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Biochemical Pharmacology (v.79, #3)

Editorial Advisory Board (pp. iii).

Graphical Abstracts Contents (pp. e1-e7).

Human DEAD-box protein 3 has multiple functions in gene regulation and cell cycle control and is a prime target for viral manipulation by Martina Schröder (pp. 297-306).

The human DEAD-box RNA helicase DDX3 has been implicated to play a role in the whole repertoire of processes regulating gene expression, including transcription, splicing, mRNA export and translation. It has also been suggested to be involved in cell cycle control and the regulation of apoptosis. In addition, DDX3 was recently shown to be part of innate immune signalling pathways and to contribute to the induction of anti-viral mediators, such as type I interferon. Interestingly, DDX3 appears to be a prime target for viral manipulation: at least four different viruses, namely Hepatitis C virus (HCV), Hepatitis B virus (HBV), Human Immunodeficiency Virus (HIV) and poxviruses, encode proteins that interact with DDX3 and modulate its function. HIV and HCV seem to co-opt DDX3 and require it for their replication. It has therefore been suggested that DDX3 could be a novel target for the development of drugs against these two viruses, both of which still pose major global health threats. However, in the light of the apparent multifunctionality of DDX3 in the cell, drug development strategies targeting DDX3 will have to be carefully evaluated. This review summarises the available data on the cellular functions of DDX3 and discusses their manipulation by the different viruses known to target DDX3. Understanding the viral strategies for manipulating or co-opting DDX3 in functional and molecular detail can provide valuable insights for the development of strategies to therapeutically target DDX3.

Keywords: Abbreviations; 4eBP; eIF4e-binding protein; aa; amino acid; Cdc; cell division cycle; CRM-1; chromosome maintenance region-1; DDX3; DEAD-box protein 3; eIF; eukaryotic initiation factor; EJC; Exon junction complex; GSK; glycogen synthase kinase; HBV; Hepatitis B virus; HCC; hepatocellular carcinoma; HCV; Hepatitis C virus; HIV; Human Immunodeficiency Virus; HLA; human leukocyte antigen; IAP; inhibitor of apoptosis; IFN; interferon; IkB; inhibitor of nuclear factor-kB; IKK; IB-kinase; IRES; internal ribosome entry site; IRF; interferon regulatory factorMAVSmitochondrial antiviral signalling; mRNA; messenger RNA; NES; nuclear export signal; NPC; nuclear pore complex; NS3/4a; non-structural 3/4a; PABP; polyA-binding protein; PRR; pattern recognition receptor; RIG; retinoic-acid inducible gene; RLH; RIG-like helicase; RNP; ribonucleoprotein; rRNA; ribosomal RNA; siRNA; small interfering RNA; snRNA; small nuclear RNA; Sp1; specificity protein 1; TAP; tip-associated protein; TBK; TANK-binding kinase; TLR; Toll-like receptor; TRAIL-R; TNF-related apoptosis-inducing ligand-receptor; TRIF; TIR-domain containing adaptor inducing IFNβ; UTR; untranslated region; VACV; vaccinia virusDEAD-box helicase; Gene expression regulation; Hepatitis virus; Viral immune evasion; Cell growth control; Type I interferon

Interaction of benzopyranone derivatives and related compounds with human concentrative nucleoside transporters 1, 2 and 3 heterologously expressed in porcine PK15 nucleoside transporter deficient cells. Structure–activity relationships and determinants of transporter affinity and selectivity by Chunmei Wang; Surekha Pimple; John K. Buolamwini (pp. 307-320).

Unlike the major equilibrative nucleoside transporters, there is a dearth of potent specific inhibitors of concentrative nucleoside transporters (CNTs). We investigated the interaction of benzopyranone derivatives and related compounds with human (h) CNTs in newly established PK15NTD transfectant cells stably expressing hCNT1 or hCNT2, and previously established PK15NTD/hCNT3 cells. Flavones exhibited the highest inhibitory activity against hCNT2 and hCNT3, whereas the most potent selective inhibitor of hCNT1 was a coumarin derivative. hCNT3 was the only transporter that exhibited moderate sensitivity to the chalcones tested. The most active compound was 6-hydroxy-7-methoxyflavone, which was hCNT3-specific with an IC₅₀ of 0.57±0.20μM, and over 40-fold more potent than the standard CNT inhibitor, phloridzin (IC₅₀ of 25±3.5μM). The SAR (Structure–Activity Relationship) shows that high potency against all three hCNTs is conferred by the presence of hydroxyl substituents at both the 7- and 8-positions of flavones and isoflavones. CoMFA (Comparative Molecular Field Analysis) and CoMSIA (Comparative Molecular Similarity Indices Analysis) 3D-QSAR (three-Dimensional Quantitative Structure–Activity Relationship) modeling indicated that electrostatic and hydrophobic properties were the most influential for interactions between the flavonoids and hCNT1, while electrostatic, hydrophobic and hydrogen bond donor properties were predominate for interactions with hCNT2 and hCNT3. The 3D-QSAR results also suggested possible commonalities in hydrogen bonding interactions of flavonoids and nucleosides, suggesting similarities between the hCNT-binding sites of the two classes of compounds. We report the most potent and selective non-nucleoside CNT inhibitors to date; which may serve as research tools and/or leads for further inhibitor development.

Keywords: Concentrative nucleoside transporters; Benzopyranone derivatives; CoMFA; CoMSIA; Structure–activity relationships

Inhibition of PDE3, PDE4 and PDE7 potentiates glucocorticoid-induced apoptosis and overcomes glucocorticoid resistance in CEM T leukemic cells by Hongli Dong; Christof Zitt; Cornelia Auriga; Armin Hatzelmann; Paul M. Epstein (pp. 321-329).

This paper concerns the targeting of cyclic nucleotide phosphodiesterases (PDEs) to induce apoptosis and overcome glucocorticoid resistance of leukemic cells.Stimulation of the cAMP signaling pathway has been shown to induce apoptosis and augment the effects of glucocorticoids in inducing apoptosis in leukemic cells. We recently reported that in primary B cell chronic lymphocytic leukemic (B-CLL) cells, apoptosis could be induced by stimulating the cAMP signaling pathway with a phosphodiesterase4 (PDE4) inhibitor alone; while in contrast, in the CEM T leukemic cell line, PDE4 inhibitors alone were ineffective, and concurrent stimulation of adenylyl cyclase was required to see effects [Tiwari et al. (2005) ]. We report here that in the CEM and Jurkat T leukemic cell lines, the most abundantly expressed PDEs are PDE3B, PDE4A, PDE4D, PDE7A, and PDE8A. Selective inhibition of PDE3, PDE4 or PDE7 alone produces little effect on cell viability, but inhibition of all three of these PDEs together dramatically enhances glucocorticoid-induced apoptosis in CEM cells, and overcomes glucocorticoid resistance in a glucocorticoid-resistant CEM cell line. These studies indicate that for some leukemic cell types, a desired therapeutic effect may be achieved by inhibiting more than one form of PDE.

