In Escherichia coli, two genetically distinct forms of SSADHs had been described that are essential for preventing accumulation of toxic levels of succinic semialdehyde (SSA) in cells.In mammals succinic semialdehyde dehydrogenase (SSADH) plays an essential role in the metabolism of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) to succinic acid (SA). Deficiency of SSADH in humans results in elevated levels of GABA and γ-Hydroxybutyric acid (GHB), which leads to psychomotor retardation, muscular hypotonia, non-progressive ataxia and seizures. E coli gabD gene by X-ray crystallographic studies and compare these data with the structure of human SSADH. In the E. coli SSADH structure, electron density for the complete NADP+ cofactor in the binding sites is clearly evident; these data in particular revealing how the nicotinamide ring of the cofactor is positioned in each active site.Here we structurally characterise SSADH encoded by the E. coli SSADH permits this enzyme to use NADP+, whereas in contrast the human enzyme utilises NAD+. Furthermore, the structure of E. coli SSADH gives additional insight into human mutations that result in disease.Our structural data suggest that a deletion of three amino acids in  The products of the gab operon (which comprises gabT (γ-aminobutyrate transferase), gabD (SSADH), gabP (GABA permease) and gabC (a regulatory gene) sad gene encodes for a NAD+ dependent SSADH (EC 1.2.1.16 and shares 32% identity with gabD) and is an orphan genesad gene is induced by exposure to exogenous SSA and functions primarily to prevent its accumulation in the cell. Furthermore, the sad gene product may also enable growth on putrescine as the nitrogen source In Burkholderia pseudomallei, without a substrate or cofactor (PDB ID: 3ifg and 3ifh), was deposited into the PDB by the Seattle Structural Genomic Centre for Infectious Disease.Recently, the structure of human SSADH has been published gabD gene product (NADP+-dependant) SSADH from E. coliE. coli SSADH is also redox regulated, furthermore it reveals that the bacterial SSADH is structurally suited for NADP+, rather than NAD+ (as utilised by its human counterpart).Despite these recent studies, the structural information of SSADH and its interactions with cofactor remains scarce. Here we report a 2.3 Å X-ray crystal structure of the E. coli SSADH was purified as a tetrameric molecule  which is in agreement with the previous description in the literature 1H NMR, as shown in mK of the purified enzyme is 16.94±2.2 µM and maxV is 40.92±1.3 µM. The enzyme activity measured in the presence of NADP+ is approximately 20-fold higher than that measured in the presence of NAD+ (data not shown) as described previously Recombinant E. coli SSADH reveals four monomers  in the asymmetric unit  and 13 α helices (1–13). The secondary structure assignments used in this report are as shown in + is bound to the active site giving rise to a binary complex, whereas the structure of the N-terminal His-tag was not resolved.The SSADH monomer  adopts a+ via two tandem Rossmann folds in a (βα)4β formation. This is a variation of the classic Rossmann fold 2β motif (residues 148–208) forms the first β strand of the second (βα)2β motif (residues 205–254). The oligomerisation domain comprises an elongated 3-stranded β-sheet (the B-sheet), which interacts with two other monomers in the final tetrameric assembly.The catalytic domain consists of a central 7 stranded β-sheet (the D-sheet) flanked by 2 helices on one side and 3 helices on the other. The catalytic loop (residues 282–290) is located adjacent to the cofactor binding site. The cofactor binding domain interacts with NADP2 buried in the dimer interface  is formed by domain swapping; such that strand s3B of the oligomerisation domain in monomer A forms β-sheet hydrogen bonding with strand s7D of the catalytic domain in monomer B to make a 10 stranded β-sheet . The scrnterface .2 buried in the interface and 10 H-bonds  and superposes with a root-mean-square deviation of 0.79 Å over 472 Cα  and inactive  state has recently been determined  E. coli . These dn of DTT . InteresE. coli SSADH reveals two major regions of structural variation: the first involves the cofactor binding site  resembles the canonical ALDH fold Comparison of human and +  and the active site residues  of human SSADH structures (PDB ID: 2w8o and 2w8q)+ results in formation of the thioacyl enzyme intermediate and NAD(P)H. Lastly, the conserved E254 residue acts as a general base to deprotonate a water molecule prior to its attack on the thioacyl enzyme intermediate resulting in formation of SA and regeneration of the C288 residue. The general base, E254, in all monomers can be modelled in two alternative conformations according to the electron density. It is suggested that the two conformations of E254 is likely to be associated with different stages of the catalytic process, with one conformation “a” tion “a”  being foE. coli SSADH structure, electron density for the cofactor in the binding sites is clearly evident . In human SSADH, 261RKN263  to catalyse the deacylation process.Amongst the well defined cofactor crystal structures, three different conformations of NAD(P)E. coli SSADH numbering in parentheses). The majority (25 mutations) give rise to truncations, deletions, insertions as well as splice site mutants . All these residues superpose well between the human and E. coli structures . The DNA sequence of the PCR product was shown to have a single amino acid change to that of the gabD gene sequence in the database, which is thought to represent a naturally occurring variant in E. coli, the SSADH cDNA was excised from the recombinant pCR® -Blunt vector using the restriction enzymes EcoRI and HindIII, then ligated into pRSETc/His_TEV plasmid as previously described E. coli BL21(DE3) pLysS cells and the transformant was stored at −80°C. For expression of SSADH recombinant protein, transformed E. coli BL21(DE3) cells were propagated in 2YT growth medium in the presence of 100 µg/mL ampicillin and 36 µg/ml chloramphenicol at 37°C to A600 = 0.6 followed by induction at 16°C with 0.5 mM IPTG for 18 hours.The cDNA encoding SSADH was isolated from Harvested cells were treated with lysozyme (1 mg/ml) at 4°C for 30 min and lysed in lysis buffer containing 300 mM NaCl, 10 mM imidazole, 5 mM β-mercaptoethanol, 0.01% triton X-100, 50 mM Tris (pH 8.0) by sonication on ice. Clarified cell lysate was loaded onto a Nickel Chelating Sepharose Fast Flow column , the His-tagged SSADH protein was eluted in 150 mM NaCl, 250 mM imidazole, 5 mM β-mercaptoethanol, 50 mM Tris (pH 8.0). Purified His-tagged SSADH were further purified using a S200 16/60 size exclusion column  pre equilibrated with 100 mM NaCl, 10 mM β-mercaptoethanol, 5% glycerol, 30 mM Tris (pH 7.5) for all experiments described in this paper.+ and SSA 0–400 mM at 30°C. The rate at which NADP+ was converted to NADPH was monitored fluorometrically (excitation: 355 nm and emission: 460 nm). SSADH oxidative inhibition (H2O2) assays were carried out by incubating various concentrations of H2O2 with SSADH (1 µg/ml) in a Na phosphate buffer (100 mM pH 8.4), containing 0.75 mM EDTA for 1 hour at room temperature, the reactions were terminated by adding 5.0 mM methionine. To reverse the effect of the H2O2, 10 mM DTT was added to H2O2 treated SSADH and incubated for a further 10 minutes. After the addition of 2.0 mM NADP+ and 0.15 mM SSA, the activity of H2O2 treated SSADH was measured spectrophotomically at 340 nm at 30°C.Enzyme kinetics were carried out using purified SSADH (2 µg/ml) in a Na phosphate buffer (100 mM pH 8.0), containing 1.1 mM NADP1H NMR spectroscopic analysis was carried out with a Bruker DPX 400 MHz spectrometer using purified SSADH in the presence of 125 µM SSA for 240 min as above.Pure SSADH was concentrated to 6 mg/mL and buffer exchanged into 20 mM NaCl, 10 mM β-mercaptoethanol, 5% glycerol, 1.0 mM NADP, 1.0 mM succinic semialdehyde, 30 mM Tris (pH 7.5) using a 10 KDa molecular weight cut off concentrator (Millipore).Crystals of SSADH were grown using hanging drop vapour diffusion by mixing 2.0 µL of protein (6 mg/mL) with 1.0 µL of reservoir solution containing 0.2 M ammonium tartrate, 26–31% polyethylene glycol 3350, 10 mM β-mercaptoethanol and 0.1 M Tris (pH 7.2–7.5). Crystals were dehydrated for 48 hours in 0.2 M ammonium tartrate, 32% polyethylene glycol 3350, 10 mM β-mercaptoethanol and 0.1 M Tris (pH 7.2–7.5) after three days. Crystals were soaked in reservoir solution supplemented with 5% 2-methyl-2,4-pentanediol for 5 minutes then soaked for a further 5 minutes in reservoir solution supplemented with 10% 2-methyl-2,4-pentanediol for cryoprotection before freezing in liquid nitrogen for data collection. Data were collected at the Australian Synchrotron High-throughput protein crystallography (PX1) beam.P4212 with unit cell dimensions of a = 151.88 Å, b = 151.88 Å, c = 165.77 Å, α = β = γ = 90.0°. These data are consistent with four molecules in the asymmetric unit. These data were merged and scaled using MOSFLM free with neither a sigma nor a low-resolution cut-off applied to the data. Summaries of the statistics are provided in SSADH crystals diffracted to 2.3 Å resolution and belong to the space group The structure of SSADH was solved using molecular replacement and the program PHASER A ‘mixed’ model consisting of conserved side chains with all other non-alanine/glycine residues truncated at Cγ atom was then created using the SCRWL server Rfree. Model building and structural validation was performed using COOT Rfree reached 28%. The presence of each water molecule was manually validated. The NADP+ moiety was modelled into the density using PHENIX ligandfit th percentile; Refinement and model building preceded using one molecule (chain A) in the asymmetric unit, with the other chains built using non-crystallographic symmetry operators. Maximum likelihood refinement using REFMACCoordinates and structure factors have been deposited in the Protein Data Bank with accession number 3jz4.http://hdl.handle.net/102.100.100/42) Diffraction image datasets and refinement logs are available online on TARDIS  substrate alone in phosphate buffer which contains succinic semialdehyde (X), 4,4-dihydroxybutanoic acid (Y) and succinic acid (Z). (b) Succinic acid alone with phosphate buffer, showing only the singlet peak of succinic acid (Z). For SSADH enzyme assay, incubation of succinic semialdehyde and SSADH in the presence of NADP+ at 0 min (c) and 240 min (d) showing all substrates  has been converted to succinic acid (Z), NADP+ is marked with W.(0.21 MB TIF)Click here for additional data file.Figure S22-sheet or a cylinder representing an Î±-helix. The secondary structure elements are coloured according the Alignment of E. coli SSADH with human SSADH. Conserved residues have been highlighted according to the following, polar (green), non-polar (yellow), acidic (red) and basic (blue). The secondary structure (E. coli SSADH above the sequence and human below the sequence) has been marked with either an arrow designating a Î(1.46 MB TIF)Click here for additional data file.Figure S3SSADH activity in an oxidised and reduced environment. The first column shows the untreated activity of SSADH. In the second column the E. coli SSADH enzyme was oxidised by incubating it with 200 Î¼M H2O, which has 11–13% of untreated SSADH activity. In the third column the addition of 10 mM DTT to previously oxidised enzyme, which rescued the inhibition to 80% that of the normal activity.(0.16 MB TIF)Click here for additional data file.Figure S4A–F. Human point mutations causing a dramatic loss of acticvity  mapped onto the E. coli SSADH structure. A cartoon representation of E. coli SSADH monomer A  showing the 17 point mutations (magenta spheres) that map to the mature human protein. A small region of monomer B catalytic domain (dark grey) and monomer C oligomerisation domain (light grey) have been included to illustrate the proximity of point mutations with regard to dimer and tetramer interfaces. Labelled, dashed red boxes highlight the area of E. coli SSADH that have been enlarged for analysis. A–F) Show the equivalent E. coli SSADH residues (magenta sticks) to the human point mutants and their interactions (black dashed lines) within their immediate surrounds with other residues (orange sticks). A–B) Mutations in this region would be anticipated to disrupt dimer and tetramerisation, while C) we would anticipate decreased stability in this region due to loss of stabilising interactions with the N255S mutation. D) A loss of NAD/P+ binding efficiency would be expected with the addition of a large negatively charged residue, G268E, into the positively charged adenosine-ribose binding pocket of SSADH. E) We anticipate an overall loss of structural integrity of this loop region from either of the mutations G409D (loss of +/+ backbone conformation) and P382L/Q (disruption of aromatic ring stacking interactions with F419). F) The introduction of a large charged residue (N335K) in a buried surface on the catalytic loop would be anticipated to greatly disrupt the structural integrity and catalytic ability of SSADH.(2.71 MB TIF)Click here for additional data file.Table S1Intermolecular contacts with respect to monomer A.(0.18 MB DOC)Click here for additional data file.Table S2Residues involved in significant NADP+ binding using Molprobity (0.07 MB DOC)Click here for additional data file.


