T317 phosphorylation of the p38 substrate mitogen-activated protein kinase-activated protein kinase 2 (MK2) correlates with muscle weight in atrophic and hypertrophic denervated muscle and may influence the nuclear and cytoplasmic distribution of p38 and/or MK2. The present study investigates expression and phosphorylation of p38, MK2 and related proteins in cytosolic and nuclear fractions from atrophic and hypertrophic 6-days denervated skeletal muscles compared to innervated controls.rodent/27human, Hsp25/27) and Hsp70 protein expression were studied semi-quantitatively using Western blots with separated nuclear and cytosolic fractions from innervated and denervated hypertrophic hemidiaphragm and atrophic anterior tibial muscles. Unfractionated innervated and denervated atrophic pooled gastrocnemius and soleus muscles were also studied.Expression and phosphorylation of p38, MK2, Hsp25 (heat shock protein25No support was obtained for a differential nuclear/cytosolic localization of p38 or MK2 in denervated hypertrophic and atrophic muscle. The differential effect of denervation on T317 phosphorylation of MK2 in denervated hypertrophic and atrophic muscle was not reflected in p38 phosphorylation nor in the phosphorylation of the MK2 substrate Hsp25. Hsp25 phosphorylation increased 3-30-fold in all denervated muscles studied. The expression of Hsp70 increased 3-5-fold only in denervated hypertrophic muscles.The study confirms a differential response of MK2 T317 phosphorylation in denervated hypertrophic and atrophic muscles and suggests that Hsp70 may be important for this. Increased Hsp25 phosphorylation in all denervated muscles studied indicates a role for factors other than MK2 in the regulation of this phosphorylation. It is likely that skeletal muscle atrophy (muscle wasting) and hypertrophy (muscle growth) are caused by alterations in the balance between protein synthesis and degradation. It has been suggested that one pathway which plays a role in this balance is the p38 mitogen-activated protein kinase  pathway . p38 hasIn vertebrates four isoforms of p38 have been identified: p38α  and SAPK2a), p38β , p38γ  and p38δ,  . Activatrodent/27human) [One substrate of p38 is mitogen-activated protein kinase-activated protein kinase 2  with proposed roles in actin remodeling, cell migration, development and regulation of the cell cycle . In the 27human) , suggest27human) . Phospho27human) ,30. Over27human) .Hsp70, another heat shock protein that has been shown to prevent skeletal muscle atrophy when overexpressed ,33, was Denervation of skeletal muscles generally causes atrophy of the muscles but the hemidiaphragm becomes transiently hypertrophic following denervation -48. We hAll results reported are based on data from 4 sets of 8 animals generating 8 denervated anterior tibial muscles with 8 contralateral innervated controls, 8 denervated pooled gastrocnemius and soleus muscles with 8 contralateral innervated controls, 8 denervated hemidiaphragm muscles and 8 innervated control hemidiaphragms from separate animals.Six days after denervation hemidiaphragm muscles were hypertrophic with a wet weight of 40.3 ± 1.5 mg compared to 29.6 ± 0.6 mg for innervated controls . Six days after denervation anterior tibial muscles were atrophic with a wet weight of 44.7 ± 1.8 mg compared to 60.8 ± 1.9 mg for innervated controls . Six days after denervation pooled gastrocnemius and soleus muscles were atrophic with a wet weight of 149.1 ± 4.4 mg, compared to innervated controls with a wet weight of 198.3 ± 5.7 mg .Successful separation of nuclear and cytosolic fractions was confirmed using Western blots for Lamin A/C and superoxide dismutase 1 (SOD-1), respectively. Very little nuclear protein (Lamin A/C) was present in cytosolic fractions and only a small amount of cytosolic protein (SOD-1) was present in the nuclear fraction  and Hsp25 were increased about 0.6-fold, 1.3-fold and 3.2 fold, respectively, in cytosolic fractions  and Hsp25 were increased about 0.2-fold, 1.4-fold and 30-fold, respectively, whereas the expression of phosphorylated MK2 (T317) decreased about 0.7-fold in cytosolic fractions following denervation  become atrophic following denervation, unfractionated homogenates of pooled gastrocnemius and soleus muscles were examined. In atrophic 6-days denervated pooled gastrocnemius and soleus muscles changes in the expression of total and phosphorylated p38, MK2 and Hsp25 were similar to those seen in denervated atrophic anterior tibial muscle. Expression of total p38 was unchanged whereas MK2 protein expression decreased about 0.5-fold and Hsp25 protein expression increased about 0.6-fold. Phosphorylated p38, MK2 (T205) and Hsp25 increased about 1.2-fold, 1.3-fold and 12-fold, respectively, whereas phosphorylated MK2 (T317) decreased about 0.3-fold following denervation Figure .The mean expression level of total p38 protein Figure a was 86.The mean expression level of total MK2 protein Figure c was 51.The mean expression level of total Hsp25 protein Figure f was 157Hsp70 expression was studied since it has recently been reported to play a role in the phosphorylation of MK2 by p38 (see Background). Unlike the other proteins studied Hsp70 was more abundant in nuclear than in cytosolic fractions of innervated as well as 6-days denervated muscles. In atrophic 6-days denervated anterior tibial muscles the expression of Hsp70 was slightly increased (about 0.3-fold) in the nuclear fraction compared to innervated controls. In 6-days denervated hypertrophic hemidiaphragm the expression of Hsp70 was increased about 5-fold and 2.9-fold in cytosolic and nuclear fractions, respectively  was not reflected in p38 phosphorylation. Thus, in denervated atrophic pooled gastrocnemius and soleus muscles p38 phosphorylation was increased Figure b whereasThe discrepancies between p38 phosphorylation and MK2 T317 phosphorylation may also be due to different isoforms of p38 responding differently to denervation. The antibodies used in the present study do not differentiate between the different p38 isoforms and while all p38 isoforms appear to be expressed to some extent in skeletal muscle -59 p38γ Following denervation the level of Hsp25 was increased in all muscles studied, as was the level of phosphorylated Hsp25. Similar to the discordance between p38 phosphorylation and MK2 T317 phosphorylation, the level of MK2 T317 phosphorylation was not reflected in Hsp25 phosphorylation in atrophic muscle. In atrophic anterior tibial muscles as well as in atrophic pooled gastrocnemius and soleus muscles Hsp25 phosphorylation was increased Figures g despiteThe present study confirms a differential response of MK2 T317 phosphorylation to denervation in hemidiaphragm  and hind-limb muscle  and suggests that factors other than p38 may be important for this differential response, e.g. Hsp70. The study further shows that, despite the differential response of MK2 T317 phosphorylation to denervation in the different muscles, Hsp25 phosphorylation was increased in all denervated muscles studied indicating a role for other kinases and/or phosphatases in the regulation of Hsp25 phosphorylation in denervated muscle.All experiments were performed on adult male NMRI mice . Before surgery the animals were anaesthetized by inhalation of isoflurane. Denervation of either the left hind-limb or the left hemidiaphragm was performed by sectioning and removing a few mm of either the sciatic nerve or the phrenic nerve as described previously . While a2, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol (DTT), pH 7.9 with 1% Halt™ Protease and Phosphatase Inhibitor Cocktail from Thermo Scientific, Rockford, IL). The homogenized tissue was vortexed for 15 s, put on ice for 10 min, vortexed again for 15 s and centrifuged at 16,000 g for 15 s. The supernatant cytosolic extract was immediately frozen (-80°C) for subsequent analyses. The nuclear pellet was resuspended on ice in a high salt nuclear extraction buffer  at a ratio of 4 μl of nuclear extraction buffer per mg muscle wet weight. Preparations were incubated on ice for 30 min and vortexed for 10 s every 5 min before being centrifuged at 16,000 g for 6 min. The supernatant nuclear extract was then removed and frozen (-80°C) for subsequent analyses. Protein concentrations for each fraction were obtained using the Bradford assay [Mouse hemidiaphragm and anterior tibial muscles were used for nuclear and cytosolic protein extraction. The method used was slightly modified from . The musrd assay .Pooled gastrocnemius and soleus muscles were used for preparing protein extracts of unfractionated muscle homogenates. The muscles were homogenized in 2 ml of a buffer containing 100 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% sodium deoxycholate with 1% Halt™ Protease and Phosphatase Inhibitor Cocktail from Thermo Scientific . The homogenate was then centrifuged at 16,000 g for 10 min. The supernatant was recovered and the pellet was resuspended in 2 ml of buffer and re-centrifuged. The supernatants were combined and the protein concentration was determined using the Bradford assay .Western blots were prepared essentially as described in . Ten to The expression levels of total and phosphorylated proteins were studied semi-quantitatively using data from Western blots. Measured levels of total and phosphorylated proteins were expressed without normalization to any specific protein. No loading controls were used and any difference in protein quantifications, pipetting steps, protein transfers etc. are included in the variations of the data sets.http://rsb.info.nih.gov/ij/, 1997-2007). Results were obtained in uncalibrated units.Image analysis was performed using the gel plotting macro of the program ImageJ . Student’s t-test was used for statistical comparisons of normally distributed data . Statistical significance for data that was not normally distributed was determined using the Wilcoxon matched pairs test  and Mann-Whitney test (hemidiaphragm muscles).The authors declare no competing interests.The work presented here was carried out in collaboration between all authors. KE designed the study and carried out the studies on cytosolic and nuclear fractions, most of the protein expression studies, statistical analyses and drafted the manuscript. AKF carried out the studies on total and phosphorylated p38, MK2 and Hsp25 in 6-days denervated atrophic pooled gastrocnemius and soleus muscles. MN and ST conceived of the study, participated in the design, statistical analyses and drafting of the manuscript. All authors have read and approved the final manuscript.


