It was previously designated as the putative HPV type FADI3 by sequencing of a subgenomic FAP amplicon. We obtained the complete genome by combined methods including rolling circle amplification (RCA), genome walking through an adapted method for detection of integrated papillomavirus sequences by ligation-mediated PCR (DIPS-PCR), long-range PCR, and finally by cloning of four overlapping amplicons. Phylogenetically, the HPV154 genome clustered together with members of the proposed species Gammapapillomavirus 11, and demonstrated the highest identity in L1 to HPV136 (68.6%). The HPV154 was detected in 3% (2/62) of forehead skin swabs from healthy children. In addition, the different detection sites of 62 gammapapillomaviruses were summarized in order to analyze their tissue tropism. Several of these HPV types have been detected from multiple sources such as skin, oral, nasal, and genital sites, suggesting that the gammapapillomaviruses are generalists with a broader tissue tropism than previously appreciated. The study expands current knowledge concerning genetic diversity and tropism among HPV types in the rapidly growing gammapapillomavirus genus.The novel human papillomavirus type 154 (HPV154) was characterized from a wart on the  Beta- and Gammapapillomavirus (β-PV and γ-PV). Their prevalence and natural history, for example acquisition soon after birth, reflects a commensalic interaction with the immunocompetent host Gammapapillomavirus.The papillomaviruses (PVs) are small viruses with icosahedral symmetry and a circular, double stranded genome crena ani of a three-year-old boy. The index sample was negative for HPV by PCR using MGP primers HPV154 was isolated from a wart on the The FAP amplicon represented a putative novel type, as the partial L1 ORF was below 90% of sequence identity to any other known PV type The four clones were submitted to the International Reference Center for Human Papillomaviruses at the German Cancer Research Center, Heidelberg, Germany, and the compiled sequence was verified and officially designated as HPV154 (GenBank JN211193).The genome of HPV154 comprised 7286 bp with a GC content of 37.9%. The most closely related type was HPV136, isolated from the oral cavity 2CX29/30CX2C) that were separated by 36 amino acids, which are conserved among PVs HPV154 demonstrated the classical genomic organization of PVs, with seven ORFs identified . The put6GGT). Near the E2BS proximal to E6, several E1 binding sites (AACAAT) or related AT-rich sequences were identified and probably represent the origin of replication The early polyadenylation site (AATAAA) for processing of early mRNAs, was located at the 5′ end of the L2 ORF, while the late polyadenylation site was downstream of L1 within the upstream regulatory region (URR). The URR was relatively short, being 517 bp, and contained six putative E2 binding sites  by real-time PCR. One of the samples showed a viral load of 0.093 genomes and the other 1.3 genomes per human cell .The phylogenetic relationships of HPV154 were inferred based on the complete genomes of 91 HPV-sequences . HPV154 In order to increase knowledge of the biological niches of gammapapillomaviruses, we enumerated the isolation sources (complete HPV-genomes) and detection sites of all HPV types from this genus . The γ-Pcrena ani of a three-year-old boy we characterized the novel HPV154, which was added to the rapidly growing genus Gammapapillomavirus. The genome of the HPV154 demonstrated the typical genomic organization of papillomaviruses and was lacking the E5 ORF, as with γ- β- and µ-HPVs From a wart on the HPV154 demonstrated the typical genomic organization of papillomaviruses, but lacked the E5 ORF as in all known members of the β- γ- and µ-PV genera Concerning the presented phylogenetic tree, HPV154 was placed with high support along with members of the currently proposed γ-PV11 species http://www.ictvonline.org/codeOfVirusClassification.asp), but among a majority of the γ-PV species it is difficult to find distinctive characteristics besides sequence identity. Although species definition for γ-PV is currently based mainly on sequence identity Accordingly, a more stringent/divergent limit of 68.5% identity allowed us to split the proposed γ-PV11 into three putative novel species. However, the proposed γ-PV11 With regard to the isolation sites enumerated in However, our approach to summarize isolation sites of gammapapillomaviruses described in reports provides only a suggestive mode to study the tropism of these viruses. In order to perform a rigorous evaluation of the tropism for each HPV type, a random population study with samples collected from several sites is needed.In this study we successfully characterized the novel HPV154, thereby expanding knowledge about the diversity and tropism of HPV types in the rapidly growing γ-PV genus. In addition, we suggest that gammapapillomaviruses are generalist with broad tissue tropism. We expect that improved amplification methods will promote the discovery of additional HPV types of the γ-PV genus, and will shed light onto its apparent wider tropism compared to other papillomavirus genera.Ethical approval for this study was granted by the Ethical Committee of Lund University (LU 106-01). The samples were obtained with written informed consent from the parents of the minors.A swab suspension of 200 µl was processed using the automated MagNA Pure LC with the Total Nucleic acid kit (Roche), and eluted in 100 µl.Sample adequacy was assessed by testing 5 µL of the sample for the human β-globin gene with a real-time PCR The eluted total DNA was subjected to rolling-circle amplification (RCA) using illustra TempliPhi 100 Amplification Kit , basically following the manufacturer’s instructions, but in a slightly modified form. As indicated elsewhere 2, 0.2 mM each dNTP, 1.25 U of Taq polymerase . The cycling conditions were 94°C 5 min; 45 cycles of 94°C 15 s, 50°C 20 s, 72° 3 min; 72° 5 min. A second PCR  using 2 µl of the linear PCR as a template was made using a nested viral-specific primer (primer sequence available upon request) combined with the adapter-specific “AP1” primer (ggccatcagtcagcagtcgtag) in the same conditions as the linear PCR but with 0.5 µM of both primers. The cycling conditions were 94°C 5 min; 35 cycles of 94°C 15 s, 55°C 20 s, 72°C 3 min; 72°C 5 min. The PCR products were evaluated by electrophoresis, and the longest product (from the different restriction enzymes) was selected, cloned using a TOPO-TA cloning kit (Invitrogen) according to the manufacturer’s instructions, and sequenced at MWG (Germany). The novel sequence was used to design primers with Oligo 7 software  in order to carry out successive steps of linear/exponential PCR.The RCA product (∼0.5 µg) was digested in order to be used as input for DIPS-PCR Primers were designed in the L2 ORF from a multiple alignment of HPV sequences  related to the known FAP region of HPV154. The degenerated primer “L2 gamma F” (tttgratwtgaaaatcccgccttt) was used in conjunction with the HPV154 specific primer “PV77 FADI3 R” (gacctgtgctaccgactccaag). The cycling conditions were 94°C for 5 min; 40 cycles of 94°C 15 s, 52°C 20 s, 72°C 1 min and a final extension of 72° 7 min. The PCR product was purified with an Illustra Microspin S-300 HR column  and sequenced with a nested specific primer.A second set of degenerated primers was designed for the E1 ORF. The primers ‘E1 gamma Fnew’ (gacagtggdatwkddgaagatgaa) and ‘E1 gamma Rnew’ (ttcatcttcddmwatdccactgtc), were combined with ‘FADI3 −2 nes R' and ‘FADI3 +2 nes F’, respectively . One microliter of RCA-pre-amplified template was subjected to Long Range PCR  amplification in 1x PCR buffer 2 (Roche), 0.5 mM each dNTP, 0.7 U of Enzyme mix . The cycling conditions were 94°C 2 min; 10 cycles of 94°C 15 s, 50°C 1 min, 68°C 4 min and 30 cycles of 94°C 15 s, 59°C 30 s, 68°C 4 min. The product was electrophoresed and the bands excised from the agarose gel, purified with a QIAquick Gel Extraction kit (Qiagen) and cloned using a TOPO-TA cloning kit (Invitrogen), according to the manufacturer’s instructions.2 and 1.24 U of enzyme in a final volume of 50 µl. The cycling was 94°C 2 min; 40 cycles 94°C 15 s, 55°C 20 s, 72°C 2 min increasing 5 s/cycle; 72°C 10 min. The product was purified and cloned as indicated before.The remainder of the genome was amplified using an Expand High Fidelity PCR system  with 2.5 µl of the RCA amplified DNA (diluted 1∶100) as a template; 0.3 µM of each primer, 3.5 mM MgClForehead samples from children from three age groups, one month (23), one year (19), and four years old (20), were obtained from a previous study 2 of 0.99. The PCR efficiency calculated from slope was 97.5%. Similarly, in order to calculate the number of cells analyzed per sample, the β-globin gene was amplified with PC03 and PC04 primers in a 25 µl PCR reaction containing 2.5 µL template 2 of 0.99. The PCR efficiency calculated from slope was 97.5%. For the calculations of number of human cell per sample, the copy number of β-globin was divided by two. We assumed that each human cell carries two β-globin gene copies and that the diploid genome equivalent contains ∼6.6 pg DNA. No-template controls of water samples were tested of both the HPV154 PCR and the human β-globin gene PCR. All samples were analyzed triplicate. Coefficient of variation was calculated for each triplicate measurement of viral copy number per human cell. In the quantitative PCR, negative controls showed no Ct values.The number of viral genomes per cell was quantified by carrying out two separate quantitative real-time PCR assays to amplify a part of the HPV154 L2 gene and the human β-globin gene. For the quantification of HPV154 the primers and PCR conditions were identical to that used for screening of samples from children as described above. Quantification was extrapolated from a linear regression standard curve obtained from serial dilutions of 100,000 to 100 copies per PCR of HPV154 plasmid DNA clone L,  in a bachttp://genome.unipro.ru). Putative proteins from ORFs were generated by UGENE and they were then searched for similarities with other proteins using BLASTp (http://blast.ncbi.nlm.nih.gov). Proteins were analyzed for unique domains with ScanProsite (http://www.expasy.ch/prosite) and SMART http://pfam.sanger.ac.se/search) General sequence handling and feature identification were carried out using the UGENE software v1.11.5 , from which 723 unique genomes remained after dereplication using Usearch v6.0.307


This favors the persistence of Thr286 autophosphorylated state of CaMKII in a CaMKII/phosphatase conjugate system in vitro. Overall our data indicate that the NR2B- bound state of CaMKII attains unique biochemical properties which could help in the efficient functioning of the proposed molecular switch supporting synaptic memory.Calcium/calmodulin dependent protein kinase II (CaMKII) is implicated to play a key role in learning and memory. NR2B subunit of N-methyl-D-aspartate receptor (NMDAR) is a high affinity binding partner of CaMKII at the postsynaptic membrane. NR2B binds to the T-site of CaMKII and modulates its catalysis. By direct measurement using isothermal titration calorimetry (ITC), we show that NR2B binding causes about 11 fold increase in the affinity of CaMKII for ATPγS, an analogue of ATP. ITC data is also consistent with an ordered binding mechanism for CaMKII with ATP binding the catalytic site first followed by peptide substrate. We also show that dephosphorylation of phospho-Thr Owing to its unique autoregulatory ability, CaMKII is implicated to play a major role in the molecular mechanisms underlying learning and memory. In the postsynaptic compartment, Caa. From a separate set of experiments, we also present data to reveal how NR2B favours the persistence of Thr286 autophosphorylated form of CaMKII. The implications of these findings for the efficient functioning of the CaMKII-PP1 switch are discussed.In the present study, by a direct measurement of binding affinity using isothermal titration calorimetry (ITC), we show that the affinity of the ATP analogue, ATPγS, for CaMKII increases significantly in the presence of NR2B as shown by the change in value of the association constant, KWe have previously reported that CaMKII shows enhanced activity at low [ATP] in the presence of saturating concentrations of non-phosphorylatable GST-NR2B (S1303A) a = 