bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of Ferredoxin and Flavodoxins in Bacillus subtilis Fatty Acid Desaturation
Authors: Chazarreta-Cifre Lorena , Martiarena Leticia , de Mendoza Diego , Altabe Silvia G.
Published: June 2011

The
Bacillus subtilis
acyl lipid desaturase (Δ5-Des) is an iron-dependent integral membrane protein able to selectively introduce double bonds into long-chain fatty acids. In the last decade since its discovery, the molecular mechanism of Δ5-Des expression has been studied extensively. However, the mechanism of desaturation, which must rely on unknown bacterial proteins for electron transfer, has not yet been explored. The
B. subtilis
genome encodes three proteins that can act as potential electron donors of Δ5-Des, ferredoxin (Fer) and two flavodoxins (Flds) (YkuN and YkuP), which are encoded by the
ykuNOP
operon. Here we report that the disruption of either the
fer
gene or the
ykuNOP
operon decreases the desaturation of palmitic acid by ∼30%. Nevertheless, a
fer ykuNOP
mutant abolished the desaturation reaction almost completely. Our results establish Fer and the two Flds as redox partners for Δ5-Des and suggest that the Fer and Fld proteins could function physiologically in the biosynthesis of unsaturated fatty acids in
B. subtilis
. Although Flds have extensively been described as partners in a number of redox processes, this is the first report describing their role as electron donors in the fatty acid desaturation reaction.
Self-Transmissibility of the Integrative and Conjugative Element ICEPm1between Clinical Isolates Requires a Functional Integrase, Relaxase, and Type IV Secretion System
Authors: Flannery Erika L. , Antczak Samantha M. , Mobley Harry L. T.
Published: June 2011

Integrative and conjugative elements (ICEs), which are chromosomal mobile elements, can conjugatively transfer between bacteria. Recently, we identified a genomic island ofProteus mirabilis, a common agent of catheter-associated urinary tract infection (UTI), that possesses all the properties consistent with an ICE. This element, designated ICEPm1, is highly conserved in other causative agents of UTI, suggesting its mobility. We demonstrate that ICEPm1can actively excise from the chromosome in a clonal population of bacteria and that this excision is integrase dependent. Although inP. mirabilisHI4320, ICEPm1is annotated as integrated into the phenylalanine tRNA genepheV, we show that ICEPm1can integrate into eitherpheVorpheU. We determined that ICEPm1transfers at a frequency of 1.35 × 10−5transconjugants/donor to ICEPm1-deficientP. mirabilisusing plate mating assays with clinical isolates. Insertional inactivation of a putative integrase gene on ICEPm1decreased transfer frequencies of ICEPm1to below the limit of detection. Mutation of the relaxase of ICEPm1also eliminates transfer and demonstrates that this element is indeed self-transmissible and not transferred intrans, as are some mobilizable genomic islands. Together, these findings clearly demonstrate that ICEPm1can actively excise from the chromosome in an integrase-dependent manner, dynamically integrate into both phenylalanine tRNA genes, and transfer into clinical strains using its own conjugation machinery.
Coevolution of ABC Transporters and Two-Component Regulatory Systems as Resistance Modules against Antimicrobial Peptides in Firmicutes Bacteria
Authors: Dintner Sebastian , Staroń Anna , Berchtold Evi , Petri Tobias , Mascher Thorsten , Gebhard Susanne
Published: June 2011

In
Firmicutes
bacteria, ATP-binding cassette (ABC) transporters have been recognized as important resistance determinants against antimicrobial peptides. Together with neighboring two-component systems (TCSs), which regulate their expression, they form specific detoxification modules. Both the transport permease and sensor kinase components show unusual domain architecture: the permeases contain a large extracellular domain, while the sensor kinases lack an obvious input domain. One of the best-characterized examples is the bacitracin resistance module BceRS-BceAB of
Bacillus subtilis
. Strikingly, in this system, the ABC transporter and TCS have an absolute mutual requirement for each other in both sensing of and resistance to bacitracin, suggesting a novel mode of signal transduction in which the transporter constitutes the actual sensor. We identified over 250 such BceAB-like ABC transporters in the current databases. They occurred almost exclusively in
Firmicutes
bacteria, and 80% of the transporters were associated with a BceRS-like TCS. Phylogenetic analyses of the permease and sensor kinase components revealed a tight evolutionary correlation. Our findings suggest a direct regulatory interaction between the ABC transporters and TCSs, mediating communication between both components. Based on their observed coclustering and conservation of response regulator binding sites, we could identify putative corresponding two-component systems for transporters lacking a regulatory system in their immediate neighborhood. Taken together, our results show that these types of ABC transporters and TCSs have coevolved to form self-sufficient detoxification modules against antimicrobial peptides, widely distributed among
Firmicutes
bacteria.
A Sulfite Respiration Pathway from Thermus thermophilus and the Key Role of Newly Identified Cytochrome
c
550
Authors: Robin Sylvain , Arese Marzia , Forte Elena , Sarti Paolo , Giuffrè Alessandro , Soulimane Tewfik
Published: June 2011

Sulfite, produced for instance during amino acid metabolism, is a very reactive and toxic compound. Various detoxification mechanisms exist, but sulfite oxidoreductases (SORs) are one of the major actors in sulfite remediation in bacteria and animals. Here we describe the existence of an operon in the extreme thermophilic bacterium
Thermus thermophilus
HB8 encoding both a SOR and a diheme
c
-type cytochrome. The
in vitro
analysis clearly showed that the newly identified cytochrome
c
550
acts as an acceptor of the electrons generated by the SOR enzyme during the oxidation of sulfite. The electrons are then rapidly shuttled via cytochrome
c
552
to the terminal
ba
3
- and
caa
3
-type oxidases, thereby unveiling a novel electron transfer pathway, linking sulfite oxidation to oxygen reduction in
T. thermophilus
: sulfite → SOR
HB8
→ cytochrome
c
550
→ cytochrome
c
552
→
ba
3
oxidase/
caa
3
oxidase → O
2
. The description of the complete pathway reveals that electrons generated during sulfite oxidation by the SOR are funneled into the respiratory chain, participating in the energy production of
T. thermophilus
.
Proteins Involved in Formation of the Outermost Layer of Bacillus subtilis Spores
Authors: Imamura Daisuke , Kuwana Ritsuko , Takamatsu Hiromu , Watabe Kazuhito
Published: June 2011

