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Biochemical Pharmacology (v.80, #2)

Editorial Advisory Board (pp. iii).

Graphical Abstracts Contents (pp. e1-e4).

The biochemical basis for the anti-inflammatory and cytoprotective actions of ethyl pyruvate and related compounds by Kenneth K. Kao; Mitchell P. Fink (pp. 151-159).

Pyruvate is an important metabolic intermediate, and also is an effective scavenger of hydrogen peroxide and other reactive oxygen species (ROS). Pharmacological administration of pyruvate has been shown to improve organ function in animal models of oxidant-mediated cellular injury. However, pyruvate is relatively unstable in aqueous solutions, which could limit the therapeutic potential of this compound. Ethyl pyruvate (EP), a simple derivative of pyruvic acid, is also an ROS scavenger, but seems to exert pharmacological effects, such as suppression of inflammation, which are at least quantitatively different and in some instances are qualitatively distinct from those exerted by pyruvate anion. Treatment with EP has been shown to improve survival and/or ameliorate organ dysfunction in a wide variety of pre-clinical models of acute illnesses, such as severe sepsis, acute pancreatitis and stroke. Using other animal models, some studies have demonstrated that more prolonged treatment with EP can ameliorate inflammatory bowel disease or slow the rate of growth of malignant tumors. In a clinical trial of patients undergoing cardiac surgery, treatment with EP was shown to be safe, but it failed to improve outcome. The true therapeutic potential of EP and related compounds remains to be elucidated. In this review, some of the biochemical mechanisms, which might be responsible for the pharmacological effects of EP, are discussed.

Keywords: ROS; Inflammation; NF-kappaB; Glyoxalase; Janus-associated kinase

Associated proteins: The universal toolbox controlling ligand gated ion channel function by Tanguy Araud; Susan Wonnacott; Daniel Bertrand (pp. 160-169).

Associated proteins interacting with the ligand gated channels regulate the physiological and pharmacological properties of these integral membrane proteins allowing the cell to fine tune receptor activity.Ligand gated ion channels are integral multimeric membrane proteins that can detect with high sensitivity the presence of a specific transmitter in the extracellular space and transduce this signal into an ion flux. While these receptors are widely expressed in the nervous system, their expression is not limited to neurons or their postsynaptic targets but extends to non-neuronal cells where they participate in many physiological responses. Cells have developed complex regulatory mechanisms allowing for the precise control and modulation of ligand gated ion channels. In this overview the roles of accessory subunits and associated proteins in these regulatory mechanisms are reviewed and their relevance illustrated by examples at different ligand gated ion channel types, with emphasis on nicotinic acetylcholine receptors. Dysfunction of ligand gated ion channels can result in neuromuscular, neurological or psychiatric disorders. A better understanding of the precise function of associated proteins and how they impact on ligand gated ion channels will provide new therapeutic opportunities for clinical intervention.

Keywords: Abbreviations; LGICs; ligand gated ion channels; GluR; glutamate receptor; GlyR; glycine receptor; GABA; _A; R; γ-amino butyric acid receptor; P2X; purinergic; 5HT; ₃; R; 5-hydroxytryptamine 3 receptor; nAChR; nicotinic acetylcholine receptor; NMDA; N-methyl-; d; -aspartate; SLURP; secreted Ly-6/uPAR-related proteinLigand gated ion channels; Associated proteins; Activation properties; Desensitization; Subunit assembly; Receptor trafficking; Allosteric modulator

Syrbactin class proteasome inhibitor-induced apoptosis and autophagy occurs in association with p53 accumulation and Akt/PKB activation in neuroblastoma by Crystal R. Archer; Dana-Lynn T. Koomoa; Erin M. Mitsunaga; Jérôme Clerc; Mariko Shimizu; Markus Kaiser; Barbara Schellenberg; Robert Dudler; André S. Bachmann (pp. 170-178).

