In addition, we determined the expression of P2Y6,11 receptors in the dorsal spinal cord in presence of the selective P2Y6,11 receptors antagonists. Furthermore, we evaluated the participation of spinal microglia and astrocytes in the pronociceptive role of P2Y6,11 receptors.The participation of spinal P2X receptors in neuropathic pain is well recognized. However, the role of P2Y receptors has been less studied. The purpose of this study was to investigate the contribution of spinal P2Y6  and P2Y11  receptor antagonists reduced tactile allodynia in spinal nerve ligated rats. Nerve injury increased the expression of P2Y6,11 receptors at 7, 14 and 21 days after injury. Furthermore, intrathecal administration of MRS2578 (100 μM/day) and NF340 (30 μM/day) for 3 days significantly reduced spinal nerve injury-induced increase in P2Y6,11 receptors expression, respectively. Spinal treatment (on day 14 after injury) with minocycline (100 μg/day) or fluorocitrate (1 nmol/day) for 7 days reduced tactile allodynia and spinal nerve injury-induced up-regulation in Iba-1 and GFAP, respectively. In addition, minocycline reduced nerve injury-induced up-regulation in P2Y6,11 receptors whereas that fluorocitrate diminished P2Y11, but not P2Y6, receptors up-regulation. Intrathecal treatment (on day 21 after injury) with the selective P2Y6  and P2Y11  receptor agonists produced remarkable tactile allodynia in nerve ligated rats previously treated with minocycline or fluorocitrate for 7 days.Spinal administration of the selective P2Y6 is present in spinal microglia while P2Y11 receptors are present in both spinal microglia and astrocytes, and both receptors are up-regulated in rats subjected to spinal nerve injury. In addition, our data suggest that the spinal P2Y6 and P2Y11 receptors participate in the maintenance of neuropathic pain.Our data suggest that spinal P2Y The ptization -24.1 receptors mRNA is up-regulated in rat dorsal root ganglion neurons after peripheral axotomy of the sciatic nerve [1 and P2Y2 receptors mRNAs are up-regulated in mouse L2/L3 dorsal root ganglion neurons after nerve transection [12 receptors are up-regulated in spinal microglia after peripheral nerve injury [6, P2Y13 and P2Y14 mRNAs are up-regulated in microglia in the ipsilateral spinal cord following nerve injury [In comparison to P2X receptors, the contribution of P2Y receptors to neuropathic pain has been less studied. At the moment, there is limited evidence to suggest that P2Y receptors play a role in neuropathic pain. However, data have shown that P2Yic nerve . Likewisnsection . P2Y rece injury . Moreovee injury . Furthere injury .6 and P2Y11 receptors in the maintenance of neuropathic pain in the rat by pharmacological and molecular methods. Our data suggest that spinal P2Y6 is present in spinal microglia while P2Y11 receptors are present in both spinal microglia and astrocytes, and both receptors are up-regulated in rats subjected to spinal nerve injury. In addition, our data suggest that the spinal P2Y6 and P2Y11 receptors participate in the maintenance of neuropathic pain.Due to the limited information about the participation of P2Y receptors in neuropathic pain, we decided to study the role of spinal P2Y6  and dose-dependently tactile allodynia in the spinal nerve injured rats.Spinal nerve injury produced tactile allodynia in the ipsilateral hind paw, which was evident by a significant decrease in the 50% paw withdrawal threshold response as compared to the naive group Figure A/C. As e6  increase in the expression of P2Y6 and P2Y11 receptors at 7 and 14 days  increase of P2Y6  and P2Y11  receptor antagonists, but not vehicle, significantly (p < 0.05) reversed spinal nerve ligation-induced increase in P2Y6  of the selective P2Yp < 0.05) increase in the expression of microglia and astrocytes markers Iba-1  reduced spinal nerve injury-induced increase in P2Y6 , repeated intrathecal treatment with minocycline , repeated intrathecal treatment with minocycline  administration (endogenous agonist) shows antiallodynic effects after partial sciatic nerve ligation [6 receptor agonist selectivity as UDP also activates the P2Y14 receptor with higher affinity than for P2Y6 receptors [6 (PSB0474) and P2Y11 (NF546) receptor agonists produces long-lasting tactile allodynia which argues against the antinociceptive effect of spinal P2Y6 receptors. Regarding NF340, this seems to be the first report demonstrating the antinociceptive effect of this compound suggesting that spinal P2Y11 receptors play an important role in the maintenance of neuropathic pain. The contribution of P2Y6 receptors seems to be different from that of P2Y11 receptors as the P2Y6 receptor antagonist produced a peak in the antiallodynic effect in about 1–2 h and then decayed rapidly. In contrast, the P2Y11 receptor antagonist produced a modest but sustained antiallodynic effect. This discrepancy could be due to differences in the pharmacokinetics or pharmacodynamics of the tested drugs. Taken together, our data suggest the participation of spinal P2Y6,11 receptors in the maintenance of neuropathic pain.This study demonstrates that intrathecal administration of MRS2578 and NF340 partially reverses spinal nerve injury-induced tactile allodynia. Since these drugs are selective P2Y6 and P2Y16[11 receptor6[11, suggestligation . This di6,11 receptors are expressed in the dorsal spinal cord of naïve rats. Previous studies indicate that P2Y6 receptor mRNA is found in rat microglia [11 receptors have only been described in humans. Currently there are no reports of a cloned P2Y11 receptor in rats. However, this receptor has been recently reported in epithelial cells [6 receptor is expressed and completely functional in the spinal cord. In addition, our results suggest the presence of a P2Y11–like receptor in the rat spinal cord. Besides their presence in naïve animals, P2Y6,11 receptors expression in the ipsilateral dorsal spinal cord was up-regulated at 7 and 14 days after spinal nerve ligation. These results partially agree with a previous study showing that P2Y6 receptor mRNA is up-regulated in the ipsilateral spinal cord at 3 days following peripheral nerve injury [6 receptor (PCR versus WB). In the case of P2Y11 receptors, this is the first report about their increased expression. To further reinforce the participation of spinal P2Y6,11 receptors in the maintenance of neuropathic pain in rats, we demonstrated that intrathecal administration of the P2Y6,11 receptor antagonists reverses spinal nerve injury-induced tactile allodynia and P2Y6,11 receptors up-regulation. Admittedly, the mechanisms for the suppressive effects of P2Y6,11 receptor antagonists on the up-regulation of these receptors in the dorsal spinal cord after nerve injury are unclear. However, it is likely that blockade of both receptors may lead to a fall in central sensitization that then could promote the reduction in P2Y6,11 receptors expression. Clearly, further experiments which fall beyond the scope of our study will be required to confirm our suggestion.In support to our pharmacological study, we demonstrated by western blot that P2Yicroglia . On the al cells , primaryal cells , lacrimaal cells , neutropal cells , and braal cells  of rats.e injury . This ti6,11 receptors in the dorsal spinal cord whereas that treatment with minocycline reduced spinal nerve damage-induced P2Y6,11 receptors enhanced expression. Thus, our data seem to point out that spinal P2Y6,11 receptors are present in activated microglia and they participate in the maintenance of neuropathic pain in the rat. In support of this, Kobayashi and coworkers reported the enhanced presence of P2Y6,13,14 receptors mRNA in spinal microglia following peripheral nerve injury [6 mRNA suggesting that activated microglia leads to an enhanced expression of P2Y6 receptors and neuropathic pain [It is accepted that spinal microglia and astrocytes release several pro-inflammatory mediators in the dorsal horn following nerve damage ,24. We ce injury . Furtherhic pain .11, but not P2Y6, receptors up-regulation suggesting that activated astrocytes are over-expressing P2Y11 receptors and this phenomenon contributes to maintenance of neuropathic pain. In support of this, P2Y11 receptors have been found in astrocytes [6,11 receptors produced tactile allodynia once microglia and astrocytes have been previously blocked suggests that spinal P2Y6,11 receptors may participate promoting neuropathic pain at least partially in a microglia- or astrocytes-independent way. Supporting this, P2Y6, but not P2Y11, receptors mRNA have been found in dorsal root ganglion [6,11 receptors are present in microglia while P2Y11 receptors are present only in astrocytes.We showed that fluorocitrate reduces spinal P2Ytrocytes . Howeverganglion ,40,41. TWe found that repeated injections (for 7 days) of minocycline or fluorocitrate produced an antiallodynic effect in neuropathic rats. These data suggest that microglia and astrocytes play an important role in the maintenance phase of neuropathic pain. These data agree with previous observations showing that repeated treatment with minocycline reverses thermal hyperalgesia and mechanical allodynia -46. In c6,11 receptors are present in spinal microglia while P2Y11 receptors are present only in astrocytes. Both receptors are up-regulated in rats subjected to spinal nerve injury. Our data suggest that the spinal P2Y6 and P2Y11 receptors participate in the maintenance of neuropathic pain.Our data suggest that spinal P2YSince previous experiments in our conditions found no differences between male and female rats , all expRats were prepared according to the method of . Briefly6,11 receptor antagonists (μM/10 μl). The antiallodynic effect was evaluated for the following 6 h in both conditions.Chronic catheterization of the spinal subarachnoid space was performed as described . Seven obiscarbonylimino))-bis, N,N"-1,4-butanediylbis[N'-(3-isothiocyanatophenyl)thiourea (MRS2578) and 4,4'-carbonylimino))bis tetrasodium salt (NF340) were purchased from Tocris Bioscience . The purinergic drugs mentioned above were selected based on relevant receptor selectivity and efficacy. These included: (i) selective P2Y6 and P2Y11 receptors agonists PSB0474 [6 and P2Y11 receptors antagonists MRS2578 [6,11 receptor antagonists were chosen based in previous studies [3-(2-Oxo-2-phenylethyl)-uridine-5'-diphosphate disodium salt (PSB0474), 4,4'- and DL-fluorocitric acid barium salt  were purchased from Sigma-Aldrich . PSB0474, NF340 and NF546 were dissolved in 0.9% saline solution. MRS2578 and minocycline was dissolved in 20% DMSO. Fluorocitrate was dissolved initially in 2 M HCl (0.1 μmol/μL) and then diluted in 0.01 M PBS to attain a final concentration of 0.1 nmol/μL (pH 6.0). The vehicle control for fluorocitrate was 0.1% 2 M HCl in 0.01 M PBS (pH 6.0) .6,11 receptors as well as Iba-1 and GFAP protein levels in the lumbar ipsilateral and contralateral dorsal spinal cord. For this purpose, naïve and ligated rats were sacrificed by decapitation. The lumbar region of the spinal cord (L4–L6) was sectioned to