Figure 4: Carvedilol Activates AKT, Prevents the Doxorubicin-dependent Loss of β

(A) Lysates from β1AR-overexpressing cardiomyocytes treated for 24 h with vehicle, 10 μM doxorubicin alone, or 10 μM doxorubicin plus 1 μM carvedilol were probed for β1AR and β2AR expression, protein kinase B (AKT) and ERK phosphorylation, and caspase-3 cleavage (with β-actin immunoreactivity used as a loading control). Results are representative of data obtained in 4 separate experiments on separate culture preparations. (B) Lysates from cardiomyocytes treated with 0.1 μM isoproterenol or 1 μM carvedilol for the indicated intervals were probed for ERK and AKT phosphorylation, with immunoreactivity for the cognate protein serving as a loading control. A representative experiment is depicted on top, with maximal agonist-dependent phosphorylation responses for ERK (at 5 min, n = 5) or AKT (at 30 min, n = 3) quantified at the bottom, with the basal value in each individual experiment set to 100% as a reference (open bar) and agonist-dependent increases in immunoreactivity expressed relative to basal (shaded bars). Abbreviations as in Figures 1 and 3

Figure 3: Doxorubicin Treatment Leads to a Decrease in β

(A) β1AR-overexpressing cardiomyocyte cultures were treated for 24 h with doxorubicin (Dox) in the absence or presence of 1 μM carvedilol as indicated, and lysates were probed for β1AR expression, with β-actin immunoreactivity used as a loading control. A representative experiment is depicted on the left, with effects of doxorubicin (10 μM) and/or carvedilol on the abundance of the larger full-length (FL) and smaller truncated β1AR species quantified on the right (n = 7; *p (B) Cardiomyocytes were pretreated for 24 h with vehicle or 10 μM doxorubicin and then challenged for 5 min with vehicle or isoproterenol (1 μM, unless indicated otherwise). Doxorubicin treatment resulted in a significant decrease in isoproterenol-dependent cyclic adenosine monophosphate (cAMP) accumulation and extracellular regulated kinase (ERK) phosphorylation (p < 0.05; n = 4). Abbreviations as in Figure 1

Figure 1: H

(A) Abcam and Santa Cruz (SC) anti–β1-adrenergic receptor (β1AR) antibodies were used to detect native or heterologously overexpressed β1ARs in cardiomyocytes. (B to D) Lysates from cardiomyocyte cultures treated with the specified H2O2 concentrations or 1 μM isoproterenol (for the indicated time intervals) were probed with the SC anti-β1AR antibody to track endogenous β1AR expression (with β-actin immunoreactivity serving as a protein loading control). Results in D are from a single set of gels exposed for a uniform duration, with white space inserted where data from 2 regions of the same gel were merged for presentation purposes. All results in A to D were replicated in 3 to 5 separate experiments. (E) βAR density (as defined by saturation binding experiments with 125I-iodocyanopindolol) in β1AR overexpressing cardiomyocytes treated with vehicle or 0.1 mM H2O2 for 30 min (n = 3)

Figure 2: The H

(A and C) Cardiomyocytes were pretreated for 1 h with 10 μM GF109203X (GFX), 10 μM PP1, 10 μM H89, 10 μM Gö6976, 0.1 μM propranolol (prop), 1 μM isoproterenol (Iso), 1 μM carvedilol (carv), 10 μM pindolol (pin), 10 μM timolol (tim), 10 μM atenolol (aten), or 10 μM metoprolol (met) as indicated and then challenged with vehicle or 0.1 mM H2O2 for 60 min (unless indicated otherwise). (B) Treatment with vehicle or the indicated concentrations H2O2 (in the absence or presence of 1 μM carvedilol) followed a 24-h pre-incubation with 100 ng/ml pertussis toxin (PTX). Experiments in A to C were performed in parallel on cardiomyocyte cultures that did or did not overexpress the β1AR transgene to compare stimulus-induced changes in native rat β1ARs (tracked with SC anti-β1AR antibody) and heterologously overexpressed human β1ARs (tracked with SC and/or Abcam anti-β1AR antibodies as indicated). Because β-actin immunoreactivity was not altered by β1AR overexpression, a single β-actin blot from uninfected cultures is depicted in the figures as a protein loading control. H2O2 and carvedilol-dependent changes in endogenous or heterologously overexpressed β1AR immunoreactivity are quantified in A, right (n = 6). (D) β1AR-overexpressing cardiomyocytes were treated for 24 h with a panel of β1AR ligands (at concentrations stipulated in A). Effects on β1AR transgene abundance are depicted on top, with results for 3 separate experiments on different culture preparations quantified on the bottom (*p (gray bars) were normalized to the level of the truncated β1AR species in the corresponding vehicle-treated culture (black bar), which was set to 100%. (E) β1AR-overexpressing cardiomyocytes were pretreated for 24 h with vehicle, 1 μM carvedilol, or 5 μM mitoTempo (mitoT, Sigma-Aldrich, St. Louis, Missouri) and then challenged with 100 μM H2O2 as indicated. Lysates were probed for β1AR immunoreactivity with β-actin immunoreactivity included as a loading control. The experiment is representative of data obtained in 4 separate experiments on different culture preparations. (F) Lysates from cardiomyocytes treated for 1 h with vehicle or 100 μM H2O2 (following a 1-h pretreatment with vehicle or 1 μM carvedilol as indicated) were probed for cyclic adenosine monophosphate binding response element–protein (CREB) phosphorylation and CREB protein expression. All immunoblotting data represent results obtained in 3 to 5 separate experiments. Abbreviations as in Figure 1

