Gunsup Lee

High dose concentration administration of ascorbic acid inhibits tumor growth in BALB/C mice implanted with sarcoma 180 cancer cells via the restriction of angiogenesis.

To test the carcinostatic effects of ascorbic acid, we challenged the mice of seven experimental groups with 1.7 × 10-4 mol high dose concentration ascorbic acid after intraperitoneal administrating them with sarcoma S-180 cells. The survival rate was increased by 20% in the group that received high dose concentration ascorbic acid, compared to the control. The highest survival rate was observed in the group in which 1.7 × 10-4 mol ascorbic acid had been continuously injected before and after the induction of cancer cells, rather than just after the induction of cancer cells. The expression of three angiogenesis-related genes was inhibited by 0.3 times in bFGF, 7 times in VEGF and 4 times in MMP2 of the groups with higher survival rates. Biopsy Results, gene expression studies, and wound healing analysis in vivo and in vitro suggested that the carcinostatic effect induced by high dose concentration ascorbic acid occurred through inhibition of angiogenesis.

Expressions of Oxidative Stress-Related Genes and Antioxidant Enzyme Activities in Mytilus Galloprovincialis (Bivalvia, Mollusca) Exposed to Hypoxia

BackgroundIn this study, we investigated transcription and enzyme level responses of mussels Mytilus galloprovincialis exposed to hypoxic conditions. Genes for catalase (CAT), cytochrome P450, glutathione S-transferase (GST), metallothionein, superoxide dismutase (SOD), cytochrome c oxidase subunit 1 (COX-1), and NADH dehydrogenase subunit 2 were selected for study. Transcriptional changes were investigated in mussels exposed to hypoxia for 24 and 48 h and were compared to changes in control mussels maintained at normal oxygen levels. Activities of CAT, GST, and SOD enzymes, and lipid peroxidation (LPO) were also investigated in mussels following exposure to hypoxia for 24, 48, and 72 h.ResultsRelative to the control group, the CAT activity decreased in all hypoxia treatments, while the activity of GST significantly increased in mussels exposed to hypoxia for 24 and 48 h, but decreased in those exposed for 72 h. The LPO levels were significantly higher in mussels in the 24- and 48-h hypoxia treatments than those in the control mussels, but there was no significant change in the SOD activities among all hypoxia treatments. Messenger RNA levels for the CAT, cytochrome P450, GST, metallothionein, and SOD genes were not significantly affected by hypoxic conditions for 48 h, but the expressions of the COX-1 and NADH dehydrogenase subunit 2 genes were significantly repressed in mussels in both the 24- and 48-h exposure treatments.ConclusionsThese results demonstrate the transcriptional stability and changes among several genes related to oxidative stress under oxygen-depletion conditions in M. galloprovincialis and provide useful information about the modulation of antioxidant enzyme activities induced by hypoxia in a marine animal.

Ascorbic acid alleviates toxicity of paclitaxel without interfering with the anticancer efficacy in mice

Paclitaxel is used extensively as a chemotherapeutic agent against a broad range of tumors but often leads to the early termination of treatment due to severe toxic side effects. In this study, we hypothesized that ascorbic acid could reduce the toxic side effects without interfering with the anticancer effect of paclitaxel. To demonstrate this, we examined the effect of the combinational treatment of ascorbic acid and paclitaxel using H1299 (a non-small cell lung cancer cell line) and BALB/c mice implanted with or without sarcoma 180 cancer cells. In H1299 cells, the anticancer effects of the combinational treatment with paclitaxel and ascorbic acid were up to 1.7-foldhigher than those of single-agent paclitaxel treatment. In addition, it was shown that the viability of the HEL299 normal cells was up to 1.6-fold higher with the combinational treatment than with paclitaxel treatment alone. In vivo mouse experiments also showed that mice co-treated with paclitaxel and ascorbic acid did not exhibit the typical side effects induced by paclitaxel, such as a reduction in the numbers of white blood cells and red blood cells and the level of hemoglobin (P < .05). The analysis of cancer-related gene expression by quantitative real-time polymerase chain reaction and immunohistochemistry revealed that the combinational treatment suppressed cancer cell multiplication. Taken together, these results suggest that combinational chemotherapy with ascorbic acid and paclitaxel not only does not block the anticancer effects of paclitaxel but also alleviates the cytotoxicity of paclitaxel in vivo and in vitro.