Syrbactins: Novel proteasome inhibitor class inhibits cancer cells and induces apoptosis and autophagy.Syrbactins belong to a new class of proteasome inhibitors which include syringolins and glidobactins. These small molecules are structurally distinct from other, well-established proteasome inhibitors, and bind the eukaryotic 20S proteasome by a novel mechanism. In this study, we examined the effects of syringolin A (SylA) and glidobactin A (GlbA) as well as two synthetic SylA-analogs (SylA-PEG and SylA-LIP) in human neuroblastoma (SK-N-SH), human multiple myeloma (MM1.S, MM1.RL, and U266), and human ovarian cancer (SKOV-3) cells. While all four syrbactins inhibited cell proliferation in a dose-dependent manner, GlbA was most potent in both dexamethasone-sensitive MM1.S cells (IC₅₀: 0.004μM) and dexamethasone-resistant MM1.RL cells (IC₅₀: 0.005μM). Syrbactins also inhibited the chymotrypsin-like proteasome activity in a dose-dependent fashion, and GlbA was most effective in SK-N-SH cells (IC₅₀: 0.015μM). The GlbA-promoted inhibition of proteasomal activity in SK-N-SH cells resulted in the accumulation of ubiquitinated proteins and tumor suppressor protein p53 and led to apoptotic cell death in a time-dependent manner. GlbA treatment also promoted the activation of Akt/PKB via phosphorylation at residue Ser⁴⁷³ and induced autophagy as judged by the presence of the lipidated form of microtubule-associated protein 1 light chain 3 (LC3) and autophagosomes. Collectively, our data suggest that syrbactins belong to a new and effective proteasome inhibitor class which promotes cell death. Proteasome inhibition is a promising strategy for targeted anticancer therapy and syrbactins are a new class of inhibitors which provide a structural platform for the development of novel, proteasome inhibitor-based drug therapeutics.

Keywords: Apoptosis; Autophagy; Neuroblastoma; Proteasome inhibitors; Syrbactins

Functional characterization of gefitinib uptake in non-small cell lung cancer cell lines by Maricla Galetti; Roberta R. Alfieri; Andrea Cavazzoni; Silvia La Monica; Mara Bonelli; Claudia Fumarola; Paola Mozzoni; Giuseppe De Palma; Roberta Andreoli; Antonio Mutti; Marco Mor; Marcello Tiseo; Andrea Ardizzoni; Pier Giorgio Petronini (pp. 179-187).

High cell density and acidity affect gefitinib active uptake into NSCLC cell lines and may be critical determinants of intracellular gefitinib levels and hence of inhibition of EGFR autophosphorylation.Gefitinib, an inhibitor of epidermal growth factor receptor tyrosine kinase, has been developed and approved for treatment of advanced non-small cell lung cancer (NSCLC).In this study, we investigated the uptake of gefitinib in gefitinib-sensitive and -resistant NSCLC cell lines. The transport system was temperature-dependent, indicative of an active process and sodium- and potential-independent. Moreover, high cell densities and low extracellular pH significantly reduced the uptake of gefitinib. Inhibitors of the human organic cation transporter 1 (hOCT1) significantly decreased gefitinib uptake; however, gefitinib was not a substrate for hOCT1 or hOCT2 in overexpressing HEK293 cells. Interestingly, gefitinib significantly reduced uptake of the hOCT prototypical substrate MPP suggesting that gefitinib may exert an inhibitory effect on the intracellular accumulation of drugs transported by hOCT1 and hOCT2.After 15min of treatment at 1μM (the maximum plasma concentration of gefitinib obtained at the clinically relevant dose) gefitinib accumulated within the cell in resistant-cell lines at concentrations similar or even higher than in gefitinib-sensitive cells tending to rule out an alteration in drug uptake as a mechanism of resistance to gefitinib treatment.Moreover, our results suggest that the extrusion of lactate by crowded cells may contribute in decreasing the pH, which in turn can influence the uptake of gefinitib and as a result the inhibition of EGFR autophosphorylation.

Keywords: Lung cancer; EGFR; Gefitinib; Uptake

Multivalent dendrimeric and monomeric adenosine agonists attenuate cell death in HL-1 mouse cardiomyocytes expressing the A₃ receptor by Athena M. Keene; Ramachandran Balasubramanian; John Lloyd; Asher Shainberg; Kenneth A. Jacobson (pp. 188-196).

