• Journal of Microbiology and Biotechnology
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• v.27 no.11
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• pp.1999-2009
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• 2017

The secretion efficiency of a protein in a Sec-type secretion system is mainly determined by an N-terminal signal peptide and its combination with its cognate protein. Five signal peptides, namely, two synthetic Sec-type and three Bacillus licheniformis alpha-amylase-derived signal peptides, were compared for periplasmic expression of the human growth hormone (hGH) in E. coli. Based on in silico predictions on the signal peptides' cleavage efficiencies and their corresponding mRNA secondary structures, a number of amino acid substitutions and silent mutations were considered in the modified signal sequences. The two synthetic signal peptides, specifically designed for hGH secretion in E. coli, differ in their N-terminal positively charged residues and hydrophobic region lengths. According to the mRNA secondary structure predictions, combinations of the protein and each of the five signal sequences could lead to different outcomes, especially when accessibility of the initiator ATG and ribosome binding sites were considered. In the experimental stage, the two synthetic signal peptides displayed complete processing and resulted in efficient secretion of the mature hGH in periplasmic regions, as was demonstrated by protein analysis. The three alpha-amylase-derived signal peptides, however, were processed partially from their precursors. Therefore, to achieve efficient secretion of a protein in a heterologous system, designing a specific signal peptide by using a combined approach of optimizations of the mRNA secondary structure and the signal peptide H-domain and cleavage site is recommended.

• The Korean Journal of Physiology and Pharmacology
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• v.26 no.6
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• pp.469-478
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• 2022

WNT signaling plays an important role in cardiac development, but abnormal activity is often associated with cardiac hypertrophy, myocardial infarction, remodeling, and heart failure. The effect of WNT signaling on regulation of atrial natriuretic peptide (ANP) secretion is unclear. Therefore, the purpose of this study was to investigate the effect of Wnt agonist 1 (Wnta1) on ANP secretion and mechanical dynamics in beating rat atria. Wnta1 treatment significantly increased atrial ANP secretion and pulse pressure; these effects were blocked by U73122, an antagonist of phospholipase C. U73122 also abolished the effects of Wnta1-mediated upregulation of protein kinase C (PKC) β and γ expression, and the PKC antagonist Go 6983 eliminated Wnta1-induced secretion of ANP. In addition, Wnta1 upregulated levels of phospho-transforming growth factor-β activated kinase 1 (p-TAK1), TAK1 banding 1 (TAB1) and phospho-activating transcription factor 2 (p-ATF2); these effects were blocked by both U73122 and Go 6983. Wnta1-induced ATF2 was abrogated by inhibition of TAK1. Furthermore, Wnta1 upregulated the expression of T cell factor (TCF) 3, TCF4, and lymphoid enhancer factor 1 (LEF1), and these effects were blocked by U73122 and Go 6983. Tak1 inhibition abolished the Wnta1-induced expression of TCF3, TCF4, and LEF1 and Wnta1-mediated ANP secretion and changes in mechanical dynamics. These results suggest that Wnta1 increased the secretion of ANP and mechanical dynamics in beating rat atria by activation of PKC-TAK1-ATF2-TCF3/LEF1 and TCF4/LEF1 signaling mainly via the WNT/Ca²⁺ pathway. It is also suggested that WNT-ANP signaling is implicated in cardiac physiology and pathophysiology.

• Korean Journal of Veterinary Research
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• v.39 no.5
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• pp.930-937
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• 1999

Atrial natriuretic peptide(ANP) is a hormone with potent natriuretic, diuretic and relaxing properties on vascular smooth muscle. Specific chemical modulator in response for the ANP secretion has not been found yet. Therefore, we have investigated the role of $Ca^{2+}$ responsible for the regulation of ANP induced by protein kinase C(PKC) on mechanically stretch-induced ANP secretion in the rat atria. The results obtained were as follows ; 1. ANP secretion and ANP concentration were increased to more in $Ca^{2+}$-free buffer than in the Kreb-Henseleit buffer on mechanically stretch-induced ANP secretion(p < 0.05), but extracellular fluid translocation(ECF) was not significant. Phorbol 12-myristate 13-acetate(PMA, $10^{-7}M$) induced ANP secretion and ANP concentration in $Ca^{2+}$-free buffer shown to more accentuate on mechanically stretch-induced ANP secretion than in the $Ca^{2+}$-free buffer(p < 0.05), but ECF translocation was not significant. 2. In the presence of ryanodine($3{\times}10^{-6}M$), PMA($10^{-7}M$) induced ANP secretion and ANP concentration in the Kreb-Henseleit buffer were shown to more increase on mechanically stretch-induced ANP secretion than in the ryanodine($3{\times}10^{-6}M$) with the Kreb-Henseleit buffer(p < 0.05), but ECF translocation was not significant. 3. In the presence of ryanodine($3{\times}10^{-6}M$), PMA($10^{-7}M$) induced ANP secretion and ANP concentration in the $Ca^{2+}$-free buffer was shown to more increase on mechanically stretch-induced ANP secretion than in the ryanodine($3{\times}10^{-6}M$) with the $Ca^{2+}$-free buffer on mechanically induced ANP secretion(p < 0.05), but ECF translocation was not significant. The results suggest that PKC-induced ANP secretion may not be related to the change of $Ca^{2+}$ on mechanically induced ANP secretion in the rat atria.