A Nucleic-Acid Hydrolyzing Single Chain Antibody Confers Resistance to DNA Virus Infection in HeLa Cells and C57BL/6 Mice

Viral protein neutralizing antibodies have been developed but they are limited only to the targeted virus and are often susceptible to antigenic drift. Here, we present an alternative strategy for creating virus-resistant cells and animals by ectopic expression of a nucleic acid hydrolyzing catalytic 3D8 single chain variable fragment (scFv), which has both DNase and RNase activities. HeLa cells (SCH7072) expressing 3D8 scFv acquired significant resistance to DNA viruses. Virus challenging with Herpes simplex virus (HSV) in 3D8 scFv transgenic cells and fluorescence resonance energy transfer (FRET) assay based on direct DNA cleavage analysis revealed that the induced resistance in HeLa cells was acquired by the nucleic acid hydrolyzing catalytic activity of 3D8 scFv. In addition, pseudorabies virus (PRV) infection in WT C57BL/6 mice was lethal, whereas transgenic mice (STG90) that expressed high levels of 3D8 scFv mRNA in liver, muscle, and brain showed a 56% survival rate 5 days after PRV intramuscular infection. The antiviral effects against DNA viruses conferred by 3D8 scFv expression in HeLa cells as well as an in vivo mouse system can be attributed to the nuclease activity that inhibits viral genome DNA replication in the nucleus and/or viral mRNA translation in the cytoplasm. Our results demonstrate that the nucleic-acid hydrolyzing activity of 3D8 scFv confers viral resistance to DNA viruses in vitro in HeLa cells and in an in vivo mouse system.

RNA virus accumulation is inhibited by ribonuclease activity of 3D8 scFv in transgenic Nicotiana tabacum

Plant viruses continue to cause diseases on economically important crops. Therefore, numerous attempts to produce virus resistant plants have been reported by using the mechanisms such as host mediated protection and virus mediated protection. Here, a novel strategy of targeting viral RNA itself, rather than viral gene products, is presented to generate virus-resistant transgenic plants. A catalytic single chain variable antibody, 3D8 scFv, which has RNase activities, was functionally expressed in the cytosol of Nicotiana tabacum. We found that progenies of the transgenic tobacco plant acquired complete resistances against four ss-RNA tobamoviruses and one cucumovirus tested without viral accumulation and delayed onset of disease symptoms. The results showed that the resistance observed in 3D8 scFv transgenic plants was caused by the RNase activity of 3D8 scFv itself, not by RNA-mediated gene silencing mechanism. Taken together, we suggested that newly gained resistance of the 3D8 scFv transgenic plants to five ss-RNA viruses most likely resulted from the RNase activity of 3D8 scFv.

A nucleic acid hydrolyzing recombinant antibody confers resistance to curtovirus infection in tobacco

A catalytic single chain variable antibody (scFv), 3D8 scFv, which has DNase activities, was functionally expressed in Nicotiana tabacum. The subcellular localization of the GFP-fused 3D8 indicated that the 3D8 protein was expressed in the cytosol of the N. tabacum protoplasts. Progenies of the transgenic tobacco plants exhibited complete resistance against two single stranded (ss) DNA geminiviruses, including the Beet curly top virus and the Beet severe curly top virus, without viral accumulation or disease symptoms. We presented a novel strategy for targeting the viral DNA itself in a sequence non-specific manner, rather than the viral proteins or RNAs, in order to generate virus-resistant transgenic plants. No noticeable adverse effects on the growth and reproduction of the transgenic plants were observed. Our results demonstrated that targeting viral DNA is an effective strategy for protecting plants from ssDNA viruses.

Purification and Biochemical Characterization of Insoluble Acid Invertase (INAC-INV) from Pea Seedlings

