• Biomedical Science Letters
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• v.2 no.2
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• pp.175-185
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• 1996

The mitogen-activated protein(MAP) kinase signal transduction pathway represents an important mechanism by which mitogen, such as serum and PMA, regulate cell proliferation and differentiation. Target substrates of the MAP kinase are located within several compartments containing plasma membranes and nucleus. We now report that serum addition induces proliferation of the P388 murine leukemia cell, but PMA does not, while both serum and PMA treatment cause translocation of the MAP kinase, mainly p42$^{mapk}$ isoform, from cytosol into the nucleus, which was monitored by immunoblot analysis using polyclonal anti-ERK1 antibodies. We investigated whether the MAP kinase was capable of phosphorylating c-Jun protein and GST-fusion proteins, the P562$^{kk}$N-terminal peptides (1-77 or 1-123 domain) of the T cell tyrosine kinase, using the partially purified MAP kinase by SP-sephadex C-50, phenyl superose and Mono Q column chromatography. We found that the partially purified MAP kinase was able to phosphorylate c-Jun protein and the GST-fusion protein expressed using E.coli DH5$\alpha$ which is transformed with pGEX-3Xb plasmid vector carrying of p562$^{kk}$N-terminal peptide-encoding DNA. These results imply that tyrosine kinase receptor/Ras/Raf/MAP kinase pathway is a major mechanism for mitogen-induced cell proliferation in P388 murine leukemia cell and that the various MAP kinase isoforms may have their own target substrates located in distinct subcellular compartments.

• Journal of Life Science
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• v.20 no.2
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• pp.202-207
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• 2010

Ras genes are responsible for up to 30% of human tumor mutations and are composed of three isoforms: H-Ras, K-Ras and N-Ras. The post-translational modification of the CAAX motif of the Ras protein is essential in Ras actions. In the present study, we studied the effects of novel farnesyl transferase inhibitors (FTIs), YH3938 and YH3945, on the actions of oncogenic mutants of H-Ras, K-Ras and N-Ras. YH3938 and YH3945 completely reverted the proliferation and morphology of oncogenic H-Ras-transformed Rat2 cells, but not of oncogenic K-Ras-transformed Rat2 cells. Oncogenic N-Ras-transformed Rat2 cells were slightly affected. Activation of SRE promoters by oncogenic H-Ras and N-Ras, but not by K-Ras, were inhibited by treatment with YH3938 and YH3945. Using bandshift analysis, YH3938 suppressed the processing of oncogenic H-Ras and N-Ras, but not that of oncogenic K-Ras protein. YH3945 only inhibited the processing of H-Ras. From these results, we conclude that YH3938 and YH3945 specifically inhibit actions of oncogenic H-Ras through inhibition of its farnesylation, that YH3938 also inhibits N-Ras activity in a dose-dependent manner, and that these drugs have no effect on oncogenic K-Ras activity.

• Journal of Ginseng Research
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• v.42 no.1
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• pp.50-56
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• 2018

Background: Saponins from Panax japonicus (SPJ) are the most abundant and main active components of P. japonicus, which replaces ginseng roots in treatment for many kinds of diseases in the minority ethnic group in China. Our previous studies have demonstrated that SPJ has the effects of anti-inflammation through the mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-${\kappa}B$) signaling pathways. The present study was designed to investigate whether SPJ can modulate intestinal tight junction barrier in aging rats and further to explore the potential mechanism. Methods: Aging rats had been treated with different doses (10 mg/kg, 30 mg/kg, and 60 mg/kg) of SPJ for 6 mo since they were 18 mo old. After the rats were euthanized, the colonic samples were harvested. Levels of tight junctions (claudin-1 and occludin) were determined by immunohistochemical staining. Levels of proinflammatory cytokines (interleukin-$1{\beta}$ and tumor necrosis factor-${\alpha}$) were examined by Western blot. NF-${\kappa}B$ and phosphorylation of MAPK signaling pathways were also determined by Western blot. Results: We found that SPJ increased the expression of the tight junction proteins claudin-1 and occludin in the colon of aging rats. Treatment with SPJ decreased the levels of interleukin-$1{\beta}$ and tumor necrosis factor-${\alpha}$, reduced the phosphorylation of three MAPK isoforms, and inhibited the expression of NF-${\kappa}B$ in the colon of aging rats. Conclusion: The studies demonstrated that SPJ modulates the damage of intestinal epithelial tight junction in aging rats, inhibits inflammation, and downregulates the phosphorylation of the MAPK and $NF-{\kappa}B$ signaling pathways.

