• Asian Pacific Journal of Cancer Prevention
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• v.16 no.18
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• pp.8177-8186
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• 2016

The present study was aimed at determining the effects of alkylating and oxidative stress inducing agents on a newly identified variant of DNA polymerase beta ($pol{\beta}{\Delta}_{208-304}$) specific for ovarian cancer. $Pol{\beta}{\Delta}_{208-304}$ has a deletion of exons 11-13 which lie in the catalytic part of enzyme. We compared the effect of these chemicals on HeLa cells and HeLa cells stably transfected with this variant cloned into in pcDNAI/neo vector by MTT, colony forming and apoptosis assays. $Pol{\beta}{\Delta}_{208-304}$ cells exhibited greater sensitivity to an alkylating agent and less sensitivity towards $H_2O_2$ and UV when compared with HeLa cells alone. It has been shown that cell death in $Pol{\beta}{\Delta}_{208-304}$ transfected HeLa cells is mediated by the caspase 9 cascade. Exon 11 has nucleotidyl selection activity, while exons 12 and 13 have dNTP selection activity. Hence deletion of this part may affect polymerizing activity although single strand binding and double strand binding activity may remain same. The lack of this part may adversely affect catalytic activity of DNA polymerase beta so that the variant may act as a dominant negative mutant. This would represent clinical significance if translated into a clinical setting because resistance to radiation or chemotherapy during the relapse of the disease could be potentially overcome by this approach.

• Journal of Animal Science and Technology
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• v.51 no.1
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• pp.25-32
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• 2009

This study was conducted to determine the effects of probiotics supplementation on growth performance, nutrient digestibility, occurrence of diarrhea and immune response in weaning pigs. Treatments were 1) NC (basal diet), 2) PC (basal diet + 0.12% avilamycin) and 3) A (basal diet + 0.2% Aspergillus oryzae), 4) B (basal diet + 0.2% Lactobacillus casei), 5) C (basal diet + 0.2% Bacillus subtilis), 6) D (basal diet + 0.2% Lactobacillus crispatus). A total of 120 pigs ($7.16\pm0.01$ kg average body weight, weaned at $23\pm3$days of age) were assigned to 6 treatments, 5 replicates and 4 pigs per pen in a randomized complete block (RCB) design. During the whole experimental period, body weight (P<0.01), average daily gain (ADG; P<0.01), and average daily feed intake (ADFI; P<0.05) of treatment PC were higher than other treatments. However, the probiotics treatments tended to increase ADG and G:F ratio compared to treatment NC. The G:F ratio in treatment A (Aspergillus oryzae) was similar to treatment PC during the whole experimental period (P<0.05). The supplementation of probiotics in the diet of weaning pig did not change nutrient digestibility (dry matter, crude protein, crude fat and ash) and nitrogen retention of weaning pigs. In blood urea nitrogen (BUN) concentration, treatment B had lower value than other treatments at 2 and 4 weeks (P<0.05). Treatments PC and C tended to decrease diarrhea score than other treatments (P=0.18). At 3h after lipopolysaccharide (LPS) injection, treatments NC and PC had higher count of $CD^{4+}$ T-cells than probiotics treatments, and treatment C showed the lowest value (P<0.01). There were no differences on count of $CD^{8+}$ T-cells and $CD^{4+}:CD^{8+}$ ratio among all treatments (P>0.10). These results suggest that the dietary probiotics are likely able to improve the growth performance, occurrence of diarrhea and immune response although they do not have similar effects like antibiotics in weaning pigs.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.28 no.5
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• pp.375-395
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• 2006

