Streptomyces species are a major source of antibiotics. They usually grow slowly at their optimal temperature and fermentation of industrial strains in a large scale often takes a long time, consuming more energy and materials than some other bacterial industrial strains . Most thermophilic Streptomyces species grow fast, but no gene cloning systems have been developed in such strains.S. coelicolor), moderately thermophilic (growing at both 30°C and 50°C) Streptomyces strains, detection of one linear and three circular plasmids in them, and sequencing of a 6996-bp plasmid, pTSC1, from one of them. pTSC1-derived pCWH1 could replicate in both thermophilic and mesophilic Streptomyces strains. On the other hand, several Streptomyces replicons function in thermophilic Streptomyces species. By examining ten well-sporulating strains, we found two promising cloning hosts, 2C and 4F. A gene cloning system was established by using the two strains. The actinorhodin and anthramycin biosynthetic gene clusters from mesophilic S. coelicolor A3(2) and thermophilic S. refuineus were heterologously expressed in one of the hosts.We report here the isolation of 41 fast-growing (about twice the rate of Streptomyces species. Although just a few plasmids and one antibiotic biosynthetic gene cluster from mesophilic Streptomyces were successfully expressed in thermophilic Streptomyces species, we expect that by utilizing thermophilic Streptomyces-specific promoters, more genes and especially antibiotic genes clusters of mesophilic Streptomyces should be heterologously expressed.We have developed a gene cloning and expression system in a fast-growing and moderately thermophilic  Streptomyces species are high G+C, Gram-positive bacteria that are a major source of natural products, producing about half of all known microbial antibiotics [Streptomyces coelicolor A3(2) is the genetically most studied Streptomyces species from the in vivo through in vitro to in silico phases and is an excellent model for studying antibiotic production and differentiation [Escherichia coli into A3(2), the closely related S. lividans, with no such barrier and cured of indigenous plasmids  grow slowly at their optimal temperature  - grows exponentially with a doubling time of about 2.2 h on SMM medium at 28°C, see ref [Streptomyces strains to produce and accumulate antibiotics at a good yield on an industrial scale.ibiotics . Membersibiotics . Streptontiation . Mainly nd SLP3: ), has bend SLP3: . However see ref ). It takStreptomyces strains have been studied for a long time. Some earlier described thermophilic Streptomyces species  have been employed for expression of some genes or antibiotic biosynthetic gene clusters from thermophilic Streptomyces species [Fast-growing, thermophilic ofuscus: ,8) were ofuscus: ,10. Numeptomyces ,12. Mostmophilic ,12. Howe to 68°C ; the optc strain . Growth perature ; for exa at 50°C . Thermopibiotics , such asibiotics , alpha-aibiotics , granatiibiotics  and anthibiotics . Since t species -22.S. coelicolor) and moderately thermophilic (growing at both 30°C and 50°C) Streptomyces strain, and successful heterologous expression of antibiotic biosynthetic gene clusters from both thermophilic and mesophilic Streptomyces species.We report here the development of a gene cloning system in a fast-growing . As summarized in Table Streptomyces species have been isolated from composts, soil and sewage [To isolate thermophilic d sewage , as welld sewage . Our resStreptomyces, 16S rRNA genes were amplified, cloned and sequenced. The sequences displayed high homology (97-99%) to those of known mesophilic Streptomyces and/or thermophilic Streptomyces species. A phylogenetic tree was drawn by using a neighbor-joining method [Streptomyces species. As shown in Figure Streptomyces species . Moderately thermophilic Streptomyces species form at least two distinct clades [S. megasporus and S. thermodiastaticus, respectively. The phylogenetic tree of the 11 newly isolated strains reveals more clades . Thus strains 4F and 2C were classified in the genus Streptomyces.Like typical Streptomyces strains (S. coelicolor M145 and S. venezuelae ISP5230) grew at 30°C and 37°C. 4F and 2C grew well at 45°C and 50°C but poorly at 55°C, while M145 and ISP5230 could not grow at 45°C and 50°C (data not shown). Thus, 4F and 2C were concluded to be moderately thermophilic Streptomyces strains.As shown in Figure Streptomyces strains at 30°C and 37°C  in liquid medium with a diluted inoculum [Strains 4F and 2C grew on MS medium at 37°C and 45°C faster than the mesophilic inoculum . The douGU271942), with 72% G+C, resembling that of a typical Streptomyces genome : [Streptomyces or Mycobacterium genes  of Streptomyces plasmids pIJ101 [We detected three circular plasmids, 7-kb pTSC1, from X4-3, 7.5-kb pTSC2 from X3-3, and 40-kb pTSC3 as well as 16-kb linear pTSL1 from T6-1-4. The complete nucleotide sequence of the circular pTSC1 consisted of 6996 bp (GenBank accession number r A3(2): ; seven os pIJ101  and pSNAs pIJ101 .Streptomyces protoplasts with slight modifications (see Methods), pTSC1-derived pCWH1  could be used in strains 2C and 4F. No antibacterial activity  was detected in the two strains (unpublished data). Thus, we found two promising cloning hosts, 2C and 4F.Followed the standard protocols of preparation and transformation of Streptomyces, several mesophilic Streptomyces vectors were employed for transformation experiments. As shown in Table S. lividans ZX7 [4/μg DNA). A Streptomyces integrating plasmid, pSET152 [E. coli into many thermophilic Streptomyces strains (14 of 22 strains). Thus, pTSC1-derived pCWH1 can replicate in both thermophilic and mesophilic Streptomyces strains. On the other hand, several Streptomyces replicons, including circular plasmids pIJ101, pFP1 and pFP11 and linear plasmid pFRL2, were able to propagate in the thermophilic Streptomyces strains 2C and 4F, but no transformants were obtained for circular plasmids SCP2 and SLP1 and linear plasmids SAP1 and pSHK1.Since 2C and 4F were classified in the genus dans ZX7  at high  pSET152 , could b6 and 1.3 × 106) were obtained in 2C with pIJ702 from both 2C itself and the largely restriction-free S. lividans ZX7. Low frequencies of transformation (8 × 101 and 3 × 102) were obtained in 4F with pIJ702 from 2C and ZX7, although a high frequency (1.2 × 105) was obtained with plasmid DNA from the strain itself. These results indicated that strain 2C showed essentially no restriction barrier to the introduction of foreign double-stranded DNA from other Streptomyces species, whereas strain 4F had a strong restriction barrier. The evaluation of restriction barriers needs much more experimental data to be supported.Comparing the transformation frequencies of pIJ702 from different hosts in 2C and 4F, as shown in Table Streptomyces plasmids functioned in thermophilic Streptomyces, we chose a phage phiC31-derived integrating plasmid pSET152 [Streptomyces strains. By using PCR with eight primers from the actinorhodin biosynthetic genes (sco5085-5092), we found that no bands for strains 4F and 2C were detected on agarose gel after electrophoresis of the PCR products, indicating no such genes in the strains. We cloned the complete actinorhodin biosynthetic gene cluster from S. coelicolor A3(2) in an integrating plasmid (see Methods), and the resulting plasmid, pCWH74, was introduced by conjugation into eight newly isolated strains, including 4F and 2C. PCR amplification experiments with eight paired primers from SCO5085 to SCO5092 confirmed the presence of the actinorhodin genes in the clones of 4F and 2C. Blue pigment was observed for strain 4F on both R2YE and MS media at 30 and 37°C after growth for 1 d, but no blue pigment was seen at 45°C. 2C with the actinorhodin gene cluster did not produce visible blue pigment on R2YE or MS media. To confirm that the blue pigment was actinorhodin, 4F containing pCWH74 was cultured in R2YE liquid medium lacking KH2PO4 and CaCl2 and the supernatant was treated with KOH and scanned at 640 nm [S. coelicolor A3(2) (data not shown). Thus the actinorhodin biosynthetic gene cluster from the mesophilic S. coelicolor A3(2) was heterologously expressed in strain 4F at low temperature (30 and 37°C), but not at high temperature (45°C).Since several mesophilic  pSET152  which ist 640 nm . The sam2PO4 and CaCl2, and 1 ml culture was harvested in a time-course. As shown in Figure 2PO4 and CaCl2 at 30°C showed that their biomass values were similar from 20 to 120 hours (data not shown). Thus, better growth of M145 and 4F in TSB medium , but not at 30 or 37°C [E. coli into strain 4F. PCR amplification experiments confirmed the presence of the anthramycin genes in the clone of 4F. After culturing in AP1 medium at 30, 37 and 47°C for 24 h, mycelium was extracted, dried and re-dissolved in MeOH. Thin-layer chromatography, followed by a bio-assay by overlaying with LB agar containing as indicator strain a Bacillus sp., revealed a zone of growth inhibition on 4F at 47°C, but no inhibition zone was found at 30 and 37°C (data not shown). A spot on a TLC plate was further purified for HPLC-MS analysis. As shown in Figure S. refuineus subsp. thermotolerans was heterologously expressed in strain 4F. We introduced the same cosmid 024COA-3 containing the anthramycin gene cluster into strain 2C, but no transformants were obtained. To see if strain 2C might be a better host than 4F, more antibiotic biosynthetic gene clusters should be tested.Expression of the anthramycin biosynthetic genes of  or 37°C . An inteStreptomyces species could be developed. Two antibiotic biosynthetic gene clusters from mesophilic and thermophilic Streptomyces were heterlogously expressed in one strain. We expect that by utilizing thermophilic Streptomyces-specific promoters, more genes and especially antibiotic genes clusters of mesophilic Streptomyces should be heterologously expressed.This study shows that by isolation of new strains and testing several plasmids, a host-vector system in a fast-growing and moderately thermophilic E. coli DH5α and PCR amplification followed Sambrook et al. [Streptomyces culture, plasmid isolation and preparation of protoplasts and transformation of Streptomyces lividans ZX7 followed Kieser et al. [E. coli ET12567/pUZ8002 into thermophilic Streptomyces strains followed Bierman et al. [KpnI-treated pTSC1 was cloned in pBluescript II SK to obtain pCWH100 and was sequenced by primer-walking at Shanghai Invitrogen Inc. Sequence comparisons were done with software from the National Center for Biotechnology Information http://www.ncbi.nlm.nih.gov/BLAST. The complete nucleotide sequence of pTSC1 was deposited in the GenBank database under no. GU271942.Strains used in this work are listed in Table k et al. . Streptor et al. . Plasmidn et al. . KpnI-tr3 2 g, MgSO4.7H2O 0.05 g, FeSO4.7H2O 0.01 g, CaCO3 0.02 g, agar 18 g, H2O to 1000 ml, pH7.2) [Streptomyces strains were cultured in TSB  liquid medium at 45°C for 1 d and genomic DNA was isolated followed the Kirby mix procedure [Samples of garden soil, weed compost and swine manure were collected from Shanghai city, Hunan, Hubei and Fujian provinces in the summers of 2005 and 2006. The samples were dried at 100°C for 1 h and cultivated on SC medium   at 50°C 2O to 1000 ml, pH7) medium covered with cellophane disks. After 2 days incubation at 42°C, the cells were fixed with fresh 2% glutaraldehyde (pH7.2) and 1% osmium tetroxide. Spores were examined with a JSM-6360LV scanning electron microscopy (Jeol).Strains were inoculated on MS  and cultured at 30, 37, 45 and 50°C. 1 ml culture was harvested at each time-point and wet mycelium was harvested by centrifugation at 12000 rpm for 5 min. After drying for 