Ctenocephalides felis antigens. 214 dogs from 22 veterinary clinics were included. These clinics were located as follows, from North to South: Hokkaido, Aomori, Fukushima, Tochigi, Saitama, Chiba, Tokyo (Tama-City and Ota-ku), Kanagawa, Gifu, Niigata, Kyoto, Nara, Osaka, Hyogo, Kagawa, Ehime, Hiroshima, Yamaguchi, Fukuoka, Kumamoto and Kagoshima. 110 dogs (51.4%) were seropositive for flea-specific IgE. No differences were associated with gender or breed. This survey confirms that flea infestation in dogs is a common problem in Japan. It especially shows that the infestation also occurs in Northern Japan where fleas are considered uncommon by the vet.In Japan, an epidemiological survey was performed in dogs from October to December 2008 by using a quantitative measurement method for antigen-specific IgE towards specific  Fleas are the most common ectoparasites of both dogs and cats throughout the world  as well as the degree of infestation cannot be established.et al. cloned a major 18-kD allergen, called Cte f 1 in the international nomenclature , Kanagawa, Gifu, Niigata, Kyoto, Nara, Osaka, Hyogo, Kagawa, Ehime, Hiroshima, Yamaguchi, Fukuoka, Kumamoto and Kagoshima .The allergen-specific IgE quantitative measurement was performed by Animal Allergy Clinical Laboratories, Inc , according to a procedure previously published  . Howeveri.e. when the IgE against house dust mites (Dermatophagoides farinae and/or Dermatophagoides pteronyssinus) was higher than flea-IgE, were excluded from this study.Cross-reactivity of IgE against house dust mites may influence flea-IgE titers. Therefore, all the cases with coincidental sensitization to house dust mites, The statistical analysis used was the Steel-Dwass’ test with IgE values according to gender, breed  and area with a significance level of p < 0.05. This survey was undertaken from October to December 2008, which only covers the end of the entire flea season. As it was not conducted throughout the whole year, a season analysis per region could not be conducted.Positive rates of anti-flea IgE in areas examined in this study were 51.4% (110/214). Cases with anti-flea IgE were found in all 22 areas surveyed in this study. The prevalence of seropositive dogs from Northern to Southern Japan was: 30% in Hokkaido (3/10 dogs), 60% in Aomori (6/10 dogs), 10% in Fukushima (1/10 dogs), 60% in Tochigi (6/10 dogs), 80% in Saitama (8/10 dogs), 40% in Chiba (4/10 dogs), 50% in Tama-City, Tokyo (4/8 dogs), 70% in Ota-Ku, Tokyo (7/10 dogs), 50% in Kanagawa (5/10 dogs), 10% in Gifu (1/10 dogs), 10% in Niigata (1/10 dogs), 70% in Kyoto (7/10 dogs), 80% in Nara (8/10 dogs), 50% in Osaka (3/6 dogs), 70% in Hyogo (7/10 dogs), 30% in Hiroshima (3/10 dogs), 60% in Yamaguchi (6/10 dogs), 40% in Kagawa (4/10 dogs), 40% in Ehime (4/10 dogs), 30% in Fukuoka (3/10 dogs), 60% in Kumamoto (6/10 dogs) and 90% in Kagoshima (9/10 dogs). Significant differences were present between areas, regarding positive rates or mean IgE values at p < 0.05 .Table IIIn all, 51.4% of dogs living in Japan were seropositive for anti-flea IgE meaning that they were either infested by fleas several weeks prior to survey dates or that they remained infested.As expected, we did not find any significant differences in gender and breed. Significant differences were present among areas, but the interpretation of the data is difficult due to the low number of samples. It will be necessary to include more dogs in a future study, to collect samples throughout a whole year in order to evaluate the seropositivity during the four seasons, and to take into account the impact of regular or occasional anti-flea treatments.In this time-limited study, we confirmed that dogs were often infested and bitten by fleas in Japan. We found solid proof that fleas are present in cold Northern areas, through the positivity rate of dogs that haven’t travelled around the country, despite the fact that vets and pet owners believe that fleas cannot be found in those regions. Climate change may explain the higher flea prevalence in the North, as it has also been shown for other insects. In Hokkaido, cockroaches and beetles were rarely found several decades ago but they have now increased significantly in numbers . This su


Dictyostelium, STATc is a key regulator of the transcriptional response to hyperosmotic stress. Its phosphorylation and consequent activation is controlled by two signaling branches, one cGMP- and the other Ca2+-dependent, of which many signaling components have yet to be identified. The STATc stress signalling pathway feeds back on itself by upregulating the expression of STATc and STATc-regulated genes. Based on microarray studies we chose two tyrosine-kinase like proteins, Pyk3 and Phg2, as possible modulators of STATc phosphorylation and generated single and double knock-out mutants to them. Transcriptional regulation of STATc and STATc dependent genes was disturbed in pyk3−, phg2−, and pyk3−/phg2− cells. The absence of Pyk3 and/or Phg2 resulted in diminished or completely abolished increased transcription of STATc dependent genes in response to sorbitol, 8-Br-cGMP and the Ca2+ liberator BHQ. Also, phospho-STATc levels were significantly reduced in pyk3− and phg2− cells and even further decreased in pyk3−/phg2− cells. The reduced phosphorylation was mirrored by a significant delay in nuclear translocation of GFP-STATc. The protein tyrosine phosphatase 3 (PTP3), which dephosphorylates and inhibits STATc, is inhibited by stress-induced phosphorylation on S448 and S747. Use of phosphoserine specific antibodies showed that Phg2 but not Pyk3 is involved in the phosphorylation of PTP3 on S747. In pull-down assays Phg2 and PTP3 interact directly, suggesting that Phg2 phosphorylates PTP3 on S747 in vivo. Phosphorylation of S448 was unchanged in phg2− cells. We show that Phg2 and an, as yet unknown, S448 protein kinase are responsible for PTP3 phosphorylation and hence its inhibition, and that Pyk3 is involved in the regulation of STATc by either directly or indirectly activating it. Our results add further complexities to the regulation of STATc, which presumably ensure its optimal activation in response to different environmental cues.Cellular adaptation to changes in environmental osmolarity is crucial for cell survival. In  Dictyostelium discoideum cells are faced with a hypertonic environment a complex response is triggered. It starts with signal sensing and transduction and leads to changes in cell shape, the cytoskeleton, transport processes, metabolism and gene expression D. discoideum, is an important mediator of the transcriptional response to hyperosmolarity as it regulates 20% of the induced genes Virtually all cells, even individual cells in multi-cellular organisms, are subjected to changes in the osmotic environment that are sometimes extremely rapid. In order to survive cells have to sense these changes and elicit an appropriate response that allows them to adapt. When D. discoideum does not encode a JAK ortholog. However, the genome encodes a large number of tyrosine-kinase like (TKL) proteins which constitute STATc kinase candidates 2+, respectively, and ultimately cause phosphorylation of STATc on Tyr922 − strain. Furthermore, hyperosmotic stress-induced tyrosine phosphorylation of STATc was still observed in a D. discoideum mutant, wherein both known guanylate cyclases (GCA and sGC) were disrupted 2+ branch in response to hyperosmolarity In mammals, STAT1 and STAT3 are normally activated by cytokines but can also be activated by hyperosmotic stress 2+ levels induced phosphorylation of S448 and S747 of PTP3 and inhibited its enzymatic activity Another essential player in the complex regulation of STATc is the constitutively active protein tyrosine phosphatase 3 (PTP3) pyk3 and phg2, a double knock-out mutant of both genes, as well as GFP-STATc, Myc-PTP3 and Myc-Phg2 overexpressing Ax2 wild-type and mutant cells with respect to transcriptional regulation, STATc and PTP3 phosphorylation, and STATc nuclear translocation. We present evidence that both TKL proteins modulate STATc-signalling and that Phg2 but not Pyk3 acts as a PTP3 inhibitor.Here we characterise two further modulators of the STATc signalling cascade, the TKL proteins Pyk3 and Phg2, which were selected based on previous microarray results and BLAST searches D. discoideum Ax2 wild-type and mutant cells , that contained 40 µg/ml dihydrostreptomycinsulfate and in the case of mutant strains in addition 10 µg/ml blasticidin or 10 µg/ml G418, on plates (90 mm diameter) or in Erlenmeyer flasks with shaking at 160 rpm 6 cells/ml. For treatment with 200 mM sorbitol, 20 mM 8-Br-cGMP, 30 µM BHQ or 5 µM thapsigargin, cells were washed twice with Soerensen phosphate buffer , resuspended in the same buffer at 2×107 cell/ml, and developed under shaking at 200 rpm for 4 hours. Treatment was performed for 5 to 15 minutes depending on the assay.nt cells  were groDictyostelium mutant strains were generated in the AX2 background. Gene replacement constructs were generated in the pLBPLP vector where the blasticidin resistance (bsr) cassette is flanked by loxP sites − construct, a PCR amplified fragment of 507 bp  was cloned into the BamHI and PstI sites and a 670 bp fragment  into the HindIII and SalI sites of the vector. Upon homologous recombination of the ‘restriction enzyme’ linearized targeting construct, the genomic region of the phg2 gene encompassing the TKL domain was replaced by the bsr cassette  was cloned into the BamHI and PstI sites and a 656 bp fragment  into the HindIII and SalI sites of the vector. Upon homologous recombination, the bsr cassette of the targeting construct replaced 2.37 kbp of the pyk3 gene thereby disrupting part of the TKL domain . The two knock-out mutants were exchanged between the two laboratories and compared in some of the real time PCR and STATc phosphorylation experiments. The plasmids for expression of Myc-PTP3 and GFP-STATc in D. discoideum cells have been described elsewhere All cassette . SimilarL domain . To geneEscherichia coli expression vector pGEX-6-P-1 . The fusion proteins were expressed in E. coli XL1-blue, purified using glutathione agarose beads, released from the beads through cleavage with PreScission protease, and used for the immunization of rabbits . For pull-down assays amino acids 207–1159 of Phg2 were expressed, purified, and eluted with glutathione. For in vitro PTP3 phosphorylation we used bacterially expressed GST-PTP3ΔCS For generation of Pyk3- and Phg2-specific polyclonal antibodies (pAbs), cDNA sequences encoding amino acids 1020–1309 of Pyk3 and 656–706 of Phg2 were amplified by PCR and cloned into the 5 cells were separated by SDS-PAGE and electrophoretically transferred to a nitrocellulose membrane with a wet blot apparatus at 10 V overnight. Phg2 and Pyk3 polyclonal antibodies were used at a 1∶100 and 1∶40,000 dilutions, respectively. GFP was detected with mAb K3-184-2 922-phosphorylated STATc with mAb CP22 448- and Ser747-phosphorylated PTP3 were monitored by using specific PTP3 phospho S448 and S747 antibodies SDS-PAGE and Western blotting were essentially performed as described −, phg2−, and pyk3−/phg2− cells ectopically expressing GFP-STATc 6 cells were collected, washed twice with Soerensen phosphate buffer, resuspended in 100 µl of the same buffer, and allowed to settle for 15 min on acid-washed glass coverslips (18 mm diameter). The cells were treated with 100 mM sorbitol for 0 to 8 minutes, immediately fixed with pre-chilled (−20°C) methanol in a petri dish, and incubated at −20°C for 10 min. The coverslips were then washed three times with 500 µl PBG , mounted onto glass slides with the cells facing downwards, sealed with gelvatol, and stored in the dark at 4°C overnight. Images of fixed cells were recorded with the inverted fluorescence microscope (LEICA DMI 6000 B) at 488 nm excitation and 500–550 nm emission and processed using the Leica MM AF software.Fluorescence microscopy studies were carried out using Ax2 wild-type, pyk3− cells. 