Keywords: Leukemia; Phosphodiesterase; cAMP; Glucocorticoids

Design of curcumin-loaded PLGA nanoparticles formulation with enhanced cellular uptake, and increased bioactivity in vitro and superior bioavailability in vivo by Preetha Anand; Hareesh B. Nair; Bokyung Sung; Ajaikumar B. Kunnumakkara; Vivek R. Yadav; Rajeshwar R. Tekmal; Bharat B. Aggarwal (pp. 330-338).

Encapsulation of curcumin into nanoparticles (NP) enhances its cellular uptake (panel A) and its apoptotic effects (panel B).Curcumin, a yellow pigment present in the spice turmeric ( Curcuma longa), has been linked with antioxidant, anti-inflammatory, antiproliferative, anticancer, antidiabetic, antirheumatic, and antiviral effects, but its optimum potential is limited by its lack of solubility in aqueous solvents and poor oral bioavailability. We employed a polymer-based nanoparticle approach to improve bioavailability. Curcumin was encapsulated with 97.5% efficiency in biodegradable nanoparticulate formulation based on poly (lactide-co-glycolide) (PLGA) and a stabilizer polyethylene glycol (PEG)-5000. Dynamic laser light scattering and transmission electron microscopy indicated a particle diameter of 80.9nm. This curcumin, renamed from hereon “as curcumin (NP)”, was characterized for its biological activity. In vitro curcumin (NP) exhibited very rapid and more efficient cellular uptake than curcumin. Estrase staining revealed that curcumin (NP) was at least as potent as or more potent than curcumin in inducing apoptosis of leukemic cells and in suppressing proliferation of various tumor cell lines. When examined by electrophoretic gel shift mobility assay, curcumin (NP) was more active than curcumin in inhibiting TNF-induced NF-κB activation and in suppression of NF-κB-regulated proteins involved in cell proliferation (cyclin D1), invasion (MMP-9), and angiogenesis (VEGF). In mice, curcumin (NP) was more bioavailable and had a longer half-life than curcumin. Overall we demonstrate that curcumin-loaded PLGA nanoparticles formulation has enhanced cellular uptake, and increased bioactivity in vitro and superior bioavailability in vivo over curcumin.

Keywords: Abbreviations; NF-κB; nuclear factor-kappaB; TNF; tumor necrosis factor; VEGF; vascular endothelial growth factorNanoparticles; Apoptosis; Inflammation; TNF-alpha

ABT-737 overcomes Bcl-2 mediated resistance to doxorubicin–DNA adducts by Michal Ugarenko; Abraham Nudelman; Ada Rephaeli; Ken-Ichi Kimura; Don R. Phillips; Suzanne M. Cutts (pp. 339-349).

Bcl-2 overexpressing HL-60 cells are resistant to doxorubicin–DNA adduct forming treatments. The addition of the Bcl-2 inhibitor ABT-737 was able to overcome this resistance and induce apoptosis.Doxorubicin is an anthracycline anticancer agent that functions primarily by inhibiting topoisomerase II, but also forms covalent DNA adducts depending on the cellular availability of formaldehyde. The combination of formaldehyde-releasing prodrugs (such as AN-9) with doxorubicin has been shown to result in synergistic doxorubicin–DNA adduct formation and synergistic apoptosis in HL-60 leukemic cells, offering the potential for lower concentrations of doxorubicin to be used clinically in order to minimize side-effects. However, the overexpression of Bcl-2 confers resistance to doxorubicin/AN-9 DNA adduct forming treatments, thus limiting the therapeutic potential of this drug combination. The small molecule inhibitor, ABT-737, which binds to and inhibits Bcl-2, Bcl-xL and Bcl-w, was used in combination with doxorubicin/AN-9 treatments to overcome resistance to doxorubicin–DNA adducts in Bcl-2 overexpressing HL-60 cells (HL-60/Bcl-2). The combination treatment of doxorubicin and AN-9 (and all single agent controls) failed to induce an apoptotic response in HL-60/Bcl-2 cells, however, the addition of low nanomolar (sub-lethal) concentrations of ABT-737 was able to greatly increase apoptosis levels. Various control compounds were used to demonstrate that the mechanism of cell kill in response to the ‘triple treatment’ (doxorubicin, AN-9 and ABT-737) is dependent on DNA adduct formation. Therefore, the ability of ABT-737 to inhibit Bcl-2 renders previously resistant HL-60 cancer cells highly sensitive to doxorubicin–DNA adducts, leading to a classical apoptotic response. In conclusion, the data obtained provides promising evidence that the anticancer activity of doxorubicin–DNA adducts can be substantially enhanced in Bcl-2 overexpressing cancers with the use of the small molecule Bcl-2 inhibitor, ABT-737.

Keywords: Abbreviations; AML; acute myelogenous leukemia; CDK; cyclin-dependent kinase; CLL; chronic lymphocytic leukemia; CPM; counts per minute; DTT; dithiothreitol; FACS; fluorescence-activated cell sorting; PEG; poly-ethylene glycol; SCLC; small-cell lung carcinoma; WT; wild-typeDoxorubicin; AN-9; ABT-737; Drug–DNA adducts; Bcl-2

Oleanane triterpenoid CDDO-Me inhibits growth and induces apoptosis in prostate cancer cells through a ROS-dependent mechanism by Dorrah Deeb; Xiaohua Gao; Hao Jiang; Branislava Janic; Ali S. Arbab; Yon Rojanasakul; Scott A. Dulchavsky; Subhash C. Gautam (pp. 350-360).

CDDO-Me, a synthetic triterpenoid derived from oleanolic acid, is a promising anticancer agent that has shown strong activity against a wide variety of cancer types in vitro and in vivo. We have previously shown that CDDO-Me induces apoptosis in prostate cancer cells irrespective of their hormonal status. To further understand the proapoptotic mechanism of CDDO-Me, we investigated the role of reactive oxygen species (ROS) in mediating the apoptosis inducing activity of CDDO-Me in LNCaP and PC-3 prostate cancer cell lines. Here, we show that CDDO-Me induces ROS generation from both nonmitochondrial and mitochondrial sources, which is associated with the induction of apoptosis as characterized by increased annexin V-binding, cleavage of PARP-1 and procaspases-3, -8, -9, loss of mitochondrial membrane potential and release of cytochrome c. In addition, CDDO-Me inhibited cell survival Akt, NF-κB and mTOR signaling proteins. The inhibition of ROS generation by N-acetylcysteine (NAC) or by overexpression of antioxidant enzymes glutathione peroxidase (GPx) and superoxide dismutase-1 (SOD-1) prevented CDDO-Me-induced apoptosis. Pretreatment with NAC blocked annexin V-binding, cleavage of PARP-1 and procaspases-3, -8, -9, loss of mitochondrial membrane potential and release of cytochrome c by CDDO-Me. NAC also prevented the inhibition of constitutively active Akt, NF-κB and mTOR by CDDO-Me. Together, these data indicate that ROS plays an essential role in the induction of apoptosis by CDDO-Me in prostate cancer cells.