To investigate the outermost structure of the
Bacillus subtilis
spore, we analyzed the accessibility of antibodies to proteins on spores of
B. subtilis
. Anti-green fluorescent protein (GFP) antibodies efficiently accessed GFP fused to CgeA or CotZ, which were previously assigned to the outermost layer termed the spore crust. However, anti-GFP antibodies did not bind to spores of strains expressing GFP fused to 14 outer coat, inner coat, or cortex proteins. Anti-CgeA antibodies bound to spores of wild-type and CgeA-GFP strains but not
cgeA
mutant spores. These results suggest that the spore crust covers the spore coat and is the externally exposed, outermost layer of the
B. subtilis
spore. We found that CotZ was essential for the spore crust to surround the spore but not for spore coat formation, indicating that CotZ plays a critical role in spore crust formation. In addition, we found that CotY-GFP was exposed on the surface of the spore, suggesting that CotY is an additional component of the spore crust. Moreover, the localization of CotY-GFP around the spore depended on CotZ, and CotY and CotZ depended on each other for spore assembly. Furthermore, a disruption of
cotW
affected the assembly of CotV-GFP, and a disruption of
cotX
affected the assembly of both CotV-GFP and CgeA-GFP. These results suggest that
cgeA
and genes in the
cotVWXYZ
cluster are involved in spore crust formation.
Comparative Analysis of the
lux
Operons in Aliivibrio logei KCh1 (a Kamchatka Isolate) and Aliivibrio salmonicida
Authors: Manukhov Ilya V. , Khrul'nova Svetlana A. , Baranova Ancha , Zavilgelsky Gennadii B.
Published: June 2011

Here we provide a molecular description of a new psychrophilic strain, KCh11, of marine luminescent bacteria classified as
Aliivibrio logei
. We sequenced the entire
lux
operon of
A. logei
KCh1 and showed that it is substantially similar to the
lux
operon of
Aliivibrio salmonicida
. It was demonstrated that the reduced production of bioluminescence in
A. salmonicida
is most likely defined by a specific defect in its
luxD
gene.
Rule-based decision support system in the biopsy diagnosis of glomerular diseases
Authors: Gupta Ruchika , Sharma Alok , Singh Sompal , Dinda Amit K
Published: June 2011

Aims

To develop a rule-based decision support system (RDSS) to assist in the diagnosis of glomerular diseases on the basis of clinical, histological and immunohistological findings.

Methods

The computer software for the RDSS was written by one of the authors. The prototypical clinical and histomorphological features, including immunofluorescence findings of various glomerular diseases, were selected by three pathologists with experience in renal pathology. For every case included, all the relevant features were fed into the RDSS in the form of one or more vectors. Diagnosis was achieved by the RDSS on the basis of shortest Euclidean distance between the vector of features of a particular ‘test’ case and vectors of the prototypical features. The diagnoses rendered by the RDSS were compared with the final diagnosis signed out by the renal pathologists; the percentage of correct diagnoses was calculated for the RDSS.

Results

A total of 612 cases of glomerular diseases were included in the analysis. The RDSS developed in this study gave the correct diagnosis in 83.2% of the cases included. Of all the cases, membranous glomerulonephritis and lupus nephritis were most frequently diagnosed accurately. However, immunotactoid glomerulonephritis was misdiagnosed due to the lack of ultrastructural findings in the study.

Conclusion

The decision support system developed in the present study holds promise, given the high accuracy in diagnosis of glomerular diseases on the basis of clinical, histological and immunofluorescence features. Such a system may prove useful not only for diagnosis but also for postgraduate teaching and self-assessment. Results need to be confirmed in further larger studies.
Evaluation of the clinical efficacy of minimally invasive procedures for breast cancer screening at a teaching hospital
Authors: Verenhitach Beatriz Daou , Elias Simone , Patrocínio Ana Claudia , Nazário Afonso Celso Pinto , Waizberg Angela
Published: June 2011

Aims

To assess the clinical efficacy of diagnostic procedures for breast cancer at a teaching hospital using internal auditing tools and quality control measures.

Methods

A retrospective assessment of 500 patients who underwent core needle biopsy (wide-bore needle biopsy; WBN) of palpable or non-palpable breast nodes that were submitted for at least one cytological examination (fine needle aspiration (FNA) cytology and/or imprint of a WBN specimen). For statistical analysis the auditing tool and quality control proposed by the National Health Service breast screening programme was utilised.

Results

For FNA, full specificity, positive predictive value, inadequate rates and suspicious rates were satisfactory while absolute sensitivity, complete sensitivity, false negatives and false positives were unsatisfactory. For imprint, absolute sensitivity, complete sensitivity, inadequate rate from cancers and suspicious rates were satisfactory, and the remaining indicators were unsatisfactory. WBN displayed the best performance with absolute sensitivity, complete sensitivity, false negative, suspicious rates, full specificity and predictive value showing satisfactory results and only one unsatisfactory result (false positive).

