Inhibition of gamma-secretase presents a direct target for lowering Aβ production in the brain as a therapy for Alzheimer's disease (AD). However, gamma-secretase is known to process multiple substrates in addition to amyloid precursor protein (APP), most notably Notch, which has limited clinical development of inhibitors targeting this enzyme. It has been postulated that APP substrate selective inhibitors of gamma-secretase would be preferable to non-selective inhibitors from a safety perspective for AD therapy.In vitro assays monitoring inhibitor potencies at APP γ-site cleavage , and Notch ε-site cleavage, in conjunction with a single cell assay to simultaneously monitor selectivity for inhibition of Aβ production vs. Notch signaling were developed to discover APP selective gamma-secretase inhibitors. In vivo efficacy for acute reduction of brain Aβ was determined in the PDAPP transgene model of AD, as well as in wild-type FVB strain mice. In vivo selectivity was determined following seven days x twice per day (b.i.d.) treatment with 15 mg/kg/dose to 1,000 mg/kg/dose ELN475516, and monitoring brain Aβ reduction vs. Notch signaling endpoints in periphery.in vivo safety of ELN475516 was demonstrated in the 7d repeat dose study in wild-type mice, where a 33% reduction of brain Aβ was observed in mice terminated three hours post last dose at the lowest dose of inhibitor tested. No overt in-life or post-mortem indications of systemic toxicity, nor RNA and histological end-points indicative of toxicity attributable to inhibition of Notch signaling were observed at any dose tested.The APP selective gamma-secretase inhibitors ELN318463 and ELN475516 reported here behave as classic gamma-secretase inhibitors, demonstrate 75- to 120-fold selectivity for inhibiting Aβ production compared with Notch signaling in cells, and displace an active site directed inhibitor at very high concentrations only in the presence of substrate. ELN318463 demonstrated discordant efficacy for reduction of brain Aβ in the PDAPP compared with wild-type FVB, not observed with ELN475516. Improved in vivo activity of ELN318463 suggests that the potency of gamma-secretase inhibitors in AD transgenic mice should be corroborated in wild-type mice. The discovery of ELN475516 demonstrates that it is possible to develop APP selective gamma-secretase inhibitors with potential for treatment for AD.The discordant  The principal pathological features of Alzheimer's disease (AD), comprised of neurofibrilary tangles and amyloid plaques, are posited by the amyloid cascade hypothesis -3 to be Immunotherapy targeting Aβ has been demonstrated to modify amyloid ,5 as welin vivo is a well-documented small molecule target for lowering brain, CSF, and plasma Aβ peptide [Pharmacological inhibition of gamma-secretase  peptide -18, and  peptide ,19-22. G peptide , as well peptide . Consequ peptide -27. Howe peptide -30, that peptide -33, and  peptide . Support peptide -38 as we peptide  of NotchApproaches to managing gastro-intestinal side effects of first generation GSIs via intermittent dosing ,41 or glin vitro selectivity of our lead compound, ELN475516 translates into improved in vivo safety in a mouse model that is sensitive to histological and molecular end-points associated with inhibition of Notch signaling.The pharmacological and genetic evidence cited above validate gamma-secretase as a target for lowering Aβ production as well as non-target organ side effects due to inhibition of Notch signaling. Together, the observations support the hypothesis that APP selective gamma-secretase inhibitors offer one approach toward potentially safer gamma secretase targeted therapeutics for AD. Toward that end, we report here the discovery of novel APP selective inhibitors of gamma-secretase discovered from a high throughput screen of a chemical library enabled by novel assays for comparing APP and Notch cleavage by gamma-secretase. We confirmed that the improved et al. [et al. [ELN46719 is the 2-hydroxy-valeric acid amide analog of LY411575  . ELN318463 was described by Zhao et al. , and ELN [et al. .in vitro digestion of NotchΔE substrate by gamma-secretase enzyme, but not the uncleaved NotchΔE substrate, was confirmed by Western blot assay. The sequence of this epitope is conserved between human and mouse Notch1.Notch intracellular domain (NICD) neo-epitope monoclonal antibody (mAb) 9F3 was generated by immunizing mice with VLLSRGGC  coupled to maleimide activated sheep anti-mouse IgG. Spleenocytes from the highest antibody titer mouse were fused with mouse myeloma cells. Hybridomas were screened against VLLSRGGC and counter screened against GCGVLLSR. Monoclonal antibody 9F3 only recognized VLLSRGGC peptide. The specificity of 9F3 for recognition of the amino-terminal neoepitope of NICD product, revealed from E. coli expressed MBP-APPc125sw fusion protein substrate [ubstrate , was purNotchΔEΔC substrate: A Notch fusion protein was constructed so as to be analogous to the APP C99 fragment as described [Z11886). The amino-terminus of the fusion protein begins at residue #1711 of mouse Notch (numbered from initiator methionine residue), and extends carboxy-terminal to residue #1809, followed by in-frame fusion with HA epitope tag and six histidine residues prior to the stop codon. The two carboxy-terminal epitope tags are separated by an "LE" di-peptide spacer. The epitope tags were incorporated to facilitate ELISA detection of the NICD cleavage product using anti-HA antibody, and purification of the fusion protein substrate from E.coli lysates using nickel column affinity chromatography, respectively. Following overnight expression of the fusion protein in E.coli, the substrate is purified on a nickel-sepharose column from lysates. Purity of the 16 kD Notch fusion protein substrate eluted from the nickel-affinity column is confirmed by SDS-PAGE. Fractions containing the desired protein were pooled, adjusted to 0.5 to 0.6 mg/mL final concentration in 3M Guanidine-HCl, 0.1% Triton X-100 with 20 mM DTT, and stored at -80°C in aliquots until use in the enzyme reaction. This protein is not stable over long periods, and will aggregate even in the above storage buffer at -80°C. Hence, a new batch is prepared every three months to ensure assay quality. The substrate is desalted over a NAP-25 column immediately prior to use, protein concentration is determined by BCA assay , and diluted 20X for incubation with enzyme.escribed . SpecifiNICD ELISA standard: A human NICD standard was produced as a fusion protein from a construct in the bacterial expression vector pCal-kc . The expression construct was modified to encode an initiating methionine followed by an enterokinase cleavage at the N-terminus, fused in frame to the gamma-secretase product NICD , an 8 residue spacer comprising the sequence PAAAAAA, and an HA epitope tag fused in frame with vector derived kemptide peptide, thrombin cleavage site and carboxy-terminal calmodulin binding peptide. The fusion protein was produced in E.coli, affinity purified using a calmodulin affinity resin and assayed for protein concentration. The protein was then cleaved with enterokinase in order to produce the free N-terminus of the NICD product. The enterokinase cleaved protein was separated by SDS-PAGE on a 12% Tris-glycine gel. Enterokinase cleavage products are detected by Western blotting using an antibody against the HA-tag. Densitometry was used to measure the efficiency of the enterokinase cleavage. The estimated concentration of enterokinase cleaved NICD standard is calculated . The standard was stored at -40°C. On the day of each assay, an aliquot of the NICD standard is serially diluted 1:1 in casein diluent to generate a standard curve with 0 to 200 ng/mL NICD.2, 0.2 mM CaCl2 + protease inhibitors, 5 mL/g cell pellet), resuspended in an equal volume of hypotonic buffer (homogenization buffer minus sucrose), subjected to freeze thaw , and re-centrifuged at 100,000 x g for 20 minutes at 4°C. The membrane pellet was snap frozen on dry ice, and stored at -80°C until extraction. Thawed membranes were washed in 1 M carbonate buffer  at 4°C for 15 minutes, centrifuged 100,000 X g as explained above, and resuspended in hypotonic wash buffer. Washed membrane pellets were resuspended in 0.5X final volume of solubilization buffer  and extracted in detergent containing 1.49 mg/mL BigCHAP (ICN), diluted fresh 1:10 from 50X stock (149 mg/mL in H2O), at 4°C for 1.5 h. Detergent extracts were centrifuged at 100,000 x g as explained above, and supernatant containing solubilized gamma-secretase aspirated for WGA chromatography. Peak eluate fractions were combined, and stored in 10 mM Hepes, pH 7.5, 1 M N-acetyl-D-glucosamine, 1 mM MgCl2, 0.2 mM CaCl2, 0.6 mg/mL TypeV brain extract , 5.6 mg/mL BigCHAP.An enriched detergent solubilized gamma-secretase enzyme preparation from IMR-32 neuroblastoma cells was obtained by membrane fractionation of crude homogenates using ultracentrifugation, followed by lectin affinity chromatography. Briefly, a 100,000 g membrane pellet of post nuclear superantant from IMR32 frozen cell pellets was homogenized in buffer , 1 mM MgClin vitro gamma-secretase assay was designed to measure the specific proteolytic cleavage of an APP substrate (MBP-APPc125Sw fusion protein) at the Aβ40 site, or cleavage of Notch fusion protein at the Notch ε-site (corresponding to N-terminus of NICD). The substrates were incubated in separate reactions, in triplicate, with an enriched preparation of the enzyme from IMR-32 cells, and the amount of specific product formed was measured using a sandwich ELISA comprised of an Aβ40-specific capture antibody, 2G3, and a biotinylated Aβ17-28 reporter antibody, 6H9 for APP substrate, or Notch ε-site specific antibody 9F3, specific for the amino-terminus of the NICD (beginning at V1755 of human Notch protein), as capture and anti-HA epitope tag antibody as the reporter for Notch substrate. Incubation of triplicate reactions in the presence of a range of inhibitor concentrations enabled determination of a dose-response for enzyme inhibition by the test compound and calculation of an IC50 value. Data analysis was performed using XLfit software package .The 2PO4.H2O, 8 mM Na2HPO4.7H2O, 8 mM NaN3, 150 mM NaCl, 0.05% (volume/volume) Triton X-405 0.6% (w/v) BSA, (globulin free).Recombinant APP or Notch fusion protein substrates (purified as described above) were incubated in reaction buffer  at 20 μg/mL or 30 ug/mL, respectively, with approximately 0.4 mg/mL enzyme preparation  for 2 h at 37°C in the presence of protease inhibitors  in the presence of a concentration range of test inhibitor compounds. Reactions were quenched with 0.1% SDS for 10 minutes at room temperature, followed by addition of equal volume of specimen diluent  and incubated overnight along with Aβ1 to 40 standards . The ELISA was developed the following day by sequential one hour incubations with biotinylated 6H9 reporter antibody  for one hour at room temperature, and Streptavidin-Alkaline Phosphatase Conjugate , 1:1,000 in specimen diluent, one hour room temperature. The ELISA was developed using fluorescent alkaline phosphatase substrate, and the plate is read at 360 nm excitation/460 nm emission, gain approximately 50, in a CytoFluor 4000 . The plates were washed 3X each with Tris-buffered saline/0.05% Tween-20 between incubations with reporter antibody, detecting antibody, and fluorescent substrate.in vitro gamma/Notch reactions were diluted 1:1 with casein diluent buffer containing 500 mM NaCl, 0.02% TritonX-100 and plated onto Notch ε-cleavage site specific neoepitope mAb 9F3  coated immulon plates, for capture of the NICD reaction product overnight at 4°C. NICD standards, prepared as described above, were serially diluted 2X over a concentration range from a starting concentration of 50 ng/mL for establishing a standard curve. The NICD reaction product and standards captured on the plate are detected with biotinylated-HA antibody , followed by alkaline phosphatase conjugated streptavidin . The plate was washed between each incubation step with Tris-buffered saline containing 0.1% Tween-20. The alkaline phosphatase reaction product is detected by incubation with 100 μl/well Fluorescent Substrate A for 15 minutes at room temperature, and detected using Cytofluor plate reader at 360 nm excitation, 460 nm emission.Quenched sw/NotchΔE/CBF) were