Shortening and removal of the polyadenylate poly(A) tail of mRNA, a process called deadenylation, is a key step in mRNA decay that is mediated through the CCR4-NOT (carbon catabolite repression ...4-negative on TATA-less) complex. In our investigation of the regulation of mRNA deadenylation in the heart, we found that this complex was required to prevent cell death. Conditional deletion of the CCR4-NOT complex components Cnot1 or Cnot3 resulted in the formation of autophagic vacuoles and cardiomyocyte death, leading to lethal heart failure accompanied by long QT intervals. Cnot3 bound to and shortened the poly(A) tail of the mRNA encoding the key autophagy regulator Atg7. In
-depleted hearts,
expression was posttranscriptionally increased. Genetic ablation of
, but not
, increased survival and partially restored cardiac function of
or
knockout mice. We further showed that in
-depleted hearts, Atg7 interacted with p53 and modulated p53 activity to induce the expression of genes encoding cell death-promoting factors in cardiomyocytes, indicating that defects in deadenylation in the heart aberrantly activated Atg7 and p53 to promote cell death. Thus, mRNA deadenylation mediated by the CCR4-NOT complex is crucial to prevent Atg7-induced cell death and heart failure, suggesting a role for mRNA deadenylation in targeting autophagy genes to maintain normal cardiac homeostasis.
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The formation of blood clots in blood vessels causes severe ischemic diseases such as cerebral infarction and myocardial infarction. While searching for microbial products that ...increase fibrinolytic activity using an in vitro fibrin degradation assay, we found malformin A1, a disulfide form of cyclo(–d-Cys-d-Cys-l-Val-d-Leu-l-Ile–), as an active compound. In this study, we synthesized malformin derivatives using a solid-phase peptide synthesis method and evaluated their fibrinolytic activity and cytotoxicity. Reduction of the disulfide bond and linearization of the cyclic peptide frame decreased the pro-fibrinolytic activity. Substitution of a branched-chain amino acid with lysine resulted in loss of activity. However, protection of the amino group in the lysine derivatives by the tert-butoxycarbonyl (Boc) group rescued the inactivity. Furthermore, the phenylalanine derivatives also exhibited a similar pro-fibrinolytic effect compared to malformin A1. These results suggest that the disulfide bond, the cyclic peptide frame, and the bulky hydrophobic side chains play a crucial role in the pro-fibrinolytic activity of malformin. The effective dose of the active derivatives for the in vitro fibrin degradation showed similar ranges (1–5μM), while the order of cytotoxic potency for the active derivatives was as follows: Phe-derivatives>BocLys-derivatives>malformin A1>reduced form. These results showed no correlation between pro-fibrinolytic activity and cytotoxicity, suggesting the possibility of the synthesis for non-toxic malformin derivatives possessing the activity.
Glechoma hederacea L. (Labiatae) has been used in folk medicine to treat various ailments for centuries. We investigated the effects of G. hederacea extract on melanogenesis in B16 melanoma cells. It ...significantly reduced both the cellular melanin content and tyrosinase activity in a concentration-dependent manner. An MTT assay did not reveal any obvious cytotoxicity. Furthermore, we found that G. hederacea extract decreased tyrosinase and microphthalmia-associated transcription factor protein expression, but did not inhibit tyrosinase-related protein-1 and tyrosinase-related protein-2 expression. RT-PCR analysis indicated that the antimelanogenic effect of G. hederacea extract might be due to inhibition of tyrosinase gene transcription. Moreover, this effect is regulated via suppression of microphthalmia-associated transcription factor protein expression. Our data indicate that G. hederacea extract inhibits melanin synthesis in B16 melanoma cells but is not cytotoxic. Hence it might prove a useful therapeutic agent for treating hyperpigmentation and an effective component of whitening cosmetics.
