Eung Chil Choi

Differential effects of annexins I, II, III, and V on cytosolic phospholipase A2 activity: specific interaction model.

Annexins (ANXs) are a family of proteins with calcium‐dependent phospholipid binding properties. Although inhibition of phospholipase A2 (PLA2) by ANX‐I has been reported, the mechanism is still controversial. Previously we proposed a ‘specific interaction’ model for the mechanism of cytosolic PLA2 (cPLA2) inhibition by ANX‐I [Kim et al., FEBS Lett. 343 (1994) 251–255]. Here we have studied the cPLA2 inhibition mechanism using ANX‐I, N‐terminally deleted ANX‐I (ΔANX‐I), ANX‐II, ANX‐II2P112, ANX‐III, and ANX‐V. Under the conditions for the specific interaction model, ANX‐I, ΔANX‐I, and ANX‐II2P112 inhibited cPLA2, whereas inhibition by ANX‐II and ANX‐III was negligible. Inhibition by ANX‐V was much smaller than that by ANX‐I. The protein–protein interactions between cPLA2 and ANX‐I, ΔANX‐I, and ANX‐II2P112 were verified by immunoprecipitation. We can therefore conclude that inhibition of cPLA2 by specific interaction is not a general function of all ANXs, and is rather a specific function of ANX‐I. The results are consistent with the specific interaction model.

Synthesis of highly antibacterial nanocrystalline trivalent silver polydiguanide.

Highly monodispersed nanoparticles of a trivalent silver polydiguanide complex are synthesized by oxidation of the monovalent silver, followed by stabilization of the oxidized higher-valent metal through complexation with a polydiguanide ligand in a reverse microemulsion at room temperature. The synthesized nanoparticles have excellent photostability and displayed superior antibacterial activity toward Gram-positive and Gram-negative prokaryotes of clinical interest in vitro compared to silver sulfadiazine. These nanoparticles may serve as a new generation antibacterial metallopharmaceutical in wound care.

Further modification of the Hodge test to screen AmpC β-lactamase (CMY-1)-producing strains of Escherichia coli and Klebsiella pneumoniae

Cefoxitin-resistant Escherichia coli and Klebsiella pneumoniae are widespread in Korea. Significant proportions of them are considered to be CMY-1 producers. For effective screening of CMY-1 producers, the Hodge test was modified by using a cefoxitin disk and the performance was evaluated. The sensitivity and specifity of the test were 100% and 94.9%, respectively. The test was easier to perform than the three-dimensional extract test. This modified test should be suitable for screening CMY-1-producing strains of E. coli and K. pneumoniae.

Downregulation of lamin A by tumor suppressor AIMP3⁄p18 leads to a progeroid phenotype in mice

Although AIMP3/p18 is normally associated with the macromolecular tRNA synthetase complex, recent reports have revealed a new role of AIMP3 in tumor suppression. In this study, we generated a transgenic mouse that overexpresses AIMP3 and characterized the associated phenotype in vivo and in vitro. Surprisingly, the AIMP3 transgenic mouse exhibited a progeroid phenotype, and the cells that overexpressed AIMP3 showed accelerated senescence and defects in nuclear morphology. We found that overexpression of AIMP3 resulted in proteasome‐dependent degradation of mature lamin A, but not of lamin C, prelamin A, or progerin. The resulting imbalance in the protein levels of lamin A isoforms, namely altered stoichiometry of prelamin A and progerin to lamin A, appeared to be responsible for a phenotype that resembled progeria. An increase in the level of endogenous AIMP3 has been observed in aged human tissues and cells. The findings in this report suggest that AIMP3 is a specific regulator of mature lamin A and imply that enhanced expression of AIMP3 might be a factor driving cellular and/or organismal aging.

Inhibition of cytopathic effect of human immunodeficiency virus-1 by water-soluble extract ofGanoderma lucidum.

To examine components ofGanoderma lucidum for anti-human immunodeficiency virus (HIV) activity, the aqueous extracts of its basidiocarps were separated into high-molecular-weight (HMF) and low-molecular-weight (LMF) fractions. These fractions were used in XTT [2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide] antiviral assay which can quantitatively measure cytopathic effects of HIV-1 on CEM, human T lymphoblastoid cell line. The CEM cell line added with serial diluted HMF or LMF was cultured in the absence or presence of HIV-1. The results showed that the LMF of the aqueous extract strongly inhibited cytopathic effect of the target cell induced by HIV-1. When two-fold serially diluted LMF ranging from 0.97 μg/ml to 125.00 μg/ml was added to the virus-free culture system, no toxicity on the target cells was detected in all the concentrations tested. However, when it was added to the HIV-infected culture system, the viabilities of the target cell reached a plateau recovering its viabilities to 71.7% and 82.5% in experiment-1 and-2 at 15.60 μg/ml, respectively. The cell viabilities were then gradually decreased but maintained at more than 50% above 31.20 μg/ml concentration. On the contrary, HMF did not prevent any HIV-induced cytopathic effect at any concentrations tested on this cell line. From these results, negligible toxicities were observed by both HMF and LMF ofG. lucidum, and recovery of cell viability in HIV infected target cell was induced only by LMF of the carpophores.

