Requirement for the N-Terminal Coiled-Coil Domain for Expression and Function, but not Subunit Interaction of, the ADPR-Activated TRPM2 Channel

Transient receptor potential melastatin 2 (TRPM2) proteins form multiple-subunit complexes, most likely homotetramers, which operate as Ca2+-permeable, nonselective cation channels activated by intracellular ADP-ribose (ADPR) and oxidative stress. Each TRPM2 channel subunit is predicted to contain two coiled-coil (CC) domains, one in the N-terminus and the other in the C-terminus. Our recent study has shown that the C-terminal CC domain plays an important, but not exclusive, role in the TRPM2 channel assembly. This study aimed to examine the potential role of the N-terminal CC domain. Domain deletion dramatically reduced protein expression and abolished ADPR-evoked currents but did not alter the subunit interaction. Deletion of both CC domains strongly attenuated the subunit interaction, confirming that the C-terminal CC domain is critical in the subunit interaction. Glutamine substitutions into individual hydrophobic residues at positions a and d in the heptad repeats to disrupt the CC formation had no effect on protein expression, subunit interaction, or ADPR-evoked currents. Mutation of Ile658 to glutamine, which did not perturb the CC formation, decreased ADPR-evoked currents without affecting protein expression, subunit interaction, or membrane trafficking. These results collectively suggest the requirement for the N-terminal CC domain for protein expression and function, but not subunit interaction, of the TRPM2 channel

ISO observations of the interacting galaxy Markarian 297: with the powerful supernova remnant 1982aa

Markarian (Mkn) 297 is a complex system with two interacting galaxies. Observations were made with ISO using ISOCAM, ISOPHOT and LWS. We present ISOCAM maps at 6.7, 7.7, 12 and 14.3 microns which, with PHT-S spectrometry of the central interacting region, probe the dust obscured star formation and dust properties. ISOCAM reveals that the strongest emission region in the four MIR bands is completely unremarkable at visible and near-IR (e.g. 2MASS) wavelengths, and does not coincide with the nuclear region of either colliding galaxy. It shares this striking characteristic with the overlap region of the colliding galaxies in the Antennae (NGC 4038, 4039), the intragroup region of Stephan's Quintet, and IC 694 in the interacting system Arp 299. At 15 microns, the hidden source in Mkn 297 is, respectively, 14.6 and 3.8 times more luminous than the hidden sources in the Antennae (NGC 4038/4039) and Stephan's Quintet. Numerical simulations indicate that we see the Mkn 297 interaction about 1.5 x 10e8 years after the collision. ISOCAM shows knots and ridges of emission. The 14.3/7.7 micron ratio map implies widespread strong star formation. Strong emission features were detected in the ISOPHOT spectrum, while [OI], [OIII] and [CII] emission lines were seen with LWS. Using data from the three instruments, luminosities and masses for two dust components were determined. The total infrared luminosity is approximately 10e11 L_sol, marginally a LIRG. A 1979 supernova generated one of the most powerful known radio remnants (SN 1982aa) close to the strongest MIR source and identified with star forming region 14 in the optical. This exceptional supernova explosion may have been accompanied by a GRB, and a search for a GRB in this direction in contemporaneous satellite data is recommended.Comment: Accepted for publication in Astronomy and Astrophysics Updated to better use recent SN/GRB work and tune terminology in Sec. 4.

Signalling mechanisms mediating Zn2+-induced TRPM2 channel activation and death cell in microglial cells

Excessive Zn2+ causes brain damage via promoting ROS generation. Here we investigated the role of ROS-sensitive TRPM2 channel in H2O2/Zn2+-induced Ca2+ signalling and cell death in microglial cells. H2O2/Zn2+ induced concentration-dependent increases in cytosolic Ca2+ concentration ([Ca2+]c), which was inhibited by PJ34, a PARP inhibitor, and abolished by TRPM2 knockout (TRPM2-KO). Pathological concentrations of H2O2/Zn2+ induced substantial cell death that was inhibited by PJ34 and DPQ, PARP inhibitors, 2-APB, a TRPM2 channel inhibitor, and prevented by TRPM2-KO. Further analysis indicate that Zn2+ induced ROS production, PARP-1 stimulation, increase in the [Ca2+]c and cell death, which were suppressed by chelerythrine, a protein kinase C inhibitor, DPI, a NADPH-dependent oxidase (NOX) inhibitor, GKT137831, a NOX1/4 inhibitor, and Phox-I2, a NOX2 inhibitor. Furthermore, Zn2+-induced PARP-1 stimulation, increase in the [Ca2+]c and cell death were inhibited by PF431396, a Ca2+-sensitive PYK2 inhibitor, and U0126, a MEK/ERK inhibitor. Taken together, our study shows PKC/NOX-mediated ROS generation and PARP-1 activation as an important mechanism in Zn2+-induced TRPM2 channel activation and, TRPM2-mediated increase in the [Ca2+]c to trigger the PYK2/MEK/ERK signalling pathway as a positive feedback mechanism that amplifies the TRPM2 channel activation. Activation of these TRPM2-depenent signalling mechanisms ultimately drives Zn2+-induced Ca2+ overloading and cell death

Cisplatin-DNA adduct formation in patients treated with cisplatin-based chemoradiation: lack of correlation between normal tissues and primary tumor

Contains fulltext : 69595.pdf (publisher's version ) (Closed access)PURPOSE: In this study, the formation of cisplatin-DNA adducts after concurrent cisplatin-radiation and the relationship between adduct-formation in primary tumor tissue and normal tissue were investigated. METHODS: Three intravenous cisplatin-regimens, given concurrently with radiation, were studied: daily low-dose (6 mg/m(2)) cisplatin, weekly 40 mg/m(2), three-weekly 100 mg/m(2). A (32)P-postlabeling technique was used to quantify adducts in normal tissue [white blood cells (WBC) and buccal cells] and tumor. RESULTS: Normal tissue samples for adduct determination were obtained from 63 patients and tumor biopsies from 23 of these patients. Linear relationships and high correlations were observed between the levels of two guanosine- and adenosine-guanosine-adducts in normal and tumor tissue. Adduct levels in tumors were two to five times higher than those in WBC (P<0.001). No significant correlations were found between adduct levels in normal tissues and primary tumor biopsies, nor between WBC and buccal cells. CONCLUSIONS: In concurrent chemoradiotherapy schedules, cisplatin adduct levels in tumors were significantly higher than in normal tissues (WBC). No evidence of a correlation was found between adduct levels in normal tissues and primary tumor biopsies. This lack of correlation may, to some extent, explain the inconsistencies in the literature regarding whether or not cisplatin-DNA adducts can be used as a predictive test in anticancer platinum therapy

Substrate cycling based fluorometric assay for dihydroxyacetone phosphate.

International audienceA sensitive fluorometric assay for the determination of dihydroxyacetone phosphate (DHAP) is reported here. DHAP is reduced to l-glycerol-3-phosphate with NADH-dependent α-glycerophosphate dehydrogenase. DHAP recycling is provided by oxidation reaction catalysed by α-glycerophosphate oxidase to release hydrogen peroxide. The reaction of hydrogen peroxide with Amplex® Red reagent under horseradish peroxidase catalysis leads to the fluorescent product resorufin. The limit of detection of DHAP is estimated at 1 pmol which is roughly 2250 fold more sensitive than the usual DHAP assay based on the detection of NADH by spectrophotometry. This assay is ready-to-use for automated medium-throughput screening

Circadian variation of the energetic and hormonal response to a meal

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