Keywords: Abbreviations; CDDO; 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid; CDDO-Me; C-28 methyl ester derivative of CDDO; H; ₂; DCFDA; 6-carboxy-2,7-dichlorodihydrofluroscein diacetate; DHE; dihydroethidium bromide; ROS; reactive oxygen species; PARP-1; poly-ADP-ribose polymerase-1; NF-κB; nuclear factor kappa B; mTOR; mammalian target of rapamycin; NAC; N-acetylcystein; SOD-1; superoxide dismutase-1; GPx; glutathione peroxidaseCCDO-Me; Prostate cancer; ROS; Apoptosis; Mitochondrial depolarization; Cell survival signaling

Apoptotic effects of mahanine on human leukemic cells are mediated through crosstalk between Apo-1/Fas signaling and the Bid protein and via mitochondrial pathways by Kaushik Bhattacharya; Suman K. Samanta; Rakshamani Tripathi; Asish Mallick; Sarmila Chandra; Bikas C. Pal; Chandrima Shaha; Chitra Mandal (pp. 361-372).

Apo-1 (Fas/CD95), a cell surface receptor, triggers apoptosis after binding to its physiological ligand, Apo-1L (FasL/CD95L). This study reports that mahanine, purified from the leaves of Murraya koenigii, has a dose- and time-dependent anti-proliferative activity in acute lymphoid (MOLT-3) and chronic myeloid (K562) leukemic cell lines and in the primary cells of leukemic and myeloid patients, with minimal effect on normal immune cells including CD34⁺ cells. Leukemic cells underwent phosphatidylserine externalization and DNA fragmentation, indicating mahanine-induced apoptosis. An increase in reactive oxygen species suggests that the mahanine-induced apoptosis was mediated by oxidative stress. A significant drop in the Bcl2/Bax ratio, the loss of mitochondrial transmembrane potential as well as cytochrome c release from the mitochondria to the cytosol suggested involvement of the mitochondrial pathway of apoptosis. Cytochrome c release was followed by the activation of caspase-9, caspase-3 and caspase-7, and cleavage of PARP in both MOLT-3 and K562 cells. In MOLT-3 cells, formation of the Fas-FasL-FADD-caspase-8 heterotetramer occurred, leading to the cleavage of Bid to its truncated form, which consequently resulted in formation of the mitochondrial transmembrane pore. The incubation of MOLT-3 cells with mahanine in the presence of caspase-8 inhibitor or FasL-neutralizing NOK-2 antibody resulted in the decrease of mahanine-induced cell death. Mahanine was also a potent inhibitor of K562 xenograft growth, which was evident in an athymic nude mice model. In summary, these results provide evidence for involvement of the death receptor-mediated extrinsic pathway of apoptosis in the mahanine-induced anticancer activity in MOLT-3 cells, but not in K562 cells, which are deficient in Fas/FasL.

Keywords: Abbreviations; BSA; bovine serum albumin; DAB; diaminobenzidine; DISC; death-inducing signaling complex; EtOH; ethanol; FCS; fetal calf serum; FITC; fluorescein isothiocyanate; H; ₂; DCFDA; 5-(and-6)-chloromethyl-2′7′-dichlorodihydrofluorescein diacetate acetyl ester; HPLC; high-performance liquid chromatography; JC-1; 5,5′-6,6′-tetracholoro-1,1′-3,3′-tetraethylbenzimidazolylcarbocyanine iodide; MS; mass spectrometry; NAC; N-acetyl; l; -cysteine; NMR; nuclear magnetic resonance; PARP; poly ADP ribose polymerase; PBMC; peripheral blood mononuclear cells; PI; propidium iodide; PS; phosphatidylserine; ROS; reactive oxygen species; TDT; terminal deoxy transferase; TUNEL; terminal deoxynucleotidyltransferase enzyme-mediated dUTP end labeling; UCB; umbilical cord bloodFas/FasL; Bid; Leukemia; Mahanine; Extrinsic apoptosis

Jnk signaling pathway-mediated regulation of Stat3 activation is linked to the development of doxorubicin resistance in cancer cell lines by Ju-Hwa Kim; Seok Chul Lee; Jungsil Ro; Han Sung Kang; Hyung Sik Kim; Sungpil Yoon (pp. 373-380).

We sought to identify altered transcription factors (Stat, AP1, and NF-kB) or signal proteins (Erk1/2, p38, Akt, Jnk, Jak, and c-Src) in cancer cell lines whose growth was arrested by doxorubicin (DOX) treatment. Jnk1 was the only signal protein to be activated. DOX increased Stat3 phosphorylation, nuclear localization, and transcriptional activity. Jnk1 activation appeared to be required for Stat3 activity. Stat3 activity via the Jnk pathway was conserved in other cell lines originating from other organs. Transcriptional activity of Stat3 was increased in cells surviving DOX treatment suggesting that Stat3 activation contributed to the resistance to cytotoxicity. To better understand the role of Stat3 in Jnk1 activation, we investigated its effect on the viability of DOX-treated cells. Co-treatment with DOX and Jnk inhibitor negatively correlated with the viability of cancer cells and reduced Stat3 activity. Taken together, these results indicate that Stat3 activation via the Jnk pathway promotes the resistance of cancer cells to DOX.

Keywords: Abbreviations; DOX; doxorubicin; phospho; phosphorylation; Stat; signal transducer and activator of transcription; Jnk; c-Jun N-terminal kinase; AP1; activator protein-1; NF-kB; nuclear factor-kappa B; Erk; RAS/extracellular signal-regulated kinase; Jak; Janus kinase; FBS; fetal bovine serum; siRNA; small interfering RNA; PBS; phosphate buffered saline; S.D.; standard deviationDoxorubicin; Stat3; Jnk1; SP600125; DOX resistance; cancer

Modulation of human UMP/CMP kinase affects activation and cellular sensitivity of deoxycytidine analogs by Jieh-Yuan Liou; Hui-Ru Lai; Chih-Hung Hsu; Wei-Ling Chang; Mei-Ju Hsieh; Yu-Chun Huang; Yung-Chi Cheng (pp. 381-388).

Deoxycytidine analogs are an important class of clinically active antiviral and anticancer agents. The stepwise phosphorylation of these analogs to triphosphate metabolites is crucial for biological action. Human UMP/CMP kinase (UMP/CMPK; cytidylate kinase; EC 2.7.4.14) is thought to be responsible for phosphorylation of UMP, CMP, and dCMP and may also play an important role in the activation of pyrimidine analogs. However, no evidence has verified this notion in intact cells. In this study we explored the functional roles of UMP/CMPK in natural pyrimidine synthesis and metabolism of deoxycytidine analogs, as well as 5-FU in HeLa S3 and HCT8 cells. The amounts of UMP/CMPK protein in different cell lines correlated with UMP, CMP, and dCMP kinase activities and amounts of UMP/CMPK RNA. Modulation of UMP/CMPK by overexpression or down-regulation had no impact on natural pyrimidine nucleotides and cell growth. However, down-regulating UMP/CMPK expression by siRNA led to a decrease in the formation of the triphosphate metabolites, resulting in cellular resistance to these analogs. More diphosphate and triphosphate metabolites of deoxycytidine analogs were detected and cellular sensitivity to these agents was increased in the UMP/CMPK-overexpressing cells. This study indicates that the second step enzyme (UMP/CMPK) is responsible for the phosphorylation of pyrimidine analogs and also has an impact on cellular sensitivity to these analogs in those cell lines.

