Ubiquitin Protein Ligase Ring2 Is Involved in S‐phase Checkpoint and DNA Damage in Cells Exposed to Benzo[a]pyrene

Previous studies in our laboratory demonstrated that Ring2 may affect DNA damage and repair through pathways other than through regulating the expression of the nucleotide excision repair protein. In a series of experiments using wild‐type cell (16HBE and WI38) and small interfering RNA (siRNA) Ring2 cells exposed to benzo[a]pyrene (BaP), we evaluated the cell cycle and DNA damage. The benzo(a)pyrene‐7,8‐dihydrodiol‐9,10‐epoxide (BPDE–DNA) adduct assay demonstrated that in vitro exposure to BaP increased DNA damage in a time‐ and dose‐dependent manner in wild‐type and siRNA Ring2 cells. Analysis of covariance showed that a decrease of Ring2 caused DNA hypersensitivity to BaP. Flow cytometry results and proliferating cell nuclear antigen levels indicated that inhibition of Ring2 attenuated the effect of BaP on S‐phase arrest. Taken together, these data implied that the lower proportion of cells in the S phase induced by inhibition of Ring2 may play an important role in DNA hypersensitivity to BaP.     

Binding of 1α,25‐dihydroxyvitamin D3 to annexin II: Effect of vitamin D metabolites and calcium

We have recently reported that annexin II serves as a membrane receptor for 1α,25‐(OH)₂D₃ and mediates the rapid effect of the hormone on intracellular calcium. The purpose of these studies was to characterize the binding of the hormone to annexin II, determine the specificity of binding, and assess the effect of calcium on binding. The binding of [¹⁴C]‐1α,25‐(OH)₂D₃ bromoacetate to purified annexin II was inhibited by 1α,25‐(OH)₂D₃ in a concentration‐dependent manner. Binding of the radiolabeled ligand to annexin II was markedly diminished by 1α,25‐(OH)₂D₃ at 24 μM, 18 μM, and 12 μM and blunted by 6 μM and 3 μM. At a concentration of 12 μM, 1β,25‐(OH)₂D₃ also diminished the binding of [¹⁴C]‐1α,25‐(OH)₂D₃ bromoacetate to annexin II, but cholecalciferol, 25‐(OH)D₃, and 24,25‐(OH)₂D₃ did not. Saturation analyses of the binding of [³H]‐1α,25‐(OH)₂D₃ to purified annexin II showed a K_D of 5.5 × 10⁻⁹ M, whereas [³H]‐1β,25‐(OH)₂D₃ exhibited a K_D of 6.0 × 10⁻⁹ M. Calcium, which binds to the carboxy terminal domain of annexin II, had a concentration‐dependent effect on [¹⁴C]‐1α,25‐(OH)₂D₃ bromoacetate binding to annexin II, with 600 nM calcium being able to inhibit binding of the radiolabeled analog. The inhibitory effect of calcium was prevented by EDTA. Homocysteine, which binds to the amino terminal domain of annexin II, had no effect on the binding of the bromoacetate analog to the protein. The data indicate that 1α,25‐(OH)₂D₃ binding to annexin II is specific and suggest that the binding site may be located on the carboxy terminal domain of the protein. The ability of 1β,25‐(OH)₂D₃ to inhibit the binding of [¹⁴C]‐1α,25(OH)₂D₃ bromoacetate to annexin II provides a biochemical explanation for the ability of the 1β‐epimer to inhibit the rapid actions of the hormone in vitro. J. Cell. Biochem. 80:259–265, 2000. © 2000 Wiley‐Liss, Inc.     

CARBON SOURCE DEPENDENCE OF THE RATIO OF δ‐AMINOLEVULINIC ACID BIOSYNTHESIS VIA THE C5 AND SHEMIN PATHWAYS IN EUGLENA GRACILIS (EUGLENOPHYCEAE)1

