• BMB Reports
• /
• v.45 no.3
• /
• pp.183-188
• /
• 2012

Participates in actin remodeling through Rac and receptor endocytosis via Rab5. Here, we used yeast two-hybrid system with Eps8 as bait to screen a human brain cDNA library. ITSN2 was identified as the novel binding factor of Eps8. The interaction between ITSN2 and Eps8 was demonstrated by the in vivo co-immunoprecipitation and colocalization assays and the in vitro GST pull-down assays. Furthermore, we mapped the interaction domains to the region between amino acids 260-306 of Eps8 and the coiled-coil domain of ITSN2. In addition, protein stability assays and immunofluorescence analysis showed ITSN2 overexpression induced the degradation of Eps8 proteins, which was markedly alleviated with the lysosome inhibitor NH4Cl treatment. Taken together, our results suggested ITSN2 interacts with Eps8 and stimulates the degradation of Eps8 proteins.

• International Journal of Oral Biology
• /
• v.37 no.3
• /
• pp.137-145
• /
• 2012

Aggregatibacter actinomycetemcomitans is the most important etiologic agent of aggressive periodontitis and can interact with endothelial cells. Monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) are chemokines, playing important roles in periodontal pathogenesis. In our current study, the effects of A. actinomycetemcomitans on the production of MCP-1 and IL-8 by human umbilical vein endothelial cells (HUVEC) were investigated. A. actinomycetemcomitans strongly induced the gene expression and protein release of both MCP-1 and IL-8 in a dose- and time-dependent manner. Dead A. actinomycetemcomitans cells were as effective as live bacteria in this induction. Treatment of HUVEC with cytochalasin D, an inhibitor of endocytosis, did not affect the mRNA up-regulation of MCP-1 and IL-8 by A. actinomycetemcomitans. However, genistein, an inhibitor of protein tyrosine kinases, substantially inhibited the MCP-1 and IL-8 production by A. actinomycetemcomitans, whereas pharmacological inhibition of each of three members of mitogen-activated protein (MAP) kinase family had little effect. Furthermore, gel shift assays showed that A. actinomycetemcomitans induces a biphasic activation (early at 1-2 h and late at 8-16 h) of nuclear factor-${\kappa}B$ (NF-${\kappa}B$) and an early brief activation (0.5-2 h) of activator protein-1 (AP-1). Activation of canonical NF-${\kappa}B$ pathway ($I{\kappa}B$ kinase activation and $I{\kappa}B-{\alpha}$ degradation) was also demonstrated in these experiments. Although lipopolysaccharide from A. actinomycetemcomitans also induced NF-${\kappa}B$ activation, this activation profile over time differed from that of live A. actinomycetemcomitans. These results suggest that the expression of MCP-1 and IL-8 is potently increased by A. actinomycetemcomitans in endothelial cells, and that the viability of A. actinomycetemcomitans and bacterial internalization are not required for this effect, whereas the activation of protein tyrosine kinase(s), NF-${\kappa}B$, and AP-1 appears to play important roles. The secretion of high levels of MCP-1 and IL-8 resulting from interactions of A. actinomycetemcomitans with endothelial cells may thus contribute to the pathogenesis of aggressive periodontitis.

• Applied Microscopy
• /
• v.23 no.1
• /
• pp.1-14
• /
• 1993

The present study was carried out to analyze the evidence of membrane recycling, and the regulation of cellular transport by dynamic changes in apical membrane area that functionally interacts with the number of cytoplasmic vesicles. Under scanning electron micrographs, turtle bladder mucosa contain three main type of cells; granular cells and carbonic anhydrase (CA)-rich cells, deviding into a and b type of epithelial cell. The granular cell is the majority cell type of the mucosa comprising 80% of the total cell number. The remaining 20% of the cells are characteristically rich in carbonic anhydrase. Uptake of HRP was detected in the most vacuoles or tubulovesicles in both type of CA-rich cells in the turtle bladder, indicating that the part of plasma membrane was internalized in the apical cytoplasmic vacuoles. It seems quite likely that CA-rich cells possess intracellular vesicles carrying proton pumps which are recycling back to the apical plasma membrane. In turtle bladder, the granular cells actively secrete large quantities of mucin and other proteins by an exocytotic mechanism in an apparently constitutive fashion. The possibility that bladder epithelial cells secrete mucin via a regulated secretory pathway has not been rigorously examined and much is still to be determined about these issues from this cell type.

