Hyun Jin Hwang

Polarization dependent translational energy release observed in the photodissociation of C2F5I at 304.7 nm

Using a state‐selective photofragment translational spectroscopy, we determined the angular distribution and the polarization dependence of the velocity distribution for the iodine atom in the ground 2P3/2 and spin–orbit excited 2P1/2 states produced in the photodissociation of C2F5I at ∼304 nm. Consistent with theoretical and experimental results on other alkyl iodides, the excited state iodine is found to be produced predominantly from the parallel 3Q0←N absorption (βlab =1.63±0.06) with a high fraction of available energy released in translation (∼67%). The substantially lower anisotropy parameter (βlab =1.08±0.03) and the polarization dependent velocity distribution observed for the ground state iodine atoms, however, suggest that they are formed from two different excited states, by direct dissociation from the 3Q1 state (∼22%) and indirect dissociation via curve crossing from the 3Q0 to 1Q state (∼78%). The dissociation along the 3Q1 state is found to release about 3.1 kcal/mol more energy in transl...

Induction of G2/M phase arrest and apoptosis by a new synthetic anti-cancer agent, DW2282, in promyelocytic leukemia (HL-60) cells

We studied the effect of DW2282-,[(S)-(+)-4-phenyl-1-[N-(4-aminobenzoyl)-indoline-5-sulfonyl-4,5-dihydro-2-imidazolone].hydrochloride], a newly developed anti-cancer agent, on cell proliferation, cell cycle progression, and induction of apoptosis in human promyelocytic leukemia (HL-60) cells. DW2282, a diarylsulfonylurea compound, was cytotoxic to HL-60 cells, with an IC(50) of 1.0 microg/mL. Treatment with DW2282 fragmented DNA in a concentration- and time-dependent manner, suggesting that these cells underwent apoptosis. Flow cytometric analysis further confirmed that DW2282-treated HL-60 cells were hypodiploid, in terms of DNA content, and were arrested at the G(2)/M phase. The cell cycle arrest was reversible upon the removal of DW2282. HL-60 cells also underwent distinct morphological changes in response to DW2282 treatment, including the appearance of elongated cells with conical tails and other apoptotic characteristics. G(2)/M phase cell cycle arrest was accompanied by a decrease in the levels of cdc2, a protein that plays a critical role for progression through the G(2)/M phase. Treatment of HL-60 cells with DW2282 was also associated with decreased levels of the anti-apoptotic protein Bcl-2, activation of caspase-3, and proteolytic cleavage of poly(ADP-ribose) polymerase. Taken together, these results demonstrate that DW2282 dramatically suppressed HL-60 cell growth by inducing apoptosis after G(2)/M phase arrest. These findings are consistent with the possibility that G(2)/M phase arrest was mediated by the down-regulation of cdc2 levels in HL-60 cells. The data also suggest that DW2282 triggered apoptosis by decreasing Bcl-2 levels and activating caspase-3 protease. These results provide important new information towards understanding the mechanisms by which DW2282 and other diarylsulfonylureas mediate their therapeutic effects.

Cytotoxicity and apoptosis induction of sodium fluoride in human promyelocytic leukemia (HL-60) cells.

The role of sodium fluoride (NaF) in cytotoxicity and induction of apoptosis was investigated by treating human promyelocytic leukemia (HL-60) cells with varying concentrations of NaF, from 0 to 250 ppm for different periods (0-72 h). At lower concentrations (0-50 ppm), no significant cytotoxicity was observed in response to NaF treatment. However, at higher concentrations (100-250 ppm), NaF reduced cell viability, and decreased DNA and protein biosynthesis capability in cultured HL-60 cells. The growth inhibitory and antiproliferative effects of NaF appear to be attributable to its induction of apoptotic cell death, as NaF induced morphological changes, internucleosomal DNA fragmentation, and increased the proportion of hypodiploid cells. NaF treatment also gradually decreased the expression of the anti-apoptotic protein Bcl-2, and increased activation of caspase-3 and cleavage of poly (ADP-ribose) polymerase. These results provides important information towards understanding the mechanism by which NaF mediates cytotoxicity and apoptosis.