isolated and expanded for characterization of Aβ secretion and NICD responsive reporter gene activity . Based on optimal Aβ secretion, and highest signal/background of reporter gene activity, a clone designated as SNC-204B8 was selected as the dual-assay stable line for subsequent profiling of APP selective γ-secretase inhibitors. Activity and selectivity of compounds for inhibition of Aβ production versus Notch signaling was determined by plating the cells in 96-well plates in media lacking ELN46719 overnight. The following day, media was replaced and supplemented one hour later with an equal volume of additional media containing test compounds over a concentration range 0 nM to 40,000 nM . The cells were treated with compounds overnight at 37°C. The following day (Day 3), conditioned media was aspirated from cells for determination of Aβ1-x levels by ELISA [In order to test the potency and selectivity of gamma-secretase inhibitors against two substrates (APP and Notch) simultaneously, we developed a dual-assay CHO cell line stably expressing APPSw, a Notch substrate lacking the ecto-domain, and a Notch responsive Luciferase reporter gene. As a first step, CHO cells stably over-expressing the APPsw were established. These cells secrete high levels of Aβ peptide into the conditioned media from endogenous β- and γ-secretase enzyme activity. A Notch intracellular domain responsive reporter gene, and the constitutive gamma-secretase Notch substrate, NotchΔE, were stably introduced into CHOsw cells in a two-step process. CHOsw cells were first stably transfected to express a NICD responsive luciferase reporter gene construct (pGL2-CBF-Luc) . Numerouby ELISA , and cel50 and approximately 10 X ED50 values in the SNC assay. Different anti-APP antibodies were used to test the compounds for effects on Aβ, total secreted APP, β-sAPP, full length-APP and APP CTFs  as noted below. Secreted Aβ in the conditioned media was quantified by ELISA using 2G3/3D6, which detects Aβ (1 to 40). The full length APP and CTF Western blots were probed with anti-APP-c-terminal specific antibody (Sigma). Western blots of the conditioned medium samples  were probed with antibody 8E5 [APP metabolites were analyzed by Western blot of SDS-PAGE fractionated cell extracts and media derived from HEK293 cells stably expressing APPsw mutation following overnight treatment with different gamma-secretase inhibitors. Equal concentrations of protein  from cell extracts were loaded for Western blot analysis. As reference GSI's we used LY411575, and the Merck active site directed isostere L-685458 for comparison with ELN-318463. Compounds were tested at two doses, approximately 1 X EDbody 8E5  which rebody 8E5 ,68 whichA displacement assay employing affinity capture of gamma-secretase complex with a biotinylated analog of active site isostere L685,458  was devead libitum. FVB mice were treated orally with a single dose (n = 7/dose) of compound as indicated in the associated Figures. The test articles were formulated at various concentrations in a 10% Solutol®/water vehicle  and dosed at 5 mL/kg via oral gavage. Control animals were dosed with vehicle. Animals were sacrificed at three hours post dose, unless indicated otherwise. Cortical brain samples were collected, frozen on dry ice and stored at -80°C until homogenization for determination of Aβ and compound levels. Thymus was collected, frozen in liquid nitrogen, stored at -80°C for analysis of Hes1 levels. Blood samples were collected by cardiac puncture, processed to plasma, frozen on dry ice and stored at -80°C until determination of compound levels by LC-MS-MS. Statistical analysis of data was performed using one-way ANOVA followed by Dunnett's.All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Elan Pharmaceuticals and conducted in accordance with its guidelines. Female, two- to three-month old, FVB/N mice were purchased from Taconic . Mice were housed four to five to a cage on a 12-hour light/dark schedule. Water and food were available N = 5 per treatment group). The test articles were formulated at various concentrations in a 10% Solutol®/water vehicle and dosed at 5 mL/kg via oral gavage. Control animals were dosed with vehicle. All animals were sacrificed at three hours post last dose. Cortical brain samples were collected, frozen on dry ice and stored at -80°C until homogenization for determination of Aβ and compound levels. Blood was collected, (via cardiac puncture), processed for CBC, or processed to plasma and frozen on dry ice and stored at -80°C until determination of compound levels. Duodenum and ileum, as well as other tissues, were collected from each treatment group and fixed in 10% formalin for histopathology evaluation. Body weight was reported as percent change from the base line and compared to vehicle control group in the one-way ANOVA Dunnett's test for statistical analysis. Relative organ weight was calculated by the absolute organ weight/body weight. Statistical analysis was done by Prism software/one-way ANOVA (and nonparametric) Dunnett's test for all toxicity endpoints. Most endpoints examined were significant at P < 0.001 except on one parameter which was P < 0.01 and is noted in the appropriate figure legend. Duodenum and ileum tissues were sectioned and double stained with H&E (hematoxylin and eosin) plus PAS (periodic acid-Schiff stain) for histopathologic evaluation.FVB mice, female, two to three months old, were treated orally with ELN475516  or LY411575 (5 mg/kg/dose) twice per day (b.i.d.) for seven days  as the capture, and 2G3  as the reporter. This assay estimates the concentration of Aβ peptides ending at amino acid 40, without specificity for amino termini upstream of residue 16. Statistical significance was determined by ANOVA.Cortical brain samples were homogenized in 5 M guanidine and AβTissues were homogenized in lysis buffer and processed for total RNA extraction using the QIAGEN Rneasy 96-well plate technology with post-column Dnase treatment. The concentration of RNA per sample was measured by a RiboGreen RNA quantitation assay (Invitrogen). Math1  or Hes1 mRNA levels were quantified by a TaqMan RT-PCR assay and results were normalized by RNA sample concentration. The sequences of the primers and probes employed in the Taqman assay were as follows: Hes1 forward primer: CGGCTTCAGCGAGTGCAT; Hes1 reverse primer: CGGTGTTAACGCCCTCACA; Hes1 probe: AACGAGGTGACCCGCTTCCTGTCC. Math1 Forward primer: AGCTGGACGCTTTGCACTTT; Math1 reverse primer: TCTGTGCCATCATCGCTGTT; Math1 Probe: 5'FAM-CAGCTTTCGAGGACCGGGCCC-TAMRA3'. All Taqman reagents were acquired from Applied Biosystems, Inc., Foster City, CA, USA.m= 358 nM, Notch substrate Km = 1.52 μM, Figure S1 in Additional file To enable discovery of novel APP selective gamma-secretase inhibitors, we developed biochemical assays for substrate cleavage followed by ELISA employing neo-epitope specific monoclonal antibodies for detection of products. A detergent solubilized, lectin affinity column enriched, enzyme preparation derived from IMR-32 neuroblastoma cells was incubated with either recombinant APP-C125, or a Notch fusion protein substrate. These substrates replicate natural processing of a site equivalent to the carboxy-terminus of Aβ40, or the S3/ε-site of Notch, respectively, with Michaelis-Menten kinetics  were characterized in secondary assays for dose response, non-specific inhibition , and selectivity for inhibition of APP cleavage versus Notch cleavage by gamma-secretase. A number of hits comprising a novel series of p-chlorobenzene-caprolactam sulfonamide compounds demonstrated concentration dependent and APP > Notch selective inhibition of gamma-secretase, as exemplified by ELN158162 , which displayed an IC0 Figure .Sw, rat NotchΔE [CBF-Luciferase reporter gene [1-x using conditioned media harvested from cells, and Notch signaling was detected using a luciferase reporter assay in cellular lysates from cells treated overnight with a concentration range of inhibitor compounds. The profile of reference non-selective gamma-secretase inhibitors compared with the HTS hit and selective lead compounds in the SNC dual cell assay corroborated the biochemical enzymatic assay data, demonstrating APP selective inhibition of cellular gamma-secretase by the screening hit and select lead compounds  MBP-C125 substrate, ELN318463 and ELN475516 were able to displace the active site isostere . These values represent an approximately 2,000X and 67X multiple over the IC50 values of the two compounds in the gammaAPP assay.Biochemical characterization of the inhibitors revealed they behave as classical GSIs in that they elevate C99 with no effects on sAPPα or sAPPβ Figure , and areIn vivo testing of a benzene caprolactam sulfonamide, ELN318463 [1-x species in PDAPP mice . The lack of a dose response in Aβx-40 species in PDAPP & FVB mice compared with Aβ1-x species in PDAPP mice can not be explained by differences in terminal compound levels in the target organ at the two doses tested, nor by assay differences. Brain levels of ELN318463 at 30 mg/kg were 0.754 μM in FVB brains and 0.69 μM in PDAPP brains, and at 100 mg/kg dose the levels were 2.7 μM in FVB brains and 1.87 μM in PDAPP brains. Discordance between Aβ1-40 and Aβx-40 in BACE KO or wild-type BACE inhibitor treated mice has been reported [1-x vs. Aβx-40, as observed by Nishitomi et al. [LN318463 , possesse Figure . In conte Figure  was equireported . Howeveri et al. , becausein vitro potency, while retaining APP selectivity of the caprolactam sulfonamide parent lead series  as well as cortical Aβx-40 in wild type FVB mice  and thymic Hes-1 mRNA (60%) , and up to a 71% reduction at 300 mg/kg Figure . A stati>300 mg/kg. Dose dependent increases in brain and plasma exposure were seen in mice at the three doses tested in this single dose study, where total exposure in the 300 mg/kg group was 24X the 30 mg/kg group, and 3X the 100 mg/kg group  at 5 mg/kg b.i.d. demonstrated 83% reduction of brain Aβ , and the minimum toxic dose is defined as the dose producing a statistically significant elevation of Math1 mRNA levels. Hence, the in-vivo selectivity of ELN475516 corroborates the improved biochemical as well as cellular selectivity for inhibition of Aβ production from APP by gamma-secretase, compared with inhibition of Notch processing and Notch signaling. Limiting N-glucuronidation precluded longer-term studies with ELN475516 in mice (or other models). However, ELND006, a lead optimized analog derived from ELN475516, was well tolerated for three and six months in a chronic PDAPP mouse study at doses which affected AD pathology in the absence of any toxicity [We assessed cell homeostasis in the intestinal tract as a more sensitive indicator of ct Table  and 9d, ct Table , the chatoxicity .in vitro. The selectivity of our lead series was confirmed in a cellular assay. Comparison of APP selectivity of non-selective reference GSIs in cell-free compared with cellular (SNC) assay reveals a basal 15-22X right-ward shift for selectivity in the cellular assay compared with the cell-free assay with reference non-selective GSI's L-685,458 and LY411575. This observed shift in selectivity between a cell-free assay which measures inhibition of substrate cleavage, versus downstream signaling events, likely reflects a difference between inhibition of Notch cleavage vs. Notch signaling. Our observation is consistent with findings that Notch signaling was only reduced 35 to 48% in cells treated with concentrations of peptidomimetic GSIs which abolished NICD formation to below detectable levels [To discover novel APP-selective inhibitors of gamma-secretase, we developed cell-free assays employing recombinant APP or Notch substrates incubated with an enriched enzyme preparation derived from IMR-32 neuroblastoma cells. We utilized this biochemical assay for APP cleavage by gamma-secretase in a high-throughput screen of a chemical library and discovered amino-caprolactam sulfonamide leads, which demonstrated APP-selective inhibition of gamma-secretase activity e levels .1-X in a concentration dependent manner in PDAPP mice; however, reduction of Aβx-40 was nominal and did not exhibit a concentration dependent effect in two mouse lines tested (PDAPP and FVB). This discordance in a concentration effect on reduction of brain Aβ species cannot be explained by differences in drug levels in