Cell proliferation assays demonstrate that neoamphimedine can inhibit Metnase-enhanced cell growth with an IC50 of 0.5 μM. Additionally, we find that the apparent Km of TopoIIα for ATP increases linearly with higher concentrations of neoamphimedine, indicating ATP-competitive inhibition, which is substantiated by molecular modeling. These findings support the continued development of neoamphimedine as an anticancer agent, particularly in solid tumors that over-express Metnase.Type IIα DNA topoisomerase (TopoIIα) is among the most important clinical drug targets for the treatment of cancer. Recently, the DNA repair protein Metnase was shown to enhance TopoIIα activity and increase resistance to TopoIIα poisons. Using  Type IIα DNA topoisomerase (TopoIIα) plays a critical role in cell cycle progression by relieving various types of topological strain induced in DNA double strands during replication, transcription, and chromosome segregation, making it a crucial target for chemotherapy in several cancers –3. The cXestospongia sp. While amphimedine is biologically inactive as an antitumor agent, neoamphimedine is known to inhibit TopoIIα and has been shown to be as effective as etoposide at inhibiting the growth of xenograft tumors in mice [in vitro [Neoamphimedine and amphimedine  are mari in mice . Though in vitro ,10.in vitro. TopoIIα-dependent ATP hydrolysis inhibition assays suggest that neoamphimedine is an ATP-competitive inhibitor and in silico molecular modeling data supports this indicating that neoamphimedine can bind to the ATPase sites of TopoIIα.In the present study we performed TopoIIα-dependent DNA decatenation and cellular proliferation assays with and without neoamphimedine and Metnase to demonstrate that neoamphimedine is able to inhibit Metnase-enhanced TopoIIα activity in vitro; however, the ability of neoamphimedine to inhibit TopoIIα mediated decatenation has not been assessed. In addition, Metnase has been shown to resist the action of TopoIIα poisons by interacting with TopoIIα and enhancing its decatenation activity in vitro. Thus, we conducted TopoIIα decatenation assays with neoamphimedine in the presence and absence of an activity-enhancing concentration of Metnase  in the presence of neoamphimedine and etoposide . HEK293-N-terminal ATPase site [N-terminal ATPase sites of TopoIIα and that neoamphimedine binds to this site, circumventing Metnase-mediated MDR [m of TopoIIα for ATP to be 390 ± 50 μM and Vmax of the catalysis to be 0.0087 ± 0.0004 pmol min−1·U−1, consistent with previously reported data [m increased linearly (R2 = 0.94) while Vmax did not seem to be dose-responsive, indicative of a competitive mode of inhibition  reveals why the position of the carbonyl group is so important  efflux pump [MDR is a major problem associated with existing clinically used TopoIIα inhibitors . The moslux pump ,19. Anotlux pump ,19,20. Llux pump ,7,21. Thin vitro as an inhibitor of TopoIIα and in vivo as an antitumor agent [50 of 2 μM and a corresponding Ki of 0.8 ± 0.3 μM. Indicative of a competitive mode of inhibition, a linear increase in the apparent Km of TopoIIα for ATP is observed with increasing neoamphimedine concentrations  in various human tumor cell lines [in vitro decatenation assays, including doxorubicin, etoposide, and ICRF-193, suggesting that neoamphimedine could overcome Metnase-dependent MDR.Neoamphimedine inhibits TopoIIα-dependent decatenation at low concentrations with 90% inhibition occurring at 30 μM . These din vitro decatenation assays  in the presence or absence of purified recombinant Metnase (200 ng) and various concentrations of neoamphimedine. Reaction mixtures were incubated at 39 °C for an optimized time of 30 min. The reactions were then separated on a 1% agarose gel containing 0.5 μg/mL ethidium bromide with decatenated and catenated DNA markers (TopoGen). Fluorescence intensity quantitative analysis was carried out using Quantity One® software [TopoIIα (TopoGen) was added to 70 ng of kinetoplast DNA  comprising small interlocked supercoiled circular DNA in reaction buffer  were described previously . A color®, Perkin Elmer).Briefly, cells were cultured in DMEM  supplemented with 10% FBS , seeded at 2000 cells/100 μL/well in 96-well plates and incubated for 24 h. Medium was then replaced with medium containing various concentrations of neoamphimedine or etoposide. After 72 h treatment, cells were fixed with 10% aqueous trichloroacetic acid for 1 h at 4 °C, and washed 5× with 200 μL water. Dry fixed cells were stained with 0.4% sulforhodamine B in 1% aqueous acetic acid for 30 min, and washed 4× with 200 μL of 1% aqueous acetic acid. Dry stained cells were treated with 10 mM unbuffered Tris-base to extract protein-bound dye and absorbance readings were measured at 535 nm using a microplate reader  in reaction buffer (without DNA and DTT) was added to the complete reaction buffer and pre-incubated for 10 min at 37 °C. Reactions were initiated by the addition of 600 μM ATP and incubated for 30 min at 37 °C. Reactions were terminated and measured as described. The IC50 value was determined and the Ki was calculated using the Cheng-Prusoff equation [® software [m and Vmax values) or the dose-response inhibition model .An ATP hydrolysis assay that quantifies inorganic phosphate through the formation of a high molar absorptivity complex with malachite green and molybdate was adapted for use in 384-well plates, with a total reaction volume of 15 μL . We firssoftware . To deteequation . Experimsoftware  was usedThe 1.87 Å crystal structure of the ATPase domain of human TopoIIα  was appin vitro, cell based, and in silico studies presented characterize neoamphimedine as an ATP-competitive inhibitor of TopoIIα. These studies show that neoamphimedine can overcome drug resistance observed with conventional TopoIIα poisons due to TopoIIα-Metnase protein interactions. Molecular modeling has elucidated the pharmacophore of neoamphimedine, most notably the position of the E ring carbonyl and the resulting hydrogen bond with Ser148. Finally, it is reasonable to conclude that Metnase likely binds TopoIIα near the DNA binding site located in the cleavage and relegation domain, which prevents or alters TopoIIα poison binding but does not block access to the N-terminal ATPase binding sites of TopoIIα, which is logical given that TopoIIα must hydrolyze ATP during catalytic function [In conclusion, the function . These r


Salmonella enterica serovar Typhimurium  infection of chickens that are more than a few days old results in asymptomatic cecal colonization with persistent shedding of bacteria. We hypothesized that while the bacterium colonizes and persists locally in the cecum it has systemic effects, including changes to metabolic pathways of skeletal muscle, influencing the physiology of the avian host. Using species-specific peptide arrays to perform kinome analysis on metabolic signaling pathways in skeletal muscle of Salmonella Typhimurium infected chickens, we have observed key metabolic changes that affected fatty acid and glucose metabolism through the 5'-adenosine monophosphate-activated protein kinase (AMPK) and the insulin/mammalian target of rapamycin (mTOR) signaling pathway. Over a three week time course of infection, we observed changes in the phosphorylation state of the AMPK protein, and proteins up and down the pathway. In addition, changes to a large subset of the protein intermediates of the insulin/mTOR pathway in the skeletal muscle were altered by infection. These changes occur in pathways with direct effects on fatty acid and glucose metabolism. This is the first report of significant cellular metabolic changes occurring systemically in chicken due to a Salmonella infection. These results have implications not only for animal production and health but also for the understanding of how Salmonella infection in the intestine can have widespread, systemic effects on the metabolism of chickens without disease-like symptoms. Salmonella are enteric bacteria, and salmonellosis is a major infectious disease around the world. A number of Salmonella serovars are responsible for human disease with symptoms ranging from gastroenteritis to sepsis [Salmonella are important zoonotic agents that infect a wide variety of animal species. Salmonella enterica serovar Typhimurium  is a rod-shaped, flagellated, aerobic, Gram-negative bacterium. Salmonella Typhimurium is infectious to humans and can cause severe gastro-intestinal pathology and typhoid fever.o sepsis . SalmoneSalmonella Typhimurium infection of day-old chickens can result in severe inflammatory responses and intestinal pathology [Salmonella Typhimurium infection in older chickens does not cause overt pathogenic symptoms [Salmonella Typhimurium may be due to a lack of a pro-inflammatory cytokine response and a release of TGF-β4 in these older birds [athology ; howeversymptoms . The lacPeptide arrays for kinome analysis have been utilized by a number of groups to study cellular signaling, ranging from the study of cancer  to bacteThe fact that alterations to the intestinal environment affect animal growth has been known for many years. Growth-promoting antibiotics are thought to carry out their function through reduced local inflammation, reduced intestinal size and, most importantly, reduced competition for nutrients in the small intestine . StudiesThe 5'-adenosine monophosphate-activated protein kinase (AMPK) enzyme is a key metabolic energy homeostasis regulator in avian species . One of The insulin signaling pathway and mammalian target of rapamycin (mTOR) signaling are extensively linked and display significant overlap, so much so that it is referred to as the insulin/mTOR signaling pathway . mTOR siSalmonella Typhimurium infection. We hypothesized that while the bacterium colonizes and persists locally in the cecum, it has systemic effects influencing the metabolism of skeletal muscle. While this may not be a direct immunological effect, a disruption in metabolism and energy resources can have a profound effect on the health of the host. Our results clearly point to alterations in AMPK phosphorylation and activity as well as significant disruptions in the insulin/mTOR signaling pathway involving numerous pathway intermediates. We subsequently confirmed these results through the use of the complementary technique of antibody microarray analysis.In this study, we used a chicken-specific global metabolic peptide array to study the changes in skeletal muscle metabolism over time following These studies were approved by the Animal Care and Use Committee (ACUC) at the Southern Plains Agricultural Research Center, Agricultural Research Service, United States Department of Agriculture (ACUC #2012007), which meets all federal requirements as defined in the Animal Welfare Act, the Public Health Service Policy, and the Humane Care and Use of Laboratory Animals.5 cfu of Salmonella Typhimurium that had been grown in tryptic soy broth with 20 ug/mL novobiocin and 25 ug/mL nalidixic acid. Control and infected animals were kept in separate isolation rooms, and cross-contamination was strictly controlled for. At 24 h, 96 h, 1 week and 3 weeks post infection, animals were sacrificed and samples were collected.Fifty 17-day-old broiler eggs, which had not been vaccinated or otherwise treated, were procured from Sanderson Farms . These eggs were incubated at 37.2°C and 92% humidity for 3 days before hatching. Forty of 50 eggs hatched after 3 days. Five days post-hatch, half were orally infected with 1 × 102 asphyxiation and muscle samples were