bluuebunny/crossref_metadata_2025 huggingface dataset:
Altered cardiac metabolic phenotype after prolonged inhibition of NO synthesis in chronically instrumented dogs
Authors: d'Agostino Chiara , Labinskyy Volodymyr , Lionetti Vincenzo , Chandler Margaret P. , Lei Biao , Matsuo Ken , Bellomo Michelle , Xu Xiaobin , Hintze Thomas H. , Stanley William C. , Recchia Fabio A.
Published: January 2006

Acute inhibition of nitric oxide (NO) synthase causes a reversible alteration in myocardial substrate metabolism. We tested the hypothesis that prolonged NO synthase inhibition alters cardiac metabolic phenotype. Seven chronically instrumented dogs were treated with Nω-nitro-l-arginine methyl ester (l-NAME, 35 mg·kg−1·day−1po) for 10 days to inhibit NO synthesis, and seven were used as controls. Cardiac free fatty acid, glucose, and lactate oxidation were measured by infusion of [3H]oleate, [14C]glucose, and [13C]lactate, respectively. After 10 days of l-NAME administration, despite no differences in left ventricular afterload, cardiac O2consumption was significantly increased by 30%, consistent with a marked enhancement in baseline oxidation of glucose (6.9 ± 2.0 vs. 1.7 ± 0.5 μmol·min−1·100 g−1, P < 0.05 vs. control) and lactate (21.6 ± 5.6 vs. 11.8 ± 2.6 μmol·min−1·100 g−1, P < 0.05 vs. control). When left ventricular afterload was increased by ANG II infusion to stimulate myocardial metabolism, glucose oxidation was augmented further in the l-NAME than in the control group, whereas free fatty acid oxidation decreased. Exogenous NO (diethylamine nonoate, 0.01 μmol·kg−1·min−1iv) could not reverse this metabolic alteration. Consistent with the accelerated rate of carbohydrate oxidation, total myocardial pyruvate dehydrogenase activity and protein expression were higher (38 and 34%, respectively) in the l-NAME than in the control group. Also, protein expression of the constitutively active glucose transporter GLUT-1 was significantly elevated (46%) vs. control. We conclude that prolonged NO deficiency causes a profound alteration in cardiac metabolic phenotype, characterized by selective potentiation of carbohydrate oxidation, that cannot be reversed by a short-term infusion of exogenous NO. This phenomenon may constitute an adaptive mechanism to counterbalance cardiac mechanical inefficiency.
Rapamycin antagonizes NF-κB nuclear translocation activated by TNF-α in primary vascular smooth muscle cells and enhances apoptosis
Authors: Giordano Arturo , Avellino Raffaella , Ferraro Paolo , Romano Simona , Corcione Nicola , Romano Maria Fiammetta
Published: January 2006

Several lines of evidence support the view that rapamycin inhibits NF-κB. TNF-α, a potent inducer of NF-κB, is released after artery injury (e.g., balloon angioplasty) and plays an important role in inflammation and restenosis. We investigated the effect of rapamycin on NF-κB activation and apoptosis in vascular smooth muscle cells (VSMCs) stimulated with TNF-α. Using EMSA, we found that TNF-α caused NF-κB nuclear translocation in VSMCs after 1 h of incubation. Rapamycin inhibited IκBα degradation, thereby preventing nuclear translocation. Activation of NF-κB was accompanied by an increase of Bcl-xL and Bfl-1/A1 proteins, detected by Western blot assay, whereas rapamycin prevented the TNF-α-induced enhancement of these antiapoptotic proteins. The extent of apoptosis of VSMCs exposed to TNF-α was significantly enhanced by rapamycin. The effect of rapamycin appeared to be independent of the phosphatidylinositol 3-kinase/Akt-protein kinase B survival pathway, because the phosphatidylinositol 3-kinase inhibitor wortmannin neither prevented IκBα degradation nor increased apoptosis of cells incubated with TNF-α. Finally, we demonstrate that the large immunophilin FK-506 binding protein FKBP51 is essential for TNF-α-induced NF-κB activation in VSMCs. Our findings show that rapamycin inhibits NF-κB activation and acts in concert with TNF-α in induction of VSMC apoptosis.
PGE2stimulates human brain natriuretic peptide expression via EP4and p42/44 MAPK
Authors: Qian Jian-Yong , Leung Alicia , Harding Pamela , LaPointe Margot C.
Published: January 2006

Brain natriuretic peptide (BNP) produced by cardiac myocytes has antifibrotic and antigrowth properties and is a marker of cardiac hypertrophy. We previously showed that prostaglandin E2(PGE2) is the main prostaglandin produced in myocytes treated with proinflammatory stimuli and stimulates protein synthesis by binding to its EP4receptor. We hypothesized that PGE2, acting through EP4, also regulates BNP gene expression. We transfected neonatal ventricular myocytes with a plasmid encoding the human BNP (hBNP) promoter driving expression of a luciferase reporter gene. PGE2increased hBNP promoter activity 3.5-fold. An EP4antagonist reduced the stimulatory effect of PGE2but not an EP1antagonist. Because EP4signaling can involve adenylate cyclase, cAMP, and protein kinase A (PKA), we tested the effect of H-89, a PKA inhibitor, on PGE2stimulation of the hBNP promoter. H-89 at 5 μM decreased PGE2stimulation of BNP promoter activity by 100%. Because p42/44 MAPK mediates the effect of PGE2on protein synthesis, we also examined the role of MAPKs in the regulation of BNP promoter activity. PGE2stimulation of the hBNP promoter was inhibited by a MEK1/2 inhibitor and a dominant-negative mutant of Raf, indicating that p42/44 MAPK was involved. In contrast, neither a p38 MAPK inhibitor nor a JNK inhibitor reduced the stimulatory effect of PGE2. Involvement of small GTPases was also studied. Dominant-negative Rap inhibited PGE2stimulation of the hBNP promoter, but dominant-negative Ras did not. We concluded that PGE2stimulates the BNP promoter mainly via EP4, PKA, Rap, and p42/44 MAPK.
Variable ECG signs of ischemia during controlled occlusion of the left and right coronary artery in humans
Authors: De Marchi Stefano F. , Meier Pascal , Oswald Philipp , Seiler Christian
Published: January 2006

