M2e-NSP4 was then evaluated side-by-side with synthetic M2e peptide for its immunogenicity and protective efficacy in a murine influenza challenge model. Here we demonstrate that M2e fused to the tetramerizing protein induces an accelerated, augmented and more broadly reactive antibody response than does M2e peptide as measured in two different assays. Most importantly, vaccination with M2e-NSP4 caused a significant decrease in lung virus load early after challenge with influenza A virus and maintained its efficacy against a lethal challenge even at very low vaccine doses. Based on the results presented in this study M2e-NSP4 merits further investigation as a candidate for or as a component of a universal influenza A vaccine.The ectodomain of the matrix 2 protein (M2e) of influenza A virus represents an attractive target for developing a universal influenza A vaccine, with its sequence being highly conserved amongst human variants of this virus. With the aim of targeting conformational epitopes presumably shared by diverse influenza A viruses, a vaccine (M2e-NSP4) was constructed linking M2e (in its consensus sequence) to the rotavirus fragment NSP4 In addition, there is the constant threat of a pandemic influenza outbreak. Currently available vaccines against seasonal epidemics are efficient as long as there is a good match between the chosen vaccine strains and circulating influenza variants. However, these vaccines target the surface molecule hemagglutinin (HA) and to a lesser extent neuraminidase (NA), and due to the continuous change of these proteins because of mutations and antibody mediated selection, these vaccines need to be updated annually Seasonal Influenza A epidemics represent a major threat to the human population worldwide causing three to five million cases of severe illness and about 250,000–500,000 deaths each year . For functional analysis, the recombinant fusion protein M2e-NSP4 was compared head-to-head to synthetic M2e peptide, which is the basis for several other M2e targeting vaccines. Here we demonstrate that the vaccine construct M2e-NSP4 is superior to M2e peptide immunization when both are formulated in the liposome based cationic adjuvant CAF-01 As already mentioned, the natural configuration of M2 is as a tetrameric structure. We were interested in constructing a vaccine based on the native structure of M2 since both monoclonal antibodies Experiments were conducted in accordance with national Danish guidelines  regarding animal experiments as approved by the Danish Animal Experiments Inspectorate, Ministry of Justice, permission numbers 2004/561–867 and 2009/561–1679.98–135 fragment of rotavirus, was chemically synthesized with optimized E. coli codons . The DNA was amplified from the pUC57 vector  and the polymerase chain reaction (PCR) product was digested with the restriction enzymes NdeI and BamHI  and ligated into a pET11a vector , which had been digested with the same enzymes. The ligation mixture was electrotransformed into B834(DE3) cells . Positive clones were confirmed by DNA sequencing . The plasmid DNA was then used for retransformation into the same cells and the cultures were grown overnight. The cultures were harvested 3–4 hours after induction with 1 mM IPTG.A construct encoding M2e, originating from the human influenza consensus sequence 600 reached 0.4–0.6. Thereafter, 1 mM IPTG was added for induction for 3–4 hours at 37°C. The cells were harvested by centrifugation at 5000×g for 5 min and resuspended in 50 mM Tris, 50 mM NaCl, pH 7.5. This was followed by centrifugation of the cells at 5000×g for 5 min. The supernatants were removed and the pellets were stored at −20°C.The bacterial cultures carrying the plasmid pET11a/M2e-NSP4 were grown in LB medium containing 50 µg/mL ampicillin until ODThe proteins were purified using nickel affinity chromatography. Specific measures were taken for purification of M2e-NSP4 as this protein only had an HVH-tag positioned at the end of the NSP4 fragment sequence. The bacteria were resuspended in 5 mL of 25 mM Tris, pH 8.0, and sonicated 3×30 s on ice. The suspensions were centrifuged at 18000 rpm × 15 min in the cold. The supernatants were applied to a Tricorn 10/50 column , which had been packed with 5 mL nickel-nitrilotriacetic acid (Ni-NTA) (Qiagen). The columns were washed with 1 column volume of 25 mM Tris, pH 8.0, and eluted with 25 mM Tris, 250 mM imidazole, pH 8.0 0–100% over 1 column volume. The relevant fractions were pooled and loaded directly on MonoQ 10/100 columns (Qiagen). The columns were washed with 1 column volume 50 mM Tris, pH 7.5, and the proteins were eluted with 0–1 M NaCl over 5 column volumes. The fractions were stored at −20°C. Analysis on the fractions was done with SDS-PAGE stained with Coommassie Blue A band corresponding to the M2e-NSP4 monomer could be visualized at 7 kDa . FurtherEndotoxin levels were determined using Kinetics Turbidimetric LAL test (Charles River); endotoxin values of the purified protein used in these experiments were ∼4 EU/mg.The M2e peptides H-MSLLTEVETPTRSEWECRCSDSSDP-OH (A/Ontario/309862/2009(swine-origin H1N1)), H-MSLLTEVETPTRNEWECRCSDSSDP-OH (A/Indonesia/560H/2006 (H5N1)), H-SLLTEVETPIRNEWGSRSNDSSDP-OH (consensus H1N1), H-SLLTEVETPTKSEWESRSNVSSDL-OH (A/equine/London/73 (H7N7)) and the NSP4 fragment H-QMDRVVKEMRRQLEMIDKLTTREIEQVELLKRIYDKL-OH were synthesized by Schafer-N .The purity of the peptides was above 80% as analysed by HPLC and mass spectrum.Female Balb/c mice were obtained from Taconic M&B , 6–8 weeks old, and housed at the Panum Institute, University of Copenhagen. Mice were always allowed to acclimatize for at least one week before entering into experiments. All animal experiments were conducted in accordance with national guidelines. Mice were immunized with the recombinant fusion protein M2e-NSP4 or M2e peptide formulated with or w/o CAF-01 adjuvant , Freund’s complete (FCA) plus incomplete adjuvant (FIA) (Sigma-Aldrich) or Alhydrogel adjuvant . Mice were immunized subcutaneously (s.c.) on days 0, 21, and 42. In some experiments, the immunization on day 42 was not performed. Blood samples were collected from the submandibular vein two weeks after each vaccination. The blood was allowed to clot prior to centrifugation, where after serum was obtained.6H8O7.H2O and 0.67 M Na2HPO4.(H2O)2 in MilliQwater, pH 5) and then incubated for 30 min at dark with 100 µL/well of substrate buffer  and 0.4 µL/mL 30% H2O2). The reaction was stopped with 0.2 M H2SO4 at 100 µL/well and the plates were read on the Multiskan® FC microplate photometer from Thermo Scientific. The antibody titer was defined as the reciprocal of the highest dilution that yielded an OD490 nm value 4 times above the mean value of the negative control wells.Nunc Maxisorb™ 96-well plates were coated overnight at 4°C with 100 µL/well of 300 ng/mL M2e peptide in 50 mM bicarbonate buffer, pH 9.6. The plates were washed 1× with PBS/Tween  containing NaCl (0.35 M) and 0.1% Tween-20, pH 7.4), and blocked for 1 hour at room temperature (RT) with a 2.5% casein solution. For ELISA, all plates were first washed 2× with PBS/Tween. The test samples and reagents were diluted in dilution buffer  at 100 µL/well and incubated for 1 hour at RT. Bound mouse antibodies were detected with HRP-labelled goat anti-mouse IgG antibodies (Fab-specific) (Sigma-Aldrich), rat anti-mouse IgG1 antibodies , rat anti-mouse IgG2a antibodies , goat anti-mouse IgM antibodies (Sigma-Aldrich) and goat anti-mouse IgA antibodies (Sigma-Aldrich). The plates were washed 2× with PBS/Tween and once with citric acid buffer (0.35 M C5 HeLa cells in 150 µL of Iscove’s Modified Dulbecco’s medium (Invitrogen) supplemented with 2-mercaptoethanol at 0.05 mM, transferrin (Sigma-Aldrich) at 0.005 mg/mL, gentamicin  at 0.05 mg/mL, and 10% fetal bovine serum (FBS) . The media was further supplemented with 1 µg/mL doxycycline (Sigma-Aldrich) for induction of Tet. After two days of incubation at 37°C in air/CO2, the media was flicked out and the cells were fixed with 0.05% glutaraldehyde (Sigma-Aldrich) for 20 min at RT. The plates were washed 2× with PBS/Tween and blocked with dilution buffer for 1 hour at RT or stored at 4°C. The test samples were diluted 1∶200 in dilution buffer at 50 µL/well and incubated for 1 hour at RT. Bound mouse antibodies were detected with HRP-labelled goat anti-mouse IgG antibodies (Fab-specific) (Sigma-Aldrich), The plates were washed 2× with PBS/Tween and 1× with citric acid buffer (0.35 M C6H8O7.H2O and 0.67 M Na2HPO4.(H2O)2 in MilliQwater, pH 5) and then incubated for 30 min at dark with 100 µL/well with substrate buffer  and 0.4 µL/mL 30% H2O2). The reaction was stopped with 0.2 M H2SO4 at 50 µL/well and the plates were read at A490 and A750 on the Multiskan® FC microplate photometer from Thermo Scientific and the difference in OD490–750 between the HeLa-M2 and the HeLa-C10 plates was recorded. The difference between OD490–750 for HeLa-M2 and HeLa-C10 was recorded as ΔOD.HeLa enzyme-linked immunosorbent assay (ELISA) plates were prepared as follows: HeLa cells transfected with M2-expressing plasmid (HeLa-M2) encoding a tetracycline response element (Tet) and HeLa cells with an empty plasmid (HeLa-C10) were obtained from Robert B. Couch 4 cells were added into wells of 96-well round bottom micro-titer plates (Sigma-Aldrich). The cells were washed twice with washing buffer . Serum samples were added diluted 1∶100 in washing buffer at 50 µL/well and incubated at 4°C for 30 min. The plates were washed twice in washing buffer and the cells were incubated with 50 µL/well of FITC-conjugated Goat anti-mouse IgG2a specific (Jackson ImmunoResearch), PE-conjugated Goat anti-mouse IgG specific (Jackson ImmunoResearch) and APC-conjugated Goat anti-mouse IgG1 specific antibodies in washing buffer at 4°C for 30 min. The cells were washed twice in washing buffer and once in PBS. The cells were resuspended in 100 µL PBS and were analysed on a FACSCalibur (BD Biosciences). Mouse sera positive for all three stains and the monoclonal antibody 14C2  specific for M2e were used for instrument settings. A minimum of 10000 live cell events were normally collected. Data were analysed using the FlowJo program, and the difference in mean fluorescence intensity (MFI) between HeLa-M2 and the HeLa-C10 cells incubated with the same serum was recorded as ΔMFI.HeLa-M2 cells and HeLa-C10 cells were maintained as stated above. After 2 days of incubation with 1 µg/mL doxycycline (Sigma-Aldrich), 5×1050  of either A/equine/London/72 (H7N7) or A/Puerto Rico/34 . Survival and weight was monitored for up to 3 weeks; mice suffering a weight loss greater than 25% of their starting weight were euthanized for humane reasons.For evaluation of clinical protection, mice immunized as described above, were anaesthetized by intraperitoneal (i.p) injection with 250–300 µL Avertin (25 mg/mL) and infected intranasally (i.n.) with approx. 