Arabidopsis suspension-cultured cells stably expressing aequorin in the plastid stroma were used as a suitable tool to dissect differential Ca2+ responses of non-green plastids versus chloroplasts to several environmental cues. 2+ homeostasis and signalling has been growing in recent years. This work has evaluated the potential participation of non-green plastids and chloroplasts in the plant Ca2+ signalling network using heterotrophic and autotrophic cell suspension cultures from Arabidopsis thaliana plant lines stably expressing the bioluminescent Ca2+ reporter aequorin targeted to the plastid stroma. Our results indicate that both amyloplasts and chloroplasts are involved in transient Ca2+ increases in the plastid stroma induced by several environmental stimuli, suggesting that these two functional types of plastids are endowed with similar mechanisms for handling Ca2+. A comparison of the Ca2+ trace kinetics recorded in parallel in the plastid stroma, the surface of the outer membrane of the plastid envelope, and the cytosol indicated that plastids play an essential role in switching off different cytosolic Ca2+ signals. Interestingly, a transient stromal Ca2+ signal in response to the light-to-dark transition was observed in chloroplasts, but not amyloplasts. Moreover, significant differences in the amplitude of specific plastidial Ca2+ changes emerged when the photosynthetic metabolism of chloroplasts was reactivated by light. In summary, our work highlights differences between non-green plastids and chloroplasts in terms of Ca2+ dynamics in response to environmental stimuli.Calcium is used by plants as an intracellular messenger in the detection of and response to a plethora of environmental stimuli and contributes to a fine-tuned internal regulation. Interest in the role of different subcellular compartments in Ca A wide array of environmental stimuli have been shown to evoke specific intracellular spatio-temporal Ca2+ signals, which are further transduced by Ca2+ sensor proteins into transcriptional and metabolic responses .Arabidopsis thaliana ecotype Columbia (Col-0) plant lines stably expressing the Ca2+-sensitive photoprotein aequorin fused to yellow fluorescent protein  were used in this study. These transgenic plant lines have been genetically transformed with plasmids encoding YA chimera targeted to different intracellular locations: (i) the plastid stroma, by fusion with the first 85 amino acids of NADPH-dependent thioredoxin reductase C at the N-terminus of YA; (ii) the surface of the plastid outer envelope, by the fusion of the full-length outer envelope protein 7 (OEP7) at the N-terminus of YA; and (iii) the cytosol (excluding the nucleus), by the presence of the nuclear export signal from the heat-stable protein kinase inhibitor introduced between the cytosolic CPK17G2A and YA. The whole expression cassettes of the different constructs were under the control of the cauliflower mosaic virus 35S promoter. All YA fusion vectors carried kanamycin resistance  medium containing 1.5% sucrose, 0.8% plant agar , and 50 µg/ml kanamycin. Cotyledons and hypocotyls from 7-day-old seedlings were transferred in axenic conditions onto callus induction medium (CIM)  supplemented with 50 µg/ml kanamycin, as described by A. thaliana cell suspension cultures stably expressing stromal aequorin were initiated from heterotrophic cell suspension cultures by gradually decreasing the supply of organic carbon in the plant cell culture medium from 3% to 0.5% (w/w) sucrose to stimulate the photosynthetic activity. Moreover, the cultures were exposed to a relatively high illumination rate (110 µmol photons m−2 s−1) under an unvaried 16/8h light/dark cycle, to further stimulate the photosynthetic activity  software. The intracellular localization of the YA chimeras in heterotrophic and autotrophic cell suspension cultures of A. thaliana was analysed using a Leica TCS SP5 II confocal laser scanning system mounted on a Leica DMI6000 inverted microscope. Excitation with the argon laser was carried out at 488nm and the emitted fluorescence was detected at 505–530nm for YFP and at 680–720nm for chlorophyll. Cells were also observed under a Leica DM5000 B microscope after staining with Lugol solution for starch granule detection. Images were acquired with a Leica DFC425 C digital camera, using the LAS software.v/Fm, where Fv is the difference between the maximal (Fm) and the basal (F0) fluorescence of chlorophyll. Chlorophyll fluorescence images were recorded using a CCD camera, and the data analysis was carried out using the FluorCam 7 software (Photon Systems Instruments).Photosynthetic activity of autotrophic cell cultures was analysed in 6-day-old cultures by placing samples in a Closed FluorCam 800 MF . Pulse amplitude modulation (PAM) fluorometric analysis expressed the photosynthetic efficiency as FA. thaliana cell suspension cultures were reconstituted overnight with 5 µM coelenterazine  on a shaker at 80rpm, at 24°C in the dark. After extensive washing, a 50 µl cell aliquot was assayed using a luminometer  to monitor intracellular Ca2+ changes in response to selected environmental stimuli. Intracellular [Ca2+] dynamics were monitored after cell samples were treated with 10mM H2O2 (oxidative stress), 0.3M NaCl , 0.6M mannitol (drought), three volumes of ice-cold cell culture medium (cold shock), and 20 µg/ml oligogalacturonides (OGs) with a degree of polymerization from 10 to 15   t-test.Data are shown as means ± SE of at least three independent experiments, and the differences between groups were assessed by Student’s in toto, permitting the analysis of a wide array of physiological processes at the cellular level. In particular, suspension-cultured cells are a valuable tool to analyse many different Ca2+-based signal transduction pathways . Lugol staining highlighted starch granules inside the plastids , and the organelle stroma. [Ca2+] in all these subcellular locations was between 100 and 200nM in resting conditions, indicating a similarly low Ca2+ level in both cytosol and plastid stroma . In the cytosolic microdomain close to the surface of the amyloplasts (outer envelope), the maximum [Ca2+] value observed was recorded at 0.35±0.02 µM after 54±4s (n = 3). In the amyloplast stroma the oxidative stress induced a slower increase in [Ca2+] that did not reach a plateau but continued to increase in the considered time interval. Interestingly, the ascending stromal Ca2+ trace matched the decrease in cytosolic [Ca2+] level, suggesting a potential involvement of plastids in the dissipation of the Ca2+ transient in the cytosol.In response to oxidative stress  and 1.37±0.21 µM with 0.6M mannitol (n=6)] than that of the corresponding [Ca2+] changes in the bulk cytosol [0.66±0.11 µM with 0.3M NaCl (n = 5) and 0.92±0.11 µM with 0.6M mannitol (n = 8)]. Interestingly, the decrease in [Ca2+] in both the bulk cytosol and cytosolic microdomain at the plastid surface temporally coincided with the increase of [Ca2+] in the stroma [peaking at 0.79±0.08 µM for 0.3M NaCl (n = 6) and 0.92±0.11 µM for 0.6M mannitol (n = 8)], again suggesting a role for non-green plastids as Ca2+ sinks involved in the modulation of intracellular Ca2+ signals.Cell treatment with salt stress  and stroma  were progressively lower, although characterized by equally fast kinetics. Indeed it is known that cold shock activates a rapid [Ca2+] increase in the cytosol due to the influx of Ca2+ from the extracellular medium and a release of the ion from the vacuole  and lasted for ~5min. A similar response was observed at the outer membrane surface subdomain, albeit with a slightly lower amplitude . OGs were even found to evoke a transient Ca2+ increase in the plastid stroma, characterized by similar kinetics but a lower amplitude . Taken together, the data obtained indicate that in resting conditions Ca2+ is kept at a similar concentration in the cytosol and in the stroma of amyloplasts in A. thaliana suspension-cultured cells, and that various environmental stresses evoke differential stromal [Ca2+] signals, specific for each stimulus. Moreover, analysis of the kinetics of the stromal Ca2+ transients demonstrated that, at least in some cases , plastids seem to be involved in the dissipation, rather than the generation, of the stimulus-induced cytosolic Ca2+ increases.OGs with a degree of polymerization from 10 to 15 . Confocal microscopy analysis showed that in the autotrophic cell line, unlike in the corresponding heterotrophic line, plastids displayed a remarkable level of chlorophyll autofluorescence, confirming that the plastids present were chloroplasts (Arabidopsis suspension-cultured cells (Supplementary Videos S1 and S2). PAM assays demonstrated the high photosynthetic efficiency of the autotrophic cell cultures . It was not possible to calculate the Fv/Fm parameter in the heterotrophic cell cultures because they contain amyloplasts, which do not possess thylakoids and therefore lack chlorophyll and photosynthetic activity.In order to compare stromal Caroplasts . It shou2+ homeostasis and signalling, autotrophic cell cultures stably expressing stromal aequorin were challenged with the same stimuli described above for heterotrophic cell cultures. The results did not highlight significant differences in the Ca2+ dynamics recorded in the stroma of non-green versus green plastids, concerning either the amplitude or the timing of the maximal Ca2+ peak . However, a clear difference in their Ca2+ response emerged when a stimulus more closely related to the specific physiology of the chloroplast was tested, that is, the light-to-dark transition at the end of the 16h light photoperiod. In this latter case, a transient [Ca2+] elevation was detected in the stroma of chloroplasts, peaking at ∼0.44 µM about 20min after light switch-off and falling back to basal values within 2h  and drought . These results indicate that, although green plastids displayed Ca2+ responses that were indistinguishable from those of non-green plastids when their photosynthetic metabolism was switched off by dark exposure , clear differences emerged after their photosynthetically active status was reactivated by 16h light exposure .To determine whether chloroplasts display differential Ca, or OGs . Howeverexposure . The eff2+ handling during signal transduction events, we monitored changes in [Ca2+] in response to different environmental stimuli in A. thaliana heterotrophic and autotrophic cell suspension cultures stably expressing aequorin targeted to the organelle stroma. The results presented in this work provide evidence that amyloplasts, that is, non-green plastids specialized in the accumulation of starch, have tightly regulated [Ca2+] inside the organelle and the ability to evoke specific Ca2+ changes in response to several environmental cues. It is known that Ca2+ plays essential regulatory roles in the physiology of plastids by affecting diverse processes that do not necessarily relate only to photosynthesis, such as plastid division and the import of nucleus-encoded proteins  were gradually delayed from the cytosol moving towards the plastid stroma, indicating a likely crucial role for plastids in shaping and switching off intracellular Ca2+ signals. The kinetics of the observed Ca2+ changes—that is, the fact that a decrease in [Ca2+] at the cytosolic surface of the outer membrane of the envelope often correlated with an increase of [Ca2+] in the stroma—support the notion that under these conditions plastids may function as Ca2+ sinks, used by the cell to dissipate cytosolic [Ca2+] signals, thus contributing to the modulation of intracellular Ca2+ signatures.To dissect the involvement of chloroplasts and non-green plastids in Caplastids , distinc2+ levels in chloroplasts and non-green plastids. Previous studies have demonstrated that a long-lasting transient [Ca2+] elevation is detected in the chloroplast stroma after a lights-off stimulus  in generating the stromal Ca2+ signal. A likely possibility is that the observed stromal Ca2+ fluxes derive from the thylakoid lumen, which may act as a mobilizable Ca2+ store. Alternatively, complexed Ca2+ may be released from the thylakoid membrane, without the involvement of Ca2+ flux-permitting transporters localized at the thylakoid membrane. Indeed, there is a plethora of Ca2+-binding proteins in the chloroplast . Interestingly, significant differences were observed upon challenge with either of the two hyperosmotic shocks simulating salinity (0.3M NaCl) and drought (0.6M mannitol), whereby more than a 2-fold higher increase in [Ca2+] was detected in light-reactivated chloroplasts. The remarkable changes in the amplitude of the stromal Ca2+ responses between light-reactivated and dark-adapted chloroplasts, but not between amyloplasts and dark-adapted chloroplasts, infer an indirect impact of light on the Ca2+-mediated transduction mechanisms of these stresses. Indeed, it has been demonstrated that drought and salinity alter the normal homeostasis of cells and cause an increased production of reactive oxygen species (ROS) that can be used by the plant as signalling molecules in retrograde signalling  and autotrophic (B) cell suspension cultures stably expressing aequorin in the stroma. (C) PAM imaging analysis of autotrophic cell suspension cultures of A. thaliana .Supplementary Fig. S2. Comparison between stromal Ca2+ dynamics in amyloplasts and dark-adapted chloroplasts after the perception of environmental stimuli.Supplementary Fig. S3. Analysis of stromal Ca2+ dynamics in terms of the difference between peak [Ca2+] and basal [Ca2+] (Δ[Ca2+]) and of the timing of the [Ca2+] peak after stimulus injection (Δt) in A. thaliana heterotrophic (grey columns) and dark-adapted autotrophic (black columns) cell suspension cultures in response to different environmental stimuli.Supplementary Fig. S4. Reactivation of photosynthetic metabolism by 16h light exposure in autotrophic A. thaliana cell cultures.Supplementary Video S1. Single-cell z-stack series of A. thaliana heterotrophic cell suspension cultures stably expressing YA targeted to the plastid stroma. Stromules, dynamic stroma-filled tubules extending from amyloplasts, are evident.Supplementary Video S2. Single-cell z-stack series of autotrophic A. thaliana cell suspension cultures stably expressing YA targeted to the chloroplast stroma. The yellow signal indicates co-localization of YFP fluorescence and chlorophyll autofluorescence.


