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Biochemical Pharmacology (v.71, #11)

Editorial Advisory Board (pp. iii).

In vitro–in vivo correlation for drugs and other compounds eliminated by glucuronidation in humans: Pitfalls and promises by John O. Miners; Kathleen M. Knights; J. Brian Houston; Peter I. Mackenzie (pp. 1531-1539).

Enzymes of the UDP-glucuronosyltransferase (UGT) superfamily are responsible for the metabolism of many drugs, environmental chemicals and endogenous compounds. Identification of the UGT(s) involved in the metabolism of a given compound (‘reaction phenotyping’) currently relies on multiple confirmatory approaches, which may be confounded by the dependence of UGT activity on enzyme source, incubation conditions, and the occurrence of atypical glucuronidation kinetics. However, the increasing availability of substrate and inhibitor ‘probes’ for the individual UGTs provides the prospect for reliable phenotyping of glucuronidation reactions using human liver microsomes or hepatocytes, thereby providing data directly relevant to drug metabolism in humans. While the feasibility of computational prediction of UGT substrate selectivity has been demonstrated, the development of easily interpretable and generalisable models requires further improvement in the datasets available for analysis. Quantitative prediction of the hepatic clearance of glucuronidated drugs and the magnitude of inhibitory interactions based on in vitro kinetic data is more problematic. Intrinsic clearance (CL_int) values generated using human liver microsomes under-predict in vivo hepatic clearance, typically by an order of magnitude. In vivo clearances of glucuronidated drugs are also generally under-predicted by CL_int values from human hepatocytes, but to a lesser extent than observed with the microsomal model. While it is anticipated that systematic analysis of the potential causes of under-prediction may provide more reliable in vitro–in vivo scaling strategies, mechanistic interpretation of in vitro–in vivo correlation more broadly awaits further advances in our understanding of the structural and cellular determinants of UGT activity.

Keywords: In vitro–in vivo correlation; UDP glucuronosyltransferase; Clearance prediction; Reaction phenotyping; Glucuronidation

The BH3-only protein, PUMA, is involved in oxaliplatin-induced apoptosis in colon cancer cells by Xinying Wang; Ming Li; Jide Wang; Chung-Man Yeung; Hongquan Zhang; Hsiang-fu Kung; Bo Jiang; Marie Chia-mi Lin (pp. 1540-1550).

Oxaliplatin, the first line chemotherapeutic of colon cancer, induces damage to tumors via induction of apoptosis. PUMA (p53 up-regulate modulator of apoptosis) is an important pro-apoptotic member of Bcl-2 family and regulated mainly by p53. Here we investigated the role of PUMA in oxalipaltin-induced apoptosis and the potential mechanism. We showed that oxaliplatin-induced PUMA expression in a time- and dose-dependent manner and suppression of PUMA expression by stable transfecting anti-sense PUMA plasmid decreased oxaliplatin-induced apoptosis in colon cancer cells. By abrogating the function of p53, we further demonstrated that the induction was p53-independent. We also found that oxaliplatin could inactivate ERK and suppression of ERK activity by its specific inhibitor (PD98059), and dominant negative plasmid (DN-MEK1) enhanced the oxaliplatin-induced PUMA expression and apoptosis in a p53-independent manner. Taken together, our data suggest that PUMA plays an important role in oxaliplatin-induced apoptosis and the induction could be both p53-dependent and p53-independent. Moreover, PUMA expression and apoptosis in oxaliplatin-treated colon cancer cells could be regulated partly by ERK inactivation. Identification of the molecular components involved in regulating the cellular sensitivity to oxaliplatin may provide potential targets for development of novel compounds that may be useful in enhancement of oxaliplatin cytotoxicity in p53 deficient colon cancer.

Keywords: Oxaliplatin; PUMA; p53; ERK; Colon cancer; Apoptosis

Cellular FLICE-like inhibitory protein (c-FLIP): A novel target for Taxol-induced apoptosis by Travis W. Day; Farhad Najafi; Ching-Huang Wu; Ahmad R. Safa (pp. 1551-1561).

