• Molecules and Cells
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• 제44권8호
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• pp.569-579
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• 2021

Cyclase-associated protein 2 (CAP2) has been addressed as a candidate biomarker in various cancer types. Previously, we have shown that CAP2 is expressed during multi-step hepatocarcinogenesis; however, its underlying mechanisms in liver cancer cells are not fully elucidated yet. Here, we demonstrated that endoplasmic reticulum (ER) stress induced CAP2 expression, and which promoted migration and invasion of liver cancer cells. We also found that the ER stress-induced CAP2 expression is mediated through activation of protein kinase C epsilon (PKCε) and the promotor binding of activating transcription factor 2 (ATF2). In addition, we further demonstrated that CAP2 expression promoted epithelial-mesenchymal transition (EMT) through activation of Rac1 and ERK. In conclusion, we suggest that ER stress induces CAP2 expression promoting EMT in liver cancer cells. Our results shed light on the novel functions of CAP2 in the metastatic process of liver cancer cells.

• Journal of Microbiology and Biotechnology
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• 제27권2호
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• pp.357-364
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• 2017

In this study, we aimed to generate a series of versatile tagging plasmids that can be used in diverse molecular biological studies of the fungal pathogen Cryptococcus neoformans. We constructed 12 plasmids that can be used to tag a protein of interest with a GFP, mCherry, $4{\times}FLAG$, or $6{\times}HA$, along with nourseothricin-, neomycin-, or hygromycin-resistant selection markers. Using this tagging plasmid set, we explored the adenylyl cyclase complex (ACC), consisting of adenylyl cyclase (Cac1) and its associated protein Aca1, in the cAMP-signaling pathway, which is critical for the pathogenicity of C. neoformans. We found that Cac1-mCherry and Aca1-GFP were mainly colocalized as punctate forms in the cell membrane and non-nuclear cellular organelles. We also demonstrated that Cac1 and Aca1 interacted in vivo by co-immunoprecipitation, using $Cac1-6{\times}HA$ and $Aca1-4{\times}FLAG$ tagging strains. Bimolecular fluorescence complementation further confirmed the in vivo interaction of Cac1 and Aca1 in live cells. Finally, protein pull-down experiments using $aca1{\Delta}$::ACA1-GFP and $aca1{\Delta}$::ACA1-GFP $cac1{\Delta}$ strains and comparative mass spectrometry analysis identified Cac1 and a number of other novel ACC-interacting proteins. Thus, this versatile tagging plasmid system will facilitate diverse mechanistic studies in C. neoformans and further our understanding of its biology.

• Journal of Microbiology and Biotechnology
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• 제16권8호
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• pp.1222-1228
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• 2006

Two kinds of Haematococcus pluvialis cells (green vegetative cells cultivated under optimal cell culture conditions and red cyst cells maintained under high light stress conditions to induce astaxanthin production) were used to investigate the protein expression profiles by two-dimensional electrophoresis, image analysis, and peptide mass fingerprinting. The cellular accumulation of astaxanthin was evident after exposure to high light intensity and reached the maximum cellular level after 78 h of high light stress. In a 2-D electrophoresis analysis, 22 proteins were upregulated over 2-fold in the red cyst cells when compared with the green vegetative cells and selected for further analysis by chemically assisted fragmentation (CAF)-MALDI-TOF sequencing to identify the protein functions. Among 22 different spots, several key enzymes specific to the carotenoid pathway, including isopentenyl pyrophosphate isomerase (IPP) and lycopene $\beta$-cyclase, appeared in H. pluvialis after exposure to high light intensity. Therefore, IPP and lycopene $\beta$-cyclase would appear to be involved with carotenoid accumulation in the cytoplasm, as these peptides were preferentially upregulated by high light intensity preceding an increase in carotenoid, and only these forms were detected in the red cyst cells.

• The Korean Journal of Physiology and Pharmacology
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• 제13권6호
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• pp.409-416
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• 2009

Acute pancreatitis is a multifactorial disease associated with the premature activation of digestive enzymes. The genes expressed in pancreatic acinar cells determine the severity of the disease. The present study determined the differentially expressed genes in pancreatic acinar cells treated with cerulein as an in vitro model of acute pancreatitis. Pancreatic acinar AR42J cells were stimulated with $10^{-8}$ M cerulein for 4 h, and genes with altered expression were identified using a cDNA microarray for 4,000 rat genes and validated by real-time PCR. These genes showed a 2.5-fold or higher increase with cerulein: lithostatin, guanylate cyclase, myosin light chain kinase 2, cathepsin C, progestin-induced protein, and pancreatic trypsin 2. Stathin 1 and ribosomal protein S13 showed a 2.5-fold or higher decreases in expression. Real-time PCR analysis showed time-dependent alterations of these genes. Using commercially available antibodies specific for guanylate cyclase, myosin light chain kinase 2, and cathepsin C, a time-dependent increase in these proteins were observed by Western blotting. Thus, disturbances in proliferation, differentiation, cytoskeleton arrangement, enzyme activity, and secretion may be underlying mechanisms of acute pancreatitis.