Acute leukemias are the most common childhood cancer in all age groups. Acute myeloid leukemias (AML) constitute about 15–20% of acute leukemias. Fatigability, pallor, fever, and bleeding are the most common presenting symptoms of AML. Hepatosplenomegaly and lymphadenopathy are commonly encountered during physical examination. In rare instances eruptions due to skin involvement and localized tumor masses (myeloid sarcoma) may be found. Myeloid sarcoma is especially seen in AML-M2 subtype. By cytogenetic analysis, in AML-M2 subtype t is often seen and it is more probable to find inversion 16 in AML-M4Eos subtype. Herein, we present a 15-year-old girl whose initial symptom was abdominal pain for three days and her pathological sign was a large abdominal mass which was verified by imaging studies and diagnosed as myeloid sarcoma by biopsy. On bone marrow examination, she had diagnosis of AML-M2 and by cytogenetic analysis inversion 16 was positive. She was treated with AML-BFM 2004 protocol and she is being followed up in remission on her ninth month of the maintenance therapy. Acute myeloid leukemia (AML) comprises 15–20% of all childhood acute leukemias. In most of the patients, fever, pallor, weight loss, and mucosal bleeding are seen. In more than half of the cases, liver, spleen, and lymph nodes are palpable [Myeloid sarcoma (chloroma and granulocytic sarcoma) is defined as a tumoral mass which is formed by immature myeloid cells in the extramedullary area. It is seen in less than 5% of all AML cases. The most common locations are head and neck region, skin, gingival region, and intracranial and paravertebral areas –6.In FAB classification, AML is classified according to myeloblasts' morphology into 10 subtypes . In 20089/L, anemia (hemoglobin: 107 g/L), and mild thrombocytopenia (platelets: 125 × 109/L). Lactate dehydrogenase level was 1144 U/L and other biochemical tests were at normal ranges. On peripheral blood smear examination 20% of cells were blasts. On bone marrow aspiration, 85% of cells were large myeloid blasts with fine chromatin and striking nucleoli  comprises 10–15% of all AML cases. To fulfill morphological diagnostic criteria, in the bone marrow, blast percentage must be over 20%, mature myeloid cell percentage must be over 10%, and monocytic component must be less than 20%. Immunophenotypically expression of CD13, CD33, CD65, CD11b, CD15, CD34, CD38, and HLA-DR is present .By cytogenetic analysis in cases of AML-M2, generally t8; 21) is present. Inv(16) and t which are abnormalities of the 16th chromosome are seen in 7-8% of AML cases –19. Inv( is preseThe prognosis of AML patients with inv16) is generally better than other AML subtypes. Because the response to chemotherapy is satisfactory, it is recommended that bone marrow transplantation should be reserved for relapsed cases 6 is gene, 9. In oIn conclusion, in children who present with a solid mass, the possibility of myeloid sarcoma should be kept in mind. Our case also supported the opinion that inv(16) is not restricted to AML-M4Eos subtype.


E. perforans was the most prevalent specie (42.73 %), followed in order by Eimiera media, E. irresidua, E. magna, and E. intestinalis with prevalences of 35.45, 34.55, 31.82, and 23.64 %, respectively. Results of the present investigation indicated that the prevalence of coccidial infection is high among the rabbit population in southwest China. This study also elucidate about the coccidial infection and emphasis to adopt control strategies in commercial rabbitories.Coccidiosis is a challenging disease of wild and domestic rabbits both, caused by Eimeria and thereby leads enormous economic losses at rabbit farms. The present study carried out to survey the prevalence and intensity of coccidial infection among the rabbits in Sichuan Province, southwest China. A total of 110 faecal samples were collected from 11 farms situated in eight main rabbits rearing administrative regions. Oocysts in faecal samples were purified, sporulated and identified according to morphological features. The overall prevalence of infection was 56.4 % (62/110), with prevalence of 64 % (47/75) for local meat breeds of rabbit and 51.4 % (18/35) for Rex Rabbits . Weanling rabbits had the highest prevalence , followed by young rabbits  and the adult rabbits showed the lowest prevalence . Concurrent infection with two to seven Eimeria species was found. In total, 9 species of Eimeria were identified from oocyst-positive samples.  Oryctolagus cuniculus) (Duszynski and Couch Eimeria stiedai) infecting the biliary ducts of the liver  from apparently healthy animals of 8 main rabbits rearing administrative regions. From each chosen population, 500 g of fresh faecal pellets were collected as one sample. All samples were stored at 4 °C and transported to the laboratory  for further analysis. Each faecal sample was homogenised in 500 ml tap water, and then 2 g of the mixture was put into 60 ml of saturated salt solution , followed in order by E. media, E. irresidua, E. magna, and E. intestinalis with prevalences of 35.45, 34.55, 31.82, and 23.64 %, respectively  obtained from the eight regions of Sichuan Province. The prevalence of coccidian oocysts in the eight regions ranged from 10.0 to 90 % (Table Coccidian oocysts were found in 64 % (47/75) of faecal samples from local meat rabbits and 51.4 % 18/35) from Rex rabbits, these values were not significantly different (P > 0.05)  was higher than in the growing rabbits (45 %) and breeding rabbits (42 %). All the differences were statistically significant (P < 0.05) , which suggests that the breed may have little influence on the prevalence of coccidiosis in rabbit production. The prevalence of coccidian oocysts in weanling rabbits (74 %) was higher than that in growing rabbits (45 %) and breeding rabbits (42 %), and the intensity of infection in young animals (weanling rabbits) was significantly higher (P < 0.05) than in adults (breeding rabbits), This could be due to lower resistance or less immunity to coccidian oocysts in young rabbits compared to elder animals as described previously (Pakandl et al. In conclusion, the present survey revealed that prevalence of rabbit coccidial infection in Sichuan Province is high. Knowledge of the prevalence of coccidiosis and current Eimeria species will help to evaluate the infection potential and control programs, and thus to minimize the economic losses in the rabbit production industry. These results also provide relevant “base-line” data for assessing the effectiveness of future control strategies against rabbit coccidiosis.


Drosophila brain. They have been involved in several crucial functions, such as learning and memory, sleep, locomotor activity, and decision making.The mushroom bodies (MBs) are the most prominent structures in adult  dunce and rutabaga, which regulate the level of cAMP, were identified more than 30 years ago, their in vivo effects on cellular and physiological mechanisms and particularly on the Ca2+-responses still remain largely unknown. In this work, performed in Drosophila, we took advantage of in vivo bioluminescence imaging, which allowed real-time monitoring of the entire MBs  simultaneously. We imaged neuronal Ca2+-activity continuously, over a long time period, and characterized the nicotine-evoked Ca2+-response. Using both genetics and pharmacological approaches to interfere with different components of the cAMP signaling pathway, we first show that the Ca2+-response is proportional to the levels of cAMP. Second, we reveal that an acute change in cAMP levels is sufficient to trigger a Ca2+-response. Third, genetic manipulation of protein kinase A (PKA), a direct effector of cAMP, suggests that cAMP also has PKA-independent effects through the cyclic nucleotide-gated Ca2+-channel (CNG). Finally, the disruption of calmodulin, one of the main regulators of the rutabaga adenylate cyclase (AC), yields different effects in the calyx/cell-bodies and in the lobes, suggesting a differential and regionalized regulation of AC. Our results provide insights into the complex Ca2+-response in the MBs, leading to the conclusion that cAMP modulates the Ca2+-responses through both PKA-dependent and -independent mechanisms, the latter through CNG-channels.The mushroom bodies (MBs), one of the main structures in the adult insect brain, play a critical role in olfactory learning and memory. Though historical genes such as  Drosophila brain. They have been involved in several crucial functions, such as learning and memory, sleep, locomotor activity, and decision making. However, although the historical genes such as dunce and rutabaga, which regulate the cAMP level, were identified more than 30 years ago, their effect on the cellular and physiological mechanisms of the Ca2+-response still remain largely unknown. Here, using an in vivo functional Ca2+-imaging approach, we describe the roles of the different components of the cAMP signaling pathways in the MBs. These results may serve as a foundation for disentangling the complex roles of cAMP in memory formation, as well as guiding new behavioral experiments that focus on CNG-channels and calmodulin.The mushroom bodies (MBs) are the most prominent structures in adult Drosophila melanogaster, demonstrated the critical role of the mushroom bodies (MBs) in olfactory learning and memory (L&M)  (rutabaga (rut)  , as wellry (L&M) , locomotry (L&M) , and decry (L&M) . In Drosormation . Genes sce (dnc) , encodinga (rut) , encodinerformed . Indeed,oneurons  or on dioneurons .in vivo effect of disturbing the cAMP signaling pathway on the Ca2+-response and on the overall cellular physiology of the Kenyon cells (KCs) still remains poorly characterized.More recently, physiological approaches using fluorescent markers to image the fly brain have begun to explore MB physiology. Except in the few studies that have investigated both the calyx and the lobes , up to nin vivo bioluminescence imaging technique recently developed  and the lobes, continuously, over a long time period. We recorded the nicotine (cholinergic)-induced Ca2+-response, employing both genetics (using mutants and/or targeted RNAi) and pharmacological approaches to manipulate different components of the cAMP signaling pathway. We show that the downregulation or upregulation of cAMP levels results in a proportional change of the Ca2+-response, while acute increase in the cAMP levels is sufficient to trigger a Ca2+-response. Finally, genetic manipulation of PKA, a cAMP effector, suggests that cAMP also has a PKA-independent effect, via the cyclic nucleotide-gated Ca2+-channel (CNG).In this work, we took advantage of the eveloped  to simulrutabaga-RNAi]: rut-RNAi(1)=VDRC-101759-KK, rut-RNAi(2)=VDRC-5569-GD, P[UAS-dunce-RNAi]: dnc-RNAi(1)=VDRC-107967-KK, dnc-RNAi(2)=NIG, P[UAS-CaM-RNAi]: cam-RNAi(1)=VDRC-102004-KK, cam-RNAi(2)= VDRC-28242-GD, P[UAS-cngc-RNAi]: cngc-RNAi(1)=VDRC-101745-KK, cngc-RNAi(2)= VDRC-28625-GD, P[UAS-cngl-RNAi]: cngl-RNAi(1)=VDRC-102411-KK, cngl-RNAi(2)=VDRC-40964-GD, from two different collections from Vienna Drosophila Resource Center (VDRC) and from R. Ueda , Mishima, Shizuoka, Japan) to knock-down the genes investigated specifically in the MBs. We use the P[UAS-Gαs*] provided by C. O’Kane  to activate the rut-AC. We overexpressed rut and dnc specifically in the MBs using the transgenic constructs UAS-rut (UAS-dnc (2+-response (data not shown). We therefore use the UAS-RNAi lines in trans-heterozygotes . All experiments were performed on females.Flies were maintained on standard medium at room temperature (24 °C). P[UAS-GFP-aequorin] (GA) transgenic flies  were use UAS-rut  and UAS- for 2 h before experiments.Preparation of flies for live 2+-response  in the MBs were monitored with an electron multiplier CCD camera  fitted onto a microscope . The setup was housed inside a tight dark box (Sciences Wares) to avoid any undesired (ambient) light contamination. We used a 20× immersion-objective lens , giving a field of view of 400 × 400 µm (512 × 512 pixels). To improve signal-to-noise ratio, data were acquired with a 0.25 s integration time (4 Hz), and 2 × 2 binning was used (1 pixel = 1.2 × 1.2 μm). To acquire and store data, each detected photon was assigned x,y-coordinates and a time point.Nicotine-induced CaAll drug applications were controlled externally using a six-way multivalves gravity perfusion system . The flow was controlled using six volumetric perfusion regulators  calibrated prior each recording session for a flow of 2 ml/min. Simultaneously, 2 ml/min of liquid were extracted from the recording chamber using a peristaltic pump  to allow a continuous flow. All tubing was bio-compliant .2O and diluted to 25 μM in Drosophila Ringers just prior experiment. Forskolin (Sigma) was prepared as a 13 mM stock in ethanol and then dissolved in Drosophila Ringers to 13 µM. IBMX (Sigma) was daily dissolved at 40 mM in 100% ethanol and diluted further in Drosophila Ringers at 200 µM final concentration. 