• Biomedical Science Letters
• /
• v.20 no.4
• /
• pp.250-255
• /
• 2014

Mycobacterium tuberculosis (MTB) resides and replicates inside macrophages. In our previous report, we reported that CD8+ T cell-mediated immune responses specific for the peptide derived from MTB RNA polymerase beta-subunit ($RpoB_{127-135}$) could be induced in TB patients expressing HLA-$A^*0201$ subtype. In order to examine whether $RpoB_{127-135}$ specific CD8+ T cells can recognize MTB infected macrophages in vitro, CD8+ T cell lines specific for $RpoB_{127-135}$ peptide were generated from peripheral blood mononuclear cells (PBMCs) of healthy HLA-$A^*0201$ subjects by in vitro immunization technique. In this study, we observed $RpoB_{127-135}$ specific CD8+ T cells could recognize and destroy macrophages infected with MTB for 2 to 4 days. $RpoB_{127-135}$ specific CD8+ T cell immune response was inducible from PBMC of healthy subjects expressing HLA-$A^*0206$ subtype, one of HLA-A2 supertype members. Next, we investigated the HLA-I processing mechanism of $RpoB_{127-135}$ peptide in MTB infected macrophages. As a result, the presentation of the MTB derived epitope peptide, $RpoB_{127-135}$, to CD8+ T cells was not inhibited by the treatment with brefeldin-A (ER-Golgi transport inhibitor) or lactacystin (proteasome inhibitor), which blocks the classical HLA-I processing pathway. However, $RpoB_{127-135}$ specific CD8+ T cell activity was blocked either by the blocking agent for the endocytosis (cytochalasin D) or by the blocking antibody (W6/32) for HLA-I molecules. Therefore, the $RpoB_{127-135}$ peptide may be processed by accessing the alternate HLA-I processing pathway. Understanding the processing and presentation mechanisms of the MTB derived proteins will help to improve the efficacy of vaccines and the efficiency of therapeutic agents for TB.

• Applied Microscopy
• /
• v.38 no.3
• /
• pp.235-243
• /
• 2008

A compact soft x-ray microscope operated in the 'water window' wavelength region ($2.3{\sim}4.4nm$) was used for observing cells with nano-scale spatial resolution. To obtain cellular imaging captured with colloidal gold nanoparticles using a compact soft x-ray microscope. The colloidal gold nanoparticles showed higher contrast and lower transmission more than 7 times than that of cellular protein on the soft x-ray wavelength region. The structure and thickness of the cell membrane of the Coscinodiscus oculoides (diatome) and red blood cells were seen clearly. The gold nanoparticles within the HT1080 and MDA-MB 231 cells were seen clearly on the soft x-ray microscopy. The gold nanoparticles were aggregated within vesicles by endocytosis.

• Asian-Australasian Journal of Animal Sciences
• /
• v.33 no.11
• /
• pp.1824-1836
• /
• 2020