Figure 5: The N-terminally Truncated β

(A) Cardiomyocytes that heterologously overexpress similar levels of FL or N-terminally truncated β1ARs (Δ2-52) (as validated according to both immunoblot analysis and radioligand binding with 125I-iodocyanopindolol, based on protocols detailed elsewhere [12]) were treated for 24 h with vehicle or doxorubicin. Lysates were probed for β1AR expression, AKT phosphorylation, and caspase-3 and poly (ADP-ribose) polymerase (PARP) cleavage, with β-actin immunoreactivity as a loading control. A representative experiment is depicted on the left; results for pAKT (n = 9), caspase-3 cleavage (n = 4), or PARP cleavage (n = 6) are quantified on the right (with all results normalized to immunoreactivity in corresponding vehicle-treated FL-β1AR expressing cardiomyocytes). (B) Effects of PTX (100 ng/ml) on AKT phosphorylation in FL-β1AR or Δ2-52-β1AR overexpressing cardiomyocytes. The data are representative of results obtained in 3 separate culture preparations. Abbreviations as in Figures 1, 2, 3, and 4

Multisegmented Se−Te−Se Hybrid Nanowires: A Building Unit with Inbuilt Block and Glue Functionality

A novel hybrid nanomaterial incorporating Te and Se components within a multisegmented nanowire morphology is synthesized through a facile aqueous phase reaction at room temperature. Te nanowires were used as templates to grow Se segments at their terminal locations. The Se−Te−Se structures obtained exhibit a self-organization property thereby enabling the formation of “nanotweezers” at elevated temperatures. The physical and chemical properties of its individual components are expected to provide interesting functionality and promising utility to these nanostructures. The inbuilt block and glue features associated with its components make it a potential building unit toward nanoarchitectures of higher sophistication

Flow chart of this study.

Flow chart of this study.</p

CTP class stratified by ALBI and PALBI grades.

CTP class stratified by ALBI and PALBI grades.</p

The Carcinogenic Significance of Reactive Intermediates Derived from 3-Acetoxy- and 5-Acetoxy-2-hydroxy-<i>N-</i>nitrosomorpholine

N-Nitroso-2-hydroxymorpholine (NHMOR), a relatively reactive metabolite of two potent carcinogens, N-nitrosodiethanolamine (NDELA) and N-nitrosomorpholine (NMOR), has been reported to not be carcinogenic. Two isomeric acetate esters of the α-hydroxynitrosamines expected to be produced from the cytochrome P450-mediated metabolism of NHMOR have been synthesized, and their hydrolytic decomposition products, hydrolysis rates, and deoxyguanosine (dG) reaction adducts have been determined. N-Nitroso-3-acetoxy-2-hydroxymorpholine was prepared in high yield from the reaction of N-nitroso-2,3-dehydromorpholine with dry peracetic acid in glacial acetic acid or by the reaction of its dimethyldioxirane-produced epoxide with glacial acetic acid. The hydrolysis of this α-acetoxynitrosamine gave acetaldehyde (10%), ethylene glycol (55%), glyoxal (95%), and acetic acid. The pH rate profile for the hydrolysis of this nitrosamine was abnormal in that it exhibited pronounced base-catalyzed hydrolysis beginning at pH 5. The mechanism of hydrolytic decomposition is proposed to involve neighboring group participation with the formation of a reactive epoxide intermediate. N-Nitroso-3-acetoxy-2-hydroxymorpholine reacted with dG to give these guanine adducts after acidic deglycosylation:  1,N2-glyoxal (65%), 7-(2-hydroxyethyl)guanine (9%), and O6-hydroxyethylguanine (3%). N-Nitroso-5-acetoxy-2-hydroxymorpholine was synthesized from 2-hydroxyethylvinylnitrosamine by its oxidative conversion to the corresponding aldehyde followed by reaction with dry peracetic acid in glacial acetic. The hydrolytic decomposition products of this nitrosamine were 2-acetoxyacetaldehyde (65%), a rearrangement product, glycol aldehyde (15%), a trace of glyoxal, and acetic acid. The pH rate profile for the hydrolysis of this acetate is similar to other α-acetoxynitrosamines in that it exhibits a pH-independent region which gives way to base-catalyzed ester hydrolysis beginning at pH 7. The lower pH (≈ 7 < 9) onset of base catalysis is proposed to involve base-catalyzed opening of the hemiacetal and intramolecular acyl transfer to give an unstable α-hydroxynitrosamine. N-Nitroso-5-acetoxy-2-hydroxymorpholine was less reactive toward dG and gave the 1,N2-etheno-dG adduct (44%). The products from both of the isomeric α-acetoxy nitrosamines were judged to arise from diazonium ions produced from unstable α-hydroxynitrosamine intermediates. The high yield of the rearrangement product 2-acetoxyacetaldehyde could explain the low carcinogenic potential of NHMOR if it is mainly α-hydroxylated at the 5 carbon. Hydroxylation of NHMOR at carbon 3 is expected to yield a carcinogenic outcome

Utility of the ALBI and PALBI grades according to palliative treatment modality.

Utility of the ALBI and PALBI grades according to palliative treatment modality.</p