get the ipsilateral section of the dorsal spinal cord. Immediately, tissues were stored at −70°C. Tissues from individual animals were homogenized in ice-cold lysis buffer , pH 7.4 for 30 min at 4°C. The protease inhibitors PMSF (1 mM), aprotinin (10 μg/mL), leupeptin (10 μg/mL), pepstatin A (10 μg/mL) and the surfactant 0.1% Triton X-100  were added to the lysis buffer immediately prior to use. The homogenate was then centrifuged  at 14,000 rpm for 10 min to remove cellular debris. The resultant supernatant was used to measure protein concentration by the Bradford’s method .Western blot analysis was used to detect changes in the expression of P2Y6 and P2Y11 receptors) μg of proteins were resolved by denaturing by 10% or 12% (for Iba-1) SDS–polyacrylamide gel electrophoresis and transferred to PVDF membranes. Membranes were incubated in 5% non-fat milk in phosphate-buffered saline (PBS) at pH 7.4  with 0.1% Tween 20 for 1 h to block nonspecific proteins. After that, they were incubated overnight at 4°C in 5% non-fat dry milk/PBS containing rabbit anti-P2Y6 , rabbit anti-P2Y11 , mouse anti-Iba-1  or rabbit anti-GFAP  antibodies. Membranes were incubated for 1 h at room temperature in 1% non-fat milk/PBS containing the horseradish peroxidase-conjugated secondary antibody . Protein signal detection was achieved with the ECL chemiluminescence system . The next day, blots were stripped and incubated with a monoclonal antibody directed against β-actin  which was used as an internal control to normalize P2Y6 and P2Y11 receptors and Iba-1 and GFAP protein expression levels. Scanning of the immunoblots was performed and the bands were quantified by densitometry using an image analysis program . In addition, control peptides for P2Y6,11 receptors were tested to determinate the bands specificity.Fifty (for Iba-1 and GFAP) or one hundred  were intrathecally administered with vehicle or increasing doses of the selective P2Y6  and P2Y11  receptor antagonists.In order to determine the role of spinal P2Y6,11 receptor antagonists produced antiallodynic effects, we next determined the expression of these receptors at the ipsilateral and contralateral dorsal spinal cord at 1, 3, 7 and 14 days after injury. Furthermore, to confirm the participation of the spinal P2Y6,11 receptors in the processing of neuropathic pain in rats, the expression of these receptors in absence and presence of the selective P2Y6,11 receptor antagonists was determined. For this, we administered the P2Y6 (100 μM) and P2Y11 (30 μM) receptor antagonists every 12 h for 3 days starting on day 12 after ligation. Rats were sacrificed on day 14 after nerve ligation.As selective P2YIn order to investigate the role of microglia and astrocytes in spinal nerve injury-induced tactile allodynia, rats were ligated and intrathecally canulated. On day 14, these animals received a daily intrathecal injection of vehicle, minocycline (100 μg/day) or fluorocitrate (1 nmol/day) for seven days. Tactile allodynia was determined 24 h after the first administration and then every other day until day 21. Rats were sacrificed on day 21 to determine the protein expression of Iba-1 and GFAP in the ipsilateral and contralateral dorsal spinal cord.6,11 receptors in the same conditions. To further confirm the participation of glial cells and spinal P2Y6,11 receptors, we next evaluated the effect of selective P2Y6  and P2Y11  receptor agonists in rats previously treated with minocycline or fluorocitrate for 7 days.As our data showed the participation of microglia and astrocytes in neuropathic pain induced by spinal nerve ligation, we next investigated the role of the spinal purinergic P2YAll behavioral results are given as the mean ± S.E.M. for six animals per group. Curves were constructed by plotting the threshold for paw withdrawal as a function of time. An increase of 50% withdrawal threshold was considered as antiallodynia.For protein expression, all results are given as the mean relative intensity ± S.E.M. for 3 animals per group.p < 0.05.One- or two-way analysis of variance, followed by the Student-Newman-Keuls was used to compare differences between groups. Differences were considered to reach statistical significance when N,N"-1,4-Butanediylbis[N'-(3-isothiocyanatophenyl)thiourea; NF340: 4,4'-carbonylimino))bis tetrasodium salt; NF546: 4,4'-carbonylimino))-bis-uridine-5'-diphosphate disodium salt; TNFα: Tumor necrosis factor α; UDP: Uridine diphosphate.ADP: Adenosine diphosphate; ATP: Adenosine triphosphate; BDNF: Brain derived neurotrophic factor; CCL-2: Chemokine ligand 2; CCL-3: Chemokine ligand 3; GFAP: Glial fibrillary acidic protein; Iba-1: Ionized calcium-binding adapter molecule-1; IL-1β: Interleukin-1β; IL-6: Interleukin-6; MRS2578: The authors declare that they have no competing interests.PB-I carried out the molecular and pharmacological studies, performed the statistical analysis and drafted the manuscript. JBP-F carried out the molecular studies. CC-D participated in the pharmacological studies. MB-H participated in the molecular and pharmacological studies. HIR-G participated in the design and coordination of the study and helped to draft the manuscript. JM participated in the design, coordination and helped to draft to manuscript. VG-S conceived the idea, participated in the design and coordination of the study. He also helped to interpret the data and to draft the final manuscript. All authors read and approved the final manuscript.PB-I, JBP-F, CC-D and MB-H are graduate students at the Department of Pharmacobiology in Cinvestav, South Campus. Mexico City, Mexico. HIR-G is a full professor at the Sección de Estudios de Posgrado e Investigación at the Escuela Superior de Medicina, Instituto Politécnico Nacional, Mexico City, Mexico. JM is a full professor at the Department of Pharmacobiology in Cinvestav, South Campus. Mexico City, Mexico. VG-S is a full professor at the Department of Pharmacobiology in Cinvestav, South Campus. Mexico City, Mexico.6,11 receptors at 21 days after injury. Western blot analysis of the P2Y6 and P2Y11 receptors expression in the ipsilateral dorsal spinal cord obtained 21 days after nerve injury from naïve and spinal nerve injured rats. Data were normalized against β-actin and are expressed as the mean ± S.E.M. of 3 independent rats. *Significantly (p < 0.05) different from the naïve group, as determined by one-way analysis of variance followed by the Student-Newman-Keuls test.Spinal nerve injury enhances the expression of P2YClick here for file6 and P2Y11 protein expression in the contralateral dorsal spinal cord. Western blot analysis of the P2Y6 (panel A) and P2Y11 (panel B) receptors expression in the contralateral dorsal spinal cord obtained from naïve (N) and spinal nerve injured rats (SNL). Data were normalized against β-actin and are expressed as the mean ± S.E.M. of 3 independent rats.Full blots of the P2YClick here for file6,11 receptor agonists. Effect of the intrathecal treatment with minocycline (green symbols) or fluorocitrate (purple symbols) in spinal nerve injured rats (panels A-D). Time course of the allodynic effect of PSB0474 and NF546 in rats previously treated with repeated intrathecal minocycline (panels A and B) or fluorocitrate (panels C and D), respectively. Data are expressed as mean ± S.E.M. for 6 animals. *Significantly (p < 0.05) different from the vehicle (Veh) group, as determined by two-way analysis of variance followed by the Student-Newman-Keuls test.Repeated intrathecal minocycline or fluorocitrate reduces tactile allodynia: Effects of P2YClick here for file


Activity of an alleged adenylylsulfate–ammonia adenylyltransferase is actually an inherent property of fragile histidine triad proteins (Fhits). We describe how our search for the transferase led to this serendipitous finding and provide basic kinetic parameters of ammonolytic reactions catalysed by human Fhit. Fhits (fragile histidine triad proteins) occur in eukaryotes but their function is largely unknown, although human Fhit is believed to act as a tumour suppressor. Fhits also exhibit dinucleoside triphosphatase, adenylylsulfatase and nucleoside phosphoramidase activities that in each case yield nucleoside 5′-monophosphate as a product. Due to the dinucleoside triphosphatase activity, Fhits may also be involved in mRNA decapping. In the present study, we demonstrate Fhit-catalysed ammonolysis of adenosine 5′-phosphosulfate, which results in the formation of adenosine 5′-phosphoramidate. This reaction has previously been associated with adenylylsulfate–ammonia adenylyltransferase (EC 2.7.7.51). Our finding shows that the capacity to catalyse ammonolysis is another inherent property of Fhits. Basic kinetic parameters and substrate specificity of this reaction catalysed by human Fhit are presented. Fragile histidine triad proteins (Fhits) attract interest of biologists and biochemists over the last two decades. . Mammalian Fhit functions both as tumour suppressor and genome ‘caretaker’  that cat2-pA), either hydrolytically to 5′-AMP (pA) and ammonia A and F-p[8-3H]A were purchased from MoravekBiochemicals. Standards of guanosine-, cytidine- and uridine-5′-phosphoramidates were kindly donated by Professor Adam Kraszewski and Dr Joanna Romanowska .CAPS [3-(cyclohexylamino)-1-propanesulfonic acid], NHa et al. . Adenosia et al. . Custom Buffer A: 20 mM potassium phosphate (pH 6.8) containing 5% glycerol and 1 mM 2-mercaptoethanol; buffer B: 50 mM potassium phosphate (pH 6.8) containing 5% glycerol and 1 mM 2-mercaptoethanol; buffer C: 50 mM Tris/HCl (pH 8.0) containing 5% glycerol and 1 mM 2-mercaptoethanol.4HCO3, 1 mM SO4-pA or 1 mM F-pA and the analysed fraction. Incubation was carried out at 30°C. After the appropriate time, depending on the purification stage, 3 μl aliquots of the mixtures were spotted on a TLC plate . The chromatogram was developed for 30–40 min in 2-propanol–25% ammonia–water [11:2:2 (v/v)] and nt spots visualized under shortwave UV light. In active fractions, the substrate was converted to NH2-pA. This qualitative approach was sufficient for making quick decisions about which fractions from one step of enzyme purification could be collected and used for a following step. Slightly modified assays are described in the legends to The mixture  used for monitoring adenylyl sulfate–ammonia adenylyltransferase activity during purification of the lupin enzyme contained 500 mM NHRates of ammonolysis of unlabelled substrates were analysed by TLC and densitometry of reaction product spots by the use of the G:Box system and the Gene Tools program. Within the range of 0.5 and 1.5 nmol of nt per spot, there was perfect linear dependence of attenuance compared with nt amount .Km for SO4-pA in the ammonolytic reaction was carried out in mixtures (25 μl) containing 200 mM CAPS/NaOH (pH 10.6), 185 mM NH3, SO4-pA in the range 0.3–1 mM and rate-limiting amounts of human Fhit (0.03 μg). At time intervals 0, 10, 20, 30 and 40 min, 3 μl of aliquots were spotted at the origin of a chromatographic plate, 2 nmol NH2-pA standard added and the plate developed for 30 min in 