Images in cardiovascular medicine. A swelling of the right neck and sudden death.
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Aneurysm↗
Biomedical subjects
Thomas Neumann
Publications and source records attributed to Thomas Neumann.
Asymptomatic recurrences of atrial fibrillation after pulmonary vein isolation.
AIMS: The purpose of this study was to determine the occurrence of asymptomatic episodes of atrial fibrillation (AF) and wrong AF perception after pulmonary vein isolation (PVI). We evaluated the success of ablation by using the following measurements: (i) clinical symptoms and duration of symptoms noticed by patients and (ii) synchronous event recording (ER). METHODS AND RESULTS: Eighty patients with paroxysmal AF underwent PVI and were provided repeatedly with a portable ER upon discharge and every 3 months for a year. The ER automatically detects arrhythmias by a detection algorithm and can also be manually triggered by the patient. In 46/80 patients (57.5%), episodes of AF were documented. Asymptomatic AF was detected in 21.3%. In 9/80 patients (11.3%), who reported clinical AF recurrence, no AF could be shown by ER. We compared patients' perception to have suffered AF episodes with the ERs and found a sensitivity of 75% and a specificity of 92%. CONCLUSION: Reliance on perception of AF by patients after PVI results in an underestimation of recurrence of the arrhythmia. We observed a maximal occurrence of silent AF or wrong perception of AF in 26/80 (32.6%) patients.
Analysis of Variance↗
T20-insensitive HIV-1 from naive patients exhibits high viral fitness in a novel dual-color competition assay on primary cells.
The relationship between sensitivity to antiviral drugs and viral fitness is of paramount importance in understanding the long-term implications of clinical resistance. Here we report the development of a novel recombinant virus assay to study entry inhibitor-resistant HIV variants using a biologically relevant cell type, primary CD4 T-cells. We have modified the replication-competent molecular clone HIV(NL4-3) to express a reporter protein (Renilla luciferase), Green Fluorescent Protein (EGFP), or Red Fluorescent Protein (DsRed2) upon infection, thus allowing quantification of replication. Luciferase-expressing virus was used to evaluate drug sensitivity, while co-infection with viruses carrying the green and red fluorescent proteins was employed in the competitive fitness assay. Using envelope proteins from three T20 insensitive variants, lower levels of resistance were observed in primary CD4 T-cells than had been previously reported for cell lines. Importantly, dual-color competition assays demonstrated comparable or higher fitness for these variants despite their reduced T20 sensitivity. We conclude that reduced sensitivity to T20 is compatible with high viral fitness in the absence of selection pressure. Thus, simultaneously measuring both resistance and viral fitness using this newly described dual-color competition assay will likely provide important information about resistant viral variants that emerge during therapy with entry inhibitors.
Amino Acid Sequence↗
Discordant effects of nicotine on endothelial cell proliferation, migration, and the inward rectifier potassium current.
The inward rectifier K+ current (K(ir)) determines the resting membrane potential of endothelial cells. Basic fibroblast growth factor (bFGF) has been shown to activate K(ir) and acts as angiogenic factor and vasodilator. In contrast, nicotine has been demonstrated to reduce endothelium-dependent vasorelaxation by increasing radical formation. Aim of the present study was to investigate whether nicotine modulates K(ir) and if this plays a role in bFGF-mediated proliferation, migration and nitric oxide (NO)-formation of endothelial cells. Using the patch-clamp technique in cultured endothelial cells of human umbilical cord veins (HUVEC), we found characteristic K(ir), which were blocked by extracellular barium (100 micromol/l). Perfusion with nicotine (1 nmol/l-10 micromol/l) revealed a dose-dependent reduction of K(ir). The simultaneous perfusion with bFGF (50 ng/ml) and nicotine (10 micromol/l) still significantly reduced K(ir) (n = 8; P < 0.01). Cell counts revealed that bFGF-mediated proliferation of HUVEC was significantly inhibited when using 1-10 micromol/l nicotine (n = 8, P < 0.01). The bFGF-induced endothelial cell migration--examined using the "Fences-Migration-Assay"--was significantly reduced by 10 mumol/l nicotine (n = 12; P < 0.05). NO-production was examined using a cGMP-Radioimmunoassay. The significant bFGF-induced increase of cGMP-levels was reduced by nicotine (n = 10; P < 0.05). Our data indicate that the modulation of K(ir) seems to be an essential pathway in the antagonistic effects of nicotine on bFGF-mediated endothelial cell growth, migration and NO-formation.