Infarct size (IS) increases with vascular occlusion time, area at risk for infarction, lack of collateral supply, absence of preconditioning, and myocardial demand for O2 supply. ECG S-T segment elevation is used as a measure of severity of ischemia and a surrogate for IS. This study in 50 patients with coronary artery disease undergoing a first 120-s balloon occlusion of a stenosis sought to determine whether S-T segment elevation, corrected for the above-mentioned variables, in the left coronary artery (LCA group, n = 36) is different from that in the right coronary artery (RCA group, n = 14) territory. After consideration of all known determinants of IS, particularly mass at risk and collateral supply, the LCA territory is more sensitive than the RCA region to a 2-min period of myocardial ischemia.
Fluorescence imaging of cardiac propagation: spectral properties and filtering of optical action potentials
Authors: Mironov Sergey F. , Vetter Frederick J. , Pertsov Arkady M.
Published: January 2006

Fluorescence imaging using voltage-sensitive dyes is an important tool for studying electrical propagation in the heart. Yet, the low amplitude of the voltage-sensitive component in the fluorescence signal and high acquisition rates dictated by the rapid propagation of the excitation wave front make it difficult to achieve recordings with high signal-to-noise ratios. Although spatially and temporally filtering the acquired signals has become de facto one of the key elements of optical mapping, there is no consensus regarding their use. Here we characterize the spatiotemporal spectra of optically recorded action potentials and determine the distortion produced by conical filters of different sizes. On the basis of these findings, we formulate the criteria for rational selection of filter characteristics. We studied the evolution of the spatial spectra of the propagating wave front after epicardial point stimulation of the isolated, perfused right ventricular free wall of the pig heart stained with di-4-ANEPPS. We found that short-wavelength (<3 mm) spectral components represent primarily noise and surface features of the preparation (coronary vessels, fat, and connective tissue). The time domain of the optical action potential spectrum also lacks high-frequency components (>100 Hz). Both findings are consistent with the reported effect of intrinsic blurring caused by light scattering inside the myocardial wall. The absence of high-frequency spectral components allows the use of aggressive low-pass spatial and temporal filters without affecting the optical action potential morphology. We show examples where the signal-to-noise ratio increased up to 150 with <3% distortion. A generalization of our approach to the rational filter selection in various applications is discussed.
Aldosterone increases voltage-gated sodium current in ventricular myocytes
Authors: Boixel Christophe , Gavillet Bruno , Rougier Jean-Sébastien , Abriel Hugues
Published: January 2006

The role of aldosterone in the pathogenesis of heart failure (HF) is still poorly understood. Recently, aldosterone has been shown to modulate the function of cardiac Ca2+and K+channels, thus playing a role in the electrical remodeling process. The goal of this work was to investigate the role of aldosterone on the cardiac Na+current ( INa). We analyzed the effects of aldosterone on INain isolated adult mouse ventricular myocytes, using the whole cell patch-clamp technique. After 24 h incubation with 1 μM aldosterone, the INadensity was significantly increased (+55%), without alteration of the biophysical properties and the cell membrane capacitance. Aldosterone (10 nM) increased the INaby 23%. In 24-h coincubation experiments, with the use of actinomycin D, cycloheximide, or brefeldin A, the effect of aldosterone on INawas abolished. Spironolactone (mineralocorticoid receptor antagonist, 10 μM) prevented the 1 μM aldosterone-dependent INaincrease, whereas RU-38486 (glucocorticoid receptor antagonist, 10 μM) did not. The action potential duration (APD) was longer in aldosterone-treated (APD90: +53%) than in control myocytes. In addition, the L-type Ca2+current was also upregulated (+48%). We performed quantitative RT-PCR measurements and Western blots to quantify the mRNA and protein levels of Nav1.5 and Cav1.2 (main channels mediating cardiac INaand ICa), but no significant difference was found. In conclusion, this study shows that aldosterone upregulates the cardiac INaand suggest that this phenomenon may contribute to the HF-induced electrical remodeling process that may be reversed by spironolactone.
Compression induced by RV pressure overload decreases regional coronary blood flow in anesthetized dogs
Authors: Gibbons Kroeker Carol A. , Adeeb Samer , Shrive Nigel G. , Tyberg John V.
Published: January 2006

