Young-Gyu Chai

Studies on the interaction of REST4 with the cholinergic repressor element-1/neuron restrictive silencer element

REST4 is a neuron specific truncated form of the transcription factor REST/NRSE derived by alternative splicing. REST4 was previously shown to block the repressor activity of REST/NRSF by forming a hetero-oligomer, Shimojo et al. [Mol. Cell. Biol. 19 (1999) 6788-6795]. A series of deletion mutants have now been used to characterize REST4 in terms of its structure and DNA binding. REST4 was found to be O-glycosylated between between residues 87 and 152. Binding of REST4 to the cholinergic RE-1/NRSE was approximately 1/10 to 1/20 as strong as full length REST/NRSF. DNA binding was enhanced by deletion of the first 86 residues and was found to require all four of the C-terminal zinc fingers as well as a twelve amino acid sequence preceding the first of these zinc fingers. REST4 can form homo-oligomers, however only the monomer was found to bind to DNA. REST4 binds to the 3 sequence of the cholinergic NRSE suggesting an anti-parallel orientation of the protein to the DNA.

Mutational analysis of aspartate residues in the transmembrane regions and cytoplasmic loops of rat vesicular acetylcholine transporter.

The vesicular acetylcholine transporter (VAChT) is responsible for the transport of the neurotransmitter acetylcholine (ACh) into synaptic vesicles using an electrochemical gradient to drive transport. Rat VAChT has a number of aspartate residues within its predicted transmembrane domains (TM) and cytoplasmic loops, which may play important structural or functional roles in acetylcholine transport. In order to identify functional charged residues, site-directed mutagenesis of rVAChT was undertaken. No effect on ACh transport was observed when any of the five aspartate residues in the cytoplasmic loop were converted to asparagine. Similarly, changing Asp-46 (D46N) in TM1 or Asp-255 (D255N) in TM6 had no effect on ACh transport or vesamicol binding. However, replacement of Asp-398 in TM10 with Asn completely eliminated both ACh transport and vesamicol binding. The conservative mutant D398E retained transport activity, but not vesamicol binding, suggesting this residue is critical for transport. Mutation of Asp-193 in TM4 did not affect ACh transport activity; however, vesamicol binding was dramatically reduced. With mutant D425N of TM11 transport activity for ACh was completely blocked, without an effect on vesamicol binding. Activity was not restored in the conservative mutant D425E, suggesting the side chain as well as the negative charge of Asp-425 is important for substrate binding. These mutants, as well as mutant D193N, clearly dissociated ACh binding and transport from vesamicol binding. These data suggest that Asp-398 in TM10 and Asp-425 in TM11 are important for ACh binding and transport, while Asp-193 and Asp-398 in TM4 and TM10, respectively, are involved in vesamicol binding.

Expression patterns of mouse repressor element-1 silencing transcription factor 4 (REST4) and its possible function in neuroblastoma.

The expression pattern of the repressor element-1 silencing transcription factor (REST) also known as the neuron-restrictive silencer factor (NRSF) and its truncated forms have been analyzed in the neuroblastoma cell lines, NS20Y and NIE115 and in NIH3T3 cells. The neuroblastoma cell lines express transcripts of REST/NRSF and its neuron-specific truncated form REST4; with REST4 being the major transcript. NIH3T3 cells express predominantly REST/NRSF, with no detectable REST4. The cellular localization of REST4, determined using a REST4-GFP fusion protein, was shown to be nuclear. Mutational analysis implicates the zinc finger domains as the nuclear-targeting signal. Analysis of reporter-gene activities in the NS20Y cell line showed that the presence of four RE-1/NRSE sequences did not affect promoter activity. However, coexpression of exogenous REST4 produces a small increase in promoter activity of the reporter plasmid, whereas expression of exogenous REST/NRSF leads to repression. In the NIH3T3 cell line, the RE-1/NRSE sequence leads to repression of reporter-gene activity, whereas introduction of exogenous REST4 leads to de-repression. These data indicate that REST4 does not act as a transcriptional repressor. However, they support a mechanism where REST4 can block the repressor activity of REST/NRSF.

Version Space Learning with DNA Molecules

Version space is used in inductive concept learning to represent the hypothesis space where the goal concept is expressed as a conjunction of attribute values. The size of the version space increases exponentially with the number of attributes. We present an efficient method for representing the version space with DNA molecules and demonstrate its effectiveness by experimental results. Primitive operations to maintain a version space are derived and their DNA implementations are described. We also propose a novel method for robust decision-making that exploits the huge number of DNA molecules representing the version space.

Comparative analysis of virulence factors secreted by Bacillus anthracis Sterne at host body temperature.

