• Toxicological Research
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• v.25 no.3
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• pp.113-118
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• 2009

Cell death induced by menadione (vitamin K-3,2-methyl-1,4-naphthoquinone) has been investigated in human promyelocytic leukemia HL-60 cells. Menadione was found to induce both apoptosis and necrosis in HL-60 cells. Low concentration ($1{\sim}$50 ${\mu}$M) of menadione induced apoptotic cell death, which was demonstrated by typical DNA ladder patterns on agarose gel electrophoresis and flow cytometry analysis. In contrast, a high concentration of menadione (100 ${\mu}$M) induced necrotic cell death, which was demonstrated by DNA smear pattern in agarose gel electrophoresis. Necrotic cell death was accompanied with a great reduction of cell viability. Menadione activated caspase-3, as evidenced by both increased protease activity and proteolytic cleavage of 116 kDa poly(ADP-ribose) polymerase (PARP) into 85 kDa cleavage product. Caspase-3 activity was maximum at 50 ${\mu}$M of menadione, and very low at 100 ${\mu}$M of menadione. Taken together, our results showed that menadione induced mixed types of cell death, apoptosis at low concentrations and necrosis at high concentrations in HL-60 cells.

• Fisheries and Aquatic Sciences
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• v.12 no.3
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• pp.185-193
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• 2009

A complete cDNA, which encodes for a myostatin-like protein (Es-MSTN), was isolated from the Chinese mitten crab, Eriocheir sinensis. Es-MSTN was composed of 2,397 nucleotides and the open reading frame (ORF) specified a protein containing 468 amino acids. Es-MSTN exhibited 32% amino acid sequence identity and 52% similarity to human myostatin. Multiple sequence alignment analysis indicated that Es-MSTN possessed the conserved proteolytic cleavage site (RXXR) for maturation of the protein and nine cysteine residues for disulfide bridges. Besides the conserved structural features, Es-MSTN also exhibits its unique characters; a longer N-terminal domain which is involved in protein folding and latent form of myostatin and absence of the cleavage site for BMP-1/tolloid family of metalloproteinase to activate mature myostatin. Phylogenetic analysis suggests that Es-MSTN showed the closely related to both vertebrate myostatin and GDF11. Es-MSTN is expressed highly in the claw muscle, leg muscle, thoracic muscle and heart, and moderately in the hindgut suggesting that Es-MSTN may play important roles in the muscle tissues. As homolog of mammalian myostatin and GDF11, Es-MSTN may be involved in development of muscular tissue and further study will help to produce high-quality seafood.

• BMB Reports
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• v.44 no.11
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• pp.735-740
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• 2011

Tunicamycin, an endoplasmic reticulum (ER) stress inducer, specifically inhibits N-glycosylation. The cyclic AMP (cAMP) response element-binding protein H (CREBH) was previously shown to be regulated by UPR-dependent proteolytic cleavage in the liver. On the other hand, the role of CREBH in other tissues is unknown. In the present study, tunicamycin increased the level of CREBH activation (cleavage) as well as mRNA expression in osteoblast cells. Adenoviral (Ad) overexpression of CREBH suppressed BMP2-induced expression of alkaline phosphatase (ALP) and osteocalcin (OC). Interestingly, the BMP2-induced OASIS (structurally similar to CREBH, a positive regulator of osteoblast differentiation) expression was also inhibited by CREBH overexpression. In addition, inhibition of CREBH expression using siRNA reversed the tunicamycin-suppressed ALP and OC expression. These results suggest that CREBH inhibited osteoblast differentiation via suppressing BMP2-induced ALP, OC and OASIS expression in mouse calvarial derived osteoblasts.