collected. Muscle samples from the thigh were removed and immediately flash frozen in liquid nitrogen to preserve kinase enzymatic activity. Samples were taken from liquid nitrogen and transferred to a −80°C freezer until further experimental procedures were conducted.At 24 h, 96 h, 1 week and 3 weeks post infection, birds were sacrificed by COAt each of the 4 time points and under each condition (infected and uninfected), 3 muscle samples from 3 different animals were taken from storage for analysis . Muscle samples were weighed to obtain a consistent 40 mg samples for the array protocol. Samples were homogenized by a hand-held Qiagen TissueRuptor  in 100 uL of lysis buffer , 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM Na3VO4, 1 mM NaF, 1 μg/mL leupeptin, 1 g/mL aprotinin and 1 mM Phenylmethylsulphonyl fluoride , unless indicated)). Following homogenization, the peptide array protocol was carried out as per Jalal et al. .The mTOR Signaling Phospho-Specific Antibody Array and antibody array assay kit were procured from Full Moon BioSystems . This technique was used as an alternative to procuring phosphospecific antibodies individually and performing several western blot assays. Protocol was carried out as per manufacturer’s instructions with the following alteration to the homogenization step: instead of using the bead and vortex homogenization indicated in the kit, the hand-held Qiagen TissueRuptor was used.Data normalization and PCA analysis was performed for both the peptide and antibody microarrays as per Li et al. . This co5 cfu of Salmonella Typhimurium. Control and infected animals were kept in separate isolation rooms, and cross-contamination was strictly controlled for. At 24 h, 96 h, 1 week and 3 weeks post infection, animals were sacrificed and samples collected. Infection status was confirmed by Salmonella Typhimurium culturing of cecal contents and feces from each animal with and without enrichment. Cultures confirmed that the infected group displayed Salmonella infection throughout the experiment, while Salmonella was not isolated from animals in the control group. Samples were snap-frozen in liquid nitrogen as quickly as possible to preserve enzymatic activity.Non-vaccinated, non-treated broiler eggs were procured and hatched. Five days post-hatch, half the chicks were orally infected with 1 × 10Chicken-specific peptide arrays designed for the study of chicken metabolic signaling pathways were used to analyze 24 thigh muscle samples. The results from the three animals from each condition (infected and uninfected) and time point were averaged together to provide a representative result  for each time point were input into the Search Tool for the Retrieval of Interacting Genes (STRING) database [To account for any changes in phosphorylation state that were not due to the infection, the results at each time point were corrected using their respective time-matched controls. All peptides that showed statistically significant differential phosphorylation  corrected)  Table . Of partp-value 5.77 × 10-15), adenyl nucleotide binding  and ATP binding . At 96 h, the top two results were adenyl ribonucleotide binding and adenyl nucleotide binding . At 1 week, one of the top results was ATP binding . At 3 weeks, the top result was AMP-activated protein kinase activity , and the fourth and fifth results were adenyl ribonucleotide binding  and adenyl nucleotide binding  respectively. These results strongly implicated a central role for signaling events associated with the ATP, ADP and/or AMP levels, all of which play a role as energy currency in cells.Using STRING GO analysis, the Molecular Function results generated for each time point were very similar. The most significant results related to adenyl phosphate moiety binding. Note that all the following results were FDR corrected. At 24 h, the top three results were adenyl ribonucleotide binding  over control at the 24 h time point. Also, AMPK is known to inhibit protein synthesis through phosphorylation-mediated inhibition of eukaryotic elongation factor-2 kinase (EF2K) at S398 but not S78 or S366 [p 0.017) at the 24 h time point. This was the same time point that showed the greatest AMPKα phosphorylation. The EF2K S78 peptide target was also present on the peptide array, but since this site phosphorylation is not affected by AMPK in vivo [Other phosphorylation events provided further evidence for the early activation of AMPK. Calcium/calmodulin-dependent protein kinase II (CaMK2) is known to affect AMPK phosphorylation . Results or S366 . Peptide in vivo  it showeThe later time points showed a reduced phosphorylation of AMPKα. One of the effects of reduced AMPK activity is a decrease in fatty acid oxidation and an increase in fatty acid and cholesterol synthesis . We saw p 0.042) increase in phosphorylation of S1255 of ACC1 at the 24 h time point. This change indicates early inhibition of fatty acid synthesis. At later time points, this phosphorylation site showed no significant phosphorylation indicating no inhibition of this enzyme and the potential for fatty acid synthesis. PPAR gamma co-factor 1 (PGC-1) is an enzyme which is present in adipose cells and skeletal muscle, and it coordinates fatty acid catabolism and thermogenesis [p-value 0.03). With the elimination of PGC-1 activity, the signal to initiate fatty acid catabolism would be lost. Both the loss of PGC-1 and the loss of inhibition ACC1 lead to increased fatty acid stores in the muscle. The above results can all proceed from a change in AMPK activity over time brought about by the Salmonella Typhimurium infection. The result of this infection-induced AMPK change may be a disruption of the normal metabolic functions within the muscle of the birds.Acetyl-coenzyme A carboxylase 1 (ACC1) is an enzyme which is involved in long chain fatty acid synthesis. ACC1 activity is inhibited by phosphorylation at two sites: S80 (S79 in mice), which was not present on the peptide array, and S1263, ortholog of S1255 in chicken [ogenesis . At the Salmonella Typhimurium infection over time: mitogen-activated protein kinase (MAPK) signaling and phosphoinositide 3-kinase (PI3K)/Akt  signaling. The majority of the peptides that had significant changes over multiple time points are part of the MAPK branch. Raf, mitogen-activated protein kinase kinase kinase 3 (MEKK3), mitogen-activated protein kinase kinase 4 (MKK4), mitogen-activated protein kinase kinase 6 (MKK6) and TAB1 are all part of MAPK-related signaling. In the PI3K branch at the 3 week time point, the phosphoinositide 3-kinase regulatory subunit 1 (PIK3R1) is activated by the phosphorylation ofY528 [Based on the pathway and GO results described previously insulin signaling appeared to be affected by the infection. The KEGG insulin pathway showed a statistically significant change due to the infection at all time points Table , and theThe function of insulin and its signaling pathway is intimately related to glucose metabolism. Indeed, one of the major functions of insulin is to stimulate the uptake of glucose by cells. Again considering the peptides that display significant differential phosphorylation at multiple time points, we see several peptides involved in glucose metabolism with a consistent change in response Figure . EnolaseSalmonella Typhimurium infection has a number of effects on the insulin/mTOR pathway. Effects can be seen at various branch points including MAPK signaling, PI3K signaling and glucose related signaling. Considering the specific peptides described which showed differential phosphorylation over the course of the study, it is clear why insulin, mTOR, glycolysis/gluconeogesis and MAPK pathways were implicated by the KEGG pathway analysis generated by the STRING database  is shown in Table p value 9.50 × 10-10) and mTOR signaling pathway . GO biological processes indicated insulin receptor signaling pathway , and GO molecular function showed ATP binding . All of these results were in agreement with the peptide array data. In addition, specific results such as the dephosphorylation of AMPK, activation of insulin receptor, activation of Akt (S374) and shutting down of the mTOR pathway  are in agreement with peptide array results.Analysis of the antibody array data via the STRING data based showed that the top two KEGG pathways were insulin signaling pathway  and a decrease in glycolysis. As can be observed in the phosphorylation states of several glucose related proteins Figure  and the d Figure . The numSalmonella induced muscle metabolism changes. The peptide array data at the later time points showed that the PI3K/Akt pathway appears active from the level of the receptor to Akt; however, mTOR and proteins downstream appear inactive. The phosphorylation of proline-rich AKT1 substrate 1 (PRAS40)  by Akt2 at T246, shown on the antibody array, provides further evidence for active insulin related signaling. PRAS40/AKT1S1 is an inhibitor of mTOR, but phosphorylation at T246 inhibits PRAS40/AKT1S1 inhibition of mTOR [Salmonella Typhimurium have gone systemic and are altering metabolic signaling in the peripheral muscle.Results from the antibody microarray provide a further characterization of  of mTOR . As indi of mTOR . These d of mTOR , all resSalmonella, though dangerous from a food safety perspective, was not a detriment to the health and growth of commercial chickens [Salmonella Typhimurium infection may alter muscle metabolism:It had been assumed that an infection with chickens . However1. A transient response to early infection may activate stress responses, including the mobilization of energy by AMPK. Following the establishment of infection, either the stress response may cease or AMPK may become desensitized to the signal resulting in the inverse response of fatty acid synthesis. This desensitization of AMPK has been observed previously in response to repeated exercise .Salmonella would almost certainly disrupt the normal population balance of the gut and alter these signals [Salmonella infection of the chicken gut must be exerting effects on the other bacteria inhabiting the system. Salmonella infection has been shown previously to cause changes in the microbial ecology of the gastrointestinal tract of mice [Salmonella infection does not result in obvious negative consequences to an avian host, it may disrupt natural beneficial bacteria. It is possible that these are the same or similar beneficial bacteria that thrive following animal treatment with growth enhancing antibiotics. Following antibiotic treatment of food animals, gut microbiota populations are disrupted and those bacteria beneficial to growth are able to thrive [Salmonella out-competing the beneficial bacteria. The infection at later time points causes an increase in fatty acid synthesis, a negative consequence in terms of meat-animal production. For many years, there has been evidence of an effect on chicken adiposity due to infections [2. The normal gut microbiota provides a large, complex assortment of signals, metabolites and stimulations to the host. An infection of  signals . Ample e signals . A persi of mice . A previ of mice . Even tho thrive . In our fections .Salmonella Typhimurium infection has been shown to cause large scale changes in host hormone metabolism [Salmonella Typhimurium showed changes in a variety of metabolites including those involved in fatty acid and glucose metabolism. In addition, it was found that significant changes in eicosanoid metabolism intermediates occurred due to Salmonella Typhimurium. Eicosanoids are fatty acid derived hormones which