Biomedical subjects

So Young Kim

Publications and source records attributed to So Young Kim.

At least 19 recordsLinked to original sources

PubMed2006

Expression, purification and transduction of PEP-1-botulinum neurotoxin type A (PEP-1-BoNT/A) into skin.

Botulinum neurotoxin A (BoNT/A) has been used therapeutically to treat muscular hypercontractions and sudomotor hyperactivity and it has been reported that BoNT/A might have analgesic properties in headache. PEP-1 peptide is a known carrier peptide that delivers full-length native proteins in vitro and in vivo. In this study, a BoNT/A gene were fused with PEP-1 peptide in a bacterial expression vector to produce a genetic in-frame PEP-1-BoNT/A fusion protein. The expressed and purified PEP-1-BoNT/A fusion proteins were efficiently transduced into cells in a time- and dose-dependent manner when added exogenously in a culture medium. In addition, immunohistochemical analysis revealed that PEP-1-BoNT/A fusion protein efficiently penetrated into the epidermis as well as the dermis of the subcutaneous layer, when sprayed on mice skin. These results suggest that PEP-1-BoNT/A fusion protein provide an efficient strategy for therapeutic delivery in various human diseases related to this protein.

Administration, Topical↗

PubMed2006

PEP-1-SOD fusion protein efficiently protects against paraquat-induced dopaminergic neuron damage in a Parkinson disease mouse model.

Parkinson disease (PD) is a common neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra (SN). However, the mechanism of the pathology of PD still remains poorly understood. Because the administration of the herbicide paraquat triggers selective dopaminergic neuronal cell death, exposure of mice to this herbicide is one valuable model for studying the pathological aspects of PD. In this study, we investigated the protective effects of PEP-1-SOD in vitro and in vivo under exposure to the herbicide paraquat. The viability of neuronal cells treated with paraquat was markedly increased by transduced PEP-1-SOD. When the PEP-1-SOD fusion protein was injected intraperitoneally into mice, a completely protective effect against dopaminergic neuronal cell death in the SN was observed. This protective effect was synergistically increased when the PEP-1-SOD was cotransduced with Tat-alpha-synuclein. These results suggest that PEP-1-SOD provides a strategy for therapeutic delivery in various human diseases related to reactive oxygen species, including PD.

Animals↗

PubMed2006

Transduced Tat-alpha-synuclein protects against oxidative stress in vitro and in vivo.

Parkinson's disease (PD) is a common neurodegenerative disorder and is characterized by the progressive loss of dopaminergic neurons in the substantia nigra. Although many studies showed that the aggregation of alpha-synuclein might be involved in the pathogenesis of PD, its protective properties against oxidative stress remain to be elucidated. In this study, human wild type and mutant alpha-synuclein genes were fused with a gene fragment encoding the nine amino acid transactivator of transcription (Tat) protein transduction domain of HIV-1 in a bacterial expression vector to produce a genetic in-frame WT Tat-alpha-synuclein (wild type) and mutant Tat-alpha-synucleins (mutants; A30P and A53T), respectively, and we investigated the protective effects of wild type and mutant Tat-alpha-synucleins in vitro and in vivo. WT Tat-alpha-synuclein rapidly transduced into an astrocyte cells and protected the cells against paraquat induced cell death. However, mutant Tat-alpha-synucleins did not protect at all. In the mice models exposed to the herbicide paraquat, the WT Tat-alpha-synuclein completely protected against dopaminergic neuronal cell death, whereas mutants failed in protecting against oxidative stress. We found that these protective effects were characterized by increasing the expression level of heat shock protein 70 (HSP70) in the neuronal cells and this expression level was dependent on the concentration of transduced WT Tat-alpha-synuclein. These results suggest that transduced Tat-alpha-synuclein might protect cell death from oxidative stress by increasing the expression level of HSP70 in vitro and in vivo and this may be of potential therapeutic benefit in the pathogenesis of PD.

Animals↗

PubMed2006

Immunohistochemical studies of human ribosomal protein S3 (rpS3).

