The mammalian target of rapamycin (mTOR) is known to regulate cell proliferation and growth by controlling protein translation. Recently, it has been shown that mTOR signaling pathway is involved in long-term synaptic plasticity. However, the role of mTOR under different pain conditions is less clear. In this study, the spatiotemporal activation of mTOR that contributes to pain-related behaviors was investigated using a novel animal inflammatory pain model induced by BmK I, a sodium channel-specific modulator purified from scorpion venom. In this study, intraplantar injections of BmK I were found to induce the activation of mTOR, p70 ribosomal S6 protein kinase (p70 S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) in rat L5-L6 spinal neurons. In the spinal cord, mTOR, p70 S6K and 4E-BP1 were observed to be activated in the ipsilateral and contralateral regions, peaking at 1–2 h and recovery at 24 h post-intraplantar (i.pl.) BmK I administration. In addition, intrathecal (i.t.) injection of rapamycin – a specific inhibitor of mTOR – was observed to result in the reduction of spontaneous pain responses and the attenuation of unilateral thermal and bilateral mechanical hypersensitivity elicited by BmK I. Thus, these results indicate that the mTOR signaling pathway is mobilized in the induction and maintenance of pain-activated hypersensitivity. Buthus martensi Karsch (BmK) and have been shown to be specific modulators of the voltage gated sodium channel (VGSC)  supplemented with 1 mM sodium vanadate, 2 mM leupeptin, 2 mM aprotinin, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM DTT, and 2 mM pepstatin A. After centrifugation at 14,000 rpm for 15 min, the supernatant which contained the total cellular protein extract was collected and stored at -70°C. The protein concentration was determined using the Brandford assay .Western blotting experiments were performed in order to confirm the temporal phosphorylation level of p-mTOR/p-p70 S6K/p-4E-BP1 following BmK I administration. Briefly, thirty micrograms of protein extracts were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a PVDF membrane. Nonspecific binding was blocked by incubation in 5% nonfat milk overnight at 4°C. The membranes were incubated with the following primary antibodies which were diluted in PBS-T containing 5% nonfat milk for 5 h at room temperature: rabbit polyclonal antibody against p-mTOR /rabbit Inevitably, the antibody against p-4E-BP1  may cross-react with 4E-BP2 and 4E-BP3 when phosphorylated at equivalent sites.All histochemical and blotting data were calculated by an observer blind for treatment.All results were expressed as mean ± S.E.M. (standard error of the mean). Data of spontaneous response behaviors and western blots between each groups were compared by Owo-way ANOVA followed by a Bonferroni’s post hoc test. Two-way ANOVA followed by a Bonferroni’s post hoc test was used to analyze the effect of rapamycin or CCI-779 on the PWTL and PWMT values after BmK I administration.Immunostaining was performed to evaluate whether the level of phosphorylated mTOR, p70 S6K and 4E-BP1 had changed following BmK I administration. In saline injected rats, the intensity of p-mTOR immunostaining in the spinal cord was low Figure , meanwhiIn addition, western blotting experiments were performed to confirm the immunostaining results. In naive rats, limited activation of mTOR, as well as 4E-BP1 and p70 S6K, was found on each side of the spinal cord. In response to BmK I injection, the amount of p-mTOR on the injured side increased rapidly and then peaked at 2 h  suppressed the spontaneous flinching behaviors significantly Figure . The supWestern blots revealed a significant inhibition of 200 μM rapamycin on mTOR cascades in dorsal spinal cord compared with vehicle and saline Figure . I.t. raLikewise, CCI-779 showed remarkable inhibitory effect on BmK I-induced spontaneous responses Figure A-C and hPost-treatment with rapamycin was also found to be able to attenuate BmK I-induced hypersensitivity. Bilateral mechanical hypersensitivity and unilateral thermal hypersensitivity could be suppressed by post-treatment with rapamycin  significantly Figure . NeitherIn the physiological condition, the level of phosphorylated mTOR, 4E-BP1/2 and p70 S6K were extremely low and they were only detected in spinal cord dorsal horn neurons, but not in astrocytes or microglia . In the Astrocytes are believed to contribute to the maintenance of chronic pain since the activation of astrocytes is delayed under conditions of peripheral injury ,42. In oIn BmK I-induced pain, we did not find high amounts of p-p70 S6K was co-labeled with glial cells. Moreover, capsaicin and carrageenan induced activation of S6 protein (the substrate of p70 S6K) was found primarily in spinal neurons but not glial cells, which is confirmed by the results presented ,47. HoweAlthough, most of previous studies supported the view that mTOR cascades were involved in pain sensation, the opinion was still conflicting regarding to the activation modes in different animal models. Géranton et al. reported that activated mTOR in SNI model was found in a subpopulation of A-fiber dorsal root ganglion neurons and in spinal lamina I/III projection neurons , while AAs with previous studies ,31, a raIn the current study rapamycin (CCI-779 as well) has no effect on lifting and licking behavior induced by BmK I, whereas rapamycin significantly attenuated licking and biting but not on lifting and flinching elicited by formalin . The seeSpinal synaptic plasticity is indispensable in the sensitization to peripheral noxious stimuli and generation of chronic pain . ProteinThe administration method of a therapeutic agent/molecule is a key factor of consideration. Clinically, the post-treatment phase is more relevant. Pre- and post-administration of rapamycin has shown excellent anti-nociceptive effect on both electrophysiological and behavioral studies in spared nerve injury (SNI) and formalin model ,46,49. IHowever, the contribution of mTOR signaling pathway on thermal hyperalgesia is still controversial. While the mTOR inhibitor CCI-779 has no effect on spinal nerve ligation evoked thermal hypersensitivity , rapamycIn combination with previous results, including electrophysiological recording ,58, behaIn summary, this work revealed that mTOR signaling pathway was dramatically activated on both sides of the rat spinal cord in response to the peripheral injection of BmK I. Both pre- and post-treatment of rapamycin showed a remarkable inhibition on spinal mTOR activity, and thereby displayed anti-nociceptive effect. The present study suggested that the mTOR-dependent signaling pathway, at least partially, contributes to BmK I-induced persistent pain. The mTOR signaling pathway might be a novel target to alleviate persistent and chronic pain.The authors declare that they have no conflict of interest.FJ carried out the immunohistochemical staining, participated in western blotting, animal behavioral studies and drafted the manuscript. XYP participated in experimental design. QSN conceived and carried out behavioural experiments. LMH participated in western blotting. MC participated in immunohistochemical double labeling. YHJ conceived the project. All authors read and approved the final manuscript.Immunostaining of p-mTOR was performed by using two individual antibodies from Cell Signaling Technology . A&B, animal received i.pl. injection of saline and pre-administration of Rapamycin (i.t.). C&D, animal received i.pl. injection of saline and pre-administration of saline (i.t.). E&F, animal received i.pl. injection of BmK I and pre-administration of saline (i.t.). G&H, animal received i.pl. injection of BmK I and pre-administration of Rapamycin (i.t.).Click here for fileImmunostaining of p-4E-BP1 was performed by using two individual antibodies: antibody 1 catalog BS5047 from Bioworld Technology and antibody 2 catalog #2855 from CST). A&B, animal received i.pl. injection of saline and pre-administration of Rapamycin (i.t.). C&D, animal received i.pl. injection of saline and pre-administration of saline (i.t.). E&F, animal received i.pl. injection of BmK I and pre-administration of saline (i.t.). G&H, animal received i.pl. injection of BmK I and pre-administration of Rapamycin (i.t.).Click here for fileActivated p70 S6K was stained by two individual antibodies: antibody 1 catalog sc-7984-R from Santa Cruz and antibody 2 catalog #9204 from CST). A&B, animal received i.pl. injection of saline and pre-administration of Rapamycin (i.t.). C&D, animal received i.pl. injection of saline and pre-administration of saline (i.t.). E&F, animal received i.pl. injection of BmK I and pre-administration of saline (i.t.). G&H, animal received i.pl. injection of BmK I and pre-administration of Rapamycin (i.t.).Click here for fileIllustration of quantitative assessment of double labeling staining. Selected (A) mTOR cascade positive microphotograph and (C) double labeled microphotograph (the same position as shown in A). (B) and (D) Software assisted evaluation of A & B: blue regions were defined as positive signals. (E) The measuring area, as well as the high magnified images in Figure Click here for file


Caenorhabditis elegans (C. elegans) has become a widely used model to explore the effect of food constituents on health as well as on life-span extension. The results imply that besides essential nutrients several flavonoids are able to impact the aging process. What is less investigated is the bioavailability and biotransformation of these compounds in C. elegans. In the present study, we focused on the soy isoflavone genistein and its metabolism in the nematode as a basis for assessing whether this model system mimics the mammalian condition.C. elegans was exposed to 100 µM genistein for 48 hours. The worm homogenate was extracted and analyzed by liquid chromatography (LC). 11 metabolites of genistein were detected and characterized using LC electrospray ionization mass spectrometry. All genistein metabolites formed by C. elegans were found to be sugar conjugates, primarily genistein-O-glucosides. The dominant metabolite was identified as genistein-7-O-phosphoglucoside. Further interesting metabolites include two genistein-di-O-glycosides, a genistein-O-disaccharide as well as a genistein-O-phosphodisaccharide.C. elegans leading to conjugative metabolites which are not known for mammals. The metabolism of genistein in mammals and in C. elegans differs widely which may greatly impact the bioactivity. These differences need to be appropriately taken into consideration when C. elegans is used as a model to assess possible health or aging effects.Our study provides evidence for a novel biotransformation pathway in  Metabolic transformation plays a key role in mammalian and non-mammalian defense against xenobiotics, which might exert beneficial or harmful effects on biological systems. Biotransformation generally includes phase I reactions in which functional groups are either introduced to the xenobiotic or exposed, followed by conjugation reactions with water-soluble moieties. These chemical modifications change the physicochemical properties of the xenobiotic which in turn alters also its biological and physiological effects. Consequently, detailed information on the metabolism of a foreign compound is crucial for a reliable assessment of potential benefits but also risks associated with its exposure. When using an animal model to investigate the biological activity of a substance it is important to keep in mind that its response, especially its metabolism, may differ between the model system and that of humans.Caenorhabditis elegans (C. elegans) have become an important tool in systems biology research. The genome of C. elegans is fully sequenced and many physiological functions found in mammals have been identified in the nematode as well. Due to the relatively high degree of complexity and the small total number of 959 somatic cells C. elegans provides a controllable experimental system employed in developmental research, cell physiology and aging Non-mammalian model organisms such as the nematode C. elegans has become a widely used model to explore the effect of food constituents on health as well as on life-span extension. The results imply that essential nutrients like tocopherols but also plant secondary compounds from fruit, vegetables and herbs, especially polyphenols, are able to impact the aging process. Such effects on life span of C. elegans have recently been described for example