Solid-phase genetic engineering with DNA immobilized on a gold surface

A novel method for immobilizing large DNA fragments on a solid surface was developed. A mixed self-assembled monolayer of thiolated single-stranded DNA with inert alkanethiol was generated on a gold (Au) surface through the Au-S reaction. Surface-tethered DNA generated by this method was compatible with various genetic engineering techniques, including hybridization, polymerization, restriction enzyme digestion and ligation. Kinetic control of surface coverage of immobilized DNA was critical for optimizing genetic engineering techniques on solid-phase. Multi-step reaction schemes utilizing various genetic engineering techniques described above were employed for solid-phase gene assembly. We were able to immobilize DNA fragments of up to 1180 bp on a solid surface. Furthermore, we showed that these immobilized genes can be regenerated by PCR. The present work suggests that these types of assembled genes can be used to store and regenerate genes on solid-phase.

Photodissociation dynamics of iodobenzene by state-selective photofragment translational spectroscopy

State-selective photofragment translational spectroscopy is used to probe the detailed nature of the photodissociation dynamics of iodobenzene at 304 nm. Simultaneous determination of the recoil speed, the spatial anisotropy, and the final state of the iodine fragment reveals that three dissociation channels with different dynamical characteristics compete in the photodissociation of iodobenzene at 304 nm. Based on the observed energy partitioning between the internal and translational modes and the dissociation time t d determined from the spatial anisotropy by using a rotational depolarization model, the three dissociation channels are assigned as follows. Two fast dissociation channels, which result in formation of I * ( 2 P 1/2 ) (t d = 0.4 ps, quantum yield Φ = 0.005 ± 0.002) and high velocity 1( 2 P 3/2 ) (t d = 0.3 ps, Φ=0.70 ± 0.04), are due to a parallel transition to the repulsive 3 Q 0 (n,σ * ) state in the C-I bond, followed by dissociation along the same state or curve crossing to the 1 Q 1 state respectively. A slow dissociation channel (t d = 0.5 - 1.4 ps, Φ = 0.30 ± 0.04) which produces low velocity I( 2 P 3/2 ) is due to a parallel transition to the triplet π,π * state(s) in the phenyl ring that is predissociated by the repulsive ν,σ * state(s). The dissociation times determined in the present work are in excellent agreement with those of the recent femtosecond real-time measurements by Cheng et al. at 278 nm (P. Y. Cheng, D. Zhong and A. H. Zewail, Chem. Phys. Lett. 237 (1995) 399).

Identification and Functional Analysis of Salmon Annexin 1 Induced by a Virus Infection in a Fish Cell Line

ABSTRACT In this study, we investigated changes in protein expression of fish cells induced by infection of infectious pancreatic necrosis virus (IPNV) using two-dimensional electrophoresis and matrix-assisted laser desorption-time of flight proton motive force analysis and identified a novel type of salmon annexin 1 that is induced in fish cells by infection with IPNV. Northern blotting showed that this annexin is overexpressed in IPNV-infected cells compared to control cells, and further analysis revealed that it has a 1,509-bp full-length cDNA sequence with an open reading frame encoding 339 amino acids (GenBank accession no. AY944135). Amino acid sequence analysis revealed that this protein belongs to the annexin 1 subfamily. By applying RNA interference, the mRNA levels of salmon annexin 1 were suppressed and, under these conditions, apoptosis of IPNV-infected cells was significantly increased. While small interfering RNA (siRNA) treatment did not affect the levels of the viral proteins significantly until 10 h postinfection, it reduced the titer of extracellular virus to 25% of that of a scrambled siRNA-treated control. These data provide evidence of an antiapoptotic function for salmon annexin 1 that is important for IPNV growth in cultured cells.

