is most likely due to changed eNOS phosphorylation, which is supported by decreases in Akt expression and phosphorylation. By reducing NO, chronic hypoxia may accentuate endothelial dysfunction in cardiovascular disease.
Cigarette smoke irreversibly modifies glutathione in airway epithelial cells
Authors: van der Toorn Marco , Smit-de Vries Maria P. , Slebos Dirk-Jan , de Bruin Harold G. , Abello Nicolas , van Oosterhout Antoon J. M. , Bischoff Rainer , Kauffman Henk F.
Published: August 2007

In patients with chronic obstructive pulmonary disease (COPD), an imbalance between oxidants and antioxidants is acknowledged to result in disease development and progression. Cigarette smoke (CS) is known to deplete total glutathione (GSH + GSSG) in the airways. We hypothesized that components in the gaseous phase of CS may irreversibly react with GSH to form GSH derivatives that cannot be reduced (GSX), thereby causing this depletion. To understand this phenomenon, we investigated the effect of CS on GSH metabolism and identified the actual GSX compounds. CS and H2O2 (control) deplete reduced GSH in solution [Δ = −54.1 ± 1.7 μM ( P < 0.01) and −39.8 ± 0.9 μM ( P < 0.01), respectively]. However, a significant decrease of GSH + GSSG was observed after CS (Δ = −75.1 ± 7.6 μM, P < 0.01), but not after H2O2. Exposure of A549 cells and primary bronchial epithelial cells to CS decreased free sulfhydryl (-SH) groups (Δ = −64.2 ± 14.6 μM/mg protein, P < 0.05) and irreversibly modified GSH + GSSG (Δ = −17.7 ± 1.9 μM, P < 0.01) compared with nonexposed cells or H2O2 control. Mass spectrometry (MS) showed that GSH was modified to glutathione-aldehyde derivatives. Further MS identification showed that GSH was bound to acrolein and crotonaldehyde and another, yet to be identified, structure. Our data show that CS does not oxidize GSH to GSSG but, rather, reacts to nonreducible glutathione-aldehyde derivatives, thereby depleting the total available GSH pool.
Feedlot dust stimulation of interleukin-6 and -8 requires protein kinase Cε in human bronchial epithelial cells
Authors: Wyatt Todd A. , Slager Rebecca E. , DeVasure Jane , Auvermann Brent W. , Mulhern Michael L. , Von Essen Susanna , Mathisen Tracy , Floreani Anthony A. , Romberger Debra J.
Published: August 2007

Individuals exposed to dusts from concentrated animal feeding operations report increased numbers of respiratory tract symptoms, and bronchoalveolar lavage samples from such individuals demonstrate elevated lung inflammatory mediators, including interleukin (IL)-8 and IL-6. We previously found that exposure of bronchial epithelial cells to hog barn dusts resulted in a protein kinase C (PKC)-dependent increase in IL-6 and IL-8 release. We hypothesized that cattle feedlot dusts would also generate bronchial epithelial interleukin release in vitro. To test this, we used interleukin ELISAs and direct PKC isoform assays. We found that a dust extract from cattle feedlots [feedlot dust extract (FLDE)] augments PKC activity of human bronchial epithelial cells in vitro. A 5–10% dilution of FLDE stimulated a significant release of IL-6 and IL-8 at 6–24 h in a PKC-dependent manner vs. control medium-treated cells. An increase in PKCα activity was observed with 1 h of FLDE treatment, and PKCε activity was elevated at 6 h of FLDE exposure. The PKCα inhibitor, Gö-6976, did not inhibit FLDE-stimulated IL-8 and IL-6 release. However, the PKCε inhibitor, Ro 31-8220, effectively inhibited FLDE-stimulated IL-8 and IL-6 release. Inhibition of FLDE-stimulated IL-6 and IL-8 was confirmed in a dominant-negative PKCε-expressing BEAS-2B cell line but not observed in a PKCα dominant negative BEAS-2B cell line. These data support the hypothesis that FLDE exposure stimulates bronchial epithelial IL-8 and IL-6 release via a PKCε-dependent pathway.