Pulmonary artery constriction (PAC), a model of right ventricular (RV) pressure overload, flattens or inverts the septum and may flatten the left ventricular (LV) free wall. Finite element (FE) analysis predicts that such deformations may cause substantial compression. This study tests the hypothesis that deformation-induced myocardial compressive stress impedes coronary blood flow (CBF). Colored microspheres (∼2 × 106) were injected into the left atrium of 13 open-chest, anesthetized dogs under control conditions and during PAC, which decreased the end-diastolic transseptal pressure gradient (LV − RV) from 1.6 ± 1.3 to −3.4 ± 1.7 mmHg. Septal and LV deformation was assessed with the use of two-dimensional echocardiography, and by FE analysis, the hydrostatic component of stress was assessed. Postmortem, a 2.5-cm wide, LV equatorial ring was divided into 16 endocardial and epicardial samples. PAC decreased CBF in the FE-predicted compression zones, areas with the greatest compression having the greatest reductions in CBF. During PAC, compression reached a maximum of 25.3 ± 1.8 mmHg on the (LV) endocardial sides of the RV insertion points, areas that saw CBF decrease from 1.05 ± 0.08 to 0.68 ± 0.05 ml·min−1·g−1 ( P < 0.001), more than 30%. CBF decreased (from 1.08 ± 0.07 to 0.81 ± 0.07 ml·min−1·g−1; P < 0.001) on the RV side of the midseptum, an area with as much as 16.0 ± 1.0 mmHg of compression. Overall, average compressions of 10 mmHg decreased CBF by ∼30%. We conclude that acute RV pressure overload deforms the septum and LV and induces compressive stresses that reduce CBF substantially. This may help explain why some patients with pulmonary hypertension and no critical coronary disease have chest discomfort indistinguishable from angina pectoris.
Genetic ablation of caveolin-1 modifies Ca2+spark coupling in murine arterial smooth muscle cells
Authors: Cheng Xiaoyang , Jaggar Jonathan H.
Published: January 2006

L-type, voltage-dependent calcium (Ca2+) channels, ryanodine-sensitive Ca2+release (RyR) channels, and large-conductance Ca2+-activated potassium (KCa) channels comprise a functional unit that regulates smooth muscle contractility. Here, we investigated whether genetic ablation of caveolin-1 (cav-1), a caveolae protein, alters Ca2+spark to KCachannel coupling and Ca2+spark regulation by voltage-dependent Ca2+channels in murine cerebral artery smooth muscle cells. Caveolae were abundant in the sarcolemma of control (cav-1+/+) cells but were not observed in cav-1-deficient (cav-1−/−) cells. Ca2+spark and transient KCacurrent frequency were approximately twofold higher in cav-1−/−than in cav-1+/+cells. Although voltage-dependent Ca2+current density was similar in cav-1+/+and cav-1−/−cells, diltiazem and Cd2+, voltage-dependent Ca2+channel blockers, reduced transient KCacurrent frequency to ∼55% of control in cav-1+/+cells but did not alter transient KCacurrent frequency in cav-1−/−cells. Furthermore, although KCachannel density was elevated in cav-1−/−cells, transient KCacurrent amplitude was similar to that in cav-1+/+cells. Higher Ca2+spark frequency in cav-1−/−cells was not due to elevated intracellular Ca2+concentration, sarcoplasmic reticulum Ca2+load, or nitric oxide synthase activity. Similarly, Ca2+spark amplitude and spread, the percentage of Ca2+sparks that activated a transient KCacurrent, the amplitude relationship between sparks and transient KCacurrents, and KCachannel conductance and apparent Ca2+sensitivity were similar in cav-1+/+and cav-1−/−cells. In summary, cav-1 ablation elevates Ca2+spark and transient KCacurrent frequency, attenuates the coupling relationship between voltage-dependent Ca2+channels and RyR channels that generate Ca2+sparks, and elevates KCachannel density but does not alter transient KCacurrent activation by Ca2+sparks. These findings indicate that cav-1 is required for physiological Ca2+spark and transient KCacurrent regulation in cerebral artery smooth muscle cells.
Midbrain vlPAG inhibits rVLM cardiovascular sympathoexcitatory responses during electroacupuncture
Authors: Tjen-A-Looi Stephanie C. , Li Peng , Longhurst John C.
Published: January 2006

The periaqueductal gray (PAG) is an important integrative region in the regulation of autonomic outflow and cardiovascular function and may serve as a regulatory center as part of a long-loop pathway during somatic afferent stimulation with acupuncture. Because the ventrolateral PAG (vlPAG) provides input to the rostral ventrolateral medulla (rVLM), an important area for electroacupuncture (EA) regulation of sympathetic outflow, we hypothesized that the vlPAG plays a role in the EA-related modulation of rVLM premotor sympathetic neurons activated during visceral afferent stimulation and autonomic excitatory reflexes. Cats were anesthetized and ventilated, and heart rate and mean blood pressure were monitored. Stimulation of the splanchnic nerve by a pledget of filter paper soaked in bradykinin (BK, 10 μg/ml) every 10 min on the gallbladder induced consistent cardiovascular reflex responses. Bilateral stimulation with EA at acupoints over the pericardial meridian (P5-6) situated over the median nerve reduced the increases in blood pressure from 34 ± 3 to 18 ± 5 mmHg for a period of time that lasted for 60 min or more. Unilateral inactivation of neuronal activity in the vlPAG with 50–75 nl of kainic acid (KA, 1 mM) restored the blood pressure responses from 18 ± 3 to 36 ± 5 mmHg during BK-induced gallbladder stimulation, an effect that lasted for 30 min. In the absence of EA, unilateral microinjection of the excitatory amino acid dl-homocysteic acid (DLH, 4 nM) in the vlPAG mimicked the effect of EA and reduced the reflex blood pressure responses from 35 ± 6 to 14 ± 5 mmHg. Responses of 21 cardiovascular sympathoexcitatory rVLM neurons, including 12 that were identified as premotor neurons, paralleled the cardiovascular responses. Thus splanchnic nerve-evoked neuronal discharge of 32 ± 4 spikes/30 stimuli in six neurons was reduced to 10 ± 2 spikes/30 stimuli by EA, which was restored rapidly to 28 ± 4 spikes/30 stimuli by unilateral injection of 50 nl KA into the vlPAG. Conversely, 50 nl of DLH in the vlPAG reduced the number of action potentials of 5 rVLM neurons from 30 ± 4 to 18 ± 4 spikes/30 stimuli. We conclude that the inhibitory influence of EA involves vlPAG stimulation, which, in turn, inhibits rVLM neurons in the EA-related attenuation of the cardiovascular excitatory response during visceral afferent stimulation.
Distinct transcriptional regulation of long-chain acyl-CoA synthetase isoforms and cytosolic thioesterase 1 in the rodent heart by fatty acids and insulin
Authors: Durgan David J. , Smith Justin K. , Hotze Margaret A. , Egbejimi Oluwaseun , Cuthbert Karalyn D. , Zaha Vlad G. , Dyck Jason R. B. , Abel E. Dale , Young Martin E.
Published: January 2006