The ratio of two biosynthetic pathways was estimated, the C5 and Shemin pathways, to δ‐aminolevulinic acid (ALA, a biosynthetic intermediate of tetrapyrrole) from the ¹³C‐enrichment ratios (¹³C‐ER) at the carbon atoms of chl a (after conversion to methyl pheophorbide a) biosynthesized by Euglena gracilis G. A. Klebs when l ‐[3‐¹³C]alanine was used as a carbon source. On the basis of these estimations, we confirmed that ALA was efficiently biosynthesized via both the C5 and Shemin pathways in the plastids of E. gracilis, and we determined that the ratio of ALA biosynthesis via the Shemin pathway was increased in the ratio of 14%–67%, compared with that in our previous d ‐[1‐¹³C]glucose feeding experiment ( Iida et al. 2002 ). This carbon source dependence of the contributions of the two biosynthetic pathways might be related to activation of gluconeogenesis by the amino acid substrate. The methoxy carbon of the methoxycarbonyl group at C‐13² of chl a was labeled with the ¹³C‐carbon of l ‐[methyl‐¹³C]methionine derived from l ‐[3‐¹³C]alanine via [2‐¹³C]acetyl coenzyme A (CoA), through the atypical tricarboxylic acid (TCA) cycle, gluconeogenesis, and l‐ [3‐¹³C]serine. The phytyl moiety of chl a was also labeled on C‐P2, C‐P3¹, C‐P4, C‐P6, C‐P7¹, C‐P8, C‐P10, C‐P11¹, C‐P12, C‐P14, C‐P15¹, and C‐P16 from ¹³C‐isoprene (2‐[1,2‐methyl,3‐¹³C₃]methyl‐1,3‐butadiene) generated from l ‐[3‐¹³C]alanine via [2‐¹³C]acetyl CoA.     

Estimation of the Oxidative Stress and Molecular Damage Caused by 1‐Butyl‐3‐Methylimidazolium Bromide Ionic Liquid in Zebrafish Livers

The present study investigated the toxic effects of 1‐butyl‐3‐methylimidazolium bromide ([C₄mim]Br) in zebrafish livers after exposure to 5–40 mg/L of [C₄mim]Br on days 7, 14, 21, and 28. The results showed that low levels of [C₄mim]Br exposure activated the superoxide dismutase (SOD) activity during early periods of exposure. However, high concentrations (20–40 mg/L) of [C₄mim]Br caused the inhibition of SOD, which, accordingly, led to excess reactive oxygen species and malondialdehyde. The present results indicate that [C₄mim]Br causes oxidative stress in the livers of both male and female zebrafish. Additionally, the DNA damage revealed that [C₄mim]Br has a genotoxic effect on zebrafish livers, even when the concentration of [C₄mim]Br is low (5 mg/L). The DNA damage was demonstrated by quantifying the DNA strand breakage.     

IDENTIFICATION AND EXPRESSION ANALYSIS OF TWO 14‐3‐3 PROTEINS IN THE MOSQUITO Aedes aegypti,AN IMPORTANT ARBOVIRUSES VECTOR

The 14‐3‐3 proteins are evolutionarily conserved acidic proteins that form a family with several isoforms in many cell types of plants and animals. In invertebrates, including dipteran and lepidopteran insects, only two isoforms have been reported. 14‐3‐3 proteins are scaffold molecules that form homo‐ or heterodimeric complexes, acting as molecular adaptors mediating phosphorylation‐dependent interactions with signaling molecules involved in immunity, cell differentiation, cell cycle, proliferation, apoptosis, and cancer. Here, we describe the presence of two isoforms of 14‐3‐3 in the mosquito Aedes aegypti, the main vector of dengue, yellow fever, chikungunya, and zika viruses. Both isoforms have the conserved characteristics of the family: two protein signatures (PS1 and PS2), an annexin domain, three serine residues, targets for phosphorylation (positions 58, 184, and 233), necessary for their function, and nine alpha helix‐forming segments. By sequence alignment and phylogenetic analysis, we found that the molecules correspond to ? and ζ isoforms (Aeae14‐3‐3ε and Aeae14‐3‐3ζ). The messengers and protein products were present in all stages of the mosquito life cycle and all the tissues analyzed, with a small predominance of Aeae14‐3‐3ζ except in the midgut and ovaries of adult females. The 14‐3‐3 proteins in female midgut epithelial cells were located in the cytoplasm. Our results may provide insights to further investigate the functions of these proteins in mosquitoes.     