3 LDViral loads were determined on 100 ng purified total RNA by duplex QRT-PCR using the Brilliant QRT-PCR Master Mix (Stratagene). Mice challenged with a lethal dose of PR8 were sacrificed by cervical dislocation at day 3 post infection. Lungs were removed and snap-frozen in either liquid nitrogen or dry ice. The tissues were homogenized in 3.6 mL lysis buffer  using a polytron. The tissue lysates were centrifuged twice at 3500 RPM for 10 min., and the supernatants were transferred to new tubes in between and after the second centrifugation. The clear tissue lysates were stored at −80°C. RNA from the homogenized tissues was extracted according to the standard kit extraction protocol from Nucleospin RNA II kit . RNA samples were quantified at 260 nm and qualitatively analysed by the 260/280 nm ratio. The samples were stored at −80°C before further use.AGATGAGTCTTCTAACCGAGGTCG 3′ and 5′ TGCAAAAACATCTTCAAGTCTCTG 3′) and the housekeeping gene murine glyceraldehyde-3-phosphate dehydrogenase (mGAPDH) (5′ CAATGTGTCCGTCGTGGA 3′ and 5′ GATGCCTGCTTCACCACC 3′). The following probes were used, M2: FAM-TCAGGCCCCCTCAAAGCCGA-BHQ-1, mGAPDH: HEX-CGCCTGGAGAAACCTGCCAAGTAT-BHQ-1. The primers and the probes were synthesized by TAG Copenhagen A/S. The primer and probe sequences for the M gene had been previously published by Spackman et alThe primers used were directed against the M gene .E. coli. The fusion partner we have chosen has an easily predictable coiled-coil sequence. Coiled-coil sequences are known to be specific with respect to the number of subunits, and both the full-length native NSP4 as well as the coiled-coil region we have used as fusion partner for M2e are known to form stable tetramers A construct containing the consensus sequence of M2e To compare the immunogenicity of the recombinant fusion protein to that of free peptide, mice were immunized with 10 µg of antigen on days 0, 21 and 42, and sera collected 2 weeks after each immunization were analyzed for M2e-specific IgG antibodies by ELISA . As stanIn the same experiment we examined the requirement for linking M2e to the NSP4 fragment, acquiring an oligomerized construct, in order to obtain the same potency as seen with M2e-NSP4 immunization. Mice were immunized with a mix of 10 µg M2e and 10 µg of the NSP4 fragment and serum samples taken 2 weeks after each immunization were analyzed by ELISA. It was evident that linkage significantly increased the immunogenicity to M2e .The immunogenicity of the M2e-NSP4 vaccine was further investigated by comparing immunization with 100 µg M2e to 50 µg of M2e-NSP4. Increasing the M2e peptide dose ten-fold led to a marked increase in the antibody titer, which now matched that in mice immunized with the linked construct . HoweverNext, the isotypes of the M2e and M2e-NSP4 M2e induced antibodies after the third immunization were determined by ELISA. The main isotypes that were induced by both M2e and M2e-NSP4 immunization were IgG1, and to a lesser extent IgG2a . M2e-NSPTo test the efficacy of the CAF-01 adjuvant in comparison to other adjuvants commonly used in mouse experiments, the two vaccines were formulated with either CAF-01, AlOH, or FCA plus FIA . FormulaIn the case of unpredicted influenza outbreaks, it might be of importance to spare on the available antigen. It was therefore investigated if antigen sparing by either lowering the antigen dose to 1 µg while preserving the immunization schedule or limiting the number of doses to two while maintaining the antigen dose at 10 µg would affect the potency of either vaccine. As can be seen in The results presented above showed that antibodies induced by M2e as well as M2e-NSP4 immunization could react with synthetic consensus M2e peptide. It was next investigated whether the induced antibodies would also recognize the presumably native M2 protein, as expressed on transfected HeLa cells (HeLa-M2 cells). HeLa-M2 cells and HeLa-C10 cells  were grown in 96-well plates in media supplemented with doxycycline for two days. The cells were then fixed and blocked, followed by incubation with individual serum samples (diluted 1∶200) obtained two weeks after the third immunization. 4 cells/well were added into 96-well plates. Serum samples (diluted 1∶100) were added next, followed by staining with isotype specific immunoconjugates. As expected, a significantly higher total IgG ΔMFI was observed for the M2e-NSP4 immunized mice  and survival were monitored during 16 days post challenge . It was The trivalent influenza vaccine that includes the viral surface proteins HA and NA, is the primary vaccine in use against seasonal influenza epidemics. This vaccine is quite efficient at inducing neutralizing antibodies, which can then protect against the viral strains circulating during a specific season. However, a vaccine that would not have to be updated every season would have major economical and health benefits, particularly in developing countries where annual vaccination is not easily applicable. Recent studies have led to the discovery and targeting of highly conserved regions in the HA protein showing significant protective potential 98–135 fragment of rotavirus, giving a fusion protein (M2e-NSP4) in which the tetramerizing NSP4 fragment would structurally replace the transmembrane section of the native M2 protein. This would allow for four M2e fragments to aggregate and thus mimic the native viral M2e conformation. When compared to M2e peptide immunization in side-by-side analysis and with both vaccines formulated in CAF-01 adjuvant, it was clearly revealed that M2e-NSP4 was more immunogenic and could be used at lower doses than the M2e peptide without loosing protective efficacy against closely related influenza A virus challenge. At higher antigen doses, both vaccines induced similar M2e peptide specific antibody responses, though the early reduction of the lung viral titers in PR8-challenged M2e-NSP4 immunized mice compared to M2e peptide vaccinated mice could indicate that the more efficient recognition of native M2 protein in the former mice might be associated with better clinical protection.In this study we characterized and analyzed the potential of a M2 vaccine construct intended to mimick the native structure of M2e. Thus, consensus sequence M2e was fused to the NSP4As antibodies recognizing conformational epitopes might prove to be more relevant in relation to in vivo protection, two assays were used to analyze the reactivity of the induced antibodies to native M2 protein as expressed on HeLa-M2 cells. It has previously been demonstrated that assays using native M2 protein as a basis for the assessment of vaccine-induced antibodies provide more relevant information than M2e-peptide based assays et al. the cross-reactivity of antibodies induced by M2e-carrier constructs based on M2e sequences from the consensus or the PR8 sequence were tested against several M2e-peptides, and no cross-reactivity was observed with avian M2e-peptides that differed by 3–4 aa from the consensus sequence et al. et al., which was of the H1N1 subtype, H7N7 is considered to be a highly pathogenic subtype For a universal vaccine to be of any real relevance as an add-on vaccine to the normal seasonal vaccine and/or as an interim pandemic influenza vaccine it needs to be cross-protective. Several attempts have been made to induce cross-reactive and therefore cross-protective antibodies based on the highly conserved M2e sequence. Although the M2e sequence is almost invariant within human influenza A viruses there are several aa differences between the human consensus sequence and avian sequences To conclude, in this study we show that M2e-NSP4 is superior to M2e-peptide immunization in inducing antibodies against the native M2 protein. Additionally, the M2e-NSP4 construct is very cheap and easy to produce in large quantities. In the case where both vaccines were equally good at providing full protection against lethal challenge with a virus differing from the vaccine sequence by one aa, only M2e-NSP4 immunized mice presented with a significantly reduced early virus load in the lungs. Moreover, following a reduction in either the number of vaccine doses or the amount of antigen used for each immunization, M2e-NSP4 was clearly superior at inducing protective immunity. Finally, M2e-NSP4 immunization induced antibodies that were significantly more cross-reactive to non-consensus M2e peptides and tended to afford better protection against a virus differing by five aa from the M2e consensus sequence.Figure S1Dose/response comparison of M2e and M2e-NSP4 immunization. Groups of Balb/c mice (n = 9–10) were immunized at days 0, 21, and 42 with 10 or 100 µg of M2e peptide or 10 and 50 µg of M2e-NSP4, both formulated in CAF-01. Serum samples were obtained 14 days after each vaccination, i.e. at days 14 (1°vacc), 35 (2°vacc), and 56 (3°vacc) after primary vaccination and analyzed in an ELISA for reactivity against M2e peptide. Results of individual sera are presented; group median titres are indicated by horizontal bars. *denotes a p-value <0.05. The results are representative of two experiments.(TIF)Click here for additional data file.Figure S2Comparison of adjuvants. Groups of Balb/c mice (n = 5) were immunized with 10 µg M2e peptide or M2e-NSP4 formulated in CAF-01, FIA + FCA, or AlOH at day 0, 21, and 42. Serum samples obtained on day 56 after primary vaccination were analyzed in ELISA for reactivity against M2e peptide. Results of individual sera are presented; group median titres are indicated by horizontal bars. *denotes a p-value <0.05. The data shown are representative of two experiments.(TIF)Click here for additional data file.Figure S3Reactivity of sera from M2e and M2e-NSP4 immunized mice against HeLa cells expressing M2 protein. Balb/c mice were immunized with 10 µg M2e peptide or M2e-NSP4 formulated in CAF-01 at days 0, 21, and 42. Binding of antibodies to HeLa cells expressing M2e (HeLa-M2) and non-expressing control cells (HeLa-C10) was analyzed by flowcytometry using sera harvested on day 56 after primary vaccination, representative histograms are presented. Binding of the M2e-specific monoclonal antibody 14C2 served as a positive control.(TIF)Click here for additional data file.Figure S4Evaluation of dose of M2e-NSP4 vaccine required for in vivo protection. Balb/c mice (n = 4–5) were immunized three times with 3 weeks interval with either 10 µg M2e-NSP4 with or w/o CAF-01, or 1 or 0.1 µg M2e-NSP4 in CAF-01. Control mice were left untreated. Around 6 weeks after the last immunization the mice were infected with 3 LD50 of influenza virus PR8. The mice were monitored daily with regard to survival (A) and loss of body weight (B).(TIF)Click here for additional data file.