Keywords: Abbreviations; 5-FU; 5-fluorouracil; 5-FUR; 5-fluorouridine; araC; 1-β-; d; -arabinofuranosylcytosine; dC; β-; d; -2′-deoxycytidine; Gem; 2′,2′-difluorodeoxycytidine, gemcitabine; MP; monophosphate; DP; diphosphate; TP; triphosphate; dCK; deoxycytidine kinase; CMPK; CMP kinase; dCMPK; dCMP kinase; UMPK; UMP kinase; NDPK; nucleoside diphosphate kinase; dNTP; deoxynucleoside triphosphateUMP/CMP kinase; Deoxycytidine analogs; Down-regulation; Overexpression; Drug sensitivity

Investigation of anti-leukemia molecular mechanism of ITR-284, a carboxamide analog, in leukemia cells and its effects in WEHI-3 leukemia mice by Yen-Fang Wen; Jai-Sing Yang; Sheng-Chu Kuo; Chrong-Shiong Hwang; Jing-Gung Chung; Hsi-Chin Wu; Wen-Wen Huang; Jia-Hua Jhan; Chung-Ming Lin; Hui-Jye Chen (pp. 389-398).

ITR-284, a potent anti-leukemia agent of carboxamide derivative, has been shown to inhibit the proliferation of leukemia cells. In this study, the underlying molecular mechanisms in vitro and anti-leukemia activity in vivo of ITR-284 were investigated. ITR-284 reduced the cell viability and induced apoptosis in HL-60 and WEHI-3 leukemia cells. Following exposure of cells to 30nM of ITR-284, there is a time-dependent decrease in the mitochondrial membrane potential (Δ Ψ_m) and an increase in the reactive oxygen species (ROS). ITR-284 treatment also caused a time-dependent increase of Fas/CD95, cytosolic cytochrome c, cytosolic active form of caspase-8/-9/-3, cytosolic Apaf-1 and Bax, and the decrease of Bcl-2. However, the ITR-284-induced caspase-8/-9 and -3 activities can be blocked by pan-caspase inhibitor (Z-VAD-FMK). In addition, the anti-leukemia effects of ITR-284 in vivo were further evaluated in BALB/c mice inoculated with WEHI-3 cells. Orally treatment with ITR-284 (2 and 10mg/kg/alternate day for 7 times) increased the survival rate and prevented the loss of body weight in leukemia mice. The enlargement of spleen and infiltration of immature myeloblastic cells into spleen red pulp were significantly reduced in ITR-284-treated mice compared with control mice. Moreover, ITR-284 application can enhance the anti-leukemia effect of all-trans retinoic acid (ATRA). These results revealed that ITR-284 acted against both HL-60 and WEHI-3 in vitro via both intrinsic and extrinsic apoptotic signaling pathways, and exhibited an anti-leukemic effect in a WEHI-3 orthotopic mice model of leukemia.

Keywords: ITR-284; Anti-leukemia activity; Apoptosis; Caspase; Orthotopic mice model of leukemia

Chronic inhibition of farnesyl pyrophosphate synthase attenuates cardiac hypertrophy and fibrosis in spontaneously hypertensive rats by Liang Li; Guo-Ping Chen; Yin Yang; Yang Ye; Lei Yao; Shen-Jiang Hu (pp. 399-406).

Farnesyl pyrophosphate synthase (FPPS), an essential enzyme in the mevalonate pathway, was reported to be upregulated in young spontaneously hypertensive rats (SHR) when compared with Wistar–Kyoto (WKY) rats, and this was accompanied by development of left ventricular hypertrophy. Five-week-old rats were daily gavaged with vehicle or an FPPS inhibitor (alendronate, 1 or 10mg/kg) and blood pressures was monitored by the tail-cuff method every other week. Twelve weeks of alendronate treatment attenuated the left ventricular weight to body weight ratio (LVW/BW), hydroxyproline content, collagen deposition in the interstitia, and gene expression of atrial natriuretic peptide, B-type natriuretic peptide, and procollagen type I/III in the SHR left ventricle, all of which were significantly higher in SHRs than in WKY rats. Furthermore, long-term treatment with an FPPS inhibitor significantly reduced RhoA activation, ERK phosphorylation, and TGF-β1 expression in the SHR left ventricle, all of which were upregulated more in SHRs than in WKY rats. In conclusion, chronic treatment with an FPPS inhibitor attenuates the development of cardiac hypertrophy and fibrosis, and the suppression of ERK1/2 phosphorylation and TGF-β1 expression with inhibition of RhoA activation may be an important mechanism.

Keywords: Farnesyl diphosphate synthase; Alendronate; Cardiac hypertrophy; Spontaneously hypertensive rat

Identification of a novel 12-nucleotide insertion polymorphism in the promoter region of ADRA2B: Full linkage with the 9-nucleotide deletion in the coding region and influence on transcriptional activity by Pierre-Antoine Crassous; Régis Blaise; Amélie Marquette; Amir Snapir; Mika Scheinin; Hervé Paris; Stéphane Schaak (pp. 407-412).

The α_2B-adrenoceptor (α_2B-AR) mediates vasoconstriction and a common polymorphism (+901 Ins/Del), located in the coding region of the human α_2B-AR gene (ADRA2B), has been demonstrated to affect receptor function in vitro. In this study, we have identified a novel polymorphism corresponding to the insertion of 12-nucleotides (GGGACGGCCCTG) at position −4825 relative to the start codon (−4825 del/ins) in the far upstream region of the ADRA2B promoter. The genotyping of 71 unrelated Finnish individuals showed that the −4825 ins polymorphism is common and in complete linkage with the Del polymorphism at position +901 and a G/C substitution at position −98. Transfection of various cell lines with luciferase constructs containing a 5.5kb fragment of the ADRA2B promoter region indicated that the 12-nucleotide insertion at −4825 resulted in a large reduction of transcriptional activity. Electrophoretic mobility shift assays with oligonucleotide probes corresponding to the two ADRA2B alleles demonstrated that the region where the −4825 del/ins variation occurs binds nuclear proteins and that the 12-nucleotide insertion affects the pattern of bound transcription factors. Altogether, the present findings show that the previously identified +901 Del polymorphism is linked with a variation in the ADRA2B promoter that affects transcriptional activity in vitro. The molecular mechanisms underlying this effect are still unclear but a possible impact of the −4825 ins polymorphism on α_2B-AR expression would merit to be examined in vivo as a diminution of promoter activity may limit the functional consequences of the +901 Del polymorphism.

Keywords: Adrenoceptor; ADRA2B promoter; Polymorphism; Transcription; Haplotype

N-acetyl-l-cysteine (NAC) inhibits virus replication and expression of pro-inflammatory molecules in A549 cells infected with highly pathogenic H5N1 influenza A virus by Janina Geiler; Martin Michaelis; Patrizia Naczk; Anke Leutz; Klaus Langer; Hans-Wilhelm Doerr; Jindrich Cinatl Jr. (pp. 413-420).