The molecular mechanism(s) responsible for channeling long-chain fatty acids (LCFAs) into oxidative versus nonoxidative pathways is (are) poorly understood in the heart. Intracellular LCFAs are converted to long-chain fatty acyl-CoAs (LCFA-CoAs) by a family of long-chain acyl-CoA synthetases (ACSLs). Cytosolic thioesterase 1 (CTE1) hydrolyzes cytosolic LCFA-CoAs to LCFAs, generating a potential futile cycle at the expense of ATP utilization. We hypothesized that ACSL isoforms and CTE1 are differentially regulated in the heart during physiological and pathophysiological conditions. Using quantitative RT-PCR, we report that the five known acsl isoforms ( acsl1, acsl3, acsl4, acsl5, and acsl6) and cte1 are expressed in whole rat and mouse hearts, as well as adult rat cardiomyocytes (ARCs). Streptozotocin-induced insulin-dependent diabetes (4 wk) and fasting (≤24 h) both dramatically induced cte1 and repressed acsl6 mRNA, with no significant effects on the other acsl isoforms. In contrast, high-fat feeding (4 wk) induced cte1 without affecting expression of the acsl isoforms in the heart. Investigation into the mechanism(s) responsible for these transcriptional changes uncovered roles for peroxisome proliferator-activated receptor-α (PPARα) and insulin as regulators of specific acsl isoforms and cte1 in the heart. Culturing ARCs with oleate (0.1–0.4 mM) or the PPARα agonists WY-14643 (1 μM) and fenofibrate (10 μM) consistently induced acsl1 and cte1. Conversely, PPARα null mouse hearts exhibited decreased acsl1 and cte1 expression. Culturing ARCs with insulin (10 nM) induced acsl6, whereas specific loss of insulin signaling within the heart (cardiac-specific insulin receptor knockout mice) caused decreased acsl6 expression. Our data expose differential regulation of acsl isoforms and cte1 in the heart, where acsl1 and cte1 are PPARα-regulated genes, whereas acsl6 is an insulin-regulated gene.
Pleiotropic role of VEGF-A in regulating fetal pulmonary mesenchymal cell turnover
Authors: Majka S. , Fox K. , McGuire B. , Crossno J. , McGuire P. , Izzo A.
Published: January 2006

Tight regulation of VEGF-A production and signaling is important for the maintenance of lung development and homeostasis. VEGF null mice have provided little insight into the role of VEGF during the later stages of lung morphogenesis. Therefore, we examined the in vitro effects of autocrine and paracrine VEGF-A production and the inhibition of VEGF-A signaling on a Flk-1-negative subset of fetal pulmonary mesenchymal cells (pMC). We hypothesized that VEGF-A receptor signaling regulates turnover of fetal lung mesenchyme in a cell cycle-dependent manner. VEGF receptor blockade with SU-5416 caused cell spreading and decreased proliferation and bcl-2 localization. Nuclear expression of the cell cycle inhibitory protein, p21, was increased with SU-5416 treatment, and p27 was absent. Autocrine VEGF production by pMC resulted in proliferation and p21/p27-dependent contact inhibition. In contrast, exogenous VEGF-A increased cell progression through the cell cycle. Selective activation of Flt by placental growth factor demonstrated the importance of this receptor/kinase in the VEGF-A responsiveness of pMC. The expression and localization of the survival factor bcl-2 was dependent on VEGF. These results provide evidence that VEGF-A plays a critical role in the regulation of fetal pulmonary mesenchymal proliferation, survival, and the subsequent development of normal lung architecture through bcl-2 and p21/p27-dependent cell cycle control.
TNF-α and IFN-γ inversely modulate expression of the IL-17E receptor in airway smooth muscle cells
Authors: Lajoie-Kadoch Stéphane , Joubert Philippe , Létuvé Séverine , Halayko Andrew J. , Martin James G. , Soussi-Gounni Abdellilah , Hamid Qutayba
Published: January 2006

