- Delayed Degradation and Impaired Dendritic Delivery of Intron-LackingEGFP-Arc/Arg3.1mRNA inEGFP-ArcTransgenic Mice. [Journal Article]
- FMFront Mol Neurosci 2017; 10:435
- Arcis a unique immediate early gene (IEG) whose expression is induced as synapses are modified during learning. Newly-synthesizedArcmRNA is rapidly transported throughout dendrite...
Arcis a unique immediate early gene (IEG) whose expression is induced as synapses are modified during learning. Newly-synthesizedArcmRNA is rapidly transported throughout dendrites and localizes near recently activated synapses.ArcmRNA levels are regulated by rapid degradation, which is accelerated by synaptic activity in a translation-dependent process. One possible mechanism is nonsense-mediated mRNA decay (NMD), which depends on the presence of a splice junction in the 3'UTR. Here, we test this hypothesis using transgenic mice that expressEGFP-Arc. Because the transgene was constructed fromArccDNA, it lacks intron structures in the 3'UTR that are present in the endogenousArcgene. NMD depends on the presence of proteins of the exon junction complex (EJC) downstream of a stop codon, soEGFP-Arc mRNAshould not undergo NMD. Assessment ofArcmRNA rundown in the presence of the transcription inhibitor actinomycin-D confirmed delayed degradation ofEGFP-ArcmRNA.EGFP-ArcmRNA and protein are expressed at much higher levels in transgenic mice under basal and activated conditions butEGFP-ArcmRNA does not enter dendrites efficiently. In a physiological assay in which cycloheximide (CHX) was infused after induction ofArcby seizures, there were increases in endogenousArcmRNA levels consistent with translation-dependentArcmRNA decay but this was not seen withEGFP-ArcmRNA. Taken together, our results indicate: (1)ArcmRNA degradation occurs via a mechanism with characteristics of NMD; (2) rapid dendritic delivery of newly synthesizedArcmRNA after induction may depend in part on prior splicing of the 3'UTR.
- Common traffic routes for imported spermine and endosomal glypican-1-derived heparan sulfate in fibroblasts. [Journal Article]
- ECExp Cell Res 2018 Feb 07
- Import of the polyamine spermine from the extracellular environment depends on the presence of cell surface heparan sulfate proteoglycans, such as glypican-1. This proteoglycan is internalized by end...
Import of the polyamine spermine from the extracellular environment depends on the presence of cell surface heparan sulfate proteoglycans, such as glypican-1. This proteoglycan is internalized by endocytosis, releases its heparan sulfate chains in endosomes by a nitric oxide-, copper- and amyloid precursor protein-dependent mechanism, then penetrates the membrane and is transported to the nucleus and then to autophagosomes. This process is spontaneous or induced by ascorbate depending on the growth-state of the cell. Here, we have explored possible connections between the heparan sulfate traffic route and spermine uptake and delivery in wild-type and Tg2576 mouse fibroblasts. Cells were examined by deconvolution immunofluorescence microscopy. The antibodies used were specific for spermine, glypican-1-derived heparan sulfate, Rab7, nucleolin and a marker for autophagosomes. Endogenous immunostainable spermine was primarily associated with autophagosomes. When spermine synthesis was inhibited, imported spermine appeared in Rab7-positive endosomes. When ascorbate was added, heparan sulfate and spermine were transported to the nucleus where they colocalized with nucleolin. Spermine also appeared in autophagosomes. In a pulse-chase experiment, heparan sulfate and spermine were first arrested in late endosomes by actinomycin D treatment. During the chase, when arrest was abolished, heparan sulfate and spermine were both transported to the nucleus and targeted nucleolin. In amyloid precursor protein-/--fibroblasts, ascorbate failed to induce release of heparan sulfate and spermine remained in the endosomes. We propose that cell surface glypican-1 carries spermine to the endosomes and that the released heparan sulfate carries spermine across the membrane into the cytosol and then to the nucleus.
- Early pregnancy following multidrug regimen chemotherapy in a gestational trophoblastic neoplasia patient: A case report. [Case Reports]
- MMedicine (Baltimore) 2017; 96(51):e9221
- CONCLUSIONS: Pregnancy soon after chemotherapy can be viable with rigorous prenatal care.
- Antifungal effects of actinomycin D on Verticillium dahliae via a membrane-splitting mechanism. [Journal Article]
- NPNat Prod Res 2018 Jan 31; :1-5
- Antifungal bioassays led to the isolation of actinomycins D and A1from Streptomyces luteus TRM45540 collected from Norpo in Xinjiang, and these compounds were identified by nuclear magnetic resonance...
