• BMB Reports
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• v.51 no.11
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• pp.602-607
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• 2018

Aberrant expression of microRNAs (miRNAs) plays important roles in carcinogenesis and tumor progression. However, the expression and biological role of miR-301b in triple-negative breast cancer (TNBC) remains unclear. Here we aimed to evaluate the roles and mechanisms of miR-301b in TNBC cells. miR-301b expression was assessed in TNBC specimens and cell lines by quantitative Real-Time PCR (qRT-PCR). TNBC cells were transfected with miR-301b mimics, inhibitors or Cylindromatosis (CYLD) small interfering RNA (siRNA) using Lipofectamine 2000. The functional roles of miR-301b were determined by cell proliferation, colony formation, and apoptosis assays. Western blots and qRT-PCR were used to measure the expression of mRNAs and proteins in the cells. We found that miR-301b was upregulated in TNBC specimens and cell lines. Overexpression of miR-301b promoted cell proliferation in TNBC cells, while inhibited the apoptosis induced by 5-FU. CYLD was downregulated by miR-301b at both mRNA and protein levels in TNBC cells. Dual-luciferase report assay confirmed that miR-301b downregulated CYLD by direct interaction with the 3'-untranslated region(3'-UTR) of CYLD mRNA. $NF-{\kappa}B$ activation was mechanistically associated with miR-301b-mediated downregulation of CYLD. However, inhibition of miR-301b reversed all the effects of miR-301b. In conclusion, miR-301b plays an oncogenic role in TNBC possibly by downregulating CYLD and subsequently activating $NF-{\kappa}B$ p65, and this may provide a novel therapeutic approach for TNBC.

• Korean Journal of Biological Psychiatry
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• v.12 no.2
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• pp.136-142
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• 2005

Object:The intracellular action of the antidepressant, venlafaxine, was studied in C6-gliomas using heat shock protein 70(HSP70) immunocytochemistry and HSP70 Western blots because HSP70 is associated with stress and depression. Methods:To examine how the glucocorticoid affects the expression of HSP70 in nerve cells, the rat C6 glioma cell was treated with dexamethasone for 6 hours. In addition, venlafaxine was administered to the experimental groups of C6 glioma cells for 1, 6, 24, and 72 hours each, after which the expression of HSP70 was investigated. Finally, venlafaxine and dexamethasone were simultaneously administered to the experimental groups for 1, 6, 24, and 72 hours, followed by an investigation of the expression of HSP70. Results:The short term(1 hour) venlafaxine treatment significantly increased the level of HSP70 expression. The short term treatment of venlafaxine with dexamethasone also increased the level of HSP70 expression but this reduction was not statistically significant. The long term(72 hours) venlafaxine with dexamethasone treatment significantly reduced the level of HSP70 expression. The long term treatment of venlafaxine also reduced the level of HSP70 expression but this reduction was not statistically significant. Dexamethasone(10uM, 6hours) did not affect the level of HSP70 expression compared with controls. Conclusion:Venlafaxine increases the expression of HSP70 at short term treatment, but prolonged treatment with dexamethasone suppresses the expression of HSP70.

• Journal of Microbiology and Biotechnology
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• v.18 no.9
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• pp.1590-1598
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• 2008

