bluuebunny/crossref_metadata_2025 huggingface dataset:
Spectroscopic and DNA‐binding characterization of the isolated heme‐bound basic helix–loop–helix‐PAS‐A domain of neuronal PAS protein 2 (NPAS2), a transcription activator protein associated with circadian rhythms
Authors: Mukaiyama Yuji , Uchida Takeshi , Sato Emiko , Sasaki Ai , Sato Yuko , Igarashi Jotaro , Kurokawa Hirofumi , Sagami Ikuko , Kitagawa Teizo , Shimizu Toru
Published: April 2006

Neuronal PAS domain protein 2 (NPAS2) is a circadian rhythm‐associated transcription factor with two heme‐binding sites on two PAS domains. In the present study, we compared the optical absorption spectra, resonance Raman spectra, heme‐binding kinetics and DNA‐binding characteristics of the isolated fragment containing the N‐terminal basic helix–loop–helix (bHLH) of the first PAS (PAS‐A) domain of NPAS2 with those of the PAS‐A domain alone. We found that the heme‐bound bHLH‐PAS‐A domain mainly exists as a dimer in solution. The Soret absorption peak of the Fe(III) complex for bHLH‐PAS‐A (421 nm) was located at a wavelength 9 nm higher than for isolated PAS‐A (412 nm). The axial ligand trans to CO in bHLH‐PAS‐A appears to be His, based on the resonance Raman spectra. In addition, the rate constant for heme association with apo‐bHLH‐PAS (3.3 × 107 mol−1·s−1) was more than two orders of magnitude higher than for association with apo‐PAS‐A (< 105 mol−1·s−1). These results suggest that the bHLH domain assists in stable heme binding to NPAS2. Both optical and resonance Raman spectra indicated that the Fe(II)–NO heme complex is five‐coordinated. Using the quartz‐crystal microbalance method, we found that the bHLH‐PAS‐A domain binds specifically to the E‐box DNA sequence in the presence, but not in the absence, of heme. On the basis of these results, we discuss the mode of heme binding by bHLH‐PAS‐A and its potential role in regulating DNA binding.
Albumin Regeneration in Liver Support—Comparison of Different Methods
Authors: Mitzner Steffen , Klammt Sebastian , Stange Jan , Schmidt Reinhart
Published: April 2006

Abstract:  Albumin is the most abundant human plasma protein. Among many other functions it is an important transporter of hydrophobic internal and external substances such as intermediate and end products of metabolism and drugs. In liver failure the albumin binding capacity is decreased because of a disproportion between available albumin molecules caused by decreased hepatic synthesis and hydrophobic toxins because of decreased hepatic clearance. The resulting increase in plasma and tissue concentrations of these substances is associated with multiple organ dysfunctions frequently seen in severe liver failure. The scope of the present article is to compare different liver support strategies with regard to their ability to regenerate the patients albumin pool by removing albumin‐bound toxins. Most prominent technique in this group is the molecular adsorbent recirculating system (MARS). It will be compared with single pass albumin dialysis (SPAD), fractionated plasma separation and adsorption system (FPSA, Prometheus), and plasma perfusion/bilirubin adsorption with special regard to efficacy and selectivity.
Review Article: Clinical Experience With Prometheus
Authors: Rifai Kinan , Manns Michael P
Published: April 2006

Abstract:  Prometheus is a new extracorporeal liver support device which facilitates the combined removal of both albumin‐bound and water‐soluble toxins based upon the method of fractionated plasma separation and adsorption (FPSA). The pilot trial included 11 patients with acute‐on‐chronic liver failure and concomitant renal failure. Prometheus therapy was found to be safe except for a reversible decrease of blood pressure. In three patients, clotting of the secondary system occurred. Prometheus treatment significantly improved blood levels of protein‐bound (conjugated bilirubin, bile acids, ammonia) and water‐soluble (creatinine, urea) substances. Thus, Prometheus might be a new therapeutic option in patients with severe hepatorenal syndrome. Furthermore, there is some preliminary experience with Prometheus in the treatment of refractory cholestatic pruritus and in successful bridging to liver transplantation. In order to compare extraction capacities of Prometheus and the molecular adsorbent recirculating system (MARS), five patients were crossover‐treated with both systems. Prometheus resulted in significantly higher reduction ratios of bilirubin, ammonia and urea. Another study closely monitored whether the device causes an unselective removal. Neither important cytokines nor coagulation factors were found to be removed. In conclusion, Prometheus seems to be a new therapeutic option in artificial liver support. A significant improvement of the biochemical milieu was already observed after two treatments. The potential to remove protein‐bound and water‐soluble substances has been shown without signs of a significant unselective removal.
Sorbent Suspensions vs. Sorbent Columns for Extracorporeal Detoxification in Hepatic Failure
Authors: Ash Stephen R , Sullivan Thomas A , Carr David J
Published: April 2006

Abstract:  Hepatic failure is a significant medical problem which has been unsuccessfully treated by hemodialysis. However, similar therapies using recirculated dialysate regenerated by sorbents in place of single‐pass dialysate have been beneficial in treating acute‐on‐chronic liver failure. The advantages of sorbent‐based treatments include some selectivity of toxin removal and improved removal of protein‐bound toxins. Activated carbon has been extensively used in detoxification systems, but has often had insufficient toxin capacity. Powdered activated carbon, because of its large surface area, can provide greater binding capacity for bilirubin and other toxins than granular carbon commonly used in detoxifying columns. Methods of using powdered carbon in extracorporeal blood treatment devices are reviewed in the present paper, including liver dialysis and a new sorbent suspension reactor (SSR); and the abilities and limitations of the SSR and columns to process protein solutions are discussed.
Fluidized Bed Adsorbent Systems for Extracorporeal Liver Support
Authors: Falkenhagen Dieter , Brandl Martin , Hartmann Jens , Kellner Karl‐Heinz , Posnicek Thomas , Weber Viktoria
Published: April 2006