Multivalent dendrimeric conjugates of GPCR ligands may have increased potency or selectivity in comparison to monomeric ligands, a phenomenon that was tested in a model of cytoprotection in mouse HL-1 cardiomyocytes. Quantitative RT-PCR indicated high expression levels of endogenous A₁ and A_2A adenosine receptors (ARs), but not of A_2B and A₃ARs. Activation of the heterologously expressed human A₃AR in HL-1 cells by AR agonists significantly attenuated cell damage following 4h exposure to H₂O₂ (750μM) but not in untransfected cells. The A₃ agonist IB-MECA (EC₅₀ 3.8μM) and the non-selective agonist NECA (EC₅₀ 3.9μM) protected A₃ AR-transfected cells against H₂O₂ in a concentration-dependent manner, as determined by lactate dehydrogenase release. A generation 5.5 PAMAM (polyamidoamine) dendrimeric conjugate of a N⁶-chain-functionalized adenosine agonist was synthesized and its mass indicated an average of 60 amide-linked nucleoside moieties out of 256 theoretical attachment sites. It non-selectively activated the A₃AR to inhibit forskolin-stimulated cAMP formation (IC₅₀ 66nM) and, similarly, protected A₃-transfected HL-1 cells from apoptosis-inducing H₂O₂ with greater potency (IC₅₀ 35nM) than monomeric nucleosides. Thus, a PAMAM conjugate retained AR binding affinity and displayed greatly enhanced cardioprotective potency.

Keywords: Abbreviations; ADAC; N; ⁶; -[4-[[[4-[[[(2-aminoethyl)amino]carbonyl]methyl]-anilino]carbonyl]methyl]phenyl]adenosine; AF488; Alexa-Fluor; ^®; 488; CHAPS; 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate; CHO; Chinese hamster ovary; Cl-IB-MECA; 2-chloro-; N; ⁶; -(3-iodobenzyl)-5′-; N; -methylcarboxamidoadenosine; DMEM; Dulbecco's modified Eagle medium; DMF; N,N; -dimethylformamide; DMSO; dimethyl sulfoxide; ED; ethylenediamine; EDC; N; -(3-dimethylaminopropyl)-; N; ′-ethylcarbodiimide; EDTA; ethylenediaminetetraacetic acid; FBS; fetal bovine serum; [; ³; H]CGS21680; 2-[; p; -(2-carboxyethyl)phenylethyl-amino]-5′-; N; -ethylcarboxamido-adenosine; GPCR; G protein-coupled receptor; HEK; human embryonic kidney; HEPES; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; [; ¹²⁵; I]AB-MECA; [; ¹²⁵; I]-4-aminobenzyl-5′-; N; -methylcarboxamidoadenosine; IB-MECA; N; ⁶; -(3-iodobenzyl)-5′-; N; -methylcarboxamidoadenosine; MALDI-TOF; matrix assisted laser desorption/ionization time-of-flight; MES; 2-(; N; -morpholino)ethanesulfonic acid; MS; mass spectrometry; NMR; nuclear magnetic resonance; PAMAM; polyamidoamine; qRT-PCR; quantitative real-time polymerase chain reactionCardioprotection; Nucleoside; G protein-coupled receptor; Dendrimers; Polymeric drugs; HL-1 cells

An activated renin–angiotensin system maintains normal blood pressure in aryl hydrocarbon receptor heterozygous mice but not in null mice by Nan Zhang; Larry N. Agbor; Jason A. Scott; Tyler Zalobowski; Khalid M. Elased; Alicia Trujillo; Melissa Skelton Duke; Valerie Wolf; Mary T. Walsh; Jerry L. Born; Linda A. Felton; Jian Wang; Wei Wang; Nancy L. Kanagy; Mary K. Walker (pp. 197-204).

