• Korean Journal of Fisheries and Aquatic Sciences
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• v.28 no.2
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• pp.209-218
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• 1995

Bacteriophage T7 gene 2.5 protein, a single-stranded DNA binding protein, has been implicated in T7 DNA replication, recombination, and repair. Purified gene 2.5 protein has been shown to interact with the phage encoded gene 5 protein (DNA polymerase) and gene 4 proteins (helicase and primase) and stimulates their activities. Genetic analysis of T7 phage defective in gene 2.5 shows that the gene 2.5 protein is essential for T7 DNA replication and growth. T7 phage that contain null mutants of gene 2.5 were constructed by homologous recombination. These mutant phage $(T7\Delta2.5)$ cannot grow in Escherichia coli. After infection of E. coli with $T7\Delta2.5$, host DNA synthesis is shut off, and $T7\Delta2.5$ DNA synthesis is reduced to less than $1\%$ of wild-type phage DNA synthesis (Kim and Richardson, 1993, Proc. Natl. Aca. Sci. USA, 90, 10173-10177). A truncated gene 2.5 protein $(GP2.5-\Delta21C)$ deleted the 21 carboxyl terminal amino acids was constructed by in vitro mutagenesis. $GP2.5-\Delta21C$ cannot substitute for wild-type gene 2.5 protein in vivo; the phage are not viable and exhibit less than $1\%$ of the DNA synthesis observed in wild-type phage-infected cells. $GP2.5-\Delta21C$ has been purified to apparent homogeneity from cells overexpressing its cloned gene. Purified $GP2.5-\Delta21C$ does not physically into「act with T1 gene 4 protein as measured by affinity chromatography and immunoblot analysis. The mutant protein cannot stimulate T7 gene 4 protein activity on RNA-primed DNA synthesis and primer synthesis. These results suggest that C-terminal domain of gene 2.5 protein is essential for protein-protein interactions.

• Toxicological Research
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• v.17
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• pp.47-57
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• 2001

Alzheimer's disease (AD) is the most common cause of dementia in the elderly, and its pathology is characterized by the presence of numerous numbers of senile plaques and neurofibrillary tangles. Several genetic and transgenic studies have indicated that excess amount of $\beta$-amyloid protein (A$\beta$) is produced by mutations of $\beta$TEX>$\beta$-amyloid precursor protein and causes learning impairment. Moreover, $A\beta$ has a toxic effect on cultured nerve cells. To prepare AD model animals, we have examined continuous (2 weeks) infusion of $A\beta$ into the cerebral ventricle of rats. Continuous infusion of $A\beta$ induces learning impairment in water maze and passive avoidance tasks, and decreases choline acetyltransferase activity in the frontal cortex and hippocampus. Immunohistochemical analysis revealed diffuse depositions of $A\beta$ in the cerebral cortex and hippocampus around the ventricle. Furthermore, the nicotine-evoked release of acetylcholine and dopamine in the frontal cortex/hippocampus and striatum, respectively, is decreased in the $A\beta$-infused group. Perfusion of nicotine (50 $\mu\textrm{M}$) reduced the amplitude of electrically evoked population spikes in the CA1 pyramidal cells of the control group, but not in those of the $A\beta$-infused group, suggesting the impairment of nicotinic signaling in the $A\beta$-infused group. In fact, Kd, but not Bmax, values for ［$^3H$］ cytisine binding in the hippocampus significantly increased in the $A\beta$-infused rats. suggesting the decrease in affinity of nicotinic acetylcholine receptors. Long-term potentiation (LTP) induced by tetanic stimulations in CA1 pyramidal cells, which is thought to be an essential mechanism underlying learning and memory, was readily observed in the control group, whereas it was impaired in the $A\beta$-infused group. Taken together, these results suggest that $A\beta$ infusion impairs the signal transduction mechanisms via nicotinic acetylcholine receptors. This dysfunction may be responsible, at least in part, for the impairment of LTP induction and may lead to learning and memory impairment. We also found the reduction of glutathione- and Mn-superoxide dismutase-like immunoreactivity in the brains of $A\beta$-infused rats. Administration of antioxidants or nootropics alleviated learning and memory impairment induced by $A\beta$ infusion. We believe that investigation of currently available transgenic and non-transgenic animal models for AD will help to clarify the pathogenic mechanisms and allow assessment of new therapeutic strategies.