The interleukin-17B receptor (IL-17BR) is expressed in a variety of tissues and is upregulated under inflammatory conditions. This receptor binds both its cognate ligand IL-17B and IL-17E/IL-25, a novel cytokine known to promote Th2 responses. The present study shows that airway smooth muscle cells express IL-17BR in vitro and that its expression is upregulated by TNF-α and downregulated by IFN-γ. Our data indicate that TNF-α upregulates IL-17BR mainly through nuclear factor-κB as assessed with the IκB kinase 2 inhibitor AS-602868. In addition, both IFN-γ and dexamethasone are able to antagonize a TNF-α-induced IL-17BR increase in mRNA expression. The mitogen-activated protein kinase kinase inhibitor U0126 totally reversed the inhibition observed with IFN-γ, suggesting the involvement of the extracellular signal-regulated kinase pathway in this effect. In addition, on stimulation with IL-17E, airway smooth muscle cells increase their expression of ECM components, namely procollagen-αI and lumican mRNA. Furthermore, immunohistochemical analysis of biopsies from asthmatic subjects reveals that this receptor is abundant in smooth muscle layers. This is the first report showing IL-17BR receptor in structural cells of the airways. Our results suggest a potential proremodeling effect of IL-17E on airway smooth muscle cells through the induction of ECM and that its receptor is upregulated by proinflammatory conditions.
Manganese and iron transport across pulmonary epithelium
Authors: Heilig Elizabeth A. , Thompson Khristy J. , Molina Ramon M. , Ivanov Alexander R. , Brain Joseph D. , Wessling-Resnick Marianne
Published: January 2006

Pathways mediating pulmonary metal uptake remain unknown. Because absorption of iron and manganese could involve similar mechanisms, transferrin (Tf) and transferrin receptor (TfR) expression in rat lungs was examined. Tf mRNA was detected in bronchial epithelium, type II alveolar cells, macrophages, and bronchus-associated lymphoid tissue (BALT). Tf protein levels in lung and bronchoalveolar lavage fluid did not change in iron deficiency despite increased plasma levels, suggesting that lung Tf concentrations are regulated by local synthesis in a manner independent of body iron status. Iron oxide exposure upregulated Tf mRNA in bronchial and alveolar epithelium, macrophages, and BALT, but protein was not significantly increased. In contrast, TfR mRNA and protein were both upregulated by iron deficiency. To examine potential interactions with lung Tf, rats were intratracheally instilled with54Mn or59Fe. Unlike59Fe, interactions between54Mn and Tf in lung fluid were not detected. Absorption of intratracheally instilled54Mn from the lungs to the blood was unimpaired in Belgrade rats homozygous for the functionally defective G185R allele of divalent metal transporter-1, indicating that this transporter is also not involved in pulmonary manganese absorption. Pharmacological studies of54Mn uptake by A549 cells suggest that metal uptake by type II alveolar epithelial cells is associated with activities of both L-type Ca2+channels and TRPM7, a member of the transient receptor potential melastatin subfamily. These results demonstrate that iron and manganese are absorbed by the pulmonary epithelium through different pathways and reveal the potential role for nonselective calcium channels in lung metal clearance.
Maternal smoking during pregnancy increases the risk of recurrent wheezing during the first years of life (BAMSE)
Authors: Lannerö Eva , Wickman Magnus , Pershagen Goran , Nordvall Lennart
Published: January 2006

Background

Exposure to cigarette smoking during foetal and early postnatal life may have implications for lung health. The aim of this study was to assess the possible effects of such exposure in utero on lower respiratory disease in children up to two years of age.

Methods

A birth cohort of 4089 newborn infants was followed for two years using parental questionnaires. When the infant was two months old the parents completed a questionnaire on various lifestyle factors, including maternal smoking during pregnancy and after birth. At one and two years of age information was obtained by questionnaire on symptoms of allergic and respiratory diseases as well as on environmental exposures, particularly exposure to environmental tobacco smoke (ETS). Adjustments were made for potential confounders.

Results

When the mother had smoked during pregnancy but not after that, there was an increased risk of recurrent wheezing up to two years' age, ORadj = 2.2, (95% CI 1.3 – 3.6). The corresponding OR was 1.6, (95% CI 1.2 – 2.3) for reported exposure to ETS with or without maternal smoking in utero. Maternal smoking during pregnancy but no exposure to ETS also increased the risk of doctor's diagnosed asthma up to two years of age, ORadj = 2.1, (95% CI 1.2 – 3.7).

Conclusion

Exposure to maternal cigarette smoking in utero is a risk factor for recurrent wheezing, as well as doctor's diagnosed asthma in children up to two yearsof age.
Phenotypical and functional characterization of alveolar macrophage subpopulations in the lungs of NO2-exposed rats
Authors: Garn Holger , Siese Anette , Stumpf Sabine , Wensing Anka , Renz Harald , Gemsa Diethard
Published: January 2006

Background

Alveolar macrophages (AM) are known to play an important role in the regulation of inflammatory reactions in the lung, e.g. during the development of chronic lung diseases. Exposure of rats to NO2has recently been shown to induce a shift in the activation type of AM that is characterized by reduced TNF-α and increased IL-10 production. So far it is unclear, whether a functional shift in the already present AM population or the occurrence of a new, phenotypically different AM population is responsible for these observations.

Methods

AM from rat and mice were analyzed by flow cytometry for surface marker expression and in vivo staining with PKH26 was applied to characterize newly recruited macrophages. Following magnetic bead separation, AM subpopulations were further analyzed for cytokine, inducible NO synthase (iNOS) and matrix metalloproteinase (MMP) mRNA expression using quantitative RT-PCR. Following in vitro stimulation, cytokines were quantitated in the culture supernatants by ELISA.