Objective: On the hypothesis that grazing of cattle prompts organs to secrete or internalize circulating microRNAs (c-miRNAs) in parallel with changes in energy metabolism, we aimed to clarify biological events in adipose, skeletal muscle, and liver tissues in grazing Japanese Shorthorn (JSH) steers by a transcriptomic approach. Methods: The subcutaneous fat (SCF), biceps femoris muscle (BFM), and liver in JSH steers after three months of grazing or housing were analyzed using microarray and quantitative polymerase chain reaction (qPCR), followed by gene ontology (GO) and functional annotation analyses. Results: The results of transcriptomics indicated that SCF was highly responsive to grazing compared to BFM and liver tissues. The 'Exosome', 'Carbohydrate metabolism' and 'Lipid metabolism' were extracted as the relevant GO terms in SCF and BFM, and/or liver from the >1.5-fold-altered mRNAs in grazing steers. The qPCR analyses showed a trend of upregulated gene expression related to exosome secretion and internalization (charged multivesicular body protein 4A, vacuolar protein sorting-associated protein 4B, vesicle associated membrane protein 7, caveolin 1) in the BFM and SCF, as well as upregulation of lipolysis-associated mRNAs (carnitine palmitoyltransferase 1A, hormone-sensitive lipase, perilipin 1, adipose triglyceride lipase, fatty acid binding protein 4) and most of the microRNAs (miRNAs) in SCF. Moreover, gene expression related to fatty acid uptake and inter-organ signaling (solute carrier family 27 member 4 and angiopoietin-like 4) was upregulated in BFM, suggesting activation of SCF-BFM organ crosstalk for energy metabolism. Meanwhile, expression of plasma exosomal miR-16a, miR-19b, miR-21-5p, and miR-142-5p was reduced. According to bioinformatic analyses, the c-miRNA target genes are associated with the terms 'Endosome', 'Caveola', 'Endocytosis', 'Carbohydrate metabolism', and with pathways related to environmental information processing and the endocrine system. Conclusion: Exosome and fatty acid metabolism-related gene expression was altered in SCF of grazing cattle, which could be regulated by miRNA such as miR-142-5p. These changes occurred coordinately in both the SCF and BFM, suggesting involvement of exosome in the SCF-BFM organ crosstalk to modulate energy metabolism.

• Journal of Life Science
• /
• v.25 no.3
• /
• pp.263-268
• /
• 2015

Coelomocytes are specialized cells that continually and nonspecifically scavenge fluid from the body cavity through endocytosis in Caenorhabditis elegans. Our previous study revealed that coelmocytes were specifically required for dietary-restriction-induced longevity in C. elegans. In the present study, we examined the effect of coelomocyte ablation on the response to environmental stressors and reproduction in C. elegans. Coelomocytes were ablated using diphtheria toxin specifically expressed in coelomocytes. After exposing worms to 20 J/cm²/min of ultraviolet irradiation in vivo, the survival of the worms was monitored daily. To examine their response to heat stress, their survival after 10 h of 35℃ heat shock was measured. Oxidative stress was induced using paraquat, and the susceptibility to oxidative stress was compared between wild-type control and coelomocyte-ablated worms. The total number of progeny produced was counted, and the time-course distribution of the progeny was determined. The worms with ablated coelomocytes showed reduced resistance to ultraviolet irradiation, but the ablation of coelomocytes had no effect on their response to heat or oxidative stress. The number of progeny produced during the gravid period was significantly decreased in the coelomocyte-ablated worms. These findings suggest that coelomocytes specifically modulate the response to ultraviolet irradiation and are required for normal reproduction in C. elegans. The findings could contribute to understanding of the mechanisms underlying dietary-restriction-induced longevity.

• Journal of Periodontal and Implant Science
• /
• v.36 no.4
• /
• pp.863-878
• /
• 2006