Cell Movement↗
Quercetin-induced induction of the NO/cGMP pathway depends on Ca2+-activated K+ channel-induced hyperpolarization-mediated Ca2+-entry into cultured human endothelial cells.
Quercetin is one of the dietary-derived flavonoids that are held responsible for the beneficial effects of red wine drinking in coronary artery disease known as the "French paradox". We examined whether quercetin modulates endothelial function by influencing Ca2+-activated K+ channels with large conductance (BK(Ca)) in cultured human endothelial cells. Membrane potential and intracellular Ca2+ concentrations of cultured human endothelial cells derived from umbilical cord veins (HUVEC) were measured using the fluorescence dyes DiBAC, and FURA-2, respectively. NO production was examined using a cGMP radioimmunoassay. HUVEC proliferation was analyzed by cell counts and thymidine incorporation. A dose-dependent hyperpolarization of HUVEC was recorded when quercetin was added (5-100 micromol/L). The maximum effect (50 micromol/L) was significantly reduced by the addition of the highly selective BK(Ca) inhibitor iberiotoxin (100 nmol/L), but not by blockers of other Ca2+-activated K+ channels (n = 30; p < 0.05). This BK(Ca)-induced hyperpolarization caused a transmembrane Ca2+ influx, because the quercetin-induced increase of intracellular Ca2+ was blocked by iberiotoxin, or by applying 2-aminoethoxydiphenylborate (100 micromol/L)--an inhibitor of capacitative Ca2+ entry (n = 30; p < 0.05). Quercetin-induced cGMP levels were significantly reduced by the eNOS-inhibitor l-NMMA (300 micromol/L), and by iberiotoxin (n = 10; p < 0.05). Endothelial proliferation was significantly reduced by 56 % when cells were incubated with quercetin (n = 12; p < 0.05). This effect was due to the increased NO production, because it was reversed when the cells were treated with a combination of quercetin and l-NMMA. In conclusion quercetin improves endothelial dysfunction by increasing NO synthesis involving BK(Ca)-dependent membrane hyperpolarization-induced capacitative Ca 2+ entry. Increased NO production is responsible for the quercetin-dependent inhibition of endothelial proliferation.
Calcium↗
A decrease in pulmonary vein diameter after radiofrequency ablation predicts the development of severe stenosis.
A decrease in ostial pulmonary vein (PV) diameter was observed in patients on the day after radiofrequency ablation of atrial fibrillation (AF). This study examined whether a relative reduction in PV diameter on day 1 (RRPVD1) after the procedure predicts the late development of severe PV stenosis (PVS). The study included 104 consecutive patients (mean age = 55 years, range 46-61, 34 women) with drug refractory AF. Pulmonary vein diameter was measured using MR angiography (MRA) on the day before and on day 1 after the ablation procedure. The MRA was repeated every 3 months after the procedure. Severe PVS was defined as a >70% diameter reduction from the initial ostial diameter. The cut-off of RRPVD1 was prespecified as 25% decrease in initial diameter. The data are presented as medians and interquartile range. A total of 357 PV were treated. The RRPVD1 was 0.0% (0.0-11.1%). Severe PVS was found in 18 PV during a follow-up of 12 months (range 6-13). The log-rank analysis confirmed a strong association between a RRPVD1 >/=25% and the development of PVS (hazard ratio: 7.1; 95% confidence interval 3.8-13.5, P < 0.0001). By multivariate Cox regression model, after adjustment of procedure variables, RRPVD1 was the strongest predictor of development of severe PVS. RRPVD1 >/=25% was a strong independent predictor of development of severe PVS.
Aged↗
Invasive optimization of cardiac resynchronization therapy: role of sequential biventricular and left ventricular pacing.