for the proanthocyanidin fraction derived from blueberries, epigallocatechin gallate from green tea, or quercetin derivatives from onions C. elegans is thus very limited and so far, only a few reports have addressed this question The aging trajectory is controlled by genetic mechanisms involving signal transduction pathways that are evolutionarily conserved in yeasts, worms, flies and mammals including humans C. elegans and observed indications for a substantial metabolism C. elegans. Very recently Fischer et al. reported that genistein affect the immunity of the worm by acting antiestrogenic via a reduced expression of the estrogen-responsive vitellogenin-genes We recently assessed for four structurally related flavonoids the apparent bioavailability in C. elegans as a basis for assessing whether this model system mimics the mammalian condition.In mammalian systems the metabolism of genistein is well described and reveals an impressive complexity O-β-d-glucoside) was bought from Plantech UK , sophoricoside (genistein-4′-O-β-d-glucoside) was from ChromaDex  and genistein-4′,7-di-O- β-D-glucoside was purchased from Apin Chemicals Ltd. . Maltodextrin DE 4.0–7.0 was purchased from Sigma Aldrich  and cyclodextrin glucanotransferase was provided by Amano Enzyme Europe Ltd. . All solvents used were of analytical grade. A stock solution of genistein was prepared using ethanol∶Tween 80   as a solvent.Genistein was purchased from Sigma Aldrich  with a purity >99.0% as determined by LC/photodiode array detection (DAD). Genistin  and Escherichia coli (E. coli) strain OP50 were obtained from the Caenorhabditis Genetics Center (CGC), University of Minnesota, USA. The nematodes were grown at 20°C on nematode growth medium (NGM) agar plates as previously described E. coli OP50 as food source in order to prevent any bacterial transformation of genistein. Bacteria were grown overnight, concentrated 5-fold by centrifugation and heat killed at 65°C for 30 min according to The nematodes were grown on NGM plates containing 100 µM of genistein for 48 hours. After exposure, worms were washed five times with 0.1% bovine serum albumin (w/v) to remove all adhering genistein. The washed worm pellets were frozen in liquid nitrogen until analysis. For analysis, worm samples were thawed and treated with a FastPrep homogenizer (4×15 sec) using 1-mm silica spheres with intermediate cooling periods on ice . A 50 µl aliquot of the homogenate was mixed with 150 µl of methanol and the suspension was centrifuged . An aliquot of the supernatant was subsequently diluted with water (1∶1) and analyzed by LC-DAD and LC-MS.The LC-DAD analyses were performed on a Shimadzu LC system equipped with a controller (CBM-20A), a degasser (DGU-20A3), two pumps (LC-20AD), an autosampler (SIL-20AC HT), a column oven (CTO-20AC) and a DAD (SPD-M20A). The LC system was controlled by the software LCsolution 1.24. LC separation was carried out on a YMC Pack Hydrosphere C18 column  equipped with a Phenomenex SecurityGuard . Eluent A was ammonium acetate  and eluent B was acetonitrile. A linear gradient was used with a flow rate of 0.8 ml/min and the following elution profile: 0–5 min isocratic with 10% B, 5–16 min from 10% to 20% B, 16–30 min from 20% to 42% B, 30–31 min from 42% to 95% B, 31–36 min isocratic with 95% B, 36–37 min from 95% to 10% B and 37–47 min isocratic with initial conditions. The column oven and the DAD flow cell were adjusted to 40°C. The injection volume was 20 µl. The chromatograms were recorded from 200 to 400 nm and the 260 nm trace was used to monitor the analytes.The LC-MS analyses were performed on two systems. The first system was an ABSciex QTrap 5500 mass spectrometer equipped with a Shimadzu LC system, which consisted of a controller (CBM-20A), a degasser (DGU-20A5), two pumps (LC-30AD), an autosampler (SIL-30AC) and a column oven (CTO-20AC). The LC-MS system was controlled by the software Analyst 1.5.2. The LC conditions were the same as described above. The electrospray ionization (ESI) source was operated in the negative mode using the following parameters: Curtain gas (CUR) 20 psi, ion spray voltage (IS) −3500 V, ion source gas-1 (GS 1) 60 psi, ion source gas-2 (GS 2) 40 psi, ion source gas-2 temperature (TEM) 600°C. The MS parameters were adjusted as follows: scan rate 20,000 Da/sec, declustering potential (DP) −250 V, entrance potential (EP) −10 V. The MS full scans (enhanced MS mode) were performed with a scan range from 100 to 755 m/z and a collision energy voltage (CE) of −10 V. The MS/MS measurements (enhanced product ion mode) were executed with a collision energy voltage (CE) of −40 V and collision energy spread of 30 V. Nitrogen was used as collision gas.The second LC-MS system was an Agilent 6540 QToF mass spectrometer equipped with an Agilent 1290 Infinity LC system, which consisted of a controller, a degasser, a binary pump, an autosampler, a column oven and a DAD. The system was controlled by the software MassHunter B.03.01 Build 3.1.346.0. The LC conditions were the same as described above. The jet stream ESI source was operated in the negative mode and the MS parameters were adjusted as follows: Nebulising pressure 50 psi, gas temperature 350°C, gas flow 10 l/min, capillary voltage (VCap) 3500 V, skimmer voltage 65 V, fragmentor voltage 175 V, Oct 1 RF Vpp voltage 750 V, scan range from 100 to 1100 m/z, scan rate 1 spectrum/sec. The MS/MS experiments were performed with a CID voltage of 30 V. Nitrogen was used as collision gas.C. elegans converted the isoflavone genistein to at least 11 metabolites which were detected and characterized by LC-DAD and LC-MS analysis. C. elegans exposed to 100 µM genistein for 48 h. The peaks (denoted as M1 to M11) were numbered according to their order of elution. All peaks that were not present in control incubations without genistein were assumed to be metabolites and characterized by interpretation of their MS, MS/MS and MS-TOF data. In sum, all metabolites detected were identified as genistein sugar conjugates. In the following, the mass spectrometric data of each metabolite will be discussed and explained.10 and 11 were identified as genistein-7-O-β-D-glucoside and genistein-4′-O-β-D-glucoside, respectively using reference standard compounds. Both MS spectra measured in the negative mode exhibit a high intensity ion at m/z 431 assigned to the [M-H]− ion. The signals at m/z 269 and m/z 268 correspond to the genistein aglycone fragment in form of the anion (Y−) and the radical anion (Y-H)•− respectively, indicating the loss of a glucose moiety via heterolytic (−162 Da) or homolytic (−163 Da) cleavage of the glycosidic bond O-β-D-glucoside and of metabolite M10 are shown in Metabolite 8 and 9 additionally exhibited [M-H]− ions at m/z 431 indicating two further O-glycosylated genistein metabolites comprising a hexose moiety. Because of the similarity of their mass spectra to those of M10 and M11, we assume that these metabolites are genistein-7- and genistein-4′-O-hexoside stereoisomers, carrying a different hexose sugar moiety, e.g. galactose instead of glucose. The presence of a genistein-5-O-glucoside seems less likely because the 5-OH group of the A-ring is involved in intramolecular hydrogen bond formation with the 4-carbonyl group of the isoflavone C-ring m/z 431 precursor with the four genistein-O-monohexoside metabolites M8, M9, M10, and M11.Two minor metabolites defined as peak 1 and 3 appeared with low abundance. Both produced a molecular [M-H]− ion at m/z 593 indicating a genistein molecule plus two monosaccharide units. In both cases the molecular ion was confirmed by precursor ion scans for m/z 431, a fragment ion in both peak mass spectra. The detected ions were m/z 593 proving the [M-H]− ion, m/z 653 for the corresponding solvent adduct [M+CH3COO]− ion as well as m/z 629 and 631 for the respective chloride ion adducts [M+ 35Cl]− and [M+ 37Cl]−. Besides the m/z 431 ion, the product ion mass spectra of m/z 593 further show the characteristic genistein aglycone ions at m/z 269 and m/z 268 , which corresponds to the characteristic fragment mass of an anhydro-disaccharide unit, is preferred -linked maltoside, we propose that M7 most likely is a genistein-O-β-glucosyl-(1→6)-glucoside (genistein-gentiobioside) or a genistein-O-β-glucosyl-(1→2)-glucoside as these two linkages are the most common ones found in nature. The fragmentation pattern of genistein-di-O-hexosides compared to genistein-O-disaccharides is depicted in Metabolite M5 with a molecular [M-H]− ion at m/z 511, which was confirmed by precursor ion experiments scanning for m/z 269, the genistein anion. In addition, the corresponding sodium [M-2H+Na]− and potassium [M-2H+K]− adduct ions at m/z 533 and m/z 549, respectively, were observed , whereas the ion at m/z 241.0126 arose vice versa by the neutral loss of a genistein molecule (−270 Da). The ion peak at m/z 241 further indicates that the phosphate group is linked to the hexose moiety (e.g. in O-6 position of the hexose) since the MS/MS/MS spectrum of m/z 241 showed product ions at m/z 79 [PO3]− and m/z 97 [H2PO4]− . We therefore conclude that the main metabolite formed is a genistein-7-O-(6″-O-phospho)-β-D-glucoside. The proposed fragmentation pathway is depicted in The main metabolite detected was observed Table 1.6 and 2 occurred in minor amounts and exhibited MS and MS/MS spectra analog to those of M5. Treatment of M6 with acid phosphatase yielded in genistein-4′-O-β-D-glucoside suggesting peak M6 to be genistein-4′-O-(6″-O-phospho)-β-D-glucoside. The third genistein-O-monophosho-hexoside M2 was so far not further characterized being only present in trace amounts.Metabolite m/z 673 peak M4 showed the highest molecular ion of all metabolites detected. This was confirmed by precursor ion experiments and detection of the corresponding sodium [M-2H+Na]− and potassium [M-2H+K]− adduct ions at m/z 695 and m/z 711, respectively , whereas the ion at m/z 403 results from a neutral loss of genistein (−270 Da) .Our studies on the metabolism of the isoflavone genistein in C. elegans were identified as sugar conjugates, primarily genistein-O-glucosides. Similar glucosylation reactions of flavonoids in non-mammalian systems are primarily reported for some bacteria species, especially aerobic or facultative anaerobic rod-shaped bacteria such as Bacillus subtilis, Bacillus cereus, Xanthomonas campestris and Lactobacillus delbrueckiiC. elegans where a glucose moiety was introduced into the anthelmintic drug albendazole as a xenobiotic response and a variety of quercetin glycosides including sulfate-glycosyl derivatives were recently found by Surco-Laos et al. C. elegans.All genistein metabolites formed by C. elegans was reliably identified as a 7-O-phosphoglucoside. To our knowledge, this is the first time that phosphoglucosides as biotransformation reaction products are identified in C. elegans. By the high concentrations found, it seems that this is the predominant conjugation pathway in the worms. Since we observed both, the genistein-7-O-glucoside and the corresponding phosphoglucoside, we speculate that the formation may occur in a two-step reaction. C. elegans expresses a variety of UDP-glycosyltransferases which could catalyze the generation of an O-glucoside, followed by phosphorylation of the glucose moiety by the action of a hexokinase . To identify the relevant enzymes involved in this pathway will be challenging.In our study, the dominant genistein metabolite in N2 xokinase Figure 7O-disaccharides and phosphorylated genistein-O-disaccharides were only formed in minor amounts. However, disaccharide conjugates are a new metabolite class as well and have never been reported as products of the C. elegans xenobiotic defense system. The pathway of their formation is equally unclear. We have not identified any hydroxylated genistein metabolites in worms known to be formed by cytochrome P450 enzymes in mammalian species (data not shown). However, even in mammals this reaction is only a side-pathway since genistein already possesses three hydroxyl groups.Genistein-C. elegans and humans. In contrast to the formation of phosphoglucosides and glucosides described here for C. elegans, mammalian systems appear to metabolize genistein to glucuronides, sulfates and mixed sulfoglucuronides exclusively. O-desmethylangolensin, and 2-(4-hydroxyphenyl)-propionic acid which influence the bioactivity dramatically. In C. elegans none of these microbial metabolites were detected.C. elegans which is proposed to be responsible for lifespan prolongation of the nematode in the presence of pathogenic bacteria, whereas genistein acts in an antiestrogenic manner, diminishes the resistance against the pathogen and reduces lifespan under the same conditions O-sulfate of daidzein exerted a much higher activity than daidzein itself O-sulfation acts in an opposite way and reduced the estrogenic activity substantially. However, not only estrogenicity has been shown to be affected by the type of conjugation. Daidzein sulfoconjugates are also described as potent inhibitors of sterol sulfatase while daidzein does not affect this enzyme Very recently, Fischer et al. made the interesting observation that daidzein exhibits an estrogenic effect in C. elegans leading to the formation of glucoside- and phosphoglucosides derivatives which so far have not been identified in a mammalian system. The metabolism of genistein in mammals and C. elegans therefore shows huge differences which may greatly impact the bioactivity. These differences need to be appropriately taken into consideration when C. elegans is used as a model to assess possible health or aging effects.In conclusion, our study provides evidence for a novel biotransformation pathway in