• Development and Reproduction
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• v.5 no.2
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• pp.123-129
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• 2001

When mammalian oocytes undergo maturation, cumulus cells surrounding the oocyte exhibit remodeling of their structure known as cumulus expansion. Many molecules including hyaluronic acid participate in this remodeling. The present study aimed to investigate a possible existence of matrix metalloproteinases(MMPs) in the extracellular matrix(ECM) of human oocyte-cumulus complex. ECM was extracted from the human oocyte-cumulus complex. Gelatin gel zymogram of ECM exhibited 7 gelatinases having molecular weight of 300kDa, 240kDa, 200kDa, 180kDa, 116kDa, 97kDa, and 84kDa. This gelatinase profile was very different from that of ovarian mural granulosa cell extract or white blood cell extract, indicating that the oocyte-cumulus complex donating ECM was free from other than cumulus cells. When ethylenediaminetetraacetic acid or 1', 10'-phenanthroline was added to the reaction buffer during zymographic development, almost gelatinase activities were abolished, suggesting that they were MMPs. Following incubation of ECM in the presence of aminophenylmercuric acetate, an activator of proMMPs, 4 gelatinases of 240kDa, 180kDa, 97kDa, and 84kDa disappeared with the concomitant appearance of 80kDa, 65kDa, and 60kDa gelatinases. Based upon these observation, it is suggested that ECM of the human oocyte-cumulus complex consists of gelatinases, presumed to be MMP-2 and MMP-9 isoforms.

• Development and Reproduction
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• v.2 no.1
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• pp.21-27
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• 1998

The present study was performed to analyze the gene expressions of pituitary adenylate cyclase-activating polypeptide(PACAP) and its receptor in the rat uterus, a candidate for novel extrahypothalamic source and target. The PACAP cDNA fragments corresponding to the common exon region which is found in both the rat hypothalamus and testis were produced from all tissue samples including the rat uterus by reverse transcriptionpolymerase chain reaction (RT-PCR). No PCR product was amplified from the rat hypothalamic, pituitary, ovarian and uterine samples when the 5' primer corresponding to the testis-specific exon 1 region was used, while the predicted size of product was detected from the testis sample. RT-PCR using the uterine RNA and specific primers for the PACAP receptor yielded products with predicted sizes. Transcripts for the rat uterine PACAP receptor were identified as type I isoforms with hip-hop and hip- or hop-type inserts. After pregnant mare's serum gonadotropin (15 IU) treatment of immature rats (day 25), the level of PACAP mRNA was increased in 24 h and 48 h group, and was declined to the lowest in 72 h group. The present study shows the presence of transcripts for PACAP and its receptor isoform in the rat uterus. These finding ssuggest that the uterine PACAP ight act as a novel autocrine and/or paracrine factor via its specific receptors on the reglulation of rat uterine function and physiology during the reproductive cycle.

• Journal of fish pathology
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• v.26 no.1
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• pp.19-30
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• 2013

Megalocytivirus is a major fish pathogen in marine aquaculture of Asian countries including Korea. Despite of many species affected by this pathogen, little is known interaction between megalocytivirus and the fish immune system. One of the cyclooxygenase isoforms, named COX-2, is playing an important role in immune regulation, and distinct from COX-1 isoform of constitutive activity. COX-2 enzyme is induced by various inflammatory signals, including injection of lipopolysaccharide or infection by pathogenic agents. We cloned COX-2 gene in rock bream using degenerated primers designed from reported sequences of other fish species in PCR followed with 5'- and 3'-end RACE-PCR. The full length of cDNA of rbCOX2 (rock bream COX-2) gene are 2655 bp and that translates into 609 amino acids. The rbCOX-2 genomic organization are found to span 10 exons separated by 9 introns. We also studied if the experimental infection of rock bream with megalocytivirus could affect the expression of COX-2 gene. When injected with LPS, expression of the COX-2 gene was reached peak level at 1 day post injection and showed 13.10 fold increased level compared with that of control. While, when injected with megalocytivirus, we were not able to find significantly increased COX-2 gene expression different from that of control. Cloned and analyzed COX-2 gene in rock bream will help to understand defence mechanisms in fish after viral infection and will also support the development of the measures for treatment and prevention of viral infection.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.11
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• pp.1599-1607
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• 2004

This study examined the effects of pre-slaughter fasting, chasing stress and chiller ageing on objective meat quality, and their relations to the proteome profile of longissimus muscle using 20 male Korean native black pigs. Treatments were composed of two levels of pre-slaughter feed withdrawal, two levels of pre-slaughter stress and four chiller ageing times. A 15 min chasing stress immediately prior to slaughter significantly (p<0.05) decreased detectable levels of $\mu$-calpain activity during rigor development and chiller ageing, but did not have any direct effect on objective meat quality. On the other hand, pigs fed until the morning of slaughter resulted in significantly (p<0.05) higher hunter L* value and cooking loss than those which received an 18 h feed withdrawal prior to slaughter. Cooking loss and hunter L* value were constant during 7 d of chiller ageing, followed by significant increases at 14 d. The fed animals showed a significantly (p<0.05) higher hunter a* value at both 3 and 7 d, while the other group maintained a stable redness for 7 d. WB-shear force was not affected by the pre-slaughter treatments, but had significant (p<0.05) linear reduction from 1 to 7 d. A gelbased proteome analysis was performed on selected animals for low and high hunter L* values at 1 d. Ten and five spots had greater than two-fold spot densities for the low and high hunter L* groups, respectively. The ten spots included chain A, deoxyribounclease I complex with actin, heat shock protein 27 kDa, a protein similar to cardiac $Ca^{2+}$ release channel, and myosin heavy chain, while the five spots included chain A aldehyde dehydrogenase, glycerol-3 phosphate dehydrogenase, and hemoglobin alpha chain. In general, feeding until the morning of slaughter resulted in more desirable meat color, but appeared to reduce palatability due to increased cooking loss. Proteome analysis demonstrated that various proteins were concomitantly involved in the determination of final meat color. The most noticeable observation in the current study was that various isoforms for a particular protein differed in degradation and/or expression rate depending on meat quality.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.13
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• pp.5249-5252
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• 2014