Purpose: Lingual nerve (LN) damage may be caused by either tumor resection or injury such as wisdom tooth extraction, Although autologous nerve graft is sometimes used to repair the damaged nerve, it has the disadvantage of necessity of another operation for nerve harvesting. Moreover, the results of nerve grafting is not satisfactory. The nerve growth factor (NGF) is well-known to play a critical role in peripheral nerve regeneration and its local delivery to the injured nerve has been continuously tried to enhance nerve regeneration. However, its application has limitations like repeated administration due to short half life of 30 minutes and an in vivo delivery model must allow for direct and local delivery. The aim of this study was to construct a well-functioning $rhNGF-{\beta}$ adenovirus for the ultimate development of improved method to promote peripheral nerve regeneration with enhanced and extended secretion of hNGF from the injured nerve by injecting $rhNGF-{\beta}$ gene directly into crush-injured LN in rat model. Materials and Methods: $hNGF-{\beta}$ gene was prepared from fetal brain cDNA library and cloned into E1/E3 deleted adenoviral vector which contains green fluorescence protein (GFP) gene as a reporter. After large scale production and purification of $rhNGF-{\beta}$ adenovirus, transfection efficiency and its expression at various cells (primary cultured Schwann cells, HEK293 cells, Schwann cell lines, NIH3T3 and CRH cells) were evaluated by fluorescent microscopy, RT-PCR, ELISA, immunocytochemistry. Furthermore, the function of rhNGF-beta, which was secreted from various cells infected with $rhNGF-{\beta}$ adenovirus, was evaluated using neuritogenesis of PC-12 cells. For in vivo evaluation of efficacy of $rhNGF-{\beta}$ adenovirus, the LNs of 8-week old rats were exposed and crush-injured with a small hemostat for 10 seconds. After the injury, $rhNGF-{\beta}$ adenovirus($2{\mu}l,\;1.5{\times}10^{11}pfu$) or saline was administered into the crushed site in the experimental (n=24) and the control group (n=24), respectively. Sham operation of another group of rats (n=9) was performed without administration of either saline or adenovirus. The taste recovery and the change of fungiform papilla were studied at 1, 2, 3 and 4 weeks. Each of the 6 animals was tested with different solutions (0.1M NaCl, 0.1M sucrose, 0.01M QHCl, or 0.01M HCl) by two-bottle test paradigm and the number of papilla was counted using SEM picture of tongue dorsum. LN was explored at the same interval as taste study and evaluated electro-physiologically (peak voltage and nerve conduction velocity) and histomorphometrically (axon count, myelin thickness). Results: The recombinant adenovirus vector carrying $rhNGF-{\beta}$ was constructed and confirmed by restriction endonuclease analysis and DNA sequence analysis. GFP expression was observed in 90% of $rhNGF-{\beta}$ adenovirus infected cells compared with uninfected cells. Total mRNA isolated from $rhNGF-{\beta}$ adenovirus infected cells showed strong RT-PCR band, however uninfected or LacZ recombinant adenovirus infected cells did not. NGF quantification by ELISA showed a maximal release of $18865.4{\pm}310.9pg/ml$ NGF at the 4th day and stably continued till 14 days by $rhNGF-{\beta}$ adenovirus infected Schwann cells. PC-12 cells exposed to media with $rhNGF-{\beta}$ adenovirus infected Schwann cell revealed at the same level of neurite-extension as the commercial NGF did. $rhNGF-{\beta}$ adenovirus injected experimental groups in comparison to the control group exhibited different taste preference ratio. Salty, sweet and sour taste preference ratio were significantly different after 2 weeks from the beginning of the experiment, which were similar to the sham group, but not to the control group.

• Food Science and Preservation
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• v.18 no.1
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• pp.124-129
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• 2011

PC12 neuronal cell-protective effects of hot water extracts of guava fruit and leaf were evaluated. Total phenolic levels in fruit and leaf were 11.75 and 293.25 mg/g, respectively. Gallic acid, the predominant phenoic, was detected in both extracts. Intracellular reactive oxygen species (ROS) accumulation after $H_2O_2$ treatment was significantly reduced when the hot water extract of guava leaf was added to cell medium, compared to PC12 cells treated with $H_2O_2$ only. In a cell viability assay using 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl- tetrazoliumbromide (MTT), the hot water extracts of fruit and leaf protected against $H_2O_2$-induced neurotoxicity. The leaf extract was more effective in terms of inhibition of lactate dehydrogenase (LDH) release into medium, compared to the fruit extract. These in vitro data suggest that hot water extracts of guava fruit and leaf may be useful in treatment of neurodegenerative conditions such as Alzheimer's disease.