It is known that by binding to the FAS-associated death domain (FADD) protein and/or caspases-8 and -10 at the level of the death-inducing signaling complex (DISC), cellular FLICE-like inhibitory protein (c-FLIP) can prevent apoptosis triggered by death-inducing ligands. We investigated whether the c-FLIP splice variants, c-FLIP long [c-FLIP(L)] and c-FLIP short [c-FLIP(S)], play a role in Taxol-induced apoptosis. Our results showed that low Taxol concentrations triggered caspase-8- and caspase-10-dependent apoptosis in the CCRF-HSB-2 human lymphoblastic leukemia cell line, and induced the down-regulation of c-FLIP(S) and c-FLIP(L). Taxol decreased the expression of c-FLIP by a post-transcriptional and caspase-independent mechanism. To explore the distinct functions of the c-FLIP variants in Taxol-induced apoptosis, we transfected the cells with expression vectors carrying c-FLIP(L) and c-FLIP(S) in the sense orientation or c-FLIP(S) in the antisense orientation [c-FLIP(S)-AS]. Caspases-8 and -10 were more efficiently activated in the c-FLIP(S)-AS strain treated with 5–50nM Taxol, which revealed that c-FLIP regulates Taxol-induced apoptosis by interacting with these caspases. Furthermore, our data showed that increased expression of c-FLIP(L) or c-FLIP(S) reduced apoptosis at 5–50nM Taxol concentrations suggesting that both isoforms of c-FLIP prevent Taxol-induced apoptosis. These results revealed that Taxol induces apoptosis by down-regulating c-FLIP(S) and c-FLIP(L) expression.

Keywords: Abbreviations; MT; microtubule; MAPK; smitogen-activated protein kinases; PTK; protein tyrosine kinases; TNF-α; tumor necrosis factor-α; TNFR1; tumor necrosis factor-α receptor 1; TRAIL; tumor necrosis factor-related apoptosis-inducing ligand; AIF; apoptosis-inducing factor; IAP; an inhibitor of apoptosis protein; Apaf1; apoptotic proteinase activating factor-1; PKC; protein kinase C; PARP; poly(ADP-ribose) polymerase; DFF45; DNA fragmentation factor-45; ROS; reactive oxygen species; c-FLIP; cellular FLICE-like inhibitory protein; PTP; permeability transition pore; PI; propidium iodideTaxol; Apoptosis; Caspase-8; Caspase-10; C-FLIP; Death receptors

Synergism between staurosporine and drugs inducing endoplasmic reticulum stress by Federico Cusinato; Isabella Pighin; Sisto Luciani; Lucia Trevisi (pp. 1562-1569).

Drugs causing endoplasmic reticulum or mitochondrial dysfunction may trigger apoptosis in eukaryotic cells. The thiol reagent dithiothreitol (DTT) belongs to the first group whereas the protein kinases inhibitor staurosporine acts on mitochondria. Since the endoplasmic reticulum and the mitochondrial pathways of apoptosis may converge in common steps, we examined the possibility of synergism between these two drugs. Using the activation of caspase-3 as indicator of apoptosis, we found that in two cell lines, Jurkat and Mono-Mac 6, staurosporine and DTT elicited apoptosis with a different pattern: staurosporine acted rapidly and at nanomolar concentrations while DTT acted slowly and at higher concentrations (1mM). When staurosporine and DTT were combined, the proapoptotic action was increased. This was confirmed examining late apoptotic events such as the translocation of phosphatidylserine across the plasma membrane and the cleavage of the antiapoptotic protein Mcl-1. The use of subthreshold DTT concentrations and isobologram analysis demonstrated the synergic nature of the interaction. Tunicamycin, a drug that, like DTT, inhibits protein folding in the endoplasmic reticulum also increased the proapoptotic effect of staurosporine. In agreement with the interplay between the mitochondrial and the endoplasmic reticulum pathways it was found that both staurosporine and DTT induced cytochrome c release. Furthermore, 90min incubation with DTT did not induce caspase-4 activation while staurosporine alone or in combination with DTT stimulated caspase-4 activity. We conclude that staurosporine is more active in cells undergoing endoplasmic reticulum stress. This synergism may warrant evaluation to establish whether the anticancer activity of staurosporine is also enhanced.