It has been postulated that fetal vascular abnormalities in aryl hydrocarbon receptor null ( ahr^−/−) mice may alter cardiovascular homeostasis in adulthood. We tested the hypothesis that blood pressure regulation in adult heterozygous mice ( ahr^+/−) would be normal, compared to ahr^−/− mice, since no vascular abnormalities have been reported in the heterozygote animals. Mean arterial blood pressure (MAP) was measured using radiotelemetry prior to and during treatment with inhibitors of the autonomic nervous system, nitric oxide synthase (NOS), angiotensin converting enzyme (ACE), or endothelin-1 A receptor (ET_A). Also, indices of renin–angiotensin system (RAS) activation were measured. ahr^+/− and ahr^−/− mice were normotensive and hypotensive, respectively, compared to wild-type ( ahr^+/+) littermates. Responses of all genotypes to autonomic nervous system inhibition were normal. ahr^+/− mice responded normally to NOS inhibition, while the responses of ahr^−/− mice were significantly blunted. In contrast, ahr^+/− mice were significantly more responsive to inhibition of ACE, an ET_A antagonist, or both, while ahr^−/− mice were significantly less responsive to ACE inhibition and more responsive to an ET_A antagonist. ahr^+/− mice also exhibited significant increases in plasma renin and ACE activity, plasma sodium, and urine osmolality, indicative of RAS activation. Thus, normotension in ahr^+/− mice appears to be maintained by increased RAS and ET-1 signaling, while hypotension in ahr^−/− mice may result from decreased RAS signaling. In conclusion, despite the lack of overt fetal vascular abnormalities in ahr^+/− mice, the loss of a single ahr allele has a significant effect on blood pressure regulation.

Keywords: Aryl hydrocarbon receptor; Heterozygote; Blood pressure; Vasocontraction; Angiotensin II

Carbonylation of myosin heavy chains in rat heart during diabetes by Chun-Hong Shao; George J. Rozanski; Ryoji Nagai; Frank E. Stockdale; Kaushik P. Patel; Mu Wang; Jaipaul Singh; William G. Mayhan; Keshore R. Bidasee (pp. 205-217).

Cardiac inotropy progressively declines during diabetes mellitus. To date, the molecular mechanisms underlying this defect remain incompletely characterized. This study tests the hypothesis that ventricular myosin heavy chains (MHC) undergo carbonylation by reactive carbonyl species (RCS) during diabetes and these modifications contribute to the inotropic decline. Male Sprague–Dawley rats were injected with streptozotocin (STZ). Fourteen days later the animals were divided into two groups: one group was treated with the RCS blocker aminoguanidine for 6 weeks, while the other group received no treatment. After 8 weeks of diabetes, cardiac ejection fraction, fractional shortening, left ventricular pressure development (+d P/d t) and myocyte shortening were decreased by 9%, 16%, 34% and 18%, respectively. Ca²⁺- and Mg²⁺-actomyosin ATPase activities and peak actomyosin syneresis were also reduced by 35%, 28%, and 72%. MHC-α to MHC-β ratio was 12:88. Mass spectrometry and Western blots revealed the presence of carbonyl adducts on MHC-α and MHC-β. Aminoguanidine treatment did not alter MHC composition, but it blunted formation of carbonyl adducts and decreases in actomyosin Ca²⁺-sensitive ATPase activity, syneresis, myocyte shortening, cardiac ejection fraction, fractional shortening and +d P/d t induced by diabetes. From these new data it can be concluded that in addition to isozyme switching, modification of MHC by RCS also contributes to the inotropic decline seen during diabetes.

Keywords: Rat; Streptozotocin; Diabetes; Myosin heavy chain; Contractility; Carbonyl adducts; Reactive carbonyl species

Obaculactone suppresses Th1 effector cell function through down-regulation of T-bet and prolongs skin graft survival in mice by Fangyuan Gong; Yan Shen; Qi Zhang; Yang Sun; Jiayu Tang; Feifei Tao; Qiang Xu (pp. 218-225).

Allograft rejection is a predominantly Th1 immune response. In this study, we showed that obaculactone, a natural compound derived from citrus fruit, prolonged skin graft survival in mice when treated after but not before transplantation. Furthermore, obaculactone inhibited alloantigen-specific production of Th1 cytokine IFN-γ as well as proinflammatory cytokine IL-2, TNFα and IL-6. In parallel, IL-10 production was markedly up-regulated. Obaculactone significantly enhanced the percentage of CD4⁺CD25⁺Foxp3⁺ Treg cells in the CD4⁺ splenocytes without any effect on their inhibitory function. In vitro and in vivo tests showed obaculactone down-regulated T-bet expression in Th1 effector cells. Taken together, the unique immunomodulatory properties might qualify obaculactone as a putative, therapeutic compound for the treatment of Th1-driven diseases, including transplant rejection.