Abstract:  Acute liver failure based on acute‐on‐chronic liver failure (AoCLF) or on acute severe damage of the liver caused by different etiologies includes complex mechanisms resulting in severe disturbances of principle liver functions. In order to compensate the liver’s function of detoxification as efficiently as possible, fluidized bed absorbent systems have been designed. In these systems, small particles with specific adsorption properties for toxins related to acute liver failure are applied. A special technology based on adsorbents in suspension has been developed under the guidance of our group and is prepared for clinical application during the coming year. This technology is called microspheres‐based detoxification system (MDS) and is based on microadsorbents with a diameter of 1–10 µm which are recirculated in suspension. The safety of the MDS is guaranteed by the use of fluorescently labeled magnetic microparticles, which in case of a membrane‐leakage are detected in the blood circuit by an optical system equipped with a magnetic trap. In vitro tests with two kinds of microadsorbents (a combination of a hydrophobic neutral resin and an anion exchange resin) showed excellent efficiency of the system with respect to adsorption capacity as well as to the kinetics of elimination of albumin‐bound substances (e.g. unconjugated bilirubin or cholic acid) and of non‐protein‐bound substances (e.g. phenol). Moreover, using a plasma filter or the Albuflow filter as membrane filters in the blood circuit, the MDS technology offers the possibility to remove inflammatory mediators such as tumor necrosis factor‐α (TNF) by additional use of specific adsorbents.
Is the Volume of the Parathyroid Gland a Predictor of Maxacalcitol Response in Advanced Secondary Hyperparathyroidism?
Authors: Tominaga Yoshihiro , Inaguma Daijyo , Matsuoka Susumu , Tahara Hideki , Kukita Kazutaka , Kurihara Satoshi , Onoda Noritaka , Tsuruta Yoshinari , Tsutsui Syuichi , Ohta Kazumichi , Kuwahara Morimasa , Tanaka Motoko , Nishizawa Yoshiki ,   (PTG study group)
Published: April 2006

Abstract:  We evaluated the relationship between the volume of parathyroid glands estimated by ultrasonography (US) and response of 22‐oxa calcitriol (Maxacalcitol, OCT) in patients with secondary hyperparathyroidism (2HPT) to evaluate whether the volume can be a predictor of the OCT response. Eleven institutes participated in this study. Ninety‐four patients with advanced 2HPT were enrolled. The volume of the parathyroid glands were estimated by US before and 6 months after OCT treatment. The response of OCT treatment was classified into three groups (Group A: i‐PTH < 300 pg/mL; Group B: 300 pg/mL ≤ i‐PTH < 500 pg/mL; Group C: i‐PTH ≥ 500 pg/mL). Forty‐eight patients were in Group A, 28 patients in Group B, and 18 patients in Group C. The PTH levels at the beginning and 6 months were 458.3–199.1 pg/mL (P < 0.0001) in Group A, 524.6–403.2 pg/mL (P = 0.007) in Group B and 736.7–613.6 pg/mL (ns) in Group C, respectively. The volume of the largest gland in Group B was significantly larger than that in Group A (96.2 vs. 343.2 mm3: P < 0.001). Clinical factors affecting response of OCT was evaluated by logistic regression analysis and only the volume of the largest gland was a significant factor. In the patients whose volume was less than 300 mm3, the OCT response was significantly effective. We conclude that the glandular volume of the largest parathyroid gland estimated by US can be a useful factor to predict the OCT response in patients with moderate or severe renal HPT.
Characterization of lymphangiogenesis in a model of adult skin regeneration
Authors: Rutkowski Joseph M. , Boardman Kendrick C. , Swartz Melody A.
Published: April 2006

To date, adult lymphangiogenesis is not well understood. In this study we describe the evolution of lymphatic capillaries in regenerating skin and correlate lymphatic migration and organization with the expression of matrix metalloproteinases (MMPs), immune cells, the growth factors VEGF-A and VEGF-C, and the heparan sulfate proteogylcan perlecan, a key component of basement membrane. We show that while lymphatic endothelial cells (LECs) migrate and organize unidirectionally, in the direction of interstitial fluid flow, they do not sprout into the region but rather migrate as single cells that later join together into vessels. Furthermore, in a modified “shunted flow” version of the model, infiltrated LECs fail to organize into functional vessels, indicating that interstitial fluid flow is necessary for lymphatic organization. Perlecan expression on new lymphatic vessels was only observed after vessel organization was complete and also appeared first in the distal region, consistent with the directionality of lymphatic migration and organization. VEGF-C expression peaked at the initiation of lymphangiogenesis but was reduced to lower levels throughout organization and maturation. In mice lacking MMP-9, lymphatics regenerated normally, suggesting that MMP-9 is not required for lymphangiogenesis, at least in mouse skin. This study thus characterizes the process of adult lymphangiogenesis and differentiates it from sprouting blood angiogenesis, verifies its dependence on interstitial fluid flow for vessel organization, and correlates its temporal evolution with those of relevant environmental factors.
Lack of activation of molecular forms of the BNP system in human grade 1 hypertension and relationship to cardiac hypertrophy
Authors: Belluardo Paola , Cataliotti Alessandro , Bonaiuto Lorena , Giuffrè Eliana , Maugeri Egle , Noto Paola , Orlando Giovanna , Raspa Giuseppa , Piazza Brigida , Babuin Luciano , Chen Horng H. , Martin Fernando L. , McKie Paul M. , Heublein Denise M. , Burnett John C. , Malatino Lorenzo S.
Published: April 2006