2-propanol–25% ammonia–water [11:2:2 (v/v)]. Nt spots were visualized under shortwave UV light. Initial rate for each substrate concentration was estimated by densitometry (see above).Km for F-pA in the ammonolytic reaction was carried out in mixtures (25 μl) containing 200 mM CAPS/NaOH (pH 10.6), 185 mM NH3 and labelled F-p[3H]A in the range 0.62–5 mM and rate-limiting amounts of human Fhit (0.03 μg). At time intervals 0, 20, 40, 60 and 120 min, 3 μl of aliquots were spotted at the origin of a chromatographic plate, 2 nmol NH2-pA added and the plate developed for 30 min in 2-propanol–25% ammonia–water [11:2:2 (v/v)]. Nt spots were visualized under shortwave UV light. Spots of labelled NH2-pA were excised, immersed in scintillation cocktail and radioactivity determined. Radioactivity in 3 μl of aliquots ranged between 60000 and 430000 cpm.Estimation of Km for NH3 in the ammonolysis of F-p[3H]A was carried out in mixtures containing 200 mM CAPS/NaOH (pH 10.6), 1 mM F-p[3H]A, NH3 in the range 5.7–153 mM and rate-limiting amounts of human Fhit (3 μg). To estimate initial rates for each concentration of NH3, 3 μl of aliquots were withdrawn at time intervals 0, 10, 20, 40 and 80 min, spotted at the origin of a chromatographic plate and 2 nmol NH2-pA added. The plate was developed for 30 min in 2-propanol–25% ammonia–water [11:2:2 (v/v)] and nt spots visualized under shortwave UV light. Spots containing labelled NH2-pA were excised, immersed in scintillation cocktail and radioactivity determined. Radioactivity in 3 μl of aliquots was 85000 cpm. In each case, the Km values were calculated from Eadie–Hofstee plots (v compared with v/[S]).Estimation of SDS/PAGE was conducted in 16% gels and native PAGE in 12% gels according to Laemmli . After nThis was performed at the proteomics facility of the Institute of Biochemistry and Biophysics, Polish Academy of Sciences in Warsaw.g) and the transferase activity was precipitated from the resulting supernatant with ammonium sulfate (50%–70% saturation). The precipitate was dissolved in buffer B and the resulting solution dialysed against this buffer. After centrifugation (15 min at 20000 g), the dialysate was applied to a DEAE-Sephacel column (5×15 cm) equilibrated with the same buffer. The column was washed with 1.5 l of buffer B followed by a linear gradient of 0–0.5 M KCl in the same buffer . Of the three active peaks , obtained from yellow lupin seeds, was extracted with 700 ml of buffer A. The slurry was centrifuged  dinucleoside triphosphatase.Protein from the gel shown in A. thaliana Fhits and T. brucei HIT-45 protein do catalyse ammonolysis of SO4-pA. Detailed studies on this reaction were carried out with the recombinant human Fhit.This serendipitous finding switched our study towards various Fhit proteins that have been known as dinucleoside triphosphatases (EC 3.6.1.29). We demonstrated that recombinant human and 4-pA, reaction 4, the ammonolysis of adenosine 5′-phosphorofluoridate (F-pA), reaction 5 and of diadenosine triphosphate (ApppA), reaction 6. It was also shown that these conversions were Fhit-dependent. In addition to increasing amounts of NH2-pA in the incubations containing SO4-pA or F-pA, we observed the slow accumulation of AMP (pA), which is a product of the Fhit-catalysed hydrolysis of NH2-pA, reaction 2 and F-pA, reaction 7 [As shown in 4-pNs) of the four canonical ribonucleosides , relative rates were: SO4-pC (91), SO4-pG (47) and SO4-pU (56). Kinetic parameters of the ammonolytic reactions 3 and 5 were as follows: Km for SO4-pA, 2.4±0.3 mM; Km for F-pA, 1.6±0.2 mM; and Km for NH3 estimated for the ammonolysis of F-pA, 60±15 mM. The hydrolytic reactions 2, 7 and 8 had pH optima between 6.5 and 7 [3 at a pH of around 10.6. (Compare the lanes involving SO4-pA in 4-pAp (known as PAPS), was not a substrate of the Fhit-catalysed ammonolysis.We also found that human Fhit could catalyse the ammonolysis of all the 5′-phosphosulfates .A possible mechanism for Fhit-catalysed ammonolysis of these substrates most probably involves attack by NHbstrates ,23. At h4-pA conversions and the biological role of Fhits. Speculating on this issue, we see at least the following ways by which the novel Fhit capacity could affect general metabolism: Fhits may compete for and control the cellular level of SO4-pA which in the reaction catalysed by adenylylsulfate kinase (EC 2.7.1.25) is converted to SO4-pAp, the main donor of sulfate moiety to polysaccharides or various low-molecular compounds. In plants, SO4-pA can be a substrate of the adenylylsulfate reductase (EC 1.8.4.9) that catalyses the glutathione-involving reaction leading to sulfite [2-pA in the revealed reaction 4 or hydrolyses it in reaction 2. Thus Fhits may be involved in distribution (channelling) of ammonia; the molecule that, in addition to conversions on the main metabolic pathways involving carbamoyl phosphate biosynthesis, has been recognized as a diffusible regulator of autophagy in human cells [2-pA should be considered as a novel signal molecule. Recently, this idea has gained support from experiments performed in our laboratory on Arabidopsis seedlings [2-pA added to the growth medium induced in these seedlings activity of several genes that encode enzymes catalysing key reactions of phenylpropanoid pathways and caused accumulation of lignins, anthocyanins and salicylic acid, the compounds that appear in the plant cells in response to stress. Future experiments will answer the question whether NH2-pA behaves as a signal molecule also in mammalian and human cells.In conclusion, our finding shows first of all that the activity of an alleged adenylylsulfate–ammonia adenylyltransferase is actually an inherent property of Fhits and secondly, adds a new facet to the catalytic promiscuity of an enzyme that so far has been shown to catalyse reactions assigned as dinucleoside triphosphate hydrolase (EC 3.6.1.29), reaction 1, adenylylsulfate sulfohydrolase (EC 3.6.2.1), reaction 8 and nucleoside phosphoramidate hydrolase (EC 3.9.1.-), reaction 2. . Very recently, Fhits have been demonstrated to catalyse also fluorolysis of nucleoside 5′-phosphoramidates and nucleoside 5′-phosphosulfates, reactions 9 and 10 . Whether sulfite . Fhit maEC 2.7.1. is conveeedlings . Micromo


The accumulation pattern of 90Sr was examined for L. minor root and fronds separately over a seven-day time period and was subsequently used in a dynamic dosimetric model to calculate β-radiation dose rates. Exposing L. minor plants for seven days to a 90Sr activity concentration of 25 up to 25,000 kBq·L−1 resulted in a dose rate between 0.084 ± 0.004 and 97 ± 8 mGy·h−1. After seven days of exposure, root fresh weight showed a dose dependent decrease starting from a dose rate of 9.4 ± 0.5 mGy·h−1. Based on these data, an EDR10 value of 1.5 ± 0.4 mGy·h−1 was estimated for root fresh weight and 52 ± 17 mGy·h−1 for frond fresh weight. Different antioxidative enzymes and metabolites were further examined to analyze if β-radiation induces oxidative stress in L. minor.In the following study, dose dependent effects on growth and oxidative stress induced by β-radiation were examined to gain better insights in the mode of action of β-radiation induced stress in plant species. Radiostrontium ( It is produced with high yields as a byproduct of the fission of uranium and plutonium and is thus a common waste product from nuclear activities [90Sr in the environment [90Sr was accidently released during nuclear meltdown in Fukushima in 2011 [90Sr decays, it releases moderate energy β-particles and forms yttrium-90, which in turn emits very strong energetic β-particles within 64 h to stable zirconium. Since most of the energy from such β-particles is absorbed by surface tissues, the bioaccumulation of these radionuclides can potentially produce deleterious effects to the health of living organisms [Anthropogenic activities have led to enhanced releases of fission products from nuclear facilities into the environment. Many of these fission products are radionuclides that are short-lived γ- and β-emitting isotopes, which rapidly lose their radioactivity. However, some radionuclides have a physical half-life that takes several years and therefore can contribute to a long-lasting contamination of the biosphere . Among tironment . More re in 2011  and in s in 2011 . Geochem in 2011 ,6. As 9090Sr accumulation in edible plants, in particular crops [i.e., to decontaminate soils contaminated with low levels of 90Sr [90Sr have, to date, not been studied in detail in plants [90Sr exposure levels [Radioecological research on β-emitting isotopes in plants has mainly focused on ar crops ,8,9,10 o of 90Sr ,12,13,14 of 90Sr ,16,17. Hn plants . Only one levels . Relative levels . A transe levels . High lee levels ,23. These levels . Other ae levels ,26. HoweL. minor [90Sr accumulates, it can subsequently deliver a considerable β-radiation dose to the plant organs.Although Sr is not essential for plant metabolism, it bears a chemical analogy with the essential plant macronutrient Ca. In terrestrial plants, the uptake of Ca occurs mainly via Ca channels in the roots . CalciumL. minor ,32. OverLemna minor was selected as a biological model to study irradiation effects following β-radiation exposure. This free-floating vascular macrophyte develops roots and has the potential to take up nutrients through both fronds and roots. L. minor is to date the only freshwater plant model for testing metal biomonitoring and other toxic chemicals in freshwater ecosystems [L. minor to β-radiation by using 90Sr as β-emitting isotope. First, the uptake pattern of 90Sr was characterized over a seven-day time period to construct a dynamic dosimetric model to determine internal and external exposures rates. Afterwards, L. minor plants were exposed to different 90Sr concentrations in order to study possible dose dependent relationships. The classical growth related endpoints were evaluated and compared with antioxidative defense system endpoints. Our findings contribute to a better insight into the antioxidant defense system in plants following β-radiation. osystems ,34. ThisL. minor plants were continuously exposed for seven days to four different activity concentrations from 25 up to 25,000 kBq·L−1 to study dose dependent relationships of β-radiation. In order to constitute a maximal 90Sr uptake and accumulation, L. minor plants were exposed in a medium containing 0.2 mM Ca. The 90Sr concentration ratios (CR) to frond and root expressed on fresh and dry weigh basis are shown in DW of the highest tested activity concentration was slightly lower compared with other tested activity concentrations, no significant differences in CRDW were observed between tested Sr activity concentrations from non-exposed plants . It was found that the 90Sr content was similar in both plant organs (fronds and roots) indicated by equivalent CRDW for each tested activity concentration. In contrast, the CRs expressed on plant fresh weight (CRFW) and used in dosimetry calculations, were remarkably lower in the roots compared to the fronds. The CRFW in this study were approximately 60 and 30 Bq·kg−1 FW/Bq·L−1 for fronds and roots respectively. As for CRDW, no concentration-dependent differences in CRs were observed for the tested activity concentrations. 