can have systemic effects. It is possible that the muscle metabolism changes we have observed here are a result of changes in hormones, specifically eicosanoids, brought about by the intestinal infection of the host. cPLA2 phosphorylation is evidence of eicosanoid hormone influence. cPLA2 is an enzyme that catalyzes an early step in the biosynthesis of eiconsaoid hormones including the prostaglandins [3. Another possible cause of systemic changes in the host is changes in hormones and/or hormone metabolism. tabolism . Metabolglandins . We see Salmonella infection, either through altered gut microbiota populations or altered gut absorption, alters these neuronal signals, it could affect metabolism in the entire body. A lack of nutrient absorption could signal the body to engage energy stores in the muscle, adipose cells or the liver, for example. Any or all of the potential Salmonella effects on the gut described above could affect skeletal muscle metabolism in the ways observed in this study.4. There are numerous nervous system connections between the brain and gut. These connections can influence metabolism throughout the body, including glucose metabolism . If a SaSalmonella Typhimurium infection, and that these alterations change over time. Early activation of AMPK indicates an energy-producing catabolic response, while late AMPK deactivation would lead to an anabolic response which appears biased toward fatty acid metabolism. The disregulation of the mTOR/insulin signaling pathway points to altered glucose metabolism throughout the time course of the study, with a strong inhibition of glycogenesis at the later time points. This study also adds to the growing body of evidence pointing to the importance of gut bacterial populations in systemic host functions.In summary, this study shows that skeletal muscle metabolism is altered following Salmonella enterica serovar Typhimurium; AMPK: 5'-adenosine monophosphate-activated protein kinase; mTOR: mammalian Target of rapamycin; ACUC: Animal Care and Use Committee; GO: Geneontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; STRING: Search Tool for the Retrieval of Interacting Genes; FDR: False discovery rate; CaMK2: Calcium/calmodulin-dependent protein kinase II; EF2K: Eukaryotic elongation factor-2 kinase; cPLA2: cytosolic phospholipase A2; CPT1A: carnitine Palmitoyltransferase 1A; ACC1: Acetyl-coenzyme A carboxylase 1; PGC-1: PPAR gamma co-factor 1; MAPK: Mitogen-activated protein kinase; PI3K: Phosphoinositide 3-kinase/Akt; MEKK3: Mitogen-activated protein kinase kinase kinase 3; MKK4: Mitogen-activated protein kinase kinase 4; MKK6: Mitogen-activated protein kinase kinase 6; PIK3R1: Phosphoinositide 3-kinase regulatory subunit 1; ENO1: Enolase 1; PFKM: 6-phosphofructokinase, muscle type; PYGL: glycogen phosphorylase; PGM2L1: Phosphoglucomutase 2-like 1; PRAS40 or AKT1S1: AKT1 substrate 1; PTEN: Phosphatase and tensin homolog; FOXO1: Forkhead box protein O1.Salmonella Typhimurium: The authors declare that they have no competing interests.MK and RA conceived the experiment and wrote the manuscript. RA carried out the experiment and analyzed the data. SN designed the peptide array. All authors read and approved the final manuscript.Peptide array results. Complete peptide array results for all 300 peptides on the array and all 4 time points. Results represent the average of 3 separate animals combined to produce a representative result. Protein and Target Amino Acid show the protein name represented by the peptide and phosphorylated amino acid residue printed onto the array. Human Accession number indicates the human equivalent protein. Fold change is the relative phosphorylation compared to the time-matched control. P-value is the statistical significance of the differential phosphorylation.Click here for file


Inhibition kinetics showed non competitive inhibition of the WT and competitive inhibition of the C-less. The presence of carnitine or acylcarnitines during the incubation of the proteoliposomes with omeprazole increased the inhibition. Using site-directed Cys mutants it was demonstrated that C283 and C136 were essential for covalent inhibition. Molecular docking of omeprazole with CACT indicated the formation of both covalent interactions with C136 and C283 and non-covalent interactions in agreement with the experimental data.The effect of omeprazole on the mitochondrial carnitine/acylcarnitine transporter has been studied in proteoliposomes. Externally added omeprazole inhibited the carnitine/carnitine antiport catalysed by the transporter. The inhibition was partially reversed by DTE indicating that it was caused by the covalent reaction of omeprazole with Cys residue(s). Inhibition of the C-less mutant transporter indicated also the occurrence of an alternative non-covalent mechanism. The IC A similar mechanism of interaction of omeprazole was also described for the plasma membrane carnitine (OCTN2) transport system of rat kidney. The drug causes inactivation of the transporter, by forming mixed disulphides with Cys residues of the protein Mitochondria represent important targets of pharmacological compounds. Indeed, exogenous substances interacting with mitochondrial enzymes or transporters may influence the entire cell metabolism. Among many proteins which have been shown to interact with pharmacological compounds l-[methyl-3H]carnitine 99% pure, 85 Ci/mmol, from Scopus Research BV, Wageningen The Netherlands, egg-yolk phospholipids (l-α-phosphatidylcholine from fresh turkey egg yolk), 1,4-piperazinediethanesulfonic acid (Pipes), Triton X-100, cardiolipin, l-carnitine and N-dodecanoylsarcosine (sarkosyl) from Sigma-Aldrich. All other reagents were of analytical grade.Sephadexes G-50, G-75 and G-200 were purchased from Pharmacia, E. coli C0214. Bacterial overexpression, isolation of the inclusion body fraction, solubilization and purification of the wild-type CACT and mutant CACT proteins were performed as described previously The previously constructed pMW7-WTratCACT recombinant plasmid containing the wild-type (WT) CACT cDNA was used to introduce the mutations in the CACT protein The recombinant proteins were reconstituted into liposomes as described previously 3H]carnitine to proteoliposomes and terminated by the addition of 1.5 mM NEM. In controls, the inhibitor was added together with the labeled substrate, according to the inhibitor stop method 3H]carnitine with a specific radioactivity of 10 nCi/nmol, for 60 min at 25°C. Then, the external radioactivity was removed by passing again the proteoliposomes through Sephadex G-75 as described above except that this chromatography was performed at 4°C to minimize the loss of internal substrate during the chromatography. Transport (efflux) was started by adding external buffer or substrate and stopped at the appropriate time interval. The transport assay temperature was 25°C. Finally, each sample of proteoliposomes (100 µl) was passed through a Sephadex G-50 column to separate the external from the internal radioactivity. Efflux activity was expressed as intraliposomal cpm. Activation of omeprazole was obtained as follows: 25 mM omeprazole in ethanol was diluted 10 fold in HCl 0.1 M and incubated for 15 min. Then, the pH was adjusted to pH 6.0 by Tris powder.Transport at 25°C was started by adding 0.1 mM carnitine uptake into proteoliposomes containing 15 mM carnitine, (carnitine/carnitine homologous antiport) in the presence of externally added 30 µM omeprazole activated by pre-incubation at pH 1.0 , the experimental data resembled those obtained with the C-less protein (not shown). The IC50 of the WT CACT were also determined for pantoprazole (6.4±0.33 µM) and lansoprazole (80±23 µM) (experiments not shown). Inhibition kinetic studies were further performed on omeprazole to obtain data on the localization of the omeprazole binding respect to the substrate binding site. The dependence of the antiport rate on the external carnitine concentration was studied in the absence or presence of activated omeprazole. The data obtained on the WT and C-less CACT, analyzed in double reciprocal (Lineweaver-Burk) plot are shown in To characterize the effect of omeprazole caused by the two different mechanisms of interaction with the transporter, kinetic experiments were performed on the WT or C-less protein. The dependence of the inhibition on the concentration of omeprazole was studied. The antiport rate, measured as uptake of 100 µM carnitine. To this aim, proteoliposomes were loaded with [3H]carnitine by transporter-mediated exchange equilibration . After this procedure the proteoliposomes contained internal 15 mM [3H]carnitine. In the absence of external substrate some efflux of intraliposomal radioactivity was detected  for the top scoring complexes suggest the following rank of affinity towards CACT: omeprazole ≃ pantoprazole > lansoprazole  solutions, with a top docking-score of −6.64 kcal/mol for the non-covalently bound omeprazole molecule. Through this procedure, we demonstrated the possibility that CACT could bind at the same time two omeprazole molecules: one covalently bound to C283 and another non-covalently bound inside the CACT central cavity. This solution is depicted in Three LowModeMD simulations were performed in order to evaluate the possibility that activated omeprazole could covalently bind to CACT cysteines, C89, C136 and C238. We analyzed 250 solutions for each simulation, and counted the number of solutions with strain energy lower than 2 kcal/mol versus the conformation with the lowest energy. We also evaluated the interaction potential energies between omeprazole and CACT of the lowest energy solutions for the three simulations (one for each of the studied cysteines). From our computations, C283 seems the most likely to covalently bind to omeprazole, since 9 conformations out of 250 showed a strain energy lower than 2 kcal/mol and the best solution had an energy of −29.9 kcal/mol. An intermediate reactivity can be hypothesized for C89, with 3 conformations under 2 kcal/mol and potential omeprazole-CACT interaction energy of −23.6 kcal/mol. The least reactive should be C136, with just 2 solutions under 2 kcal/mol and potential omeprazole-CACT interaction energy of −19.3 kcal/mol. From our http://dailymed.nlm.nih.gov/dailymed/drugInfo.cfm?id=4761]. Moreover it has been demonstrated that omeprazole can enter the cells in the activated form leading to toxicity at gastric level due to oxidative damage caused by the interaction and oxidation of Cys residues of proteins 2O2In the present study, the proteoliposome system reconstituted with the recombinant mitochondrial CACT has been used for investigating the inhibition of the transporter by omeprazole. The compound forms S-S mixed disulphide(s) with critical Cys residues or week interactions with the substrate binding site, which are observed as irreversible or competitive inhibition, respectively. The WT transporter contains thiol groups of Cys exposed towards the extraliposomal environment, which corresponds to the cytosolic side of the transporter In the light of the data presented, the described experimental approach may have on the one hand utility for predicting side interaction of pump inhibitor analogues with the CACT and, hence, improve their safety. On the other hand such tools could be useful for searching specific inhibitors of the CACT, which are of interest, for example, as cardioprotective agents, as it was previously described for mildronate which plays benefits under ischemic conditions