We evaluated relationships among two circulating molecular forms of brain natriuretic peptide (BNP32 and NT-proBNP), severity of hypertension (HTN), and cardiac hypertrophy in subjects with mild, moderate, and severe HTN. We prospectively studied 78 patients (43 males; mean age 51.4 ± 11 yr) with essential HTN and 28 age- and sex-matched controls. BNP32 and NT-proBNP were measured by radioimmunoassay. In grade 1 HTN, BNP32 was not elevated and NT-proBNP was reduced ( P = 0.030) compared with controls. However, log-transformed values of BNP32 and NT-proBNP were both increased with severity of HTN from grade 1 to 3 ( P <0.0001 and P = 0.003, respectively). By multivariate analysis, log BNP32 was independently predicted by age (β = 0.210, P = 0.026) and HTN grade (β = 0.274, P = 0.004), whereas log NT-proBNP was independently predicted by sex (β = 0.235, P = 0.012) and HTN grade (β = 0.218, P = 0.0023). Two forms of BNP were measured in normal subjects and patients with essential HTN. In grade 1 HTN, BNP32 was unchanged and NT-proBNP was significantly reduced compared with controls. As severity increased in humans with grade 1 to 3 HTN, both BNP32 and NT-proBNP levels were increased while not being affected by the presence of left ventricular hypertrophy. The lack of activation of BNP32 together with the reduction of NT-proBNP in grade 1 HTN may represent an impaired response of the BNP system in the early phase of HTN. The later activation of both forms of BNP may be a late compensatory effect, because it correlates with severity of HTN rather than cardiac hypertrophy/remodeling.
Vascular reactivity and endothelial NOS activity in rat thoracic aorta during and after hyperbaric oxygen exposure
Authors: Hink Jonas , Thom Stephen R. , Simonsen Ulf , Rubin Inger , Jansen Erik
Published: April 2006

Accumulating evidence suggests that hyperbaric oxygen (HBO) stimulates neuronal nitric oxide (NO) synthase (NOS) activity, but the influence on endothelial NOS (eNOS) activity and vascular NO bioavailability remains unclear. We used a bioassay employing rat aortic rings to evaluate vascular NO bioavailability. HBO exposure to 2.8 atm absolute (ATA) in vitro decreased ACh relaxation. This effect remained unchanged, despite treatment with SOD-polyethylene glycol and catalase-polyethylene glycol, suggesting that the reduction in endothelium-derived NO bioavailability was independent of superoxide production. In vitro HBO induced contraction of resting aortic rings with and without endothelium, and these contractions were reduced by the NOS inhibitor Nω-nitro-l-arginine. In addition, in vitro HBO attenuated the vascular contraction produced by norepinephrine, and this effect was reversed by Nω-nitro-l-arginine, but not by endothelial denudation. These findings indicate stimulation of extraendothelial NO production during HBO exposure. A radiochemical assay was used to assess NOS activity in rat aortic endothelial cells. Catalytic activity of eNOS in cell homogenates was not decreased by HBO, and in vivo HBO exposure to 2.8 ATA was without effect on eNOS activity and/or vascular NO bioavailability in vitro. We conclude that HBO reduces endothelium-derived NO bioavailability independent of superoxide production, and this effect seems to be unrelated to a decrease in eNOS catalytic activity. In addition, HBO increases the resting tone of rat aortic rings and attenuates the contractile response to norepinephrine by endothelium-independent mechanisms that involve extraendothelial NO production.
Biphasic changes in cardiac excitation-contraction coupling early in chronic alcohol exposure
Authors: Aistrup Gary L. , Kelly James E. , Piano Mariann R. , Wasserstrom J. Andrew
Published: April 2006

Although the negative inotropic effects of both acute and chronic ethanol (EtOH) exposure are well known, little is known concerning the acute-to-chronic transition of such effects. In this study, our objective was to address this question by detailing the effects that acute EtOH exposure induces on cellular excitation-contraction (EC) coupling and, subsequently, comparing whether and how such changes translate to the early chronic EtOH condition in a rat model known to develop alcohol-induced cardiomyopathy. Acute EtOH exposure, as formerly reported, indeed induced dose-dependent negative inotropic changes in cellular EC coupling, manifest as reductions in cell shortening, Ca2+ transient amplitude, Ca2+ decay rate, and sarcoplasmic reticulum Ca2+ content of isolated rat ventricular cardiac myocytes. Supplementary to this, we found Ca2+ spark character not to be significantly affected by acute EtOH exposure. In contrast, the results obtained from cardiac myocytes isolated from rats fed a diet containing ∼9% (vol/vol) EtOH for 1 mo revealed changes in these parameters reflecting positive inotropy, whereas at 3 mo, these parameters again reflected negative inotropy similar but not identical to that induced by acute EtOH exposure. No significant changes were evident at either 1- or 3-mo chronic EtOH administration in echocardiographic parameters known to be perturbed in alcoholic cardiomyopathy (ACM), thus indicating that we were examining an asymptomatic stage in chronic EtOH administration consistent with an acute-to-chronic transition phase. Continued study of such transition-phase events should provide important insight into which molecular-cellular components of EC coupling play pivotal roles in EtOH-induced disease processes, such as ACM.
Paradoxical coronary microcirculatory constriction during ischemia: a synergic function for nitric oxide and endothelin
Authors: Kusmic Claudia , Lazzerini Guido , Coceani Flavio , Barsacchi Renata , L'Abbate Antonio , Sambuceti Gianmario
Published: April 2006