Conclusions

Based on an overall analysis, WBN displayed the highest clinical efficacy compared with FNA and imprint, and demonstrated adequate safety for confirming the appropriate diagnosis and management of patients, ensuring the efficacy of the service.
Locked nucleic acid probes for enhanced detection of FLT3 D835/I836, JAK2 V617F and NPM1 mutations
Authors: Warshawsky Ilka , Mularo Frank
Published: June 2011

Aims

Detecting low-level clinically significant cancer-relevant somatic mutations can be difficult. Several technologies exist for detecting minority mutations. One method is locked nucleic acid (LNA) PCR. In this study, LNA probes were used to enhance the sensitivity for detecting FLT3 D835/I836 tyrosine kinase domain (TKD) mutations, the JAK2 V617F mutation and insertion mutations in the nucleophosmin 1 gene.

Methods

PCR was performed with and without LNA probes using DNA known to contain FLT3 D835/I836 TKD, JAK2 V617F and NPM1 mutations. FLT3 D835/I836 TKD mutations were detected following EcoRV restriction enzyme digestion and capillary electrophoresis. The JAK2 V617F mutation was detected by melt-curve analysis. NPM1 insertions were detected by capillary electrophoresis.

Results

The detection of FLT3 D835/I836, JAK2 V617F and NPM1 mutations was enhanced approximately 10–50-fold using LNA probes. Rare JAK2 double mutants gave abnormal blocking patterns with the LNA probe.

Conclusions

Adding LNA probes to existing assays is a simple way to enhance and confirm the detection of mutations, especially those at low levels.
Acute rosiglitazone treatment is cardioprotective against ischemia-reperfusion injury by modulating AMPK, Akt, and JNK signaling in nondiabetic mice
Authors: Morrison Alex , Yan Xiaoyan , Tong Chao , Li Ji
Published: June 2011

Rosiglitazone (RGZ), a peroxisome proliferator-activated receptor (PPAR)-γ agonist, has been demonstrated to possess cardioprotective properties during ischemia-reperfusion. However, this notion remains controversial as recent evidence has suggested an increased risk in cardiac events associated with long-term use of RGZ in patients with type 2 diabetes. In this study, we tested the hypothesis that acute RGZ treatment is beneficial during I/R by modulating cardioprotective signaling pathways in a nondiabetic mouse model. RGZ (1 μg/g) was injected intravenously via the tail vein 5 min before reperfusion. Myocardial infarction was significantly reduced in mice treated with RGZ compared with vehicle controls (8.7% ± 1.1% vs. 20.2% ± 2.5%, P < 0.05). Moreover, isolated hearts were subjected to 20 min of global, no-flow ischemia in an ex vivo heart perfusion system. Postischemic recovery was significantly improved with RGZ treatment administered at the onset of reperfusion compared with vehicle ( P < 0.001). Immunoblot analysis data revealed that the levels of both phospho-AMP-activated protein kinase (Thr172) and phospho-Akt (Ser473) were significantly upregulated when RGZ was administered 5 min before reperfusion compared with vehicle. On the other hand, inflammatory signaling [phospho-JNK (Thr183/Tyr185)] was significantly downregulated as a result of RGZ treatment compared with vehicle ( P < 0.05). Intriguingly, pretreatment with the selective PPAR-γ inhibitor GW-9662 (1 μg/g iv) 10 min before reperfusion significantly attenuated these beneficial effects of RGZ on the ischemic heart. Taken together, acute treatment with RGZ can reduce ischemic injury in a nondiabetic mouse heart via modulation of AMP-activated protein kinase, Akt, and JNK signaling pathways, which is dependent on PPAR-γ activation.
Direct regulation of membrane type 1 matrix metalloproteinase following myocardial infarction causes changes in survival, cardiac function, and remodeling
Authors: Zavadzkas Juozas A. , Mukherjee Rupak , Rivers William T. , Patel Risha K. , Meyer Evan C. , Black Laurel E. , McKinney Richard A. , Oelsen J. Marshall , Stroud Robert E. , Spinale Francis G.
Published: June 2011

The membrane type 1 matrix metalloproteinase (MT1-MMP) is increased in left ventricular (LV) failure. However, the direct effects of altered MT1-MMP levels on survival, LV function, and geometry following myocardial infarction (MI) and the proteolytic substrates involved in this process remain unclear. MI was induced in mice with cardiac-restricted overexpression of MT1-MMP (MT1-MMPexp; full length human), reduced MT1-MMP expression (heterozygous; MT1-MMP+/−), and wild type. Post-MI survival was reduced with MT1-MMPexp and increased with MT1-MMP+/− compared with WT. LV ejection fraction was lower in the post-MI MT1-MMPexp mice compared with WT post-MI and was higher in the MT1-MMP+/− mice. In vivo localization of MT1-MMP using antibody-conjugated microbubbles revealed higher MT1-MMP levels post-MI, which were the highest in the MT1-MMPexp group and the lowest in the MT1-MMP+/− group. LV collagen content within the MI region was higher in the MT1-MMPexp vs. WT post-MI and reduced in the MT1-MMP+/− group. Furthermore, it was demonstrated that MT1-MMP proteolytically processed the profibrotic molecule, latency-associated transforming growth factor-1-binding protein (LTBP-1), and MT1-MMP-specific LTBP-1 proteolytic activity was increased by over fourfold in the post-MI MT1-MMPexp group and reduced in the MT1-MMP+/− group, which was directionally paralleled by phospho-Smad-3 levels, a critical signaling component of the profibrotic transforming growth factor pathway. We conclude that modulating myocardial MT1-MMP levels affected LV function and matrix structure, and a contributory mechanism for these effects is through processing of profibrotic signaling molecules. These findings underscore the diversity of biological effects of certain MMP types on the LV remodeling process.
Persistent sympathoexcitation long after submaximal exercise in subjects with and without coronary artery disease
Authors: Wang Norman C. , Chicos Alexandru , Banthia Smriti , Bergner Daniel W. , Lahiri Marc K. , Ng Jason , Subačius Haris , Kadish Alan H. , Goldberger Jeffrey J.
Published: June 2011

