Biomedical subjects

Tae-Wan Kim

Publications and source records attributed to Tae-Wan Kim.

At least 19 recordsLinked to original sources

PubMed2006

Presenilin mutations linked to familial Alzheimer's disease cause an imbalance in phosphatidylinositol 4,5-bisphosphate metabolism.

Phosphatidylinositol 4,5-bisphosphate (PIP2) is an important cellular effector whose functions include the regulation of ion channels and membrane trafficking. Aberrant PIP2 metabolism has also been implicated in a variety of human disease states, e.g., cancer and diabetes. Here we report that familial Alzheimer's disease (FAD)-associated presenilin mutations cause an imbalance in PIP2 metabolism. We find that the transient receptor potential melastatin 7 (TRPM7)-associated Mg2+ -inhibited cation (MIC) channel underlies ion channel dysfunction in presenilin FAD mutant cells, and the observed channel deficits are restored by the addition of PIP2, a known regulator of the MIC/TRPM7 channel. Lipid analyses show that PIP2 turnover is selectively affected in FAD mutant presenilin cells. We also find that modulation of cellular PIP2 closely correlates with 42-residue amyloid beta-peptide (Abeta42) levels. Our data suggest that PIP2 imbalance may contribute to Alzheimer's disease pathogenesis by affecting multiple cellular pathways, such as the generation of toxic Abeta42 as well as the activity of the MIC/TRPM7 channel, which has been linked to other neurodegenerative conditions. Thus, our study suggests that brain-specific modulation of PIP2 may offer a therapeutic approach in Alzheimer's disease.

Alzheimer Disease↗

PubMed2006

Simple procedures for the construction of a robust and cost-effective cell-free protein synthesis system.

In this study, as a part of our efforts to improve the robustness and economical feasibility of cell-free protein synthesis, we developed a simple method of preparing the cell extracts used for catalyzing cell-free protein synthesis reactions. We found that the high-speed centrifugation, pre-incubation, and dialysis steps of the conventional procedures could be omitted without losing the translational activity of the resulting cell extract. Instead, a simple centrifugation step at low speed (12,000 RCF for 10 min) followed by a brief period of incubation was sufficient for the preparation of an active extract to support cell-free protein synthesis with higher productivity and consistency. Compared to the present standard procedures for the preparation of the S30 extract, the overall cost of the reagents and processing time were reduced by 80 and 60%, respectively.

Cell-Free System↗

PubMed2006

Rapid production of milligram quantities of proteins in a batch cell-free protein synthesis system.

We developed a cell-free protein synthesis system that produces more than 1mg/ml of recombinant proteins in two hours. A basal system that supports the stable maintenance of ATP and amino acids was constructed by using high concentrations of CP (100 mM) and amino acids (3 mM). Approximately 0.6 mg/ml of protein was produced during the batch incubation of the basal system. We found that the accumulation of inorganic phosphate reduces the concentration of free magnesium ions and that there exists a critical concentration of magnesium at which the protein synthesis is halted. Based on this finding, we attempted to extend the duration of the protein synthesis by keeping the magnesium concentration sufficiently high throughout the reaction period. The protein synthesis reaction continued for at least 2 h when the reaction was repeatedly supplemented with magnesium, and approximately 1.2 mg/ml of active CAT or GFP was produced. The simple, fast, and highly productive cell-free protein synthesis system described herein should offer a versatile platform for the preparation of protein molecules in various post-genomic efforts.

Adenosine Triphosphate↗

PubMed2006

Phase domain of the cubic Im3m mesoporous silica in the EO106PO70EO106-butanol-H2O system.