Aims:u2002 For the analysis of virulence factors produced and secreted by Bacillus anthracis vegetative cells during mammalian host infection, we evaluated the secretome of B. anthracis Sterne exposed to host‐specific factors specifically to host body temperature.

Time-lapse, single cell based confocal imaging analysis of caspase activation and phosphatidylserine flipping during cellular apoptosis

Abstract Apoptosis is an important phenomenon for investigating the efficacy of anti-cancer drug candidates. The conventional assays for cellular apoptosis, such as enzyme-linked immunosorbent assay, absorbance monitoring for the activity of caspase, and flow cytometric assay, have focused only on biochemical events. We investigated the staurosporine (STS)-induced apoptosis of the murine macrophage RAW-264.7 cell using a cell based bioimaging technique. Using time-lapse confocal microscopy, we monitored caspase-3 activation during apoptosis by imaging the translocation of green fluorescent protein from the cytosol to the nuclei. Five hours after 1 μM STS treatment, caspase-3 was observed to be activated and membrane blebbing was observed simultaneously. Also, the loss of phosphatidylserine (PS) asymmetry in the phospholipid bilayer of plasma membrane during early apoptosis was monitored by imaging annexin-V labeled with fluorescein isocyanate binding to the externalized PS at various concentrations of STS. Moreover, disintegration of the plasma membrane during late apoptosis was confirmed using a nuclear dye, propidium iodide. The single cell based bioimaging data agreed well with those of the biochemical assays for caspase activation and morphological observation for membrane integrity.

DNA Implementation of Theorem Proving with Resolution Refutation in Propositional Logic

Theorem proving is a classical AI problem having a broad range of applications. Since its complexity grows exponentially with the size of the problem, many researchers have proposed methods to parallelize the theorem proving process. Here, we use the massive parallelism of molecular reactions to implement parallel theorem provers. In particular, we show that the resolution refutation proof procedure can be naturally and efficiently implemented by DNA hybridization. Novel DNA encoding schemes, i.e. linear encoding and hairpin encoding, are presented and their effectiveness is verified by biochemical experiments.

A Lab-on-a-Chip Module for Bead Separation in DNA-Based Concept Learning

Affinity separation with magnetic beads is an important and widely used technique for DNA computing. We have designed and implemented an experimental lab-on-a-chip module for affinity-bead separation for DNA-based concept learning. Magnetic beads with DNA-probe sequences immobilized on their surface were used to select target strands, and these beads are restrained in the channel by a permanent magnet on top of the module. The separation process consists of two steps, i.e. hybridization and denaturation. We confirmed the separation process by a mixed solution that contains FITC modified strands, and measured the yield by UV spectrophotometer. The experimental results demonstrate a successful separation of the mixed DNA.

RCA-Based Detection Methods for Resolution Refutation

In molecular resolution refutation, the detection of empty clauses is important. We propose a rolling circle amplification-based detection method for resolution refutation. The rolling circle amplification (RCA) technique is known to be able to distinguish and amplify circular DNA. In this paper, we describe the representation of clauses and the RCA-based detection method. Bio-lab experiments show the basic idea for this method is correct.

NCAM140 and pCREB Expression after Tianeptine Treatment of SH-SY5Y Cells

Objective Antidepressants Modulate Neuronal Plasticity. Tianeptine, An Atypical Antidepressant, Might Be Involved In The Restoration Of Neuronal Plasticity; It Primarily Enhances The Synaptic Reuptake Of Serotonin. Ncam140 Is Involved In Neuronal Development Processes, Synaptogenesis And Synaptic Plasticity. We Investigated The Effect Of Tianeptine On The Expression Of Ncam140 And Its Downstream Signaling Molecule In The Human Neuroblastoma Cell Line Sh-sy5y. Methods NCAM protein expression was measured in human neuroblastoma SH-SY5Y cells that were cultivated in serum-free media and treated with 0, 10, or 20 µM tianeptine for 6, 24, or 72 hours. NCAM140 expression in the tianeptine treatment group was confirmed by Western blot, and quantified through measurement of band intensity by absorbance. CREB and pCREB expression was identified after treatment with 20 µM tianeptine for 6, 24, and 72 hours by Western blot. Results Compared to cells treated for 6 hours, cells treated with 0 or 10 µM tianeptine for 72 hours showed a significant increase in NCAM140 expression and cells treated with 20 µM tianeptine showed a significant increase after 24 and 72 hours. The pCREB level in cells treated with 20 µM tianeptine increased in time-dependent manner. Conclusion Our findings indicated that the tianeptine antidepressant effect may occur by induction of NCAM140 expression and CREB phosphorylation.