There is an increased risk of cardiac events after exercise, which may, in part, be mediated by the sympathoexcitation that accompanies exercise. The duration and extent of this sympathoexcitation following moderate exercise is unknown, particularly in those with coronary artery disease (CAD). Twenty control subjects (mean age, 51 years) and 89 subjects with CAD (mean age, 58 years) underwent two 16-min bicycle exercise sessions followed by 30–45 min of recovery. Session 1 was performed under physiological conditions to peak workloads of 50–100 W. In session 2, parasympathetic blockade with atropine (0.04 mg/kg) was achieved at end exercise at the same workload as session 1. RR interval was continually recorded, and plasma catecholamines were measured at rest and selected times during exercise and recovery. Parasympathetic effect, measured as the difference in RR interval with and without atropine, did not differ between controls and CAD subjects in recovery. At 30 and 45 min of recovery, RR intervals were 12% and 9%, respectively, shorter than at rest. At 30 and 45 min of recovery, plasma norepinephrine levels were 15% and 12%, respectively, higher than at rest. A brief period of moderate exercise is associated with a prolonged period of sympathoexcitation extending >45 min into recovery and is quantitatively similar among control subjects and subjects with CAD, with or without left ventricular dysfunction. Parasympathetic reactivation occurs early after exercise and is also surprisingly quantitatively similar in controls and subjects with CAD. The role of these autonomic changes in precipitating cardiac events requires further evaluation.
IRAG and novel PKG targeting in the cardiovascular system
Authors: Schlossmann Jens , Desch Matthias
Published: June 2011

Signaling by nitric oxide (NO) determines several cardiovascular functions including blood pressure regulation, cardiac and smooth muscle hypertrophy, and platelet function. NO stimulates the synthesis of cGMP by soluble guanylyl cyclases and thereby activates cGMP-dependent protein kinases (PKGs), mediating most of the cGMP functions. Hence, an elucidation of the PKG signaling cascade is essential for the understanding of the (patho)physiological aspects of NO. Several PKG signaling pathways were identified, meanwhile regulating the intracellular calcium concentration, mediating calcium desensitization or cytoskeletal rearrangement. During the last decade it emerged that the inositol trisphosphate receptor-associated cGMP-kinase substrate (IRAG), an endoplasmic reticulum-anchored 125-kDa membrane protein, is a main signal transducer of PKG activity in the cardiovascular system. IRAG interacts specifically in a trimeric complex with the PKG1β isoform and the inositol 1,4,5-trisphosphate receptor I and, upon phosphorylation, reduces the intracellular calcium release from the intracellular stores. IRAG motifs for phosphorylation and for targeting to PKG1β and 1,4,5-trisphosphate receptor I were identified by several approaches. The (patho)physiological functions for the regulation of smooth muscle contractility and the inhibition of platelet activation were perceived. In this review, the IRAG recognition, targeting, and function are summarized compared with PKG and several PKG substrates in the cardiovascular system.
Enhanced endothelin-1 system activity with overweight and obesity
Authors: Weil Brian R. , Westby Christian M. , Van Guilder Gary P. , Greiner Jared J. , Stauffer Brian L. , DeSouza Christopher A.
Published: June 2011

Endothelin (ET)-1-mediated vasoconstrictor tone contributes to the development and progression of several adiposity-related conditions, including hypertension and atherosclerotic vascular disease. The aims of the present study were to determine 1) whether endogenous ET-1 vasoconstrictor activity is elevated in overweight and obese adults, and, if so, 2) whether increased ET-1-mediated vasoconstriction contributes to the adiposity-related impairment in endothelium-dependent vasodilation. Seventy-nine adults were studied: 34 normal weight [body mass index (BMI) < 25 kg/m2], 22 overweight (BMI ≥ 25 and < 30 kg/m2), and 23 obese (BMI ≥ 30 kg/m2). Forearm blood flow (FBF) responses to intra-arterial infusion of ET-1 (5 pmol/min for 20 min) and selective ET-1 receptor blockade (BQ-123, 100 nmol/min for 60 min) were determined. In a subset of the study population, FBF responses to ACh (4.0, 8.0, and 16.0 μg·100 ml tissue−1·min−1) were measured in the absence and presence of selective ET-1 receptor blockade. The vasoconstrictor response to ET-1 was significantly blunted in overweight and obese adults (∼70%) compared with normal weight adults. Selective ET-1 receptor blockade elicited a significant vasodilator response (∼20%) in overweight and obese adults but did not alter FBF in normal weight adults. Coinfusion of BQ-123 did not affect FBF responses to ACh in normal weight adults but resulted in an ∼20% increase ( P < 0.05) in ACh-induced vasodilation in overweight and obese adults. These results demonstrate that overweight and obesity are associated with enhanced ET-1-mediated vasoconstriction that contributes to endothelial vasodilator dysfunction and may play a role in the increased prevalence of hypertension with increased adiposity.
Understanding Guyton's venous return curves
Authors: Beard Daniel A. , Feigl Eric O.
Published: June 2011