Results

In untreated rats the majority of AM showed a low expression of the surface antigen ED7 (CD11b) and a high ED9 (CD172) expression (ED7-/ED9high). In contrast, NO2exposure induced the occurrence of a subpopulation characterized by the marker combination ED7+/ED9low. Comparable changes were observed in mice and by in vivo labeling of resident AM using the dye PKH26 we could demonstrate that CD11b positive cells mainly comprise newly recruited AM. Subsequent functional analyses of separated AM subpopulations of the rat revealed that ED7+cells showed an increased expression and production of the antiinflammatory cytokine IL-10 whereas TNF-α production was lower compared to ED7-AM. However, iNOS and IL-12 expression were also increased in the ED7+subpopulation. In addition, these cells showed a significantly higher mRNA expression for the matrix metalloproteinases MMP-7, -8, -9, and -12.

Conclusion

NO2exposure induces the infiltration of an AM subpopulation that, on the one hand may exert antiinflammatory functions by the production of high amounts of IL-10 but on the other hand may contribute to the pathology of NO2-induced lung damage by selective expression of certain matrix metalloproteinases.
Cluster analysis of networks generated through homology: automatic identification of important protein communities involved in cancer metastasis
Authors: Jonsson Pall F , Cavanna Tamara , Zicha Daniel , Bates Paul A
Published: January 2006

Background

Protein-protein interactions have traditionally been studied on a small scale, using classical biochemical methods to investigate the proteins of interest. More recently large-scale methods, such as two-hybrid screens, have been utilised to survey extensive portions of genomes. Current high-throughput approaches have a relatively high rate of errors, whereas in-depth biochemical studies are too expensive and time-consuming to be practical for extensive studies. As a result, there are gaps in our knowledge of many key biological networks, for which computational approaches are particularly suitable.

Results

We constructed networks, or 'interactomes', of putative protein-protein interactions in the rat proteome – the rat being an organism extensively used for cancer studies. This was achieved by integrating experimental protein-protein interaction data from many species and translating this data into the reference frame of the rat. The putative rat protein interactions were given confidence scores based on their homology to proteins that have been experimentally observed to interact. The confidence score was furthermore weighted according to the extent of the experimental evidence, giving a higher weight to more frequently observed interactions. The scoring function was subsequently validated and networks constructed around key proteins, identified as being highly up- or down-regulated in rat cell lines of high metastatic potential. Using clustering methods on the networks, we have identified key protein communities involved in cancer metastasis.

Conclusion

The protein network generation and subsequent network analysis used here, were shown to be useful for highlighting key proteins involved in metastasis. This approach, in conjunction with microarray expression data, can be extended to other species, thereby suggesting possible pathways around proteins of interest.
Gene selection and classification of microarray data using random forest
Authors: Díaz-Uriarte Ramón , Alvarez de Andrés Sara
Published: January 2006

Background

Selection of relevant genes for sample classification is a common task in most gene expression studies, where researchers try to identify the smallest possible set of genes that can still achieve good predictive performance (for instance, for future use with diagnostic purposes in clinical practice). Many gene selection approaches use univariate (gene-by-gene) rankings of gene relevance and arbitrary thresholds to select the number of genes, can only be applied to two-class problems, and use gene selection ranking criteria unrelated to the classification algorithm. In contrast, random forest is a classification algorithm well suited for microarray data: it shows excellent performance even when most predictive variables are noise, can be used when the number of variables is much larger than the number of observations and in problems involving more than two classes, and returns measures of variable importance. Thus, it is important to understand the performance of random forest with microarray data and its possible use for gene selection.

Results

We investigate the use of random forest for classification of microarray data (including multi-class problems) and propose a new method of gene selection in classification problems based on random forest. Using simulated and nine microarray data sets we show that random forest has comparable performance to other classification methods, including DLDA, KNN, and SVM, and that the new gene selection procedure yields very small sets of genes (often smaller than alternative methods) while preserving predictive accuracy.

Conclusion

Because of its performance and features, random forest and gene selection using random forest should probably become part of the "standard tool-box" of methods for class prediction and gene selection with microarray data.
Application of machine learning in SNP discovery
Authors: Matukumalli Lakshmi K , Grefenstette John J , Hyten David L , Choi Ik-Young , Cregan Perry B , Van Tassell Curtis P
Published: January 2006

Background

Single nucleotide polymorphisms (SNP) constitute more than 90% of the genetic variation, and hence can account for most trait differences among individuals in a given species. Polymorphism detection software PolyBayes and PolyPhred give high false positive SNP predictions even with stringent parameter values. We developed a machine learning (ML) method to augment PolyBayes to improve its prediction accuracy. ML methods have also been successfully applied to other bioinformatics problems in predicting genes, promoters, transcription factor binding sites and protein structures.

Results

The ML program C4.5 was applied to a set of features in order to build a SNP classifier from training data based on human expert decisions (True/False). The training data were 27,275 candidate SNP generated by sequencing 1973 STS (sequence tag sites) (12 Mb) in both directions from 6 diverse homozygous soybean cultivars and PolyBayes analysis. Test data of 18,390 candidate SNP were generated similarly from 1359 additional STS (8 Mb). SNP from both sets were classified by experts. After training the ML classifier, it agreed with the experts on 97.3% of test data compared with 7.8% agreement between PolyBayes and experts. The PolyBayes positive predictive values (PPV) (i.e., fraction of candidate SNP being real) were 7.8% for all predictions and 16.7% for those with 100% posterior probability of being real. Using ML improved the PPV to 84.8%, a 5- to 10-fold increase. While both ML and PolyBayes produced a similar number of true positives, the ML program generated only 249 false positives as compared to 16,955 for PolyBayes. The complexity of the soybean genome may have contributed to high false SNP predictions by PolyBayes and hence results may differ for other genomes.