1–6×107 cells were treated with 200 mM sorbitol for 15 minutes and lysed in 1 ml mNP40 lysis buffer ) for 10 min on ice. The cell lysate was centrifuged at 7,000× g for 10 min, the supernatant was pre-cleared by incubation with Dynabeads Protein G (Life technologies™) for 30 min at 4°C with rotation, followed by centrifugation at 500× g for 1 min. The cleared supernatant was incubated for 30 min at 4°C with 2 µg Myc antibody (mAb 9E10) and then for further 60 min with the Dynabeads Protein G. After incubation the Dynabeads were washed four times with EDTA free mNP40-buffer. 5–10 µg bacterially expressed and purified GST-Phg2 were released from glutathione agarose beads through incubation with 20 mM glutathione, and added in pull-down buffer  and 0.2 mM ATP) to the Dynabeads followed by incubation for 2 h at 4°C. For reverse pulldown myc-PTP3 was immunopurified from phg2− cells as above and eluted from the Dynabeads by a pH shift . The eluted Myc-PTP3 was then incubated in pull-down buffer together with 50 µl GST-Phg2 coated glutathione agarose beads. In both protocols the beads were washed 5–10 times with washing buffer , eluted in 50 µl 3 x SDS sample buffer, boiled for 3 minutes, and the proteins were separated by SDS-PAGE followed by Western blot analysis using anti Myc, anti Phg2, and anti GST-antibodies.Myc-PTP3 was expressed in phg2− cells  a positive transcriptional feedback loop, ii) their STATc-dependent differential regulation, and iii) the presence of a tyrosine-kinase-like catalytic domain, we considered Pyk1, Pyk3, Phg2, and the MORN kinase as potential upstream regulators of STATc. The Pyk2 kinase was excluded from further analysis because its genetic ablation has been shown to cause only a one minute delay in sorbitol-induced STATc activation D. discoideum proteome with a human JAK protein sequence (NP_002218) revealed highest sequence similarity with Pyk3. Furthermore, it was previously shown that Phg2 interacts with Rap1-GTP which has been linked to the response to sorbitol − knock-out strain was generated by Tsuyoshi Araki (University of Dundee). We exchanged the two Pyk3 knock-out mutants and in all experiments where we used them in parallel we obtained similar results (data not shown).The −, pyk3−, phg2−, and pyk3−/phg2− strains were treated with 200 mM sorbitol or left untreated, RNA was isolated, reverse transcribed, and quantitative real time PCR was performed for dstc, phg2, pyk3, ptpc, and abcB1 (ABC transporter B family protein) as control. abcB1 was previously found to be differentially regulated independently of STATc in response to osmotic stress abcB1 were upregulated in Ax2 wild-type cells but not differentially regulated in the STATc− cells. This is consistent with previous microarray and quantitative real time PCR results , which causes an increase in cytosolic Ca2+ levels. The real time PCR results showed that only dstc but not the other genes were upregulated under these conditions in Ax2 wild-type cells, but not in the mutants (phg2 in Ax2 and pyk3− cells and for pyk3 in Ax2 wild-type cells. We obtained similar results when we treated the cells with thapsigargin (data not shown). In summary, these results show that activation of both, the cGMP and the Ca2+, branches are needed for a full transcriptional response of STATc and the investigated STATc dependent genes. They also show that expression of STATc can be induced by a rise in cGMP as well as Ca2+, while induction of phg2, pyk3 and ptp3 needs activation of the cGMP branch. Interestingly, the observed fold changes in response to sorbitol in AX2 cells were generally higher than the sum of these changes in response to 8-Br-cGMP and BHQ. This is very obvious for pyk3 where we see a smaller upregulation in response to 8-Br-cGMP as compared to sorbitol and a downregulation in response to BHQ. The discrepancy in the fold change in response to sorbitol versus activation of the single branches can be explained if we assume a crosstalk between the two branches where e.g. activation of the cGMP branch would somehow exert an inhibitory effect on the Calcium branch. Alternatively, sorbitol might activate yet another, so far unknown signalling pathway.It has been previously shown that two signalling branches converge on STATc in response to hypertonicity, which are under control of the second messengers cGMP and Cativation . We next mutants . The folD. discoideum cells are exposed to 200 mM sorbitol STATc is activated by phosphorylation on Tyr922. In the absence of hyperosmotic conditions STATc phosphorylation can also be induced by 8-Br-cGMP and BHQ −, phg2−, and pyk3−/phg2− cells. For quantitation of phospho- and total STATc we used the STATc phosphorylation-specific monoclonal antibody CP22 and the total STATc antibody 7H3 − and phg2− cells showed significantly lower levels of phosphorylated STATc and the pyk3−/phg2− double mutant displayed a further decrease but not a complete abolishment of STATc phosphorylation . These results exclude Pyk3 as a specific inhibitor of PTP3 and suggest that it is involved in the phosphorylation of STATc as a regulated activator. This could in principle be analysed using an immunoprecipitation-kinase assay but, given that any difference might be expected to be only two-fold, it would be a difficult task.To directly study phosphorylation of PTP3 on S448 and S747 we generated Myc-PTP3 overexpressing Ax2 wild-type, pyk3rylation . The abs− cells were treated with sorbitol and Western blot analyses using the phospho-specific PTP3 antibodies were performed. We observed a similar increase in Myc-PTP3 S448 phosphorylation in both strains upon sorbitol treatment  proteins of which 66 are encoded in the genome − cells five genes encoding TKLs were already differentially regulated 15 min after exposure to sorbitol and their differential expression was dependent on the presence of STATc  Pyk2 is either constitutively active, as proposed by Araki et al − strain in an Ax4 background Our work aimed at the identification of protein kinases apart from Pyk2, which are involved in STATc regulation. Based on the early differential regulation in our microarray approach, their suspected catalytic activity and previously published results, we chose Pyk3 and Phg2 for further investigation. Pyk3 was originally identified by screening of a λgt11 expression library with a phospho-tyrosine specific antibody − cells . Our res2 via a novel binding site, which mediates membrane localization − mutant STATc still became activated and translocated to the nucleus, albeit with a significant delay . Therefore, we postulate that the reduced level of p-STATc in the pyk3− strain in response to sorbitol and 8-Br-cGMP but not BHQ is due to the presence of another, as yet unidentified, STATc protein kinase (SPK) downstream of cGMP. The exact target and action of Pyk3 has still to be elucidated and STATc is an attractive candidate. The STATc phosphorylation patterns  The phg2− strain was generated by replacement of a 2.9 kbp genomic fragment of the phg2 gene that contained part of the tyrosine kinase domain coding sequence with the targeting construct containing the blasticidin resistance cassette flanked by loxP recombination sites. The targeting construct with the blasticidin resistance cassette is shown on top, the genomic sequence of phg2 before the recombination event in the middle, and after the recombination event at the bottom. BamHI, PstI, HindIII, SalI: DNA restriction enzyme sites that were used for the generation of the targeting construct. A8P: Actin 8 promoter. A15T: Actin 15 terminator. Bsr: blasticidin resistance gene. The illustration does not reflect the true scale of actual parts of the disruption vector. (B) The pyk3− strain was generated by replacement of a 2.37 kbp genomic fragment of the pyk3 gene with the targeting strategy as described for phg2−. The pyk3−/phg2− double knock-out strain was generated by transforming the pyk3− strain after transient expression of the cre recombinase (restored blasticidin sensitivity) with the targeting construct for phg2 (shown at the bottom).(PDF)Click here for additional data file.Figure S2Domain structure of D. discoideum Pyk3 and Phg2. The protein sequences of Pyk3 (DDB_G0289001) and Phg2 (DDB_G0283699) were analysed with SMART (http://smart.embl-heidelberg.de/) to elucidate their domain architecture. STYKc: possible dual-specificity Ser/Thr/Tyr protein kinase domain; green rectangles: coiled coil regions; lilac rectangles: segments of low compositional complexity; aa: amino acids.(PDF)Click here for additional data file.Figure S3−, phg2− and pyk3−/phg2− knock-out strains.Analysis of the generated pyk3 (A) Reverse transcription PCR using primer pairs that amplify small fragments of the 3′-end of the pyk3, phg2, and dstc cDNA, respectively. dstc was used as a positive control. (B) Western blot analysis of total cell lysates of Ax2 wild-type, pyk3−, phg2−, and pyk3−/phg2− cells. Proteins were separated by SDS-PAGE, transferred to nitrocellulose and Western blot analyses with polyclonal pyk3 and phg2 antibodies were performed. Severin was used as loading control and detected with a monoclonal severin antibody (mAb 101-460-2). Please note that the weak band in the pyk3− strain (*) is due to cross-reaction of the Pyk3 antibody. A similar cross-reaction with this antibody was also seen in the independently generated pyk3− mutant in the lab of J.G. Williams (data not shown).(PDF)Click here for additional data file.Figure S4−, phg2−, and pyk3−/phg2− cells.Western Blot analysis of GFP-STATc expression in Ax2, pyk3 The expression of GFP-STATc was monitored by Western blot analysis of total cell lysates with a monoclonal GFP antibody (mAb K3-184-2). Actin was used as loading control and detected with a monoclonal actin antibody (mAb Act1-7).(PDF)Click here for additional data file.Figure S5−, phg2−, and pyk3−/phg2− cells.Immunofluorescence analysis of GFP-STATc nuclear translocation in GFP-STATc expressing Ax2, pyk3 (A) Ax2/GFP-STATc, (B) pyk3−/GFP-STATc, (C) phg2−/GFP-STATc, (D) pyk3−/phg2−/GFP-STATc. Log phase cells were washed twice with Soerensen buffer, transferred to coverslips and allowed to settle for 15 minutes. After treatment with 100 mM sorbitol for 0, 2, 3, 4, 5 and 8 minutes, cells were fixed with methanol and the nuclear translocation of GFP-STATc was analysed with a fluorescence microscope. Top: Exemplary images. Scale bar: 10 µm. Bottom: Quantification of GFP-STATc nuclear translocation in Ax2 wild-type and mutant cells. For each time point, we analysed 150 cells per experiment. The number of cells showing either clear cytosolic (black bar) beginning nuclear (grey bar) or prominent nuclear (white bar) localization of GFP-STATc was determined and the percentage of cells in each of these three categories was calculated. The error bars depict standard deviations of two independent experiments.(PDF)Click here for additional data file.Figure S6Overexpression of Phg2 causes a strong increase in STATc protein levels. Ax2 wild-type and Ax2/Myc-Phg2 expressing cells were either treated with 200 mM sorbitol for 15 min or left untreated and total cell lysates were prepared. Proteins were separated by SDS-PAGE and transferred to nitrocellulose. Western blot analysis was performed with antibodies specific for total (7H3) and tyrosine phosphorylated STATc (CP22), for Myc-Phg2 (mAb 9E10), and for endogenous Phg2 . Actin was used as loading control and detected with an actin-specific antibody (mAb Act 1–7).(PDF)Click here for additional data file.