The antioxidant N-acetyl-l-cysteine (NAC) had been shown to inhibit replication of seasonal human influenza A viruses. Here, the effects of NAC on virus replication, virus-induced pro-inflammatory responses and virus-induced apoptosis were investigated in H5N1-infected lung epithelial (A549) cells. NAC at concentrations ranging from 5 to 15mM reduced H5N1-induced cytopathogenic effects (CPEs), virus-induced apoptosis and infectious viral yields 24h post-infection. NAC also decreased the production of pro-inflammatory molecules (CXCL8, CXCL10, CCL5 and interleukin-6 (IL-6)) in H5N1-infected A549 cells and reduced monocyte migration towards supernatants of H5N1-infected A549 cells. The antiviral and anti-inflammatory mechanisms of NAC included inhibition of activation of oxidant sensitive pathways including transcription factor NF-κB and mitogen activated protein kinase p38. Pharmacological inhibitors of NF-κB (BAY 11-7085) or p38 (SB203580) exerted similar effects like those determined for NAC in H5N1-infected cells. The combination of BAY 11-7085 and SB203580 resulted in increased inhibitory effects on virus replication and production of pro-inflammatory molecules relative to either single treatment. NAC inhibits H5N1 replication and H5N1-induced production of pro-inflammatory molecules. Therefore, antioxidants like NAC represent a potential additional treatment option that could be considered in the case of an influenza A virus pandemic.

Keywords: ROS; NAC; Cytokines; H5N1; Apoptosis

Synergistic anti-inflammatory effects of low doses of curcumin in combination with polyunsaturated fatty acids: Docosahexaenoic acid or eicosapentaenoic acid by Constance Lay Lay Saw; Ying Huang; Ah-Ng Kong (pp. 421-430).

Inflammatory response plays an important role not only in the normal physiology but also in the pathology such as cancers. As chronic inflammations are associated with malignancies, it is important to prevent inflammation-mediated neoplastic formation, promotion and/or progression. One possible intervention will be using cancer chemopreventive agents such as curcumin (CUR), a potent anti-inflammatory and anti-oxidative stress compound. Polyunsaturated fatty acids (PUFA) such as docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA) are potent anti-inflammatory agents by decreasing the production of inflammatory eicosanoids, cytokines, and reactive oxygen species (ROS). The present study aims at examining whether CUR with DHA or EPA would have synergistic anti-inflammatory effects in RAW 264.7 cells. Non-toxic concentrations of single and combination of the compounds were investigated at 6, 12 and 24h. The nitric oxide (NO) suppression effects were most prominent at 24h. All the combinations of CUR and DHA or EPA with lower concentrations of CUR 5μM and 25μM of DHA or EPA were found to have synergistic effects in suppressing LPS-stimulated NO and endogenous NO levels. Importantly, very low doses of CUR 2.5μM and DHA or EPA of 0.78μM could synergistically suppress the LPS-induced prostaglandin E₂ (PGE₂). The combinations were also found to suppress iNOS, COX-2, 5-lipoxygenase (5-LOX) and cPLA₂ but induce HO-1. Taken together, the present study clearly shows the synergistic anti-inflammatory as well as anti-oxidative stress effects of CUR and PUFA.

Keywords: Abbreviations; CUR; curcumin; DHA; docosahexaenoic acid; EPA; eicosapentaenoic acid; PUFA; polyunsaturated fatty acids; Nrf2; nuclear factor-erythroid 2-related factor 2; ARE; antioxidant response element; NF-κB; nuclear factor-kappa-B; LPS; lipopolysaccharide; COX; cyclooxygenase; LOX; lipoxygenase; iNOS; inducible nitric oxide synthase; HO-1; hemeoxygenase-1; ROS; reactive oxygen species; cPLA; ₂; cytosolic phospholipase A; ₂; PGE; ₂; prostaglandin E; ₂; qRT-PCR; quantitative reverse-transcriptase-polymerase chain reaction; CI; combination indexCurcumin; Docosahexaenoic acid; Eicosapentaenoic acid; Nuclear factor-erythroid 2-related factor 2; Synergism; Inflammation

Src-mediated regulation of inflammatory responses by actin polymerization by Joo Young Kim; Yong Gyu Lee; Mi-Yeon Kim; Se Eun Byeon; Man Hee Rhee; Jongsun Park; David R. Katz; Benjamin M. Chain; Jae Youl Cho (pp. 431-443).

Actin cytoskeleton disruption by cytochalasin B mainly blocked the NF-κB-mediated production of inflammatory mediators via the inhibition of Src kinase activity.Although the role of the actin cytoskeleton has become increasingly elucidated, the role of actin polymerization in inflammatory processes remains poorly understood. Here, we examine the role of the actin cytoskeleton during LPS-mediated inflammatory events in RAW264.7 cells and peritoneal macrophages. We observed that actin cytoskeleton disruption by cytochalasin B and siRNA to cytoplasmic actin strongly down-regulated LPS-mediated inflammatory responses such as NO production, PGE₂ release, and TNF-α secretion. Actin cytoskeleton disruption by cytochalasin B down-regulated a series of signaling cascades including PI3K, Akt, and IKK, but not MAPKs, necessary for NF-κB activation without down-regulating total forms of the proteins as assessed by measuring their phosphorylation levels. In particular, cytochalasin B significantly inhibited LPS-induced both phosphorylation and kinase activity of Src without altering total level, implying that Src may be a potential pharmacological target of actin cytoskeleton rearrangement. Moreover, the direct association of Src with actin was actin polymerization-dependent according to immunoprecipitation analysis performed with a GFP-actin wild type and HA-tagged Src. Therefore, our data suggest that actin cytoskeleton rearrangement may be a key event during the regulation of inflammatory responses that modulates the activity of Src and its downstream signaling molecules.

Keywords: Actin cytoskeleton; Inflammation; Cytochalasin B; Src kinase; NF-κB

Structural constraints and the importance of lipophilicity for the mitochondrial uncoupling activity of naturally occurring caffeic acid esters with potential for the treatment of insulin resistance by Hoda M. Eid; Diane Vallerand; Asim Muhammad; Tony Durst; Pierre S. Haddad; Louis C. Martineau (pp. 444-454).

Caffeic acid phenethyl ester (CAPE) has recently been shown to potently stimulate glucose uptake in cultured skeletal muscle cells through the AMPK pathway and therefore to have anti-diabetic potential. We report here that CAPE increases glucose uptake in C2C12 muscle cells by 225±21% at 50μM, and that activation of AMPK is a consequence of the metabolic stress resulting from an uncoupling-type disruption of mitochondrial function (complete uncoupling at 50μM). We also observe that the therapeutic potential of CAPE is offset by its high potential for toxicity. The purpose of this study was therefore to identify other active caffeic acid derivatives, evaluate their ratio of activity to toxicity, and elucidate their structure–activity relationship. Twenty naturally occurring derivatives were tested for glucose-uptake stimulating activity in C2C12 cells following 18h of treatment and for uncoupling activity in isolated rat liver mitochondria. Cytotoxicity was assessed in C2C12 cells by the release of lactate dehydrogenase over 18h. In addition to CAPE, four compounds were identified to be active, both stimulating glucose uptake and uncoupling isolated mitochondria. Activity required that the caffeic acid moiety be intact and that the compound not contain a strongly ionized group. Both activity and toxicity were found to be well-correlated to predicted lipophilicity. However, two compounds exhibited little to no toxicity while still stimulating glucose uptake by 65–72%. These results support a therapeutic potential for this family of compounds and provide the framework for the design of alternatives to Metformin with an optimized balance of safety and activity.