• BMB Reports
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• v.54 no.5
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• pp.246-252
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• 2021

The Golgi complex plays a central role in protein secretion by regulating cargo sorting and trafficking. As these processes are of functional importance to cell polarity, motility, growth, and division, there is considerable interest in achieving a comprehensive understanding of Golgi complex biology. However, the unique stack structure of this organelle has been a major hurdle to our understanding of how proteins are secreted through the Golgi apparatus. Herein, we summarize available relevant research to gain an understanding of protein secretion via the Golgi complex. This includes the molecular mechanisms of intra-Golgi trafficking and cargo export in the trans-Golgi network. Moreover, we review recent insights on signaling pathways regulated by the Golgi complex and their physiological significance.

• Journal of Microbiology and Biotechnology
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• v.14 no.1
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• pp.128-133
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• 2004

The secretion capacity of two E. coli strains (JM109 and AF1000) was evaluated through the expression of two human proinsulin fusion proteins using the translocation signal sequence from Staphylococcal protein A (SpA). Although a 7 to 11-fold difference in the expression levels was attained by the use of different promoters (SpA and malK promoters) and copy-number vectors (700 and 50 copies per cell), the maximum translocation rates for all the systems were around 140,000 amino acids $cell^{-1} min^{-1}$. Moreover, the secretion capacity was found to be independent of the size of the exiting peptide and its translational rate.

• Journal of Microbiology and Biotechnology
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• v.25 no.7
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• pp.963-977
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• 2015

The well-characterized gram-positive bacterium Bacillus subtilis is an outstanding industrial candidate for protein expression owing to its single membrane and high capacity of secretion, simplifying the downstream processing of secretory proteins. During the last few years, there has been continuous progress in the illustration of secretion mechanisms and application of this robust host in various fields of life science, such as enzyme production, feed additives, and food and pharmaceutical industries. Here, we review the developments of Bacillus subtilis as a highly promising expression system illuminating strong chemical- and temperatureinducible and other types of promoters, strategies for ribosome-binding-site utilization, and the novel approach of signal peptide selection. Furthermore, we outline the main steps of the Sec pathway and the relevant elements as well as their interactions. In addition, we introduce the latest discoveries of Tat-related complex structures and functions and the countless applications of this full-folded protein secretion pathway. This review also lists some of the current understandings of ATP-binding cassette transporters. According to the extensive knowledge on the genetic modification strategies and molecular biology of Bacillus subtilis, we propose some suggestions and strategies for improving the yield of intended productions. We expect this to promote striking future developments in the optimization and application of this bacterium.

• Journal of Microbiology
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• v.35 no.3
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• pp.213-221
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• 1997

The secretion of the alkaliphilic Bacillus sp. S-1 extracellular pullulanase involves translocation across the cytoplasmic membrane of the Gram-positive bacterial cell envelope. Translocation of the intracellular pullulanase PUL-I, was traced to elucidate the mechanism and pathway of protein secretion from an alkaliphilic Bacillus sp. S-1. Pullulanase could be slowly bue quantitatively released into the medium during growth of the cells in medium contianing proteinase K. The released pullulanase lacked the N-terminal domain. The N-terminus is the sole membrane anchor in the pullulanase protein and was not affected by proteases, confirming that it is not exposed on the cell surface. Processing of a 180,000M$\_$r/ pullulanase to a 140,000M$\_$r/ polypeptide has been demonstrated in cell extracts using antibodies raised against 140,000M$\_$r/ extracellular form. Processing of the 180,000 M$\_$r/ protein occured during the preparation of extracts in an alkaline pH condition. A modified rapid extraction procedure suggested that the processing event also occured in vivo. Processing apparently increased the activity of pullulanase. The western blotting analysis with mouse anti-serum against 140-kDa extracellular pullulanase PUL-E showed that PUL-I is processed into PUL-X via intermediate form of PUL-E. Possible explanationa for the translocation are discussed.

