Biomedical subjects

Jin-Soo Kim

Publications and source records attributed to Jin-Soo Kim.

At least 19 recordsLinked to original sources

PubMed2006

Engineering of GAL1 promoter-driven expression system with artificial transcription factors.

We isolated and characterized artificial transcription factors (ATFs) that functionally activate GAL1 promoter in yeast. These ATFs transformed the yeast galactose-dependent GAL1 promoter system into a galactose-independent one. The ATFs were identified by screening a combinatorial library of zinc finger-containing transcription factors for components that activated the transcription of a reporter gene under the control of a truncated GAL1 promoter from which the GAL4p-binding sites were deleted. We also showed that these ATFs activate transcription from GAL1 promoter by binding directly to specific sequence elements in the promoter.

Base Sequence↗

PubMed2006

One-step selection of artificial transcription factors using an in vivo screening system.

Gene expression is regulated in large part at the level of transcription under the control of sequence-specific transcriptional regulatory proteins. Therefore, the ability to affect gene expression at will using sequence-specific artificial transcription factors would provide researchers with a powerful tool for biotechnology research and drug discovery. Previously, we isolated 56 novel sequence-specific DNA-binding domains from the human genome by in vivo selection. We hypothesized that these domains might be more useful for regulating gene expression in higher eukaryotic cells than those selected in vitro using phage display. However, an unpredictable factor, termed the "context effect", is associated with the construction of novel zinc finger transcription factors--- DNA-binding proteins that bind specifically to 9-base pair target sequences. In this study, we directly selected active artificial zinc finger proteins from a zinc finger protein library. Direct in vivo selection of constituents of a zinc finger protein library may be an efficient method for isolating multi-finger DNA binding proteins while avoiding the context effect.

Cells, Cultured↗

PubMed2006

Artificial transcription factors increase production of recombinant antibodies in Chinese hamster ovary cells.

A randomized library that encodes for artificial zinc finger protein transcription factors (ZFP-TF) was constructed and screened for components that increased production of a monoclonal antibody (mAb-72) in Chinese hamster ovary (CHO) cells. One of these ZFP-TF, LK52, increased mAb-72 production approximately 10-fold at approximately 60% transduction efficiency; a mutated version of LK52, however, did not boost mAb-72 production. LK52 also increased production of other mAbs in CHO cells. These results demonstrate that ZFP-TF libraries can be used to identify components that improve antibody production in CHO cells.

Animals↗

PubMed2006

Dissociation/association properties of a dodecameric cyclomaltodextrinase. Effects of pH and salt concentration on the oligomeric state.

As an effort to elucidate the quaternary structure of cyclomaltodextrinase I-5 (CDase I-5) as a function of pH and salt concentration, the dissociation/association processes of the enzyme were investigated under various pH and salt conditions. Previous crystallographic analysis of CDase I-5 indicated that it existed exclusively as a dodecamer at pH 7.0, forming an assembly of six 3D domain-swapped dimeric subunits. In the present study, analytical ultracentrifugation analysis suggested that CDase I-5 was present as a dimer in the pH range of 5.0-6.0, while the dodecameric form was predominant at pH values above 6.5. No dissociation of the dodecamer was observed at pH 7.0 and the above. Gel filtration chromatography showed that CDase I-5 dissociated into dimers at a rate of 8.58 x 10(-2) h(-1) at pH 6.0. A mutant enzyme with three histidine residues (H49, H89, and H539) substituted with valines dissociated into dimers faster than the wild-type enzyme at both pH 6.0 and 7.0. The tertiary structure indicated that the effect of pH on dissociation of the oligomer was mainly due to the protonation of H539. Unlike the pH-dependent process, the dissociation of wild-type CDase I-5 proceeded very fast at pH 7.0 in the presence of 0.2-1.0 M of KCl. Stopped-flow spectrophotometric analysis at various concentrations of KCl showed that the rate constants of dissociation (kd) from dodecamers into dimers were 5.96 s(-1) and 7.99 s(-1) in the presence of 0.2 M and 1.0 M of KCl, respectively.

Chromatography, Gel↗

PubMed2005

Suppression of vascular endothelial growth factor expression at the transcriptional and post-transcriptional levels.