Both screening hits inhibited PKD isoforms at about 100 nM and were ATP-competitive inhibitors. Analysis of several related kinases indicated that 1-NA-PP1 was highly selective for PKD as compared to IKK-16. SAR analysis showed that 1-NA-PP1 was considerably more potent and showed distinct substituent effects at the pyrazolopyrimidine core. 1-NA-PP1 was cell-active, and potently blocked prostate cancer cell proliferation by inducing G2/M arrest. It also potently blocked the migration and invasion of prostate cancer cells, demonstrating promising anticancer activities on multiple fronts. Overexpression of PKD1 or PKD3 almost completely reversed the growth arrest and the inhibition of tumor cell invasion caused by 1-NA-PP1, indicating that its anti-proliferative and anti-invasive activities were mediated through the inhibition of PKD. Interestingly, a 12-fold increase in sensitivity to 1-NA-PP1 could be achieved by engineering a gatekeeper mutation in the active site of PKD1, suggesting that 1-NA-PP1 could be paired with the analog-sensitive PKD1M659G for dissecting PKD-specific functions and signaling pathways in various biological systems.The emergence of protein kinase D (PKD) as a potential therapeutic target for several diseases including cancer has triggered the search for potent, selective, and cell-permeable small molecule inhibitors. In this study, we describe the identification,  Following the discovery of Gleevec, it was widely demonstrated that highly potent and specific small molecule inhibitors of kinases exhibit remarkable clinical efficacy and reduced toxicity PKD is a key signaling component of the diacylglycerol (DAG) signaling network. It is a primary target of DAG and a downstream effector of protein kinase C (PKC). There are several conserved structural motifs in PKD, such as a C1 domain that binds DAG and modulates PKD localization, and a PH domain that exerts an autoinhibitory function on the kinase domain. In intact cells, PKD is directly phosphorylated by DAG-responsive PKCs on the two conserved serine residues in the activation loop of the catalytic domain, which leads to its activation. PKD often exhibits sustained activity upon activation which is mainly maintained through autophosphorylation in vitro and in intact cells. Particularly, CRT0066101 was found to potently block the growth of pancreatic tumor xenografts in vivo in mice in vivo stability and general toxicity issues with the current set of known inhibitors. Therefore, it is imperative to continue the search for novel PKD inhibitory chemotypes that demonstrate attractive target selectivity, improved pharmacokinetic profiles, and greater in vivo efficacy.In the past several years, significant progress has been made in the development of potent and specific small molecule PKD inhibitors. CID755673 and analogs in vitro and in cells, and improved metabolic profiles in vitro and in vivo applications.We have previously identified both ATP-competitive and -noncompetitive PKD inhibitors that are distinct in structure to other reported inhibitors in vitro PKD1 inhibitory activity of these compounds was evaluated based on their ability to inhibit the recombinant PKD1 protein at 1 µM concentration in a radiometric PKD kinase assay. The percent PKD1 inhibition was calculated as the percent inhibition of the total PKD1 kinase activity in the absence of inhibitors (DMSO). Sixteen compounds were identified as primary hits  in the radiometric PKD1 assay , 133.4+/−3.6 nM (n = 3), 109.4+/−6.8 nM (n = 3), respectively, while IKK-16 similarly inhibited the PKD isoforms with an IC50 of 153.9+/−7.7 nM (n = 2) for PKD1, 115.0+/−7.1 nM (n = 2) for PKD2, and 99.7+/−3.0 nM (n = 2) for PKD3. These results indicate that both 1-NA-PP1 and IKK 16 are potent pan-PKD inhibitors.The To gain a better understanding of the mode of action for 1-NA-PP1 and IKK-16, we examined the effects of increasing concentrations of ATP on PKD1 inhibition. Lineweaver-Burk plots were generated by plotting the reciprocal of reaction velocities (1/v) against the reciprocal of ATP concentrations (1/[ATP]) at different compound concentrations. The points were fitted by linear regression. As shown Next, we determined the specificity of 1-NA-PP1 and IKK-16 for PKD by examining their activity for several functionally or structurally related kinases, including PKCα, PKCδ and CAMKIIα. No significant inhibitory activities for PKC isoforms were detected at 0.1, 1 and 10 µM concentrations of 1-NA-PP1, in contrast to IKK-16 which showed a concentration-dependent inhibition for both PKCα and PKCδ and >50% inhibition at 10 µM concentration (50 of 138.7±33.2 nM (n = 3) (http://kir.fccc.edu/) as described (Table S1) Further insights on the specificity of 1-NA-PP1 could be gained by evaluating the kinome scan data on 1-naphthylmethyl PP1 (1-NM-PP1), where 1-NM-PP1, along with 178 known kinase inhibitors, was profiled against a panel of 300 recombinant human protein kinases at a single concentration  (n = 3) Fig. 3D,d]pyrimidine core structure and determine the biological response to these modifications, as well as to potentially identify more PKD subtype selective analogs. We considered 4 types of variations, as shown in The goal of our synthetic studies was to modify the substituents on the 1-NA-PP1 pyrazolopyrimidine 456N-iodosuccinimide 2B(OH)2 led to the chlorinated derivative, and the chlorine group was reduced by a catalytic hydrogen transfer process to give product 1 with R4 = H (File S1.Vilsmeier–Haack reaction of barbituric acid in vitro radiometric kinase assay was used to screen an 80 compound library for PKD1 inhibitory activity at 1 µM concentration. 1.2 µM of a HDAC5 peptide 32P] ATP (Perkin Elmer Life Sciences), 25 µM ATP, 50 ng purified recombinant PKD1 in 50 µL kinase buffer containing 50 mM Tris-HCl, pH 7.5, 4 mM MgCl2 and 10 mM β-mercaptoethanol. The reaction was incubated at 30°C for 10 minutes and 25 µL of the reaction was spotted on Whatman P81 filter paper. The filter paper was washed 3 times in 0.5% phosphoric acid, air dried and counted using Beckman LS6500 multipurpose scintillation counter. Percent PKD1 inhibition was graphed using GraphPad Prism software 5.0.An 32P]ATP, 20 µM ATP, 50 ng of purified PKCα or PKCδ and 5 µg of myelin basic protein 4–14, 0.25 mg/mL bovine serum albumin, 0.1 mg/mL phosphatidylcholine/phosphatidylserine (80/20%) (1 µM), 1 µM phorbol dibutyrate in 50 µL of kinase buffer containing 50 mM Tris-HCl, pH 7.5, 4 mM MgCl2 and 10 mM β-mercaptoethanol. For the CAMK assay, 50 ng of CAMKIIα and 2 µg syntide-2 substrate in 50 µL kinase buffer were incubated with 0.1 mM MgCl2, 1 µCi of [γ-32P] ATP, 70 µM ATP. 0.5 mM CaCl2 and 30 ng/µL calmodulin were preincubated for 15 min on ice and then added in the kinase reaction. The reactions were incubated at 30°C for 10 min and 25 µL of the reaction was spotted on Whatman P81 filter paper. The filter paper was washed 3 times in 0.5% phosphoric acid, air dried and counted using Beckman LS6500 multipurpose scintillation counter.The PKC kinase assay was carried out by co-incubating 1 µCi [γ-2 at 37°C. Western blot analysis was carried out as previously reported The prostate cancer cell lines  were obtained from the American Type Culture Collection (ATCC). LNCaP and DU145 prostate cancer cells were maintained in RPMI 1640, while PC3 cells were maintained in Ham's F-12 medium, supplemented with 10% fetal bovine serum (FBS) and 1000 units/L penicillin, and 1 mg/mL streptomycin in 5% COPC3 cells were seeded into 96-well plates (3000 cells/well) and allowed to attach overnight. Cells were then incubated in media containing 0.7–100 µM inhibitors for 72 h. 3--2,5-diphenyltetrazolium bromide methyl thiazolyl tetrazolium (MTT) solution was prepared at 2 mg/mL concentration in PBS, sterilized by filtering through a 0.2 µm filter, and wrapped in foil to protect from light. 50 µL MTT solution was added to each well and incubated for 4 h at 37°C. Then, media was removed and 200 µL DMSO was added to each well. The plate was mix by shaking for 5 min and the optical density was determined at 570 nm.Proliferation of PC3 cells was measured by counting the number of viable cells upon trypan blue staining as previously described PC3 or DU145 cells were grown to confluence in 6-well plates. Migration was initiated by scraping the monolayer with a pipette tip, creating a “wound.” The indicated concentration of compound was added to the media, and the wound was imaged immediately under an inverted phase-contrast microscope with 10× objective. After 24 h, a final image was taken. The wound gap was measured, and % wound healing was calculated. The average % wound healing was determined based on at least 6 measurements of the wound gap.4 cells/ml) in RPMI containing 0.1% fetal bovine serum (FBS) were seeded into the top chamber of BioCoat control inserts (pore size 8 µm) or BioCoat Matrigel invasion inserts with Matrigel-coated filters (BD Pharmingen). To stimulate invasion, media in the lower chamber of the insert contained 20% FBS. Inhibitors were added at 30 µM concentration to both the upper and lower chambers, and cells were incubated for 22 h. After incubation, noninvasive cells were removed using a cotton swab, and invasive cells were fixed in 100% methanol and stained with 0.4% hematoxylin. After staining, cells were counted under a microscope (200× magnification). The percentage invasion was determined by cell counts in 5 fields of the number of cells that invaded the Matrigel matrix relative to the number of cells that migrated through the control insert.DU145 cells  at 100 MOI. After 24 h, cells were replated in migration chambers and incubated with vehicle or 30 µM 1-NA-PP1 for 20 h. The number of cells that migrated was determined by cell counts in 6 fields. Percentage migration was calculated as the percent of the Adv-null treated with vehicle DMSO (set to 100%). The experiment was repeated three times and data from a representative experiment are shown.(TIF)Click here for additional data file.File S1The Synthesis of New Pyrazolopyrimidine Inhibitors of Protein Kinase D. Experimental details and spectroscopic data for 1-NA-PP1 analogs.(DOCX)Click here for additional data file.Table S1http://kir.fccc.edu/). The kinase activity was determined using a radiometric HotSpot assay which directly measures kinase catalytic activity toward a specific substrate.The specificity of 1-NM-PP1 in a kinome scan. The activity of 1-NM-PP1, along with 178 known kinase inhibitors, was profiled against a panel of 300 recombinant human protein kinases at a concentration of 0.5 µM in the presence of 10 µM ATP. Data were extracted at a cut-off of <50% residual kinase activity from the Kinase Inhibitor Resource (KIR) online tool ((DOCX)Click here for additional data file.