BACKGROUND: Aim of this invasive study was to characterize and quantify changes in left ventricular (LV) systolic function due to sequential biventricular pacing (BV) as compared to right atrial triggered simultaneous BV (BV(0)), LV, and right ventricular (RV) pacing in patients with congestive heart failure (CHF). METHODS: In 22 CHF patients, all in sinus rhythm, temporary multisite pacing was performed prior to implantation of a permanent system. LV systolic function was evaluated invasively by the maximum rate of LV pressure increase (dP/dt(max)). Sequential BV pacing was performed with preactivation of either ventricle at 20-80 ms. RESULTS: In comparison to RV pacing, LV and BV(0) pacing increased dP/dt(max) by 33.9 +/- 19.3% and 34.0 +/- 22.6%, respectively (P < 0.001). In 9 patients, optimized sequential BV pacing further improved dP/dt(max) by 8.5 +/- 4.8% compared to BV(0) (range 3.3-17.1, P < 0.05). In 10 patients exhibiting a PR interval < or =200 ms, LV pacing was either superior (n = 6) or equal to BV(0) pacing (n = 4). In these 10 patients, LV pacing yielded a 7.4 +/- 8.0% higher dP/dt(max) than BV(0) pacing (P < 0.05). CONCLUSIONS: Using sequential BV pacing, generally with LV preactivation, moderate improvements in LV systolic function can be achieved in selected patients. Baseline PR interval may aid in the selection of the optimum cardiac resynchronization therapy (CRT) mode, favoring LV pacing in patients with a PR interval < or =200 ms.
Aged↗
Usefulness of atrial fibrillation burden as a predictor for success of pulmonary vein isolation.
BACKGROUND: The definition of symptomatic improvement after pulmonary vein isolation (PVI) is controversial. We assessed primarily the usefulness and predictive accuracy (PA) of AF (atrial fibrillation) burden (AFB) within the first 3 months after the procedure as an early predictor for curative success as well as reduction of symptoms. METHODS: We studied 100 pts (62 men, mean age: 54 +/- 9 years, LVEF: 57 +/- 9%, paroxysmal AF, 85, persistent AF, 15) who underwent PVI. RF ablation was used in 63 patients. The combination of cryoablation and RF (Hybrid therapy) was used in the remaining 37 patients, in 19 patients of whom additionally linear lesions were performed. All patients were followed up 3 months after PVI (the blanking period) and every 3 months thereafter. The significance of early recurrence of AF (ERAF) was separately analyzed for RF and for hybrid therapy arm. Quality of life (QoL) was assessed using the physical composite summary score (PCS) and mental composite summary score (MCS). AFB was dichotomized at: 0, 0.5, 1, 6, 12, and 24 hours. RESULTS: ANOVA revealed significant differences between PCS at baseline and after PVI in patients without recurrence and with those with postablation AFB <12 hours. There was no difference between PCS in patients with AFB > or =12 hours within a 3 months interval and PCS at baseline. ERAF occurred in 35% of patients (16 patients, AFB > or =12 hours). Symptomatic ERAF occurred in 29 patients (17 patients AFB > or =12 hours). After the blanking period, documented AF recurrence was found in 50 patients (17 patients, AFB > or =12 hour). Symptomatic late recurrence was found in 47 patients (31 patients with AFB > or =12 hours). Absence of ERAF was revealed to be predictive for long-term success and symptomatic ERAF was found to be predictive for symptomatic AF recurrence (P <0.0001). CONCLUSION: AF recurrence with AFB > or =12 hours within 3 months is associated with a missed improvement of QoL. Symptomatic ERAF predicts recurrence independently of therapy.
Analysis of Variance↗
Percutaneous pulmonary vein stenting for the treatment of severe stenosis after pulmonary vein isolation.