Gene expression is regulated at the transcriptional and post-transcriptional levels. Therefore, in order to achieve a high level of silencing, which includes minimizing any residual expression of a target gene, suppression at both the transcriptional and post-transcriptional levels is required. In this study, we describe a new method for highly efficient gene silencing that combines zinc finger protein-mediated transcriptional repression and small interfering RNA (siRNA)-mediated inhibition of post-transcriptional events. To measure the amount of gene expression under various conditions, we used a luciferase reporter gene that was driven by a variety of promoters, including that of the human vascular endothelial growth factor-A (VEGF-A) gene. We also measured expression of the endogenous VEGF-A gene. Inhibition of gene expression by each of the two individual technologies was effective, but in-depth analyses revealed residual expression of the target gene. The combination of specific zinc finger transcription factors and siRNAs greatly enhanced the silencing of the human VEGF-A gene, not only when cells were grown in the presence of normal amounts of oxygen but also under conditions of hypoxic stimulation. These results suggest that a bi-level approach to the silencing of VEGF-A expression may be clinically beneficial as part of a cancer treatment protocol.

Cell Hypoxia↗

PubMed2005

Phenotypic alteration and target gene identification using combinatorial libraries of zinc finger proteins in prokaryotic cells.

We have developed a method with prokaryotic organisms that uses randomized libraries of zinc finger-containing artificial transcription factors to induce phenotypic variations and to identify genes involved in the generation of a specific phenotype of interest. Combining chromatin immunoprecipitation experiments and in silico prediction of target DNA binding sequences for the artificial transcription factors, we identified ubiX, whose down-regulation correlates with the thermotolerance phenotype in Escherichia coli. Our results show that randomized libraries of artificial transcription factors are powerful tools for functional genomic studies.

Amino Acid Sequence↗

PubMed2005

Development of strain-specific PCR primers based on a DNA probe Fu12 for the identification of fusobacterium nucleatum subsp. nucleatum ATCC 25586T.

The objective of this study was to assess the strain-specificity of a DNA probe, Fu12, for Fusobacterium nucleatum subsp. nucleatum ATCC 25586T (F. nucleatum ATCC 25586T), and to develop sets of strain-specific polymerase chain reaction (PCR) primers. Strain-specificity was tested against 16 strains of F. nucleatum and 3 strains of distinct Fusobacterium species. Southern blot hybridization revealed that the Fu12 reacted exclusively with the HindIII-digested genomic DNA of F. nucleatum ATCC 25586T. The results of PCR revealed that three pairs of PCR primers, based on the nucleotide sequence of Fu12, generated the strain-specific amplicons from F. nucleatum ATCC 25586T. These results suggest that the DNA probe Fu12 and the three pairs of PCR primers could be useful in the identification of F. nucleatum ATCC 25586T, especially with regard to the determination of the authenticity of the strain.

Blotting, Southern↗

PubMed2004

Induction and characterization of taxol-resistance phenotypes with a transiently expressed artificial transcriptional activator library.

Phenotype-based functional genomic methods are useful for the identification of genes that are related to a particular biological function or disease. Essential to this approach is the ability to regulate the expression of selected genes. Artificial transcription factors (ATFs) are key molecular tools that selectively regulate gene expression in vivo. Here, we use an ATF library to identify genes that participate in rendering a cell resistant to the drug Taxol, a potent anti-cancer drug that binds to tubulin and inhibits cell division. The library, which encodes ATFs that activate (rather than inhibit) transcription, was introduced into a HeLa cell line, and Taxol-resistant cells were selected. After eight rounds of selection, we identified two ATFs that significantly increased the level of Taxol resistance (TR) in HeLa cells. Gene expression microarray experiments using these ATFs identified 37 co-regulated genes, including genes already known to participate in TR. This study demonstrates that ATF libraries can be used to induce phenotypic alterations in eukaryotic cells and then identify specific genes that are associated with the phenotype of choice.

Antineoplastic Agents, Phytogenic↗

PubMed2004

Unilateral periodic limb movement in sleep after supratentorial cerebral infarction.

We report a patient with involuntary right leg movements during sleep, which developed after acute ischemic stroke in the corona radiata. Nocturnal polysomnography confirmed the diagnosis of periodic limb movement in sleep (PLMS). Although the pathogenesis of PLMS is still controversial, it has been proposed that the loss of cortical or subcortical inhibition resulting from the pyramidal tract lesion might cause PLMS. The present case provides evidence to support this hypothesis.

Adult↗

PubMed2003

Phenotypic alteration of eukaryotic cells using randomized libraries of artificial transcription factors.