90Sr uptake in L. minor roots and fronds was monitored over a seven-day period in order to enable more correct dose assessment estimations. With the tested 90Sr activity concentration used in this experiment, the activity of 90Sr found in roots increased rapidly during the first 8 h, then much slower during the next 72 h before it tended to stabilize (90Sr uptake capacity at equilibrium phase (at which the net uptake is zero) of the L. minor fronds and 97% ± 4% of the roots was accumulated after 72 h of exposure. These time-dependent uptake parameters and the respective CRFW were considered to calculate accurate dose rates for L. minor organs (fronds and roots) exposed over a seven-day exposure. Calculating internal and external dose rates for roots and fronds separately, the exposure of L. minor plants to 90Sr activity concentration from 25 up to 25,000 kBq·L−1 resulted in a total dose rate ranging from 0.084 ± 0.004 and 97 ± 8 mGy·h−1 for the L. minor plants . The growth endpoints measured on root fresh and dry weight were significantly affected at 9.4 ± 0.5 mGy·h−1. At the highest tested dose rate, root fresh and dry weight was inhibited for 63% ± 1.5% and 57% ± 0.4%, respectively  for frond area was 95 ± 7, and 154 ± 15 and 152 ± 13 mGy·h−1 for fresh weight and frond number, respectively. The EDR10-values for root biomass were 1.5 ± 0.4 and 1.0 ± 0.5 mGy·h−1 for root fresh and dry weight, respectively. Growing L. minor plants in stable Sr concentrations up to 60 nM, the equivalent nominal concentration at 90Sr activity concentration of 25,000 kBq·L−1, did not result in a significant difference in growth rate (data not shown). As such, observed growth effects are expected to be linked to 90Sr induced radiotoxicity and not Sr chemotoxicity.Frond number, frond area and frond and root fresh and dry weight were measured for ectively . The derL. minor plants, antioxidative enzyme activities including CAT, GR, SOD, APX, GPOD and SPOD were analyzed  each having tissue-specific dose conversion coefficients. The calculated total dose ranged between 0.084 ± 0.004 mGy·h−1 up to 97 ± 8 mGy·h−1, of which the lowest dose rate was of the same order of magnitude as the IAEA threshold dose rate. After seven days of exposure, the second highest dose rate tested (9.4 ± 0.5 mGy·h−1) had a negative impact on root growth. According to the EDR values, the root related endpoints were about twenty times more sensitive compared to frond related endpoints. Highly sensitive stress responses on L. minor root growth were also observed in metal and pesticide toxicity. Following this, L. minor root growth/length has been suggested as an alternative bioassay for toxicity testing [−1). From the available literature dealing with effect studies [90Sr or β-radiation induced effects in aquatic plants, emphasizing a large data gap for aquatic plants [−1 with 90Sr revealed a 36% reduction in life span, while another study with daphnids using 90Sr, showed a decrease of 26% in fertility rate after exposure to 0.8 mGy·h−1 [L. minor root growth was affected. Since β-particles are proposed to have an RBE factor of 1 (or 3 for low-energy β-radiation), it is generally accepted that radiological effects caused by γ-radiation can also be adopted for making radiation comparisons. From a 14-year field experiment in the Canadian Boreal forest, it has been found that the most sensitive of plant species studied, pine trees, died within 2–3 years when exposed to a dose rate of 5–10 mGy·h−1 and within two weeks at an estimated dose rate of 250–300 mGy·h−1 for pine trees close to the damaged reactor in Chernobyl [50 values ranged between 250 and 500 mGy·h−1 for L. minor fronds, suggesting that L. minor is more radioresistant than pine trees. Garnier-Laplace, et al. [10 values on population related endpoints. Considering the most sensitive EDR10 endpoint obtained in this study (1.0 ± 0.5 mGy·h−1 for root dry weight), approximately 50% of the selected organisms from the SSD curve were more radioresistant in comparison to L. minor.To obtain accurate dose rate estimates,  testing ,43,44. A studies ,46, no rc plants ,45. Howe mGy·h−1 . These dhernobyl ,49. In tArabidopsis plants [L. minor plants following β-radiation exposure, some features of the ROS scavenging biosynthetic pathways involving both enzymatic and non-enzymatic processes were studied. There were minor changes in total ASC content or redox status, although an enhanced activity of APX was found. Increased APX activity with a stable ASC content was also found in other duckweed studies under chemical stress [2O2 from the cytosol and chloroplasts [Oxidative stress related genes are among the most represented genes in responding to radiation in s plants ,52. Therl stress . Under sroplasts . Similar−1. An increased GSH content was also found in other plants tissues suffering from oxidative stress to compensate ascorbate oxidation [−1 corroborate the hypothesis that L. minor plants have difficulty in controlling the production of ROS under high radiation stress. In addition to APX, also CAT increased at levels below the EDR10 value. Increased CAT activity is supposed to be an adaptive trait helping to reduce toxic levels of H2O2 in peroxisomes [S. capillata exposed to 65 µGy·h−1 with γ-radiation. L. minor plants exposed to radiofrequency radiation and in A. thaliana where increased expression levels of CAT1 and CAT3 genes were observed at 50 mGy·h−1 [Stipa capillata (65 µGy·h−1) did not show any response [−1 for seven days. However, since growth was clearly hampered at the highest dose rate, available energy supplies might be used for other metabolic pathways to neutralize β-radiation stress in L. minor plants.The concentration of the other antioxidant studied, GSH, significantly increased at the highest tested dose rate of 97 mGy·hxidation ,56,57. Txidation . Also inxidation . Howeveroxisomes . Sensiti mGy·h−1 ,59,60. T mGy·h−1 ,54. For response ,60. AlsoLemna minor cv. Blarney plants were obtained from Dr. M. Jansen  and cultured aseptically in 250 mL glass Erlenmeyer flasks containing half-strength Hütner medium [−2·s−1) at 24 °C. Plants were sub-cultured every 10–12 days by transferring three plants to 100 mL of fresh growth medium. To obtain sufficient and homogenous plant population, 1 week before the experiment a preculture was initiated with five mature plants per pot (3–4 fronds) in 100 mL of fresh medium.r medium  under co90Sr treatment, 3 healthy duckweed plants were chosen randomly and transferred to 250 mL PC containers (VWR) containing 25 mL experiment nutrient solution. For the 90Sr uptake experiments, modified K-medium (0.4 mM Ca and (in mg·L−1) 889 KNO3, 95 Ca(NO3)2·4H2O, 500 MgSO4·7H2O, 9 Na-EDTA, 3 tartaric acid, 1.86 H3BO3, 0.22 ZnSO4·7H2O, 0.12 Na2MoO4·2H2O, 0.08 CuSO4·5H2O, 3.62 MnCl2·4H2O, 5.4 FeCl3·6H2O with lower KH2PO4 concentrations (0.5 mg·L−1) [−190Sr, added as SrCl2  for 6 biological replicates. For the 90Sr effect experiments, a modified Steinberg medium (0.2 mM Ca and (in mg·L−1) 350 KNO3, 50 Ca(NO3)2·4H2O, 0.09 KH2PO4, 100 MgSO4·7H2O, 1.5 Na-EDTA, 0.12 H3BO3, 0.18 ZnSO4·7H2O, 0.044 Na2MoO4·2H2O, 0.18 MnCl2·4H2O, 0.76 FeCl3·6H2O [−190Sr for 9 biological replicates. Since L. minor plants exposed in Steinberg medium had the highest Sr90 uptake, and hence highest dose rate, this medium was selected for all effect experiments. The growth of the plants did not differ between both exposure media. The chemical concentration of Sr at the highest tested activity level amounted to 54 nM. Plants were exposed in a growth chamber in sterile environment under continuous light (80 to 100 µmol/s m2) at 24 °C during 90Sr treatment. Plants were harvested after 7 days of exposure by washing in 1 mM Pb(NO3)2 and twice in distilled water for 10 min in order to discriminate 90Sr accumulation from 90Sr adsorption on root and frond surface. A significant difference was found between plants washed with Pb-solution compared to non-washed or washed with distilled water . On the other hand, it was confirmed that there was no significant difference in washing the L. minor plants with a solution containing an excess of Pb (1 mM) with plants washed in a high Ca-solution (10 mM. data not shown). Fresh weight was determined by weighing all collected plant material from all individual pots after quickly dry patting with clean tissue. For the 90Sr effect experiments, plants from 3 pots were further dried for 2 days at 60 °C (humidity < 5%) for dry weight determination and Sr uptake measurement. Plants from the remaining 6 pots were, after fresh weight determination, snap frozen in liquid nitrogen for storage at −80 °C for biochemical analysis.For the  mg·L−1) ) was use90Sr uptake experiments, all 6 individual pots were used. Digested samples were then diluted 10-fold in dH2O, vortexed and diluted again 4-fold in scintillation cocktail . Samples (5 mL) were taken from each pot at the end of the experiment and brought to 20 mL with the same scintillation cocktail. After careful mixing of sample and scintillation fluid, the 90Sr activity was measured for 60 min by β-liquid scintillation counting . The counting efficiency was determined using a dilution series of 90Sr with known activities. No difference was found between concentrations in samples from growth medium before and after the experimental period indicating that 90Sr was dissolved during the exposure period (data not shown). Concentration Ratios (CR) are expressed as the ratio of 90Sr activity in the plant organ (root and frond) (in Bq·kg−1) and the external solution 90Sr content (expressed in Bq·L−1). Concentration ratios were calculated on a organ fresh weight (CRFW) and organ dry weight (CRDW) basis. CRs were calculated for roots and fronds.All collected plant material from 3 individual pots was dry-ashed in a muffle furnace, and subsequently digested in 0.1 M HCl. For L. minor plants were split in two functional units for dose calculations. The internal and external dose conversion coefficients  were calculated by the ERICA tool for root and shoot separately [L. minor plant had the following dimensions: 0.25 mm thick, 3 mm wide and 3.5 mm deep for frond and the average root length determined at 90 mm long with a diameter of 0.15 mm. Within the Tier 2 assessment, DCCs can be calculated for any organism by entering the required radionuclide and the organism’s dimensions (the mass and the three axes that define the ellipsoid used as its simplified representation). The geometries for both organs were separately entered in the software and the DCCs obtained were in agreement with the DCCs obtained from Biermans et al. [−5 and 8.82 × 10−5 µGy·h−1/Bq·kg−1·FW for frond and root, respectively, and external DCCs were 5.58 × 10−4 and 5.63 × 10−4 µGy·h−1/Bq·kg−1·FW.Dosimetry of radionuclides in species with small sizes requires special attention for accurate dose rate calculations