n-butanol has been chosen as an organic additive in the SiO(2)-EO(106)PO(70)EO(106)-H(2)O system at low HCl concentrations, for the generation of large-pore mesoporous silicas with easily tailored textural and structural properties. As opposed to previous reports on syntheses of cubic mesoporous silica that are usually performed in a very narrow composition range, we report now the possibility of preparing large-pore cagelike mesoporous silicas in a wide range of synthesis mixture compositions. Particularly, the cubic Imm silica with large interconnected cagelike pores (SBA-16) can easily be synthesized with controlled pore sizes and wall thicknesses, depending upon the synthesis mixture composition. The primary mesopore volume of the SBA-16 cages can be tuned from 0.27 to 0.56 cm(3) g(-)(1), and the mesopore size is shown to range from 4.7 to 7.2 nm, by performing a simple adjustment of the starting mixture composition. With the synthesis parameters varied, we describe the first complete diagram of the product phase domains obtained for silica mesophases in a SiO(2)-EO(106)PO(70)EO(106)-butanol-H(2)O system. Other ordered mesophases also observed in this system are the face-centered cubic Fmm silica mesophase and a 2D hexagonal-like mesostructure. Importantly here, the use of a low acid catalyst concentration regime allowed the preparation of silica mesophases in almost thermodynamically controlled conditions because of slow condensation kinetics of the inorganics. Such conditions enabled the introduction of n-butanol as the phase-controlling agent in the system, providing efficient tuning of the mesophase topology. The description of the phase domains provides a future basis for the design of large-pore mesoporous silicas with tailored textural and structural properties. Mesoporous samples obtained within the composition ranges of the phase domains are characterized by powder X-ray diffraction (PXRD) and nitrogen physisorption measurements.

Journal Article↗

PubMed2006

Radical scavenging and anti-inflammatory activity of extracts from Opuntia humifusa Raf.

Opuntia humifusa Raf. (O. humifusa Raf.) is a member of the Cactaceae family. To determine the antioxidative and anti-inflammatory effects of this herb, various solvent fractions (methanol, hexane, chloroform, ethyl acetate, butanol, and water) prepared from the leaves of cacti were tested using DPPH (2,2-diphenyl-l-picrylhydrazyl radical) and xanthine oxidase assays, and nitric oxide (NO)-producing macrophage cells. We found that O. humifusa Raf. displayed potent antioxidative and anti-inflammatory activity. Thus, all solvent fractions, except for the water layer, showed potent scavenging effects. The scavenging effect of the ethyl acetate fraction was higher than that of the other fractions, with IC50 values of 3.6 and 48.2 microg mL(-1). According to activity-guided fractionation, one of the active radical scavenging principles in the ethyl acetate fraction was found to be quercetin. In contrast, only two fractions (chloroform and ethyl acetate) significantly suppressed nitric oxide production from the lipopolysaccharide (LPS)-activated RAW264.7 cells. In addition, chloroform and ethyl acetate fractions significantly blocked the expression of inducible nitric oxide synthetase (iNOS) and interleukin-6 (IL-6) from the RAW264.7 cells stimulated by LPS. Moreover, ethyl acetate fractions significantly blocked the expression of IL-1beta from the RAW264.7 cells stimulated by LPS. Therefore, the results suggested that O. humifusa Raf. may modulate radical-induced toxicity via both direct scavenging activity and the inhibition of reactive species generation, and the modulation of the expression of inflammatory cytokines. Finally, O. humifusa Raf. may be useful as a functional food or drug against reactive species-mediated disease.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed2006

Oxalate enhances protein synthesis in cell-free synthesis system utilizing 3-phosphoglycerate as energy source.

Herein, we report our results showing that the productivity of cell-free protein synthesis can be enhanced through the regulation of the in vitro metabolism of an energy source. In a reaction mixture utilizing 3-phosphoglycerate (3PG) as an energy source, the supply of ATP was significantly enhanced when the reaction mixture was supplied with sodium oxalate, a potent inhibitor of phosphoenolpyruvate synthetase (PPS). The productivity of protein synthesis was also increased by approximately 70% upon the addition of oxalate. It was presumed that this enhancement in ATP supply resulted from the prevention of the pyruvate --> PEP reaction, which causes nonproductive ATP consumption. For the initial presence of 2.1 mM sodium oxalate, approximately 720 microg/ml chloramphenicol acetyltransferase (CAT) was produced after 3 h of incubation at 37 degrees C.

Adenosine Triphosphate↗

PubMed2006

Alteration of nitrergic neuromuscular transmission as a result of acute experimental colitis in rat.