Head and Neck squamous cell carcinoma (HNSCC) is a human lethal cancer with clinical, pathological, phenotypical and biological heterogeneity. Caner initiating cells (CICs), which are responsible for tumor growth and coupled with gain of epithelial-mesenchymal transition (EMT), have been identified. Previously, we enriched a subpopulation of head and neck cancer initiating cells (HN-CICs) with up-regulation of CD133 and enhancement of EMT. Others demonstrate that Src kinase interacts with and phosphorylates the cytoplasmic domain of CD133. However, the physiological function of CD133/Src signaling in HNSCCs has not been uncovered.in vitro malignant properties including cell migration/cell invasiveness/anchorage independent growth, and in vivo tumor growth by nude mice xenotransplantation assay. In opposite, overexpression of CD133 enhanced the stemness properties and tumorigenic ability of HNSCCs. Lastly, up-regulation of CD133 increased phosphorylation of Src coupled with EMT transformation in HNSCCs, on the contrary, silence of CD133 or treatment of Src inhibitor inversely abrogated above phenotypic effects, which were induced by CD133 up-regulation in HNSCCs or HN-CICs.Herein, we determined the critical role of CD133/Src axis modulating stemness, EMT and tumorigenicity of HNSCC and HN-CICs. Initially, down-regulation of CD133 significantly reduced the self-renewal ability and expression of stemness genes, and promoted the differentiation and apoptotic capability of HN-CICs. Additionally, knockdown of CD133 in HN-CICs also lessened both Our results suggested that CD133/Src signaling is a regulatory switch to gain of EMT and of stemness properties in HNSCC. Finally, CD133/Src axis might be a potential therapeutic target for HNSCC by eliminating HN-CICs. Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancers in the world and one of causes of cancer-related death due to therapy resistance Accumulating data demonstrate that tumor formation is driven by a subpopulation of cells that exhibit self-renewal capacity–the purported cancer stem cells (CSCs) or cancer initiating cells (CICs) CD133 (prominin-1), a 5-transmembrane glycoprotein, was originally recognized as a hematopoietic stem cells marker EMT, a cellular transformation that converts adherent epithelial cells into migratory mesenchymal cells, is critical for embryonic development and tumorigenic progression of cancer cells and cancer metastasis Src, a classical non-receptor tyrosine kinase with the potential to cause cell transformation including uncontrolled proliferation and loss of contact inhibition, becomes activated by interacting with the stimulated membrane receptors in vitro and in vivo. Ultimately, we demonstrate the significance of CD133/Src signaling on EMT process in HNSCC.The detailed molecular mechanisms involved in the regulatory links between EMT and stem cell–related genes such as CD133 and Src are still poorly understood. Herein, we demonstrate a critical role of CD133 in the enhancement of stemness, gain of EMT, and promoting tumorigenicity of HN-CICs. Additionally, down-regulation of CD133 or inhibition of CD133 induced Src activation lessens stemness properties and tumorigenicity of HNSCCs both 4 to 1×105 live cells/10-mm dishes, and the medium was changed every other day until the tumor sphere formation was observed in about 4 weeks Two HNSCC cell lines, SAS 5′-ACCGTCTAGAATGGCCCTCGTACTCGGCTCCCTGTTGCTG-3′ and 5′- ATCAAAGCTTATTGAAGCTGTTCTGCAGGTGAAGAG- tgcc-3′. Lentivirus production was performed by co-transfection of plasmid DNA mixture with lentivector plus helper plasmids (VSVG and Gag-Pol) into 293T cells  using Lipofectamine 2000 . The lentivirus M.O.I titer is determined by flow cytometry (average of 5×104 and 2×105 TU/ml). To generate the stable cell lines, sub-confluent HNSCC cells were infected with lentivirus in the presence of 8 µg/ml polybrene . The green fluorescence protein (GFP), which was co-expressed in lentiviral-infected cells, was served as a selection marker to indicate the successfully infected HNSCCs. Stable CD133-overexpressing HNSCC cell lines were further purified by cell sorting with GFP positive cells  and then cloned into pCDH1-MCS1-EF1-copGFP . The sequences of oligos used for CD133 PCR amplification are ve cells . The pCDSh-CD133-1:5′-AAAAGGACAAGGCGTTCACAGATTTGGATCCAAATCTGTGAACGCCTTGTCC-3′; Sh-CD133-2:5′-AAAAGGATACACCCTACTTACTATTGGATCCAATA GTAAGTAGGGTGTATCC-3′) were synthesized and cloned into pLVRNAi to generate a lentiviral expression vector. Sh-Luc:5′-CCGGACTTACGCTGAGTACTTCGAA CTCGAGTTCGAAGTACTCAGCGTAAGTTTTTTG-3′ was utilized for experimental control. Lentivirus production was performed as above. Stable pLV-RNAi expressed HNSCC cell lines were further purified by cell sorting with GFP positive cells . The method of cloning the double-stranded shRNA sequence is described in the manufacturer's protocol. Lentiviral vectors expressing shRNA that targets human CD133 . Hoechst 33342 dye was then added at a final concentration of 5 µg/ml in the presence or absence of fumitremorgin C (FTC)  and was incubated at 37°C for 90 min with intermittent shaking. The cells were washed with ice-cold HBSS with 2% FBS and centrifuged at 4°C, and re-suspended in the same buffer. Propidium iodide at a final concentration of 2 µg/ml was added for gating viable cells. The Hoechst 33342 dye was excited at 357 nm and its fluorescence was dual-wavelength analyzed . Analyses were done on a FACS Vantage  For side population analysis, single HNSCC cells suspension at 1×10Apoptotic cells were detected with an Annexin V-APC kit  according to manufacturer's guidelines. After staining, the cells incubated with 20 µg/ml propidum iodide (PI) were analyzed by FACS Calibur apparatus  5 cells were plated into the top chamber of a transwell  with a porous membrane (8.0 µm pore size). Cells were plated in medium with lower serum (0.5% FBS), and medium supplemented with higher serum (10% FBS) was used as a chemoattractant in the lower chamber. The cells were incubated for 24 h at 37°C and cells that did not migrate through the pores were removed by a cotton swab. Cells on the lower surface of the membrane were stained with Hoechst 33258 (Sigma-Aldrich) to show the nuclei; fluorescence was detected at a magnification of 100x using a fluorescence microscope . The number of fluorescent cells in a total of five randomly selected fields was counted. In vitro cell invasion analysis was described previously For transwell migration assays, 2×104 cells was added, and the dishes were incubated at 37°C for 4 weeks. Plates were stained with 0.005% Crystal Violet then the colonies were counted. The number of total colonies with a diameter ≥100 µm was counted over five fields per well for a total of 15 fields in triplicate experiments.Each well (35 mm) of a six-well culture dish was coated with 2 ml bottom agar (Sigma-Aldrich) mixture  FCS, 0.6% (w/v) agar). After the bottom layer was solidified, 2 ml top agar-medium mixture  FCS, 0.3% (w/v) agar) containing 2×103)  =  (Length × Width 2)/2.All the animal practices in this study were approved and in accordance with the Institutional Animal Care and Use Committee (IACUC) of National Yang-Ming University, Taipei, Taiwan . Stable sh-Luc and sh-CD133 HN-CICs or control GFP and CD133-overexpresiing HNSCCs mixed with Matrigel  (1:1) were injected subcutaneously into BALB/c nude mice (6–8 weeks). Tumor volume (TV) was calculated using the following formula: TV   was used for statistical analysis. Student's +) was also decreased in sh-CD133 infected HN-CICs by FACS analyses (+/PI+ cells (We have identified a subpopulation of head and neck cancer initiating cells (HN-CICs) with enhanced stemness properties from HNSCC cells (HNSCCs) by sphere formation assay analyses . Furtheranalyses . We alsoanalyses  but the analyses  in CD133I+ cells . Togethein vitro malignant properties including abilities of cell migration, matrigel invasion and anchorage independent growth of HN-CICs, single cell suspension of control- or CD133- knockdown HN-CICs were plated onto Transwell chamber  and 97 to 91% (SAS), respectively . The twoanalyses . In addianalyses  and signanalyses . We alsoanalyses . Next, wanalyses . Of note or cancer initiating cells (CICs), within tumor tissues posses stemness properties + cells isolated from brain tumors exhibit CICs properties, are refractory to chemo- or radiotherapy, and promote angiogenesis to facilitate the brain tumor growth + CICs in solid tumors characterized confer resistance to chemotherapy + CICs may explain the failure of cancer management, and will provide us with new therapeutic approaches CD133, a cell surface marker of hematopoietic stem cells and endothelial progenitors, has been proposed to be involved in the angiogenesis as well as cancer tumorigenicity n vivo tumorigenic properties of HN-CICs  SAS and (B) OECM1, were infected with lentivirus co-expressing GFP and CD133. Afterward virus transduction, GFP positive HNSCCs were sorted according to the expression of GFP to isolate stable HNSCCs. GFP positive HNSCC cells demonstrated the successful lentivirus infection. (C) Single cell of HN-CICs (derived from SAS cells) were infected with lentivirus co-expressing GFP and small hairpin RNA targeting CD133. The successful infected cells were sorted by flow cytometry according to the co-expression of GFP protein.(TIF)Click here for additional data file.Figure S2Expression of Oct4 and CK18 in tumors derived from CD133 ovexpressing HNSCCs or CD133 stable knockdown HN-CICs. Tumors derived from control HN-CICs (sh-Luc) or CD133-knockdown SAS-HN-CICs, and SAS cells were collected, sectioned and stained with Hematoxylin and anti-Oct4 (A) or anti-CK18 (B) as described. Arrows indicate the positive staining. (C) Tissue sections of control-GFP and CD133-overexpressing SAS cells xenograft tumor were stained with CK18 or Oct4.(TIF)Click here for additional data file.Figure S3Src kinase activation promotes EMT, tumorigenicity and stemness. (A) Expression of p-Src, E-cadherin and Vimentin in HNSCCs transfected with either Src-overexpressing or control vector was examined by western blot. The amount of GAPDH protein was referred as loading control. (B) Anchorage-independent growth of control-vecor or Src-overexpressing HNSCCs was analyzed . (C) Sphere formation ability of control or Src-overexpressing HNSCCs was examined under the serum-free defined selection medium . (D) Representative tumor growth of control- or Src-overexpressing (Srcover) HNSCCs in the subcutaneous space of recipient mice . (E) SAS HN-CICs (5*105 cells) were subcutaneously injected into both backs of nude mice and allowed to develop tumors to a size around 0.2 cm3 (12 days). On day 12, 15, and 18 after the inoculation of HN-CIC cells, PP2 (10 µM) was injected into the right back tumors whereas DMSO was injected into the left back as negaive control .(TIF)Click here for additional data file.Figure S4Effect of CD133Y828F mutant on p-Src activity. (A) DNA sequencing confirmed the CD133Y828F mutation, which encodes CD133Y828F mutant protein. (B) 293A cells transfected with plasmids, control-GFP, CD133 wild type (CD133over) or CD133Y828F (CD133Y828F), respectively, were collected (under no serum culture condition). The protein levels of p-Src, total Src and CD133 were examined by immunoblot.(TIF)Click here for additional data file.