since the range of high-energy β-radiation in biological matter exceeds the geometrical size of species. Therefore, parately . The estparately . An avers et al. . The obt90Sr is taken up yet by the plants, a time dependent dose rate approach was designed with the following assumptions: (i) The uptake ratio of 90Sr for both frond and root were given by their CRFW, which reached equilibrium phase at harvest time; (ii) The rate of uptake for all tested activity concentrations followed the same equation as obtained by the time course illustrated by −1) is given by Equations (1) and (2), respectively:totalD is the total dose (µGy), mAC is the activity concentration measured in medium (Bq·L−1), int organDCC and ext organDCC are the internal and external dose conversion coefficients, respectively, for root or frond, unweighted for radiation quality (µGy·h−1/Bq·kg−1·FW), max organCR is the organ specific CRFW at equilibrium phase (Bq·kg−1·FW/Bq·L−1). β is the slope of the fit from the 7-day uptake experiment given by t is the time of the exposure experiment which is set at 7 days. The ext frondDCC was divided by two since L. minor floats on water surface and therefore, only half of the fronds are surrounded by contaminated liquid medium. The total dose rate of the whole plant was calculated as the cumulative dose rate from frond and root separately, including organ-sized correction factors. The correction factors were the average fresh weight ratios for fronds and roots for each dose rate condition; (iii) Since L. minor exhibits an exponential growth with an average reproduction rate of 0.45 ± 0.02 fronds per day [L. minor plants during this seven days treatment, meaning that not all plants were equally exposed with respect to exposure time. However, since the offspring of contaminated organisms are also subjected to equal dose rates, this dynamic approach considers the total population of L. minor as one organism with a treatment time of seven days, as in accordance with the OECD guidelines for the evaluation of chemical toxicants; (iv) 90Sr radionuclides were distributed homogeneously in both organs. However, since the morphologic dimensions of fronds and roots of L. minor fall within the range of the path length of a β-particle, this effect can be considered as negligible in this dosimetric approach.Since at the start of the experiment, no  per day ,65, the 90Sr treatment condition and control.Growth parameters, in terms of relative frond number, relative frond area and fresh weight were analyzed according to OECD guidelines . Images g at 4 °C for 10 min. The supernatant was kept on ice and used freshly for determination of enzyme capacities. All measurements were performed at room temperature (20–22 °C) based on spectrophotometric assays, as previously described [2O2 solution. The absorbance at 240 nm was monitored kinetically. To determine the GPX capacity, 150 μL 0.1 M phosphate buffer, 10 μL sample extract and 40 μL guajacol mastermix, consisting of 90 mM guaiacol and 163 mM H2O2 mixed on a 1:1 ratio were added in each well of a plastic 96-well plate. Subsequently, the appearance of tetraguajacol was monitored kinetically at 436 nm. SPX capacity was assessed in 96-well UV-plates. In each well, 155 μL 0.1 TRIS (pH 7.5), 20 μL 98 mM H2O2, 20 μL plant extract and 5 μL syringaldazine (SAZ) were added. The appearance of oxidized SAZ was monitored kinetically at 530 nm. For the determination of the GR capacity, 165 μL TRIS-EDTA buffer  (pH 8), 7 μL GR mastermix (1:1 mix of 82 mM GSSG and 6 mM NADPH) and 28 μL were added in each well of a 96-well UV-plate. The decrease of NADPH, used for the reduction of GSSG, was followed kinetically at 340 nm. The APX capacity was determined in UV-cuvettes using the Ultrospec 2000 UV/VIS Spectrophotometer . In each cuvette, 665 μL HEPES-EDTA  buffer, 100 μL 30 mM Na-ascorbate, 35 μL 196 mM H2O2 and 200 μL sample extract were added. Subsequently, the appearance of dehydroascorbate (DHA) was measured spectrophotometrically at 298 nm. The activity of SOD was measured on the Ultrospec 2000 UV/VIS Spectrophotometer (Pharmacia Biotech) in plastic cuvettes. In each cuvette, 580 μL KH2PO4 buffer (pH 7.8), 100 μL 1 mM EDTA, 100 μL 0.5 mM xanthine, 100 μL 0.1 mM cytochrome C, 100 μL extract and 20 μL xanthine oxidase was added. The extent of cytochrome C reduction (measured at 550 nm) was compared to this in a blank, not containing any sample extract. Then, the relative inhibition of cytochrome C reduction by the sample extract was calculated as a measure of SOD capacity. At least 3 biological replicas were analyzed in triplicate . Statistical analysis was performed on the measured data before expressing them relative to controls.Frozen plant tissue (50–80 mg·FW) was homogenized under frozen conditions with liquid nitrogen using two tungsten carbide beads  of 3-mm diameter in a Retsch Mixer Mill MM400 at 30 Hz for 3 min after a spatula of insoluble polyvinylpyrrolidone (PVP) was added. Hereafter, 400 µL ice-cold 0.1 M Tris-HCl buffer (pH 7.8) containing 1 mM EDTA and 1 mM dithiothreitol was added to the frozen homogenized tissue, the mixture was vortexed and subsequently centrifuged at 20,000× escribed . Briefly2PO4 (pH 5.6) was added to 300 μL of the supernatant. Subsequently, the pH of all samples was adjusted to 4.5 using 200 mM NaOH. For the measurement of total AsA, samples were incubated with 25 μM DTT and 120 mM NaH2PO4 (pH 7.5) for 15 min at 20 °C to fully reduce the AsA pool. Subsequently, the pH of the samples was adjusted to pH 5.5, the optimal pH for ascorbate oxidase, using 200 mM HCl. Further, all measurements were performed as described by Queval and Noctor [Oxidized and reduced forms of ascorbate and glutathione were measured spectrophotometrically using a plate-reader assay, as described by . Frozen d Noctor . Oxidized Noctor . At leasp-value < 0.05) were found, a Tukey post hoc test was applied to further discriminate between significantly different groups. Student’s unpaired two-tailed t-test was used for single comparisons. The dose response curves were modeled using the three parameter log-logistic drm equation from the drc package available in the software package R  [50 parameters were −0.99 and 238 for frond area, −1.02 and 391 for frond number, −0.96 and 509 for frond fresh weight, −0.92 and 732 for frond fresh weight, −0.67 and 39 for root fresh weight and −0.5 and 51 for root dry weight in the equation model.All data have been presented as mean values ± standard error (SE). Statistical analysis was performed with the open-source software package R . Normal distribution was tested with a Shapiro–Wilk test, Barlett’s test was used to test for homoscedasticity. To identify any statistical differences between treatments, a one-way ANOVA was performed. When significant differences ( < 0.05) . MaximalL. minor plants. Submerged roots of L. minor plants were more radiosensitive to β-radiation compared to the floating fronds. All considered growth related endpoints showed a negative growth at the highest tested dose rate, although only a mild stimulation of the antioxidative defense system was observed. L. minor plants relied mainly on catalase to counteract the ROS generation following β-radiation, although some compounds of the ASC-GSH cycle were also activated, as they possessed higher levels of GSH at the highest tested dose rate level. The regulation of these antioxidative enzymes at the transcriptional level might be crucial in further examinations to support the observed biochemical changes.In conclusion, our data demonstrated for the first time that β-radiation provokes a negative growth in


We expected that the high specific activity of [18F]flutemetamol would make it an attractive small animal Aβ imaging agent.The purpose of the study was to evaluate the applicability of 18F]flutemetamol uptake in the mouse brain was evaluated in vivo at 9 to 22 months of age with an Inveon Multimodality PET/CT camera . Retention in the frontal cortex (FC) was evaluated by Logan distribution volume ratios (DVR) and FC/cerebellum (CB) ratios during the late washout phase (50 to 60 min). [18F]flutemetamol binding to Aβ was also evaluated in brain slices by in vitro and ex vivo autoradiography. The amount of Aβ in the brain slices was determined with Thioflavin S and anti-Aβ1−40 immunohistochemistry.[18F]flutemetamol retention in the FC increased from 9 to 18 months. In younger mice, DVR and FC/CB50-60 were 0.88 (0.81) and 0.88 (0.89) at 9 months (N = 2), and 0.98 (0.93) at 12 months (N = 1), respectively. In older mice, DVR and FC/CB50-60 were 1.16 (1.15) at 15 months (N = 1), 1.13 (1.16) and 1.35 (1.35) at 18 months (N = 2), and 1.05 (1.31) at 21 months (N = 1). In Tg2576 mice, DVR and FC/CB50-60 showed modest increasing trends but also high variability. In APPswe-PS1dE9 mice, DVR and FC/CB50-60 did not increase with age. Thioflavin S and anti-Aβ1−40 positive Aβ deposits were present in all transgenic mice at 19 to 22 months, and they co-localized with [18F]flutemetamol binding in the brain slices examined with in vitro and ex vivo autoradiography.In APP23 mice, [18F]flutemetamol retention in the brain was detected in old APP23 mice in vivo. However, the high specific activity of [18F]flutemetamol did not provide a notable advantage in Tg2576 and APPswe-PS1dE9 mice compared to the previously evaluated structural analogue [11C]PIB. For its practical benefits, [18F]flutemetamol imaging with a suitable mouse model like APP23 is an attractive alternative.Increased [ Sev. Sev11C]l models -8]. Due. Due11C] imaging -[11].18F]flutemetamol (2-(3-[18F]fluoro-4-(methylamino)phenyl)-1,3-benzothiazol-6-ol, [18F]3′F-PiB, [18F]GE067) is a 18F-labelled analogue of [11C]PIB developed and marketed by GE Healthcare . [18F]flutemetamol PET has been shown to successfully differentiate between patients with AD and healthy control subjects and to perform similarly to [11C]PIB in this respect [18F]flutemetamol has shown high test-retest reliability and high specificity  203137, GE Healthcare).. [1. [118F]fty  and senty  in the ty  mouse models of AD. We expected that the very high specific activity of [18F]flutemetamol (>1 TBq/μmol) and thus lower injected mass to transgenic animals would be beneficial in small animal Aβ imaging. We also expected physical characteristics and practical benefits of the 18F-radionuclide to make [18F]flutemetamol an attractive preclinical Aβ imaging agent for preclinical studies.The present demonstration of principle study evaluated the applicability of [18F]flutemetamol was produced with a FASTLab® Synthesizer , following analogous procedures to those described in the patent, WO 2007/020400 A1 ‘Fluorination process of anilide derivatives and benzothiazole fluorinate derivatives as in vivo imaging agents’. The specific radioactivity of [18F]flutemetamol was >1 TBq/μmol at synthesis completion (N = 34). Radiochemical purity exceeded 92%.flutemetamol binding was detected using digital autoradiography (25 μm resolution).escribed  with 20-N = 11) of [18F]flutemetamol. After the tracer had distributed for 