The human ribosomal protein S3 (rpS3) was expressed in E. coli using the pET-15b vector and the monoclonal antibodies (mAbs) were produced and characterized. A total of five hybridoma cell lines were established and the antibodies recognized a single band of molecular weight of 33 kDa on immunoblot with purified rpS3. When the purified rpS3 was incubated with the mAbs, the UV endonuclease activity of rpS3 was inhibited up to a maximum of 49%. The binding affinity of mAbs to rpS3 determined by using a biosensor technology showed that they have similar binding affinities. Using the anti-rpS3 antibodies as probes, we investigated the cross-reactivities of various other mammalian brain tissues and cell lines, including human. The immunoreactive bands on Western blots appeared to be the same molecular mass of 33 kDa in all animal species tested. They also appear to be extensively cross-reactive among different organs in rat. These results demonstrated that only one type of immunologically similar rpS3 protein is present in all of the mammalian brain tissues including human. Furthermore, these antibodies were successfully applied in immunohistochemistry in order to detect rpS3 in the gerbil brain tissues. Among the various regions in the brain tissues, the rpS3 positive neurons were predominantly observed in the ependymal cells, hippocampus and stantia nigra pars compacta. The different distributions of rpS3 in brain tissues reply that rpS3 protein may play an important second function in the neuronal cells.

Animals↗

PubMed2006

A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen.

To enable arrayed or pooled loss-of-function screens in a wide range of mammalian cell types, including primary and nondividing cells, we are developing lentiviral short hairpin RNA (shRNA) libraries targeting the human and murine genomes. The libraries currently contain 104,000 vectors, targeting each of 22,000 human and mouse genes with multiple sequence-verified constructs. To test the utility of the library for arrayed screens, we developed a screen based on high-content imaging to identify genes required for mitotic progression in human cancer cells and applied it to an arrayed set of 5,000 unique shRNA-expressing lentiviruses that target 1,028 human genes. The screen identified several known and approximately 100 candidate regulators of mitotic progression and proliferation; the availability of multiple shRNAs targeting the same gene facilitated functional validation of putative hits. This work provides a widely applicable resource for loss-of-function screens, as well as a roadmap for its application to biological discovery.

Animals↗

PubMed2006

Tat-mediated protein transduction of human brain pyridoxine-5-P oxidase into PC12 cells.

Pyridoxine-5-P oxidase catalyses the terminal step in the biosynthesis of pyridoxal-5-P, the biologically active form of vitamin B6 which acts as an essential cofactor. Here, a human brain pyridoxine-5-P oxidase gene was fused with a gene fragment encoding the HIV-1 Tat protein transduction domain (RKKRRQRRR) in a bacterial expression vector to produce a genetic in-frame Tat-pyridoxine-5-P oxidase fusion protein. Expressed and purified Tat-pyridoxine-5-P oxidase fusion protein transduced efficiently into PC12 cells in a time- and dose-dependent manner when added exogenously to culture media. Once inside the cells, the transduced Tat-pyridoxine-5-P oxidase protein showed catalytic activity and was stable for 48 h. Moreover, the formation of pyridoxal-5-P was increased by adding exogenous Tat-pyridoxine-5-P oxidase to media pre-treated with the vitamin B6 precursor pyridoxine. In addition, the intracellular concentration of pyridoxal-5-P was markedly increased when Tat-pyridoxal kinase was transduced together with Tat-pyridoxine-5-P oxidase into cells.These results suggest that the transduction of Tat-pyridoxine-5-P oxidase fusion protein presents a means of regulating the level of pyridoxal-5-P and of replenishing this enzyme in various neurological disorders related to vitamin B6.

Animals↗

PubMed2006

Lens-sparing vitrectomy for stage 4 and stage 5 retinopathy of prematurity.

PURPOSE: To describe the results of lens-sparing vitrectomy for the correction of retinal detachment associated with retinopathy of prematurity (ROP) and its associated complications. METHODS: Seventeen patients who underwent a lens-sparing vitrectomy for stage 4 and stage 5 ROP with plus disease at Seoul National University Children' s Hospital between 1999 and 2003 were enrolled in this study. The patients who had bilateral retinal detachment of ROP underwent a lens-sparing vitrectomy in one eye and a scleral buckling surgery or lensectomy-vitrectomy in the other eye. The patients who had a retinal detachment in one eye and a regressed ROP in the other eye underwent unilateral lens-sparing vitrectomies. A review of their preoperative clinical findings (including the status of retinal detachment and plus disease), post-operative results, and any complications encountered was performed. RESULTS: In 17 patients, the postoperative success rate of lens-sparing vitrectomy was 58.8%. However, lens-sparing vitrectomy as a treatment for stage 5 ROP (25.0%) produced more negative post-operative results than it did when used to treat either those for stage 4a (75,0%) or 4b (66.7%) ROP. Among the 10 eyes in which the retina was attached, form vision was shown in six eyes, light could be followed by three eyes, and no light perception was present in one eye. Intra- and post-operative complications included retinal break formation, cataracts, vitreous hemorrhages, and glaucoma in patients with stages 4b and stage 5 ROP. CONCLUSIONS: Lens-sparing vitrectomy resulted in encouraging surgical outcomes in the correction of retinal detachment of ROP, especially in stage 4 patients. Therefore, a lens-sparing vitrectomy for stage 4 ROP patient may be beneficial, although it is still associated with some intra- and post-operative complications.