Blastochloris viridis possess Tyr-L162 located mid-way between the special pair chlorophyll (P) and the heme (heme3). While mutation of the tyrosine does not affect the kinetics of electron transfer from heme3 to P, recent time-resolved Laue diffraction studies reported displacement of Tyr-L162 in response to the formation of the photo-oxidized P+•, implying a possible tyrosine deprotonation event. pKa values for Tyr-L162 were calculated using the corresponding crystal structures. Movement of deprotonated Tyr-L162 toward Thr-M185 was observed in P+• formation. It was associated with rearrangement of the H-bond network that proceeds to P via Thr-M185 and His-L168.Photosynthetic reaction centers from  The resulting P680+• is reduced by D1-Tyr161 (YZ) through electron transfer events from the Mn4CaO5 cluster Blastochloris viridis, the corresponding chlorophyll pair is the bacteriochlorophyll b (BChlb) PL/M pair P960 (P). The photo-oxidized P+• state that is generated as a consequence of electronic excitation of P960 can be reduced by electron transfer from the nearest heme group (heme3) in the adjacent tetraheme subunit. The role of a highly conserved residue, Tyr-L162, has been long discussed In biological systems, tyrosine residues often play an important role in functioning as a redox active group and mediating electron transfer. In photosystem II (PSII), electronic excitation of the chlorophyll +• was very recently reported in the light-exposed crystal structure (light structure) with respect to the dark-state structure (dark structure) in time-resolved Laue diffraction analysis. Wöhri et al. interpreted that negatively charged and deprotonated Tyr-L162 was attracted to the P+• positive charge Ka value of Tyr-L162 (pKa(Tyr-L162)) is neither experimentally measured nor explicitly calculated in Ref. On the other hand, displacement of Tyr-L162 by 1.3 Å toward PBlastochloris viridis at higher resolutions, so far only the crystal structure by Wöhri et al. Ka(Tyr-L162) in the original protein geometry of the photoactivated form and ii) what residues/groups contribute to downshift pKa(Tyr-L162). As a driving force of the tyrosine deprotonation, the P+• state formation is definitely a key factor. However, there are also other titratable residues in the neighborhood of P. It is unclear whether protonation state changes of other titratable residues may occur in response to the P+• formation, or whether deprotonation of other titratable residues compensate for the influence of P+• on pKa(Tyr-L162).Although there are crystal structures of bRC from +• state formation, pKa(Tyr-L162) were calculated using the corresponding protein crystal structures, by solving the linear Poisson-Boltzmann equation with consideration of the protonation states of all titratable sites in the entire bRC protein. Using this approach, one will be able to sufficiently consider the equilibrium in protonation states of all titratable groups in bRC Ka(Tyr-L162) in the protein environment.To evaluate the energetics of Tyr-L162 deprotonation in the P+• state. As a consequence, deprotonated Tyr-L162 moved further toward Thr-M185 (Tyr-L162-OThr-M185) was 2.7 Å in the resulting geometry with deprotonated Tyr-L162 (Ydeprot position), which was 0.6 Å shorter than that in the light structure (Ylight position). Although the resulting Ydeprot position was not exactly identical to the Ylight position, this result implies that Tyr-L162 deprotonation leads to tyrosine movement from the one in the dark structure (Ydark position) to the Ylight position. Interestingly, the OTyr-L162-OThr-M185 distance obtained with deprotonated Tyr-L162 is 2.7 Å (in the Ydeprot structure).To investigate the possible presence of deprotonated tyrosine, Tyr-L162 was treated in its deprotonated form, and its geometry was energetically optimized with CHARMM in the PThr-M185 : the H-b0 and protonated Tyr-L162 state (P0 Y) to the photo-oxidized P+• and deprotonated Tyr-L162 state (P+• Y−). In the P0 Ydark state (Tyr-L162-OThr-M185 distance = 4.4 Å). The hydroxyl H atom of Thr-M185, in turn, is oriented to the Nδ site of His-L168 (OThr-M185–NHis-L168 distance = 4.3 Å), forming the O-HTyr-L162 ••• O-HThr-M185 ••• NHis-L168 network over Tyr-L162, Thr-M185, and His-L168.In addition to Tyr-L162 movement, a striking rearrangement in the H-bond network containing P and Tyr-L162 was observed in the transition from the initial uncharged Prk state , left, t0 Ydark state, orientation of the H-bond network is completely different in the P+• Ydeprot− state, since the hydroxyl OH group of Thr-M185 is subject to forming an H-bond with the deprotonated Tyr-L162 (OTyr-L162-OThr-M185 distance = 2.7 Å) to stabilize the negative charge  was calculated to be 22 in the P0 Ydark state (Ka(Tyr-L162) value of 22, which is even higher than that in aqueous solution (∼10), is mainly due to the presence of acidic residues in the bRC that upshift pKa(Tyr-L162), e.g., Asp-M182, Glu-C254, Asp-L155, and Glu-M171 (Ka (Tyr-L162) and thus does not energetically allow deprotonated Tyr-L162 formation.prk state , indicatGlu-M171 . The pre0 Ydark state, P+• Y− state formation leads to a drastic decrease in pKa (Tyr-L162). In particular, the P+• Ydeprot− state possesses the deprotonated Tyr-L162 since pKa (Tyr-L162) = 6.7 (Ka (Tyr-L162):In contrast to the PKa (Tyr-L162) are Thr-M185 and a water molecule. They alter the H-bond pattern with respect to Tyr-L162 in response to the P+• Ydeprot− state formation (Ka (Tyr-L162) by 8 and 4 in the P+• Ydeprot− state (relative to the P0 Ydark state), respectively  by 4.2 (2.7 from PA and 1.5 from PB) in the P+• Ydeprot− state (+• on pKa(Tyr-L162) did not essentially differ in the Ydark, Ylight, and Ydeprot positions ; 2X5V) For performing computations of bRC from deprot position, see the later part), atomic coordinates for the minimum set of relevant residues, i.e., Tyr-L162, Thr-M185, and a water molecule (HOH M 2001 in PDB: 2X5V) were released and geometrically optimized , and protonated with neutral charge (YH)). The atomic charges of BChlb and bacteriopheophytin b (BPheob) were determined from the electronic wave functions obtained with the density functional (DFT) module (B3LYP) in Gaussian03 +•, a unit positive charge was distributed with a ratio of PA+•/PB+• = 2/1 derived from ENDOR studies Atomic partial charges of the amino acids were adopted from the all-atom CHARMM22 b, BPheob, and quinones) were kept in their neutral charge state. Hemes in the cytochrome c subunit were kept in the reduced state. The ensemble of the protonation patterns was sampled by the Monte Carlo method with Karlsberg Karlsberg online manual, http://agknapp.chemie.fu-berlin.de/karlsberg/). The dielectric constants were set to pε = 4 inside the protein and wε = 80 for water. All computations were performed at 300 K, pH 7.0, and an ionic strength of 100 mM. The LPB equation was solved using a 3-step grid-focusing procedure at the resolutions 2.5 Å, 1.0 Å, and 0.3 Å. The Monte Carlo sampling for a redox active group yielded the probabilities [Aox] and [Ared] of the two redox states of the molecule A.The present computation is based on the electrostatic continuum model by solving the linear Poisson-Boltzmann (LPB) equation with the MEAD program Ka values of a target site (e.g. pKa(Tyr-L162)), the electrostatic energy difference was calculated between the two protonation states, protonated and deprotonated, in a reference model system using a known experimentally measured pKa value. The difference in the pKa value of the protein relative to the reference system was added to the known reference pKa value. Experimentally measured pKa values employed as references are 12.0 for Arg, 4.0 for Asp, 9.5 for Cys, 4.4 for Glu, 10.4 for Lys, 9.6 for Tyr Ka value was evaluated using the Henderson-Hasselbalch equation. A bias potential was applied to obtain an equal amount of both protonation states ([protonated] = [deprotonated]), yielding the pKa value as the resulting bias potential.To obtain absolute pKa of titratable residues are equivalent to those of the redox potential for redox-active groups, although in the latter case, the Nernst equation is applied instead of the Henderson-Hasselbalch equation Ka computations is directly comparable to that obtained for recent computations Ka computation can be estimated to be about 0.2 pH units. Systematic errors typically relate to specific conformations that may differ from the given crystal structures.The procedures to compute pTable S1deprot), Thr-M185, and a water molecule.Energetically minimized atomic coordinates of Tyr-L162 (Y(DOC)Click here for additional data file.Table S2b.Atomic partial charge of BChl(DOC)Click here for additional data file.Table S3b.Atomic partial charge of BPheo(DOC)Click here for additional data file.