A paradoxical microcirculatory constriction has been observed in hearts of patients with ischemia, secondary to coronary stenosis. Here, using the isolated mouse heart (Langendorff), we examined the mechanism of this response, assuming involvement of nitric oxide (NO) and endothelin-1 (ET-1) systems. Perfusion pressure was maintained at 65 mmHg for 70 min ( protocol 1), or it was reduced to 30 mmHg over two intervals, between the 20- and 40-min marks ( protocol 2) or from the 20-min mark onward ( protocol 3). In protocol 1, coronary resistance (CR) remained steady in untreated heart, whereas it progressively increased during treatment with the NO synthesis inhibitor NG-nitro-l-arginine methyl ester (l-NAME) (2.7-fold) or the ETAantagonist BQ-610 (2.8 fold). The ETBantagonist BQ-788 had instead no effect by itself but curtailed vasoconstriction to BQ-610. In protocol 2, hypotension raised CR by 2.2-fold. This response was blunted by reactive oxygen species (ROS) scavengers (mannitol and superoxide dismutase plus catalase) and was converted into vasodilation by l-NAME, BQ-610, or BQ-788. Restoration of normal pressure was followed by vasodilation and vasoconstriction, respectively, in untreated and treated preparations. In protocol 3, CR progressively increased with hypotension in the absence but not presence of l-NAME or BQ-610. We conclude that the coronary vasculature is normally relaxed by two concerted processes, a direct action of NO and ET-1 curtailing an ETB2-mediated tonic vasoconstriction through ETAactivation. The negative feedback mechanism on ETB2subsides during hypotension, and the ensuing vasoconstriction is ascribed to ET-1 activating ETAand ETB2and reactive nitrogen oxide species originating from ROS-NO interaction.
Relationship between muscle sympathetic nerve activity and systemic hemodynamics during nitric oxide synthase inhibition in humans
Authors: Charkoudian N. , Joyner M. J. , Barnes S. A. , Johnson C. P. , Eisenach J. H. , Dietz N. M. , Wallin B. G.
Published: April 2006

Large interindividual differences exist in resting sympathetic nerve activity (SNA) among normotensive humans with similar arterial pressure (AP). We recently showed inverse relationships of resting SNA with cardiac output (CO) and vascular adrenergic responsiveness that appear to balance the influence of differences in SNA on blood pressure. In the present study, we tested whether nitric oxide (NO)-mediated vasodilation has a role in this balance by evaluating hemodynamic responses to systemic NO synthase (NOS) inhibition in individuals with low and high resting muscle SNA (MSNA). We measured MSNA via peroneal microneurography, CO via acetylene uptake and AP directly, at baseline and during increasing systemic doses of the NOS inhibitor NG-monomethyl-l-arginine (l-NMMA). Baseline MSNA ranged from 9 to 38 bursts/min (13 to 68 bursts/100 heartbeats). l-NMMA caused dose-dependent increases in AP and total peripheral resistance and reflex decreases in CO and MSNA. Increases in AP with l-NMMA were greater in individuals with high baseline MSNA ( PANOVA < 0.05). For example, after 8.5 mg/kg of l-NMMA, in the low MSNA subgroup ( n = 6, 28 ± 4 bursts/100 heartbeats), AP increased 9 ± 1 mmHg, whereas in the high-MSNA subgroup ( n = 6, 58 ± 3 bursts/100 heartbeats), AP increased 15 ± 2 mmHg ( P < 0.01). The high-MSNA subgroup had lower baseline CO and smaller decreases in CO with l-NMMA, but changes in total peripheral resistance were not different between groups. We conclude that differences in CO among individuals with varying sympathetic traffic have important hemodynamic implications during disruption of NO-mediated vasodilation.
Activation of AMPK α- and γ-isoform complexes in the intact ischemic rat heart
Authors: Li Ji , Coven David L. , Miller Edward J. , Hu Xiaoyue , Young Martin E. , Carling David , Sinusas Albert J. , Young Lawrence H.
Published: April 2006

AMP-activated protein kinase (AMPK) plays a key role in modulating cellular metabolic processes. AMPK, a serine-threonine kinase, is a heterotrimeric complex of catalytic α-subunits and regulatory β- and γ-subunits with multiple isoforms. Mutations in the cardiac γ2-isoform have been associated with hypertrophic cardiomyopathy and pre-excitation syndromes. However, physiological regulation of AMPK complexes containing different subunit isoforms is not well defined and is important for an understanding of the function of this signaling pathway in the intact heart. We evaluated the kinase activity associated with heart AMPK complexes containing specific α- and γ-subunit isoforms of AMPK in an in vivo rat model of regional ischemia. Left coronary artery occlusion activated the immunoprecipitated α1-isoform (6-fold, P < 0.01) and α2-isoform (9-fold, P < 0.01) in the ischemic left ventricle compared with sham controls. The degree of α-subunit activation depended on the extent of ischemia and paralleled echocardiographic contractile dysfunction. The regulatory γ1- and γ2-isoforms were expressed in the heart. The γ1- and γ2-isoforms coimmunoprecipitated with α1- and α2-isoforms in proportion to α-subunit content. γ1-Isoform immunocomplexes accounted for 70% of AMPK activity and AMPK phosphorylation (Thr172) in hearts. Ischemia similarly increased AMPK activity associated with the γ1- and γ2-isoform complexes threefold ( P < 0.01 for each). Thus AMPK catalytic α1- and α2-isoforms are activated by regional ischemia in vivo in the heart, irrespective of the regulatory γ1- or γ2 -isoforms to which they are complexed. Despite the pathophysiological importance of γ2-isoform mutations, γ1-isoform complexes account for most of the AMPK activity in the ischemic heart.
Role of 12-lipoxygenase in volatile anesthetic-induced delayed preconditioning in mice
Authors: Tsutsumi Yasuo M. , Patel Hemal H. , Huang Diane , Roth David M.
Published: April 2006

Delayed cardiac protection mediated by 12-lipoxygenase (12-LO) expression and activity has been linked to opioid receptor stimulation. The role of 12-LO in volatile anesthetic-induced delayed cardiac protection has not been determined. We tested the hypothesis that expression and activity of 12-LO mediate delayed cardiac protection induced by isoflurane in the mouse heart in vivo. Mice were pretreated with 1.4% isoflurane for 30 min and allowed to recover for 1, 12, or 24 h. Immunoblot analysis showed isoflurane significantly enhanced 12-LO protein expression at 12 and 24 h after isoflurane exposure, and this induction of 12-LO was confirmed by immunohistochemistry of whole heart sections at 24 h. The induced protein expression appeared to be localized to intercalated disc regions adjoining adjacent cardiac myocytes. Additionally, mice ± isoflurane (24 h previously) were subjected to 30 min coronary artery occlusion followed by 2 h of reperfusion in the presence and absence of a 12-LO inhibitor. Isoflurane reduced infarct size (27.1 ± 2.2% of the area at risk; n = 8) compared with the control group (44.6 ± 3.6%, n = 8). Baicalein (3 mg/kg), a selective 12-LO inhibitor, blocked the delayed protective effects of isoflurane pretreatment on infarct size (40.6 ± 3.6%, n = 8). These data suggest that 12-LO is an important mediator of isoflurane-induced delayed preconditioning.
L-type calcium channel α-subunit and protein kinase inhibitors modulate Rem-mediated regulation of current
Authors: Crump Shawn M. , Correll Robert N. , Schroder Elizabeth A. , Lester William C. , Finlin Brian S. , Andres Douglas A. , Satin Jonathan
Published: April 2006