• Molecules and Cells
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• v.28 no.3
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• pp.167-174
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• 2009

Genistein has been reported to potentiate glucose-stimulated insulin secretion (GSIS). Inhibitory activity on tyrosine kinase or activation of protein kinase A (PKA) was shown to play a role in the genistein-induced potentiation effect on GSIS. The aim of the present study was to elucidate the mechanism of genistein-induced potentiation of insulin secretion. Genistein augmented insulin secretion in INS-1 cells stimulated by various energygenerating nutrients such as glucose, pyruvate, or leucine/glutamine (Leu/Gln), but not the secretion stimulated by depolarizing agents such as KCl and tolbutamide, or $Ca^{2+}$ channel opener Bay K8644. Genistein at a concentration of $50{\mu}M$ showed a maximum potentiation effect on Leu/Gln-stimulated insulin secretion, but this was not sufficient to inhibit the activity of tyrosine kinase. Inhibitor studies as well as immunoblotting analysis demonstrated that activation of PKA was little involved in genistein-induced potentiation of Leu/Gln-stimulated insulin secretion. On the other hand, all the inhibitors of $Ca^{2+}$/calmodulin kinase II tested, significantly diminished genistein-induced potentiation. Genistein also elevated the levels of $[Ca^{2+}]_i$ and phospho-CaMK II. Furthermore, genistein augmented Leu/Gln-stimulated insulin secretion in CaMK II-overexpressing INS-1 cells. These data suggest that the activation of CaMK II played a role in genistein-induced potentiation of insulin secretion.

• Biomolecules & Therapeutics
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• v.20 no.3
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• pp.346-351
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• 2012

Cells show various stress signs when they are challenged with severe physiological problems. Majority of such cellular stresses are conveyed to endoplasmic reticulum (ER) and unfolded protein response (UPR) serves as typical defense mechanism against ER stress. This study investigated an interaction between ER stress agents using macropage cell line Raw 264.7. When activated by lipopolysaccharide (LPS), the cell lines showed typical indicators of ER stress. Along with molecular chaperones, the activation process leads to the production of additional inflammatory mediators. Following activation, the macrophage cell line was further treated with TUN and characterized in terms of chaperone expression and cytokine secretion. When treated with TUN, the activated macrophage cell leads to increased secretion of IL-6 although expression of ER stress markers, GRP94 and GRP78 increased. The secretion of cytokines continued until the addition of BFA which inhibits protein targeting from ER to Golgi. However, secretion of cytokines was ceased upon dual treatments with BFA and TG. This result strongly implies that cells may differently deal with various polypeptides depending on the urgency in cellular function under ER stress. Considering IL-6 is one of the most important signal molecules in macrophage, the molecule might be able to circumvent ER stress and UPR to reach its targeting site.

• The Korean Journal of Physiology
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• v.25 no.1
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• pp.27-35
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• 1991

Generally, it has been known that cholecystokinin (CCK) release into the plasma is under cholinergic control, but secretin release is not. Thus in anesthetized dogs we studied the effect of atropine $(50\;{\mu}g/kg\;followed\;by\;50\;{\mu}g/kg/hr)$ on pancreatic secretion and plasma concentrations of bioactive CCK and immunoreactive secretin in response to intraduodenal perfusion of sodium oleate (1, 3 and 9 mmol/hr). The volume, protein output and bicarbonate output of the secretion were increased by sodium cleats and this oleate-induced secretion was decreased significantly by atropine administration. However the increased plasma CCK and secretin levels by sodium oleate were not changed by atropine. These results indicate that atropine suppressed sodium oleate-induced pancreatic secretion through inhibiting cholinergic mechanism directly rather than decreasing the release of pancreatic secretory hormones. In another set of experiments, bilateral cervical vagi were stimulated electrically to observe the changes of pancreatic secretion and the above two plasma hormone levels in the presence or absence of atropine. In the vagally stimulated dogs, the volume, protein output and bicarbonate output of the pancreatic secretion were increased significantly. Both plasma secretin and CCK were concomitantly released significantly by vagal stimulation. Atropine significantly depressed the pancreatic secretory response as well as the release of these two pancreatic secretory hormones. Therefore, we conclude that in the presence of atropine the depressed pancreatic response to vagal stimulation is at least, in part, due to decreased release of endogenous CCK and secretin. In the vagally stimulated animals, however, the involvement of direct cholinergic influence on pancreatic exocrine gland remains to be answered.