the two mouse lines, nor by assay differences, as discussed above. We are not able to explain the basis for this discrepancy; however, our observation suggests efficacy determination of GSI's in AD transgene models should be corroborated in wild type mice .The benzene caprolactam sulfonamide ELN381463  lowered in vivo activity against Aβ1-X in PDAPP transgene as well as Aβx-40 in wild type mice, retaining favorable 123X APP-selective inhibition of gamma-secretase. The potency and cellular selectivity of ELN475516 compares favorably with recently disclosed GSI-953 [50 values for inhibition of Aβ production in the SNC assay of 8.0 nM, and 1.13 nM, respectively. The novel sulfonamide inhibitors we describe exhibit all the signatures associated classical gamma-secretase inhibitors: elevation of C99 levels and equipotent inhibition of Aβ40 and Aβ42 production without affecting secreted APP levels. These properties are consistent with those reported for a novel thiophene sulfonamide gamma-secretase inhibitor, GSI-953 [50 for Aβ inhibition by the compound. This novel observation suggests that occupancy of the substrate binding site on gamma-secretase by APP induces a conformational change bringing the sulfonamide binding site and isostere binding  sites in close proximity, enabling displacement of isostere by sulfonamide at supra-physiological concentrations. Although we did not test the effect of other substrates in the competitive displacement assay, it will be interesting to compare the effect, for example, of Notch substrate on displacement of active site isostere by test GSI's in future studies.Lead optimization of the caprolactam sulfonamide enabled discovery of a novel pyrazolylazabicyclo(3.3.1)nonane sulfonamide, ELN475516 . ELN475 GSI-953  and BMS- GSI-953 ,79, whic GSI-953 . In bind GSI-953 , as well GSI-953 . Interesin vivo selectivity >65 based on dose as well as terminal compound concentration in plasma. For this study, a minimum efficacious dose was defined as that dose effecting >25% reduction at study termination . Terminal plasma compound levels at the highest dose tested were 12.3 μM, a 17-fold multiple over the cellular IC50 for inhibition of Notch signaling. The utility of the mouse as a model for screening of lead gamma-secretase inhibitor candidates against Notch endpoints has been previously described [et al. to avoid potential transgene model specific influences on in vivo inhibitor potency against Aβ endpoints. For example, substrate expression levels [in vitro potencies of gamma-secretase inhibitors. If substrate levels, or PS1 isoform contributes to in vivo potency shifts, the effect could potentially confound estimates of in vivo selectivity in an APP and/or PS1FAD transgene mouse model. Consistent with the influence of APP expression levels on inhibitor potency, we observed a 33% reduction of brain Aβx-40 in wt FVB mice at three hours post treatment with 30 mg/kg GSI-953, compared with 5 mg/kg producing a similar magnitude effect in Tg2567 mice [The improved selectivity of ELN475516 was tested in a mouse seven-day repeat dose model. Relative to LY411575, a reference non-selective gamma-secretase inhibitor, ELN475516 demonstrated significant reduction of brain Aβ at the lowest dose tested , with no overt signs of toxicity at the highest dose tested  at the end of the study, indicating an n levels , as welln levels ,82-84 ha567 mice , althougin vivo active (bicycle(4:2:1) nonane sulfamide GSI's [It is noteworthy that all substrate selective GSIs described to date are based on sulfonamide pharmacophore nonane sulfonamide. ELN475156 represents a validated foundation for further lead optimization to discover APP selective second generation GSIs with improved safety and drug like properties suitable for chronic AD therapy [The molecular basis of sulfonamide based GSIs APP selectivity remains to be fully elucidated. We have reported that one contributor of selectivity may be the more potent inhibition of PS1 gamma-secretase compared with PS2 gamma-secretase  by sulfo therapy ,103,104.in vitro and in vivo selectivity of ELN475516 demonstrates that discovery of APP selective GSI's is feasible, and that APP selective GSI's offer potentially safer candidates as therapeutics for Alzheimer's disease.Our results showing discordance in reduction of brain Aβ between PDAPP and wild-type FVB mice following treatment with ELN318463 highlight the importance of evaluating GSI's for potency and selectivity in non-transgene models of Alzheimer's disease. The R)-1-(((4-chlorophenyl)sulfonyl)amino)ethyl)-5-fluorobenzenebutanoic acid; CHES: 2-(N-Cyclohexylamino)ethanesulfonic acid; CHO: Chinese hamster ovary; CSF: cerebrospinal fluid; CTF: carboxy-terminal fragment; DTT: dithiothreitol; DMSO: dimethyl-sulfoxide; ELISA: enzyme like immunoadsorbent assay; ELN158162: 4-Chloro-N-ethoxy)benzyl)-N-((3R)-2-oxoazepan-3-yl)benzenesulfonamide; ELN-318463: N-(4-Bromobenzyl)-4-chloro-N-((3R)-2-oxoazepan-3-yl)benzenesulfonamide; ELN-475516: 10-((4-Chlorophenyl)sulfonyl)-4,5,6,7,8,9-hexahydro-1H-4,8-epiminocycloocta(c)pyrazole; FAD: familial Alzheimer's disease; GSI: gamma-secretase inhibitor; HA: hemagglutnin antigen; H&E, hematoxylin and eosin; HEK, human embryonic kidney; Hes, hairy enhancer-of-split; HTS: high through-put screen; KO: knock-out,; L-685,458: tert-Butyl -6-((S)-1-((S)-1-amino-1-oxo-3-phenylpropan-2-ylamino)-4-methyl-1-oxopentan-2-ylamino)-5-benzyl-3-hydroxy-6-oxo-1-phenylhexan-2-ylcarbamate; LC: liquid chromatography; mAb: monoclonal antibody; LY 411575, (S)-2-((S)-2--2-hydroxyacetamido)-N-((S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzoazepin-7-yl)propanamide; MES, 2(N-Morpholino)ethanesulfonic acid; Math: mouse atonal homolog; MS: mass spectrometry; Nct: Nicastrin; NICD: Notch Intracellular domain; PAGE: polyacrylamide gel electrophoresis; PAS: periodic acid-Schiff stain; PDAPP: platelet derived growth factor promoter driven AD transgene mouse; sAPP: secreted APP; sAPPβ: secreted APP product of BACE cleavage; SNC: a stable CHO cell line co-expressing APPSw, rat NotchΔE, and an NICD responsive CBF-Luciferase reporter gene; PS: presenilin; RT-PCR: reverse-transcriptase polymerase chain reaction; WGA: wheat-germ agglutnin.Aβ: amyloid beta-peptide; AD: Alzheimer's disease; APP: amyloid precursor protein; APPsw: Swedish familial Alzheimer's disease mutant isoform of APP: BACE: beta-APP cleaving enzyme; BMS299897: 2- stock in Elan, and are inventors on patent filings resulting from the work described herein.GSB, EFB, JB, MSD, JH, SH, LHL, MNM, LM, MLN, KQ, PS, JAT, AWG, JMS, AWK, GS, MAP, SBF, and DS participated in research design. SH , AL, DLA, X-HC, MSD, TE, EG, KH, TH, JJJ, PSK, DK, RLH, ML, JM, MNM, SM, JLM, RM, MLN, HN, LN, LR, CMS, JS, FS, BS, KT, PT, XMY, MY, JW and YZ conducted experiments. GSB, SH, EFB, AL, DLA, JB, RB, MB, X-HC, MSD, EG, JH, KH, TH, JJJ, PSK, LHL, ML, MNM, JLM, RM, LM, MLN, LN, KQ, CMS, PS, BS, JAT, XMY, YZ, AWG, JMS, AWK, DS and GS performed data analysis. GSB, SH, EFB, MB, JH, LHL, AWG, DN and DS wrote, or contributed to writing of manuscript.In vitro enzyme kinetics of gamma-secretase preparation on APP and Notch substratesFigure S1. . The results show that the in vitro assay displays Michaelis-Menten kinetics for substrate.Click here for fileFigure S2. Neo-epitope specificity of Notch antibody recognizing Notch Intracellular domain.Click here for fileFigure S3. Equipotent inhibition of Aβ40 and Aβ42 by gamma-secretase inhibitors in a cellular assay.Click here for fileFigure S4. Flow-chart of displacement assay with active-site binding affinity ligand in the presence or absence of added APP substrate plus competing sulfonamide.Click here for fileFigure S5. Evolution of sulfonamide structure activity relationship from caprolactam sulfonamides to fused bi-cyclic sulfonamides.Click here for file


Dpt-specific IgG antibodies were purified by ammonium sulfate precipitation followed by protein-G affinity chromatography. Purity was checked by SDS-PAGE and immunoreactivity by slot-blot and immunoblot assays. The blocking activity was evaluated by inhibition ELISA. The electrophoretic profile of the ammonium sulfate precipitated fraction showed strongly stained bands in ligand fraction after chromatography, compatible with molecular weight of human whole IgG molecule. The purity degree was confirmed by detecting strong immunoreactivity to IgG, negligible to IgA, and no reactivity to IgE and IgM. Dpt-specific IgG fraction was capable of significantly reducing levels of IgE anti-Dpt, resulting in 35%–51% inhibition of IgE reactivity to Dpt in atopic patients sera. This study showed that allergen-specific IgG antibodies purified from mite-allergic patients sera block the IgE recognition of Dermatophagoides pteronyssinus antigens. This approach reinforces that intermittent measurement of serum allergen-specific IgG antibodies will be an important objective laboratorial parameter that will help specialists to follow their patients under SIT.One of the purposes of specific immunotherapy (SIT) is to modulate humoral immune response against allergens with significant increases in allergen-specific IgG levels, commonly associated with blocking activity. The present study investigated  Dermatophagoides pteronyssinus, play an important role to induce respiratory allergic diseases, particularly asthma and allergic rhinitis, in genetically predisposed individuals [Allergic diseases are considered one of the major health problems worldwide and constitute a breakdown in the immune tolerance against natural exposure to environmental antigens . Among tividuals , 3. Receividuals . The immividuals .Several studies have been performed focusing on the development of new treatments beyond the pharmacotherapy already established, aiming to relieve the symptoms caused by exacerbated responses of the organism against allergens . Allergein vitro the blocking capability of specific IgG antibodies purified from mite-allergic patients sera on the IgE reactivity to D. pteronyssinus.Considering that a successful SIT correlates with decreasing of clinical symptoms and increasing of allergen-specific IgG antibody levels, the aim of this study was to investigate Dermatophagoides pteronyssinus (Dpt) allergen extract from a panel of standardized aeroallergens  as follows: house dust mites ; cockroaches (Blattella germanica and Periplaneta americana); mold ; and pet danders (Felis domesticus and Canis familiaris). The exclusion criteria were positive results in rheumatoid factor assay , the use of antihistamines in the previous week to the skin test, and previous or current immunotherapy. Thirty-six patients, male and female, aged 18 to 60, with clinical history of allergic rhinitis (atopic group) were recruited from the Laboratory of Allergy and Clinical Immunology, Federal University of Uberlandia, Uberlandia, MG, Brazil. As inclusion criteria, patients should have positive skin prick test (SPT) to at least Fifteen volunteers, healthy subjects, male and female, aged 18 to 60, were selected based on the absence of clinical history or symptoms of allergic rhinitis and negative SPT to a panel of standardized aeroallergens (nonatopic group). In parallel, blood samples (10 mL) were collected from all individuals, and the serum was stored at −20°C until serological assays. The study was approved by the Ethics Committee in Human Research of the Federal University of Uberlandia, and written informed consent was obtained from all participants.