Xenopus tropicalis embryos with antisense morpholino oligonucleotides targeting XtEMSY impairs gastrulation movements, disrupts dorsal structures, and kills embryos by tailbud stages. Consistent with these observations, regional markers such as Xbra, Chd, Gsc, Shh, Sox3 and Sox17 are downregulated. In contrast to these regional markers, expression of p53 is upregulated in such embryos, and at later stages Bax expression is elevated and apoptotic cells can be detected. Our results demonstrate that EMSY has an essential role in development and they provide an in vivo loss-of-function model that might be used to explore the biochemical functions of this protein in more detail.EMSY interacts directly with BRCA2 and links the BRCA2 pathway to sporadic breast and ovarian cancer. It also interacts with BS69 and HP1b, both of which are involved in chromatin remodelling, and with NIF-1 and DBC-1 in the regulation of nuclear receptor-mediated transcription. Here we investigate the function of EMSY during amphibian development, and in doing so provide the first loss-of-function analysis of this protein. Injection of  The EMSY protein is upregulated in breast and ovarian cancers Xenopus tropicalis genome contains a gene that is highly homologous to mammalian EMSY (XtEMSY). A ‘short’ form of XtEMSY is expressed throughout early development and is co-expressed with mRNAs encoding interacting proteins such as BRCA2, BS69 and HP1b. Use of antisense morpholino oligonucleotides directed against XtEMSY disrupts gastrulation and causes a downregulation of genes including Gsc and Shh. At later stages we observe an upregulation of p53, which may underlie the elevated levels of apoptosis we observe and, through its ability to interact with the TGF-β signal transduction pathway (5), may exacerbate the defects in embryonic patterning.In this study, in an effort to gain further insights into the functions of EMSY, we address its role during early development, and in so doing provide the first loss-of-function analysis of EMSY. The Embryo generation and manipulation were carried out as described Antisense morpholino oligonucleotides (MOs) were obtained from GeneTools. Embryos were injected with 15 ng antisense morpholino oligonucleotides at a concentration of 10 ng/nl in water. MOs had the following sequences. XtEMSY MO1: 5′-CCACACCACCGGCATCCTGGCCTCT-3′; XtEMSY mMO1: 5′-CCACACCACCGGCATCCTGGCCTCT-3′, XtEMSY MO2: 5′-TGGCCTCTCCTCCACAGAGCGCCCT-3′; XtEMSY mMO2: 5′-TGGCCTCTCCTCCACAGAGCGCCCT-3′; Xtp53MO: 5′-GCCGGTCTCAGAAGAAGGTCCCATG-3′.XtEMSY forward: 5′-GCCAGGATGCCGGTGGTG-3′; reverse: 5′-GCGTTTATTCCAGGGATCCTCTG-3′. EF1α forward: 5′-TGGACACGTAGATTCTGG-3′; reverse: 5′-CAGCAACAATCAGGACAG-3′.RNA extraction was carried out using TRIZOL reagent (Invitrogen) according to the manufacturer's instructions (except that RNA was LiCl precipitated a second time at the end of the protocol). cDNA synthesis was carried out using SuperscriptII (Invitrogen) and random hexamer primers. The following primers were used in RT-PCR: in situ hybridisation was carried out as described X. tropicalis cDNA collection. They were digested with EcoRI and transcribed with T7 RNA polymerase. Xbra: Tgas071i19; Chd: Tgas143n13; EMSY: Tgas029a21; Gsc: Tneu077f20; p53: Tneu034a23; Rad51: Tegg126n13; Shh: Tneu023n04; Sox3: Tgas079n14; Sox17a: Tgas079e22.Whole mount TUNEL staining was carried out as described ODC forward: 5′-GCCATCGTGAAGACTCTCTCCC-3′; reverse: 5′-TTCGGGTGATTCCTTGCCAC-3′. p53 forward: 5′-AAACTTTGCGGAGTTTTCAGAG-3; reverse: 5′-GGTGGAGTATGTGCAGGTAACA-3′. Bax forward: 5′-GAGCCTTGGTGCTGCAGGGG-3′; reverse: 5′-GGAGCCTGGGAATAGCGCCC-3′. Xbra forward: 5′-ATCAAACACAACCCCTTTGC-3′; reverse: 5′-CGAGCGGTGGTTTCTTAGAG-3′. Chd forward: 5′-AACTGCCAGGACTCATGGATG-3′; reverse: 5′-GGCAGGATTTAGAGTTGCTTC-3′. Gsc forward: 5′-GTTTTCAGCCAGGAGAGAGAGA 3′; reverse: 5′-ATGTTGTCAATGCTGAACATGC-3′. Shh forward: 5′-AGCCTTTGATGTAATTGGCTTC-3′; reverse: 5′-AATCTTTCCTTCGTATCGACCA-3′.Real-time RT-PCR was performed using a Roche Lightcycler 480. The following primer pairs were used: Xenopus orthologue of human EMSY we performed sequence searches with the full-length human EMSY sequence using the University of California Santa Cruz (UCSC) genome browser , the X. tropicalis full-length EST database (http://informatics.gurdon.cam.ac.uk/online/xt-fl-db.html) http://www.ncbi.nlm.nih.gov) X. tropicalis locus that is orthologous to human EMSY on scaffold_609 between positions 307237 and 325312. Our analysis of the X. tropicalis EMSY locus indicates that it consists of 22 exons, the first 21 of which are predicted to generate a protein of 1291 amino acids which is 78% identical to its 1322 amino acid human orthologue  in X. tropicalis embryos (data not shown). This apparent discrepancy may derive from differences between truncated human EMSY and the short form of XtEMSY, from differences between in vitro and in vivo systems, or from differences between adult and embryonic cells.Although over-expression of a truncated form of human EMSY in 184-hTert cells causes chromosomal abnormalities In an effort to inhibit XtEMSY function, we turned to antisense morpholino oligonucleotides (MOs) Our attempts to rescue the XtEMSY MO phenotype by injection of mRNA encoding the short form of XtEMSY did not succeed (data not shown). It is possible that injected mRNA does not spread rapidly enough throughout the embryo, or that XtEMSY protein is unstable and does not persist to the appropriate stages.in situ hybridisation to examine the expression of genes including Xbra (expressed in mesoderm), Chordin (Chd: chordamesoderm), Goosecoid , Sonic hedgehog (Shh: chordamesoderm), Sox3 (neurectoderm) and Sox17a (endoderm)  a–x. ExprXbra, Chordin, Goosecoid and Sonic hedgehog .EMSY was discovered in a screen for BRCA2 interactors and it links the BRCA2 pathway to sporadic breast and ovarian cancer stage 18 c. p53 inGsc and Shh, we note that expression of the apoptosis associated gene BaxAlthough p53 upregulation is unlikely to influence directly the decrease in expression of genes such as XtEMSY, the orthologue of human EMSY, is expressed as a short isoform during early development of X. tropicalis and is co-expressed with genes encoding proteins that interact with its human homologue, suggesting that XtEMSY might be involved in transcriptional regulation, chromatin remodelling and DNA repair.X. tropicalis disrupts gastrulation and axial development. Expression of the regional markers Xbra, Chd, Gsc, Shh, Sox3 and Sox17a is reduced at gastrula and tailbud stages, although their spatial expression patterns are little affected. In contrast, p53 is upregulated during gastrula stages, as is Bax at neurula stages. The upregulation of these genes may underlie the apoptosis observed in XtEMSY knockdown embryos p53 or Bax disrupts gastrulation or causes the loss of regional markers, because expression of the two genes increases only after these phenotypes are detectable, and injection of an antisense morpholino oligonucleotide directed against X. tropicalis p53 cannot rescue the embryos. Furthermore, upregulation of p53 activates the expression of mesodermal markers rather than inhibiting their transcription Loss of EMSY function in XtEMSY function in the spatio-temporal control of gene transcription including epigenetic regulation and epigenomic remodelling (determining cell fate in this case) versus its role in replication, similar to Polycomb protein group protein-containing complexes, which modulate whether chromatin is in a closed or open state (in conjunction with Top2) independently from their role in replication (in conjunction with Top1). This type of work is usually only addressed in studies based on cell lines, addressing in this case ‘stem cellness’, transit amplification and differentiation. However through our initial characterisation we have the first opportunity to take this type of analysis in vivo.Using our current study as a platform, in future work we will aim to investigate Xenopus morphant system as a technique for discovering the potential functions of genes found in mammals for which the loss-of-function models yield no obvious phenotype.Finally this work also demonstrates the usefulness of the


Spinal cord injury (SCI) often results in permanent functional loss. This physical trauma leads to secondary events, such as the deposition of inhibitory chondroitin sulfate proteoglycan (CSPG) within astroglial scar tissue at the lesion.in situ gelling hydrogel combined with lipid microtubes that slowly released constitutively active (CA) Cdc42, Rac1, or Brain-derived neurotrophic factor (BDNF). Treatment with BDNF, CA-Cdc42, or CA-Rac1 reduced the number of GFAP-positive astrocytes, as well as CSPG deposition, at the interface of the implanted hydrogel and host tissue. Neurofilament 160kDa positively stained axons traversed the glial scar extensively, entering the hydrogel-filled cavity in the treatments with BDNF and CA-Rho GTPases. The treated animals had a higher percentage of axons from the corticospinal tract that traversed the CSPG-rich regions located proximal to the lesion site.We examined whether local delivery of constitutively active (CA) Rho GTPases, Cdc42 and Rac1 to the lesion site alleviated CSPG-mediated inhibition of regenerating axons. A dorsal over-hemisection lesion was created in the rat spinal cord and the resulting cavity was conformally filled with an Local delivery of CA-Cdc42, CA-Rac1, and BDNF may have a significant therapeutic role in overcoming CSPG-mediated regenerative failure after SCI. Physical injury to the spinal cord results in the damage and death of neurons and axons, leading to loss of both sensory and motor neuronal function. Injury triggers a cascade of events, including the migration of macrophages and activation of resident glial cells, microglia and astrocytes in situ gelling hydrogel system However, as more than one ‘inhibitory’ entity exists after spinal cord injury (SCI), we examined a strategy to alter the intrinsic axonal responses to inhibitory cues using an in vitro, neurites extend through inhibitory, CSPG-rich regions in vivo, axonal regeneration was enhanced in vivo after SCI. Therefore, to our knowledge this is the first study to provide controlled release of Cdc42 and Rac1 in vivo.Rho GTPases, Cdc42 and Rac1, are responsible for the filopodial and lamellopodial extension of axonal growth cones in vivoDistinct from altering growth cone sensitivity to inhibitory signals, it is important to promote neuronal survival and stimulate axon growth. Neurotrophins, such as BDNF, neurotrophin-3 (NT-3), and nerve growth factor (NGF), are neuroprotective in vivo was used to deliver CA-Cdc42, CA-Rac1, and BDNF after SCI. Our results demonstrated that there was in vivo release for at least 2 weeks, and all three treatments had an effect on the reduction of reactive astrocytes and CSPG deposition, and axonal growth within the inhibitory CSPG-rich regions. CA-Cdc42 significantly reduced the amount of reactive astrocytes and CSPG deposition, and treatment with CA-Rac1 and BDNF had a statistically higher number of axons growing within inhibitory CSPG-rich regions compared to the untreated control.In this study, an agarose hydrogel scaffold embedded with lipid microtube delivery vehicles that provide local, sustained bioavailability All animals were handled in strict accordance with good animal practices as defined by the relevant national and/or local guidelines. All animal procedures were approved (Protocol ID A04016) by the Georgia Institute of Technology's Institutional Animal Care and Use (IACUC) Committee.8,9PC, Avanti Polar Lipids, Alabaster, AL) was dissolved in 70% ethanol at a concentration of 1 mg/mL. The lipid was placed in a water bath  programmed to decrease from 50 to 20°C over 48 h and then stored at room temperature to facilitate self-assembly of lipid microtubes. Trehalose (18.9 mg/mL) was added to the microtubule solution and then dehydrated using a rotary evaporator. For the various treatments, 1 mg of dehydrated microtubes were loaded with BDNF , CA-Cdc42, or CA-Rac1 and the loading concentrations were 500 µg/mL, 500 µg/mL and 230 µg/ml, respectively. The protein loaded microtubes were mixed into 2.6% agarose (w/v) at a 1∶1 volume ratio.Lipid microtubes were fabricated