The photodissociation reaction dynamics of CF3I at 304nm (Q0+3, Q11←Q0+3, and Q13)

The photodissociation of CF3I at 304nm has been studied using long time-delayed core-sampling photofragment translational spectroscopy. Due to its capability of detecting the kinetic energy distribution of iodine fragments with high resolution, it is able to directly assign the vibrational state distribution of CF3 fragments. The vibrational state distributions of CF3 fragments in the I*(P1∕22) channel, i.e., Q0+3 state, have a propensity of the ν2′ umbrella mode with a maximum distribution at the vibrational ground state. For the I(P3∕22) channel, i.e., Q11←Q0+3, the excitation of the ν2′ umbrella mode accounts for the majority of the vibrational excitation of the CF3 fragments. The 1 ν1′ (symmetric CF stretch) +nν2′ combination modes, which are associated with the major progression of the ν2′ umbrella mode, are observed for the photodissociation of CF3I at the I channel, i.e., Q13 state. The bond dissociation energy of the CI bond of CF3I is determined to be D0(CF3–I)⩽53.62±0.5kcal∕mol (18754±175cm−1) b...

pTOC-KR: a positive selection cloning vector based on the ParE toxin

Several prokaryotic cloning vectors have been developed to clone foreign DNA in bacteria. The insertion inactivation of β-galactosidase activity, for instance, is a common screening method of identifying recombinant DNA molecules in many vectors (1,2). However, this system poses several problems. For one, many vectors can self-ligate and give false transformants. Although β-galactosidase is widely used as a screening marker, it is limited to use with specially mutated Escherichia coli hosts for α-complementation. The color of the colonies is also hard to distinguish when the blue/white selection system is used. This system also requires expensive reagents: 5-bromo4-chloro-3-indolyl-β-D-galactosidase (X-Gal) and isopropylthio-β-D-thiogalactopyranoside (IPTG). To address these problems, several positive selection vector systems have been developed based on the toxinantitoxin systems parD (Kis/Kid) of plasmid R1 (3) and on the CcdAB system of plasmid F (4). Similar positive selection cloning vectors using the transcriptional factor GATA-1 (5) or a cellulase gene (CelA) as screening marker (6) have also been developed. Nonetheless, the application of these systems is also limited; in most cases, they are host-limited and their cloning

A radioimmunoassay method for detection of DNA based on chemical immobilization of anti-DNA antibody

High selectivity provided by biomolecules such as antibodies and enzymes has been exploited during the last two decades for development of biosensors. Of particular importance are efficient immobilization methods for biomolecules in order to preserve their biological activities. In this study, we have evaluated immobilization strategies for an anti-DNA antibody on a self-assembled monolayer of ω-functionalized thiols. The antibody was immobilized via peptide bond formation between the primary amines in the antibody and the carboxyl groups on the self-assembled monolayer. The peptide bond coupling was achieved by activating COOH groups on the surface through N-Hydroxysuccimide (NHS)-ester formation, followed by acylation of NH2 group in the antibody. DNA binding activity of the immobilized antibody was examined by counting β emission from 35S-labeled DNA.

Construction of a pTOC-T vector using GST-ParE toxin for direct cloning and selection of PCR products

Using linker insertion mutations, we determined the most stable region of the parE gene which encodes a toxic protein (ParE) that inhibits growth of Escherichia coli. The toxicity of ParE was sustained until a 144 bp linker was inserted into this region. We have developed a 3′ T-overhang vector based on these characteristics of the GST-ParE toxin, and named pTOC-T. Because pTOC-T uses a post-segregational killing system, all transformants grown up on the plates can be considered as recombinants containing foreign DNA. pTOC-T not require X-Gal, IPTG or other substrates for selection. This T-vector using a positive selection system can be applied to various E. coli strains such as XL1-Blue, BL21, DH5α, JM109, and JM110.