This compound represents the first completely deuterated terpene obtained by fermentation. Despite a few previous reports in the literature the operability of this approach to fully deuterated compounds is still surprising, because the strong kinetic isotope effect of deuterium is known to slow down all metabolic processes in living organisms. Potential applications of completely labelled compounds from natural sources in structure elucidation, biosynthetic or pharmacokinetic investigations are discussed.During growth on fully deuterated medium the volatile terpene [ Streptomyces griseus is a producer of three terpenes, 2-methylisoborneol , (+)-caryolan-1-ol (2), and (+)-1-epi-cubenol -carreaction . For bioreaction . A drawbreaction . For 13Cextracts –9. Deuteextracts , stereosextracts , hydrideextracts , or deprextracts , the sitextracts , allowinernative –17. Furternative  may alteernative . At highernative –23. For ernative . Our curStreptomyces griseus was grown on agar plates containing completely deuterated minimal medium  or normal 65.GYM. While the agar plate cultures on normal 65.GYM were fully grown within two days, the cultures grown on DMM agar required eight to ten days of incubation until fully grown, reflecting the significantly slowed metabolism due to a strong deuterium kinetic isotope effect. After full growth of the culture the volatiles emitted by S. griseus were collected on charcoal traps by use of a closed-loop stripping apparatus (CLSA) -epi-cubenol in this work are not only a curiosity, but are also of interest to natural product chemists for the elucidation of biosynthetic pathways. Interesting options for their applications in biosynthetic studies include the use of non-labelled compounds in an inverse feeding experiment PO4: 0.5 g, MgSO4·7[2H2]O: 0.2 g, FeSO4·7[2H2]O: 0.01 g, agar: 12.0 g, [2H2]O: 1000 mL) to obtain [2H26]-3. DMM liquid culture medium was devoid of agar and CaCO3. Deuterated [2H8]glycerol (99% 2H) was obtained from Eurisotop , deuterium oxide (99.9% 2H) was obtained from Deutero GmbH , and all deuterated hydrated salts were obtained by dissolving the non-deuterated analogs in a large excess of deuterium oxide (100 equiv) followed by lyophilization. Agar that is composed of ca. 1 wt % of hydrogen atoms that are exchangable with water was added as the only unlabelled compound. The added agar (12 g L−1) is equal to ca. 1 mL of water (0.1% of the medium).Headspace analysis. For analysis of the volatiles liquid cultures were inoculated and incubated at 28 °C for 2 d (65.GYM) or 8–10 d (DMM). From these cultures 1 mL was transferred onto agar plates containing the same medium, and incubation was continued for 2 d (65.GYM) or 7–10 d (DMM). The volatile products emitted by the agar plate cultures were collected by use of a closed-loop stripping apparatus (CLSA) [s (CLSA) . BrieflyGC–MS. GC–MS analyses were carried out on an Agilent 7890A connected with an Agilent 5975C inert mass detector fitted with a HP5-MS fused silica capillary column . Instrumental parameters were (1) inlet pressure 77.1 kPa, He 23.3 mL min−1, (2) injection volume 1.5 μL, (3) transfer line 300 °C, and (4) electron energy 70 eV. The operation mode was splitless  and the carrier gas was He at 1.2 mL min−1. The GC was programmed as follows: 5 min at 50 °C increasing with 5 °C min−1 to 320 °C. Retention indices (I) were determined from a homologous series of n-alkanes (C8–C38).


Bacillus selenitireducens MLS10 exhibited inorganic phosphate (Pi)-dependent hydrolysis of kojibiose at the rate of 0.43 s−1. No carbohydrate acted as acceptor for the reverse phosphorolysis using β-d-glucose 1-phosphate (βGlc1P) as donor. During the search for a suitable acceptor, we found that Bsel_2816 possessed hydrolytic activity on βGlc1P with a kcat of 2.8 s−1; moreover, such significant hydrolytic activity on sugar 1-phosphate had not been reported for any inverting phosphorylase. The H218O incorporation experiment and the anomeric analysis during the hydrolysis of βGlc1P revealed that the hydrolysis was due to the glucosyl-transferring reaction to a water molecule and not a phosphatase-type reaction. Glycerol was found to be the best acceptor to generate 2-O-α-d-glucosylglycerol (GG) at the rate of 180 s−1. Bsel_2816 phosphorolyzed GG through sequential Bi-Bi mechanism with a kcat of 95 s−1. We propose 2-O-α-d-glucopyranosylglycerol: phosphate β-d-glucosyltransferase as the systematic name and 2-O-α-d-glucosylglycerol phosphorylase as the short name for Bsel_2816. This is the first report describing a phosphorylase that utilizes polyols, and not carbohydrates, as suitable acceptor substrates.The glycoside hydrolase family (GH) 65 is a family of inverting phosphorylases that act on α-glucosides. A GH65 protein (Bsel_2816) from  TherefoBacillus selenitireducens is an anaerobic haloalkaliphilic bacterium that is often isolated from the bottom sediments B. selenitireducens MLS10 strain, which was isolated from the anoxic mud of a saline lake, has been published (GenBank accession No. CP001791.1). B. selenitireducens MLS10 possesses three genes encoding possible inverting phosphorylases belonging to GH65. We have revealed the activities of two of them: one (locus: Bsel_2056) is a maltose phosphorylase that produces derivatives of maltose possessing a branch at position 2 d-Glucose 1-phosphate (βGlc1P) disodium salt was purchased from Tokyo Chemical Industry . β-Phosphoglucomutase (βPGM), βGlc1P bis(cyclohexylammonium) salt, and d-glucose 1,6-bisphosphate (Glc16bP) were obtained from Sigma-Aldrich Chemicals . Kojibiose, glucose 6-phosphate dehydrogenase , thio-NAD+, and Glucose C-II Test Wako were purchased from Wako Pure Chemicals . All other chemicals used are of reagent grade.β-http://clustalw.ddbj.nig.ac.jp/index.php?lang=ja). A phylogenetic tree was drawn from the results obtained with the neighbor-joining method using a TreeView version 1.6.6 (http://taxonomy.zoology.gla.ac.uk/rod/rod.html).Multiple alignments of the amino acid sequences of GH65 proteins with known activities with that of Bsel_2816 were performed using the ClustalW version 2.1 on the DDBJ sever  with the following primers based on the genomic sequence: 5′-aaaccatgggccatgaaattggagaacatc-3′ as the forward primer containing the NcoI site (underlined) and 5′-tttctcgaggcgggacttggtgatgcg-3′as the reverse primer containing the XhoI site (underlined). The amplified gene was purified using the FastGene Gel/PCR Extraction Kit , digested by NcoI and XhoI , and inserted into pET28a (+)  to add a His6-tag at the C-terminus of the recombinant protein. The expression plasmid was propagated in Escherichia coli DH5α (Toyobo), purified with the FastGene Plasmid Mini Kit , and verified by sequencing .A gene (GenBank ID: ADI00307.1) encoding Bsel_2816 was amplified by PCR from the genomic DNA of et al.bsel_2816 gene was used as template for PCR to obtain the DNA fragment containing the mutation. Pairs of the mutagenic primers  were used. The parental strands in the PCR products were digested with DpnI, which specifically digests methylated and hemimethylated DNA, and the amplified DNA were retained. The nicked plasmid DNA incorporating the desired mutation was transformed into the E. coli DH5α. The expression plasmid for the mutants was purified with the FastGene Plasmid Mini Kit , and verified by sequencing (Operon Biotechnologies).Site-directed mutagenesis was carried out by the method of Braman E. coli BL21 (DE3) (Novagen) transformants harboring the expression plasmid for Bsel_2816 or its mutants were grown at 37°C in 200 ml of Luria-Bertani medium  containing 50 µg/ml of kanamycin up to an absorbance (Abs) of 0.6 at 600 nm. The expressions were induced by 0.1 mM isopropyl β-d-thiogalactopyranoside and continued at 18°C for 24 h. Wet cells, harvested by centrifugation at 20,000×g for 20 min, were suspended in 50 mM HEPES–NaOH buffer (pH 7.0) containing 500 mM NaCl (buffer A) and 10% glycerol. The suspended cells were disrupted by sonication  and the supernatant, collected by centrifugation at 20,000×g for 20 min, was applied to a HisTrap HP column  equilibrated with buffer A containing 10 mM imidazole using an ÄKTA prime system . After a wash with buffer A containing 22 mM imidazole and the following elution using a 22−400 mM imidazole linear gradient in buffer A, the fractions containing the recombinant protein (Bsel_2816) were pooled, dialyzed against 10 mM HEPES–NaOH buffer (pH 7.0), and concentrated . The concentration of the wild-type, E475Q mutant, and E475A mutant was determined spectrophotometrically at 280 nm using a theoretical extinction coefficient of ε = 124,110 M−1cm−1 on the basis of amino acid sequences TLC was performed on a TLC plate . After spotting the samples, the plate was developed once with acetonitrile–water (4∶1 v/v) or twice with acetonitrile–water (9∶1 v/v). The TLC plates were then soaked in 5% sulfuric acid–methanol solution and heated in an oven until the spots of the carbohydrates became sufficiently visible.505 was measured after incubation for 60 min at 37°C. The concentration of βGlc1P was determined by using the βPGM/G6PDH method +, 20 µg/ml Glc16bP, and 10 mM MgCl2 in 50 mM MOPS–NaOH buffer (pH 7.0). An aliquot of sample was mixed with the same volume of the working reagent and the increase in the Abs400 was measured after incubation for 60 min at 37°C.The concentration of Glc was determined by using a glucose oxidase/peroxidase kit, the Glucose C-II Test Wako. Two volumes of the coloring reagent of the kit were added to one volume of the sample. The increase in the AbsTo examine the specificity on α-linked glucobioses, each reaction mixture containing each disaccharide , 10 mM inorganic phosphate (Pi), and 29 µM Bsel_2816 in 50 mM MES–NaOH buffer (pH 6.0) was incubated at 30°C for 3 h. The products were analyzed by TLC. The reaction rate for the cleavage of kojibiose was determined by quantifying the Glc liberated in the reaction mixtures containing various concentrations of kojibiose (1–10 mM) and Pi (0–10 mM) in 40 mM sodium acetate buffer (pH 5.5) at 30°C. An aliquot of the reaction mixture was mixed with the same volume of dimethyl sulfoxide to stop the enzymatic reaction, followed by the enzymatic quantification of Glc. The kinetic parameters were calculated by curve-fitting the experimental data to the theoretical P in 50 mM MES–NaOH buffer (pH 6.0) was reacted with Bsel_2816 at 30°C. After the reaction, the products were analyzed by TLC.To determine the acceptor specificity of Bsel_2816 in the reverse phosphorolysis, each reaction mixture containing 10 mM each acceptor and 10 mM βGlc1P , 1 M glycerol, and 0.25 mg/ml Bsel_2816 was incubated at 30°C for 18 h. After terminating the reaction by boiling, an aliquot (3 ml) was deionized with Amberlite MB-3 , applied on an Amberlite IRA400 column , and eluted with H2O to remove the reducing sugars. The fractions containing the product were collected, concentrated to 9 ml, and loaded on a Toyopearl HW40F column  equilibrated with distilled water. The product was eluted by distilled water at a flow rate of 5 ml/min. The fractions containing the product were collected, concentrated, and lyophilized to be syrup (298 mg). The one-dimensional (1H and 13C) and two-dimensional  NMR spectra of the product were recorded in D2O with 2-methyl-2-propanol as an internal standard at 298 K using the Bruker Avance 500 spectrometer . The product was determined to be 2-O-α-d-glucosylglycerol (GG).A reaction mixture (5 ml) containing 500 mM βGlc1P produced during the enzymatic reaction from 10 mM GG and 10 mM Pi in 40 mM HEPES–NaOH buffer (pH 8.0) at 30°C. An aliquot of the reaction mixture was mixed with the same volume of dimethyl sulfoxide to stop the enzymatic reaction, followed by the enzymatic quantification of βGlc1P. The hydrolytic activity on βGlc1P was determined by measuring the increase in Glc released during the enzymatic reaction from 10 mM βGlc1P in 40 mM MES–NaOH buffer (pH 6.0) or 40 mM HEPES–NaOH buffer (pH 7.5) at 30°C. An aliquot of the reaction mixture was mixed with the same volume of dimethyl sulfoxide to stop the enzymatic reaction, followed by the enzymatic quantification of Glc. The synthetic activity of GG was determined by measuring the increase in Pi in