Nogo protein, encoded by gene reticulon-4 (RTN4), includes three major isoforms by different splicing, named Nogo-A Nogo-B and Nogo-C. Nogo proteins play an important role in the apoptosis of cells, especially in tumor cells. RTN4 single nucleotide polymorphisms (SNPs) can influence the efficiency of transcription and translation thus being related with an individual's predisposition to cancer. The CAA insertion/deletion polymorphism (rs34917480) within RTN4 3'-UTR has been reported to be associated with many cancer types. In order to investigate the relationship between this polymorphism and susceptibility to non-small cell lung cancer (NSCLC) in the Chinese population, we conducted the present case-control study including 411 NSCLC patients and 471 unrelated healthy controls. The genotype distributions were significantly different between cases and controls (p=0.014). We found that the del allele could significantly increase NSCLC risk (ins/ins vs ins/del: p=0.007, OR 1.46, 95%CI=1.11-1.93; dominant model: p=0.004, OR 1.47, 95%CI=1.13-1.92 and allele model: p=0.008, OR 1.35, 95%CI=1.08-1.67). This association was stronger in participants over 60 years old, males and smokers. We therefore conclude that the CAA insertion/deletion polymorphism (rs34917480) contributes to non-small cell lung cancer risk in Chinese population. Age, sex and environmental exposure are also related to carcinogenic effects of rs34917480.

• Reproductive and Developmental Biology
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• v.35 no.2
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• pp.151-157
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• 2011

Clinical arthritis is typically divided into rheumatoid arthritis (RA) and osteoarthritis (OA). Arthritis-induced muscle weakness is a major problem in aged people, leading to a disturbance of balance during the gait cycle and frequent falls. The purposes of the present study were to confirm fiber type-dependent expression of muscle atrophy markers induced by arthritis and to identify the relationship between clinical signs and expression of muscle atrophy markers. Mice were divided into four experimental groups as follows: (1) negative control (normal), (2) positive control (CFA+acetic acid), (3) RA group (CFA+acetic acid+type II collagen), and (4) aging-induced OA group. DBQA/1J mice (8 weeks of age) were injected with collagen (50 ${\mu}g/kg$), and physiological (body weight) and pathological (arthritis score and paw thickness) parameters were measured once per week. The gastrocnemius muscle from animals in each group was removed, and the expression of muscle atrophy markers (MAFbx and MuRF1) and myosin heavy chain isoforms were analyzed by reverse transcription-polymerase chain reaction. No significant change in body weight occurred between control groups and collagen-induced RA mice at week 10. However, bovine type II collagen induced a dramatic increase in clinical score or paw thickness at week 10 (p<0.01). Concomitantly, the expression of the muscle atrophy marker MAFbx was upregulated in the RA and OA groups (p<0.01). A dramatic reduction in myosin heavy chain (MHC)-$I{\beta}$ was seen in the gastrocnemius muscles from RA and OA mice, while only a slight decrease in MHC-IIb was seen. These results suggest that muscle atrophy gene expression occurred in a fiber type-specific manner in both RA- and OA-induced mice. The present study suggests evidence regarding why different therapeutic interventions are required between RA and OA.

• The Journal of the Korean Society for Microbiology
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• v.35 no.2
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• pp.97-108
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• 2000

Two-dimensional gel electrophoresis (2-DE) is an essential tool of proteomics to analyse the entire set of proteins of an organism and its variation between organisms. Helicobacter pylori was tried to identify differences between strains. As the first step, whole H. pylori was lysed using high concentration urea contained lysis buffer [9.5 M Urea, 4% CHAPS, 35 mM Tris, 65 mM DTT, 0.01% SDS and 0.5% Ampholite (Bio-Rad, pH 3-10)]. The extract ($10\;{\mu}g$) was rehydrated to commercially available immobilised pH gradient (IPG) strips, then the proteins were separated according to their charges as the first dimensional separation. The IPG strips were placed on Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) to separate according to molecular mass of the proteins as the second dimension. The separated protein spots were visualised by silver staining in order to compare different expression of proteins between strains. Approximately 120 spots were identified in each mini-protein electrophoresised gel, furthermore about 65 to 75 spots were regarded as identical proteins in terms of pI value and molecular weight between strains used. In addition, distinct differences were found between strains, such as 219-1, Y7 and Y14, CH150. Two representative strains were examined using strips which had pH range from 4 to 7. This strips showed a number of isoforms which were considered large spots on pH range 3-10. Furthermore, the rest of spots on pH 4-7 IPG strips appeared very distinctive compared to broad range IPG strips. 2-DE seems to be an excellent tool for analysing and identifying variations between H. pylori strains.