Misun Won

Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe.

We report the construction and analysis of 4,836 heterozygous diploid deletion mutants covering 98.4% of the fission yeast genome providing a tool for studying eukaryotic biology. Comprehensive gene dispensability comparisons with budding yeast—the only other eukaryote for which a comprehensive knockout library exists—revealed that 83% of single-copy orthologs in the two yeasts had conserved dispensability. Gene dispensability differed for certain pathways between the two yeasts, including mitochondrial translation and cell cycle checkpoint control. We show that fission yeast has more essential genes than budding yeast and that essential genes are more likely than nonessential genes to be present in a single copy, to be broadly conserved and to contain introns. Growth fitness analyses determined sets of haploinsufficient and haploproficient genes for fission yeast, and comparisons with budding yeast identified specific ribosomal proteins and RNA polymerase subunits, which may act more generally to regulate eukaryotic cell growth.

Inactivation kinetics of Listeria monocytogenes, Salmonella enterica serovar Typhimurium, and Campylobacter jejuni in ready-to-eat sliced ham using UV-C irradiation

To investigate the applicability of UV-C irradiation on the inactivation of foodborne pathogenic bacteria in ready-to-eat sliced ham, UV-C treatment was evaluated. Irradiation dose required for 90% reduction of the populations of Listeria monocytogenes, Salmonellaenterica serovar Typhimurium, and Campylobacter jejuni were determined to be 2.48, 2.39, and 2.18J/m(2). Ready-to-eat sliced hams were inoculated with the pathogens and irradiated with UV-C light of 1000, 2000, 4000, 6000, and 8000J/m(2). Microbiological data indicated that foodborne pathogen populations significantly (p<0.05) decreased with increasing UV-C irradiation. In particular, UV-C irradiation at 8000J/m(2) reduced the populations of L. monocytogenes, S. Typhimurium, and C. jejuni in the ham by 2.74, 2.02, and 1.72logCFU/g. The results indicate that UV-C irradiation can be used as a microbial inactivation method for ready-to-eat sliced ham, and inactivation kinetics of the foodborne pathogens fit the Weibull model better than the first-order kinetics model.

A novel benzimidazole analogue inhibits the hypoxia-inducible factor (HIF)-1 pathway.

Hypoxia-inducible factor (HIF)-1 is a therapeutic target in solid tumors. We report the novel benzimidazole analogue AC1-004, obtained from a chemical library using an HRE-dependent cell-based assay in colorectal carcinoma HCT-116 cells. The accumulation of hypoxia-induced HIF-1alpha was inhibited by compound AC1-004 in various cancer cells, including HCT-116, MDA-MB435, SK-HEP1, and Caki-1. Further, AC1-004 down-regulated VEGF and EPO, target genes of HIF-1, and inhibited in vitro tube formation of HUVEC, suggesting its potential inhibitory activity on angiogenesis. Importantly, AC1-004 was found to regulate the stability of HIF-1alpha through the Hsp90-Akt pathway, leading to the degradation of HIF-1alpha. An in vivo antitumor study demonstrated that AC1-004 reduced tumor size significantly (i.e., by 58.6%), without severe side effects. These results suggest the benzimidazole analogue AC1-004 is a novel HIF inhibitor that targets HIF-1alpha via the Hsp90-Akt pathway, and that it can be used as a new lead in developing anticancer drugs.

Antioxidant activities of distiller dried grains with solubles as protein films containing tea extracts and their application in the packaging of pork meat.

Distiller dried grains with solubles (DDGS) as protein (DP) films were prepared. Additionally, to prepare anti-oxidant films, green tea extract (GTE), oolong tea extract (OTE), and black tea extract (BTE) were incorporated into the DP films. Consequently, the incorporation of the tea extracts did not alter the physical properties of the films much, whereas the antioxidant activities, such as ABTS and DPPH radical scavenging activities were observed. To apply the DP films containing tea extracts to food packaging, pork meat was wrapped with the films and stored at 4 °C for 10 d. During storage, the pork meat wrapped with the DP films containing GTE, OTE, and BTE had less lipid oxidation than did the control. Among the tea extracts, the DP film containing GTE had the greatest antioxidant activity. These results indicate that the DP films containing green tea extracts can be utilized as an anti-oxidative packaging material for pork meat.

RhoB induces apoptosis via direct interaction with TNFAIP1 in HeLa cells.