The effects of weight concentration and molecular weight of PPTA, as well as operating temperature, on dynamic viscoelasticity of the PPTA-H2SO4 LC solution system are discussed. The transition from a biphasic system to a single-phase LC occurs in the weight concentration range of SWNTs from 0.1% to 0.2%, in which complex viscosity reaches the maximum at 0.2 wt% and the minimum at 0.1 wt%, respectively, of SWNTs. With increasing SWNT weight concentration, the endothermic peak temperature increases from 73.6 to 79.9 °C. The PPTA/SWNT/H2SO4 solution is in its plateau zone and storage modulus (G′) is a dominant factor within the frequency (ω) range of 0.1–10 rad/s. As ω increases, the G′ rises slightly, in direct proportion to the ω. The loss modulus (G″) does not rise as a function of ω when ω < 1 s−1, then when ω > 1 s−1 G″ increases faster than G′, yet not in any proportion to the ω.We have studied the dynamic scanning of liquid-crystalline (LC) poly( Nematic sulfuric acid LC solution of PPTA, referred to as PPTA-H2SO4, with a weight concentration range of 19–20% when heated to 80–95 °C has been used to fabricate high-strength fibers by dry-jet wet-spinning process [2SO4 LC solution have been reported [2SO4 LC solution system in the spinning process. We then carried out the dynamic scanning of the PPTA-H2SO4 LC solution system with a flat plate rotational rheometer and discussed the effects of weight concentration and molecular weight of PPTA, as well as temperature, on such viscoelastic properties as complex viscosity (η*), storage modulus (G′), loss modulus (G″), and loss-tangent (tanδ). Our results shown in this paper provide some general fundamental insight for the LC spinning process.A liquid-crystalline (LC) solution is an orientationally ordered liquid that displays fluidic characteristics of the liquid state while the molecules still maintain a directional order. There are three main classes of LC phases, ch phase . Poly have been shown to possess extraordinary mechanical, electrical and thermal properties –15. Polyl curves . It is t2.2.1.2SO4 LC solution are shown in ω), i.e., its η* decreases as ω increases, which results in the alignment of PPTA chains in parallel. With increasing ω, the density of junction points decreases since the crosslinks break significantly faster than they form owing to their shear-thinning behavior [G″ and tanδ of PPTA-H2SO4 LC solution would increase as ω rises. At a high shearing rate, nearly all macromolecular chains orient, implying that G′ hardly follows the variation of frequency, since in this regard the first normal stress difference hardly follows frequency variation [η*, G′, G″, and tanδ. On the other hand, as the temperature increases, thermal motion of macromolecular chains increases, flow resistance decreases, and rheological curves of η*, G′, G″, and tanδ move down.Effects of the variation of weight concentrations of PPTA  and temperatures  on rheological curves of the PPTA-Hbehavior . Such chariation .However,2.2.2SO4 LC solution at 19.5 wt% of PPTA as a function of wM  and temperature . All four rheological parameters  increase with increasing wM of polymers. Also, the larger wM is, the longer relaxation time (λ) appears, meaning that oriented molecular chains upon shearing cannot resile, thus showing more distinct shear-thinning behaviour. This is greatly advantageous to obtain high degree of orientation in spinning process and hence obtain fibers with higher tenacity and modulus. Moreover, the larger wM is, the more entanglement crosslinks are generated, meaning that the dynamic process of disentanglement and entanglement would again take place at less shear rate [wM of PPTA would be ideally neither high (difficult for molding) nor low (accelerating the relaxation).Supramolecular structure is key to affecting the rheological characterization of the polymer system. ear rate ,25. Henc2.3.2SO4 dopes is plotted in η* first gradually increases with increasing SWNT wt%, and then decreases abruptly when beyond ∼0.1 SWNT wt%, and arises again when beyond 0.2 SWNT wt% to reach about the same viscosity for three temperature-dependent curves. In general, this concentration dependent viscosity behavior of PPTA/SWNT/H2SO4 solution resembles that of SWNT/H2SO4 solution. As the SWNT concentration was below 0.1 wt%, the solution viscosity increased accordingly with increasing SWNT wt%. This can be considered as normal behavior of an isotropic solution. When SWNT concentration was exceeded 0.1 wt%, the viscosity decrease at increasing SWNT concentration can be considered as a unique behavior of anisotropic solution. It is caused by the liquid crystal behavior where SWNT are aligned upon shear and form domains of highly ordered structures.Variation of complex viscosity as a function of SWNT weight concentration (SWNT wt%) in PPTA/SWNT/H2SO4 exhibit roughly similar phase behavior as rod-like polymer solutions [2SO4 system. A biphasic region of mixed isotropic and anisotropic phase existed between 0.1 wt% and 0.2 wt%. Beyond 0.2 wt%, the solution became an increasingly anisotropic-rich phase, and the solution viscosity increased again with increasing SWNT wt%.It has been revealed in rheology and microscopy studies that SWNTs in Holutions ,26. We holutions . The LC olutions . For a bolutions ,28. Thus2.4.η*) as opposed to shearing frequency (ω) of PPTA/SWNT/H2SO4 dope is shown in Temperature effect on complex viscosity  The η* of PPTA/H2SO4 dope decreases as temperature increases from 75 to 85 °C. Such behavior is often observed among conventional polymers. (2) The η* of PPTA/SWNT (98/2 wt%)/H2SO4 dope decreases as temperature increases from 80 to 85 °C, while it becomes nearly independent of temperature ranging between 75 °C and 80 °C. (3) The η* of PPTA/SWNT (97/3 wt%)/H2SO4 dope decreases slightly as temperature increases from 80 to 85 °C, but increases slightly as temperature increases from 75 to 80 °C.All three samples composed of pure PPTA and PPTA coupled with different portion of SWNT exhibit an obvious shear-thinning behavior through the entire 2SO4 dopes. The DSC thermogram , the G″ does not rise, while in other regions (ω > 1 s−1), G″ rises faster than G′, but not in proportion to frequency.G′ and G″, a logarithmic plot of G′ and G″ as a function of frequency comprising three zones: terminal, plateau and transition zone, has been proposed [2SO4 dope is in its plateau zone within the frequency range from 0.1 to 10 rad/s [®, for example, supports this argument [2SO4 dope seems to exhibit more intermolecular interaction and chain rigidity, thus giving rise to the plateau-zone behavior [According to the relative magnitude of proposed . Referre10 rad/s . Rod-likargument . As SWNTbehavior .3.3.1.3 was prepared by mixing the fuming sulfuric acid (25 wt%) with concentrated sulfuric acid (98 wt%). The concentration of sulfuric acid was measured by acid-base titration. The SWNTs used in this study was acquired from Shenzhen Nanotechnologies Co., Ltd. and an average diameter of <2 nm. Finally, SWNTs were dried under vacuum overnight at 80–100 °C. Dispersions were prepared by mixing with a magnetic stir bar for 3 days in an anhydrous environment in a glovebox which was nitrogen atmosphere at room temperature [Sulfuric acid with 0.2 wt% excess SOperature . The PPTperature ,37. All 2SO4 LC solutions were prepared with four different molecular weight polymers w, at different temperatures  and with different weight concentrations . For example, into a 250 mL three-neck flask equipped with a mechanical stirrer and a nitrogen inlet/outlet, were placed 34.01 g of PPTA and 140.4 g of H2SO4 at 75 °C. The mixture was then stirred for 2.5 h and opalescence was observed during this step. Likewise, the LC solutions of PPTA/SWNT/H2SO4, in which PPTA was kept at 19.5 wt% and PPTA/SWNT is 99/1 wt%, were prepared in a 250 mL three-neck flask. SWNTs (0.3401 g) were dispersed in H2SO4 (140.4 g) under an inert and anhydrous atmosphere with mechanical stirring and at room temperature for at least 3 days [The PPTA-Ht 3 days . Into th3.2.2SO4 LC and PPTA/SWNT/H2SO4 dope solutions were performed with dynamical scanning on an ARES rotational rheometer  at four temperatures  in the frequency range of 0.01–100 rad·s−1 and 0.1–10 rad·s−1, respectively. Testing fixtures parallel plates (50 mm) were made of stainless steel 316 to avoid corrosion. In order to prevent water exchange between the sample and environment, the sample surface between the plate and the cone was coated with a mineral oil [ω) on rheological behaviors such as η*, G′, G″ and tanδ have been examined.Subsequently, the PPTA-Heral oil ,39. The 3.3.2SO4 dope was carried out on a differential scanning calorimeter (DSC) (Mettler Toledo 822) under a N2 atmosphere at a heating rate of 5 °C/min ranging from 25 to 90 °C.Thermal analysis on the PPTA/SWNT/H4.2SO4 LC solution exhibits a typical shear-thinning behavior. Our rheology studies are summarized as follows. (1) As PPTA wt% increases, rheological curves of η*, G′, G″ and tanδ all move up. By contrast; (2) as temperature rises, rheological curves of η*, G′, G″ and tanδ all move down. Finally, (3) η*, G′, G″ and tanδ all increase as wM of PTTA increase.We have shown that PPTA-H2SO4 LC solution, the protonation of SWNTs in superacids enables their good dispersion to form homogeneous PPTA/SWNT/H2SO4 dope solution. The dope solution exhibits a remarkable nematic LC phase. Our findings are multi-folds. (1) For all temperature and concentration of SWNT, the complex viscosity of PPTA/SWNT/H2SO4 dope shows an obvious shear-thinning behavior, and the slope of complex viscosity versus frequency on a logarithmic plot is about the identical at ca. −0.91. The DSC thermogram displays multiple phase transitions between monotetic and the liquid state, as well as isotropic and nematic phase. As concentration of SWNT increase, G′ and G″ rise sharply at various temperatures. (2) For all temperature and concentration of SWNT, the elastic characteristic such as G′ of PPTA/SWNT/H2SO4 dope is a dominant factor within the frequency range from 0.1 to 10 rad/s. At sufficiently high concentrations of SWNT, approximately 0.2 wt%, and at 85 °C, a novel type of single-phase nematic liquid crystal is formed. This allows the dope solution to produce superior-performance fibers by dry-jet wet-spinning process.When SWNTs are added into PPTA-H