Delayed contrast enhanced (DE) cardiac MR imaging (cMRI) has become the gold standard for the in vivo detection and quantification of myocardial infarcts (MI). Up to now, cMRI had to be performed in a closed bore environment due to technical constraints of the existing open low-field MR scanners. The introduction of an open 1.0T MR-system with high gradient performance has paved the way for cardiac imaging, which may be beneficial in critically ill and claustrophobic patients as well as during stress examinations and MR-guided interventions.The aim of this study was to evaluate image quality, scar/myocardium-contrast and assess feasibility of delayed contrast enhanced cMRI using a 1) breath hold (BH) 3 dimensional (3D) Inversion Recovery (IR), 2) BH phase-sensitive IR (PSIR), and 3) a respiratory triggered (RT) PSIR sequence at an open MR-system.41 patients  with known or suspected MI underwent viability imaging 10 min after i.v. application of Gd-DTPA (0.2 mmol/kg BW) at an open MRI system . DE cMRI with complete coverage of the left-ventricular myocardium in short-axis (SA) slices was acquired using three different sequences and approaches: 1) A conventional k-space segmented 3D T1-weighted inversion-recovery-prepared gradient-echo sequence  was performed. The inversion delay time TI was iteratively adjusted to null the signal from normal myocardium. Additionally, a fast multislice single-shot 3D PSIR sequence was performed in 2) BH technique  respiratory triggered .Image quality of DE was rated on a 4-point grading scale. Contrast (C=SIscar/SImyo) was calculated and area of DE was assessed in % of LV mass.There was no statistical significant difference in image quality using BH 3D IR, BH PSIR and RT PSIR , respectively. No infarcts were missed and agreement between the three approaches regarding infarct extent  was high. Contrast for 3D BH IR, BH PSIR and RT PSIR was 10.4+/-3.3 and 11.8+/-3.0, and 12.6+/-1.9, respectively.Delayed contrast enhanced cMRI is feasible at an open 1.0T MRI system in a clinical setting and offers excellent image quality. Respiratory triggered PSIR findings correlate well with breath hold 3D IR and PSIR sequences for the detection and extent of myocardial infarcts.None.


We have established 17 Y chromosome consomic (Y-consomic) mouse strains in an inbred DH/Sgn strain. In this study, based on investigations in four different genetic backgrounds, we proved that the Y chromosome of the inbred mouse KK/Ta strain is associated with reduced body size.KK-+/+ and DH-Chr YSJL-+/+ strains, and the DH-Chr YKK-+/+ strain was the lightest among the 17 Y-consomic strains. In the DH-Chr Y-Dh/+ background , although Y chromosome substitution did not significantly alter the body weight, the DH-Chr YKK-Dh/+ strain was the lightest among the 17 Y-consomic-Dh/+ strains. In the (B6.Cg-Ay × DH-Chr Y) F1-+/+ background, Y chromosome substitution significantly decreased the body weight and length in the (B6.Cg-Ay × DH-Chr YKK) F1 hybrids. In the (B6.Cg-Ay × DH-Chr Y) F1-Ay/+ background (Ay causes obesity and promotes linear growth), Y chromosome substitution significantly decreased body weight and length in the (B6.Cg-Ay × DH-Chr YKK) F1-Ay/+ hybrids.In the DH-Chr Y-+/+ background, Y chromosome substitution significantly decreased the body weight in DH-Chr YDh and Ay, the autosomal dominant mutations, both of which are known to have substantial effects on body size. These results suggest that there are Y-linked genes that control the body size in mice.A body-size-reducing effect of the Y chromosome of the KK/Ta mouse strain was observed irrespective of genetic background. The effect was observed in the presence of  KK strain was lighter than other Y-consomic strains [We have established 17 Y chromosome consomic (hereafter Y-consomic) mouse strains in an inbred DH/Sgn (hereafter DH) strain. There was a wide spectrum of variation in body weight and testis weight among the Y-consomic mouse strains . Thus, iDh/+ genotypes at the dominant hemimelia (Dh) locus on chromosome 1, each Y-consomic strain includes both +/+ and Dh/+ mice. Some skeletal elements are lost in Dh/+ mice; therefore, Dh/+ mice are usually lighter than +/+ littermates (see Methods for details). We next analyzed Y-consomic strains with the Dh mutation. We further investigated the Y-consomic strains in combination with Ay, the obesity mutation. The Ay allele at the agouti locus on chromosome 2 is known to cause obesity and promote linear growth in mice (see Methods for details). When the males of each Y-consomic strain were crossed with females of the B6.Cg-Ay strain, the F1 generation consisted of Ay  and non-Ay  mice. We analyzed the F1-+/+ and F1-Ay hybrids. This analysis allowed us to evaluate the effect of the Y chromosome in obese (F1Ay) animals as well as in addition to non-obese (F1 non-Ay) animals in the same genetic background. Thus, we investigated the effect of the Y chromosome in the presence of autosomal dominant mutations, both of which substantially affected body size.First, we analyzed Y-consomic strains in the DH strain background. Because DH includes both +/+ and A (Y chromosome from A/J strain), DH-Chr YAKR (AKR/J), DH-Chr YB6 (C57BL/6J), DH-Chr YBALB (BALB/cA), DH-Chr YC3H (C3H/HeJ), DH-Chr YCAST (CAST/EiJ), DH-Chr YCBA (CBA/N), DH-Chr YCF1 (CF1/Sgn), DH-Chr YDBA (DBA/2J), DH-Chr YDDD (DDD/Sgn), DH-Chr YDH , DH-Chr YKK (KK/Ta), DH-Chr YRF (RF/J), DH-Chr YRR (RR/Sgn), DH-Chr YSJL (SJL/J), DH-Chr YSS (SS/Sgn), and DH-Chr YSWR (SWR/J). B6.Cg-Ay strain was purchased from the Jackson Laboratory  and maintained at the National Institute of Agrobiological Sciences . Each Y-consomic strain included Dh/+ and +/+ mice with respect to the genotype at the Dh locus. Dh causes visceral and skeletal malformations of various degrees of severity [Dh are expressed more severely in Dh/Dh than in Dh/+ animals. Because Dh/Dh mice die shortly after birth owing to their visceral abnormalities, only heterozygous Dh/+ mice were available for this study. The skeletal malformations in Dh/+ mice are worth mentioning. In Dh/+ mice, the number of lumbar vertebrae is reduced to five, as opposed to six in +/+ mice. Loss of the hallux  is commonly observed in Dh/+ mice. However, triphalangy of the hallux  is also commonly observed. Polydactyly is sometimes observed and is associated with an additional phalange on the hallux . Although the fibula is rarely affected, various lengths of the distal part of the tibia are frequently lost. Thus, Dh is associated essentially with reduction of skeletal elements. Dh/+ mice were distinguished from +/+ mice by the presence of hindlimb malformation, and the Dh/+ genotype was confirmed by the absence of the spleen on laparotomy. The Y-consomic strains in a DH background are hereafter designated as DH-Chr Y-+/+ and DH-Chr Y-Dh/+ for convenience.The following Y-consomic strains were used in this study: DH-Chr Yseverity . VisceraAy × ♂DH-Chr Y-+/+) F1 hybrids. The Ay allele at the agouti locus causes obesity and promotes linear growth in mice [Ay mice, the Ay allele is associated with a large deletion, causing agouti gene expression to be aberrantly controlled by the unrelated Raly gene promoter and leading to its ectopic overexpression [Ay mice have a yellow coat color and develop maturity-onset obesity. The yellow coat makes it possible to visually distinguish Ay mice from non-Ay mice. Obesity in Ay mice is believed to be a consequence of the agouti proteins serving as a constitutive antagonist of the melanocortin 3 receptor (MC3R) and melanocortin 4 receptor (MC4R) by mimicking the action of the agouti-related protein [Ay mice are heavier and longer than their non-Ay littermates. Importantly, mice homozygous for the Ay allele are embryonic lethal; therefore, living Ay mice are invariably heterozygotes. The F1 Y-consomic mice were designated as F1-+/+ and F1Ay/+. Strain designations and numbers of mice used in this study are summarized in Table We also investigated  and F1-+/+ and F1-Ay/+ hybrids were fed a CRF-1 . We are uncertain whether or not the difference in the lot of diet might have any impacts on body weights and/or body sizes of mice. All animal experiments were performed in accordance with guidelines approved by the Institutional Animal Care and Use Committee of NIAS.All mice were maintained in a specific-pathogen-free facility with a regular light cycle (12 h light and 12 h dark) and controlled temperature (23 ± 1°C) and humidity (50%). Food and water were freely available throughout the experimental period. DH-Chr Y-+/+ and DH-Chr Y-Dh/+ strains and at the age of 16 weeks for F1-+/+ and F1-Ay/+ hybrids, the mice were weighed on an electric balance to the nearest 0.01 g after 4 h fasting. For F1-+/+ and F1-Ay/+ hybrids, the anal–nasal length and tail length of each mouse were measured to the nearest 0.01 mm with digital calipers. Body length was defined as the anal–nasal length.At the age of 80 days for DH-Chr Y-+/+ and DH-Chr Y-Normality of distribution of the trait data for each Y-consomic strain was tested by the Shapiro–Wilk W test . If the trait values did not follow a normal distribution, they were normalized using the Box–Cox transformation.Statistical comparison of two groups was performed by the Student’s t-test. Effects of Y chromosome substitution were assessed using Dunnett’s multiple-comparison tests with the background DH strain as a reference. P <0.05 was considered statistically significant.Dh/+ (B) strains. As expected, average body weight was significantly higher in +/+ strains  than in Dh/+ strains (25.29 ± 0.12 g). Body weight showed bell-shaped distribution curves in both mice. Strictly, the distribution of body weight in DH-Chr Y-Dh/+ strains followed a normal distribution but that of DH-Chr Y-+/+ strains did not. Therefore, Box–Cox transformation was applied to the DH-Chr Y-+/+ strains before subsequent analyses. Figure Dh/+ strains. In the DH-Chr Y-+/+ background, Y chromosome substitution significantly decreased body weight in DH-Chr YSJL-+/+ and DH-Chr YKK-+/+ strains. The DH-Chr YKK-+/+ strain was the lightest among the DH-Chr Y-+/+ strains. In the DH-Chr Y-Dh/+ background, although Y chromosome substitution did not significantly alter the body weight, the DH-Chr YKK-Dh/+ strain was the lightest among the strains. The difference between DH-Chr YDH and DH-Chr YC3H strains was not statistically significant in both +/+ and Dh/+ backgrounds.Figure 1-+/+ (A) and 218 F1-Ay/+ (B) hybrids. As expected, average body weight was significantly higher in Ay/+ mice  than in +/+ mice (33.98 ± 0.24 g). The distribution of body weight in F1-Ay/+ hybrids followed a normal distribution but that of F1-+/+ hybrids did not. Therefore, Box–Cox transformation was applied to the F1-+/+ hybrids before subsequent analyses. Figure 1-+/+ and F1-Ay/+ hybrids. In the F1-+/+ and F1-Ay/+ backgrounds, Y chromosome substitution significantly decreased body weight in the (B6.Cg-Ay × DH-Chr YKK) F1-+/+ and (B6.Cg-Ay × DH-Chr YKK) F1-Ay/+ hybrids, respectively. Although average body weight of the (B6.Cg-Ay × DH-Chr YC3H) F1 hybrids was the highest in +/+ background, the difference between (B6.Cg-Ay × DH-Chr YDH) F1-+/+ and (B6.Cg-Ay × DH-Chr YC3H) F1-+/+ hybrids was not statistically significant.Figure 1-+/+ (A) and 218 F1-Ay/+ (B) hybrids (body length was measured only in F1 strains). Average body length was significantly greater in Ay/+ mice  than in +/+ mice (101.79 ± 0.16 mm). The distribution of body length in F1-+/+ hybrids followed a normal distribution but that of F1-Ay/+ hybrids did not. Therefore, Box–Cox transformation was applied to the F1-Ay/+ hybrids before subsequent analyses. Figure 1-+/+ and F1-Ay/+ hybrids. In the F1-+/+ and F1-Ay/+ backgrounds, Y chromosome substitution significantly decreased body length in the (B6.Cg-Ay × DH-Chr YKK) F1-+/+ and (B6.Cg-Ay × DH-Chr YKK) F1-Ay/+ hybrids, respectively. Although average body length of the (B6.Cg-Ay × DH-Chr YC3H) F1 hybrids was the greatest in +/+ background, the difference between (B6.Cg-Ay × DH-Chr YDH) F1-+/+ and (B6.Cg-Ay × DH-Chr YC3H) F1-+/+ hybrids was not statistically significant.Figure Body size is probably determined by multiple genes under the influence of non-genetic factors such as nutritional condition. To identify Y-linked gene polymorphisms associated with body size, it is essential to unify autosomal effects and to minimize non-genetic environmental influences. Thus, Y-consomic mouse strains are desirable and essential tools for investigating the effect of the Y chromosome on body size. There are several reports on the association of the Y chromosome with adult male height in humans, but the results are still contradictory ,17.Shox is not pseudoautosomal but autosomal. Therefore, the effect of the Y chromosome on body size observed in this study should not be attributed to Shoxy. Thus, it was suggested that there are other genes on the Y chromosome that influence body size in mice. We have genotyped Y-linked SNPs and other gene polymorphisms in these Y-consomic strains [It is a fact that many reports on body size have been obtained in human studies . For exa strains . HoweverKK strain becomes smaller than that of the other Y-consomic strains. Analysis of growth curves will be useful for this purpose. Because the difference was apparent at 80 days at the latest, the effect of the Y chromosome is expected to manifest earlier. The effect may already be apparent during the fetal period because the effect of Y chromosome on fetal growth rate has been hypothesized [As a next step, it is crucial to determine at what age the body size of DH-Chr Ythesized . CompariDh and Ay, the autosomal dominant mutations, both of which are known to have substantial effect on body size. These results suggest that there are Y-linked genes that control body size in mice.A body-size-reducing effect of the Y chromosome of the KK/Ta mouse strain was observed irrespective of genetic background. The effect was observed in the presence of The author declares that he has no competing interests.JS designed the research, carried out experiments for data collection, analyzed the data, and wrote the manuscript.


