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        Differential Localisation of PARP-1 N-Terminal Fragment in PARP-1+/+ and PARP-1-/- Murine Cells

        Jeremy Skepper,Ida Rachel Rajiah 한국분자세포생물학회 2014 Molecules and cells Vol.37 No.7

        Human PARP family consists of 17 members of which PARP-1 is a prominent member and plays a key role in DNA repair pathways. It has an N-terminal DNA-binding domain (DBD) encompassing the nuclear localisation signal (NLS), central automodification domain and C-terminal catalytic domain. PARP-1 accounts for majority of poly-(ADP-ribose) polymer synthesis that upon binding to numerous proteins including PARP itself modulates their activity. Reduced PARP-1 activity in ageing human samples and its deficiency leading to telomere shortening has been reported. Hence for cell survival, maintenance of genomic integrity and longevity presence of intact PARP-1 in the nucleus is paramount. Although localisation of full-length and truncated PARP-1 in PARP-1 proficient cells is well documented, subcellular distribution of PARP-1 frag-ments in the absence of endogenous PARP-1 is not known. Here we report the differential localisation of PARP-1 N-terminal fragment encompassing NLS in PARP-1+/+ and PARP-1-/- mouse embryo fibroblasts by live imaging of cells transiently expressing EGFP tagged fragment. In PARP-1+/+ cells the fragment localises to the nuclei presenting a granular pattern. Furthermore, it is densely packaged in the midsections of the nucleus. In contrast, the fragment localises exclusively to the cytoplasm in PARP-1-/- cells. Flou-rescence intensity analysis further confirmed this observation indicating that the N-terminal fragment requires endogenous PARP-1 for its nuclear transport. Our study illustrates the trafficking role of PARP-1 independently of its enzymatic activity and highlights the possibility that full-length PARP-1 may play a key role in the nuclear transport of its siblings and other molecules.

      • KCI등재

        Neuronal Activity-Dependent Regulation of MicroRNAs

        심수언,Joseph Bakes,강봉균 한국분자세포생물학회 2014 Molecules and cells Vol.37 No.7

        MicroRNAs are non-coding short (~23 nucleotides) RNAs that mediate post-transcriptional regulation through sequence-specific gene silencing. The role of miRNAs in neuronal development, synapse formation and synaptic plasticity has been highlighted. However, the role of neuronal activity on miRNA regulation has been less focused. Neuronal activity-dependent regulation of miRNA may fine-tune gene expression in response to synaptic plasticity and memory formation. Here, we provide an overview of miRNA regulation by neuronal activity including high-throughput screening studies. We also discuss the possible molecular mechanisms of activity-dependent induction and turnover of miRNAs.

      • KCI등재

        Abnormal Astrocytosis in the Basal Ganglia Pathway of Git1−/− Mice

        임수연,마원 한국분자세포생물학회 2015 Molecules and cells Vol.38 No.6

        Attention deficit/hyperactivity disorder (ADHD) is one of the most common neurodevelopmental disorders, affecting approximately 5% of children. However, the neural mechanisms underlying its development and treatment are yet to be elucidated. In this study, we report that an ADHD mouse model, which harbors a deletion in the Git1 locus, exhibits severe astrocytosis in the globus pallidus (GP) and thalamic reticular nucleus (TRN), which send modulatory GABAergic inputs to the thalamus. A moderate level of astrocytosis was displayed in other regions of the basal ganglia pathway, including the ventrobasal thalamus and cortex, but not in other brain regions, such as the caudate putamen, basolateral amygdala, and hippocampal CA1. This basal ganglia circuit-selective astrocytosis was detected in both in adult (2-3 months old) and juvenile (4 weeks old) Git1−/− mice, suggesting a developmental origin. Astrocytes play an active role in the developing synaptic circuit; therefore, we performed an immunohistochemical analysis of synaptic markers. We detected increased and decreased levels of GABA and parvalbumin (PV), respectively, in the GP. This suggests that astrocytosis may alter synaptic transmission in the basal ganglia. Intriguingly, increased GABA expression colocalized with the astrocyte marker, GFAP, indicative of an astrocytic origin. Collectively, these results suggest that defects in basal ganglia circuitry, leading to impaired inhibitory modulation of the thalamus, are neural correlates for the ADHD-associated behavioral manifestations in Git1−/− mice.