• Molecules and Cells
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• v.21 no.3
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• pp.395-400
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• 2006

Glutathione S-transferase P1 (GSTP1) plays an important role in detoxification and the metabolism of xenobiotics. Here we show that GSTP1 also regulates the MEKK1-MKK7 signaling pathway. Over-expression of GSTP1 in HEK293 cells inhibited both ${\Delta}MEKK1$- and etoposide-induced apoptosis, and inhibited procaspase-3 activation and PARP cleavage. MEKK1- induced apoptosis requires both its kinase activity and proteolytic cleavage. ${\Delta}MEKK1$ activity was inhibited by over-expression of GSTP1 in vivo and MEKK1 kinase activity was also inhibited by GSTP1 in vitro when assayed with bacterially-expressed MKK7(KM) protein as substrate. GSTP1 inhibition of etoposide-induced cell apoptosis was mainly due to its ability to suppress MEKK1 kinase activity. The glutathione-conjugating activity of GSTP1 was essential for the above effects. These findings provide insight into the mechanism by which GSTP1 protects cells from genotoxin-induced apoptosis.

• The Plant Pathology Journal
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• v.23 no.4
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• pp.245-250
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• 2007

The complete genome sequence of Zucchini yellow mosaic virus stain A (ZYMV-A) isolated from a hollyhock (Althaea rosea) was determined by using RT-PCR with a series of primer sets. The virus genome consisted of 9593 nucleotides (nt), excluding the poly(A) tract at 3' terminus of the virus genome, with 5' and 3' untranslated region of 139 and 211 nt, respectively. The deduced polyprotein of ZYMV-A consisted of 3080 amino acid (aa) residues and was 351 kDa in molecular weight. All proteolytic cleavage sites of the polyprotein of ZYMV-A were compared with those of ZYMV strains, which showed the cleavage sites were conserved among ZYMV strains. The HC-Pro contained the KITC and PTK motifs, and the DAG motif was located at CP ORF of ZYMV-A, suggesting that ZYMV-A is aphid-transmissible. Phylogenetic tree analysis based on the complete genome among ZYMV strains or CP ORFs with other potyviruses showed ZYMV strains formed a distinct group. These results clearly confirmed that ZYMV-A was another distinct strain in ZYMV population at molecular level.

• Animal cells and systems
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• v.7 no.1
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• pp.1-9
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• 2003

Programmed cell death, or apoptosis, is one of the most studied areas of modern biology. Apoptosis is a genetically regulated process, which plays an essential role in the development and homeostasis of higher organisms. Mitochondria, known to play a central role in regulating cellular metabolism, was found to be critical for regulating apoptosis induced under both physiological and pathological conditions. Mitochondria are a major source of reactive oxygen species (ROS) but they can also serve as its target during the apoptosis process. Release of apoptogenic factors from mitochondria, the best known of which is cytochrome c, leads to assembly of a large apoptosis-inducing complex called the apoptosome. Cysteine pretenses (called caspases) are recruited to this complex and, following their activation by proteolytic cleavage, activate other caspases, which in turn target for specific cleavage a large number of cellular proteins. The redox regulation of apoptosis during and after cytochrome c release is an area of intense investigation. This review summarizes what is known about the biological role of ROS and its targets in apoptosis with an emphasis on its intricate connections to mitochondria and the basic components of cell death.

• Korean Journal of Microbiology
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• v.38 no.3
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• pp.153-161
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• 2002

Sox4, a transcription factor, consists of three functional domains: an HMG-box domain as a DNA binding domain, serine rich region as a transactivation domain and glycine rich region (GRR), an unknown functional domain. Although Sox4 is known to be functionally involved in heart, B-cell and reproductive system development, its physiological function remains to be elucidated. We used pGEX expression system to develop a simple and rapid method for purifying Sox4 protein in suitable forms for biochemical studies of their functions. Unexpectedly, we observed that full-length Sox4 appears to be protease-sensitive during expression and purification in E. coli. To map the protease-sensitive site in Sox4, we generated various constructs with each of functional domains of Sox4 and purified as the GST-Sox4 fusion proteins using glutathione beads. We found that the specific cleavage site for the proteolytic enzyme, which exists in E. coli, is localized within the novel GRR of Sox4. Our study suggest that the GRR of Sox4 may a target for the cellular protease action and this cleavage in the GRR may be involved in regulating physiological function of Sox4. Additionally, our study may provide a useful method for investigating the proteolytic cleavage of the target molecule in E. coli.