INTRODUCTION: Pulmonary vein stenosis (PVS) is a potential complication of pulmonary vein isolation (PVI) using radiofrequency energy. The aim of our study was the evaluation of the severity and long-term outcome of primary angioplasty and angioplasty with pulmonary vein stenting for PVS. METHODS AND RESULTS: Twelve patients with 15 PVS (greater than 70% stenosis) were prospectively evaluated. Primary dilation of the stenosis was performed because of clinical symptoms (10 patients) and/or the lung perfusion scans showed a significant perfusion defect (11 patients). Magnetic resonance imaging and lung perfusion scans performed before, directly after, during 3-month, and 6-month follow-up. In the stenting group additional multislice CT-scans directly after, during 6-month, and 12-month follow-up were performed. Within 2 months after primary balloon angioplasty, the PV size parameters were significantly reduced (P < 0.001) with recurrence of PVS in 11 of 15 PVs (73%). Pulmonary vein stenting in 8 patients and 11 PVs resulted in no vein stenosis during 12-month follow-up. Normalization of lung perfusion was noted in 8 of 12 patients. We observed 2 patients with hemoptysis during PV dilation, as severe complications with potential life-threatening character. CONCLUSION: PVS stenting seems to be superior to balloon angioplasty and effective at least over a period of 12 months in treating acquired PVS after pulmonary vein isolation.
Angioplasty, Balloon↗
Basic fibroblast growth factor-induced endothelial proliferation and NO synthesis involves inward rectifier K+ current.
OBJECTIVE: Inward rectifier K+ currents (K(ir)) determine the resting membrane potential and thereby modulate essential Ca2+-dependent pathways, like cell growth and synthesis of vasoactive agents in endothelial cells. Basic fibroblast growth factor (bFGF) acts as a vasodilatator and angiogenic factor. Therefore, we investigated the effect of bFGF on K(ir) and assessed the role in proliferation and nitric oxide (NO) formation of endothelial cells. METHODS AND RESULTS: Using the patch-clamp technique, we found characteristic K(ir) in human umbilical cord vein endothelial cells (HUVEC), which were dose-dependently blocked by barium (10 to 100 micromol/L). Perfusion with bFGF (50 ng/mL) caused a significant increase of K(ir), which was blocked by 100 micromol/L barium (n=18, P<0.01). The bFGF-induced HUVEC proliferation was significantly inhibited when using 50 to 100 micromol/L barium (n=6; P<0.01). NO production was examined using a cGMP radioimmunoassay. bFGF caused a significant increase of cGMP levels (n=10; P<0.05), which were blocked by barium. CONCLUSIONS: Modulation of K(ir) plays an important role in bFGF-mediated endothelial cell growth and NO formation.
Barium↗
Land use-related chemical composition of street sediments in Beijing.
BACKGROUND: More than 10 million people are currently living in Beijing. This city faces severe anthropogenic air pollution caused by an intense vehicle increase (11% per year in China), coal combusting power plants, heavy industry, huge numbers of household and restaurant cookers, and domestic heating stoves. Additionally, each year dust storms are carrying particulate matter from the deserts of Gobi and Takla Makan towards Beijing, especially in spring. Other geogenic sources of particulate matter which contribute to the air pollution are bare soils, coal heaps and construction sites occurring in and around Beijing. Streets function as receptor surfaces for atmospheric dusts. Thus, street sediments consist of particles of different chemical compositions from many different sources, such as traffic, road side soils and industry. METHODS: Distributions and concentrations of various chemical elements in street sediments were investigated along a rural-urban transect in Beijing, China. Chemical elements were determined with X-ray fluorescence analysis. Factor analysis was used to extract most important element sources contributing to particulate pollution along a main arterial route of the Chinese capital. RESULTS AND DISCUSSION: The statistical evaluation of the data by factor analysis identifies three main anthropogenic sources responsible for the contamination of Beijing street sediments. The first source is a steel factory in the western part of Beijing. From this source, Mn, Fe, and Ti were emitted into the atmosphere through chimneys and by wind from coal heaps used as the primary energy source for the factory. The second source is a combination of traffic, domestic heating and some small factories in the center of Beijing discharging Cu, Pb, Zn and Sn. Calcium and Cr characterize a third anthropogenic element source of construction materials such as concrete and mortar. Beside the anthropogenic contamination, some elements like Y, Zr, Nb, Ce, and Rb are mainly derived from natural soils and from the deserts. This is supported by mineral phase analysis, which showed a clear imprint of material in road dusts coming from the West-China deserts. CONCLUSIONS: Our results clearly show that the chemical composition of urban road dusts can be used to identify distinct sources responsible for their contamination. The study demonstrates that the chemistry of road dusts is an important monitor to assess the contamination in the urban environment. Chemical composition of street sediments in Beijing comprises the information of different sources of atmospheric particles. RECOMMENDATIONS AND OUTLOOK: This study is only a small contribution to the understanding of substance fluxes related to Beijing's dust. More effort is required to assess Beijing's dust fluxes, since the dust harms the living quality of the inhabitants. Especially the measurable superimposing of long scale transported dust from dry regions with the anthropogenic polluted urban dust makes investigations of Beijing's dust scientifically valuable.
