Ribosomal protein L30 belongs to the L7Ae family of RNA-binding proteins, which recognize diverse targets. L30 binds to kink-turn motifs in the 28S ribosomal RNA, L30 pre-mRNA, and mature L30 mRNA. L30 has a noncanonical function as a component of the UGA recoding machinery that incorporates selenocysteine (Sec) into selenoproteins during translation. L30 binds to a putative kink-turn motif in the Sec Insertion Sequence (SECIS) element in the 3’ UTR of mammalian selenoprotein mRNAs. The SECIS also interacts with SECIS-binding protein 2 (SBP2), an essential factor for Sec incorporation. Previous studies showed that L30 and SBP2 compete for binding to the SECIS in vitro. The SBP2:SECIS interaction has been characterized but much less is known about how L30 recognizes the SECIS.Here we use enzymatic RNA footprinting to define the L30 binding site on the SECIS. Like SBP2, L30 protects nucleotides in the 5’ side of the internal loop, the 5’ side of the lower helix, and the SECIS core, including the GA tandem base pairs that are predicted to form a kink-turn. However, L30 has additional determinants for binding as it also protects nucleotides in the 3’ side of the internal loop, which are not protected by SBP2. In support of the competitive binding model, we found that purified L30 repressed UGA recoding in an in vitro translation system, and that this inhibition was rescued by SBP2. To define the amino acid requirements for SECIS-binding, site-specific mutations in L30 were generated based on published structural studies of this protein in a complex with its canonical target, the L30 pre-mRNA. We identified point mutations that selectively inhibited binding of L30 to the SECIS, to the L30 pre-mRNA, or both RNAs, suggesting that there are subtle differences in how L30 interacts with the two targets.This study establishes that L30 and SBP2 bind to overlapping but non-identical sites on the SECIS. The amino acid requirements for the interaction of L30 with the SECIS differ from those that mediate binding to the L30 pre-mRNA. Our results provide insight into how L7Ae family members recognize their cognate RNAs. Eukaryotic ribosomal protein L30 is a component of the large ribosomal subunit. L30 has no prokaryotic ortholog but the gene is essential in yeast . Cryo-elIn vitro studies support a model in which the two proteins bind to the SECIS element independently, and likely sequentially  ATP  and T4 PNK (NEB) at 20 units/pmole RNA. The transcripts were gel purified on 8% acrylamide (19:1)/7M urea gels and eluted in 10 mM Tris HCl, pH 7.5, 1 mM EDTA, pH 8, 300 mM NaAc, pH 5.5 at 4°C overnight. Purified RNA was stored in 10 mM Tris HCl, pH 7.5 at -20°C.Plasmid DNAs were linearized and used as templates for in vitro transcription. For UV cross-linking and REMSA analyses, RNAs were synthesized in the presence of [αRecombinant rat L30 was expressed in BL21 Star (DE3) cells (Invitrogen). IPTG was added to 1 mM and the culture was grown at 37°C for 3 hr. The cells were harvested at 4°C, and the pellets washed with 1X PBS and frozen on dry ice. Purification buffers (PB) contained 20 mM HEPES-HCl, pH 8.0 and the indicated amounts of NaCl and imidazole. The frozen cell pellet was re-suspended in 20 ml of PB/0.2 M NaCl/20 mM imidazole, to which 60 kU rlysozyme (Novagen), 500 U of Benzonase Nuclease (Novagen) and two Complete, Mini, EDTA-free Protease Inhibitor Cocktail tablets (Roche) were added. The cells were further lysed by sonication on ice. The insoluble material was removed by two centrifugations of the lysate at ~16,000 × g for 15 min at 4°C. The cell lysate was filtered through a 0.45 μM filter and passed across a HisTrap TM FF crude column  using an AKTA Purifier (UPC-900) . The resin was sequentially washed with PB/0.2 M NaCl/20 mM imidazole, PB/1 M NaCl/20 mM imidizole, and PB/0.2M NaCl/40 mM imidazole. The protein was eluted with PB/0.2 M NaCl/250 mM imidazole and collected in 1 or 2 ml fractions. Fractions containing the pure protein were combined and dialyzed against 1 L of PB/0.2 M NaCl buffer for 2 hr at 4°C, which was then repeated with fresh buffer for an additional 2 hr at 4°C. The protein was dialyzed against PB/0.2 M NaCl containing 50% glycerol for 18 hr at 4°C. The dialyzed protein was aliquoted and stored at −70°C. Recombinant SBP2-CT was purified as previously described [32P-labeled PHGPx SECIS RNA was heated to 95°C and slow cooled to room temperature. The RNA (2.5 nM) was incubated in L30 binding buffer , 10% glycerol, and 50 ng/μL yeast tRNA) with or without rat L30 protein (0.75 or 1.5 μM as indicated) at 30°C for 15 min. Reactions were cooled to room temperature over a 2 min period and then placed at 22°C for 2-5 min. The indicated amounts of RNase T1, A, or V1 (Ambion) were added to the appropriate samples and incubated at 22°C for 5 min. Enzymatic reactions were quenched with 20 μL Inactivation/Precipitation buffer (Ambion) and purified according to manufacturer’s directions. Samples were resuspended in 10 μL of dye-less loading buffer , heat-denatured at 95°C for 5 min, and separated in a denaturing 8% (19:1) polyacrylamide/7 M urea gel. The dried gels were visualized with a phosphorimager or on film.End-labeled 32P-labeled PHGPx SECIS RNA (2.5 nM) in 1X Sequencing Buffer (Ambion) supplemented with 50 ng/μL yeast tRNA. The RNAs were incubated at 50°C for 5 min, cooled to 22°C and the indicated amounts of RNase T1, A, or V1 added. The samples were incubated, quenched, and purified as described above. Alkali ladders were prepared by incubating end-labeled 32P-labeled PHGPx SECIS RNA (2.5 nM) in 100 mM NaOH, 2 mM EDTA, pH 8.0, and 2 μg/μL yeast tRNA at 37°C for 3 min, to which 0.2 M Tris HCl, pH 8.0  was added. The samples were frozen on dry ice and combined with an equal volume of denaturing loading buffer .Sequencing ladders were prepared by incubating end-labeled 32P-labeled PHGPx SECIS or U40C mutant RNAs (10 fmol) were incubated in buffer containing 0.7X PBS, 11 mM DTT, 250 ng/μL yeast tRNA and RNAguard (Amersham). The indicated amounts of L30 were added last and the reactions incubated at 37°C for 30 min. The samples were irradiated on ice at 254 nm for 10 min in Costar 96-well polystyrene plates  using a Bio-Rad GS Genelinker. The RNA was digested with 20 U of RNase A (Fermentas) at 37°C for 1 hr. The samples were separated on a 15% SDS–PAGE (37.5:1) gel. The gels were dried and visualized using a phosphorimager.The In vitro transcribed RNAs (100 ng) were added to an in vitro translation reaction (25 μL) containing rabbit reticulocyte lysate (Promega), complete amino acid mix (Promega), Protector RNase Inhibitor (Roche), and 70 nM purified recombinant SBP2-CT protein. Purified recombinant L30 protein was added as indicated. Reactions were incubated at 30°C for 30 min and placed on ice for 15 min. Aliquots of the translation products (2.5 μL) were then added to the luciferase substrate (50 μL) in a well of a Microlite 1 microtiter plate (Thermoscientific). Luminescence was measured in 10 sec intervals using a Victor3 Multilabel Counter (Perkin Elmer).32P-labeled SECIS RNA (10 fmol) was incubated in L30 binding buffer , 10% glycerol, 50 ng/μL yeast tRNA, and 0.2 U/μL Protector RNase Inhibitor (Roche)) with the indicated final concentration of L30 protein at 30°C for 15 min. Samples were transferred to ice and then separated in non-denaturing polyacrylamide gels, either 6% (19:1) or 8% (29:1), in 0.5X TBE gel at 4°C. The dried gels were visualized with a Phosphorimager or on film. The 32P-labeled yeast L30 RNA was incubated in 350 mM KCl, 30 mM Tris HCl, 8.0, and 10 mM DTT at 60°C and allowed to slow cool to room temperature as previously described [The escribed . The binData were analyzed using GraphPad Prism software. The results are expressed as means +/- standard error of the mean (S.E.M). Statistical significance is indicated as * (P < 0.05), ** (P < 0.01), or *** (P < 0.001).PHGPx: Phospholipid Hydroperoxide Glutathione Peroxidase; REMSA: RNA Electrophoretic Mobility Shift Assay; RRL: Rabbit reticulocyte lysate; SBP2: SECIS-binding protein 2; Sec: Selenocysteine; SECIS: Selenocysteine insertion sequence; SelN: Selenoprotein N; TR1: Thioredoxin Reductase 1.The authors declare they have no competing interests.AB, TF, and DD conceived and designed the experiments, and analyzed data. AB, TF, and TA performed experiments. AB and DD wrote the manuscript. All authors read and approved the final manuscript.Oligonucleotides used in this study. Click here for file


Amphibacillus sp. CGTase was purified to homogeneity up to 22.1 fold by starch adsorption and anion exchange chromatography with a yield of 44.7%. The purified enzyme was a monomeric protein with an estimated molecular weight of 92 kDa using SDS-PAGE. Catalytic activities of the enzyme were found to be 88.8 U mg−1 protein, 20.0 U mg−1 protein and 11.0 U mg−1 protein for cyclization, coupling and hydrolytic activities, respectively. The enzyme was stable over a wide pH range from pH 5.0 to 11.0, with a maximal activity at pH 8.0. CGTase exhibited activity over a wide temperature range from 45 °C to 70 °C, with maximal activity at 50 °C and was stable at 30 °C to 55 °C for at least 1 h. Thermal stability of the purified enzyme could be significantly improved in the presence of CaCl2. Km and Vmax values were estimated using soluble starch as a substrate to be 1.7 ± 0.15 mg/mL and 100 ± 2.0 μmol/min, respectively. CGTase was significantly inhibited in the presence of Co2+, Zn2+, Cu2+, Hg2+, Ba2+, Cd2+, and 2-mercaptoethanol. To the best of our knowledge, this is the first report of CGTase production by Amphibacillus sp. The achieved high conversion of insoluble raw corn starch into cyclodextrins (67.2%) with production of mainly β-CD (86.4%), makes Amphibacillus sp. NPST-10 desirable for the cyclodextrin production industry.Screening for cyclodextrin glycosyltransferase (CGTase)-producing alkaliphilic bacteria from samples collected from hyper saline soda lakes , resulted in isolation of potent CGTase producing alkaliphilic bacterium, termed NPST-10. 