Cardiac voltage-gated L-type Ca channels (CaV) are multiprotein complexes, including accessory subunits such as CaVβ2 that increase current expression. Recently, members of the Rad and Gem/Kir-related family of small GTPases have been shown to decrease current, although the mechanism remains poorly defined. In this study, we evaluated the contribution of the L-type Ca channel α-subunit (CaV1.2) to CaVβ2-Rem inhibition of Ca channel current. Specifically, we addressed whether protein kinase A (PKA) modulation of the Ca channel modifies CaVβ2-Rem inhibition of Ca channel current. We first tested the effect of Rem on CaV1.2 in human embryonic kidney 293 (HEK-293) cells using the whole cell patch-clamp configuration. Rem coexpression with CaV1.2 reduces Ba current expression under basal conditions, and CaVβ2a coexpression enhances Rem block of CaV1.2 current. Surprisingly, PKA inhibition by 133 nM H-89 or 50 μM Rp-cAMP-S partially relieved the Rem-mediated inhibition of current activity both with and without CaVβ2a. To test whether the H-89 action was a consequence of the phosphorylation status of CaV1.2, we examined Rem regulation of the PKA-insensitive CaV1.2 serine 1928 (S1928) to alanine mutation (CaV1.2-S1928A). CaV1.2-S1928A current was not inhibited by Rem and when coexpression with CaVβ2a was not completely blocked by Rem coexpression, suggesting that the phosphorylation of S1928 contributes to Rem-mediated Ca channel modulation. As a model for native Ca channel complexes, we tested the ability of Rem overexpression in HIT-T15 cells and embryonic ventricular myocytes to interfere with native current. We find that native current is also sensitive to Rem block and that H-89 pretreatment relieves the ability of Rem to regulate Ca current. We conclude that Rem is capable of regulating L-type current, that release of Rem block is modulated by cellular kinase pathways, and that the CaV1.2 COOH terminus contributes to Rem-dependent channel inhibition.
Increased arterial load alters aortic structural and functional properties during embryogenesis
Authors: Lucitti Jennifer L. , Visconti Richard , Novak Jacqueline , Keller Bradley B.
Published: April 2006

As in the adult dorsal aorta, the embryonic dorsal aorta is an important determinant of cardiovascular function, and increased stiffness may have secondary effects on cardiac and microcirculatory development. We previously showed that acutely and chronically increased arterial load via vitelline artery ligation (VAL) increases systemic arterial stiffness. To test the hypothesis that local dorsal aortic stiffness also increases, we measured aortic pulse-wave velocity (PWV) and assessed the active and passive properties (stress and strain) of isolated aortic segments. PWV along the dorsal aorta increased acutely and chronically after VAL. Analysis of isolated aortic active properties suggests that load-exposed aortas experienced higher stress, but not strain, at similar intraluminal pressures. When smooth muscle tone was relaxed, strain decreased in VAL vessels, whereas stress became similar to control vessels. Immunohistochemical analysis revealed that although aortic smooth muscle α-actin content was similar between groups, more cell layers expressed smooth muscle α-actin, and myocyte cell shape was markedly rounder in VAL embryos. Additionally, aortic and perivascular collagen type I and III content significantly increased in load-exposed VAL vessels. Increased production of these proteins is consistent with the observed increase in aortic PWV and decreased strain in VAL passive aortic segments. Thus the embryonic dorsal aorta is sensitive to increased arterial load and adapts by altering its material properties via changes in collagen content.
Effects of adenosine on myocardial glucose and palmitate metabolism after transient ischemia: role of 5′-AMP-activated protein kinase
Authors: Jaswal Jagdip S. , Gandhi Manoj , Finegan Barry A. , Dyck Jason R. B. , Clanachan Alexander S.
Published: April 2006

Loss of cardioprotection by adenosine in hearts stressed by transient ischemia may be due to its effects on glucose metabolism. In the absence of transient ischemia, adenosine inhibits glycolysis, whereas it accelerates glycolysis after transient ischemia. Inasmuch as 5′-AMP-activated protein kinase (AMPK) is implicated as a regulator of glucose and fatty acid utilization, this study determined whether a differential alteration of AMPK activity contributes to acceleration of glycolysis by adenosine in hearts stressed by transient ischemia. Studies were performed in working rat hearts perfused aerobically under normal conditions or after transient ischemia (two 10-min periods of ischemia followed by 5 min of reperfusion). LV work was not affected by adenosine. AMPK phosphorylation was not affected by transient ischemia; however, phosphorylation and activity were increased nine- and threefold, respectively, by adenosine in stressed hearts. Phosphorylation of acetyl-CoA carboxylase and rates of palmitate oxidation were unaltered. Glycolysis and calculated proton production were increased 1.8- and 1.7-fold, respectively, in hearts with elevated AMPK activity. Elevated AMPK activity was associated with inhibition of glycogen synthesis and unchanged rates of glucose uptake and glycogenolysis. Phentolamine, an α-adrenoceptor antagonist, which prevents adenosine-induced activation of glycolysis in stressed hearts, prevented AMPK phosphorylation. These data demonstrate that adenosine-induced activation of AMPK after transient ischemia is not sufficient to alter palmitate oxidation or glucose uptake. Rather, activation of AMPK alters partitioning of glucose away from glycogen synthesis; the increase in glycolysis may in part contribute to loss of adenosine-induced cardioprotection in hearts subjected to transient ischemia.
Cell signaling underlying the pathophysiology of pneumonia
Authors: Prince Alice S. , Mizgerd Joseph P. , Wiener-Kronish Jeanine , Bhattacharya Jahar
Published: April 2006