10 min in a vacuum, the pellet was weighed with a fine balance (min. 10 mg). Growth curves were drawn with an average of three weighings at each time-point.About 1.5 × 10Streptomyces strains 2C and 4F followed standard Streptomyces protocols [9 spores were inoculated into 50-ml YEME liquid medium  at 45°C for ~7 h. Mycelium was harvested, washed once with 10.3% sucrose, and 1 mg/ml lysozyme solution in P buffer was added at 30°C (ca. 15 min for 2C and 30 min for 4F) to make protoplasts. After transformation, regeneration of protoplasts was achieved on R2YE medium at 45°C for ca. 9 h, to be selected by antibiotics.Protoplast preparation, regeneration and transformation of the thermophilic rotocols ,45 with Streptomyces strains were measured by electrophoresis with known DNA markers (i.e. 1-kb supercoiled ladder and sequenced circular/linear plasmids). pQC156 [Streptomyces selection markers melC/tsr was cloned in an E.coli plasmid pSP72. KpnI-treated pTSC1 was cloned in pQC156 to obtain pCWH1. The mesophilic Streptomyces replicons, including circular plasmids SCP2 [Streptomyces strains.Plasmids used in this work are listed in Table . pQC156  containiids SCP2 , pFP1 anids SCP2 , linear ids SCP2 , pFRL2 [ids SCP2  and pSHKids SCP2 , and intids SCP2 , were clE.coli replication origin and two cos sites of Supercos1 [Streptomyces selection markers melC/tsr genes [S. coelicolor A3(2). A 3.4-kb XbaI/NheI fragment containing the phage фC31 integrase gene of pSET152 was cloned in a XbaI site of N7-85. The resulting plasmid, pCWH74, was introduced by conjugation from E. coli into thermophilic Streptomyces strains [2SO4 0.25 g, MgCl2.6H2O 10.12 g, glucose 10 g, Difco Casaminoacids 0.1 g, trace element solution 2 ml, Difco yeast extract 5 g, TES 5.73 g, agar 22 g, H2O to 1000 ml, after autoclave and add 0.5% KH2PO4 5 ml, 5 M CaCl2.2H2O 4 ml, 20% L-proline 15 ml, 1N NaOH 7 ml) and MS media at 30, 37 and 45°C to detect blue actinorhodin pigment. To quantitate the production of actinorhodin, about 1 × 106 spores of M145 and 4F containing pCWH74 were inoculated into 50 ml R2YE liquid medium (lacking KH2PO4 and CaCl2) at 30 and 37°C; 1 ml culture was harvested in a time-course and treated with KOH, whereupon absorption at OD640 indicated actinorhodin production [pHAQ31  containeupercos1  and Stre strains , which woduction .E. coli into strain 4F [et al. [2O to 1000 ml, pH7) medium at 47°C for 24 h, mycelium was extracted, dried and re-dissolved in MeOH. Anthramycin was first isolated on a HPLC column (Zorbax eclips 1.8 μm XDB-C18) and then mass spectrometry was performed using 6520 Agilent Accurate-Mass Q-TOF LC/MS. Anthramycin was separated by using a Zorbax eclips 1.8 μm XDB-C18 with a linear water-acetonitrile gradient containing 10 mM ammonium acetate (0.2 ml/min). The electrospray needle of the mass spectrometer was at 4000 V, the voltage of the skimmer was set to 65 V, Oct RF Vpp750V, collision ev 45 V, nebulizer pressure at 45 psig, and drying gas N2 350°C 9 L/min.An integrating cosmid, 024COA-3, containing the whole anthramycin biosynthetic gene cluster  (kindly provided by Prof. Brian Bachmann) was introduced by conjugation from train 4F . Detecti [et al. . After cWHC designed and performed all the experiments. ZJQ was involved in project design, and prepared the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest.Predicted ORFs of plasmid pTSC1. Detailed information and possible functions of the eight ORFs of pTSC1.Click here for file


The in situ growth of CIS nanocrystals has been realized by solvothermally treating nanoporous TiO2 film in ethanol solution containing InCl3 · 4H2O, CuSO4 · 5H2O, and thioacetamide with a constant concentration ratio of 1:1:2. InCl3 concentration plays a significant role in controlling the surface morphology of CIS layer. When InCl3 concentration is 0.1 M, there is a layer of CIS flower-shaped superstructures on TiO2 film, and CIS superstructures are in fact composed of ultrathin nanoplates as ‘petals’ with plenty of nanopores. In addition, the nanopores of TiO2 film are filled by CIS nanocrystals, as confirmed using scanning electron microscopy image and by energy dispersive spectroscopy line scan analysis. Subsequently, HSC with a structure of FTO/TiO2/CIS/P3HT/PEDOT:PSS/Au has been fabricated, and it yields a power conversion efficiency of 1.4%. Further improvement of the efficiency can be expected by the optimization of the morphology and thickness of CIS layer and the device structure.Inorganic/organic heterojunction solar cells (HSCs) have attracted increasing attention as a cost-effective alternative to conventional solar cells. This work presents an HSC by  The power conversion efficiency of DSSCs with organic solvent-based electrolyte has been reported to exceed 11%[The quest and demand for clean and economical energy sources have increased interest in the development of various solar cells, such asar cells-6, organar cells, and dyear cells-12. Amonar cells-12. The ceed 11%,13,14. H2 films, two typical approaches have been developed. The first and most common route is the in situ synthesis of the nanocrystals on TiO2 film, for example, by chemical bath deposition[2 substrates by direct adsorption[ex situ approach could achieve better control over the sizes and electronic properties of nanocrystals but suffers from low surface coverage and poor electronic coupling[For reducing the cost, the use of inorganic semiconductor nanocrystals instead of Ru-based dyes in DSSCs has attracted an enormous interest-18. Semiposition or by suposition. This meposition. The secsorption or linkesorption. This excoupling. Up to ncoupling,22,25, Ccoupling,26, and coupling-29. Unfo2/Sb2S3/P3HT has reached 5%[2S3 nanocrystals are non-toxic compared with Cd/Pb-based semiconductors. These facts show the great potentiality of all-solid HSCs, which also encourages to further achieve other kind of robust, efficient, and cheap HSCs without toxic component.On the other hand, for avoiding the sealing problem in DSSCs, many attempts have been made to substitute liquid electrolytes with quasi-solid electrolytes or solidached 5%, which i2, abbreviated as CIS) has a small direct bandgap of 1.5 eV that matches well the solar spectrum, a large absorption coefficient (α = 5 × 105 cm−1), and low toxicity. It has been regarded to be a promising light-absorbing material for film solar cells[2 film to construct DSSCs with liquid electrolyte[in situ growth of CIS on TiO2 film has also been realized, by electrodeposition[2 film for the construction of all-solid HSCs. In this paper, we report a facile one-step solvothermal route for the in situ growth CIS nanocrystals on nanoporous TiO2 film. The effects of reagent concentration on the surface morphology of CIS have been investigated. The all-solid HSC with the structure of FTO/compact-TiO2 /nanoporous-TiO2/CIS/P3HT/PEDOT:PSS/Au is fabricated, and it exhibits a relatively high conversion efficiency of 1.4%.Copper indium disulfide . TiO2 (P25) was obtained from Degussa. Transparent conductive glass  was purchased from Wuhan Geao Instruments Science & Technology Co., Ltd . P3HT was bought from Guanghe Electronic Materials Co., Ltd. . The poly(3-4-ethylenedioxythiophene) doped with poly(4-stylenesulfonate) (PEDOT:PSS) solution  was obtained from Aldrich .All of the chemicals were commercially available and were used without further purification. Titanium butoxide, petroleum ether, TiCl2 layer was first deposited onto the FTO glass as follow[V/V), taken out carefully, hydrolyzed in air for 30 min, and sintered in oven for 30 min at 450°C.A part of FTO glass was chemically etched away in order to prevent direct contact between the two electrodes. A compact (about 100-nm thick) TiOs follow. FTO gla2 films were prepared by the doctor-blading technique with TiO2 (P25) colloidal dispersion and subsequently sintered at 450°C for 30 min, according to a previous study[4 (20 mM in water) solution for 12 h. It was then washed with deionized water and ethanol, dried with air, and sintered again at 450°C for 30 min.Then, nanoporous TiOus study. This fiin situ grown on nanoporous TiO2 films by a solvothermal process. In a typical process, thioacetamide  was added into a 12 mL ethanol solution containing InCl3 · 4H2O (0.01 M) and CuSO4 · 5H2O (0.01 M) under magnetic stirring, until a clear solution was formed. The resulting solution was transferred into a Teflon-lined stainless steel autoclave with 30-mL capacity. Subsequently, FTO/compact-TiO2/nanoporous-TiO2 film as the substrate was vertically immersed into the solution. Lastly, the autoclave was kept in a fan-forced oven at 160°C for 12 h. After air-cooling to room temperature, CIS film on non-conductive glass side was scraped off, while CIS film on nanoporous TiO2 film side was washed with deionized water and absolute ethanol successively, and dried in air. For comparison, the effects of InCl3 · 4H2O concentrations  on the morphologies CIS layer were investigated. The concentration ratio of InCl3 · 4H2O, CuSO4 · 5H2O, and thioacetamide was maintained constant (1:1:2) for all the cases.CIS layer was 2/CIS with 3,000 rpm for 60 s. Then, in order to improve the contact between P3HT and gold, a PEDOT:PSS solution diluted with two volumes of methanol was introduced onto TiO2/CIS/P3HT layer by spin-coating at 2,000 rpm for 30 s[2/CIS/P3HT/PEDOT:PSS layer was then annealed at 90°C for 30 min in a vacuum oven. Gold layer as the back contact was prepared by magnetron sputtering with a metal mask, giving an active area of 16 mm2 for each device. The resulting HSC has a structure of FTO/compact-TiO2/nanoporous-TiO2/CIS/P3HT/PEDOT:PSS/Au.The P3HT solution  was spin-coated onto TiOfor 30 s. In ordeλ = 1.5418 Å). An ultraviolet/visible (UV-vis) spectrophotometer  was used to carry out the optical measurements. The photocurrent density/voltage curves of HSC was measured under illumination (100 mW cm−2) using a computerized Keithley Model 2400 Source Meter unit  and a 300-W xenon lamp  serving as the light source.The sizes and morphologies of the sample were investigated by field emission scanning electron microscopy . During SEM measurement, energy dispersive spectroscopy  line scan was also performed to locate and determine the distribution of different layer in the composite film. The X-ray diffraction  measurement was carried out using a Cu Kα radiation source  colloidal dispersion  as the reactant, and the solution was solvothermally treated at 160°C for 12 h. It has been found that reactant concentrations play a significant role in the controlled growth of CIS films in our previous study[3 concentration: 0.01, 0.03, 0.1 M) on the surface morphologies of CIS layer were investigated by SEM observation. Figure 3 concentration. When InCl3 concentration is low (0.01 or 0.03 M), a large amount of high-ordered potato chip-shaped CIS nanosheet arrays are densely packed and uniformly covered over the entire surface of FTO/compact-TiO2/nanoporous-TiO2 film  and TiO2 film , the diffraction peaks at 27.8°, 46.5°, and 55.1° are assigned to (112), (204)/(220), and (312)/(116) planes of CIS, respectively, which are consistent with our previous study[2/CIS film was measured using a UV-vis spectrometer, as shown in Figure Furthermore, the phase and optical property of TiO2/nanoporous-TiO2/CIS film by the spin-coating process  of 0.573 V, a short-circuit current density (Jsc) of 4.36 mA/cm2, and a fill factor (FF) of 0.561, yielding an overall energy conversion efficiency (η) of 1.40%. This conversion efficiency has been greatly improved, compared with that  of TiO2/P3HT hybrid HSCs in the absence of dye or PCBM[2, CIS, and P3HT  as well as the data analysis. All authors read and approved the final manuscript.