μg/well; Hollister-Stier Laboratories, Spokane, WA, USA), blocked with phosphate-buffered saline  containing 0.05% Tween 20 and 1% bovine serum albumin (PBS-T-BSA) for IgE and PBS-T-BSA 0.1% for IgG1 and IgG4, and then incubated with serum samples diluted 1 : 2 (IgE), 1 : 10 (IgG1), or 1 : 5 (IgG4) for 2 hours at 37°C. After washing, plates were incubated with biotinylated secondary antibodies as anti-human IgE , Gaithersburg, MD, USA), anti-human IgG1 , or anti-human IgG4  for 1 hour at 37°C and subsequently with streptavidin peroxidase . The assay was developed with ABTS-peroxidase substrate system (KPL), and optical density (OD) values were determined at 405 nm. Antibody levels were expressed as ELISA index (EI) according to the following formula: EI = OD test sample/cutoff, where the cutoff was established as the mean OD value of negative control sera plus 3 standard deviations. EI values >1.2 were considered to be positive in order to exclude borderline reactivity values close to EI = 1.0.All serum samples were assessed by enzyme linked immunosorbent assay (ELISA) for measuring levels of IgE, IgG1, and IgG4 to Dpt as previously described , 15, witFive serum samples of each patient group were selected to constitute the Dpt-specific (atopic) and nonspecific (nonatopic) serum pools. The selection criteria were based on the highest (EI > 2.0) and lowest (EI < 1.0) values of reactivity to both IgG1 and IgG4 to Dpt allergen in atopic and nonatopic groups, respectively. Thus, these criteria would allow obtaining a maximal diversity in antigen/epitope recognition, according to different seroreactivity profiles and intensity of reaction observed in immunoenzymatic assays, favouring the identification of biological phenomena rather than individual immune responses. Initially, the serum albumin from specific and nonspecific pools was partially depleted by salting-out precipitation using 40% ammonium sulfate . The supµm; Bio-Rad Laboratories Inc., Hercules, CA, USA) were coated with serum pools, S 40% and P 40% fractions, and bovine serum albumin (BSA) as negative control, using the Mini Protean II Multiscreen Apparatus (Bio-Rad). After blocking with PBS-T plus 5% skim milk, membranes were incubated with detection antibodies labeled with peroxidase , biotin , and monoclonal antibody to IgE . After incubation with streptavidin peroxidase  or anti-mouse IgG/peroxidase , when appropriate, the membranes were revealed with DAB tablets (Sigma). Bands were analyzed using the ImageJ 1.46 software .Nitrocellulose membranes , previously equilibrated with binding buffer . Samples were diluted 1 : 1 in binding buffer, applied to the column, and washed with at least 10 volumes of binding buffer. Total IgG was eluted in elution buffer , and 1 mL fractions were collected. After pH neutralization with 1 M Tris-HCl, pH 9.0, absorbance was read at 280 nm. Values of absorbance and pH of each fraction prior to neutralization were used to build the chromatographic profile. The purity of total IgG obtained from the affinity chromatography was checked using SDS-PAGE on 8% gels as described above. To confirm the immunoreactivity of the eluted fractions, immunoblots were performed for detection of IgG, IgA, IgE, and IgM antibodies. Briefly, the fractions previously separated on 8% SDS-PAGE were electrotransferred onto nitrocellulose membranes, and blotting efficiency was validated by reversible Ponceau S staining. Membranes were blocked with 5% skim milk in PBS-T. After blocking, blots were incubated with the respective detection antibodies to IgG, IgA, IgE, and IgM as described in slot-blot assays and revealed with DAB. After monitoring the eluted fractions by 8% SDS-PAGE, the samples containing IgG were pooled, dialyzed, and concentrated against PBS by using Amicon system. Protein concentration was determined using the Lowry method . μg/well), blocked with PBS-T-BSA 0.1%, and subsequently incubated with specific and nonspecific IgG fractions diluted from 40 to 2.5 μg/well. Subsequent steps were similar to the ELISA for detection of Dpt-specific IgE, IgG1, and IgG4 as described above.An indirect ELISA was carried out to determine the optimal concentrations of specific and nonspecific IgG fractions through detection of IgG1 and IgG4 anti-Dpt as well as residual specific IgE as previously described , 15, witμg/well), blocked with PBS-T-BSA, and incubated with the optimal concentration of specific or nonspecific IgG fractions for 1 h at 37°C. Then, the serum pools from atopic patients were diluted 1 : 2, incubated for 1 h at 37°C, and followed by incubation with biotinylated anti-human IgE . Subsequent steps were similar to the ELISA for detection of IgE anti-Dpt as described above. Results were reported as absorbance values at 405 nm and inhibition percentage as follows: % inhibition = 1 − (DO inhibited/DO uninhibited) × 100 [To evaluate the blocking activity of the Dpt-specific IgG fractions on IgE reactivity to Dpt allergen, an inhibition ELISA was developed by using three serum pools of atopic patients with different positivity for Dpt-specific antibody classes as follows: pool I ; pool II ; and pool III . Briefly, plates were coated with Dpt (2 d) × 100 .t-test. Differences between the groups were analyzed by one-way ANOVA using the Bonferroni posttest (IgE reactivity and inhibition ELISA). Correlation between the levels of antibody classes was analyzed by the Spearman correlation test. Values of P < 0.05 were considered statistically significant. Statistical analysis was performed using GraphPad Prism version 5.0 (GraphPad Software Inc.). Comparison between levels of IgE, IgG1, and IgG4 antibodies to Dpt within the groups was analyzed by the Mann-Whitney test. Differences in slot-blot data were determined by Student's D. pteronyssinus, and D. farinae (97%) than to Blomia tropicalis (64%) and to other aeroallergens (<54%) (P < 0.0001). The atopic and nonatopic groups were comparable regarding the sex and age. The demographic and clinical characteristics of the study subjects are shown in D. pteronyssinus were higher in atopic patients than in nonatopic subjects , although the positivity was similar between groups. In contrast, levels and positivity of IgG4 anti-Dpt were similar between the groups. Significant positive correlations were found between Dpt-specific IgE and IgG1  or IgG4 , with slightly higher number of double-positive patients for IgE and IgG4 (65%) than for IgE and IgG1 anti-Dpt (56%) (Levels of IgE to pt (56%) .To select the serum samples with the highest and lowest concomitant IgG1 and IgG4 reactivity within the atopic and nonatopic groups, respectively, levels of IgG1 and IgG4 were compared as shown in P < 0.01) as shown in Figures Total human IgG purification was performed in two steps. Firstly, Dpt-specific and nonspecific serum pools were partly purified by 40% ammonium sulfate precipitation, obtaining the S 40% and P 40% fractions. The immunoglobulin profile in these fractions was verified by slot-blot, showing that all analyzed classes  were more concentrated in P 40% than S 40% fractions , showing that the predominant antibody subclass in purified specific IgG fractions was IgG1, as expected.To determine the optimal concentration of Dpt-specific and nonspecific IgG fractions to be used in inhibition ELISA, an indirect ELISA was performed to detect levels of IgG1 and IgG4 anti-Dpt in these fractions. As shown in Figures e cutoff . For theP < 0.0001) (P < 0.0001). However, the IgE reactivity was significantly lower in the presence of specific fractions than in nonspecific IgG fractions (P < 0.0001). When the percentage of inhibition for IgE reactivity was evaluated, the specific IgG fractions were able to inhibit ≥50% IgE reactivity for the pools I and II, and above 35% inhibition for the pool III  of atopic patients selected with basis on the positivity for the three antibody classes: pool I ; pool II ; and pool III  . For the 0.0001) . As show 0.0001) . A similpool III . It is known that the final purpose of allergen-specific immunotherapy is to modulate the immunological profile against allergens and that the benefits achieved are long-lasting even though the therapy is discontinued . Increasin vitro tests represent a main tool to elucidate the role of different types of immunoglobulins in the allergic response. In the present study, this approach was performed initially through purification of Dpt-specific IgG fractions from serum pools of mite-allergic patients and subsequently, evaluation of its blocking capability on the IgE reactivity by inhibition ELISA assays. The characterization, quantification, and evaluation of the blocking capability of IgG antibodies on the allergen-IgE interaction using D. pteronyssinus and D. farinae as relevant sensitizing agents in this region. IgE levels and seropositivity in atopic patients found in this study resembled those found in our previous work evaluating the levels of IgE, IgA, and IgG4 antibodies to D. pteronyssinus and to its major allergens, Der p1 and Der p2, in samples of serum and saliva from allergic and nonallergic children [ D. pteronyssinus [Blomia tropicalis [B. tropicalis [The recruitment of participants for the atopic group of this study was based on clinical history of allergic rhinitis and positive SPT to at least Dpt allergen extract. A previous study in patients of the Triângulo Mineiro region, Brazil , showed children . Althougnyssinus  and Blomopicalis , reinforopicalis , supportopicalis , 24.Considering the highest and lowest concomitant IgG1 and IgG4 reactivities to Dpt in atopic and nonatopic patients, respectively, serum samples were selected to obtain the specific and nonspecific serum pools. These serum pools were partially purified by salting-out precipitation using 40% ammonium sulfate and then submitted to affinity chromatography to get the specific and nonspecific IgG fractions. These techniques of immunoglobulin purification have been widely used with high quality and integrity of recovered antibodies , 26. TheThe electrophoretic profile after affinity chromatography showed strongly stained bands in the ligand fractions, compatible with the molecular weight of the human whole IgG molecule. Although other bands were visible, they can be considered degradation products since they were stained in immunoblot assays for detection of total IgG. The purification was considered successful because a strong reactivity to IgG was detected, whereas IgA reactivity was negligible, and no reactivity was found for IgE and IgM. Although recent studies have been looking for new clarifications concerning the role of IgD in regulation of immune system , 29, we It is well known that SIT leads to an increase in the allergen-specific IgG production –33, withIn the present study, we obtained Dpt-specific IgG fractions purified from serum pools of atopic patients that were not under any immunotherapy. In addition, the IgG1 and IgG4 levels to Dpt were measured in these fractions to warrant the presence of high levels of these specific blocking antibodies. A low level of residual IgE antibody was detected in the chosen concentration of specific IgG fraction, suggesting a probable crossreactivity with the biotinylated anti-human IgE antibody used in ELISA, since no IgE reactivity was detected in this purified IgG fraction in immunoblot assays using another secondary antibody. The blocking activity of the allergen-specific IgG fraction on the IgE reactivity to Dpt allergen extract was then evaluated by inhibition ELISA. Allergen-specific IgG fraction was capable of reducing levels of IgE anti-Dpt, resulting in 35%–51% inhibition of IgE reactivity to Dpt in the three serum pools tested. Also, we verified that the presence of specific IgG1 or IgG4 or both subclasses together with specific IgE in the tested serum pools did not interfere with the IgE blocking capability of these specific IgG fractions. Considering that the inhibition phenomena of IgE binding seen for the specific IgG fractions include both IgG1 and IgG4 subclasses, it was not possible to attribute a more protective role to IgG4 compared to IgG1 in the design of the present study. However, as the IgG1/IgG4 ratio in the specific IgG fractions was higher than 1.0, it may be speculated that the specific IgG1 antibody could also play a protective role. Further studies should be conducted using absorption methods or purification of specific IgG1 or IgG4 fractions from patients that are single positive for each IgG subclass to evaluate separately the role of each subclass of specific IgG fraction. This blocking role of allergen-specific IgG antibodies has been recently investigated in mouse models through passive immunization with specific IgG antibodies for prevention and treatment of allergy to major birch and grass pollen allergens . The autAltogether, our results showed that allergen-specific IgG antibodies purified from mite-allergic patient sera using available and standardized methodology are able to inhibit IgE reactivity to Dpt allergen extract. This approach reinforces that the intermittent measurement of serum allergen-specific IgG antibodies will be an important objective laboratorial parameter that will help specialists to follow their patients under allergen-specific immunotherapy.