Keywords: Obaculactone; Allorejection; Th1 effector cell; T-bet; Treg cell

Photodynamic therapy targeting neuropilin-1: Interest of pseudopeptides with improved stability properties by Noémie Thomas; Marlène Pernot; Régis Vanderesse; Philippe Becuwe; Ezatul Kamarulzaman; David Da Silva; Aurélie François; Céline Frochot; François Guillemin; Muriel Barberi-Heyob (pp. 226-235).

New peptidases-resistant pseudopeptides (aTWLPPR, Aψ[CH₂NH]TWLPPR) and corresponding conjugated photosensitizers were tested, demonstrating a molecular affinity for NRP-1 (A) and no degradation in plasma in vivo 4h after intravenous injection (B).The general strategy developed aims to favor the vascular effect of photodynamic therapy by targeting tumor vasculature. Since angiogenic endothelial cells represent an interesting target to potentiate this vascular effect, we previously described the conjugation of a photosensitizer to a peptide targeting neuropilins (NRPs) over-expressed specially in tumor angiogenic vessels and we recently characterized the mechanism of photosensitization-induced thrombogenic events. Nevertheless, in glioma-bearing nude mice, we demonstrated that the peptide moiety was degraded to various rates according to time after intravenous administration. In this study, new peptidases-resistant pseudopeptides were tested, demonstrating a molecular affinity for NRP-1 and NRP-2 recombinant chimeric proteins and devoid of affinity for VEGF receptor type 1 (Flt-1). To argue the involvement of NRP-1, MDA-MB-231 breast cancer cells were used, strongly over-expressing NRP-1 receptor. We evidenced a statistically significant decrease of the different peptides-conjugated photosensitizers uptake after RNA interference-mediated silencing of NRP-1. Peptides-conjugated photosensitizers allowed a selective accumulation into cells. In mice, no degradation was observed in plasma in vivo 4h after intravenous injection by MALDI-TOF mass spectrometry. This study draws attention to this potential problem with peptides, especially in the case of targeting strategies, and provides useful information for the future design of more stable molecules.

Keywords: Abbreviations; AcOH; acetic acid; Ahx; 6-aminohexanoic acid; a.u.; arbitrary units; a.i.; arbitrary intensity; CHAPS; 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; DCC; dicyclohexylcarbodiimide; DIEA; N,N; -diisopropylethylamine; a.i.; arbitrary intensity; DMF; dimethylformamide; DLI; drug-light interval; EDTA; ethylene diamine tetra acetic acid; FBS; fetal bovine serum; Flt-1; fms-like tyrosine kinase 1; Fmoc; 9-fluorenyl-methoxy-carbonyl; HOBt; 1-hydroxybenzotriazole; HPLC; high performance liquid chromatography; HUVEC; human umbilical vein endothelial cells; i.v.; intravenous; MALDI-TOF; matrix assisted laser desorption ionisation-time-of-flight; NaBH; ₃; CN; sodium cyanoborohydride; NHS; N; -hydroxysuccinimide; NRP; neuropilin; NRP-1; neuropilin-1; NRP-2; neuropilin-2; PDT; photodynamic therapy; PEG; polyethylene glycol; PMSF; phenylmethylsulphonyl fluoride; RER; relative expression ratio; RNAi; RNA interference; RP-HPLC; reverse phase-high performance liquid chromatography; RT-PCR; reverse transcription-polymerase chain reaction; s.d.; standard deviation; siRNA; small interfering RNA; tBu; tertio-butyl; TBTU; 2-(1; H; -benzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium tetrafluoroborate; TFA; trifluoroacetic acid; TIS; triethylsilane; TLC; thin layer chromatography; TPC; 5-(4-carboxyphenyl)-10,15,20-triphenyl-chlorin; VEGF; vascular endothelial growth factor; Wt; wild typeTargeted photodynamic therapy; Pseudopeptide; Neuropilin-1; siRNA; Affinity; In vivo stability

Extracellular calcium-sensing receptor mediates human bronchial epithelial wound repair by Javier Milara; Manuel Mata; Adela Serrano; Teresa Peiró; Esteban J. Morcillo; Julio Cortijo (pp. 236-246).