C462AMdm2 mouse model to characterize in further detail the role of Mdm2's RING domain in the control of p53. Here, we show in vivo that the Mdm2C462A protein not only fails to suppress p53, but compared to the complete absence of Mdm2, Mdm2C462A actually enhances p53 transcriptional activity toward p53 target genes p21/CDKN1A, MDM2, BAX, NOXA, and 14-3-3σ. In addition, we found that Mdm2C462A facilitates the interaction between p53 and the acetyltransferase CBP/p300, and it fails to heterodimerize with its homolog and sister regulator of p53, Mdmx, suggesting that a fully intact RING domain is required for Mdm2's inhibition of the p300-p53 interaction and for its interaction with Mdmx. These findings help us to better understand the complex regulation of the Mdm2-p53 pathway and have important implications for chemotherapeutic agents targeting Mdm2, as they suggest that inhibition of Mdm2's E3 ubiquitin ligase activity may be sufficient for increasing p53 activity in vivo, without the need to block Mdm2-p53 binding.The p53 transcription factor and tumor suppressor is regulated primarily by the E3 ubiquitin ligase Mdm2, which ubiquitinates p53 to target it for proteasomal degradation. Aside from its ubiquitin ligase function, Mdm2 has been believed to be capable of suppressing p53's transcriptional activity by binding with and masking the transactivation domain of p53. The ability of Mdm2 to restrain p53 activity by binding alone, without ubiquitination, was challenged by a 2007 study using a knockin mouse harboring a single cysteine-to-alanine point mutation (C462A) in Mdm2's RING domain. Mouse embryonic fibroblasts with this mutation, which abrogates Mdm2's E3 ubiquitin ligase activity without affecting its ability to bind with p53, were unable to suppress p53 activity. In this study, we utilized the  The p53 tumor suppressor protein is frequently mutated in cancer, with approximately 50% of cancers containing mutations that inactivate p53 itself, and many of the remaining cancers thought to harbor mutations that otherwise inactivate the p53 tumor suppressor pathway in vitro and/or overexpression studies supported the idea that Mdm2 binding alone, without ubiquitination, could suppress p53's transactivational activity Mdm2 has long been thought to inactivate p53 in two ways: by ubiquitinating p53 to induce its degradation, and by binding with p53 to conceal its transactivation domain. Mdm2 serves as an E3 ubiquitin ligase that conjugates a chain of ubiquitin molecules onto p53, targeting p53 for proteasome-mediated degradation C462A/C462AMdm2 (hereafter referred to as m/mMdm2), the separate contributions of Mdm2's E3 ubiquitin ligase activity and its ability to bind with p53 could be analyzed in vivo under conditions of endogenous protein expression. Using mouse embryonic fibroblast (MEF) cells obtained from this model, Itahana et al. showed that the Mdm2C462A protein was capable of binding with p53 yet could not ubiquitinate p53 nor elicit its degradation A recent study by Itahana et al. C462A indeed interacts with p53 on the p21 promoter and that Mdm2C462A fails to suppress transcription of multiple p53 targets, including p21, Mdm2, Bax, Noxa, and 14-3-3σ. Interestingly, we found that Mdm2-p53 binding alone, without ubiquitination, not only fails to inhibit p53, but actually further enhances p53 activity toward each of these targets compared to the complete absence of Mdm2. Finally, we show that binding of Mdm2C462A to p53 enhances the interaction between p53 and the acetyltransferase CBP/p300, suggesting a mechanism for the enhanced p53 activity.While this work suggested that Mdm2-p53 binding alone, without ubiquitination, is not capable of inhibiting p53's activity, two issues became apparent: first, the expression of only one p53 target, aside from Mdm2 itself, was examined, and second, it was not shown that the mutant Mdm2 retained the ability to interact with p53 while on a target gene promoter. The study here aimed to address these concerns and further characterize the contribution of Mdm2's RING domain in suppressing p53. We show that Mdm2C462A on p53's transcriptional activity in vivo using MEF cells. Mice harboring the Mdm2C462A mutation are not viable due to unchecked p53 activity ER), in which p53 is fused with a portion of the estrogen receptor protein, rendering it inactive until treatment with the estrogen mimic, 4-hydroxytamoxifen (4-OHT) m/mMdm2; ER/−p53 mice are viable, and MEF cells from these mice can be used for studies requiring both mutant Mdm2 and active p53, as 4-OHT can be added to cultured MEF cells to induce p53 activation. To assess the effect of the C462A mutation on p53 activity, MEF cells from +/+Mdm2; ER/−p53, −/−Mdm2; ER/−p53, and m/mMdm2;ER/−p53 mice were treated with 4-OHT to reactivate p53 and were lysed after zero, 12, or 24 hours. RNA was isolated from each sample and subjected to RT-PCR to assess transcription of the p53 targets Mdm2, p21, Bax, Noxa, and 14-3-3σ. Transcription of these genes was elevated in the mutant MEFs compared to wild-type cells, confirming Itahana et al.'s finding that the RING C462A mutation renders Mdm2 unable to suppress p53 activity. Surprisingly, however, these p53 targets were expressed to a greater extent in MEFs expressing Mdm2C462A than in Mdm2-null cells. Transcription was elevated in m/mMdm2 MEFs compared to +/+Mdm2 and −/−Mdm2 MEFs at both the 12-hour and 24-hour time points for the five p53 targets examined  analysis was carried out to assess p53-Mdm2 binding on the p21 promoter in m/m;p53ER/−Mdm2 MEFs cells. m/m;p53−/−Mdm2 cells were included as a negative control. The cells were incubated with 4-OHT for 24 hours to activate p53, and formaldehyde was applied to crosslink proteins to DNA. The cells were lysed, sonicated to shear DNA, and immunoprecipitated with p53 antibody or IgG (negative control). A subset of each sample was resolved by SDS PAGE and western blotting, while another portion was subject to reverse crosslinking and PCR targeting the p21 (CDKN1A) promoter   . Westernull MEFs . These dm/mMdm2 MEFs compared to Mdm2-null MEFs. p53 activity can be dramatically increased by acetylation, and p53 is well-known to be acetylated by its transcription cofactor, the acetyltransferase CBP/p300 +/+;p53ER/−Mdm2, m/m;p53ER/−Mdm2, and m/m;p53−/−Mdm2 were lysed with 0.1% NP-40 lysis buffer, immunoprecipitated with p53 antibody, resolved by SDS-PAGE, and blotted for p300 and p53. The presence of the C462A mutation greatly enhanced the interaction between p53 and p300, as evidenced by a stronger band representing p300 after immunoprecipitating p53-containing complexes in the m/m;p53ER/−Mdm2 MEFs compared to the +/+;p53ER/−Mdm2 MEFs. No p300 was immunoprecipitated in the p53-null negative control sample  for Mdm2 in +/+Mdm2;ER/−p53 and m/mMdm2;ER/−p53 MEF cells. We found that the C462A RING mutation disrupts the interaction between Mdm2 and Mdmx , not only is it unable to inhibit p300-mediated acetylation of p53, but the monomeric Mdm2 further enhances this acetylation beyond the basal level found in Mdm2-null cells. This may be a result of monomeric Mdm2 bridging together p300 and p53, as Mdm2 is known to interact with both of these proteins C462A on the p300-p53 interaction is mediated by its ability to heterodimerize with Mdmx, or by another mechanism.How might disruption of Mdm2's RING domain enhance the p53-p300 interaction? Is it due to lack of E3 ubiquitin ligase activity, or is it caused by another essential function of the RING domain? E3 ubiquitin ligase activity is not likely to be essential for inhibiting p53-p300 binding, as Mdm2's sister protein, Mdmx, lacks E3 ligase activity yet has been shown to inhibit p300-mediated acetylation of p53. It is possible that another function of the RING domain influences the interaction. One hypothesis is that formation of an Mdm2-Mdmx heterodimer may be necessary for Mdm2 to inhibit p300, as the RING domain was shown to mediate this heterodimerization and Mdmx , indicatNonetheless, these data show that Mdm2-p53 binding alone is not sufficient for inhibiting p53 activity or p53's interaction with the acetyltransferase p300, enhancing our understanding of the complex regulation of the p53 tumor suppressor pathway.Mice were generated, maintained, and genotyped as described previously RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. cDNA was synthesized using SuperScript III (Invitrogen). Quantitative real-time PCR was performed with SYBR Green using the Applied Biosystems 7900HT Fast Real-Time PCR System, and data was collected and exported with SDS 2.2.2. Relative expression was calculated using GAPDH as an internal control.ER, 100 nM 4-hydroxytamoxifen  dissolved in ethanol was added to the culture medium.MEF cells were cultured in a 37°C incubator with 5% CO2 in DMEM supplied with 10% fetal bovine serum (Gibco) and penicillin (100 IU/ml)/streptomycin (100 µg/ml). To activate p53Cells were lysed in 0.1% NP-40 buffer for immunoprecipitation and 0.5% NP-40 buffer for straight western blotting. Procedures and conditions for immunoprecipitation and immunoblotting were described previously 2, 10 mM KCl, 0.5 mM DTT). Tubes were rotated at 4°C for 30 min and spun down at 13,000 rpm for 5 min. Proteins not crosslinked to chromatin were removed, and pellets were resuspended in Lysis Buffer B . Lysates were sonicated for 10 seconds at 1 min intervals a total of 4 times using a Branson Digital SONIFIER®  and Branson Sound Enclosure (Model SSE-1). Samples were centrifuged for 10 min at 13,000 rpm at 4°C. The supernatant was collected, the protein concentration was measured, and the supernatant was mixed with an equal volume of dilution buffer . Samples were precleared with Protein G Agarose with Salmon Sperm DNA  for 30–60 min at 4°C with agitation. Samples were spun down at 3000 rpm for 2–5 min at 4°C, and supernatant was collected, with 5% reserved for use as a loading control for western blotting. Samples were incubated with antibodies overnight (goat anti-p53 FL393 or goat IgG) using 0.6 µg of antibody per 1 mL sample. 20 µL of Protein G Agarose/Salmon Sperm DNA was added and samples were incubated for 1 hr, spun at 3000 rpm at 4°C for 3 min, and washed sequentially with the following buffers at 4°C: TSE I , TSE II , TSE III , and twice with TE . A portion of beads was resuspended in 1× sample buffer for western blotting. For anticrosslinking and PCR, samples were eluted 3× with 75 µL of elution buffer , vortexed briefly, and incubated at room temperature for 15 min with rotation. Eluates were pooled (200 µL) and 8 µL of 5 M NaCl added. Anticrosslinking was performed at 65°C for 6 h to overnight. Samples were treated with 4 µL of 0.5 M EDTA, 4 µL of 2 M Tris-HCl pH 6.8, 2 µL of 10 mg/ml Proteinase K, 2 µL of 10 mg/ml RNAse A, and incubated for 1 h at 45 C. DNA was recovered with a QiaQuick PCR Purification Kit (Qiagen) and eluted with 50 µL of 10 mM Tris-HCl pH 8.5. PCR was carried out using the following primers for p21 promoter: Promoter mp21 F1  and Promoter mp21 R1  MEF cells were crosslinked using 1% formaldehyde for 10 min at 37°C and washed with PBS. Crosslinking was stopped with 0.125 M glycine in PBS, cells were washed in PBS, centrifuged for 5 min at 1200 rpm, and pellets were resuspended in Lysis Buffer A (10 mM Hepes pH 7.5, 0.5% NP40, 1.5 mM MgCl