Keywords: Naturally occurring phenolic compounds; Mitochondrial energy transduction; Uncoupling of oxidative phosphorylation; Adenosine monophosphate (AMP)-activated protein kinase signaling pathway; Glucose uptake; Insulin resistance

Role of basolateral efflux transporter MRP4 in the intestinal absorption of the antiviral drug adefovir dipivoxil by Xin Ming; Dhiren R. Thakker (pp. 455-462).

Adefovir dipivoxil is a diester prodrug of the antiviral drug adefovir, with much greater oral bioavailability than adefovir. Evidence shows that the prodrug is metabolized to adefovir in the enterocytes during intestinal absorption. However, it is unknown how the highly charged and hydrophilic adefovir crosses the basolateral membrane in the intestine. This study determines the role of specific basolateral transporter(s) in the egress of adefovir across the basolateral membrane when formed from adefovir dipivoxil in Caco-2 cells, a model for intestinal epithelium. Multidrug resistance-associated protein 4 (MRP4) plays an important role in renal secretion of adefovir. Immunofluorescence images showed that MRP4 is localized in the basolateral membrane of Caco-2 cells. This localization was further confirmed by Western blotting of the apical and basolateral membrane fractions that were isolated by a novel method involving biotinylation of respective membrane proteins and affinity enrichment. MRP4-knockdown Caco-2 cells were produced by stable transfection with MRP4-specific siRNA expression plasmid. These cells showed reduced MRP4 protein expression and corresponding reduction in the basolateral egress of adefovir when adefovir dipivoxil was dosed on the apical side. A comparison of these data with the reduction in the basolateral egress of adefovir by the general MRP inhibitor indomethacin established that MRP4, among MRPs, plays a predominant role in the basolateral egress of adefovir in Caco-2 cells. The results highlight the importance of MRP4 in oral absorption of adefovir dipivoxil, and suggest that significant drug–drug interactions can occur if an MRP4 inhibitor is co-administered with adefovir dipivoxil.

Keywords: Abbreviations; ABC; ATP binding cassette; DMEM; Dulbecco's Modified Eagle Medium; EMEM; Eagle's minimum essential medium; FBS; Fetal bovine serum; HBSS; Hank's balanced salt solution; HEPES; N-hydroxyethylpiperazine-N′-2-ethanesulfonate; h; human; MDCKII; Madin–Darby Canine Kidney II; MRP; multidrug resistance-associated protein; NEAA; nonessential amino acids; P-gp; P-glycoproteinIntestinal absorption; Basolateral efflux; Caco-2 cells; MRP4; Adefovir dipivoxil

Werner's syndrome helicase participates in transcription of phenobarbital-inducible CYP2B genes in rat and mouse liver by Antoine Amaury Lachaud; Sacha Auclair-Vincent; Laurent Massip; Étienne Audet-Walsh; Michel Lebel; Alan Anderson (pp. 463-470).

CYP2B10 mRNA level is reduced in mice that carry a homozygous deletion in the Werner helicase domain.Werner's syndrome (WS) is a rare human autosomal recessive segmental progeroid syndrome clinically characterized by atherosclerosis, cancer, osteoporosis, type 2 diabetes mellitus and ocular cataracts. The WRN gene codes for a RecQ helicase which is present in many tissues. Although the exact functions of the WRN protein remain unclear, accumulating evidence suggests that it participates in DNA repair, replication, recombination and telomere maintenance. It has also been proposed that WRN participates in RNA polymerase II-dependent transcription. However no promoter directly targeted by WRN has yet been identified. In this work, we report mammalian genes that are WRN targets. The rat CYP2B2 gene and its closely related mouse homolog, Cyp2b10, are both strongly induced in liver by phenobarbital. We found that there is phenobarbital-dependent recruitment of WRN to the promoter of the CYP2B2 gene as demonstrated by chromatin immunoprecipitation analysis. Mice homozygous for a Wrn mutation deleting part of the helicase domain showed a decrease in basal and phenobarbital-induced CYP2B10 mRNA levels compared to wild type animals. The phenobarbital-induced level of CYP2B10 protein was also reduced in the mutant mice. Electrophoretic mobility shift assays showed that WRN can participate in the formation of a complex with a specific sequence within the CYP2B2 basal promoter. Hence, there is a WRN binding site in a region of DNA sequence to which WRN is recruited in vivo. Taken together, these results suggest that WRN participates in transcription of CYP2B genes in liver and identifies the first physical interaction between a specific promoter sequence and WRN.

Keywords: Abbreviations; Ab; antibody; CAR; constitutive androstane receptor; C/EBPα; CCAAT/enhancer binding protein α; ChIP; chromatin immunoprecipitation; EMSA; electrophoresis mobility shift assay; FXR; farnesoid X receptor; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; HPRT; hypoxanthine phosphoribosyl transferase; LXR; liver X receptor; PXR; pregnane X receptor; NF1; nuclear factor 1; oligo; oligonucleotide; PB; phenobarbital; PBRU; phenobarbital response unit; PBREM; phenobarbital responsive enhancer module; PEPCK; phosphoenolpyruvate carboxykinase; Pol II; RNA polymerase II; RXR; retinoid X receptor; TBS; Tris buffered saline; WS; Werner's syndrome; WRN; WS proteinPhenobarbital-inducible genes; Werner's syndrome; Chromatin immunoprecipitation; Gene regulation; Transcription; Liver

Binding thermodynamics at the human cannabinoid CB₁ and CB₂ receptors by Stefania Merighi; Carolina Simioni; Stefania Gessi; Katia Varani; Pier Andrea Borea (pp. 471-477).

The thermodynamic parameters Δ G°, Δ H° and Δ S° of the binding equilibrium of agonists and antagonists at cannabinoid CB₁ and CB₂ receptors were determined by means of affinity measurements at different temperatures and van’t Hoff plots were constructed. Affinity constants were measured on CHO cells transfected with the human CB₁ and CB₂ receptors by inhibition assays of the binding of the cannabinoid receptor agonist [³H]-CP-55,940. van’t Hoff plots were linear for agonists and antagonists in the temperature range 0–30°C. The thermodynamic parameters for CB₁ receptors fall in the ranges 17≤Δ H°≤59kJ/mol and 213≤Δ S°≤361kJ/mol for agonists and −52≤Δ H°≤−26kJ/mol and −12≤Δ S°≤38kJ/mol for antagonists. The thermodynamic parameters for CB₂ receptors fall in the ranges 27≤Δ H°≤48kJ/mol and 234≤Δ S°≤300kJ/mol for agonists and −19≤Δ H°≤−17kJ/mol and 43≤Δ S°≤74kJ/mol for antagonists. Collectively, these data show that agonist binding is always totally entropy-driven while antagonist binding is enthalpy and entropy-driven, indicating that CB₁ and CB₂ receptors are thermodynamically discriminated. These data could give new details on the nature of the forces driving the CB₁ and CB₂ binding at a molecular level. Enthalpy, entropy, free energy and binding affinity for each ligand to its receptor can all be assessed and therefore the optimal binding profile discovered. Carrying out these binding investigations as early as possible in the discovery process increases the probability that a lead compound will become a successful pharmaceutical compound.

