• Genomics & Informatics
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• 제17권1호
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• pp.2.1-2.12
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• 2019

Chronic obstructive pulmonary disease (COPD) is a type of progressive lung disease, featured by airflow obstruction. Recently, a comprehensive analysis of the transcriptome in lung tissue of COPD patients was performed, but the heterogeneity of the sample was not seriously considered in characterizing the mechanistic dysregulation of COPD. Here, we established a new transcriptome analysis pipeline using a deconvolution process to reduce the heterogeneity and clearly identified that these transcriptome data originated from the mild or moderate stage of COPD patients. Differentially expressed or co-expressed genes in the protein interaction subnetworks were linked with mitochondrial dysfunction and the immune response, as expected. Computational protein localization prediction revealed that 19 proteins showing changes in subcellular localization were mostly related to mitochondria, suggesting that mislocalization of mitochondria-targeting proteins plays an important role in COPD pathology. Our extensive evaluation of COPD transcriptome data could provide guidelines for analyzing heterogeneous gene expression profiles and classifying potential candidate genes that are responsible for the pathogenesis of COPD.

• International Journal of Stem Cells
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• 제17권3호
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• pp.298-308
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• 2024

Sine oculis homeobox 1 (Six1) is an important factor for embryonic development and carcinoma malignancy. However, the localization of Six1 varies due to protein size and cell types in different organs. In this study, we focus on the expression and localization of Six1 in male reproductive organ via bioinformatics analysis and immunofluorescent detection. The potential interacted proteins with Six1 were also predicted by protein-protein interactions (PPIs) and Enrichr analysis. Bioinformatic data from The Cancer Genome Atlas and Genotype-Tissue Expression project databases showed that SIX1 was highly expressed in normal human testis, but low expressed in the testicular germ cell tumor sample. Human Protein Atlas examination verified that SIX1 level was higher in normal than that in cancer samples. The sub-localization of SIX1 in different reproductive tissues varies but specifically in the cytoplasm and membrane in testicular cells. In mouse cells, single cell RNA-sequencing data analysis indicated that Six1 expression level was higher in mouse spermatogonial stem cells (mSSCs) and differentiating spermatogonial than in other somatic cells. Immunofluorescence staining showed the cytoplasmic localization of Six1 in mouse testis and mSSCs. Further PPIs and Enrichr examination showed the potential interaction of Six1 with bone morphogenetic protein 4 (Bmp4) and catenin Beta-1 (CtnnB1) and stem cell signal pathways. Cytoplasmic localization of Six1 in male testis and mSSCs was probably associated with stem cell related proteins Bmp4 and CtnnB1 for stem cell development.

• BMB Reports
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• 제40권2호
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• pp.270-276
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• 2007

We have cloned a novel mouse zinc finger protein gene Znf313 by rapid amplification of cDNA ends (RACE) according to the homologue of human ZNF313 gene. The cDNA is 2,163 base pairs (bp) in length and encodes a 229 amino acids (aa) protein with a $C_3HC_4$ ring finger domain and three $C_2H_2$ domains. 89% and 93% nucleotide (nt) and aa sequence identity is observed with its human homologue. Revealed by Northern blot and RT-PCR, full mRNA consists of 2.16 kb and widely expresses in tissues as a single transcript, most abundantly in heart, liver, kidney and testis. The expression of Znf313 in testis is detected in all development stages. Western blot analysis also reveals that Znf313 is expressed in the tissues. Immunohistochemical staining and subcellular localization demonstrate that Znf313 is expressed both in the cytoplasm and nucleus whereas predominantly localized in the nucleus. Present data suggests that Znf313 gene might play a fundamental role in gene transcription and regulation in organism and relates to spermatogenesis.