In RNAi transformants, Kin5 was severely downregulated and disappeared from the cilia, but cilia did not resorb, although tip structure was affected. After deciliation of the knockdown cell, cilia regrew and cells swam, which suggested that Kin5 is not responsible for the trafficking of axonemal precursors to build the cilium, but could be transporting molecules that act in ciliary signal transduction, such as guanine nucleotide exchange proteins (GEFs). Gef1 is a Tetrahymena ciliary protein, and current coimmunoprecipitation and immunofluorescence studies showed that it is absent in regrowing cilia of the knockdown cells lacking ciliary Kin5. We suggest that one important cargo of Kin5 is Gef1 and knockdown of Kin5 results in cell lethality.A critical process that builds and maintains the eukaryotic cilium is intraflagellar transport (IFT). This process utilizes members of the kinesin-2 superfamily to transport cargo into the cilium (anterograde transport) and a dynein motor for the retrograde traffic. Using a novel RNA Tetrahymena cilia may contain evolutionary precursors of important metazoan signaling pathways ChlamydomonasTetrahymena is probably represented by the motor proteins, Kin1 and Kin2 TetrahymenaC. elegans homodimeric IFT motor protein that is responsible for building the distal segments of sensory cilia in that organism Chlamydomonas IFT complex proteins Recent studies suggest that Tetrahymena IFT motor possibly involved in the intraciliary transport of membrane-associated signaling molecules toward the cilium tip. To do this, we proposed to alter the amount of Kin5 produced in the cell and observe how the phenotype is altered. Typically, Tetrahymena contains a somatic macronucleus and a germline micronucleus. Using biolistic transformation and a mutant construct based on the work of Cassidy-Hanley et al. KIN5 locus. However, after phenotypic assortment, we failed to generate a somatic knockout, which indicated that this was probably an essential gene whose knockout would lead to lethality. We then decided to investigate whether RNAi production for gene knockdown of KIN5 was feasible in Tetrahymena.We wanted to explore the function of Kin5 as a KIN5 knockdown by an inducible short hairpin RNA (shRNA), with relevant conditions and controls. We characterize the KIN5 knockdown phenotype by cell survival studies to confirm that KIN5 is an essential gene, and we discuss the difficulties this presents in the knockdown experiments. We present data to indicate that Kin5 has novel intraciliary transport cargos, some probably related to the placement of membrane and signaling molecules within the cilium, rather than to the transport of components of the motile axoneme, and we discuss similarities and possible differences between the functions of Kin5 and Osm3 or Kif17.In this study, we define a successful technology for shRNA into the genome. T. thermophila expresses two redundant β-tubulin genes, BTU1 and BTU2. The pMTT1-BICH3 construct designed by Gorovsky's laboratory is able to target the IAG48[G1] sequence behind the powerful inducible metallothionein MTT1 promoter to the BTU1 coding region by biolistic transformation IAG48[G1] sequence can be replaced by other sequences of interest that will be transcribed when a heavy-metal divalent cation such as Cd2+ is added. To introduce an inducible RNAi construct into the macronuclear genome, we replaced the IAG48[G1] sequence with a sequence which when transcribed would fold to produce a short double-stranded RNA. A short sequence (K5KOAs.40) based on 19 nucleotides from the KIN5 stalk coding region was chosen, which was unique to the KIN5 gene as verified by a blast search of the Tetrahymena genome (http://www.tigr.org/tigr-scripts/tgi/T_index.cgi?species=t_thermophila). We then designed a construct containing the coding sequence, a loop of nine bases, the antisense sequence of the coding region and a termination codon . All further experiments were performed in Tris under starvation conditions. A non-ciliary control RNA (PGM1) was examined simultaneously with KIN5 RNA.The metallothionein gene, 2+ in the absence of transformation, both KIN5 RNA and PGM1 RNA remained qualitatively unchanged for 7 h and both messages persisted for at least 24 h  of the Sec7 family, cloned in Paramecium. An antibody to the PH domain of the Paramecium protein , we could demonstrate specific knockdown of the mRNA of interest, while a control transcript was unaffected. The inverse construct from the 19-nt sequence had no effect. This methodology should be applicable to shRNA production for knockdown of other genes of interest in Tetrahymena. A similar conclusion was reached by Howard-Till and Yao For this study, we developed a method of knockdown of a specific macronuclear transcript in shRNA induction, following Nilsson 2+. Presumably, under growth conditions, more MTT1 protein will be synthesized to transport Cd2+ out of the cytoplasm. We found that exposure to 5.0 µg/ml Cd2+ produced a knockdown, but was highly toxic, leading to cell death within a few hours, even in control cells. Cells transformed with an RNAi control construct (Inv2) at an order of magnitude lower concentration of Cd2+ (0.5 µg/ml) were unaffected for at least 12 h, suggesting that there was a little Cd2+ toxicity at this time. In contrast, only about 10% of the cells transformed with the KO construct survived, whether survival was measured by number of motile cells or by cell counts, which is consistent with the production of shRNA for KIN5 knockdown by 12 h exposure to 0.5 µg/ml Cd2+. We attribute lethality mainly to the near absence of Kin5 in the cell, rather than to Cd2+ toxicity under these conditions. Lower concentrations of Cd2+ did not induce shRNA for up to 24 h. There is a window of Cd2+ concentration and time of exposure that maximizes shRNA production and minimizes Cd2+ toxicity. For essential proteins, like Kin5, it is important to demonstrate survival in control populations with the Cd2+ conditions chosen. Therefore, we used 0.5 µg/ml Cd2+and 12 h exposure as standard conditions, but flexibility should be possible.In our experiments, we transformed and selected cells grown to stationary phase in growth medium (proteose peptone), but for Tetrahymena, Kin1 and Kin2 are motors of the heterotrimeric kinesin-2 whose individual knockout has little effect, but whose combined knockout leads to a resorption of cilia Chlamydomonas Fla10 and Fla10h The anterograde transport of materials within many cilia and flagella is powered by at least two different motor complexes; a heterotrimeric kinesin-2 and a homodimeric kinesin-2. In kin1/kin2 knockout phenotype, the KIN5 knockdown evidently does not cause ciliary resorption. Although forward progression is affected, cilia generally remain motile, until just before cell death. The disappearance implies that Kin5-based transport turns over recurrently to bring new motors, and presumably their cargos, into the cilium. Furthermore, the cargo transported by Kin5 cannot be critical for the maintenance of ciliary structure, except for perhaps at the cilium tip, or for motility, as demonstrated when cells are deciliated after knockdown. When RNAi for KIN5 is induced, even though KIN5 message remains, the cell regrowing the cilia end up with little or no Kin5. These cilia are motile. In Tetrahymena, Kin5 must at least in part be involved in a pathway of intraciliary transport where the cargo must presumably not include the axonemal precursors necessary for the building of the cilium.Kin5, the presumed homodimeric kinesin-2 characterized previously C. elegans, unlike Tetrahymena, the orthologous homodimeric kinesin-2, Osm3, and the heterotrimeric kinesin are redundant in building the cilia-based sensillum body Tetrahymena, but unlike C. elegans, transfection with dominant negative Kif3a results in complete loss of cilia, whereas transfection with dominant negative Kif17 does not produce ciliary resorption. CNG channels are clustered at the distal ends of olfactory cilia KIN5 may produce some ciliary shortening because Kin5 is necessary to build the mature cilia tip. However, this remains to be quantitated and the kinetics of Kin5 synthesis and turnover within the cilium remain to be defined.In Tetrahymena cilium. Gef1 could be a cargo of Kin5 that facilitated concentration of targeted membrane receptors. Based on immunolocalization and sequence homology, Gef1 is a PH domain-containing GEF that is itself probably a ciliary membrane peripheral protein. Gef1 and Kin5 coimmunoprecipitate and in untransformed, uninduced or Inv2 cells induced with Cd2+, Gef1 and Kin5 localize together along the cilia. When cells are deciliated in the absence of KIN5 shRNA induction, Gef1 and Kin5 reappear in a similar punctate pattern as new cilia grow. However, when cilia are examined after induction of KIN5 shRNA so that Kin5 is no longer seen, Gef1 is still found along the ciliary membrane. When these cells are deciliated, cilia regrow without Gef1. These results suggest that Gef1 is released from the Kin5 transport apparatus to become resident in the ciliary membrane independent of further transport. Gef1 remains to be fully characterized, but a GEF is also found resident in the connecting cilium of mammalian photoreceptors as well as in ciliated epithelial tissues Tetrahymena, would be localized along the ciliary membrane, where it might operate to facilitate release of other cargo, particularly membrane channels and receptors, imported into the cilium.One viable hypothesis is that, like Kif17, and perhaps Osm3, Kin5 transports specific membrane proteins such as guanylyl cyclase GATCCCTTGACGCCACAAAAAAGATTCAAGAGATCTTTTTGTGGCGTCAAGTGAA) and 1 µg of pK5KOAs.40a anti-sense (GGAACTGCGGTGTTTTTTCTAAGTTCTCTAGAAAAACACCGCAGTTCACTTTCGA) oligonucleotides were mixed together in a 50 µl volume, heated at 95°C for 2 min, and incubated at 37°C for 2.5 h. A similar reaction was done for the inverse control using pInv2s sense (GATCCAGAAAAAACACCGCAGTTCTTCAAGAGAGAACTGCGGTGTTTTTTCTTGAA) and pInv2a anti-sense (GTCTTTTTTGTGGCGTCAAGAAGTTCTCTCTTGACGCCACAAAAAAGAACTTTCGA) oligonucleotides. 5 µl of the oligonucleotide mix were ligated with 5 µl of pMTT1-BICH3 (HindIII/BamHI digested and gel isolated to remove IAG48[G1]). CaCl2 competent E coli (DH5α) were transformed with the ligation mix. After plasmid minipreps (Qiagen), restriction digests and PCR were used to verify correct insertion.20 µg of pMTT1-BICH3  was digested with HindIII and BamHI and resolved on a 1% low-melt agarose . The ∼6.0 KB vector was gel isolated away from the ∼1.5 KB of IAG48[G1] sequence using the Qiaquick Gel Isolation Kit  in a final elution volume of 50 µl. 1 µg of pK5KOAs.40s sense  using 1 µl of genomic DNA as follows: 95°C – 2 min; 95°C – 45 sec, 57°C – 1 min, 72°C – 1 min for 35 cycles; 72°C – 10 min; 4°C hold. 5 µl of the reaction products were resolved on a 1% agarose gel. Cells incorporating the RNAi construct were designated K5KOAs.40 (KO cells); cells incorporating the inverse construct were Inv2 cells.20 µg of each vector was digested with KpnI/SacII (New England Biolabs) and phenol∶chloroform purified. 