Child, Preschool↗

PubMed2005

Transduced human copper chaperone for Cu,Zn-SOD (PEP-1-CCS) protects against neuronal cell death.

Reactive oxygen species (ROS) contribute to the development of various human diseases. Cu,Zn-superoxide dismutase (SOD) is one of the major means by which cells counteract the deleterious effects of ROS. SOD activity is dependent upon bound copper ions supplied by its partner metallochaperone protein, copper chaperone for SOD (CCS). In the present study, we investigated the protective effects of PEP-1-CCS against neuronal cell death and ischemic insults. When PEP-1-CCS was added to the culture medium of neuronal cells, it rapidly entered the cells and protected them against paraquat-induced cell death. Moreover, transduced PEP-1-CCS markedly increased endogenous SOD activity in the cells. Immunohistochemical analysis revealed that it prevented neuronal cell death in the hippocampus in response to transient forebrain ischemia. These results suggest that CCS is essential to activate SOD, and that transduction of PEP-1-CCS provides a potential strategy for therapeutic delivery in various human diseases including stroke related to SOD or ROS.

Animals↗

PubMed2005

Human brain pyridoxal-5'-phosphate phosphatase: production and characterization of monoclonal antibodies.

We cloned and expressed human pyridoxal-5\'-phosphate (PLP) phosphatase, the coenzymatically active form of vitamin B6, in Escherichia coli using pET15b vector. Monoclonal antibodies (mAb) were generated against purified human brain PLP phosphatase in mice, and four antibodies recognizing different epitopes were obtained, one of which inhibited PLP phosphatase. The binding affinities of these four mAbs to PLP phosphatase, as determined using biosensor technology, showed that they had similar binding affinities. Using the anti-PLP phosphatase antibodies as probes, we investigated their cross-reactivities in various mammalian and human tissues and cell lines. The immunoreactive bands obtained on Western blots had molecular masses of ca. 33 kDa. Similarly fractionated extracts of several mammalian cell lines all produced a single band of molecular mass 33 kDa. We believe that these PLP phosphatase mAbs could be used as valuable immunodiagnostic reagents for the detection, identification, and characterization of various neurological diseases related to vitamin B6 abnormalities.

Animals↗

PubMed2005

Eckol isolated from Ecklonia cava attenuates oxidative stress induced cell damage in lung fibroblast cells.

We have investigated the cytoprotective effect of eckol, which was isolated from Ecklonia cava, against oxidative stress induced cell damage in Chinese hamster lung fibroblast (V79-4) cells. Eckol was found to scavenge 1,1-diphenyl-2-picrylhydrazyl radical, hydrogen peroxide (H(2)O(2)), hydroxy radical, intracellular reactive oxygen species (ROS), and thus prevented lipid peroxidation. As a result, eckol reduced H(2)O(2) induced cell death in V79-4 cells. In addition, eckol inhibited cell damage induced by serum starvation and radiation by scavenging ROS. Eckol was found to increase the activity of catalase and its protein expression. Further, molecular mechanistic study revealed that eckol increased phosphorylation of extracellular signal-regulated kinase and activity of nuclear factor kappa B. Taken together, the results suggest that eckol protects V79-4 cells against oxidative damage by enhancing the cellular antioxidant activity and modulating cellular signal pathway.

Animals↗

PubMed2005

Amino acids mediate mTOR/raptor signaling through activation of class 3 phosphatidylinositol 3OH-kinase.