Pharmacological interventions to enhance fibrinolysis are effective for treating thrombotic disorders. Utilizing the in vitro U937 cell line-based fibrin degradation assay, we had previously found a ...cyclic pentapeptide malformin A
(MA
) as a novel activating compound for cellular fibrinolytic activity. The mechanism by which MA
enhances cellular fibrinolytic activity remains unknown. In the present study, we show that RSK1 is a crucial mediator of MA
-induced cellular fibrinolysis. Treatment with rhodamine-conjugated MA
showed that MA
localizes mainly in the cytoplasm of U937 cells. Screening with an antibody macroarray revealed that MA
induces the phosphorylation of RSK1 at Ser380 in U937 cells. SL0101, an inhibitor of RSK, inhibited MA
-induced fibrinolytic activity, and CRISPR/Cas9-mediated knockout of RSK1 but not RSK2 suppressed MA
-enhanced fibrinolysis in U937 cells. Synthetic active MA
derivatives also induced the phosphorylation of RSK1. Furthermore, MA
treatment stimulated phosphorylation of ERK1/2 and MEK1/2. PD98059, an inhibitor of MEK1/2, inhibited MA
-induced phosphorylation of RSK1 and ERK1/2, indicating that MA
induces the activation of the MEK-ERK-RSK pathway. Moreover, MA
upregulated the expression of urokinase-type plasminogen activator (uPA) and increased uPA secretion. These inductions were abrogated in RSK1 knockout cells. These results indicate that RSK1 is a key regulator of MA
-induced extracellular fibrinolytic activity.
Cadmium is a toxic pollutant with occupational and environmental significance, due to its diverse toxic effects. Supramolecules that conjugate and decontaminate toxic metals have potential for use in ...treatment of cadmium intoxication. In addition, metal‐coordinating ability has been postulated to contribute to the cytotoxic effects of anti‐tumor agents such as cisplatin or bleomycin. Thiacalixarenes, cyclic oligomers of p‐alkylphenol bridged by sulfur atoms, are supramolecules known to have potent coordinating ability to metal ions. In this study, we show that cadmium‐coordinated thiacalix4arene tetrasulfate (TC4ATS‐Cd) exhibits an anti‐proliferative effect against T‐cell leukemia cells. Cadmium exhibited cytotoxicity with IC50 values ranging from 36 to 129 μM against epithelia‐derived cancer cell lines, while TC4ATS‐Cd elicited no significant cytotoxicity (IC50 > 947 μM). However, a number of T‐cell leukemia cell lines exhibited marked sensitivity to TC4ATS‐Cd. In Jurkat cells, toxicity of TC4ATS‐Cd occurred with an IC50 of 6.9 μM, which is comparable to that of 6.5 μM observed for cadmium alone. TC4ATS‐Cd induced apoptotic cell death through activation of caspase‐3 in Jurkat cells. In a xenograft model, TC4ATS‐Cd (13 mg/kg) treatment significantly suppressed the tumor growth of Jurkat cells in mice. In addition, TC4ATS‐Cd‐treated mice exhibited significantly less cadmium accumulation in liver and kidney compared to equimolar cadmium‐treated mice. These results suggest that cadmium‐coordinated supramolecules may have therapeutic potential for treatment of T‐cell leukemia.
We found that thiacalixarene‐cadmium complex has an anti‐proliferative effect against T‐cell leukemia cells, with no cytotoxicity in epithelia‐derived cancer cells in vitro and minimum accumulations of cadmium in mice. In xenograft tumor model, cadmium‐complex treatment significantly suppressed tumor growth of Jurkat cells in mice. These results suggest that thiacalixarene‐cadmium complex may have therapeutic potential for treatment of T‐cell leukemia.
Malformin A1, a cyclopentapeptide of fungal origin, enhances cellular fibrinolytic activity depending on the existence of a cofactor in blood plasma. However, the nature of this cofactor remains ...unknown. Here, we report that vitronectin acts as a plasma cofactor of malformin A1. We purified the cofactor from bovine plasma by activity-based fractionation, and confirmed that vitronectin in conjunction with plasminogen supports the activity of malformin A1 to promote the fibrinolytic activity of U937 cells. Malformin A1 action was abolished by Arg-Gly-Asp peptide (a competitor of vitronectin–integrin binding), wortmannin (an inhibitor of signaling kinases), and cytochalasin B (an inhibitor of actin polymerization). Changes in actin organization and a decrease in filopodia were observed in cells treated with malformin A1 and plasma. A focal localization of plasminogen on the cell surface was augmented by malformin A1, whereas the amount of cell-surface-bound plasminogen was minimally altered by the treatment. Our results suggest the involvement of cytoskeletal reorganization via vitronectin signaling in the cellular fibrinolytic activity-enhancing action of malformin A1.