Nitric oxide (NO) is a non-adrenergic, non-cholinergic neurotransmitter found in the enteric nervous system that plays a role in a variety of enteropathies, including inflammatory bowel disease. Alteration of nitrergic neurons has been reported to be dependent on the manner by which inflammation is caused. However, this observed alteration has not been reported with acetic acid-induced colitis. Therefore, the purpose of the current study was to investigate changes in nitrergic neuromuscular transmission in experimental colitis in a rat model. Distal colitis was induced by intracolonic administration of 4 % acetic acid in the rat. Animals were sacrificed at 4 h and 48 h postacetic acid treatment. Myeloperoxidase activity was significantly increased in the acetic acid-treated groups. However, the response to 60 mM KCl was not significantly different in the three groups studied. The amplitude of phasic contractions was increased by Nomega-nitro-L-arginine methyl ester (L-NAME) in the normal control group, but not in the acetic acid-treated groups. Spontaneous contractions disappeared during electrical field stimulation (EFS) in normal group. However, for the colitis groups, these contractions initially disappeared, and then reappeared during EFS. Moreover, the observed disappearance was diminished by L-NAME; this suggests that these responses were NO-mediated. In addition, the number of NADPH-diaphorase positive nerve cell bodies, in the myenteric plexus, was not altered in the distal colon; whereas the area of NADPH-diaphorase positive fibers, in the circular muscle layer, was decreased in the acetic acidtreated groups. These results suggest that NO-mediated inhibitory neural input, to the circular muscle, was decreased in the acetic acid-treated groups.

Acetic Acid↗

PubMed2005

Adsorption and structural properties of ordered mesoporous carbons synthesized by using various carbon precursors and ordered siliceous P6mm and Ia3d mesostructures as templates.

Adsorption and structural properties of inverse carbon replicas of two ordered siliceous P6mm and Ia3d mesostructures have been studied by nitrogen adsorption, powder X-ray diffraction, and transmission electron microscopy. These carbon replicas were prepared by filling the pores of SBA-15 and KIT-6 siliceous templates with various carbon precursors followed by carbonization and silica dissolution. Sucrose, furfuryl alcohol, acenaphthene, mesophase pitch, and petroleum pitch were used to obtain inverse carbon replicas of SBA-15 and KIT-6. While structural properties of the resulting ordered mesoporous carbons are mainly determined by the hard template used, their adsorption properties depend on the type of the carbon precursor.

Journal Article↗

PubMed2005

Cell-free synthesis of recombinant proteins from PCR-amplified genes at a comparable productivity to that of plasmid-based reactions.

The functional stability of mRNA is one of the crucial factors affecting the efficiency of cell-free protein synthesis. The importance of the stability of mRNA in the prolonged synthesis of protein molecules becomes even greater when the cell-free protein synthesis is directed by PCR-amplified DNAs, because the linear DNAs are rapidly degraded by the endogenous nucleases and, thus, the continuous generation of mRNA molecules is limited. With the aim of developing a highly efficient cell-free protein synthesis system directed by PCR products, in this study, we describe a systematic approach to enhance the stability of mRNA in cell-free extracts. First, exonuclease-mediated degradation was substantially reduced by introducing a stem-loop structure at the 3'-end of the mRNA. The endonucleolytic cleavage of the mRNA was minimized by using an S30 extract prepared from an Escherichia coli strain that is deficient in a major endonuclease (RNase E). Taken together, through the retardation of the endonucleolytic and exonucleolytic degradations of the mRNA molecules, the level of protein expression from the PCR-amplified DNA templates becomes comparable to that of conventional plasmid-based reactions. The enhanced productivity of the PCR-based cell-free protein synthesis enables the high-throughput generation of protein molecules required for many post-genomic applications.

Cell Extracts↗

PubMed2005

Cell-free synthesis and in situ isolation of recombinant proteins.

We present a method for rapid expression and isolation of recombinant proteins. Cell-free protein synthesis in the presence of affinity beads enables in situ isolation of translation products, which simplifies the procedures for the preparation of purified protein samples. In the present study, we have made an attempt to carry out in situ isolation of histidine-tagged proteins by using Ni-NTA magnetic agarose beads. The presence of Ni-NTA beads gave no drastic effects on the efficiency of protein synthesis and successfully captured the synthesized proteins. Purified proteins were obtained after subsequent washing and elution steps. In particular, most of the endogenous bead-binding proteins were removed by pre-treating S30 extract with affinity beads and the purity of the target proteins was enhanced up to 95%. The methods described here will provide a basis for fast and convenient preparation of purified proteins from multiple genetic sequences.

Cell-Free System↗

PubMed2005

Effect of temperature and ATP supply on the efficiency of programmed nonsense suppression.