30 min, the mice were sacrificed by cardiac puncture. The brain was immediately dissected, rapidly frozen in chilled isopentane, and cut to 20-μm cryosections at the levels of the FC and CB. Sections were subsequently exposed to an imaging plate for approximately 4 h. Plates were read with a Fuji BAS-5000 analyser (resolution 25 μm). Regions of interest (ROI) were drawn onto the FC and CB with the Aida Image Analyzer analysis program . ROIs were drawn onto at least ten sections per animal, and mean intensity ratios were calculated between the FC and the CB. In addition, intensity profiles were drawn across the cortex that contained Aβ deposits, and Aβ plaque-to-background ratios were calculated for TG mice.Mice were anaesthetized with isoflurane and intravenously injected with 9.1 ± 1.7 MBq  using ex vivo tissue counting to better understand the observed binding in vivo. Mice anaesthetized with isoflurane were injected with 5.1 ± 0.9 MBq of [18F]flutemetamol, and the tracer was allowed to distribute for 60 min. Mice were sacrificed by cardiac puncture, and the brain was rapidly removed. We dissected the forebrain, midbrain, hindbrain, olfactory bulb, medulla, eyes, Harderian glands and salivary glands, and we collected samples of FC, CB, medulla and cranial bone. 18F-radioactivity was measured with a gamma counter . Measurements were decay-corrected, the background was subtracted and results were expressed as the percentage of injected dose per gram of tissue (% ID/g).We further investigated flutemetamol retention compared to young TG animals and WT control animals was observed in the FC of aged APP23 mice in visual assessment of the PET images . The FC/CB ratios clearly increased over time in old APP23 mice. On the contrary, in Tg2576 and APPswe-PS1dE9 mice, the ratio was less than 1 in the later time frames.Increased 8FflutemeN = 2) and 0.98 . In older TG mice, DVRs were 1.16 , 1.13 and 1.35 , and 1.05 . DVRs for WT animals were 0.83  and 0.98 . The DVRs of the Tg2576 mice showed an increasing trend, reaching 1.24 and 1.11 (N = 2) at 22 months . In contrast, the APPswe-PS1dE9 mice tended to have lower DVRs  than WT mice  at 19 months.Logan DVRs calculated for the FCs of all TG and WT animals are presented in Figure 50-60 ratios calculated for the FCs of all TG and WT animals are presented in Figure 50-60 ratios increased with age in APP23 mice. The FC/CB50-60 values were 0.81 and 0.89  and 0.93  in younger TG mice, increasing to 1.15 , 1.16 and 1.35  and 1.31  in older TG mice. In strain-matched WT mice, FC/CB50-60 values were 0.86  and 0.99 . In Tg2576 mice, FC/CB50-60 values were 0.80 ± 0.07 (N = 4) at 12 months  and 0.89 and 0.99 (N = 2) at 22 months . In APPswe-PS1dE9 mice, FC/CB50-60 remained almost stable from 0.87 ± 0.08 (N = 3) at 12 months  to 0.95 and 0.90 (N = 2) at 19 months . The FC/CB50-60 ratios agreed well with the Logan DVR values in APP23 mice , but similar agreement was not seen in Tg2576  or APPswe-PS1dE9 mice  . This is even at its worst ten times less than previously reported with [11C]PIB  at 19 months, but plaque composition appeared to be different from the other TG models, with only small fibrillar deposits that could bind ThS and related Aβ imaging agents. In Tg2576 mice, modest increases in DVR and FC/CB50-60 ratio were seen as the individual transgenic mice aged. In a previous study with [11C]PIB and Tg2576 mice, no such trend was seen [18F]flutemetamol did bind to Aβ in the cortical sections from Tg2576 animals, both ex vivo and in vitro, consistent with previous ex vivo results [in vivo binding ratios. We concluded that Tg2576 and APPswe-PS1dE9 mouse models are not suited to small animal imaging studies with tracers that shared binding sites with [11C]PIB and [18F]flutemetamol, due to their plaque structure or low plaque load.Varying results have been found in several small animal imaging studies with FDDNP in 13 to 15-month-old Tg2576 mice [18F]FDDNP binding was insufficient and that insufficient spatial resolution and partial volume effects (PVE) limited the precision of measurements in small VOIs [18F]florbetaben, Aβ deposition was longitudinally followed in the APPswe mouse model from age 13 to 20 months, and the results were consistent with our results with [18F]flutemetamol in APP23 mice. In that study, the PET results agreed well with histopathological brain Aβ, and differentiation was further improved when a PVE correction was applied [18F]florbetapir was shown to increase from age 3 to 8 months; however, no further increase was detected at 12 months compared to the baseline scan at 3 months [18F]florbetaben . This difference further supports the higher importance of the chosen mouse model over high specific activity of the tracer for successful imaging results.Our study is difficult to compare with previous small animal amyloid imaging PET studies that used other 576 mice . The autall VOIs . However applied ,28]. Us. Us18F-l3 months . At the Bq/μmol, ) and florbetaben. Only TG mice that were 16 and 20 months old showed positive BPNDs  AZD4694 AZD4694 ,30]. Mo. Mo18F-l18F]florbetaben and [18F]florbetapir studies, the FC/CB ratios observed in our study with [18F]flutemetamol with ex vivo autoradiography  were higher than that observed with in vivo PET (1.3 at 30 to 35 min p.i.) for old APP23 mice. Adding PVE correction could further improve quantitation of [18F]flutemetamol PET data, especially in animals with still only modest plaque load. Also, spillover from tissues close to the brain could introduce errors to the VOI-based analysis method in mice. We observed high radioactivity in close proximity to the brain in vivo, and subsequent ex vivo tissue counting experiments confirmed that significant radioactivity was located outside of the brain, inside the nasal cavity. In addition, 18F-radioactivity concentrations detected in the mouse cranial bone (0.83 ± 0.13% ID/g at 30 min) were higher than those previously reported in rats (0.04 ± 0.01% ID/g at 30 min [18F]flutemetamol defluorination could be taking place in mice than in rats, reflecting more active metabolism. However, spillover from these structures would presumably lead to similar overestimation in the brains of all three TG mouse models.Imaging of small structures in the mouse brain, of a size close to the spatial resolution of the PET scanner, will inevitably be affected by PVEs; thus, measurable signals will be less precise than those achievable with digital autoradiography. Consistent with previous [18F]flutemetamol PET to small animal imaging in three different transgenic models even with limited statistical power, rather than in only one transgenic model with higher sample size and better statistical power. This demonstration of principle type of study was still able to show clear increasing trend in [18F]flutemetamol binding in aging APP23 mice and the lack of binding in Tg2576 and APPswe-PS1dE9 mice. Another limitation was that we could not perform ex vivo studies for all of the female mice imaged in vivo; however, the brain sections from these mice were used for in vitro binding experiments, and additional ex vivo studies were performed later, with aged males. Male APP23 mice were reported to show slightly slower deposition of Aβ plaques compared to females flutemetamol, similarly to its structural analogue [11C]PIB, is more dependent on which AD mouse model is used than its high specific activity. In this study, Aβ deposition in the brain could only be followed in vivo in APP23 mice. However, for its practical benefits, such as more efficient use of the produced tracer batches, [18F] flutemetamol imaging with a suitable mouse model like APP23 is an attractive alternative.AD: Alzheimer's diseaseAβ: beta-amyloidFC: frontal cortexDVR: distribution volume ratioCB: cerebellum11C]PIB: [11C]Pittsburgh compound B[PET: positron emission tomographyTG: transgenicWT: wild-typeAPP: amyloid precursor proteinCT: computed tomographyVOI: volume of interestROI: region of interestThS: Thioflavin SThe authors declare that they have no competing interests.in vitro PET experiments. FL participated in the immunohistochemical stainings and performance of PET experiments. OE performed the radiochemical synthesis of [18F]flutemetamol. MS and GF participated in the design of the animal studies and revised the manuscript critically. MS drafted and revised the manuscript critically for its intellectual content. OS, JR and MHS contributed to the conception, design and coordination of the study, revised the manuscript critically for its intellectual content and gave final approval for the published version. MHS helped to draft the manuscript. All authors read and approved the final manuscript.AS contributed to the design of the study, acquired, analysed and interpreted the PET data and histological and immunohistochemical stainings. AS drafted the manuscript. JR participated in the design of the study and carried out


Saccharomyces cerevisiae, ~3% of the lysine transfer RNA acceptor 1 (tRK1) pool is imported into mitochondria while the second isoacceptor, tRK2, fully remains in the cytosol. The mitochondrial function of tRK1 is suggested to boost mitochondrial translation under stress conditions. Strikingly, yeast tRK1 can also be imported into human mitochondria in vivo, and can thus be potentially used as a vector to address RNAs with therapeutic anti-replicative capacity into mitochondria of sick cells. Better understanding of the targeting mechanism in yeast and human is thus critical. Mitochondrial import of tRK1 in yeast proceeds first through a drastic conformational rearrangement of tRK1 induced by enolase 2, which carries this freight to the mitochondrial pre-lysyl-tRNA synthetase (preMSK). The latter may cross the mitochondrial membranes to reach the matrix where imported tRK1 could be used by the mitochondrial translation apparatus. This work focuses on the characterization of the complex that tRK1 forms with human enolases and their role on the interaction between tRK1 and human pre-lysyl-tRNA synthetase (preKARS2).In yeast  Mitochondria are the centres of critical cellular processes, such as oxidative phosphorylation, apoptosis, fatty acids, amino acids and Fe–S cluster metabolisms. Despite this central role, mitochondria present genomes only encoding a fistful of proteins and RNAs dedicated to oxidative phosphorylation or mitochondrial translation. The regulation of mitochondrial activities thus relies on nuclear-encoded factors, which need to be imported in a concerted way in order to tune mitochondrial activity with cytosolic conditions ,2. The pSaccharomyces cerevisiae, tRNALys acceptor 1 (tRK1) is imported despite the presence of a mitochondrial encoded tRNALys (tRK3) . To. To19]. eviously . Upon adpacities . A mock-dK) of 2.0 ± 0.5 µM (data not shown). Therefore, human enolase isozymes are capable of interacting with yeast tRNALys with the same affinity as yeast enolase Eno2p [dK decreased from 300 nM to less than 20 nM).The interaction of purified human enolase isoforms with labeled tRK1 was tested by electrophoretic mobility shift