16S rDNA sequence analysis identified the isolate as  Cyclodextrin glycosyl transferase  is an important industrial enzyme, unique in its ability to convert starch and related glycans into non-reducing, cyclic malto-oligosacchrarides called cyclodextrins (CDs) via a cyclization reaction, an intramolecular transglycosylation reaction ,4. The pMost CGTases convert starch into a mixture of α-, β- and γ-CD in different ratios, and depending on the main cyclodextrin produced, CGTases are classified as α-, β- or γ-CGTases . Among tAlkaliphilic microorganisms have attracted much interest in the past few decades because of their ability to produce extracellular enzymes that are active and stable at high pH values ,10. The w/v) phenolphthalein, resulted in isolation of a few isolates showing halo zones around their margins, suggesting possibility of CGTase production by those strains [E. coli numbering. According to the phylogenetic analysis, NPST-10 is a member of the genus Amphibacillus with similarity of 98%. Hence, it was designated as Amphibacillus sp. strain NPST-10. The 16S rDNA gene sequence was deposited in the GeneBank under Accession No. JX028596. The genus Amphibacillus was first proposed in 1990 by Niimura et al. [Amphibacillus xylanus [Amphibacillus fermentum, Amphibacillus tropicus [Amphibacillus sediminis [Amphibacillus sp.Screening of alkaline water and sediment samples collected from Wadi Natrun hyper saline soda lakes for isolation of CGTase producing alkaliphilic bacteria, using rich alkaline agar medium containing 0.02%  was less than that recently reported for Bacillus sphaericus strain 41, (0.3:1.0), respectively [In addition to cylization activity of the purified ectively . Althougectively .Bacillus coagulans [Bacillus sphaericus strain 41 [The molecular weight of the purified denatured CGTase was estimated to be 92 kDa  by SDS-Poagulans  and as 5train 41 .The temperature profile of the enzyme was estimated by measurement of the enzyme activity at various temperatures . CGTase Amphibacillus sp. NPST-10 CGTase was studied. It was found that the thermal stability of CGTase was enhanced by about 1.3 fold in the presence of calcium ion, and slightly enhanced in the presence of maltodextrins or starch  at 50 °C. The kinetic parameters, urk plot . The Km ectively . The low CGTases –24,37.Amphibacillus sp. NPST-10 CGTase showed higher activity using raw cornstarch than soluble starch . However, the cyclization activity using maltodextrin, short oligosaccharides (17 glucose residues) was the highest. The raw starch has a compact crystalline structure that is not easily degraded, and therefore CGTases with high activity toward raw starch is highly recommended for industrial production of CDs [The sources of starch could affect the CGTase activity, which is probably due to the differences in the starch granules structure and properties among various starch sources . Hence, n of CDs .w/v) of soluble starch as a substrate, at 50 °C and pH 8.0. A maximum yield of CDs with about 67.2% conversion of soluble starch into CDs could be obtained after 4 h and the products ratio at that point was 10.2% α-CD, 86.4% β-CD and 3.4% γ-CD. The production yield and ratio of the different CDs formed by CGTases are dependent not only on the microbial source producing the enzyme, but also on the nature of the substrate and reaction conditions, such as temperature, pH and reaction time [Amphibacillus sp. NPST-10 CGTase could be considered as β-CGTase. Of the three kinds of CDs, β-CD is of most practical use because its inclusion complexes are easily prepared and more stable. The size of the β-CD polar cavity is optimum for many molecules such as drugs and preservatives. Furthermore, β-CD is easily separated from the reaction mixtures due to its low solubility in water [The production of CDs was analyzed by HPLC using pure CGTase and 1%  phenolphthalein, as an indicator of cyclodextrin production [2CO3 (15 g/L), agar (15 g/L) and 300 μL trace elements solution. The trace elements solution contained: CaCl2·2H2O (1.7 g/L), FeSO4·7H2O (1.3 g/L), MnCl2·4H2O (15.4 g/L), ZnSO4·7H2O (0.25 g/L), H3BO3 (2.5 g/L), CuSO4·5H2O (0.125 g/L), Na2MoO4 (0.125 g/L), Co(NO3)2·6H2O (0.23 g/L) and 2.5 mL 95%–97% H2SO4. Na2CO3 and trace elements solutions were autoclaved separately before addition to the medium. Sediment and water samples were suspended and serially diluted in a 10% (w/v) NaCl solution prepared in 50 mM glycine-NaOH buffer, pH 10. Aliquots (200 μL) of different dilutions were spread on the alkaline agar medium and incubated at different temperatures, for several days. Formation of halo zone around the colonies, resulted from the production of phenolphthalein-cyclodextrin inclusion complexes, was considered as an initial indication of CGTase activity [Isolation of CGTase-producing alkaliphilic bacteria was carried out using rich alkaline agar medium containing 0.02% , , 1 μL forward primer (10 μM), 1 μL reverse primer (10 μM), 5 μL DNA template (200 ng) and 18 μL nuclease-free water. The PCR reaction was run for 35 cycles in a DNA thermal cycler under the following thermal profile: Initial denaturation at 95 °C for 5 min, denaturation at 95 °C for 1 min, primers annealing at 52 °C for 1 min and extension at 72 °C for 1.5 min. The final cycle included extension for 10 min at 72 °C to ensure full extension of the products. PCR products were run in agarose gel electrophoresis and purified using a QIAquick gel extraction kit , and sequenced using an automated sequencer . The obtained 16S-rDNA gene sequence of the isolate was aligned with reference 16S-rDNA sequences of the European Microbiological Laboratory (EMBL), GenBank  and the data base of Japan (dbj) using the BLAST algorithm available in NCBI homepage  [The selected strain was identified using 16S rRNA gene sequence analysis. The bacterial isolate was grown overnight in 5 mL alkaline broth medium. Total DNA was extracted using DNeasy Blood & Tissue Kits  according to the manufacturer’s instructions. Eubacterial-specific forward primer: 16F27 (5′-AGA GTT TGA TCC TGG CTC AG-3′), and reverse primer: 16R1525 (5′-AAG GAG GTG ATC CAG CCG CA-3′) were used to amplify 16S rDNA gene . PCR amprmation) .g for 15 min at 4 °C, and cell-free supernatant was filtered through a 0.45-μm pore-size membrane filter and used as source of crude CGTase.Colonies of the selected strain were transferred to 250 mL Erlenmeyer flasks containing 50 mL of alkaline liquid culture medium with the same composition as the solid medium, except for the presence of agar and dye, and incubated for overnight at 30 °C under orbital shaking (120 rpm). This culture was used to inoculate (2%) one liter Erlenmeyer shaking flask containing 250 mL of the same medium and cultivated under the same conditions for approximately 48 h. Cells and insoluble materials were removed by centrifugation at 6000w/v) and 1 M, respectively, and kept at 4 °C with continuous gentle agitation for 1 h to allow enzyme adsorption. The mixture was then centrifuged at 5000g for 10 min and the pellet was washed twice with cold ammonium sulphate solution (1 M) to remove any unbound proteins. In order to eluate the adsorbed CGTase from the corn starch, the residue was incubated with 200 mL of 50 mM Tris-HCl buffer, pH 8.0, containing 1 mM β-CD, for 30 min at 37 °C with shaking followed by centrifugation to give eluate 1. The elution was repeated with 80 mL of the same β-CD solution to give eluate 2. The eluates (1 and 2) were pooled (280 mL), dialysed against 50 mM Tris-HCl buffer (pH 8.0) at 4 °C. This eluate was lyophilized using freeze dryer and the solid materials were resuspended in 5 mL of 50 mM Tris-HCl buffer (pH 8). The sample was centrifuged and filtered through a 0.45-μm pore-size membrane filter. The concentrated sample was applied to glass column (1.5 × 20 cm) containing DEAE-cellulose, pre-equilibrated with 50 mM Tris-HCl buffer (pH 8). The column was washed with the same buffer and proteins were eluted using a linear 0–1 M NaCl gradient in the same buffer at flow rate of 1.5 mL/min. Fractions (2 mL) were collected and absorbance was monitored at 280 nm. Fractions containing CGTase activity were pooled and dialyzed against 50 mM Tris-HCl buffer overnight at 4 °C. All purification steps were performed in cold room.CGTase was purified using two steps including corn starch adsorption and ion exchange chromatography. Insoluble corn starch and ammonium sulfate were added to one liter of cell-free supernatant to concentrations of 5% (w/v) starch solution prepared in 50 mM Tris-HCl buffer, pH 8 was pre-incubated at 50 °C for 5 min. One hundred micro liter of enzyme sample was added to the reaction mixture and after incubating for 20 min at 50 °C, the reaction was quenched by adding 375 μL of 0.15 M NaOH. Subsequently, 100 μL of 0.02% (w/v) phenolphthalein prepared in 5 mM Na2CO3 was added, and after standing at room temperature for 15 min, the color intensity was measured at 550 nm. One unit of CGTase activity was defined as the amount of enzyme releasing 1 μmol of β-CD per min under the defined assay conditions. A calibration curve was made using 0.001–0.5 μmol of β-CD in 50 mM Tris-HCl (pH 8). Protein concentration was determined according to the method described by Bradford [Cyclization activity of CGTase was measured as β-CD forming activity according to a method previously described with some modification . Seven hBradford  with bovd-glucose.Hydrolytic activity of CGTase was performed by incubation of 750 μL 1% starch solution, prepared in 50 mM Tris-HCl buffer (pH 8), for 30 min at 50 °C. To terminate the enzymatic reactions, the mixtures were boiled for 5 min. The release of reducing sugars was followed by dinitrosalicylic acid method . 0.1 mL Coupling activity of the purified CGTase was measured according to the disappearance of β-CD in the presence of glucose using modification a protocol described previously . Enzyme The molecular weight of the purified enzyme was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), . SDS-PAGw/v) soluble starch, 0.02% (w/v) phenolphthalein and 1% (w/v) agar. After agar solidification, it was incubated for 1 h at 50 °C, and the CGTase activity was seen as a colorless band on a red background due to formation of stable colorless CD-phenolphthalein inclusion complex [w/v) soluble starch in 50 mM Tris-HCl buffer (pH 8), and incubated for 1 h at 50 °C. Then, the starch solution was decanted and iodine solution was poured on the gel. The starch-degrading activity was seen as a clear band on dark blue background.For CGTase activity staining, the enzyme samples were applied to 10% native PAGE. After gel electrophoresis, the gel was washed twice with distilled water, and twice with 50 mM glycine-NaOH buffer, pH 8. Then, the gel was immersed in 50 mM glycine-NaOH buffer (pH 10) containing 1% ( complex . For detw/v) of starch or maltodextrin, respectively, at varying temperatures from 50 °C to 70 °C for 1 h prior of determination of the residual enzyme activities under the standard assay conditions.The effect of temperature on the cyclization activity of CGTase was examined at various temperatures ranging from 35 °C to 70 °C under the standard assay conditions described above. The thermostability of the purified CGTse was determined by incubating the enzymes in 50 mM Trise-HCl (pH 8) at different temperatures (35 °C to 70 °C) and at various times interval (10–180 min), aliquots of enzyme were withdrawn and assayed for residual cyclization activity under the standard assay conditions. The thermal stability of the enzyme was further investigated in the presence of 1 mM calcium salt, 1% , 50 mM Trise-HCl (pH 7.0 and 8.0), 50 mM glycine-NaOH buffer (9.0 and 10.0) and 50 mM carbonate-bicarbonate buffer (11.0 and 12.0), respectively. In addition, the pH stability of purified enzyme was investigated by incubating the purified enzymes in buffers with different pH values for 1 h at room temperature, and the residual cyclization activities were assayed under the standard assay conditions. All experiments and enzyme assays were performed in triplicate and the mean values were reported.2+, Ca2+, Zn2+, Mn2+, Mg2+, Cd2+, Na+, Ba2+, Hg2+, Ni2+, Co2+, EDTA, 2-mercaptoethanol, α-, β-, and γ-CD.The effects of different metal ions and some inhibitors on the CGTase activity were investigated by addition of the test ions and reagents to reaction mixtures at final concentration of 1 mM or 10 mM. The test ions and reagents included CuKm and Vmax, were estimated using Michaelis-Menten equation and double reciprocal plot known as Lineweaver-Burk plot [Kinetic studies were performed by measuring the cyclization activity of CGTase at various concentrations of soluble starch ranging from 0% to 1.5 mg/mL. The kinetic constants, urk plot .w/v) corn starch, and at defined time intervals the reaction was stopped by placing the samples in a boiling water bath for 5 min. The reducing sugars, in the reaction mixture, were measured as previously reported [The enzyme reaction was carried out at 50 °C, pH 8.0 using 1%  with predominant formation of β-CD (86.4%), making this enzyme favorable for industrial application. Further work on cloning of cgtase gene from Amphibacillus sp. NPST-10 and analysis of CDs production by immobilized enzyme are in progress.In this study, we report purification and characterization of a novel CGTase from