Chemical diversity of protein molecules can be expanded through in vitro incorporation of unnatural amino acids in response to a nonsense codon. Chemically misacylated tRNAs are used for tethering unnatural amino acids to a nonsense-mutated target codon (nonsense suppression). In the course of experiments to introduce S-(2-nitrobenzyl)cysteine (NBC) into a targeted location of human erythropoietin, we found that NBC incorporates more efficiently at lower temperatures. In addition, at a fixed reaction temperature, more NBC was incorporated with a reduced supply of ATP. Since the rate of peptide elongation was remarkably higher at the elevated temperature or with enhanced supply of ATP, these results indicate that the efficiency of nonsense suppression is inversely correlated to the peptide elongation rate. Therefore, maximal yield of nonsense-suppressed proteins is obtained at a compromised elongation rate. The present result will offer a primary guideline to optimize the reaction conditions for in vitro production of protein molecules containing unnatural amino acids.

Anticodon↗

PubMed2005

Notch interferes with the scaffold function of JNK-interacting protein 1 to inhibit the JNK signaling pathway.

The transmembrane protein Notch is cleaved by gamma-secretase to yield an active form, Notch intracellular domain (Notch-IC), in response to the binding of ligands, such as Jagged. Notch-IC contributes to the regulation of a variety of cellular events, including cell fate determination during embryonic development as well as cell growth, differentiation, and survival. We now show that Notch1-IC suppresses the scaffold activity of c-Jun N-terminal kinase (JNK)-interacting protein 1 (JIP1) in the JNK signaling pathway. Notch1-IC physically associated with the JNK binding domain of JIP1 and thereby interfered with the interaction between JIP1 and JNK. JIP1 mediated the activation of JNK1 induced by glucose deprivation in mouse embryonic fibroblasts, and ectopic expression of Notch1-IC inhibited JNK activation and apoptosis triggered by glucose deprivation. Taken together, these findings suggest that Notch1-IC negatively regulates the JNK pathway by disrupting the scaffold function of JIP1.

Adaptor Proteins, Signal Transducing↗

PubMed2005

P75 neurotrophin receptor regulates expression of neural cell adhesion molecule 1.

Our recent transcriptome profiling studies suggest that presenilin 1 (PS1) regulates expression of neural cell adhesion molecule (Ncam1) through p75 neurotrophin receptor. To better understand regulation of Ncam1 transcript and protein levels by p75, we performed a series of in vitro and in vivo experiments. The combined results suggest that p75 receptor is required for both resting and NGF-induced Ncam1 expression. Activation of TrkA receptors alone does not upregulate Ncam1. The normal Ncam1 expression depends on the relative ratio of TrkA and p75 receptors, and p75 extracellular domain is necessary for baseline Ncam1 expression. NGF-induced Ncam1 expression is dependent on the presence of an intact palmitoylation site within p75 receptor. Finally, we show that the expression of Ncam1 is altered in brains of two transgenic mouse lines that express familial Alzheimer's disease (FAD)-linked PS1 variants, suggesting that expression of dominantly inherited mutant PS1 genes interferes with the normal Ncam1 expression via the p75 signaling pathway.

Alzheimer Disease↗

PubMed2005

MCM-48-like large mesoporous silicas with tailored pore structure: facile synthesis domain in a ternary triblock copolymer-butanol-water system.

Assembly of mesostructured silica using Pluronic P123 triblock copolymer (EO(20)-PO(70)-EO(20)) and n-butanol mixture is a facile synthesis route to the MCM-48-like ordered large mesoporous silicas with the cubic Iad mesostructure. The cubic phase domain is remarkably extended by controlling the amounts of butanol and silica source correspondingly. The extended phase domain allows synthesis of the mesoporous silicas with various structural characteristics. Characterization by powder X-ray diffraction, nitrogen physisorption, scanning electron microscopy, and transmission electron microscopy reveals that the cubic Iad materials possess high specific surface areas, high pore volumes, and readily tunable pore diameters in narrow distribution of sizes ranging from 4 to 12 nm. Moreover, generation of complementary pores between the two chiral channels in the gyroid Iad structure can be controlled systematically depending on synthesis conditions. Carbon replicas, using sucrose as the carbon precursor, are obtained with either the same Iad structure or I4(1)/a (or lower symmetry), depending on the controlled synthesis conditions for silica. Thus, the present discovery of the extended phase domain leads to facile synthesis of the cubic Iad silica with precise structure control, offering vast prospects for future applications of large-pore silica materials with three-dimensional pore interconnectivity.

Journal Article↗

PubMed2005

Complementary DNA cloning, genomic characterization and expression analysis of a mammalian gene encoding histidine-rich calcium binding protein.