The airway epithelium routinely undergoes damage that requires repair to restore epithelial barrier integrity. Cell migration followed by proliferation are necessary steps to achieve epithelial repair. Calcium-sensing receptor (CaSR) is implicated in cell migration and proliferation processes. Thus we hypothesized that CaSR mediates lung epithelial wound repair. We detected CaSR expression in human lung and in well-differentiated human bronchial epithelial cells (HBEC). To test the CaSR functionality, HBEC loaded with fura-2 were stimulated with extracellular Ca²⁺ ([Ca²⁺]_out) which resulted in a concentration-dependent intracellular Ca²⁺ ([Ca²⁺]_i) increase (potency∼5.6mM [Ca²⁺]_out). Furthermore, increasing [Ca²⁺]_out induced phosphorylation of the extracellular signal-regulated kinase (ERK1/2) which was blocked by siRNA-CaSR and the specific inhibitor of CaSR, NPS2390.Epithelial repair after mechanical injury of differentiated HBEC was a process dependent of [Ca²⁺]_out since it accelerated wound repair and HBEC proliferation being highest at 5mM [Ca²⁺]_out. Furthermore, U73122 (an inhibitor of phospholipase C (PLC)) and PD 98059 (an inhibitor of ERK1/2) as well as siRNA-CaSR and NPS2390 partially inhibited wound repair and HBEC proliferation. On the other hand, mechanical injury produced an [Ca²⁺]_i wave propagation that was partially inhibited by siRNA-CaSR, NPS2390 and the extracellular Ca²⁺ chelator EGTA, which suggest a link of CaSR between cell–cell communication and wound repair in differentiated HBEC. Our data, for the first time, shows that CaSR plays an important role in airway epithelial repair, which may help to develop novel regenerative therapeutics allowing the rapid repair of lung damaged epithelium.

Keywords: Calcium-sensing receptor; Airway epithelial wound repair; Migration; Proliferation

α-Bisabolol induces dose- and time-dependent apoptosis in HepG2 cells via a Fas- and mitochondrial-related pathway, involves p53 and NFκB by Wei Chen; Jie Hou; Yan Yin; Jongchol Jang; Zhongliang Zheng; Handong Fan; Guolin Zou (pp. 247-254).

In this study, the apoptotic effect of α-bisabolol, a sesquiterpene, against human liver carcinoma cell line HepG2 was investigated. MTT assay showed α-bisabolol could effectively induce cytotoxicity in several human cancer cell lines (PC-3, Hela, ECA-109 and HepG2). The results of nuclei morphology examination, DNA fragmentation detection, flow cytometry analysis and cleavage of poly(ADP-ribose) polymerase and caspases indicated α-bisabolol might induce dose- and time-dependent apoptosis in HepG2 cells. Western blot data also showed a cascade activation of caspases-8,-9,-3 and promoted expression of Fas, implying caspase-8 might function as an upstream regulator, and the Fas-related pathway might be involved in this process. Preparation of mitochondrial/cytosol fraction followed with immunoblot analysis showed the release of chromosome c from mitochondria, down-regulated expression of Bcl-2 and translocation of Bax, Bak and Bid, suggesting the mitochondrial-related pathway might be involved in α-bisabolol-induced apoptosis either. Detection of accumulation of nuclear wild-type p53 and up-regulated expression of NFκB indicated these two key regulator with transcriptional decision-making function in various signaling pathways might also play a role in α-bisabolol-induced apoptosis in HepG2 cells.

Keywords: α-Bisabolol; Apoptosis; Extrinsic; Intrinsic; HepG2

Involvement of natural killer T cells in halothane-induced liver injury in mice by Linling Cheng; Qiang You; Hao Yin; Michael P. Holt; Cynthia Ju (pp. 255-261).