The rats were euthanized 7 days after IOP elevation for histologic evaluation and at 1, 3, and 7 days after IOP elevation to immunostain for the P2X7 receptor and neuron-specific class III β-tubulin in the retina. Changes in P2X7 receptor expression were measured in total retina extracts using western blot analysis. Quantitative real-time PCR was also performed using the entire retina to determine whether the P2X7 receptor is involved in upregulating tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 at 1, 2, and 3 days after the IOP was elevated.After the IOP was elevated to 90 mmHg for 1 h, the rats were subsequently administered oxidized adenosine triphosphate (OxATP) and brilliant blue G (BBG) as P2X7 immunoreactivity in the RGCs increased after IOP elevation, with the peak occurring from day 1 through day 3. Protein levels of P2X7 receptor were significantly increased 1, 2, and 3 days after IOP elevation. The messenger ribonucleic acid expression of the P2X7 receptor, TNF-α, IL-1β, and IL-6 was significantly upregulated in the retina after IOP elevation, and was suppressed by treatment with OxATP.RGC density and the inner plexiform layer thickness significantly decreased 7 days after IOP elevation, but were dose-dependently preserved when treated with OxATP or BBG. P2X7 receptor is upregulated in the retina after IOP elevation, leading to RGC death. Upregulation of TNF-α, IL-1β, and IL-6 might be involved in this mechanism of RGC death. Furthermore, P2X7 antagonists may prevent RGC death after IOP elevation.These results suggest the expression of the P2X P2X7 receptors may affect neuronal cell death through their ability to regulate the processing and release of interleukin (IL)-1β, a key mediator in neurodegeneration and chronic inﬂammation [7 receptors may be involved in the release of tumor necrosis factor (TNF)-α, IL-1β, and IL-6 from microglia and mast cells during mitosis, inflammation, and proliferation [P2Xammation . Other sferation -7.7 receptors in retinal ganglion cells (RGCs) [7 receptors might be involved in RGC death in vitro and in vivo through intracellular calcium increase [7 receptors is related to RGC death remains unknown. Further, regarding cells other than RGCs, several studies have found an association between P2X7 receptors and TNF-α and several interleukins in apoptosis [Several studies have demonstrated the expression of P2Xs (RGCs) -10. Otheincrease -13. Howepoptosis ,15.The focus of neuroprotective therapy in glaucoma has been preventing progressive RGC damage by intervening in neuronal death pathways. Several animal models, including those for acute and chronic intraocular pressure (IOP) elevation, optic nerve axotomy, and optic nerve crush, have been used for studies of neuroprotection in glaucoma .7 receptor is involved in retinal neuronal loss, especially in the ganglion cell layer (GCL), after acute IOP elevation. First, we examined the effects of P2X7 antagonists—oxidized adenosine triphosphate (OxATP) [In the present study, we aimed to determine whether the P2X (OxATP)  and bril (OxATP) —on IOP eFor this study, we used 10- to 12-week-old adult male Wistar rats . The care of the animals and the experimental procedures conformed to the guidelines for the Care and Use of Laboratory Animals by the Institute for Laboratory Animal Research and the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. Unless otherwise noted, the chemicals used in this study were purchased from Sigma-Aldrich .A 30 G infusion cannula was inserted into the anterior chamber of the left eye under systemic anesthesia with intraperitoneal pentobarbital (35 mg/kg bodyweight). This infusion cannula was connected to a bottle of phosphate-buffered saline  through a pressure transducer  for continuous monitoring of actual IOPs. The IOP was artificially elevated to 90 mmHg for 60 min by increasing the height of the bottle. Red reflux from the fundus confirmed that complete retinal ischemia had not occurred at that IOP level. In a sham control eye, the IOP was maintained at 15 mmHg for 60 min. Immediately after the IOP elevation was completed, 5 µl of PBS or 5 µl of OxATP (1–100 µM) or BBG (0.3–300 nM) dissolved in PBS was injected intravitreally from the pars plana through a 30-gauge needle on a Hamilton syringe .A total of 60 eyes from 60 rats were used for histologic analysis in this study. Eight eyes were used for the sham control, i.e., eight eyes were treated with PBS alone. Four, six, four, and six eyes were treated with OxATP at 3, 10, 30, and 100 µM, respectively; and four, seven, seven, and six eyes were treated with BBG at 0.3, 3, 30, and 300 nM, respectively. Seven days after IOP elevation, the rats were euthanized with inhalation of 100% carbon dioxide. The eyes were enucleated, fixed in Davidson’s solution overnight, rinsed with PBS, and embedded in paraffin. Transverse sections of the retina (3 µm thick) were cut with the optic nerve head (ONH) in the center, and stained with hematoxylin and eosin. For the analysis, four light photomicrographs (200X magnification) representing a 600 µm field were obtained approximately 1 mm from the center of the ONH in each retinal slice using a light microscope . A masked examiner (SL) then counted all GCL cells in the entire field, without excluding displaced amacrine cells, because it has been reported that the density of these cells does not affect the evaluation of RGC loss in experimental glaucoma . The mas7 receptor in the retina, immunohistochemistry was performed using a rabbit primary antibody for the intracellular domain of the P2X7 receptor . For identifying the expression of the P2X7 receptor in neurons or microglia/macrophages, double immunostaining was performed for the P2X7 receptor and neuron-specific class III β-tubulin (TUJ1) or CD68. For this, mouse Alexa Fluor 488–labeled anti-TUJ1 monoclonal antibody  and mouse anti-CD68  were used. The sections were blocked in 5% normal goat serum and 2% bovine serum albumin (BSA) for 1 h, and then incubated with a rabbit anti-P2X7 receptor antibody (1:200) and mouse anti-TUJ1 (1:100) or anti-CD68 (1:50) antibody at 4 °C overnight. Then, the samples were incubated for 2 h at room temperature with fluorescently labeled secondary antibodies . In addition, for visualizing the expression of TNF-α or IL-1β in microglia/macrophages in the retina, double immunostaining of CD68 with TNF-α or IL-1β was performed. After rats were perfused through the heart with saline under deep anesthesia followed by 4% paraformaldehyde in PBS (pH 7.4), the eyes were enucleated, fixed, and embedded, followed by cutting into frozen sections as described above. Primary antibodies for TNF-α  and IL-1β  were used. After blocking with 2% BSA for 1 h, the sections were incubated overnight with a mouse anti-CD68 antibody (1:100) as well as a goat anti-TNF-α (1:50) or rabbit anti-IL-1β (1:500) antibody at 4 °C. Next, the samples were incubated for 2 h at room temperature in secondary antibodies . As a negative control, the sections were incubated only with solvent instead of diluted solution of primary antibodies. The nuclei were stained with 4’,6-diamino-2-phenylindole dihydrochloride (1:500), and the images were acquired using a fluorescence microscope .For the immunohistochemical analyses in this study, three eyes from three rats were used for each condition: normal eyes and the eyes on days 1, 2, 3, and 7 after IOP elevation, as well as the eyes treated with OxATP at 30 µM just after IOP elevation. Rats were euthanized at 1, 2, 3, and 7 days after IOP elevation, as described. The eyes were enucleated, fixed in 4% paraformaldehyde in PBS overnight, rinsed with 30% sucrose, embedded in Tissue-Tek ornithine carbamoyltransferase , and snap-frozen in liquid nitrogen. Then, 8-µm-thick frozen sections were cut with a cryostat . For visualizing the expression of the P2X7 receptor antibody  or anti-α-tubulin antibody , and washed with TBS-T. Then blocking with 5% skim milk in TBS-T was done for 1 h, followed by incubation with the peroxidase-conjugated secondary antibodies  for 2 h at room temperature and washing with TBS-T. Immunoblots were developed with an enhanced chemiluminescence plus western blotting detection system . The densities of the bands of proteins were quantified with a luminescent image analyzer . The amount of protein expression was quantified using the equipped software (Multi Gauge version 3.0), and the blots were then reprobed with α-tubulin to confirm equal loading of protein into each well.Three eyes of three rats were used for each condition: normal eyes, the eyes on days 1, 2, and 3 after IOP elevation as well as the sham control eyes on day 1. The entire retina was isolated from each eye after the rat was euthanized, and homogenized with a mechanical homogenizer in five pellet volumes of lysis buffer containing 50 mM Tris-hydrochloride (pH 7.6), 150 mM NaCl, 20 mM EDTA, 0.1% sodium dodecyl sulfate, 1.0% Nonidet P40, 1.0% sodium deoxycholate, 1.0 mM phensxylmethanesulfonulfluoride, 10 μM aprotinin, 10 μM leupeptin, and 10 μM pepstatin A. The suspension was centrifuged, and the supernatant was used to determine the protein concentration with a DC protein assay reagent . Samples containing 5 μg of protein were run on a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel and electroblotted onto a polyvinyldene difluoride membrane (Bio-Rad). Blots were blocked for 1 h with 5% skim milk in Tris buffered saline containing 0.1% Tween-20 , incubated for 2 days at 4 °C with a rabbit anti-P2XFor RNA extraction in this study, 28 eyes of 28 rats were used. Four eyes were used for each condition , with the exception of five eyes used only for day 2 after IOP elevation. At 1, 2, and 3 days after IOP elevation, the rats were euthanized as described above. The entire retina was removed from each eye, and total RNA was isolated from each retina using the PureLink RNA Mini kit (Life Technologies), according to the manufacturer’s protocol. Zirconia balls  and Tissue Lyser  were used to homogenize the retina, and the total RNA quality and quantity were assessed using a BioSpectrometer . For critical RNA purification, DNase I  was used. RNA was reverse-transcribed into cDNA using Thermal Cycler PCR System 9700  and SuperScript VILO Master Mix (Life Technologies), according to the manufacturer’s protocol, except that 16 μl of the RNA sample was used for 4 μl of the Master Mix.7 receptor Rn00570451_m1, TNF-α Rn01525859_g1, IL-1β Rn00580432_m1, and IL-6 Rn01410330_m1. For determining the cycle threshold (Ct) values, the threshold level of the fluorescence was set manually during the early phase of PCR amplification. The relative quantities of mRNAs were calculated using the 2−ΔΔCt analysis method [Quantitative real-time PCR (qPCR) analysis was performed using StepOne Plus System, TaqMan Fast Advanced Master Mix, and Assay-by-Design primers and probes (Applied Biosystems), according to the manufacturer’s instructions. The primer/probes used were as follows: P2Xs method , with β-t test or Mann–Whitney U-test.Data are represented as the means±standard deviation (SD) or standard error of the mean (SEM). Statistical analysis was performed with an unpaired Student 7 antagonists—OxATP and BBG (7 antagonists significantly increased GCL cell survival and preserved the IPL thickness (p<0.05, Cell loss in the GCL and thinning of the IPL were observed in the eye treated with IOP elevation and PBS, compared with the sham control eye. However, these effects seemed to be ameliorated by intravitreal injections of the P2X and BBG . The GCL p<0.05, . No sign7 receptor was upregulated in cells of the GCL, IPL, and INL, compared with the normal retina. Double staining of TUJ1 and the P2X7 receptor showed that the expression of P2X7 was also upregulated in the TUJ1-positive cells in the GCL on days 1, 2, and 3 after IOP elevation (7 receptor was suppressed by treatment with OxATP (7 receptor was observed in CD68-positive cells, especially in the GCL (On days 1, 2, 3, and 7, the expression of the P2Xlevation . Upregulth OxATP . On days the GCL . The imm the GCL .7 receptor were significantly increased on days 1, 2, and 3 after IOP elevation compared to the sham control (p<0.01, p<0.05, p<0.01 and p<0.01, p<0.01, p<0.05, respectively; The protein levels of the intracellular and extracellular P2X7 receptor mRNA was significantly increased on days 1 and 2 after IOP elevation—treatment with a P2X7 antagonist decreased its expression in a dose-dependent manner (p<0.05, 7 antagonist (p<0.05, 7 antagonist (p<0.05, The expression of P2X p<0.05, . The exp p<0.05, . The exp p<0.05, .7 receptor is upregulated in the retina after IOP elevation and that P2X7 antagonists ameliorated the IOP elevation–induced neuronal loss in the GCL. Our results also suggest that upregulation of TNF-α, IL-1β, and IL-6 might be involved in the mechanism underlying P2X7 receptor-mediated damage.The results of the current study revealed that the expression of the P2XIn the present study, we used an artificial IOP elevation rat model, since the damage is primarily induced in the inner retina in rats, similar to the scenario observed in human glaucoma . In a pr7 receptor in neurons as well as microglia/macrophages in the GCL of a rat model of acute IOP elevation. To the best of our knowledge, this has not been previously reported. Several studies have reported the involvement of this receptor in regulating aqueous humor outflow [7 receptor after acute IOP elevation.In this study, immunohistochemical and quantitative real-time PCR analyses revealed the upregulation of the P2X outflow  and the  outflow -30. More outflow . In addi outflow . Taken tOur results also indicate that IOP elevation resulted in increased levels of retinal TNF-α, IL-1β, and IL-6, especially in microglia/macrophages in the GCL, which peaked 2 days after IOP elevation. Ischemia has been reported to result in increased levels of retinal TNF-α, which may play a deleterious role in retinal injury ,34. Stud7 antagonists—OxATP and BBG—may play an important role in preventing cell death induced by IOP elevation in various ocular diseases, including glaucoma. Based on the present study, it is not clear whether P2X7 antagonists exert neuroprotective effects directly on RGC, indirectly through inhibition of microglia/macrophage’s release of inflammatory mediators, or both. Inflammatory cytokines, including TNF-α and IL-1β, have been shown to contribute directly to the retinal injury [7 antagonists in optic nerve crush injury [7 antagonists rescued RGCs in culture. Recently, P2X7 receptor activation was shown to mediate RGC death in a human retina model of ischemic neurodegeneration [7 antagonists might be direct and indirect.Our current results suggest that the P2Xl injury ,38. We ph injury  althoughneration . Anotherneration . In addineration . Taken t7 receptor, although we showed the involvement of inflammatory responses by TNFα, IL-1β, and IL-6, which were probably induced by upregulation of this receptor [7 receptor-activated NACHT, LLR, and PYD domains-containing protein 3 inflammasome formation and the consequent IL-1β release from glia preserve neuronal viability [Many mechanisms might be responsible for RGC injury induced by elevated IOP. The excessive pressure can damage the RGC soma directly, but it can also initiate damage by compressing RGC axons, which may interfere with intra-axonal transport of prosurvival molecules, such as trophic factors. Alternatively, pressure-induced compression of the retinal blood vessels can cause mild ischemia in retinal tissues. One limitation of our study is that we did not clarify the exact mechanisms underlying IOP elevation–induced damage involving the P2Xreceptor ,46. It wiability . Anotheriability . Further7 antagonist application have not yet been tested. For clinical applications, the effects of other methods, including instillation, should be investigated in the future. In addition, the safety of the P2X7 antagonist OxATP for human eyes should be evaluated. BBG is a dye used clinically to stain the internal limiting membrane in vitreoretinal surgeries, and its toxicity to a human Müller cell line has already been examined. Kawahara et al. [Another limitation is that other methods of P2Xa et al.  observed7 receptor in the retina, especially in neurons and microglia/macrophages in the GCL, is upregulated after IOP elevation and that P2X7 antagonists may prevent neuronal loss in the GCL. In addition, TNF-α, IL-1β, and IL-6 might be involved in this mechanism.In conclusion, the current study suggests that the expression of the P2X


Arachis hypogaea type 2 DGAT (AhDGAT2) genes were cloned from the peanut cultivar ‘Luhua 14’ using a homologous gene sequence method and rapid amplification of cDNA ends. To understand the role of AhDGAT2 in triacylglycerol biosynthesis, two AhDGAT2 nucleotide sequences that differed by three amino acids were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli Rosetta (DE3). Following IPTG induction, the isozymes (AhDGAT2a and AhDGAT2b) were expressed as 64.5 kDa GST fusion proteins. Both AhDGAT2a and AhDGAT2b occurred in the host cell cytoplasm and inclusion bodies, with larger amounts in the inclusion bodies. Overexpression of AhDGATs depressed the host cell growth rates relative to non-transformed cells, but cells harboring empty-vector, AhDGAT2a–GST, or AhDGAT2b–GST exhibited no obvious growth rate differences. Interestingly, induction of AhDGAT2a–GST and AhDGAT2b–GST proteins increased the sizes of the host cells by 2.4–2.5 times that of the controls (post-IPTG induction). The total fatty acid (FA) levels of the AhDGAT2a–GST and AhDGAT2a–GST transformants, as well as levels of C12:0, C14:0, C16:0, C16:1, C18:1n9c and C18:3n3 FAs, increased markedly, whereas C15:0 and C21:0 levels were lower than in non-transformed cells or those containing empty-vectors. In addition, the levels of some FAs differed between the two transformant strains, indicating that the two isozymes might have different functions in peanuts. This is the first time that a full-length recombinant peanut DGAT2 has been produced in a bacterial expression system and the first analysis of its effects on the content and composition of fatty acids in E. coli. Our results indicate that AhDGAT2 is a strong candidate gene for efficient FA production in E. coli.Diacylglycerol acyltransferase (DGAT) is the rate-limiting enzyme in triacylglycerol biosynthesis in eukaryotic organisms. Triacylglycerols are important energy-storage oils in plants such as peanuts, soybeans and rape. In this study,  Arabidopsis, rice, and other plant species Diacylglycerol acyltransferase (DGAT) is the rate-limiting enzyme of the Kennedy pathway for synthesizing triacylglycerols (TAGs) in eukaryotes. DGAT genes have been identified in a wide range of organisms Arabidopsis DGAT1 gene in tobacco and yeast greatly enhanced the TAG content of the transformed lines Ricinus communis DGAT2 (RcDGAT2) has a strong preference for hydroxyl FAs containing diacylglycerol (DAG) substrates, the levels of which increased from 17% to nearly 30% when RcDGAT2 was expressed in ArabidopsisRicinus seeds, RcDGAT2 expression was 18-fold higher than in leaves, whereas RcDGAT1 expression differed little between seeds and leaves. Hence, RcDGAT2 probably plays a more important role in castor bean seed TAG biosynthesis than RcDGAT1 Olea europaea is responsible for most TAG deposition in seeds, while OeDGAT2 may be a key mediator of higher oil yields in ripening mesocarps Much research has focused on DGATs because of their important roles in TAG synthesis. Overexpression of such genes can greatly increase the oil content of transgenic organisms. For example, overexpression of the et al. expressed the N-terminal 116 amino acid residues of Brassica napus (oilseed rape) DGAT1 as a His-tagged protein in Escherichia coli(1–116)His6 interacted with long chain acyl-CoA and displayed enhanced affinity for erucoyl (22:1cisΔ13)-CoA over oleoyl (18:1cisΔ9)-CoA E. coli with similar results Vernicia fordii) in E. coli has been achieved Recombinant proteins can be used as alternatives to endogenous ones to study their structures and functions or to make high-titer antibodies that recognize them. Because most DGATs are integral membrane proteins, they are difficult to express and purify in heterologous expression systems Arachis hypogaea  is one of the most economically-important oil-producing crops, so the fact that peanut DGATs have not been extensively studied is surprising. Saha et al. identified a soluble DGAT3 from immature peanut cotyledons and expressed its full length in