The symposium addressed the burgeoning interest in fundamental mechanisms underlying the onset of pneumonia. Bacteria exploit the lung's innate immune mechanism, resulting in pathophysiological cell signaling. As a consequence inflammation develops, leading to pneumonia. New mechanisms have been identified by which bacteria or bacterial products in the airway induce cross-compartmental signaling that leads to inflammatory consequences. The speakers addressed activation of the transcription factor, NF-κB occurring as a consequence of bacterial interactions with specific receptors, such as the Toll-like receptors and the TNF receptor 1 (Prince), or as a consequence of cytokine induction (Mizgerd). Also considered were mechanisms of bacterial virulence in the clinical setting (Wiener-Kronish) and the role of alveolar-capillary signaling mechanisms in the initiation of lung inflammation.
A novel bronchial ring bioassay for the evaluation of small airway smooth muscle function in mice
Authors: Liu John Q. , Yang Dennis , Folz Rodney J.
Published: April 2006

Advances in our understanding of murine airway physiology have been hindered by the lack of suitable, ex vivo, small airway bioassay systems. In this study, we introduce a novel small murine airway bioassay system that permits the physiological and pharmacological study of intrapulmonary bronchial smooth muscle via a bronchial ring (BR) preparation utilizing BR segments as small as 200 μm in diameter. Using this ex vivo BR bioassay, we characterized small airway smooth muscle contraction and relaxation in the presence and absence of bronchial epithelium. In control BRs, the application of mechanical stretch is followed by spontaneous bronchial smooth muscle relaxation. BRs pretreated with methacholine (MCh) partially attenuate this stretch-induced relaxation by as much as 42% compared with control. MCh elicited a dose-dependent bronchial constriction with a maximal tension ( Emax) of 8.7 ± 0.2 mN at an EC50of 0.33 ± 0.02 μM. In the presence of nifedipine, ryanodine, 2-aminoethoxydiphenyl borate, and SKF-96365, Emaxto MCh was significantly reduced. In epithelium-denuded BRs, MCh-induced contraction was significantly enhanced to 11.4 ± 1.0 mN with an EC50of 0.16 ± 0.04 μM ( P < 0.01). Substance P relaxed MCh-precontracted BR by 62.1%; however, this bronchial relaxation effect was completely lost in epithelium-denuded BRs. Papaverine virtually abolished MCh-induced constriction in both epithelium-intact and epithelium-denuded bronchial smooth muscle. In conclusion, this study introduces a novel murine small airway BR bioassay that allows for the physiological study of smooth muscle airway contractile responses that may aid in our understanding of the pathophysiology of asthma.
AMP-activated protein kinase protects against anti–epidermal growth factor receptor-Pseudomonas exotoxin A immunotoxin-induced MA11 breast cancer cell death
Authors: Andersson Yvonne , Le Hang , Juell Siri , Fodstad Øystein
Published: April 2006

We have shown previously that our 425.3PE immunotoxin inhibits protein synthesis and induces apoptosis in human breast cancer cells. In attempts to further elucidate the intracellular pathways implicated in its cellular effects, we found that the immunotoxin induced an initial stress response, which rapidly caused an imbalance in the cellular energy status with an increase in reactive oxygen species. The AMP-activated protein kinase (AMPK), a sensor of increased cellular AMP/ATP ratio, was activated by 425.3PE. An immunotoxin-induced activation of c-Jun NH2-terminal kinase (JNK) preceded and overlapped caspase-mediated cleavage of the α-subunit of AMPK in a time- and dose-dependent manner. The JNK activation occurred already at a dose level too low to induce any detectable changes in the apoptotic machinery or protein synthesis. In contrast, cycloheximide, even at a concentration causing a 90% inhibition of protein synthesis, did neither affect the ATP level nor activate JNK and AMPK. Pretreatment of the cells with the specific AMPK inhibitor compound C and JNK inhibitor SP600125 blocked activation of AMPK and JNK, respectively, and subsequently sensitized the cells to 425.3PE-induced cell death. Whereas the antioxidant N-acetyl-l-cysteine blocked the generation of reactive oxygen species and activation of JNK and AMPK, it did not block immunotoxin-induced apoptosis. Together, the results show that 425.3PE induces several parallel signaling events, observed initially as an early activation of survival pathways, protecting the cells against the toxic effects of the immunotoxin, followed by subsequent apoptosis induction and protein synthesis inhibition. Conceivably, therapeutic manipulation of the signaling intermediates AMPK and JNK might provide a means to maximize the anticancer effects of the 425.3 immunotoxin. [Mol Cancer Ther 2006;5(4):1050–9]
Organ-specific expression profiles of rat mammary gland, liver, and lung tissues treated with targretin, 9-cis retinoic acid, and 4-hydroxyphenylretinamide
Authors: Wang Yian , Yao Ruisheng , Maciag Anna , Grubbs Clinton J. , Lubet Ronald A. , You Ming
Published: April 2006