For this compound comparable cytotoxic action on non-cancer murine fibroblast cells was detected (IC50=16.5 µM). The obtained results indicate that appropriate lipophilization of the quercetin molecule could improve its cytotoxic action in cells, probably due to its enhanced bioavailability.Quercetin is a natural polyphenol with proven health beneficial activities. In this study 15 new quercetin derivatives were prepared with the aim to enhance their bioavailability. Modification of their physicochemical properties could herewith improve the action in cells. The prepared compounds were tested for their antioxidant and cytotoxic activity. The ability to scavenge free radicals as well as ferric reducing antioxidant power of the new derivatives was not better than that of unmodified quercetin. But for acetylated esters a better cytotoxic activity was found on human cervical cancer cells HeLa than for the initial molecule. The best effect revealed chloronaphtoquinone quercetin (IC In 2007, 28 131 new cancer cases were registered in Slovakia. The incidence and mortality of cancer in recent years in the Slovak population is displaying a raising trend. For this rapid increase and large occurrence mainly prostate and colorectal cancers are responsible in males. In females the dramatic increase in cancer cases was caused by breast, female genital organs and lung cancers , vegetables , soybeans and different herbs . Flavonoids have for decades been known for their broad spectrum of identified biological activities. As a part of medicinal herbs they were used for treatment purposes from ancient times. Later experiments and studies have shown that they dispose of many health beneficial properties, e.g. anti-inflammatory, cardioprotective, anti-allergic or hepatoprotective effects , adult bovine serum (BOS), penicillin-streptomycin solution, thiazolyl blue tetrazolium bromide, 2,2’-diphenyl-1-picrylhydrazyl (DPPH), 2,4,6-tris(2-pyridyl)-S-triazine (TPTZ) and dimethyl sulfoxide (DMSO) for HPLC were purchased from Sigma-Aldrich (DE). All other solvents and reagents were supplied from local companies  and were of analytical grade.et al., Quercetin structure was modified using simple condensation reactions or selective protection procedures and subsequent acylation with acylchlorides (Veverka 2 and 95%-air atmosphere were in Dulbecco's modified eagle medium supplemented with 10% (vol/vol) inactivated bovine serum, penicillin G and streptomycin (100 mg/l). Before a uniform monolayer of cells was formed, cells were freed from the surface of the culture flask by a 0.25% solution of trypsin and were subcultivated two to three times a week. The cells were plated on the cultivation flask (surface 25 cm2) at a density of 4×104 HeLa cells and 6×104 NIH-3T3 cells per ml medium and incubated for 24 h prior to the experiments.The human tumor cell line HeLa and murine fibroblast NIH-3T3 cells were obtained from the ATCC . The cells grown at 37 °C in humidified 5%-CO50) were expressed as the concentration of the derivative tested to react with one half of the DPPH? and were calculated by SigmaPlot.We used the simple spectrophotometric method according to Yen and Chen  to deter50) were expressed as the concentration of the derivative tested reducing one half of ferric complexes and were calculated by SigmaPlot.The antioxidant reducing power of the derivatives tested was performed using the FRAP method according to Benzie and Strain . Ferric in vitro using the mitochondrial cytotoxic test according to Jantová et al. (3 HeLa or NIH-3T3 cells per well. Samples were dissolved in DMSO (stock solution 10 mM) and subsequently diluted in medium to the final concentration of 5 µM to 100 µM (concentration of DMSO 0.5%) and after 24 h they were added to the cells. Microplates were cultivated for 72 h in thermostat at 37 °C and 5% CO2 atmosphere. After incubation thiazolyl blue tetrazolium bromide  was pipetted to each well and left to incubate for further two hours. Then the medium with MTT solution was removed. Formazan crystals in viable cells were dissolved in the lysis solution (4 mM HCl and 0.1% Nonidet P40 in ethanol). Microplates were shaken 15 min at 1500 rpm. Absorbance was measured at 540 nm and reference wavelength at 740 nm . Each value is the mean of 6 wells with standard deviation. Inhibition activity was expressed as percentages of control with DMSO. For each compound IC50 values were calculated by SigmaPlot.The cytotoxic effect of the compounds prepared on cells was detected á et al.  with mod50) are shown in We chose two simple spectrophotometric methods to investigate antioxidant properties of the prepared quercetin derivatives. As reference standard synthetic analogue of vitamin E (trolox) was used. The 50% effective doses of samples (IC2 (IC50=27 µM), chloronaphtoquinone quercetin 5 (IC50=28.4 µM) and tri(diacetylcaffeoyl) quercetin 11 (IC50=35.4 µM), which were as effective as trolox and about one half less effective than quercetin. A certain activity was determined for diquercetin 1 (IC50=58.8 µM), di(tetraacetylquinoyl) quercetin 9 (IC50=63.7 µM), di(prenylferuloyl) quercetin 14 (IC50=82.8 µM) and di(diprenylcaffeoyl) quercetin 8 (IC50=160.9 µM). The derivatives 3, 4, 6, 7, 10, 12, 13, 15 were inactive at the concentrations tested. Their IC50 value was greater than 200 µM.Of the screened samples the best ability to bind free radical DPPH? were monochloropivaloyl quercetin 2 (IC50=12.5 µM), which was as effective as quercetin and three times more effective than the standard antioxidant trolox. Certain activity was found for chloronaphtoquinone quercetin 5 (IC50=64.5 µM), diquercetin 1 (IC50=103.1 µM), di(tetraacetylquinoyl) quercetin 9 (IC50=112.5 µM) and di(prenylferuloyl) quercetin 14 (IC50=183.3 µM). The other compounds tested were inactive up to 200 µM concentration .In the second antioxidant test ferric reducing ability (FRAP) of the samples was evaluated. From 50) for quercetin and its derivatives on HeLa and NIH-3T3 cells. The values were obtained from the MTT test. The highest cytotoxic effect on human cancer cell line HeLa was induced by chloronaphtoquinone quercetin 5 (IC50=13.2 µM), tri(diacetylcaffeoyl)quercetin 11 (IC50=16.5 µM), di(tetraacetylquinoyl) quercetin 9 (IC50=19.5 µM) and pentaacetyl quercetin 6 (IC50=29.6 µM). The cytotoxic effect of these derivatives on cancer cells was higher than the cytotoxicity of quercetin. Certain cytotoxicity was observed in monochloropivaloyl quercetin 2 (IC50=43.4 µM) and diquercetin 1 (IC50=64.0 µM). Eight derivatives tested were inactive, IC50 values were greater than 50 µM for the derivatives 7, 8, 10, 12–15 and IC50 was greater than 100 µM for derivative 4.11 (IC50=10.6 µM), pentaacetyl quercetin 6 (IC50=15.5 µM), di(tetraacetylquinoyl) quercetin 9 (IC50=16.1 µM) and chloronaphtoquinone quercetin 5 (IC50=16.5 µM). Certain cytotoxicity was observed in tri quercetin 3 (IC50=20.3 µM), monochloropivaloyl quercetin 2 (IC50=26.8 µM) and diquercetin 1 (IC50=32.9 µM). Derivatives 4, 7, 8, 10–14 and the derivative 15 were inactive, IC50 values were greater than 100 and 50 µM, respectively.The highest cytotoxicity on the non-cancer cell line NIH-3T3 was initiated by tri(diacetylcaffeoyl) quercetin 5 to the medium reduced HeLa and NIH-3T3 viable cell numbers. The three highest concentrations tested  had an acute effect manifested by cell degeneration (NIH-3T3) or total inhibition of cell proliferation (HeLa) after 72 h of treatment. The cytotoxic effect of chloronaphtoquinone quercetin 5 at concentrations 5 and 10 µM was directly proportional to the concentration and time of influence. Inhibition of cell proliferation was 94.54% and 71.88% (for 25 µM), 97.89% and 85.24% (for 50 µM) and 97.72% and 93.66% (for 100 µM) for HeLa and NIH-3T3 cells, respectively   . Of the et al. quercetin 11, di(tetraacetylquinoyl) quercetin 9 and pentaacetyl quercetin 6 were more effective than quercetin. The comparison of the structures of quercetin derivatives and their cytotoxic effect showed that acetylated esters were the most effective derivatives.The cytotoxic screening of quercetin derivatives on HeLa and NIH-3T3 cells demonstrated that chloronaphtoquinone quercetin et al., et al.  induced morphologic changes which resulted within 72 h in cell rounding and cell separation from the cultivation surface.Light microscopy revealed that both concentrations studied of chloronaphtoquinone quercetin According to the obtained data we can conclude also that the antioxidant activity was not in correlation with the cytotoxic action. Although structure modification lowered the antioxidant properties of quercetin, cancer cell inhibitory activities were enhanced mainly in the case of compound acetylation.50=13.2 µM for HeLa cells, IC50=16.5 µM for NIH-3T3 cells).The best effects were found for chloronaphtoquinone quercetin. This compound inhibited the growth of non-cancer and cancer cells at comparable level (IC