as previously described Eighty microliters of 500 µg/mL BDNF was loaded into 2 mg of lipid microtubes by incubating the BDNF solution with dehydrated microtubes. The BDNF loaded microtubes were mixed into 2.6% agarose to obtain a microtubule concentration of 8.33 mg/mL in 1.3% concentration of agarose. Forty microliters of the BDNF loaded microtubes and agarose mixture was added into well of a 96 well plate, allowed to gel, and 150 µL of PBS was added to the above mixture. The 96 well plate was placed in a 37°C oven and after the first 24 hours, the 150 µL of phosphate buffered saline (PBS) was removed and replaced with 150 µL of fresh PBS. This method was followed every 48 hours for the following 13 days. The retrieved PBS solutions were stored in −20°C until they were ready to be analyzed for BDNF content. The BDNF released from the microtubes was quantified using the BDNF ELISA Sandwich Kit (Chemicon) and then the standard and sample wells were placed in the Synergy HT Micro Detection Microplate Reader  and the absorbance was read at 450 nm. A standard curve was obtained from the absorbances of the BDNF standards to determine the concentrations of the BDNF released in the PBS.To permit visualization and characterization of BDNF's diffusion through spinal cord tissue, Rhodamine was conjugated to BDNF. The protocol provided with the EZ-Label™ Rhodamine Protein Labeling Kit  that allows Rhodamine to be conjugated to most biomolecules using primary amines was followed to conjugate the Rhodamine to BDNF (BDNF/Rhodamine). The BDNF/Rhodamine was diluted to 500 µg/mL and then loaded to the microtubes as described above. The agarose/BDNF/Rhodamine loaded microtubes mixture was injected into the spinal cord as described below. After 2 weeks the spinal cords were retrieved and processed, as described in further detail below.TAT Rho GTPase fusion proteins were prepared as previously described Male Sprague-Dawley (Charles River Laboratory) rats weighing 190–230 grams were anesthetized with Nembutal®. The skin and muscle were opened to expose the thoracic vertebrae T8–T10 and the underlying spinal cord was exposed. A modified dorsal-over hemisection was made by creating a 2 mm×2 mm×1.5 mm deep cavity thereby removing the dorsal portion of the corticospinal tract (CST). Four microliters of a protein loaded microtubes/agarose solution was injected into the cavity. A cooling system was used to gel the agarose scaffold containing the microtubes loaded with protein Four weeks post-injury, biotinylated dextran amine  was bilaterally injected into the motor cortex to trace the axons in the corticospinal tract (CST). The animals were anesthetized with 2% isoflurane and maintained through the surgery at 0.5% isoflurane. The skin and the periosteum were opened to expose the skull. Three injection sites, located within each hemisphere 2 mm from the midline and 1 mm apart from each site, were used to inject 0.5 µL of BDA at each site over 2 min. Six weeks post-injury (two weeks after BDA injection), the animals were anesthetized with ketamine (1 mL/kg), xylazine (0.17 mL/kg), and Acepromazine (0.37 mL/kg), transcardially perfused with 4% paraformaldehyde, the spinal cords were retrieved, and incubated in 4% paraformaldehyde overnight and then stored in PBS containing 0.01% sodium azide.Serial sagittal sections (25 µm) of the spinal cords were cut on a cryostat  and mounted onto glass slides. To visualize reactive astrocytes and microglia/macrophages, and labeled axons, a triple-stain was performed using anti-rabbit glial fibrillary acidic protein (GFAP) (Chemicon), ED-1 , and BDA. The sections were incubated in 4% goat serum in 0.5% Triton-X 100 for 1 hour. GFAP (1∶1000) and ED-1 (1∶1000) in 4% goat serum and 0.5% Triton-X 100 were added to the sections and incubated at 4°C overnight. The secondary antibodies, Alexa Fluor 350 goat anti-rabbit IgG (1∶200 dilution) , Alexa Fluor 594 goat anti-mouse (1∶200 dilution) (Molecular Probes), and Alexa Fluor 488 Streptavidin (1∶200) were added for GFAP, ED-1, and BDA, respectively, and allowed to incubate for 1 hour at room temperature. The sections were rinsed twice with 0.1M PBS and covered with glass coverslips.A triple stain was also done with BDA, GFAP and CS-56, an antibody to identify growth inhibitory CSPGs . Spinal cord sections were incubated overnight at 4°C with anti-GFAP and CS-56 (1∶250) in 4% goat serum and 0.5% Triton-X 100. The secondary antibodies, Alexa Fluor 350 goat anti-rabbit (1∶200), Alexa Fluor 594 goat anti-mouse IgM (1∶200), and Alexa Fluor 488 Streptavidin (1∶200) were added as described above. In addition, to help visualize the axons and neurons at the injury site a double stain with BDA and anti-mouse Neurofilament 160 kDa  was performed as described above. An anti-rabbit calcitonin gene related peptide  was used to identify sensory fibers around the lesion site. All images were taken on the Zeiss Axioskop 2 Plus microscope .The methodology to quantify the fluorescent intensity of GFAP and CS-56 images has been previously published + stained boundary. Ten sections that were located at the epicenter of the lesion were used to determine the lesion area. The lesion area was measured using ImagePro software and the average was then calculated for each of the conditions.The lesion area was identified as the cavity surrounded by the GFAP+ cells in and around the lesion site was quantified. Two 10× images were taken of each section using an Olympus digital camera attached to the Zeiss Axioskop 2 Plus microscope. ImagePro software  was used to count the number of cells that were present. Two parameters, (1) the fluorescent intensity and (2) cell diameter, were kept constant for all the images of the different conditions when counting the cells.The number of ED-1+ axons were imaged at 10× on the Nikon Eclipse 80i upright microscope using a Microfire CCD camera  that interfaced with the Neurolucida software  to obtain a montage of each section. The images were used to measure axon distance from the lesion site for all of the conditions. The distance between the 3 or more closest axons, with dystrophic endbulbs, and the lesion site was measured using ImagePro Software.Spinal cord sections stained for BDA+ axons, from 0 to 1 mm, 1 to 2 mm, 2 to 3 mm, and 3 to 4 mm, proximal to the injury site was counted using 10× montage images. The percent of axonal outgrowth was determined by the following equation:The beginning of the proximal side of the lesion was marked as 0 mm and every millimeter was marked and ‘binned’ up to a distance of 4 mm rostral to the lesion site. The number of axons 4 mm proximal to the lesion site was considered to be the original number of axons in the CST and is written as total number of axons at 4mm in the equation below. The number of BDA * 100+ axons were placed in three categories: (1) axons that stopped before the CS-56+ inhibitory region, (2) axons crossed the proximal interface of the inhibitory region but stopped within the inhibitory region, and (3) axons that crossed the distal interface of the inhibitory region and grew past it; and counted. A percent of axons in each region was determined by the following equation:CSPGs are present in the glial scar around the lesion area at high levels in the immediate vicinity of the lesion, but extending up to 400–500 microns away from the lesion border. The fluorescent intensity values of CS-56 stained regions were used to identify these ‘inhibitory regions’. To determine the degree to which regenerating axons where able to overcome CSPG-mediated inhibition, BDA *100The axons that had stopped within the inhibitory region mentioned in the section above were then measured to determine the distance extended after crossing the proximal interface. The shortest total distance extended by the axons within the inhibitory region was measured and then averaged. The length of all the axons that stopped their outgrowth within this region was measured and then averaged for each condition. The percent of the distance extended by the axons within the CS-56 region was calculated by the following equation: *100GFAP, CS-56, and NF-160: The area under the curve of the overall average intensity profiles was determined for four different distance ‘bins’: 0 to 100 µm, 100 to 200 µm, 200 to 300 µm, and 300 to 400 µm from the interface extending into the spinal cord. The area under the curve of the profiles was statistically compared by ANOVA and the post-hoc test Tukey's test (P<0.05). To determine if there was significant difference amongst the conditions and between different groups, ANOVA and Tukey's test (P<0.05) was performed for all other analysis.in vitro release assay for BDNF loaded microtubes was performed. Approximately 3 µg of BDNF was released within the first 24 h. A cumulative of 4 µg of BDNF was released within the first 3 days. An + cells (green) overlap with the BDNF-Rhodamine. The image shows some co-localization demonstrating engulfment of the BDNF-Rhodamine by the ED-1+ cells. The BDNF-Rhodamine was double labeled with DAPI to visualize cell nuclei (blue) in Lipid microtubes loaded with BDNF/Rhodamine embedded in the agarose were injected into the spinal cord cavity. For this experiment only, the spinal cords were retrieved two weeks post-injury and sectioned. BDNF/Rhodamine can be seen in the spinal cord tissue between 1 to 2 mm proximal to the lesion site . Insets Six weeks post injury, regions with reactive astrocytes (high GFAP intensity) are generally correlated with increased CSPG deposition (CS-56 intensity) . Untreat2, which was significantly larger than all of the treated groups, BDNF, CA-Cdc42, and CA-Rac1 that had an area approximately 0.7 mm2  were identified proximal to the lesion site in the treated and control groups. The area was considered to be inhibitory if the CS-56 pixel intensity values were within the range of those in the glial scar bordering the lesion . The numin situ gelling hydrogel allows the lesion cavity to be conformally filled. Second, the lipid microtubes allow slow release of therapeutics from the hydrogel over time. Third, this strategy allows neuroprotective or axonal migration stimulators to be delivered locally over time.This study was performed to determine whether utilizing the hydrogel/microtubule scaffold delivery system to deliver BDNF, CA-Cdc42, or CA-Rac1 would decrease the sensitivity of growth cones to CSPGs, and promote axonal growth through CSPG-rich regions at the lesion site. First, the use of the in situ gelling hydrogels that released CA-Cdc42, CA-Rac1, or BDNF over time. The sustained release profile of BDNF was characterized in vivo. Two weeks post-implantation, BDNF/Rhodamine is present around the lesion site, demonstrating at least 2 week release, as well as diffusion 2 mm from the lesion site , a downstream effector of Rho, phosphorylates GFAP In addition to the initial injury, secondary injury leads to the increase in the lesion size. It is important that the therapeutic strategies do not enhance lesion size. In a study using anti-transforming growth factor-β, although the antibody reduced astorycytosis, the lesion size increased + axons were present in the treated animals. CGRP+ sensory axons infiltrated the inhibitory glial scar and the scaffold compared to the untreated and agarose controls. In vivo studies have shown that BDNF helps stimulate axonal outgrowth of sensory fibers Numerous NF-160+ axons were not located within the scaffold-filled lesion, in the treatment conditions axons were present within the glial scar, in the inhibitory CSPG-rich regions, which was significantly increased from the untreated and agarose controls. The Rho GTPases, as well as BDNF, influence actin cytoskeleton dynamics by aiding in actin polymerization. These treatments may have reduced retraction or altered dieback of axons in the treated conditions to allow axons to extend toward the lesion site despite the presence of the CSPG inhibitory regions. Rather than promote actin polymerization, CA-Cdc42, CA-Rac1, and BDNF may have hindered actin depolymerization reducing the amount of axonal retraction. Therefore, the axons in the treated groups remained closer to the lesion site compared to the controls.The effect of BDNF, CA-Cdc42, and CA-Rac1 on axonal infiltration into the glial scar and scaffold-filled lesion was analyzed using the anterograde tracer BDA. Although BDACSPG inhibitory regions, identified by the fluorescent intensity of CS-56 were observed proximal to the glial scar in the spinal cord at similar intensity values. When the astrocytes become reactive after injury, these cells deposit CSPGs creating a glial scar. The presence of the CSPG-rich inhibitory regions and the effects of the Rho GTPases and BDNF on axonal growth proximal to the lesion site, suggests that it is imperative to deliver therapeutics that block inhibitory signals not only into the cavity, but also to areas proximal to the lesion site, thereby increasing the number of axons extending towards and possibly through the lesion site.