We have developed a method in which randomized libraries of zinc finger-containing artificial transcription factors are used to induce phenotypic variations in yeast and mammalian cells. By linking multiple zinc-finger domains together, we constructed more than 100,000 zinc-finger proteins with diverse DNA-binding specificities and fused each of them to either a transcription activation or repression domain. The resulting transcriptional regulatory proteins were expressed individually in cells, and the transfected cells were screened for various phenotypic changes, such as drug resistance, thermotolerance or osmotolerance in yeast, and differentiation in mammalian cells. Genes associated with the selected phenotypes were also identified. Our results show that randomized libraries of artificial transcription factors are useful tools for functional genomics and phenotypic engineering.

Animals↗

PubMed2003

Caveolin-3 expression during early chicken development.

Caveolin-3, a protein that is correlated with caveolae, is found in muscle cells, especially during their differentiation. Although the distribution of caveolin-3 has been studied in cases such as adult and late embryonic mammalians, the expression of caveolin-3 has not been clearly defined during chicken development. In this study, we detected intense caveolin-3 immunoreactivity (IR) as early as embryonic day 4 (E4), most of the signals were localized within the neural tube and myotome. While IRs in the brain occurred in radial glia at E6, these intensities were reduced to an almost undetectable level at E8. In the case of muscle cells, the exclusive localization of caveolin-3 in the cytoplasmic membrane was detected even at E11, much earlier than in mammalian muscle tissues. Although the caveolin-3 IR pattern was similar to that reported by previous studies, we found some interesting mismatches in the case of avian tissues. Although we are unable to explain caveolin-3 expression patterns in the early embryonic stages, this study could provide a basis for further study on the function of caveolin-3 in avian embryogenesis.

Animals↗

PubMed2003

Human zinc fingers as building blocks in the construction of artificial transcription factors.

We describe methods for generating artificial transcription factors capable of up- or downregulating the expression of genes whose promoter regions contain the target DNA sequences. To accomplish this, we screened zinc fingers derived from sequences in the human genome and isolated 56 zinc fingers with diverse DNA-binding specificities. We used these zinc fingers as modular building blocks in the construction of novel, sequence-specific DNA-binding proteins. Fusion of these zinc-finger proteins with either a transcriptional activation or repression domain yielded potent transcriptional activators or repressors, respectively. These results show that the human genome encodes zinc fingers with diverse DNA-binding specificities and that these domains can be used to design sequence-specific DNA-binding proteins and artificial transcription factors.

DNA↗

PubMed2003

Characteristics of salt-fermented sauces from shrimp processing byproducts.

A salt-fermented sauce from shrimp processing byproducts (heads, shells, and tails) was prepared and characterized. Three types of sauces were prepared; sauce C, with 30 g of salt/100 g of byproduct (high salt); sauce E, with 30 g of salt and 0.2 g of sodium erythorbate (high salt); and sauce L, with 20 g of salt, 0.2 g of sodium erythorbate, 6 g of sorbitol, 0.5 mL of lactic acid, and 5 mL of ethanol (low salt). Sauces C and E showed higher exopeptidase activities than sauce L, whereas sauce L showed the highest endopeptidase activity. After 3 months of fermentation, the amino N content of sauce increased from 150-200 to 500-600 mg/100 g and the nonprotein nitrogen content increased from 300 to 950-1050 mg/100 g. Volatile basic nitrogen content increased significantly from 18 to 60 mg/100 g. The total carotenoids retained in sauces C, E, and L were 26.3, 76.2, and 73%, respectively, thus indicating that the addition of sodium erythorbate to sauces E and L retarded oxidation. Water activities of sauces C, E, and L were 0.753, 0.751, and 0.773, respectively. According to the omission test, the taste of sauces was influenced by the content of free amino acids, mainly glutamic acid and aspartic acid. All three sauces examined showed a 35% higher total amino acid content than commercial salt-fermented shrimp sauces. Therefore, shrimp processing byproducts may lend themselves to the preparation of high-quality salt-fermented sauces.

Adenosine Triphosphate↗

PubMed2003

Current status of Parkinson's disease treatment in Korea.