Gangliosides, the glycosphingolipids carrying one or several sialic acid residues, are located on the outer leaflet of the plasma membrane in glycolipid-enriched microdomains, where they interact with molecules of signal transduction pathways including receptors tyrosine kinases (RTKs). The role of gangliosides in the regulation of signal transduction has been reported in many cases and in a large number of cell types. In this review, we summarize the current knowledge on the biosynthesis of gangliosides and the mechanism by which they regulate RTKs signaling. D3 and GD2 are over-expressed in neuroectoderm-derived tumors such as melanoma, neuroblastoma, and breast cancer, in which they mediate cell proliferation, migration, tumor growth, and angiogenesis . Nor. Nor1]. ogenesis . Ganglioogenesis . Quantitative or qualitative  modifications of gangliosides can affect GEMs architecture and functions . AmongstUGCG gene (M3 synthase), ST8Sia I (GD3 synthase) and ST8Sia V (GT3 synthase), all being highly specific for glycolipid substrates . Th. ThUGCG bstrates . LacCer bstrates  and a losyndrome . The GD3ubstrate . Howeverescribed ,20. The cceptors . LacCer,ectively . Furtherynthase)  and the ynthase) , both acynthase) ,25. The ynthase) ,27 and Synthase) , and theynthase)  or V [29B4GALNACT1 gene is essentially expressed in embryonic tissue and in adult brain, lung and testis. By contrast, ST3GAL5 is ubiquitously expressed in human tissues [N-glycosylation, phosphorylation, and dephosphorylation. For example, protein kinases PKA and PKC can activate the GM2/GD2 synthase while inhibiting the activity of ST3Gal II or GM1a/GD1b synthase [The first steps of gangliosides synthesis take place in the cis/median-Golgi and the later steps in the trans-Golgi and trans-Golgi network . The reg tissues ,32,33. Gsynthase ,35,36.Receptor tyrosine kinases (RTKs) are key proteins involved in the control of cellular processes such as survival, proliferation, differentiation, migration and invasion. Fifty-eight RTKs have been identified in Humans. They all share a similar structural organization comprising of an extracellular domain containing the ligand-binding site, a unique transmembrane domain, and a cytoplasmic region containing the tyrosine kinase activity . UsuallyA number of growth factor receptors, including receptors for epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), nerve growth factor (NGF), hepatocyte growth factor (HGF), and insulin, were demonstrated to be regulated by gangliosides. RTKs are localized in GEMs with other lipid rafts associated proteins including integrins, tetraspanins, or plexins. Within lipid rafts, RTKs signaling can be negatively or positively regulated by gangliosides by either direct or indirect interactions ,42. ChanM3 is able to bind to the extracellular domain and inhibit the kinase activity of EGFR in a variety of cell lines. The effect of gangliosides on EGF-dependent tyrosine phosphorylation of EGFR was first demonstrated in human epidermoid carcinoma cell line A431 [M3 added exogenously to cells in culture was shown to inhibit EGFR autophosphorylation [M3 (II3NeuNH2LacCer) enhances serine phosphorylation independently of receptor-receptor interaction [M3 in A431 cells by PDMP (d-threo-1-phenyl-2-decannoylamino-3-morpholino-1-propanol), which inhibits the GlcCer synthase, increased EGFR autophosphorylation upon EGF stimulation [M3 directly interacts with EGFR on a site distinct from the EGF-binding site [M3 and terminal GlcNAc residues on EGFR N-glycans [M3 binding to EGFR is enhanced after glycosidase-treatment that exposes N-glycan terminal GlcNAc, whereas GM3 does not bind to EGFR from ManIB-knocked down cells that accumulates high mannose-type  N-glycans [M3 on EGFR [M3 was also shown to suppress murine hepatoma cell motility by inhibiting EGFR phosphorylation and the downstream PI3K/Akt signaling pathway [Several studies have shown that Gine A431 . GM3 addrylation ,46 whereeraction ,48. Simimulation . GM3 diring site  through -glycans ,52. GM3  on EGFR . GM3 was pathway . M3 and the tetraspanin tumor suppressor CD82 induce synergistic inhibition of migration Hepa1-6 cells by reducing EGFR phosphorylation [M3 inhibits the structural transition from inactive EGFR to signaling EGFR dimer, by preventing the autophosphorylation of the intracellular kinase domain in response to ligand binding [D3 synthase in CHO-K1 cells induces cell surface expression of GD3 and decreases EGFR phosphorlation and Erk2 activation upon EGF stimulation [M3, GM1, GD1a, and GT1b treatment were also reported in human neuroblastoma cells [D1a promotes the ligand-independent EGFR dimerization and enhances EGFR-mediated activation of the mitogen-activated protein kinase (MAPK) signaling pathway [M1 to GD1a [More recently, it has been reported that Grylation . By reco binding . In paramulation . Inhibitma cells . In norm pathway . Accordi to GD1a . M3 on FGFR activation and tyrosine phosphorylation was first demonstrated in cultured retinal glial cells [M3 with FGFR was hinted by confocal microscopy analysis in human lung embryonic fibroblast WI38, showing co-localization of GM3 and FGFR in the GEM fraction [M3 depletion by GlcCer synthase inhibition enhances tyrosine phosphorylation of FGFR, activates PI3K/Akt pathway and increases the interactions of FGFR with α3/α5/β1 integrins [M3 within the ganglioside-enriched microdomains. FGFR participates in many developmental, homeostatic and healing processes including neurogenesis, axon growth, differentiation, and neuronal survival . The negal cells . The intfraction . Moreoventegrins . This deM1 ganglioside regulates neurotrophins receptors both in vivo and in cell cultures [M1 to cell culture enhances NGF/TrkA signaling and protects neuronal cells from serum deprivation-induced apoptosis [M1 by the transfection of β3GalT4 cDNA, the enzyme that converts GM2 in GM1, inhibited NGF-induced TrkA dimerization and phosphorylation as well as the downstream pathway [M1 in PC12 was due to the high concentration of GM1 at the plasma membrane in β3GalT4 expressing cells that modulated membrane fluidity, impeding the NGF receptor localization within the lipid rafts [D3 synthase gene into PC12 cells resulted in the over-expression of GD1b and GT1b. These gangliosides triggered a conformational change of TrkA that formed a constitutively active dimer, activating its downstream signal pathways, including Erk1/2 and PI3K/Akt, and leading to a marked enhancement of cell proliferation [It has been clearly demonstrated that Gcultures ,66,67. Ipoptosis . On the  pathway . Accordiid rafts . In paraferation ,70.2+, GM3, and GM2 form heterodimers that specifically interact with tetraspanin CD82, thus impairing the trans-phosphorylation of c-Met receptor, the recruiting of Grb2 and the activation of PI3K/Akt and MEK/Erk pathways [D3 synthase in MDA-MB-231 breast cancer cells induced the cell surface accumulation of b- and c- series gangliosides including GD3, GD2, and GT3 [D2 was found to be involved in the activation of c-Met, and the subsequent activation of MEK/Erk and PI3K/Akt signaling pathways, leading to enhanced cell migration and proliferation. This was shown by competition assays using anti-GD2 mAb that inhibited c-Met phosphorylation  efficiently reduced both GD2 expression and c-Met phosphorylation. Of importance, the GD2-dependent activation of c-Met occurred in the absence of HGF [D1a that belongs to the a-series, was shown to inhibit HGF-induced motility and scattering of mouse osteosarcoma cell variant FBJ-LL cells through the suppression of phosphorylation of c-Met [In HCV29 bladder epithelial cells, motility and growth are modulated by the expression of a-series gangliosides. In the presence of Capathways ,71. Simipathways . The exp and GT3 ,74. Of trylation , demonste of HGF . On the of c-Met . M1, GM2, GM3, GD1a, GD1b, GD3, and GT1b), only GM3 did not inhibit the dimerization of PDGFR [M3 antibody was found to inhibit PDGF receptor activation in T51B liver epithelial cells [M1 was the most studied. In human glioma cells, GM1 treatment resulted in reduced phosphorylation of specific tyrosine residues of the cytoplasmic tail of PDGFR [M1-dependent inhibition of the receptor [M1 inhibition of PDGFR seems to be rather due to the exclusion of the receptor from glycolipid-enriched microdomains [M1 levels at the plasma membrane [Various gangliosides were shown to inhibit PDGF-dependent tyrosine phosphorylation of PDGFR in several cell types including Swiss 3T3 , human gof PDGFR  but coulal cells . Amongstof PDGFR . Howeverreceptor . Indeed,odomains . Recentlodomains  regulatemembrane . D1a increases VEGF-induced proliferation and migration [M3 is implicated in the decrease of VEGFR-2 phosphorylation and subsequent inhibition of Akt downstream signaling pathway in HUVECs [M3 decreases VEGF-induced VEGFR-2 activation by blocking receptor dimerization and the binding of VEGF to VEGFR-2 through a GM3-specific interaction with the extracellular domain of VEGFR-2 [M3 in CT-2A malignant mouse astrocytoma cells using GM2/GD2 synthase shRNA reduces tumor-induced angiogenesis [D1a, increases GM3 content while reducing GD1a and reduces growth, VEGF gene and protein expression, and vascularity [M3 in lipid rafts on the surface of podocytes (kidney glomerular pericytes), promoting adhesion and rapid actin reorganization [Several pieces of evidence have suggested that gangliosides also modulate tumor angiogenesis by controlling the activation of VEGF receptors FLT1 (VEGFR-1) and FLK1/KDR (VEGFR-2). It has been shown that ganglioside enrichment in human umbilical vein vascular endothelial cells (HUVEC) induces VEGFR dimerization and autophosphorylation at very low VEGF concentrations  and icubigration . GM3 is n HUVECs ,89. It w VEGFR-2 . In contogenesis . Moreovecularity . Finallynization ,93.M3 has been described as a negative regulator of insulin signaling, partially responsible for insulin resistance. In 3T3-L1 adipocytes, insulin resistance induced by tumor necrosis factor (TNF) is accompanied by an increased expression of GM3 synthase activity and GM3 ganglioside [M3 leads to the dissociation of insulin receptor (IR) from caveolae [M3 inhibits the IR substrate 1 (IRS-1) phosphorylation and IR signaling pathway [M3 synthase mutant mice that show an enhanced IR phosphorylation and a heightened sensitivity to insulin [M3 level in the liver, improving insulin sensitivity [M3 interacts with a lysine residue of IR beta-subunit localized above the transmembrane domain and induces the dissociation of the IR-caveolin-1 complex, which is essential for insulin signaling [M1 and GM2 were also shown to inhibit IR phosphorylation in in vitro assay [Gglioside . The inccaveolae . Moreove pathway ,96. Simi insulin . In parasitivity . It was ignaling . Finallyro assay .M3 or GM1 can be considered as negative regulators of RTKs signaling whereas disialogangliosides including GD2, GD1a, or GD1b mostly activated RTKs-mediated signal transduction. However, the molecular mechanisms by which gangliosides regulate RTKs remain poorly understood. Direct interactions between carbohydrate moiety of gangliosides and RTKs have been clearly identified as demonstrated for GM3 inhibition of EGFR, but direct carbohydrate-carbohydrate interactions cannot explain the different observed effects. Gangliosides regulation of RTKs also involved the reorganization of GEMs due to the change in ganglioside composition that induces the dissociation of RTKs from glycolipid-enriched microdomains, resulting in a reduced phosphorylation of the receptors as it has been demonstrated for insulin receptor. Indirect interactions with other GEMs associated transmembrane proteins including integrins and tetraspanins, can also be involved in the regulation of RTKs by gangliosides, as it has been demostrated for c-Met receptor. In parallel, the regulation of RTKs by gangliosides is highly depending on the carbohydrate moiety of gangliosides as shown for c-Met receptor, which is activated by GD2 whereas GD3 has no effect. The fine recognition of the glycan part of gangliosides should involve membrane lectin domains, able to discriminate between subtle changes in ganglioside glycans. The use of emergent technologies such as glycan arrays and photocrosslinking should enable to identification of such lectin domains [To conclude, it is now clear that gangliosides regulate RTKs within glycolipid-enriched microdomains either by inhibiting the dimerization and autophosphorylation of the receptors induced by specific ligands, or activating receptors signaling without ligand binding. Moreover, the activation or inhibition of RTKs is dependent on the glycan structure of gangliosides and cellular context. From a general point of view, monosialogangliosides, such as G domains ,102. Fin