L-arabinose isomerases catalyse the isomerization of L-arabinose into L-ribulose at insight biological systems. At industrial scale of this enzyme is used for the bioconversion of D-galactose into D-tagatose which has many applications in pharmaceutical and agro-food industries. The isomerization reaction is thermodynamically equilibrated, and therefore the bioconversion rates is shifted towards tagatose when the temperature is increased. Moreover, to prevent secondary reactions it will be of interest to operate at low pH. The profitability of this D-tagatose production process is mainly related to the use of lactose as cheaper raw material. In many dairy products it will be interesting to produce D-tagatose during storage. This requires an efficient L-arabinose isomerase acting at low temperature and pH values.L-arabinose isomerase from Shewanella sp. ANA-3 was cloned and overexpressed in Escherichia coli. The purified protein has a tetrameric arrangement composed by four identical 55 kDa subunits. The biochemical characterization of this enzyme showed that it was distinguishable by its maximal activity at low temperatures comprised between 15-35°C. Interestingly, this biocatalyst preserves more than 85% of its activity in a broad range of temperatures from 4.0 to 45°C. Shewanella sp. ANA-3 L-arabinose isomerase was also optimally active at pH 5.5-6.5 and maintained over 80% of its activity at large pH values from 4.0 to 8.5. Furthermore, this enzyme exhibited a weak requirement for metallic ions for its activity evaluated at 0.6 mM Mn2+. Stability studies showed that this protein is highly stable mainly at low temperature and pH values. Remarkably, T268K mutation clearly enhances the enzyme stability at low pH values. Use of this L-arabinose isomerase for D-tagatose production allows the achievement of attractive bioconversion rates of 16% at 4°C and 34% at 35°C.The gene encoding the Shewanella sp. ANA-3 L-arabinose isomerase. Determination of the biochemical properties demonstrated that this enzyme was highly active at low temperatures. The generated T268K mutant displays an increase of the enzyme stability essentially at low pH. These features seem to be very attractive for the bioconversion of D-galactose into D-tagatose at low temperature which is very interesting from industrial point of view.Here we reported the purification and the biochemical characterization of the novel  L-Arabinose isomerase, hereafter L-AI, (EC 5.3.1.4) converts L-arabinose into L-ribulose within living systems [D-galactose isomerase due to its capacity to isomerize D-galactose into D-tagatose in vitro [D-tagatose have become of a great interest as sweeteners [D-Tagatose has not only a very low caloric value of 1.5 kcal/g (only 30% of the energy content of sucrose), but has also been reported to promote weight loss [D-tagatose are very expensive. Therefore, biological methods using L-arabinose isomerase as a catalyst are being developed due to its low cost [, EC 5.3..4 converin vitro ,3. Over eeteners . D-Tagatght loss . In factlow cost .Arthrobacter, Lactobacillus, Mycobacterium, Klebsiella and Gluconobacter are able to convert D-galactitol into D-tagatose [D-galactitol. That is why studies are now investigating a new bioprocess, which consists in converting D-galactose into D-tagatose using L-AIs. These studies have shown that the reaction equilibrium shifts towards D-tagatose production with increased temperatures, and that the majority of L-AIs are thermoactive biocatalysts requiring metal ions for their maximal thermostability and activity [L-AIs deriving from thermophilic strains such as Bacillus stearothermophilus US100, Thermotoga maritima, Thermotoga neapolitana, Geobacillus thermodenitrificans and Thermus sp. were identified [D-galactose to D-tagatose during products storage, which must be done at low temperatures, require an L-AI that acts not only at acidic pH but also at low temperatures. To satisfy these requirements, screening of novel L-AIs has been done. As a result, many L-AIs were identified and characterized to be functional at relatively high pH and at high temperatures [L-AIs from Alicyclobacillus acidocaldarius, Bacillus licheniformis, Lactobacillus plantarum NC8 and Lactobacillus sakei 23 K strains were described to be acid-tolerant. Molecular determinants for the catalytic efficiency were explored by homology modeling followed by site-directed mutagenesis [B. stearothermophilus US100 [D-tagatose production. Nevertheless, it remains of interest to set out a stable L-AI active at even lower temperatures.Many micro-organisms such as tagatose . Howeveractivity ,10-14. Uactivity ,15. For entified ,13,16-18agenesis ,20. Protus US100  and at aus US100 . On the us US100 . This doL-AI isolated from Shewanella sp. ANA-3. The activity and stability of the enzyme at low temperatures is discussed in comparison with previously described L-AIs. Site-directed mutagenesis has been performed to generate the T268K mutant and explore its biochemical properties.Here we report the cloning, over-expression, purification and biochemical characterization of an Shewanella sp. ANA-3 chromosomal DNA as template and the two oligonucleotides. DNA was ligated into the pGEMT-Easy vector and then transferred into the Escherichia coli BL21 (DE3) host strain. Numerous white colonies were observed and subsequently analyzed with the appropriate restriction endonucleases; a recombinant clone carrying the araA gene under the control of T7 promoter was obtained (pMR37). Then, the araA gene was sub-cloned in pET-15b under control of the T7 promoter leading to the pMR38 plasmid. Monitoring of liquid culture (BL21/pMR38 strain), followed by an L-AI activity test in the crude extract, showed an activity of 26 U/mg, confirming the molecular cloning and the expression of Shewanella sp. ANA-3 araA under the control of the T7 promoter. This latter gene encodes a 500 amino-acid residues protein with a calculated molecular mass of 55.3 kDa. Inspection of the amino-acid sequence multiple alignment of this protein with several other L-AIs indicated that Shewanella sp. ANA-3 L-AI displays the highest sequence identity (54.5%) with L-AI from the acidophilic strain A. acidocaldarius. Other enzymes such as those from B. licheniformis, L. plantarum NC8 and L. sakei 23 K, share 50.5%, 40.7% and 38.7% sequence identity, with Shewanella sp. ANA-3 L-AI, respectively  amplification using Shewanella sp. ANA-3 L-AI is a tetramer composed by four monomers of 55-kDa. Previous studies showed that all L-AIs have a similar, homotetrameric quaternary structure [E. coli L-AI which forms a homohexamer [L-AI 3D-structure is that of E. coli [D-galactose binding and/or catalysis. Shewanella sp. ANA-3L-AI presented a relatively high specific activity of 164 U/mg making it more active than most L-AIs from mesophilic bacterial origin  and T. neapolitana (119 U/mg) or lactic acid bacteria as L. sakei (218 U/mg) [After an over-night liquid cell culture of the BL21/pMR38 strain, the protein crude extract was subjected to fractioned precipitation with ammonium sulfate Figure . The subtructure ,13-15,18ohexamer . Present E. coli , and fur18 U/mg) .Shewanella sp. ANA-3 L-AI was optimally active within a large range of temperatures from 15 to 35°C, and highly active at low temperatures preserving nearly 90% to 95% of its optimal activity at 4 to 10°C, respectively. At 40 to 45°C, the protein retained 90% to 85% of its activity, respectively, whereas at higher temperatures (> 45°C) the relative activity rapidly decreased. This pronounced activity at low temperatures distinguishes it from all previously described L-AIs  showed a 74% decrease in the activity. Amongst several metal ions only Mnd Figure , at a co+ Figure . AnalysiEnzyme stability is a crucial criterion for industrial applications. Our study showed that without addition of metal ions this enzyme was fully stable at temperatures below 35°C. At 40°C the enzyme upheld more than 80% of its activity after two hours, and at 45 and 50°C the enzyme stability decreased with half-lives of 90 and 40 min, respectively Figure .2+ the enzyme was completely stable for 1 hour at 40°C and preserved 90% of its activity after two hours of incubation. Addition of manganese clearly improved the thermal stability at 45 and 50°C, the enzyme retained more than 50% of its initial activity after two hours  when compared to the wild-type enzyme  identified Lys269 in olerance . This hyolerance . Guided e Figure . By site005 identShewanella sp. ANA-3 L-AI showed that it has Km values of 33.7 and 52.1 mM for L-arabinose and D-galactose, respectively. Furthermore the Vmax was 164 mmole/s/mg for L-arabinose and 44 mmole/s/mg for D-galactose. To further explore the efficiency of this enzyme to produce D-tagatose, we carried out D-galactose bioconversion at different temperatures. As shown in Table D-tagatose at low temperature. In addition, analysis of Table Determination of the kinetic parameters of the L-AIs are thermoactive and not acid-tolerant. Generating a new acid-tolerant L-AI being highly active at low temperatures would allow the efficient bioconversion of D-galactose into D-tagatose in dairy products during fermentation and/or storage.Such enzyme features are very attractive from an industrial point of view. In