Keywords: Abbreviations; ACEA; arachidonyl-2′-chloroethylamide; AM 251; N; -(piperidin-1-yl)-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-1; H; -pyrazole-3-carboxamide; AM 281; N; -(morpholin-4-yl)-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-1; H; -pyrazole-3-carboxamide; AM 630; 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1; H; -indol-3-yl](4-methoxyphenyl)methanone(6-iodopravadoline); CHO; Chinese hamster ovary; CP-55,940; (1; R; ,3; R; ,4; R; )-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol; hCHO-CB; ₁; CHO cells transfected with human CB; ₁; cannabinoid receptor; hCHO-CB; ₂; CHO cells transfected with human CB; ₂; cannabinoid receptor; 2-Fl-AEA; arachidonyl-2′-fluoroethylamide; JWH-015; (2-methyl-1-propyl-1H-indol-3-yl)-1-naphthalenylmethanone; WIN 55212; (; R; )-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenyl-methanonemesylateBinding mechanisms; Binding thermodynamics; Drug development; Enthalpy–entropy compensation; Pharmacokinetics

Bradykinin-related peptides in the venom of the solitary wasp Cyphononyx fulvognathus by Gisele Picolo; Miki Hisada; Analuê B. Moura; Maurício F.M. Machado; Juliana M. Sciani; Isaltino M. Conceição; Robson L. Melo; Vitor Oliveira; Maria Teresa R. Lima-Landman; Yara Cury; Katsuhiro Konno; Mirian A.F. Hayashi (pp. 478-486).

Biochemical and pharmacological characterization of novel bradykinin-related peptides (BRP) named Cd-146, Thr6-BK, fulvonin and cyphokinin isolated from the venom of the solitary wasp Cyphononyx fulvognathus is described.Bradykinin (BK) and its related peptides are widely distributed in venomous animals, including wasps. In fact, we have previously purified a novel BK-related peptide (BRP) named Cd-146 and the threonine⁶-bradykinin (Thr⁶-BK) from the venom of the solitary wasp Cyphononyx fulvognathus. Further survey of this same wasp venom extract allowed the structural characterization of two other novel BRPs, named here as fulvonin and cyphokinin. Biochemical characterization performed here showed that although the high primary structure similarity observed with BK, these wasp peptides are not good substrates for angiotensin I-converting enzyme (ACE) acting more likely as inhibitors of this enzyme. In pharmacological assays, only those more structurally similar to BK, namely cyphokinin and Thr⁶-BK, were able to promote the contraction of guinea-pig ileum smooth muscle preparations, which was completely blocked by the B₂ receptors antagonist HOE-140 in the same way as observed for BK. Only fulvonin was shown to potentiate BK-elicited smooth muscle contraction. Moreover, the 2 new wasp BRPs, namely fulvonin and cyphokinin, as well as Cd-146 and Thr⁶-BK, showed hyperalgesic effect in the rat paw pressure test after intraplantar injection. This effect was shown here to be due to the action of these peptides on BK receptors, since the hyperalgesia induced by both Cd-146 and fulvonin was blocked by B₁ receptor antagonist, while the effect of both cyphokinin and Thr⁶-BK was reversed by B₂ antagonist. This data give support to a better understanding of the function and targets of the kinin-related peptides widely found in several insect venoms.

Keywords: Abbreviations; BK; bradykinin; ACE; angiotensin I-converting enzyme; Thr; ⁶; -BK; threonine; ⁶; -bradykinin; FRET; fluorescence resonance energy transfer; Fmoc; N; -9-fluorenylmethoxycarbonyl; TFA; trifluoroacetic acid; HPLC; high performance liquid chromatography; CPY; carboxypeptidase Y; APM; aminopeptidase M; CPB; carboxypeptidase B; CID; collision-induced dissociation; PSD; post-source decay; HOE-140; icatibantSolitary wasp venom; Bradykinin; Ileum contraction; ACE inhibition; Hyperalgesia

TCDD-induced cyclooxygenase-2 expression is mediated by the nongenomic pathway in mouse MMDD1 macula densa cells and kidneys by Bin Dong; Noriko Nishimura; Christoph F. Vogel; Chiharu Tohyama; Fumio Matsumura (pp. 487-497).

Cyclooxygenase-2 (Cox-2), which plays a critical role in hydronephrosis in mouse neonates, is induced by TCDD in mouse MMDD1 macula densa cells and kidneys through a newly discovered nongenomic pathway.Cyclooxygenase-2 (Cox-2) plays a critical role in TCDD-induced hydronephrosis in mouse neonates. In this study we found that induction of Cox-2 by TCDD in MMDD1, a mouse macula densa cell line, is accompanied with a rapid increase in the enzymatic activity of cytosolic phospholipase A2 (cPLA2) as well as activation of protein kinases. Calcium serves as a trigger for such an action of TCDD in this cell line. These observations indicate that the basic mode of action of TCDD to induce the rapid inflammatory response in MMDD1 is remarkably similar to those mediated by the nongenomic pathway of aryl hydrocarbon receptor (AhR) found in other types of cells. Such an action of TCDD to induce Cox-2 in MMDD1 was not affected by “DRE decoy oligonucleotides” treatment or by introduction of a mutation on the DRE site of Cox-2 promoter, suggesting that this route of action of TCDD is clearly different from that mediated by the classical genomic pathway. An in vivo study with Ahr^nls mouse model has shown that TCDD-induces Cox-2 and renin expression in the kidneys of the Ahr^nls mice as well as Ahr^+/− mice, but not in the Ahr^−/− mice, indicating that this initial action of TCDD in mouse kidney does not require the translocation of AhR into the nucleus, supporting our conclusion that induction of Cox-2 by TCDD in mouse kidney is largely mediated by the nongenomic pathway of TCDD-activated AhR.

Keywords: Abbreviations; AhR; aryl hydrocarbon receptor; ARNT; aryl hydrocarbon receptor nuclear translocator; Cox-2; cyclooxygenase-2; cPLA2; cytosolic phospholipase A2; CYP1A1; cytochrome P450 1A1; CYP1B1; cytochrome P450 1B1; DRE; dioxin responsive element; ERK; extracellular signal-regulated kinase; IGFBP-1; insulin-like growth factor binding protein-1; NKCC2; Na–K–2Cl co-transporter; ROMK; renal outer medullary potassium channel; TCDD; 2,3,7,8-tetrachlorodibenzo-p-dioxinTCDD; Cyclooxygenase-2; Nongenomic; Aryl hydrocarbon receptor

Identification of a nitric oxide-dependent hypotensive effect of anticoagulation factor II from the venom of Agkistrodon acutus by Dengke Shen; Xiaolong Xu; Liyun Zhang; Hao Wu; Lili Peng (pp. 498-506).