the reaction mixture containing 10 mM βGlc1P and 10 mM glycerol in 40 mM HEPES–NaOH buffer (pH 7.5) at 30°C by using the method of Lowry and Lopez The phosphorolytic activity of GG was determined by quantifying βGlc1The effects of pH on the phosphorolytic, hydrolytic, and synthetic activities of 212 nM Bsel_2816 were measured under the standard conditions by substituting each 40 mM reaction buffer into the following 40 mM buffers: sodium citrate (pH 3.0–5.5), Bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane–HCl (pH 5.5–7.0), HEPES–NaOH (pH 7.0–8.5), and glycine-NaOH (pH 8.5–10.5). The thermal and pH stabilities were evaluated by measuring the residual synthetic activity under the standard conditions after the incubation of Bsel_2816  at temperatures 30–90°C for 15 min in 10 mM HEPES–NaOH buffer (pH 7.0) containing 0.1% bovine serum albumin and at the various pH values at 4°C for 24 h, respectively.P assay. The reaction mixtures (150 µl) were prepared in the wells of a 96-well microtiter plate with various concentrations of GG (1.0–20 mM) and Pi (0.5–10 mM), and 5.2 nM Bsel_2816 in 40 mM HEPES–NaOH buffer (pH 8.0) containing 2.5 U/ml βPGM, 2.5 U/ml G6PDH, 2 mM thio-NAD+, 5 µg/ml Glc16bP, and 5 mM MgCl2. The reaction was carried out in a temperature-controlled microplate reader  at 30°C, and the Abs400 was continuously monitored at 30-s intervals without stopping the reaction. The kinetic parameters were calculated by curve-fitting the experimental data to the theoretical A kinetic analysis of the phosphorolytic reaction of GG was performed using the continuous βGlc1P were determined under the standard conditions with 5.2 nM Bsel_2816 and various concentrations of βGlc1P (0.1–10 mM). The kinetic analysis of the synthetic reaction with glycerol as acceptor was carried out under the standard conditions (5.2 nM Bsel_2816) and various concentrations of glycerol (1.0–100 mM) or βGlc1P (ranging from 0.1 to 10 mM) with 10 mM or 100 mM of each opposite substrate, respectively. The kinetic parameters were calculated by curve-fitting the experimental data with the Michaelis–Menten The initial velocities of the hydrolytic reaction on βGlc1P by glycerol was examined in the reaction mixture containing 10 mM βGlc1P and various concentrations of glycerol (0–100 mM) in 40 mM HEPES–NaOH buffer (pH 7.5) and the increase in the concentration of Glc was quantified. The rate of the release of Glc was regressed with the concentration of glycerol to determine the dissociation constant of glycerol using the The inhibition of the hydrolysis of βGlc1P bis(cyclohexylammonium) salt  was dissolved in 1 ml of H2O and applied to a Dowex 50 column  to remove the counter-ion (cyclohexylamine). An appropriate amount of 1 M NaOH was added to the decationized solution containing free βGlc1P (5 ml) adjusting pH to 6.0, followed by concentration to 1 ml by a centrifuge evaporator to obtain 100 mM βGlc1P–Na buffer (pH 6.0). An aliquot of the 100 mM βGlc1P–Na buffer (20 µl) was mixed with 70 µl of H218O  or unlabeled H2O as a control. The hydrolysis of βGlc1P was started by adding 10 µl of 29 µM Bsel_2816 in 4 mM MES–NaOH buffer (pH 6.0) to the solution. After the incubation at 30°C for 4 h, βGlc1P was completely hydrolyzed into Glc and Pi. The solution was lyophilized, dissolved in 50% methanol, and subjected to negative and positive ESI-MS analysis by using a Bruker APEX II 70e Fourier transformed cyclotron resonance mass spectrometer .βGlc1P was analyzed by HPLC P was performed in the reaction mixture containing 50 mM βGlc1P and 13.8 µM Bsel_2816 prepared in 20 mM MES–NaOH buffer (pH 6.0) at 30°C. After the reaction for 1.5 and 25 min, aliquots (10 µl each) of the reaction mixtures were immediately injected into the HPLC and separated with a TSK amide-80 column  by isocratic mode with 80% acetonitrile at a flow rate of 1.5 ml/min.The anomeric configuration of the glucose generated during the hydrolysis of βGlc1bsel_2816 gene cloned from the genomic DNA of B. selenitireducens MLS10 was expressed as a His6-tag fusion protein in E. coli BL21 (DE3) to investigate its enzymatic properties. Purified protein (approximately 14 mg) was obtained from the cell lysate of 200 ml culture. The purified Bsel_2816 migrated in SDS-PAGE as a single protein band with an estimated molecular mass of approximately 87 kDa, in agreement with the theoretical molecular weight of 86,259. However, this native molecular mass was determined to be 174 kDa by gel filtration, suggesting that Bsel_2816 was a homodimeric enzyme.The phylogenetic tree analysis showed that Bsel_2816 is distantly related with the kojibiose phosphorylase and nigerose phosphorylase . In thisP was not detected at all. The optimum pH for the cleavage of kojibiose was determined to be 5.5. The measurement of the reaction rate with various concentrations of kojibiose and Pi revealed that the reaction proceeded with a sequential Bi Bi mechanism, similarly to other inverting phosphorylases, with the following kinetic parameters: KmA, 8.9 mM; KiA, 41 mM; KmB, 0.19 mM; kcat 0.43 s−1, where A and B represent kojibiose and Pi, respectively  including the known substrates of the GH65 enzymes was examined. Bsel_2816 cleaved kojibiose at a slow rate only in the presence of Pi. To our surprise, Glc was detected as the sole product and βGlc1ectively . The read-glucose, d-mannose, d-allose, d-galactose, d-altrose, d-talose, and d-fructose), pentoses , aminosugars , uronic acids , deoxyhexoses , derivatives of d-glucose , and disaccharides ] as acceptors and βGlc1P as the donor. No transfer product was detected on TLC with any sugar acceptors that were examined. However, the consumption of βGlc1P and the concomitant generation of Glc were detected with all the acceptors. The concomitant generation of Glc and Pi was detected during the reaction in the absence of the acceptor molecule, clearly indicating that Bsel_2816 hydrolyzed βGlc1P. The rates of the hydrolysis of 10 mM βGlc1P in the presence and in the absence of 10 mM Glc were identical. The optimum pH for the hydrolysis was determined to be 6.0. The kinetic parameters of the hydrolysis of βGlc1P at pH 6.0 and pH 7.5 were determined to be as follows: Km, 0.041 and 0.16 mM; kcat, 2.8 and 1.7 s−1, respectively.To determine the substrates of Bsel_2816 as a GH65 phosphorylase, the reverse phosphorolysis was examined with various sugars [hexoses  and 277.03 m/z corresponding to [H3PO4 + Glc − H]−  were detected. In the H218O reaction, no signal of [2H3PO4 − H (18O)]−  and [2H3PO4 − H (218O)]−  was detected, clearly indicating that 18O was not incorporated in the Pi during the hydrolysis. On the other hand, the signal at 277.03 m/z decreased and a new signal at 279.04 m/z corresponding to [H3PO4 + Glc − H (18O)]− appeared, indicating that 18O was incorporated into either Pi or Glc. In positive MS  accompanied by a signal at 205.06 m/z corresponding to [Glc+Na(18O)]+ indicated that 18O was incorporated into Glc. It is concluded that the anomeric CO bond was cleaved during the hydrolysis of βGlc1P, indicating that the hydrolysis was a glycosidase-type reaction and not a phosphatase-type reaction  were used to identify the suitable acceptor. Only the hydrolysis of βGlc1P was detected with all the alcohols examined (data not shown). Among polyols, the consumption of βGlc1P was obviously faster with glycerol than with others  indicates an α-d-glucoside. The HMBC cross signals indicate the glycosyl linkage with the position 2 of glycerol. Thus, the product was identified to be GG.Because no acceptor was found within sugars, various alcohols  and polyols  on TLC  as the short name for Bsel_2816.In the presence of glycerol, the synthesis of GG was much faster than the hydrolysis of βGlc1P . In the P  with the) on TLC . We conftectable . Herein,Bsel_2816 was stable up to 35°C during 15-min incubation, and it was stable at pH between 5.5−10.5 at 4°C for 24 h (data not shown). The optimum pHs for the phosphorolytic, synthetic, and hydrolytic reactions were pH 8.0, pH 7.5, and pH 6.0, respectively (data not shown).i presented a series of lines intersecting at a point  were determined at pH 7.5. The Km and kcat values with glycerol were 5.7 mM and 163 s−1, respectively. The Km value of glycerol agreed with the Kd value determined by the inhibition of hydrolysis of βGlc1P (7.6 mM). The Km and kcat values of βGlc1P were 0.45 mM and 180 s−1, respectively.The kinetic parameters of the substrates in the synthetic reaction /base (for reverse phosphorolysis) catalyst We have determined that Bsel_2816 is a GGP. The reactions catalyzed by GG are summarized in Km values 1–10 mM with their main acceptors. Structural analyses of various phosphorylases have revealed that these enzymes recognize their acceptor molecules through their pyranose rings (or furanose ring in the case of sucrose phosphorylase) by a number of hydrogen bond interactions to obtain their strict acceptor specificities with the Km values. The Km value of GGP with glycerol was 5.7 mM, similarly to that of other phosphorylases, even though glycerol is a rather small molecule and does not adopt any rigid conformation.First, any other phosphorylase reported exhibits its highest phosphorolytic activity on the glycosyl linkages bound with sugar residues . ContrarP. Other inverting phosphorylases do not hydrolyze sugar 1-phosphates Second, the other feature of GGP is the hydrolysis of βGlc1P occurs through the same catalytic mechanism as the reverse phosphorolysis, not through a phosphatase-type reaction. The hydrolysis is explainable as a glucosyl transfer reaction from βGlc1P to a water molecule with the anomeric inversion. The phosphate-dependent hydrolysis of kojibiose is not considered to be a direct hydrolysis. It is explained as follows: GGP phosphorolyzes kojibiose at a very slow rate to form βGlc1P that is immediately hydrolyzed to generate Glc and Pi by the same enzyme. In this mechanism, the α-anomer of Glc is considered to be generated during the hydrolysis, indicating a retaining hydrolysis. Unfortunately, the anomeric analysis during the hydrolysis of kojibiose was unsuccessful because of its very slow reaction. On the other hand, the hydrolytic reaction was much slower than the phosphorolysis and the synthesis of GG, suggesting that the hydrolytic activity may be negligible in its in vivo role.To investigate the mechanism of the hydrolysis, we determined the linkage cleaved in the hydrolysis, the anomeric configuration of the hydrolytic product, and the catalytic residue of the enzyme. Our results prove that the hydrolysis of βGlc1P) by UhgbMP belonging to GH130, a β-1,4-mannooligosaccharide phosphorylase from unknown human gut bacterium, has been reported P, suggesting that the enzyme initially hydrolyzed αMan1P to generate mannose. The resultant mannose was not detected because it readily acted as the acceptor molecule to form β-1,4-mannobiose, which was a better acceptor than mannose to generate the polymer. This enzyme may share the same hydrolytic mechanism with GGP.Recently, indication of faint hydrolytic activity on α-mannnose 1-phosphate . The role of GGP is unclear but it may be involved in the biosynthesis of GG as well as in its cleavage. B. selenitireducens possesses potassium ion-dependent trehalose phosphorylase, even though the strain does not have either intracellular or extracellular biosynthetic pathways of trehalose B. selenitireducens has one more GH65 enzyme, maltose phosphorylase P. These three enzymes may relate with the production of GG and trehalose in the cytoplasm of B. selenitireducens as solutes for the control of the cytoplasmic osmotic pressure.GG has been reported as a compatible solute for the control of the cytoplasmic osmotic pressure of cyanobacteria Aspergillus oryzae) GG is often isolated from the traditional Japanese food, fermented with “koji” Click here for additional data file.Figure S2NMR spectra of the product from glycerol. The product was identified to be 2-O-α-d-glucopyranosylglycerol. The spectra were taken in D2O at 298 K with 2-methyl-2-propanol as an internal standard using Bruker Avance 500 spectrometer. (A) 1H-NMR spectrum; (B) 13C-NMR (proton decupling and DEPT135); (C) DQF-COSY; (D) HSQC; (E) HMBC.(PDF)Click here for additional data file.