Conclusion

A machine learning (ML) method was developed as a supplementary feature to the polymorphism detection software for improving prediction accuracies. The results from this study indicate that a trained ML classifier can significantly reduce human intervention and in this case achieved a 5–10 fold enhanced productivity. The optimized feature set and ML framework can also be applied to all polymorphism discovery software. ML support software is written in Perl and can be easily integrated into an existing SNP discovery pipeline.
A new experimental approach for studying bacterial genomic island evolution identifies island genes with bacterial host-specific expression patterns
Authors: Wilson James W , Nickerson Cheryl A
Published: January 2006

Background

Genomic islands are regions of bacterial genomes that have been acquired by horizontal transfer and often contain blocks of genes that function together for specific processes. Recently, it has become clear that the impact of genomic islands on the evolution of different bacterial species is significant and represents a major force in establishing bacterial genomic variation. However, the study of genomic island evolution has been mostly performed at the sequence level using computer software or hybridization analysis to compare different bacterial genomic sequences. We describe here a novel experimental approach to study the evolution of species-specific bacterial genomic islands that identifies island genes that have evolved in such a way that they are differentially-expressed depending on the bacterial host background into which they are transferred.

Results

We demonstrate this approach by using a "test" genomic island that we have cloned from the Salmonella typhimurium genome (island 4305) and transferred to a range of Gram negative bacterial hosts of differing evolutionary relationships to S. typhimurium. Systematic analysis of the expression of the island genes in the different hosts compared to proper controls allowed identification of genes with genera-specific expression patterns. The data from the analysis can be arranged in a matrix to give an expression "array" of the island genes in the different bacterial backgrounds. A conserved 19-bp DNA site was found upstream of at least two of the differentially-expressed island genes. To our knowledge, this is the first systematic analysis of horizontally-transferred genomic island gene expression in a broad range of Gram negative hosts. We also present evidence in this study that the IS200 element found in island 4305 in S. typhimurium strain LT2 was inserted after the island had already been acquired by the S. typhimurium lineage and that this element is likely not involved in the integration or excision of island 4305.

Conclusion

The "clone-and-transfer" approach of evolutionary study identifies genes whose expression patterns indicate the existence of genera-specific regulatory mechanisms that influence the expression of horizontally-transferred DNA sections. The results provide key information that can be used to facilitate the identification of these regulatory mechanisms.
Development of bile duct bezoars following cholecystectomy caused by choledochoduodenal fistula formation: a case report
Authors: Moghaddam Jamal Akhavan , Amini Mohsen , Adibnejad Soheil
Published: January 2006

Background

The formation of bile duct bezoars is a rare event. Its occurrence when there is no history of choledochoenteric anastomosis or duodenal diverticulum constitutes an extremely scarce finding.

Case presentation

We present a case of obstructive jaundice, caused by the concretion of enteric material (bezoars) in the common bile duct following choledochoduodenal fistula development. Six years after cholecystectomy, a 60-year-old female presented with abdominal pain and jaundice. Endoscopic retrograde cholangiopancreatography demonstrated multiple filling defects in her biliary tract. The size of the obstructing objects necessitated surgical retrieval of the stones. A histological assessment of the objects revealed fibrinoid materials with some cellular debris. Post-operative T-tube cholangiography (9 days after the operation) illustrated an open bile duct without any filling defects. Surprisingly, a relatively long choledochoduodenal fistula was detected. The fistula formation was assumed to have led to the development of the bile duct bezoar.

Conclusion

Bezoar formation within the bile duct should be taken into consideration as a differential diagnosis, which can alter treatment modalities from surgery to less invasive methods such as more intra-ERCP efforts. Suspicions of the presence of bezoars are strengthened by the detection of a biliary enteric fistula through endoscopic retrograde cholangiopancreatography. Furthermore, patients at a higher risk of fistula formation should undergo a thorough ERCP in case there is a biliodigestive fistula having developed spontaneously.
Systemic chemotherapy with doxorubicin, cisplatin and capecitabine for metastatic hepatocellular carcinoma
Authors: Park Se Hoon , Lee Yuna , Han Sang Hoon , Kwon So Young , Kwon Oh Sang , Kim Sun Suk , Kim Ju Hyun , Park Yeon Ho , Lee Jeong Nam , Bang Soo-Mee , Cho Eun Kyung , Shin Dong Bok , Lee Jae Hoon
Published: January 2006

Background

Although numerous chemotherapeutic agents have been tested, the role of systemic chemotherapy for hepatocellular carcinoma (HCC) has not been clarified. New therapeutic strategies are thus needed to improve outcomes, and we designed this study with new effective drug combination.

Methods

Twenty-nine patients with histologically-confirmed, metastatic HCC received a combination chemotherapy with doxorubicin 60 mg/m2 and cisplatin 60 mg/m2 on day 1, plus capecitabine 2000 mg/m2/day as an intermittent regimen of 2 weeks of treatment followed by a 1-week rest.

Results

The median age was 49 years (range, 32–64) and 19 patients were hepatitis B virus seropositive. Child-Pugh class was A in all patients and 4 had Zubrod performance status of 2. The objective response rate was 24% (95% CI 9–40) with 6 stable diseases. The chemotherapy was generally well tolerated despite one treatment-related death.