• Applied Biological Chemistry
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• v.46 no.2
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• pp.84-89
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• 2003

In order to find a chemical agent which is able to modulate the activity of $H^+-ATPase$, microsomal preparation was obtained from the root tissue of tomato plant and the effect of $La^{3+}$ was measured. The activities of plasma and vacuolar membrane $H^+-ATPase$ were analyzed by the inhibited activities using their specific inhibitors, vanadate and $NO_3-$, respectively. $La^{3+}$ inhibited microsomal ATPases in a dose-dependent manner and the inhibitory effect of $La^{3+}$ was suppressed by both vanadate and $NO_3-$, implying that $La^{3+}$ inhibits both plasma and vacuolar membrane $H^+-ATPase$. The Ki. values of $La^{3+}$which inhibit 50% of the activities of plasma and vacuolar membrane $H^+-ATPase$ were 57 and $78\;{\mu}M$, respectively. The $H^+-ATPase$ of the leaky microsomes made by the treatment of Triton X-100 were also inhibited by $La^{3+}$, suggesting that $La^{3+}$ directly inhibits both enzymes. Meanwhile, the inhibitory effect of $La^{3+}$ was decreased by increasing the concentration of ATP, The effect of ATP was also concentration-dependent and 7 mM ATP completely removed the inhibitory effect of $La^{3+}$. These results imply that $La^{3+}$ inhibits both plasma and vacuolar membrane $H^+-ATPases$ by decreasing the binding affinity of ATP and $La^{3+}$ can be used to control the activity or root $H^+-ATPases$.

• Journal of Embryo Transfer
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• v.28 no.2
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• pp.157-162
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• 2013

Methoxychlor (MXC) was developed to be a replacement for the banned pesticide DDT. HPTE [2,2-bis (p-hydroxyphenyl)-1,1,1-trichloroethane], which is an in vivo metabolite of MXC, has strong oestrogenic and anti-androgenic effects. MXC and HPTE are thought to produce potentially adverse effects by acting through oestrogen and androgen receptors. Of the two, HPTE binds to sex-steroid receptors with greater affinity, and it inhibits testosterone biosynthesis in Leydig cells by inhibiting cholesterol side-chain cleavage enzyme activity and cholesterol utilisation. In a previous study, MXC was shown to induce Leydig cell apoptosis by decreasing testosterone concentrations. I focused on the effects of MXC on male mice that resulted from interactions with sex-steroid hormone receptors. Sex-steroid hormones affect other organs including the kidney and liver. Accordingly, I hypothesised that MXC can act through sex-steroid receptors to produce adverse effects on the testis, kidney and liver, and I designed our experiments to confirm the different effects of MXC exposure on the male reproductive system, kidney and liver. In these experiments, I used pre-pubescent ICR mice; the puberty period in ICR mice is from postnatal day (PND) 45 to PND60. I treated the experimental group with 0, 100, 200, 400 mg MXC/kg b.w. delivered by an intra-peritoneal injection with sesame oil used as vehicle for 4 weeks. At the end of the experiment, the mice were sacrificed under anaesthesia. The testes and accessory reproductive organs were collected, weighed and prepared for histological investigation. I performed a chemiluminescence immune assay to observe the serum levels of testosterone, LH and FSH. Blood biochemical determination was also performed to check for other effects. There were no significant differences in our histological observations or relative organ weights. Serum testosterone levels were decreased in a dose-dependent manner; a greater dose resulted in the production of less testosterone. Compared to the control group, testosterone concentrations differed in the 200 and 400 mg/kg dosage groups. In conclusion, I observed markedly negative effects of MXC exposure on testosterone concentrations in pre-pubescent male mice. From our biochemical determinations, I observed some changes that indicate renal and hepatic failure. Together, these data suggest that MXC produces adverse effects on the reproductive system, kidney and liver.