RhoB, a tumor suppressor, has emerged as an interesting cancer target, and extensive studies aimed at understanding its role in apoptosis have been performed. In our study, we investigated the involvement of RhoB‐interacting molecules in apoptosis. To identify RhoB‐interacting proteins, we performed yeast‐two hybrid screening assays using RhoB as a bait and isolated TNFAIP1, a TNFα‐induced protein containing the BTB/POZ domain. The interaction between RhoB and TNFAIP1 was demonstrated in vivo through coimmunoprecipitation studies and in vitro binding assays. RFP‐TNFAIP1 was found to be partially colocalized with EGFP‐RhoB. The partial colocalization of RhoB and TNFAIP1 in endosomes suggests that RhoB‐TNFAIP1 interactions may have a functional role in apoptosis. TNFAIP1 elicited proapoptotic activity, while simultaneous expression of RhoB and TNFAIP1 resulted in a dramatic increase in apoptosis in HeLa cells. Furthermore, knockdown of RhoB using siRNA clearly rescued cells from apoptosis induced by TNFAIP1. This finding suggests that interactions between RhoB and TNFAIP1 are crucial for induction of apoptosis in HeLa cells. The observation of increased SAPK/JNK phosphorylation in apoptotic cells and the finding that a JNK inhibitor suppressed apoptosis indicates that SAPK/JNK signaling may be involved in apoptosis induced by RhoB‐TNFAIP1 interactions. In conclusion, we found that RhoB interacts with TNFAIP1 to regulate apoptosis via a SAPK/JNK‐mediated signal transduction mechanism.

Identification of malate dehydrogenase 2 as a target protein of the HIF-1 inhibitor LW6 using chemical probes

Hypoxia-inducible factor (HIF) regulates tumor angiogenesis and metastasis in response to low oxygen tension. In the presence of oxygen, HIF-1a is rapidly degraded through the ubiquitin–proteasome pathway. In hypoxic conditions, stabilized HIF-1a dimerizes with HIF-1b. The HIF-1a/b heterodimer binds to hypoxia response elements (HRE) in gene promoters and induces the expression of target genes involved in angiogenesis, metastasis, glycolysis, cell proliferation, and resistance to apoptosis. Increased expression of HIF-1a in many solid tumors correlates with aggressive tumor growth, therapeutic resistance, and a poor clinical outcome. HIF-1a shifts the metabolism from oxidative phosphorylation to anaerobic glycolysis. Therefore, HIF-1a is an important therapeutic target for cancer. We previously synthesized and evaluated aryloxyacetylamino benzoic acid analogues. LW6 (1 in Figure 1A) potently inhibited HIF-1a accumulation by degrading HIF1a without affecting the HIF-1a mRNA levels during hypoxia. LW6, which is commercially available, has been used in various studies as an HIF-1a inhibitor. However, the molecular target of LW6 remains unknown. To identify a drug target, chemical biological methods such as activity-based probes (ABPs), photoaffinity labeling, biotinylation, and click conjugation have been used. Herein, we identify the molecular target of 1 using chemical probes. Cellular images and direct protein interactions of 1 were examined in living cells with a series of chemical probes (2–6), which were designed using the structure– activity relationship (SAR) of 1. Synthesis and characterization data for these probes are available in the Supporting Information. The distribution of drug molecules within subcellular compartments can provide information about the mechanism of drug action. The intracellular localization of LW6 was visualized through click chemistry with probe 2, containing an acetylene group, in colon cancer HCT116 cells (Figure 1A). Both 1 and 2 suppressed HIF-1a accumulation (Figure 1B) and HRE-luciferase activity (Figure 1A; Supporting Information, Figure S12). Subsequently, the cellular localization of probe 2 was determined by a click reaction with an azidelinked Alexa Fluor 488 molecule. Notably, copper-catalyzed azide–alkyne cycloadditions (click reactions) are highly specific and efficient bio-orthogonal reactions to visualize intracellular probe distribution. We found that compound 2 was localized primarily in the cytoplasm (Figure 1C). The colocalization of compound 2 (3 mm) with the mitochondriaselective probe, MitoTracker (500 nm), indicated that 2 is specifically localized in the mitochondria, whereas the localization of an adamantyl-free probe 4 was not observed (Figure S13). The mitochondrial localization of probe 2 was Figure 1. Biological activities and cellular localization of a chemical probe for LW6. A) Formula of 1 and its clickable probe 2. B) Inhibitory effects of 1 and probe 2 on HIF-1a accumulation were determined by immunoblot analysis. C) Localization of probe 2 (3 mm, green) was detected through a click reaction using azide-linked Alexa Fluor 488 in HCT116 cells. Mitochondria were selectively stained with the MitoTracker probe (red). Nuclei (blue) were stained with 4,6-diamidino-2phenylindole (DAPI). D) Competitive binding of probe 2 (3 mm) to its target molecules in the presence or absence of 1 (10 mm). Scale bars = 20 mm.

Reactive oxygen species-mediated activation of the Akt/ASK1/p38 signaling cascade and p21Cip1 downregulation are required for shikonin-induced apoptosis