8Br-cAMP (Sigma) was dissolved at 20 mM in 100% ethanol and diluted further in Drosophila Ringers at 200 µM final concentration. All drugs were applied using the previously described perfusion system.To stimulate the flies, we used either acetylcholine or nicotine. To investigate the roles of cAMP pathway, forskolin and IBMX were used. Nicotine (Sigma) was prepared as a 10 mM stock solution in Ht test with a control window of 240 data points (30 s) corresponding to the recorded resting phase before nicotine application. The response of the KCs was considered (quantified) between the time we obtained more than six of 10 consecutive p values above 0.025 (starting response) and more than six of 10 consecutive p values below 0.025 (end of response).The duration and the total photons (TP) were determined using an automated statistical analysis of the signal script developed at the laboratory . Briefly, a sliding window of 20 data points (5 s) was compared using http://www.r-project.org/). The dataset was analyzed via one-way ANOVA followed by a planned comparison on the predicted means to compare the level of the selected effect using the Benjamini-Hochberg's with a rank transformation (We used the Photon Viewer (2.1) software (Science Wares) written in LabView 7.1  to analyze the imaging data. Nicotine-induced bioluminescence signals are presented as photons/s (within the ROI). Image recordings were obtained from five to 15 flies for each genotype. All statistics were done using InVivoStat (2.1) software , a biostormation .Drosophila, as in mammals, the olfactory integration network is composed of at least two successive integration nodes (synapses) linked by nerve bundles. The odor, transduced by the olfactory receptors neurons (ORNs), is first integrated in the antennal lobe glomeruli , which triggers a Ca2+-response when applied to dissected brains, as reported previously . A 1 min application of nicotine  evoked a typical response pattern in the MBs. The response started in the CCB and propagated into the axonal projections at the level of the MB lobes , and peaked at approximately 9 s , which were recorded and then shown to share the same characteristics as the CS trans-heterozygotes flies , while the β/β’ and γ lobes are considered altogether as the medial lobes (ML) in this study. Moreover, due to the position of the fly’s head and the recording angle, the VLs partly overlapped with the peduncles of the MBs. Thus, in order to avoid any bias in subsequent analysis, we only quantified the response in the CCB and ML. In summary, the first component of the response corresponds to the calyx (dendritic branches), whereas the second component corresponds to the cell bodies E. The r2+-response is still only partly documented. Thus, in order to determine the cAMP pathway’s role in the MB Ca2+-response modulation, we disrupted it using two different complementary strategies: mutations and targeted MB-specific RNAi . We first tested two different mutants of the rut gene encoding AC: the loss of function 1rut, the response was reduced to ∼44% compared to the control. Altogether, these data show that a defect in rut-AC activity, resulting in decreased cAMP, leads to an overall Ca2+-response decrease in the MBs, without modifying its general kinetic proprieties.The cAMP pathway is a ubiquitous signaling pathway involved in several other critical processes, such as apoptosis  and cellrutabaga using the Gal4-UAS system  and independent of any physiological regulation. To assess the effect of acute AC stimulation, we pharmacologically induced cAMP production using forskolin, an AC stimulator  . Importantly, in the CCB, the amplitude of the two components was significantly increased by 244%, while the amplitude increase was even greater in the ML (300% increase). TP increased by 240% in the CCB and 282% in the ML  A. The kelopment . Next, w the MBs B, whichn the ML J−M. Dur1dnc, we used two additional independent approaches. First, we undertook a second RNAi (RNAi-2) experiment using a different construct (from NIG Japan). This experiment resulted in a very similar Ca2+-response pattern , did not increase response duration in the CCB, whereas chronic activity, using genetic approaches such as Gαs* and dnc-RNAi, increased this duration. These results suggest the existence of different molecular mechanisms in Ca2+ modulation in acute versus long-term cAMP increase. In order to further investigate the effect of a dnc disturbance, we overexpressed the gene using the UAS-dnc construct (cDNA), which is commonly used to rescue dnc mutations (rut mutation) with a TP, which reduces to 15% of the control response in the CCB and 16% in the ML , the duration was also severely reduced. This result is similar to the two independent RNAis and the pharmacology, while completely opposite to what was observed in 1dnc mutation.Since the above RNAi results contradicted those obtained from  pattern C to tha pattern B constr pattern J−M. The pattern , to bloc pattern . In the response E. This response J−M. Durutations . In thesutations F  (MP4 = 2.89 Mb).10.1523/ENEURO.0054-14.2015.video.1rut and dnc double-deficient lines by combining the mutation 2080rut with dnc-RNAi(1), and rut-RNAi(1) with dnc-RNAi(2)  G,H. In -RNAi(2) J,K, whi-RNAi(2) G,H. Therut AC by Ca2+-calmodulin (CaM) (supposedly in association with the nAchR odor response) present in the CCB, and the G-protein (Gs) coupled to a metabotropic receptor (associated with neuromodulators released by reward or nociceptive pathways) present in the MB lobes  and the conditioned stimulus  . TheoretMB lobes . Despitee memory . Hence, response A,B. Theresponse G,H. Theresponse I,J. Therut AC, CaM is known to interact with several Ca2+-regulated proteins through notably the Ca2+/calmodulin-dependent protein kinase II (CaMKII) (caki (renamed recently CASK)   and cakily CASK) , as welle and TP C. Howeve and TP H and the and TP C.dnc-RNAi or IBMX in a CaM-deficient context, thus combining the disruption of the degradation of cAMP with a decreased endogenous production of cAMP due to the deficit of Ca2+/CaM stimulation on rut. In both cases, these combinations failed to restore the control response level in the CCB (dnc-RNAi+CaM-RNAi = 36%)  G−J. HowDrosophila has been related with strong L&M phenotypes . In cngc-RNAi(1), the TP was decreased, but the overall response followed similar kinetics as controls (two components in the CCB and a delayed response in the ML) (cngc-RNAi(2) had a disrupted waveform: the ML response peaked before the CCB and was followed by a low-activity tail. In addition, it was narrower than the control response. The CCB response in the second RNAi was only made of one component, which had a longer rising time. These different effects between the two RNAi constructs may potentially be due to different expression levels.In order to explore the cAMP-dependent, PKA-independent effect in MB response modulation, we looked for direct potential targets of cAMP. Amongst them, the CNGs are a class of channels that can be opened by cAMP or cGMP . These c cg42260 . For ourand cngl . CNGC haand cngl . It is eand cngl . We usedenotypes A,C. Bot the ML) . cngc-RNMovie 3In vivo bioluminescence imaging of Ca2+-responses in the KCs induced by nicotine application on a fly expressing the cngc-RNAi(1). Left, Control-CS fly. Right, A fly expressing the cngc-RNAi(1). We remark that the level and the duration of activity are importantly reduced in this fly. Each frame represents 1 s of light accumulation and is shifted by 250 ms, seen at 25 frames/s. The light emission is coded in pseudocolors (2-6 photons/pixel) (MP4 = 2.89 Mb).10.1523/ENEURO.0054-14.2015.video.3cngc knocked-down background using three independent methods. First, we coexpressed Gαs* with RNAi(1) ;Gαs* flies, except for the response in the ML, which was decreased compared to controls, but similar to the cngc-RNAi(1) response. Next, we applied 8Br-cAMP to cngc-RNAi(2)  E, whichorskolin G led to-RNAi(2) I flies,UAS-mC* and UAS-R*. With the coexpression of UAS-mC* (the constitutively activated PKA subunit) and cngc-RNAi(1), the nicotinic responses are very close to those of the mC* flies, both in duration and TP. However, the double transgenes (cngc-RNAi(1);UAS-mC*) show some slight modification of their kinetics (UAS-R* and cngc-RNAi. While double-transgenic flies show a kinetic phenotype very close to what was observed with cngc-RNAi(1) alone (A vs M), their TP and response duration remained at the same value as observed in both single-transgenic flies (which were themselves very similar to each other)  using cngc knockdown.Finally, we investigated the physiological effects of the combination of the depletion of CNGC and the PKA transgenic construct: kinetics K, whichkinetics B. We th1) alone , A vs M,h other) . The rescngl, is another cAMP/cGMP-sensitive channel that is expressed at high levels in the central brain of Drosophila (2+ (cngl that gave roughly similar results (cngl-RNAi(2) (cngl-RNAi(2) flies with either forskolin or 8Br-cAMP had a strikingly similar effect on the responses (H and J vs F). The CCB amplitude was restored, but the rising phase was longer than controls, increasing the duration of the response. The response in the ML was significantly decreased and delayed compared to controls. TP in the CCB was between 398% and 509% higher than in flies containing cngl-RNAi alone , but can be supplemented in the CCB by enhancement of another class of channels sensitive to cAMP. However, CNGL seems to be essential for propagating the response to the ML. A noticeable feature of both CNGs is that their phenotype is more severe than that observed in the rut-deficient files, suggesting that either other ACs are involved in CNG stimulation or they play a similar role as the voltage-gated cation channels through their voltage-sensitive characteristics.CNG-like (CNGL), encoded by osophila . It sharosophila . This reosophila . No datahila  F. SimilAi alone , and in cngc, we combined the cngl-RNAi with UAS-mC* and UAS-R*. As observed with cngc-RNAi(1);UAS-mC* flies, the response of the cngl-RNAi(2);UAS-mC* flies were very similar to UAS-mC* flies, with few modifications of the kinetic property of the response; the response amplitude seemed to be lower than in mC*  using cngl knockdown.Finally, as previously performed with n in mC* L flies,n in mC* C,D, givic flies A,B. TheB and ML B,N, 7. 