A rexinoid, targretin, and two retinoids, 9-cis retinoic acid (9cRA) and 4-hydroxyphenylretinamide (4HPR), were examined for their effects on gene expression in rat mammary gland, liver, and lung tissues. The chemopreventive effects of these agents have largely been attributed to their ability to interact with retinoic acid receptors (RAR) and/or retinoid X receptors (RXR). Targretin interacts with the RXR receptors. 9cRA interacts with both the RAR and RXR receptors, whereas 4HPR has a moderate affinity primarily for RAR γ. Based on previous studies on mammary chemoprevention, targretin (150 mg/kg diet), 9cRA (100 mg/kg diet), and 4HPR (782 mg/kg diet), were administered to rats continually in their diet for 7 days. Tissue- and agent-specific expression differences were determined by comparing tissues from treated rats with those from rats given a control diet. There were significantly more changes associated with targretin than 9cRA or 4HPR. Only a limited number of expression changes were found with 4HPR treatment. For each organ, targretin- and 9cRA-treated tissues clustered closely together, whereas 4HPR-treated tissues clustered with the tissues from the control diet group. In contrast to 9cRA treatment, targretin treatment altered genes that involved fatty acid metabolism and modulation of various cytochromes P450 in the liver, clearly demonstrating the very disparate nature of these two retinoids. These expression signatures could provide useful pharmacodynamic biomarkers for retinoid treatment and chemoprevention. [Mol Cancer Ther 2006;5(4):1060–72]
Targeted chemotherapy against intraperitoneally disseminated colon carcinoma using a cationized gelatin–conjugated HVJ envelope vector
Authors: Mima Hidetoshi , Yamamoto Seiji , Ito Makoto , Tomoshige Ryuji , Tabata Yasuhiko , Tamai Katsuto , Kaneda Yasufumi
Published: April 2006

The hemagglutinating virus of Japan envelope (HVJ-E; Sendai virus) vector derived from inactivated HVJ particles can be used to deliver DNA, proteins, and drugs into cells both in vitro and in vivo. HVJ-E is capable of delivering bleomycin, an anticancer drug, to various cancer cell lines, thereby producing 300-fold greater cytotoxicity than administration of bleomycin alone. In a mouse model of peritoneally disseminated colon cancer, we injected HVJ-E containing the luciferase gene into the peritoneum. Unexpectedly, luciferase gene expression was not observed within the tumor deposits or any organs. However, when combined with cationized gelatin (CG), CG-HVJ-E produced a high level of luciferase gene expression primarily within the tumor deposits. Forty-eight hours after introducing colon cancer cells into the peritoneum of experimental mice, CG-HVJ-E with or without bleomycin was injected into the abdominal cavity. Following six injections of bleomycin-incorporated CG-HVJ-E, complete responses were observed in 40% of the mice examined. All of the mice that received either empty CG-HVJ-E or bleomycin alone died within 40 days of having cancer cells introduced into the peritoneum. When the mice with complete responses were rechallenged with colon cancer cells from the same cell line, no tumors developed. Thus, CG-HVJ-E may suppress peritoneal dissemination of cancer. [Mol Cancer Ther 2006;5(4):1021–8]
Effect of combined DNA repair inhibition and G2 checkpoint inhibition on cell cycle progression after DNA damage
Authors: Sturgeon Christopher M. , Knight Zachary A. , Shokat Kevan M. , Roberge Michel
Published: April 2006

In response to DNA damage, cell survival can be enhanced by activation of DNA repair mechanisms and of checkpoints that delay cell cycle progression to allow more time for DNA repair. Inhibiting both responses with drugs might cause cancer cells to undergo cell division in the presence of lethal amounts of unrepaired DNA. However, we show that interfering with DNA repair via inhibition of DNA-dependent protein kinase (DNA-PK) reduces the ability of checkpoint inhibitors to abrogate G2 arrest and their radiosensitizing activity. Cells exposed to the DNA-PK inhibitor AMA37, DNA-PK-deficient cells, and nonhomologous end joining–deficient cells all enter prolonged G2 arrest after exposure to ionizing radiation doses as low as 2 Gy. The checkpoint kinase Chk2 becomes rapidly and transiently overactivated, whereas Chk1 shows sustained overactivation that parallels the prolonged accumulation of cells in G2. Therefore, in irradiated cells, DNA repair inhibition elicits abnormally strong checkpoint signaling that causes essentially irreversible G2 arrest and strongly reduces the ability of checkpoint kinase inhibitors to overcome G2 arrest and radiosensitize cells. Variable levels of proteins controlling DNA repair have been documented in cancer cells. Therefore, these results have relevance to the development of DNA-PK inhibitors and G2 checkpoint inhibitors as experimental therapeutic approaches to enhance the selective killing of tumor cells by radiotherapy or DNA-damaging chemotherapeutic agents. [Mol Cancer Ther 2006;5(4):885–92]
Inhibition of phosphorylation of the colony-stimulating factor-1 receptor (c-Fms) tyrosine kinase in transfected cells by ABT-869 and other tyrosine kinase inhibitors
Authors: Guo Jun , Marcotte Patrick A. , McCall J. Owen , Dai Yujia , Pease Lori J. , Michaelides Michael R. , Davidsen Steven K. , Glaser Keith B.
Published: April 2006

The properties of several multitargeted receptor tyrosine kinase inhibitors have been studied for their inhibition of colony-stimulating factor-1 receptor (CSF-1R) signaling. A structurally novel, multitargeted tyrosine kinase inhibitor (ABT-869), imatinib (STI571), and four compounds currently in clinical development (AG013736, BAY 43-9006, CHIR258, and SU11248) were tested for inhibition of CSF-1R signaling in both the enzymatic and cellular assays. ABT-869 showed potent CSF-1R inhibition in both the enzyme and cell-based assays (IC50s < 20 nmol/L). In contrast to a previous report, we have found that imatinib has activity against human CSF-1R in both assays at submicromolar concentrations. In enzyme assays, we have found that the inhibition of CSF-1R by both ABT-869 and imatinib are competitive with ATP, with Ki values of 3 and 120 nmol/L, respectively. SU11248 is a potent inhibitor of CSF-1R in the enzyme assay (IC50 = 7 nmol/L) and inhibits receptor phosphorylation in the cellular assay (IC50 = 61 nmol/L). AG013736 was also a potent inhibitor of CSF-1R in both assays (enzyme, IC50 = 16 nmol/L; cellular, IC50 = 21 nmol/L), whereas BAY 43-9006 is less potent in the enzyme assay (IC50 = 107 nmol/L) than in the cellular system (IC50 = 20 nmol/L). In contrast, we found that CHIR258 had less activity in the cellular assay (IC50 = 535 nmol/L) relative to its enzymatic potency (IC50 = 26 nmol/L). These results show the use of a cell-based assay to confirm the inhibitory activity of lead compounds and drug candidates, such as ABT-869, against the CSF-1R protein in situ. [Mol Cancer Ther 2006;5(4):1007–13]
Curcumin induces caspase-3-dependent apoptotic pathway but inhibits DNA fragmentation factor 40/caspase-activated DNase endonuclease in human Jurkat cells
Authors: Sikora Ewa , Bielak-Żmijewska Anna , Magalska Adriana , Piwocka Katarzyna , Mosieniak Grazyna , Kalinowska Magdalena , Widlak Piotr , Cymerman Iwona A. , Bujnicki Janusz M.
Published: April 2006