Cell proliferation and differentiation are critical processes in a developing fetal rat brain, during which programmed cell death (PCD) also plays an important role. One of the decisive factors for PCD is Bcl-2 family proteins, where Bax induces cell death, whereas Bcl-2 acts as an inhibitor of PCD. As maternal drinking is known to cause fetal alcohol syndrome (FAS) or malformation of the fetal brain during pregnancy, the objective of the present study was to investigate whether maternal ethanol exposure alters the PCD-related Bax and Bcl-2 protein expression during fetal brain development. Pregnant female rats were orally treated with 10% ethanol and the subsequent expressions of the Bax and Bcl-2 proteins examined in the fetal brain, including the forebrain, midbrain, and hindbrain, from gestational day (GD) 15.5 to GD 19.5, using Western blots, in situ hybridization, and immunohistochemistry. With regard to the ratio of Bcl-2 to Bax proteins (Bcl-2/Bax), the Bax protein was dominant in the forebrain and midbrain of the control GD 15.5 fetuses, except for the hindbrain, when compared with the respective ethanol-treated groups. Moreover, Bcl-2 became dominant in the midbrain of the control GD 17.5 fetuses when compared with the ethanol-treated group, representing an alternation of the natural PCD process by ethanol. Furthermore, a differential expression of the Bcl-2 and Bax proteins was found in the differentiating and migrating zones of the cortex, hippocampus, thalamus, and cerebellum. Thus, when taken together, the present results suggest that ethanol affects PCD in the cell differentiation and migration zones of the prenatal rat brain by modulating Bax and Bcl-2 expression in an age- and area-dependent manner. Therefore, this is the first evidence that ethanol may alter FAS-associated embryonic brain development through the alteration of Bax and Bc1-2 expression.

• BMB Reports
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• v.38 no.6
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• pp.703-708
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• 2005

We cloned and expressed human pyridoxal-5'-phosphate (PLP) phosphatase, the coenzymatically active form of vitamin $B_6$, in Escherichia coli using pET15b vector. Monoclonal antibodies (mAb) were generated against purified human brain PLP phosphatase in mice, and four antibodies recognizing different epitopes were obtained, one of which inhibited PLP phosphatase. The binding affinities of these four mAbs to PLP phosphatase, as determined using biosensor technology, showed that they had similar binding affinities. Using the anti-PLP phosphatase antibodies as probes, we investigated their cross-reactivities in various mammalian and human tissues and cell lines. The immunoreactive bands obtained on Western blots had molecular masses of ca. 33 kDa. Similarly fractionated extracts of several mammalian cell lines all produced a single band of molecular mass 33 kDa. We believe that these PLP phosphatase mAbs could be used as valuable immunodiagnostic reagents for the detection, identification, and characterization of various neurological diseases related to vitamin $B_6$ abnormalities.

• BMB Reports
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• v.39 no.2
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• pp.208-215
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• 2006

The human ribosomal protein S3 (rpS3) was expressed in E. coli using the pET-I5b vector and the monoclonal antibodies (mAbs) were produced and characterized. A total of five hybridoma cell lines were established and the antibodies recognized a single band of molecular weight of 33 kDa on immunoblot with purified rpS3. When the purified rpS3 was incubated with the mAbs, the UV endonuclease activity of rpS3 was inhibited up to a maximum of 49%. The binding affinity of mAbs to rpS3 determined by using a biosensor technology showed that they have similar binding affinities. Using the anti-rpS3 antibodies as probes, we investigated the cross-reactivities of various other mammalian brain tissues and cell lines, including human. The immunoreactive bands on Western blots appeared to be the same molecular mass of 33 kDa in all animal species tested. They also appear to be extensively cross-reactive among different organs in rat. These results demonstrated that only one type of immunologically similar rpS3 protein is present in all of the mammalian brain tissues including human. Furthermore, these antibodies were successfully applied in immunohistochemistry in order to detect rpS3 in the gerbil brain tissues. Among the various regions in the brain tissues, the rpS3 positive neurons were predominantly observed in the ependymal cells, hippocampus and substantia nigra pars compacta. The different distributions of rpS3 in brain tissues reply that rpS3 protein may play an important second function in the neuronal cells.

• Journal of Nutrition and Health
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• v.44 no.3
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• pp.196-202
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• 2011