+ axons through the hydrogel scaffold, a reduction in GFAP and CS-56 fluorescent intensity, thereby reducing astrocytes reactivity and CSPG deposition, and a higher percentage of BDA+ axonal extension towards the lesion site. This suggests that the Rho GTPases and BDNF may influence the inflammatory response possibly affecting the macrophage/microglia and astrocyte responses and neuronal axonal extension. The double combination of CA-Cdc42/CA-Rac1 or delivery of CA-Cdc2, CA-Rac1, and BDNF in combination was not performed in this study due to cytotoxic effects that were observed in the neurons when CA-Cdc42 and CA-Rac1 were transduced together + and CGRP+ axonal growth also demonstrates that local delivery of Rho GTPases and BDNF promotes axonal infiltration through CSPG-rich regions and into the lesion site filled with the hydrogel substrate after SCI. These findings suggest that there exists a significant potential for strategies aimed at modulating Rho GTPases alone, and in combination with each other or BDNF to overcome CSPG-mediated inhibition after SCI.Quantification of GFAP, CS-56, BDA, and NF-160 stains demonstrated a significant difference between the treatments and the control groups. Treatment with CA-Cdc42, CA-Rac1, and BDNF demonstrated NF-160


Aim. The aim of the present study was to investigate the effect of cornel iridoid glycoside (CIG) on tau hyperphosphorylation induced by wortmannin (WT) and GF-109203X (GFX) and the underlying mechanisms. Methods. Human neuroblastoma SK-N-SH cells were preincubated with CIG  for 24 h and then exposed to 10 µM WT and 10 µM GFX for 3 h after washing out CIG. Immunohistochemistry was used to observe the microtubular cytoskeleton of the cultured cells. Western blotting was used to measure the phosphorylation level of tau protein, glycogen synthase kinase 3β (GSK-3β), and protein phosphatase 2A (PP2A). The activity of PP2A was detected by a biochemical assay. Results. Preincubation of CIG significantly attenuated the WT/GFX-induced tau hyperphosphorylation at the sites of Thr205, Thr212, Ser214, Thr217, Ser396, and PHF-1 and improved the damage of morphology and microtubular cytoskeleton of the cells. CIG did not prevent the decrease in p-AKT-ser473 and p-GSK-3β-ser9 induced by WT/GFX. However, CIG significantly elevated the activity of PP2A by reducing the demethylation of PP2A catalytic subunit (PP2Ac) at Leu309 and the ratio of PME-1/LCMT in the WT/GFX-treated cells. The results suggest that CIG may be beneficial to the treatment of AD.  Microtubule associated protein tau is mainly expressed in neurons and it is involved in modulating microtubule assembly and stabilizing the neuronal cytoskeleton . The funβ (GSK-3β) is an important serine-threonine kinase that phosphorylates glycogen synthase in the glycogen synthesis pathway. Protein phosphatase 2A (PP2A) is a major phosphatase implicated in tau phosphorylation. Studies have shown that PP2A activity is decreased in AD brains [β and PP2A signaling pathways were reported to be interconnected. Activation of GSK-3β through simultaneous inhibition of phosphatidylinositol-3 kinase (PI3K) and protein kinase C (PKC) in rats or cells induces tau hyperphosphorylation [β. Activation of GSK-3β can inhibit PP2A by increasing the inhibitory Leu309-demethylation involving upregulation of protein phosphatase methylesterase-1 (PME-1) and inhibition of leucine carboxyl methyltransferase (LCMT) [The phosphorylation of tau is regulated by the activities of various kinases and phosphatases. Glycogen synthase kinase 3D brains . GSK-3β rylation –10. Coade (LCMT) . PME-1 aCornus officinalis Sieb. et Zucc is a member of the Cornaceae family. Use of this herb was first recorded in Shen Nong's Materia Medica about 2000 years ago in China. In traditional Chinese medicine, Cornus officinalis is used to tonify the liver and the kidney for the treatment of vertigo, aching back, spontaneous emission, and sweating. Clinically, Cornus officinalis is also used to treat AD combining with other herbs [Cornus officinalis. The purity of CIG was 71.19% determined by RP-HPLC assay, in which morroniside accounted for 67% and loganin 33% [er herbs , 13. Coranin 33% . Previouanin 33% . CIG alsanin 33% . Specifianin 33% . All finβ, to induce AD-like tau hyperphosphorylation, and investigated the effects and mechanisms of CIG on tau phosphorylation.In the present study, we treated the human neuroblastoma SK-N-SH cells with wortmannin and GF-109203X, the classical activator of GSK-3Cornus officinalis as described previously and the purity of CIG was 71.19% determined by RP-HPLC assay, in which morroniside accounted for 67% and loganin 33% [Cornel iridoid glycoside (CIG) was extracted from anin 33% .The primary antibodies used in this study are listed in 2 and 95% air at 37°C, and the culture medium was replaced every 3 days. SK-N-SH cells were treated for 24 h with different concentrations of CIG  and then exposed to 10 μM wortmannin and GF-109203X in serum-free DMEM for 3 h after washing out CIG.Human neuroblastoma SK-N-SH cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and were kept in a humidified atmosphere of 5% COβ-tubulin (1 : 300 dilution) overnight at 4°C. Cells were washed and incubated with Oregon Green 488-conjugated goat anti-rabbit IgG secondary antibody (1 : 500) and visualized with a laser confocal microscope .Cells growing on glass coverslips were fixed for 30 min at room temperature in 4% paraformaldehyde. Cell membranes were blocked in 3% bovine serum albumin for 1 h at room temperature and incubated with rabbit polyclonal Total protein was extracted from cell lysates using RIPA buffer. Three volumes of cell homogenate were added to one volume of sample buffer then boiled for 5 min. The protein concentration was measured by RC-DC protein assay according to manufacturer's instructions . The proteins were separated by 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene fluoride membrane . The blots were then probed with primary antibodies and then incubated with the corresponding anti-mouse or anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence kit . Densitometric quantification of the protein bands was analyzed by TINA software .μL) were incubated with a chemically synthesized phosphopeptide in the half area 96-well plate. After incubating at 37°C for 30 min, the reaction was stopped by adding 50 μL of Molybdate Dye/Additive mixture and then the fluorescence intensity of the samples was measured under a 630 filter set.PP2A activity was measured according to the PP2A assay protocol . In brief, the extracts of cell samples were centrifuged to remove particulate matter, and then endogenous free phosphate was removed with gel columns. Enzyme samples  followed by Tukey's post-hoc test using SPSS16.0 software. A probability of μM wortmannin/GF-109203X (WT/GFX) for 3 h. Compared with WT/GFX-treated group, preincubation of CIG (100 and 200 μg/mL) for 24 h with SK-N-SH cells prevented the damage induced by WT/GFX and improved the morphology of cells.β-tubulin to visualize the structures. In the normal group, we found that β-tubulin (green) was distributed in cell bodies and processes homogeneously, and CIG did not affect the microtubular cytoskeleton of the normal cells. After being exposed to 10 μM WT/GFX for 3 h, β-tubulin disappeared and the processes of cells were retracted, indicating that WT/GFX caused disintegration of the microtubular cytoskeleton. Preincubation of CIG (100 and 200 μg/mL) with SK-N-SH cells for 24 h improved the structures of microtubular cytoskeleton compared with WT/GFX-induced model group . Pretreatment of CIG (100 and 200 μg/mL) for 24 h significantly attenuated the WT/GFX-induced tau hyperphosphorylation at Thr205, Thr212, Ser214, Thr217, and Ser396/404   .β is the pivotal kinase involving the formation of tau phosphorylation in AD brains. GSK-3β activity is regulated by Ser9 phosphorylation. Akt (protein kinase B) is upstream kinase in phosphorylating GSK-3β at Ser9 and inhibiting the activity of GSK-3β [β at Ser9 (the inactivated form) in the cell extracts. The results displayed that the levels of phosphorylated Akt at Ser473 and phosphorylated GSK-3β at Ser9 were significantly decreased in the cells exposed to 10 μM WT/GFX for 3 h (P < 0.05), suggesting that GSK-3β activity may be increased after PI3K/Akt inhibition. However, the pretreatment of CIG for 24 h did not alter the phosphorylation levels of both GSK-3β-ser9 and Akt-ser473 compared with WT/GFX model group. There was no obvious difference in the expression of GSK-3β and Akt among control, WT/GFX model, and CIG-treated groups  affects the activity of PP2A [μM WT/GFX treatment for 3 h decreased PP2A activity in SK-N-SH cells compared with the control group (P < 0.05), and the pretreatment with 100 μg/mL CIG significantly enhanced PP2A activity compared with the WT/GFX model group (P < 0.05) (P < 0.05) but was significantly decreased in CIG-treated groups compared with the model group  , which may enhance the demethylation level of PP2Ac. Pretreatment with CIG  significantly decreased the ratio of PME-1/LCMT compared with WT/GFX model group (P < 0.01)  . Althoug Figures . It is k Figures . The incCornus officinalis, inhibited hyperphosphorylation of tau via inhibiting PP2Ac demethylation.Tau hyperphosphorylation has been reported to play a pivotal role in AD pathology . In the β and downregulation of PP2A have been proposed to be involved in the abnormal tau phosphorylation in AD. The inactive form of GSK-3β which is phosphorylated at Ser9 is increased in AD brains [β activity by phosphorylating GSK-3β at Ser9 site. Phosphoinositol-3 kinase (PI3K) activates Akt, which inhibits GSK-3β by phosphorylating its Ser9 residue [β phosphorylates tau at many sites, with Thr205, Thr212, Ser214, Thr217, and Ser396/404 being the favorable sites in cells [β activity by increasing the phosphorylation at Ser9 and did not alter the phosphorylation level of AKT compared with WT/GFX model group.The cytoskeleton is a cellular structure that provides neuronal morphology and whose essential components are the microtubules. The cytoskeleton is important in the formation of axon and dendrites, which are involved both in transport and neurotransmission . TubulinD brains . Wortmanin cells . In the β and PP2A signaling pathways may be interconnected [β inhibits the activity of PP2A via upregulating the demethylation of PP2A catalytic subunit (PP2Ac) at Leu309. Activation of GSK-3β by wortmannin increases the level of demethylation of PP2Ac at Leu309 [PP2A is the major protein phosphatase in the brain that removes phosphate residues from tau, thereby stopping the ability of tau to inhibit microtubule assembly and to self-assemble into paired helical filaments and neurofibrillary tangles . PP2A haonnected , 33. GSKt Leu309 . In thisβ induces the demethylation of PP2A at Leu309 by increasing the protein level of PME-1 and decreasing the protein level of LCMT [Leucine carboxyl methyltransferase (LCMT) and protein phosphatase methylesterase-1 (PME-1) can catalyze the methylation and demethylation of PP2A, respectively . Upregul of LCMT , 34. In In conclusion, our findings provide the evidence in the first time that cornel iridoid glycoside (CIG) attenuates tau hyperphosphorylation by increasing the activity of PP2A. The mechanism of CIG may be related to decreasing the ratio of PME-1/LCMT, thus reducing the demethylation of PP2Ac at Leu309. The results suggest that CIG may be beneficial to the treatment of AD.