• Microbiology and Biotechnology Letters
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• v.40 no.2
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• pp.92-97
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• 2012

Antimicrobial peptides (AMPs) are important components of living organisms acting against Gram-negative and Gram-positive bacterial and fungal pathogens. Cathelicidin human peptides have a variety of biological activities that can be used in clinical applications. AMPs are not produced naturally in large quantities, and chemical synthesis is also economically impractical, especially for long peptides. Therefore, as an alternative, heterologous expression of AMPs by recombinant techniques has been studied as a means to reduce production costs. E. coli is an excellent host for the expression of AMPs, as well as other recombinant proteins, because of the low cost involved and its easy manipulation. However, overexpression of AMPs in E. coli has been shown to cause difficulties resulting from the toxicity of the subsequently produced AMPs. Therefore, fusion expression was theorized to be a solution to this problem. In this study, AMPs were expressed as fused proteins with the glutathione S-transferase (GST) binding protein to protect against the toxicity of AMPs when expressed in E. coli. The LL37, and hybrid gaegurin and LL37 (GGN4(1-16)-LL37(17-32), which we designated as GL32, peptides were expressed as GST-fusion proteins in E. coli and the fusion proteins were then purified by affinity columns. The purified peptides were obtained by removal of GST and were confirmed by western blot analysis. The purified antimicrobial peptides then demonstrated antimicrobial activities against Gram-negative and Gram-positive bacterial strains.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.1
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• pp.179-184
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• 2014

Chemoresistance of breast cancer to doxorubicin is mediated mainly through activation of NF-kB and over expression of HER2. Curcumin and its analogues (PGV-0 and PGV-1) exert cytotoxic effects on T47D breast cancer cells. Suppression of NF-kB activation is suggested to contribute to this activity. The present study aimed to explore the effects of curcumin, PGV-0, and PGV-1 singly and in combination with doxorubicin on MCF-7/Dox cells featuring over-expression of HER2. In MTT assays, curcumin, PGV-0, and PGV-1 showed cytotoxicity effects against MCF-7/Dox with IC50 values of $80{\mu}M$, $21{\mu}M$, and $82{\mu}M$ respectively. These compounds increased MCF-7/Dox sensitivity to doxorubicin. Cell cycle distribution analysis exhibited that the combination of curcumin and its analogues with Dox increased sub G-1 cell populations. Curcumin and PGV-1 but not PGV-0 decreased localization of p65 into the nucleus induced by Dox, indicating that activation of NF-kB was inhibited. Molecular docking of curcumin, PGV-0, and PGV-1 demonstrated high affinity to HER2 at ATP binding site. This interaction were directly comparable with those of ATP and lapatinib. These findings suggested that curcumin, PGV-0 and PGV-1 enhance the Dox cytotoxicity to MCF-7 cells through inhibition of HER2 activity and NF-kB activation.

• Korean Journal of Weed Science
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• v.18 no.4
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• pp.314-324
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• 1998

This study was carried out to investigate the herbicidal action characteristics of EK3143 and EK3150, newly synthesized cyclohexanedione analogues, and their inhibitory effects on the acetyl-CoA carboxylase activity isolated from various plants. Sethoxydim, a typical cyclohexanedione herbicide, had a high herbicidal activity against only grasses with growth inhibition, leaf chlorosis and necrosis. EK3143 and EK3150 had a lower effect against grasses but a higher effect against broadleaf plants such as black nightshade and velvetleaf than sethoxydim had. Low rate application of EK3143 induced leaf bleaching(whitening), while high rate application induced the same symptoms as those of sethoxydim. EK3150 induced more significant whitening, additionally accompanying a curling of meristem part in broadleaf plants. A consistant tendency was observed between the growth inhibition against grasses and the senstivity of grass ACCase to these compounds, but not between herbicidal activities against broadleaf plants and the senstivity of dicotyledonous ACCase; i.e. EK3150, which had higher herbicidal activity against black nightshade and velvetleaf than EK3143 had in greenhouse, rather showed lower ACCase inhibition. In EK3150-treated barnyardgrass, growth inhibition occurred prior to decrease of photosynthetic pigments, in contrast to that of norflurazon and EK3140. These results suggest that EK3143 and EK3150 may have two target sites, ACCase and an unknown site on fatty acid synthesis related to growth inhibition and bleaching respectively, and their symptoms would be appeared in different degree by a differential binding affinity between two action sites.