4 µl of the digest were mixed with 40 µl of prepared gold particles for biolistic transformation of 4 cells/ ml. CdCl2 was added to a final concentration, either in a range from 0 to 0.5 µg/ml or 5.0 µg /ml. Aliquots of motile cells were studied. For ciliary regrowth experiments, deciliation was performed by shear. Cells were routinely examined microscopically for the presence of motility and cilia, and then placed in new 2XPP. Return of motility and beating cilia were monitored for up to 2 h.KO and Inv2 cells were grown in 15 ml 2XPP for two days, spun down, resuspended in 5 ml starvation media , incubated overnight without shaking, and diluted to 2.0×10Cell survival was measured by counting the cells in the motile population by hemocytometer. One to three readings were taken at each time point and a percentage survival calculated by comparison to t = 0. To ensure that the populations were composed of viable cells, motility was measured as the number of cell crossings at a pre-determined line in a one minute-time period. One to four readings were taken at t = 0 and every four hrs up to 24 hrs and calculated as a percentage corresponding to t = 0.KIN5 primers  or PGM1 primers  in a total reaction volume of 25 µl. The reaction conditions were performed on the Geneamp PCR System 2400 (Perkin Elmer) as follows: 50°C – 30 min, 95°C – 15 min; 95°C – 45 sec, 57°C – 1 min, 72°C – 1 min for 35 cycles; 72°C – 10 min; 4°C hold. 5 µl of the reactions were resolved on a 1% agarose gel. Within the linear range of product production as described by the kit, the amount of product produced is at least qualitatively proportional to the amount of message, which was confirmed by gel analysis of repeated multiple replications of the PCR experiments.Total RNA was isolated from equal number of cells at various time points using an RNeasy mini prep kit (Qiagen). Afterwards, 1 µl was used as template for the One-Step RT PCR kit (Qiagen) using either T. thermophila K5KOAs.40 and Inv2cells were incubated at 0.5 µg/ml CdCl2, spun down at different time points and resuspended in PHEM buffer (50 mM PIPES 50 mM HEPES 1 mM EGTA 2 mM MgS04) and an equal volume of Buffer A  containing 2 mM AMP-PNP (Sigma-Aldrich). After incubating for 5 min at room temperature, 1/20 volume of 10% Triton X-100 was added for 30 min. The cells were spun down (1100 g for 3 min) and washed 2× with TBST . For immunofluorescence microscopy, cells were incubated in the primary antibody for Kin5 -HCl pH 8.0, 150 mM NaCl, 0.05% Tween-20, 3% bovine serum albumin, 5 mM CaCl2), incubated with Cy3 or Cy5 secondary antibody  at 1∶100 dilution for 15 min and washed once with Buffer B. Controls omitted primary antibody incubation. Gef1 is a ciliary protein defined by a rabbit polyclonal antibody (Gef1 Ab) based on a peptide sequence (IQLMGRFDLDEEKDT) from the PH domain of a cloned Paramecium ciliary protein, PSec7 4Tetrahymena cells or cell equivalents  were used. Samples were run on a 7.5% SDS-PAGE gel and transferred to a nitrocellulose membrane  and blocked with blotto  for one hour. The membrane was incubated overnight in blotto+affinity-purified K5T1 antibody (1∶100) or Gef1 Ab (1∶500). The membrane was washed three times in TBST  for 5 min each and incubated with anti-rabbit secondary antibody (1∶4000 in blotto) conjugated with alkaline phosphatase (Sigma-Aldrich). After washing in TBST for 5 min each, the membrane was exposed to NBT/BCIP .For western blotting, cilia fractions were prepared by shear or by dibucaine deciliation. After pelleting by centrifugation, membrane/matrix and axoneme fractions were prepared by demembranation in Triton ×100. Ciliary membranes were precipitated from the Triton supernate with 10%TCA and pelleted at 16000 g for 10 min. Normally, 15–20 µl samples derived from diluted packed cells calculated as about 10T. thermophila (SB255) was employed. 15 ml of T. thermophila SB255 at stationary phase were spun down (1100 g for 3 min) and resuspended in 1.0 ml lysis buffer . After incubation on ice for 30 min, the tube was spun (13200 RPM for 1 min) in a table-top centrifuge to remove cell debris. 100 µl of K5T1 antibody or Gef1 antibody was added and incubated at 4°C with gentle shaking overnight. 100 µl of Protein-A coated sepharose beads (10% v/v) were added and incubated at 4°C for one h. The tube was centrifuged (13200 RPM for 1 min) to pellet the beads which were washed twice with 350 µl lysis buffer and resuspended in 50 µl lysis buffer. After brief vortexing, 8 µl was used for Western blot with the opposite antibody (K5T1 Ab used for the Gef1 immunoprecipitate and the Gef1 Ab for the K5T1 immunoprecipitate).To ensure clean results with protein immunoprecipitation, a mucocyst-less strain of


PREPL-C2ORF34 gene pair, whose transcription relies on the synergic cooperation of two transcription factors binding to an intergenic bidirectional minimal promoter.Many mammalian genes are organized as bidirectional (head-to-head) gene pairs with the two genes separated only by less than 1 kb. The transcriptional regulation of these bidirectional gene pairs remains largely unclear, but a few studies have suggested that the two closely adjacent genes in divergent orientation can be co-regulated by a single transcription factor binding to a specific regulatory fragment. Here we report an evolutionarily conserved bidirectional gene pair, known as the PREPL is present primarily in brain and heart, C2ORF34 is ubiquitously and abundantly expressed in almost all tissues. Genomic analyses revealed that these two non-homologous genes are adjacent in a head-to-head configuration on human chromosome 2p21 and separated by only 405 bp. Within this short intergenic region, a 243-bp GC-rich segment was demonstrated to function as a bidirectional minimal promoter to initiate the transcription of both flanking genes. Two key transcription factors, NRF-2 and YY-1, were further identified to coordinately participate in driving both gene expressions in an additive manner. The functional cooperation between these two transcription factors, along with their genomic binding sites and some cis-acting repressive elements, are essential for the transcriptional activation and tissue distribution of the PREPL-C2ORF34 bidirectional gene pair.While This study provides new insights into the complex transcriptional mechanism of a mammalian head-to-head gene pair which requires cooperative binding of multiple transcription factors to a bidirectional minimal promoter of the shared intergenic region. In prokaryotic genomes, genes within operons usually have much shorter intergenic distances than genes at the borders of transcription units ,2. The dPACRG-PARK2 gene pair, the highly homologous mouse surfeit Surf1-Surf2 gene pair, and the non-homologous mouse Sars2-Mrps12 gene pair, in which the expression of two genes is delicately regulated by a transcription factor: in these cases N-myc, YY-1, and NF-Y, respectively dCTP by random prime labeling system . Pre-hybridization and hybridization of the membrane was performed in ULTRAhyb solution (Ambion) at 42°C. The hybridized membrane was washed twice with 2× SSC and 0.1% (w/v) SDS for 20 min at 42°C followed by washed twice with 0.25× SSC and 0.1% (w/v) SDS (w/v) for 20 min at 65°C. The hybridization signal was detected by exposure to X-ray film . The signal was stripped off and rehybridized with ACTB as an internal control.Human 12 major tissue polyA+ blot (HB-2010) was purchased from OriGene . To increase the probe specificity and sensitivity in the recognition of all possible splicing variants, a 735-bp probe spanning from exon 9 to exon 14 of the PREPL (5'-CAATCAATGAAGGGCTGGTCTAACT-3') or for C2ORF34 (5'-GGTCCAGAAACAGGCAGTCAGCACACATAACA-3') under the following conditions: 1 cycle at 95°C for 5 min; 40 cycles at 95°C for 1 min, 55°C for 1 min and 72°C for 1 min; and 1 cycle at 72°C for 10 min. For the second round of PCR, the DNA amplified during the first round of PCR was diluted 1:100 and reamplified using a forward 5'-RACE inner primer provided by the manufacturer with either a reverse inner PREPL primer (5'-CCAGTGAAGGCAGGCCGGAGAAC-3') or C2ORF34 primer (5'-GGTCCAGAAACAGGCAGTCAGCACACATAACA-3') under the following conditions: 1 cycle at 95°C for 5 min; 40 cycles at 95°C for 1 min, 57°C for 1 min and 72°C for 1 min; and 1 cycle at 72°C for 10 min. The desired PCR products were isolated from agarose gels and cloned into a pGEM-T Easy vector  followed by transformation into competent Top10 E. coli cells. Two to three independent clones were then selected, purified, and sequenced with generic sequencing primer to determine the 5'-ends of C2ORF34 and PREPL transcripts.The 5'-RACE analysis on the human fetal brain was carried out by two consecutive PCR reactions with the use of First Choice RNA ligase-mediated RACE kit (Ambion). In the first round of PCR, at least five reverse primers were attempted for each gene in a trial-and-error fashion until successful PCR amplification occurred. In brief, the PCR was performed using a forward 5'-RACE outer primer provided by the manufacturer and the most appropriated and specific reverse primer for . In brief, for all the genes  that were found to be clustered on human chromosome 2p21, at least three features were examined and compared in each genome. These features included chromosomal localization, orientation, and distance between the two adjacent genes. All nucleotide sequences, in particular those for C2ORF34 and PREPL, were downloaded from the NCBI databases and aligned using Vector NTI software .The comparisons were performed based on the data obtained from genome assemblies of the human NCBI Build 36.2, the mouse NCBI Build 37.1, the rat RGSC 3.4, the dog NCBI Build 2.1, and the chicken NCBI Build 2.1 databases E. coli cells. Four individual clones for each tissue sample were randomly selected and subjected to sequencing by the core facility of the National Health Research Institutes.Genomic DNAs used for bisulfate analysis were purchased from Biochain  including samples of normal brain , normal colon  and normal testis  tissues. The CpG methylation status was analyzed using the Zymo EZ DNA Methylation-Gold Kit  according the manufacturer's instructions. In brief, 1 μg of each genomic DNA was treated with the CT Conversion reagent supplied within this kit under the following conditions: 98°C for 10 min, 64°C for 4 h, and then at 4°C until use. All bisulfite-converted DNA samples were purified by the Zymo-Spin IC column with 600 μl M-Binding buffer, 100 μl M-Wash buffer, 200 μl M-Desulphonation buffer, 200 μl M-Wash buffer, and then eluted with 10 μl M-Elution buffer. The samples were then PCR-amplified using the forward primer (5'-CCACATAATTCCTCCTCTT-3') and reverse primer (5'-TCCACATCTCTCTCATCCTCTTTTC-3') under following condition: 1 cycle of 95°C for 10 min; 30 cycles of 95°C for 30 sec, 50°C for 30 sec, and 72°C for 40 sec; and 1 cycle of 72°C for 10 min. The amplified products were cloned into a pGEM-T Easy vector (Promega) followed by transformation into competent Top10 C2ORF34 and PREPL were amplified by PCR using ExTag polymerase (TaKaRa) with a series of forward and reversed primers (listed in Table PREPL (exon 1a) or C2ORF34 (exon 1) are numbered as +1 and -406, respectively. Thus, the first intergenic nucleotide in front of the PREPL transcriptional start site is numbered as -1, and the intergenic nucleotide right before the transcriptional start site of C2ORF34 is numbered as -405.All restriction enzymes were purchased from Takara. The genomic DNA of peripheral blood lymphocytes was isolated from a healthy donor using the QIAamp Blood kit  as described by the manufacturer. Various intergenic fragments between 5 cells per well and allowed to grow overnight. Cells were then co-transfected, in triplicate, with 3 μl of Lipofectamine 2000 (Gibco Invitrogen) and a mixture of 525 ng of