      • KCI등재

        Odorant Stimulation Promotes Survival of Rodent Olfactory Receptor Neurons via PI3K/Akt Activation and Bcl-2 Expression

        김소연,유승준,Gabriele V Ronnett,김은경,문제일 한국분자세포생물학회 2015 Molecules and cells Vol.38 No.6

        Olfactory stimulation activates multiple signaling cascades in order to mediate activity-driven changes in gene expression that promote neuronal survival. To date, the mechanisms involved in activity-dependent olfactory neuronal survival have yet to be fully elucidated. In the current study, we observed that olfactory sensory stimulation, which caused neuronal activation, promoted activation of the phosphatidylinositol 3’-kinase (PI3K)/Akt pathway and the expression of Bcl-2, which were responsible for olfactory receptor neuron (ORN) survival. We demonstrated that Bcl-2 expression increased after odorant stimulation both in vivo and in vitro. We also showed that odorant stimulation activated Akt, and that Akt activation was completely blocked by incubation with both a PI3K inhibitor (LY294002) and Akt1 small interfering RNA. Moreover, blocking the PI3K/Akt pathway diminished the odorantinduced Bcl-2 expression, as well as the effects on odorant- induced ORN survival. A temporal difference was noted between the activation of Akt1 and the expression of Bcl-2 following odorant stimulation. Blocking the PI3K/Akt pathway did not affect ORN survival in the time range prior to the increase in Bcl-2 expression, implying that these two events, activation of the PI3K pathway and Bcl-2 induction, were tightly connected to promote post-translational ORN survival. Collectively, our results indicated that olfactory activity activated PI3K/Akt, induced Bcl-2, and promoted long term ORN survival as a result.

      • KCI등재

        Role of Sphingolipids and Metabolizing Enzymes in Hematological Malignancies

        Kazuyuki Kitatani,Toshiro Okazaki,Makoto Taniguchi 한국분자세포생물학회 2015 Molecules and cells Vol.38 No.6

        Sphingolipids such as ceramide, sphingosine-1-phosphate and sphingomyelin have been emerging as bioactive lipids since ceramide was reported to play a role in human leukemia HL-60 cell differentiation and death. Recently, it is well-known that ceramide acts as an inducer of cell death, that sphingomyelin works as a regulator for microdomain function of the cell membrane, and that sphingosine-1- phosphate plays a role in cell survival/proliferation. The lipids are metabolized by the specific enzymes, and each metabolite could be again returned to the original form by the reverse action of the different enzyme or after a long journey of many metabolizing/synthesizing pathways. In addition, the metabolites may serve as reciprocal biomodulators like the rheostat between ceramide and sphingosine-1-phosphate. Therefore, the change of lipid amount in the cells, the subcellular localization and the downstream signal in a specific subcellular organelle should be clarified to understand the pathobiological significance of sphingolipids when extracellular stimulation induces a diverse of cell functions such as cell death, proliferation and migration. In this review, we focus on how sphingolipids and their metabolizing enzymes cooperatively exert their function in proliferation, migration, autophagy and death of hematopoetic cells, and discuss the way developing a novel therapeutic device through the regulation of sphingolipids for effectively inhibiting cell proliferation and inducing cell death in hematological malignancies such as leukemia, malignant lymphoma and multiple myeloma.

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        A Tale of Two Models: Mouse and Zebrafish as Complementary Models for Lymphatic Studies

        김준대,진석원 한국분자세포생물학회 2014 Molecules and cells Vol.37 No.7

        Lymphatic vessels provide essential roles in maintaining fluid homeostasis and lipid absorption. Dysfunctions of the lymphatic vessels lead to debilitating pathological conditions, collectively known as lymphedema. In addition, lymphatic vessels are a critical moderator for the onset and progression of diverse human diseases including metastatic cancer and obesity. Despite their clinical impor-tance, there is no currently effective pharmacological therapy to regulate functions of lymphatic vessels. Recent efforts to manipulate the Vascular Endothelial Growth Factor-C (VEGFC) pathway, which is arguably the most important signaling pathway regulating lymphatic endo-thelial cells, to alleviate lymphedema yielded largely mixed results, necessitating identification of new targetable signaling pathways for therapeutic intervention for lym-phedema. Zebrafish, a relatively new model system to investigate lymphatic biology, appears to be an ideal model to identify novel therapeutic targets for lymphatic biology. In this review, we will provide an overview of our current understanding of the lymphatic vessels in verte-brates, and discuss zebrafish as a promising in vivo model to study lymphatic vessels.