2+-response continuously, over long time periods, with high sensitivity. This probe was previously used to study odor-induced Ca2+-response in ORNs  to demonstrate that the nicotine-induced Ca2+-response is proportional to the level (higher or lower) of cAMP. The downregulation through the two alleles of rut, or via two different rut-RNAis, leads to an ∼50% Ca2+-response decrease in both the CCB and lobes. Similarly, the overexpression of dnc leads to even a stronger phenotype, suggesting the PDE activity in the response modulation is of critical importance. Conversely, the upregulation using two independent dnc-RNAis or by targeted overexpression of rut and a constitutively active G-protein subunit (Gαs*) significantly increases the Ca2+-response. Unexpectedly, however, the response is decreased with 1dnc, a contrasting result compared to the other strategies used to increase cAMP. These contradictory results are likely due to defects accumulated during fly development. Indeed, it was reported that dnc, which is expressed at various developmental stages, has diverse roles in cells and notably affects the survival of KCs throughout development, leading to smaller CCB in adult flies . Thus, it is possible that these additional noncharacterized ACs could be active in the MB lobes or the CCB.We also use a pharmacological strategy to upregulate the level of cAMP. Again, either increasing its synthesis by directly stimulating adenylyl cyclase using forskolin or diminishing its degradation using IBMX yields similar results: a huge increase of the Caophobism  or sleepophobism . Researciscussed , a numbe2+-activity in the MBs. In contrast, chronic dysregulation  did not induce any spontaneous Ca2+-activity. These results suggest that chronic misregulation of cAMP can be  compensated by other mechanisms  in neurons, while an acute modification is not compensated, and therefore is sufficient to trigger a response in the KCs. We also found that a chronic upregulation of cAMP in the calyx led to a prolonged response , possibly through the effect of PKA, while acute upregulation (by pharmacology: IBMX and forskolin) does not affect the duration. This suggests that the effect might be, at least partially, through the CNGs. Interestingly, it was reported that KCs cultured from late stage pupae showed spontaneous Ca2+-transients in a cell autonomous fashion , which induce chronic modifications of the pathway; and on the other hand, we used the pharmacological approach, which corresponds to an acute effect (stimulation or blockade). Interestingly, an acute increase of cAMP synthesis, or reducing its degradation by pharmacological approaches (forskolin or IBMX), induced Ca fashion . In addi fashion . Consequ2+-response both in CCB and lobes. Moreover, it significantly prolongs the response duration by up to 444 s in the CCB (more than fourfold). In contrast, blocking the regulatory subunit using a dominant negative form (UAS-R*) decreases the Ca2+-response. However, the latter also delays the response in the CCB. PKA likely acts by phosphorylating the K+-channels  revealed that cAMP can act by itself, and thus represents its PKA-independent effect. To corroborate these results, we increased the production of cAMP in PKA-blocked flies, which indeed increased the Ca2+-response . Furthermore, the knockdown of the different CNGs, which decreased the Ca2+-response, suggests that these channels play a crucial role in KC responsiveness. Moreover, cAMP supplementation is never sufficient to restore the kinetic properties of the response in a CNG-deficient context. The different response patterns displayed after cAMP enhancement in a CNG knockdown context suggest that the two different CNGs are playing segregated and sequential roles in cAMP-dependent regulation of MBs responsiveness: CNGC seems to play an early role on the overall response, while CNGL seems to play its role mainly in the medial lobe . The overactivation of PKA in CNG-deficient context  does not restore the kinetic proprieties of the responses but completely restores the quantitative parameters (TP and duration) observed with an overactivation of the PKA alone, suggesting that CNGs are not required for PKA’s modulation of the response but are critical for its kinetics. Inversely, the blockade of the PKA activity in CNG-deficient flies  yields to a CNG phenotype, confirming that the CNG-dependent modulation of Ca2+-response is independent of the PKA.PKA is the best known effector of cAMP. The constitutive activation of the catalytic subunit strikingly increases the Cachannels  and/or rchannels ; 1995b. rut-AC is a Ca2+/calmodulin-dependent enzyme , is consistent with the canonical model involving CaM directly in the rut stimulation. However, the unexpected striking decreased Ca2+-response in the ML due to the increase of the cAMP (IBMX or dnc-RNAi) combined with CaM knockdown, suggesting that another partner, hypothetically coregulated by cAMP (directly or through PKA) and/or CaM (directly or through CaMKII and/or CASK), could play a role in the ML regulation by inhibiting the Ca2+-response when cAMP is increased in absence of the CaM. Interestingly, the Ca2+-response of the forskolin in the CaM-RNAi  combined to either the forskolin or the IBMX or dnc-RNAi C−E, whiCaM-RNAi C resemb context F,H,J, s systems . Therefo systems , as wella larvae .2+-response between the CCB and the lobes makes calmodulin a new and interesting candidate for alternative AC activation and so potentially in relation to the coincidence detector hypothesis. However, the L&M effect of the knockdown of calmodulin, specifically within the MBs, would first have to be determined.The differential Ca


Acting as Ca2+/H+ counter-transporter, PMCA transports large quantities of protons which may affect organellar pH homeostasis. PMCA exists in four isoforms (PMCA1-4) but only PMCA2 and PMCA3, due to their unique localization and features, perform more specialized function. Using differentiated PC12 cells we assessed the role of PMCA2 and PMCA3 in the regulation of intracellular pH in steady-state conditions and during Ca2+ overload evoked by 59 mM KCl. We observed that manipulation in PMCA expression elevated pHmito and pHcyto but only in PMCA2-downregulated cells higher mitochondrial pH gradient (ΔpH) was found in steady-state conditions. Our data also demonstrated that PMCA2 or PMCA3 knock-down delayed Ca2+ clearance and partially attenuated cellular acidification during KCl-stimulated Ca2+ influx. Because SERCA and NCX modulated cellular pH response in neglectable manner, and all conditions used to inhibit PMCA prevented KCl-induced pH drop, we considered PMCA2 and PMCA3 as mainly responsible for transport of protons to intracellular milieu. In steady-state conditions, higher TMRE uptake in PMCA2-knockdown line was driven by plasma membrane potential (Ψp). Nonetheless, mitochondrial membrane potential (Ψm) in this line was dissipated during Ca2+ overload. Cyclosporin and bongkrekic acid prevented Ψm loss suggesting the involvement of Ca2+-driven opening of mitochondrial permeability transition pore as putative underlying mechanism. The findings presented here demonstrate a crucial role of PMCA2 and PMCA3 in regulation of cellular pH and indicate PMCA membrane composition important for preservation of electrochemical gradient.Plasma membrane Ca According to the chemiosmotic model, ΔΨm and ΔpH are thermodynamically equivalent to power ATP synthesis 2+ to enter the matrix 2+ in the matrix activate dehydrogenases of Krebs cycle, modulate the activity of electron transport chain and stimulate the respiratory rate 2+ overload may activate permeability transition pore (mPTP) formation allowing ions to leave the mitochondrion, thereby triggering cell death Acting as the energetic centers, they shape signaling pathways, control propagation of Ca2+ uptake in intact cells was observed at low cytosolic Ca2+ concentrations ranging from 150 to 300 nM 2+)c stimulate matrix acidification and result in ΔpH drop what is suggested to decrease oxygen consumption 2+/H+ counter-transport with a 1∶1 stoichiometry, the extrusion of Ca2+ generates large quantities of protons that are transmitted to mitochondrial matrix leading to pH decrease 2+ and protons have opposite effects on many cellular processes, the role of PMCA in the regulation of calcium homeostasis may be of fundamental importance for preservation of cellular energy.Mitochondrial Ca2+ extrusion systems for synaptic formation 2+ homeostasis following neural excitation. The existence of PMCA2 is expected to provide neuronal cells with higher sensitivity to even subtle (Ca2+)c changes. This specificity of PMCA2, which is further highlighted by its interaction with specific partners 2+ demands. The role of PMCA3 is much less understood. However, distribution, kinetic properties and scarce studies including our previous work on PC12 cells suggest that it should be also considered as an important Ca2+ player in differentiation process.PMCA exists in four isoforms PMCA1-4. Pumps 1 and 4 are ubiquitously distributed and perform a “housekeeping” role whereas the location of 2 and 3 isoforms is restricted to only some tissues where they perform more specialized functions mito and pHcyto but only in PMCA2-downregulated line increased ΔpH was observed in steady-state conditions. Also, we demonstrated that PMCA2 and PMCA3 were primarily responsible for Ca2+-dependent pHmito and pHcyto decreases and accompanying ΔpH drop during KCl stimulations. In PMCA2-downregulated cells, Ca2+ overload led to dissipation of mitochondrial membrane potential, a phenomenon that was blocked by cyclosporin and bongkrekic acid suggesting the involvement of mitochondrial permeability transition pore. Our findings point out that neuro-specific PMCA isoforms are important regulators of cellular pH in steady-state conditions and may also shape Ca2+-dependent pH changes during depolarization events.To study the potential role of neuro-specific PMCA isoforms in regulation of cellular pH, we used differentiated PC12 lines with experimentally downregulated PMCA2 or PMCA3. Due to possessing of several features characteristic for sympathetic-like neurons All reagents, if not separately mentioned, were purchased from Sigma-Aldrich (Germany). The PC12 rat pheochromocytoma cell line was obtained from ATCC (USA) and from Sigma-Aldrich (Germany). RPMI 1640 medium was from PAA (Austria). Calf and horse sera were from BioChrom (UK). Maxima SYBR Green Master Mix was from Fermentas (Canada). M-MLV Reverse Transcriptase, Trizol, Alexa Fluor 488, MitoTracker Red 580, MitoTracker Green TM, Fura-2 AM and SNARF were from Life Technologies (USA). Total RNA isolation kit was from Epicentre Biotech. (USA). Protein Assay Kit was from Bio-Rad (USA). TurboFect transfection regent was from Thermo Scientific. Primary antibodies against GFP, GAPDH, PMCA2 and PMCA3 were from Santa Cruz Biotech. (USA). Paq5000 polymerase was from Stratagene (USA). Phosphothioate oligodeoxynucleotides were from IDT (USA). Primers were synthesized in Institute of Biochemistry and Biophysics (Poland). mitoSypHer construct was kindly donated by Dr. Nicolas Demaurex.pcDNA3.1(+) vectors carrying the antisense oligonucleotides directed to either PMCA2 or PMCA3 were used to establish a stably-transfected PC12 lines as described in The model of stable transfection section.PC12 cells transient transfection with antisense probes described in 3 cells were seeded in a 96-well plate and incubated with WST-1 solution in a 1∶10 ratio for 4°C at 37°C. The absorbance of samples was spectrophotometrically measured at 450 nm.Cellular viability was assessed using WST-1 assay. 