Protein Ser/Thr Phosphatase PPP1CC2 is an alternatively spliced isoform of PPP1C that is highly enriched in testis and selectively expressed in sperm. Addition of the phosphatase inhibitor toxins okadaic acid or calyculin A to caput and caudal sperm triggers and stimulates motility, respectively. Thus, the endogenous mechanisms of phosphatase inhibition are fundamental for controlling sperm function and should be characterized. Preliminary results have shown a protein phosphatase inhibitor activity resembling PPP1R2 in bovine and primate spermatozoa.Here we show conclusively, for the first time, that PPP1R2 is present in sperm. In addition, we have also identified a novel protein, PPP1R2P3. The latter was previously thought to be an intron-less pseudogene. We show that the protein corresponding to the pseudogene is expressed. It has PPP1 inhibitory potency similar to PPP1R2. The potential phosphosites in PPP1R2 are substituted by non-phosphorylable residues, T73P and S87R, in PPP1R2P3. We also confirm that PPP1R2/PPP1R2P3 are phosphorylated at Ser121 and Ser122, and report a novel phosphorylation site, Ser127. Subfractionation of sperm structures show that PPP1CC2, PPP1R2/PPP1R2P3 are located in the head and tail structures.The conclusive identification and localization of sperm PPP1R2 and PPP1R2P3 lays the basis for future studies on their roles in acrosome reaction, sperm motility and hyperactivation. An intriguing possibility is that a switch in PPP1CC2 inhibitory subunits could be the trigger for sperm motility in the epididymis and/or sperm hyperactivation in the female reproductive tract. PPP1CC undergoes alternative splicing, giving rise to a ubiquitous isoform PPP1CC1 and a testis-enriched and sperm specific-isoform, PPP1CC2 . T. T34]. TMass spectrometry data of the same immunoprecipitates showed that PPP1CC2 was also present. Five peptides out of 10 MS/MS spectra were identified, that matched PPP1CC2 with 15% coverage and a protein MASCOT score higher than 200 . The results show that PPP1CC2 binds to PPP1R2/PPP1R2P3 in human spermatozoa  and CBC502 antibodies Figure . ResultsFurthermore, tail and head preparations were also obtained using a sucrose gradient. In this separation although heads are kept intact, tails are demembranated and only the axoneme structure is maintained, thus all soluble proteins present in the tail are removed. The results clearly demonstrate that PPP1R2 and PPP1R2P3, as well as PPP1CC2, are present in the spermatozoa tail and head and in similar amounts Figure B.Western blotting of human sperm heat-treated extracts revealed, depending on the samples, two bands migrating slower than the purified PPP1R2 and PPP1R2P3 Figure A. To invPPP1R2 mRNAs of 1.2 kb, 1.4–1.7 kb, 2.4–2.7 kb and 4 kb have been found in rat and rabbit testis phosphorylase a as substrate. The substrate was prepared from phosphorylase b  using [γ-32P]ATP  and phosphorylase kinase  as previously described . An apprEjaculated sperm were collected from healthy donors by masturbation into an appropriate sterile container. Spermograms were performed by experienced technicians and only samples with normal parameters were used . For allFor the preparation of heads and tails, washed sperm were briefly sonicated and the detachment checked by phase contrast microscopy (PH) using an Olympus IX81 epifluorescence microscope, equipped with appropriate software . Sperm were directly applied onto a sucrose step gradient  to separate heads from tails, by centrifuging at 5000 g for 1 hr and fractions corresponding to tails and heads collected. Subsequently fractions were centrifuged at 16000 g, 10 min, resulting in a pellet free of sucrose and proteins were dissolved in 1% SDS. For immunocytochemistry, washed sperm was used directly on the coverslips. Human testicular biopsy was also prepared using 1% SDS and the same protocol as for sperm samples, but previously homogenized using a tissue homogenizer. Testicular biopsy was collected in Centro Hospitalar de Coimbra, Portugal during a procedure to collect organs for transplantation from a brain death 35-years-old adult man and the biopsy was diagnosed as “normal spermatogenesis” based on histopathological analysis.Written informed consent, for the use of sperm samples, was obtained from the donors for publication and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. The study was conducted in accordance with the guidelines of the “Helsinki Declaration”.It is permitted the dissection of corpses, or parts of them, of national citizens, stateless persons and foreign residents in Portugal, as well as extraction of parts, tissues or organs when the deceased has explicitly declared in life's will that his body can be used for purposes of teaching and scientific research.”).Testicular biopsies for research purposes are covered by the legislation of the Portuguese Constitution  were phosphorylated by GSK3β or CK2  or both kinases as previously described . The phoRIPA supernatant sperm extracts were pre-cleared using dynabeads protein G . A direct immunoprecipitation approach was performed with 1 μg of sheep anti-PPP1R2 or rabbit anti-PPP1R2 pre-incubated with Dynabeads® Protein G during 1 hr at 4ºC with rotation. After incubation, pre-cleared sperm extracts were applied to the antibody-dynabeads complex and incubated overnight with rotation at 4ºC. After washing three times with 1× PBS in 3% BSA for 10 min with rotation at 4ºC, beads were resuspended in loading buffer and boiled.For mass spectrometry analysis, immunoprecipitates were resolved by 10% SDS-PAGE along with purified positive controls. Gels were stained with Coomassie blue colloidal . In brief, gels were fixed with a fixation solution (40% methanol and 10% acetic acid) during 1 hr, washed with distilled water and then transferred to the Coomassie blue colloidal staining solution for 1 hr. After staining, the gels were washed with distilled water and afterwards destained with 25% methanol until bands were visualized.4HCO3) at pH 7.8. Resulting peptides were extracted once with 100 μl of 1% formic acid (FA), and twice with 100 μl of 5% FA, 50% acetonitrile (ACN). Extracts were combined and ACN was removed in vacuo. For LC-MS analysis, a final volume of 40 μl was prepared by addition of 1% FA. Electrospray tandem mass spectrometry (ESI-MS/MS) was performed on an Orbitrap Velos instrument . Fragment ions were generated by low-energy collision-induced dissociation (CID) on isolated ions with a fragmentation amplitude of 0.5 V. MS spectra were summed from four individual scans ranging from m/z 300–1500 with a scanning speed of 8.100 (m/z)/s. MS/MS spectra were a sum of two scans ranging from m/z 100–2800 at a scan rate of 26.000 (m/z)/s. Generated data were imported to ProteinScape™, a proteomics data platform  and analyzed using MASCOT  search algorithm with search parameters as follows: precursor ion tolerance of 1.2 and 0.3 Da for MS/MS spectra. Proteins were considered to be identified if the Mascot score (ProteinScape™) was higher than 65.Bands were excised directly from the gel using a spatula and completely destained. In-gel digestion was performed overnight at 37°C with trypsin  in 10 mM HCl and 50 mM ammonium hydrogen carbonate . Immunoprecipitates and extracts were resolved by 10% SDS-PAGE. Proteins were subsequently electrotransferred onto nitrocellulose membranes and immunodetected with the appropriate antibodies, using ECL detection . The primary antibodies used in this study included sheep polyclonal anti-PPP1R2 (1:100), the rabbit CBC502 , the rabbit CBC3C  and the loading controls, mouse monoclonal anti-β-tubulin  and mouse monoclonal acetylated-α-tubulin . The secondary antibodies used were horseradish peroxidase-conjugated anti-rabbit (1:5000), anti-sheep (1:1000) and anti-mouse (1:5000) IgGs for ECL detection .An aliquot of washed diluted sperm (25 μl) was placed onto a glass coverslip pre-coated with 100 μg/ml poly-L-ornithine, and dried at room temperature, in a six-well plate containing one coverslip per well. To each well 1 ml of 4% paraformaldehyde in 1× PBS was gently added and left to stand for 10 min. Subsequently, sperm sample was washed twice with 1 ml 1× PBS for 10 min. For permeabilization, 1 ml of 1:1 methanol/acetone solution was added for 2 min and then the samples washed twice with 1 ml 1× PBS for 10 min and blocked for 1 hr with 3% BSA in 1× PBS, before incubation with primary antibodies  for 2 hrs at room temperature. After three washes with 1× PBS, the fluorescently labeled secondary antibodies anti-rabbit Texas-Red, 1:300  and anti-sheep FITC, 1:50  were added and the coverslips incubated for 2 hrs. Finally, three washes with 1× PBS were performed and coverslips were mounted on microscope glass slides with one drop of anti-fading reagent containing DAPI for nucleic acid staining . Images were acquired using an Olympus IX81 epifluorescence microscope and digital camera, equipped with the appropriate software .The human sperm heat-stable extracts (hsPPP1R2) or hsPPP1R2 plus 10 ηg of recombinant PPP1R2 were acetone precipitated and the pellets were resuspended in 250 μl of 2D rehydration solution (8 M Urea/ 2 M Thiourea/ 2% CHAPS/ 0.002% of bromophenol blue) and supplemented with 2.5 μl of IPG buffer (in the 4–7 pH range) and 14 mg of DTT. The samples were pipetted into a strip holder and the electrophoresis was started . After the second dimension on 12% gels, samples were analyzed by Western blot.The hsPPP1R2 extracts were incubated overnight with either protein tyrosine phosphatase 1B  at 37ºC, calf intestinal phosphatase , Herts, UK) at 37ºC or PPP1CC1, at 30ºC, with the respective assay buffers. The reactions were stopped with the addition of 4× SDS loading buffer and analyzed by Western blot.GSK3: Glycogen synthase kinase 3; CK2: Casein kinase 2; PPP1: Phosphoprotein phosphatase 1; PPP1C: Catalytic subunit of PPP1; PPP1R2: PPP1 regulator 2; PPP1R2P3: PPP1R2 pseudogene 3; IPTG: Isopropyl β-D-1-thiogalactopyranoside; ECL: Enhanced chemiluminescence; RIPA: Radioimmunoprecipitation buffer; PH: Phase contrast microscopy; FA: Formic acid; ACN: Acetonitrile; ESI-MS/MS: Electrospray mass spectrometry; CID: Low-energy collision-induced dissociation; FITC: Fluorescein isothiocyanate; DAPI: 4',6-diamidino-2-phenylindole; DTT: Dithiothreitol; IPG: Immobilized pH gradient; EST: Expressed sequence tag; MGC: Mammalian gene collection; HEK293: Human embryonic kidney 293 cells; pI: Isoelectric point; aa: Amino acid; IP: Immunoprecipitation.The authors declare that they have no competing interests.Performed the sequence alignment: LKG. Conceived and designed the experiments: LKG, MFe, APV, WW, MF and EFCS. Performed the experiments: LKG, MFe, APV, WW and TM. Analyzed the data: LKG, MFe, APV, WW and TM. Contributed with reagents/materials/analysis tools: KM, SV, DLB, OABCS, MF and EFCS. Wrote the first draft: LKG. Edited the first draft: MFe, SV, DLB, OABCS and MF. All authors read and approved the final manuscript.