A protein complex present at the junctional sarcoplasmic reticulum (SR) membrane is implicated in the Ca(2+) release process during muscle contraction. The histidine-rich Ca(2+)-binding protein (HRC) is an emerging component associated into the SR protein complex. We cloned cDNAs for rat and monkey HRCs, showing a conserved sequence organization in common with other mammalian HRCs. Genomic analysis revealed that each mammalian HRC gene is present as a single copy in the genome, consisting of 6 exons and 5 introns. Developmental expression analysis using mouse embryos and postnatal hearts demonstrated that Hrc transcription begins at 12.5 days postcoitum and its level increases gradually, reaching an adult level in the range 5-20 days after birth. Comparing the Hrc gene and other SR genes, we found that the timing and pattern of gene expression vary among the SR genes and the full-level expression of these genes is achieved in the heart after postnatal day 20. Collectively, our study provides comprehensive information about the structure and expression of the mammalian HRC gene, together with the comparative expression data of the related SR genes.

Amino Acid Sequence↗

PubMed2005

Design and fabrication of diffractive optical elements by use of gray-scale photolithography.

Diffractive optical elements (DOEs) are key components in the miniaturization of optical systems because of their planarity and extreme thinness. We demonstrate the fabrication of DOEs by use of gray-scale photolithography with a high-energy-beam sensitive glass photomask. We obtained DOE lenses with continuous phase profiles as small as 800 microm in diameter and 5.9 microm in the outermost grating pitch by selecting a suitable optical density for each height level and optimizing the process variables. Microlenses patterned with eight levels and replicated by UV embossing with the polymer master mold showed a diffraction efficiency of 81.5%, which was sufficiently high for the devices to be used as optical pickups. The effects of deviations in diffraction efficiency between the DOE height and profile design were analyzed.

Journal Article↗

PubMed2005

Increase in neurokinin-1 receptor-mediated colonic motor response in a rat model of irritable bowel syndrome.

AIM: Irritable bowel syndrome (IBS) is a functional bowel disorder. Its major symptom is bowel dysmotility, yet the mechanism of the symptom is poorly understood. Since the neurokinin-1 receptor (NK1R)-mediated signaling in the gut is important in the control of normal bowel motor function, we aimed to investigate whether the NK1R-mediated bowel motor function was altered in IBS, using a rat IBS model that was previously reported to show colonic dysmotility in response to restraint stress. METHODS: IBS symptoms were produced in male Sprague-Dawley rats by inducing colitis with acetic acid. Rats were left to recover from colitis for 6 d, and used for experiments 7 d post-induction of colitis. Motor activities of distal colon were recorded in vitro. RESULTS: The contractile sensitivity of isolated colon to a NK1R agonist (Sar9,Met(O2)11)-substance P (1-30 nmol/L) was higher in IBS rats than that in normal rats. After the enteric neurotransmission was blocked by tetrodotoxin (TTX, 1 micromol/L), the contractile sensitivity to the NK1R agonist was increased in normal colon but not in IBS rat colon. The NK1R agonist-induced contraction was not different between the two groups when the agonist was challenged to the TTX-treated colon or the isolated colonic myocytes. A nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 micromol/L) augmented the NK1R agonist-induced contraction only in normal rat colon. CONCLUSION: These results suggest that the NK1R-meidated colonic motor response is increased in IBS rats, due to the decrease in the nitrergic inhibitory neural component.

Acetic Acid↗

PubMed2005

Model-guided microarray implicates the retromer complex in Alzheimer's disease.

Although, in principle, gene expression profiling is well suited to isolate pathogenic molecules associated with Alzheimer's disease (AD), techniques such as microarray present unique analytic challenges when applied to disorders of the brain. Here, we addressed these challenges by first constructing a spatiotemporal model, predicting a priori how a molecule underlying AD should behave anatomically and over time. Then, guided by the model, we generated gene expression profiles of the entorhinal cortex and the dentate gyrus, harvested from the brains of AD cases and controls covering a broad age span. Among many expression differences, the retromer trafficking molecule VPS35 best conformed to the spatiotemporal model of AD. Western blotting confirmed the abnormality, establishing that VPS35 levels are reduced in brain regions selectively vulnerable to AD. VPS35 is the core molecule of the retromer trafficking complex and further analysis revealed that VPS26, another member of the complex, is also downregulated in AD. Cell culture studies, using small interfering RNAs or expression vectors, showed that VPS35 regulates Abeta peptide levels, establishing the relevance of the retromer complex to AD. Reviewing our findings in the context of recent studies suggests how downregulation of the retromer complex in AD can regulate local levels of Abeta peptide.

Adult↗