Here, we focus on the family member H11/HspB8 and its herpes simplex virus type 2 (HSV-2) homologue ICP10PK, and discuss the possible impact of this relationship on human disease. H11/HspB8 and ICP10PK are atypical protein kinases. They share multi-functional activity that encompasses signaling, unfolded protein response (UPR) and the regulation of life cycle potential. In melanocytes H11/HspB8 causes growth arrest. It is silenced in a high proportion of melanoma prostate cancer, Ewing's sarcoma and hematologic malignancies through aberrant DNA methylation. Its restored expression induces cell death and inhibits tumor growth in xenograft models, identifying H11/HspB8 as a tumor suppressor. This function involves the activation of multiple and distinct death pathways, all of which initiate with H11/HspB8-mediated phosphorylation of transforming growth factor β-activated kinase 1 (TAK1). Both ICP10PK and H11/HspB8 were implicated in inflammatory processes that involve dendritic cells activation through Toll-like receptor-dependent pathways and may contribute to the onset of autoimmunity. The potential evolutionary relationship of H11/HspB8 to ICP10PK, its impact on human disorders and the development of therapeutic strategies are discussed. Small heat shock proteins (sHsp) also known as HspB are a large family of widely expressed proteins that contain a 90 residues domain known as  The 10 ystallin . They haystallin . Currentystallin , 5. HereTo the extent of our knowledge, HSV-2 is the only human viral pathogen that encodes Hsp homologues. One of these is UL14 that has 27% sequence identity with the protein-binding domain of Hsp70, but lacks ATPase activity. Like Hsp70, UL14 undergoes nuclear translocation after heat shock, it appears to aid in protein folding, and it has antiapoptotic activity . The secβ-phosphates, and the two conserved charged residues that, respectively, constitute catalytic motifs II (Lys) and III (Glu) [α-crystallin motif [ICP10PK is a serine/threonine kinase that is fused in frame to the large subunit of the HSV-2 ribonucleotide reductase  . PhylogeII (Glu) , 12. TheII (Glu) –13. It iin motif  that est14C-labeled ATP analogue FSBA, and binding is specifically competed by another ATP analogue, AMP-PNP, (iii) treatment with epidermal growth factor activates the kinase activity of a chimeric protein consisting of the ligand-binding domain of the epidermal growth factor receptor and the PK domain of ICP10, and (iv) the ICP10 kinase activity favors Mn2+ ions, does not require monovalent cations, and is not inhibited by zinc sulfate, all of which are properties distinct from those of the putative contaminant [Significantly, despite its overall similarity to HSV-2, HSV-1 has an R1 protein that contains a poorly conserved additional N-terminal domain, which lacks kinase activity , 8. Whiltaminant , 12. In taminant . ICP10PK is required for HSV-2 growth and latency reactivation –19. Thisα-crystallin motif in these ICP10PK functions is still unknown. ICP10PK behaves as a constitutively activated growth factor receptor that activates multiple survival/proliferative pathways . Ras actE2IG1 gene expressed in estrogen-treated breast cancer cells [α-crystallin sequence, which is believed to provide sine qua non evidence of evolutionary relationship, even when characteristic residues in the sequence have been lost [12q24.1-12q24.31 [12q24.23. Our original data [β-catenin phosphorylation at the transcriptional activity site Ser552 [H11/HspB8 was cloned in our laboratory using ICP10PK as a probe and called H11 , 32. It er cells . H11/Hsper cells , 31. It 12q24.31  and its nal data , confirmnal data , indicatnal data , 37. In e Ser552 . Conseque Ser552 , 38. H11e Ser552  and in he Ser552 . H11/Hsp2+ ions, like ICP10PK [N-glycosylation site (amino acid 138), suggestive of potential structural similarity [Confocal microscopy confirmed that H11/HspB8, like ICP10PK, is associated with the plasma membrane . However ICP10PK , 36. Kin ICP10PK  but not  ICP10PK . Like ICmilarity , 36. Sigα) by phosphorylation of its α-subunit. Induction of ER-resident proteins is mediated by activated transcription factor 6 (ATF6) and inositol requiring kinase 1 (IRE1) receptors. ATF6 activates the transcription of molecular chaperones such as BiP, and IRE1 induces the synthesis of a potent X-box-binding protein 1 (XBP1) and consequently activates the transcription of ER-associated degradation proteins such as EDEM (reviewed in [Over 30% of cellular proteins are synthesized in the endoplasmic reticulum (ER). Delayed or impaired protein folding causes misfolded proteins accumulation thereby triggering ER stress that is characterized by distension and loss of homeostasis. To overcome the toxicity associated with the accumulation of misfolded proteins and/or their aggregation, the ER induces a cascade of reactions called the unfolded protein response (UPR). Cellular response to UPR restores normal cell function/survival and includes (i) translational attenuation, (ii) induction of ER-resident proteins that assist in protein folding, notably chaperones, (iii) induction of ER-associated degradation machinery, and (iv) ER enlargement to accommodate the large load of unfolded proteins. Translational attenuation is mediated by the double-stranded RNA-activated protein-kinase-like endoplasmic reticulum kinase (PERK) signaling pathway that reduces the activity of the ribosomal initiating factor (eIF2iewed in ). If theβ-crystallin, HspB3, and HspB7 [β-crystallin mutants associated with the development of desmin-related cardiomyopathy or myofibrillar myopathy [β-peptides (A 1–42 and A 1–40). However, it only inhibits the death of cardiovascular cells induced by Dutch-type A 1–40 mutants and the role that chaperone activity and/or UPR inhibition plays in protection is still poorly understood [H11/HspB8 has basic chaperone activity. Original studies reported its interaction with HspB1 (HSP27)  but thisnd HspB7 . Other pnd HspB7 , Ddx20, nd HspB7 , and β-cmyopathy . H11/Hspmyopathy  and bindderstood . Two misderstood  apparentderstood . P < 0.001 versus PBS), preserved neuromuscular junctions, and inhibition of MN degeneration . Inhibition of mSOD1 aggregate formation   that is ormation  is assocormation . The conε-isoform of PKC) and inducible nitric oxide synthase [Theoretical predictions and results of circular dichroism spectroscopy studies suggest that H11/HspB8 is an intrinsically disordered protein (IDP) . IDPs arsynthase –58. Howesynthase . In thessynthase . The conβ-catenin phosphorylation at the transcriptional activity site Ser552 and the resulting inhibition of the cell-cycle regulatory proteins cyclin E/Cdk2 [In human melanocytes, H11/HspB8 causes growth arrest through n E/Cdk2 . Accordin E/Cdk2 . Signifin E/Cdk2 . H11/Hspn E/Cdk2  and in hn E/Cdk2  and its n E/Cdk2 –39. In mn E/Cdk2 . While tn E/Cdk2 . Howevern E/Cdk2 . The cell-cycle regulatory potential of H11/HspB8 in normal cells, its dysfunctional state in cancer cells, and its ability to induce tumor cell death upon restored expression identify H11/HspB8 as a tumor suppressor. Also characteristic of tumor suppressors is the cell-type specificity of the H11/HspB8 effects and the finding that it can undergo single-site transforming mutation. Indeed, one of the H11/HspB8 missense mutants, W51C, causes anchorage-independent growth and it overrides the death-inducing capacity of the wild-type H11/HspB8  through β-activated kinase 1 (TAK1). TAK1 is not activated by the missense H11/HspB8 mutants P173H and W51C, which do not cause cell death. Growth arrest is through the ability of activated TAK1 (pTAK1) to bind and phosphorylate β-catenin  of transforming growth factor -catenin  promotin-catenin . The out-catenin . This reβ and IL-18, indicating that caspase-1 functions independent of the inflammasome-mediated death pathway (pyroptosis) [The death pathways initiated by pTAK1 include p38MAPK/caspases-3/7-dependent apoptosis, which is a major component of cell death in one of the studied melanoma cultures/xenografts A375) and previously unrecognized death pathways in the other studied melanoma culture/xenografts (A2058) 5 and pre. In A205optosis) . Indeed,optosis) . The cleα (κB [κB induces TNF-α by binding kB promoter elements [H11/HspB8 was implicated in inflammatory processes based on its ability to activate dendritic cells through a Toll-like receptor-4- (TLR4-) dependent pathway . Its resα , which lα ) that acα  of its multivariate activities remain largely unknown. Its similarity to ICP10PK, which is expressed by a common viral pathogen, underscores the evolutionary importance of these functions, particularly as it pertains to human disorders and therapeutic strategies. Future investigation should include the potential interaction of H11/HspB8 with ATP-dependent chaperones and their co-chaperones, as well as different components of proteasomal and autophagosomal systems. Further analysis of the signaling pathways modulated by H11/HspB8 should emphasize the contribution of the cell type, the criteria that define new methylation patterns in some tumors, and the genetic diversity of distinct tumor lines. Better understanding of the ability of H11/HspB8 to modulate regulatory protein families (namely Bcl-2 and IAP), its mutation frequency, and its contribution to disease should also be emphasized. The mechanism of the H11/HspB8-induced death of tumor cells other than melanoma (namely prostate) and the role of TAK1 need further investigation, as does the contribution of H11/HspB8 to oncolytic virotherapy. Because H11/HspB8 is involved in different cellular processes, it appears to be an attractive therapeutic target. The use of H11/HspB8 as a marker for the selection of patients to be included in Aza-C treatment protocols should be further explored, particularly within the context of panels of hypermethylated genes and combinatorial therapy that includes PI3-K inhibition. The identification of hypermethylated genes that may contribute to H11/HspB8-induced cell death is particularly important. Finally, better understanding of the inflammatory potential of H11/HspB8 and the contribution that molecular mimicry with ICP10PK may have towards the development of autoimmune disorders should also be given further attention. Such understanding will provide important new tools to advance the development of much needed therapeutic strategies.