BCR to ABL resulting in deregulated ABL-tyrosine kinase activity. The constitutively activated BCR/ABL-kinase “escapes” the auto-inhibition mechanisms of c-ABL, such as allosteric inhibition. The ABL-kinase inhibitors (AKIs) Imatinib, Nilotinib or Dasatinib, which target the ATP-binding site, are effective in Ph + leukemia. Another molecular therapy approach targeting BCR/ABL restores allosteric inhibition. Given the fact that all AKIs fail to inhibit BCR/ABL harboring the ‘gatekeeper’ mutation T315I, we investigated the effects of AKIs in combination with the allosteric inhibitor GNF2 in Ph + leukemia.Chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive (Ph+) acute lymphatic leukemia (Ph + ALL) are caused by the t, which fuses The efficacy of this approach on the leukemogenic potential of BCR/ABL was studied in Ba/F3 cells, primary murine bone marrow cells, and untransformed Rat-1 fibroblasts expressing BCR/ABL or BCR/ABL-T315I as well as in patient-derived long-term cultures (PDLTC) from Ph + ALL-patients.Here, we show that GNF-2 increased the effects of AKIs on unmutated BCR/ABL. Interestingly, the combination of Dasatinib and GNF-2 overcame resistance of BCR/ABL-T315I in all models used in a synergistic manner.Our observations establish a new approach for the molecular targeting of BCR/ABL and its resistant mutants using a combination of AKIs and allosteric inhibitors. BCR/ABL results in a deregulated and constitutively activated tyrosine kinase, which is responsible for the induction of the phenotype of Ph + leukemia. BCR/ABL constitutively activates several signaling pathways leading to uncontrolled proliferation and inhibition of apoptosis. The expression of BCR/ABL is sufficient for the initiation and maintenance of early stage CML and the “CML-like disease” in mice [Chronic myeloid leukemia (CML) and 30% of adult acute lymphatic leukemia (ALL) are characterized by the Philadelphia chromosome (Ph+), which is the cytogenetic correlate of the 9;22) chromosomal translocation. The t leads to the fusion of the inase ABL. BCR/ABL2 chromosSelective targeting of BCR/ABL by ABL-kinase inhibitors (AKI) such as Imatinib, Nilotinib or Dasatinib, all competitive ATP-analogues, leads to durable cytogenetic and molecular remissions in the majority of CML patients in the early chronic phase of the disease. However, unsatisfactory responses in advanced disease stages, resistance and long-term tolerability of BCR/ABL inhibitors represent major clinical problems. In fact, advanced CML and Ph + ALL respond only transiently to AKIs ,4. SeconThe activation status of wild-type c-ABL is finely regulated by several regulation signals. Myristoylation of the N-terminus of c-ABL is involved in the regulation of the ABL kinase activity. The N-terminus of ABL is myristoylated, and the myristate residue binds to a hydrophobic pocket in the kinase domain - the myristoyl-binding pocket (MBP) – in a process called “capping”. The “capping” leads to conformational changes that allow the intramolecularly docking of the “SRC homology 2 domain” to the kinase domain. Hence, c-ABL adopts an auto-inhibited conformation. The absence of an N-terminal myristoylated domain activates c-ABL consistent with its auto-regulatory role. In the context of the t, the N-terminal auto-inhibitory “Cap” region is substituted by the BCR portion of the fusion protein. The absence of the “Cap” region allows the BCR/ABL to “escape” auto-inhibition contributing to the constitutive activation of its kinase activity .We have recently shown that the allosteric inhibition increases the sensitivity of BCR/ABL-T315I towards the inhibition of oligomerization most likely by interfering with the overall confirmation of the kinase . Given tThe cDNAs encoding BCR/ABL and BCR/ABL-T315I have been previously described (3). All retroviral expression vectors used in this study were based on the bi-cistronic PINCO vector.-5 M β–mercaptoethanol , 200 mg/mL Fe –saturated human apo–transferrin , 0.6% human serum albumin , 2.0 mM L–glutamine and 20 mg/mL cholesterol (Sigma) [The Ba/F3 and Rat-1 cells were obtained from the German Collection of Microorganisms and Cell Cultures  and were maintained as previously described (3). Ph + ALL patient derived long term cultures (PDLTCs) expressing BCR-ABL-T315I (KÖ) were obtained from a patient enrolled in the German Multi-Center Study Group for acute lymphatic leukemia of the adult (GMALL 07/2003) upon informed and written consent  and were (Sigma) . Prolife+/lin- HSPCs were isolated from 8- to 12-week-old female C57BL/6 N mice  after euthanization by CO2 asphyxiation. Bone marrow (BM) was harvested from the femur and tibia by flushing the bones with a syringe and a 26-gauge needle. Sca1+ cells were purified by immunomagnetic beads using MACS cell separation columns according to the manufacturer’s instructions . Prior to subsequent use, the purified cells were pre-stimulated for 2 days in DMEM supplemented with 10% FCS , 1% L-Glutamine, 1% Penicillin/Streptomycin, mIL-3 (20 ng/mL), mIL-6 (20 ng/mL) and mSCF (100 ng/mL) .Sca15 target cells were seeded per well. Infection efficiency was measured after 48 h by determining the percentage of GFP positive cells using flow cytometry.Ecotropic retroviral supernatants were obtained after transfection of Phoenix packaging cells as described earlier . For inf3 transduced Rat-1 cells were suspended in “top-agar” (DMEM supplemented with 10% FCS and 0.25% bacto-agar) (1 ml per well) and stacked in the wells. Colonies were counted after 15 days of incubation at 37°C and 5% CO2. For focus formation assays, 4x104 transduced Rat-1 cells were plated per well of a 24-well plate. Foci were stained after 15 days using 1% crystal violet (Sigma).Soft agar and focus formation assays were performed using untransformed Rat-1 fibroblasts retro virally transduced with PINCO vectors harboring unmutated BCR/ABL or BCR/ABL-T315I. Six-well plates were filled with DMEM supplemented with 10% FCS and 0.5% bacto-agar  (2 ml per well). Then, 5x10+ cells were plated at 5x103 cells/mL in methyl-cellulose either with mIL-3 (20 ng/mL), mIL-6 (20 ng/mL) and mSCF (100 ng/mL) or without cytokines . The number of colony forming units (CFUs) was determined 10 days after plating and normalized according to the transduction efficiency.At day 5 post-infection, Sca1Western blot analysis was performed according to widely accepted protocols. The following antibodies were used: anti-ABL (α-ABL) , anti-phosphorylated ABL specific for the phosphorylated tyrosine residue 245 (α-p-ABL-Y245) , anti-BCR (α-BCR) (St. Cruz Biotechnology), anti-phosphorylated BCR specific for the phosphorylated tyrosine residue 177 (α-p-BCR-Y177), anti-Crkl, and anti-phosphorylated Crkl .Differences in response rates towards different concentrations of a single inhibitor or inhibitors in combination were analyzed by Student′s t-tests. Statistical analyses were performed using the GraphPad Prism  software package. Evaluation of the character of the combined effects was performed according to the three dimensional model of Prichard and Shipman using MacSynergy software .Unmutated BCR/ABL can be efficiently inhibited not only by AKIs but also by allosteric inhibitors such as GNF-2 or GNF-5 ,11,12. TTaken together, these data suggest that the inhibition effect of Dasatinib is enhanced by GNF-2 in cells expressing unmutated BCR/ABL.The major clinical challenge in Ph + leukemia is the drug resistance due to the “gatekeeper” mutation T315I. T315I confers a nearly global resistance to all molecular therapy approaches that target BCR/ABL. Neither GNF-2 nor AKIs have any effect on cells transformed by BCR/ABL-T315I. To analyze whether the combination of allosteric inhibition with AKIs is able to inhibit BCR/ABL-T315I, we exposed Ba/F3 cells expressing BCR/ABL-T315I to increasing concentrations of Dasatinib  and GNF-2 . Cytotoxicity and proliferation were assessed by the XTT assay. Here, we show that only the combination of GNF-2 and Dasatinib inhibited BCR/ABL-T315I-dependent cell growth with a very high synergy index of 186  and in 6-well plates for the focus formation assay (contact inhibition). Colonies and “foci” stained with crystal violet were counted after 15 days. As shown in Figure We have shown recently that GNF-2 inhibits the transformation potential of unmutated BCR/ABL but not of BCR/ABL-T315I in untransformed fibroblasts . TherefoThese data indicate that the combination of allosteric inhibitors with AKIs inhibits the transformation potential of BCR/ABL-T315I.+ HSPCs with BCR/ABL-T315I, and plated the cells in methyl-cellulose with increasing concentrations of GNF-2 and Dasatinib. As shown in Figure To further confirm the synergistic effect of the combination of Dasatinib and GNF-2, we extended our investigation to a model of primary murine hematopoietic stem and progenitor cells expressing BCR/ABL. We studied the effects of the drug combination on the colony formation by BCR/ABL cells in semi-solid medium in the presence or absence of cytokines . We transduced Sca1These data demonstrate that mHPSCs expressing the “gatekeeper” mutation T315I can be targeted efficiently by the combination of GNF-2 and Dasatinib.The major therapeutic challenge in Ph + leukemia is to efficiently treat patients with BCR/ABL harboring the T315I mutation. The T315I mutation is the most resistant to inhibition because of a combination of several factors, including steric hindrance of drug binding, loss of a key hydrogen-bonding interaction with the T315 side-chain hydroxyl group exploited by Imatinib, Nilotinib and Dasatinib and potentially through increasing aberrant intrinsic kinase activity accompanied by aberrant substrate phosphorylation ,14. UnfoGNF-2 and its analogues are non-ATP competitive ABL kinase inhibitors, which bind to the MBP in the kinase domain. It seems that the binding of GNF-2 to the MBP stabilizes the protein in an inhibited conformation resulting in a structural reorganization of ABL that disrupts the catalytic machinery located in the ATP-binding region . Thus, oAn additive but not synergistic effect was shown for the combination of Nilotinib with GNF-2 or GNF-5 on BCR/ABL-T315I-related resistance. The stronger effects may be attributed to the fact that Dasatinib, originally developed as a SRC-kinase inhibitor, not only inhibits the BCR/ABL kinase but also targets a broader range of kinases compared to Nilotinib, the spectrum of which is mainly limited to ABL, c-KIT and PDGFR . An addi+ leukemia. In fact, it not only overcomes the resistance mediated by the “gatekeeper” mutation T315I but also increases the response of unmutated BCR/ABL to AKI. In the clinical setting, this feature could contribute to a more efficient use of AKI at a lower dosage in “normally” responsive patients and the possibility to further increase dosage in patients early in the progression of disease, in the absence of BCR/ABL mutations, for whom dosage escalation is still a therapeutic option.Our data further establish allosteric inhibition as alternative or additional molecular therapy approach for the treatment of PhThe results presented here contribute to the further development of allosteric inhibition for the molecular targeting of both unmutated BCR/ABL and BCR/ABL harboring the multi-resistance mutation T315I.Resistance and long-term tolerability of BCR/ABL inhibitors represent the major therapeutic challenge in Philadelphia Chromosome-positive (Ph+) leukemia. Advanced Ph + leukemia respond only transiently to ABL kinase inhibitors (AKI). Resistance is mostly caused by the acquisition of point mutations in BCR/ABL. The “gatekeeper” mutation T315I confers resistance against all available molecular therapy approaches. Conformational changes by allosteric inhibition increases the response of both unmutated BCR/ABL and BCR/ABL-T315I towards inhibition of oligomerization. Therefore we investigated whether the conformational changes induced by the allosteric inhibition also enhances the response towards the AKI Dasatinib in clinically relevant models of Ph + leukemia. Allosteric inhibition not only increased the response of unmutated BCR/ABL to Dasatinib but also contributed to overcome resistance of BCR/ABL-T315I in a synergistic manner in all models used.Therefore allosteric inhibition may contribute to the optimization of the therapy of patients with both unmutated BCR/ABL or harboring resistance mutations such as the T315I.The authors declare that they have no competing interests.Afsar Ali Mian, Anna Metodieva both authors have to be considered first authors.AAM carried out the studies on transduced Ba/F3 cells and participated in drafting the manuscript. AM performed the transformation assays on Rat-1 fibroblasts and on the murine HSCs. SB generated the PDLTCs harboring the BCR/ABL-T315I and carried out the experiments on these cells. MK and NR participated in the elaboration of the data. JM and YN participated in the design of the study and performed the statistical analysis. OGO participated in the design and coordination of the study. MR conceived of the study and wrote the paper. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/12/411/prepub