5×102, 10 mM glucose, 2.2 mM CaCl2, 10 mM NaHCO3 and 1 mM KH2PO4 at pH 7.4 and cells were loaded with 10 µM SNARF (with 0.01% pluronic acid) for 40 min at 37°C. Simultaneous pHmito/pHcyto measurements were performed in a thermostatic chamber at 37°C on TCS SP5 laser scanning confocal microscope equipped with DM6000 CFS system, DFC 360FX camera, HCX PL APO 63× objective and LAS AF Lite software (Leica). Fluorescence imaging was done with the tandem resonant scanner . SNARF and mitoSypHer were excited using argon laser low-intensity light (488 nm). The fluorescence emitted in the range of 500–530 nm was collected for mitoSypHer whereas fluorescence in two separate channels (620–765 nm and 560–600 nm) was collected for SNARF. At the end of each experiment, fluorescence changes were calibrated to absolute mitochondrial and cytosolic pH using nigericin (5 µg/ml) and monensin (5 µM) in pH 9.5–10.0 (20 mM N-methyl-D-glutamine), pH 8.0–9.0 (Tris), pH 7.0–7.5 (HEPES) or pH 5.5–6.5 (MES), as described in +/Ca2+ exchanger) in KCl-evoked pH changes was assessed in a loading buffer with 0–5 mM Na+. Ca2+- free solution contained 1 mM EDTA instead of 2.2 mM CaCl2.For mitoSypHer/SNARF dual imaging, cells expressing mitoSypHer were adhered to poly-L-lysine coated coverslips and differentiated for 2 days. Then, the culture medium was changed into a buffer containing 20 mM HEPES, 131 mM NaCl, 5 mM KCl, 1 mM MgClpH measurements section. When recording simultaneously with mitoSypHer, Fura-2 was alternately excited at 340 and 380 nm for 0.3 s through a 505 nm dichroic long pass filter and 535 nm emission filter. When recording only Fura-2, the dye was excited at 340 and 380 nm (0.3 s) through a 430 nm long way pass dichroic filter and a 510 nm bandpass emission filter. The mitoSypHer was alternately excited at 430 and 480 nm for 0.3 s through a 505 nm dichroic long pass filter and imaged with a 535 nm band pass emission filter. The contour of single cells was taken to define region of interest (ROI) from which the fluorescence was recorded. Background fluorescence was automatically subtracted from all measurements. Ratiometric images of pHmito and Ca2+ were acquired using fluorescent Axiovert S100 TV inverted microscope (Carl Zeiss) equipped with a 40× Plan Neofluar objective and attached to a cooled CCD camera .Cells expressing mitoSypHer were adhered to poly-L-lysine coated slides and loaded with 10 µM Fura-2 for 1 h at 37°C. After several washes, cells were placed in a buffer composed as described in 3 differentiated cells seeded on poly-L-lysine coated glass LabTek II chamber slides were fixed for 30 min with 3.8% paraformaldehyde and permeabilized with 0.1% Triton X-100 for 10 min at 4°C. Fixed cells were then blocked with 6% bovine serum albumin (BSA), overnight incubated with monoclonal anti-GFP antibody (1∶100) at 4°C and probed with secondary antibodies conjugated to Alexa Fluor 488 (1∶1000) for 2 h at room temperature. Next, mitochondria were stained for 15 min with MitoTracker Red 580 (40 nM). Images were taken on TCS S5 confocal laser scanning microscope with 63× objective (Leica). In a separate experiment, mitochondrial mass was determined with MitoTracker Green. In this method cells were first loaded with 150 nM MitoTracker Green FM for 30 min at 37°C and then fixed as described above. The average fluorescence intensity after background subtraction was measured with TCS S5 microscope accompanying software (Leica). Raw images were processes with CorelDraw Graphics Suite 11.Confocal microscopy was used to analyze SypHer mitochondrial targeting and mitochondrial mass. ∼103VO4 and protease inhibitor cocktail and lysed for 30 min on ice. Then, lysates were centrifuged at 10 000× g for 5 min and supernatants were boiled for 5 min in Laemmli buffer. Total protein content was quantified using Bio-Rad Protein Assay.Scraped cells were resuspended in RIPA buffer supplemented with 1 mM PMSF, 2 mM Na20 µg of total cellular proteins were resolved on a 10% SDS-PAGE and transferred onto nitrocellulose membrane using semi-dry method. Membranes were first blocked with 6% BSA in TBS-T buffer  for 1 h at room temperature and then probed overnight at 4°C with primary antibodies against GFP (1∶1000), PMCA2 (1∶750), PMCA3 (1∶750) and GAPDH (1∶1000). Following several washes with TBS-T, membranes were incubated with appropriate secondary antibodies (1∶5000) coupled with alkaline phosphatase at room temperature for 4 h. Bands were visualized using Sigma Fast BCIP/NBT used according to the manufacturer's instructions. Blots were scanned and the bands intensity was measured using GelDocEQ with Quantity One 1-D Analysis Software version 4.4.1 (Bio-Rad).−ΔΔCt method Total cellular RNA was extracted using Trizol reagent according to the procedure provided by the manufacturer. 1 µg of isolated RNA was subsequently used for cDNA synthesis with oligo(dT) primers in a 20 µl reaction mixture containing M-MLV reverse transcriptase. The cDNA templates were used to quantify gene expression level using Maxima SYBR Green Master Mix in the following conditions: 15 min at 95°C followed by 40 cycles at 95°C for 15 s, 60°C for 30 s and 72°C for 30 s. PCR reactions were performed in an AbiPrism 7000 sequence detection system (Applied Biosciences). For each PCR amplicon, a melting curve was run. The relative fold change after normalization to Gapdh expression was calculated using a comparative 2Conventional PCR used to estimate the efficiency of mitoSypHer transfection was carried out using Paq5000 polymerase in the following conditions: 5 min at 95°C followed by 30 cycles at 95°C for 1 min, 50°C for 1 min, 72°C for 2 min with a final extension at 72°C for 10 min in T300 thermocycler (Biometra) using cDNA obtained as described above. PCR products after staining with ethidium bromide were analyzed under UV light in GelDocEQ system (Bio-Rad). The primers used in PCR reactions are listed in 2PO4, 2 µM rotenone (to inhibit electron backflow to complex I), 1 µg/ml oligomycin (to maintain constant ATP/ADP ratio) and mitochondria suspended at ∼1 mg/ml. Before exposure to 10 µM CaCl2, mitochondria were energized with 5 mM succinate for 2 min. Cyclosporin (1 µM), bongkrekic acid (10 µM) or atractylate (20 µM) was added just prior to succinate. Swelling was assessed by changes in light scattering monitored spectrophotometrically at 520 nm under a continuous stirring of mitochondrial suspension.Mitochondria were isolated as described in m) was measured with TMRE (tetra-methyl-rhodamine-ethyl ester), which accumulates in mitochondrial matrix according to the Nernst equation p) was measured with DiSBAC2 trimethine oxonol). For estimation, cells were loaded in a dark with 25 nM TMRE or 1 µM DiSBAC2 for 30 min at 37°C in a buffer A containing 20 mM HEPES, pH 7.4, 2 mM CaCl2, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM glucose and analyzed by FACScan Becton Dickinson flow cytometer using an accompanying software. Cells incubated with 0.1% DMSO, used as a solvent for TMRE, were monitored to record background fluorescence, which was later subtracted from the TMRE recordings. Changes in ΔΨm were monitored in resting cells and at selected points following 59 mM KCl treatment . The reliability of TMRE to be used for ΔΨm measurement was confirmed by the pre-incubation with either 6 µM oligomycin or 1 µM FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) for 10 min. The influence of ΔΨp on mitochondrial TMRE uptake was assessed by 5 min preincubation of the cells with Ca2+-free buffer A containing 59 mM KCl before loading with 25 nM TMRE. Cyclosporin A (1 µM), bongkrekic acid (10 µM) or FK-506 (10 µM) were added to the culture medium 1 h before 25 nM TMRE loading.Mitochondrial membrane potential . TMRE-loaded cells (25 nM) were illuminated at 535 nm through a 15 nm band-pass filter for 2 min and following 30 s depolarization with 1 µM FCCP. Fluorescence at 580 nm was recorded with equipped CCD camera . Digital camera and monochromator were controlled by TILL Vision 4.0 imaging software, which was also used for data collection and processing. All procedures were performed at 37°C.P-value <0.05 was considered as statistically significant.The data are shown as means ± SEM of n separate experiments (n≥3). Statistical analyses were done using STATISTICA 8.0 (StatSoft). Normally distributed data were analyzed with one-way ANOVA with Tukey's post-hoc test. In other cases, Kruskal-Wallis non-parametric ANOVA with post-hoc Dunn's test was applied. The expression of PMCA2 or PMCA3 mRNA was mostly abolished (∼60% decrease) by an antisense mRNA targeted against it, but was not changed in mock-transfected cells . Similarex = 430 and λex = 485. To verify if PMCA2 or PMCA3 reduction could affect mitoSypHer spectral properties we performed in situ calibration, which showed a Hill slope of 0.95±0.1, 0.92±0.16 and 0.89±0.09 for C, _2 and _3 lines, respectively , followed by _3 (7.49±0.02) and control cells (7.41±0.03). In overall, the cytosolic pH was lower than mitochondrial in each line measured, consistent with chemiosmotic coupling hypothesis and experimental data mito−pHcyto) was higher in _2 line.First, we tested the properties of mitoSypHer in our modified differentiated PC12 cells: mock transfected control line (C), PMCA2-downregulated line (_2) or PMCA3-downregulated line (_3). When transfected, mitoSypHer was expressed at comparable level in all examined PC12 lines  and effiectively . The swile cells  using mi2+ loads, PC12 lines expressing mitoSypHer were loaded with Fura-2 for simultaneous recording of (Ca2+)c and pHmito. Depolarizing concentration of KCL was chosen because (i) it can mimic action potential-driven activation of voltage-dependent Ca2+ channels (VDCCs) and Ca2+ release from intracellular stores and (ii) PMCA is largely responsible for (Ca2+)i restoration after such stimulation 2+ elevations evoked by repetitive treatment with 59 mM KCl were in parallel with mitochondrial acidification c transients by 60±18% and by 32±13% in _2 and _3, respectively, during each stimulation the absolute pHmito response, in relation to control, was reduced by 54±6% in _2 line and by 35±11% in _3 line.To evaluate the effects of PMCA2 or PMCA3 reduction on pH changes during KCl-evoked Cafication , howevermito and pHcyto  to inhibit sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and with 2-APB (2-Aminoethoxydiphenyl borate), an inhibitor of store-operated calcium channels and IP3 receptor. Under low extracellular Ca2+, Tg will deplete the ER and 2-APB will block its repletion through store-operated calcium entry (SOCE), when Ca2+ will be restituted. As shown, in 2+ together with KCl showed a massive Ca2+ influx and concomitant decrease in pHmito with the magnitude comparable to non-inhibitory conditions. Moreover, we observed that PMCAs downregulation slowed down Ca2+ clearance following extracellular Ca2+ removal, and the pH recovery was delayed until (Ca2+)c was nearly at the resting level c influx and similar profiles of (Ca2+)c and ΔpH changes were observed in conditions with or without Tg c decrease upon the removal of extracellular Ca2+. La3+ (5 mM), which is known to inhibit PMCA, added during second stimulation blocked Ca2+ clearance and resulted in mitochondrial alkalinization even under low extracellular K+. We also inhibited PMCA by reducing the availability of H+ to be exchanged with cytosolic Ca2+, by increasing extracellular pH up to 9 c clearance, pointing out inhibition of PMCA under high extracellular pH. Also, alkaline pH completely attenuated KCl-induced pHmito decrease in all lines. Because all the conditions that inhibited PMCA every time decreased cellular acidification, PMCA may be considered as a main source of intracellular H+ during KCL-evoked Ca2+ loads. Thus, markedly decreased Ca2+-induced pH response in _2 and _3 lines can be attributed to diminished level of PMCA2 and PMCA3 isoforms.Further, we attempted to verify NCX role by treating cells with Tg and replacing Naa buffer . The pH  up to 9 . This ma2+)c transients was shown in this study to occur in parallel with cytosolic pH drop, we attempted to evaluate if the electron transport chain (ETC) may regulate cytosolic H+ influx to the matrix. We first blocked ETC with rotenone (inhibitor of complex I) or KCN (inhibitor of complex IV). Application of inhibitors alone caused immediate decrease in pHmito in all lines, matching the pHmito response during first KCl stimulation before the inhibitors were added , which is