• 한국생물공학회:학술대회논문집
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• 2004.07a
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• pp.1-20
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• 2004

Polychlorinated biphenyls (PCBs), one a group of persistent and widespread environmental pollutants, have been considered to be involved in immunotoxicity, carcinogenesis, and apoptosis. However, the toxic effects and physical properties of a PCB congener are dependent on the structure. In the present study, we investigate the toxic effects and action mechanisms of PCBs In cells. Among the various congeners tested, 2,2',4,6,6'-PeCB-pentachlorobiphenyl (PeCB), a highly ortho-substituted congener having negligible binding affinity for aryl hydrocarbon receptor (AhR), caused the most potent toxicity and specific effects in several cell types. 2,2',4,6,6'-PeCB induced apoptotic cell death of human monocytic cells, suggesting that PCB-induced apoptosis may be linked to immunotoxicity. In addition, 2,2',4,6,6'-PeCB induced mitotic arrest by interfering with mitotic spindle assembly in NIH3T3 fibroblasts, followed by genetic instability which triggers p53 activation. Which suggests that 2,2',4,6,6'-PeCB may be involved in cancer development by causing genetic instability through mitotic spindle damage. On the other hand, 2,2',4,6,6'-PeCB increased cyclooxygenase-2 (COX-2) involved in cell survival through ERK1/2 MAPK and p53 in Rat-1 fibroblasts and mouse embryonic fibroblasts, triggering compensatory mechanism for abating its toxicity. Taken together, these results demonstrate that PCB congeners of different structure have distinct mechanism of action and 2,2',4,6,6'-PeCB causes several toxicity as well as compensatory mechanism in cells.

• Toxicological Research
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• v.21 no.4
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• pp.347-353
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• 2005

Eukaryotic initiation factor 4E (elF4E) is a key element for cap-dependent protein translation controlled by affinity between elF4E and 4E-binding protein 1 (4E-BP1). Rapamycin can also affect protein translation by regulating 4E-BP1 phosphorylation. Tobacco-specific nitrosamine, 4(N-methyl-N-nitrosamino )-1-(3-pyridyl)-1-butanone (NNK) is a strong lung carcinogen, but its precise lung cancer induction mechanism remains unknown. Relative roles of cap-dependent and -independent protein translation in terms of NNK-induced lung carcinogenesis were elucidated using normal human bronchial epithelial cells. NNK concentrations applied in this study did not decrease cell viability. Addition of NNK restored rapamycin-induced decrease of protein synthesis and rapamycin-induced phosphorylation of 4E-BP1, and increased expression levels of mTOR, ERK1/2, p70S6K, and Raf-1 in a concentration-dependent manner. NNK also caused perturbation of normal cell cycle progression. Taken together, NNK might cause toxicity through the combination of restoration of 4E-BP1 phosphorylation and increase of elF4E as well as mTOR protein expression, interruption of Raf1/ERK as well as the cyclin G-associated p53 network. Our data could be applied towards elucidation of the molecular basis for lung cancer treatment.

• Journal of Life Science
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• v.28 no.1
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• pp.99-104
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• 2018

Streptococcus pneumoniae is one of the major pathogens in community-acquired diseases, and it contains several factors that promote its pathogenesis, including pneumolysin (PLY). PLY is a member of the cholesterol-dependent cytolysin family, which attacks cholesterol-containing membranes, thereby forming ring-shaped pores. Thus, it is a major key target for vaccines against pneumococcal disease. We cloned the PLY gene from S. pneumoniae D39 and inserted it into the pQE-30 vector. Recombinant PLY (rPLY) was overexpressed in Escherichia coli M15 and purified by $Ni^{2+}$ affinity chromatography. Similarly, a PLY-EGFP fusion gene was produced by inserting the EGFP gene at the 3' end of the PLY gene in the same vector, and the recombinant protein was purified. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) showed that both recombinant proteins were purified. rPLY exhibited significant hemolytic activity against 1% human red blood cells (RBCs). Complete hemolysis was obtained at 500 ng/ml, and 50% hemolysis was found with a 240 ng/ml concentration. In contrast, rPLY-EGFP did not show hemolytic activity. However, rPLY-EGFP did bind the RBC membrane, indicating that rPLY-EGFP lost hemolytic activity via EGFP fusion, while retaining its membrane-binding ability. These data suggest that PLY's C terminus is important for its hemolytic activity. Therefore, these two recombinant proteins can be extremely useful for investigating the toxin mechanism of PLY and cell damage during pneumonia.