Mesenchytraeus solifugus and related species, are the largest glacially obligate metazoans. As one component of cold temperature adaptation, ice worms maintain atypically high energy levels in an apparent mechanism to offset cold temperature-induced lethargy and death. To explore this observation at a mechanistic level, we considered the putative contribution of 5′ adenosine monophosphate deaminase (AMPD), a key regulator of energy metabolism in eukaryotes. We cloned cDNAs encoding ice worm AMPD, generating a fragment encoding 543 amino acids that included a short N-terminal region and complete C-terminal catalytic domain. The predicted ice worm AMPD amino acid sequence displayed conservation with homologues from other mesophilic eukaryotes with notable exceptions. In particular, an ice worm-specific K188E substitution proximal to the AMP binding site likely alters the architecture of the active site and negatively affects the enzyme's activity. Paradoxically, this would contribute to elevated intracellular ATP levels, which appears to be a signature of cold adapted taxa.Glacier ice worms,  Mesenchytraeus solifugus  -αdCTP (Perkin-Elmer). Approximately 500 000 phage from a λTriplex2 whole animal ice worm cDNA library were plated and screened at high stringency, as described [λ plaques were cored from agar plates with a glass pipette and subjected to Cre-lox-mediated in vivo excision (Clontech). Plasmid DNA was purified using a Wizard Plus SV Minipreps DNA Purification System (Promega). Following restriction endonuclease digestion with Eco  R1, Xba  I, and Hin d III, inserts ≥600 bp were DNA sequenced . DNA sequence chromatograms were analyzed by Vector NTI 10.3.0 (Invitrogen Corporatioon) and GenBank BLAST analysis [Mesenchytraeus solifugus  (EU624492); Enchytraeus albidus  (EU624493); Lumbriculus variegatus (EU624494).Independent probes comprising ice worm AMPD cDNAs were constructed with a Prime-a-Gene labeling kit (Promega), incorporating escribed . Positivanalysis . Linear analysis . Newly sAMPD-specific forward and reverse primers  were employed to amplify a fragment of AMP deaminase from genomic DNA and cDNA encoding the ice worm-specific K188E substitution. Genomic DNA was extracted from ice worm specimens collected from Byron Glacier, Alaska, USA, as described , and preDrosophila melanogaster  sequence served as default in “animal” comparisons, and E. albidus  was default in clitellate comparisons. Amino acid compositions, molecular weights, and hydrophobicities were calculated with ProtParam [Positions that contained an identical amino acid in both ice worm and a mesophilic counterpart were excluded from the comparison. In variable positions , the most abundant amino acid was chosen; in the absence of a dominant residue, the rotParam .Arabidopsis thaliana  adenosine 5′-monophosphate deaminase  in complex with coformycin 5′ phosphate  and the catalytic Zinc ion  was used as a template structure to generate the 3D model of ice worm AMPD. This was the only AMPD crystal structure available at PDB, but the sequence identity between ice worm and A. thaliana  AMPD  is sufficient for supporting model accuracy. The presence of coformycin 5′ phosphate is unlikely to alter the AMPD 3D structure since coformycin 5′ phosphate, a well-known AMPD and ADA transition state analogue inhibitor [The Protein Database  was emplnhibitor , is strunhibitor . Ice wornhibitor  and PyMonhibitor  to highlnhibitor .”Arabidopsis thaliana  (PDB ID 2A3L chain A) and M. solifugus  AMP deaminase linear amino acid sequences, respectively . Swiss Model in the ExPASy server [AMP deaminase mutant models were prepared by introducing K480E and E188K substitutions in the y server , 24 was y server , and wasE. albidus, L. variegatus), and retrieved from GenBank for Homo sapiens  (Chordata), D. melanogaster  (Arthropoda), Caenorhabditis elegans  (Nematoda) and the plant Arabidopsis thaliana, from which the only AMPD structural model was available [D. melanogaster  (~72%), and most distant from Homo sapiens  (isoform 3) and A. thaliana   was assembled with overlapping fragments obtained by degenerate PCR, RACE-PCR, and library screening. The combined sequence encoded 543 amino acids of AMPD, including a 69 amino acid N-terminal region, and a 474 amino acid C-terminal catalytic domain containing active sites for AMP binding and regulatory binding sites for the allosteric effectors ATP and inorganic phosphate . Homologvailable . AMPD hoC. elegans  displayed a larger number of nonconservative changes than M. solifugus, consistent with its deeply rooted ancestry , while ice worm AMPD encodes T. By these criteria, 26 ice worm-specific substitutions (16 nonconservative changes) were identified in the linear ice worm AMPD sequence, all of which occurred in the C-terminal catalytic region . Among iancestry .β strands, displayed an incomplete triose-phosphate isomerase (TIM) (α/β)8 barrel fold, with the active site located on the C-terminal side of the imperfect barrel, in a cleft surrounded by multiple helices and loops  , β sheets  and linker regions   . Inorganic phosphate is considered as one of the primary physiological inhibitors of AMPD , 32, andA. thaliana  .To gain a perspective on the types of amino acid substitutions favored in ice worm AMPD, its amino acid sequence was compared to “consensus” sequences  from clitellates and other mesophilic eukaryotes . In comp-strands . FinallyCold adapted enzymes typically display gains in flexibility at the expense of thermal stability –35. Amonα-helical segments [Apart from a slight increase in charged amino acids in ice worm AMPD with respect to its animal counterparts, increased polarity, reductions in side chain volumes and charged residues , and decreased core hydrophobicity  are all segments , and sevA. thaliana  AMPD suggests that ice worm residue 188 does not function in such a faculty. Rather, K188E is 10–15 Åfrom the AMP binding pocket and interacts with a loop of decreased flexibility (owing to P176) responsible for coordinating AMP. In fact, comparison of the A. thaliana  structure  could introduce additional destabilizing forces owing to negative-negative repulsion. Since the substitution lies adjacent to the active site, the effect of repelling charges may indeed introduce flexibility to the enzyme's active site, contributing to its catalytic efficiency at low temperatures. However, another explanation for the nonconservative K188E substitution should be considered. Bae and Phillips  and Fieltructure  with thePD model  supportsPD model . Due to The quest to understand the molecular adaptations of proteins to cold environments usually centers on the assumption that the psychrophilic protein should function more efficiently than its mesophilic counterpart at lower temperatures. For ice worm AMPD, however, this may not necessarily be the case. The reaction catalyzed by the AMPD represents a branch-point in the energy-generating adenylate catabolic pathway regulating the availability of adenosine nucleotides: blocking this pathway reduces the depletion of the total adenine pool and thus the metabolically expensive requirement for the de novo synthesis of ATP . Ice worThus the putative mechanism by which ice worms increase their cellular ATP levels involves a predicted reduction in the ability to remove AMP from the adenylate pool, and subsequent activation of ATP synthetic processes as a direct consequence of increased AMP levels , 11. Our


SLC19A2 and SLC19A3 genes, respectively). In this study we examined the effect of chronic exposure of pancreatic acinar cells in vitro (pancreatic acinar 266-6 cells) and in vivo (wild-type and transgenic mice carrying the SLC19A2 and SLC19A3 promoters) to the cigarette smoke component 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) on physiological and molecular parameters of the thiamin uptake process. The results show that chronic exposure of 266-6 cells to NNK  leads to a significant inhibition in thiamin uptake. The inhibition was associated with a significant decrease in the level of expression of THTR-1 and -2 at the protein and mRNA levels as well as in the activity of SLC19A2 and SLC19A3 promoters. Similarly chronic exposure of mice to NNK  leads to a significant inhibition in thiamin uptake by freshly isolated pancreatic acinar cells, as well as in the level of expression of THTR-1 and -2 protein and mRNA. Furthermore, activity of the SLC19A2 and SLC19A3 promoters expressed in transgenic mice were significantly suppressed by chronic exposure to NNK. The effect of NNK on the activity of the SLC19A2 and SLC19A3 promoters was not mediated via changes in their methylation profile, rather it appears to be exerted via an SP1/GG and SP1/GC cis-regulatory elements in these promoters, respectively. These results demonstrate, for the first time, that chronic exposure of pancreatic acinar cells to NNK negatively impacts the physiological and molecular parameters of thiamin uptake by pancreatic acinar cells and that this effect is exerted, at least in part, at the level of transcription of the SLC19A2 and SLC19A3 genes.Thiamin is indispensable for the normal function of pancreatic acinar cells. These cells take up thiamin via specific carrier-mediated process that involves thiamin transporter-1 and -2 (THTR-1 and THTR-2; products of  Thiamin (vitamin B1) is indispensable for the normal function and health of pancreatic cells due to its involvement in oxidative energy (sugar) metabolism and ATP production in the mitochondria SLC19A2 gene The pancreas is an important organ of the digestive system, and diseases of this organ lead to significant morbidity and mortality. Thiamin deficiency negatively impacts both the exocrine  via transport across cell membranes. We have recently characterized the mechanism of thiamin uptake by pancreatic acinar (as well as beta/islets) cells and showed the involvement of specific and regulated carrier-mediated process SLC19A2 and SLC19A3 knockout mouse models, we also showed that both of the thiamin transporters -1 and -2 (THTR-1 and THTR-2) are involved in thiamin uptake by pancreatic acinar cells http://www.ebi.ac.uk/gxa/qrs?gprop_0=&gnot_0=&gval_0=slc&fact_1=&fexp_1=UP_DOWN&fmex_1=&fval_1=smoking&view=list&searchMode=simple), oxidative stress in vitro (mouse-derived pancreatic acinar 266-6 cells) and in vivo (wild-type and transgenic mice that carry the human SLC19A2 and SLC19A3 promoters) on thiamin uptake by these cells. The results showed that this compound causes a significant inhibition in thiamin uptake by pancreatic acinar cells and that this inhibition is mediated, at least in part, at the level of transcription of the SLC19A2 and SLC19A3 genes.Pancreatic cells maintain high level of thiamin 3H]-Thiamin  was obtained from American Radiolabeled Chemicals . Nitrocellulose filters (0.45-µm pore size) were from Millipore (Fisher Scientific). Unlabeled thiamin and other chemicals including molecular biology reagents were from commercial vendors and were of analytical grade. Oligonucleotide primers used in this study were synthesized by Sigma Genosys . labeled or unlabeled thiamin was added to initiate uptake. The