Ceranib‐2‐induced suicidal erythrocyte death

Ceramide is known to trigger apoptosis of nucleated cells and eryptosis of erythrocytes. Eryptosis is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Besides ceramide, stimulators of eryptosis include increase of cytosolic Ca²⁺‐activity ([Ca²⁺]_i) and oxidative stress. Ceramide is degraded by acid ceramidase and inhibition of the enzyme similarly triggers apoptosis. The present study explored, whether ceramidase inhibitor Ceranib‐2 induces eryptosis. Flow cytometry was employed to quantify phosphatidylserine‐exposure at the cell surface from annexin‐V‐binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo3‐fluorescence, reactive oxygen species (ROS) from DCF dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was estimated from hemoglobin concentration in the supernatant. A 48 h exposure of human erythrocytes to Ceranib‐2 significantly increased the percentage of annexin‐V‐binding cells (≥50 μM) and the percentage of hemolytic cells (≥10 μM) without significantly modifying forward scatter. Ceranib‐2 significantly increased Fluo3‐fluorescence, DCF fluorescence and ceramide abundance. The effect of Ceranib‐2 on annexin‐V‐binding was not significantly blunted by removal of extracellular Ca²⁺. Ceranib‐2 triggers phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to increase of ceramide abundance and induction of oxidative stress, but not dependent on Ca²⁺ entry. Copyright © 2016 John Wiley & Sons, Ltd.     

Benzo(a)pyrene diolepoxide-haemoglobin and albumin adducts at low levels of benzo(a)pyrene exposure

A biomonitoring study was conducted to simultaneously measure individual benzo(a)pyrene (BaP) exposure in 50 office employees, not occupationally exposed to polycyclic aromatic hydrocarbons (PAH), using personal samplers and the formation of (+) r-7, t-8-dihyroxy-t-9,t-10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE) adducts to haemoglobin (BPDE–Hb) and serum albumin (BPDE–SA). The population enrolled was exposed to an average of 0.58 ± 0.46 ng BaP m^?3 (mean ± SD). The concentration of BaP collected from smokers' samples was double that from non-smokers (P = 0.007). BPDE adducts to Hb and SA were quantified as BaP tetrols released from hydrolysis of macromolecules and measured by high-resolution gas chromatography–negative ion chemical ionization–mass spectrometry. BPDE–Hb adducts were detected in 16% of the population and BPDE–SA adducts in 28%. Smoking did not affect adduct formation. When BaP personal monitoring data were used as the criterion of exposure, no correlation was found with the presence and the levels of BPDE–Hb and BPDE–SA adducts. Undetected sources of PAH, such as the diet, might markedly alter the exposure profile depicted by individual air sampling and affect the frequency and levels of protein biomarkers. This is the first comparative analysis of BPDE–Hb and BPDE–SA adducts, providing reference values for these biomarkers in a general urban population. However it is difficult to establish which biomarkers would be the more relevant in assessing low BaP exposure, due to undetectable factors such as dietary PAHs, that might have influenced the results to some degree.     

Responses of the Antioxidant System in QGY‐7701 Cells to the Cytotoxicity and Apoptosis Induced by 1‐Octyl‐3‐methylimidazolium Chloride

This study aims to evaluate the cytotoxicity and responses of the cellular antioxidant system of 1‐octyl‐3‐methylimidazolium chloride ([C₈mim][Cl]) on human hepatocarcinoma QGY‐7701 cells. The results show that [C₈mim][Cl] can inhibit QGY‐7701 cell growth and decrease their viabilities in a dose‐dependent manner. The results also reveal that [C₈mim][Cl] exposure can induce apoptosis in the [C₈mim][Cl]‐treated QGY‐7701 cells. In addition, the results of biochemical assays show that [C₈mim][Cl] exposure causes overproduction of reactive oxygen species (ROS), inhibits superoxide dismutase (SOD) and catalase (CAT) activities, decreases reduced glutathione (GSH) content, and increases the cellular malondialdehyde (MDA) level. These results suggest that ROS‐mediated oxidative stress may be responsible for the apoptosis induced by [C₈mim][Cl] in QGY‐7701 cells. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:330‐336, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21495     

Formation of DAN-binding products from isolated benzo[a]pyrene metabolites in rat liver nuclei

Liver nuclei from 3-methylcholanthrene-treated rats in the presence of NADPH metabolized 3- and 9-hydroxybenzo[a]pyrene and 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene to products that bound to DNA. Maximal binding was obtained with the dihydrodiol which was approximately 3-fold that with 9-hydroxybenzo[a]pyrene, and 60-fold that with 3-hydroxybenzo[a]pyrene, as substrates. Both 4,5-dihydro-4,5-dihydroxybenzo[a]pyrene and 9,10-dihydro-9,10-dihydroxybenzo[a]pyrene were also extensively metabolized by the nuclear fraction but did not give rise to DNA-binding products.The available evidence suggests that the DNA binding species derived from 9-hydroxy-benzo[a]pyrene is 9-hydroxy-benzo[a]pyrene-4,5-oxide and from 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene, as previously observed in different systems, 7,8-dihydro-7,8-dihydroxy-benzo[a]pyrene-9,10-oxide.     