Here we used intravital microscopy to measure the contributions of sympathetic receptors, particularly α2+]i) in femoral arteries were determined by intravital fluorescence microscopy in mice expressing a Myosin Light Chain Kinase (MLCK) based calcium-calmodulin biosensor. Pharmacological agents were applied locally to the femoral artery to determine the contributions of vascular receptors to tonic contraction and [Ca2+]i,.Diameter and intracellular calcium (i. Chloroethylclonidine  had no effect. Abolition of sympathetic nerve activity  reduced basal tone by 90%.In the anesthetized animal, femoral arteries were constricted to a diameter equal to 54% of their passive diameter (i.e. tone = 46%). Of this total basal tone, 16% was blocked by RS79948 (0.1 µM) and thus attributable to α1A- and α1D-adrenoceptors elevates [Ca2+]i and accounts for at least 55% of the tone.Tone of mouse femoral arteries in vivo is almost entirely sympathetic in origin. Activation of α Systemic application of receptor blockers or agonists or genetic ablation of receptor subtypes inevitably involve receptors located elsewhere than the vasculature. Similarly, many of the factors potentially influencing vascular tone in vivo will be lost when arteries are removed from the animal for study.The sympathetic nervous system (SNS) plays a major role in maintaining arterial blood pressure, through its effects on the heart, blood vessels, kidneys and adrenal glands. In rats, total block of autonomic ganglionic transmission results in a rapid fall in arterial blood pressure, due acutely to a decrease in total peripheral vascular resistance 1-adrenoceptor subtypes in basal-state tone of femoral arteries of anesthetized mice in vivo. These mice express a FRET-based genetically encoded Ca2+/Calmodulin biosensor molecule, based on smooth muscle myosin light chain kinase (MLCK) specifically on smooth muscle cells. The fluorescence in smooth muscle cells of the artery walls provides 1) quantitative measurement of smooth muscle cell [Ca2+], and 2) a precise measurement of artery diameter. The preparation we developed 1-adrenoceptor subtype to femoral artery tone in vivo by using the α1-adrenoceptor selective antagonist prazosin 1A-adrenoceptor selective antagonist RS100329 1D-adrenoceptor selective antagonist BMY7378 1B-adrenoceptor alkylating agent, chloroethylclonidine In the present study therefore, we utilized a new experimental model, the exMLCK optical biosensor mouse 2+-calmodulin through changes in FRET (Forster Resonance Energy Transfer) between cyan (CFP) and yellow (YFP) fluorescent proteins. All mice were maintained on 12∶12-h light/dark schedule at 22–25°C and 45–65% humidity and fed ad libitum on a standard rodent diet and tap water. A total of 29  mice were used, ages of 16–20 weeks, weights 28–32 grams.All experiments were approved by the Institutional Animal Care and Use Committee of the University of Maryland, School of Medicine, MD. The transgenic mouse line  was the same as used previously 2. During the surgical procedure and the subsequent experiment anesthesia was maintained with 1.5% isoflurane in O2. After induction of anesthesia, mice were placed in a supine position on a custom made temperature-controlled platform set to maintain core temperature of animals at 37–38°C.Anesthesia was induced with 1–5% isoflurane  in O3, 4.9 KCl, 2.0 CaCl2, 2.0 MgSO4, 1.2 KHPO4, 11.5 glucose, and 10.0 HEPES . Solutions containing elevated KCl were made by replacing the NaCl with KCl on an equimolar basis. Experiments in which a “zero”-calcium solution was used, the solution had the same composition as the standard PSS with the omission of CaCl2 and the addition of Na2EGTA (2 mM). Superfusion was at 2 ml/min, 35°C monitored continuously by a temperature probe . The arteries were continuously exposed to antagonist for 10 minutes before arterial diameter was recorded using fluorescent edges of the blood vessel. For CEC treatment, arterial segments were incubated with CEC for 10 min at 35°C followed by washing in PSS for 30 min Hair from the hind limb region was removed using a depilatory agent. Under microscopic observation, the femoral artery was exposed via a cutaneous incision in the upper thigh. The underlying connective tissue above the artery was lightly dissected, taking care to avoid severing nerves. After exposing the femoral artery in this way, the animal was moved to the stage of a fluorescence microscope and superfusion of the artery was begun, with the standard physiological salt solution containing  112.0 NaCl, 25.7 NaHCOin vivo, an Olympus MVX10 MacroView microscope   was used. Excitation illumination was via a xenon arc lamp . For measurements of arterial diameter, the biosensor was excited at 426–446 nm. Emission was collected at 455–485 nm (CFP) and 520–550 nm (YFP) with a charge-coupled device . Total optical zoom was set such that an effective imaging of 1.0–2.0 µm/pixel was established. Acquisition of images was set at 1.0 Hz. The microscope was equipped with an image splitter equipped with the appropriate filters for CFP/YFP FRET microscopy  and a sensitive CCD video camera . The camera was controlled and images acquired using HCImage .For imaging femoral arteries 0.9, c. 310 µm) were mounted in a four-channel wire myograph  for isometric tension measurement and were maintained in gassed PSS at 35°C. After incubation for 1 hr, the vessels were then normalized, that is, the resting tension–internal circumference (IC) relation was determined for each vessel segment 0.9, where IC0.9 = 0.9IC100 and IC100 is the internal circumference of the vessel under an effective resting transmural pressure (ERTP) of 100 mmHg (13.3 kPa). ERTP was calculated from the Laplace equation ). Lab View software was used for acquisition. At 30 min after normalization, the vessels were exposed to 60 mM KCl solution twice followed by 10 µM noradrenaline in the presence of 60 mM KCl solution. The arteries were considered viable if the equivalent transmural pressure produced by 60 mM KCl was >100 mmHg (13.3 kPa) Arterial segments of 2 mm length , desmethylimipramine  and corticosterone acetate  were added to the PSS before each CRC. Results are expressed as mean±s.e.m., n being the number of animals.After equilibration, three to four concentration–response curves (CRC) to PE were obtained in each femoral arterial segment (30 min between each CRC). Preliminary experiments showed no significant time-dependent changes in sensitivity. The first CRC was taken as control and subsequent curves were obtained after incubating the vessels with increasing concentrations of the same antagonist for 30 min. To characterize αin vivo, there is no way to clamp the [Ca2+]i concentration reliably due to blood flow. For calcium calibration curves arteries were allowed to stabilize in Krebs solution, followed by incubation of the vessel in a high relaxing (HR) solution for 10–20 min before permeabilization. The high relaxing (HR) and pCa solutions were used during and following permeabilization of the mesenteric vessels were designed to maintain a desired free Ca2+ concentration, using a Ca2+-EGTA buffering system. The composition of the solutions was calculated with the MAXC Computer Program for calculating free Ca2+ concentrations. The HR solution (pCa >9) was composed of the following chemicals (in mM): 53.28 KCl, 6.81 MgCl2, 0.025 CaCl2, 10.0 EGTA, 5.4 Na2ATP, and 12.0 creatine phosphate. The composition of the pCa 4.5 solution was similar to HR, except for the following differences (in mM): 33.74 KCl, 6.48 MgCl2, and 9.96 CaCl2. The pH of the HR and pCa 4.5 solutions were adjusted to 7.1 with KOH, and ionic strength was held constant (0.15). Solutions containing a desired free Ca2+ concentration between pCa 9 and 4.5 were achieved by mixing appropriate volumes of the HR and pCa 4.5 solutions based on the Bathe algorithm. All solutions contained the protease inhibitors leupeptin (1 µg/ml), pepstatin A (2.5 µg/ml), and PMSF (50 µM). Mesenteric arteries were permeabilized with α-toxin from staphylococcus aureus by incubating the vessel segment with 1000 U/ml α-toxin in HR for 1 hour at room temperature. After permeabilization, segments were washed with HR solution and force was allowed to stabilize. The pCa-tension relationship was then determined by bathing the permeabilized vessels in solutions of sequentially increasing Ca2+ concentrations, ranging from pCa 8.5 to 4.5, while recording force for 5 min in each solution or until stabilized. The Ca2+ induced alteration in tension was expressed as a percent relative to the basal tension at pCa 9. The changes in FRET ratios and force, measured with [Ca2+] ranging from 1 nM to 50 µM at excitation wavelength of 426–446 nm, is characterized by a normalized sigmoid curve fit. The calcium calibration graph was plotted with the normalized FRET ratio on the y-axis and calcium concentration on the x-axis. The EC50 and Hill Coefficient extracted from the curve were 6.05 (pCa) and 1.4 respectively. Rmin and Rmax were calculated by exposing the artery to 0Ca2+ (2 mM EGTA and 1 µM acetylcholine) and KCl (60 mM) respectively.Third order mesenteric arterial segments from exMLCK FRET biosensor mice, 2–3 mm in length were dissected Dissected segments of femoral artery, 1–2 mm in length, were transferred to a recording chamber, where their ends were mounted on glass pipettes (tip diameter 60–100 µm) and secured by 10-0 Ethilon ophthalmic nylon sutures . One pipette was attached to a servo-controlled pressure-regulating device , whereas the other was attached to a closed stopcock to study the pressure-dependent effects in the absence of intraluminal flow. The intraluminal pressure was set to 70 mmHg and was continuously superfused with gassed PSS at 35°C. During the entire process, the arteries that developed significant leaks were discarded. Measurements of arterial wall position from transmitted light images were recorded at 2 images/sec with a Nikon ×20 objective.50 . The pEC50 and Emax values were calculated using the Graphpad Prism software program, which fits CRCs to the four parameter logistic equation below:Agonist potency is expressed as the pEC(logEC50-X)P)], where X is the logarithm of the molar concentration of agonist, Y is the response and P is the Hill slope. Antagonist affinity was expressed either as pKB or pIC50 values. When three different concentrations of the antagonist were used, pKB values were obtained from the x-intercept of the plot of log (r−1) vs. log(B), where r is the ratio of the agonist EC50 in the presence and absence of antagonist and B is the molar concentration of antagonist B. When one concentration of antagonist was used to obtain the affinity, estimated pKB values were calculated from the Schild equation B = -log[(B)/(r-1)]. The change in arterial diameter (in vivo) was calculated and expressed as a fractional diameter based on full passive diameter with 0[Ca2+]. Antagonist potencies were also expressed as mean pIC50 values (the negative logarithm of the concentration of antagonist producing 50% inhibition of the prazosin-sensitive component of the vascular tone). Best fit pEC50 and Emax values obtained from nonlinear regression of CRC (described above) and other mean values were compared by an unpaired t-test for two groups or by repeated measures one-way analysis of variance (ANOVA) followed by Newman-Keuls multiple comparison test (three or more groups) after checking for normality (Kolmogorov–Smirnov test).Y = Bottom+ethyl]-8-azaspiro[4.5]decane-7,9-dione (BMY7378) . -5,8,8a,9,10,11,12,12a,13,13a-dechydro-3-methoxy-12-(ethylsulfonyl)-6H-isoquino 2+] and 2 mM EGTA). Basal ‘tone’ was thus 46±1% (100%–54%). We found no significant difference in femoral artery tone in vivo between female and male mice (16–30 weeks old). Basal tone in females = 46.48±1.49% (n = 15) and males = 46.63±1.25% (n = 11), P>0.05 (un-paired t-test). To determine the component of this tone that might be due to autonomic nervous system activity, we blocked autonomic ganglionic transmission with systemically applied hexamethonium . This caused a vasodilation of femoral arteries nearly to PD, and a reduction of arterial blood pressure, as we have reported previously in vivo is revealed as neurogenic. Myogenic tone In the anesthetized animal, femoral arteries were constricted to a diameter equal to 54±1% (n = 29) of their passive diameter  and prazosin  reduced vascular tone by 16±3% (n = 12) and 46±4% (n = 9), respectively. Subsequent addition of both BIBP 3226  and Suramin  also had significant effect on vascular tone . A summary of the effects on femoral artery tone in vivo of combined block of adrenergic, P2X, and NPY1 receptors is shown in The contribution to vascular tone of several neurotransmitter receptors that might be involved in sympathetic neurogenic tone examined Fig. 1. 