uptake reaction was terminated by adding 1 ml ice-cold K–R buffer, and the cell suspension was placed on nitrocellulose filters under negative pressure by use of a vacuum manifold , washed with 5 ml of ice-cold K–R buffer, and dissolved in scintillation fluid. Counting or radioactivity and protein determination were done as described above.Wild-type and transgenic mice carrying the full-length human Western blot analysis was performed on whole cell lysate prepared from 266-6 cells and from mice primary pancreatic acinar cells chronically exposed to NNK and their respective controls as described previously Total RNA (5 µg) was isolated from 266-6 cells (treated with NNK and their controls) and from primary acinar cells of NNK treated mice and their pair-fed controls, then treated with DNase I (Invitrogen) enzyme to prevent genomic DNA contamination. The DNase I-treated samples were subjected to cDNA synthesis using iScript cDNA synthesis kit . The coding region of mice THTR-1, THTR-2, and ARPO, were PCR amplified using gene-specific primers  for quanSLC19A2 and SLC19A3 full-length, minimal and the mutated minimal promoter-luciferase reporter constructs utilized in this study were generated previously Renilla transfection control plasmid Renilla luciferase-thymidine kinase (pRL-TK) . Transfection was performed with lipofectamine 2000 reagent (Invitrogen) according to manufacturer’s instructions. On the second posttransfection day the cells transfected with SLC19A2 and SLC19A3 constructs were exposed to NNK (3 µM) for 24 h as described above. At the end of 24 h of NNK exposure, Renilla-normalized firefly luciferase activity was determined by using the Dual Luciferase Assay system (Promega).The SLC19A2 and SLC19A3 promoters was assessed by bisulfite sequencing. Full-length promoter–luciferase constructs of SLC19A2 and SLC19A3 were transiently transfected in 266-6 cells and were exposed to NNK as described above and DNA was isolated using wizard Genomic DNA purification kit (Promega). The total DNA was digested with BamHI or KpnI for SLC19A2 and SLC19A3 respectively. Bisulfite reactions were performed using EpiTect Bisulfite Kit (Qiagen) under conditions that allowed for complete conversion of unmethylated cytosines to uracil, but not 5-methylcytosines. Subsequently, bisulfite-treated DNA was amplified using primers designed by MethPrimer SLC19A2 and SLC19A3. Primer sequences were designed to contain no CG dinucleotides and are listed in Methylation status of the t-test was used for statistical analysis, and P<0.05 was considered statistically significant.Uptake data with mouse primary pancreatic acinar cells presented in this paper are mean ± SE of at least three separate experiments from different mice and are expressed as percentage relative to simultaneously performed controls. Uptake with the 266-6 cells is also mean ± SE from at least three separate experiments. Carrier-mediated thiamin uptake was determined by subtracting uptake by simple diffusion from total uptake. Protein, RNA and luciferase activity determinations were performed from at least three sets of samples prepared at different occasions. The Student’s P<0.01) in carrier-mediated thiamin uptake by cells chronically treated with NNK compared to controls (P<0.05 for both) reduction in the level of expression of THTR-1 and THTR-2 proteins in cells chronically exposed to NNK compared to controls  on the initial rate of thiamin (15 nM) uptake. The result showed a significant inhibition  reduction in the level of expression of THTR-1 and THTR-2 mRNA in 266-6 cells chronically exposed to NNK compared to controls  reduction in the activity of both SLC19A2 and SLC19A3 promoters as a result of exposure to NNK  inhibition in the initial rate of thiamin uptake by pancreatic acinar cells from the NNK-treated mice compared to those from pair-fed controls (P<0.05 for THTR-1 and 0.01 for THTR-2) reduction in the level of expression of THTR-1 and THTR-2 proteins in pancreatic acinar cells of mice chronically exposed to NNK compared to pair-fed controls  reduction of expression of the THTR-1 and THTR-2 messages in pancreatic acinar cells of mice chronically treated with NNK compared to pair-fed controls  the effect of chronic exposure of mice to NNK on the level of expression of THTR-1 and THTR-2 mRNA. The results showed a significant  lower activity of the SLC19A2 and SLC19A3 promoters in pancreatic acinar cells of transgenic mice chronically exposed to NNK compared to activity of transgenic controls  an observed/expected CpG ratio over 0.6, 2) the percentage of G plus C over 50%, and 3) a window size of at least 100 bp. Our Methprimer analysis identified one CpG island of 536 bp for SLC19A2 and three CpG island of 112, 108 and 114 bp for SLC19A3. We then used the results of the Methprimer analysis to design primers that did not contain CpG dinucleotide sequences (SLC19A2 and SLC19A3 promoters as a result of chronic exposure to NNK. These findings suggest that other mechanism(s) are involved in mediating the inhibitory effects of chronic exposure to NNK on activity of the SLC19A2 and SLC19A3 promoters.One of the mechanisms through which a given condition affects transcriptional activity of a particular gene is via epigenetic mechanisms like DNA methylation . In general, hypo-methylation of the DNA surrounding a gene promoter is believed to activate the gene, while hyper-methylation silences the gene equences  and amplSLC19A2 and SLC19A3 promoters are involved in mediating the NNK effect on activity of these promoters. Thus, we examined and compared the effect of chronic exposure to NNK on the activity of the full-length vs the minimal SLC19A2 and SLC19A3 promoters in pancreatic acinar 266-6 cells. The results showed NNK to cause a similar degree of inhibition in activity of the full-length and the minimal SLC19A2 and SLC19A3 promoters  to be important for its activity SLC19A3 promoter, the cis regulatory element SP-1/GC-box binding site was found to be important for its activity SLC19A2 and SLC19A3 promoters. The results showed that while mutating the GKLF/SP-1 and NF-1 binding sites did not interfere with the inhibitory effect of NNK on activity of the SLC19A2 minimal promoter, mutating SP-1/GG-box site lead to a complete disappearance of the inhibitory effect  are involved in mediating the inhibitory effects of NNK on activity of the SLC19A2 and SLC19A3 promoters. To shed light on this other mechanism(s), we determined if the minimal regions of the SLC19A2 and SLC19A3 promoters needed for basal activity of the respective gene are involved in mediating can also mediate the NNK inhibitory effect. For this, we examined and compared the effect of chronic exposure to NNK on the activity of the minimal and full-length SLC19A2 and SLC19A3 promoters expressed in 266-6 cells and found a similar degree of inhibition with both constructs, suggesting involvement of the minimal regions in mediating the NNK inhibitory effect. Knowing that the minimal region of the SLC19A2 promoter contains the cis-elements GKLF/SP-1, NF-1 and SP-1/GG box binding sites and that these elements are important for its function SLC19A2 promoter activity. The results showed the disappearance of the inhibitory effect of NNK on the SLC19A2 promoter only when the SP-1/GG-box site was mutated, suggesting involvement of this site in mediating the NNK effect. Similarly the SLC19A3 minimal promoter is known to have an SP-1(SP-3)/GC-box binding site that is important for its activity SLC19A3 promoter activity, which suggest involvement of the site in mediating the NNK effect.It is well established that gene transcription can be modulated by epigenetic mechanisms like DNA methylation SLC19A2 and SLC19A3 genes.In conclusion, this study demonstrates for the first time that chronic exposure of pancreatic acinar cells to NNK negatively impacts the physiological and molecular parameters of thiamin uptake by these cells and this effect is exerted, at least in part, at the level of transcription of the


The metastasis-associated tyrosine phosphatase PRL-3/PTP4A is upregulated in numerous cancers, but the mechanisms modulating PRL-3 activity other than its expression levels have not been investigated. Here we report evidence for both Src-dependent tyrosine phosphorylation of PRL-3 and Src-mediated regulation of PRL-3 biological activities. We used structural mutants, pharmacological inhibitors and siRNA to demonstrate Src-dependent phosphorylation of endogenous PRL-3 in SW480 colon cancer cells. We also demonstrated that PRL-3 was not tyrosine phosphorylated in SYF mouse embryo fibroblasts deficient in Src, Yes and Fyn unless Src was re-expressed. Further, we show that platelet-derived growth factor (PDGF) can stimulate PRL-3 phosphorylation in a Src-dependent manner. Finally, we show that PRL-3-induced cell motility, Matrigel invasion and activation of the cytoskeleton-regulating small GTPase RhoC were abrogated in the presence of the phosphodeficient PRL-3 mutant Y53F, or by use of a Src inhibitor. Thus, PRL-3 requires the activity of a Src kinase, likely Src itself, to promote these cancer-associated phenotypes. Our data establish a model for the regulation of PRL-3 by Src that supports the possibility of their coordinate roles in signaling pathways promoting invasion and metastasis, and supports simultaneous use of novel molecularly targeted therapeutics directed at these proteins. SYF and H1299 cells were maintained in 10% CO2 in Dulbecco's modified Eagle medium with high glucose (DMEM-H) supplemented with 10% fetal calf serum. To generate mammalian expression plasmids (pSBP-PRL-3) encoding fusion proteins that could be pulled down using streptavidin-Sepharose , coding sequences for PRL-3 and mutants were cloned into pSBP, a pcDNA3-based vector into which we had previously inserted streptavidin-binding protein (SBP). All transfections were performed on 50–60% confluent cells plated in 100 mm dishes, with 1 µg each of the indicated plasmid(s), using the transfection reagent TransIT-LT1 (Mirus) according to the manufacturer's protocol.SW480 colon adenocarcinoma cells and H1299 nonsmall cell lung cancer cells were obtained from the UNC-Lineberger Comprehensive Cancer Center Tissue Culture Facility. Src/Yes/Fyn-deficient (SYF) mouse embryo fibroblast (MEF) cells For bacterial expression, PRL-3 or mutants were cloned into pGEX2T  fused to glutathione-S-transferase (GST). To generate SW480 cells stably expressing PRL-3 or mutants, coding sequences were cloned into pBABE-HAII. pUSE-Src was obtained from Upstate Biotechnology . Western blot analyses were done with the following antibodies: anti-PRL-3 , pY99 anti-phosphotyrosine , anti-Src antibody (32G6) and anti-phospho-Src family Tyr 416 antibody  and anti-RhoC  To evaluate endogenous PRL-3 phosphorylation, cells were treated for 1 h with 100 mM pervanadate. Pervanadate was made fresh by adding 1 µL of 30% hydrogen peroxide to 200 µL of 50 mM sodium orthovanadate in water. Following treatment, cells were lysed in RIPA  +100 mM pervanadate and protease inhibitors. PRL-3 antibody  was diluted 1:250 in lysates containing 200-500 µg total protein. Samples were