Air Pollutants↗
Statins prevent oxidized low-density lipoprotein- and lysophosphatidylcholine-induced proliferation of human endothelial cells.
The proliferation of endothelial cells is induced by oxidized low-density lipoprotein (oxLDL) and its major component, lysophosphatidylcholine (LPC). The aim of this study was to investigate the effect of statins on the proliferation of endothelial cells derived from human umbilical cord veins (HUVEC). Cerivastatin, simvastatin and fluvastatin caused a dose-dependent inhibition of endothelial cell growth (n=12; P<.01) when using cell counts and [3H]-thymidine incorporation, respectively. The strongest inhibition of HUVEC proliferation was achieved at statin concentrations of 0.1 micromol/l (cerivastatin), 2.5 micromol/l (simvastatin) and 1 micromol/l (fluvastatin). Cell counts were significantly reduced from 22937+/-280.6 (control) to 7791+/-133.6 (cerivastatin), 7292+/-146.6 (simvastatin) and 6792+/-135.5 (fluvastatin) (n=12; P<.01). Interestingly, cell proliferation induced by oxLDL (10 microg/ml) and LPC (20 micromol/l) could be effectively prevented using statins at concentrations between 0.01 and 0.1 micromol/l (cerivastatin), 1 and 2.5 micromol/l (simvastatin) and 0.25 and 1 micromol/l (fluvastatin). This effect of the statins was abolished by preincubation with mevalonate (500 micromol/l). Our results demonstrate an interesting direct effect of statins on the proliferation of human endothelial cells induced by oxLDL and LPC, which may be beneficial to prevent vascular effects of these atherogenic lipids.
Cell Division↗
Lysophosphatidylcholine-induced modulation of Ca(2+)-activated K(+)channels contributes to ROS-dependent proliferation of cultured human endothelial cells.
Proliferation of endothelial cells plays a crucial role in the process of atherosclerotic plaque destabilization. The major component of oxidized low-density lipoprotein lysophosphatidylcholine (LPC) has been shown to promote endothelial proliferation by increasing the production of reactive oxygen species (ROS). Since K(+) channels are known to control the cell cycle, we investigated the role of Ca(2+)-activated K(+) channels (BK(Ca)) in the regulation of LPC-induced endothelial proliferation and ROS generation. A significant increase of cell growth induced by LPC (20 micromol/l; cell counts (CCs): +87%, thymidin incorporation: +89%; n = 12, P < 0.01) was observed, which was inhibited by the BK(Ca) inhibitor iberiotoxin (IBX; 100 nmol/l), by the NAD(P)H-oxidase inhibitor diphenyleneiodonium (5 micromol/l) and by transfection with antisense (AS) oligonucleotides against NAD(P)H oxidase, whereas N(G)-monomethyl-l-arginine (l-NMMA) further increased LPC-induced cell growth. Using the patch-clamp technique a significant increase of BK(Ca) open-state probability (control: 0.004 +/- 0.002; LPC: 0.104 +/- 0.035; n = 21, P < 0.05) by LPC was observed. Using dichlorofluorescein fluorescence microscopy a significant increase of ROS induced by LPC was reported, that was blocked by IBX and Ca(2+) antagonists. Intracellular Ca(2+) measurements revealed a capacitative Ca(2+) influx caused by LPC. Bioactivity of nitric oxide (NO) was measured using a [(3)H]-cGMP radioimmunoassay. LPC significantly decreased acetylcholine-induced NO synthesis. LPC significantly increased cGMP levels in endothelial cells transfected with AS, which was blocked by IBX. In conclusion, our results demonstrate that LPC activates BK(Ca) thereby increasing ROS production which induces endothelial proliferation. In addition LPC-induced BK(Ca)-activation contributes to increased cGMP levels, if ROS production is prevented by AS.
Calcium↗
Dose-dependent activation of Ca2+-activated K+ channels by ethanol contributes to improved endothelial cell functions.