Based on observations that as cardiac output (as determined by an artificial pump) was experimentally increased the right atrial pressure decreased, Arthur Guyton and coworkers proposed an interpretation that right atrial pressure represents a back pressure restricting venous return (equal to cardiac output in steady state). The idea that right atrial pressure is a back pressure limiting cardiac output and the associated idea that “venous recoil” does work to produce flow have confused physiologists and clinicians for decades because Guyton's interpretation interchanges independent and dependent variables. Here Guyton's model and data are reanalyzed to clarify the role of arterial and right atrial pressures and cardiac output and to clearly delineate that cardiac output is the independent (causal) variable in the experiments. Guyton's original mathematical model is used with his data to show that a simultaneous increase in arterial pressure and decrease in right atrial pressure with increasing cardiac output is due to a blood volume shift into the systemic arterial circulation from the systemic venous circulation. This is because Guyton's model assumes a constant blood volume in the systemic circulation. The increase in right atrial pressure observed when cardiac output decreases in a closed circulation with constant resistance and capacitance is due to the redistribution of blood volume and not because right atrial pressure limits venous return. Because Guyton's venous return curves have generated much confusion and little clarity, we suggest that the concept and previous interpretations of venous return be removed from educational materials.
Role of Pyk2 in cardiac arrhythmogenesis
Authors: Lang Di , Glukhov Alexey V. , Efimova Tatiana , Efimov Igor R.
Published: June 2011

Proline-rich tyrosine kinase 2 (Pyk2) is a nonreceptor protein kinase regulated by intracellular Ca2+, CaMK, and PKC and can be activated by different stress signals involved in heart failure. However, Pyk2 has not been investigated in the human heart, and the functional role of Pyk2 signaling at the whole heart level has not been elucidated. We hypothesize that Ca2+-dependent activation of Pyk2 is involved in cardiac electrophysiology. We examined the expression of Pyk2 in nonfailing versus ischemic and nonischemic failing human hearts ( n = 6 hearts/group). To investigate Pyk2 function, we optically mapped perfused hearts from wild-type (WT; n = 7) and knockout (Pyk2−/−; n = 8) mice during autonomic stimulation. Experiments were done in control mice and after 1 wk of transverse aortic constriction. We used the Illumina beadarray approach for transcriptional profiling of WT and Pyk2−/− mouse ventricles. Western blot analysis revealed a doubling of Pyk2 activation in nonischemic failing versus nonfailing human hearts. In mouse hearts, we observed a much higher probability of ventricular tachyarrhythmia during ACh perfusion in Pyk2−/− versus WT mice. Parasympathetic stimulation resulted in a dose-dependent decrease of atrial action potential duration (APD) in both WT and Pyk2−/− mice, whereas in ventricles it induced APD shortening in Pyk2−/− mice but not in WT mice. Deficiency of Pyk2 abolished ACh-induced prolongation of atrioventricular delay in Pyk2−/− mouse hearts but did not affect heart rate. Lower mRNA and protein levels of sarco(endo)plasmic reticulum Ca2+-ATPase 2 and higher mRNA levels of Na+/Ca2+ exchanger 1 were detected in Pyk2−/− hearts compared with WT hearts. The transverse aortic constriction protocol did not change the phenotype. In conclusion, our results indicate a protective role of Pyk2 with respect to ventricular tachyarrhythmia during parasympathetic stimulation by regulation of gene expression related to Ca2+ handling. We hypothesize that activation of Pyk2 in the human heart during heart failure may contribute to protection against arrhythmia.
A three-dimensional insight into the complexity of flow convergence in mitral regurgitation: adjunctive benefit of anatomic regurgitant orifice area
Authors: Chandra Sonal , Salgo Ivan S. , Sugeng Lissa , Weinert Lynn , Settlemier Scott H. , Mor-Avi Victor , Lang Roberto M.
Published: June 2011

Mitral effective regurgitant orifice area (EROA) using the flow convergence (FC) method is used to quantify the severity of mitral regurgitation (MR). However, it is challenging and prone to interobserver variability in complex valvular pathology. We hypothesized that real-time three-dimensional (3D) transesophageal echocardiography (RT3D TEE) derived anatomic regurgitant orifice area (AROA) can be a reasonable adjunct, irrespective of valvular geometry. Our goals were to 1) to determine the regurgitant orifice morphology and distance suitable for FC measurement using 3D computational flow dynamics and finite element analysis (FEA), and ( 2) to measure AROA from RT3D TEE and compare it with 2D FC derived EROA measurements. We studied 61 patients. EROA was calculated from 2D TEE images using the 2D-FC technique, and AROA was obtained from zoomed RT3DE TEE acquisitions using prototype software. 3D computational fluid dynamics by FEA were applied to 3D TEE images to determine the effects of mitral valve (MV) orifice geometry on FC pattern. 3D FEA analysis revealed that a central regurgitant orifice is suitable for FC measurements at an optimal distance from the orifice but complex MV orifice resulting in eccentric jets yielded nonaxisymmetric isovelocity contours close to the orifice where the assumptions underlying FC are problematic. EROA and AROA measurements correlated well ( r = 0.81) with a nonsignificant bias. However, in patients with eccentric MR, the bias was larger than in central MR. Intermeasurement variability was higher for the 2D FC technique than for RT3DE-based measurements. With its superior reproducibility, 3D analysis of the AROA is a useful alternative to quantify MR when 2D FC measurements are challenging.
Chronic resveratrol treatment restores vascular responsiveness of cerebral arterioles in type 1 diabetic rats
Authors: Arrick Denise M. , Sun Hong , Patel Kaushik P. , Mayhan William G.
Published: June 2011