Keywords: Abbreviations; Ac-LEVD-AMC; Ac-; l; -leucyl-; l; -glutamyl-; l; -valyl-; l; -aspartyl-7-amino-4-methylcoumarin; BiP; binding protein; CHAPS; 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; DTT; dithiothreitol; CHOP; C/EBP homologous protein; FITC; fluorescein 5-isothiocyanate; PIPES; piperazine-1,4-bis(2-ethanesulfonic acid); PMSF; phenylmethanesulfonyl fluoride; Z-DEVD-R110; rhodamine 110 bis-(; N; -CBZ-; l; -aspartyl-; l; -glutamyl-; l; -valyl-; l; -aspartic acid amide)Jurkat cells; Mono-Mac 6 cells; Dithiothreitol; Staurosporine; Tunicamycin; Apoptosis

Decreased polyphenol transport across cultured intestinal cells by a salivary proline-rich protein by Kuihua Cai; Ann E. Hagerman; Robert E. Minto; Anders Bennick (pp. 1570-1580).

Tannins are polyphenols commonly found in plant-derived foods. When ingested they can have various harmful effects, but salivary proline-rich proteins (PRPs) may provide protection against dietary tannins. The aim of this study was to investigate whether basic PRPs, a major family of salivary proteins, can prevent intestinal absorption of tannin. To do so it was necessary first to characterize transport of pentagalloyl glucose (5GG), a hydrolysable tannin, across cultured epithelial cells. Using human intestinal epithelial cells (Caco-2 cells) it was found that a partial degradation of 5GG occurred during transepithelial transport resulting in the presence of 5GG as well as tetra- and trigalloyl glucose and glucose in the receiving compartment. The sodium-dependent glucose transporter SGLT1 played a role in apical (mucosal) to basolateral (serosal) transport and transport in the opposite direction was dependent on the multidrug resistance-associated protein MRP2. An increased uptake from the apical compartment was seen when the basolateral receiving solution was human serum rather than a balanced salt solution. Transport both in apical–basolateral and basolateral–apical directions was reduced when 1B4, a human basic PRP, was added to the 5GG-containing medium. This decrease closely paralleled the formation of insoluble 5GG–1B4 complexes. It appears that the formation of insoluble tannin–protein complexes diminishes the uptake of 5GG and its metabolites. There is little evidence of other biological activities of basic PRPs so in contrast to other salivary proteins they may exert a biological function in the intestines.

Keywords: Abbreviations; PRP; proline-rich protein; 3GG; trigalloyl glucose; 4GG; tetragalloyl glucose; 5GG; pentagalloyl glucose; DMEM; Dulbecco's minimum modified eagle's medium; TFA; trifluoroacetic acid; DMF; dimethylformamide; HATU; O; -(7-azobenzotriazo-1-yl)-1,1,3,3-tetramethyl uranium hexafluorophosphate; HPLC; high performance liquid chromatography; TEER; transepithelial electric resistance; HBSS; Hank's balanced salt solution; HEPES; (; N; -2)hydroxyethylpiperazine-; N; ′-2-ethanesulfonic acid; SGLT-1; sodium glucose transporter-1; MRP 2; multidrug resistance-associated protein 2Salivary proline-rich protein; Hydrolysable tannin; Pentagalloyl glucose; Caco-2 cells; Intestinal absorption; Flavonoid

The transcription factor Cdx2 regulates the intestine-specific expression of human peptide transporter 1 through functional interaction with Sp1 by Jin Shimakura; Tomohiro Terada; Yutaka Shimada; Toshiya Katsura; Ken-ichi Inui (pp. 1581-1588).