To investigate the biologic effect of an 810 nm diode laser on the induction of heat shock protein 70 (Hsp70) in choroid-retinal endothelial cells in vitro.Cultured rhesus macaque choroid-retinal endothelial cells (RF/6A) were irradiated using an 810 nm diode laser . Cell viability was assessed by annexin V- fluorescein isothiocyanate (FITC) and propidium iodide flow cytometric assay. Hsp70 expression was determined by western blot at 6, 12, 18, 24, and 48 h following laser exposure. Intracellular distribution of Hsp70 was examined by immunofluorescence staining.2), above which there was significant cell death. Under this power, Hsp70 expression elevated obviously and was stronger at 600–1,000 mW power settings . The expression of Hsp70 peaked at 12–18 h postirradiation, and returned to baseline by 48 h. Immunofluorescence staining indicated the induced Hsp70 expression in both the cytoplasm and the nucleus.The laser-induced cell injury threshold was found to be at a power of 1,100 mW power (fluence, 84.08 J/cmSubthreshold 810 nm diode laser exposure can induce Hsp70 hyperexpression from 12 to 18 h postirradiation in cultured choroid-retinal endothelial cells without obvious cell death. The results could be useful for investigating and designing more effective laser therapies. Choroidal neovascularization (CNV) causes severe vision loss in several eye disorders such as age-related macular degeneration, ocular histoplasmosis, and high myopia . MinimalTTT, which is a long-exposure, large spot size, low-irradiance, near-infrared diode 810 nm) laser protocol, slowly increases tissue temperature to approximately 4–10 °C above basal levels -7. Howev10 nm lasHeat shock proteins (Hsps) are a group of ubiquitous, highly conserved proteins in all organisms, and are classified according to their molecular mass. Elevated Hsps can be triggered by a variety of stressful stimuli and protect cells against stress. The 70 kDa Hsp (Hsp70) is particularly known to be induced by thermal, ischemic, and oxidative stress. Acting as a molecular chaperone, Hsp70 can assist in the refolding of denatured proteins and inhibit improper protein aggregation -12. MoreIn this study, we focus on Hsp70 expression induced by 810 nm diode laser exposure in cultured choroid-retinal endothelial cells. We aim to elaborate on the conditions in which the protein is hyperexpressed, as well as the threshold at which cellular damage occurs.2 humidified atmosphere at 37 °C. Prior to laser irradiation, cells were seeded in 24-well culture plates (4×104 cells/well) to grow to reach about 80% confluence (about 3 days).RF/6A rhesus macaque choroid-retinal endothelial cells were obtained from the Shanghai Institutes for Biologic Sciences  and were grown as monolayers in RPMI 1640 culture medium  supplemented with 15% fetal bovine serum (Hyclone) plus 100 U/ml penicillin/streptomycin mixture  in a 5% CO2PO4, 7 mM Na2HPO4; pH 7.4) and then transferred to no phenol red RPMI 1640 medium  without fetal bovine serum. The no phenol red medium was used to cover the cells during exposure to avoid absorption of laser energy by the culture medium. Following irradiation, fresh culture medium was added and the cells were incubated for an additional period for measurements. The control group was treated in the same manner, except the laser irradiation. Another group without any treatment was defined as the normal group.Irradiation was performed using an 810 nm diode laser , delivered through a 200 µm fiber-optic system with different power outputs for 60 s in continuous wave mode. The distance between the laser source and the cell monolayer was adjusted to achieve a 10 mm diameter laser spot , adequatFirst, we observed the morphological change of RF/6A cells after laser exposure under an inverted microscope , and then identified the power required to cause significant cell death by annexin V-fluorescein isothiocyanate (annexin V-FITC) and propidium iodide (PI) flow cytometric assay (threshold application). Using a spot diameter of 10 mm and a power setting of 1,100 mW or greater, 60 s of laser irradiation produced significant cell death. Then, the induced Hsp70 expression was studied at various subthreshold power settings, using decrements of 100 mW .Flow cytometry using Annexin V-FITC and PI double staining  was employed to detect cell viability. Following 6 h of incubation after exposure, cells were washed with PBS and resuspended in 60 µl binding buffer. annexin V-FITC (3.8 µl) and 3.8 µl PI were sequentially added to the cell solution. After incubation for 10 min in the dark, the stained cell solution was diluted with another 120 µl binding buffer and directly analyzed using the FACSCalibur system (BD Biosciences).g at 4 °C for 30 min and the supernatant was collected for protein quantification using the bicinchoninic acid assay kit . Equal amounts of protein (10–15 µg per lane) were separated by 10% sodium dodecyl sulfate PAGE and transferred onto a nitrocellulose membrane . After blocking with 5% skimmed milk in 10 mmol/l Tris-buffered saline with 0.1% Tween-20 for 1 h, the blotted membrane was incubated with mouse monoclonal anti-Hsp70 antibody  overnight at 4 °C. After washing three times with 10 mmol/l Tris-buffered saline with 0.1% Tween-20, the membrane was incubated with IRDye 800CW goat anti-mouse secondary antibody  for 1 h at room temperature. The signals were detected using an Odyssey Infrared Imaging System (LI-COR). Band intensity was analyzed by Quantity One software and compared with the internal standard glyceraldehyde-3-phosphate dehydrogenase .Based on the previous in vivo experimental data -20, we fCells were prepared in triplicate on 0.1% gelatin-coated glass coverslips and treated with the laser, as described above. At 18 h after irradiation, adherent cells were fixed in 4% paraformaldehyde for 15 min, blocked with 10% goat serum containing 0.5% Triton X-100 in PBS for 30 min, and then incubated with mouse monoclonal anti-Hsp70 antibody  overnight at 4 °C. After three rinses with PBS, the samples were incubated with Cy3-conjugated goat anti-mouse secondary antibody  for 1 h at room temperature in the dark. The slides were then washed in PBS and stained with 300 nM diamidinophenylindole  for 10 min followed by three rinses with PBS. After mounting with fluorescent mounting media , the slides were observed under a fluorescence microscope .t test. Statistical significance was defined as p<0.05.All experiments were repeated at least three times. Statistical analysis was performed using SPSS 16.0 software. All error bars represent standard error of the mean (SEM). Differences between the laser-treated and control groups were evaluated by the Student paired The morphology of RF/6A cells treated with laser power lower than 1,100 mW was similar to that of the control . HoweverWe performed a flow cytometry experiment of double staining with Annexin V-FITC and PI to determine the cell viability and the laser injury threshold. The cells were distinguished into the following four groups : those tIn the control group, the percentage of viable cells was 95.54%±0.34%. After laser treatment with 900 mW, 1,000 mW, or 1,100 mW power, the percentages of viable cells were 94.54%±0.82%, 93.63%±0.50%, and 68.44%±2.66%, respectively . Signifi2, 2) laser power for observation of Hsp70 expression at different recovery times. Hsp70 expression at 1 and 3 h after laser exposure were similar to that of control (data not shown). The maximum expression of Hsp70 after laser exposure occurred at 12–18 h , and gradually dropped to baseline at 48 h. Statistical analysis exhibited that Hsp70 expressions at 12, 18, and 24 h were all significantly higher than control . Moreover, the results showed that 1,100 mW power induced about 31.56%±2.66% cell death. As the laser spot in our experiment only covered half a well, the actual percentage of 1,100 mW laser induced cell death could be more than 50%. In addition, the inverted microscopy showed a large number of apoptotic or necrotic cells in samples treated with 1,100 mW power. Therefore, we assumed that this laser power of 1,100 mW  corresponded to the laser injury threshold.In animal models, the laser power that induced retinal whitening was thought to be the photocoagulation threshold -21. In o2). With these parameters, Hsp70 expression was highly induced, especially at 600–1,000 mW power settings . As indicated by immunofluorescence staining, induced expression of Hsp70 was present in both the cytoplasm and nucleus. Although the exposure conditions for Hsp70 induction in cultured cells in our study differed from those observed in vivo, their proportions of fluences for Hsp70 hyperexpression to fluences for the injury threshold are similar, about 60%–80% [After the cellular damage threshold was identified, a range of subthreshold powers from 400 to 1,000 mW was applied for detection of Hsp70 expression  applied for 60 s can stimulate Hsp70 production in cultured choroid-retinal endothelial cells without obvious cell damage. The Hsp70 expression was found to peak at 12 to 18 h following laser exposure, and returned to baseline at about 48 h. These results may facilitate designing more effective laser therapies.In summary, our study demonstrated that an 810 nm diode laser with subthreshold power (fluence lower than 84.08 J/cm


V7/KCNQ channels are widely expressed in neurons and they have multiple important functions, including control of excitability, spike afterpotentials, adaptation, and theta resonance. Mutations in KCNQ genes have been demonstrated to associate with human neurological pathologies. However, little is known about whether KV7/KCNQ channels are expressed in oligodendrocyte lineage cells (OLCs) and what their functions in OLCs.KV7/KCNQ channels expression in rat primary cultured OLCs by RT-PCR, immunostaining and electrophysiology. KCNQ2-5 mRNAs existed in all three developmental stages of rat primary cultured OLCs. KV7/KCNQ proteins were also detected in oligodendrocyte progenitor cells  of rat primary cultures and cortex slices. Voltage-clamp recording revealed that the IM antagonist XE991 significantly reduced KV7/KCNQ channel current (IK(Q)) in OPCs but not in differentiated oligodendrocytes. In addition, inhibition of KV7/KCNQ channels promoted OPCs motility in vitro.In this study, we characterized KV7/KCNQ channels were functionally expressed in rat primary cultured OLCs and might play an important role in OPCs functioning in physiological or pathological conditions.These findings showed that K So far, homomeric compositions are shown for KV7.2-5 subunits; heteromeric compositions are represented by KV7.2+3, KV7.3+4 and KV7.3+5 channels V7 channels are composed of KV7.2 and KV7.3 subunits V7.2 subunits V7.5 subunits in some neurons V7.4 subunits are predominantly expressed in the auditory and vestibular systems, but also probably contribute to KV7 channels in central dopaminergic neurons V7 channels have profound effects on neuronal excitability V7.2 construct, strongly enhances repetitive firing and even effects