8.29×104±1.06×104 M−1, ΔH = −3428±379.6 cal/mol, ΔS = 10.8 cal/mol*K  or GST-NR2A (S1291A). It was very interesting to find that the presence of NR2B sequence caused an increase in the level of autophosphorylated CaMKII when compared to that in the presence of the homologous NR2A sequence  indicating that the presence of NR2B segment inhibited dephosphorylation of phospho-Thr286-CaMKII by PP1 m values a values obtained in the present study is however higher (∼11 fold) compared to the differences in Km values for ATP obtained by enzyme kinetics (∼6 fold) The Ka value obtained for titration by ATPγS in the absence of any peptide is smaller by more than an order of magnitude compared to that in the presence of peptide substrate , protease inhibitor cocktail, β-mercaptoethanol, calmodulin purified from bovine testes, etc. were from Sigma-Aldrich, USA. ATPγS was from Roche or Sigma. PD-10 desalting columns, calmodulin-Sepharose, glutathione-Sepharose, etc. were from Amersham/GE Healthcare, USA. Reduced glutathione was from Sisco Research Laboratories, India or Calbiochem, USA. Sf21 cells in 175 cm2 flasks were infected with the stock of recombinant baculovirus encoding WT-α-CaMKII. The infected cells were harvested 72 hours post infection. Purification of the expressed protein was carried out as explained earlier. Each batch of purification had insect cell pellets from 15 flasks. The cell pellets were resuspended in lysis buffer containing 50 mM PIPES, pH 7.0, 5% betaine, 1 mM EGTA, 1 mM EDTA and 1× complete protease inhibitor cocktail (Sigma).Expression of α-CaMKII in insect cells was carried out as described before In the first step of purification the lysate was loaded onto a 75 ml bed volume phosphocellulose cation exchanger column pre-equilibrated with equilibration buffer . The bound protein was eluted with elution buffer . The eluate having CaMKII activity was used for affinity purification on CaM-Sepharose column as described before 2. The flow-through was collected and was reloaded once. A high salt wash with equilibration buffer containing 1 M NaCl was given followed by wash with equilibration buffer before elution in the buffer having 40 mM Hepes pH 7.3, 0.5 M NaCl, 5% glycerol, and 3.5 mM EGTA.A 20 ml bed volume CaM-sepharose column was used for affinity purification. Equilibration buffer contained 40 mM Hepes, pH 7.3, 0.1 M NaCl, 10% glycerol and 2 mM CaClE.coli as described before The GST fusion proteins were expressed in BL21 DE3 strain of 2 and 15 mM MgCl2) and 0.5 mM β-mercaptoethanol required in the final titration experiments were added to the purified CaMKII before concentrating the protein using Amicon Ultra centrifugal devices with a molecular weight cut-off of 100 kDa. The dodecameric α-CaMKII with a molecular mass of approximately 600 kDa, will be retained by the filter during concentration. The filtrate obtained by this method will have all the constituents except the enzyme and can thereafter be used to reconstitute the ligand solution.We adopted a simplified procedure in which the ionic constituents  method.2+/CaM (2 mM/27 U per µl), and 3.2 µM of either GST-NR2B (S1303A) or GST-NR2A (S1291A). This was used as the enzyme source for the assay. Assay had a final concentration of 2 mM CaCl2 and 2.7 U/µl CaM. [γ-32P] ATP at concentrations ranging from 0.2 µM–100 µM was used to carry out the assay. The phosphorylated bands were detected from the autoradiogram and were quantified by densitometry using QuantityOne software.Kinetic analyses were carried out as described earlier 2, at least 4 U/µl calmodulin, 0.5 mM β-mercaptoethanol, 3.5 mM EGTA and 15 mM MgCl2 except for the blank titration  experiments were done using a VP-ITC system . All the experiments presented were conducted at 20°C while trials at 30°C were also done. The buffer used was 40 mM Hepes, pH 7.3, 0.5 M NaCl, 5% glycerol, 4 mM CaClitration  in whichTm 7.0 software. Before analysis of the data, the heat changes accompanying ATPγS binding to CaMKII alone were subtracted from the individual data of enthalpy change accompanying ATPγS binding to NR2B or NR2A saturated CaMKII. The data were fit to single binding site model. From the curve, values for stoichiometry of binding (N), association constant (Ka) and enthalpy of binding (ΔH) were obtained. Change in entropy (ΔS) was obtained using the equation :(ΔGb = ΔHb−TΔS), where ΔGb = −RTlnKa; R and T represent the gas constant and the absolute temperature (in Kelvin), respectively.Data obtained from the titrations was analyzed using Origin2 and 27 U/µl CaM was carried out before the assay. The reaction mix for assay was similar to that of the autophosphorylation reaction described earlier except for the addition of phosphatase, PP1 2, 0.4 mM EGTA, 1.3 mM CaCl2, 6.7 U/µl CaM, 0.2 mg/ml BSA, 1 mM MnCl2, 0.9 µM of ATP, 0.2 µM CaMKII and either 1.6 µM GST-(S1291A)-NR2A or 1.6 µM GST-(S1303A)-NR2B in the presence or absence of 3.75 units of PP1 in a total volume of 20 µl. The reaction duration was mostly 5 minutes, but 1 minute, and 15 minutes durations were also tried. The reactions were started by the addition of ATP and were stopped by the addition of 5× SDS sample buffer. The reaction samples were resolved in a 10% SDS-PAGE and western blotting was carried out to monitor Thr286 autophosphorylation. A mouse monoclonal anti-phospho-Thr286-α-CaMKII primary antibody was used in conjunction with alkaline phosphatase conjugated secondary antibody. Experiments using γ-32P-ATP were also performed. The reaction samples were resolved in a 10% SDS-PAGE gel which was later dried and exposed to phosphor screen and was subsequently scanned in a BioRad PhosphorImager.The reactions were carried out in the presence of non-phosphorylatable GST-NR2A or GST-NR2B. A preincubation step having 0.8 µM CaMKII, 6.4 µM of either GST-NR2B (S1303A) or GST-NR2A (S1291A), 2 mM CaClAutophosphorylation reaction was carried out as mentioned above for 30 seconds without including phosphatase or the GST fusion proteins and the reaction was terminated by adding 10 µM staurosporine, a kinase inhibitor. After stopping the autophosphorylation reaction, aliquots of the autophosphorylated CaMKII were incubated with GST-NR2B (S1303A) or GST-NR2A (S1291A) separately, in the same buffer. The dephosphorylation reaction in these samples was initiated by the addition of PP1 and the reaction was allowed for 30 minutes. The reactions were stopped by quick freezing by transferring to −80°C followed by addition of 5× SDS sample buffer.Figure S1A) Representative SDS-PAGE showing purified α-CaMKII (1 µg). B) SDS-PAGE of purified and concentrated GST-NR2A and GST-NR2B used for ITC experiments. Molecular sizes are indicated in kDa. 16 µg of purified GST-NR2A and 15 µg of purified GST-NR2B were loaded.(TIF)Click here for additional data file.Figure S2286 of α-CaMKII in the presence of GST-NR2B (S1303A) in the CaMKII/phosphatase coupled system. Autoradiogram of autophosphorylated CaMKII (32P-labeled) is shown. The duration of the reaction was 1 minute. Reactions were started by addition of 0.7 µM [γ-32P] ATP as described in methods. Data represents at least three similar experiments.Enhancement in the level of phospho-Thr(TIF)Click here for additional data file.Figure S32, 50 µM pNPP, 0.34 µM GST-(S1291A)-NR2A or 0.27 µM GST-(S1303A)-NR2B and 2.5 U of PP1. The experiment was carried out in a 96 well plate. The reaction mixture was incubated for 10 minutes at 30°C. The reaction was stopped by the addition 0.5 M EDTA and the absorbance was measured at 405 nm wavelength in an automated microplate reader. The activity was unaffected in the presence of GST fusion proteins but was significantly reduced by the phosphatase inhibitor, okadaic acid.PP1 activity assay using pNPP (para-Nitrophenyl Phosphate) hydrolysis to investigate the effect of GST fusion proteins on the activity of PP1. A 50 µl reaction was set up which had 1× PP1 buffer , 1 mM MnCl(TIF)Click here for additional data file.


Zinc concentrates at excitatory synapses, both at the postsynaptic density and in a subset of glutamatergic boutons. Zinc can modulate synaptic plasticity, memory formation and nociception by regulating transmitter receptors and signal transduction pathways. Also, intracellular zinc accumulation is a hallmark of degenerating neurons in several neurological disorders. To date, no single zinc extrusion mechanism has been directly localized to synapses. Based on the presence of a canonical PDZ I motif in the Zinc Transporter-1 protein (ZnT1), we hypothesized that ZnT1 may be targeted to synaptic compartments for local control of cytosolic zinc. Using our previously developed protocol for the co-localization of reactive zinc and synaptic proteins, we further asked if ZnT1 expression correlates with presynaptic zinc content in individual synapses.Here we demonstrate that ZnT1 is a plasma membrane protein that is enriched in dendritic spines and in biochemically isolated synaptic membranes. Hippocampal CA1 synapses labelled by postembedding immunogold showed over a 5-fold increase in ZnT1 concentration at synaptic junctions compared with extrasynaptic membranes. Subsynaptic analysis revealed a peak ZnT1 density on the postsynaptic side of the synapse, < 10 nm away from the postsynaptic membrane. ZnT1 was found in the vast majority of excitatory synapses regardless of the presence of vesicular zinc in presynaptic boutons.Our study has identified ZnT1 as a novel postsynaptic density protein, and it may help elucidate the role of zinc homeostasis in synaptic function and disease. Zn2+ may act as a co-transmitter at certain glutamatergic synapses, participate in neuronal signal transduction, modulate memory formation and nociception, or promote neurodegeneration upon brain insults [2+ are found among cellular compartments owing to the coordinated actions of two families of zinc transporter proteins, Slc30a (ZnT1-10) and Slc39 (ZIP1-14). Whereas ZnTs export Zn2+ away from the cytosol into organelles or the extracellular space, ZIPs shuttle Zn2+ in opposite direction [2+ is estimated to be in the subnanomolar range, but Zn2+ transients in neurons have been reported following strong depolarization or oxidation [2+ is common in degenerating neurons in models of epilepsy, ischemia or Parkinson’s disease [2+ into glutamatergic vesicles via ZnT3 [2+ homeostasis at synapses, but their identity remains elusive. One candidate protein is ZnT1 [2+, confers resistance against Zn2+ toxicity, and it is expressed in several brain regions [2+ by combining immunogold electron microscopy with zinc histochemistry [2+[2+ and ZnT1 expression.Homeostasis of ionic or labile zinc . Particle distribution along the axis perpendicular to the synaptic junction revealed a main peak on the cytoplasmic side of the postsynaptic membrane   or absence (ZN-) of vesicular Zns in CA1 . Indeed, heavily , we founThe present results provide compelling evidence that ZnT1 is preferentially expressed in synaptic membrane domains in rodent hippocampus (mouse and rat), and identify ZnT1 as a novel PSD protein. ZnT1 was found in the somatodendritic compartment in region CA1, it co-localized with PSD proteins GluR1 and SynGAP in dendritic spine-like structures, and it was enriched in synaptic membrane fractions compared with extrasynaptic membrane. High-resolution immunogold electron microscopy confirmed and extended these results, showing that ZnT1 densities were over 5-fold higher in postsynaptic junction membranes than in other membrane domains, including extrasynaptic spine membrane, shafts, boutons, somata or astrocytes. Furthermore, the ZnT1 particle distribution across the synapse, peaking within 10 nm inside the postsynaptic membrane, suggests that ZnT1 is an integral transmembrane protein at the PSD, which is consistent with the predicted 6 hydrophobic regions in its aminoacid sequence . The fac2+. This widespread distribution implies a general physiological role for ZnT1 at synapses independently of the actions of ZnT3 or vesicular Zn2+. Accordingly, organizing synaptic scaffolds appear particularly sensitive to intracellular Zn2+, leading to changes in PSD morphology upon zinc chelation [2+[2+ liberated in dendrites from redox-sensitive metalloproteins, such as metallothionein-3 or PKC [2+ following seizures [2+ transport, such as modulation of MAPK or Ca2+ signaling [Notably, ZnT1 was not restricted to the subset of synapses containing