H⁺/peptide cotransporter 1 (PEPT1, SLC15A1) localized at the brush-border membranes of intestinal epithelial cells plays important roles in the intestinal absorption of small peptides and a variety of peptidemimetic drugs. We previously demonstrated that transcription factor Sp1 functions as a basal transcriptional regulator of human PEPT1. However, the factor responsible for the intestine-specific expression of PEPT1 remains unknown. In the present study, we investigated the effect of the intestinal transcription factors on the transcription of the PEPT1 gene and found that only Cdx2 markedly trans-activated the PEPT1 promoter. However, the promoter region responsible for this effect lacked a typical Cdx2-binding sequence, but instead, possessed some Sp1-binding sites. In vitro experiments using Caco-2 cells showed that (1) mutation of the Sp1-binding site diminished the effect of Cdx2, (2) co-expression of Cdx2 and Sp1 synergistically trans-activated the PEPT1 promoter and (3) Sp1 protein was immunoprecipitated with Cdx2 protein. These results raise the possibility that Cdx2 modulates the PEPT1 promoter by interaction with Sp1. The significance of Cdx2 in vivo for PEPT1 regulation was shown by the determination of mRNA levels of Cdx2 and PEPT1 in human tissue. In gastric samples, some with intestinal metaplasia, the levels of PEPT1 and Cdx2 mRNA were highly correlated. Taken together, the present study suggests that Cdx2 plays a key role in the transcriptional regulation of the intestine-specific expression of PEPT1, possibly through interaction with Sp1.

Keywords: Abbreviations; Cdx2; caudal-related homeobox protein 2; ChIP; chromatin immunoprecipitation; GAPDH; glyceraldehydes-3-phosphate dehydrogenase; HNF; hepatocyte nuclear factor; PEPT1; H+/peptide cotransporter 1; TBS; Tris-buffered salinePEPT1; Cdx2; Sp1; Transporter; Caco-2; Intestinal metaplasia

Distribution of tocopheryl quinone in mitochondrial membranes and interference with ubiquinone-mediated electron transfer by Wolfgang Gregor; Katrin Staniek; Hans Nohl; Lars Gille (pp. 1589-1601).

α-Tocopherol (Toc) is an efficient lipophilic antioxidant present in all mammalian lipid membranes. This chromanol is metabolized by two different pathways: excessive dietary Toc is degraded in the liver by side chain oxidation, and Toc acting as antioxidant is partially degraded to α-tocopheryl quinone (TQ). The latter process and the similarity between TQ and ubiquinone (UQ) prompted us to study the distribution of TQ in rat liver mitochondrial membranes and the interference of TQ with the activity of mitochondrial and microsomal redox enzymes interacting with UQ. In view of the contradictory literature results regarding Toc, we determined the distribution of Toc, TQ, and UQ over inner and outer membranes of rat liver mitochondria. Irrespective of the preparation method, the TQ/Toc ratio tends to be higher in mitochondrial inner membranes than in outer membranes suggesting TQ formation by respiratory oxidative stress in vivo. The comparison of the catalytic activities using short-chain homologues of TQ and UQ showed decreasing selectivity in the order complex II (TQ activity not detected)>Q_o site of complex III>Q_i site of complex III>complex I∼cytochrome b₅ reductase>cytochrome P-450 reductase (comparable reactivity of UQ and TQ). TQ binding to some enzymes is comparable to UQ despite low activities. These data show that TQ arising from excessive oxidative degradation of Toc can potentially interfere with mitochondrial electron transfer. On the other hand, both microsomal and mitochondrial enzymes contribute to the reduction of TQ to tocopheryl hydroquinone, which has been suggested to play an antioxidative role itself.