Mycelial protoplast isolation and regeneration of Lentinus lepideus

Generation of fungal protoplast is essential for fusion and transformation systems. Protoplast fusion offers great potential for the improvement of industrially important microorganisms. To establish conditions for the protoplast isolation and regeneration of the mycelia of Lentinus lepideus, various enzymes and osmotic stabilizers were examined. To investigate suitable medium for the culture of L. lepideus, the mycelia were grown in ten different media at 28 degrees C for 10 days. Among them potato dextrose agar (PDA) medium was found to be the best for colony growth. When Novozym 234, cellulase and beta-glucuronidase were added to the mycelia in combination or alone, Novozym 234 alone at the concentration of 10 mg/ml was the most effective for the protoplast yield. Purified spherical protoplasts of the mycelia were osmotically hypersensitive and further incubation of the mycelia with the lytic enzyme resulted in the older parts of the hyphae swollen. When we applied various osmotic stabilizers at the fixed concentration of 0.6 M on the protoplasts, the yields of protoplasts were increased until 4-hr incubation. However application of sucrose or MgSO4 led to further protection of protoplasts after that time and reached a plateau on 5- and 7-hr incubations, respectively. The suitable incubation time and optimal pH with the lytic enzyme for the maximum release of protoplasts were 6 hrs of incubation and pH 5, respectively. When we examined various osmotic stabilizers for the regeneration of the protoplast, the complete medium containing 0.6 M sucrose induced highest hyphal growth with regeneration frequency of 3.28%.

Metallopharmaceuticals based on silver(I) and silver(II) polydiguanide complexes: activity against burn wound pathogens

OBJECTIVES The in vitro pharmacodynamics of silver(I) and silver(II) complexes of a polydiguanide ligand, chlorhexidine, were assayed to examine the value of the bactericidal endpoint as an alternative means of evaluating their antibacterial activities against burn wound pathogens. METHODS Synthesis of silver(I) chlorhexidine [Ag(I)CHX] was accomplished by in situ precipitation of the complex from a feebly acidic or neutral aqueous solution of AgNO(3) and chlorhexidine, whereas silver(II) chlorhexidine [Ag(II)CHX] was synthesized by oxidation of Ag(I), followed by complexation of the oxidized metal with chlorhexidine. Their antibacterial potencies were assessed in vitro by determining the MICs and MBCs for four Gram-positive and four Gram-negative bacteria. Time-kill assays using three different concentrations of these agents were also performed. RESULTS The MICs of Ag(I)CHX and Ag(II)CHX were much lower than those of chlorhexidine, AgNO(3) and silver sulfadiazine. The time-kill study provided quantitative information on actual times required to reach the bactericidal endpoint using a particular concentration of the active agent. The lethality rates of Ag(I)CHX and Ag(II)CHX against the tested bacteria were 2× to 8× faster than those of chlorhexidine or AgNO(3) at a concentration equal to or 4× MIC. CONCLUSIONS Ag(I)CHX and Ag(II)CHX showed superior antibacterial activity and faster killing kinetics compared with chlorhexidine and AgNO(3). These complexes may serve as new-generation antibacterial agents in wound care.

Glial Cell-Specific Regulation of the JC Virus Early Promoter by Histone Deacetylase Inhibitors

ABSTRACT The human polyomavirus JC virus is the etiologic agent of the fatal disease demyelinating progressive multifocal leukoencephalopathy. Although multiple transcription factors have been shown to interact with the JC virus promoter and regulate transcriptional activity, their relevance to cell specificity remains elusive. To investigate whether chromatin structure controls glial cell-specific expression of JC virus early genes, glial and nonglial cells were transfected with a reporter plasmid containing the JC virus early promoter and then treated with the histone deacetylase (HDAC) inhibitors trichostatin A (TSA) and sodium butyrate. TSA and butyrate induced 20- to 30-fold activation of the JC virus promoter in nonglial cells, whereas less than 2-fold induction was observed in glial cells. These results indicate that the JC virus early promoter might be highly suppressed in nonglial cells by hypoacetylated chromatin and activated by hyperacetylation. In support of this, chromatin immunoprecipitation assays demonstrated acetylation of the JC virus promoter region in U87MG cells but no acetylation in HeLa cells. In addition, treatment of HeLa cells with TSA induced hyperacetylation of the JC virus promoter, whereas minimal induction was seen in U87MG cells. Deletional and site-directed mutational analyses revealed that the enhancer region and Sp1 binding site upstream of the TATA box were important for TSA-mediated activation. We confirmed TSA-mediated activation of the JC virus promoter in the context of natural chromatin structure in stable cell lines. Thus, it appears that chromatin structure may control JC virus transcription in a cell-specific manner.

Studies on intergeneric protoplast fusion and nuclear transfer betweenGanoderma lucidum andCoriolus versicolor

Stable intergeneric hybrids involving allodiploid were obtained through protoplast fusion and nuclear transfer between the auxotrophic mutants of two basidiomycetes,Ganoderma lucidum andCoriolus versicolor.

An antitumor component ofLaetiporus sulphureus and its immunostimulating activity

A protein-polysaccharide fraction was prepared from the carpophores ofLaetiporus sulphureus. This fraction suppressed growth of sarcoma 180 in A-strain mice when administeredi.p. To investigate the mechanism of antitumor action of this fraction, plaque assay was conducted by administratingi.p. to the mice at a dose level of 50mg/kg for five days. Ten days later, the mice were immunized with 1×107 sheep red blood cells, The number of hemolytic plaque forming cells was significantly greater than that of the control mice. Three monosaccharides and fifteen amino acids were identified in the protein-polysaccharide fraction.