fact, until now the majority of characterized L-AI from psychrotolerant Shewanella sp. ANA-3 is highly active at a wide pH range and converts efficiently D-galactose into D-tagatose even at low temperatures. These biochemical properties are of interest for industrial purposes. Indeed, isomerization of D-galactose at acid pH prevents the formation of unwanted side-products and high stability at low temperatures is convenient for food industry applications. The T268K mutation did not change enzyme behavior regarding temperature but clearly enhanced the enzyme activity and stability at low pH values.The D-galactose into D-tagatose in milk and dairy products such as yoghurts. In order to improve the suitability of this biocatalyst for industrial applications it is important to increase its affinity to D-galactose. For this purpose crystallization followed by the determination of the three-dimensional structure of this protein in complex with substrates by X-ray crystallography in combination with protein engineering are underway. Such data will allow improving the efficiency of other food-grade L-AIs.Further work will target the overexpression of this particular enzyme in a food-grade system such as lactic acid bacteria. Given the attractive properties of the enzyme it would be of high interest to employ it as a model for optimization of the bioconversion of Shewanella sp. ANA-3 strain was routinely grown as reported by Saltikov et al. [Escherichia coli BL21 (DE3) were used in this study as host strains. Culture of different E. coli strains was done in Luria Bertani (LB) medium. These media were supplemented, when necessary, with ampicillin (100 μg/ml) and IPTG  at 160 μg/ml. The pGEMT-Easy (Promega) and pET-15b plasmids (Novagen) were used according to the manufacturer's instructions.v et al. . Escheri® PCR System 9700 (Applied Biosystems). The amplification reaction mixtures (100 μl) contained High Fidelity Taq amplification buffer, 20 pmol of each primer, 100 ng of DNA template, and 10 units of High-Fidelity Taq DNA polymerase (Invitrogen). The cycling parameters were 94°C for 5 min, followed by 40 cycles of 94°C for 30 s, 55°C for 60 s, and 72°C for 120 s. The amplified fragment containing the Shewanella sp. ANA-3 araA gene was sequenced using an automated DNA sequencer (Applied Biosystems).Genomic DNA was prepared as reported by Johnson . PreparaShewanella sp. ANA-3 araA gene, we designed two primers based on the complete genome available in the NCBI databank. Oligonucleotide sequences were F-araA 5'GATAAGCACTATTTGCGTAAGCATG3' and R-araA 5' TTATTGCTGCGCAGAATTATAAACCTCTTGCTAAACCG3'. Chromosomal DNA isolated from Shewanella sp. ANA-3 strain was used as a template and High-Fidelity Taq DNA polymerase for amplification. The PCR product was purified using QIAquick Gel Extraction Kit (QIAGEN®) by following the manufacturer's instructions. Subsequently, the resulting fragments were cloned in pGEMT-Easy Vector (Promega) and transformed into E. coli BL21 (Invitrogen) competent cells. Recombinant white clones were selected on LB agar medium added with ampicillin (100 μg/ml), IPTG (160 μg/ml) and X-gal (100 μg/ml). Then, the araA gene was subcloned in the pET-15b (Novagen) under the control of the T7 promoter with six histidines at the N-terminal end of the protein. The T268K L-AI mutant was generated by using a site-directed mutagenesis Kit purchased from Stratagene (used as recommendation by supplier) and oligonucleotides T268Kd 5'AACCTCACTGGCATGAAAGGATTACCCGGACTG3' and T268Kr 5' CAGTCCGGGTAATCCTTTCATGCCAGTGAGGTT 3'.Aiming the amplification of the L-AI amino-acid sequence was done using the program ClustalW [The multiple alignment of the ClustalW  and the ClustalW .Escherichia coli strains, overexpressing Shewanella sp. ANA-3 L-AI, were grown in LB medium with ampicillin (100 μg/ml). From this culture a second one was launched under the same conditions at 200 rpm and 37°C. The culture was supplemented with IPTG (0.5 mM) at OD600 nm of 1.0 and subsequently incubated at 37°C and 220 rpm for over-night. Cells were harvested by centrifugation (6500 × g for 10 min at 4°C) and the pellets were re-suspended in 100 mM MES buffer at pH 5.5 supplemented with one Complete™ protease inhibitor cocktail tablet (Roche®). Cell disruption was carried out, twice, using a French Press at 1000 psi. For the purification, crude cell extract from the E. coli BL21 (DE3) strain was centrifuged . Proteins precipitated between 60 and 70% ammonium sulphate saturation, suspended in 100 mM MES buffer pH 5.5, concentrated and desalted in centrifugal micro-concentrators  with a 30 kDa cut-off membrane. Then, an affinity chromatography was done using IMAC column on the ÄKTA Purifier FPLC system (Amersham Pharmacia Biotech) equilibrated with 100 mM MES buffer pH 5.5. Proteins were eluted at a flow rate of 3 ml/min using a linear imidazole gradient ranging from 0 to 500 mM in the same MES buffer with imidazole (500 mM). Fractions containing L-AI activity were pooled and the final purification phase was performed using size exclusion chromatography on a Sephacryl S-200 column .The recombinant L-AI was determined by size exclusion chromatography.Protein concentration was determined using Bradford's method with bovine serum albumin as standard . The proL-AI activity was measured by determining the amount of formed L-ribulose or D-tagatose. Under standard conditions, the reaction mixture contained 100 μl of enzyme (L-AI) preparation at a 1 mg/ml, 50 mM of L-arabinose  and 100 mM MES buffer (pH 5.5) in a final volume of 1 ml. The reaction was stopped by heating the mixture at 99°C for 2 min. The amount of L-ribulose (or D-tagatose) was colorimetrically determined using the cystein-carbazol-sulphuric-acid method and the absorbance was measured at 560 nm [D-Tagatose production was also detected with high-performance ionic chromatography (HPLC) using a Polypore CA column (250 × 4.6 mm). The products were separated by isocratic elution with water at a flow rate of 0.3 ml/min and detected with a refractive index detector . Solutions of 5 g/l D-galactose and 5 g/l D-tagatose were used as standards.t 560 nm . D-TagatL-AI activity was defined as the amount of enzyme catalyzing the formation of 1 μmol keto-sugar per min under the above-specified conditions.One unit of The effect of temperature on the activity was studied by incubating the purified protein at temperatures from 4 to 55°C. The enzyme pH profile was obtained at pH values between 3.0 and 8.5 . Determination of the enzyme stability as a function of temperature and pH was carried out by measuring the residual activity at periodic intervals.Shewanella sp. ANA-3 L-AI was purified as described in the enzyme purification section but without addition of metal ions to the crude extract and dialyzed against 100 mM MES buffer (pH 5.5) containing 10 mM EDTA for 48 h at 4°C (buffer renewed twice). Subsequently, we twice dialyzed the EDTA-treated enzyme against 100 mM MES buffer (pH 5.5). Thereafter, the enzyme was pre-incubated during 10 min in solutions containing metal ions  followed by L-AI activity assays under standard conditions. The optimal metal ion concentrations were monitored by incubating the enzyme samples at various concentrations followed by activity assays.For sample preparation, each mineralized sample was employed at a final concentration evaluated to 10 mg/ml. A Perkin-Elmer Analyst 200 atomic absorption , equipped with a deuterium lamp background correction system, was used for metal binding quantification. Hollow cathode lamps (Perkin-Elmer) were used as primary radiation source. Analytical measurements were based on time average absorbance, under conditions recommended by the manufacturer.2+ and 1 to 800 mM substrate . Samples were incubated at 35°C and the amount of keto-sugar generated (L-ribulose or D-tagatose) was determined by the cysteine-carbazole-sulfuric acid method.Kinetic parameters were determined on the basis of Lineweaver-Burk plots. Assays were carried out in 100 mM MES buffer (pH 5.5), 0.6 mM MnD-galactose into D-tagatose was monitored in a solution containing 100 mM MES buffer (pH 5.5), 0.6 mM Mn2+ and 50 mM D-galactose. Kinetic conversion of D-galactose was investigated for 7 h at 4, 10, 20, 30 and 35°C. Samples were taken periodically and the concentration of generated D-tagatose was determined as described earlier and confirmed by HPLC as indicated in the enzyme assay section.Bioconversion of L-AI: L-arabinose isomerase; MES: 2(-N-morpholino)ethanesulfonic acid; EDTA: Ethylenediaminetetraacetic acid; HPLC: High-performance liquid chromatography; IPTG: Isopropyl β-D-thiogalactopyranoside; PCR: Polymerase Chain Reaction; RT: Retention Time; SDS-PAGE: Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis.The authors declare that they have no competing interests.MR participated in the design of the study, experiments analysis and writing the manuscript. GB participated in the molecular biology experiments. IR participated in the biochemical experiments. SB participates in the construction of mutant and expression of proteins. EM participated in the design of the work. RH participated in the conception of mutant. AN participated in the design of the work and helped in drafting the manuscript. All authors read and approved the final manuscript.