Invertase (EC 3.2.1.26) catalyzes the hydrolysis of sucrose into D-glucose and D-fructose. Insoluble acid invertase (INAC-INV) was purified from pea (Pisum sativum L.) by sequential procedures entailing ammonium sulfate precipitation, ion exchange chromatography, absorption chromatography, reactive green-19 affinity chromatography, and gel filtration. The purified INAC-INV had a pH optimum of 4.0 and a temperature optimum of 45 °C. The effects of various concentrations of Tris-HCl, HgCl(2), and CuSO(4) on the activities of the purified invertase were examined. INAC-INV was not affected by Tris-HCl and HgCl(2). INAC-INV activity was inhibited by 6.2 mM CuSO(4) up to 50%. The enzymes display typical hyperbolic saturation kinetics for sucrose hydrolysis. The K(m) and V(max) values of INAC-INV were determined to be 4.41 mM and 8.41 U (mg protein)(-1) min(-1), respectively. INAC-INV is a true member of the β-fructofuranosidases, which can react with sucrose and raffinose as substrates. SDS-PAGE and immunoblotting were used to determine the molecular mass of INAC-INV to be 69 kDa. The isoelectric point of INAC-INV was estimated to be about pH 8.0. Taken together, INAC-INV is a pea seedling invertase with a stable and optimum activity at lower acid pH and at higher temperature than other invertases.

Identification of nickel response genes in abnormal early developments of sea urchin by differential display polymerase chain reaction.

Bioassays and biomarkers have been previously developed to assess the effects of heavy metal contaminants on the early life stages of the sea urchin. In this study, malformation in the early developmental processes was observed in sea urchin (Strongylocentrotus intermedius) larvae exposed to 10 ppm Ni for over 30 h. The most critical stage at which the triggering of nickel effects takes place is thought to be the blastula stage, which occurs after fertilization in larval development. To investigate the molecular-level responses of sea urchin exposed to heavy metal stress and to explore the differentially expressed genes that are induced or repressed by nickel, differential display polymerase chain reaction (DD-PCR) was used with sea urchin mRNAs. The malformation-related genes expressed in the early life stages of the sea urchin were cloned from larvae exposed to 10 ppm of nickel for 15 h, and accessed via DD-PCR. Sequence analysis results revealed that each of the genes evidenced high homology with EGF2, PCSK9, serine/threonine protein kinase, apolipophorin precursor protein, and MGC80921 protein/transcript variant 2. This result may prove useful in the development of novel biomarkers for the assessment of heavy metal stresses on sea urchin embryos.

Agroinoculation of Nicotiana benthamiana with cloned honeysuckle yellow vein virus isolated from Lonicera japonica

A new honeysuckle yellow vein geminivirus (HYVV) isolate (2,763 nucleotides) and the HYVV-β satellite (1,227 nucleotides) from Korea were cloned and characterized from symptomatic leaves of Lonicera japonica with a yellow net mosaic appearance. Phylogenetic analysis of HYVV and 13 other begomoviruses revealed that HYVV has the highest nucleotide sequence homology to HYVV-UK2. Grafting challenge of a virus-free L. japonica (scion) on an HYVV-infected L. japonica (stock) resulted in the appearance of typical HYVV disease symptoms on the newly developed leaves of the scion. Two pMon521 plasmids containing 1.3 copies and 2.0 copies of the cloned HYVV isolate, respectively, were infectious and produced disease symptoms in Nicotiana benthamiana after agroinoculation. We conclude that the causal agent of yellow net mosaic in L. japonica is a new Korean isolate of HYVV, based on sequence comparisons, agroinoculation-induced disease symptoms, electron microscopy, and phylogenetic analysis.

Proteomic analysis of tumor tissue in CT-26 implanted BALB/C mouse after treatment with ascorbic acid

Tumor establishment and penetration consists of a series of complex processes involving multiple changes in gene expression and protein modification. Proteome changes of tumor tissue were investigated after intraperitoneal administration of a high concentration of ascorbic acid in BALB/C mice implanted with CT-26 cancer cells using two-dimensional gel electrophoresis and mass spectrometry. Eighteen protein spots were identified whose expression was different between control and ascorbic acid treatment groups. In particular, eukaryotic translation initiation factor 3 subunit 1, nucleophosmin, latexin, actin-related protein 2/3 complex subunit 5, M2-type pyruvate kinase, vimentin, tumor protein translationally-controlled 1, RAS oncogene family Ran, plastin 3 precursor, ATPase, Rho GDT dissociation inhibitor β, and proteasome activator subunit 2 expression were quantitatively up-regulated. The increase in the level of these proteins was accompanied by an increase in mRNA level. The cytoskeleton protein actin, vimentin, and tumor protein translationally-controlled 1 showed quantitative expression profile differences. A change in actin cytoskeleton distribution, functionally relevant to the proteome result, was observed after treatment with ascorbic acid. These results suggest a previously undefined role of ascorbic acid in the regulation of cytoskeleton remodeling in tumor tissues.