Epigallocatechin gallate (EGCG), or epigallocatechin 3-gallate, is the ester of epigallocatechin and gallic acid, and is a type of catechin. EGCG may be therapeutic for many disorders including diabetics and some types of cancer. However it is unknown whether EGCG can induce transdifferentiation of pancreatic cells in pancreatitis. The aim of this study was to investigate the effects of EGCG on the expression of pancreatic regenerating related markers in pancreatic AR42J cells, a model of pancreatic progenitor cells. AR42J cells, differentiated with betacellulin and activin A, were cultured with/without EGCG in a time-dependent manner. Cell growth rate, levels of mRNA, and protein expression were examined with the MTT assay, quantitative PCR, and Western blots, respectively. The results showed that AR42J cell growth rates were inhibited by EGCG in a dose-dependent manner. mRNA and protein expression of amylase, insulin and neurogenin 3 (ngn 3) increased in AR42J cells treated with EGCG. Additionally, we demonstrated that the signal transduction pathway of mitogen-activated protein (MAP) kinase is active in EGCG-treated AR42J cells. ERK and JNK phosphorylation decreased in cells treated with EGCG but not p38 phosphorylation. Activation of the p38 MAP kinase pathway was confirmed by specific MAP kinase pathways inhibitors: U0126 for ERK, SP600126 for JNK, and SB203580 for p38. Activated p38 phosphorylation was inhibited by the specific p38 inhibitor SB203580 but p38 phosphorylation was inhibited with increased EGCG treatment. The ERK and JNK MAP kinase pathways were not affected by EGCG treatment. Although further studies are needed, these results suggest that EGCG affects the induction of pancreatic cell regeneration by increasing the ngn 3 protein and mRNA expression and activating the p38 MAP kinase pathway.

• Journal of Periodontal and Implant Science
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• v.37 no.1
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• pp.35-44
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• 2007

Periodontal ligament (PDL) is the connective tissue located between the tooth root and alveolar bone. In a previous study, PDLs22 was isolated as a PDL-specific gene by using subtractive hybrid-ization between cultured PDL fibroblasts and gingival fibroblasts. It was also suggested that PDLs22 plays important roles in the development, differentiation and maintenance of periodontal tissues. However, little is known about functional study of PDLs22 using recombinant protein in PDL fibroblast differentiation and periodontium formation. In this study, in order to produce the PDLs22 recombinat protein, PDLs22 expression vector were constructed and expressed its protein in various host cell and temperature conditions. The results were as follows: 1. PDLs22 protein was not strongly expressed In the induction system using pRSET-PDLs22 construct. 2. When the BL21(DE3) pLysS was used as a expression host, PDLS22 protein was strongly ex-pressed in the induction system using pHCEIIBNd-PDLs22 construct. 3. The PDLs22 protein was recognized at a molecular weight of 28 kDa in western blots. 4. Almost of the expressed PDLs22 protein was not soluble and observed like as inclusion body. 5. The protein solubility was not improved after modification of induction time and temperature during PDLs22 protein production. In this study, the system for the PDLs22 protein production was connstructed. However, the re-results suggest that further studies will be needed to produce the considerable amount of PDLs22 re-combinat protein, which can use for the periodontal regeneration.

• Asian-Australasian Journal of Animal Sciences
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• v.15 no.11
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• pp.1653-1658
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• 2002

The objectives of this study were to examine calpain activity and meat tenderness by three different feeding patterns in Korean native cattle (KNC). Total forty-five animals were assigned each fifteen in long term restriction feeding (LTFR), long-term restriction feeding and hormone treatment (LTFR-tH), and short term non-restriction feeding (STFNR), respectively. Concentrate was restricted based on body weight in exp 1 and 2. However, it was fed ad libitum in exp. 3. Hormonal implantation was made with $M-PO^{TM}$ for bulls and with $F-TO^{TM}$ for heifers at 18, 20, 22 months of age in exp. 2. Animals were purchased (3-5 month old) from local cattle market and managed in two local farms and university research unit at three different years. Animals were slaughtered at 24 months for long-term trial and at 18 month for short term-trial. Loin and tender loin muscle was used for calpain activity and meat quality. Calpain proteolytic system was not changed by treatment. However, calpastatin activity was low in short-term trial. The calpain and calpastatin activity is reciprocal relationship, therefore, the high calpain activity may effect on quality grade. The shear force value was decreased as the processing of aging after postmortem. On the other hand, the cooking loss was significantly higher in short-term than in long-term trial, and then gradually decreased by the aging. Hormone implants to increase meat yield influenced to calpastatin activity more powerfully than calpain activity to meat tenderness. In meat color-a*, there was not significant difference in loin. Meat color-b* was decreased as postmortem aging time increased in tenderloin. Western blots were done to learn whether these proteins are degraded during postmortem storage and whether this degradation temporally parallels the decrease of shear force value. Vinculin was detected at 0 day and 1 day and degraded after 3 day. In conclusion, Calpain activity was affected slightly on meat tenderness. But meat tenderness was influenced by calpastatin, more effectively.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.20
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• pp.8651-8656
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• 2014

