Yong-Hak Kim

EFFECTS OF PH ON THE DEGRADATION OF PHENANTHRENE AND PYRENE BY MYCOBACTERIUM VANBAALENII PYR-1

The effects of pH on the growth of Mycobacterium vanbaalenii PYR-1 and its degradation of phenanthrene and pyrene were compared at pH 6.5 and pH 7.5. Various degradation pathways were proposed in this study, based on the identification of metabolites from mass and NMR spectral analyses. In tryptic soy broth, M. vanbaalenii PYR-1 grew more rapidly at pH 7.5 (μ′=0.058 h−1) than at pH 6.5 (μ′=0.028 h−1). However, resting cells suspended in phosphate buffers with the same pH values displayed a shorter lag time for the degradation of phenanthrene and pyrene at pH 6.5 (6 h) than at pH 7.5 (48 h). The one-unit pH drop increased the degradation rates four-fold. Higher levels of both compounds were detected in the cytosol fractions obtained at pH 6.5. An acidic pH seemed to render the mycobacterial cells more permeable to hydrophobic substrates. The major pathways for the metabolism of phenanthrene and pyrene were initiated by oxidation at the K-regions. Phenanthrene-9,10- and pyrene-4,5-dihydrodiols were metabolized via transient catechols to the ring fission products, 2,2′-diphenic acid and 4,5-dicarboxyphenanthrene, respectively. The metabolic pathways converged to form phthalic acid. At pH 6.5, M. vanbaalenii PYR-1 produced higher levels of the O-methylated derivatives of non-K-region phenanthrene- and pyrene-diols. Other non-K-region products, such as cis-4-(1-hydroxynaphth-2-yl)-2-oxobut-3-enoic acid, 1,2-dicarboxynaphthalene and benzocoumarin-like compounds, were also detected in the culture fluids. The non-K-region polycyclic aromatic hydrocarbon oxidation might be a significant burden to the cell due to the accumulation of toxic metabolites.

Two polycyclic aromatic hydrocarbon o-quinone reductases from a pyrene-degrading Mycobacterium.

Polycyclic aromatic hydrocarbon (PAH) o-quinone reductase (PQR) plays a crucial role in the detoxification of PAH o-quinones by reducing them to catechols. Two constitutive PQRs were found in cell extracts of a pyrene-degrading Mycobacterium sp. strain PYR100. The enzymes had an activity towards 9,10-phenanthrenequinone (PQ) and/or 4,5-pyrenequinone (PyQ), and the relative amounts varied with the pH of the culture media. PQR1, containing an FAD cofactor, was a monomer (20.1 kDa), and PQR2, with no flavin cofactor, was a homodimer (26.5 kDa subunits). There was no homology between the N-terminal sequences of PQR1 and PQR2. Dicumarol and quercetin inhibited PQR2 more strongly than PQR1. PQR1 had much lower specificity constants (k(cat)/K(m), 10(5)M(-1)s(-1)) for menadione (0.80) and PQ (5.19) than PQR2 (13.9 for menadione and 176 for PQ). Additionally, PQR2 exhibited a broad substrate specificity with high specificity constants for 1,4-naphthalenequinone, 1,2-naphthalenequinone, and PyQ.

Numerical and Genetic Analysis of Polycyclic Aromatic Hydrocarbon-Degrading Mycobacteria

Ability to degrade high molecular weight polycyclic aromatic hydrocarbons (PAHs) has been found in diverse species of fast-growing mycobacteria. This study included several PAH-degrading mycobacteria from heavily contaminated sites and an uncontaminated humus soil in the Natural Park, Schwäbische Alb, Germany. The numerical analysis with a total of 131 tests showed that isolates from humus soil and contaminated sites had similar substrate utilization patterns for primary alcohols from ethanol to pentanol, 1,4-butanediol, benzyl alcohol, hexadecane, ethyl acetate, fluoranthene, phenanthrene, and pyrene as the sole carbon and energy (C/E) sources. Significant differences between the two subgroups isolated from humus soil and contaminated sites were observed in the utilization of polyalcoholic sugars, including adonitol, D-arabitol, L-arabitol, erythritol, inositol, rhamnose, sorbitol, and xylitol. Among isolates from humus soil, strain PYR100 showed high similarity in 16S rDNA sequence with M. vanbaalenii strain PYR-1 (=DSM 7251, 100%) and M. austroafricanum ATCC 33464 (99.9%). In addition to the numerical analysis, the 16S–23S intergenic spacer sequence was useful for discriminating between the closely related strains PYR100 and PYR-1 (98% similarity). The patterns of the variable V2 and V3 regions in the ribosomal RNA gene corresponding to Escherichia coli positions 179 to 197 and 1006 to 1023, respectively, were useful for dividing fast-growing and thermosensitive PAH-degrading mycobacteria into ten subgroups consistent with the phylogenetic positions.