Shikonin derivatives exert powerful cytotoxic effects, induce apoptosis and escape multidrug resistance in cancer. However, the diverse mechanisms underlying their anticancer activities are not completely understood. Here, we demonstrated that shikonin-induced apoptosis is caused by reactive oxygen species (ROS)-mediated activation of Akt/ASK1/p38 mitogen-activated protein kinase (MAPK) and downregulation of p21Cip1. In the presence of shikonin, inactivation of Akt caused apoptosis signal-regulating kinase 1 (ASK1) dephosphorylation at Ser83, which is associated with ASK1 activation. Shikonin-induced apoptosis was enhanced by inhibition of Akt, whereas overexpression of constitutively active Akt prevented apoptosis through modulating ASK1 phosphorylation. Silencing ASK1 and MKK3/6 by siRNA reduced the activation of MAPK kinases (MKK) 3/6 and p38 MAPK, and apoptosis, respectively. Antioxidant N-acetyl cysteine attenuated ASK1 dephosphorylation and p38 MAPK activation, indicating that shikonin-induced ROS is involved in the activation of Akt/ASK1/p38 pathway. Expression of p21Cip1 was significantly induced in early response, but gradually decreased by prolonged exposure to shikonin. Overexpression of p21Cip1 have kept cells longer in G1 phase and attenuated shikonin-induced apoptosis. Depletion of p21Cip1 facilitated shikonin-induced apoptosis, implying that p21Cip1 delayed shikonin-induced apoptosis via G1 arrest. Immunohistochemistry and in vitro binding assays showed transiently altered localization of p21Cip1 to the cytoplasm by shikonin, which was blocked by Akt inhibition. The cytoplasmic p21Cip1 actually binds to and inhibits the activity of ASK1, regulating the cell cycle progression at G1. These findings suggest that shikonin-induced ROS activated ASK1 by decreasing Ser83 phosphorylation and by dissociation of the negative regulator p21Cip1, leading to p38 MAPK activation, and finally, promoting apoptosis.

Inactivation of Escherichia coli O157:H7, Salmonella typhimurium, and Listeria monocytogenes on Stored Iceberg Lettuce by Aqueous Chlorine Dioxide Treatment

Inactivation of Escherichia coli O157:H7, Salmonella typhimurium, and Listeria monocytogenes in iceberg lettuce by aqueous chlorine dioxide (ClO(2)) treatment was evaluated. Iceberg lettuce samples were inoculated with approximately 7 log CFU/g of E. coli O157:H7, S. typhimurium, and L. monocytogenes. Iceberg lettuce samples were then treated with 0, 5, 10, or 50 ppm ClO(2) solution and stored at 4 degrees C. Aqueous ClO(2) treatment significantly decreased the populations of pathogenic bacteria on shredded lettuce (P < 0.05). In particular, 50 ppm ClO(2) treatment reduced E. coli O157:H7, S. typhimurium, and L. monocytogenes by 1.44, 1.95, and 1.20 log CFU/g, respectively. The D(10)-values of E. coli O157:H7, S. typhimurium, and L. monocytogenes in shredded lettuce were 11, 26, and 42 ppm, respectively. The effect of aqueous ClO(2) treatment on the growth of pathogenic bacteria during storage was evaluated, and a decrease in the population size of these pathogenic bacteria was observed. Additionally, aqueous ClO(2) treatment did not affect the color of lettuce during storage. These results suggest that aqueous ClO(2) treatment can be used to improve the microbial safety of shredded lettuce during storage.

Upregulation of RhoB via c-Jun N-terminal kinase signaling induces apoptosis of the human gastric carcinoma NUGC-3 cells treated with NSC12618

RhoB expression is reduced in most invasive tumors, with loss of RhoB expression correlating significantly with tumor stage. Here, we demonstrate that upregulation of RhoB by the potent anticancer agent NSC126188 induces apoptosis of NUGC-3 human gastric carcinoma cells. The crucial role of RhoB in NSC126188-induced apoptosis is indicated by the rescue of NUGC-3 cells from apoptosis by knockdown of RhoB. In the presence of NSC126188, c-Jun N-terminal kinase (JNK) signaling was activated, and the JNK inhibitor SP600125 reduced RhoB expression and suppressed the apoptosis of NUGC-3 cells. Knockdowns of mitogen-activated protein kinase kinase (MKK) 4/7, JNK1/2 and c-Jun downregulated RhoB expression and rescued cells from apoptotic death in the presence of NSC126188. The JNK inhibitor SP600125 suppressed transcriptional activation of RhoB in the presence of NSC126188, as indicated by a reporter assay that used luciferase under the RhoB promoter. The ability of NSC126188 to increase luciferase activity through both the p300-binding site and the inverted CCAAT sequence (iCCAAT box) suggests that JNK signaling to upregulate RhoB expression is mediated through both the p300-binding site and the iCCAAT box. However, the JNK inhibitor SP600125 did not inhibit the upregulation of RhoB by farnesyltransferase inhibitor (FTI)-277. The p300-binding site did not affect activation of the RhoB promoter by FTI-277 in NUGC-3 cells, suggesting that the transcriptional activation of RhoB by NSC126188 occurs by a different mechanism than that reported for FTIs. Our data indicate that NSC126188 increases RhoB expression via JNK-mediated signaling through a p300-binding site and iCCAAT box resulting in apoptosis of NUGC-3 cells.

Isolation of a new member of DnaJ-like heat shock protein 40 (Hsp40) from human liver

A new member of Hsp40, HLJ1, consisting of 337 amino acids, was cloned from a human liver cDNA library. The deduced amino acid sequence of HLJ1 has an 84% homology (69% identity) with that of HDJ-1 isolated from human placenta. Northern analysis showed that expression of the HLJ1 gene is heat-inducible and its transcription shows some degree of preference in heart, skeletal muscle, and pancreas.