The objective of the current study was to determine the clinical significance of junctional adhesion molecule A (JAM-A) in patients with non-small cell lung cancer (NSCLC) and the biological function of JAM-A in NSCLC cell lines. We showed that JAM-A is predominantly expressed in cell membranes and high expression of JAM-A occurred in 37% of lung tumor specimens compared to corresponding normal tissues. High expression of JAM-A was significantly correlated with TNM stage (P = 0.021), lymph node metastasis (P = 0.007), and decreased overall survival (P = 0.02), In addition, we observed that silencing JAM-A by small interfering RNA inhibited tumor cell proliferation and induced cell cycle arrest at the G1/S boundary. Western blotting analysis revealed that knockdown of JAM-A decreased the protein levels of cyclin D1, CDK4, 6, and P-Rb. Thus, JAM-A plays an important role in NSCLC progression. Similar clinical- or in vitro-generated data were reported by others involving endometrial carcinoma in vitro-, and in vivo-generated data sets were recently reported in breast cancer and epidermoid carcinoma Junctional adhesion molecule A (JAM-A) is a type I transmembrane glycoprotein that belongs to the immunoglobulin superfamily. JAM-A is widely distributed in tissues, including placenta, lungs, liver, kidneys, pancreas, heart, brain, intestines, and lymph nodes This study was conducted with the approval of the Institutional Review Board of the China Medical University. Written informed consent was obtained from all NSCLC patients and all clinical investigations were conducted according to the principles promulgated in the Declaration of Helsinki.Eighty-two NSCLC samples were obtained from the First Affiliated Hospital of China Medical University between 2002 and 2009; 42 samples underwent follow-up studies. None of the patients received radiotherapy or chemotherapy before surgical resection. The histologic diagnosis and grade of differentiation of the tumors were defined by evaluation of hematoxylin and eosin-stained tissue sections according to the World Health Organization guidelines of classification. All 82 specimens were re-evaluated with respect to histologic subtype, differentiation status, and tumor stage. Squamous cell carcinoma and adenocarcinoma were identified in 36 and 46 of the 82 cases, respectively. Lymph node metastases were observed in 31 patients. Tumors were classified into stages I (n = 45), II (n = 30), and III (n = 17) according to the p-TNM staging system of the International Union Against Cancer (7th edition). Overall survival was defined as the period of time from initial diagnosis to death or the date of last follow-up.HBE, A549, H1299, H157, and H460 cell lines were obtained from the American Type Culture Collection . SPC and LK2 cell lines were purchased from the Shanghai Cell Bank of Chinese Academy of Science. The cells were cultured in RPMI-1640  containing 10% fetal calf serum , 100 IU/ml of penicillin , and 100 mg/ml of streptomycin (Sigma). Cells were grown on sterile tissue culture dishes and passaged every 2 days using 0.25% trypsin (Invitrogen).Surgically-excised tumor specimens were fixed with 10% neutral formalin, embedded in paraffin, and 4-µm thick sections were prepared. Immunostaining was performed using the ElivisionTM plus Polyer HRP (mouse/rabbit) IHC Kit . The sections were deparaffinized in xylene, rehydrated in graded alcohol series, and boiled in 0.01 M EDTA buffer (pH 9.0) for 20 minutes in a boiler. Endogenous peroxidase activity was blocked using hydrogen peroxide (0.3%), which was followed by incubation with normal goat serum to reduce non-specific binding. Tissue sections were incubated with JAM-A rabbit monoclonal antibody . Rabbit immunoglobulin, at the same concentration as the antigen-specific antibody (Maixin) was used as a negative control. Staining for all primary antibodies was performed at 4°C overnight. ElivisionTM plus Polyer HRP was used as the secondary antibody. After washing, the sections were incubated with 3, 3′-diaminobenzidine tetrahydrochloride to develop the peroxidase reaction. Counterstaining of the sections was done with hematoxylin, which were then dehydrated in ethanol before mounting. Two independent investigators examined all tumor slides randomly. Five views were examined per slide, and 100 cells were observed per view at 400X magnification. Immunostaining of JAM-A was scored following a semi-quantitative scale by evaluating representative tumor areas; the intensity and percentage of cell membranes had higher immunostaining than the control cells. Membrane staining of the tumor cells was considered to be positive immunostaining. The intensity of JAM-A membranes staining was also scored as 0 (no staining), 1 (weak), 2 (moderate), and 3 (high). Percentage scores were assigned as follows: 1, 1%–10%; 2, 11%–50%; 3, 51%–80%; and 4, 81%–100% −ΔΔCt method, where ΔΔCt = (Ct gene–Ct reference) sample– (Ct gene–Ct reference) calibrator. Primer sequences of JAM-A were 5′-GTG AAG TTG TCC TGT GCC TAC TC-3′ (forward) and 5′-ACC AGT TGG CAA GAA GGT CAC C-3′ (reverse). Primer sequences of β-actin were 5′- TGC CTA GAT CAA GAA GCA G -3′ (forward) and 5′- TGC CTA GAT CAA GAA GCA G -3′ (reverse). Three independent experiments were conducted in triplicate under identical conditions.Quantitative real-time PCR was performed using SYBR Green PCR master mix  in a total volume of 20 ul on a 7900HT Fast Real-Time PCR System (Applied Biosystems) as follows: 95°C for 30 seconds; 40 cycles at 95°C for 5 seconds; and 60°C for 30 seconds. A dissociation step was performed to generate a melting curve to confirm the specificity of the amplification. β-actin was used as the reference gene. The relative levels of gene expression were represented as ΔCt = Ct gene–Ct reference and the fold change of gene expression were calculated by the 2Small Interfering RNA (siRNA) for JAM-A was synthesized by Genepharma . The sequences of siRNA were as follows: 5′- GAA GUG AAG GAG AAU UCA ATT-3′ (sense); and 5′-UUG AAU UCU CCU UCA CUU CTT-3′ (antisense). The sequences of non-targeting siRNA (negative control) used as a negative control were as follows: 5′-UUC UCC GAA CGU GUC ACG UTT-3′ (sense); and 5′-ACG UGA CAC GUU CGG AGA ATT-3′ (antisense). Cells were seeded in a 6-well plate 24 h before transfection experiments. The cells were transfected with 100 pmol JAM-A siRNA or negative control siRNA using Lipofectamine2000  according to the manufacturer’s protocol. Following transfection, the protein and mRNA levels of JAM-A were assessed 48 h later. Three independent experiments were conducted under identical conditions.Total protein from cells was extracted in lysis buffer  and quantified using the bicinchoninic acid (BCA) method (Beyotime). Sixty µg of protein was separated by SDS–PAGE (10%) and transferred to polyvinylidene fluoride (PVDF) membranes , After blocking with 5% BSA in Tris-buffered saline-Tween 20 , membranes were incubated at 4°C overnight with the following primary antibodies: JAM-A ; and anti-P-Rb, anti-P27, anti-P21, anti-cyclin A, anti-cyclin B, anti-cyclin D1, anti-CDK4, anti-CDK6,anti-AKT1/2, and anti-P-AKT Thr 308 . After incubation with peroxidase-coupled anti-mouse or rabbit IgG  at 37°C for 2 h, bound proteins were visualized using ECL  and detected using BioImaging Systems . The relative protein levels were calculated based on GAPDH as the loading control. Three independent experiments were conducted under identical conditions.6 cells were washed twice with chilled incubation buffer (2% BSA in PBS) and centrifuged, then the cells were resuspended in 100 uL of incubation buffer and incubated with or without FITC-conjugated mouse anti-human JAM-A antibody  or FITC-conjugated mouse IgG1, κ isotype control antibody  on ice for 30 min in the dark. The cells were then washed twice with incubation buffer and processed for flow cytometric analysis. Data were analyzed using FlowJo 7.6.1 software. Three independent experiments were conducted in triplicate under identical conditions.Cells were trypsinized and 1×103 cells/100 ul/well in 96-well culture plates overnight. Each subsequent day, cells were treated with 20 ul (5 mg/ml)/well of MTT solution during the last 4 h of the culture. The culture media was replaced with DMSO (150 ul/well). The optical density of the wells was measured at 490 nm using a microplate reader. Three independent experiments with 5 internal replicates per experiment were conducted under identical conditions.A cell proliferation assay was performed using MTT (Sigma) according to the manufacturer’s protocol. Briefly, H1299 and A549 cells were transiently transected with JAM-A siRNA or negative control siRNA. After 24 h, the cells were trypsinized and seeded at a concentration of 3×10For colony formation assays, cells were transfected with JAM-A or negative control siRNA for 48 h, then plated into 6-well culture plates (1000 per well) and incubated for 12 days. Plates were washed with PBS and stained with Giemsa. The number of colonies with >50 cells was counted. The colonies were manually counted using a microscope. Three independent experiments were conducted in triplicate under identical conditions.Cells  were seeded into 6-well culture plates, synchronized after serum starvation for 24 h, then transfected with the indicated amounts of siRNA. Cells were harvested, fixed in 75% ethanol 48 h after transfection, washed with PBS, and stained in 5 mg/ml of propidium iodide in PBS supplemented with RNase A  for 30 min at room temperature. Data were collected using BD systems. Three independent experiments were conducted under identical conditions.SPSS  was used for all analyses. The chi-squared test was used to determine the correlation between JAM-A expression and clinicopathologic characteristics. Kaplan–Meier curves were used for survival analysis, and log-rank was determined based on the differences. Student’s t-test was used to compare other data. P-values were based on two-sided statistical analysis, and a p<0.05 was considered to indicate statistical significance.We analyzed the expression and distribution of JAM-A in 82 NSCLC specimens and the corresponding normal tissues using immunohistochemistry. JAM-A was predominantly expressed in cell membranes; high expression of JAM-A was found in 37% of lung tumor specimens , which wWe detected JAM-A expression in normal bronchial epithelial (HBE) cells and six lung cancer cell lines by Western blot and real-time PCR analysis. H1299 and A549 cells exhibited high levels of expression of JAM-A, HBE cells exhibited moderate levels of expression of JAM-A, and H157, H460, SPC, and LK2 exhibited low levels of expression of JAM-A . BecauseTo determine whether or not JAM-A takes part in modulating lung cancer cell growth, we used siRNA to knockdown JAM-A expression in H1299 and A549 cell lines. Compared with negative control siRNA-transfected cell lines, JAM-A-specific siRNA effectively suppressed total JAM-A protein levels , JAM-A mWe next sought to investigate possible mechanisms whereby JAM-A knockdown can decrease cell proliferation. We analyzed the cell cycle by fluorescence-activated cell sorting (FACS), and showed that the percentage of cells in the G1 phase was increased  proliferation by inhibiting PI3K-Akt-β-catenin signal cascade activation JAM-A is expressed in several kinds of tumors and is implicated in tumor progression. The expression of JAM-A protein in NSCLC tissues and the relationship with various clinicopathologic factors has not been fully examined. Furthermore, the exact role of JAM-A in lung cancer progression is unclear. In this study we examined the pattern of expression and the biological function of JAM-A in NSCLC. We showed that JAM-A is predominantly localized in cell membranes, and the expression of JAM-A in lung cancer tissues is higher than corresponding normal lung tissues. The high level of expression of membrane JAM-A was significantly associated with advanced pTNM stage, lymph node metastases, and reduced overall survival in lung cancer patients. Taken together, our results revealed that JAM-A is a potential oncogenic protein in NSCLC and might serve as a negative predictor of survival in NSCLC patients.To further explore the potential function of JAM-A in lung cancer cells, we used siRNA to knockdown JAM-A expression in H1299 and A549 cell lines, which express relatively high levels of surface JAM-A. Proliferation and colony formation in the two cell lines was significantly suppressed after silencing JAM-A. Most of the proliferative factors influence cell growth by affecting cell cycle progression, thus we analyzed the cell cycle and showed that JAM-A knockdown cells had higher levels of G1 phase and lower S phase than control cells. Thus, JAM-A silencing inhibited the G1-to-S transition in cell cycle progression, which might elucidate the mechanism of JAM-A on lung cancer cell proliferation. To determine the potential mechanism of JAM-A on cell cycle regulation, we examined the effect of JAM-A knockdown on cell-cycle related molecules. We analyzed the expression of cyclin A, cyclin B, cyclin D1, CDK4, CDK6, P21, P27, and P-RB, and found that the levels of expression of cyclin D1, CDK4, CDK6, and P-RB were decreased after JAM-A knockdown. It has been previously demonstrated that P-RB is responsible for a major G1 checkpoint, blocking S-phase entry and cell growth by physically associating with E2F factors and blocking activation of gene expression that encodes products necessary for S-phase progression. The protein complex comprised of cyclin D1, CDK4, and CDK6 will phosphorylate P-RB and promote P-RB inactivation. Continued phosphorylation on P-RB by various CDKs leads to the release of E2F, thereby facilitating the activation of genes critical for S-phase progression Published studies indicate that JAM-A restricts IEC proliferation by inhibiting PI3K-Akt-β-catenin activation in vivo, in vitro, or using a genetic mammary tumor model. Comparing all of the aforementioned studies, we speculate that JAM-A function is context-dependent. For example, Naik et al. The role of JAM-A in tumor growth and dissemination remains a controversial issue. Naik et al. In conclusion, our study has demonstrated for the first time that high expression of JAM-A in NSCLC tissues is positively correlated with NSCLC progression. JAM-A can promote NSCLC cell proliferation through cell cycle regulation.