During the evolution of metazoans and the rise of systemic hormonal regulation, the insulin-controlled class 1 phosphatidylinositol 3OH-kinase (PI3K) pathway was merged with the primordial amino acid-driven mammalian target of rapamycin (mTOR) pathway to control the growth and development of the organism. Insulin regulates mTOR function through a recently described canonical signaling pathway, which is initiated by the activation of class 1 PI3K. However, how the amino acid input is integrated with that of the insulin signaling pathway is unclear. Here we used a number of molecular, biochemical, and pharmacological approaches to address this issue. Unexpectedly, we found that a major pathway by which amino acids control mTOR signaling is distinct from that of insulin and that, instead of signaling through components of the insulin/class 1 PI3K pathway, amino acids mediate mTOR activation by signaling through class 3 PI3K, hVps34.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Cytoprotective effect of tectorigenin, a metabolite formed by transformation of tectoridin by intestinal microflora, on oxidative stress induced by hydrogen peroxide.

In the present study, the antioxidative properties of tectorigenin, a metabolite formed by transformation of tectoridin by intestinal microflora, were investigated. Tectorigenin was found to scavenge intracellular reactive oxygen species, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, and thus prevented lipid peroxidation. The radical scavenging activity of tectorigenin protected the viability of Chinese hamster lung fibroblast (V79-4) cells exposed to hydrogen peroxide (H2O2) via activation of extracellular signal regulated kinase (ERK) pathway. Furthermore, tectorigenin reduced the apoptotic cells formation and cell cycle arrest at G2/M phase induced by H2O2. Tectorigenin increased the activities of cellular antioxidant enzymes like superoxide dismutase, catalase, glutathione peroxidase, and also increased their protein level. Taken together, these findings suggest that tectorigenin protected V79-4 cells against H2O2 damage, by enhancing the antioxidative activity and by activating ERK pathway.

Animals↗

PubMed2005

Enhanced transduction of Cu,Zn-superoxide dismutase with HIV-1 Tat protein transduction domains at both termini.

The human immunodeficiency virus type 1 (HIV-1) Tat protein transduction domain (PTD) is responsible for highly efficient protein transduction across plasma membranes. In a previous study, we showed that Tat-Cu,Zn-superoxide dismutase (Tat-SOD) can be directly transduced into mammalian cells across the lipid membrane barrier. In this study, we fused the human SOD gene with a Tat PTD transduction vector at its N- and/or C-terminus. The fusion proteins (Tat-SOD, SOD-Tat, Tat-SOD-Tat) were purified from Escherichia coli and their ability to enter cells in vitro and in vivo compared by Western blotting and immunohistochemistry. The transduction efficiencies and biological activities of the SOD fusion protein with the Tat PTD at either terminus were equivalent and lower than the fusion protein with the Tat PTD at both termini. The availability of a more efficient SOD fusion protein provides a powerful vehicle for therapy in human diseases related to this anti-oxidant enzyme and to reactive oxygen species.

Animals↗

PubMed2005

Transduced Tat-SOD fusion protein protects against ischemic brain injury.

Reactive oxygen species (ROS) are implicated in reperfusion injury after transient focal cerebral ischemia. The antioxidant enzyme, Cu,Zn-superoxide dismutase (SOD), is one of the major means by which cells counteract the deleterious effects of ROS after ischemia. Recently, we reported that when Tat-SOD fusion protein is transduced into pancreatic beta cells it protects the beta cells from destruction by relieving oxidative stress in ROS-implicated diabetes (Eum et al., 2004). In the present study, we investigated the protective effects of Tat-SOD fusion protein against neuronal cell death and ischemic insults. When Tat-SOD was added to the culture medium of neuronal cells, it rapidly entered the cells and protected them against paraquat-induced cell death. Immunohistochemical analysis revealed that Tat-SOD injected intraperitoneally (i.p.) into mice has access to various tissues including brain neurons. When i.p. injected into gerbils, Tat-SOD prevented neuronal cell death in the hippocampus in response to transient fore-brain ischemia. These results suggest that Tat-SOD provides a strategy for therapeutic delivery in various hu-man diseases, including stroke, related to this anti-oxidant enzyme or to ROS.

Animals↗

PubMed2005

Inactivation of brain myo-inositol monophosphate phosphatase by pyridoxal-5'-phosphate.