Decreased dilation of cerebral arterioles via an increase in oxidative stress may be a contributing factor in the pathogenesis of diabetes-induced complications leading to cognitive dysfunction and/or stroke. Our goal was to determine whether resveratrol, a polyphenolic compound present in red wine, has a protective effect on cerebral arterioles during type 1 diabetes (T1D). We measured the responses of cerebral arterioles in untreated and resveratrol-treated (10 mg·kg−1·day−1) nondiabetic and diabetic rats to endothelial (eNOS) and neuronal (nNOS) nitric oxide synthase (NOS)-dependent agonists and to a NOS-independent agonist. In addition, we harvested brain tissue from nondiabetic and diabetic rats to measure levels of superoxide under basal conditions. Furthermore, we used Western blot analysis to determine the protein expression of eNOS, nNOS, SOD-1, and SOD-2 in cerebral arterioles and/or brain tissue from untreated and resveratrol-treated nondiabetic and diabetic rats. We found that T1D impaired eNOS- and nNOS-dependent reactivity of cerebral arterioles but did not alter NOS-independent vasodilation. While resveratrol did not alter responses in nondiabetic rats, resveratrol prevented T1D-induced impairment in eNOS- and nNOS-dependent vasodilation. In addition, superoxide levels were higher in brain tissue from diabetic rats and resveratrol reversed this increase. Furthermore, eNOS and nNOS protein were increased in diabetic rats and resveratrol produced a further increased eNOS and nNOS proteins. SOD-1 and SOD-2 proteins were not altered by T1D, but resveratrol treatment produced a decrease in SOD-2 protein. Our findings suggest that resveratrol restores vascular function and oxidative stress in T1D. We suggest that our findings may implicate an important therapeutic potential for resveratrol in treating T1D-induced cerebrovascular dysfunction.
In vivo bioluminescence for tracking cell fate and function
Authors: de Almeida Patricia E. , van Rappard Juliaan R. M. , Wu Joseph C.
Published: June 2011

Tracking the fate and function of cells in vivo is paramount for the development of rational therapies for cardiac injury. Bioluminescence imaging (BLI) provides a means for monitoring physiological processes in real time, ranging from cell survival to gene expression to complex molecular processes. In mice and rats, BLI provides unmatched sensitivity because of the absence of endogenous luciferase expression in mammalian cells and the low background luminescence emanating from animals. In the field of stem cell therapy, BLI provides an unprecedented means to monitor the biology of these cells in vivo, giving researchers a greater understanding of their survival, migration, immunogenicity, and potential tumorigenicity in a living animal. In addition to longitudinal monitoring of cell survival, BLI is a useful tool for semiquantitative measurements of gene expression in vivo, allowing a better optimization of drug and gene therapies. Overall, this technology not only enables rapid, reproducible, and quantitative monitoring of physiological processes in vivo but also can measure the influences of therapeutic interventions on the outcome of cardiac injuries.
Characterization of Ath29, a major mouse atherosclerosis susceptibility locus, and identification of Rcn2 as a novel regulator of cytokine expression
Authors: Manichaikul Ani , Wang Qian , Shi Yu Lena , Zhang Zhimin , Leitinger Norbert , Shi Weibin
Published: June 2011

Ath29 is an atherosclerosis susceptibility locus on chromosome 9 identified in an intercross between C57BL/6 (B6) and C3H/HeJ (C3H) apolipoprotein E-deficient (apoE−/−) mice. This locus was subsequently replicated in two separate intercrosses that developed early or advanced atherosclerotic lesions. The objective of this study was to characterize Ath29 through construction and analysis of a congenic strain and identify underlying candidate genes. A congenic line was constructed by introgressing the chromosomal segment harboring Ath29 from C3H.apoE−/− into B6.apoE−/− mice. Congenic mice developed significantly smaller early and advance atherosclerotic lesions than B6.apoE−/− mice. Microarray analysis revealed 317 genes to be differentially expressed in the aorta of congenic mice compared with B6.apoE−/− mice. Pathway analysis of these genes suggested the Ca2+ signaling pathway to be implicated in regulating atherosclerosis susceptibility. Rcn2 is located underneath the linkage peak of Ath29 and involved in Ca2+ signaling. Multiple single-nucleotide polymorphisms between B6 and C3H mice were detected within and surrounding Rcn2 with one single-nucleotide polymorphism falling within an upstream cAMP response element. Immunostaining demonstrated its expression in atherosclerotic lesions. Knockdown of Rcn2 with small interfering RNAs resulted in significant reductions in both baseline and oxidized phospholipid-induced VCAM-1 and monocyte chemoattractant protein-1 expression by endothelial cells. Ath29 is confirmed to be a major atherosclerosis susceptibility locus affecting both early and advanced lesion formation in mice, and Rcn2 is identified as a novel regulator of cytokine expression.
Visualizing calcium responses to acetylcholine convection along endothelium of arteriolar networks in Cx40BAC-GCaMP2 transgenic mice
Authors: Bagher Pooneh , Davis Michael J. , Segal Steven S.
Published: June 2011

Acetylcholine evokes endothelium-dependent vasodilation subsequent to a rise in intracellular calcium. Despite widespread application in human and animal studies, calcium responses to intravascular ACh have not been visualized in vivo. Microiontophoresis of ACh in tissue adjacent to an arteriole activates abluminal muscarinic receptors on endothelial cells within a “local” region of diffusion, but it is unknown whether ACh released in such fashion gains access to the flow stream resulting in further actions downstream. To test this hypothesis and provide new insight into calcium signaling in vivo, we studied the cremaster muscle microcirculation of anesthetized male Cx40BAC-GCaMP2 transgenic mice ( n = 22; 5–9 mo; 33 ± 1 g) expressing the fluorescent calcium sensor GCaMP2 selectively in arteriolar endothelial cells. Submaximal ACh stimuli were delivered using microiontophoresis (1-μm pipette tip, 500 nA). With stimulus duration <500 ms or with the micropipette positioned within one vessel diameter (∼30 μm) away from an arteriole, endothelial cell calcium fluorescence was restricted to the region of ACh diffusion (<200 μm). In contrast, with the micropipette tip positioned immediately adjacent to an arteriole or within its lumen, calcium fluorescence encompassed entire networks downstream. The velocity of downstream calcium signaling in response to ACh increased with centerline velocity of fluorescent tracer microbeads ( r2 > 0.99; range: <1 mm/s to >10 mm/s). Diverting arteriolar blood flow into a side branch redirected downstream fluorescence responses to ACh; occluding flow abolished responses. Blocking luminal muscarinic receptors (intravascular glycopyrrolate; 6 μg/kg) inhibited downstream responses reversibly. Through visualizing the actions of a “local” ACh stimulus on endothelial cell calcium fluorescence in vivo, the present findings illustrate that transmural diffusion and convection of an agonist can activate entire networks of arteriolar endothelial cells concomitant with its delivery in the flow stream.
Adiponectin mediates cardioprotection in oxidative stress-induced cardiac myocyte remodeling
Authors: Essick Eric E. , Ouchi Noriyuki , Wilson Richard M. , Ohashi Koji , Ghobrial Joanna , Shibata Rei , Pimentel David R. , Sam Flora
Published: June 2011