E. coli, where the recombinant protein had high levels of DGAT activity but no wax ester synthase activity E. coli. This is the first time that a full-length recombinant DGAT2 protein from peanut has been successfully expressed in E. coli, and the first evaluation of its effects on the growth and FA content of the transformed E. coli strains studied.TCTTACACCAGCAACAAGGAAA 3′ and AhD2-A: 5′ GACCAAAGCAGAAAACAGGAAC 3′)  that successfully amplified a 197-bp fragment of the gene. The 20 µL PCR mixture contained 1 µL cDNA, 1 µL of each primer (10 µM), 2 µL PCR buffer (10×), 2 µL dNTPs (2.5 mM each), and 1 unit of Pyrococcus furiosus (Pfu) DNA polymerase (Invitrogen). The reaction was denatured at 94°C for 5 min; followed by 30 cycles of 30 s at 94°C, 30 s at 50°C, and 30 s at 72°C; then 10 min at 72°C. PCR was performed in a PCR Thermal Cycler Dice-TP600 . The AhDGAT2 fragment was purified using a MinElute™ Gel Extraction Kit , cloned into a pMD18-T vector (Takara), and sequenced.Total RNA (5 µg) from peanut cultivar ‘Luhua 14’ pods obtained 25 days after flowering (DAF) was reverse-transcribed into first-strand cDNAs using a cDNA synthesis kit  in a 20 µL reaction volume. Examination of the conserved domains of soybean GmDGAT2 and RcDGAT2 nucleotide sequences enabled us to design a pair of primers (AhD2-S: 5′ TCTTACACCAGCAACAAGGAAA 3′) and AhD2-3I (5′ CCCTCTTGGATAATGGCTACAGTTG 3′), and AhD2-5O (5′ ACTGTAGCCATTATCCAAGAGGG 3′) and AhD2-5I (5′ TTTCTTTGTTGCTGGTGTAA 3′). PCRs were performed according to the manufacturer's protocol. The fragments were sequenced and assembled into a full-length sequence.The full-length AhDGAT2 from ‘Luhua 14’ was cloned using a SMART™ RACE cDNA Amplification Kit . Total RNA (1 µg) from the 25-DAF peanut pods was used for cDNA synthesis following the manufacturer's protocol. Rapid amplification of cDNA ends (RACE) primers were based on the sequence of the AhDGAT2 fragment described above as follows: AhD2-3O (5′ TCAACAGCCACCGAATCCA 3′ and AhD2-FA: 5′ TAAAACAAGGAAGGGTGCCA 3′). The 20 µL PCR volume comprised 1 µL cDNA, 1 µL of each primer (10 µM), 2 µL PCR buffer (10×), 4 µL dNTPs (2.5 mM each), and 1 unit of Pfu DNA polymerase. The reaction was denatured at 94°C for 5 min; followed by 30 cycles of 30 s at 94°C, 30 s at 60°C, and 1 min 20 s at 72°C; then 10 min at 72°C. The full length fragment (AhDGAT2 ORF) was purified from an agarose gel and cloned into a pMD18-T vector for sequencing.Based on the full-length sequence of the AhDGAT2 gene, its full-length open reading frame (ORF) was amplified with gene-specific primers (AhD2-FS: 5′ http://www.cbs.dtu.dk/services/TMHMM/), conserved domains were found using the Conserved Domain Database (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) at the National Center for Biotechnology Information (NCBI), and putative functional motifs were identified using PROSCAN (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_proscan.html). We also predicted the two- and three-dimensional structures of the genes using phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index).Translations of the full-length ORF sequences were analyzed for structural motifs. Transmembrane helices were predicted using TMHMM , which was also used to reconstruct a phylogenetic tree using the CGCGGATCCATGGAAGATCGAGGGAACGT 3′), DGAT2b-S2 (5′ CGCGGATCCATGAAGTCCGAGGGAACGT 3′) and DGAT2-A2 (5′ CCGCTCGAGTCAGACAATTCTCAACTCAAGG 3′) primers, after which the PCR products were restriction digested with BamHI and XhoI then ligated into the similarly-digested expression vector pGEX-4T-1  for expression as GST-tagged fusion proteins. The constructs were transformed into the E. coli Rosetta (DE3) strain  following the manufacturer's directions.AhDGAT2a and AhDGAT2b ORFs were amplified using DGAT2a-S2 , and the pellets were sonicated for 10 min in homogenization buffer (3–4 mL/g wet cells) containing 20 mM Tris-HCl, pH 7.4, 200 mM NaCl, 10 mM β-mercaptoethanol, 1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, and 1∶100–1∶500 dilution of protease inhibitor cocktail . Cell debris, inclusion bodies, and protein aggregates in the homogenate were removed by two consecutive centrifugations . Samples were treated with 0.5 M NaOH and protein concentrations were determined with the Bradford method . The supernatant and the pellet were evaluated by SDS-PAGE (10%) and visualized by staining with Coomassie brilliant blue. Furthermore, the expression locations of the recombinant proteins were determined by purifying cytoplasmic and inclusion body fractions using a B-PER GST Fusion Protein Purification Kit . Protein expression was verified by a western blot analysis using an anti-GST tag monoclonal antibody .E. coli strains and strains transformed with either empty vector, AhDGAT2a–GST, or AhDGAT2b–GST were cultured in Luria broth (LB) medium at 37°C at an initial density of OD600 = 0.05. IPTG  was added after 2.0 h for protein induction. OD600 measurements were conducted every hour for 8 h using an Evolution 300 spectrophotometer . Each treatment was examined in triplicate.To examine how transformation with DGAT2 affected bacterial growth rate, wild-type (WT) Rosetta (DE3) E. coli cells were smeared onto glass slides and heat fixed, after which a drop of crystal-violet Gram staining solution was added. Cells were stained for ∼1 min, washed three times in distilled water, air-dried, then visualized under a light microscope  at 1000× magnification under an oil immersion objective. Over 100 cells were observed at each time point in each of three replicates.We also evaluated the morphological effects of transformation on these four cell types. At 0, 2, 4, and 6 h after IPTG induction, WT and transformed strains were cultured in liquid LB for 2 h, after which the fusion proteins were induced with 1.0 mM IPTG for 6 h. Isochronous WT cultures and un-induced transformed strains were used as controls. After induction, thalli were collected and freeze-dried for FA analysis. The whole procedure was repeated for a total of two replicates.Thalli were accurately quantified and soaked in 2.0 mL 2% sulfuric acid in dry methanol for 16 h at room temperature followed by 80 min at 90°C to convert the FAs into FA methyl esters (FAMEs). Supelco™ 37 Component FAME Mix  was added as an internal standard. After addition of 2.0 mL of distilled water and 3.0 mL of hexane, the FAMEs were extracted for analysis with a 6890N gas chromatograph . Lipids were measured with a programmed temperature method. An initial column temperature of 140°C was maintained for 5 min, then increased to 240°C at a rate of 4°C/min and held for 10 min. Injection and detector temperatures were 240°C and 260°C, respectively. Two microliters of the sample were injected into the column. FAMEs were identified by comparison of their retention times with those of known standards. The results were analyzed using Chrom Perfect® LSi system software  with the FAME Mix peak area used as the reference.Fatty acid content was computed as the absolute content (mg/g) using the gas chromatograph area counts for the different FAMEs. The FAME quantity in a sample was used to calculate the oil content using the following equation:s is the weight of the internal standard added to a sample, Ai is the area count of an individual FAME, As is the area count of the corresponding FAME in the internal standard, and M is the weight of the E. coli extract used.Where MDifferences in cell volumes, growth rates, and FA content among the four strains when induced and uninduced were statistically analyzed by one-way analysis of variance (ANOVA) using SPSS 16.0 .Medicago truncatula), to which they exhibited high similarity (91.02%). They were also quite similar to EaDGAT2 from Euonymus alatus .By employing a homologous gene sequence method with RACE, we identified two isozymes of AhDGAT2 from the peanut cultivar ‘Luhua 14’ and designated them AhDGAT2a and AhDGAT2b . We identified 14 nucleotide differences between them , but theE. coli.The two AhDGAT2s had the same predicted internal structures. Using TMHMM, we identified two potential transmembrane helices, at amino acid positions 40–62 and 67–82 , suggestE. coli, and the recombinant AhDGAT2–GST proteins, containing a GST-tag at the N-terminus, were induced with IPTG at 25°C or 37°C. Recombinant AhDGAT2 expression at 25°C was somewhat lower than that at 37°C (data not shown), so we selected 37°C as the optimal induction temperature. Cells induced at 37°C were collected after 0, 2, 4, and 6 h and the recombinant proteins extracted for SDS–PAGE analysis  strain relative to the WT strain . The same phenomenon was observed in E. coli cells harboring AhDGAT2a–GST or AhDGAT2b–GST. The AhDGAT2a–GST and AhDGAT2b–GST vector-transformed cells increased markedly in size after IPTG addition, by ∼2.4–2.5 times (P<0.01)  . DiffereE. coli strains . The quantities of C12:0, and C18:3n3 showed significant increases only under IPTG induction compared with either the WT or empty-vector transformed strains (P<0.01 or 0.05). The levels of C15:0 decreased significantly relative to the WT in both uninduced (P<0.01) and induced (P<0.05) AhDGAT2a–GST and AhDAGT2b–GST strains (P<0.01) and increased significantly in the induced AhDGAT2a strain , C18:2n6t (P<0.01), CLA-c9t11 (P<0.01) (P<0.05) (P<0.01) (P<0.05)  , and C22(P<0.05)  were sig(P<0.01)  and sign(P<0.05) .E. coli systems, remain an effective way of producing large quantities of a variety of fusion proteins. For example, Lu et al. introduced four distinct genetic changes into the E. coli genome to achieve a high level of FA production (2.5 g/L) E. coli that lacked FA production, and found that the transformed E. coli strains synthesized approximately 0.2 g/L of free FAs E. coli MG1655 and found that the FA (C16) levels from the recombinant strains were 1.23–2.41 times higher than those from the wild type E. coli cell line that produced 4.5 g/L/day FAs E. coli, the transformants showed high levels of DGAT activity and formation of labeled TAG Increasing energy costs and environmental concerns have compelled the production of sustainable renewable fuels and chemicals. In recent years, biofuels have received significant attention and investment E. coli Rosetta (DE3) and showed that the FA content of the transformants was significantly higher than in the WT or empty-vector transformed strains ) changes over time E. coli strains would increase with longer induction times, but our study clearly demonstrated the potential of AhDGAT2 for efficient FA production in E. coli.In this study, we overexpressed two types of AhDGAT2 in E. coli DH5α strain forms long filamentous cells upon caffeine exposure E. coliBacteria have evolved sophisticated systems to maintain their morphologies. However, in certain environments, rod-shaped bacteria can become more filamentous E. coli strains changed their general morphology from short rods to filamentous structures Click here for additional data file.Table S1Original data and data analysis.(XLS)Click here for additional data file.