Antifungal bioassays led to the isolation of actinomycins D and A1from Streptomyces luteus TRM45540 collected from Norpo in Xinjiang, and these compounds were identified by nuclear magnetic resonance spectroscopy. The antifungal activity of actinomycin D was higher than that of actinomycin A1. Actinomycin D clearly inhibited the spore germination, hyphal growth and biomass accumulation of Verticillium dahliae in a dose-dependent manner. Flow cytometric analysis with propidium iodide, total ergosterol measurement, cell leakage and scanning electron microscopy experiments demonstrated that the plasma membrane of this fungus was damaged by actinomycin D, resulting in swollen cells and cellular content leakage. Transmission electron microscopy revealed that parts of the plasma membrane infolded after being treated with actinomycin D. The antifungal activity of actinomycin D damaged the fungal plasma membrane of V. dahliae via a membrane-splitting mechanism, which provided new insights into the functional mechanism of actinomycin D.
- P2X7 Nucleotide and EGF Receptors Exert Dual Modulation of the Dual-Specificity Phosphatase 6 (MKP-3) in Granule Neurons and Astrocytes, Contributing to Negative Feedback on ERK Signaling. [Journal Article]
- FMFront Mol Neurosci 2017; 10:448
- Extracellular signal-regulated kinases 1 and 2 (ERK1/2) play a central role in the intracellular signaling of P2X7 nucleotide receptors in neurons and glial cells. Fine spatio-temporal tuning of mito...
Extracellular signal-regulated kinases 1 and 2 (ERK1/2) play a central role in the intracellular signaling of P2X7 nucleotide receptors in neurons and glial cells. Fine spatio-temporal tuning of mitogen-activated protein (MAP) kinases is essential to regulate their biological activity. MAP kinase phosphatases (MKPs) are dual specificity protein phosphatases (DUSPs) that dephosphorylate phosphothreonine and phosphotyrosine residues in MAP kinases. This study focuses on how DUSP, DUSP6/MKP3, a phosphatase specific for ERK1/2 is regulated by the P2X7 nucleotide receptor in cerebellar granule neurons and astrocytes. Stimulation with the specific P2X7 agonist, BzATP, or epidermal growth factor (EGF) (positive control for ERK activation) regulates the levels of DUSP6 in a time dependent manner. Both agonists promote a decline in DUSP6 protein, reaching minimal levels after 30 min yet recovering to basal levels after 1 h. The initial loss of protein occurs through proteasomal degradation, as confirmed in experiments with the proteasome inhibitor, MG-132. Studies carried out with Actinomycin D demonstrated that the enhanced transcription of the Dusp6 gene is responsible for recovering the DUSP6 protein levels. Interestingly, ERK1/2 proteins are involved in the biphasic regulation of the protein phosphatase, being required for both the degradation and the recovery phase. We show that direct Ser197 phosphorylation of DUSP6 by ERK1/2 proteins could be part of the mechanism regulating their cytosolic levels, at least in glial cells. Thus, the ERK1/2 activated by P2X7 receptors exerts positive feedback on these kinase's own activity, promoting the degradation of one of their major inactivators in the cytosolic compartment, DUSP6, both in granule neurons and astrocytes. This feedback loop seems to function as a common universal mechanism to regulate ERK signaling in neural and non-neural cells.
- Anti-Müllerian hormone levels in patients with gestational trophoblastic neoplasia treated with different chemotherapy regimens: a prospective cohort study. [Journal Article]
- OOncotarget 2017 Dec 26; 8(69):113920-113927
- CONCLUSIONS: Chemotherapy for GTN affects the ovarian reserve, with substantial differences between chemotherapy protocols. The results improve our understanding of ovarian toxicity and support the use of fertility preservation strategies.
- Determining Genome-wide Transcript Decay Rates in Proliferating and Quiescent Human Fibroblasts. [Journal Article]
- JVJ Vis Exp 2018 Jan 02; (131)
- Quiescence is a temporary, reversible state in which cells have ceased cell division, but retain the capacity to proliferate. Multiple studies, including ours, have demonstrated that quiescence is as...
Quiescence is a temporary, reversible state in which cells have ceased cell division, but retain the capacity to proliferate. Multiple studies, including ours, have demonstrated that quiescence is associated with widespread changes in gene expression. Some of these changes occur through changes in the level or activity of proliferation-associated transcription factors, such as E2F and MYC. We have demonstrated that mRNA decay can also contribute to changes in gene expression between proliferating and quiescent cells. In this protocol, we describe the procedure for establishing proliferating and quiescent cultures of human dermal foreskin fibroblasts. We then describe the procedures for inhibiting new transcription in proliferating and quiescent cells with Actinomycin D (ActD). ActD treatment represents a straightforward and reproducible approach to dissociating new transcription from transcript decay. A disadvantage of ActD treatment is that the time course must be limited to a short time frame because ActD affects cell viability. Transcript levels are monitored over time to determine transcript decay rates. This procedure allows for the identification of genes and isoforms that exhibit differential decay in proliferating versus quiescent fibroblasts.