BACKGROUND: Regular moderate alcohol (EtOH) intake seems to protect against both coronary artery disease and ischemic stroke, whereas the risk increases with heavy EtOH consumption. Effects of EtOH on endothelial cell function may be relevant to these disparate effects. Potassium channels play an important role in the regulation of endothelial cell functions. Therefore, we investigated whether Ca-activated K channels (BKCa) are modulated by EtOH. Furthermore, we examined whether EtOH-induced changes of endothelial nitric oxide (NO) formation and cell proliferation are due to BKCa activation. METHODS: The patch-clamp technique was used to investigate BKCa activity in cultured human umbilical vein endothelial cells (HUVEC). NO formation was analyzed by using the fluorescence dye 4,5-diaminofluorescein. Endothelial proliferation was examined by using cell counts and measuring [H]thymidine incorporation. RESULTS: EtOH dose-dependently (10-150 mmol/liter) modulated BKCa-activity, with the highest increase of open-state probability at a concentration of 50 mmol/liter (n = 13; p < 0.05). Inside-out recordings revealed that this effect was due to direct BKCa activation, whereas open-state probability was not changed in cell-attached recordings after pertussis toxin preincubation. EtOH (10 and 50 mmol/liter) caused a significant increase of NO levels, which was blocked by the highly selective BKCa inhibitor iberiotoxin (100 nmol/l; n = 30; p < 0.05). Higher concentrations of EtOH (100 and 150 mmol/liter) significantly reduced NO synthesis (n = 30; p < 0.05). Both methods revealed a significant increase of HUVEC proliferation, which was inhibited by iberiotoxin (n = 30; p < 0.05). At a concentration of 150 mmol/liter, EtOH caused a significant reduction of endothelial proliferation. CONCLUSIONS: EtOH directly activates BKCa in HUVEC, leading to an increase of endothelial proliferation and production of NO. These results indicate a possible beneficial effect of low-dose EtOH on endothelial function, whereas higher concentrations must be considered as harmful.
Cell Proliferation↗
Risk stratification using heart rate turbulence and ventricular arrhythmia in MADIT II: usefulness and limitations of a 10-minute holter recording.
BACKGROUND: We evaluated the usefulness of heart rate turbulence (HRT) parameters and frequency of ventricular premature beats (VPBs) for risk-stratifying postinfarction patients with depressed left ventricular function enrolled in Multicenter Automatic Defibrillator Trial II (MADIT II). METHODS: In 884 MADIT II patients, 10-minute Holter monitoring at enrollment was used to evaluate HRT parameters and frequency of VPBs. The primary endpoints were defined as all-cause mortality in patients randomized to conventional treatment and as appropriate therapy for ventricular tachycardia or fibrillation in patients randomized to implantable cardioverter defibrillator (ICD) therapy. RESULTS: The median turbulence slope was lower in patients who died in comparison to survivors in the conventional arm (2.3 vs 4.5 ms/RR; P < 0.05); but it was not a significant predictor of mortality after adjustment for clinical covariates (age, ejection fraction, beta-blocker use, and BUN levels). There was no association between HRT parameters and arrhythmic events in ICD patients. Conventionally treated patients who died and ICD patients who had appropriate ICD therapy had significantly more frequent VPBs than those without such adverse events. After adjustment for clinical covariates, frequent VPBs>3/10 min were associated with death in the conventional arm (HR = 1.63; P = 0.070) and were predictive for appropriate ICD therapy in the ICD arm (HR = 1.75; P = 0.003). CONCLUSION: In postinfarction patients with severe left ventricular dysfunction, frequent VPBs are associated with increased risk of mortality and with appropriate ICD therapy. HRT obtained from 10-min Holter ECG showed a trend toward the association with mortality in univariate analysis but HRT parameters were not predictive of the outcome in multivariate analyses.
Adult↗
The K+-channel opener NS1619 increases endothelial NO-synthesis involving p42/p44 MAP-kinase.