Purpose: To study the expression of angiogenin-2 (Ang-2) and its receptor Tie-2 in colorectal cancer and discuss the possible mechanisms behind this process. Materials and Methods: Using the streptavidin-peroxidase (SP) immunohistochemical method, paraffin sections from 100 colorectal cancer samples and 10 samples from tumor-adjacent normal tissue (> 2 cm from the edge of the gross tumor) were tested for protein expression of Ang-2, Tie-2, PI3K, and AKT. Reverse transcription-polymerase chain reaction and Western blots were further used to measure expression of the 4 genes and proteins in 20 freshly-resected colorectal cancer samples and tumor-adjacent normal tissues. Results: In colorectal cancer tissues, the expression of the Ang-2, Tie-2, PI3K, and AKT genes and their proteins was significantly higher than in tumor-adjacent normal tissues. Protein expression in poorly-differentiated adenocarcinoma was higher than that in well and moderately differentiated adenocarcinoma. According to Duke's classification, the protein expression in Stages C and D was significantly higher than that in Stages A and B. In the group with lymphatic metastasis, the protein expression was higher than that without lymphatic metastasis. Conclusions: In colorectal cancer, the expression of the Ang-2, Tie-2, PI3K, and AKT genes and their proteins is markedly higher than those in tumor-adjacent normal tissues. No correlation was observed between protein expression and gender, location, or histologic type. Correlations did exist between protein expression and differentiation level, stage of Duke's classification, and lymphatic metastasis; in colorectal cancer tissues with lower differentiation levels, higher stages of Duke's classification, and lymphatic metastasis, the expression of all 4 proteins was higher. The study of their expression patterns and relationships with aggression and metastasis will provide a valuable experimental foundation for assessing prognosis and targeted therapy of colorectal cancer.

• BMB Reports
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• v.34 no.5
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• pp.484-488
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• 2001

STATs are proteins with a dual function: signal transducers in the cytoplasm and transcriptional activators in the nucleus. Among the six known major STATs (STAT1-6), STAT3 has been implicated in the widest range of signaling pathways that regulate cell growth and differentiation. As a part of our on-going investigation on the pleiotropic functions of STAT proteins, we examined the role of STAT3 as a molecular adaptor that links diverse cell growth signaling pathways. We observed that STAT3 can be specifically activated by multiple cytokines, such as IL-3, in transformed fibroblasts and IL-4 or IFN-$\gamma$ in primary immune cells, respectively. The selective activation of STAT3 in H-ras-transformed NIH3T3 cells is associated with an increased expression of phosphoserioe STAT3 in these cells, compared to the parental cells. Notably phosphoresine-STAT3 interacts with oncogenic ras, shown by immunoprecipitation and Western blots. The results suggest the role of STAT3 in rasinduced cellular transformation as a molecular adaptor linking the Jak/STAT and Ras/MAPK pathways. In primary immune cells, IL-4 and IFN-$\gamma$ each induced (in addition to the characteristic STAT6 and STAT1 homodimers) the formation of STAT3-containing complexes that bind to GAS probes, which correspond to the $Fe{\varepsilon}$ Rll and $Fe{\gamma}$ RI promoter sequences, respectively. Since IL-4 and IFN-$\gamma$ are known to counter-regulate the expression of these genes, the ability of STAT3 to form heterodimeric complexes with STAT6 or STAT1 implies its role in the fine-tuned control of genes that are regulated by IL-4 and IFN-$\gamma$.