Anticoagulation factor II (ACF II) not only binds with FXa to prolong the clotting time, but also induces hypotension in rats through an endothelium-dependent vasodilation.Anticoagulation factor II (ACF II) isolated from the venom of Agkistrodon acutus is a member of the coagulation factor IX/coagulation factor X-binding protein (IX/X-bp) family. ACF II forms a 1:1 complex with activated coagulation factor X in a Ca²⁺-dependent fashion and thereby blocks the amplification of the coagulation cascade. In the present study, we have investigated the effect of ACF II on the mean arterial blood pressure (MABP) and heart rate (HR) in anaesthetized rats. The results indicate that ACF II induces a dose-dependent response in rats with a short fast drop of MABP followed by an increase and then a longer lasting slight decrease in MABP, but does not obviously affect HR. ACF II-induced hypotension is significantly blocked by the nitric oxide (NO) synthase inhibitor N-omega-l-arginine methyl ester (l-NAME). ACF II produces a concentration-dependent relaxation of rat aortic rings with functional-endothelium. The ACF II-induced vasodilatation is completely inhibited by removal of endothelium and significantly inhibited by pretreatment withl-NAME. These observations demonstrate that ACF II induces hypotension through an endothelium-dependent vasodilation, which is strongly mediated by the release of NO from endothelium. ACF II exhibits high anticoagulation activity in vivo based on activated partial thromboplastin time assay. Therefore, ACF II is so far identified as the first unique bifunctional protein in the IX/X-bp family that has both anticoagulant and hypotensive effects on the blood of rats through different pathways.

Keywords: Abbreviations; ACF I; anticoagulation factor I; ACF II; anticoagulation factor II; FIX; coagulation factor IX; FIXa; activated coagulation factor IX; FX; coagulation factor X; FXa; activated coagulation factor X; IX/X-bp; coagulation factor IX/coagulation factor X-binding protein; habu IX/X-bp; habu coagulation factor IX/factor X-binding protein; habu IX-bp; habu coagulation factor IX-binding protein; Deinagkistrodon X-bp; Deinagkistrodon; coagulation factor X-binding protein; SNP; sodium nitroprusside dihydrate; l; -NAME; N-omega-; l; -arginine methyl ester; PPT; plasma prothrombin time; APTT; activated partial thromboplastin time; NO; nitric oxide; eNOS; endothelial nitric oxide synthase; nNOS; neuronal NO synthase; PE; phenylephrine; BP; blood pressure; MABP; mean arterial blood pressure; HR; heart rate Agkistrodon acutus; Anticoagulation factor II; Hypotensive effect; Nitric oxide; N-omega-; l; -arginine methyl ester; Endothelium cell

Crystal structures of oxime-bound fenamiphos-acetylcholinesterases: Reactivation involving flipping of the His447 ring to form a reactive Glu334–His447–oxime triad by Andreas Hörnberg; Elisabet Artursson; Rikard Wärme; Yuan-Ping Pang; Fredrik Ekström (pp. 507-515).

Organophosphorus insecticides and nerve agents inhibit the vital enzyme acetylcholinesterase by covalently bonding to the catalytic serine residue of the enzyme. Oxime-based reactivators, such as [(E)-[1-[(4-carbamoylpyridin-1-ium-1-yl)methoxymethyl]pyridin-2-ylidene]methyl]-oxoazanium dichloride (HI-6) and 1,7-heptylene- bis- N, N′-2-pyridiniumaldoxime dichloride (Ortho-7), restore the organophosphate-inhibited enzymatic activity by cleaving the phosphorous conjugate. In this article, we report the intermolecular interactions between Mus musculus acetylcholinesterase inhibited by the insecticide fenamiphos (fep- mAChE) and HI-6 or Ortho-7 revealed by a combination of crystallography and kinetics. The crystal structures of the two oxime-bound fep- mAChE complexes show that both oximes interact with the peripheral anionic site involving different conformations of Trp286 and different peripheral-site residues (Tyr124 for HI-6 and Tyr72 for Ortho-7). Moreover, residues at catalytic site of the HI-6-bound fep- mAChE complex adopt conformations that are similar to those in the apo mAChE, whereas significant conformational changes are observed for the corresponding residues in the Ortho-7-bound fep- mAChE complex. Interestingly, flipping of the His447 imidazole ring allows the formation of a hydrogen bonding network among the Glu334–His447–Ortho-7 triad, which presumably deprotonates the Ortho-7 oxime hydroxyl group, increases the nucleophilicity of the oxime group, and leads to cleavage of the phosphorous conjugate. These results offer insights into a detailed reactivation mechanism for the oximes and development of improved reactivators.The Glu334–His447–Ortho-7 triad can function as an activator by deprotonating the hydroxyl oxygen of the oxime prior to reactivation of organophosphorus-inhibited acetylcholinesterase.

Keywords: Abbreviations; AChE; acetylcholinesterase; h; AChE; Homo sapiens; acetylcholinesterase; m; AChE; Mus musculus; acetylcholinesterase; CAS; catalytic active site; HI-6; [(E)-[1-[(4-carbamoylpyridin-1-ium-1-yl)methoxymethyl]pyridin-2-ylidene]methyl]-oxoazanium dichloride; OP; organophosphate; Ortho-7; 1,7-heptylene-; bis; -; N; ,; N; ′-2-pyridiniumaldoxime dichloride; PAS; peripheral anionic site; sarin; isopropyl methylphosphonofluoridateAcetylcholinesterase; Crystallography; Histidine-flip; Oxime; Organophosphorus compound

Mechanisms involved in phosphatidylinositol 3-kinase pathway mediated up-regulation of the mu opioid receptor in lymphocytes by Han Liu; Hui Li; Liyuan Guo; Mengsen Li; Chaoying Li; Shanshan Wang; Wei Jiang; Xinhua Liu; Michael A. McNutt; Gang Li (pp. 516-523).

Despite the substantial progress made in understanding initiation expression of the MOR gene in lymphocytes, the signal pathway associated with MOR gene transcription remains to be better defined. As the phosphatidylinositol 3-kinase (PI3K)/AKT pathway can mediate diverse biological responses and is crucial for optimal immune responses and lymphocyte development, this study was undertaken to delineate the role of PI3K/AKT signaling in expression of the MOR gene in CEM ×174 cells. The data show that morphine treatment enhanced the level of phosphorylated, rather than un-phosphorylated, PI3K and AKT, which were synchronously recruited to membrane. The levels of PTEN and p53 which are negative regulators of these signal molecules were reduced, and as a result, the interaction between PTEN and p53 was completely interrupted. With morphine treatment, the levels of both cytoplasmic and nuclear E2F1 which is the downstream effecter of AKT were elevated and the interaction of E2F1with YY1, rather than Sp1, was also increased. Subsequently, E2F1 triggered the transcription of the MOR gene through its enhanced ability to bind the element in promoter region of the MOR gene. All responses to morphine were abolished by naloxone, which is an antagonist of MOR, or by LY294002, an inhibitor of PI3K, implying specific involvement of PI3K/AKT. These results strongly suggest that the PI3K/AKT pathway plays a critical role in the transfer of signal from morphine stimuli to the machinery by which MOR gene transcription is initiated.

Keywords: Morphine; Mu opioid receptor; PI3K; Signaling; Gene expression; Regulation

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Biochemical Pharmacology (v.79, #3)