• 대한한의학회지
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• 제21권4호
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• pp.193-203
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• 2000

Objectives : Hindered barrier function of vascular endothelium has been implicated in the initiation and progression of degenerative vascular diseases such as atherosclerosis. In this study, the effect of Sunghyangchungisan(SHCS) as a protectant against oxidant-induced destruction of endothelial barrier function was assessed. Methods : Toward this end, endothelial cells derived from the human umbilical vein were cultured as monolayers on permeable membrane filters. Endothelial permeability was monitored by measuring transendothelial electrical resistance and movement of low density lipoprotein (LDL) across the endothelial monolayer. Results : Along with increased movement of LDL, $H_2O_2$-induced increase in endothelial permeability was paralleled by a decrease in transendotheliaI electrical resistance. The effect of $H_2O_2$ was mimicked by phorbol 12-myristate 13-acetate (PMA), a potent activator of proteinkinase C. Calphostin-C, a protein kinase C inhibitor, effectively blocked the increase in endothelial permeability induced by $H_2O_2$ or PMA, indicating that activation of protein kinase C is associated with the $H_2O_2-induced$ permeability change. SHCS effectively protected the endothelial monolayer against $H_2O_2-induced$ increase in permeability, whereas, it did not affect PMA-induced change. Forskolin, a potent activator of adenylyl cyclase, antagonized $H_2O_2$ to increase endothelial permeability. In addition, in ${H_2O_2}-treated$ cens, intracenular cAMP concentration was significantly decreased, indicating that impaired cAMP production as well as activation of proteinkinase C is a mechanism underlying ${H_2O_2}>-induced$$H_2O_2$ with regard to its effect on intracellular cAMP content. However, SHCS itself did not affect resting cAMP concentration in endothelial cells. Conclusions : These results suggest that SHCS might operate as an effective protectant against oxidant-induced destruction of endothelial barrier function. The mechanism does not appear to involve direct interaction with protein kinase C- or cAMP-associated signaling mechanism.

• Journal of Microbiology and Biotechnology
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• 제27권2호
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• pp.262-270
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• 2017

Yersinia enterocolitica is a well-known foodborne pathogen causing gastrointestinal infections worldwide. The strain Y. enterocolitica FORC_002 was isolated from the gill of flatfish (plaice) and its genome was sequenced. The genomic DNA consists of 4,837,317 bp with a GC content of 47.1%, and is predicted to contain 4,221 open reading frames, 81 tRNA genes, and 26 rRNA genes. Interestingly, genomic analysis revealed pathogenesis and host immune evasion-associated genes encoding guanylate cyclase (Yst), invasin (Ail and Inv), outer membrane protein (Yops), autotransporter adhesin A (YadA), RTX-like toxins, and a type III secretion system. In particular, guanylate cyclase is a heat-stable enterotoxin causing Yersinia-associated diarrhea, and RTX-like toxins are responsible for attachment to integrin on the target cell for cytotoxic action. This genome can be used to identify virulence factors that can be applied for the development of novel biomarkers for the rapid detection of this pathogen in foods.

• The Korean Journal of Physiology
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• 제29권2호
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• pp.243-250
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• 1995

A possible potentiation between cholecystokinin (CCK) and secretin in amylase secretion from isolated rat pancreatic acini was investigated. Combined treatment of acini with secretin and CCK at low concentrations, which are known to be physiological, resulted in enzyme secretion larger than the arithmetic sum of their separate effects. Such a potentiating effect also occurred between secretin and A23187 (Ca ionophore), between forskolin (adenylate cyclase activator) and CCK, and between forskolin and A23187. Staurosporin (protein kinase C inhibitor) and W7 (calmodulin antagonist) inhibited markedly the potentiated amylase release induced by the agonists, but KT5720 (protein kinase A inhibitor) did not affect the potentiated amylase release. Therefore, we concluded that the action of CCK in a physiological concentration is potentiated by secretin in a physiological concentration range and vice versa, and that the intracellular mechanism necessary for the potentiation is associated with $Ca^{2+}$. However, it is uncertain what mechanisms are involved in potentiation of amylase release after CAMP and $Ca^{2+}$.