Myo-inositol monophosphate phosphatase (IMPP) is a key enzyme in the phosphoinositide cell-signaling system. This study found that incubating the IMPP from a porcine brain with pyridoxal-5'-phosphate (PLP) resulted in a time-dependent enzymatic inactivation. Spectral evidence showed that the inactivation proceeds via the formation of a Schiff's base with the amino groups of the enzyme. After the sodium borohydride reduction of the inactivated enzyme, it was observed that 1.8 mol phosphopyridoxyl residues per mole of the enzyme dimer were incorporated. The substrate, myo-inositol-1-phosphate, protected the enzyme against inactivation by PLP. After tryptic digestion of the enzyme modified with PLP, a radioactive peptide absorbing at 210 nm was isolated by reverse-phase HPLC. Amino acid sequencing of the peptide identified a portion of the PLP-binding site as being the region containing the sequence L-Q-V-S-Q-Q-E-D-I-T-X, where X indicates that phenylthiohydantoin amino acid could not be assigned. However, the result of amino acid composition of the peptide indicated that the missing residue could be designated as a phosphopyridoxyl lysine. This suggests that the catalytic function of IMPP is modulated by the binding of PLP to a specific lysyl residue at or near its substrate-binding site of the protein.

Amino Acids↗

PubMed2005

Production and characterization of monoclonal antibodies against human ceruloplasmin.

Ceruloplasmin (CP) is the major plasma antioxidant and copper transport protein. Monoclonal antibodies (mAbs) against human CP were produced and characterized. A total of five hybridoma cell lines were established (CP2, CP10, CP20, CP25, CP30). From the epitope mapping analysis, two subgroups of mAbs recognize different peptide fragments were identified. When the purified CP was incubated with the mAbs, the ferroxidase activity of CP was inhibited up to a maximum 57 %. Immunoblotting with various tissue homogenates indicated that all the mAbs specifically recognize a single protein band of 130 kDa. They also appear to be extensively cross-reactive among different mammalian including human and avian sources. These results demonstrated that only one type of immunologically similar CP is present in all of the mammalian tissues including human. The CP mAbs could be of great benefit to design the diagnostic kit for CP-related diseases such as Wilson's disease.

Animals↗

PubMed2005

Tat-mediated protein transduction of human brain pyridoxal kinase into PC12 cells.

Pyridoxal kinase (PK) catalyses the phosphorylation of vitamin B6 to pyridoxal-5'-phosphate (PLP). A human brain PK gene was fused with a gene fragment encoding the HIV-1 Tat protein transduction domain (RKKRRQRRR) in a bacterial expression vector to produce a genetic in-frame Tat-PK fusion protein. The expressed and purified Tat-PK fusion proteins transduced efficiently into PC12 cells in a time- and dose-dependent manner when added exogenously in culture media. Once inside the cells, the transduced Tat-PK proteins showed catalytic activity and are stable for 48 h. The intracellular concentration of PLP, which is known as a biologically active form of vitamin B6, was increased by pre-treatment of Tat-PK to the PC12 cells. Those results suggest that the transduction of Tat-PK fusion protein can be one of the ways to regulate the PLP level and to replenish this enzyme in the various neurological disorders related to vitamin B6.

Animals↗

PubMed2004

In vivo protein transduction: biologically active intact pep-1-superoxide dismutase fusion protein efficiently protects against ischemic insult.

Reactive oxygen species (ROS) are implicated in reperfusion injury after transient focal cerebral ischemia. The antioxidant enzyme Cu,Zn-superoxide dismutase (SOD) is one of the major means by which cells counteract the deleterious effects of ROS after ischemia. Recently, we reported that denatured Tat-SOD fusion protein is transduced into cells and skin tissue. Moreover, PEP-1 peptide, which has 21 amino acid residues, is a known carrier peptide that delivers full-length native proteins in vitro and in vivo. In the present study, we investigated the protective effects of PEP-1-SOD fusion protein after ischemic insult. A human SOD gene was fused with PEP-1 peptide in a bacterial expression vector to produce a genetic in-frame PEP-1-SOD fusion protein. The expressed and purified fusion proteins were efficiently transduced both in vitro and in vivo with a native protein structure. Immunohistochemical analysis revealed that PEP-1-SOD injected intraperitoneally (i.p.) into mice can have access into brain neurons. When i.p.-injected into gerbils, PEP-1-SOD fusion proteins prevented neuronal cell death in the hippocampus caused by transient forebrain ischemia. These results suggest that the biologically active intact forms of PEP-1-SOD provide a more efficient strategy for therapeutic delivery in various human diseases related to this antioxidant enzyme or to ROS, including stroke.

Animals↗