vesicular Znhelation . It is tation [2+ or for r3 or PKC ,19. In kseizures . Additioignaling ,22, or tignaling . Given tZnT1 is the first zinc transporter protein shown to concentrate at the PSD. The presence of ZnT1 in the vast majority of excitatory synapses in hippocampus suggests an unanticipated basic role for ZnT1 in synaptic function that warrants further investigation.Rabbit anti-ZnT1 antibodies were obtained from William F. Silverman (Ben-Gurion University). The specificity of the antibody, raised against the carboxyl-tail of mouse ZnT1 , was pre2, rinsed in PB, incubated in 0.5% NaBH4 in PB for 15 min, and blocked in 10% NGS, 1% BSA and 0.2 M glycine in PBS for 2 hr. Slices were incubated in rabbit anti-ZnT1 antibody (1:500) at 4°C for 2 d, then rinsed and incubated in biotinylated goat anti-rabbit IgG (1:200) at 4°C for 8 hr, rinsed again, and incubated in streptavidin-biotinylated HRP (1:300). The HRP was developed in 0.05% DAB and 0.001% H2O2 in Tris–HCl buffer.All procedures followed present regulations for animal care and handling and were approved by the University of Barcelona Ethical Committee. BALB/c mice (2 month-old) were anesthesized and perfused with heparinized saline, and 4% formaldehyde/0.1% glutaraldehyde in 0.1 M phosphate buffer . Brains were postfixed for 2 hr, cryoprotected in 30% sucrose in PB, frozen, and cut into 40 μm-thick sections. Brain slices were freeze-thawed twice with liquid NHippocampi were collected from postnatal day 1 pups, digested with 10 U/ml papain in Hank’s buffer, dissociated, and plated in Neurobasal A with supplements on poly-D-lysine-coated coverslips. Cultures were fixed with 4% formaldehyde, 4% sucrose in PB, and then 100% methanol for 10 min at 4°C. Tyramide-Cyanine 3 signal amplification was used according to manufacturer instructions (Perkin Elmer) to detect binding of anti-GluR1  or anti-SynGAP  antibodies. Immunolabeling with anti-ZnT1 antibodies (1:500) was detected with goat anti-rabbit Alexa488 IgG. Images were captured using an Olympus Fluoview FV600 confocal microscope and x63 objectives. Z-series of 4 consecutive optical sections (0.6 μm) were acquired using sequential scanning and Kalman filter.Hippocampi were homogenized in 10 volumes of buffer containing 10 mM HEPES, pH 7.5, 0.32 M sucrose, 2 mM EDTA, 2 mM EGTA, 5 mM DTT, and protease inhibitor cocktail tablet (Roche), followed by centrifugation at 800 g for 10 min. Supernatants were saved as the cytoplamic fraction. Pellets were resuspended in homogenization buffer plus 0.5% Nonidet P-40, and centrifuged again to obtain crude nuclear fractions. These nuclear fractions were resuspended in extraction buffer containing 10 mM HEPES, pH 7.5, 400 mM NaCl, 10% glycerol, 0.5% Nonidet P-40, 2 mM EDTA, 2 mM EGTA, 5 mM DTT, and protease inhibitor cocktail. Suspensions were rocked for 30 min on ice and centrifuged at 16,700 g for 10 min. Resulting supernatants were used as nuclear extracts. The cytoplasmic fraction was centrifuged  to obtain the crude synaptosomal pellet, resuspended with 0.5% Triton, rocked, and centrifuged at 21,000 g for 40 min. The PSD-enriched pellet was resuspended in LB. The supernatant containing non-PSD membranes was precipitated with -20°C acetone. Equal protein amounts were separated by SDS-PAGE in 8% gels. PVDF membranes were blocked with 5% BSA, 0.1% Tween-20 in Tris-buffered saline (TBS), and incubated overnight at 4°C with ZnT1 antibodies , or 1 h at RT with PSD-95  or actin  antibodies. Proteins were detected with HRP-conjugated secondary antibodies and visualized by chemiluminescence.2+ staining and epoxy embedding, we used our previous protocol (Sindreu’03). Briefly, two rats were perfused with 0.1% Na2S and 2.5% glutaraldehyde in PB, followed by 1% formaldehyde, 2.5% glutaraldehyde and 0.2% picric acid. Slices were stained with the Neo-Timm’s method , post-fixed using an osmium-free protocol  and flat-embedded in epoxy resin . Lowicryl or epoxy sections (60–70 nm) were collected on gold grids (200 mesh), incubated in 0.5% NaBH4 in 50 mM Tris-buffered saline  for 10 min, rinsed in 0.005% Tergitol NP-10 in TBS (TBST), blocked in 10% NGS, 2% BSA and 50 mM glycine in TBST, and reacted with anti-ZnT1 antibodies (1:200) overnight. Grids were rinsed in TBST and TBST/pH 8.2, and incubated in 10 nm gold-conjugated goat anti-rabbit F(ab)2 fragments  for 2 h. Grids were rinsed and counterstained.For acrylic embedding, brains from two Wistar rats (250–300 g) were perfused with 4% formaldehyde, 0.2% glutaraldehyde and 0.2% picric acid in PB, pH 7.4. Vibratome slices were cryoprotected in glycerol, frozen in liquid propane, freeze-substituted in 0.5% uranyl acetate in methanol at -90°C, infiltrated with Lowicryl HM20 at -45°C, and polymerized with UV light. For Zn2+ and ZnT1 was studied in synapses located within 4 μm from the net surface of the vibratome slice to avoid false negative counts [Electron micrographs were taken at × 30,000 magnification, recorded by a CCD camera (Gatan Inc.) and analysed with Canvas software . The CA1 stratum radiatum was examined by random systematic sampling of visual fields (1/3) at 10–200 μm from the pyramidal cell layer. Synapses were defined as those structures showing synaptic vesicles in the presynaptic element and a postsynaptic membrane specialization. Labeling at the synaptic junction was analysed in transversely cut synaptic profiles. To assess the transversal location of synaptic gold particles, we measured the distance from the outer margin of the postsynaptic junctional membrane to the center of each gold particle in a line perpendicular to the membrane. Particles were considered ‘synaptic’ when found within a radius of 30 nm from the postsynaptic junctional membrane, and ‘extrasynaptic’ when located further away. Similarly, a 30 nm limit was used to differentiate between surface and intracellular labeling. To assess the tangential location of particles, the distance of each gold particle from the centre of the PSD was normalized to the half-length of the PSD profile. PSD lengths shorter than 200 nm were excluded for tangential analysis. To calculate the surface density, gold particles in each membrane compartment were counted in digital EM pictures, and effective membrane lengths measured. Double staining for vesicular Zne counts . T-test The authors declare they have no competing interests.CS performed experiments. CS, AB, XA and JPC analyzed the data. CS wrote the manuscript with input from all authors. All authors read and approved the final manuscript.


Pre-incubation of MSCs with HDL increased cell viability, reduced apoptotic indices and resulted in parallel decreases in reactive oxygen species (ROS) in comparison with control MSCs. Each of the beneficial effects of HDL on MSCs was attenuated by inhibiting the PI3K/Akt pathway. Preconditioning with HDL resulted in higher MSC survival rates, improved cardiac remodeling and better myocardial function than in the MSC control group. Collectively, these results suggest that HDL may protect against H2O2-induced apoptosis in MSCs through activation of a PI3K/Akt pathway, and by suppressing the production of ROS.The therapeutic effect of transplantation of mesenchymal stem cells (MSCs) in myocardial infarction (MI) appears to be limited by poor cell viability in the injured tissue, which is a consequence of oxidative stress and pro-apoptotic factors. High density lipoprotein (HDL) reverses cholesterol transport and has anti-oxidative and anti-apoptotic properties. We, therefore, investigated whether HDL could protect MSCs from oxidative stress-induced apoptosis. MSCs derived from the bone marrow of rats were pre-incubated with or without HDL, and then were exposed to hydrogen peroxide (H An increasing number of studies have described transplantation of mesenchymal stem cells (MSCs) from bone marrow as a strategy for cardiac repair following myocardial infarction . HoweverIt is widely reported that high density lipoprotein (HDL) lowers the risks associated with ischemic cardiovascular diseases . HDL has2O2), which is widely used as an oxidant [in vitro to induce a form of cellular damage similar to oxidative stress.We, therefore, investigated whether HDL could protect MSCs against oxidative stress-induced apoptosis. Additionally, we also examine the effect of HDL on the MSCs senescence, which was regarded to be induced by oxidative stress and to influence cell therapeutic potential considerably. Previously, the effects of HDL on endothelial progenitor cells , embryon oxidant ,15, was To investigate the effect of HDL on the biological activity of MSCs, we performed flow cytometry and MTS to detect cell differentiation and cell viability respectively. We observed that the MSCs (Passage 4), used in our experiments, expressed typical MSC-related cell surface antigens, which were positive for CD29, CD90 and negative for CD45, CD34; and that the incubation with HDL (100 μg/mL) for 24 h did not exhibit a notably impact on the characteristic phenotypes of MSCs . Meanwhi2O2, which was employed to induce the cell apoptosis, MSCs was cultured with different concentration of H2O2 for 24 h and a MTS assay was performed afterwards. It was found that H2O2 impaired the viability of MSCs in a concentration-dependent manner over the tested concentration range  for 24 h and then cultured with 400 μM of H2O2 for an additional 24 h. TUNEL assay showed that incubation with 100 μg/mL HDL did not affect the apoptosis of MSCs (HDL group vs. Control group: (6.83 ± 1.35)% vs. (7.93 ± 1.76)%, p > 0.05). However, H2O2 significantly increased MSCs apoptosis (H2O2 group vs. Control group: (47.37 ± 7.53)% vs. (7.93 ± 1.76)%, p < 0.01), which was remarkably attenuated after preconditioning with HDL (HDL + H2O2 group vs. H2O2 group: (28.77 ± 6.91)% vs. (47.37 ± 7.53)%, p < 0.05) (2O2-stimulated MSCs compared to controls ((267.4 ± 25.3)% vs. (100 ± 12.2)%, p < 0.01). The increased caspase-3 activity was ameliorated by pre-incubation with HDL (HDL + H2O2 group vs. H2O2 group: (130.9 ± 19.7)% vs. (267.4 ± 25.3)%, p < 0.05) % vs. (58.6 ± 6.8)%, p < 0.05) (In order to investigate the effects of HDL on H < 0.05) . Caspase < 0.05) . Similar < 0.05) .2O2-stimulated replicative exhaustion. Senescence-associated β-Galactosidase  staining showed that there were no significant differences in the percentages of SA-β-Gal positive cells between the HDL group and the Control group ((102.3 ± 13.7)% vs. (100 ± 12.4)%, p > 0.05). Similarly, there was nearly identical in those between the H2O2 group and the HDL+H2O2 group ((356.2 ± 25.8)% vs. (367.9 ± 22.37)%, p > 0.05), although there was a significant increment compared the H2O2 group with the Control one (p < 0.05) (INK4a between the HDL group and the Control ((1.06 ± 0.13) vs. (1.00 ± 0.10), p > 0.05), and between the H2O2 group and the HDL + H2O2 group ((3.62 ± 0.19) vs. (3.40 ± 0.26), p > 0.05). Likewise, it was found that the expression of p16INK4a in the H2O2 group statistically increased than that in the Control one (p < 0.05)  . Further < 0.05) . Therefovs. Control group: (283.8 ± 25.1)% vs. (100 ± 24.71)%, p < 0.05) and was still present at 24 h (24-h group vs. Control group: (197.5 ± 19.37)% vs. (100 ± 24.71)%, p < 0.05) (vs. Control group: (12.37 ± 5.19)% vs. (100 ± 24.28)%, p < 0.05) (vs. Control group: (97.6 ± 6.8)% vs. (100 ± 5.1)%, p > 0.05) (vs. Control group: (106.4 ± 13.5)% vs. (100 ± 11.3)%, p > 0.05) (2O2-stimulated cells. This resulted in decreased cell viability as measured by MTS assay (HDL + H2O2 + LY294002 group vs. HDL + H2O2 + DMSO group: (51.7 ± 7.1)% vs. (83.6 ± 7.8)%, p < 0.05) (2O2 + LY294002 group vs. HDL + H2O2 + DMSO group: (286.3 ± 16.7)% vs. (149.5 ± 21.8)%, p < 0.05)  , suggest < 0.05) . Conside > 0.05)  and apop > 0.05) , which w < 0.05)  and incr < 0.05) , consistp > 0.05) (2O2 group than in the Control one ((435 ± 48)% vs. (100 ± 15)%, p < 0.05). HDL pretreatment restored intracellular ROS generation induced by H2O2 (HDL + H2O2 group vs. H2O2 group: (156 ± 25)% vs. (435 ± 48)%, p < 0.05), and the protective effect disappeared when the PI3K/Akt pathway was inhibited by LY294002 (HDL + H2O2 + DMSO group vs. HDL + H2O2 + LY294002 group: (172 ± 37)% vs. (482 ± 57)%, p < 0.01).The influence of the PI3K/Akt pathway in ROS production based on LY294002 inhibition and assessed using fluorescent probe-DHE demonstrated that the percentage of ROS negative cells with low