• 농업과학연구
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• 제44권2호
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• pp.145-180
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• 2017

Alternanthera mosaic virus (AltMV) is a member of the genus Potexvirus which has been known for less than twenty years, and has been detected in Australasia, Europe, North and South America, and Asia. The natural host range to date includes species in at least twenty-four taxonomically diverse plant families, with species in at least four other families known to be infected experimentally. AltMV has been shown to differ from Potato virus X (PVX), the type member of the genus Potexvirus, in a number of ways, including the subcellular localization of the Triple Gene Block 3 (TGB3) protein and apparent absence of interactions between TGB3 and TGB2. Differences between AltMV variants have allowed identification of viral determinants of pathogenicity, and identification of residues involved in interactions with host proteins. Infectious clones of AltMV differing significantly in symptom severity and efficiency of RNA silencing suppression have been produced, suitable either for high level protein expression (with efficient RNA silencing suppression) or for Virus-Induced Gene Silencing (VIGS; with weaker RNA silencing suppression), demonstrating a range of utility not available with most other plant viral vectors. The difference in silencing suppression efficiency was shown to be due to a single amino acid residue substitution in TGB1, and to differences in subcellular localization of TGB1 to the nucleus and nucleolus. The current state of knowledge of AltMV biology, including host range, strain differentiation, host interactions, and utility as a plant viral vector for both protein expression and VIGS are summarized.

• Molecules and Cells
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• 제45권11호
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• pp.855-867
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• 2022

For proper function of proteins, their subcellular localization needs to be monitored and regulated in response to the changes in cellular demands. In this regard, dysregulation in the nucleocytoplasmic transport (NCT) of proteins is closely associated with the pathogenesis of various neurodegenerative diseases. However, it remains unclear whether there exists an intrinsic regulatory pathway(s) that controls NCT of proteins either in a commonly shared manner or in a target-selectively different manner. To dissect between these possibilities, in the current study, we investigated the molecular mechanism regulating NCT of truncated ataxin-3 (ATXN3) proteins of which genetic mutation leads to a type of polyglutamine (polyQ) diseases, in comparison with that of TDP-43. In Drosophila dendritic arborization (da) neurons, we observed dynamic changes in the subcellular localization of truncated ATXN3 proteins between the nucleus and the cytosol during development. Moreover, ectopic neuronal toxicity was induced by truncated ATXN3 proteins upon their nuclear accumulation. Consistent with a previous study showing intracellular calcium-dependent NCT of TDP-43, NCT of ATXN3 was also regulated by intracellular calcium level and involves Importin α3 (Imp α3). Interestingly, NCT of ATXN3, but not TDP-43, was primarily mediated by CBP. We further showed that acetyltransferase activity of CBP is important for NCT of ATXN3, which may acetylate Imp α3 to regulate NCT of ATXN3. These findings demonstrate that CBP-dependent acetylation of Imp α3 is crucial for intracellular calcium-dependent NCT of ATXN3 proteins, different from that of TDP-43, in Drosophila neurons.

• Journal of Korean Neurosurgical Society
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• 제41권1호
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• pp.30-38
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• 2007

Objective : To elucidate the role of aquaporin-4[AQP4] in cerebral edema formation, we studied the expression and subcellular localization of AQP4 in astrocytes after focal cerebral ischemia. Methods : Cerebral ischemia were induced by permanent middle cerebral artery[MCA] occlusion in rats and estimated by the discoloration after triphenyltetrazolium chloride[TTC] immersion. Change of AQP4 expression were evaluated using western blot. Localization of AQP4 was assessed by confocal microscopy and its interaction with ${\alpha}-syntrophin$ was analyzed by immunoprecipitation. Results : After right MCA occlusion, the size of infarct and number of apoptotic cells increased with time. The ratio of GluR1/GluR2 expression also increased during ischemia. The polarized localization of AQP4 in the endfeet of astrocytes contacting with ventricles, vessels and pia mater was changed into the diffuse distribution in cytoplasm. The interactions of AQP4 and Kir with ${\alpha}-syntrophin$, an adaptor of dystrophin complex, were disrupted by cerebral ischemia. Conclusion : The deranged spatial buffering function of astrocytes due to mislocalized AQP4/Kir4.1 channel as well as increased assembly of $Ca^{2+}$ permeable AMPA receptors might contribute to the development of edema formation and the excitotoxic neuronal cell death during ischemia.