2-adrenoceptors activated by neurally released NA could affect the contribution of α1-adrenoceptors to vascular tone in vivo, the experiments to determine the α1-adrenoceptor subtypes involved were all carried out in the presence of RS79948 (0.1 µM). In vivo, local prazosin  produced concentration dependent inhibition of vascular tone . To determine the α1-adrenoceptor subtype based on affinity to prazosin, femoral arteries were isolated and mounted on a wire myograph for isometric force recording and the effect of prazosin on the concentration-response curves to phenylephrine (PE) was determined. Prazosin  significantly inhibited vascular tone with a pIC50 value of 7.4±0.1. The fractional diameter increased by 0.14±0.01 (P<0.001) with 0.1 µM RS 100329. BMY 7378, specific for α1D-adrenoceptors  also inhibited vascular tone significantly with a pIC50 value of 7.2±0.2 and an increase in fractional diameter by 0.12±0.01 (P<0.001) with 0.1 µM BMY7378. The combination of RS100329 (0.1 µM) and BMY7378 (0.1 µM) effected a change in fractional diameter of 0.25±0.03  had no significant effect on vascular tone, but higher doses of CEC  did have a small effect, increasing fractional diameter by 0.07±0.01 .We examined the contributions of α3 n = 5, Fig. 3C 2+]. The inhibition of vascular tone using α1-adrenoceptor antagonists was also evident in the changes in intracellular calcium calculated by the changes in CFP and YFP ratios in ex-MLCK biosensor animals. Prazosin (0.1 µM) decreased [Ca2+]i by 0.18±0.03 µM (2+]i by 0.15±0.02 µM (2+]i by 0.11±0.03 µM (2+]i. (not shown).The presence of the genetically encoded biosensor in the arterial smooth muscle provided not only a convenient way to measure artery diameter, but also provided a measurement of smooth muscle intracellular i. The small effect on vascular tone with CEC at 10 µM that was observe may be attributable to α1A/D-adrenoceptors since at that concentration, CEC, also alkylates α1A/D-adrenoceptors Finally, α1A- and α1D-adrenoceptors is maintenance of sympathetic neurogenic tone in mouse femoral artery in vivo. Consistent with this, α1A-adrenoceptors play a major role in tonic maintenance of mouse blood pressure 1D-adrenoceptors in mediating nerve mediated responses are well documented in rat caudal artery where the authors suggested that α1D-adrenoceptors are restricted to the junctional region by neuronal activity, but if the nerves are lost, these receptor subtypes spread from the postsynaptic region along the smooth muscle 1D-adrenoceptors in rat femoral arteries 1D-adrenoceptors are known to play a modulatory role which is constitutively active in contractile tone in rat conductance arteries 1D-adrenoceptors are also involved in nerve mediated responses in rat 1D-adrenoceptor knock out models have directly shown that α1D-adrenoceptor participates in the regulation of systemic blood pressure 1A- and α1D- in neurogenic tone of mouse femoral arteries in vivo.When RS100329 (0.1 µM) and BMY7378 (0.1 µM) were used in combination, the prazosin (0.1 µM) sensitive response was completely eliminated. This suggests the major role from αFigure S1(TIF)Click here for additional data file.Figure S2(TIF)Click here for additional data file.Figure S3(TIF)Click here for additional data file.Figure S4(TIF)Click here for additional data file.Table S1(DOC)Click here for additional data file.


Glutamatergic synapse maturation is critically dependent upon activation of NMDA-type glutamate receptors (NMDARs); however, the contributions of NR3A subunit-containing NMDARs to this process have only begun to be considered. Here we characterized the expression of NR3A in the developing mouse forebrain and examined the consequences of NR3A deletion on excitatory synapse maturation. We found that NR3A is expressed in many subcellular compartments, and during early development, NR3A subunits are particularly concentrated in the postsynaptic density (PSD). NR3A levels dramatically decline with age and are no longer enriched at PSDs in juveniles and adults. Genetic deletion of NR3A accelerates glutamatergic synaptic transmission, as measured by AMPAR-mediated postsynaptic currents recorded in hippocampal CA1. Consistent with the functional observations, we observed that the deletion of NR3A accelerated the expression of the glutamate receptor subunits NR1, NR2A, and GluR1 in the PSD in postnatal day (P) 8 mice. These data support the idea that glutamate receptors concentrate at synapses earlier in NR3A-knockout (NR3A-KO) mice. The precocious maturation of both AMPAR function and glutamate receptor expression are transient in NR3A-KO mice, as AMPAR currents and glutamate receptor protein levels are similar in NR3A-KO and wildtype mice by P16, an age when endogenous NR3A levels are normally declining. Taken together, our data support a model whereby NR3A negatively regulates the developmental stabilization of glutamate receptors involved in excitatory neurotransmission, synaptogenesis, and spine growth. In early postnatal development, the formation and maturation of excitatory synapses play critical roles in the proper wiring of neuronal networks required for learning and memory. The balance between synapse stabilization and elimination is highly sensitive to changes in the complement of synaptic proteins. The subunit composition of NMDA- and AMPA-type glutamate receptors (NMDARs and AMPARs) is particularly important for defining ionotropic glutamate receptor-mediated synaptic transmission. Synaptic activity and sensory experience modify synaptic function, in part by promoting the transition between ‘immature’ and ‘mature’ forms of NMDARs (from predominantly NR2B- to NR2A-containing) in the postsynaptic density (PSD) and by the synaptic incorporation of AMPARs. These changes regulate the stabilization of the PSD, the subsequent decline in functional plasticity of the synapse, and the spine growth associated with synapse maturation NMDAR activation is crucial for synaptic strengthening and weakening Maximal NR3A expression coincides with a period during which many synapses are being formed, stabilized, or eliminated Given the importance of NR3A for synaptic function and memory formation, here we sought to further investigate how NR3A regulates the transition from immature to mature synapses. We aimed to define the subcellular localization of NR3A-containing receptors at peak expression levels (∼P8 in mouse forebrain), and to establish the consequences of deleting NR3A on PSD proteins. We found that NR3A is synaptically targeted during early development, and this preferential expression at the PSD is lost with age. The genetic deletion of NR3A causes NMDARs and AMPARs to concentrate prematurely at synapses, and results in enhanced AMPAR currents in CA1 hippocampal pyramidal neurons. However, these measures in NR3A-KO mice return to WT control levels at an age when endogenous NR3A expression is normally declining (∼P16 in mice). Our results support a model by which NR3A expression inhibits glutamatergic synaptic transmission, thus providing a molecular brake to limit the premature development of forebrain synapse size, strength, and the ability to form lasting long-term memories during early development Animal use in this study was approved by the Institutional Animal Care and Use Committee of the University of North Carolina at Chapel Hill (protocol #10-115.0) in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Homozygous WT and NR3A-KO offspring were selected from a mixed heterozygote colony and then maintained on a C57BL/6J background as separate homozygous breeding colonies. The generation and genotyping procedure of NR3A-KO mice have been previously described Brains were removed from mice at postnatal (P) days 8, 16, and >40 (range from P40-55). Forebrain tissue was rapidly dissected, frozen on dry ice, and stored at −80°C until use.g for 10 min) to yield the postnuclear supernatant (PNS) fraction. Synaptosomal pellets were lysed in hypo-osmotic solution, layered on a discontinuous gradient consisting of 0.32, 0.8, 1.0, and 1.2 M sucrose in 4 mM HEPES, pH 7.4, and subjected to density centrifugation . The synaptic plasma membrane (SPM) fraction was collected from the 1.0/1.2 M sucrose interface, resuspended in 0.5% Triton X-100-containing buffer, and centrifuged to obtain the postsynaptic density (PSD) fraction. Complete protease inhibitors , and phosphatase inhibitor mixtures 1 and 2  were added to all buffers. Procedures were performed on ice and/or in a cold room and fractions were stored at −80°C. Protein concentrations were determined by BCA Assay . The following fractions were collected in this study: PNS, whole homogenate, postnuclear supernatant; CYT, cytosol; LM, light membranes; P2, crude synaptosomes; P3, lysed synaptic membranes; SPM, purified synaptic plasma membranes; TSF, Triton-soluble fraction; PSD, postsynaptic densities.One to three forebrains from each genotype, NR3A-KO or WT, were pooled for each fraction (n = 5–10 fractions/antibody for each age). Biochemical fractions were prepared as described Increasing amounts (1–15 µg) of total protein from each fraction were loaded in wells of 4–12% or 8% tris-glycine Novex gels (Invitrogen), resolved by SDS-PAGE, and transferred to nitrocellulose membranes. Blotting (Bio-Rad) and Odyssey system imaging and quantification (LI-COR) were carried out following manufacturers' protocols. The following antibodies were used at optimized concentrations: goat anti-NR1 , rabbit anti-NR3A , goat anti-NR2B , rabbit anti-NR2A , rabbit anti-GluR1 , mouse anti-PSD-95 , mouse anti-synaptophysin , mouse anti-β-tubulin , Alexa Fluor 680-labeled donkey anti-goat IgG , Alexa Fluor 680-labeled goat anti-mouse IgG , and IRDye 800-labeled donkey anti-rabbit IgG . All samples were run multiple times.2PO4, 1.25; NaHCO3, 26; sucrose, 75; d-(+)-glucose, 10; ascorbic acid, 1.3; MgCl2, 7; and CaCl2, 0.5) and bubbled with 95% O2 and 5% CO2. The hippocampus was dissected and then 350 µm coronal slices were prepared using a vibrating microtome . Slices were allowed to recover for 20 min in a 35°C submersion chamber filled with oxygenated artificial cerebrospinal fluid (ACSF) -glucose, 20) and then kept at room temperature for at least 40 min until use.Mice were anesthetized with sodium pentobarbital and euthanized upon disappearance of corneal reflexes. Brains were rapidly removed and immersed in ice-cold dissection buffer , with pH adjusted to 7.2 and osmolarity adjusted to 300 mOsm by the addition of sucrose. Hippocampal CA1 pyramidal cells were voltage-clamped in the whole-cell configuration using a patch-clamp amplifier , and data were acquired and analyzed using pCLAMP 9.2 software (Molecular Devices). Changes in series resistance were monitored throughout the experiment by giving a test pulse and measuring the amplitude of the capacitive current. Only cells with series resistance <18 MΩ were included for analysis. Input resistance was monitored throughout the experiment by measuring the amplitude of the steady-state current, filtered at 2 kHz, evoked from a test pulse. Only cells with <30% change in Rinput and Rseries were included for analysis. EPSCs were evoked from a stimulating electrode  by stimulation (200 µs every 15 sec) of the stratum radiatum. For AMPA input-output curves, EPSCs were recorded at −80 mV and two trials at the same stimulus intensity were averaged to produce a measurement of AMPAR current at a given stimulus intensity. AMPAR currents were pharmacologically isolated by modifying the standard ACSF to contain picrotoxin  and d,l-APV , and this bath solution was perfused at 2 ml/min at 30°C. A minimum of three animals were used per experimental group.Patch pipettes were pulled from thick-walled borosilicate glass. Open tip resistances were 2–7 MΩ when pipettes were filled with an internal solution containing (in mM): cesium hydroxide, 107; t-tests or one-way analyses of variance (ANOVAs) (for multiple group comparisons), followed by between-group comparisons with Tukey-Kramer tests . Significance was placed at p<0.05.Calculations of signal intensity per microgram protein were determined from multiple wells on each gel for each target antigen and then averaged across multiple gels. Fraction means per genotype were either presented as immunoreactive (IR) units/µg protein or normalized to mean control values and expressed as % of control or % of maximum. Error bars represent mean ± SEM. Statistical evaluations were performed using either two-tailed student's To examine the subcellular localization of glutamate receptor subunit proteins in forebrain neurons, we performed immunoblot analysis on various fractions extracted from WT and NR3A-KO mice  of peak expression  = 17.988, p = 0.0001; SPM, F = 5.397, p = 0.0153; PSD, F = 23.612, p<0.0001). The developmental profile we observed for NR3A is similar to that reported previously  = 8.973, p = 0.0015). However, NR3A levels in PSD fractions decrease significantly at juvenile and young adult ages  = 23.612, p<0.0001; P16% of maximum = 70.8±8.4, p<0.05; >P40% of maximum = 32.9±3.1, p<0.0001) and PSD enrichment is lost after P8  = 2.682, p = 0.1033; >P40, F = 3.034, p = 0.080). These data indicate that not only do NR3A levels decline dramatically during development, but also the privileged access of NR3A-containing NMDARs to the PSD is lost with age.Because NMDAR subunit composition and localization determines synaptic functions, we further probed how the subcellular expression of NR3A changes with age. Our study focused on the PNS, SPM, and PSD fractions taken from P8 , P16 (juvenile), and age >P40 (young adult) mice. We found that NR3A levels decrease 50–70% from P8 through adulthood in all fractions  = 74.691, p<0.0001; P16, F = 44.507, p<0.0001; >P40, F = 29.234, p<0.0001; NR1 comparison by fractions: PNS, F = 1.636, p = 0.2258; SPM, F = 1.404, p = 0.2727; PSD, F = 0.3277, p = 0.7250.).Because NR1 is required for all functional NMDARs  = 32.582, p<0.0001; P16, F = 77.483, p<0.0001; >P40, F = 31.964, p<0.0001; NR2B comparison by fractions: PNS, F = 2.304, p = 0.1302; SPM, F = 2.617, p = 0.1021; PSD, F = 1.046, p = 0.3730).Similarly, NR2B showed abundant expression during early life and minimal change with age  = 11.725, p = 0.0004; P16, F = 14.377, p = 0.0004; >P40, F = 39.610, p<0.0001; NR2A comparison by fractions: PNS, F = 12.549, p = 0.0005; SPM, F = 53.233, p<0.0001; PSD, F = 28.548, p<0.0001). These increases in protein levels indicate that NR2A is significantly regulated across developmental ages, consistent with previous observations In contrast, we found that NR2A levels are very low during early development and increase dramatically after the first week of life, as expected  = 30.729, p<0.0001; P16, F = 6.454, p = 0.0113; >P40, F = 12.991, p = 0.0005; GluR1 comparison by fractions: PNS, F = 57.918, p<0.0001; SPM, F = 13.702, p = 0.0004; PSD, F = 40.306, p<0.0001). The preferential activity-dependent recruitment of GluR1-containing AMPARs is thought to result from LTP during the early phases of synapse maturation In line with other studies % of control = 138.6±13.4, n = 8–9, p = 0.033; data re-plotted from % of control = 150.4±16.3, n = 7–8, p = 0.047; % of control = 142.4±12.9, n = 8, p = 0.046). Both NR2A and GluR1 WT expression levels are very low at this age , and their increased levels in mutant PSDs indicate that the loss of NR3A promotes the early concentrations at synapses of both NR2A-NMDARs and GluR1-AMPARs. No differences were observed in total receptor protein fractions . These data also indicate that NR3A limits synaptic expression of AMPARs as well as NMDARs. The increase of NR2A in the NR3A-KO PSD fraction may point to a critical role of NR3A in preventing NR2A from being targeted to the PSD.To examine the effects of genetic deletion of NR3A on the composition of glutamate receptor subunits, we measured changes in protein levels of synapse maturation markers in NR3A-KO and WT mice over development. We found that both NMDAR and AMPAR subunit abundance in PSD fractions are significantly increased in P8 knockouts. Immunoblot analysis of synaptic membranes revealed that, like NR1 .Because the NR2B subunit is also highly expressed in immature forebrains during the early postnatal period, we examined whether any changes occurred for NR2B subunit expression in the NR3A-KO forebrain. However, in contrast to the pronounced upregulation of NR1, NR2A, and GluR1, we observed no discernible changes in NR2B expression levels . Although there appears to be a trend towards increased levels at older ages, this effect did not achieve statistical significance. The normalizationof the phenotype was maintained in more mature mice , further confirming that the premature concentration of synapse maturation markers, NR1, NR2A, and GluR1 in the absence of NR3A is a transient effect. Again, changes in NR2B levels were not detected . The accelerated expression of NMDAR and AMPAR subunits in immature NR3A-KO mice, along with the strong presence of NR3A proteins in the developing but not adult rodent brain, support a model in which NR3A negatively regulates glutamate receptor expression during normal postnatal CNS development.To determine whether the precocious concentration of glutamate receptor subunits in NR3A-KO mice is transient or sustained, we next examined mice at P16 and P40–55, the period during and after which synaptic refinements are completed, and when endogenous NR3A levels are declining. Interestingly, we found no significant differences between NR3A-KO and WT mice at P16 for NR1, NR2A, and GluR1 protein levels  NR3A-containing NMDARs shift from a synaptic to a peri-/extrasynaptic location with development, and (2) NR3A suppresses glutamate receptor activity during early life to prevent premature synaptic strengthening and stabilization. Our data show that NR3A is expressed at high levels and enriched within the postsynaptic density (PSD) during early postnatal life (P8). However, NR3A levels undergo a striking age-dependent decline and are no longer enriched in the PSD fraction after P8. Glutamate receptor subunit levels are increased in PSDs from neonatal NR3A-KO mice, and this effect is normalized soon afterward. This transient enhancement of NMDAR and AMPAR expression is accompanied by accelerated functional AMPAR responses that also return to WT levels in juvenile mice. These results support the idea that NR3A-containing NMDARs restrict the synaptic expression of NR1, NR2A, and GluR1. Because concentration of these glutamate receptor subunits and the development of AMPAR currents are key measures of synapse maturation, our data indicate that the absence of NR3A results in synapses that mature earlier than their WT counterparts. Such changes might be expected if, in the narrow temporal window of its highest expression, NR3A plays a regulatory role as a molecular brake for the functional and structural changes associated with synapse stabilization.Our data suggest that NR3A levels not only decline with age, but NR3A also undergoes redistribution from the PSD to other biochemical compartments. In older animals, NR3A is relatively more abundant in the SPM fraction (which includes TSF and PSDs, We do not know whether increased synaptic localization of NR1, NR2A, and GluR1 in week-old NR3A-KO mice could be due to a change in transcription, translation, and/or a redistribution of glutamate receptors. This question will take additional experiments to answer, especially given that there is often discrepancy between transcript and protein levels It is not yet known how NR3A-containing receptors might be acting outside the PSD. Additional detailed ultrastructural analyses of NR3A localization Intriguingly, NR3A directly suppresses NMDAR function Synapses in many CNS regions undergo significant remodeling during postnatal brain development, although the molecules and signaling pathways responsible for the elimination of inappropriate synapses and the maintenance and strengthening of appropriate connections are still largely unknown NR3A expression at synapses is dependent on a number of factors, including its unique intracellular interactions and association with other NMDAR subunits. Unlike NR2 subunits If NR3A is responsible for delaying the stabilization of NMDARs and the insertion of AMPARs at the synapse, the prolonged absence of NR3A would be expected to cause an increase in the fraction of mature synapses. This idea is consistent with the notion that activity-dependent NR3A removal by PACSIN may ‘unsilence’ synapses through the insertion of more mature NMDAR subtypes that trigger AMPAR insertion Behavioral studies have only begun to address the long-term implications of NR3A in the refinement of neural circuits capable of learning and storing memories