      • KCI등재

        Suppression of ASK (AtSK32), a Clade III Arabidopsis GSK3, Leads to the Pollen Defect during Late Pollen Development

        동향순,노일섭,이한길,허윤강 한국분자세포생물학회 2015 Molecules and cells Vol.38 No.6

        Arabidopsis Shaggy-like protein kinases (ASKs) are Ara-bidopsis thaliana homologs of glycogen synthase kinase 3/SHAGGY-like kinases (GSK3/SGG), which are com-prised of 10 genes with diverse functions. To dissect the function of ASKβ (AtSK32), ASKβ antisense transgenic plants were generated, revealing the effects of ASKβ down-regulation in Arabidopsis. Suppression of ASKβ expression specifically interfered with pollen development and fertility without altering the plants’ vegetative phenotypes, which differed from the phenotypes reported for Arabidopsis plants defective in other ASK members. The strength of these phenotypes showed an inverse correlation with the expression levels of ASKβ and its co-expressed genes. In the aborted pollen of ASKβ antisense plants, loss of nuclei and shrunken cytoplasm began to appear at the bicellular stage of microgametogenesis. The in silico analysis of promoter and the expression characteristics implicate ASKβ is associated with the expression of genes known to be involved in sperm cell differentiation. We speculate that ASKβ indirectly affects the transcription of its co-expressed genes through the phosphorylation of its target proteins during late pollen development.

      • KCI등재

        Oncomodulin/Truncated Protamine-Mediated Nogo-66 Receptor Small Interference RNA Delivery Promotes Axon Regeneration in Retinal Ganglion Cells

        Zhili Cui,Jun Kang,Dan Hu,Jian Zhou,Yusheng Wang 한국분자세포생물학회 2014 Molecules and cells Vol.37 No.8

        The optic nerve often suffers regenerative failure after injury, leading to serious visual impairment such as glaucoma. The main inhibitory factors, including Nogo-A, oligodendrocyte myelin glycoprotein, and myelin-associated glycoprotein, exert their inhibitory effects on axonal growth through the same receptor, the Nogo-66 receptor (NgR). Oncomodulin (OM), a calcium-binding protein with a molecular weight of an ~12 kDa, which is secreted from activated macrophages, has been demonstrated to have high and specific affinity for retinal ganglion cells (RGC) and promote greater axonal regeneration than other known polypeptide growth factors. Protamine has been reported to effectively deliver small interference RNA (siRNA) into cells. Accordingly, a fusion protein of OM and truncated protamine (tp) may be used as a vehicle for the delivery of NgR siRNA into RGC for gene therapy. To test this hypothesis, we constructed OM and tp fusion protein (OM/tp) expression vectors. Using the indirect immunofluorescence labeling method, OM/tp fusion proteins were found to have a high affinity for RGC. The gel shift assay showed that the OM/tp fusion proteins retained the capacity to bind to DNA. Using OM/tp fusion proteins as a delivery tool, the siRNA of NgR was effectively transfected into cells and significantly down-regulated NgR expression levels. More importantly, OM/tp-NgR siRNA dramatically promoted axonal growth of RGC compared with the application of OM/tp recombinant protein or NgR siRNA alone in vitro. In addition, OM/tp-NgR siRNA highly elevated intracellular cyclic adenosine monophosphate (cAMP) levels and inhibited activation of the Ras homolog gene family, member A (RhoA). Taken together, our data demonstrated that the recombinant OM/tp fusion proteins retained the functions of both OM and tp, and that OM/tp-NgR siRNA might potentially be used for the treatment of optic nerve injury.

      • KCI등재

        Nuclease Delivery: Versatile Functions of SLX4/FANCP in Genome Maintenance

        김용환 한국분자세포생물학회 2014 Molecules and cells Vol.37 No.8

        As a scaffold, SLX4/FANCP interacts with multiple proteins involved in genome integrity. Although not having reco¬gnizable catalytic domains, SLX4 participates in diverse genome maintenance pathways by delivering nucleases where they are needed, and promoting their cooperative execution to prevent genomic instabilities. Physiological importance of SLX4 is emphasized by the identification of causative mutations of SLX4 genes in patients diagnosed with Fanconi anemia (FA), a rare recessive genetic disor¬der characterized by genomic instability and predisposi-tion to cancers. Recent progress in understanding functional roles of SLX4 has greatly expanded our knowl¬edge in the repair of DNA interstrand crosslinks (ICLs), Holliday junction (HJ) resolution, telomere homeostasis and regulation of DNA damage response induced by replication stress. Here, these diverse functions of SLX4 are reviewed in detail.

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