Ki  but does not require the determination of the arterial input function or metabolite correction. In this initial study, we show that Patlak-P (a) mimics Patlak Ki images in terms of visual assessment and target-to-background (TB) ratios of regions of elevated uptake, (b) has higher visual contrast and  better image quality than SUV, and (c) may have an important role in improving radiotherapy planning, therapy monitoring, and neurometabolism studies.Currently, kinetic analyses using dynamic positron emission tomography (PET) experience very limited use despite their potential for improving quantitative accuracy in several clinical and research applications. For targeted volume applications, such as radiation treatment planning, treatment monitoring, and cerebral metabolic studies, the key to implementation of these methods is the determination of an arterial input function, which can include time-consuming analysis of blood samples for metabolite correction. Targeted kinetic applications would become practical for the clinic if blood sampling and metabolite correction could be avoided. To this end, we developed a novel method (Patlak-P) of generating parametric maps that is identical to Patlak  The major issue in calculating Ki is determining the complete time course of the AIF, which requires either (a) approximately 1 hour of invasive blood samples or (b) some combination of blood samples and an initial cardiac acquisition as we've done in this study. Though our protocol reduces the total number of blood samples needed, it is still intrusive and requires metabolite correction for FLT and other tracers including methionine. Equation  states tWe addressed this problem by making a simple, yet novel assumption regarding the late-time sequence of AIFs. Fitting AIFs with a triple exponential function is an established method in reducing the effect of noise on kinetic calculations . Howevert0, which differs from the usual Patlak view. By evaluating the integral and performing algebraic rearrangement, , Ki, λ (AIF decay constant), and V. Inspection of (P(t = ∞), or P∞, is equivalent to P(t0) + α and is directly proportional to Ki  if we assume that the integral of the AIF is constant in space at late times. The global scalar constant, the total integrated activity (iAIF∞), is the sum of two terms: the total integrated activity from 0 to t0 (iAIF0) and the total integrated activity from t0 to infinity, (AIF0/λ). Thus, we now have a framework for obtaining parametric images that reflect the behavior of Patlak Ki without needing to determine an AIF or correct for metabolites. In addition, we have a basis for determining Ki given a priori knowledge of the input function such as population-based AIFs for FDG (see Appendix).Equation  states tP∞ maps is most dependent upon accurate estimates of λ, a global parameter and the only nonlinear term in (P(t0) and α are linear terms and are easily calculated using linear least squares given knowledge of λ. To this end, we propose a strategy where λ would be determined first from nonlinear regression of a large region of interest (ROI) using the Levenberg-Marquardt algorithm [However, accurate calculation of  term in . In contlgorithm . By doinλ and P∞, respectively, with weights equal to the product of frame duration (Δt) and the radioactive decay factor (DF). Since the biodistribution of tracers at late times changes minimally, the nonlinear noise properties of OSEM were assumed to have a minimal effect on predicting a simple, accurate weighting scheme for nonlinear and linear regression. Thus, we employed weighted nonlinear and linear regression to determine P∞ is just a measure of activity at very late times, it is easily set in the framework of SUV. Thus, we propose a new quantity, SUV∞, which better reflects the kinetic behavior of irreversible tracers such as FDG. The application of SUV∞ to treatment monitoring will be explored in the discussion section.Because P as a surrogate for Patlak Ki in radiotherapy planning required two major comparisons of tumors and other regions of elevated uptake derived from Patlak Ki, SUV∞, and SUV maps, target-to-background (TB) ratios and qualitative assessments of image quality. TB ratios were calculated from manual segmentations of the target region and a nearby background region that presented the largest challenge to target visualization. In this way, TB ratios quantitatively reflected the ability of each technique to delineate the target from all nearby structures. The metrics for qualitative assessment were visual contrast, delineation of target boundary, and presence/absence of well-known structural detail such as tooth “holes” and the cerebellum. In our view, validating Patlak-In an effort to maintain analytical cohesion, we decided to combine select quantitative and qualitative findings for each patient and present them in their own subsections. A final subsection was used to summarize general trends.  Tumor volumes derived from the three methods were not directly compared, because (a) there is no general consensus for which segmentation algorithm to use ; (b) dosAs stated previously, the purpose of TB ratios was to compare how each technique is able to delineate tumors or other regions of elevated uptake from the background and adjacent structures. A representative example of how target and background definitions were defined is illustrated in Both FDG and FLT acquisitions of Patient 1 show a region with elevated uptake (REU) near the base of the mouth . P and Patlak have higher contrast than SUV and in general appear to be of higher quality because their backgrounds are diminished. These findings are quantitatively confirmed through TB ratios: Patlak-P (3.45), Patlak (3.77), and SUV (2.98). In addition, the REU appears to be slightly smaller in Patlak and Patlak-P images. Images generated from the FDG-PET scans are similar to their FLT counterparts, but the contrast differences are slightly less pronounced: Patlak-P (2.736), Patlak (2.481), and SUV (2.270). In this case, however, the Patlak-P parametric map has slightly higher contrast than Patlak. Inspection of P generated parametric maps with higher contrast and visual quality than SUV. Furthermore, this increased contrast led to sharper boundaries between regions of high uptake and their surrounding areas in Patlak and Patlak-P images, as evidenced in how well each technique resolved the teeth and bony structures (arrows) in FLT-PET . An overall analysis of image quality showed that parametric maps generated by the three methods follow the prior examples. For both FDG and FLT studies of this patient, Patlak and Patlak- Figures  and the  Figures . In partAn REU was seen in both FDG and FLT images of Patient 2 near the upper row of teeth .P FLT images have higher contrast than either Patlak or SUV, which is mirrored by TB ratios of the REU: Patlak-P (5.64), Patlak (4.37), and SUV (3.71). Patlak-P's high TB ratio is explained by increased diffusivity of the REU in Patlak and SUV images and increased background reduction in the Patlak-P image. In this study, however, the increase of image contrast is paired with a slight decrease in image quality.Inspection of P image, though both are superior to SUV in both respects. The TB ratios do not mirror the relative performance of SUV: 1.92 (Patlak-P), 2.661 (Patlak), and 1.95 (SUV). It is more than likely that the TB ratio for SUV is improperly elevated, because the background ROI , 1.43 (Patlak), and 1.36 (SUV) for the tumor nodule and 4.639 (Patlak-P), 2.912 (Patlak), and 2.77 (SUV) for the tumor cross-section  by applying Patlak-P possess higher contrast and image quality than SUV for the tumor and the cerebellum. These visual findings do not reflect calculated TB ratios, which do not vary significantly between techniques: 1.71 (Patlak-P), 1.59 (Patlak), and 1.69 (SUV). TBSUV, however, is artificially inflated because, as before, the background region in the SUV image  TB ratios calculated by Patlak-P and Patlak were statistically similar (P = 0.125); (b) TB ratios calculated by Patlak and SUV were not statistically similar (P = 0.004); (c) TB ratios calculated by Patlak-P and SUV were not statistically similar (P = 0.002). P and Patlak images were higher than SUV images. There was one instance where the Patlak image possessed significantly higher contrast than the Patlak-P image (2-FDG), and two instances where the Patlak-P image possessed significantly higher contrast than the Patlak image (2-FLT and 3-FLT). In all of these cases, images with significantly enhanced contrast appeared slightly noisier.  In general, visual contrast observed in Patlak-P counterparts, which impacted delineation of teeth, bony structures, the cerebellum, and REU/tumor boundaries. This effect was often emphasized around small structures  better image quality than SUV images. These findings show that Patlak or Patlak-P images may have significant bearing on radiotherapy planning. In particular, improved resolution of tumors from surrounding structures may help dose-planning physicians deliver a lethal dose to tumors while preserving more normal tissue. We performed this feasibility study to assess the possible impact of Patlak-λ and was likely the proximate cause of discrepancies between the TB ratios of Patlak and Patlak-P in three studies. Since we only applied the Levenberg-Marquardt algorithm with a single initial guess in this study, determination of λ can be made more robust using several methods. The easiest method is to execute the Levenberg-Marquardt algorithm with multiple initial guesses and accept the parameter set with the lowest “energy” as the global minimum. In fact, it has been shown that dynamic PET studies benefit from this technique [λ will increase the statistical similarity  between Patlak and Patlak-P.Nonlinear regression is a more difficult problem than linear regression, because solutions of nonlinear regression are not guaranteed to be unique. This added variability increases the sensitivity of echnique . We do nechnique  or genetechnique , becauseechnique . We expet0. However, by choosing t0 to be approximately 15–20 minutes after-injection, as was the case in this feasibility study, the Patlak assumption of minimal dephosphorylation may be better upheld than if t0 was delayed further. For example, if t0 was chosen to be 2 hours after injection, even a small k4, depending on k2 and k3 values, may decrease the extrapolated uptake at infinity and, thus, underestimate P∞ as a correlate for Ki. Therefore, if the objective is to mimic Ki, then t0 = 15–20 minutes after-injection may be the most suitable. However, as is shown in several delayed-time PET publications [t0) until 30–60 minutes after-injection may be the better option.Since this was a retrospective analysis, we could not choose ications , 34, if Ki parametric maps, rather than static images, would alter radiotherapy planning. Though, Visser et al. [Ki maps in radiotherapy planning and monitoring [P would be better suited for this application than Patlak, because it is easier to implement [To our knowledge, there has not been a study on how using Patlak r et al.  do give r et al. , it woulnitoring , 21, 36,mplement  and lessP for radiotherapy planning. However, Patlak-P has the potential to impact other areas of research and clinical projects. The first application area we propose, therapy monitoring, is closely associated with radiotherapy planning. Early indication of treatment efficacy can be very beneficial [In this preliminary work, our primary focus was an initial validation of Patlak-neficial , 12–18; r) between percent change in SUV (%SUV) and therapeutic outcome from 0.77 to 0.88, 0.85, and 0.97. Since the first correction should be equivalent to using SUV∞ instead of SUV, it would be logical to conclude that Patlak-P may have bearing on treatment monitoring. Furthermore, two investigations by Dimitrakopoulou-Strauss et al. [Ki and SUV; (b) tumor staging may benefit from serial observations of Patlak Ki from two different tracers (18FDG and 68Ga-BZH3); (c) diagnosis of suspicious nodules may be improved by combining TAC slope and static uptake. Thus, it is possible that the combination of SUV and SUV∞ may improve the accuracy of outcome prediction and tumor staging relative to SUV. Though SUV is the current standard in treatment monitoring, it has two major weaknesses: (a) it does not account for unmetabolized tracer, and (b) it crudely approximates the integrated input function with several normalization factors . The quantitative effect of these weaknesses on prediction of therapeutic outcome from serial measurements of SUV was assessed by Freedman et al.  using ses et al. , 18 and s et al.  have shoKi and P∞ that allows all observations, independent of tracer or application, made previously regarding SUV and Ki to remain partially or wholly true for SUV∞ and Ki. For the unique case of FDG, which has well-characterized population-based input functions and minimal metabolites, Ki values may be directly calculated from P∞ and λ (see Appendix).We emphasize here that it is the proportional relationship between P may have a significant impact on neurometabolism studies. However, in contrast to radiotherapy