The health insurance system in Korea has been under governmental control, and has covered whole population since 1989. About 97% of Korean people have benefits from this system, while 3% are covered by a Medical Aid system for poor people. According to the statistical yearbook of Health Insurance Review in Korea, the number of claims under Parkinson's Disease, in whole country increased steeply from 44619 in 1995 to 96229 in 2000. If each patient were to take medication for the full duration of their insurance coverage, the average number of visits per year would be 11 in 2000. Thus the number of PD patients calculated using these statistics is about 8600. The prevalence is 19/100000, which is much lower than that in Japan. Considering the significant increase in the number of claims in last few years, however, there are still many PD patients who do not receive an accurate diagnosis and appropriate treatment. The other factor influencing these statistics is the fact that patients were able to buy their medications in drug stores without prescription, i.e. without visiting a hospital (since 2001 this has no longer been allowed). Almost all PD medications are available in Korea. There are several forms of levodopa drugs. Dopamine agonists include bromocriptine, pergolide, dihydroergocriptine, ropinirol, and pramipexole. Other parkinsonian medications are amantadine, benztropin, trihexyphenydil, deprenyl, and entacapone. Recently multicentre clinical trials were performed using ropinirol and entacapone, which showed reliable results. Currently deep brain stimulation is available in a number of medical institutes.

Alzheimer Disease↗

PubMed2003

Toward a functional annotation of the human genome using artificial transcription factors.

We have developed a novel, high-throughput approach to collecting randomly perturbed gene-expression profiles from the human genome.A human 293 cell library that stably expresses randomly chosen zinc-finger transcription factors was constructed, and the expression profile of each cell line was obtained using cDNA microarray technology.Gene expression profiles from a total of 132 cell lines were collected and analyzed by (1) a simple clustering method based on expression-profile similarity, and (2) the shortest-path analysis method. These analyses identified a number of gene groups, and further investigation revealed that the genes that were grouped together had close biological relationships. The artificial transcription factor-based random genome perturbation method thus provides a novel functional genomic tool for annotation and classification of genes in the human genome and those of many other organisms.

Antigens, Neoplasm↗

PubMed2003

Custom DNA-binding proteins and artificial transcription factors.

Expression of the genome is primarily regulated at the level of transcription by gene-specific transcription factors, which recognize specific DNA sequences to activate or inhibit transcription. The ability to control gene expression at will would provide scientists with a powerful tool for biotechnology and drug-discovery research. Over the last decade or so, researchers have made great strides in our understanding of the structures and mechanisms of action of naturally occurring transcription factors. Such research has revealed that members of the Cys2-His2 zinc finger family of transcription factors consist of functional modules that recognize a wide variety of DNA sequences. This review describes recent advances in the development of novel methods to design and construct artificial transcription factors to control gene expression at will. The applications of artificial transcription factors in the areas of medicine and biotechnology are discussed.

Animals↗

PubMedDate not supplied

Analysis of the effect of aging on the response to hypoxia by cDNA microarray.

Little is known about the detail of hypoxia-responsive gene expression patterns in advanced age, even though aging is thought to be partially associated with a decreased response to hypoxia. In the present study, we identified several hypoxia-inducible genes and investigated the effect of aging on hypoxic gene expression profiles using cDNA microarray analysis of young/old human diploid fibroblasts. Of 7458 genes in the microarray, we found that genes involved in angiogenesis, defense against oxidative stress, and transcription regulation are severely impaired in senescent cell, which is consistent with the fact that aged cells have attenuated responses to various stimuli.

Cells, Cultured↗

PubMedDate not supplied

Identification and use of zinc finger transcription factors that increase production of recombinant proteins in yeast and mammalian cells.

Randomized ZFP-TF libraries could induce a specific phenotype without detailed knowledge about the phenotype of interest because, theoretically, the libraries could modulate any gene in the target organism. We have developed a novel method for enhancing the efficiency of recombinant protein production in mammalian and microbial cells using combinatorial libraries of zinc finger protein transcription factors. To this end, we constructed tens of thousands of zinc finger proteins (ZFPs) with distinct DNA-binding specificities and fused these ZFPs to either a transcriptional activation or repression domain to make transcriptional activators or repressors, respectively. Expression vectors that encode these artificial transcription factors were delivered into Saccharomyces cerevisiae or HEK 293 cells along with reporter plasmids that code for human growth hormone (hGH) or SEAP (secreted alkaline phosphatase) (for yeast or HEK, respectively). Expression of the reporter genes was driven by either the cytomegalovirus (CMV) or SV40 virus promoters. After transfection, we screened the cells for increased synthesis of the reporter proteins. From these cells, we then isolated several ZFP-transcription factors (ZFP-TFs) that significantly increased hGH or SEAP synthesis and subjected these regulatory proteins to further characterization. Our results show that randomized ZFP-TF libraries are useful tools for improving the yield of heterologous recombinant protein both in yeast and mammalian cells.

Alkaline Phosphatase↗