Porphyromonas gingivalis는 치주질환을 야기하는 독성세균으로서, 구강상피세포에 p. gingivalis가 감염되었을 때, 세포형태에 변화를 초래함으로 인해 방어기작이 작동하게 된다. 치주질환과 관련되어 생성된 활성 산소종의 소거에 관여하는 항산화성분은 p. gingivalis 이 감염된 구강상피세포에서 그 분포와 발현수준이 달라지리라 예상된다. 따라서 이번 연구에서는 구강상피세포(KB 세포)에 p. gingivalis가 감염되었을 때 야기되는 활성산소종과 이를 소거하는 역할을 하는 항산화단백들의 역할들을 규명하고자 하였다. 활성산소종 형성을 조절하는 NADPH oxidase 중 NOX4와 Rac1 전사체는 구강상피세포에서 p. gingivalis세균에 의해 증가하였으며 $gp91^{phox}$, Rac2, $p47^{phox}$와 $p67^{phox}$는 세균에 의한 변화가 관찰되지 않았다. 반면에 $p40^{phox}$ 전사체는 감소하는 경향을 보였다. NOX1 전사체는 p. gingivalis 처리 30분 후 감소하였다가 60분 후에는 다시 증가하는 양상을 보였다. 같은 시간에 NOX 활성화 단백인 NOXA1은 감소하고, NOX 구성단백질인 NOXO1은 증가하는 경향을 보였다. p. gingivalis가 감염된 구강상피세포를 방어하는 항산화단백 발현수준을 평가한 결과, SOD1, 2, 3 모두 p. gingivalis 처리시간에 따라 증가하는 양상을 보였다. GPx 발현 양상도 SOD와 유사하게 나타났다. $H_2O_2$를 소거하는 Prx는 감염된 KB 세포에서 Prx4와 Prx5가 4-6배 증가하는 것을 알 수 있었다. 반면 endocytosis 과정 중 $H_2O_2$ 생산은 변화되지 않았다. 이번 연구의 결과, p. gingivalis의 감염은 KB 세포의 NOX4와 Rac1의 NADPH oxidase 발현을 증가시켰으며, NOX1은 NOXA1과 NOXO1의 조절에 의해 영향을 받음을 알 수 있었다. 또한 항산화기작으로는 SOD, GPx, Prx가 증가하였는데, 이것은 Prx4와 Prx5가 중요한 역할을 할 것을 시사하였다.

• Korean Journal of Food Science and Technology
• /
• v.47 no.2
• /
• pp.246-253
• /
• 2015

In this study, we evaluated the lipoprotein lipase (LPL) inhibitory activity of 11 water extracts derived from Cinnamomum cassia Blume, Sarcodon aspratus, Cordyceps militaris, Crataegus pinnatifida Bunge, Corni fructus, Allium cepa, Coix lacryma-jobi, Plantago asiatica L., Lentinus edodes, Rosa rugosa, and Foeniculum fructus. The results of the LPL secretion and activity assay showed Sarcodon aspratus (NE) extract have an LPL secretion inhibitory acitivity. The cause of reduction in LPL secretion after NE treatment was investigated using molecular biology methods. NE treatment affected the LPL content in cells, but did not affect LPL mRNA expression. It also increased the mRNA expression level of sortilin-related receptor LDLR class A (SorLA), a receptor that induces endocytosis and intracellular trafficking of LPL. Finally, cell fractionation revealed that NE treatment induced the expression of CCAAT-enhancer-binding protein beta ($C/EBP{\beta}$), a SorLA transcription factor, in the nuclei of 3T3-L1 adipocytes. These results show that NE's anti-obesity effect involves inhibition of LPL secretion through $C/EBP{\beta}$-mediated induction of SorLA expression.

• Journal of Life Science
• /
• v.34 no.8
• /
• pp.594-607
• /
• 2024

Plastic products have long been widely used in both industrial and household applications. However, tiny plastic particles derived from plastic products, such as microplastics (MPs) and nanoplastics (NPs), can infiltrate the human body through inhalation, ingestion, or skin contact. Once inside cells via endocytosis, MPs and NPs (MNPs) can trigger autophagy, but lysosomal dysfunction can block autophagic flux. Accumulating in the cytoplasm, these particles induce cellular stress, including oxidative stress from free radicals, mitochondrial dysfunction, and increased inflammatory response. Meanwhile, cellular senescence is a hallmark of aging and is defined as the stable termination of the cell cycle in response to cell damage and stress. In particular, the accumulation of oxidative stress, a key factor in inducing cellular senescence, induces the expression of major senescence markers. Senescent cells increase the secretion of senescence-associated secretory phenotype, including inflammatory cytokines and chemokines. Despite growing interest in how MNPs induce cellular senescence, there remains a gap regarding their onset and therapeutic targets. Therefore, this review focuses on identifying recent research trends on how MNPs induce cellular aging in key human cell types and proposes future research directions to overcome these challenges.