• 대한의생명과학회지
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• 제10권4호
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• pp.375-383
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• 2004

It has been reported that osteoporosis induced by the deficiency of estrogens in menopause is associated with the unbalance of Ca/sup 2+/ and Pi levels. Proximal tubule is very important organ to regualte Ca/sup 2+/ and Pi level in the body. However, the effect of estrogens on Ca/sup 2+/ and Pi regulation was not elucidated. Thus, we examined the effect of 17-β estradiol (E₂) on Ca/sup 2+/ and Pi uptake in the primary cultured rabbit renal proxiaml tubule cells. In the present study, E₂(> 10/sup -9/M) decreases Ca/sup 2+/uptake and stimulates Pi uptake over 3 days. E₂-induced decrease of Ca/sup 2+/ uptake and stimulation of Pi uptake were blocked by actinomycin D (a gene transcription inhibitor), cycloheximide (a protein synthesis inhibitor). tamoxifen, and progesterone (estrogen receptor antagonists). E₂-induced decrease of Ca/sup 2+/ uptake and stimulation of Pi uptake were blocked by SQ22536 (an adenylate cyclase inhibitor), Rp-cAMP (a cAMP antagonist), and PKI (a protein kinase A inhibitor). Indeed, E₂ increased cAMP formation. In addition, E₂-induced decrease of Ca/sup 2+/ uptake and stimulation of Pi uptake were blocked by staurosporine, H-7, and bisindolylmaleimide I (protein kinase C inhibitors) and E₂ translocated PKC from cytoslic fraction to membrane fraction. In conclusion, E₂ decreased Ca/sup 2+/ uptake and stimulated Pi uptake via cAMP and PKC pathway in the PTCs.

• BMB Reports
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• 제45권12호
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• pp.724-729
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• 2012

In this study, we evaluated the anti-melanogenesis effects of Caffeoylserotonin (CaS) in B16 melanoma cells. Treatment with CaS reduced the melanin content and tyrosinase (TYR) activity in B16 melanoma cells in a dose-dependent manner. CaS inhibited the expression of melanogenesis-related proteins, including microphthalmia-associated transcription factor (MITF), TYR, and tyrosinase-related protein-1 (TRP-1), but not TRP-2. ${\alpha}$-MSH is known to interact with melanocortin 1 receptor (MC1R) thus activating adenylyl cyclase and increasing intracellular cyclic AMP (cAMP) levels. Furthermore, cAMP activates extracellular signal-regulated kinase 2 (ERK2) via phosphorylation, which phosphorylates MITF, thereby targeting the transcription factor to proteasomes for degradation. The CaS reduced intracellular cAMP levels to unstimulated levels and activated ERK phosphorylation within 30 min. The ERK inhibitor PD98059 abrogated the suppressive effect of CaS on ${\alpha}$-MSH-induced melanogenesis. Based on this study, the inhibitory effects of CaS on melanogenesis are derived from the downregulation of MITF signaling via the inhibition of intracellular cAMP levels, as well as acceleration of ERK activation.

• 생명과학회지
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• 제21권4호
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• pp.486-493
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• 2011

Nitric oxide (NO) donors는 heme oxygenase-1 (HO-1)의 강력한 유도제이다. 그러나 NO donors에 의한 HO-1의 발현이 NO donor에 의해 방출되는 NO에 의한 직접적인 영향인지는 불분명하다. 본 연구에서 흰쥐의 무릎으로부터 분리 배양한 관절연골세포에서 HO-1의 발현에 NO donors의 영향을 조사하였다. NO donors (SIN-1, SNAP 그리고 SNP)는 HO-1의 mRNA와 단백질의 합성을 크게 증가시켰다. 그리고 NO의 표적 분자인 guanylate cyclase와 protein kinase G의 관련성을 살펴본 결과, NO donors에 의한 Nrf2와 HO-1의 발현증가와는 무관한 것으로 보였다. 흥미롭게도, NO scavenger인 carboxy-PTIO와 SOD mimetic TEMPOL은 NO donors에 의한 HO-1의 발현을 억제하였다. 게다가, peroxynitrite scavenger인 MnTBAP에 의해서도 Nrf2와 HO-1의 발현이 완전히 억제되었다. Peroxynitrite는 NO와 superoxide의 반응에 의해 세포 내에서 자연적으로 형성되는 물질이므로 peroxynitrite가 관절연골세포에서 HO-1의 발현에 직접적인 영향을 주는지를 관찰하였다. 관절연골세포에 peroxynitrite를 처리한 결과, 시간과 농도 의존적으로 Nrf2와 HO-1의 발현을 크게 증가시켰다. 본 실험 자료 는 NO donors에 의한 HO-1의 발현증가는 방출되는 NO의 직접적인 영향이라기 보다는 NO와 superoxide의 반응으로 형성되는 peroxynitrite에 의해 유도된다는 것을 시사한다.