Deciphering these complex responses remains difficult because relationships between AR subtypes and various end-effectors  remain to be identified. A1AR stimulation is associated with the production of 20–hydroxyeicosatetraenoic acid (20–HETE) and activation of protein kinase C (PKC). 20–HETE and PKC can inhibit large conductance Ca2+/voltage-sensitive K+ (BK) channels that regulate smooth muscle contraction. We tested the hypothesis that activation of A1AR inhibits BK channels via a PKC-dependent mechanism. Patch clamp recordings and Western blots were performed using aortae of wild type (WT) and A1AR knockout (A1KO) mice. There were no differences in whole-cell K+ current or α and β1 subunits expression between WT and A1KO. 20–HETE (100 nmol/L) inhibited BK current similarly in WT and A1KO mice. NECA , a nonselective AR agonist, increased BK current in myocytes from both WT and A1KO mice, but the increase was greater in A1KO . This suggests that A1AR signaling negatively regulates BK channel activity. Accordingly, CCPA (2–chloro–N(6)-cyclopentyladenosine; 100 nmol/L), an A1AR-selective agonist, inhibited BK current in myocytes from WT but not A1KO mice . Gö6976 (100 nmol/L), a PKCα inhibitor, abolished the effect of CCPA to inhibit BK current (99 ± 3% of control). These data lead us to conclude that, in aortic smooth muscle, A1AR inhibits BK channel activity and that this occurs via a mechanism involving PKCα.Adenosine receptors (AR; A These AR subtypes play important roles in vascular reactivity, as A1AR and A3AR contract smooth muscle, whereas A2AAR and A2BAR relax smooth muscle  (Miyata and Roman Adenosine exerts its effects through four G-protein coupled receptors: the known adenosine receptor (AR) subtypes are AFredholm . It is w2+ concentration. Large conductance, Ca2+/voltage-sensitive K+ (BK) channels participate in this electromechanical coupling   mice  are on C57BL/6 background. A1KO mice were backcrossed four generations with C57BL/6 ; genotypes were confirmed by polymerase chain reaction. C57BL/6 (WT) mice  were bred in-house. Equal number of males and females of 14–18 weeks of age were used in our studies, as no gender differences were observed. The Institutional Animal Care and Use Committee of West Virginia University provided regulatory oversight and protocols followed guidelines set forth in The Guide for the Care and Use of Laboratory Animals  20 Tris-HCl, 150 NaCl, 1 Na2EDTA, 1 EGTA, 2.5 sodium pyrophosphate,1 beta-glycerophosphate, and 1 Na3VO4; plus 1% NP-40, 1% sodium deoxycholate, and 1 μg/mL leupeptin. Samples were vortexed and then centrifuged for 10 min at 13,800 g at 4°C. Protein was measured using the Bradford dye procedure with bovine serum albumin as a standard . The protein extract was divided into aliquots and stored at −80°C. Samples  were loaded on slab gels , separated by Sodium dodecyl sulfate polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes (Hybond-ECL). Protein transfer was confirmed by visualization of prestained molecular weight markers (Bio-Rad). Membranes were blocked with 5% nonfat dry milk and incubated with primary antibody. A 1:5000 primary antibody dilution used for BK α and β1 subunits , while 1:10,000 dilutions were used for secondary antibody and β-actin.Aortae from WT and A1KO mice aortae were digested in a physiological saline solution containing (mg/mL) 2 collagenase type-II, 1 soybean trypsin inhibitor, 1 bovine serum albumin, and 1 elastase for 30 min at 37°C. Single cells were liberated by passing the tissue through the tip of a fire-polished Pasteur pipette. The suspension was passed through a 100 μm nylon mesh and spun for 10 min at 10,000 g. The pellet was resuspended in low Ca2+ physiological saline solution and cells were stored on ice for use within 8 h. Cells were allowed to attach to glass coverslip, which was then transferred to the recording chamber. Solutions flowed into the recording chamber by gravity at a rate of 2-3 mL/min and the chamber had a volume of 0.2–0.3 mL. BK channel currents were recorded at room temperature from whole-cell patches as described previously  140 KCl, 1 MgCl2, 1 EGTA and 0.281 CaCl2 (pCa 7), 10 HEPES, 1 Mg-ATP, 0.1 Na-GTP, and 5 Tris; pH 7.1. pClamp software and an Axopatch 200B amplifier were used . Currents were low pass filtered at 1 kHz and digitized at 5 kHz.Wild type and An = 1. Current–voltage relationships were analyzed by two-way repeated measures analysis of variance (ANOVA). This was followed with Bonferroni post hoc test to determine where differences existed. When only two values were compared  an unpaired t-test was used. P < 0.05 was considered significant in all tests.Data are expressed as mean ± SEM from n number of mice, because the treatment level  is on a per mouse basis. For patch clamp experiments, that means results from all cells (≥3) from a single mouse aorta were averaged to represent 1KO . The group data are shown in 1KO mice. BK α and β1 subunit proteins migrated at 100 and 25 kDa, respectively. There were no differences observed in the two protein levels between genotypes ; this was decreased to 51.6 ± 10.3 pA/pF by 20-HETE  and this was decreased to 41.4 ± 7 pA/pF by 20-HETE  is a nonselective adenosine receptor agonist and can activate multiple AR subtypes simultaneously. Whole-cell recordings showed prominent BK current in smooth muscle cells from WT and A1KO mice. Caffeine (5 mmol/L) was used as a positive control to release Ca2+ and increase BK current in both WT and A1KO aortic smooth muscle cells (1KO aortic myocytes was significantly increased by 10 μmol/L NECA (1KO smooth muscle cells was 52 ± 15% (n = 7); this was significantly higher than the response to NECA in smooth muscle cells from WT mice  and decreased with the application of CCPA to 42.8 ± 6.2 pA/pF (1KO mice (1KO mice was 55.2 ± 7.7 and 54.1 ± 3.9 pA/pF (n = 4) in the absence or presence of 100 nmol/L CCPA, respectively (1KO mice.2–chloro–N(6)-cyclopentyladenosine , an A WT mice A and B. .2 pA/pF C. That i1KO mice D and E. ectively F. That i1AR activation, 20-HETE production, and mediates contraction of smooth muscle. We determined if inhibition of PKCα affected regulation of BK current by A1AR activation in smooth muscle cells from WT and A1KO mice. When PKCα was inhibited with Gö6976 (100 nmol/L) in WT smooth muscle cells, subsequent addition of CCPA (100 nmol/L) was no longer able to inhibit current  that A1AR stimulation is associated with 20-HETE production and activation of PKC  There were no differences in whole-cell K+ current in aortic smooth muscle cells from WT and A1KO mice, nor were there any differences in the expression of pore-forming α or regulatory β1 subunit proteins. (2) Inhibition of BK current by 20-HETE was similar in aortic smooth muscle cells from WT and A1KO mice. (3) NECA, a nonselective AR agonist increased BK current in aortic smooth muscle cells from both WT and A1KO mice, but the increase was greater in smooth muscle cells from mice lacking the A1AR. (4) CCPA, an A1AR-selective agonist, inhibited BK current in smooth muscle cells from WT, but not A1KO, mice. (5) Inhibition of PKCα with Gö6976 abolished the effect of CCPA to inhibit BK current in smooth muscle cells from WT mice. Together, these data lead us to conclude that, in aortic smooth muscle, A1AR stimulation inhibits BK channel activity and that this occurs via a mechanism involving PKCα.We tested the hypothesis that activation of A2+-sensitive pore, while β subunits can modify many characteristics including pharmacology and Ca2+-sensitivity. We observed no difference in the expression of α or β 1 BK subunits  at a single concentration (100 nmol/L). However, our previously published data indicate that PKC α  is the isoform most abundant in mouse smooth muscle  in the A1KO. Unpublished results from our lab suggest that A2A receptor expression is upregulated in A1KO mice and this might also be a factor in the larger responses to NECA. However, studies investigating the effect of A2A receptor signaling on BK current have not been performed and represent a limitation of this study. There is evidence showing that in the rat preglomerular vessels A2AAR, through EETs, activate BK channels mediate vasodilation  has negative effects  are known to play a role in modulating BK channel activity. Whether ROS activates or inhibits BK channel is debatable. For instance, studies have shown that hydrogen peroxide . Specifically, MAP in conscious mice was 85 ± 1 and 97 ± 2 mmHg for WT and A1KO mice, respectively (P < 0.05). In addition to changes in salt and water balance and renal hemodynamics, these data support the idea that A1 receptors couple to smooth muscle contraction and increases in total peripheral resistance (Brown et al. 1KO mice was indistinguishable from WT. Specifically, Lee et al. (1KO mice, respectively. When mice are made hypertensive with Ang II infusions, the blood pressure is less in A1KO mice, reinforcing the idea that A1 receptors couple to smooth muscle contraction and increased total peripheral resistance (Lee et al. There is no clear consensus on mean arterial pressure in WT and An et al.  generatee et al.  report t+ channels (Dart and Standen ATP channel activity are generally well accepted (Sharifi Sanjani et al. 2+-dependent K+ current in smooth muscle cells from the rat mesenteric artery that may be mediated by BK channels (Li and Cheung Adenosine, via multiple receptor subtypes, contracts and relaxes vascular smooth muscle through several mechanisms, including the regulation of K1AR leads to signaling via PLC-βIII, PKC-α leading to ERK1/2 phosphorylation. However, this study demonstrates that adenosine receptor signaling converges on BK channels in vascular smooth muscle. The study is novel in showing a relationship between A1AR; Cyp4a product, 20-HETE; PKC-α and BK channel in WT and A1KO aortic myocytes. However, this can be extrapolated to understand vasomotor responses in resistance vessels, but this will require direct investigation, outside the scope of this study. This conduit artery does not contribute to resistance that regulates blood flow, but results from this cell type provide insight into signaling mechanisms that may also be present in smaller arteries and arterioles. From our data, we conclude that A1AR signaling inhibits BK channels via 20-HETE and PKCα.Our lab has previously shown in the coronary artery smooth muscle cells that activation of A


