Zobrazení zdroje stránky Pacewoodward2161

Carbon monoxide (CO) remains the most common cause of human poisoning. The consequences of CO poisoning include cardiac dysfunction, brain injury, and death. CO causes toxicity by binding to hemoglobin and by inhibiting mitochondrial cytochrome c oxidase (CcO), thereby decreasing oxygen delivery and inhibiting oxidative phosphorylation.&nbsp; We have recently developed a CO antidote based on human neuroglobin (Ngb-H64Q-CCC). This molecule enhances clearance of CO from red blood cells in vitro and in vivo. Herein, we tested whether Ngb-H64Q-CCC can also scavenge CO from CcO and attenuate CO-induced inhibition of mitochondrial respiration. Heart tissue from mice exposed to 3% CO exhibited a 42% ± 19% reduction in tissue respiration rate and a 33% ± 38% reduction in CcO activity compared with unexposed mice. [https://www.selleckchem.com/products/d-lin-mc3-dma.html selleck inhibitor] Intravenous infusion of Ngb-H64Q-CCC restored respiration rates to that of control mice correlating with higher electron transport chain CcO activity in Ngb-H64Q-CCC-treated compared with PBS-treated, CO-poisoned mice. Further, using a Clark-type oxygen electrode, we measured isolated rat liver mitochondrial respiration in the presence and absence of saturating solutions of CO (160 µM) and nitric oxide (NO; 100 µM). Both CO and NO inhibited respiration, and treatment with Ngb-H64Q-CCC (100 and 50 µM, respectively) significantly reversed this inhibition. These results suggest that Ngb-H64Q-CCC mitigates CO toxicity by scavenging CO from carboxyhemoglobin, improving systemic oxygen delivery, and reversing the inhibitory effects of CO on mitochondria. We conclude that Ngb-H64Q-CCC, or other CO scavengers, demonstrate potential as antidotes that reverse the clinical and molecular effects of CO poisoning. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.Nucleoside analogs are a valuable experimental tool. Incorporation of these molecules into newly synthesized DNA (i.e. pulse-labeling) is used to monitor cell proliferation or to isolate nascent DNA. Some of the most common nucleoside analogs used for pulse-labeling of DNA in cells are the deoxypyrimidine analogs 5-ethynyl-2'-deoxyuridine (EdU) and 5-ethynyl-2'-deoxycytidine (EdC). Click chemistry enables conjugation of an azide molecule tagged with a fluorescent dye or biotin to the alkyne of the analog, which can then be used to detect incorporation of EdU and EdC into DNA. The use of EdC is often recommended because of the potential cytotoxicity associated with EdU during longer incubations. Here, by comparing the relative incorporation efficiencies of EdU and EdC during short 30-min pulses, we demonstrate significantly lower incorporation of EdC than of EdU in non-infected human fibroblast cells or in cells infected with either human cytomegalovirus (HCMV) or Kaposi's sarcoma-associated herpesvirus (KSHV). Interestingly, cells infected with herpes simplex virus type-1 (HSV-1) incorporated EdC and EdU at similar levels during short pulses. Of note, exogenous expression of HSV-1 thymidine kinase (TK) increased the incorporation efficiency of EdC. These results highlight the limitations when using substituted pyrimidine analogs in pulse-labeling and suggest that EdU is the preferable nucleoside analog for short pulse-labeling experiments, resulting in increased recovery and sensitivity for downstream applications. This is an important discovery that may help to better characterize the biochemical properties of different nucleoside analogs with a given kinase, ultimately leading to significant differences in labeling efficiency of nascent DNA. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.Coenzyme Q (Qn) is a vital lipid component of the electron transport chain that functions in cellular energy metabolism and as a membrane antioxidant. In the yeast Saccharomyces cerevisiae, coq1-coq9 deletion mutants are respiratory incompetent, sensitive to lipid peroxidation stress, and unable to synthesize Q6 The yeast coq10 deletion mutant is also respiratory deficient and sensitive to lipid peroxidation, yet continues to produce Q6 at an impaired rate. Thus, Coq10 is required for the function of Q6 in respiration and as an antioxidant and is believed to chaperone Q6 from its site of synthesis to the respiratory complexes. In several fungi, Coq10 is encoded as a fusion polypeptide with Coq11, a recently identified protein of unknown function required for efficient Q6 biosynthesis. Because "fused" proteins are often involved in similar biochemical pathways, here we examined the putative functional relationship between Coq10 and Coq11 in yeast. We used plate growth and Seahorse assays and LC-MS/MS analysis to show that COQ11 deletion rescues respiratory deficiency, sensitivity to lipid peroxidation, and decreased Q6 biosynthesis of the coq10Δ mutant. Additionally, immunoblotting indicated that yeast coq11Δ mutants accumulate increased amounts of certain Coq polypeptides and display a stabilized CoQ synthome. These effects suggest that Coq11 modulates Q6 biosynthesis, and that its absence increases mitochondrial Q6 content in the coq10Δcoq11Δ double mutant. This augmented mitochondrial Q6 content counteracts the respiratory deficiency and lipid peroxidation sensitivity phenotypes of the coq10Δ mutant. This study further clarifies the intricate connection between Q6 biosynthesis, trafficking, and function in mitochondrial metabolism. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.Endorepellin, the C-terminal fragment of the heparan sulfate proteoglycan perlecan, influences various signaling pathways in endothelial cells by binding to VEGFR2. In this study, we discovered that soluble endorepellin activates the canonical stress signaling pathway&nbsp; consisting of PERK, eIF2α, ATF4 and GADD45α. Specifically, endorepellin evoked transient activation of VEGFR2 which in turn phosphorylated PERK at Thr980. Subsequently, PERK phosphorylated eIF2αat Ser51, thereby upregulating its downstream effector proteins ATF4 and GADD45a. RNAi-mediated knockdown of PERK or &nbsp;eIF2α abrogated the endorepellin-mediated upregulation of GADD45α, the ultimate effector protein of this stress signaling cascade. To functionally validate these findings, we utilized an ex vivo model of angiogenesis. Exposure of the aortic rings embedded in 3D fibrillar collagen to recombinant endorepellin for 2-4 h activated PERK and induced GADD45a vis-à-vis vehicle-treated counterparts. Similar effects were obtained with the established cellular stress inducer tunicamycin.