DNA consisting of 500 ng of the tested pGL3 promoter constructs and 25 ng of the pRL-TK Renilla luciferase reporter plasmid (Promega) in a serum-free medium. The pGL3-promoter plasmid containing the SV40 promoter was used as the positive control. After a 2-h transfection at 37°C, the medium was replaced with fresh culture medium supplemented with 10% FBS and cells were incubated for another 24 h. After incubation, cells were harvested and lysed in Passive Lysis Buffer (Promega) at 4°C for 30 min. Cellular debris was removed after centrifugation at 10,000 × g for 5 min at 4°C. The chemiluminescence of the two different luciferases was measured using a SIRIUS luminometer . The transfection efficiency and cell viability were normalized with the pRL-TK plasmid containing the Renilla luciferase gene under the control of the TK promoter. Relative luciferase activity of each clone was normalized to the pGL3-promoter vector containing a firefly luciferase gene driven by the weak SV40 promoter. The experiments were performed in triplicate and repeated independently at least three times.For luciferase reporter assays, the cells from different cell lines were first seeded in 12-well culture plates at a density of 1 × 10Nuclear extracts were prepared from cultured U87MG and H4 cells using nuclear extraction kit  according to the manufacturer's specifications. Cells grown to 80–90% confluence in 10 cm culture plates were washed twice with an appropriate volume of ice-cold PBS. After complete removal of PBS by centrifugation, cells were treated with 1 mL of Buffer A working reagent containing 1 mM DTT, 10 μl protease inhibitor, 10 μl phosphatase inhibitor I, and 10 μl phosphatase inhibitor II for 10 min at 4°C. Cells were released from the bottom of the culture plate by using a sterile cell scraper and transferred into a 1.5 mL microcentrifuge tube with 14,000 × g for 5 min at 4°C. The remnant pellet was re-suspended with 100 μl Buffer B working reagent containing 1 mM DTT, 1 μl protease inhibitor, 1 μl phosphatase inhibitor I, and 1 μl phosphatase inhibitor II on ice for 60 min with vortex every 10 min. Nuclear proteins were subsequently obtained by centrifugation at 14,000 × g for 5 min at 4°C.Electrophoretic mobility shift assays were performed by using the EMSA gel shift kit (Panomics) according to the manufacturer's instructions. In brief, the EMSA probe for YY-1 binding assay was synthesized by PCR reaction using the forward primer (5'-ACTCGCATCTCCTTGGCAACCAGGG-3') and the reverse primer (5'-GGCCCTGGTTGCCAAGGAGATGCG-3') under the following conditions: 1 cycle at 95°C for 5 min; 30 cycles at 95°C for 30 sec, 68°C for 30 sec, and 72°C for 20 sec; and 1 cycle at 72°C for 10 min. The EMSA probe for NRF-2 binding analysis was synthesized with the forward primer (5'-CCCGAATTCCGCATTTCCGGTACTCGCATC-3') and the reverse primer (5'-GATGCGAGTACCGGAAATGCGGAATTCGGG-3') under the following conditions: 1 cycle at 95°C for 5 min, 70°C for 30 sec, and 60°C for 30 sec. With the same PCR condition, the mutant probe of NRF-2(A) site was generated using the forward primer (5'-CCCGAATTAAGCATTTCCGGTACTCGCATC-3') and the reversed primer (5'-GATGCGAGTACCGGAAATGCTTAATTCGGG-3'), and the mutant probe of NRF-2(B) was synthesized with forward primer (5'-CCCGAATTCCGCACTTAATGTACTCGCATC-3') and reversed primer (5'-GATGCGAGTACATTAAGTGCGGAATTCGGG-3'). The specific hot probes were prepared by labeling the same oligonucleotide probes with biotin. The DNA-protein binding reaction was performed in supplemented binding buffer containing 1 μl of probe, 5 μg of nuclear extracts, and 1 μl poly(dI-dC) at 15°C for 30 min. For competition studies, the nuclear extracts were incubated with 1- or 10-fold molar excess of unlabelled competitor oligonucleotides. For supershift analysis, the same amounts of nuclear extracts were preincubated with 2 μl of either anti-NRF-2 , anti-YY-1 , or anti-NF-1  antibody at 15°C for 15 min prior to the binding of the probe. All the DNA-protein complexes were analyzed on native 6% (w/v) polyacrylamide gels in 0.5× TBE buffer  at 5 mA for 7 to 12 h depending on the fragment size. The gels were then transferred onto nylon membranes, detected using streptavidin-HRP with a chemiluminescent substrate, and visualized by exposure to Kodak Biomax MR films.PREPL . The C2ORF34 gene was examined with the forward primer (5'-CGCCGAGGGAATACTTTAAACCAG-3') and reverse primer (5'-GGGCAGGACGAGCAACAAGCAGAGT-3') under following condition 1 cycle at 95°C for 5 min; 30 cycles at 95°C for 30 sec, 67.5°C for 30 sec, and 72°C for 30 sec; and 1 cycle at 72°C for 10 min. ACTB (β-actin) serving as internal control was amplified using the primer set 5'-GATGATGATATCGCCGCGCT-3' and 5'-TGGGTCATCTTCTCGCGGTT-3' under the following conditions: 1 cycle at 95°C for 5 min; 28 cycles at 95°C for 30 sec, 64°C for 30 sec, and 72°C for 30 sec; and then 1 cycle at 72°C for 10 min.Total RNAs were extracted from various cell lines using TRIZOL reagent (Gibco Invitrogen) followed by DNase (Ambion) treatment to eliminate potential genomic DNA contamination. The quality and integrity of the RNAs were examined by agarose gel electrophoresis. Complementary DNA (cDNA) was synthesized from 1 μg of total RNA using oligo d(T)-anchor primer  and M-MLV reverse transcriptase (Ambion) according to the manufacturer's instructions. After a 2-h incubation at 42°C, the reaction mixtures were heat inactivated at 70°C for 10 min and then applied as templates to amplify cDNAs specific for 7 cells from each cell line were incubated with 1% formaldehyde fixation solution for 10 min at room temperature and then quenched with glycine. The nuclei were fully resuspended in 1 mL lysis buffer with 5 μl protease inhibitor cocktail and 1 μl PMSF. The lysates were sonicated 3 times  at 4°C using Bioruptor . Following sonication, the chromatin fragments of 200~1,000 bp with a major size of 500 bp were yielded. The sonicated chromatin was pre-cleaned with protein G agarose beads with gentle agitation at 4°C for 2 h followed by centrifugation at 5,000 × g for 1 min. One-tenth volume of the sample was diluted in a ChIP dilution buffer  and stored at 4°C as the input fraction; the remaining solutions were divided into several aliquots and incubated with 5 μg each of specific antibodies including anti-NRF-2 , anti-YY-1 , anti-histone 3 (Upstate), and control rabbit anti-IgG (Upstate) antibodies. The protein-DNA-antibody complexes were incubated at 4°C for overnight with gentle rotation and then immunoprecipitated by protein G agarose slurry for 2 h at 4°C. The chromatin immune complexes were washed sequentially with 1 mL of low-salt buffer, high-salt buffer, LiCl buffer, and TE buffer before being eluted twice with 100 μl of elution buffer (1% SDS and 0.1 M NaHCO3). The cross-linked antibody-TF-DNA complexes were reversed by the addition of 8 μl of 5 M NaCl and heated at 65°C overnight. After removal of RNA and protein by the treatments with RNase A and proteinase K, the remnant DNA and input control were purified using the EZ ChIP kit. The precipitated DNAs were subjected to PCR in attempt to amplify the NRF-2 and YY-1 binding elements under the following conditions: 1 cycle of 95°C for 5 min; 40 cycles of 95°C for 30 sec, 68°C for 30 sec, and 72°C for 20 sec; and 1 cycle of 72°C for 5 min with the forward primer (5'-CGCGCTCTCCCCGAATT-3') and reverse primer (5'-TTGGGTTCCTGGGTTCTTTAGTC-3'). The amplified fragments were then resolved electrophoretically on a 2% (w/v) agarose gel and verified by DNA sequencing.The ChIP assays were performed using the EZ ChIP kit  according to the manufacturer's instructions. For each assay, 3 × 10PREPL: prolyl oligopeptidase-like; C2ORF34: chromosome 2 open reading frame 34; FBS: fetal bovine serum; RACE: rapid amplification of cDNA ends; TSSs: transcriptional start sites; RT-PCR: reverse transcription-polymerase chain reaction; ChIP: chromatin immunoprecipitation assay.CCH designed and performed the experiments and analyzed the results. WSWC supervised the project and oversaw the experiments. Both authors read and approved the final manuscript.PREPL splice variantVerification of the newly identified . The genomic organization and transcribed mRNA of PREPL are illustrated in the upper panel, with each exon numbered in bold. The designed primers (indicated by arrows) for RT-PCR include the common forward primer (Exon 1a-F) derived from the alternative exon 1a of PREPL, and a set of reversed primers  derived from exons 4, 6, 8, 10, 13 and 14, respectively. Bottom left: the amplified PCR products were detected on a 1.5% (w/v) agarose gel confirming the presence of the identified PREPL splice variant. Bottom right: the nucleotide sequences of the primers.Click here for filePREPL-C2ORF34 bidirectional gene pair in normal tissuesCpG methylation pattern of the . Upper panel: The GC content from position -1,100 to +636 is calculated using Methprimer software with a 200-bp sliding window, and with a ratio of observed versus expected CpGs greater than 0.7 with an average GC content larger than 60%. The identified 322-bp CpG island (from position -286 to -607) is indicated in light blue. Lower panel: The bisulfite-treated genomic DNAs from human normal brain, colon, and testis tissues were PCR-amplified and then cloned into pGEM-T vector for autosequencing. A total of 4 clones were selected and the methylation patterns were analyzed and presented. Each row indicates an individual sequencing clone and each vertical line represents the same CpG position. The empty circles denote the unmethylated CpG sites, and the filled circles indicate the methylated CpG sites.Click here for fileIdentification of the functional importance of the NRF-2 and YY-1 binding sites in the bidirectional minimal promoter. These additional experiments were the same as for those given in Figure 6 except for using three different cell lines: brain H4, GBM8401, and GBM8901 cells.Click here for fileSemi-quantitative PCR products from DNA prepared by ChIP assays. These additional experiments were the same as for those given in Figure 8 except for using three different cell lines: brain H4, GBM8401, and GBM8901 cells.Click here for file