Drug-induced liver injury (DILI) causes significant patient morbidity and mortality, and is the most common reason for drug withdrawals. It is imperative to gain a thorough understanding of the underlying mechanisms of DILI to effectively predict and prevent these reactions. We have recently developed a murine model of halothane-induced liver injury (HILI). The aim of the present study was to investigate the role of hepatic natural killer T (NKT) cells in the pathogenesis of HILI. The degrees of HILI were compared between WT and CD1d^−/− mice, which are deficient in NKT cells. The data revealed that CD1d^−/− mice were resistant in developing HILI. This resistance appeared to be a direct result of NKT cell depletion rather than an indirect one due to the absence of cross-talk between NKT cells and other hepatic innate immune cells. Compared with WT mice, CD1d^−/− mice exhibited a significantly lower number of hepatic infiltrating neutrophils upon halothane challenge (470,000±100,000/liver in WT vs. 120,000±31,500/liver in CD1d^−/− mice). This result in conjunction with our previous finding of an indispensable role of neutrophils in HILI strongly suggests that NKT cells play a critical role in regulating neutrophil recruitment, thereby contributing to the development of HILI. Collectively, the current study and published reports indicate that this murine model of HILI provides an experimental system for the investigation of the underlying mechanisms of DILI. In addition, this model may yield the discovery of susceptibility factors that may control the development of liver injury in patients treated with halothane and potentially other drugs.

Keywords: Abbreviations; DILI; drug-induced liver injury; HILI; halothane-induced liver injury; NKT; natural killer T; IL; interleukin; IFN; interferon; TNF; tumor necrosis factor; α-GalCer; alpha-galactosylceramide; i.p.; intraperitoneally; KCs; Kupffer cells; i.v.; intravenously; ALT; alanine transaminase; H/E; hematoxylin and eosin; CYP450 2E1; cytochrome P450 2E1; TFA; trifloroacetylchloride; FITC; fluoresceinisothiocyanate; PE; phycoerythrin; APC; allophycocyanin; polyI:C; polyinosinic–polycytidylic acidDrug; Hepatotoxicity; Neutrophils

Identification and functional analysis of genes which confer resistance to cisplatin in tumor cells by Zchong-Zcho Wu; Hsing-Pang Lu; Chuck C.-K. Chao (pp. 262-276).

The present model illustrates that cisplatin activates the expression of cisplatin resistance (CPR) genes in treated cancer cells. Upregulation of CPR genes may help cancer cells to survive against cisplatin stress, whereas inhibition of CPR gene activity with short-hairpin RNA (shRNA) may lead to apoptosis.The efficacy of cisplatin during cancer chemotherapy is often impaired by the emergence of cancer cells which become resistant to chemotherapeutic agents. While various mechanisms have been proposed to explain resistance to cisplatin, the genes involved in this process still remain unclear. By using DNA microarrays, we performed a genome-wide analysis of cisplatin-resistant HeLa cells in order to identify genes involved in resistance. We identified nine genes ( NAPA, CITED2, CABIN1, ADM, HIST1H1A, EHD1, MARK2, PTPN21, and MVD), which were consistently upregulated in two cisplatin-resistant HeLa cell lines. The upregulated genes, here referred to as cisplatin resistance genes ( CPR), were further analyzed for their ability to modify the response of HEK293 cells to cisplatin. Short-hairpin RNA (shRNA) knockdown of CPR genes, individually or in combination, was shown to sensitize HEK293 cells to cisplatin, but not to vincristine or taxol, suggesting that CPR genes may be involved specifically in cisplatin resistance. Among the treatments performed, shRNA knockdown of NAPA was the most efficient treatment able to sensitize cells to cisplatin. Furthermore, shRNA knockdown of a single CPR gene was sufficient to partially reverse acquired cisplatin resistance in HeLa cells. Sensitization to cisplatin following knockdown of CPR genes was also observed in the tumorigenic cell lines Sk-ov-3, H1155, and CG-1. Based on these results, we propose that the CPR genes identified here may represent potential candidates for novel target therapies aimed at preventing resistance to cisplatin during chemotherapy.