Synthesis and Fungicidal Activities of New 1,2,4‐Triazolo[1,5‐a]pyrimidines

A series of new acetohydrazone‐containing 1,2,4‐triazolo[1,5‐a]pyrimidine derivatives were designed and synthesized for the purpose of searching for novel agrochemicals with higher fungicidal activity. Their in vitro fungicidal activities against Rhizoctonia solani were evaluated, and the most promising compound, 2‐[(5,7‐dimethyl[1,2,4]triazolo[1,5‐a]pyrimidin‐2‐yl)sulfanyl]‐2′‐[(2‐hydroxyphenyl)methylidene]acetohydrazide ( 2‐17 ), showed a lower EC₅₀ value (5.34 μg ml^?1) than that of commercial carbendazim (EC₅₀=7.62 μg ml^?1). Additionally, compound 2‐17 was also found to display broad‐spectrum fungicidal activities, and its EC₅₀ value (4.56 μg ml^?1) against Botrytis cinereapers was very similar to that of carbendazim. Qualitative structure–activity relationships (QSARs) of the synthesized compounds were also discussed.     

DNA‐PK suppresses a p53‐independent apoptotic response to DNA damage

p53 is required for DNA damage‐induced apoptosis, which is central to its function as a tumour suppressor. Here, we show that the apoptotic defect of p53‐deficient cells is nearly completely rescued by inactivation of any of the three subunits of the DNA repair holoenzyme DNA‐dependent protein kinase (DNA‐PK). Intestinal crypt cells from p53 nullizygous mice were resistant to radiation‐induced apoptosis, whereas apoptosis in DNA‐PK_cs/p53, Ku80/p53 and Ku70/p53 double‐null mice was quantitatively equivalent to that seen in wild‐type mice. This p53‐independent apoptotic response was specific to the loss of DNA‐PK, as it was not seen in ligase IV (Lig4)/p53 or ataxia telangiectasia mutated (Atm)/p53 double‐null mice. Furthermore, it was associated with an increase in phospho‐checkpoint kinase 2 (CHK2), and cleaved caspases 3 and 9, the latter indicating engagement of the intrinsic apoptotic pathway. This shows that there are two separate, but equally effective, apoptotic responses to DNA damage: one is p53 dependent and the other, engaged in the absence of DNA‐PK, does not require p53.     

Diverse modes of 5′‐[4‐(aminoiminomethyl)phenyl]‐[2,2′‐bifuran]‐5‐carboximidamide (DB832) interaction with multi‐stranded DNA structures

The modes of binding of 5′‐[4‐(aminoiminomethyl)phenyl]‐[2,2′‐Bifuran]‐5‐carboximidamide (DB832) to multi‐stranded DNAs: human telomere quadruplex, monomolecular R‐triplex, pyr/pur/pyr triplex consisting of 12 T*(T·A) triplets, and DNA double helical hairpin were studied. The optical adsorption of the ligand was used for monitoring the binding and for determination of the association constants and the numbers of binding sites. CD spectra of DB832 complexes with the oligonucleotides and the data on the energy transfer from DNA bases to the bound DB832 assisted in elucidating the binding modes. The affinity of DB832 to the studied multi‐stranded DNAs was found to be greater (K_ass ≈ 10⁷M^?1) than to the duplex DNA (K_ass ≈ 2 × 10⁵M^?1). A considerable stabilizing effect of DB832 binding on R‐triplex conformation was detected. The nature of the ligand tight binding differed for the studied multi‐stranded DNA depending on their specific conformational features: recombination‐type R‐triplex demonstrated the highest affinity for DB832 groove binding, while pyr/pur/pyr TTA triplex favored DB832 intercalation at the end stacking contacts and the human telomere quadruplex d[AG₃(T₂AG₃)₃] accommodated the ligand in a capping mode. Additionally, the pyr/pur/pyr TTA triplex and d[AG₃(T₂AG₃)₃] quadruplex bound DB832 into their grooves, though with a markedly lesser affinity. DB832 may be useful for discrimination of the multi‐sranded DNA conformations and for R‐triplex stabilization. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 8–20, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Resolution of 1‐n‐Butyl‐3‐Methyl‐3‐Phospholene 1‐Oxide With TADDOL Derivatives and Calcium Salts of O,O'‐Dibenzoyl‐(2R,3R)‐ or O,O'‐di‐p‐Toluoyl‐(2R,3R)‐tartaric Acid