fluorescence intensity were similarly observed among the Control, the HDL and the LY294002 groups  . Cells wThe above results showed that the inhibition of PI3K/Akt pathway attenuated the protective effect of HDL and the decreased ROS, indicating that Akt activation was partly upstream of ROS generation. Excessive production of ROS has been shown to damage stem cells through caspase-mediated pathways . Therefoin vivo (6 in 100 μL) were respectively transplanted into the infarcted heart of rats. It was discovered that the number of GFP+ MSCs per HP in the HDL-MSCs group was 2.58-fold greater than that in the MSCs group (p < 0.05). Furthermore, when we transplanted the male MSCs to the female hearts, it was revealed that the sry DNA level in the HDL-MSCs group was approximately 2.83-fold higher than that in the MSCs group (p < 0.05), indicating that HDL preconditioning could enhance the survival of MSCs after transplantation.The poor viability of the donor cell, typically <1% at 4 days after transplantation, has been reported in the previous study regarding MSCs-base therapy . Therefoin vivo . After ap < 0.05); and that LVIDd was 40.9% higher and LVIDs was 90.0% higher in the PBS than in the Sham group (both p < 0.05). Furthermore, compared with PBS group, transplantation of MSCs improved LVEF and LVFS by 13.0% and 21.6%, respectively (both p < 0.05). Injection of HDL-MSCs enhanced the LVEF and LVFS by an additional 13.0% and 20.1%, respectively (both p < 0.05). Transplantation of MSCs decreased the LVIDd and LVIDs by 19.5% and 23.3%, respectively compared to the PBS group (both p < 0.05), and transplantation of HDL-MSCs further decreased LVIDd and LVIDs by approximately 12.3% and 30.0% (both p < 0.05), respectively.The effect of MSCs preconditioned with HDL on cardiac function was observed by transthoracic echocardiography four weeks after experimentally induced MI in rats . It indiet al.[We noticed that Kiya Y et al. had prev7 implanted MSCs in infarcted porcine hearts yielded only limited enhancement of LVEF [In the recent literature, the effect of transplantation of MSCs on myocardial repair was reported as confined for the low viability of graft cells ,22. For  of LVEF . A serie of LVEF  but had 2O2in vitro. The mechanisms involved activation of PI3K/Akt pathway, which was consistent with findings from previously reported studies [in vivo, supporting the findings of previous researches showing that the administration of HDL preserved the post-ischemic cardiac function [Therefore, we explored whether improving the in-situ physiological environment of MSCs could enhance cells viability and prevent oxidative stress-induced apoptosis after transplantation or mobilization. Epidemiological studies have consistently shown an inverse correlation between HDL levels and outcomes in ischemic heart diseases . We ende studies ,22. We afunction .2O2 treatment expectably spurred a considerable replicative exhaustion in MSCs; however, MSCs senescence could not be reversed by HDL preconditioning, which indicated that the effectiveness of HDL on the MSCs therapeutical potential in vivo was independent on the influence in cellular aging, while mainly through enhancement in cellular anti-apoptotic property. Indeed, the elucidation of this mechanism is especially critical for physicians engaged in cell-based therapy, whom must be sure that MSCs are devoid of chromosome alterations and that there are no signs of cellular senescence.Beside apoptosis, cellular senescence had been proved to be the other main outcome following oxidative stress stimuli , which cin vivo.The clinical significance of our results is the possible benefit of HDL in patients with myocardial infarction in terms of improving the survival of transplanted MSCs. Elevation of HDL levels in patients, at high risk of cardiovascular disease may also be of vital clinical importance in preventing cardiovascular events. However, we did not identify the specific components of HDL that were responsible for its anti-oxidant and anti-apoptotic effects in the present study, for HDL is a lipoprotein, containing HDL2 and HDL3, with a density ranged from 1.063 to 1.210 g/mL . This wiAll animal procedures were undertaken in accordance with the Guidelines for the Care and Use of Laboratory Animals, published by the National Academy Press . The experiments were approved by the Animal Care and Use Committee of Zhongshan Hospital, Fudan University.2. The culture medium was changed every 3–4 days. Non-adherent hematopoietic cells were removed during this process, and the adherent, spindle-shaped MSCs were cultured and expanded. Passage 4 cells were used for subsequent experiments.MSCs were isolated from four-week-old Sprague-Dawley (SD) rats weighing 100 to 120 g by flushing femurs and tibias with Dulbecco’s modified Eagle’s medium , 10% Fetal Bovine Serum  and penicillin/streptomycin. The bone marrow (BM) solution was cultured at 37 °C in an atmosphere of 5% CO2. The medium was removed, replaced with fresh medium and incubated for a further 24 h. Cells were then sorted by flow cytometry for GFP to ensure that a homogenous population of GFP+ MSCs was obtained (data were not shown).An adenoviral vector carrying a green fluorescent protein (GFP) (GeneChem) for cell transduction was used to establish and identify a stable population of MSCs. Transduction was facilitated by transfecting Passage 4 MSCs with purified adenovirus cells that had been exposed to 7 μg/mL polybrene. The transfected cells were incubated for 24 h at 37 °C with 5% CO2O2 (0 to 500 μM) or HDL  for 24 h. The experiments were undertaken in four groups: a control group; a HDL group (MSCs incubated with HDL 100 μg/mL for 24 h); a H2O2 group (incubation with 400 μM H2O2 for 24 h); a HDL + H2O2 group . For inhibitor experiments, three additional groups were set as follows: MSCs were pre-incubated with PI3K inhibitor  or vehicle (DMSO) for 1 h, and then successively treated with HDL and H2O2 as above; the third group was pre-cultured with LY294002 alone. The concentrations of HDL, H2O2 and LY294002 used in these experiments were determined from pilot studies and based on similar methods described in the literature [MSCs were incubated for 6 h with DMEM supplemented with 1% FBS prior to experimental interventions. The starved MSCs were then respectively exposed to Hterature ,29.H-tetrazolium, inner salt (MTS) assay. Cells were cultured in 24-well plates and treated according to the protocol. After 24 h, Cell Titer 96® Aqueous One Solution Reagent (Promega) was added to each well according to the manufacturer’s instructions. Cell viability was evaluated after 2-h incubation with this reagent, by testing absorbance at 490 nm using a plate reader.MSC viability was assessed using a 3--5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-22O2 and exposed to TdT enzyme at 37 °C for 1 h. The cells were then incubated with digoxigenin-conjugated nucleotide substrate at 37 °C for 30 min. Nuclei exhibiting DNA fragmentation were identified after staining with 3,3-diamino benzidine (DAB) for 5 min.Cell apoptosis was investigated using an In Situ Cell Death Detection kit (Roche). In brief, MSCs were fixed in 3.7% buffered formaldehyde, pretreated with 3.0% HThe MSCs were counterstained with hematoxylin and viewed by light microscopy. Five fields at high magnification (×200) were randomly chosen to assess the apoptosis index, which was calculated as a percentage of labeled cells to total nuclei.Caspase-3 activity was determined in MSC lysates (200 μg) using Caspase-3/CPP32 Fluorometric Assay kit (BioVision). The fluorogenic CPP32/caspase-3 substrate was labeled with the fluorochrome 7-amino-4-methyl coumarin. In this assay, the amount of fluorescence produced upon cleavage was proportional to the amount of caspase-3 activity present in the sample.MSCs cultured in six-well plates were rinsed with phosphate-buffered saline (PBS), fixed and then incubated with freshly prepared senescence-associated β-Galactosidase  staining solution  at 37 °C overnight. Five fields at high magnification (×200) were randomly chosen to count the number of blue cells . At least 500 cells in each group were examined and the percentages of blue cells presented.INK4a. Following a 30 min wash, the membranes were incubated for 1 h with a secondary antibody conjugated to horseradish peroxidase-conjugated IgG (Jackson). Protein expression was determined using an enhanced chemiluminescence system and quantified by densitometry .MSCs from each group were rinsed with cold PBS and lysed using RIPA buffer containing 1 mM phenylmethanesulfonylfluoride on ice for 30 min. The lysates were transferred to 1.5 mL Eppendorf tubes and centrifuged at 4 °C and 15,000 rpm for 30 min. The protein component was separated by 12% SDS-PAGE and then transferred to polyvinylidene fluoride membranes (Millipore). The membranes were blocked in 5% skimmed milk for 2 h at room temperature, and were incubated overnight at 4 °C with anti-phospho-Akt , anti-Akt , anti-p16The production of intracellular ROS was measured using an oxidation-sensitive fluorescent dihydroethidium (DHE) probe (Vigorous). The assay was based on the principle that DHE crosses cell membranes and is rapidly oxidized in the presence of ROS resulting in the formation of a highly fluorescent form of oxidative ethidium.In this assay MSCs were washed three-times with PBS and incubated with 10 μM DHE in phenol-red-free MEM medium (Invitrogen) at 37 °C in the dark. After 15 min incubation, the cells were rinsed and the medium was replaced. The fluorescence level, as an indicator of ROS production, was detected using fluorescence microscopy. The excitation and emission filters were 488 nm and 590 nm, respectively.6 in 100 μL, derived from male rats) (MSCs group) or MSCs preconditioned with HDL (HDL-MSCs group) were intramyocardially injected into female rats 10 min after ligation-induced infarction. The MSCs were injected into 3 to 5 injection sites into the anterior aspect of the viable myocardium that bordered the area of infarction. For the experiments concerning cardiac remodeling, two additional groups were set as follows: rats received intramyocardial injection of 100 μL of PBS without MSCs (PBS group) or received thoracotomy only but were not ligated (Sham group).The MI model was developed in SD rats 220 ± 30 g), as previously described [0 ± 30 g,in vivo, rats were sacrificed four days after cell implantation. Hearts were harvested and sliced transversally above the ligation suture. Tissue fixation was minimized to reduce autofluorescence. The sample was embedded at optimum cutting temperature and cut by a cryostat from the base (above the ligation) towards the apex until the ligation suture was reached. Beginning at this point, 10 μm sections were collected throughout the entire lesion. The sections were stained using DAPI , and the number of GFP+ MSCs was estimated under fluorescent microscopy at a magnification of ×200.To observe the donor MSCs survival + MSCs were counted and averaged.Three photomicrographs were taken from the infarct border zone of each section. Six sections were selected for counting, giving a total of 18 high power fields per rat. The numbers of GFPsry DNA levels on Day 4 after cell transplantation. Rats were sacrificed and hearts were excised, frozen in liquid nitrogen and powdered. DNA purification was performed using Genomic DNA Isolation kit (Qiagen). The concentration of the purified DNA was determined by spectrophotometry. Real-time PCR was performed using Takara SYBR Premix Ex TaqTM in a Bio-Rad iQ5 optical module. Primers for amplification of rat Y chromosome sry and β-actin genes were synthesized (Invitrogen) and were listed as follows: sry, forward, 5′-GAG GCA CAA GTT GGC TCA ACA-3′; reverse, 5′-CTC CTG CAA AAA GGG CCT TT-3′. β-actin, forward, 5′-CCA TTG AAC ACG GCA TTG-3′; reverse, 5′-TAC GAC CAG AGG CAT ACA-3′. The cycling conditions were: 30 s at 95 °C, followed by 40 cycles of 5 s at 95 °C, 30 s at 59.5 °C, 30 s at 72 °C. Melting curves were acquired at the end of the reaction by gradually raising the temperature by 1 °C/min from 59.5 °C to 95 °C over a time period of 35 min.Real-time polymerase chain reaction (PCR) was performed for quantification of the male Cardiac remodeling and left ventricular function were assessed by transthoracic echocardiography 4 weeks after MI, using a Vevo 770 high-resolution imaging system  with a 17.5-MHz probe. After the induction of light general anesthesia, hearts were imaged in two dimensional and M-modes. The recordings were obtained in the para-sternal long-axis view at the level of the greatest LV diameter. The LV internal end-diastolic diameter (LVIDd) and LV internal end-systolic diameter (LVIDs) were measured from M-mode recordings according to the leading-edge method. All echocardiographic measurements were averaged from at least five separate cardiac cycles.t-test. Comparisons between more than two groups were performed using one-way analysis of variance (ANOVA) with Bonferroni’s correction. Values of p < 0.05 were considered statistically significant.Statistical analysis was undertaken using SPSS version 16.0. All data were expressed as means and standard errors (SE). Between-group of differences was analyzed using Student’s 2O2-induced apoptosis by activation of the PI3K/Akt pathway and down-regulation of ROS generation; and that preconditioning with HDL promoted MSCs survival after cell transplantation, thereby preserving cardiac function. This observation provides an experimental basis for improving the prognosis of patients with myocardial infarction through elevation of HDL levels. The HDL-based therapy may hold the promise of reducing the incidences of such left ventricular remodeling and heart failure after myocardial damage.In summary, we demonstrated that HDL protected MSCs from H