postnatal brain development V7.2 or KV7.3 lead to benign familial neonatal seizures V7.5 V7.4 are associated with progressive hearing loss The KCNQ gene family encodes five voltage-gated delayed rectifier KV 3.12+ channel + channels were linked to cell migration. Kv7.1 has been reported to regulate invasiveness of stem-like cell types V channel promotes migration of intestinal epithelial cells V10.1 is involved in adhesion and viability of CHO cells V11.1 participates in tumor cells invasion V1.3 suppresses the motility and activation of effector memory T (Tem) cells + channels, Kv1.1–Kv1.6 + (Kir) channels Kir2.1, Kir1.1 and Kir4.1 V7 channels is still unknown. In this paper, we studied the expression and function of KV7 channels in OLCs.Oligodendrocytes are generated from oligodendroglial progenitor cells (OPCs) which proliferate in the subventricular zone and migrate to formative white matter regions, where they further proliferate, differentiate, and form myelin sheaths around axons −A2B5+NG2+ OPCs, multipolar O4+GalC− IOs, and complex process bearing MBP+GalC+ MOs −A2B5+NG2+ OPC cultures   . In diffMBP+ MOs . As KCNQMBP+ MOs  left. KCMBP+ MOs  left. InMBP+ MOs  left. NoMBP+ MOs  right.V7.2-5 in cultured OLCs was further confirmed by immunostaining. The antibodies of anti-NG2, anti-O4 and anti-MBP were used to identify OPCs, IOs, and MOs in cultures respectively. The staining for KV7.2-5 was on the soma and processes of OPCs. The immunofluorescence signals were positive in both the cytoplasm and the cell membrane of OPCs   to activate KV7 channels + channels that activate in this membrane potential region V7 channels (K(Q) was measured as the difference between the instantaneous current at the onset of hyperpolarization and the steady-state current at the end of voltage command K(Q) from 17 OPCs. The mean IK(Q) amplitude was voltage dependent and the maximal IK(Q) amplitude (61.16±6.32 pA) was measured at −40 mV. The deactivation time constant of IK(Q) was determined by fitting the current curves measured at each voltage with a single exponential function. K(Q) as a function of voltage (n = 17). The mean IK(Q) deactivation time constant was 292.94±26.79 ms (−30 mV), 246.94±24.97 ms (−40 mV), 152.85±17.59 ms (−50 mV) and 131.89±14.18 ms (−60 mV), indicating that it was voltage dependent. Note that the deactivation time constant was a linear function of voltage (correlation coefficient r = 0.93) and was shorter at more negative membrane potentials which means at these potentials, IK(Q) was deactivated faster. We also recorded IK(Q) in IOs and MOs and the inward deactivation relaxation currents were almost not existed. (K(Q) mainly were obtained from OPCs.In order to determine the Kchannels . The ampexisted. . ConsequM) in native neurons and currents from artificial expressed KV7 channels K(Q) was monitored with a 1-s-long hyperpolarizing voltage stepped from a holding potential of −20 to −40 mV. XE991 (10 µM) reduced about 46.1±6.8% IK(Q) in OPCs. Higher concentration of XE991 (30 µM) inhibited IK(Q) more than a half (by 67.4±5.6%) (K(Q) amplitude versus the log concentration of XE991 from 54 OPCs. The mean inhibition of IK(Q) by XE991 was 19.5±3.1% (at 1 µM), 33.3±5.7% (at 3 µM) and 80.1±4.2% (at 100 µM). The mean data was fitted with the Hill equation (see METHODS). The IC50 for XE991 was 13.3 µM and the power term n (Hill slope), which is related to the steepness of curve was 0.63. The goodness of fit R2 was 0.99. In contrast, the currents in IOs and MOs were very insensitive to XE991 (XE991 has been shown to be a potent and selective inhibitor for M current (I.4±5.6%) . Fig. 6Eto XE991 .V7 channel proteins have different sensitivities to TEA K(Q) in OPCs. The voltage protocol used to measure IK(Q) is the same as XE991 on IK(Q) recorded in OLCs. We then applied TEA at concentrations ranging from 0.3 mM to 30 mM. TEA caused a concentration dependent reduction in IK(Q) in OPCs. K(Q) amplitude versus the log concentration of TEA from 20 OPCs. The mean inhibition of IK(Q) by TEA was 33.3%±5.1% (0.3 mM), 54.7%±2.4% (1 mM), 72.1%±5.5% (3 mM), 87%±4.6% (10 mM) and 92.8%±1.2% (30 mM). Application of 30 mM TEA completely abolished the current. The mean data was fitted with the Hill equation (see METHODS). The IC50 for TEA was 0.84 mM and the power term n (Hill slope) was 0.7. The goodness of fit R2 was 0.99.Previous studies reported that different KV7 channels, we also investigated the effect of these channels on OPCs migration, which is important for myelin development. We measured the mobility of OPCs cultured in a Boyden chamber. After 8 h incubation with 1 µM, 3 µM or 10 µM XE991 in the lower wells of the chemotaxis chambers, the number of migrated OPCs was significantly increased compared with the control group  V7.4/5, but they didn't state clearly the type of glial cells V7 channel subunits in OLCs. The mRNA of the four genes (KCNQ2-5) was detectable in OPCs. KCNQ3 and KCNQ5 mRNAs were detected strongly, and lesser abundances of mRNAs encoding KCNQ2 and KCNQ4 were observed. KCNQ2-4 mRNAs also existed in IOs with similar expression levels. In MOs, we only find very week sign of KCNQ4 mRNA. These indicated that the transcripts of KCNQ2-5 might be down regulated during the maturation of OLCs. Previous studies found that KCNQ2, 4, 5 genes have alternative splice variants V7.2-5 proteins in cultured OLCs. The signals of four proteins (KV7.2-5) were weakly detected in differentiated OLCs (IOs and MOs), while they were obviously dyed out in OPCs. In agreement with the immunostaining experiments, the inward deactivation relaxation currents almost did not exist in IOs and MOs. It is likely that the mRNAs for KV7.2-4, or only KV7.4 which were detected in IOs and MOs respectively were unable to be translated into enough Kv7 proteins to be detected by immunocytochemistry or electrophysiology method. This developmental regulation may reflect some yet unknown roles played by KV7 channels in early development of OLCs.Neuronal KV7.2, 3 and 5 proteins, but not KV7.4, in NG2+ OPCs in rat cortex slices. These results agree with the work of Kharkovets et al. V7.4 channel transcripts.However, previous work on the expression of members of the Kv channel family suggested that the precise topographical distribution of Kv channel subunits in cultured cells may not fully reproduce that obtained in situ V7 channels are composed of four homomeric or heteromeric subunits. The sensitivity to XE991 of these homomeric or heteromeric channel currents differs considerably. Homomeric KV7.2 channels have an IC50 value for XE991 inhibition which is 0.7 µM. The KV7.2+3 heteromultimer retain the sensitivity of the KV7.2 homomultimer. However, homomeric KV7.3 and homomeric KV7.5 are very insensitive to XE991 with estimated IC50 values of <50 µM and 65 µM K(Q) (by 67.4±5.6%) in OPCs. The IC50 for XE991 was 13.3 µM.Functional KV7 channel proteins also have different sensitivities to TEA K(Q) relaxations were completely inhibited by TEA with an IC50 of 0.84 mM, which is less than the IC50 for block of artificially expressed homomeric KV7.4 channels and heteromeric KV7.2/3 channels currents V7.2 currents. KV7.2-5 channel proteins were all detected in OPCs, however, their exactly expression level was unknown. The difference of pharmacological sensitivity to XE991 or TEA might due to various expression level and composition of each KV7 channel subunit in OPCs.Different KK(Q) in OPCs was found to be voltage dependent, which is similar to IM measured in sympathetic ganglion, hippocampal and dopamine neurons K(Q) amplitude in the OPCs was obtained at −40 mV with the deactivation protocol. The deactivation time constant was voltage dependent in OPCs, becoming shorter at more hyperpolarized membrane potentials, as has been observed for native IM currents in neuronal cell types K(Q) deactivation in OPCs was well fitted with a single exponential function and the value of the deactivation time constant was 152.85 ms at −50 mV. The deactivation time constant in OPCs seems to be closest to the fast component of the deactivation time constant in sympathetic neurons, which was reported to be 145 ms at −50 mV M currents in neurons have a biphasic  time course V7 channels underlying IK(Q) in OPCs.In the present study, the amplitude of I+ channels, Kv1.1–Kv1.6 + (Kir) channels Kir2.1, Kir1.1 and Kir4.1 V7 channels were expressed in OLCs, and downregulated in IOs and MOs. This developmental regulation may reflect some yet unknown roles played by KV7 channels in early OLCs development.During development, OLCs express all six members of the delayed rectifier Shaker family K+ channels may be linked to cell migration is supported by several studies V7.1 potassium channels have been implicated recently in the regulation of migration and invasiveness of stem-like cell types V7 channels are important for the regulation of OPCs migration in vitro. In our migration assay, the motility of the OPCs was promoted by the inhibition of KV7 channels. In fact, in neurons, KV7 channels can be inhibited by many endogenous factors. For example, stimulation of a variety of Gq/11-coupled neurotransmitter receptors, local changes in PIP2 concentration V7 channel 2+ waves or cell lysis V7 channels though P2Y1, and consequently promote the OPCs migration.Migration of OPCs from proliferation zones to their final position is an essential step in the development of the nervous system 4 cells/cm2) onto poly-L-lysine-precoated plates (0.1 mg/ml) and cultured in DMEM containing 10% FBS medium. After 2 hr, DMEM supplemented with 30% B104 neuroblastoma conditioned medium, 1% B27  and 1% N2  was added to the culture medium. Immature oligodendrocytes (IOs) were produced by substitution of DMEM, 30% B104, 1% B27 and 1% N2 with Neurobasal medium  containing 1% B27, 1% N2, T3 (40 ng/ml) and biotin (10 ng/ml) for 2 days, and mature oligodendrocytes (MOs) by substitution with that medium for 4 days.The animal experiments were carried out in adherence with the National Institutes of Health Guidelines on the Use of laboratory Animals and were approved by Second Military Medical University Committee on Animal Care (permission <$>\raster="rg1"<$>: SCXK-HU-2007-0003). OLCs were prepared as previously described Total RNA was extracted from adult rat forebrain or cultured OLCs using TRIZOL Reagent , followed by the treatment with DNase I RNA-free . Synthesis of cDNA was carried out with the Superscript First-Strand Synthesis System for RT-PCR . The RNA from OLCs without reverse transcription was performed PCR procedure directly as negative control, while the RNA from adult rat forebrain was as positive control. The PCR temperature profile was 94°C for 3 min, followed by 40 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min, 72°C 5 min. All primers are listed in V7.2, 3  , KV7.4  and KV7.5  at 4°C overnight. Cells were then washed and incubated with fluorescence-conjugated secondary antibodies  for 6 hours at room temperature and examined by fluorescence microscopy . For immunohistochemical analysis, animals