Keywords: Abbreviations; BHT; 3,5-di-; tert; -butyl-4-hydroxy-toluol; Cox; cytochrome; c; oxidase; Cyt; cytochrome; DETAPAC; diethylene-triamino-pentaacetic acid; FAD; flavin adenine dinucleotide; FMN; flavin mononucleotide; HPLC; high performance liquid chromatography; IM; mitochondrial inner membrane; k; _cat,app; apparent turnover number; K; _M,app; apparent (bulk phase) Michaelis constant; MAO; monoamine oxidase; MP; mitoplasts; OM; mitochondrial outer membrane; RHM; rat heart mitochondria; RLM; rat liver mitochondria; S.D.; standard deviation; SMP; submitochondrial particles; Toc; α-tocopherol; TQ; α-tocopheryl quinone; UQ; ubiquinone; V; _max; maximal velocityMitochondria; α-Tocopherol; α-Tocopheryl quinone; Ubiquinone; Respiratory complexes

Long term environmental tobacco smoke activates nuclear transcription factor-kappa B, activator protein-1, and stress responsive kinases in mouse brain by Sunil K. Manna; Thirumalai Rangasamy; Kimberly Wise; Shubhashish Sarkar; Shishir Shishodia; Shyam Biswal; Govindarajan T. Ramesh (pp. 1602-1609).

Environmental tobacco smoke (ETS) is a key mediator of several diseases. Tobacco smoke contains a mixture of over 4700 chemical components many of which are toxic and have been implicated in the etiology of oxidative stress related diseases such as chronic obstructive pulmonary disease, Parkinson's disease, asthma, cancer and cardiovascular disease. However, the mechanism of action of cigarette smoke in the onset of these diseases is still largely unknown. Previous studies have revealed that the free radicals generated by cigarette smoke may contribute to many of these chronic health problems and this study sought to address the role of environmental tobacco smoke in oxidative stress related damage in different regions of the mouse brain. In this study, male mice were exposed for 7h/day, 7 days/week, for 6 months. Our results show that tobacco smoke led to increased generation of reactive oxygen species with an increase in NF-κB activation. Gel shift analysis also revealed the elevated level of the oxidative stress sensitive proinflammatory nuclear transcription factor-kappa B and activator protein-1 in different regions of the brain of cigarette smoke exposed mice. Tobacco smoke led to activation of COX-2 in all the regions of the brain. Activation of mitogen activated protein kinase and c-Jun N-terminal kinase were also observed in various regions of brain of ETS exposed mice. Overall our results indicate that exposure to long-term cigarette smoke induces oxidative stress leading to activation of stress induced kinases and activation of proinflammatory transcription factors.

Keywords: Cigarette smoke; ROS; NF-κB; AP-1; Signal transduction

Glyoxal markedly compromises hepatocyte resistance to hydrogen peroxide by Nandita Shangari; Tom S. Chan; Marija Popovic; Peter J. O’Brien (pp. 1610-1618).

Glyoxal is an interesting endogenous α-oxoaldehyde as it originates from pathways that have been linked to various pathologies, including lipid peroxidation, DNA oxidation and glucose autoxidation. In our previous study we showed that the LD₅₀ of glyoxal towards isolated rat hepatocytes was 5mM. However, 10μM glyoxal was sufficient to overcome hepatocyte resistance to H₂O₂-mediated cytotoxicity. Hepatocyte GSH oxidation, NADPH oxidation, reactive oxygen species formation, DNA oxidation, protein carbonylation and loss of mitochondrial potential were also markedly increased before cytotoxicity ensued. Cytotoxicity was prevented by glyoxal traps, the ferric chelator, desferoxamine, and antioxidants such as quercetin and propyl gallate.These results suggest there is a powerful relationship between H₂O₂-induced oxidative stress and glyoxal which involves an inhibition of the NADPH supply by glyoxal resulting in cytotoxicity caused by H₂O₂-induced mitochondrial oxidative stress.