The aim of the study was to evaluate arterial and venous ophthalmic blood flow parameters in mild and severe preeclampsia pregnancies and in normotensive pregnancies.A total of 117 women 25 to 30 years old with singleton pregnancies 30 to 40 weeks of gestation were recruited. Among them 40 pregnant women developed severe preeclampsia, 42 mild preeclampsia, and 35 were normotensive. Using color flow mapping (CFM) and pulse-wave Doppler imaging (PWD), maximum blood flow velocity (mFV) in the right/left arterial and venous ophthalmics along with Gosling's Doppler pulsatility index (PI) [The highest mFV values (59.2 ± 4.61 and 23.6 ± 4.03 cm/second) were in the severe preeclampsia group while in the mild preeclampsia group mFV increased slightly or remained normal (35.6 ± 2.97 and 13.6 ± 0.81 cm/second). There was no mFV increase in the normotensive pregnancy group (31.5 ± 2.21 cm/second). No significant correlation was found between gestation age and mentioned hemodynamic parameters in the normotensive pregnancy group. PI values in the arterial ophthalmic in normotensive pregnant women were 2.92 ± 0.59 and the highest in all groups. In group with mild preeclampsia this parameter was 1.47 ± 0.30 and the lowest one was in patients with severe preeclampsia - 1.17 ± 0.08.In women with preeclampsia significant changes in ophthalmic hemodynamics take place - mFV in arterial and venous ophthalmics increases while PI values go down. This might be evidence of orbital hyperperfusion in preeclamptic pregnant women. Low PI values may be used as the markers of severe preeclampsia.


The massive cuff stitch (MCS) is known to be a strong suture, suitable for rotator cuff repair. We modified this technique for massive cuff tears by employing a horizontal medial mattress suture from an anchor as well as a vertically crossing transosseous suture.We included 42 patients with massive cuff tears suitable for repair: 22 were treated with the modified MCS (MCS group), and 20 with a simple transosseous suture (STS group). The range of motion (ROM), muscle strength, visual analog scale, and the Japanese Orthopaedic Association (JOA) scores were evaluated pre-operatively and 12 and 24 months post-operatively. The incidence of post-operative re-tears was examined at least 1 year post-operatively using Sugaya's classification.. 40%, P = 0.0296).The ROM, muscle strength, degree of pain, and the JOA scores were much improved after surgery in both groups, and there was no significant intergroup difference throughout the pre- and post-operative periods. In contrast, post-operative MRI revealed a significantly lower re-tear rate in the MCS group than in the STS group (9.1% vsThe techniques tested were comparable in terms of functional outcome after surgical repair of massive cuff tears; however, the modified MCS repair technique produced superior structural outcomes with a significantly lower re-tear rate. Surgical intervention for rotator cuff tears using both arthroscopic and open techniques has resulted in good outcomes -4. SucceSeveral studies have shown that hybrid double-row repairs (suture anchor + transosseous suture) are stronger than repairs performed using either a suture anchor or transosseous tunnel alone, as the tendon-to-bone attachment area is greater . The masA total of 62 patients underwent open surgery between June 2005 and April 2007 for repair of rotator cuff tears. The surgery for the first 30 patients was performed using the simple transosseous suture (STS) repair technique, and the surgery for the remaining 32 cases was performed using the modified MCS technique. The inclusion criteria were as follows: (1) the largest dimension of the ruptured site measured during surgery was >5 cm or the involvement of at least two tendons ,6, (2) iA total of 42 patients with rotator cuff tears were candidates for this study: 22 were treated with the modified MCS (MCS group), and 20 with STS repairs (STS group). The mean age at the time of surgery was 61.1 ± 6.7 years in the modified MCS group and 61.3 ± 8.4 years in the STS group. The mean time after the onset of symptoms was 3.8 ± 2.8 months in the modified MCS group and 5.1 ± 3.1 months in the STS group. A single surgeon (MG) performed all surgeries. Approval from the Institutional Review Board in our institute was obtained (#13037).Pre- and post-operative evaluations included a patient questionnaire and a physical examination performed by an independent examiner who was blinded to the study. The examination consisted of functional assessment based on the Japanese Orthopedic Association (JOA) scoring system , range oThe patients in the present study underwent magnetic resonance imaging (MRI)  at our institution both pre- and post-operatively. The MRI protocol was as described previously . All scaAll procedures were performed in beach-chair position. Skin incisions were made along Langer's lines just lateral to the coracoid process. The deltoid was split longitudinally in the raphe, and the insertion of the anterior deltoid was detached from the acromion. After skeletonizing the acromion, an acromioplasty was performed with a chisel. After mobilizing the retracted cuff and excising its margin, the ruptured cuff was sutured. A bone tunnel at the greater tuberosity corresponding to the repair site was made with a chisel. Single interrupted sutures were placed at the lateral edge of the torn cuff and passed through the bone tunnel with 2.4-mm Kirschner wire and large needle (3 cm in diameter).For the modified MCS repair, a horizontal mattress stitch was made on the inside 10–15 mm of the ruptured rotator cuff using a single-loaded bioabsorbable suture anchor  placed just medial to the rotator cuff footprint. Next, a vertical simple transosseous suture was made to cross over the horizontal suture proximally after medial tying  [Data are expressed as mean ± SD. A repeated measure analysis of variance was used for comparing pre- and post-operative parameters  in the same procedure. Mann-Whitney Austria) .P < 0.05), and the range of motion in abduction from 81.4° ± 44.3° pre-operatively to 144.8° ± 25.4° at final follow-up (P < 0.05). The internal rotation was significantly increased from 5.7 ± 2.4 vertebrae pre-operatively to 7.7 ± 2.9 vertebrae at final follow-up (P < 0.05). The external rotation was not significantly improved from baseline to final follow-up . In the STS repair group, the range of motion was significantly improved from 107.4° ± 43.3° to 149.2° ± 17.3° in elevation (P < 0.05) and from 97.1° ± 47.8° to 145.0° ± 27.6° in abduction (P < 0.05). There was no significant difference in external and internal rotation from baseline to final follow-up. The details are shown in Table In the MCS repair group, the range of motion in elevation was significantly improved from 95.2° ± 47.1° pre-operatively to 152.1° ± 15.4° at the final follow-up (P < 0.05) and from 79.3 ± 29.9% to 96.2 ± 19.6% in internal rotation at final follow-up (P < 0.05). There was no significant improvement in elevation strength or abduction strength from baseline to final follow-up. In the STS repair group, strength in elevation was significantly improved from 55.4 ± 18.3% pre-operatively to 74.2 ± 27.1% (P < 0.05), and strength in internal rotation from 85.2 ± 27.2% to 102.8 ± 21.4% at final follow-up (P < 0.05). There was no significant improvement in the external rotation strength or abduction strength between baseline and final follow-up. The details are shown in Table Muscle strength was evaluated using a hand-held dynamometer  by the sP < 0.05), the degree of pain on motion from 6.3 ± 2.0 pre-operatively to 0.5 ± 1.5 at final follow-up (P < 0.05), and the degree of pain at night from 4.0 ± 2.7 pre-operatively to 0.4 ± 1.4 at final follow-up (P < 0.05). In the STS repair group, the degree of pain at rest was significantly improved from 2.1 ± 2.6 pre-operatively to 0.4 ± 1.4 at final follow-up (P < 0.05), the degree of pain on motion from 5.9 ± 2.9 pre-operatively to 0.9 ± 1.8 at final follow-up (P < 0.05), and the degree of pain at night from 5.1 ± 2.8 pre-operatively to 0.7 ± 2.1 at final follow-up (P < 0.05). The details are shown in Table The degree of shoulder pain was evaluated by VAS . In the P < 0.05), and from 59.1 ± 7.7 pre-operatively to 89.4 ± 7.6 at final follow-up in the STS repair group (P < 0.05). The details are shown in Table The total JOA score was significantly improved in both groups: from 62.3 ± 9.6 pre-operatively to 91.9 ± 5.5 at final follow-up in the MCS repair group (There was no significant difference in the improvements between the modified MCS and STS repair groups throughout the pre- and post-operative periods.All patients underwent MRI assessment during post-operative year 1 (12–18 months) to determine the integrity of the repaired cuff, by using Sugaya's classification . In the Previous studies on massive rotator cuff tears treated by open or arthroscopic surgery have reported a wide range of re-tear rates (40%–94%): 94% by Galatz et al. , 40% by Apart from the structural outcome, the functional outcome has consistently been satisfactory in patients with large/massive cuff tears: Bigliani et al.  reportedThe MCS was developed to increase the strength of the suture-tendon interface for arthroscopic surgery; it is biomechanically similar to the modified Mason-Allen stitch . Ko et aThe original MCS consists of a vertical stitch and a horizontal mattress stitch that originate from the same suture anchor. Pullout of the vertical stitch is prevented by the tendon-grasping effect of the horizontal stitch . The maiOur data also suggest an important role of the medial mattress stitch near the suture anchor for obtaining a good structural outcome. This modified MCS is expected to provide increased bridging compression between the medial stitch and the lateral edge of the vertical transosseous stitch. Thus, our modified MCS may be thought of as a transosseous-equivalent double-row repair, the latter of which has been reported to have greater fixation in rotator cuff repair .The present study had several limitations. First, this study did not perform biomechanical testing of the modified MAC stitch. However, the structural outcome of the present series revealed a favorable outcome compared with previous studies of massive cuff tears ,2,16,17.We compared the clinical outcomes of the modified MCS stitch and the simple transosseous suture in patients with massive cuff tears. We found that the techniques were comparable in terms of functional outcome; however, the modified MCS repair technique produced a superior structural outcome with a significantly lower re-tear rate.The authors declare that they have no competing interests.The design of the study and preparation of the manuscript were done by MG, YM and KY. KN assisted in the study processes, sample collections, and preparations. TO, FH, and KN assisted in the manuscript preparation. All authors read and approved the final manuscript.