Interferon regulatory factor 2 binding protein 2 (IRF2BP2) is a muscle-enriched transcription factor required to activate vascular endothelial growth factor-A (VEGFA) expression in muscle. IRF2BP2 is found in the nucleus of cardiac and skeletal muscle cells. During the process of skeletal muscle differentiation, some IRF2BP2 becomes relocated to the cytoplasm, although the functional significance of this relocation and the mechanisms that control nucleocytoplasmic localization of IRF2BP2 are not yet known.354ARKRKPSP361 in IRF2BP2. This sequence corresponds to a classical nuclear localization motif bearing positively charged arginine and lysine residues. Substitution of arginine and lysine with negatively charged aspartic acid residues blocked nuclear localization. However, these residues were not sufficient because nuclear targeting of IRF2BP2 also required phosphorylation of serine 360 (S360). Many large-scale phosphopeptide proteomic studies had reported previously that serine 360 of IRF2BP2 is phosphorylated in numerous human cell types. Alanine substitution at this site abolished IRF2BP2 nuclear localization in C2C12 myoblasts and CV1 cells. In contrast, substituting serine 360 with aspartic acid forced nuclear retention and prevented cytoplasmic redistribution in differentiated C2C12 muscle cells. As for the effects of these mutations on VEGFA promoter activity, the S360A mutation interfered with VEGFA activation, as expected. Surprisingly, the S360D mutation also interfered with VEGFA activation, suggesting that this mutation, while enforcing nuclear entry, may disrupt an essential activation function of IRF2BP2.Here, by fusing IRF2BP2 to green fluorescent protein and testing a series of deletion and site-directed mutagenesis constructs, we mapped the nuclear localization signal (NLS) to an evolutionarily conserved sequence Nuclear localization of IRF2BP2 depends on phosphorylation near a conserved NLS. Changes in phosphorylation status likely control nucleocytoplasmic localization of IRF2BP2 during muscle differentiation. Interferon regulatory factor 2 binding protein 2 (IRF2BP2), together with the related protein IRF2BP1, were initially discovered as interacting partners to interferon regulatory factor 2 (IRF2) in a yeast two-hybrid screening assay Structurally, IRF2BP2 is encoded by 2 exons producing 3 alternatively spliced proteins IRF2BP2a of 587, IRF2BP2b of 561 and IRF2BP2c of 163 amino acids depending on the use of alternative donor (2a and 2b) and acceptor (2c) splice sites. IRF2BP2a and b isoforms have a Zinc-finger motif at their N-terminus, missing in the IRF2BP2c isoform, and a C3HC4 RING-finger motif at their C-terminus. The function of the Zinc-finger motif appears to enable homo- and hetero-dimerization between different members of the IRF2BP2 familyThe tumor suppressor p53 binds to the IRF2BP2 promoter and transactivates its expression in response to actinomycin D treatments in both cervical carcinoma (HeLa) and osteosarcoma (U2OS) 2C12 myoblasts 2C12 myoblasts is nuclear, following ischemia, IRF2BP2 is mostly cytoplasmic. This discrepancy suggests a potential mechanism for modulating IRF2BP2 translocation across the nuclear membrane.We identified IRF2BP2 as a cofactor of VGLL4 in a yeast two-hybrid screen Nucleocytoplasmic shuttling is a carefully regulated process controlling the import and export of both mRNA and proteins In silico database mining revealed that this serine residue is phosphorylated in IRF2BP2 and IRF2BP1, and sequence comparisons revealed that this motif is conserved in all members of the IRF2BP2 family of transcription factors. Thus, our study is the first to reveal a conserved functional domain controlling nuclear import in IRF2BP2.In this report, we used deletion and site-directed mutagenesis to localize a conserved functional NLS in IRF2BP2. In addition, we found that phosphorylation of serine residue 360 (S360) adjacent to the NLS is also involved in controlling the nuclear entry of the protein. GGA TCC GGC TCC TCG GACATG GCC-3′5′-  and TCT AGA GTC TCT CTC TTT TTT CAC TTT-3′5′-  and was then subcloned into TOPO vectors . To generate pEGFP-IRF2BP2a, full-length IRF2BP2a was released from TOPO-IRF2BP2a plasmid by BamHI digestion and then subcloned in pEGFP-C1 . A summary of the GFP-IRF2BP2 fusion constructs is shown in GAA TTC TGG AGC CTT GGA GGG CAC GGC5′-  - 3′ (EcoRI) and GGA TCC CCG AGT CTC TCA TTC TTT CAC CAG -3′5′-  (BamHI) and subcloned into pEGFP-C1 vector between EcoRI and BamHI sites. For generating mutant constructs, the IRF2BP2 mutants – 355 GAC GAC GAC GAC 358 (aspartic acids), TCT 360 TAT (S360A), and TCT 360 GAT (S360D) - flanked between the endogenous PstI and NcoI in IRF2BP2 were ordered from Integrated DNA Technologies  as pIDTSMART-IRF2BP2 and were subcloned in pEGFP-IRF2BP2a (Cloning details will be provided upon inquiry). Similarly, a pEGFP-IRF2BP2b construct was generated by swapping a PstI/NcoI fragment of IRF2BP2b into the pEGFP-IRF2BP2a clone. All plasmid constructs were purified by CsCl-banding and their reading frames were verified by sequencing. DsRed-Rab11 WT (Addgene plasmid 12679) and RFP-LAMP1 (Addgene plasmid 1817) expression vectors were obtained from Addgene . Expression vectors for wild type IRF2BP2, S360A and S360D mutants were generated by subcloning a full-length EcoR1 fragment from pEGFP-IRF2BP2 into the pXJ40-CMVHA vector. The mouse VEGFA promoter construct was described previously Human IRF2BP2a (AY278023) was first amplified by PCR with the primers 2C12 myoblasts and African green monkey kidney (CV1) cells were obtained from American Type Culture Collection . Cells were maintained in high glucose DMEM with 20% FBS (or 10% FBS for CV1), 100 U/ml penicillin, 100 ug/ml streptomycin. Lipofectamine 2000  and GenJet Reagent  were used to transfect CV1 and C2C12 myoblasts, respectively, according to the manufacturer's instructions. Luciferase assays were conducted as described previously CThe protein kinase A (PKA) inhibitor H89 , the inhibitor of phosphoinositide 3-kinases LY294002 , and the MEK1/MEK2 inhibitor U0126  were from Sigma-Aldrich . The calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor CK-59  and the ribosomal S6 kinase (RSK) inhibitor SL0101  were from Calbiochem . The cyclin dependent kinase inhibitor olomoucine  was from Promega .2HPO4, 0.5 mM KH2PO4, 1.3 mM KCl, 135 mM NaCl, pH 7.4), fixed in ice-cold methanol at −20°C for 10 minutes, washed thrice with 1x PBS, permeabilized by 0.5% Triton-X 100, and washed thrice with 1x PBS. Cells were then counter-stained with 4′,6-diamidino-2-pheylindole , mounted on glass slides, and visualized on a Zeiss M1 microscope.The rabbit polyclonal anti-IRF2BP2 antibody was described previously 2C12 myoblasts that becomes redistributed to the cytoplasm during muscle differentiation 2C12 myoblasts whereas a control construct fusing GFP to the TEAD4 transcription factor is localized to the nucleus of C2C12 myoblasts  was not imported into the nucleus, whereas a fragment encoded largely by exon 2 (amino acids 333–587) was localized in the nucleus . A fragm357ARKRKP364 and the other and the C-terminus we called Ex2C, 579KVKKERD587. To evaluate whether these sites are functional, the second exon was further divided into three parts, namely 333-422, 422–587, and 575–587, fused with GFP, and then transiently overexpressed in C2C12 myoblasts. Here, the 333–422 fragment was found in the nucleus  within this fragment. Bioinformatic analyses  suggeste nucleus , but the nucleus . When exnctional .2C12 myoblasts. To rule out that the S360A mutant created a cryptic nuclear export signal, leptomycin B (10 nM) was used to block CRM1-dependent nuclear export, but it did not cause nuclear retention of the GFP-IRF2BP2 S360A mutant , two amino acids downstream of the newly identified Ex2N NLS, in many human cell types . To testA mutant . Thus, cA mutant . During  nuclear .When co-expressed with the transcription factor TEAD1, we reported previously that IRF2BP2 strongly co-activates a mouse VEGFA promoter in African green monkey kidney CV1 cells, where endogenous IRF2BP2 levels are low2C12 myoblasts 24 hours post-treatment , kinase B (PKB), kinase C (PKC), kinase G (PKG), ribosomal S6 kinase (RSK), extracellular signal-regulated kianses 1/2 , calmodulin-dependent protein kinase II , and Cdc2 (S/T-P-X-R/L), we tested various inhibitors against these kinases to determine whether they would cause cytoplasmic retention of IRF2BP2. However, in no case was IRF2BP2 excluded from the nucleus of Creatment .The diagram shown in Nuclear import of IRF2BP2 depends not only on a classical nuclear localization sequence, but also on phosphorylation of serine 360. Although our studies have not identified the protein kinase responsible for this phosphorylation event, the cytoplasmic localization of IRF2BP2 in differentiating skeletal muscle cells suggests that this protein kinase might become down-regulated or inactivated. Preventing nuclear entry of IRF2BP2 by mutating S360A was sufficient to render the protein inactive to regulate VEGFA promoter activity. Surprisingly, forced nuclear localization did not make IRF2BP2 a better transactivator of VEGFA. This result indicates that nuclear localization is not sufficient to ensure transcriptional activity of IRF2BP2 and that the S360D mutation might affect the interaction of IRF2BP2 with a putative transcription cofactor or affect a transactivation function of IRF2BP2.Nuclear trafficking is a fundamental and essential mechanism to modulate import and export of RNA and proteins across the nuclear membranes within eukaryotes. This process depends heavily on thousands of protein complexes on the nuclear envelope and the import/export signals on cargo molecules. The classic NLS, composed of a cluster of arginine and lysine residues, has been extensively studied. X-ray crystallography shows that the positive charges from lysine and arginine enhance the binding affinity to negatively charged armadillo pockets of karyopherin-α Post-translational modifications are important for modulating protein structures or functions for downstream actions in response to environmental cues. One of frequently encountered examples includes phosphorylation The discovery of a single classical NLS in IRF2BP2 may help to predict the NLS in both IRF2BP1 and EAP1. The results show a striking similarity in sequence conservation, with both paralogs having a conserved NLS and a conserved phosphorylation target in an adjacent serine . Mass-spFigure S1Blocking CRM1 nuclear export did not cause retention of the S360A mutant of IRF2BP2. C2C12 myoblasts transfected with GFP-IRF2BP2 (S360A) were treated with vehicle  or leptomycin B . Scale bar, 10 µm.(TIF)Click here for additional data file.Figure S2Various protein kinase inhibitors failed to block nuclear import of endogenous IRF2BP2. C2C12 myoblasts were treated with vehicle (DMSO) or inhibitors of protein kinases for 24 hours. Scale bar, 50 µm. See (TIF)Click here for additional data file.