• Journal of Korean Neurosurgical Society
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• 제42권3호
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• pp.205-210
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• 2007

Objective : Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels mediate the hyperpolarization-activated currents (Ih) that participate in regulating neuronal membrane potential and contribute critically to pacemaker activity, promoting synchronization of neuronal networks. However, distinct regional and cellular localization of HCN channels in the brain have not been precisely defined. Aim of this study was to verify the precise cellular location of HCN1 channels in rat cerebellum to better understand the physiological role these channels play in synaptic transmission between CNS neurons. Methods : HCN1 expression in rat brain was analyzed using immunohistochemistry and electron-microscopic observations. Postsynaptic density-95 (PSD-95), otherwise known as locating and clustering protein, was also examined to clarify its role in the subcellular location of HCN1 channels. In addition, to presume the binding of HCN1 channels with PSD-95, putative binding motifs in these channels were investigated using software-searching method. Results : HCN1 channels were locally distributed at the presynaptic terminal of basket cell and exactly corresponded with the location of PSD-95. Moreover, nine putative SH3 domain of PSD-95 binding motifs were discovered in HCN1 channels from motif analysis. Conclusion : Distinct localization of HCN1 channels in rat cerebellum is possible, especially when analyzed in conjunction with the SH3 domain of PSD-95. Considering that HCN1 channels contribute to spontaneous rhythmic action potentials, it is suggested that HCN1 channels located at the presynaptic terminal of neurons may play an important role in synaptic plasticity.

• Molecules and Cells
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• 제24권1호
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• pp.76-82
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• 2007

By combining in situ hybridization (ISH) and immunocytochemistry (IC), microscopic topological localization of mRNAs and proteins can be determined. Although this technique can be applied to a variety of tissues, it is particularly important for use on neuronal cells which are morphologically complex and in which specific mRNAs and proteins are located in distinct subcellular domains such as dendrites and dendritic spines. One common technical problem for combined ISH and IC is that the signal for immunocytochemical localization of proteins often becomes much weaker after conducting ISH. In this manuscript, we report a simplified but robust protocol that allows immunocytochemical localization of proteins after ISH. In this protocol, we fix cultured cortical or hippocampal neurons with 4% paraformaldehyde (PFA), rinse briefly in PBS, and then further fix the cells with $-20^{\circ}C$ methanol. Our method has several major advantages over previously described ones in that (1) it is simple, as it is just consecutive routine fixation procedures, (2) it does not require any special alteration to the fixation procedures such as changes in salt concentration, and (3) it can be used with antibodies that are compatible with either methanol (MeOH-) or PFA-fixed target proteins. To our best knowledge, we are the first to employ this fixation method for fluorescence ISH + IC.

• Molecules and Cells
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• 제38권6호
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• pp.482-495
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• 2015

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.

• 식물조직배양학회지
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• 제27권4호
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• pp.269-282
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• 2000

Benzylisoquinoline alkaloids are a diverse group of natural products that include many pharmacologically active compounds produced in a limited number of plant families. Despite their complexity, intensive biochemical research has extended our knowledge of the chemistry and enzymology of many important benzylisoquinoline alkaloid pathways, such as those leading to the analgesic drugs morphine and codeine, and the antibiotics sanguinarine and berberine. The use of cultured plant cells as an experimental system has facilitated the identification and characterization of more than 30 benzylisoquinoline alkaloid biosynthetic enzymes, and the molecular cloning of the genes that encode at least 8 of these enzymes. The recent expansion of biochemical and molecular technologies has creat-ed unique opportunities to dissect the mechanisms involved in the regulation of benzylisoquinoline alkaloid biosynthesis in plants. Research has suggested that product accumulation is controlled by the developmental and inducible regulation of several benzylisoquinoline alkaloid biosynthetic genes, and by the subcellular compartmentation of biosynthetic enzymes and the intracellular localization and trafficking of pathway intermediates. In this paper, we review our current understanding of the biochemistry, cell biology, and molecular regulation of benzylisoquinoline alkaloid biosynthesis in plants. We also summarize our own research activities, especially those related to the establishment of protocols for the genetic transformation of benzylisoquinoline alkaloid-producing species, and the development of metabolic engineering strategies in these plants.