Reactive oxygen species (ROS) induce matrix metalloproteinase (MMP) activity that mediates hypertrophy and cardiac remodeling. Adiponectin (APN), an adipokine, modulates cardiac hypertrophy, but it is unknown if APN inhibits ROS-induced cardiomyocyte remodeling. We tested the hypothesis that APN ameliorates ROS-induced cardiomyocyte remodeling and investigated the mechanisms involved. Cultured adult rat ventricular myocytes (ARVM) were pretreated with recombinant APN (30 μg/ml, 18 h) followed by exposure to physiologic concentrations of H2O2 (1–200 μM). ARVM hypertrophy was measured by [3H]leucine incorporation and atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) gene expression by RT-PCR. MMP activity was assessed by in-gel zymography. ROS was induced with angiotensin (ANG)-II (3.2 mg·kg−1·day−1 for 14 days) in wild-type (WT) and APN-deficient (APN-KO) mice. Myocardial MMPs, tissue inhibitors of MMPs (TIMPs), p-AMPK, and p-ERK protein expression were determined. APN significantly decreased H2O2-induced cardiomyocyte hypertrophy by decreasing total protein, protein synthesis, ANF, and BNP expression. H2O2-induced MMP-9 and MMP-2 activities were also significantly diminished by APN. APN significantly increased p-AMPK in both nonstimulated and H2O2-treated ARVM. H2O2-induced p-ERK activity and NF-κB activity were both abrogated by APN pretreatment. ANG II significantly decreased myocardial p-AMPK and increased p-ERK expression in vivo in APN-KO vs. WT mice. ANG II infusion enhanced cardiac fibrosis and MMP-2-to-TIMP-2 and MMP-9-to-TIMP-1 ratios in APN-KO vs. WT mice. Thus APN inhibits ROS-induced cardiomyocyte remodeling by activating AMPK and inhibiting ERK signaling and NF-κB activity. Its effects on ROS and ultimately on MMP expression define the protective role of APN against ROS-induced cardiac remodeling.
Antecedent hydrogen sulfide elicits an anti-inflammatory phenotype in postischemic murine small intestine: role of heme oxygenase-1
Authors: Zuidema Mozow Y. , Peyton Kelly J. , Fay William P. , Durante William , Korthuis Ronald J.
Published: June 2011

We recently demonstrated that preconditioning with an exogenous hydrogen sulfide donor (NaHS-PC) 24 h before ischemia and reperfusion (I/R) causes postcapillary venules to shift to an anti-inflammatory phenotype in C57BL/6J wild-type (WT) mice such that these vessels fail to support increases in postischemic leukocyte rolling (LR) and leukocyte adhesion (LA). The objective of the present study was to determine whether heme oxygenase-1 (HO-1) is a mediator of these anti-inflammatory effects noted during I/R in mice preconditioned with NaHS. Intravital fluorescence microscopy was used to visualize LR and LA in single postcapillary venules of the murine small intestine. I/R induced marked increases in LR and LA, effects that were prevented by NaHS-PC. Treatment with the HO inhibitor tin protoporphyrin IX, but not the inactive protoporphyrin CuPPIX, just before reperfusion prevented the anti-inflammatory effects of antecedent NaHS. The anti-inflammatory effects of NaHS-PC were mimicked by preconditioning with hemin, an agent that induces HO-1 expression. We then evaluated the effect of NaHS as a preconditioning stimulus in mice that were genetically deficient in HO-1 (HO-1−/−on an H129 background with appropriate WT strain controls). NaHS-PC was ineffective in HO-1−/−mice. Our work indicates that HO-1 serves as an effector of the anti-inflammatory effects of NaHS-PC during I/R 24 h later.
Vascular reactivity to calcitonin gene-related peptide is enhanced in subtotal nephrectomy-salt induced hypertension
Authors: Supowit Scott C. , Katki Khurshed A. , Hein Travis W. , Gupta Prakash , Kuo Lih , Dickerson Ian M. , DiPette Donald J.
Published: June 2011

In subtotal nephrectomy (SN)- and salt-induced hypertension, calcitonin gene-related peptide (CGRP) plays a compensatory role to attenuate the blood pressure increase in the absence of an increase in the neuronal synthesis and release of this peptide. Therefore, the purpose of this study was to determine whether the mechanism of this antihypertensive activity is through enhanced sensitivity of the vasculature to the dilator actions of this neuropeptide. Hypertension was induced in Sprague-Dawley rats by SN and 1% saline drinking water. Control rats were sham-operated and given tap water to drink. After 11 days, rats had intravenous (drug administration) and arterial (continuous mean arterial pressure recording) catheters surgically placed and were studied in a conscious unrestrained state. Baseline mean arterial pressure was higher in the SN-salt rats (157 ± 5 mmHg) compared with controls (128 ± 3 mmHg). Administration of CGRP (and adrenomedullin) produced a significantly greater dose-dependent decrease in mean arterial pressure in SN-salt rats compared with controls (∼2.0-fold for both the low and high doses). Interestingly, isolated superior mesenteric arterioles from SN-salt rats were significantly more responsive to the dilator effects of CGRP (but not adenomedullin) than the controls (pEC50, SN-salt, 14.0 ± 0.1 vs. control, 12.0 ± 0.1). Analysis of the CGRP receptor proteins showed that only the receptor component protein was increased significantly in arterioles from SN-salt rats. These data indicate that the compensatory antihypertensive effects of CGRP result from an increased sensitivity of the vasculature to dilator activity of this peptide. The mechanism may be via the upregulation of receptor component protein, thereby providing a more efficient coupling of the receptor to the signal transduction pathways.
Ca2+-induced delayed afterdepolarizations are triggered by dyadic subspace Ca2+ affirming that increasing SERCA reduces aftercontractions
Authors: Fink Martin , Noble Penelope J. , Noble Denis
Published: June 2011