thought to reflect mitochondrial energization state. By using nonquenching concentration of TMRE (25 nM) we also determined whether PMCA2- or PMCA3-reduction may trigger depolarization during (Ca2+)c transients. First, we observed increased TMRE fluorescence intensity in _2 and _3 cells in steady-state conditions in relation to control , in parallel experiment we monitored ΔΨp using DiSBAC2. Elevated ΔΨp-related fluorescence observed in _2 and _3 lines indicated altered plasma membrane potential c in _2 and _3 lines than in control (2+)c  or oligomycin (6 µM). FCCP-induced depolarization resulted in massive decrease in TMRE signal in all lines coinciding with a slight increase in ΔΨal cells . Moreove control , whereasol (2+)c . This de2+)c elevations can affect ΔΨm, TMRE fluorescence was measured at selected time points of KCl stimulation or recovery, at which ΔpH alterations were the most pronounced: 10 min after 1st KCl stimulation, 10 min after KCl removal (1st recovery phase), 10 min after 2nd KCl stimulation and 10 min after 2nd KCl removal (2nd recovery phase). In control and _3 line, we observed only minor alterations in ΔΨm during KCl treatment but these little depolarization events did not correlate with the amplitude of (Ca2+)c transients, and were considered as insignificant. In contrast, in _2 line each KCl stimulation evoked ΔΨm depolarization with subsequent decrease in TMRE fluorescence by 51±18% in relation to resting level. c2+ extrusion, parallel acidification of cytosol and mitochondria is expected if the activity of PMCA remains unaffected. Such a phenomenon has been demonstrated in cortical neurons stimulated with glutamate + entering cytosol leading to pHmito increase. The regulation of mitochondrial pH and function by cytosolic Ca2+ transients requires the uptake of Ca2+ to mitochondria and both Ca2+-dependent alkalization or acidification of matrix have been demonstrated 2+ depends on ΔΨm-driven electrochemical Ca2+ gradient and the gradient of this ion between cytosol and mitochondria. Whether Ca2+ uptake into mitochondria is through mitochondrial uniporter or Ca2+/H+ exchanger, it should depolarize energized mitochondria  will also significantly diminish ΔΨpp, similarly to the effect reported in 2 signal can also be due to the fact that plasma membrane potential is created not by proton pump so protonophore cannot short-circuit it. Instead, FCCP equilibrates pH across plasma membrane carrying positively charged protons from cytoplasm to outside medium thus generating higher membrane potential. Treatment with oligomycin caused a moderate increase in pHmito what is consistent with slight hyperpolarization shown in Tetramethylrhodamine probes have been widely used to monitor ΔΨ2+ uptake may elevate ΔpH if Ca2+ charge is compensated by protons moving through ETC, we found ΔpH to be increased in _2 line. The observed raise in matrix pH in steady-state conditions could then result from charge compensation by the respiratory chain. It is attractive to propose that mitochondrial Ca2+ accumulation in _2 line will represent a major trigger coupling pH changes to the rate of ATP synthesis. Indeed, three matrix dehydrogenases activated by (Ca2+)m increases 2+)m responds to (Ca2+)c elevations. Alternatively, elevation in PMF could result from the inhibition of pathways that dissipate H+ gradient. It has been reported that mitochondrial Ca2+ uptake may inhibit ATP synthase mito may indicate higher capacity of mitochondria to produce ATP.In agreement with the prediction that mitochondrial Ca2+)c elevations, however with a PMCAs-dependent magnitude. Bearing in mind that PMCA regulates the amount of protons entering cytosol, downregulation of fast responsive PMCA2 or PMCA3 isoforms may explain weaker pH response in _2 and _3, even despite potentiation of Ca2+ influx in these lines during KCl stimulation. Different amplitudes of pHmito decreases between our lines could also reflect altered proton buffering capacity. Higher mitochondrial pH in _2 and _3 lines in steady-state conditions might affect ΔpH drop during KCl stimulation, as reduced buffering pH capacity of mitochondria in the alkaline pH was shown to underlie the loss of ΔpH upon treatment with Ca2+ mobilizing agents 2+)c particularly visible in _2 line. This effect is most likely due to over-activation of mitochondrial matrix dehydrogenases by Ca2+ transients but also indicate, that PMCAs-downregulated cells irreversibly lose a substantial part of Ca2+ clearing potency. This is additionally supported by the observed KCl-evoked higher Ca2+ influx in _2 and _3 lines. Our previous study demonstrated increased expression and concomitantly greater contribution of certain VDCCs to Ca2+ influx in PMCAs-deficient lines 2+ influx in _2 and _3 cells. It is now apparent that mitochondria of some cell types can accumulate large amounts of Ca2+ during membrane depolarization events 2+)c allows direct mitochondrial Ca2+ uptake following VDCCs activation and rapid uptake mode of the mitochondrial uniporter in response to extramitochondrial Ca2+ bursts. Nonetheless, even under the disturbed Ca2+ homeostasis and despite variations in the absolute cellular pH, all cell lines retained the ability to maintain positive matrix vs. cytosol gradient.In our study we detected pronounced cellular acidification associated with  reduction of their level led to lower degree of mitochondrial acidification as less protons entered cytoplasm; (2) the acidification did not require Ca2+ release from internal stores but was related to plasma membrane Ca2+ influx through VDCCs; (3) all agents used to inhibit PMCA prevented KCl-induced pH drop and markedly delayed Ca2+ clearance.Our data show that the main function of protons transport during Ca2+)c elevations and subsequent uptake by mitochondria should result in ΔΨm dissipation to restrict the availability of mitochondria to synthesize ATP. Decreases in ΔΨm have been observed in isolated mitochondria exposed to Ca2+ overload m in control and _3 lines, despite large pHmito and pHcyto drop during KCl-evoked (Ca2+)c elevations. In agreement with the statement that mitochondrial Ca2+ uptake must affect ΔΨm, the depolarization events could be too faint to be detected in control and _3 lines. In turn, ΔΨm depolarizations did occur in _2 cells in response to (Ca2+)c elevations. We can assume it could be due to higher ΔΨm – dependent mitochondrial Ca2+ uptake shown in this line, as ΔΨm – driven elevation of mitochondrial Ca2+ may itself dissipate ΔΨm2+ influx through VDCCs resulting in ΔΨm loss was shown in CA1 pyramidal cells in hippocampal slides 2+)c and then in (Ca2+)m may depolarize ΔΨm through promotion of Ca2+ cycling or by decreasing the ATP/ADP·Pi ratio due to higher ATP consumption by Ca2+-dependent ATPases. This would in turn increase proton backflow to the mitochondrial matrix, depolarizing ΔΨm and stimulating respiration. The net Ca2+ accumulation may occur through the mitochondrial uniporter which activity in neural tissue is particularly high m allowing net H+ extrusion by the ETC with the consequent increase in ΔpH. Another possible mechanism may involve Ca2+-dependent inhibition of ETC, as was demonstrated in mitochondria exposed to increasing Ca2+ concentration m changes during stimulation were due to collapsing of proton gradient or the exchange of charged molecules .c and higher Ψm-dependent accumulation of mitochondrial Ca2+, and/or decreased influx of cytosolic H+ due to PMACA knock-down, may lead to mitochondrial alkalization. It is believable as the amount of H+ entering cytosol in exchange for Ca2+ seems to depend on the kinetic properties of PMCA isoforms. This could explain why pH response observed during (Ca2+)c elevations was modulated in a manner dependent on isoform activity: the smallest response when PMCA2 was downregulated, which is regarded as the fastest reacting, followed by PMCA3 which is only slightly slower than PMCA2. However, during massive Ca2+ loads, the potentiation in Ca2+ influx observed in _2 line and, as a consequence, mitochondrial Ca2+ overload may lead to ΔΨm depolarization. Our data indicate that ΔΨm collapse was triggered by CsA-sensitive mechanism suggesting the involvement of mPTP opening as a possible underlying mechanism. Lack of signs for mPTP formation in _3 cells could indicate that the threshold Ca2+ concentration required for Ca2+-dependent mPTP opening has not been achieved although an increased Ca2+ influx during membrane depolarization was also observed in these cells. The overall data indicate that the relationship between mitochondria and PMCA is much more complex and intimate and exceeds far beyond a simple energetic connection. Our findings provide the evidence, that PMCA membrane composition might be of great importance for preservation of bioenergetic function of mitochondria. Therefore, changes in PMCA expression occurring i.e. in ageing brain or spinal cord injury In summary, we showed that PMCA2 and PMCA3 are responsible for dynamic regulation of cellular pH. In steady-state conditions, concomitant elevation of  The expression of mitoSypHer vector  and the corresponding protein content  assessed using PCR or monoclonal anti-GFP antibodies, respectively. GAPDH was used as an internal control. (B) Confocal images of mitoSypHer (green) in fixed cells labeled with MitoTracker Red (red) showing mitochondrial localization of mitoSypHer (merged). Insets show clear mitochondrial targeting of both probes. Scale bar 20 µm. (C) In situ calibration of mitoSypHer obtained by measuring changes in 485/430 ratio with increasing extracellular pH.(TIF)Click here for additional data file.Figure S22+.The induction of mitochondrial swelling in the presence of Ca Mitochondrial swelling induced by the addition of 10 µM CaCl2 was enhanced by atractylate (20 µM) but inhibited by bongkrekic acid (10 µM) or cyclosporin (1 µM). Swelling was assessed by light absorbance at 520 nm in a suspension of mitochondria. The absorbance at time 0 (before Ca2+ exposure) was taken as 100%.(TIF)Click here for additional data file.


Acute myeloid leukaemia (AML) is a heterogeneous group of haematological malignancy. In spite of recurrent chromosomal abnormalities present in a significant proportion of AML patients, more than 50% of the patients have a normal karyotype (NK-AML) and lack reliable molecular markers. NPM1 has been recently characterized as the most frequently mutated gene in AML Patients. The objective of the present study was to profile the karyotype and to assess the types of NPM1 gene mutations in AML patients in south India.in situ hybridization was performed with standard methods. Mutation screening of NPM1 mutation hotspot exon12 performed in DNA by PCR-SSCP and suspected samples were sequenced.A total of 200 de novo AML patients were investigated in this study. Cytogenetic G- banding analysis, Fluorescence Among 200 de novo AML patients 119 showed normal karyotype. Fourty out of two hundred patients showed mutations in NPM1 exon12. Most of the NPM1 mutation were detected in NK-AML (37/40) (92.5%).Three of them showed abnormal karyotype. The highest incidence of mutation was detected in AML-M1 with 47.5% followed by M5, M4, M2 and M3 .Predominant type of mutation was a type A mutation (c.860-863dupTCTG) (75%). Other type of mutations includes tetranucleotide insertion of TGCA (10%), CTGC (10%), GTCA (2.5%) and GTAG (2.5%). All the mutated cases were heterozygous in nature retaining wild type allele and have a distinct sequence in the protein c-terminal.Presence of 31% mutation in the NK-AML in the present study indicates that it is the most prevalent mutation in NK-AML patients in south India. NPM1 mutations are associated with good prognosis in AML. So predominance of NPM1 mutation in the AML especially in NK-AML patients suggests that it could be used as a reliable molecular marker for those patients.