incubated at 4°C O/N with rocking. Immune complexes were precipitated with protein A/G Sepharose, washed three times in RIPA, and resuspended in protein gel sample buffer without reducing agents prior to SDS-PAGE and western blot analysis. For treatment with PDGF, SW480 cells were starved overnight in the absence of serum. Cells were then treated with the Src inhibitor SU6656 ) or with vehicle (DMSO) for 1 h. Alternatively, Src expression was ablated using siRNA as described below, and serum starved overnight prior to PDGF treatment. Cells were then treated for 20 min with human PDGF-BB  and lysed for PRL-3 immunoprecipitation as described above.plus non-targeting siRNA pool was used as a negative control. Cells were maintained in standard conditions for five days and treated with pervanadate for 1 h prior to lysis and PRL-3 immunopreciptiation as described above.Expression of endogenous Src was reduced using an siRNA pool containing four targeting sequences . SW480 cells were transfected according to the manufacturer's protocol using a final siRNA concentration of 50 nM. The Dharmacon ON-TARGETFollowing treatment, cells were lysed in streptavidin lysis buffer . To pull down and detect SBP-tagged PRL-3, streptavidin-Sepharose beads were incubated with clarified lysates for 1 h at 4°C with rocking, washed three times with SLB and subjected to SDS-PAGE and western blot analysis using anti-PRL-3 antibody. For inhibitor studies, the Src family kinase inhibitor SU6656 was added to the culture medium 1 h prior to pervanadate treatment.E. coli bacteria, purified on glutathione beads, and subjected to in vitro phosphorylation by purified p60 c-Src . The in vitro kinase assay was carried out with ∼500 ng of each protein according to the manufacturer's protocol, except that non-radiolabelled ATP was used as the phosphate donor. Samples were then subjected to SDS-PAGE and western blot analysis for phosphotyrosine and for PRL-3.GST-tagged PRL-3 was induced from the pGEX2T vector in 4/well) were placed in the top section of the invasion chamber in the absence of serum. Medium containing 10% serum was placed in the bottom chamber to create a chemoattractant gradient. After 72 h invaded cells were stained and counted. For inhibitor studies, SU6656 was placed in both the top and bottom chambers. Data were analyzed using a two-tailed Student's t-test.The invasiveness of SW480 cells stably expressing PRL-3 or a phosphodeficient mutant (PRL-3 Y53F) was evaluated using Matrigel invasion chambers  as described previously 3 cells/plate), whose basal adhesive and motile characteristics are suitable for this assay system, were plated on glass-bottom plates  and transfected with EGFP-tagged PRL-3, PRL-3 (Y53F) or empty vector. Time-lapse images of fluorescent cells were taken at 5 min intervals over 2 h. Average velocity of individual cells was evaluated using ImageJ (http://rsb.info.nih.gov/ij/). Non-fluorescent cells were also evaluated as controls. Data were analyzed using a two-tailed Student's t-test.The effect of PRL-3 and the phospho-deficient mutant Y53F on cell motility was evaluated using a Nikon BioStation IM single cell recorder. H1299 cells  indicated a high probability of PRL-3 phosphorylation on tyrosines  was subjected to phosphorylation by purified Src. Using western blot analysis with anti-phosphotyrosine antibody, we observed that WT PRL-3 was phosphorylated on tyrosine in the presence of Src, whereas a mutant form of PRL-3 lacking all six tyrosines (designated “All_F”) was not . In neither case was GST alone phosphorylated (data not shown). Thus, PRL-3, like PRL-1, can be a direct substrate of Src tyrosine kinase activity in vitro.Many cellular proteins, including some tyrosine phosphatases, are regulated by phosphorylation on tyrosine residues yrosines . To dete was not . We obtaWe next wished to determine whether PRL-3 is also tyrosine phosphorylated in cells. We have shown previously that PRL-3 promotes motility and invasion in SW480 colon adenocarcinoma cellsin vivo, either directly or indirectly in vitro and that endogenous PRL-3 is tyrosine phosphorylated in vivo, we wished to determine the role of Src in modulating PRL-3 phosphorylation. First, to determine whether Src is sufficient to cause an increase in PRL-3 tyrosine phosphorylation, we co-transfected SW480 cells with both PRL-3 and Src, then probed for tyrosine phosphorylation of PRL-3 by western blot analysis. Src regulates some tyrosine phosphatases Src and of the related Src family kinases Yes and Fyn To confirm the importance of Src as a modulator of PRL-3 phosphorylation, we next performed a complementary experiment in which we transfected PRL-3 into SYF cells, which are MEFs that are simultaneously deficient in expression both of We observed that vanadate was not required to detect phosphorylation of PRL-3 by Src in SW480 cells  whereas in vivo. Together, these observations demonstrate that Src is both sufficient and necessary to promote phosphorylation of endogenous PRL-3 in SW480 cells, although they do not rule out the possibility that other kinases may also serve this role in other contexts. Indeed, the presence of weak but detectable PRL-3 tyrosine phosphorylation in SYF cells  inhibitor SU6656 , followed by treatment with pervanadate to reveal phosphorylation of endogenous PRL-3 as done for YF cells  suggestsTo determine whether PRL-3 could be phosphorylated in response to a physiological stimulus, we treated SW480 cells with platelet-derived growth factor (PDGF), the receptors for which contribute to aspects of tumor development To identify the site(s) of tyrosine phosphorylation on PRL-3, we first analyzed the PRL-3 sequence using NetPhos 2.0, which predicted high probabilities for phosphorylation on tyrosine residues 14, 53, and 126 . As shown in Our results show that Src kinase activity leads to the tyrosine phosphorylation of PRL-3, primarily on Y53. Both PRL-3 and Src play well-established roles in cell motility, and both are implicated by numerous correlative and functional studies in tumor invasion and metastasis We next determined whether the phosphorylation-deficient mutant of PRL-3 was still able to promote cell motility, which is a necessary component of the invasive phenotype. Because SW480 cells are not suitable for this type of analysis, we used H1299 cells, which have suitable properties of basal adhesiveness and motility. Cells were transiently transfected with EGFP-tagged PRL-3 or the Y53F mutant. The average velocities of individual cells were measured and compared to the velocities of cells expressing EGFP only. Our data show that PRL-3 is phosphorylated downstream of Src , 2 and 3in vivo. Collectively, these observations support a causal role for PRL-3 in tumor invasion and metastasis and have led to the frequent characterization of PRL-3 as a "metastasis-associated tyrosine phosphatase". Our data suggest that these biological functions of PRL-3 are regulated by a Src family tyrosine kinase, and that this may be due to Src-mediated phosphorylation of tyrosine 53, a residue that we show here is both the major site of tyrosine phosphorylation and a requirement for full PRL-3 activity. Specifically, we have shown that: 1) Src itself can phosphorylate PRL-3 directly in vitro, 2) endogenous PRL-3 is tyrosine phosphorylated, 3) Src is required for tyrosine phosphorylation of PRL-3, 4) PDGF can stimulate PRL-3 phosphorylation in a Src-dependent manner, 5) of the six tyrosines in PRL-3, Y53 is the major site of phosphorylation, 6) Y53 is required for PRL-3 promotion of motility and invasion, and 7) PRL-3 requires the activity of a Src family kinase to fully promote motility, invasion and RhoC activation. Collectively these results support a model in which Src causes phosphorylation of PRL-3 on Y53 to promote its pro-invasion functions, and suggest for the first time that the metastasis-associated tyrosine phosphatase PRL-3 may itself be regulated by post-translational modification.Numerous correlative studies in tumors of the colon, stomach, breast, pancreas and ovaries have shown that PRL-3 expression increases with tumor progression  domains, thus exposing the active site and increasing catalytic activity. Phosphorylation of LMW-PTP has also been shown potentially to create a binding site for the adapter protein Grb2 There are precedents for regulation of tyrosine phosphatases by their own tyrosine phosphorylation. PTP1B, LMW-PTP and Shp-2 are each reported to be phosphorylated on tyrosines with functional consequences of that modification It is not yet clear by what mechanism phosphorylation affects the function of PRL-3, although several possibilities exist. As with LMW-PTP, PTP1B and Shp-2, phosphorylation of PRL-3 at Y53, which lies in the second alpha-helical domain adjacent to the active site in vitro and in vivoA structural feature unique to PRL PTPs is that they form non-covalent trimers There is an increasing appreciation that signal transduction occurs in the context of complex feedback loops rather than a simple unidirectional flow of linear pathways By functionally linking PRL-3 and Src, which individually play established roles in cell motility, invasion and metastasis, our model helps to better define the biochemical mechanisms of these important aspects of tumor progression. In light of recent progress towards clinically-relevant Src inhibitors (reviewed in


Fc-mediated inhibitory activity of neutralizing antibodies has been shown to participate in HIV protection . In addition, a non-neutralizing antibody F240 was found to partially protect macaques from SHIV vaginal transmission . However, mechanisms involved in this protection need further investigations. In this study, two non-neutralizing antibodies have been selected on the basis of their Fc-mediated inhibitory functions in vitro for further analysis of their protective role on vaginal challenge in non-human primate (NHP).We have assessed and scored various in vitro HIV-inhibitory activities of non-neutralizing antibodies: Fc-mediated inhibition of macrophages through phagocytosis of immunecomplexes, ADCC in primary infected CD4+ T lymphocytes by autologous NK cells, capture of native primary virus particles. Efficacy of two anti-PID antibodies with high in vitro functional scores has been tested in NHP. The combination of two antibodies formulated in 1.6% HEC gel has been topically applied in the vagina of macaques (n=6), 1 hour before vaginal challenge with high dose (10 AID50) of SHIVSF162P3.Infection was followed by assessing viral load in the plasma.Although unable to block virus entry at mucosal site as all treated animals became persistently infected, the antibody treated macaques have significant decrease of plasma viral load .Decrease in viral load following antibody treatment strongly suggests that non-neutralizing inhibitory antibodies could interfere with early viral replication and dissemination through Fc-mediated inhibitory functions. Additional studies will be required to optimize this inhibition, and combined strategies should be developed to assess the potential synergy between neutralizing and non-neutralizing inhibitory antibodies.