Conclusion

Combination chemotherapy with doxorubicin, cisplatin and capecitabine produced modest antitumor activity with tolerable adverse effects in patients with metastatic HCC.
Hidden chromosomal abnormalities in pleuropulmonary blastomas identified by multiplex FISH
Authors: Quilichini Benoit , Andre Nicolas , Bouvier Corinne , Chrestian Marie-Anne , Rome Angelique , Intagliata Dominique , Coze Carole , Lena Gabriel , Zattara Helene
Published: January 2006

Background

Pleuropulmonary blastoma (PPB) is a rare childhood dysontogenetic intrathoracic neoplasm associated with an unfavourable clinical behaviour.

Cases presentation

We report pathological and cytogenetic findings in two cases of PPB at initial diagnosis and recurrence. Both tumors were classified as type III pneumoblastoma and histological findings were similar at diagnosis and relapse. In both cases, conventional cytogenetic techniques revealed complex numerical and structural chromosomal abnormalities. Molecular cytogenetic analysis (interphase/metaphase FISH and multicolor FISH) identified accurately chromosomal aberrations. In one case, TP53 gene deletion was detected on metaphase FISH. To date, only few cytogenetic data have been published about PPB.

Conclusion

The PPB genetic profile remains to be established and compared to others embryonal neoplasia. Our cytogenetic data are discussed reviewing cytogenetics PPBs published cases, illustrating the contribution of multicolor FISH in order to identify pathogenetically important recurrent aberrations in PPB.
Fluorescent amplified fragment length polymorphism (FAFLP) genotyping demonstrates the role of biofilm-producing methicillin-resistant periocular Staphylococcus epidermidis strains in postoperative endophthalmitis
Authors: Kenchappa Prashanth , Duggirala Aparna , Ahmed Niyaz , Pathengay Avinash , Das Taraprasad , Hasnain Seyed E , Sharma Savitri
Published: January 2006

Background

An observational case series was used to study the virulence characteristics and genotypes of paired Staphylococcus epidermidis isolates cultured from intraocular samples and from periocular environment of patients with postcataract surgery endophthalmitis.

Methods

Eight S. epidermidis isolates were obtained from three patients (2 from patients #1 and 2 and 4 from patient #3) whose vitreous and/or anterior chamber (AC) specimens and preoperative lid/conjunctiva samples were culture positive. Cultures were identified by API-Staph phenotypic identification system and genotypically characterized by Fluorescent Amplified Fragment Length Polymorphism (FAFLP) and checked for their antimicrobial susceptibility. The isolates were tested for biofilm-production and methicillin-resistance (MR) by PCR amplification of icaAB and mecA gene respectively.

Results

Four out of eight S. epidermidis strains showed multiple drug resistance (MDR). All the eight strains were PCR positive for mecA gene whereas seven out of eight strains were positive for icaAB genes. In all three patients FAFLP typing established vitreous isolates of S. epidermidis strains to be indistinguishable from the strains isolated from the patient's conjunctival swabs. However, from patient number three there was one isolate (1030b from lid swab), which appeared to be nonpathogenic and ancestral having minor but significant differences from other three strains from the same patient. This strain also lacked icaAB gene. In silico analysis indicated possible evolution of other strains from this strain in the patient.

Conclusion

Methicillin-resistant biofilm positive S. epidermidis strains colonizing the conjunctiva and eyelid were responsible for postoperative endophthalmitis (POE).
Genetic transformation of novel isolates of chicken Lactobacillusbearing probiotic features for expression of heterologous proteins: a tool to develop live oral vaccines
Authors: Mota Rodrigo M , Moreira João Luiz S , Souza Marcelo R , Fátima Horta M , Teixeira Santuza MR , Neumann Elisabeth , Nicoli Jacques R , Nunes Álvaro C
Published: January 2006

Background

The use of lactic acid bacteria as vehicles to delivery antigens to immunize animals is a promising issue. When genetically modified, these bacteria can induce a specific local and systemic immune response against selected pathogens. Gastric acid and bile salts tolerance, production of antagonistic substances against pathogenic microorganisms, and adhesive ability to gut epithelium are other important characteristics that make these bacteria useful for oral immunization.

Results

Bacteria isolated on de Man, Rogosa and Sharpe medium (MRS) from different gastrointestinal portions of broiler chicks were evaluated for their resistance to artificial gastric acid and bile salts, production of hydrogen peroxide, and cell surface hydrophobicity. Thirty-eight isolates were first typed at species level by PCR amplification of 16S-23S rRNA intergenic spacers using universal primers that anneal within 16S and 23S genes, followed by restriction digestion analyses of PCR amplicons (PCR-ARDRA). An expression cassette was assembled onto the pCR2.1-Topo vector by cloning the promoter, leader peptide, cell wall anchor and terminator sequences derived from the laminin binding S-layer protein gene of L. crispatus strain F5.7 (lbs gene). A sequence encoding the green fluorescent protein (GFP) was inserted as reporter gene, and an erythromycin resistance gene was added as selective marker. All constructs were able to express GFP in the cloning host E. coli XL1-Blue and different Lactobacillus strains as verified by FACS and laser scanning confocal microscopy.

Conclusion

Lactobacillus isolated from gastrointestinal tract of broiler chickens and selected for probiotic characteristics can be genetically modified by introducing an expression cassette into the lbs locus. The transformed bacteria expressed on its cell wall surface different fluorescent proteins used as reporters of promoter function. It is possible then that similar bacterial model expressing pathogen antigens can be used as live oral vaccines to immunize broilers against infectious diseases.