• Journal of Microbiology and Biotechnology
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• v.14 no.2
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• pp.430-434
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• 2004

Three extracellular protease-deficient Bacillus subtilis strains were transformed with the plasmid pCK98 containing the endo-$\beta$-1,4-glucanase (Eng) gene of B. subtilis BSE616. The three transformants, B. subtilis DB104 (pCK98), WB600 (pCK98) and WB700 (pCK98), produced the same high level of enzyme activity and showed similar patterns of cell growth and enzyme production. When B. subtilis DB 104 (pCK98), a two-extracellular protease deficient strain, was cultured for 22 h, almost all the secreted enzyme was found to be in the completely cleaved form by both activity staining and Western blotting studies. B. subtilis WB600 (pCK98), a six-extracellular protease-deficient strain, produced a partially cleaved form in addition to the intact form of the enzyme, although the degree of internal cleavage of the enzyme was greatly reduced. With B. subtilis WB700 (pCK98), a seven-extracellular protease-deficient strain, almost all the enzyme was produced as the intact uncleaved form. This study illustrates that a role of the V pr protease is to degrade foreign proteins produced in B. subtilis and WB700 is a suitable expression system for producing the intact form of the Eng and other foreign proteins that may lose at least part of their efficacy due to internal proteolytic cleavage.

• Molecules and Cells
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• v.24 no.2
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• pp.224-231
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• 2007

$H_2O_2$, as an example of oxidative stress, induces cardiac myocyte apoptosis. Bcl-2 family proteins are key regulators of the apoptotic response while their functions can be regulated by post-translational modifications including phosphorylation, dimerization or proteolytic cleavage. In this study, we examined the role of various protein kinases in regulating total BAD protein levels in adult rat cardiac myocytes undergoing apoptosis. Stimulation with 0.1 mM $H_2O_2$, which induces apoptosis, resulted in a marked down-regulation of BAD protein, which is attributed to cleavage by caspases since it can be restored in the presence of a general caspase inhibitor. Inhibition of PKC, p38-MAPK, ERK1/2 and PI-3-K did not influence the reduced BAD protein levels observed after stimulation with $H_2O_2$. On the contrary, inhibition of PKA or specifically $PKC{\delta}$ resulted in up-regulation of BAD. Decreased caspase 3 activity was observed in $H_2O_2$ treated cells after inhibition of PKA or $PKC{\delta}$ whereas inhibition of PKA also resulted in improved cell survival. Furthermore, addition of okadaic acid to inhibit selected phosphatases resulted in enhanced BAD cleavage. These data suggest that, during oxidative stress-induced cardiac myocyte apoptosis, there is a caspase-dependent down-regulation of BAD protein, which seems to be regulated by coordinated action of PKA, $PKC{\delta}$ and phosphatases.

• Biomedical Science Letters
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• v.21 no.2
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• pp.126-130
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• 2015

There are several studies that show hypothermia improves cellular ischemia damages on experimental and clinical bases. However, its exact molecular mechanisms are unclear. In this study, we demonstrate that hypothermia induced insulin-like growth factor 1 (IGF1) gene expression, and its expression was dramatically decreased under ischemic insults. It was also demonstrated that hypothermia activated endoplasmic reticulum (ER) stress sensors especially both the phosphorylation of $eIF2{\alpha}$ (eukaryotic translation initiation factor 2 alpha) and ATF6 (activating transcription factor-6) proteolytic cleavage. However, the factors of apoptosis and autophagy were not associated with hypothermia. We suggest that hypothermia-treated IGF1 gene expression after ischemia may show a good possibility for the development of treatments and diagnostic methods in cerebral ischemic damages.