The resolution methods applying (?)‐(4R,5R)‐4,5‐bis(diphenylhydroxymethyl)‐2,2‐dimethyldioxolane (“TADDOL”), (?)‐(2R,3R)‐α,α,α',α'‐tetraphenyl‐1,4‐dioxaspiro[4.5]decan‐2,3‐dimethanol (“spiro‐TADDOL”), as well as the acidic and neutral Ca²⁺ salts of (?)‐O,O'‐dibenzoyl‐ and (?)‐O,O'‐di‐p‐toluoyl‐(2R,3R)‐tartaric acid were extended for the preparation of 1‐n‐butyl‐3‐methyl‐3‐phospholene 1‐oxide in optically active form. In one case, the intermediate diastereomeric complex could be identified by single‐crystal X‐ray analysis. The absolute P‐configuration of the enantiomers of the phospholene oxide was also determined by comparing the experimentally obtained and calculated CD spectra. Chirality 26:174–182, 2014. © 2014 Wiley Periodicals, Inc.     

Green fluorescent protein (GFP) as a direct biosensor for mutation detection: elimination of false-negative errors in target gene expression

A new method was developed to determine the mutagenic efficacy of a suspected mutagen by employing green fluorescent protein (GFP) as a direct biosensor for mutation detection. Alterations in target gene (AcGFP1) expression after mutagen [(±)-7p,8a-dihydroxy-9a,10a-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)] treatment were measured to detect the mutagenic efficacy of the carcinogen. In contrast to mutagen treatment of the entire plasmid or cell culture, the target AcGFP1gene devoid of the plasmid backbone was exposed to BPDE (10–500 μM) to eliminate the need for an additional fusion gene. Shuttle vectors (pAcGFP-N1) were religated to the AcGFP1 gene with BPDE adducts (0–8.59 μM) and replicated in the eukaryotic host. This approach eliminated false-negative errors in target gene expression that arose from BPDE adduct formation in the residual plasmid backbone rather than in the AcGFP1 gene. Determination of the BPDE–AcGFP1 adducts allowed the quantitative mutagenic effect of the BPDE adducts on AcGFP1 gene expression to be monitored. The results obtained with flow cytometry and confocal microscopy validate our method and demonstrate efficient and direct use of GFP as a biosensor for mutation detection.     

14‐3‐3 Ligand Prevents Nuclear Import of c‐ABL Protein in Chronic Myeloid Leukemia

Here we demonstrated that the ‘loss of function’ of not‐rearranged c‐ABL in chronic myeloid leukemia (CML) is promoted by its cytoplasmic compartmentalization bound to 14‐3‐3 sigma scaffolding protein. In particular, constitutive tyrosine kinase (TK) activity of p210 BCR‐ABL blocks c‐Jun N‐terminal kinase (JNK) phosphorylation leading to 14‐3‐3 sigma phosphorylation at a critical residue (Ser¹⁸⁶) for c‐ABL binding in response to DNA damage. Moreover, it is associated with 14‐3‐3 sigma over‐expression arising from epigenetic mechanisms (promoter hyper‐acetylation). Accordingly, p210 BCR‐ABL TK inhibition by the TK inhibitor Imatinib mesylate (IM) evokes multiple events, including JNK phosphorylation at Thr¹⁸³, p38 mitogen‐activated protein kinase (MAPK) phosphorylation at Thr¹⁸⁰, c‐ABL de‐phosphorylation at Ser residues involved in 14‐3‐3 binding and reduction of 14‐3‐3 sigma expression, that let c‐ABL release from 14‐3‐3 sigma and nuclear import, and address BCR‐ABL‐expressing cells towards apoptotic death. Informational spectrum method (ISM), a virtual spectroscopy method for analysis of protein interactions based on their structure, and mathematical filtering in cross spectrum (CS) analysis identified 14‐3‐3 sigma/c‐ABL binding sites. Further investigation on CS profiles of c‐ABL‐ and p210 BCR‐ABL‐containing complexes revealed the mechanism likely involved 14‐3‐3 precluded phosphorylation in CML cells.     