Neisseria meningitidis serogroups, neisserial infections continue to cause septicaemia and meningitis across the world. This is in part due to the difficulties in developing a, cross-protective vaccine that is effective against all serogroups, including serogroup B meningococci. Although convalescent N. meningitidis patients develop a natural long-lasting cross-protective immunity, the antigens that mediate this response remain unknown. To help define the target of this protective immunity we identified the proteins recognized by IgG in sera from meningococcal patients by a combination of 2D protein gels, western blots and mass spectrometry. Although a number of outer membrane antigens were identified the majority of the antigens were cytoplasmic, with roles in cellular processes and metabolism. When recombinant proteins were expressed and used to raise sera in mice, none of the antigens elicited a positive SBA result, however flow cytometry did demonstrate that some, including the ribosomal protein, RplY were localised to the neisserial cell surface.Despite the introduction of conjugated polysaccharide vaccines for many of the  Neisseria meningitidis infection continues to cause considerable disability and mortality throughout the world. Although polysaccharide conjugate vaccines have been developed and used successfully against many of the serogroups of N. meningitidis, such a strategy has proved ineffective against group B meningococci. This serogroup now represents the majority of cases in the UK N. meningitidisNeisseria lactamica provides immunity to meningococcal disease N. meningitidis or N. lactamica) can provide long-term, cross-reactive protection, however the identity of the antigens involved remains unknown. They are unlikely to be the well characterised class 1, 2 and 5 Outer Membrane Proteins (OMPs) since these antigens do not induce a cross-reactive bactericidal immune responses in immunized volunteers Classic studies performed in the 1960's demonstrated the importance of bactericidal antibody for protection against meningococcal disease, as reviewed by Pollard and Frasch N. meningitidisN. lactamicaet al.M. tuberculosisStreptococcus pneumoniaStaphylococcus epidermidisCandida albicansN. meningitidis to identify proteins that bind IgG from acute and convalescent meningococcal patient's sera with the aim of further understanding the immune response to neisserial infection and to potentially identify new cross-protective neisserial antigens.The proteome of both the serogroup B strain, MC58 N. meningitidis L91543 were separated by 2D gel electrophoresis and western blotted with sera from acute and convalescent patients , this is a group C:2a:P1.2, and part of the ST-11, ET-37 complexes. Although such a serogroup C strain is not suited to the identification of vaccine antigens aimed at serogroup B Neisseria, it is applicable for the identification of the cross reactive antigens which this study aimed to discover. Protein for 2D gels was prepared from cultures grown for 4 h on Columbia Agar Base supplemented with 6% horse blood (CAB) at 37°C with 5% CO2. Cells were washed in PBS and proteins prepared using a ProteoPrep® Sample Extraction Kit (Sigma). Cells were resuspended in Reagent 4 and sonicated for 10 min. The cells were centrifuged for 5 min and the pellet discarded, freshly prepared Tributylphosphine (TBP) was added to the supernatant, and the mixture incubated for 1 h at room temperature. The proteins were alkylated with iodoacetamide (IAA) for 1.5 h at room temperature. OMVs were prepared using sodium deoxycholate from 4 h old CAB plate cultures according to Fredriksen et al.RC DC Protein Assay (BioRad). Zoom® 1st dimension strips (Invitrogen) were rehydrated for 18 h with 20 µg protein in 155 µl rehydration buffer , 0.5% Zoom® carrier ampholytes of an appropriate pI range , 0.002% bromophenol blue) and focused at 200 V for 15 min, 450 V for 15 min, 750 V for 15 min followed 2000 V for 140 min. Strips were alkylated with 1% DTT in equilibration buffer  for 15 min, and then reduced with 4% IAA in equilibration buffer for 15 min. Focused 1st dimension strips were loaded onto 2nd dimension NuPAGE® Novex 4–12% Bis-Tris ZOOM® gels (Invitrogen) and electrophoresed at 200 V as recommended by the manufacturers. Gels were silver stained as described by Mortz et al.Protein concentrations were determined using Four pairs of acute and convalescent sera ; two single convalescent serum (patients 5 and 6) and three samples of pooled serum, each containing five convalescent sera , were used to probe 2D gels of total neisserial proteins . OMV preReplica 2D gels were western blotted onto PVDF membrane (Roche). Blots were incubated in PBS containing 10% blocking reagent (Roche) for 30 min at room temperature, and then with patient sera at dilutions of between 1∶100 and 1∶500. Blots were washed three times for 10 min each with PBS containing 0.1% Tween® 20 (PBS-Tw), and then incubated with 1∶10,000 dilution of anti-human IgG conjugated with peroxidase (Sigma) for 30 min at room temperature followed by three 10 min washes with PBS-Tw. Blots were developed with CSPD (Roche) and exposed.4HCO3 (ABC). Gel plugs were left in 100% ACN for 10 min and then air dried. The plugs were incubated in 10 mM DTT in 50 mM ABC for 30 min at 60°C followed by 30 min at room temperature in the dark with 100 mM IAA in 100 mM ABC. The gel plugs were again washed twice in 50% ACN, 50% 400 mM ABC and again incubated for 10 min in 100% ACN and air dried. The pellet was digested for 3 h with 50 ng trypsin (Promega) in 5 µl 10 mM ABC.Developed films were overlain with replica silver stained 2D gels and spots that aligned with spots on the western blot were determined. These immunogenic spots were picked and washed twice for 20 min each with 50% acetonitrile (ACN), 50% 400 mM NHProteins were identified by Matrix-assisted laser desorption time of flight mass spectrometry (MALDI-TOF) using a Bruker Autoflex® machine. Matrix solutions were freshly prepared by dissolving α-cyano-α-hydroxycinnamic acid (α-CCA) in acetone to saturation and spotted onto either a stainless steel or AnchorChip™ target plates and allowed to dry. A 0.5 µl drop of trypsin digested sample was placed on the matrix and the peptides allowed to adsorb for 1–2 min before being removed and the samples washed with 10% formic acid. Mass spectra were acquired with between 300–1200 laser pulses in positive reflective mode with an acceleration voltage of 19 kV. Samples were calibrated with Peptide Calibration Standard (Bruker). The peptide masses were obtained from the spectra using XMASS 5.1 (Bruker) and used to search the neisserial proteins in the NCBI database using MASCOT (Matrix Science) with search criteria of 50 ppm error, fixed modifications of carbamidomethyl modified cysteine residues, variable modifications of oxidised methionines and allowing 1 missed trypsin cleavage. Protein identities were considered reliable if the expected probability was <0.05 (details of methods are available in N. meningitidis MC58 genome (http://cmr.tigr.org) were used to PCR amplify products from L91543 genomic DNA prepared according to Bjorvatin et al.4)2SO4, 2 mM MgSO4, 0.2 µM primers, 0.02 U µl−1Pwo (Roche) and 0.2 mM dNTPs. Cycle conditions were 94°C for 5 min, followed by 35 cycles of 94°C for 30 sec, 58°C for 30 sec and 72°C for 2 min, followed by 5 min at 72°C. Products were mixed with pET101-TOPO vector and transformed into TOP10 cells (Invitrogen). The correct constructs were identified by PCR and restriction digests, and the plasmids transformed in BL21 cells. The pET21b plasmid expressing factor H binding protein (fHBP), NMB1870 was kindly supplied by Chris Tang . Proteins were expressed by growing BL21 transformants to approximately OD600 0.6 and inducing with IPTG to 1 mM. Cells were incubated for 3 h, the cultures centrifuged and the pellet lysed by resuspending in BugBuster® (Novagen) with 2 µg ml−1 DNase and 50 µg ml−1 lysozyme for 20 min. Lysates were centrifuged and the cleared supernatant applied to a previously equilibrated HisSelect™ (Sigma) column. The column was washed with 50 mM NaPO4, pH 8, 0.3 M NaCl, 5 mM imadazole and the proteins eluted with 50 mM NaPO4, pH 8, 0.3 M NaCl, 250 mM imadazole. Proteins were dialysed against 1000× volumes of PBS and quantified using a 2D Quant kit (Amersham).Proteins that had been identified by MALDI analysis were cloned and expressed using the pET101-TOPO system (Invitrogen). Primers for the N and C terminals of the annotated ORFs from et al.Recombinant proteins were used to generate serum according to Giuliani −1 was added to each Maxisorp™ (Nunc) microtiter well and incubated overnight at 4°C. For whole cell ELISAs, bacteria were grown for 4 h on CAB, cells were resuspended in PBS and the OD600 adjusted to 0.05. Cells were heated at 56°C for 30 min, followed by 5 min of sonication and 50 µl added to Maxisorp™ ELISA wells and dried. All plates were washed three times in PBS-Tw and blocked for 90 min at 37°C with 50 µl PBS containing 10% blocking agent (Roche). A serial dilution of serum in PBS with 10% blocking agent was added to the wells and the plates incubated for 2 h at 37°C. Plates were washed three times with PBS-Tw and anti-mouse IgG peroxidase conjugate (Sigma) diluted 1∶10,000 in PBS with 10% blocking agent added. Plates were incubated for 1 h in the dark at 37°C and washed three times in PBS-Tw. Colour reagent, 3,3′,5,5′-Tetramethylbenzidine (TMB) was dissolved in 0.05 M phosphate-citrate buffer, pH 5 containing 10% DMSO and added to each well, the colour was allowed to develop and the reaction stopped by adding H2SO4 to 0.4 M. The OD450 of each well was measured. Titres were determined as the last dilution at which the OD450 was greater than 0.1 and more than twice the value for the equivalent dilution containing negative sera .For ELISAs with recombinant protein, 50 µl of 2 µg mlN. meningitidis, L91543, grown overnight on CAB were inoculated into Mueller Hinton broth to an OD600 0.1, and shaken at 35°C for 4 h. Cultures were diluted to OD600 0.1 and then diluted a further 1∶5,000, 10 µl was added to microtiter wells containing a 10 µl of a 1∶4 dilution of baby rabbit complement  and 20 µl of a twofold serial dilution of heat denatured (56°C for 30 min) sera. Plates were incubated for 1 h and samples plated onto CAB media. Each assay included control wells with either no sera, heat inactivated complement, or neither sera nor active complement. Positive wells were those containing 50% of the number of cells in the control wells. SBA results for recombinant proteins were compared to sera from mice immunised with PBS alone, OMVs (as a positive control for the SBA), and the known bactericidal antigen fHBP Serum Bactericidal Assays (SBAs) were carried out according to Borrow and Carlone N. meningitidis for flow cytometry were grown for 4 h on CAB, resuspended in PBS and adjusted to an OD600 of 1. A 20 µl volume of cells was centrifuged, the pellet resuspended in 10 µl heat denatured sera and incubated for 30 min at 37°C. The cells were washed three times in 100 µl PBS-Tw and resuspended in 100 µl 4% paraformaldehyde for 18 h at 4°C. Cells were washed three time in 100 µl PBS-Tw and resuspended in 20 µl anti-mouse IgG conjugated to FITC (Sigma) diluted 1∶50 in PBS. Cells were incubated for 30 min at room temperature in the dark, washed three times in PBS-Tw and resuspended in 1 ml PBS. Fluorescence was measured with a BD FACSCanto™ Flow Cytometer recording 10,000 events and the data compared to samples containing either no cells, or cells stained with sera from mice immunised with fHBP, OMV preparations or PBS alone. Data were analysed using WinMDI, recorded events were gated with reference to the control samples and the population statistics determined from triplicate experiments.Table S1Mass Spectrometry Data(0.05 MB XLS)Click here for additional data file.Table S2MS exclusion list(0.03 MB XLS)Click here for additional data file.Document S1Parameters for MS data acquisition and database searching(0.03 MB DOC)Click here for additional data file.