planning, where the main goal is to provide high-quality images to the dose-planning physician, neurometabolism studies may require quantitative measures of uptake. Fortunately, these measures are typically not required to be absolute, only relative, because uptake patterns in certain regions of the brain  are often constant across wide populations. For example, Mosconi et al. [glc) ratios between the medial temporal lobe (MTL) and pons and the posterior cingulate cortex (PCC) and pons. These relative metrics could easily be calculated using Patlak-P. Furthermore, the hallmark of AD is an atypical pattern of FDG uptake [P may be able to improve the detection of early AD, because of its high contrast and ability to resolve structural details in the brain . In this way, Patlak-P could be made more practical for targeted analyses of relatively large structures such as the brain or liver.In their work , SundaraP technique and, thus, serve as the basis for planning future validation studies and clinical investigations. Validation studies are needed to compare (a) quantitative and qualitative similarities between P∞ and Ki parametric maps for other cancer types, (b) ratios of SUV∞, SUV, Ki , and Ki determined from FDG-PET measurements taken before and after treatment and (c) intrapatient ratios of P∞ and Ki between brain regions for the assessment of neurometabolism distributions.We believe that this feasibility study of a single cancer is sufficient to provide proof of principle of the Patlak-P), which does not require any sampling data or an image-derived input function from the blood pool region, to generate parametric maps that mimic Patlak Ki. Using quantitative and qualitative comparisons, we demonstrated that Patlak-P images are similar to Patlak images and possess higher contrast, sharper boundaries, and  better image quality than SUV images. We believe that these improvements over SUV could have direct impact in radiotherapy planning, therapy monitoring, and neurometabolism studies.In this feasibility study, we demonstrated a novel method (Patlak-


However, it is not known whether DUSP1 overexpression contributes to PDAC chemoresistance. Using BxPC3 and COLO-357 human PCCs, we show that gemcitabine activates c-JUN N-terminal kinase (JNK) and p38 mitogen activated protein kinase (p38 MAPK), key kinases in two major stress-activated signaling pathways. Gemcitabine-induced JNK and p38 MAPK activation mediates increased apoptosis, but also transcriptionally upregulates DUSP1, as evidenced by increased DUSP1 mRNA levels and RNA polymerase II loading at DUSP1 gene body. Conversely, shRNA-mediated inhibition of DUSP1 enhances JNK and p38 MAPK activation and gemcitabine chemosensitivity. Using doxycycline-inducible knockdown of DUSP1 in established orthotopic pancreatic tumors, we found that combining gemcitabine with DUSP1 inhibition improves animal survival, attenuates angiogenesis, and enhances apoptotic cell death, as compared with gemcitabine alone. Taken together, these results suggest that gemcitabine-mediated upregulation of DUSP1 contributes to a negative feedback loop that attenuates its beneficial actions on stress pathways and apoptosis, raising the possibility that targeting DUSP1 in PDAC may have the advantage of enhancing gemcitabine chemosensitivity while suppressing angiogenesis.Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer with a poor prognosis that is characterized by excessive mitogenic pathway activation and marked chemoresistance to a broad spectrum of chemotherapeutic drugs. Dual specificity protein phosphatase 1 (DUSP1) is a key negative regulator of mitogen activated protein kinases (MAPKs). Yet, DUSP1 is overexpressed in pancreatic cancer cells (PCCs) in PDAC where it paradoxically enhances colony formation in soft agar and promotes  Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in the US, with an annual mortality of nearly 38,000, a median survival of 6–7 months and a five-year survival rate of 6% The resistance of PDAC to gemcitabine and many other chemotherapeutic agents is due, in part, to a wide range of genetic and epigenetic alterations which lead to abnormal activation of cell survival and anti-apoptotic pathways Abnormal activation of mitogen-activated protein kinases (MAPKs) plays a critical role in regulating cell survival and apoptosis The dual-specificity phosphatase (DUSP) family of proteins consists of 25 members We reported that DUSP1 is overexpressed in PDAC, and that antisense-mediated suppression of DUSP1 expression reduces tumor development in nude mice The effects of gemcitabine on the PCC growth were evaluated using the MTT assay. In all three cell lines, gemcitabine exerted a dose-dependent growth inhibitory effect. AsPC-1 was most resistant to gemcitabine, with an IC50 greater than 100 ng/mL, whereas BxPC-3 and COLO-357 cells were more sensitive to gemcitabine, with IC50 values of 10 ng/mL and 5 ng/mL, respectively .To evaluate the effects of gemcitabine on the activation of MAPKs, cells were incubated with gemcitabine at a concentration close to their respective IC50 s. In BxPC-3 and COLO-357, gemcitabine induced the phosphorylation of p38 MAPK and JNK, key kinases in two major stress-activated signaling pathways . GemcitaConsidering that DUSP1 is a key regulator of MAPK activities In response to various stimuli, the transcription factor AP-1 , which is the major downstream target of p38 MAPK and JNK signaling, has been shown to associate with the DUSP1 promoter and regulate its transcription The increase in DUSP1 mRNA levels could be due to enhanced transcriptional activity or posttranscriptional mRNA stability. Therefore, chromatin immunoprecipitation against RNA polymerase II, which is the core component of the transcription machinery, was next carried out, followed by Q-PCR to quantify the amount of RNA polymerase II bound to the gene body region of DUSP1. This assay allows for direct measurement of the active elongation step and reflects the transcriptional activity of the DUSP1 gene. Incubating BxPC-3 and COLO-357 cells with gemcitabine for 24 h increased the amount of RNA polymerase II loading at the gene body region of DUSP1 by 6 fold and 12 fold respectively , pointinTo investigate the effect of silencing DUSP1 on MAPK activities and gemcitabine chemosensitivity, AsPC-1, BxPC-3, and COLO-357 cells were stably transduced with lentivirus expressing shRNA against DUSP1 or non-targeting scramble control and then incubated with various gemcitabine concentrations. In all three cell lines, DUSP1 knockdown using two shRNAs increased the growth inhibitory actions of gemcitabine. Thus, silencing DUSP1 shifted the IC50 values for gemcitabine in AsPC-1, BxPC-3 and COLO-357 cells from 500 to 10 ng/ml, from 10 to 5 ng/ml, and from to 5 to 2.5 ng/ml, respectively . These rThe anti-DUSP1 antibody routinely revealed 2 closely-migrating bands on western blots –3. HowevConsidering that gemcitabine may be used in conjunction with cisplatin or 5-fluorouracil to treat locally advanced or metastatic PDAC We next sought to determine whether enhanced chemosensitivity is unique to DUSP1 silencing or a common feature of targeting any DUSP family members. Accordingly, we chose to knockdown DUSP4/MKP-2, due to its similarity to DUSP1 in terms of subcellular localization and substrate preference in vivo  and Ki67 (proliferation), as well as by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and cleaved caspase 3 immunoreactivity, both of which are markers of apoptosis. Gemcitabine slightly attenuated CD34 and Ki67 signals, while DUSP1 knockdown had a marked inhibitory effect on CD34 staining and a more moderate but highly significant inhibitory effect on Ki67 staining . DUSP1 sMAPK8, MAPK9 and MAPK10, respectively, and alternative splicing gives rise to at least ten isoforms MAPK14, MAPK11, MAPK12, and MAPK13, respectively, and there are two alternatively spliced isoforms of MAPK14JNK1, -2, and -3 are encoded by In the current study we determined that gemcitabine activated JNK and p38 MAPK, thereby inducing apoptosis in PCCs. While the role of specific isoforms was not evaluated, gemcitabine-activated JNK and p38 MAPK signaling also induced DUSP1 transcription, as evidenced by increased DUSP1 mRNA levels and increased RNA polymerase II loading at DUSP1 gene body. Moreover, shRNA-mediated inhibition of DUSP1 enhanced gemcitabine-induced JNK and p38 MAPK activation and sensitized PDAC cells to gemcitabine and cisplatin, leading to decreased cell proliferation and increased PARP and caspase 3 cleavage. These results suggest that gemcitabine-mediated activation of JNK and p38 MAPK leads to the upregulation of DUSP1, which in turn contributes to a negative feedback loop that attenuates JNK and p38 MAPK activities, thereby interfering with the beneficial actions of gemcitabine on stress pathways and apoptosis . These oDownregulation of DUSP1 suppresses the expression of angiogenic factors, such as SH2D2A and VEGF-C in non-small-cell lung cancer (NSCLC) cells, and functional assays have confirmed the role of DUSP1 in promoting tumor angiogenesis Using doxycycline-inducible knockdown of DUSP1 in established orthotopic pancreatic tumors, in the present study we determined that combining gemcitabine with DUSP1 inhibition prolonged animal survival and enhanced apoptotic cell death, compared with gemcitabine alone. In addition, DUSP1 knockdown markedly attenuated PCC proliferation and tumor angiogenesis. Our use of immune deficient mice precludes an assessment of the consequence of DUSP1 inhibition on the immune system or on macrophage number and activation within the pancreatic tumor mass. Given that DUSP1 is known to modulate macrophage proliferation and activation The mechanisms that lead to increased DUSP1 expression in PDAC are not known. It has been demonstrated, however, that hypoxia can upregulate DUSP1 transcription All animal studies were approved by the Institutional Animal Care and Use Committee of Indiana University (Permit Number: 10108). All animal studies described were conducted in accord with accepted standards of humane animal care and all efforts were made to minimize suffering.et al., AsPC-1 and BxPC-3 human pancreatic cancer cells were obtained from the American Type Culture Collection . COLO-357 cells were a gift from Dr. R. Metzger at Duke University, and were originally placed in culture by Morgan, MTT assay was performed as described Immunoblotting was done as described previously Total RNA was extracted using RNeasy purification kit . Total RNA (1 µg) was reverse transcribed using High-Capacity cDNA Reverse Transcription Kit. Taqman quantitative real-time PCR (Q-PCR) was carried out on an ABI Prism 7300 machine, and analyzed using a StepOnePlus Real-Time PCR system, all from Applied Biosystems, . All probes were pre-designed and obtained from Applied Biosystems. 18S was used as internal control. Gene expression levels were calculated using the relative ΔCt method Cambridge, MA) or control IgG. After 24 h, beads were washed and immune complexes were eluted using 100 ml of 1% SDS and 0.1 M NaHCO3TGTGGGCAACATTCCTGTAA; DUSP1 gene body reverse, CAAAGGATGGCACAGGATTT.BxPC-3 and COLO-357 cells were cross-linked with 1% formaldehyde for 10 min at 37°C. The cells were then rinsed with cold PBS, harvested and lysed with 1% SDS, 10 mM EDTA, and 50 mM Tris-HCl (pH 8.1) containing a protease inhibitor cocktail . Chromatin fragmentation was subsequently achieved by two sequential sonications (10 min each) using 30 sec on/off cycles and a Bioruptor sonicator  at the highest intensity. The soluble chromatin was diluted in buffer containing 1% Triton, 2 mM EDTA, 150 mM NaCl, and 20 mM Tris-HCl (pH 8.1) and added into Dynal magnetic beads  which were pre-incubated with anti-RNA polymerase II antibody  and non-silencing shRNA control (RHS4743), and the pGIPZ lentiviral vectors for MKP2  and non-silencing shRNA control (RHS4348) were purchased from Open Biosystems, Huntsville, AL. Packaging was performed using a second generation plasmid transfection system as previously described null mice were obtained from the In Vivo Therapeutics Core of Indiana University Simon Cancer Center. COLO-357 cells (0.5×106) were stably transduced with pTRIPZ/sh-scramble or pTRIPZ/sh-DUSP1, suspended in 50 µl sterile PBS, and injected into the subcapsular region of the pancreas Twenty four 6- to 8-week-old male NOD/SCID/IL-2RgCambridge, MA). Sections were incubated in HRP-labeled secondary antibody and staining was detected by DAB . TUNEL assay was performed using the In Situ Cell Death Detection POD Kit  according to the manufacturer’s protocol. Images were taken using an Olympus BX60 microscope  equipped with a QImaging EXI Blue camera and ImagePro software .Orthotopic tumors were fixed in 10% formalin and embedded in paraffin. H&E staining and immunohistochemistry were performed using 5 µm thick sections Statistical analysis was conducted using the GraphPad InStat software . Significance was determined using one-way ANOVA, followed by the Dunnett test to compare all groups against the corresponding control group, and the Bonferroni test for specific pairwise comparisons. Statistical significance was taken as p≤0.05.