Thieno[3,4‐c]Pyrrole‐4,6‐Dione‐Based Polymer Acceptors for High Open‐Circuit Voltage All‐Polymer Solar Cells

While polymer acceptors are promising fullerene alternatives in the fabrication of efficient bulk heterojunction (BHJ) solar cells, the range of efficient material systems relevant to the “all‐polymer” BHJ concept remains narrow, and currently limits the perspectives to meet the 10% efficiency threshold in all‐polymer solar cells. This report examines two polymer acceptor analogs composed of thieno[3,4‐c ]pyrrole‐4,6‐dione (TPD) and 3,4‐difluorothiophene ([2F]T) motifs, and their BHJ solar cell performance pattern with a low‐bandgap polymer donor commonly used with fullerenes (PBDT‐TS1; taken as a model system). In this material set, the introduction of a third electron‐deficient motif, namely 2,1,3‐benzothiadiazole (BT), is shown to (i) significantly narrow the optical gap (E _opt) of the corresponding polymer (by ≈0.2 eV) and (ii) improve the electron mobility of the polymer by over two orders of magnitude in BHJ solar cells. In turn, the narrow‐gap P2TPDBT[2F]T analog (E _opt = 1.7 eV) used as fullerene alternative yields high open‐circuit voltages (V _OC) of ≈1.0 V, notable short‐circuit current values (J _SC) of ≈11.0 mA cm⁻², and power conversion efficiencies (PCEs) nearing 5% in all‐polymer BHJ solar cells. P2TPDBT[2F]T paves the way to a new, promising class of polymer acceptor candidates.     

4S polycyclic aromatic hydrocarbon receptor (glycine N‐methyltransferase) and the aryl hydrocarbon receptor nuclear translocator (hypoxia inducible factor‐1β) interaction in Chinese hamster ovary and rat hepatoma cells: 4S PAH‐R/ARNT hetero‐oligomers?

Rat CYP1A1 promoter‐luciferase, transiently transfected wild‐type and 4S PAH receptor (glycine N‐methyl transferase, GNMT)‐transformed Chinese hamster ovary (CHO) cells were exposed to benzo[a]pyrene and assayed for luciferase activity as an indicator of CYP1A1 promoter activity. CHO cells transformed with the rat 4S PAH receptor/GNMT expression vector had twice the induction level of luciferase activity with respect to wild‐type CHO cells in concert with previously published reports that the 4S PAH receptor/GNMT mediates benzo[a]pyrene induction of CYP1A1 gene expression. Lysates of GNMT‐transformed CHO cells and wild‐type H4IIE rat hepatoma cells exposed to benzo[a]pyrene were immuno‐precipitated with anti‐GNMT antibodies, separated by SDS–polyacrylamide gel electrophoresis and transferred to PVDF membrane for Western blot analysis with anti‐aryl hydrocarbon receptor nuclear translocator (ARNT, HIF‐1β) antibodies. Results of this analysis indicated that the 4S PAH receptor/GNMT forms a hetero‐oligomer (dimer?) with ARNT/HIF‐1β which dissociates in the presence of B[a]P. These observations further indicate the role of GNMT (which has been shown to be multifunctional) and B[a]P in the induction of CYP1A1 and also a potential role of GNMT in the modulation of hypoxia inducible factor‐1 function with respect to the HIF‐1β subunit (ARNT). J. Cell. Biochem. 112: 2015–2018, 2011. © 2011 Wiley‐Liss, Inc.     

In Vitro and In Vivo Evaluation of N‐{2‐[4‐(3‐Cyanopyridin‐2‐yl)piperazin‐1‐yl]ethyl}‐3‐[11C]methoxybenz­amide,a Positron Emission Tomography (PET) Radioligand for Dopamine D4 Receptors,in Rodents

The D₄ dopamine receptor belongs to the D₂‐like family of dopamine receptors, and its exact regional distribution in the central nervous system is still a matter of considerable debate. The availability of a selective radioligand for the D₄ receptor with suitable properties for positron emission tomography (PET) would help resolve issues of D₄ receptor localization in the brain, and the presumed diurnal change of expressed protein in the eye and pineal gland. We report here on in vitro and in vivo characteristics of the high‐affinity D₄ receptor‐selective ligand N‐{2‐[4‐(3‐cyanopyridin‐2‐yl)piperazin‐1‐yl]ethyl}‐3‐[¹¹C]methoxybenzamide ([¹¹C] 2 ) in rat. The results provide new insights on the in vitro properties that a brain PET dopamine D₄ radioligand should possess in order to have improved in vivo utility in rodents.