were deeply anaesthetized with 2% pentobarbital sodium and perfused transcardially with 4% PFA in 0.1 M PBS, pH 7.4. The brain were subsequently dissected from each animal and post-fixed in the perfusing solution overnight at 4°C. Then, the tissues were cryoprotected in 20% sucrose in PBS for 24–48 h at 4°C. Cryostat sections (10 µm) were cut and mounted onto gelatin-subbed slides and stored at −20°C. For immunostaining, the protocol performed was similar to immunocytochemical analysis.For immunocytochemical analysis, OLCs on coverslips were washed with phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde (PFA) in PBS for 20 min, followed by permeabilization with 0.3% Triton X-100 in 0.1 M PBS for 10 minutes. After blocking the non-specific binding with 10% normal goat serum or 1% BSA in 0.1 M PBS, cells were incubated with primary antibodies against A2B5 , GFAP , NG2 , O4 , MBP , K2, 0.1 mM EGTA, 4 mMATP·2Na, 0.5 mM Na3·GTP, and 10 mM HEPES (pH 7.4 with KOH). The superfusate solution used to measure IK(Q) contained 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mMCaCl2, 10 mM HEPES, and 10 mM Glucose (pH 7.4 with NaOH). All experiments were done in room temperature. Drugs were applied through OctaFlow System  to the cell under recording. XE991  and TEA  was dissolved in water to store at −20°C, and diluted part per thousand using superfusate solution. Hyperpolarizing voltage steps (1 s duration) were given from a holding potential of −20 to −60 mV (in 10-mV increments).Current recordings were performed in the whole cell configuration of the patch-clamp technique using MultiClamp700A amplifier . Date were stored in a PC, and analyzed by pClamp8.02 software . The patch pipettes (6∼8 MΩ), pulled using a Narishige puller  and polished using a MF200 Microforge , were filled with solution containing 140 mM KCl, 4 mM MgClK(Q), was fitted by a single exponential function Graphing and curve fitting of data were performed with Origin 7 software . The inward relaxation current, which was attributed to deactivation of IK(Q) as a function of drug concentration plotted on a log scale. Smooth curves were fit to these data with the Hill equation Concentration-response curves for XE991 and TEA were constructed by plotting percentage inhibition of Imax is the maximal value of y (at saturation); in the fitting procedure ymax was constrained not to exceed 100%. The term k is the IC50  and n (Hill slope) is the power term related to the slope of the curve.Where x is the concentration, y is the percentage inhibition, and y5 cells in 200 µl of culture medium containing 10% FBS per well, and 600 uL DMEM containing 10% FBS were added to lower chamber. When OPCs were adherent (about 40 min later), the DMEM medium containing 10% FBS in the upper and lower chamber was replaced with serum-free DMEM supplemented with 30% B104 neuroblastoma conditioned medium, 1%B27, and 1%N2. XE991 or TEA was added to the lower chamber. After incubation for 8 h at 37°C, non-migratory cells on the upper membrane surface were removed with a cotton swab, and migratory cells invading to the underside surface of the membrane were fixed with 4% paraformaldehyde and stained with Coomassie Brilliant Blue. For quantitative assessment, the number of stained cells was counted under microscopy at 12 fields per filter in three independent experiments.To measure the motility of OPCs, Boyden chamber migration assay was performed as previously described Data from at least three independent experiments were all presented as means ± SEM. Statistical significance was evaluated with paired Student's t-test. Differences were considered significant at p<0.05.


During neuronal development, the neuroepithelial stem cells (NSCs) initially undergo proliferative divisions, later switching to neurogenic ones whereby one NSC and a post-mitotic neuron are generated. We recently showed that a member of the PRDM family of transcriptional regulators, PRDM4/SC1, recruits a type II protein arginine methyltransferase, PRMT5, to maintain the “stem-like” cellular state of the embryonic mouse cortical NSCs. However, little is known about the regulation of activity of this complex under proliferation- or differentiation-inducing growth conditions.In the present work I investigate the regulation of SC1/PRMT5-mediated methylation activity in PC12 cells treated with EGF or NGF. I present evidence that NGF down-regulates SC1/PRMT5 methyltransferase (MTase) activity and that the reduction in SC1/PRMT5 MTase activity occurs mainly in the nucleus. I suggest that high levels of SC1/PRMT5 activity are associated with the proliferative state of the cells. For growth factor treatment the serum content was lowered to 1% HS. EGF (PeproTech) was used at 20 ng/ml, NGF (PeproTech) - at 50 ng/ml. Transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. 48 h post-transfection cells were treated with the indicated growth factors, lysed and processed for the histone methyltransferase activity (HMTase) as described 2.2PC12 cells were harvested in immunoprecipitation (IP) buffer  Nonidet P-40, 300 mM NaCl) supplemented with the protease inhibitor cocktail (Sigma) and phosphatase inhibitor cocktails 1 and 2 (Sigma), processed as previously described and collected on protein A/G beads (Santa Cruz) 2.3+ pH7.5, 10 mM KCl, 1.5 mM MgCl2, 0.5 DTT) supplemented with protease (Sigma) and phosphatase inhibitors (Sigma) and pelleted by centrifugation at 1000 rpm for 5 min at 4 °C. HBA was supplemented with NP40 to a final concentration of 0.5% and added to the pellet to lyse the cells for 10 minutes are 4 °C. Nuclei were pelleted at 3000 rpm for 2 min at 4 °C and the supernatants collected as the cytosolic fraction, aliquoted and stored at −80 °C. Pellets containing nuclei were washed with HBA without NP40, followed by resuspension in buffer C  supplemented with protease and phosphatase inhibitors. Nuclear proteins were extracted for 30 min on ice with regular vortexing. The suspension was centrifuged at 4 °C for 10 min and the supernatant containing nuclear proteins collected, aliquoted and stored at −80 °C or used for HMTase assays immediately.PC12 cells treated as specified were harvested and processed for cytoplasmic and nuclear fractionation. Cells were rinsed in cold PBS on ice, scraped off the plates, resuspended in Hypotonic Buffer A (HBA) (10 mM HEPES-K3) for 10 min on ice, centrifuged at 2000 rpm for 10 min at 4 °C, the supernatants discarded, pellets washed again in half the volume of TEB and centrifuged as before. Pellets were resuspended in 0.2 N HCl and extracted overnight at 4 °C. Extracts were centrifuged at 2000 rpm for 10 min at 4 °C and the supernatants used for SDS-PAGE.Histones were extracted from PC12 cells as follows. Cells were washed twice in cold PBS, lysed in Triton extraction buffer , 2 mM phenylmethylsulfonyl fluoride (PMSF), 0.02% (v/v) NaN2.4Western blotting was performed in TBST buffer  Tween 20). For IP's and Western blotting the following antibodies were used: anti-myc , anti-H3 , anti-H4 , anti-H4R3me2s , anti-H4R3me2a , anti-TBP (Abcam 1:2000), anti-α-tubulin . The following antibodies were used for immunocytochemistry: anti-TuJ1 , anti-PRMT5 , anti-SC1/PRDM4 (a gift from Pilar Perez and Moses Chao (1:40) and our own (1:100) 2.55 cells/13 mm on glass cover slips. NSCs were cultured in DMEM supplemented with 40 ng/ml Neurotrophin-3 (NT-3) (Pepro Tech), 0.25% FCS, B27 supplement, sodium pyruvate and glutamine . Animal experiments were approved by the University College London local ethical committee and conformed to the UK Animals (Scientific Procedures) Act 1986. Project license number PPL 70/6697.Primary cortical neural stem cells were isolated from time mated mouse E10.5 embryos according to a published protocol 33.1rd panel from the top). Moreover, an in vitro HMTase assay using the immunoprecipitated proteins revealed an increase in the amount of H4R3me2s methylation consistent with the presence of PRMT5 in mycSC1 immunoprecipitated complex (rd panel) and an in vitro radioactive HMTase assay with exogenously added purified calf thymus histone mixture was performed We recently demonstrated that SC1 recruits the protein arginine methyltransferase, PRMT5, to mediate symmetric dimethylation of histone H4 on arginine 3 (H4R3me2s) 3.2Both SC1 and PRMT5 are found in the cytosol and in the nucleus NGF is known to increase asymmetrically dimethylated proteins 3.3The observations, that NGF induced a selective reduction of the nuclear SC1/PRMT5-mediated MTase activity in PC12 cells, prompted me to investigate the subcellular distribution of SC1 and PRMT5 in differentiating neurons from the developing mouse cortex. Cortical NSCs isolated from E10.5 mouse embryos were cultured in NT3-containing media for 3 days to induce neurogenesis. Early-born neurons were detected by immunostaining cells with antibodies against Tuj1. Subcellular localization of SC1 and PRMT5 in early-born neurons was determined by immunostaining cells with appropriate antibodies. Both proteins were detected in the nucleus and cytosol of neurons , suggest4Presented results demonstrate that SC1/PRMT5-mediated MTase activity is reduced by NGF, and sustained by EGF in PC12 cells. These observations are in line with the previously published report that NGF dampens PRMT5 activity in PC12 cells Our previous work identified PRMT5-mediated deposition of H4R3me2s modifications as a “signature” of proliferating embryonic cortical NSCs suggesting a role for PRMT5 in the control of NSC proliferation and “stemness” Another interesting observation is the selective, NGF-induced reduction of SC1/PRMT5-mediated MTase activity in the nucleus of PC12 cells. The decrease in the nuclear SC1/PRMT5 MTase activity is likely to result in a diminution of proteins symmetrically dimethylated by the complex within this compartment. Interestingly, histones may not be the only targets of NGF-dependent reduction of SC1/PRMT5-mediated protein methylation as no notable change in the overall H4R3me2s was observed in PC12 cells overexpressing SC1 and treated with NGF. Perhaps other nuclear proteins are also substrates of SC1/PRMT5-mediated symmetric arginine dimethylation down-regulated by NGF. This possibility is consistent with the previously published report demonstrating NGF-dependent increase in the amount of asymmetrically dimethylated proteins in the nucleus and our previous observations that asymmetric dimethylation of H4R3 is detected in the post-mitotic neurons of the developing cortex, but not in the proliferating NSCs where H4R3me2s prevails The selective down-regulation of the nuclear SC1/PRMT5-mediated MTase activity by NGF induces a localized, specific response of the cells to different growth factors, suggesting that some of the decisions between proliferation and differentiation are regulated by the magnitude of symmetric protein arginine dimethylation within the nuclear compartment. Importantly, PRMT5 has been shown to control ES cell pluripotency by methylating the cytosolic pool of histone H2A in the embryonic stem (ES) cells