Little is known about how the hemispheres interact in processing of stimuli presented at vertical midline. Processing might be mutually independent or cooperative. Here we measured target identification and visually evoked EEG potentials while stimulus streams containing two targets, T1 and T2, were either presented at vertical midline above and below fixation, or laterally, left and right. With left and right streams, potentials evoked by filler stimuli and by T2 were earlier at the right than the left visual cortex, and T2 was better identified left than right, confirming earlier results and suggesting better capabilities of the right hemisphere in this task. With streams above and below fixation, EEG potentials evoked by filler stimuli and by T2 were likewise earlier at the right than the left hemisphere, and T2 was generally identified as well as, but not better than left T2, in one target constellation even worse (T2 in lower stream preceded by T1 in upper stream). These results suggest right-hemisphere preference for this task even with stimuli at vertical midline, and no added value through hemispheric cooperation. Lacking asymmetry for T1 amidst asymmetries for filler stimuli and for T2 might indicate alternating access of the hemispheres to midline stimuli as one means of hemispheric division of labor. Visual stimuli presented in the left or right visual field activate the contralateral primary visual cortex The two visual cortices differ in their preferences of processing, i.e., either hemisphere processes certain features of stimuli better than the other cooperation: By exchanging and adding to each other's information, the two hemispheres will process midline stimuli better than either one could do alone. Another possibility is racing: Each hemisphere will process the information it has acquired (directly or via interhemispheric transmission) and the faster result will determine the output of the entire visual system. In this case, midline stimuli will be processed as well as the better hemisphere would do alone, but not better  left hemisphere may nevertheless get faster access to T2 and determine the output, and the latter alternative would speak for hemispheric cooperation.VEPs : VEPs evoked by the constant bilateral stream of filler stimuli in this task were leading by a few milliseconds in right- compared to left-hemisphere recordings T1-evoked PCN : Being a relevant event, laterally presented T1 had evoked PCN in previous studies T2-evoked PCN: Likewise being a relevant event, laterally presented T2 also evokes its PCN To summarize, the listed ERP indicators are expected to provide evidence for right-hemispheric preference in processing of filler stimuli and of T2, and possibly for left-hemispheric preference in processing of T1. Clues about cooperation vs. race among the hemispheres will be obtained by comparing the identification rates of vertical-midline to lateral stimuli.The project was approved by the ethical committee of the University of Lübeck (ref. no. 11–198). Twenty-three students  participated, aged 20 to 27 years . Informed written consent was obtained and 7 € per hour were paid. All participants reported normal or corrected-to-normal vision, normal color vision, and no history of neurological disorders. Most of them were right-handed and two were ambidextrous, with scores of 83 ±22 (range 25–100) in the Edinburgh Handedness Inventory. Five participants were rejected from analysis due to systematic eye movements toward the target , one because of high error rates in identifying T1  and another one because of high error rates in identifying T2 . Thus, 16 participants remained for analysis .2) capital letters of the Latin alphabet were rapidly (7.7/s) presented on the white background (120 cd/m2) of a 17’’ screen driven with 85 Hz at about 1.1 m from participants' face. . Letter font was Helvetica 35, thus letters were 11 mm high . Their midpoints were 16 mm off screen center (0.8°), implying distances of their inner edges from screen center of 10 mm (0.5°) for stimuli above and below center and of around 11 mm (varying between letters) for left and right stimuli. Fixation was marked by a small red cross (0.1°×0.1°) at screen center. In each trial, two targets had to be identified. The first target (T1) was a blue letter . The second target (T2) was a black digit . The filler set consisted of all letters in black. Presentation® software, version 14.5, was used for experimental control . Fixation was controlled by means of an infra-red remote eye tracker  which communicated on-line with the Presentation program.The task is illustrated in In one block of trials, the two stimulus streams were left and right from fixation ("x-axis" block), like in previous studies. In the other, "y-axis", block, the two stimulus streams were above and below fixation.th position and T1–T2 lag of 1) and 19 frames (T1 at 10th position and T1–T2 lag of 4). Then the fixation cross went off and prompts appeared on the screen to enter identities of T1 and T2, first the T1 letter on the middle row of a standard keyboard and then the T2 digit on its number pad. Some response had to be given even if the answer was not known. Then the next trial started. In each trial, filler stimuli were randomly selected with replacement from the letter set , and T1 and T2 were randomly selected from the target sets. If the eye-tracker registered a deviation of more than 0.4° from fixation at trial onset, a big red exclamation mark (font 50) was presented at center for 2 s, leading participant's gaze back to fixation and restarting the trial after another 0.5 s.Participants were seated in a comfortable armchair in a dimly lit laboratory room in front of the computer screen. The EEG recording was prepared, as detailed below. Then gaze directions were mapped to screen coordinates for use of the eye-tracker, using the calibration routine provided by the eye-tracker manufacturer where participants had to fixate nine positions on the screen in fixed sequence. Afterwards, the experimental task was started. Each trial began with onset of the fixation cross, followed after 1.8 s by the series of letters. Each pair of letters was presented for 130 ms (precisely 129.4 ms), immediately followed by the next frame. Five, seven, or nine letter pairs preceded T1 , lag between T1 and T2 (1 or 4), and T2 location (same as T1 or opposite to T1), resulting in eight equiprobable combinations that were replicated 45 times in random sequence, making up a block of 360 trials. The two stimulus streams were horizontally or vertically aligned to screen center, in other words they flanked fixation either on the x-axis or on the y-axis. The session consisted of one x-axis and one y-axis block, with their order balanced between the 16 participants of the final sample. Before the task proper, some trials were presented in slow motion for practice, with 500 ms presentation time per frame.www.easycap.de) from 60 scalp sites, including 8 midline positions from AFz to Oz and 26 pairs of symmetric left and right sites. On-line reference was Fz, data were off-line re-referenced to the nose-tip. The ground electrode was placed at Fpz. For artifact control, vertical EOG was recorded from above vs. below the right eye, horizontal EOG (hEOG) from positions next to the outer rims of the eyes. Data were amplified from DC to 250 Hz by a BrainAmp MR plus and stored at 500 Hz per channel.EEG was recorded with Ag/AgCl electrodes . After re-referencing and low-pass filtering at 20 Hz, data from each trial were split into appropriate segments for analysis of filler-evoked VEPs and of target-evoked potentials. Data were referred to the mean amplitude of the first 100 ms as baseline and were edited for artifacts, by rejecting trials with voltage differences≥150 µV, voltage steps≥30 µV, and voltages exceeding ±100 µV. By applying these criteria also to the EOG channels, segments with eye-blinks were rejected. Actually, eye-blinks rarely occur during the rapidly presented series.As a further means of editing the data for artifacts, the averaged EOG waveforms contralateral minus ipsilateral to T1 were checked, from left and right hEOG in the x-axis block and from upper and lower vEOG in the y-axis block. Any participant's data were rejected if these averaged EOG difference waveforms deviated by 8 µV from baseline within 700 ms after T1 onset, indicating eye movements≥0.6° towards T1. As mentioned above in the “Participants” section, this criterion led to excluding 5 of the originally 23 participants.Artifact-free segments were averaged over trials separately for each participant, and grand means over participants were formed for illustrating the results.For analysis of overt behavior, T1 identification rate was assessed as number of trials with correct T1 responses relative to all trials, and T2 identification rate was assessed as number of trials with correct T2 and T1 responses relative to all trials with correct T1 responses. These percentages of trials with correct responses were computed in each combination of T1 location, T1-T2 lag, and T2 location, separately for the x-axis and the y-axis block.Segments for filler-VEP analysis spanned 1000 ms, from 100 ms before onset of the first stimulus pair to 900 ms afterwards. These segments were averaged over trials separately for x- and y-axis blocks, irrespective of success in target identification and of locations and time-points of the following T1 and T2. The mean number of accepted trials was 320 (226–349). The average waveforms consisted of a series of VEPs, evoked in intervals of 130 ms by the series of stimuli. The parameter of most interest was the lag between right and left visual cortex. Conventional measurement consisted in determining the P1 and N1 peaks of the first VEP (which were large and unambiguously identifiable) as most positive peak 70–170 ms and most negative peak 110–250 ms after stimulation onset, at the twelve posterior sites PO7 & PO8, PO9 & PO10, P7 & P8, P9 & P10, PO3 & PO4, O1 & O2, and entering their latencies and amplitudes to ANOVAs. A second method took the entire length of the waveform into account. The lag was determined by shifting each participant's waveforms of 800 ms duration from left sites against their symmetric right sites in 2 ms steps within ±50 ms, and selecting the shift that rendered the largest cross-correlation Segments for T1 analysis ranged from 100 ms before to 700 ms after T1 onset and were averaged for trials with correct responses to both T1 and T2, separately for the four T1 locations . The mean number of accepted trials was 110 (56–167). To assess PCN in x-axis blocks and its possible correlate in y-axis blocks, mean amplitudes 200–220 ms measured at PO7 and PO8 were entered to ANOVA.Segments for T2 analysis likewise started 100 ms before T1 and lasted until 500 ms after T2, i.e., for 730 ms with lag 1 and for 1120 ms with lag 4. Segments were averaged for trials with correct responses to both T1 and T2, separately for the four T2 locations , the two T1-T2 relations , and the two T1–T2 lags . The mean number of accepted trials was 35 (23–45) for T2 after same-stream T1 at lag 1 and 26 (8–44) both after same- and different-stream T1 at lag 4. Numbers were lowest for T2 after other-stream T1 at lag 1, 22 (2–42). Thus, these data were affected by noise. Nevertheless, it was decided to use the full design for analysis. . To distill the T2-evoked waveform from the lag 1 averages, independent of overlapping T1-evoked potentials, the first 730 ms of the lag 4 waveforms (averaged over same-stream and stream-change T2) including T1-evoked and background-evoked activity only, were subtracted from the lag 1 averages, and the mean of the 100 ms preceding T2 onset was subtracted as baseline from the data. With lag 4 waveforms, where T1 had preceded by 520 ms, the overlap issue reduced to slow waves evoked by T1. To cope with this, the mean of 200 ms preceding T2 onset was subtracted as baseline from the data. To assess T2-evoked PCN in x-axis blocks and its possible correlate in y-axis blocks, mean amplitudes were measured between 180 ms and 280 ms after T2 onset at PO7 and PO8 and entered to analysis.p2 = (F/15)/(1+F/15). IBM SPSS statistics 20 was used. When interactions were significant, their sources were clarified by ANOVAs or t-tests on subsets of the data.ANOVA designs will be detailed in the Results section. Degrees of freedom were corrected by Greenhouse-Geisser's ε when repeated-measurement factors had three or more levels. Corrected p-values will be reported, but ε values will not be indicated, for brevity. Likewise, partial eta-squared will not be explicitly indicated, being easily derived from the reported F-values by the formula ηTarget-identification rates are compiled in F1,15 = 10.5, p = 0.006. Other effects were not significant, in particular there was no effect of T1 Location .Responses were correct in 82% of trials. T1 was better identified when T2 followed at lag 1 than at lag 4, F1,15 = 139.1, p<0.001,. Interacting with T1-T2 lag, F1,15 = 105.3, p<0.001, this effect was very large with lag 1, such that T2 was identified nearly perfectly when following T1 in the same stream, and was much smaller with lag 4, though still significant .T2 was better identified in the same stream as T1 than in the other, F3,45 = 12.8, p<0.001, interacting with Stream Change, F3,45 = 14.8, p<0.001, with Lag, F3,45 = 3.6, p = 0.03, and with Stream Change×Lag, F3,45 = 5.0, p = 0.005. To resolve these effects, the four T2 locations were compared to each other by means of t-tests for each of the four combinations of Stream Change×Lag. Of most interest, T2 Location had its effect, 15<0.6, n.s.T2 in same stream as T1, lag 1: T2 was identified equally well at each position, t15 = 6.0, p<0.001, and better above than below, t15 = 4.4, p<0.001. T2 identification did not differ between left and above, t15 = 0.5, n.s., nor between right and below, t15 = −1.0, n.s. In consequence, T2 was worse identified below than left, t15 = −4.1, p = 0.001, and better above than right, t15 = 4.5, p<0.001.T2 in opposite stream from T1, lag 1: T2 was better identified left than right, t15≤0.7, n.s., but right T2 was worse identified than at the other locations: left, t15 = −4.0, p = 0.001; above, t15 = −2.6, p = 0.02; below, t15 = −2.2, p = 0.04.T2 in same stream as T1, lag 4: Identification rates did not differ among left, above, and below, t15 = 6.6, p<0.001. Above and below did not differ, t15 = 0.9, n.s. Bottom T2 was worse identified than left T2, t15 = −2.3, p = 0.04, and better than right T2, t15 = 3.5, p = 0.003. Top T2 did not differ from left T2, t15 = −1.1, n.s., and was better identified than right T2, t15 = 4.2, p = 0.001.T2 in opposite stream from T1, lag 4: T2 was better identified left than right, tTo summarize:T2 was better identified left than right .Midline T2 was never better identified than left T2. When T1 had been in the other stream, midline-bottom T2 was even worse identified than left T2.Midline T2 was generally better identified than right T2 .Top and bottom T2 were identified equally well, except with different-stream lag 1 where T2 was much worse identified at bottom.Grand-average waveforms of the first 850 ms of each trial are displayed in F5,75 = 3.2, p = 0.04).There were no effects on P1 amplitude (except for a main effect of Electrode Pair). P1 latencies were earliest at P9&10 and latest at PO3&4 with x-axis stimuli, and were the same at all electrode pairs with y-axis stimuli  in the PO7&8, P7&8, and P9&10 pairs . Additionally, N1 was larger for stimuli on the y-axis than on the x-axis, i.e., for midline than for lateral stimuli . This effect did not interact with Hemisphere and Electrode Pair, F<0.9, n.s.N1 amplitudes were larger at right than left sites . There were no effects of Axis and no interactions with Axis or Electrode Site.N1 latencies were earlier at right than left sites  and did not differ from each other .To assess the latency difference between hemispheres by integrating more data than just the first N1 peak, the maximum cross-correlation was determined for different lags between waveforms from left and right sites. The P7&P8 pair was selected because N1 amplitudes were numerically largest  while left, right, and above did not differ from each other . The main effect of Hemisphere was not significant, F3,45 = 0.0, but its interaction with T1 Location was, F3,45 = 50.5, p<0.001, prompting separate ANOVAs for x- and y-axis, with the factors T1 Location  and Hemisphere.Grand-average waveforms of the T1-evoked potentials are displayed in F1,15 = 66.3, p<0.001, because, as could be expected, N1 was larger contralaterally than ipsilaterally to T1, reflecting the PCN effect. In detail, N1 was larger at PO8 than PO7 with left T1, t15 = 5.8, p<.001, and larger at PO7 than PO8 with right T1, t15 = 5.0, p<.001.For stimuli in the x-axis, the only effect was a strong interaction of T1 Location×Hemisphere, F1,15 = 14.0, p = 0.002). Of interest, there was a difference between hemispheres in this effect, T1 Location×Hemisphere F1,15 = 4.9, p = 0.04, because the increase from top to bottom T1 was larger at PO7  than at PO8 . This interaction could not be resolved to differences between PO7 and PO8 with either top T1, t15 = 0.8, n.s., or bottom T1, t15 =  −0.5, n.s., though.For stimuli in the y-axis, larger N1 amplitudes were evoked by bottom than top T1  and did not differ in the 232–280 ms epoch . Of importance, this effect occurred equally for both axes: When separate ANOVAs were computed for x- and y-axis, amplitudes were larger at PO8 than PO7 in the 182–230 ms epoch both with x-axis stimuli  and with y-axis stimuli . This Epoch×Hemisphere interaction was modified by Lag , by T1–T2 Stream Change , and by Lag×Stream Change . Therefore, Hemisphere effects were computed separately for either epoch in each of the four combinations of Lag×Stream Change displayed in F1,15≥5.1, p≤0.04) and did not differ in the second epoch. For Lag 1 with side change from T1, amplitudes were larger at PO8 than at PO7 in the second epoch only , which reflected the delay of waveforms in this condition.Of most interest were effects of Hemisphere. As indicated by the Epoch × Hemisphere interaction, F3,45 = 22.3, p<0.001, reflected the expected PCN with x-axis stimuli, i.e., enhanced negativity contralateral to T2: Hemisphere×T2 Location with x-axis F1,15 = 27.8, p<0.001; with y-axis F1,15 = 0.8, n.s. This PCN differed between lags, T2 Location×Hemisphere×Lag, F3,45 = 5.7, p = 0.006 , by being more distinct with lag 4  than with lag 1 .The interaction of Hemisphere with T2 Location, F3,45 = 6.4, p = 0.004, reflected larger negative amplitudes of T2 below than left, right, and above  which did not differ from each other . Further, amplitudes were larger with lag 4 than with lag 1, F1,15 = 12.1, p = 0.003, particularly but not only in the earlier epoch  and when T1 had been on the same side, F1,15 = 4.6, p = 0.049.Independently of Hemisphere, the main effect of T2 Location, In order to find clues about which hemisphere preferentially processes filler stimuli and targets presented at vertical midline in rapid serial visual presentation and about whether such stimuli are processed in a cooperative manner, better than either hemisphere could do alone, EEG potentials and target identification were compared between streams presented above and below fixation to streams presented left and right from fixation.From the N1 peak evoked by the first filler-letter frame onwards, there was a small reliable latency advantage for right over left-hemisphere waveforms. Likewise, N1 amplitudes  were considerably larger at right than left recording sites. These results suggest that the presented symmetrical frames were processed earlier and induced more activation in the right than in the left hemisphere, which seems to be specific to the present task cf. . These lN1 was generally larger for vertical than for lateral pairs. We suspect that this difference was driven by the lower stimulus of the vertical pair, because also lower targets (T1 and T2) evoked larger negative amplitudes than targets above, left, or right, whereas targets above did not differ from left or right. This effect was probably simply due to the projection of the lower visual field to visual areas nearer to the upper cortical surface, thus nearer to the recording electrodes Relevant effects on rates of T1 identification were absent, T1 being equally well identified left, right, above and below fixation. This might be due to a ceiling effect. But on the other hand, the level of 82% identification rate is still well below 100%, and T1 identification did improve beyond such levels in previous studies, when stimuli acted as temporal cues in the stream before T1 As could be expected, contralateral negativity was evoked by laterally presented T1 cf.  to the sTargets presented at midline might be processed by the left or the right hemisphere or by both. As argued in the Introduction, if both hemispheres cooperate, the result might be better than the result achieved by either hemisphere alone. T2 was reliably better identified on the left than on the right, as in previous studies This lacking redundancy gain may appear in conflict with studies that did show gains of involving both hemispheres. Yet, many of those studies presented two distinct stimuli, one in either hemifield e.g.,  whereas Top and bottom T2 generally were equally well identified except with different-stream lag 1 where bottom T2 was much worse identified than top T2.It is not a matter of course that top and bottom T2 generally did not differ. Because the upper visual field may have more direct access to the ventral pathway In contrast to T1, T2-evoked negativity was asymmetric, both with lateral and midline streams. With lateral streams, contralateral right-hemisphere negativity evoked by left T2 occurred earlier than contralateral left-hemisphere negativity evoked by right T2. This result replicated earlier findings One interpretation proposed above for the poor identification of bottom T2 after top T1 preceding at lag 1 referred to a putative closer link of the lower visual field to the left hemisphere. This interpretation implies that a left-hemisphere negative focus should be obtained in this particular condition. This was not the case, though  be transmitted to the right hemisphere, and the right part (the "_") to the left? If so, where will these two elements be integrated? The problem is not solved by assuming that the fovea projects to both hemispheres e.g.,  because This study used behavioral and neurophysiological markers of lateralized processing to investigate hemisphere-specific processing of stimuli presented at vertical midline. We arrived at the conclusion that the right-hemisphere dominance obtained in this task with left and right stimuli also applies to these stimuli when presented at midline. The asymmetries of VEPs evoked by the filler stimuli, of T1-evoked negativity, and of T2-evoked negativity, suggest that the default setting for perceiving the series of filler letters at vertical midline is by giving preference to the right hemisphere, that there is no such tendency for T1, and that then T2 is preferably processed in the right hemisphere again. Identification rates both of T1 and T2 provided no evidence for cooperation between hemispheres in identifying stimuli presented at vertical midline, rather supporting the assumption of competitive race.