Helicobacter pylori (HpSK). Site-directed mutagenesis and isothermal titration calorimetry studies revealed critical conserved residues  that interact with shikimate and are therefore involved in catalysis. Crystal structures of HpSK·SO4, R57A, and HpSK•shikimate-3-phosphate•ADP show a characteristic three-layer architecture and a conformationally elastic region consisting of F48, R57, R116, and R132, occupied by shikimate. The structure of the inhibitor complex, E114A•162535, was also determined, which revealed a dramatic shift in the elastic LID region and resulted in conformational locking into a distinctive form. These results reveal considerable insight into the active-site chemistry of SKs and a selective inhibitor-induced-fit mechanism.Shikimate kinase (SK), which catalyzes the specific phosphorylation of the 3-hydroxyl group of shikimic acid in the presence of ATP, is the enzyme in the fifth step of the shikimate pathway for biosynthesis of aromatic amino acids. This pathway is present in bacteria, fungi, and plants but absent in mammals and therefore represents an attractive target pathway for the development of new antimicrobial agents, herbicides, and antiparasitic agents. Here we investigated the detailed structure–activity relationship of SK from  Staphylococcus aureus, Enterococcus faecium, Staphylococcus pneumonia, Klebsiella species, Acinetobacter baumannii, Pseudomonas aeruginosa, Mycobacterium tuberculosis, and Escherichia coliIn recent years, major problematic bacterial infections have been described for methicillin-resistant M. tuberculosis of aroK, the gene encoding shikimate kinase , the fifth enzyme of the shikimate pathway, further suggests that this pathway is essential for antimicrobial drug discovery The shikimate pathway is comprised of seven enzymatic components that convert erythrose 4-phosphate and phosphoenolpyruvate into chorismate, for subsequent synthesis of aromatic compounds E. coli, Erwinia chrysanthemi, Campylobacter jejuni, Aquifex aeolicus and Arabidopsis thalianaM. tuberculosis and Helicobacter pylori  SK catalyzes the specific phosphorylation of the 3-hydroxyl group of shikimic acid, using ATP as a co-substrate. Several SK structures are available •ADP, which reveal that three conserved Arg residues , the side chain of D33, and the aromatic ring of F48 are involved in binding to shikimate. We also determined the X-ray structure of the E114A mutant SK-inhibitor complex using a selective inhibitor  identified from virtual docking analysis. Site-directed mutagenesis and isothermal titration calorimetry (ITC) together revealed the key binding residues and a NSC162535/induced-fit mechanism.The likelihood of HpSK as a target enzyme for potential drug and herbicide discovery prompted us to investigate the detailed structure-activity relationship of the binding pocket. Here, we report the crystal structures of HpSK·SO4 structure (1ZUI) X, where a carboxyl moiety of shikimate makes contact with R57, R116, and R132; (ii) OCORE, where two hydroxyl groups of shikimate make contact with M10, D33, G79–G81, and E114; and (iii) OLID, where a trans hydroxyl group of shikimate interacts with V44, F48, E114, and R116. Of these residues, D33, R57, G79–G81, R116, and R132 are strictly conserved among all SKs, whereas the others  are relatively conserved  experiments to evaluate the stability and unfolding of each of the generated HpSK mutants. All HpSKs revealed a single transition and fit well . Wild-tyX site, are important in catalysis. R116 is also noted to contribute to both the CX-site and OLID-site interactions. Importantly, it has been suggested that R117 of MtSK (corresponding to R116 of HpSK) may be the primary residue involved in stabilizing the pentavalent phosphorus transition-state intermediate LID subsite and the mutant F48A exhibited no detectable activity, although F48Y restored activity to 40%, indicating that the aromatic ring of F48 contributes to catalytic activity. In support of this view, we note that the aromatic ring of F48 makes contact with R57 and other nearby residues  and forms stable interactions, thus ensuring R57 is in an appropriate position to interact with shikimate. The side chain of the other OLID residue, E114, faces the solvent, and E114A retained 82% relative activity. For OCORE residues, D33A and D33E showed loss of enzymatic activity, whereas M10A retained 38% relative activity.Enzymatic analysis of R57A, R116A and R132A revealed that these mutants completely lost enzyme activity , suggestmK (MgATP) = 293±42 µM, mK (shikimate) = 135±26 µM, Vmax(MgATP) = 26±1 µmol/min/mg, Vmax(shikimate) = 16±1 µmol/min/mg; F48Y, mK(MgATP) = 231±35 µM, mK(shikimate) = 291±99 µM, Vmax(MgATP) = 17±1 µmol/min/mg, Vmax(shikimate) = 13±2 µmol/min/mg; E114A, mK(MgATP) = 143±18 µM, mK(shikimate) = 39±7 µM, Vmax(MgATP) = 26±2 µmol/min/mg, Vmax(shikimate) = 23±1 µmol/min/mg. As compared with the wild-type enzyme (mK(MgATP) = 101±17 µM, mK(shikimate) = 60±8 µM, Vmax(MgATP) = 26±1 µmol/min/mg, Vmax(shikimate) = 22±1 µmol/min/mg) We also investigated the kinetics of three mutants  that retained enzymatic activity. The apparent values for Michaelis-Menten parameters were determined as follows: M10A, The crystal structure shows that D33 forms a hydrogen bond with the 3-OH group of shikimate, which may increase the nucleophilicity of the O atom or accept the proton from the 3-OH group of shikimate. M10, on the other hand, shows limited contacts with shikimate; thus, replacement with Ala at this site did not completely eliminate the enzymatic activity. Together, our results suggest that D33, F48, R57, R116, and R132 are critical for enzymatic catalysis.4 structure (1ZUI) 50 = 4.9 µM; iK (shikimate) = 1.8 µM; iK (ATP) = 1.9 µM; 2+), a clear shikimate ITC pattern was observed, showing a high binding affinity to shikimate , revealing that the aromatic ring contributes towards binding to shikimate. Thus, the side chains of D33 and three Arg residues  as well as the aromatic ring of F48 in HpSK are critically involved in shikimate binding  involved in catalysis, based on the HpSK·shikimate·PO binding . We alsodK = 5.2 µM). With respect to the critical residues , the mutants F48A, R57A, R132A, and R132K lacked affinity for NSC162535. F48Y, however, retained binding affinity , indicating that replacement with a more conservative side chain at F48 and R57 partially rescued the binding affinity. In contrast, the mutants D33A and D33E, and R116A and R116K had measurable ITC binding profiles with comparable dK values  associated with domain movements 4 , HpSK• S3P•ADP , and R57A  , implying a disordered region.Crystal structures of HpSK and MtSK have been reported, alone and in complex with either one or two substrates/products = 27.3%) . Overall= 27.3%) . The LID4 structure, R116 also makes a hydrogen bond with the carboxyl moiety of shikimate. Based on these structures, it is likely that S3P is chelated by R57 and R132 via a hydrogen-bonding network in HpSK•shikimate•PO4 and HpSK•S3P•ADP. This would lead to a small movement in the peptide backbone, propagated through the α6 helix into the adjacent LID loop. A subsequent large conformational rearrangement of this loop would allow the side chain of R116 to bind to the phosphate group of S3P, as seen in HpSK•S3P•ADP, or possibly the γ-phosphate moiety of ATP The HpSK•S3P•ADP structure shows clear electron density for all residues. Within the binding pocket, there is a large piece of non-peptide density that can easily be modeled as the product S3P, and nearby density can be built as ADP . The finWe also determined the structure of mutant R57A  that lacfree = 26.2%; In an effort to understand the detailed structure-activity relationship of this inhibitor at atomic resolution, we attempted crystallization trials using either wild-type HpSK or a mutant prepared in this investigation. After extensive trials, only the map of the E114A·162535 crystal showed a large piece of residual density in the binding pocket, which could be modeled as NSC162535 . The fin4 and HpSK• S3P•ADP. Notably, the side chain of R116 at LID forms hydrogen bonds with an SO4 group and packs against one of the naphthalene moieties with an electron-rich π system  β-phosphate of ADP in HpSK•S3P•ADP; and (iii) NSC162535 in E114A•162535. In the MtSK•shikimate•AMPPCP structure , the Cα atom of R57 superimposes relatively well, whereas that of R132 has a small shift in the various structures . Notably4 forms  critical to interactions with shikimate, and therefore catalytic activity. Additionally, side chains from D33 and F48 were found to play important roles in binding to shikimate. Based on virtual docking analysis, we were able to design a potent inhibitor, NSC162535, for this SB pocket, which included these residues. Furthermore, we solved four HpSK structures: HpSK•SOAnalysis of these structures reveals that R57 is critical for stabilizing and maintaining the optimal environment, whereas R132 plays a critical role in chelating the ligand. The R57A mutant structure shows a notable shift of the SB domain owing to the interactions between E53 and R132, rather than those between E53 and R57 in the wild-type structure, revealing a crucial role for R57 and R132 in defining the best active-site geometry to accommodate promising substrates . In supp4, HpSK·S3P·ADP, and E114A·162535), where R116 directly contacts shikimate/S3P/NSC162535. Interestingly, ITC measurements revealed that this catalytic residue contributes substantially to shikimate binding but not to that of NSC162535. The insignificant contribution of R116 to NSC162535 binding  between the wild-type and E114A, it is conceivable that the wild-type enzyme has an analogous binding configuration with NSC162535. As a result, the unusually elastic LID segment fits this inhibitor; R116 subsequently comes into contact with the SOSite-directed mutagenesis was performed using the overlap extension PCR method A340 . The assay was carried out at 25°C in a mixture containing 100 mM Tris-HCl, pH 7.5, 50 mM KCl, 5 mM MgCl2, 1.6 mM shikimic acid, 2.5 mM ATP, 1 mM phosphoenolpyruvate, 0.1 mM NADH, 2.5 U ml−1 pyruvate kinase, and 2.7 U ml−1 lactate dehydrogenase. All assays were conducted in a 96-well microplate and analyzed with a spectrophotometer . The relative activity of wild-type HpSK was set to 100%.SK activity was determined by coupling the release of ADP from the SK-catalyzed reaction to the oxidation of NADH using pyruvate kinase (EC 2.7.1.40) and lactate dehydrogenase (EC 1.1.1.27) as coupling enzymes. Shikimate-dependent oxidation of NADH was monitored by a decrease in −1. A buffer-buffer scan was subtracted from the buffer-sample scans, and linear-polynomial baselines were drawn for each scan. Baseline-corrected thermograms were then normalized to obtain the corresponding molar heat capacity curves. Midpoint temperature (Tm) values were estimated as the temperatures corresponding to the maximum of each thermogram peak. Analysis of DSC thermograms was implemented using Origin™ Software.DSC measurements were carried out using a VP-DSC Microcalorimeter . The HpSK proteins were in 40 mM Tris-HCl (pH 7.0) containing 100 mM NaCl. The DSC experiments were performed at a concentration of 0.1 mM HpSK protein. Prior to making measurements, a baseline was established by repeated scans of the sample cell containing only buffer solution. Scans were performed from low to high temperatures, at a rate of change of temperature of 1.0°C minTitration experiments were performed by ITC using an iTC200 or VP-ITC instrument , and a 0.2-ml (iTC200) or 1.4-ml (VP-ITC) sample cell containing the macromolecule solution. All proteins were prepared in a buffer containing 20 mM potassium phosphate, pH 7.3. Prior to the experiment, samples were filtered and degassed under vacuum for 10 min in a Thermo Vac system . The sample cell was filled with sample protein solution (15 µM) or the working buffer. Procedures of iTC200 titrations with a ligand (450 µM) were as follows: an initial 1-µl injection  followed by 19 injections of 2 µl each, with 2-min intervals between injections. Similar procedures for VP-ITC titrations were also performed: an initial 2-µl injection followed by 24 injections of 10 µl each, with 3-min intervals between injections. The experiments were performed with a constant stirrer speed of 1000 rpm (iTC200) or 290 rpm (VP-ITC) at 25°C. The binding isotherms were fitted to a one-site binding model to obtain the thermodynamic parameters with the initial point discarded. Data analysis was conducted using Origin 7 software.