Keywords: Abbreviations; AGEs; advanced glycation end-products; DCFH-DA; 2′,7′-dichlorofluoroscein diacetate; DNFB; 2′,4′-dinitrofluorobenzene; DNPH; dinitrophenylhydrazine; G6PDH; glucose-6-phosphate dehydrogenase; H; ₂; O; ₂; hydrogen peroxide; ICDH; isocitrate dehydrogenase; ROS; reactive oxygen speciesα-Oxoaldehydes; Glyoxal; Oxidative stress; Cytotoxicity; Carbonylation; Reactive oxygen species; Hydrogen peroxide

Several glutathione S-transferase isozymes that protect against oxidative injury are expressed in human liver mitochondria by Evan P. Gallagher; James L. Gardner; David S. Barber (pp. 1619-1628).

The mitochondrial environment is rich in reactive oxygen species (ROS) that may ultimately peroxidize membrane proteins and generate unsaturated aldehydes such as 4-hydroxy-2-nonenal (4HNE). We had previously demonstrated the presence of hGSTA4-4, an efficient catalyst of 4HNE detoxification, in human liver mitochondria to the exclusion of the cytosol. In the present study, GSH-affinity chromatography was used in conjunction with biochemical and proteomic analysis to determine the presence of additional cytosolic glutathione S-transferases (GSTs) in human hepatic mitochondria. HPLC-subunit analysis of GSH affinity-purified liver mitochondrial proteins indicated the presence of several potential mitochondrial GST isoforms. Electrospray ionization-mass spectrometry analysis of eluted mitochondrial GST subunits yielded molecular masses similar to those of hGSTP1, hGSTA1 and hGSTA2. Octagonal matrix-assisted laser desorption/ionization time of flight mass spectrometry and proteomics analysis using MS-FIT confirmed the presence of these three GST subunits in mitochondria, and HPLC analysis indicated that the relative contents of the mitochondrial GST subunits were hGSTA1>hGSTA2>hGSTP1. The mitochondrial localization of the alpha and pi class GST subunits was consistent with immunoblotting analysis of purified mitochondrial GST. Enzymatic studies using GSH-purified mitochondrial GST fractions demonstrated the presence of significant GST activity using the nonspecific GST substrate 1-chloro-2,4-dinitrobenzene (CDNB), as well as 4HNE, δ⁵-androstene-3,17-dione (ADI), and cumene hydroperoxide (CuOOH). Interestingly, the specific mitochondrial GST activities toward 4HNE, a highly toxic α,β-unsaturated aldehyde produced during the breakdown of membrane lipids, exceeded that observed in liver cytosol. These observations are suggestive of a role of GST in protecting against mitochondrial injury during the secondary phase of oxidative stress, or modulation of 4HNE-mediated mitochondrial signaling pathways. However, other properties of mitochondrial GST, such as conjugation of environmental chemicals and binding of lipophilic non-substrate xenobiotics and endogenous compounds, remain to be investigated.

Keywords: Abbreviations; 4HNE; 4-hydroxynonenal; δ; ⁵; -ADI; δ; ⁵; -androstene-3,17-dione; CDNB; 1-chloro-2,4-dinitrobenzene; CuOOH; cumene hydroperoxide; DCNB; 1,2-dichloro-4-nitrobenzene; DTT; dithiothrietol; ECA; ethacrynic acid; ECL; enhanced chemiluminescence; EDTA; ethylenediaminetetraacetic acid; ESI-MS; electrospray ionization-mass spectrometry; GPX; glutathione peroxidase; GSH; glutathione; GST; glutathione; S; -transferase; HEPES; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HPLC; high performance liquid chromatography; LDH; lactate dehydrogenase; NADPH; nicotinamide adenine dinucleotidephosphate, reduced; NBC; nitrobutyl chloride; oMALDI; octagonal matrix-assisted laser desorption/ionization; oMALDITOF; octagonal matrix-assisted laser desorption/ionization time of flight mass spectrometry; PBS; phosphate buffered saline; TFA; trifluoroacetic acidGlutathione; Glutathione; S; -transferase; Mitochondria; Human liver