assay (EMSA). We found that all three isoforms of human enolase can form a complex with labelled tRK1 A, with ase Eno2p . When prdK decrease (from 300 to 12–25 nM)  and fixed concentration of proteins (0.5 µM of preKARS2 and 1 µM of enolase) were used. The results demonstrate that in the absence of enolase, preKARS2 binds tRK1 with a dissociation constant of 300 nM A,B. Addi2–25 nM) B. These In vitro assays of tRK1 import in yeast mitochondria have enabled identification of a set of proteins involved in the process. In Saccharomyces cerevisiae, enolase 2 (Eno2p) hijacks ~3% of the tRK1 cytosolic pool, which undertakes conformational changes resulting in the formation of a deeply remodeled secondary structure (F-form [ (F-form ), which  -RIL cells . The transformed cells were grown in LB broth to a cell density A600 = 0.6, the protein expression was induced by 0.5 mM Isopropyl β-d-1-thiogalactopyranoside. After incubation for 3 h at 30 °C, cells were harvested, treated with 1 mg/mL of lysozyme on ice for 30 min and sonicated 10 × 20 s in 50 mM sodium phosphate buffer (pH 8.0), 300 mM NaCl and 10 mM imidazole. The clarified lysate was applied to Ni-NTA beads  for 2 h at 4 °C. After washing three times with the same buffer containing 20 mM imidazole, enolases were eluted from the beads with 200 mM imidazole, dialyzed against 50 mM Tris-HCl (pH 8.0), 300 mM NaCl and 40% glycerol and stored at −20 °C. The purity of the proteins was checked by SDS-PAGE with Coomassie blue staining.Purification of recombinant preKARS2 protein was done as described previously . Plasmidin vitro using the Ribomax kit . Following transcription, the DNA template was removed by digestion with RQ1 RNase-Free DNase (Promega), and RNA was purified by 8 M urea −12% PAGE. After elution and ethanol precipitation, RNA was dephosphorylated with alkaline phosphatase  and labeled at the 5'-end with γ-32P-ATP using T4 polynucleotide kinase (Promega). For the RNA binding assay, labeled RNA was denatured at 100 °C and then allowed to slowly cool down to room temperature. The appropriate amounts of protein and labeled RNA were mixed in 20 µL of a buffer containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM MgCl2, 5 mM DTT, 10% glycerol, 0.1 mg/mL BSA and incubated at 30 °C for 15 min. The mixture was fractionated by native 8% PAGE in 0.5× Tris-borate buffer (pH 8.3) and 5% glycerol [dK from the linear regression of experimental data was done as described in [TRK1 T7 transcripts were obtained glycerol , followeglycerol . For Scaribed in .in vitro import assay was performed as in [32P 5' labeled RNA and purified proteins in import buffer: 0.6 M Sorbitol, 20 mM HEPES-KOH (pH 7), 10 mM KCl, 2.5 mM MgCl2, 5 mM DDT and 2 mM ATP. For a standard in vitro assay, 3 pmoles of labelled RNA were added to 100 µL of reaction mixture containing 0.1 mg of mitochondria . This corresponds to 100% RNA input. After incubation for 15 min at 34 °C, 50 µg/mL of RNase A  was added and the reaction further incubated for 15 min to digest all the RNA that was not imported. Mitochondria were washed three times with buffer containing 0.6 M sorbitol, 10 mM HEPES–KOH (pH 6.7) and 4 mM EDTA, then resuspended in 100 µL of the same buffer and treated with an equal volume of 0.2% digitonin (Sigma) solution to disrupt the mitochondrial outer membrane, followed by purification of mitoplasts. The mitoplast pellet was resuspended in a solution containing 100 mM CH3COONa, 10 mM MgCl2, 1% SDS and 0.05% Diethylpyrocarbonate (DEPC) and heated at 100 °C for 1 min. RNA was extracted at 50 °C using water saturated acidic phenol. RNA was precipitated with ethanol and fractionated by 12% PAGE containing 8 M urea, followed by quantification by a Typhoon-Trio scanner using Image Quant-Tools software . The amount of imported RNA was determined by comparison of the band density of protected full-sized RNA isolated from the mitoplasts after the import assay with an aliquot (2%–5%) of the RNA input.The ed as in . For thiSequences were obtained using blastp , aligned


Gloeophyllum trabeum causes a typical brown rot and is known to use reactive oxygen species in the degradation of cellulose. The extracellular Cel12A is one of the few endo-1,4-β-glucanase produced by G. trabeum. Here we cloned cel12A and heterologously expressed it in Aspergillus niger. The identity of the resulting recombinant protein was confirmed by mass spectrometry. We used the purified GtCel12A to determine its substrate specificity and basic biochemical properties. The G. trabeum Cel12A showed highest activity on β-glucan, followed by lichenan, carboxymethylcellulose, phosphoric acid swollen cellulose, microcrystalline cellulose, and filter paper. The optimal pH and temperature for enzymatic activity were, respectively, 4.5 and 50°C on β-glucan. Under these conditions specific activity was 239.2±9.1 U mg−1 and the half-life of the enzyme was 84.6±3.5 hours. Thermofluor studies revealed that the enzyme was most thermal stable at pH 3. Using β-glucan as a substrate, the Km was 3.2±0.5 mg mL−1 and the Vmax was 0.41±0.02 µmol min−1. Analysis of the effects of GtCel12A on oat spelt and filter paper by scanning electron microscopy revealed the morphological changes taking place during the process.The basidiomycete fungus  A search of the database containing characterized lignocellulolytic enzymes of fungal origin, Gloeophyllum trabeum is a brown-rot basidiomycete fungus. Brown-rot fungi consume the cellulose in wood and leave behind the brown rotted wood that is composed mostly of lignin. Brown-rot fungi use both extracellular reactive oxygen species and hydrolytic enzymes to degrade cellulose G. trabeum has been published G. trabeum does not harbor genes for GH6 and GH7 proteins and therefore do not have cellobiohydrolases. Genes predicted to encode endoglucanases in the G. trabeum genome are two GH5 endoglucanases, two GH12 proteins and one each of GH45 and GH74 proteins. Two of the genes, cel5A and cel12A, have been characterized previously G. trabeum, attractive enzymes for detailed characterization as they may possess functionality that have not been observed previously. The relatively small size, <30 kDa, and the lack of CBM of GH12 enzymes suggest that they may be able to penetrate the low-porosity wood to depolymerize cellulose. Fungal GH12 enzymes in general have not been thoroughly characterized. These considerations prompted us to investigate further the role of GtCel12A in cellulose degradation. In this study we report the expression of cel12A of G. trabeum in Aspergillus niger. We describe the purification and biochemical characterization of the recombinant enzyme. We also report the scanning electron microscopy analysis of substrates following hydrolysis by GtCel12A.Gloeophyllum trabeum strain ATCC 11539 grown in a medium containing barley and alfalfa 1% (w/v) for 32 hours at 34°C. The gene sequence was selected from GenBank (accession number HQ163778). The recombinant expression was carried out through the gene amplification from the cDNA by PCR using three primers as follows: 5′-TACTTCCAATCCAATCCATTTGACGATATGTTCCGCTTCATCTCTGCTTTGCCCTTTG-3′ (forward), 5′-ATGATGATGATGATGATGGGATCCACGCGGAACCAGCCCGCTCAAGCTGAC-3′ (reverse 1) and 5′-TTATCCACTTCCAATCCATTTGTTAATGATGATGATGATGATGGGATCCACGCGGAACCA-3′ (reverse 2).Complementary DNA was prepared by reverse transcription of total RNA isolated from Both insert and vector were amplified with specific primers designed to enable the cloning using the ligation-independent cloning method as previously described Aspergillus niger py11 (− pyrG−cspA ΔGla::hiG), derived from strain N593 (− pyrG−cspA), using the protoplast transformation method as previously described Recombinant DNA was transformed into Transformants were cultured in MM J media, which is similar to MM except that it includes 4% (w/v) maltose as the sole carbon source and the amounts of nitrogen source, salts and trace elements were increased by 4-fold. Six colonies from the transformation plates were randomly selected and the conidia were transferred to 24 wells plates containing 1 mL of MM J and grown for 6 days at 30°C without agitation. After the incubation period, the supernatant of the cultures was harvested and analyzed by SDS-PAGE to confirm positive protein production.6 conidia mL−1 and the culture was grown for 6 days at 30°C without agitation. Culture supernatant was filtered through Miracloth  and then precipitated with 80% of ammonium sulfate at 10°C. The pellet was resuspended in 50 mM sodium citrate buffer (pH 3.0) and washed several times with the same buffer. The resulting supernatant was further applied to a desalting column, Sephadex G25 . Then, the desalinated protein sample was applied to an ion-exchange SP Sepharose  column equilibrated with the same buffer as a first purification step. Proteins were eluted with a linear gradient of 0–1 M of sodium chloride in the same buffer at a flow rate of 1 mL min−1. The samples were analyzed by SDS-PAGE and the fractions containing the protein of interest were combined and concentrated using a 10 kDa cut-off Vivaspin  concentrator. In a second purification step, the concentrated volume was applied to a Superdex 75 10/30 column , equilibrated with the same buffer added of 0.2 M of sodium chloride. Fractions (1 mL) were collected at a flow rate of 1 mL min−1 and were assayed for activity on β-glucan and CMC. All chromatographic steps were performed using AKTA chromatography system  at 4°C and fractions were stored at the same temperature until further use.For protein purification, 2-L Erlenmeyers flasks containing 500 mL of MM J media were inoculated with positive transformants at 2×10To confirm the experimental molecular weight of GtCel12A, a calibration curve was assembled using a molecular exclusion column, Superdex 75 . Standard molecules were applied and the respective elution volumes were calculated. The standard molecules used were: blue dextran (2000 kDa), conalbumin (75 kDa), egg albumin (44 kDa), carbonic anhydrase (29 kDa), ribonuclease A (13.7 kDa) and aprotinin (6.5 kDa), all purchased from GE Healthcare.http://www.matrixscience.com/server.html; Matrix Science, Boston, USA). The peptide sequences were compared with the non-redundant databases of filamentous fungi proteins generated from data compiled by the Joint Genome Institute.After analysis of the protein production by SDS-PAGE, the Coomassie Blue-stained band corresponding to GtCel12A was submitted to in-gel digestion with 0.5 µg of modified trypsin for 16 hours . The tryptic peptides were extracted with a solution containing formic acid (5%) and acetonitrile (50%) and further submitted to LC/MS analysis using MicroTOF-QII  mass spectrometer. The mass spectra of peptides generated by mono-isotropic peptide masses obtained were interpreted using the Mascot search engine , filter paper , hydroxyethylcellulose, Avicel PH-101  , 4-Nitrophenyl β-D-cellobioside (4NPC), 4-Nitrophenyl β-D-glucopyranoside (4NPG), CMC, xylan from beechwood, Sigmacell 20   and phosphoric acid swollen cellulose (PASC) Substrate specificity was examined