Acropora muricata at two reef-flat sites representing contrasting environments at Réunion Island . Growth of these injured nubbins was examined in parallel, and compared to controls. Biochemical compositions of the holobiont and the zooxanthellae density were determined at the onset of the experiment, and the photosynthetic efficiency (v/FmF) of zooxanthellae was monitored during the experiment. Acropora muricata rapidly regenerated small lesions, but regeneration rates significantly differed between sites. At the sheltered site characterized by high temperatures, temperature variations, and irradiance levels, regeneration took 192 days on average. At the exposed site, characterized by steadier temperatures and lower irradiation, nubbins demonstrated fast lesion repair (81 days), slower growth, lower zooxanthellae density, chlorophyll a concentration and lipid content than at the former site. A trade-off between growth and regeneration rates was evident here. High growth rates seem to impair regeneration capacity. We show that environmental conditions conducive to high zooxanthellae densities in corals are related to fast skeletal growth but also to reduced lesion regeneration rates. We hypothesize that a lowered regenerative capacity may be related to limited availability of energetic and cellular resources, consequences of coral holobionts operating at high levels of photosynthesis and associated growth.Regeneration of artificially induced lesions was monitored in nubbins of the branching coral  Disturbances from multiple biotic and abiotic factors cause recurring losses of living tissues from coral colonies Coral colony growth and regeneration are likely closely related processes, involving calcification and tissue extension Acropora muricata at Réunion Island in contrasting natural environments. An earlier study on this species revealed a trade-off between regeneration and growth, and indicated that they were independent of size A. muricata. We suggest that rapid coral growth, promoted by a high zooxanthellae density and high photosynthetic efficiency, compromised their capacity to invest energy and cellular resources in lesion repair. We hypothesize that maintaining coral-symbiont functioning under conditions that boost zooxanthellae densities and their photosynthetic rate implies their sequestering of an important share of host resources. This may particularly involve the use of the energy-rich photosynthetic compounds produced by the Symbiodinium.The present study was designed to investigate the lesion regenerative capacity of the branching coral This experiment was conducted and corals were sampled with permission granted by the Réserve Naturelle Marine de la Réunion. All survey procedures carried out were done with proper precautions to minimize impacts to the reefs.−2) data were obtained from the French Meteorological Service on land at station close to each site (stations no. 97415590 and 97413545).Experiments were conducted on the west coast of Réunion Island  at two shallow (1∼2 m deep) reef-flat sites 11 km apart from each other: Planch’Alizé and Kiosque. At each site, the seawater surface temperature (SST) was measured at hourly intervals using calibrated underwater temperature loggers . Solar radiation , senior synonym of A. formosaMontipora circumvallata and Porites (Synaraea) rus, forming large thickets. At this site, benthic communities comprise a high proportion of dead corals covered by algal turfs . This site is further characterized by nutrient enrichment originating from N-rich groundwater seepage near the shore A. muricata is lower than at Planch’Alizé The branching coral A. muricata colonies separated by >10 m in distance. Nubbins were glued onto numbered acrylic glass tiles, mounted on racks positioned in a representative area at each native reef flat site, and left to recover for 2 months prior to the experiments. On March 8, 2008, 5 replicate nubbins from each colony were randomly selected, and artificial circular lesions of 11.5±0.6 mm in diameter and 3 mm in depth were inflicted using a grinding stone at mid-height on the side of each branch, removing all traces of living tissue. Particular care was taken to induce lesions of a constant size and depth. Nubbins were then left to heal at their respective native sites.At each site, 15 nubbins (7 cm long) were sampled from each of 5 haphazardly selected The surface area of lesions and projected vertical surface area of nubbins were monitored until April 30, 2008 and quantified with CPCe software −1) of nubbins was calculated from the difference between the initial and final projected nubbin surface area multiplied by the mean site-specific skeletal density, divided by the duration of the experiment. The calcification rate, or mass of CaCO3 deposited per unit area per day (g cm−2 d−1), was estimated for each nubbin as the product of its linear extension and skeletal density. The skeletal density (g cm−3) of nubbins was calculated as the dry weight-to-volume ratio, following Bucher et al. The relative mass increase (‰ dThe remaining undamaged nubbins (9 or 10 per colony) were used as controls, and their growth (relative increases in the buoyant mass and surface area) was monitored simultaneously.v/FmF  DNA was amplified using the primers “ITSintfor2” and “ITS2revclamp” For 3 of the 5 colonies per site, 1 undamaged nubbin was sampled before the beginning of the experiment , snap frozen in liquid nitrogen and stored at −80°C. Genomic DNA was then extracted from a subsample using a Qiagen® Blood and Tissue Kit . The internal transcribed spacer (ITS)-2 region of 3 each) using a hemocytometer at 400×magnification. The chlorophyll (Chl) a concentration was determined by spectrometric absorbance at 664 nm 2Cl2: CH3OH: H2O; 1∶2: 0.8 v/v/v) 2Cl2 and H2O (1∶1 v/v) were added to the supernatant to create a biphasic mixture. The organic (dichloromethane) phase was collected in glass bottles with Teflon caps, evaporated under nitrogen, and stored under a nitrogen atmosphere at −20°C until being analyzed. The total lipid content was then determined using thin-layer chromatography coupled with flame-ionization detection (TLC-FID) on an Iatroscan apparatus model MK6-s (with hydrogen flow of 160 mL min−1 and air flow of 2000 mL min−1) and an i-Chromstar 6.1 integration system .Tissues were removed from the skeleton using a dental jet  with recycled freshly filtered seawater reg is the maximum area that can be fully regenerated, slope is the regression slope, and t is the time in days. Since there is no natural logarithm for 0 , +1 was added to the remaining lesion size for the calculations.Regeneration of each nubbin was quantified by fitting the remaining lesion size (%) over time using a least-squared regression and an exponential decay model, allowing full recovery Conformity with parametric assumptions was visually assessed from residual plots. A potential site difference in the initial lesion size was tested using a 1-way analysis of variance (ANOVA). Possible effects of the initial lesion size on the regression slopes were analyzed using an analysis of covariance (ANCOVA) with the initial lesion size as the co-variable. A site difference in model slopes was investigated using an ANOVA, with “colony” as the random factor. For each site, mean slopes were used to simulate regeneration during 300 days. The difference in relative growth between sites and treatments was compared using a 2-way ANOVA, and Tukey’s honest significant difference (HSD) test was used for post-hoc comparisons.t-test was used to compare biochemical properties of nubbins between sites at the onset of the lesion regeneration experiment. As a parametric assumption could not be met, non-parametric Mann-Whitney U-test was used to compare v/FmF of injured and control nubbins at the beginning, middle, and end of the experiment, and was also used to assess site differences in v/FmF of injured nubbins. Friedman’s ANOVA was used to test temporal variations in v/FmF, where values obtained from the same nubbin over time were considered dependent. Results are presented as the mean ± standard deviation (SD).Welch’s Acropora muricata nubbins were consistently associated with C3 Symbiodinium. Corals from the sheltered site Planch’Alizé were characterized by a higher zooxanthellae density, Chl a concentration, and total lipid content, but also not significant lower tissue biomass and protein content compared to those from the exposed site Kiosque ( Kiosque .2 (n = 48) and did not differ between sites  or colonies . Two nubbins at Kiosque died before the onset of regeneration monitoring; these were not included in the analysis. There was no mortality of nubbins during the experimental period. The lesion size decreased rapidly over time (n = 25) and Kiosque  or initial lesion size . Increases in the relative surface area of nubbins  and treatment , with a significant interaction term observed between the two factors .The initial lesion size was 104±11 mmver time . After 5ver time . Regress nubbins  varied wp<0.001), and rates of calcification and lesion regeneration were negatively correlated . At this latter site, lesion regeneration rates were slow and not related to growth  and 0.684±0.021 (n = 50) at Planch’Alizé, and 0.682±0.020 (n = 23) and 0.690±0.016 (n = 50) at Kiosque. v/FmF did not significantly differ between sites or treatments , and no significant temporal variation was observed for injured nubbins .While the existence of a trade-off between growth and regeneration was clearly identified for some coral species A. muricata at this site is further reflected in the high lipid and low protein content of the nubbins. Indeed, lipids are mainly derived from excess carbon fixed by zooxanthellae Environmental conditions at Planch’Alizé, including low water flow, high mean SST and high SST variation, combined with nutrient enrichment from N-rich groundwater seepage At Kiosque, the tissue biomass of nubbins tended to be higher than at Planch’Alizé, although the difference was not significant. Higher tissue biomass may reflect the opportunities corals have for heterotrophic feeding  that settle inside lesions may hamper lesion regeneration as an energetically costly mechanism An understanding of key processes of corals is critical to appreciating the divergent population trajectories after exposure to disturbances


The Salton Sea is the largest inland body of water in California, with salinities ranging from brackish freshwater to hypersaline. The lake experiences high nutrient input, and its surface water is exposed to temperatures up to 40°C. Here, we report the community profiles associated with surface water from the Salton Sea. The water temperatures and pH levels of the samples were 29.9°C and 23.6°C and pH 8.09 and pH 8.20 from sites 1 and site 2, respectively. DNA was extracted using the FastDNA SPIN kit for soil from MP Biomedicals, according to the manufacturer’s protocol. The V6 to V8 region of the bacterial 16S rRNA gene was amplified using the primer set 926f/1392r and sequenced using the Roche 454 FLX+ platform . The raw pyrosequence reads were quality filtered and analyzed using QIIME version 1.7.0 (Proteobacteria (50.0% at site 1 and 52.1% at site 2) and Bacteroidetes (11.19% at site 1 and 8.50% at site 2). Spirochaetes (5.9%), Planctomycetes (5.1%), and unclassified bacteria (4.3%) were the next-most-abundant phyla for site 1, while unclassified bacteria (7.5%), Planctomycetes (4.2%), and Cyanobacteria (4.2%) were the next-most-abundant phyla for site 2.A total of 1,484 and 3,341 high-quality sequences, representing 492 and 1,043 distinct OTUs, were obtained from sites 1 and 2, respectively. A total of 46 distinct phyla were identified in this study, with 34 of the phyla shared between the two samples. The two most abundant phyla detected were Collectively, our data reveal a phylogenetic diversity and variance within the microbial communities of geospatially distinct sites from California’s largest lake, the Salton Sea.SRP033722.The DNA sequences from this metagenomic project have been deposited in the NCBI Short Read Archive under the accession no.