Chemical modification at subunit 1 of rat kidney Alpha class glutathione transferase with 2,3,5,6-tetrachloro-1,4-benzoquinone: Close structural connectivity between glutathione conjugation activity and non-substrate ligand binding by Siobhan M. O'Sullivan; Ronan M. McCarthy; Melissa A. Vargo; Roberta F. Colman; David Sheehan (pp. 1629-1636).

2, 3, 5, 6-Tetrachloro-1, 4-benzoquinone (TCBQ) is a metabolite of pentachlorophenol known to react with cysteines of glutathione transferases (GSTs). TCBQ treatment of rat kidney rGSTA1-2 and rGSTA1-1 abolishes 70–80% conjugation of glutathione (GSH) to 1-chloro-2, 4-dinitrobenzene and results in strongly correlated quenching of intrinsic fluorescence of Trp-20 ( R>0.96). rGSTA2-2 is only inhibited by 25%. Approximately 70% (rGSTA1-1) and 60% (rGSTA1-2) conjugation activity is abolished at TCBQ: GST stoichiometries near 1:1. The inactivation follows a Kitz/Wilson model with K_D of 4.77±2.5μM for TCBQ and k₃ for inactivation of 0.036±0.01min⁻¹. A single tryptic peptide labelled with TCBQ was isolated from kidney rGSTA1-2 containing Cys-17 which we identify as the site of modification. Treatment with more than stoichiometric amounts of TCBQ modified other residues but resulted in only modest further inhibition of catalysis. We interpret these findings in terms of localised steric effects on the relatively rigid α-helix 1 adjacent to the catalytic site of subunit 1 possibly affecting the Alpha class-specific α-helix 9 which acts as a “lid�? on the hydrophobic part of the active site. Homology modelling of rGSTA1-1 modified at Cys-17 of one subunit revealed only modest structural perturbations in the second subunit and tends to exclude global structural effects.

Keywords: Abbreviations; CDNB; 1-chloro-2,4-dinitrobenzene; DNPG; dinitrophenyl glutathione; GSH; reduced glutathione; GST; glutathione transferase; HNB; 2-hydroxyl-5-nitrobenzyl alcohol; MALDI-TOF; matrix-assisted laser desorption ionisation mass spectrometry with time-of-flight detection; NEM; N; -ethyl maleimide; TCBQ; 2,3,5,6-tetrachloro-1,4-benzoquinoneChemical modification; Cysteine; Glutathione transferase; Alpha class; Affinity label; Structure; Tetrachlorobenzoquinone

AMP-activated protein kinase (AMPK) activating agents cause dephosphorylation of Akt and glycogen synthase kinase-3 by Taj D. King; Ling Song; Richard S. Jope (pp. 1637-1647).

AMP-activated protein kinase (AMPK) is a key cellular sensor of reduced energy supply that is activated by increases in the cellular ratio of AMP/ATP. Phenformin and 5-aminoimidazole-4-carboxamide riboside (AICAR) are two drugs widely used to activate AMPK experimentally. In both differentiated hippocampal neurons and neuroblastoma SH-SY5Y cells we found that these two agents not only activated AMPK, but conversely greatly reduced the activating Ser/Thr phosphorylation of Akt. This blockade of Akt activity consequently lowered the inhibitory serine-phosphorylation of its substrates, glycogen synthase kinase-3α/β (GSK3α/β). An inhibitor of AMPK (Compound C) did not block dephosphorylation of Akt and GSK3. Thus, both drugs widely used to activate AMPK also caused dephosphorylation of Akt and of GSK3. The mechanism for Akt dephosphorylation caused by phenformin, but not AICAR, was due to inhibition of growth factor-induced signaling that leads to Akt phosphorylation. Stimulation of muscarinic receptors with carbachol in SH-SY5Y cells also activated AMPK and transiently caused dephosphorylation of Akt. These findings show that Akt dephosphorylation often occurs concomitantly with AMPK activation when cells are treated with phenformin or AICAR, indicating that these drugs do not only affect AMPK but also cause a coordinated inverse regulation of AMPK and Akt.