in assays using a range of 21 substrates. Substrates were prepared as 1% stock solutions in MilliQ water, except for the amorphous cellulose which was suspended at 5.7 mg mLEach assay was performed in triplicate in a final volume of 100 µL containing: 50 µL of each substrate at 1% (w/v), 1.5 µg of enzyme in 5 µL, and 45 µL of 50 mM sodium citrate buffer, pH 4.5. For control, the volume of enzyme was replaced with 5 µL of the same buffer. The reaction mixture was incubated at 5°C for 30 minutes and 100 µL of dinitrosalicilic acid (DNS) 2CO3 was added, followed by absorbance measurements at 400 nm.For reactions with 4NPG and 4NPC, after the incubation time of 30 minutes, 100 µL of 1M NaThe amount of reducing sugars was determined using the DNS method Temperature and pH profiles of the enzymatic activity were performed using β-glucan from barley at 1% (w/v) as substrate. The final reaction volume was 100 µL, which contained 0.15 µg of enzyme in 5 µL, 45 µL of each buffer of pH values ranging from 1 to 10 at 50 mM, and 50 µL of the substrate diluted in water. In the reaction control, the volume of enzyme was replaced with 5 µL of the same buffer. Initially, a screening of the optimal conditions was performed using ABP (acetate/borate/phosphate) buffer, with pH values ranging from 2 to 10. Then, different buffers were used to confirm the preliminary results observed (data not shown). All buffers were prepared at 50 mM. For pH 1, we used hydrochloric acid at 50 mM and for pH 2, hydrochloric acid equilibrated with potassium chloride. Sodium citrate buffer was used for pH 3, 3.5, 4.0, 4.5, 5.0 and 6.0, sodium phosphate for pH 7, Tris/HCl for 8 and 9, and sodium carbonate/bicarbonate for pH 10. Each assay was performed in triplicate. The experiment was performed in 96 wells PCR plates  using a thermocycler  with a temperature gradient function, and temperature ranging from 30 to 80°C for all pH values, totalizing 768 samples. After determining the optimal conditions of pH and temperature, separately, we also investigated the influence of enzyme concentration on the enzymatic assay (data not shown).Residual enzymatic activity was determined under the optimal conditions found in the previous experiment and was performed by incubating the enzyme diluted in 50 mM of sodium citrate buffer, pH 4.5 at 50°C for 168 hours. Aliquots of 5 µL were removed and assayed for activity on β-glucan from barley at 1% over a time course of 168 h.m) determination were carried out using GraphPad Prism software (version 5.0) .To investigate the effect of pH on thermal stability for GtCel12A, 10 µM of protein in different buffer solutions were mixed with 1 µL of Sypro Orange , used as the reporter dye, with a final concentration of 1/2000. All buffers used in the analysis were prepared at 50 mM. All measurements were performed in triplicate in a final volume of 20 µL. In the reaction control, the volume of enzyme was replaced with 19 µL of the same buffer. Mixtures were added to a 96-well thin wall PCR plate  and sealed with Optical-quality sealing tape  and incubated in an iCycler iQ Real-Time PCR Detection System . The temperature range tested was from 25 to 90°C, with stepwise increments of 1°C per minute and 10 s hold step for every point, followed by the fluorescence reading with excitation/emission wavelengths at 490/530 nm. Melting curve analysis and the melting temperature (T−1), 45 µL of sodium citrate buffer, pH 4.5 and 5 µL of enzyme. The final enzyme concentration used in the experiment was 30 nM.The kinetic parameters were determined using β-glucan from barley as a substrate. The reaction volume was 100 µL, which contained 50 µL of each substrate to yield final substrate concentrations of 0.6, 1.25, 2.50, 5, 10 and 15 .The 100 µL reaction mixtures were incubated in a 96 well micro plate at 50°C. Triplicates were removed after 1, 2, 5, 10, 12 and 15 min and were immediately assayed for reducing sugar end determination by DNS. The KSamples of oat spelts, a β-glucan rich substrate, and filter paper were hydrolyzed by GtCel12A for 12 hours (oat spelt and filter paper) and 24 hours (only filter paper) at 50°C and analyzed by SEM. The assays were performed in triplicate in a final volume of 100 µL, which contained 5 µg of enzyme in 5 µL, 95 µL of 50 mM sodium citrate buffer, pH 4.5 and 6 mg of oat spelts or a 0.7 cm disc of Whatman No. 1 filter paper. In the reaction control, the volume of enzyme was replaced with 5 µL of the same buffer. Following hydrolysis the substrates were washed with 2 mL of 50 mM sodium citrate buffer, pH 4.5 and dried at 37°C for 12 hours. After drying, the substrates were coated with gold in a SCD 050 sputter coater . Sample imaging was carried out using a high resolution environmental scanning electron microscope, equipped with a field emission gun (FESEM) Quanta 650 . Both the coater and the microscope were available at the National Laboratory of Nanotechnology (LNNano) in Campinas-SP, Brazil. Images were obtained under vacuum, using a 5 kV accelerating voltage and a secondary electron detector. At least 20 images per sample were obtained on different areas of the samples to ensure the reproducibility of the results.Supernatant from the culture of one of the positive transformants was used to purify the recombinant Cel12A by SP Sepharose and Superdex 75  column chromatography. From the calibration performed on the molecular exclusion column, the experimental molecular weight of the protein was estimated to be close to 26.2±0.1 kDa, very close to the mass calculated from the amino acid residues of 26.1 kDa. This suggests that Cel12A is a monomeric enzyme with few or no post-translational modifications. G. trabeum Cel12A sequence.The identity of the purified GtCel12A was confirmed by LC-MS analysis. Gloeophyllum trabeum Cel12A is quite stable enzyme under the conditions of our study, since it retained about 30% of its activity even after 120 hours of incubation at 50°C  by GtCel12A was examined by scanning electron microscopy. Following 12 hours of incubation, the control oat spelt (sample without enzyme treatment) displayed relatively uniformed size granules with smooth round surface. After hydrolysis by GtCel12A, the oat spelt showed a predominance of pitted surface with numerous small bulbous particles . The difIn Filter paper was used as a model cellulosic substrate to demonstrate the effect of GtCel12A. At low magnification , the conAt high magnification , the fibFomitopsis palustris is most active on lichenan , and is also active on barley β-glucan, pachyman , laminarin , CMC , pustulan , glucommanan from konjac and larch, and xyloglucan from tamarind  Phanerochaete chrysosporium GH12 endoglucanase is active on CMC, amorphous cellulose, and minor activity was observed on xylan and mannan Aspergillus japonicus shows highest activity on β-glucan from barley and lichenan G. trabeum was shown to be active on CMC with minor activities on birchwood xylan and PASC trabeum Cel12A has its highest activity on β-glucan under optimal conditions of pH 4.5 and 50°C. It also displays a high activity on lichenan (65% of the maximum activity), which agrees well with previous findings that shows a preference of GH12 family members for substrates with mixed linkages such as β-glucan and lichenan over polyssacharides with only β-1,4 linkages. The available results suggest that for GtCel12A and other members of GH12 family, the enzymatic activities are not limited to cellulose-based substrates Trichoderma harzianum Cel12A incorporates important residues mediating interactions of the Cellulomonas fimi CBM with the amorphous regions of cellulose G. trabeum Cel12A with none-crystalline polysaccharides. Furthermore, we and others Previous studies showed that enzymes from GH12 family are active on a relatively wide range of substrates nd generation ethanol production, which frequently involves processes run at acidic pHs.Temperature and pH profile for GtCel12A suggested that ithas an acidophilic character, since it retained about 40% of its activity even for the lowest pH tested (pH = 1) under the optimal temperature of 50°C.This feature might be of biotechnological interest, mainly for 2G. trabeum Cel12A against CMC in similar conditions, which showed a much shorter half-life of 2 hours Thermal stability assay reveals a very stable enzyme, with half-life of 84.6±3.5 hours at 50°C and retains about 30% of its activity even after 120 hours of incubation at 50°C and pH 4.5. These results differ from previous thermal stability results on testing of nd generation biofuel production, especially in Brazil and the United States Analysis by SEM showed that GtCel12A is active on filter paper, used as a model cellulosic substrate. The effect of enzyme digestion was evidenced by the degradation of the thinner and most vulnerable cellulose fibers and the emergence of defects on the fiber surface. Furthermore, analysis by SEM showed that GtCel12A is also active on oat spelt, a natural substrate rich in β-glucan. Mixed-linkage (1→3),(1→4)-β-D-glucan or β-glucans are important unbranched water-soluble polysaccharide present in plant cell walls G. trabeum is highly active on β-glucan. The enzyme is thermal stable in acidic environment. Results from SEM analysis on the effects of GtCel12A on oat spelt are consistent with the assertion that GH12 enzymes prefer mixed linkage (1→3), (1→4)-β-D-glucan as substrate. Since β-glucans are often found in abundance in grasses, these results suggest that GH12 enzymes are potentially important in the conversion of grass-derived biomass into fermentable sugars.Detailed biochemical characterization showed that Cel12A of


Amycolatopsis mediterranei DSM 40773 has been of special interest as successors of this strain are in use for the commercial production of rifamycin B. Here we present the draft genome sequence (~10 Mb) of this strain, which contains 108 contigs, 9,198 genes, and has a G+C content of 71.3%. Amycolatopsis mediterranei DSM 40773 was derived from the parent strain isolated from the soil of pine arboretum at St. Raphael, France . A total of 9,353 open reading frames (ORFs) falling into 24 clusters of orthologous groups (COGs) categories were found with COGs of the class R being most abundant. The draft genome was found to contain 9,198 genes, 9,100 coding sequences (CDSs), and 21 pseudogenes. 51 tRNA’s and 4 copies of the 16S rRNA genes were detected using RNAmmer 1.2  gene cluster of 90 kb was identical to that of nei S699 , 17 and anei U32 . Analysianei U32 , DSM 407anei U32  and the anei U32 .A. mediterranei S699 (A. mediterranei U32 (Based on average nucleotide identity, the draft genome of strain DSM 40773 was 99.99% identical to those of nei S699  and A. manei U32 . HoweverA. mediterranei S699, A. mediterranei U32, and A. rifamycinica DSM 46095 (The whole-genome sequencing of strain DSM 40773 coupled with the genome sequences of SM 46095  can now SM 46095 . FurtherJMQJ00000000. The version described in this paper is the first version, JMQJ00000000.1.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession no.