−1) in 40 mM Tris-HCl (pH 7.0) containing 100 mM NaCl was equal to the reservoir solution and equilibrated against 60 µl of reservoir solution. Initial crystallization conditions were screened using 672 different kit solutions . We observed many different crystal forms in the drops.Crystallization was performed in 96-well microplates at 20°C using an Oryx8 robotic system (Douglas Instruments Ltd). The volume of HpSK protein solution  polyethylene glycol (PEG) 8000, and 0.1 M sodium acetate buffer (pH 6.5). Crystals of HpSK·S3P·ADP, added to 5 mM shikimate and 5 mM MgATP, were obtained from 0.1 M HEPES sodium salt (pH 7.5), 0.1 M sodium acetate, 18% (w/v) PEG 8000 and 2% (w/v) 2-propanol. HpSK·R57A crystals were grown in a hanging-drop containing 0.1 M HEPES sodium salt (pH 8.0), 8% (w/v) 2-propanol and 18% (w/v) PEG4000. The best crystals of the E114A·162535 structure were obtained in a modified condition, containing 0.1 M HEPES sodium salt (pH 6.7) and 1.2 M potassium sodium tartrate tetrahydrate. Prior to data collection, crystals were dipped into Fomblin cryoprotectant oil for several seconds and then flash-frozen in a liquid nitrogen stream. The X-ray diffraction data were collected on NSRRC BL-13B1, BL-13C1, and SPring-8 BL-12B2 using an ADSC Quantum 4R CCD detector. All datasets were collected at −150°C and processed with the HKL/HKL2000 software suite The condition of HpSK·SOfree) for cross-validation of the model. 2Fo−Fc and Fo−Fc maps were produced and inspected after each cycle of refinement to revise the model manually on an interactive graphics computer with the program Coot 4), 3MUF (HpSK·S3P·ADP), 3MRS (HpSK·R57A) and 3N2E (E114A·162535) (The four structures were solved by molecular replacement with the program MOLREP ·162535) .Comparison with the six HpSK structures  and HpSK , respectively. We programmed GEMDOCK 4, HpSK·shikimate·PO4, HpSK·S3P·ADP, and E114A·162535).GEMDOCK was also used to dock NSC162535 to each of the four HpSK structures  are shown in blue and cyan, respectively. Apo form of MtSK (PDB code: 2IYT) is shown in orange, and closed-form (PDB code: 2IYQ) in yellow. The EcSK (PDB code: 1SHK) is red, and the EcSK complexed with ADP (PDB code: 2SHK) is pink. Green, brown and gray are indicated in CjSK (PDB code: 1VIA), EcoSK (PDB code: 1KAG) and AaSK (PDB code: 2PT5), respectively. .(TIF)Click here for additional data file.Figure S2Conserved residues of SKs and structure-based alignment of HpSK and MtSK. Six shikimate kinases  are aligned and shown with WebLogo program (http://weblogo.berkeley.edu/). HpSK and MtSK alignment are shown with ESPript program (http://espript.ibcp.fr/ESPript/ESPript/). The secondary structural elements are shown above the sequence. Mutants generated for the structure-activity analyses are indicated below the aligned sequence. Three arginines  belong to CX shikimate-binding subsite. OCORE consists of M10 and D33; and OLID consists of F48, E114, and R116.(TIF)Click here for additional data file.Figure S3DSC heat capacity curve of HpSK proteins.(TIF)Click here for additional data file.Figure S4Binding properties of HpSK mutants. Isothermal titration calorimetry data showing (A) the titration of shikimate into M10A mutant; (B) the titration of NSC162535 into M10A, D33A, D33E, R57K, E114A and R116A.(TIF)Click here for additional data file.Figure S5Chemical structure of NSC162535.(TIF)Click here for additional data file.Figure S6Conformational movement in the SB domain of HpSK·R57A structure. A stereo view of the superimposed binding pocket between open HpSK (blue) and R57A (magenta) structures is shown. The oxygen, nitrogen and phosphorus atoms are colored red, blue and orange, respectively. The dashed line indicates hydrogen bonds.(TIF)Click here for additional data file.Table S1DSC thermodynamic parameters for the melting of HpSK wild-type and its mutants.(DOC)Click here for additional data file.


Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae). The nucleotide sequence of the clone, designated CYP345D3, was determined. The cDNA is 1554 bp in length and contains an open reading frame from base pairs 32 to 1513, encoding a protein of 493 amino acid residues and a predicted molecular weight of 57466 Daltons. The putative protein contains the classic heme-binding sequence motif FxxGxxxCxG (residues 430–439) conserved among all P450 enzymes as well as other characteristic motifs of the cytochrome P450s. Comparison of the deduced amino acid sequence with other CYP members shows that CYP345D3 shares 91% identity with the previously published sequence of CYP345D1 from the T. castaneum genome project and the nucleotide sequence identity between them is less than 80%. Phylogenetic analysis of amino acid sequences from members of various P450 families indicated close phylogenetic relationship of CYP345D3 with CYP6 of other insects than those from mammals and amore distant relationship to P450 from other families. CYP345D3 was submitted to GenBank, accession number EU008544.A novel cDNA clone encoding a cytochrome P450 gene has been isolated from the insecticide-susceptible strain of the red flour beetle,  P450 enzymes , one of the most important enzyme systems involved in insecticide detoxification or activation, are a complex family of heme containing enzymes found in most organisms. To date, various kinds of P450 enzymes have been reported in animals, microorganisms, and plants, and classified into more than 36 families . P450 enCYP prefix, followed by a numeral for the family, a letter for the subfamily, and a number for the individual gene  was isolated  technique  was isolated from an insecticide-resistant strain of the housefly, omestica . Subsequisolated . Three nbopictus  and the echnique . Nine CY Pallens , two CYP Pallens  and ten  Pallens  fragmentechnique .Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), is a cosmopolitan and destructive pest of raw and processed cereal grains  Brewer's yeast under standard conditions . Twenty individuals were homogenized with at least 1 ml TRNzol Reagent in the glass homogenizer. The process of total RNA extraction and purification was carried out following the manufacturer°s instructions including a DNase  treatment. Finally the total RNA (A260/A280= 1.8) was dissolved in 40 µl DEPC treated H2O and stored at -80 °C. The first strand cDNA was synthesized using 2 µg of DNase-treated total RNA by RevertAid™ First Strand cDNA Synthesis Kit  with oligo(dT)-adaptors. The total volume of reverse transcriptional system was 25 µl. The reaction condition was performed according to the manufacturer's instructions and the reaction mixture was stored at -20 °C.Total RNA for the amplification of cDNA fragments and the Rapid Amplification of cDNA Ends (RACE) were isolated from the last instar larvae of www.biometra.de) using rTaqTM polymerase (Takara). There was 2 µl cDNA in the total volume of 25 µl. The PCR program included an initial denaturation step of 3 min at 94 °C and then 30 cycles were run as follows: 94 °C for 30 sec, 50 °C for 30 sec and 72 °C for 45 sec with a final extension of 10 min at 72 °C. The PCR products were separated by 1.5 % agarose gel electrophoresis and stained with ethidium bromide (EB). The band of the expected size (243 bp) was excised and the fragment was recovered with the Gel Extraction Mini Kit .The degenerate primers used in PCR were designed as described by Kasai et al.  and the www.invitrogen.com).The purified 243 bp fragment was cloned into a pMD-18-T vector (Takara). After transformation into JM109 competent cells (Takara), the DNA inserts of the recombinant clones were amplified by PCR with degenerate primers used above, and sequenced in both directions . The 3′ RACE adaptor primer 3AP (anti-sense): 5′-CTGATCTAGAGGTACCGGATCC-3′ and a genespecific primer 3GSP1 (sense): 5′-TTGAACCGGAAGTCAGATGTA-3′ were used for PCR with PrimeSTAR™ HS DNA polymerase (Takara) under the following conditions: an initial denaturation at 98 °C for 5 min, followed by 30 cycles of 98 °C for 10 sec, 55 °C for 20 sec and 72 °C for 1 min and a final extension at 72 °C for 10 min. A nested PCR was conducted under similar conditions using 1 www.clontech.com). The 5′ region was amplified using a gene-specific antisense primer 5GSP1: 5′-CCTTCCCCAAATGGCAAATACG-3′ and a 5′ RACE adaptor primer, UPM: 5′-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3′. The PCR amplification was performed with Advantage™ 2 PCR Kit (Clontech) under the following conditions: an initial denaturation at 94 °C for 3 min, Mowed by 30 cycles of 94 °C for 30 sec, 65.5 °C for 30 sec and 72 °C for 2 min and a final extension at 72 °C for 10 min. Again, a nested PCR was conducted using 1 µl of the first PCR product as template, the 5′ adaptor primer NUP: 5′-AAGCAGTGGTATCAACGCAGAGT-3′ and the gene-specific primer 5GSP1 used above. The PCR condition was identical with that described above. The PCR product was excised and sub-cloned as described above.The 5′ RACE was conducted with BD SMART™ cDNA Amplification Kit .Amplification of full-length cDNA was confirmed with NUP and FGSP1: 5′GAAAGTTTCGAAAATTGTGTG-3′. The cDNA in 5′ RACE was used as template. The PCR amplification was performed with Advantage™ 2 PCR Kit (Clontech) under the following conditions: an initial denaturation at 94 °C for 3 min, followed by 30 cycles of 94 °C for 30 sec, 55 °C for 30 sec and 72 °C for 2 min and a final extension at 72 °C for 10 min. All the gene-specific primers used in 3′ RACE and 5′ RACE was designed utilizing Primer Premier 5.0 (http://www.ncbi.nlm.nih.gov). A phylogenetic tree was constructed by MEGA version 3.1 (DNA sequence was determined using the ABI-PRISM 3730 sequencer (Invitrogen). Searching of similar sequences was performed using BlastP in the non-redundant protein sequences (nr) database of the NCBI website  is shown in n et al.  and Nelsn et al. .T. castaneum and other insect CYP6 members than families of CYP1A1, CYP2A1, CYP3A1, CYP4A1, CYP5A1 from mammals and CYP302A1, CYP314A1, CYP315A1, CYP346A1 from T. castaneum. Not surprisingly, phylogenetic analysis of amino acid sequences from members of various P450 families indicated closer phylogenetic relationship of CYP345D3 with CYP6 members from T. castaneum and other insects than from mammals and a more distant relationship to P450s from other families.The deduced amino acid sequence of CYP345D3 contains all important motifs characteristic of the P450 enzymes, particularly the CYP6 family. Using MEGA version 3.1 software , a phylohttp://www.hgsc.bcm.tmc.edu, SearchLauncher, Human Genome Center, Baylor College of Medicine, Houston, TX) for protein secondary structure prediction (Gene cloning by PCR using degenerate primers derived from conserved amino acid sequences from other species has proven to be a powerful method to obtain related DNA sequences from the target species. Although the P450 super-family has a very divergent sequence and the overall homology maybe less than 40% even within the same family, particular in insects , there aediction  indicateediction . These rT. castaneum, while the nucleotide sequences share less than 80% identity with each other. However, the CYP345D3 sequence is not present in the Tribolium assembly from the genome project. This suggsts that the Tribolium species are highly polymorphic, and the strain used here is different than that used in the genome project. Analysis of the CYP345D3 sequence, revealed the conserved functional sites. However, the real function of the sequence is still unknown.The deduced amino acid sequence of CYP345D3 shares 91% identity with probable CYP345D1 as computed from the genome of Escherichia coli and Pichia postoris can be used to identify the biochemical characteristics of this enzyme and validate the function of it. In conclusion, the isolation of the CYP345D3 sequence provides some basic knowledge to understand the P450 enzyme system in T. castaneum. However, better understanding of the system needs much further study.Molecular cloning of CYP345D3 is just the first step to study the cytochrome P450 system in the red flour beetle, which has not been very well studied relative to their detoxification of insecticides. In future research, heterogenous expression of CYP345D3 such as in