Keywords: Akt; Protein kinase B; AMPK; AMP-activated kinase; GSK3; AICAR; Phenformin

Evaluation of dichloroacetate treatment in a murine model of hereditary tyrosinemia type 1 by Chantale Langlois; Rossana Jorquera; Milton Finegold; Albert L. Shroads; Peter W. Stacpoole; Robert M. Tanguay (pp. 1648-1661).

Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disease severely affecting liver and kidney and is caused by a deficiency in fumarylacetoacetate hydrolase (FAH). Administration of 2-(2-nitro-4-trifluoro-methylbenzyol)-1,3 cyclohexanedione (NTBC) improves the HT1 phenotype but some patients do not respond to NTBC therapy. The objective of the present study was to evaluate whether administration of dichloroacetate, an inhibitor of maleyl acetoacetate isomerase (MAAI) to FAH-knockout mice could prevent acute pathological injury caused by NTBC withdrawal. DCA (0.5 and 5g/L) was given in combination with a standard diet or with a tyrosine-restricted diet. With the low-tyrosine diet body weight loss and most of hepatic and renal injuries were prevented regardless the DCA dose. The administration of DCA with a standard diet did not prevent damage nor the oxidative stress response nor the AFP induction seen in FAH-knockout mice. DCA was shown to inhibit hepatic MAAI activity to 86% (0.5g/L) and 94% (5g/L) of untreated wild-type mice. Interestingly, FAH^−/− mice deprived of NTBC (NTBC-OFF) and NTBC-treated FAH-knockout mice had similar low hepatic MAAI activity levels, corresponding to 10–20% of control. Thus the failure of DCA treatment in FAH^−/− mice seems to be attributed to the residual MAAI activity, high enough to lead to FAA accumulation and HT1 phenotype.

Keywords: Abbreviations; HTI; hereditary tyrosinemia type 1; DCA; dichloroacetate; MAAI; maleylacetoacetate isomerase; FAH; fumarylacetoacetate hydrolase; NTBC; 2-(2-nitro-4-trifluoro-methylbenzoyl)-1,3-cyclohexanedione; MAA; maleylacetoacetate; FAA; fumarylacetoacetate; MA; maleylacetone; FA; fumarylacetone; SAA; succinylacetoacetate; SA; succinylacetone; δ-ALAD; δ-aminolevulinic acid dehydratase; δ-ALA; δ-aminolevulinic acid; GSH; reduced glutathione; GSSG; oxidized glutathione; HPPD; 4-hydroxyphenylpyruvate dioxygenase; AP; alkaline phosphatase; GGT; γ-glutamyl-transferase; GC–MS; gas chromatography–mass spectrometry; γ-GCS; gamma-glutamyl cysteine synthetase; MAT; methionine adenosyl transferase; NMO; NAD(P)H:quinone oxidoreductase 1; HO-1; heme oxygenase 1; DTNB; 5,5′-dithiobis (2-nitrobenzoic acid); NADPH; nicotinamide adenine dinucleotide phosphate (reduced form); HGA; homogentisateHereditary tyrosinemia type 1; Fumarylacetoacetate hydrolase; Dichloroacetate; Maleylacetoacetate isomerase; NTBC

Corrigendum to “Niacin induces PPARγ expression and transcriptional activation in macrophages via HM74 and HM74a-mediated induction of prostaglandin synthesis pathways�? [Biochem. Pharmacol. 71 (5) (2006) 646–656] (pp. 1662-1662).

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Biochemical Pharmacology (v.71, #11)