Ca(2+)-activated K(+) channels with large conductance (BK(Ca)) have been shown to play an important role in the regulation of vascular tone. We examined the role of the p42/p44 MAP-kinase (p42/p44(MAPK)) on nitric oxide (NO) production in human endothelial cells induced by the BK(Ca)-opener NS1619. Using DiBAC-fluorescence imaging a concentration-dependent (2.5-12.5 microM) hyperpolarization induced by NS1619 was observed. A significant increase of intracellular Ca(2+)-concentration by NS1619 was seen using Fura-2-fluorescence-imaging, which was blocked by 2-APB, or reduction of extracellular Ca(2+) (n=30; p<0.05). A cGMP-radioimmunoassay was used to examine NO synthesis. NS1619 significantly increased cGMP levels which was inhibited by LNMMA, iberiotoxin, BAPTA, 2-APB, reduction of extracellular Ca(2+), PD 98059, or U0126 (cGMP (pmol/mg protein): NS1619 3.25 +/- 0.85; NS1619 + L-NMMA 0.86 +/- 0.02; NS1619 + iberiotoxin 0.99 +/- 0.09; NS1619 + BAPTA 0.93 +/- 0.29; NS1619 + 2-APB 0.99 +/- 0.31; NS1619 + Ca(2+)-reduction 1.17 +/- 0.06; NS1619 + PD98059 1.06 +/- 0.49; NS1619 + U0126 1.10 +/- 0.24; n=10; p<0.05). The phosphorylation of eNOS and p42/p44(MAPK) was examined by immunocytochemistry. Phosphorylation of p42/p44(MAPK) was significantly increased after 10 minutes of NS1619 stimulation, whereas eNOS phosphorylation was not changed over a period of 1 to 30 minutes. NS1619-induced hyperpolarization was not affected by treatment with PD 98059 or U0126. Additionally, NS1619 inhibited endothelial proliferation involving a NO-dependent mechanism. Our data demonstrate that NS1619 causes a transmembrane Ca(2+)-influx leading to an increased NO production involving p42/p44(MAPK). This rise of NO formation is responsible for the NS1619 induced reduction of endothelial cell growth.
Benzimidazoles↗
Comparative proteomic analysis of proliferating and functionally differentiated mammary epithelial cells.
Proliferation and differentiation of mammary epithelial cells are governed by hormonal stimuli, cell-cell, and cell-matrix interactions. Terminal differentiation of mammary epithelial cells depends upon the action of the lactogenic hormones, insulin, glucocorticoids, and prolactin that enable them to synthesize and secrete milk proteins. These differentiated cells are polarized and carry out vectorial transport of milk constituents across the apical plasma membrane. To gain additional insights into the mechanisms governing differentiation of mammary epithelial cells, we identified proteins whose expression distinguishes proliferating from differentiated mammary epithelial cells. For this purpose we made use of the HC11 mammary epithelial line, which is capable of differentiation in response to lactogenic hormones. Using two-dimensional gel electrophoresis and mass spectrometry, we found about 60 proteins whose expression levels changed in between these two differentiation states. Bioinformatic analysis revealed differential expression of cytoskeletal components, molecular chaperones and regulators of protein folding and stability, calcium-binding proteins, and components of RNA-processing pathways. The actin cytoskeleton is asymmetrically distributed in differentiated epithelial cells, and the identification of proteins involved in mRNA binding and localization suggests that asymmetry might in part be achieved by controlling cellular localization of mRNAs. The proteins identified provide insights into the differentiation of mammary epithelial cells and the regulation of this process.
Animals↗
Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS.
A novel MS/MS-based analysis strategy using isotopomer labels, referred to as "tandem mass tags" (TMTs), for the accurate quantification of peptides and proteins is described. The new tags are designed to ensure that identical peptides labeled with different TMTs exactly comigrate in all separations. The tags require novel methods of quantification analysis using tandem mass spectrometry. The new tags and analysis methods allow peptides from different samples to be identified by their relative abundance with greater ease and accuracy than other methods. The new TMTs permit simultaneous determination of both the identity and relative abundances of peptide pairs using a collision induced dissociation (CID)-based analysis method. Relative abundance measurements made in the MS/MS mode using the new tags are accurate and sensitive. Compared to MS-mode measurements, a very high signal-to-noise ratio is achieved with MS/MS based detection. The new tags should be applicable to a wide variety of peptide isolation methods.
Isotopes↗