In vitro oxidative stress activates poly(ADP-ribose)polymerase, decreases NAD+ level, sirtuin 1 activity and finally leads to cell death. Poly(ADP-ribose)polymerase hyperactivation contributes to cell death. In addition, poly(ADP-ribose)polymerase inhibition restores NAD+ level and sirtuin 1 activity in vitro. In vitro sirtuin 1 induction protects neurons from cell loss induced by oxidative stress. In this context, the role of sirtuin 1 and its involvement in beneficial effects of poly(ADP-ribose)polymerase inhibition were evaluated in vivo in a model of cerebral oxidative stress induced by intrastriatal infusion of malonate in rat. Malonate promoted a NAD+ decrease that was not prevented by 3-aminobenzamide, a poly(ADP-ribose)polymerase inhibitor, at 4 and 24 hours. However, 3-aminobenzamide increased nuclear SIRT1 activity/expression ratio after oxidative stress. Malonate induced a neurological deficit associated with a striatal lesion. Both were reduced by 3-aminobenzamide and SRT1720, a sirtuin 1 activator, showing beneficial effects of poly(ADP-ribose)polymerase inhibition and sirtuin 1 activation on oxidative stress consequences. EX527, a sirtuin 1 inhibitor, given alone, modified neither the score nor the lesion, suggesting that endogenous sirtuin 1 was not activated during cerebral oxidative stress. However, its association with 3-aminobenzamide suppressed the neurological improvement and the lesion reduction induced by 3-aminobenzamide. The association of 3-aminobenzamide with SRT1720, the sirtuin 1 activator, did not lead to a better protection than 3-aminobenzamide alone. The present data represent the first demonstration that the sirtuin 1 activator SRT1720 is neuroprotective during in vivo cerebral oxidative stress. Furthermore sirtuin 1 activation is involved in the beneficial effects of poly(ADP-ribose)polymerase inhibition after in vivo cerebral oxidative stress.Poly(ADP-ribose)polymerase and sirtuin 1 are both NAD Oxidative stress (OS) is involved in physiopathology of acute cerebrovascular diseases such as stroke and traumatic brain injury +-dependent nuclear enzyme that participates to DNA repair. Its excessive activation during OS contributes to energetic depletion and consequently to cell death in vitro and in vivo OS situations in vivo cerebral OS in vitro data showed that PARP activation limited other NAD+-dependent enzymes activity during OS, in particular sirtuin 1 (SIRT1), probably due to substrate depletion PARP is a NADin vitro OS-induced cell death SIRT1 is a NAD-dependent deacetylase that have numerous targets that confers it a lot of biological functions. SIRT1 plays a significant role in histone post-translational modifications and chromatin-related functions, in the regulation of p53 expression and function, and in DNA damage response In vitro studies showed that OS activates PARP, decreases NAD+ level, SIRT1 activity and finally leads to cell death In vitro PARP inhibition restored NAD pool and SIRT1 activity in cells after hydrogen peroxide treatment + preservation, thus it may allow NAD+ use for SIRT1 activity. To our knowledge, there was no information dealing with the role of SIRT1 and its involvement in beneficial effects of PARP inhibition in the consequences of an in vivo cerebral OS. In this study, we used a rodent model of cerebral OS induced by malonate administration which causes oxygen and nitrogen reactive species production + depletion, SIRT1 expression and activity. In the second part, we studied the role of SIRT1 and its implication in beneficial effects of PARP inhibition on neurological and histological OS consequences using treatment with a PARP inhibitor, a SIRT1 activator, a SIRT1 inhibitor, alone or in combination.Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health . All surgery was performed under chloral hydrate  anaesthesia, and all efforts were made to minimize suffering.Male Sprague-Dawley rats (300–330 g) were supplied by Elevage Janvier laboratories . Animals were housed under standard conditions (temperature- and light-controlled) with free access to food and water. Animal care and all the experiments were performed in compliance with the ethical approvals stipulated by the Animal Ethics Committee of Paris Descartes University (registered number: CEEA34.CML.025.11), with the French regulations and the European Communities Council Directive of September 20, 2010, 2010/63/UE, on the protection of animals for experimental use and conformed to the via the cannula using a pump (Infors AG HT type Precidor). The injection cannula was left in place for a further 5 minutes to minimize the risk of retrograde leakage of the injected solution. Sham-operated rats underwent the same surgery and were given an intrastriatal injection of the same volume of 0.9% NaCl. The scalp was sutured and the animal was returned to its cage to recover from anesthesia.Rats were anaesthetized with chloral hydrate  and placed on a stereotaxic frame. During surgery, animals were positioned on a heating blanket (Harvard) to maintain body normothermia (37.5±0.5°C). A 3 M solution of malonate  was prepared in 0.9% NaCl and pH was adjusted to 7.4. The scalp was incised and a craniotomy was performed at the following coordinates: 3.5 mm lateral to the bregma, 0 mm posterior to the bregma + determination, as this latter was depleted at these times (Data S1) and at 6 hours for SIRT1 expression and activity assessment.PARP inhibition was induced by intracerebroventricular infusion of 3-aminobenzamide (3AB) as previously described in vitro+ being decreased between 4 and 24 hours (Data S1), neurological assessments, striatal lesion, SIRT1 expression and activity were performed 6 hours after malonate infusion.For PARP inhibition, 3AB was administrated as in experiment 1. For SIRT1 activation, 0.3 µg of SRT1720 , a potent specific activator, was given i.c.v. A neurological assessment was performed in a blind fashion using 3 grading scales:the first scale evaluated contralateral sensorimotor function with items whose scores were summed and used as a neurological score the second scale was performed to evaluate motor coordination by beam-walking test as previously described the third scale measured postural asymmetry of animals using circling test These 3 scores  were summed as a global neurological score (GNS) to evaluate the global neurological function. The maximum GNS was 15 for unoperated rats.2) were measured using a computer image analysis system  and the distance between respective coronal sections (500 µm) was used to calculate the lesion volume. The brain lesion volume (mm3) was calculated by integrating the necrotic areas, corrected for edema Brain lesion was assessed by cresyl violet staining of coronal sections (50 µm) +) was measured on the left striatum as previously described + content was expressed in picomoles per milligram of wet tissue (pmol/mg).Nicotinamide adenine dinucleotide . Their brains were promptly removed, the left striatum isolated, frozen in liquid nitrogen and stored at −40°C.Nuclear proteins were extracted using a ProteoExtract Subcellular Proteome Extraction Kit , according to the supplier’s instructions, and measured by bicinchoninic acid assay. Samples were diluted in Laemmli buffer , 0.01% bromophenol blue) and heated to 95°C for 10 minutes.Samples were separated in a 6% SDS-polyacrylamide gel, transferred onto polyvinylidene fluoride (PVDF) membrane  and the free sites on membrane were blocked 20 minutes with a blocking solution  and 0.1% Tween 20 (TTBS)). Then, membranes were probed with a rabbit anti-SIRT1 primary antibody  overnight at 4°C in the blocking solution. After three washing in TTBS, membranes were incubated with donkey anti-rabbit IgG-fluorescein antibody  in the blocking solution for 1 hour at room temperature in obscurity. Then, membranes were incubated with rabbit anti-fluorescein-alkaline phosphatase antibody  following the same schedule. The SIRT1 specific immunoreactivity was visualized using the ECF Western blotting substrate , according to the supplier’s instructions. Membranes were scanned using Storm 860 apparatus  and Image Quant Software  was used for quantification. Results were expressed in arbitrary units (AU).Acetylated-histone H3, assayed by dot-blot, was used to evaluate the nuclear SIRT1 activity 2 and 1 mM dithiotreitol (DTT). Samples were incubated with 1 mg/mL of histone , used as a PAR acceptor, and 11 mM of NAD  during 10 minutes at 25°C. Addition of NAD initiated PARP reaction activity which was evaluated in presence or absence of SIRT1 inhibitor: 0.01 to 20 µM of EX527 , and SIRT1 activator: 0.001 to 100 µM of SRT1720 . Reaction was stopped by addition of 125 mM tris-HCl buffer pH 6.8 containing 130 mM DTT and 4% SDS, and samples were heated to 95°C for 10 minutes. Samples (1 µL) were quickly added in triplicate to a PVDF membrane  locally pre-wet with ethanol. The membranes were washed 5 minutes twice in bidistilled water and twice in TTBS. Unoccupied binding sites were blocked 1 hour with a blocking solution containing 5% non-fat dry milk in TTBS. Then, the membranes were incubated with a primary antibody, mouse anti-poly-ADP-ribose antibody , overnight at 4°C in blocking solution. Next day, they were washed three times with TTBS and incubated with anti-mouse IgG-peroxydase secondary antibody  for 1 hour at room temperature in the blocking solution. Membranes were washed three times and PAR production was visualized using chemioluminescence ECL Advance Western blotting detection reagent . Membranes were exposed onto Kodak Biomax Light film  which were then scanned using Gel Doc 2000 (Bio-Rad). The PAR expression was quantified using Quantity One software (Bio-Rad). Results were expressed in relative PAR production of control .Poly(ADP-ribose) (PAR) production was used as a marker of PARP activity Results were expressed as mean ± S.E.M. Figures and analyses were performed using GraphPad Prism 5.0 . Neurological scores were compared using a Kruskal-Wallis analysis followed by a Mann-Whitney test. Whenever suitable, other results were analysed using one-way ANOVA followed by a Dunnett post-hoc test or two-way ANOVA followed by a Student’s t-test. Differences were considered statistically significant when P<0.05.+ level of 119±10 pmol/mg of tissue that was not different from that of sham-operated animals receiving 3AB vehicle (105±7 pmol/mg of tissue) (+ level of sham-operated rats receiving 3AB (108±9 pmol/mg of tissue) did not differ from that of sham-operated rats receiving vehicle. Malonate led to a 50% decrease of NAD+ level at 4 h  that persisted at 24 h , demonstrating a NAD+ depletion. Treatment with 3AB did not modify NAD+ level at 4 (62±4 pmol/mg of tissue) and 24 h (51±6 pmol/mg of tissue) after malonate.Non-operated rat had a striatal NAD tissue) . The NADNon-operated rats had a nuclear SIRT1 expression of 60±12 AU that was not different from that of sham-operated animals receiving 3AB vehicle (59±3 AU) . The nucNon-operated rats had a striatal acetylated-H3 level 27±3 AU . There wNon-operated rats had a nuclear SIRT1 activity/expression ratio of 0.5±0.1 that was not different from that of sham-operated animals receiving 3AB (0.5±0.1) or its vehicle (0.5±0.1) . MalonatNon-operated rat had a GNS of 14.4±0.2 . MalonatThe association of 3AB with SRT1720, the SIRT1 activator, did not modify the neurological recovery (8.6±1.0) induced with 3AB alone (9.4±0.6). The association of 3AB with EX527, the SIRT1 inhibitor, suppressed the neurological improvement  induced with 3AB alone, showing a loss of neurological recovery when PARP and SIRT1 were both inhibited.3  and SRT1720 , showing histological protection when PARP was inhibited or SIRT1 activated. Treatment with EX527 had no effect on the striatal lesion volume (40±6 mm3), demonstrating that SIRT1 inhibition did not exacerbate the histological consequence of in vivo cerebral OS.Malonate induced a striatal lesion volume of 46±1 mm3 . This le3) induced with 3AB alone. These results showed no additive effect of PARP inhibition and SIRT1 activation. The association of 3AB with EX527, the SIRT1 inhibitor, suppressed the lesion reduction  induced with 3AB alone, showing a loss of neuroprotective effect when PARP and SIRT1 were both inhibited.The association of 3AB with SRT1720, the SIRT1 activator, did not modify the reduction of brain lesion (37±4 mmSRT1720 at the dose of 100 µM reduced about approximately 25% PAR production (73%±11%) whereas the lower doses had no effect  . These rEX527 had no effect on PAR production by PARP, whatever the dose tested  .in vivo had no effect on PARP activity in vitro.Therefore, both doses of SRT1720  and EX527  tested in vivo cerebral OS induced a NAD+ decrease in vivo and that 3AB, the PARP inhibitor, did not prevent it. However, 3AB increased SIRT1 activity after in vivo cerebral OS. SIRT1 activator, SRT1720, promoted neurological recovery and striatal lesion reduction, whereas SIRT1 inhibitor, EX527, did not worsen neurological and histological consequences of OS suggesting (1) the absence of endogenous SIRT1 activation during cerebral OS, (2) nuclear SIRT1 activation when PARP is inhibited by 3AB during cerebral OS, and (3) beneficial effect of exogenous SIRT1 activation by SRT1720 during cerebral OS. Moreover, the SIRT1 inhibitor, EX527, suppressed the beneficial effects of PARP inhibition on neurological deficit and lesion consecutive to OS. These data suggested that SIRT1 activation was implicated in beneficial effects of PARP inhibition in cerebral in vivo OS . Data are expressed as mean ± S.E.M. Differences were evaluated by one way-ANOVA followed by Dunnett test. **P<0.01 and ***P<0.001.Time course of NAD(TIFF)Click here for additional data file.