• Toxicological Research
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• v.18 no.4
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• pp.355-362
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• 2002

Pathophysiological elevation of intracellular calcium concentration ($[Ca^{2+}]_1$) in the neuron has been considered as an important responsible factor in the neuronal cell damages. However the mechanism of increase of $[Ca^{2+}]_1$ and the relationship between $[Ca^{2+}]_1$ level and cytotocixity have not been fully demonstrated. In the present study, real-time alteration of $[Ca^{2+}]_1$and cellular response (cell damages) in the pheochromocytoma cells (PC12) stimulated by glutamate were investigated. Glutamate dose dependently decreased cell viability determined propidium iodide fluorescence method and morphology change. Conversely related with cell damages, glutamate dose dependently increased the level of［Ca$^{2+}$］$_{i}$ . To investigate the mechanism of glutamate-induced increase of $[Ca^{2+}]_1$,$[Ca^{2+}]_1$, was first measured in the cell cultured in calcium free media and in the presence of dantrolene, an inhibitor of calcium release from ryanodine receptor located in endoplasmic reticulum (ER). Similar to the increase$[Ca^{2+}]_1$ in the calcium-containing media, glutamate dose dependently increased $[Ca^{2+}]_1$ in the cell cultured in free calcium media. However pretreatment (2 hr) with 20~50 $\mu\textrm{M}$ dantrolene substantial lowered glutamate-induced increase of $[Ca^{2+}]_1$, suggesting that release of calcium from ER may be major sourse of increase of $[Ca^{2+}]_1$ in PC12 cells. Dantrolene-induced inhibition of $[Ca^{2+}]_1$ resulted in recovery of cytotoxicity by glutamate. Relevance of N-methy-D-aspartate (NMDA) receptor, a type of glutamte receptor on glutamate-induced incense of $[Ca^{2+}]_1$,$[Ca^{2+}]_1$ was also determined in the cells pretreated (2 hr) with NMDA receptor antagonist MK-80l. Glutamate-induced increase of $[Ca^{2+}]_1$ was reduced by MK-801 dose dependently. Furthermore, glutamate-induced cytotoxicity was also prevented by MK-80l. These results demonstrate that glutamte increase $[Ca^{2+}]_1$ dose dependently and thereby cause cytotoxicity. The increase of $[Ca^{2+}]_1$ may release from ER, especially through ryanodine receptor and/or through NMDA receptor Alteration of calcium homeostasis through disturbance of ER system and/or calcium influx through NMDA receptor could contribute glutamate-induced cell damages.s.

• Biomedical Science Letters
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• v.12 no.2
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• pp.57-63
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• 2006

Deazaadenosine analogs such as 3-deazaadenosine (DZA), 3-deazaaristeromycin (DZAri) and ara-3-deazaadenine (DZAra-A) were developed as inhibitors of S-adenosylhomocysteine (Ado-Hcy) hydrolase (EC 3.3.1.1). These analogs were reported to induce apoptosis in human and murine leukemic cells. But, the mechanism involved in this apoptosis was not clarified yet. In the present study, we analyze the apoptosis induced by deazaadenosine analogs in human cervival cancer cell line, HeLa and the effect of Bcl-2 on this apoptosis. Whereas neither DZAri nor DZAra-A showed inhibitory effect on HeLa cell growth, DZA induced apoptosis in HeLa cells accompanied by cytochrome c release and activation of various caspases such as caspase-2,-8,-9 and -3. In HeLa-bcl-2 cell line, a stable transfectant of HeLa cell to overexpress Bcl-2, cytochrome c release, activation of all these caspases and the resulted apoptosis by DZA were completely prevented. By in vitro assay of cytochrome c release, in addition, DZA induced cytochrome c release from purified mitochondria of HeLa-pcDNA3 cells, but not HeLa-bcl-2 cells, even in the absence of cytosolic fraction. Therefore, it can be suggested that DZA might damage directly mitochondria leading to activate intrinsic pathway of caspase and thus induce apoptosis. DZA-induced apoptosis in HeLa cells may be in a bcl-2-inhibitable manner and irrelative of Ado-Hcy hydrolase.