Escherichia coli, and the two peptides were expressed in this host in inclusion bodies. Peptides were purified from inclusion bodies using a combination of anion-exchange chromatography and centrifugal filtration. The method described requires little specialized equipment and provides a facile and inexpensive procedure for production of large amounts of very pure Aβ peptides. Recombinant peptides generated using this protocol produced amyloid fibrils that were indistinguishable from those formed by chemically synthesized Aβ1–40 and Aβ1–42. Formation of fibrils by all peptides was concentration-dependent, and exhibited kinetics typical of a nucleation-dependent polymerization reaction. Recombinant and synthetic peptides exhibited a similar toxic effect on hippocampal neurons, with acute treatment causing inhibition of MTT reduction, and chronic treatment resulting in neuritic degeneration and cell loss.We report the development of a high-level bacterial expression system for the Alzheimer’s disease-associated amyloid β-peptide (Aβ), together with a scaleable and inexpensive purification procedure. Aβ(1–40) and Aβ(1–42) coding sequences together with added ATG codons were cloned directly into a Pet vector to facilitate production of Met-Aβ(1–40) and Met-Aβ(1–42), referred to as Aβ(Μ1–40) and Aβ(Μ1–42), respectively. The expression sequences were designed using codons preferred by  Subsequently, the resin was washed with buffer A (50 mL), followed by stepwise elution using 50 mL aliquots of buffer A with 50, 75, 100, 125, 150, 200, 250, 300 and 500 mm NaCl, respectively. Each aliquot was incubated with the resin for 5 min before collection under vacuum. Eluates were analyzed by SDS-PAGE and agarose gel electrophoresis, and fractions with highly pure Aβ were pooled and fractionated by centrifugation through a 30 kDa molecular mass cut-off filter. The washing and elution processes can also be performed as follows: the resin is washed with 50 mL buffer A, and then with 50 mL buffer A with 25 mm NaCl followed by three or four 50 mL aliquots of buffer A with 125 mm NaCl. Using SDS-PAGE, the peptide is then found in the first two (or first three) 125 mm aliquots, which are combined and used for centrifugal filtration.The procedures described here are for 50 mL of urea-solubilized inclusion bodies originating from 4.5 L of culture, but this process can be scaled proportionally for other amounts. The urea-solubilized inclusion bodies (50 mL) were diluted with 150 mL of 10 mm NaCl with a total gradient volume of 500 mL. Fractions were analyzed by electrophoresis on 10–20% polyacrylamide Tris-tricine gels and 1% agarose gels. In a second set of experiments, the column was equilibrated in buffer A containing 8 m urea, and the sample was eluted with a gradient of 0–300 mm NaCl in buffer A containing 8 m urea.Urea-solubilized inclusion bodies  were diluted with 150 mL of buffer A and applied to a 50 mL DEAE-cellulose column equilibrated in buffer A. The column was washed with 50 mL buffer A, followed by elution using a linear gradient from 0–300 mm citric acid). All analyses were performed in positive reflector mode, collecting data from approximately 3000 and 5000 single laser shots for MS and MS/MS analyses, respectively.Amino acid analysis was performed at the Amino Acid Analysis Center, University of Uppsala, Sweden. Sequence analysis was performed using an Applied Biosystems Procise 492 cLC sequenator  employing standard Edman chemistry, and MS analysis was undertaken using an LCQDECA LC/MS system . The MS system consisted of a Surveyor HPLC system with a diphenyl 150 × 1.0 mm column  interfaced to an LCQ-DECA electrospray ionization/ion trap mass spectrometer, and eluted using an acetonitrile/trifluoroacetic acid gradient. MALDI-TOF mass spectrometry was performed using a 4700 proteomics analyzer (Applied Biosystems). Samples were dispensed onto a MALDI sample support, and allowed to air-dry prior to addition of matrix solution , and eluted at 0.8 mL·min−1 using 50 mm ammonium acetate, pH 8.5. Fractions (0.5 mL) were collected, peak fractions pooled, and the concentration of peptide determined by absorbance at 275 nm using ε275 = 1400 m−1 cm−1.For fibrillation assays, it is essential to start with a uniform monomeric peptide sample. Solutions of monomeric Aβ were prepared by dissolving lyophilized peptides in 5 m using 50 mm ammonium acetate, pH 8.5. Peptides were then incubated in a 96-well black fluorescence plate at a final concentration of 6 or 9 μm in the presence of 10 μm ThT at 37 °C, and shaken at 700 r.p.m. using a VorTemp 56™ incubator/shaker with an orbit of 3 mm . Measurements were made at regular intervals using a SpectraMax M2 microplate reader  with excitation and emission at 440 and 480 nm, respectively. Each experimental point is the mean of the fluorescence signal of at least eight wells containing aliquots of the same solution. The morphology of Aβ aggregates formed from solutions incubated as above but in the absence of ThT and at a concentration of 50 μm was assessed by negative-contrast electron microscopy as described previously [The kinetics of fibril formation was determined using a continuous ThT assay . Solutioeviously . Briefly−1 with a 14–49% acetonitrile gradient using a Varian Pro Star HPLC system fitted with a photodiode array detector. For SDS-PAGE, samples (10 μL) were mixed with 2× sample buffer, and immediately electrophoresed on 10–20% polyacrylamide Tris-tricine gels. Proteins were stained with silver as described previously [Samples (100 μL) of peptides isolated by SEC were injected on to a CN capcell column (4.6 mm × 25 cm)  using a Varian Pro Star 410 autosampler , and eluted at 1.5 mL·mineviously .4 cells on 48-well dishes pre-coated with poly-d-lysine (50 μg·mL−1) and maintained in neurobasal medium containing 2 mm glutamine and B27 supplement without antioxidants. Half the medium was exchanged every 3 days. All media reagents were purchased from Invitrogen .Primary hippocampal neuronal cultures were prepared as described previously  with minm GuHCl, pH 8.0. Thereafter, samples were injected onto a Superdex 75 column HR 10/30 column , and eluted with 10.9 mm HEPES pH 7.4 at a flow rate of 0.8 mL·min−1. Peak fractions were then examined for absorbance at 275 nm, and the concentration of Aβ was calculated. Fractions containing monomeric peptide were diluted such that all peptides were of equal concentration. To induce peptide aggregation, samples were incubated at 37 °C and shaken at 700 r.p.m. using a VorTemp 56™ incubator/shaker with an orbit of 3 mm  until 50% of the maximal thioflavin T fluorescence had been achieved; maximal aggregation was taken as the mean plateau fluorescent signal. Peptides were then diluted with 2× neurobasal medium, and 50% of the medium of each well was replaced with an equal volume of neurobasal medium containing either Aβ(1–40), Aβ(M1–40), Aβ(1–42) or Aβ(M1–42)  and incubated for 6 h. Cell-mediated reduction of 3--2,5-diphenyltetrazolium bromide (MTT) was assessed as described previously [−1 MTT (25 μL) was added to each well, and incubation was continued for a further 2 h. Cells were then solubilized in 250 μL of 20% w/v SDS in 50% v/v N,N′-dimethylformamide, 25 mm HCl, 2% v/v glacial acetic acid, pH 4.7, and levels of reduced MTT were determined by measuring the difference in absorbance at 570 and 650 nm using a Molecular Devices Spectramax M2 microplate reader.Lyophilized peptides were resuspended and incubated for a minimum of 2 h in 5 m), and cells were fixed and used for immunocytochemical analyses.In a separate series of experiments, neurons were incubated for 4 days with each of the peptides . Neurons were then incubated with mouse monoclonal anti-MAP-2  diluted 1 : 2000 in blocking solution for 30 min. Cells were rinsed in NaCl/Pi several times and incubated in blocking serum containing anti-mouse IgG (Vectastain) for a further 30 min. Staining was developed by incubation of cells with Vectastain ABC reagent for 30 min, followed by incubation with substrate solution until colour had developed. Cells were visualized by light-phase contrast microscopy using a 40× objective lens, and captured using an SP-500 UZ digital compact camera .Neurons were fixed in 4% paraformaldehyde for 20 min at room temperature, and cells were stained for microtubule-associated protein-2 (MAP-2) using a Vectastain kit . Staining was performed according to the manufacturer’s instructions. Briefly, endogenous peroxidases were blocked in 0.3% HE. coli cells (BL21 DE3 PLysS Star) and spread on LB plates with ampicillin, kanamycin and chloramphenicol. Single colonies were picked for cultivation in liquid culture as described for Aβ alone, except that the medium contained 50 mg·L−1 ampicillin, 100 mg·L−1 kanamycin and 30 mg·L−1 chloramphenicol.Plasmids encoding MetAP-TG  and Aβ