Ca2+-induced delayed afterdepolarizations (DADs) are depolarizations that occur after full repolarization. They have been observed across multiple species and cell types. Experimental results have indicated that the main cause of DADs is Ca2+ overload. The main hypothesis as to their initiation has been Ca2+ overflow from the overloaded sarcoplasmic reticulum (SR). Our results using 37 previously published mathematical models provide evidence that Ca2+-induced DADs are initiated by the same mechanism as Ca2+-induced Ca2+ release, i.e., the modulation of the opening of ryanodine receptors (RyR) by Ca2+ in the dyadic subspace; an SR overflow mechanism was not necessary for the induction of DADs in any of the models. The SR Ca2+ level is better viewed as a modulator of the appearance of DADs and the magnitude of Ca2+ release. The threshold for the total Ca2+ level within the cell (not only the SR) at which Ca2+ oscillations arise in the models is close to their baseline level (∼1- to 3-fold). It is most sensitive to changes in the maximum sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pump rate (directly proportional), the opening probability of RyRs, and the Ca2+ diffusion rate from the dyadic subspace into the cytosol (both indirectly proportional), indicating that the appearance of DADs is multifactorial. This shift in emphasis away from SR overload as the trigger for DADs toward a multifactorial analysis could explain why SERCA overexpression has been shown to suppress DADs (while increasing contractility) and why DADs appear during heart failure (at low SR Ca2+ levels).
Enhancement of myofilament calcium sensitivity by acute hypoxia in rat distal pulmonary arteries
Authors: Weigand Letitia , Shimoda Larissa A. , Sylvester J. T.
Published: June 2011

Hypoxic contraction of pulmonary arterial smooth muscle is thought to require increases in both intracellular Ca2+concentration ([Ca2+]i) and myofilament Ca2+sensitivity, which may or may not be endothelium-dependent. To examine the effects of hypoxia and endothelium on Ca2+sensitivity in pulmonary arterial smooth muscle, we measured the relation between [Ca2+]iand isometric force at 37°C during normoxia (21% O2-5% CO2) and after 30 min of hypoxia (1% O2-5% CO2) in endothelium-intact (E+) and -denuded (E−) rat distal intrapulmonary arteries (IPA) permeabilized with staphylococcal α-toxin. Endothelial denudation enhanced Ca2+sensitivity during normoxia but did not alter the effects of hypoxia, which shifted the [Ca2+]i-force relation to higher force in E+ and E− IPA. Neither hypoxia nor endothelial denudation altered Ca2+sensitivity in mesenteric arteries. In E+ and E− IPA, hypoxic enhancement of Ca2+sensitivity was abolished by the nitric oxide synthase inhibitor Nω-nitro-l-arginine methyl ester (30 μM), which shifted normoxic [Ca2+]i-force relations to higher force. In E− IPA, the Rho kinase antagonist Y-27632 (10 μM) shifted the normoxic [Ca2+]i-force relation to lower force but did not alter the effects of hypoxia. These results suggest that acute hypoxia enhanced myofilament Ca2+sensitivity in rat IPA by decreasing nitric oxide production and/or activity in smooth muscle, thereby revealing a high basal level of Ca2+sensitivity, due in part to Rho kinase, which otherwise did not contribute to Ca2+sensitization by hypoxia.
Quantitative characterization of postnatal growth trends in proximal pulmonary arteries in rats by phase-contrast magnetic resonance imaging
Authors: Razavi Hedi , Zarafshar Shahrzad Y. , Sawada Hirofumi , Taylor Charles A. , Feinstein Jeffrey A.
Published: June 2011

Malformations of the pulmonary arteries can increase right heart workload and result in morbidity, heart failure, and death. With the increased use of murine models to study these malformations, there is a pressing need for an accurate and noninvasive experimental technique that is capable of characterizing pulmonary arterial hemodynamics in these animals. We describe the growth trends of pulmonary arteries in 13 male Sprague-Dawley rats at 20, 36, 52, 100, and 160 days of age with the introduction of phase-contrast MRI as such a technique. PCMRI results correlated closely with cardiac output measurements by ultrasound echocardiography and with fluorescent microspheres in right-left lung flow split (flow partition). Mean flow, average cross-sectional area, distensibility, and shear rates for the right and left pulmonary arteries (RPA and LPA) were calculated. The RPA was larger and received more flow at all times than the LPA ( P < 0.0001). Right-left flow split did not change significantly with age, and arterial distensibility was not significantly different between RPA and LPA, except at 160 days ( P < 0.01). Shear rates were much higher for the LPA than the RPA ( P < 0.0001) throughout development. The RPA and LPA showed different structure-function relationships but obeyed similar allometric scaling laws, with scaling exponents comparable to those of the main pulmonary artery. This study is the first to quantitatively describe changes in RPA and LPA flows and sizes with development and to apply phase-contrast MRI techniques to pulmonary arteries in rats.