• Proceedings of the Korean Society of Toxicology Conference
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• 2002.05a
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• pp.108-108
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• 2002

Mutations in the presenilin genes (PS-1 and PS-2) are linked to early onset familial Alzheimer's disease(AD), but its underlying cellular mechanisms have not been clear. 15-Deoxy-Δ12, 14-prostaglandin J2 (15-deoxy-PGJ2) is know as a naturally occurring ligand of the peroxisome proliferator-activated receptor-${\gamma}$ (PPAR-${\gamma}$).(omitted)

• Journal of the Korean Society of Food Science and Nutrition
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• v.45 no.7
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• pp.938-947
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• 2016

Antioxidant activities and neuroprotective effects of ethyl acetate fraction from Dendropanax morbifera (EFDM) against high glucose-induced oxidative stress and neurotoxicity were investigated to confirm their physiological activities. An 80% ethanolic extract of D. morbifera showed the highest contents of total phenolic compounds as well as 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and 1,1-diphenyl-2-picrylhydrazyl radical scavenging activities. The extract was fractionated using several solvents, and the ethyl acetate fraction showed the highest activities in ferric reducing/antioxidant power and malondialdehyde inhibitory assays. To evaluate the neuroprotective effect based on antioxidant activities, cell viability was assessed using PC12 and MC-IXC cells in $H_2O_2$- and high glucose-induced cytotoxic assays, respectively. EFDM evidently showed neuroprotective effects in all cells (neuron-like PC12 cells and human brain-originated neuroblastoma MC-IXC cells). Inhibitory effect of the extract on acetylcholinesterase (AChE) as an acetylcholine-hydrolyzing enzyme was performed to examine the effect on cognitive function. EFDM presented an AChE inhibitory effect. Finally, high-performance liquid chromatography analysis showed that the major phenolic compound of EFDM is probably a rutin.

• International Journal of Oral Biology
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• v.38 no.4
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• pp.169-173
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• 2013

HyPer is the genetically encoded biosensor of intracellular hydrogen peroxide ($H_2O_2$), the most stable of the reactive oxygen species (ROS) generated by living cells. HyPer has a high sensitivity and specificity for detecting intracellular $H_2O_2$ by confocal laser microscopy. However, it was not known whether high speed ratiometric imaging of $H_2O_2$ by HyPer is possible. We thus investigated the sensitivity of HyPer in detecting changes to the intracellular $H_2O_2$ levels in HEK293 and PC12 cells using a microfluorometer imaging system. Increase in the HyPer ratio were clearly evident on stimulations of more than $100{\mu}M$ $H_2O_2$ and fast changes in the HyPer ratio were observed on ratiometric fluorescent images after $H_2O_2$ treatment. These results suggest that HyPer is a potent biosensor of the fast temporal production of intracellular $H_2O_2$.

• BMB Reports
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• v.29 no.3
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• pp.200-204
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• 1996

Nerve growth factor (NGF) is literally known to promote neural differentiation and survival in several peripheral and central neurons. Thus, it is Widely believed that NGF may serve as a therapeutic agent for many types of neuronal diseases. One of the mechanisms suggested to explain the protective role of NGF is that the trophic factor can prevent the increase of intracellular calcium ions which might be responsible for neural death. To examine whether or not the calcium hypothesis works even under pathological conditions, we applied NGF to cultures deprived of glucose. Surprisingly, what was observed here is that NGF rather promoted cell death under a glucose-deprived condition. What we call the NGF paradox phenomenon occurred in a calcium concentration-dependent manner, indirectly suggesting that NGF might increase intracellular calcium ions in cells deprived of glucose. This suggestion is further supported by the fact that nifedipine, a well-known L-type calcium channel blocker, could block the cell death potentiated by NGF. Here it is still premature to propose the complete mechanism underlying the NGF paradox phenomenon. However, this study certainly indicates that NGF as a therapeutic agent for neuronal diseases should be carefully considered before use.