Proteomic analysis of breast cancer tissue reveals upregulation of actin‐remodeling proteins and its relevance to cancer invasiveness

There is an emerging interest in protein expression profiling with the aim of identifying novel diagnostic markers and therapeutic targets in breast cancer. We analyzed breast cancer tissues by 2‐D DIGE using a narrow range IPG strip (pH 5.5–6.7) after the immunodepletion of serum albumin and Ig. Sixty‐three protein spots were detected with more than ±1.8‐fold differences (p <0.05 for three technical replicates) from a set of tissue samples in which three tumor and three nontumor samples were randomly selected from six breast cancer subjects and pooled separately. Of these, 53 proteins were successfully identified by MS. Among the proteins whose levels were increased, we identified three novel WD‐repeat‐motif‐bearing proteins that have been known to be involved in actin remodeling: Arp2/3 complex subunit 2 (p34‐Arc), coronin‐1A and WD‐repeat protein 1 (Wdr1). Significantly increased amounts of p34‐Arc and coronin‐1A in breast cancer were also shown by Western blot analysis of matched tumor and nontumor tissue samples (N = 11, p <0.05), and were consistent with the mRNA levels retrieved from publicly available microarray databases. The siRNA knockdown of p34‐Arc attenuated the invasion of SK‐BR3 breast cancer cells into Matrigel. In contrast, the overexpression of coronin‐1A increased this invasive activity. Taken together, the cellular levels of p34‐Arc and coronin‐1A were linked to cancer development and migration. The data obtained from the present study provides new insight into the management of breast cancer.

Degradation of alkyl ethers, aralkyl ethers, and dibenzyl ether by Rhodococcus sp. strain DEE5151, isolated from diethyl ether-containing enrichment cultures.

ABSTRACT Twenty strains isolated from sewage sludge were found to degrade various ethers, including alkyl ethers, aralkyl ethers, and dibenzyl ether. In Rhodococcus strain DEE5151, induction of ether degradation needed substrates exhibiting at least one unsubstituted Cα-methylene moiety as the main structural prerequisite. The cleavage reaction observed with anisole, phenetole, and dibenzyl ether indicates that the initial oxidation occurs at such respective Cα positions. Diethyl ether-induced strain DEE5151 degraded dibenzyl ether via intermediately accumulated benzoic acid. Phenetole seems to be subject also to another ether-cleaving enzyme. Other strains of this group showed different enzymatic activities towards the substrate classes investigated.

FOXL2 posttranslational modifications mediated by GSK3β determine the growth of granulosa cell tumours

Approximately 97% of patients with ovarian granulosa cell tumours (GCTs) bear the C134W mutation in FOXL2; however, the pathophysiological mechanism of this mutation is unknown. Here we report how this mutation affects GCT development. Sequential posttranslational modifications of the C134W mutant occur where hyperphosphorylation at serine 33 (S33) by GSK3β induces MDM2-mediated ubiquitination and proteasomal degradation. In contrast, S33 of wild-type FOXL2 is underphosphorylated, leading to its SUMOylation and stabilization. This prominent hyperphosphorylation is also observed at S33 of FOXL2 in GCT patients bearing the C134W mutation. In xenograft mice, the S33 phosphorylation status correlates with the oncogenicity of FOXL2, and the inhibition of GSK3β efficiently represses GCT growth. These findings reveal a previously unidentified regulatory mechanism that determines the oncogenic attributes of the C134W mutation via differential posttranslational modifications of FOXL2 in GCT development.