Keywords: Abbreviations; ADR; adryamycin; CDDP; cisplatin; CPR; cisplatin resistance; DMEM; Dulbecco's modified Eagle's medium; EGTA; ethylene glycol tetra-acetic acid; ER; endoplasmic reticulum; GAPDH; glyceraldehyde 3-phosphate dehydrogenase; JNK; c-Jun N-terminal protein kinase; MAPK; mitogen-activated protein kinase; MDR; multidrug resistance; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NSF; N-ethylmaleimide-sensitive factor; PARP; poly-ADP ribose polymerase; PBS; phosphate-buffered saline; PCR; polymerase chain reaction; PMSF; phenylmethylsulfonyl fluoride; PVDF; polyvinylidene fluoride; RIPA; radio-immunoprecipitation assay; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; shRNA; short-hairpin RNA; VCR; vincristineChemotherapy; Cisplatin resistance; NAPA; CITED2; shRNA

Characterisation of the heterotrimeric presynaptic phospholipase A₂ neurotoxin complex from the venom of the common death adder ( Acanthophis antarcticus) by Benjamin Blacklow; Pierre Escoubas; Graham M. Nicholson (pp. 277-287).

While Australo-Papuan death adder neurotoxicity is generally considered to be due to the actions of reversible competitive postsynaptic α-neurotoxins, the neurotoxic effects are often poorly reversed by antivenom or anticholinesterases. This suggests that the venom may contain a snake presynaptic phospholipase A₂ (PLA₂) neurotoxin (SPAN) that binds irreversibly to motor nerve terminals to inhibit neurotransmitter release. Using size-exclusion liquid chromatography under non-reducing conditions, we report the isolation and characterisation of a high molecular mass SPAN complex, P-elapitoxin-Aa1a (P-EPTX-Aa1a), from the venom of the common death adder Acanthophis antarcticus. Using the chick biventer-cervicis nerve-muscle preparation, P-EPTX-Aa1a (44,698Da) caused inhibition of nerve-evoked twitch contractions while responses to cholinergic agonists and KCl remained unaffected. P-EPTX-Aa1a also produced significant fade in tetanic contractions and a triphasic timecourse of neuromuscular blockade. These actions are consistent with other SPANs that inhibit acetylcholine release. P-EPTX-Aa1a was found to be a heterotrimeric complex composed of α, β and γ-subunits in a 1:1:1 stoichiometry with each subunit showing significant N-terminal sequence homology to the subunits of taipoxin, a SPAN from Oxyuranus s. scutellatus. Like taipoxin, only the α-chain produced any signs of neurotoxicity or displayed significant PLA₂ enzymatic activity. Preincubation with monovalent death adder antivenom or suramin, or inhibition of PLA₂ activity by incubation with 4-bromophenacyl bromide, either prevented or significantly delayed the onset of toxicity by P-EPTX-Aa1a. However, antivenom failed to reverse neurotoxicity. Early intervention with antivenom may therefore be important in severe cases of envenomation by A. antarcticus, given the presence of potent irreversible presynaptic neurotoxins.

Keywords: Abbreviations; ACh; acetylcholine; ANOVA; analysis of variance; 4BPB; 4-bromophenacyl bromide; CBCNM; chick biventer-cervicis nerve-muscle; CCh; carbachol; ESI-Q-TOF; electrospray ionisation quadrupole time-of-flight; MALDI-TOF; matrix-assisted laser desorption ionisation time-of-flight; NSW; New South Wales; PLA; ₂; phospholipase A; ₂; P-EPTX-Aa1a; P-elapitoxin-Aa1a; RP-HPLC; reverse-phase high-pressure liquid chromatography; SPAN; snake presynaptic phospholipase A; ₂; neurotoxin; TFA; trifluoroacetic acid; t; ₉₀; time to 90% neuromuscular blockade; V; _e; elution volume; V; _o; void volumePresynaptic snake neurotoxin; SPAN; Death adder; Acanthophis antarcticus; Taipoxin; Phospholipase A; ₂

Retraction notice to “Antiviral and antiparasite properties of anl-amino acid oxidase from the Snake Bothrops jararaca: Cloning and identification of a complete cDNA sequence” [Biochem. Pharmacol. 76 (2008) 279–288] by Carolina D. Sant’Ana; Danilo L. Menaldo; Tá ssia R. Costa; Harryson Godoy; Vanessa D.M. Muller; Victor H. Aquino; Sérgio Albuquerque; Suely V. Sampaio; Marta C. Monteiro; Rodrigo G. Stá beli; Andreimar M. Soares (pp. 288-288).

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Biochemical Pharmacology (v.80, #2)