C) is a cell surface glycoprotein mainly expressed in neurons, whose misfolded isoforms generate the prion responsible for incurable neurodegenerative disorders. Whereas PrPC involvement in prion propagation is well established, PrPC physiological function is still enigmatic despite suggestions that it could act in cell signal transduction by modulating phosphorylation cascades and Ca2+ homeostasis. Because PrPC binds neurotoxic protein aggregates with high-affinity, it has also been proposed that PrPC acts as receptor for amyloid-β (Aβ) oligomers associated with Alzheimer’s disease (AD), and that PrPC-Aβ binding mediates AD-related synaptic dysfunctions following activation of the tyrosine kinase Fyn. Here, use of gene-encoded Ca2+ probes targeting different cell domains in primary cerebellar granule neurons (CGN) expressing, or not, PrPC, allowed us to investigate whether PrPC regulates store-operated Ca2+ entry (SOCE) and the implication of Fyn in this control. Our findings show that PrPC attenuates SOCE, and Ca2+ accumulation in the cytosol and mitochondria, by constitutively restraining Fyn activation and tyrosine phosphorylation of STIM1, a key molecular component of SOCE. This data establishes the existence of a PrPC-Fyn-SOCE triad in neurons. We also demonstrate that treating cerebellar granule and cortical neurons with soluble Aβ(1–42) oligomers abrogates the control of PrPC over Fyn and SOCE, suggesting a PrPC-dependent mechanizm for Aβ-induced neuronal Ca2+ dyshomeostasis.The prion protein (PrP C) is an ubiquitous cell surface protein mostly expressed in the central nervous system, particularly at synapses  or prion-infected cells have implicated PrPC in the regulation of Ca2+ signaling -sensitive glutamatergic receptors (NMDA-R), which reduces Ca2+ entry into hippocampal neurons and thus protects from glutamate excitotoxicity  after inducing store-operated Ca2+ entry , which causes the redistribution of ER luminal Ca2+ sensors  into punctate clusters that recruit and activate the plasma membrane (PM) pore-forming proteins (Orai1-3) of store-operated channels  oligomers  mice and, as control, transgenic Tg46 (PrP-Tg) mice in which the expression of PrP2, and maintained at 37°C in a humidified incubator with 5% CO2. After 48 h, cells were infected with lentiviral particles and grown as previously described in Lazzari et al. (CGN primary cultures were prepared (from 7 day-old mice killed by decapitation after anesthesia with methoxyflurane), cultured and transduced with lentiviral particles as previously described in Lazzari et al. . Primaryi et al. , except 2. Twenty four hours before infection, HeLa cells were seeded onto 13 mm-diameter glass coverslips and allowed to grow to 70–80% confluence. For the production of lentiviral particles, HEK-293T cells were seeded onto 100 mm-diameter Petri dishes at ~40% confluence and transfected 24 h after plating, as described below.HeLa and HEK-293T cells were grown in Dulbecco’s modified Eagle’s medium/High-Glucose (Euroclone), supplemented with 10% fetal bovine serum (Euroclone), 100 U/ml penicillin and 100 μg/ml streptomycin (Euroclone), and maintained at 37°C in a humidified incubator with 5% CO2+] fluctuations in specific neuronal compartments, we exploited a lentiviral expression system to transduce cells with chimeric constructs encoding the Ca2+-probe aequorin (AEQ) tagged with the influenza virus hemagglutinin (HA) epitope, and linked to sequences addressing the protein to the cytosolic domains proximal to the PM  as template, and the following primers: XbaI-mRFP (forward: CGTCTAGAATGGCCTCCTCCGAGGAC) and mRFP-BglII (reverse: GAGGCGCCGGTGGAGTG-GAGATCTCG). In the second PCR, the HA-AEQ cassette was amplified using the pCDNA1-cytAEQ plasmid  and stored at −80°C. Viral titer was assessed by infecting HeLa cells by serial viral dilutions. Minimal dilutions adequate for obtaining 100% of infected cells were used to infect primary neurons and HeLa cells. All procedures for the production and use of lentiviral particles were performed in a biosafety level-2 environment. The expression and correct cellular localization of the Ca2+ probes were ascertained by immunocytochemical procedures, as described below and reported in Supplementary Figure 2.To follow [Ca2+] range that can be reliably measured with the AEQ probes is defined by the type of AEQ (WT or mutated) and of the prosthetic group coelenterazine  or modified (Coe-n), Ottolini et al., The [Ca2+]pm, [Ca2+]cyt and [Ca2+]mit transients, cells were firstly incubated  with Coe-wt  in a modified (Ca2+-free) Krebs-Ringer buffer  containing EGTA (100 μM) for simultaneously reconstituting functional AEQ and depleting intracellular Ca2+ stores. For the reconstitution of AEQer, cells were firstly incubated  in the above Ca2+-free KRB containing EGTA (1 mM), and then incubated  in Ca2+-free KRB supplemented with EGTA (500 μM), ionomycin  and Coe-n .For measuring [Ca2+]pm, [Ca2+]cyt and [Ca2+]mit, after a 1 min-perfusion step with EGTA (100 μM)-containing KRB, SOCE was elicited by perfusing cells with Ca2+ (1 mM)-containing KRB, as previously described in Lazzari et al. ; EGTA (1 mM) and bovine serum albumin ; EGTA ; EGTA . CGN were finally stimulated for SOCE by perfusion with Ca2+ (1 mM)-containing KRB.Coverslips were then placed onto a recording chamber—equipped with a perfusion system—located into the luminometer. For the measurements of  were added to the perfusing buffer before (1 min) and during VGCC activation. Instead, the SFK inhibitors [PP2  and saracatinib ], and the negative control of PP2, PP3 , were added during the reconstitution step, and kept in the perfusion buffer throughout the entire experiment. With the exception of NiCl2, all the above molecules were dissolved in dimethylsulfoxide [DMSO 0.1% (v/v)].If needed, the inhibitors of voltage-gated Ca2+ concentration by perfusion with KRB containing digitonin  and CaCl2 (10 mM). This allowed the calibration of the recorded light signal with respect to the total AEQ content, and its conversion into Ca2+ concentration, using the algorithm and the custom-made software previously described in Brini et al.  in 2% (w/v) paraformaldehyde (Sigma) in PBS. After extensive washing in PBS, cells were permeabilized with Triton X-100 , and then incubated  with a mouse monoclonal antibody to the HA epitope .Cells were then washed in PBS, and incubated  with AlexaFluor 488-conjugated anti-mouse antibody . Finally, coverslips were washed in PBS, mounted in Mowiol 40–88  and observed with a confocal microscope system (Leica TCS SP5), which also allowed the acquisition and analysis of digital images.(1–42) peptides (Keck Laboratories) were dissolved (1 mg/ml), and incubated , in 1,1,1,3,3,3-hexafluoro-2-propanol. The suspension was divided into solvent-free (by evaporation) aliquots (50 μg) and stored (−80°C). Just before use, peptides were dissolved in NaOH , sonicated (15 min on ice), diluted with PBS , and centrifuged  to remove insoluble aggregates. After determining their concentration spectrophotometrically , Aβ(1–42) peptides were aged  to form oligomers, and then administered to CGN or cortical neurons  during the AEQ reconstitution step at a final concentration of 5 μM of monomer equivalents. Routinely, Aβ(1–42) oligomerization was tested by Western blot (WB). For SDS-PAGE, Aβ(1–42) samples (~300 ng), subjected, or not, to the aging protocol, were diluted in a buffer containing SDS [3% (w/v)], β-mercaptoethanol [1.5% (v/v)], glycerol [7.5% (w/v)], Coomassie blue G-250 [0.0125% (Serva)], Tris/HCl , and run in a urea (6M)-containing Tris-Tricine gel   .Chemically synthesized human Aβ2) in KRB  in the absence, or in the presence, of the SFK inhibitors (PP2 and saracatinib), or PP3 (see above). CGN were lysed using an ice-cold buffer containing glycerol [10% (w/v)], SDS [2% (w/v)], Tris/HCl , urea (1.8 M), Na3VO4 (5 mM), and cocktails of protease and phosphatase inhibitors (Roche). Lysates, whose protein concentration was determined by a Lowry assay kit , were then adjusted to an equal protein concentration using reducing  Laemmli sample buffer .To analyze the (activating auto-) Tyr-phosphorylation of SFK members (p-SFK) and of total neuronal proteins, CGN—treated, or not, with Aβ oligomers (see above)—were incubated  in KRB [supplemented with EGTA (100 μM) in order to deplete Ca2+ stores]— were lysed (30 min on ice) at 1 × 107 cell/ml in a buffer containing Tris-HCl , NaCl (75 mM), EGTA (5 mM), Triton X-100 [1% (w/v)], sodium deoxycholate [1% (w/v)], Na3VO4, (5 mM) and cocktails of protease and phosphatase inhibitors. Cell lysates were sonicated and centrifuged  to remove insoluble materials, and the protein content of the collected supernatant was determined by a bicinchoninic acid-based assay kit . 300 μg of the supernatant proteins were then incubated  with 1.5 μg of a monoclonal antibody to STIM1 , followed by incubation with protein A-agarose . The immunoadsorbant was centrifuged , washed twice with NaCl (100 mM), EDTA (2 mM), Na3VO4 (1 mM), and Tris-HCl , resuspended in reducing Laemmli buffer, boiled (3 min) and loaded onto Mini-protean TGX precast gels . Proteins were electro-blotted onto PVDF membranes [0.45 μm pore size (Millipore)], and immunodetected using, first, the antibody to p-Tyr , and then, after stripping membranes with a commercial kit , a rabbit polyclonal antibody to STIM1 . Immunoreactive bands were visualized as described above, and for densitometric analyses the p-Tyr band intensity was normalized to that of STIM1.CGN—incubated . Preliminarily, the correct sub-cellular localization of the differently targeted AEQ probes was confirmed by immunocytochemical approaches .Previously, comparison of primary CGN cultures obtained from WT and PrP-KO mice has demonstrated that the presence of PrP2+ transients in PM microdomains (A), the bulk cytosol (B) and the mitochondrial matrix (C) following SOCE. While these data confirm that PrP-KO CGN are exposed to higher Ca2+ transients in PM microdomains than PrP-expressing neurons . Conversely, the 125 mM K+ solution induced similar (<3 μM) PM Ca2+ peaks in both CGN types ]. We found that both inhibitors reduce Ca2+ entry stimulated by 125 mM K+ irrespective of the CGN genotype , demonstrating that p-SFK was at higher levels in PrP-KO CGN than in PrP-Tg neurons both with Ca2+-depleted stores  conditions . Albeit Fyn is particularly abundant in neurons , significantly diminish the level of p-Fyn Figure , and of s Figure . The parC-dependent control of Fyn. We focused on STIM1, one of the two ER Ca2+-sensor isoforms responsible for SOCE activation  oligomers  perturb the control of PrPC over SOCE by monitoring PM Ca2+ transients. Compared to the untreated counterpart, we found that addition of soluble Aβ(1–42) oligomers augments PM Ca2+ peaks of PrP-Tg CGN to the same value detected in untreated PrP-KO CGN  is strictly dependent on the presence of PrPC. Importantly, an identical PrPC-dependent alteration of SOCE was also observed in primary cortical neurons  oligomers to disturb SOCE only in PrP-Tg CGN was paralleled by the action on Fyn, because once again oligomeric Aβ(1–42) increases the level of active Fyn in these neurons, both under SOCE-activating  increases SOCE by impairing the PrPC-dependent downregulation of Fyn.The capacity of Aβg Figure  and basa2+ oscillations in isogenic primary CGN expressing, or not, PrPC, we report here that PrPC restricts the accumulation of Ca2+ in the cytosol and mitochondria of neurons following SOCE. PrPC accomplishes this task by limiting SOC-mediated Ca2+ entry and by increasing Ca2+ uptake by the ER, which likely depends on the capacity of PrPC to upregulate the expression of the sarco/endoplasmic reticulum Ca2+-ATPase pump . In addition to the mandatory variation in ER Ca2+ levels, SOCE is regulated by different mechanizms that include the glutathionylation and phosphorylation of STIM proteins , is highly conserved among different mammalian species, and is also present in STIM2. Further studies will clarify if Tyr361 is the actual phosphorylation site by SFK and if this is a mechanizm to functionally regulate both STIM isoforms.Mechanistically, we identified Fyn as a molecular intermediate enabling PrPC in the regulation of key cell processes, ranging from embryogenesis and neuritogenesis to, at large, neuroprotective signaling , is activated after Aβ docking to PrPC  oligomers disrupts the PrPC-Fyn-SOCE triangle because such a treatment increases activation of both Fyn and SOCE in PrP-expressing, but not in PrP-KO, CGN. Importantly, the PrPC-dependent effect of Aβ oligomers on SOCE was also observed in cortical neurons that, together with hippocampal neurons, are the preferential target of Aβ toxicity  toxicity could involve PrPC displacement from the role of sentinel against neuronal Ca2+ overload. Additional studies are needed to clarify whether this effect is consequent to a structural modification of PrPC, or a dislodgment from natural interacting partners, or a modification of the membrane lipid architecture surrounding the protein. Likewise, also the link between Fyn and PrPC in CGN needs to be elucidated. We exclude the involvement of mGluR5  results in increased activation of Fyn and SOCE. However, given that PrP-KO mice show no gross phenotype, nor overt signs of neurodegeneration, the alterations observed in this work cannot be sufficient to account for AD pathology. Nonetheless, they could act as necessary events that, combined with other (PrPC-dependent and/or PrPC-independent) insults by Aβ oligomers, eventually contribute to AD-related neuronal damage. Further studies will be necessary to demonstrate whether Aβ oligomers perturb local Ca2+ fluxes different from SOCE, in particular those arising from NMDA-R that have already been functionally associated to PrPC ; ADM, CP and AB analyzed data; AB, AFH and MCS wrote the article.This work was supported by the Italian Ministry of University and Research (PRIN/2010 to DL), the University of Padova (PRAT CPDA121988/12 to MCS), and the Australian Academy of Sciences (to AFH).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Dear Editor,Therapy-related myeloid neoplasms (t-MNs) are a unique clinical entity occurring as late complication of chemotherapy and radiotherapy administered for a primary disease . AccordiTP53 gene which is frequently mutated in t-AMLs exhibiting a potentially important role in leukemogenesis [In this study, we selected patients with therapy-related AML (t-AML) following cytotoxic treatment of malignant lymphomas as bone marrow (BM) biopsies are routinely performed during their initial staging procedures. We focused on the ogenesis . We idenogenesis . HoweverTP53 mutation in the patient’s BM obtained at the time of the lymphoma staging  which showed that the relative proportion of mutated cells increased substantially in the t-AML specimen Below is the link to the electronic supplementary material.