Molecular cloning, expression and characterization of a novel class glutathione S-transferase from the fungus Cunninghamella elegans

The structural gene for glutathione S-transferase (CeGST1-1) in the fungus Cunninghamella elegans was cloned by screening a cDNA library using a degenerate oligonucleotide probe based on the N-terminal sequence of the purified protein. Open reading frame analysis indicated that the cegst1 gene encodes a protein of 210 amino acid residues. The deduced amino acid sequence showed 25% sequence identity with the sequence of the Pi-class GST from Danio rerio (zebrafish). Similarity was also shown with the Alpha-class GST from Fasciola hepatica (liver fluke; 23% identity), the Mu class from Mus musculus (22%) and the Sigma class from Ommastrephes sloani (squid; 21%). Further screening of a cDNA library with the cegst1 gene probe revealed the presence of another GST isoenzyme (CeGST2-2) in this fungus, which shows 84% sequence identity with CeGST1-1 at the amino acid level. Reverse transcription PCR revealed that cegst2 was also expressed at the mRNA level in the fungus C. elegans. Both cegst genes were overexpressed in Escherichia coli using the expression vector pQE51, displaying specific activities with 1-chloro-2,4-dinitrobenzene of 2.04 and 0.75 micromol/min per mg of protein respectively. Both enzymes exhibited a similar substrate specificity and inhibition profile, indicating that CeGST1-1 and CeGST2-2 belong to the same GST class. Mutagenesis analysis revealed that Tyr(10) in the N-terminal region is essential for catalysis of CeGST1-1. We propose from these results that the CeGSTs are novel Gamma-class GSTs and designated as GSTG1-1 and GSTG2-2 respectively.

Voltage noise and vortex states in YBa2Cu3Ox films

Abstract The voltage-noise measurements in epitaxial YBa 2 Cu 3 O x films have been extended to the case of a high magnetic field. In a magnetic field, two noise peaks, one from the vortex motion and the other from the resistance fluctuations, were observed. The location of the vortex-motion induced peak was found to be well above the vortex-glass transition. Our interpretation is that a noise peak manifests itself in a crossover from the pinned vortex-liquid to the unpinned vortex-liquid state.

Decolorization of malachite green by cytochrome c in the mitochondria of the fungus Cunninghamella elegans

We studied the decolorization of malachite green (MG) by the fungus Cunninghamella elegans. The mitochondrial activity for MG reduction was increased with a simultaneous increase of a 9-kDa protein, called CeCyt. The presence of cytochrome c in CeCyt protein was determined by optical absorbance spectroscopy with an extinction coefficient (E(550-535)) of 19.7+/-6.3 mM(-1) cm(-1) and reduction potential of + 261 mV. When purified CeCyt was added into the mitochondria, the specific activity of CeCyt reached 440 +/- 122 micromol min(-1) mg(-1) protein. The inhibition of MG reduction by stigmatellin, but not by antimycin A, indicated a possible linkage of CeCyt activity to the Qo site of the bc1 complex. The RT-PCR results showed tight regulation of the cecyt gene expression by reactive oxygen species. We suggest that CeCyt acts as a protein reductant for MG under oxidative stress in a stationary or secondary growth stage of this fungus.

IEX-1-induced cell death requires BIM and is modulated by MCL-1.

MCL-1 (myeloid cell leukemia-1) is a distinguished and pivotal member of the pro-survival BCL-2 family of proteins, and we isolated IEX-1 (immediate early response gene X-1) as a MCL-1-interacting protein using the yeast two-hybrid system and confirmed their endogenous association in human cells. The underlying mechanisms by which IEX-1 affects cell survival and death are largely unknown. Ectopic expression of IEX-1-induced caspase-dependent apoptosis in 293T cells, and the response was significantly modulated by changes in the MCL-1 expression level in cells. Forced expression of IEX-1 was unable to induce cell death or to perturb mitochondrial membrane potential in BIM-depleted cells. Additionally, knockouts of NOXA or PUMA did not affect the activities of IEX-1, indicating that the pro-death action of IEX-1 specifically requires BIM. Our findings provide insight into a new regulatory circuit that controls cell death and survival by the coordinated action of MCL-1, IEX-1, and BIM.