Curcumin is a natural pigment that has been shown to induce cell death in many cancer cells; however, the death mode depends on the cell type and curcumin concentration. Here we show that, in Jurkat cells, 50 μmol/L curcumin severely lowers cell survival and induces initial stage of chromatin condensation. It also induces caspase-3, which is sufficient to cleave DNA fragmentation factor 45 [DFF45/inhibitor of caspase-activated DNase (ICAD)], the inhibitor of DFF40/CAD endonuclease. However, the release of DFF40/CAD from its inhibitor does not lead to oligonucleosomal DNA degradation in curcumin-treated cells. Moreover, curcumin treatment protects cells from UVC-induced oligonucleosomal DNA degradation. In biochemical experiments using recombinant DFF activated with caspase-3, we show that curcumin inhibits plasmid DNA and chromatin degradation although it does not prevent activation of DFF40/CAD endonuclease after its release from the inhibitor. Using DNA-binding assay, we show that curcumin does not disrupt the DNA-DFF40/CAD interaction. Instead, molecular modeling indicates that the inhibitory effect of curcumin on DFF40/CAD activity results from curcumin binding to the active center of DFF40/CAD endonuclease. [Mol Cancer Ther 2006;5(4):927–34]
Molecular targets for apigenin-induced cell cycle arrest and apoptosis in prostate cancer cell xenograft
Authors: Shukla Sanjeev , Gupta Sanjay
Published: April 2006

Apigenin (4′,5,7-trihydroxyflavone) is a promising chemopreventive agent abundantly present in fruits and vegetables that has been shown to promote cell cycle arrest and apoptosis in various malignant cell lines. To determine whether pharmacologic intervention with apigenin has a direct growth inhibitory effect on human prostate tumors implanted in athymic nude mice, we examined cell cycle regulatory molecules as precise molecular targets of apigenin action. Apigenin feeding by gavage to these mice at doses of 20 and 50 μg/mouse/d in 0.2 mL of a vehicle containing 0.5% methyl cellulose and 0.025% Tween 20 resulted in significant decreases in tumor volume and mass of androgen-sensitive 22Rv1 and androgen-insensitive PC-3-implanted cells. Oral intake of apigenin resulted in dose-dependent (a) increase in the protein expression of WAF1/p21, KIP1/p27, INK4a/p16, and INK4c/p18; (b) down-modulation of the protein expression of cyclins D1, D2, and E; and cyclin-dependent kinases (cdk), cdk2, cdk4, and cdk6; (c) decrease in retinoblastoma phosphorylation at serine 780; (d) increase in the binding of cyclin D1 toward WAF1/p21 and KIP1/p27; and (e) decrease in the binding of cyclin E toward cdk2 in both types of tumors. In addition, apigenin feeding resulted in stabilization of p53 by phosphorylation at serine 15 in 22Rv1 tumors, which seems to exhibit p53-dependent growth inhibitory responses. Apigenin intake by these mice also resulted in induction of apoptosis, which positively correlated with serum and tumor apigenin levels. Taken together, this is the first systematic in vivo study showing the involvement of cell cycle regulatory proteins as potential molecular targets of apigenin. [Mol Cancer Ther 2006;5(4):843–52]
Potent antitumor activity of 10-methoxy-9-nitrocamptothecin
Authors: Luo Peihua , He Qiaojun , He Xungui , Hu Yongzhou , Lu Wei , Cheng Yiyu , Yang Bo
Published: April 2006

The present data showed that 10-methoxy-9-nitrocamptothecin (MONCPT), a family of camptothecin analogues, possessed high antitumor activity in vitro and in vivo. Cytotoxicity assays showed that MONCPT was a potential and highly efficient antitumor compound with IC50 values of 0.1 to 500 nmol/L in nine tumor cell lines. The high cytotoxic potency of MONCPT was paralleled with its ability to increase the cellular accumulation of DNA damage. DNA relaxation assay also showed that MONCPT exerted high potency as a topoisomerase I inhibitor. Moreover, administration of MONCPT (5–20 mg/kg) for 15 to 17 days significantly inhibited tumor growth in human androgen–independent prostate tumor (PC3) and human non–small cell lung tumor (A549) xenografts; the inhibition rates ranged from 29.6% to 98%. The cytotoxic effect of 1,000 nmol/L of MONCPT in PC3 cells was associated with causing an arrest in G0-G1 phase, whereas that of 10 and 100 nmol/L MONCPT was relative to a persistent block in G2-M phase. Furthermore, down-regulation of CDK2, CDK4, and cyclin D1 was observed in PC3 cells treated with 1,000 nmol/L of MONCPT, whereas overexpression of CDK7, CDK1, and cyclin B1 was seen in PC3 cells treated with 10 and 100 nmol/L of MONCPT. These results suggested that cell cycle regulation might contribute to the anticancer properties of MONCPT and strongly support the further anticancer development of MONCPT. [Mol Cancer Ther 2006;5(4):962–8]