- AMP-activated protein kinase agonist N6-(3-hydroxyphenyl)adenosine protects against fulminant hepatitis by suppressing inflammation and apoptosis. [Journal Article]
- CDCell Death Dis 2018 Jan 18; 9(2):37
- Both AMP-activated protein kinase (AMPK) agonist and inhibitor have been reported to protect against fulminant hepatitis, implying that AMPK may play a complicated role in the development of fulminan...
Both AMP-activated protein kinase (AMPK) agonist and inhibitor have been reported to protect against fulminant hepatitis, implying that AMPK may play a complicated role in the development of fulminant hepatitis. In this study, we exploited whether the novel AMPK agonist N6-(3-hydroxyphenyl)adenosine (named as M1) exerted protective effects on fulminant hepatitis and whether its beneficial effects were AMPK-dependent. Results showed that intraperitoneal injection of M1 improved liver function, ameliorated liver injury and finally raised the survival rate in D-galactosamine/lipopolysaccharide (GalN/LPS)-treated mice. These beneficial effects of M1 may attribute to the suppression of pro-inflammatory cytokines production and the prevention of hepatocyte apoptosis. Furthermore, M1 pretreatment mitigated LPS-stimulated TLR4 expression and NFκB activation in murine peritoneal macrophages and prevented actinomycin D (Act D)/tumor necrosis factor α (TNFα)-induced apoptosis by promoting protective autophagy in primary hepatocytes. Additionally, M1-induced AMPK activation was responsible both for its anti-inflammatory action in macrophages and for its anti-apoptotic action in hepatocytes. To our surprise, compared with the control AMPKα1lox/lox/AMPKα2lox/loxmice, liver-specific AMPKα1 knockout (AMPKα1LS-/-) mice were more sensitive to GalN/LPS administration but not AMPKα2LS-/-mice, and the beneficial effects of M1 on acute liver failure and the production of pro-inflammatory factors were dampened in AMPKα1LS-/-mice. Therefore, our study may prove that M1 could be a promising therapeutic agent for fulminant hepatitis, and targeting AMPK may be useful therapeutically in the control of LPS-induced hepatotoxicity.
- Investigating the potential impact of dose banding for systemic anti-cancer therapy in the paediatric setting based on pharmacokinetic evidence. [Journal Article]
- EJEur J Cancer 2018; 91:56-67
- CONCLUSIONS: Based on pharmacokinetic data for these five drugs, the results generated support the implementation of NHSE dose-banding tables. Indeed, inter-patient variability in drug clearance and exposure far outweighs the impact of relatively small drug dose changes associated with dose banding.
New Search Next
- Engrailed 1 overexpression as a potential prognostic marker in quintuple-negative breast cancer. [Journal Article]
- CBCancer Biol Ther 2018 Jan 15; :1-11
- Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype characterized by poor patient prognosis and for which no targeted therapies are currently available. TNBC can be further ca...
Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype characterized by poor patient prognosis and for which no targeted therapies are currently available. TNBC can be further categorized as either basal-like (BLBC) or quintuple-negative breast cancer (QNBC). In the present study, we aimed to identify novel molecular therapeutic targets for TNBC by analyzing the mRNA expression of TNBC-related genes in publicly available microarray data sets. We found that Engrailed 1 (EN1) was significantly overexpressed in TNBC. Using breast cancer cell lines, we found that EN1 was more highly expressed in TNBC than in other breast cancer subtypes. EN1 expression was analyzed in 199 TNBC paraffin-embedded tissue samples by immunohistochemistry. EN1 protein expression was positively associated with reduced overall survival (OS) rate in patients with QNBC, but not those with BLBC. The importance of EN1 expression in QNBC cell viability and tumorigenicity was evaluated using the QNBC cell lines, HCC38 and HCC1395. Based on our data, EN1 may promote the proliferation, migration, and multinucleation of QNBC cells, likely via the transcriptional activation of HDAC8, UTP11L, and ZIC3. We also demonstrated that actinomycin D effectively inhibits EN1 activity in QNBC cells. The results of the present study suggest that EN1 activity is highly clinically relevant to the survival prognosis of patients with QNBC and EN1 is a promising potential therapeutic target for future QNBC treatment.