Neurodevelopment requires thyroid hormone, yet the mechanisms and targets of thyroid hormone action during embryonic stages remain ill-defined. We previously showed that the thyroid hormone thyroxine (T4) rapidly increases voltage-gated sodium current in zebrafish Rohon-Beard cells (RBs), a primary sensory neuron subtype present during embryonic development. Here, we determined essential components of the rapid T4 signaling pathway by identifying the involved intracellular messengers, the targeted sodium channel isotype, and the spatial and temporal expression pattern of the nongenomic αVβ3 integrin T4 receptor.Na) by perturbing specific pathways associated with nongenomic thyroid hormone signaling. We found that pharmacological blockade of protein phosphatase 1 and the mitogen-activated protein kinase p38 isoform decreased and increased tonic sodium current amplitudes, respectively, and blockade of either occluded rapid responses to acute T4 application. We next tested for the ion channel target of rapid T4 signaling via morpholino knock-down of specific sodium channel isotypes. We found that selective knock-down of the sodium channel α-subunit Nav1.6a, but not Nav1.1la, occluded T4's acute effects. We also determined the spatial and temporal distribution of a nongenomic T4 receptor, integrin αVβ3. At 24 hours post fertilization (hpf), immunofluorescent assays showed no specific integrin αVβ3 immunoreactivity in wild-type zebrafish embryos. However, by 48 hpf, embryos expressed integrin αVβ3 in RBs and primary motoneurons. Consistent with this temporal expression, T4 modulated RB INa at 48 but not 24 hpf. We next tested whether T4 rapidly modulated INa of caudal primary motoneurons, which express the receptor (αVβ3) and target (Nav1.6a) of rapid T4 signaling. In response to T4, caudal primary motoneurons rapidly increased sodium current peak amplitude 1.3-fold.We first tested which signaling pathways mediate T4's rapid modulation of sodium current (Iv1.6a. Our in vivo analyses identify molecules required for T4's rapid regulation of voltage-gated sodium current.T4's nongenomic regulation of sodium current occurs in different neuronal subtypes, requires the activity of specific phosphorylation pathways, and requires both integrin αVβ3 and Na Although thyroid hormone deficiency results in severe neurodevelopmental deficits , the undet al. . Fu. Fuv1.6a39 ± 5%)  , suggesting that T4 does not require Nav1.1l channels to increase RB INa density. In Nav1.1l morphants, the increase in INa amplitude induced by T4 actually exceeded that produced in controls , presumably because Nav1.6a channels carry the majority if not all of the remaining current [v1.6a and Nav1.1la morphants indicate that rapid T4 signaling targets Nav1.6a sodium channels.In contrast, in Na current . The effv1.6a-mediated current. We exposed Nav1.6a morphants to the thyroid hormone analog tetrac, or the αVβ3 function blocking antibody LM609. To more readily detect changes in RB INa amplitude in Nav1.6a morphants, we raised the extracellular sodium concentration to 125 mM to increase INa amplitudes. We previously showed that tetrac reduces RB INa amplitudes in wild-type embryos [v1.6a morphants, tetrac did not significantly change RB INa amplitudes compared to Nav1.6a morphants unexposed to tetrac ) or of the integrin αVβ3 block rapid T4 signaling in RBs . We next embryos . Howeverc Figure . We had  embryos . In conts Figure . The lacv1.6a sodium channel α-subunit. We next determined whether αVβ3 is present and if RBs respond rapidly to T4 at earlier stages. We reasoned that absence of integrin αVβ3 would prevent T4 from rapidly modulating RB INa. Accordingly, we tested our prediction by determining the spatial and temporal αVβ3 expression pattern.Whether T4 induced increases in sodium current occur throughout development or if T4 signaling begins at a defined developmental stage is unknown. The above results combined with our previous study  indicateTo determine when RBs express the plasma membrane T4 receptor, we performed immunocytochemistry using the LM609 antibody that specifically detects the αVβ3 dimer . As expeisl3 promoter drives GFP expression in RBs . In 48 hpf Tg(isl3:GFP) embryos  embryos. In this line, the s Figure , LM609 is Figure . These dv1.6a underlies a portion of RB INa [Na To test this prediction, we acutely applied 30 nM T4 to the spinal cords of 24 hpf embryos and recorded RB INa. In contrast to results obtained from 48 hpf embryos [Na amplitude at 24 hpf  48-hpf embryos by cell body size and ventrally projecting axons, while recording INa. In control CaP recordings, INa peak density decreased over 5 minutes by 7 ± 2%. However, CaP INa density significantly increased by 28 ± 8% (P < 0.005) after acute T4 application . Further, the data suggest that serine/threonine phosphorylation and dephosphorylation reduce and increase, respectively, RB INa amplitude. To increase RB INa amplitude, T4 may rapidly activate PP1 and/or inhibit p38. Consistent with our findings, PP1 modulates INa amplitudes in rat striatal neurons [v1.6 [et al. [Na amplitudes, similar to our results in zebrafish RBs.We propose a model in which the phosphorylation status of a particular protein or set of proteins regulates RB I neurons , which ens [v1.6 . Schiffm [et al.  found thv1.6 sodium channels, activation of p38 produces a decrease in INa [v1.6 serine, S553, revealing the Nav1.6 sodium channel as a direct p38 phosphorylation target [Na by regulating the phosphorylation state of the conserved serine residue in zebrafish Nav1.6a.Our data do not provide information about the identity of the phosphorylated protein(s). One possibility is that T4 binding to αVβ3 activates intracellular pathways that directly phosphorylate sodium channel α-subunits. In ND7/23 cells transfected with mammalian Nae in INa . Of partn target . On thisNa amplitude produced by PP1 inhibition. We previously reported that either T4 or αVβ3 blockade reduced INa by only 50%, yet 1 μM OA reduced RB INa by nearly 90%. This discrepancy could be attributed to different degrees of PP1 inhibition by 1 μM OA versus T4/αVβ3 blockade. The large decrease in INa amplitude produced by 1 μM OA could also reflect phosphorylation effects triggered by non-T4-dependent mechanisms. For example, protein kinases C and A also have effects on Nav1.6 amplitude [One result that the model does not fully account for, however, is the large reduction in RB Implitude .The important developmental roles of integrins as cell surface adhesion proteins have been well studied . Less-weet al. suggested that, in addition to αVβ3, seven other RGD integrin dimers may function as thyroid hormone receptors [Here, we focused on integrin's role as a plasma membrane receptor for thyroid hormones that traditionally signal through nuclear receptors. We focused on the integrin dimer, αVβ3, a protein that is important for neuronal migration and axon extension -41, and eceptors .Na amplitude. Although we found that T3 does not affect RB INa, previous studies show T3 can increase INa depending on cell type. For example, chronic T3 application increases INa in cultured rat hippocampal neurons [in vitro. This result was attributed to increased nuclear thyroid hormone receptor expression in hippocampus versus cortex leading to differential genomic regulation of sodium channel expression. Additionally, T3 rapidly increases INa in cultured myocytes [In addition to T4, the iodothyronine T3 binds to integrin αVβ3 and activates both ERK1/2 and phosphatidyl inositol 3-kinase . If T3 i neurons , but notmyocytes ,48 throumyocytes . Altogetv1.6a subunit. The implications for mammals are substantial because the mammalian homologue, Nav1.6, shows widespread expression in both the central and peripheral nervous systems [v1.6a plays important developmental roles for sensory neuron survival and motoneuron axon growth in zebrafish [v1.6 current during embryonic stages serves as a strategic way to regulate both structural and functional development of the nervous system.During intrauterine stages, the human embryo requires maternally provided thyroid hormone for normal development -51. Howe systems ,54 and i systems . Moreoveebrafish ,25. Takev1.6, leading to reduced excitability. Conversely, an excess of thyroid hormone would increase sodium current and potentially induce pathological hyperexcitability, associated with seizures and developmental abnormalities. Interestingly, increased expression of Nav1.6 channels are associated with epileptogenesis in mouse hippocampal neurons through mechanisms of enhanced excitability [SCN8A are somewhat resistant to seizures [v1.6 in mammals warrants further study. Altogether, T4 regulation of Nav1.6a current provides an important mechanism to influence neuronal activity and development.In the context of our results, periods of thyroid hormone deprivation during development would decrease sodium current in neurons expressing both αVβ3 and Natability , and acutability . Also ofseizures  and chilseizures . Whetherv1.6 are also present in the adult nervous system, raising the possibility that T4 acutely regulates sodium current in adults. During adult stages, Nav1.6 is the primary sodium channel isoform expressed at nodes of Ranvier [v1.6 mediated current would alter INa at nodes of Ranvier and, therefore, regulate axonal conductance. The mechanism of thyroid hormone action on adult neurons is unclear, yet alterations in Nav1.6 current could result in deficits in sensory neuron axonal conductance and could account for states of hyper- or hypo-reflexia observed in hyper- or hypothyroid patients, respectively [We focused on rapid T4 signaling in the embryonic nervous system. However, αVβ3 and Na Ranvier . T4 induectively ,61. Studectively .v1.6a sodium channel α-subunit. Our proposed pathway predicts that T4's rapid modulation of sodium current requires expression of both αVβ3 and Nav1.6a on the responding cell. Our data agree with the prediction in three ways. First, in RBs, T4 rapidly increased sodium current amplitudes at 48 but not 24 hpf, consistent with detection of αVβ3 at 48 but not 24 hpf. Second, upon knock-down of Nav1.6a protein, exogenously applied T4 no longer led to a rapid increase in RB INa amplitude. Third, another neuronal population, CaP, which expresses both αVβ3 and Nav1.6a, responded to T4 with a rapid increase in INa amplitude. Uncovering the signaling pathways and relevant proteins involved in nongenomic T4 signaling contributes to our understanding of how thyroid hormone regulates development and function of the nervous system.Our results delineate a pathway for rapid T4 signaling that is initiated by αVβ3 as a T4 receptor, transduced intracellularly by regulation of phosphorylation states, and targets the NaNa: sodium current; MAPK: mitogen-activated protein kinase; MO: morpholino; Nav: voltage-gated sodium channel; OA: okadaic acid; p38: MAPK p38 isoform; PP: protein phosphatase; RB: Rohon-Beard cell; T3: 3,3',5-triiodo-L-thyronine/triiodothyronine; T4: 3,5,3',5'-tetraiodothyronine or thyroxine; tetrac: 3,3',5,5'-tetraiodothyroacetic acid.CaP: caudal primary motoneuron; DMSO: dimethyl sulfoxide; ERK1/2: MAPK extracellular regulated kinase; GFP: green fluorescent protein; hpf: hours post-fertilization; IThe authors declare that they have no competing interests.MAY contributed to experimental design, carried out experiments, and drafted and edited the manuscript. ABR contributed to experimental design and edited the manuscript. All authors read and approved the final manuscript.