Phanerochaete chrysosporium was demonstrated in this study. The carbon monoxide difference spectra indicated induction of P450  by pentachlorophenol. The pentachlorophenol oxidation by the microsomal P450 was NADPH-dependent at a rate of 19.0±1.2 pmol min−1 (mg protein)−1, which led to formation of tetrachlorohydroquinone and was significantly inhibited by piperonyl butoxide (a P450 inhibitor). Tetrachlorohydroquinone was also found in the cultures, while the extracellular ligninases which were reported to be involved in tetrachlorohydroquinone formation were undetectable. The formation of tetrachlorohydroquinone was not detectable in the cultures added with either piperonyl butoxide or cycloheximide (an inhibitor of de novo protein synthesis). These results revealed the pentachlorophenol oxidation by induced P450 in the fungus, and it should be the first time that P450-mediated pentachlorophenol oxidation was demonstrated in a microorganism. Furthermore, the addition of the P450 inhibitor to the cultures led to obvious increase of pentachlorophenol, suggesting that the relationship between P450 and pentachlorophenol methylation is worthy of further research.The occurrence of cytochrome P450 and P450-mediated pentachlorophenol oxidation in a white rot fungus  Pentachlorophenol (PCP) has been widely applied for decades around the world, as a fungicide (mainly as wood preservative), herbicide, defoliant and detergent supplement in soaps Flavobacterium sp.) Biotransformation by microorganisms is an important fate of PCP in the environment and contaminated sites. The white rot fungi, which can decompose lignin, can effectively transform PCP and were widely studied for the treatment of PCP-contaminated soil Mucor ramosissimusMycobacterium chlorophenolicum (formerly Rhodococcus chlorophenolicus) Cytochrome P450s (P450s) are widely distributed hemoproteins involved in various steps of the biosynthesis of endogenous compounds and in the oxidative detoxification and elimination of many hydrophobic xenobiotics including pollutants, drugs and pesticides Phanerochaete chrysosporium has more than 150 P450 genes, which is one of the largest P450 contingents known to date in fungi P. chrysosporium P450 by PCP according to the carbon monoxide (CO) difference spectrum with a peak at 457 nm, and found an increase of PCP degradation with the presence of P450 inhibitor The white rot fungus PCP, pentachloroanisole (PCA), and piperonyl butoxide (PB) were obtained from Aldrich, and TCHQ was obtained from Supelco. Cycloheximide (CHI) was obtained from Roche.P. chrysosporium strain BKM-F-1767 (ATCC 24725) was maintained on Difco potato dextrose agar (PDA). Difco potato dextrose broth  was used as nutrient-rich medium.Inocula were prepared according to the method of Aiken and Logan µmol (0.25 mg) of PCP dissolved in 25 µl of acetone was added to each flask to a final concentration of 37.5 µM in the cultures. Two types of controls were prepared, one with sterile culture (autoclaved at 121°C for 20 min) and PCP and the other with intact culture but without PCP. The fungal biomass of the sterile control was approximately equivalent to that in the experimental cultures. All the flasks were incubated at 37°C in darkness (to prevent photooxidation of the PCP). The cultures were routinely examined by microscopy to ensure the absence of bacterial contamination.Then, 0.938 The flasks were incubation for another 4 days (unless otherwise stated) after addition of PCP.PCP and the metabolites were extracted according to Reddy et al. −1. UV absorbance spectra were determined with the diode array spectrophotometer (Hewlett Packard G1315A) and compared to those of standards. PCP and the quinones were detected at 238 nm and 285 nm, respectively.Reverse phase HPLC analysis of metabolites was conducted as described by Reddy et al. −1, after which the temperature was held isothermally at 300°C for 3 min. The derivatized products were identified by comparing their retention times on chromatography and their mass spectra with derivatized standards, and quantitated by using calibration curves obtained with derivatized standards.GC-ECD analysis was performed on an Agilent 6890N GC with a HP-5 capillary column (30 m×0.25 mm×0.32 µm). GC-MS analysis of derivatized metabolites was performed on a DSQ GC-MS  with a quadrupole mass filter and a VF-5 MS  capillary column (30 m×0.25 mm×0.25 µm), and ran in the electron ionization mode with electron energy of 70 eV. The injector temperature was 200°C. The column was held at 50°C for 2 min, and programmed to rise to 300°C at 10°C•ming for 10 min and filtered through 0.2 µm-pore-size filter to obtain extracellular fractions.To prepare extracellular fractions, the culture supernatants were periodically sampled during the incubation. The samples were centrifuged at 9,000 g, 15 min, twice) and the fungal pellets were washed extensively with 0.1 M sodium phosphate buffer  containing 10 mM EDTA. The microsomal fraction was then prepared according to Masaphy et al. g for 15 min twice to remove cell debris, nuclei and mitochondria. Then the supernatant was centrifuged at 105,000 g for 90 min to pellet the microsomal fraction. After washing twice with buffer B, the microsomal fraction was suspended in buffer B and stored at −80°C.To prepare microsomal fractions for P450 study, some flasks were incubated for 24 hours after addition of PCP dissolved in acetone. Non-induced and acetone-induced cultures were set up as controls for analysis of P450 induced by PCP. The mycelia were harvested by centrifugation  and a baseline spectrum was spectrophotometrically recorded in the range of 400∼500 nm . One cuvette was then gently gassed with CO at a rate of 3 ml·min−1 for 40 s, and the other one was gassed with N2 to the same extent. An equal volume (10 µl) of sodium dithionite solution (400 mg·ml−1) was accurately added into each cuvette, and then a difference spectrum was recorded. The concentrations of P450 were calculated using the extinction coefficient ε450–490 value of 91 mM−1·cm−1The P450 contents of microsomal fractions were determined by CO difference spectra as described previously 2, 1 mM NADPH, 5 mg BSA, microsomal fractions from PCP-induced cells (1∼1.4 mg of protein) and 37.5 µM PCP in 100 mM potassium phosphate buffer (pH 7.5). Three types of control reactions, no-NADPH control, sterile control and the control using microsomal fractions from non-induced cell, were run parallel. The reaction mixtures were incubated at 37°C overnight, and then extracted with ethyl acetate at pH 2, dried over sodium sulfate, evaporated under N2 and analyzed by GC-ECD after acetylation. The inhibition of P450 activity was determined with PB (a P450 inhibitor) added to the concentration of 2 mM in the reaction mixture. The decrease in the amounts of PCP transformed and TCHQ formed was measured by GC-ECD.The 1-ml assay solution contained 3 mM MgCl2+ as substrate. Laccase activity was assayed with 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonate) as substrate. One unit (U) of enzyme oxidizes 1 µmol of the substrate per min in the presence of H2O2.The activities of LiP, MnP and laccase were determined spectrophotometrically by the method of Tien and Kirk 4 (0.2 mM), and H2O2 (0.1 mM) in 50 mM sodium malonate (pH 4.5), and the second one had extracellular fractions (1 ml in the 2-ml mixture), PCP (37.5 µM), veratryl alcohol (0.1 mM), H2O2 (0.1 mM) in 20 mM sodium succinate (pH 3.0). Reactions were carried out at 25°C for 4 days. Reaction mixtures were extracted with ethyl acetate, evaporated under N2 and analyzed by GC-ECD after acetylation as described previously Two types of extracellular reaction mixtures were set according to the MnP and LiP reaction mixtures reported by Reddy et al. de novo synthesis of proteins and to inhibit protease µM), incubated for another 4 days and extracted as described above. Transformation rate of PCP and formation of PCP metabolites were analyzed by GC and compared to those controls without PB or CHI.The PDB cultures were incubated for 60 h at 37°C in the dark. Then, PB was added to some flasks to a final concentration of 2 mM, while CHI (1.46 mM) and PMSF (0.5 mM) were added to some other flasks to suppress The assays were all performed in triplicate and the mean values and standard deviations of the data were presented.µM PCP in 1 day (6.7±1.1 µM·day−1 on average), and 45±3% in 4 days (4.2±0.3 µM·day−1 on average). The average transformation efficiency was 1.37±0.23 µmol·day−1·g−1 (µmol per day per gram dry weight of biomass) in 1 day, and 0.772±0.047 µmol·day−1·g−1 in 4 days.In the PDB cultures, the fungus was able to transform 18±3% (the mean ± standard deviation of three replicates) of added 37.5 µM and 0.31±0.09 µM respectively. This result indicated that methylation was the major but not the only way of PCP biotransformation by P. chrysosporium.After concentration of the acetylated extract, the metabolites were analyzed by comparing the GC profiles  with thoP. chrysosporium which was only reported to be catalyzed by the extracellular ligninases, LiP and MnP The production of TCHQ indicated PCP oxidation by To demonstrate the existence of P450 during transformation of PCP by P. chrysosporium, CO difference spectra of microsomal fractions from PCP-induced cells were recorded. The spectra of microsomal fractions showed peaks at 450 nm , indicatin vitro transformation of PCP (−1), and 6.5±2.1 µM TCHQ was detected after 10-hour incubation. PB, a P450 inhibitor, was used at 2 mM for inhibition assay. When the mixture was added with PB, PCP transformation rate was decreased by 97% (to 0.6±0.4 nM min−1) and TCHQ was not detectable. Furthermore, PCP transformation was not obvious (at an average rate of 0.5±0.4 nM min−1) in the mixture with P450 but no NADPH. The reaction mixture without detectable P450  and the sterile control had no significant difference in PCP concentration. According to NADPH-dependent PCP transformation, the reaction rate of PCP transformation by the microsomal P450 was up to 19.0±1.2 pmol min−1 (mg protein)−1.The microsomal fraction with active P450 was utilized for n of PCP . The mic(31 folds higher than the detection limit) after incubation for 4 days −1 (about 1.1 nmol·h−1·[mg protein]−1). This is similar to the value of 1.75∼2.2 nmol·h−1·(mg protein)−1 in the case of the P450-type protein in M. chlorophenolicum−1·(nmol P450)−1 in microsomal fraction of yeast expressing the human P450 34A3 According to all the evidences described above, we conclude that P450 is involved in PCP transformation by P. chrysosporium under nutrient-rich condition. Moreover, the TCHQ produced by P450-mediated oxidation was expected to be effectively dechlorinated by P. chrysosporium according to Reddy and Gold Laugero et al. P. chrysosporium, which is consistent with previous studies 4 in fish) and can be rapidly demethylated in both rat and mouse to its more toxic precursor The present work found PCA was the major product of PCP in Phlebia radiate recently Interestingly, we found obvious increase of PCA in the cultures degrading PCP when P450 was inhibited. PCP methylation was the major source of PCA in environments