Treatment of GLUTag cells with a Ca2+/calmodulin-dependent kinaseII (CaMKII) inhibitor (KN-93) abolished D3R-stimulated GLP-1 secretion. In addition, treatment of GLUTag cells with D3R resulted in activation of CaMKII. Pre-treatment of cells with a G protein-coupled receptor (GPR) 40/120 antagonist (GW1100) also significantly decreased D3R-stimulated GLP-1 secretion. These observations suggest that D3R stimulates GLP-1 secretion in GLUTag cells, and that stimulation of GLP-1 secretion by D3R is mediated via Ca2+-CaMKII pathway, which may possibly be mediated by GPR40/120. These findings provide a possible molecular mechanism of GLP-1 secretion in intestinal L-cells mediated by foods or drugs and demonstrate a novel biological function of anthocyanins in regards to GLP-1 secretion.Glucagon-like peptide-1 (GLP-1) is an incretin hormone secreted from enteroendocrine L-cells. Although several nutrients induce GLP-1 secretion, there is little evidence to suggest that non-nutritive compounds directly increase GLP-1 secretion. Here, we hypothesized that anthocyanins induce GLP-1 secretion and thereby significantly contribute to the prevention and treatment of diabetes. Delphinidin 3-rutinoside (D3R) was shown to increase GLP-1 secretion in GLUTag L cells. The results suggested that three hydroxyl or two methoxyl moieties on the aromatic ring are essential for the stimulation of GLP-1 secretion. Notably, the rutinose moiety was shown to be a potent enhancer of GLP-1 secretion, but only in conjunction with three hydroxyl moieties on the aromatic ring (D3R). Receptor antagonist studies revealed that D3R-stimulates GLP-1 secretion involving inositol 1,4,5-trisphosphate receptor-mediated intracellular Ca Glucagon-like peptide-1 (GLP-1) secreted from enteroendocrine L-cells is one type of incretin and stimulates glucose-dependent insulin secretion and proliferation of pancreatic β-cells . Due to in vitro and in vivo and include certain fatty acids [These approaches are effective for controlling blood glucose levels in type 2 diabetic patients. However, an alternative therapeutic approach is to increase endogenous GLP-1 secretion through modulation of the secretory mechanisms in intestinal L cells using pharmaceutical agents or dietary factors. This novel therapeutic strategy may help treat diabetes and decrease the required doses of other diabetic medicines. A number of nutrients and small molecules are reported to increase GLP-1 secretion ty acids –12, as wty acids –15. Protty acids –19.We previously demonstrated that curcumin, a yellow pigment isolated from turmeric, markedly increases GLP-1 secretion in the murine GLUTag cell line . DespiteAnthocyanins are flavonoid phytopigments  that are3R)-mediated intracellular Ca2+ mobilization-Ca2+/calmodulin-dependent kinase II (CaMKII) pathway.In the present study, we demonstrated that delphinidin 3-rutinoside (D3R) significantly increased GLP-1 secretion in GLUTag cells, and clarified the structure-activity relationship using anthocyanin derivatives. Moreover, this increase was found to involve the inositol 1,4,5-trisphosphate receptor (IPKaempferia parviflora L.) using preparative ODS-HPLC. The structure of isolated Mal3R was verified by MS and NMR. The chemical structures of these anthocyanins are shown in The purity of all administered chemicals was over 98%. Commercially available purified anthocyanins  were obtained from Tokiwa Phytochemical  and Extrasynthèse . Malvidin 3-rutinoside  was isolated and purified from tuber dry powder of black ginger  supplemented with 0.5% (w/v) fatty acid-free BSA to starve the cells for 1 h. The cells were then incubated with various test compounds in glucose free Krebs-Ringer bicarbonate buffer containing 0.5% fatty acid-free BSA for 2 h [The murine GLUTag L cell line   or D3R for the indicated time periods and conditions and then lysed . SupernaCells were extracted by treatment with 0.1 M HCl to avoid degradation of cAMP, and the cAMP levels of the extract were measured using a cyclic AMP EIA kit  according to the manufacturer’s instructions.P values < 0.05.All data are expressed as the means ± SEM. Differences in the GLP-1 secretion or cytosolic cAMP concentrations of GLUTag cells were compared by the Tukey-Kramer test. In other cases, the data were analyzed by two-way ANOVA. If the interaction effect of two components (D3R and inhibitor) was significant, the Tukey-Kramer test was performed to compare the differences between the groups. For all statistical tests, values without a common letter  on the bar graphs are significantly different at We first screened various types of anthocyanins for their ability to stimulate GLP-1 secretion in GLUTag cells. The screening revealed that three anthocyanins, D3R, which contains three B-ring hydroxyl moieties and rutinose, Del (the aglycone of D3R), and Mal, which has two methoxyl moieties on the B-ring, resulted in a significant increase in the secretion of GLP-1 by GLUTag cells . Notably2+ levels via the opening of Ca2+ channels or mobilization of intracellular Ca2+ stores leads to GLP-1 secretion [2+ levels, GLUTag cells were first treated with various Ca2+ fluorescent indicators , and the intracellular fluorescence was monitored during exposure to D3R. However, the modulation of intracellular Ca2+ levels in response to D3R administration could not be detected, because the inherent fluorescence of D3R interfered with the detection of the fluorescent Ca2+ indicators (data not shown). Therefore, to indirectly elucidate the intracellular Ca2+ response of GLUTag cells to D3R, we used BAPTA-AM (intracellular Ca2+ chelator) and examined the effect of intracellular Ca2+ chelation on D3R-stimulated GLP-1 secretion. Pre-treatment of GLUTag cells with a BAPTA-AM completely blocked D3R-induced GLP-1 secretion  and IP3R. Here, GLUTag cells pre-treated with the RyR antagonist dantrolene did not canceled increased GLP-1 secretion after exposure to D3R , which is regulated via the binding of Ca2+-calmodulin, resulting in autophosphorylation of stimulatory and inhibitory sites [Caocytosis . One of ry sites . Previoury sites , 38. As ry sites , we spec3-mediated release of intracellular Ca2+ [It is known that GLP-1 secretion is stimulated via the activation of G protein-coupled receptors (GPRs) in intestinal L cells . For exalar Ca2+ , 44, whelar Ca2+ , 45.In addition, recent studies have shown that GLP-1 secretagogues are stimulated by the protein kinase C (PKC) or mitogen-activated or extracellular signal-regulated protein kinase (MEK)-extracellular signal-regulated kinase (ERK) pathways , 46–48. The effect of the GPR40/120 antagonist GW1100 on D3R-stimulated GLP-1 secretion was examined . The preIn the present study, we hypothesized that anthocyanins have multiple biological functions and aid in GLP-1 secretion, and that anthocyanins have an anti-diabetic effect through the stimulation of GLP-1 secretion. Our findings demonstrate that anthocyanins, particularly D3R, have a significant effect on GLP-1 secretion and therefore have a unique pharmacological function in the regulation of glucose homeostasis.The treatment of GLUTag cells with various anthocyanins significantly stimulated GLP-1 secretion. The results suggested that modification of the aromatic ring with at least three hydroxyl or two methoxyl moieties is essential for stimulating GLP-1 secretion. Notably, the rutinose moiety was shown to be a potent enhancer of GLP-1 secretion, but only in conjunction with three hydroxyl moieties on the aromatic ring (D3R), as rutinose alone did not stimulate GLP-1 secretion. Interestingly, Mal3R, which contains two methoxyl groups on the aromatic ring and a rutinose moiety, did not significantly stimulate GLP-1 secretion. To our knowledge, this is the first study to demonstrate that certain anthocyanins enhance GLP-1 secretion.In general, anthocyanins exhibit low bioavailability . In this2+ via endogenous Ca2+ store mobilization and/or extracellular influx from L-type Ca2+ channels results in activation of CaMKII, which may be involved in GLP-1 secretion, as well as curcumin-induced GLP-1 secretion [2+ chelator (BAPTA-AM). Furthermore, D3R-induced GLP-1 secretion was significantly inhibited by an IP3 receptor antagonist (2-APB), but not by a RyR antagonist (dantrolene) or L-type Ca2+ channel blocker (verapamil). Taken together, these results indicate that D3R stimulates intracellular Ca2+ mobilization via IP3R.To explain how D3R induces GLP-1 secretion in GLUTag cells, two molecular mechanisms are possible: 1) elevation of cytosolic Caecretion ; and 2) ecretion , 45–48. 2+-mediated signaling molecules is CaMKII, which is regulated via binding to Ca2+-calmodulin, resulting in autophosphorylation of stimulatory and inhibitory sites [2+ levels stimulates insulin secretion in β-cells [2+ elevation. Supporting this speculation, we recently showed that curcumin-induced GLP-1 secretion is regulated by the Ca2+-CaMKII pathway [2+-CaMKII pathway regulates D3R-induced GLP-1 secretion in GLUTag cells. However, the exact exocytosis mechanism of GLP-1 from L cell remains unclear. A recent study indicated that vesicle-associated membrane protein 2 (VAMP2), which is involved in vesicular transport and membrane fusion, has an essential role in GLP-1 exocytosis [One important target of Cary sites . Interes β-cells , 38, pos pathway . As showocytosis . Interesocytosis , suggest2+ mobilization via IP3R is involved in this process. Although it is also possible that D3R-stimulated GLP-1 secretion is regulated by the GPR119- or TGR5-related cAMP/PKA, PKCζ, or MEK-ERK pathways, our results do not support this conclusion.The present study raises another question regarding the primary molecular target of D3R. GPR agonists have been shown to stimulate GLP-1 secretion in intestinal L cells –43. Here2+-CaMKII pathway activation, which may be mediated by GPR40/120 .GLUTag cells were pre-treated with vehicle (0.1% DMSO) or (A) PKC inhibitor , (B) MEK inhibitor  for 15 min, followed by treatment with vehicle or D3R (100 μM) for 2 h without washing out. GLP-1 levels in the medium were measured by ELISA. Secreted GLP-1 levels are expressed as the fold change of the control levels (= 1.0). Values are expressed as the means ± SEM, n = 3. Values without a common letter (a and b) are significantly different at (TIF)Click here for additional data file.