Identification of reliable markers of chemo‐ and radiosensitivity and the key molecules that enhance the susceptibility of squamous esophageal cancer cells to anticancer treatments would be highly ...desirable. To test whether regenerating gene (REG) I expression enhances chemo‐ and radiosensitivity in esophageal squamous cell carcinoma cells, we used MTT (3‐4,5‐dimethylthiazol‐2‐yl‐2,5‐diphenyltetrazolium bromide) assays to compare the chemo‐ and radiosensitivities of untransfected TE‐5 and TE‐9 cells with those of cells stably transfected with REG Iα and Iβ. We then used flow cytometry to determine whether REG I expression alters cell cycle progression. No REG I mRNA or protein were detected in untransfected TE‐5 and TE‐9 cells. Transfection with REG Iα and Iβ led to strong expression of both REG I mRNA and protein in TE‐5 and TE‐9 cells, which in turn led to significant increases in both chemo‐ and radiosensitivity. Cell cycle progression was unaffected by REG I expression. REG I thus appears to enhance the chemo‐ and radiosensitivity of squamous esophageal cancer cells, which suggests that it may be a useful target for improved and more individualized treatments for patients with esophageal squamous cell carcinoma. (Cancer Sci 2008; 99: 2491–2495)
Oxaline and neoxaline, fungal alkaloids, were found to inhibit cell proliferation and to induce cell cycle arrest at the G
2/M phase in Jurkat cells. CBP501 (a peptide corresponding to amino acids ...211–221 of Cdc25C phosphatase), which inhibits the G
2 checkpoint, did not affect the G
2/M arrest caused by oxaline, suggesting that oxaline causes M phase arrest but not G
2 phase arrest. The Cdc2 phosphorylation level of oxaline-treated cell lysate was lower than that of the control cells, indicating that oxaline arrests the M phase. Oxaline disrupted cytoplasmic microtubule assembly in 3T3 cells. Furthermore, oxaline inhibited polymerization of microtubule protein and purified tubulin dose-dependently in vitro. In a binding competition assay, oxaline inhibited the binding of
3Hcolchicine to tubulin, but not that of
3Hvinblastine. These results indicate that oxaline inhibits tubulin polymerization, resulting in cell cycle arrest at the M phase.
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► We generated intracellular antibodies directed against GalNAc-Ts. ► A yeast two-hybrid system was used for screening of naive scFv library. ► The scFv antibodies for GalNAc-Ts were ...readily expressed in mammalian cells. ► The scFv antibodies exhibited specific binding activities inside the cells.
Mucin-type O-glycosylation is initiated by a large number of UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases (GalNAc-T). Although extensive in vitro studies using synthetic peptides as substrates suggest that most GalNAc-Ts exhibit overlapping substrate specificities, many studies have shown that individual GalNAc-Ts play an important role in both animals and humans. Further investigations of the functions of individual GalNAc-Ts including in vivo substrate proteins and O-glycosylation sites are necessary.
In this study, we attempted to generate single-chain variable fragment (scFv) antibodies to bind to GalNAc-T1, T2, T3, and T4 using a yeast two-hybrid system for screening a naive chicken scFv library. Several different scFvs were isolated against a single target GalNAc-T isoform specifically under expressed in yeast and were confirmed to be expressed in mammalian cells and to retain binding activity inside the cells. Generation of these specific antibodies provides an opportunity to modify and exploit antibodies for specific applications in investigations of GalNAc-T functions.
Wilson disease is a genetic disorder characterized by the accumulation of copper in the body by defective biliary copper excretion. Wilson disease gene product (ATP7B) functions in copper ...incorporation to ceruloplasmin (Cp) and biliary copper excretion. However, copper metabolism in hepatocytes has been still unclear. Niemann-Pick disease type C (NPC) is a lipid storage disorder and the most commonly mutated gene is
NPC1 and its gene product NPC1 is a late endosome protein and regulates intracellular vesicle traffic. In the present study, we induced NPC phenotype and examined the localization of ATP7B and secretion of holo-Cp, a copper-binding mature form of Cp. The vesicle traffic was modulated using U18666A, which induces NPC phenotype, and knock down of NPC1 by RNA interference. ATP7B colocalized with the late endosome markers, but not with the
trans-Golgi network markers. U18666A and NPC1 knock down decreased holo-Cp secretion to culture medium, but did not affect the secretion of other secretory proteins. Copper accumulated in the cells after the treatment with U18666A. These findings suggest that ATP7B localizes in the late endosomes and that copper in the late endosomes is transported to the secretory compartment via NPC1-dependent pathway and incorporated into apo-Cp to form holo-Cp.
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