Loss of the dermis zinc transporter ZIP13 promotes the mildness of fibrosarcoma by inhibiting autophagy.

Fibrosarcoma is a skin tumor that is frequently observed in humans, dogs, and cats. Despite unsightly appearance, studies on fibrosarcoma have not significantly progressed, due to a relatively mild tumor severity and a lower incidence than that of other epithelial tumors. Here, we focused on the role of a recently-found dermis zinc transporter, ZIP13, in fibrosarcoma progression. We generated two transformed cell lines from wild-type and ZIP13-KO mice-derived dermal fibroblasts by stably expressing the Simian Virus (SV) 40-T antigen. The ZIP13-/- cell line exhibited an impairment in autophagy, followed by hypersensitivity to nutrient deficiency. The autophagy impairment in the ZIP13-/- cell line was due to the low expression of LC3 gene and protein, and was restored by the DNA demethylating agent, 5-aza-2'-deoxycytidine (5-aza) treatment. Moreover, the DNA methyltransferase activity was significantly increased in the ZIP13-/- cell line, indicating the disturbance of epigenetic regulations. Autophagy inhibitors effectively inhibited the growth of fibrosarcoma with relatively minor damages to normal cells in xenograft assay. Our data show that proper control over autophagy and zinc homeostasis could allow for the development of a new therapeutic strategy to treat fibrosarcoma.

Identification and Characterization of Fungi Contaminated in the Built-In Furniture of an Apartment Home.

Fungal contamination of built-in furniture is a frequent problem in Korea when new apartment is built. However, domestic information on the contaminating fungi is very limited. This study was conducted to isolate, identify and characterize the fungi of the problem in one of the apartment houses where the fungi were claimed in the built-in furniture before the house owner moves in. Fungi present in the furniture installed in a main room, dress room, and kitchen side were visually and microscopically confirmed and purely isolated on PDA. The isolated fungi were identified by analyzing the morphological characteristics and nucleotide sequence of the ITS, calmodulin gene, and TEF-1α gene. Aspergillus creber, A. niger, A. pseudoglacus, A. ruber, Cladosporium perangustum and Penicillium commune were identified. Four out of the six fungal species were positive for at least one enzyme in six kinds of extracellular enzyme assays. When these four species (A. creber, A. niger, C. perangustum and P. commune) were inoculated onto four kinds of wood chips of furniture materials, they were able to colonize all of the wood chips. Their settlement was better at 95% humidity condition than at 30% humidity condition. Among the four species, C. perangustum caused the darkest discoloration and secreted the most number of extracellular enzymes. The four species were re-isolated from the colonized wood chips and confirmed as the problematic fungi in the built-in furniture.

Engineering of Klebsiella oxytoca for production of 2,3-butanediol via simultaneous utilization of sugars from a Golenkinia sp. hydrolysate.

The Klebsiella oxytoca was engineered to produce 2,3-butanediol (2,3-BDO) simultaneously utilizing glucose and galactose obtained from a Golenkinia sp. hydrolysate. For efficient uptake of galactose at a high concentration of glucose, Escherichia coli galactose permease (GalP) was introduced, and the expression of galP under a weak-strength promoter resulted in simultaneous consumption of galactose and glucose. Next, to improve the sugar consumption, a gene encoding methylglyoxal synthase (MgsA) known as an inhibitor of multisugar metabolism was deleted, and the mgsA-null mutant showed much faster consumption of both sugars than the wild-type strain did. Finally, we demonstrated that the engineered K. oxytoca could utilize sugar extracts from a Golenkinia sp. hydrolysate and successfully produces 2,3-BDO.

Ribosomal Intergenic Spacer 1 Based Characterization of Button Mushroom (Agaricus bisporus) Strains.

Breeding the button mushroom requires genetic information about its strains. This study was undertaken to genetically characterize four domestically bred button mushroom strains (Saea, Saejung, Saedo, Saeyeon cultivars) and to assess the possibility of using the intergenic spacer 1 (IGS1) region of rDNA as a genetically variable region in the genetic characterization. For the experiment, 34 strains of Agaricus bisporus, two strains of A. bitorquis, and one strain of A. silvaticus, from 17 countries were used. Nucleotide sequence analysis of IGS1 rDNA in these 37 Agaricus strains confirmed that genetic variations exist, not only among the four domestic strains, but also between the four domestic strains and foreign strains. Crossing two different haploid strains of A. bisporus seems to generate genetic variation in the IGS1 region in their off-spring haploid strains. Phylogenetic analysis based on the IGS1 sequence revealed all A. bisporus strains could be differentiated from A. silvaticus and A. bitorquis strains. Five genetic groups were resolved among A. bisporus strains. Saejung and Saeyeon cultivars formed a separate genetic group. Our results suggest that IGS1 could be complementarily applied in the polymorphism analysis of button mushroom.

Synergic chemoprevention with dietary carbohydrate restriction and supplementation of AMPK-activating phytochemicals: the role of SIRT1.

Calorie restriction or a low-carbohydrate diet (LCD) can increase life span in normal cells while inhibiting carcinogenesis. Various phytochemicals also have calorie restriction-mimetic anticancer properties. We investigated whether an isocaloric carbohydrate-restriction diet and AMP-activated protein kinase (AMPK)-activating phytochemicals induce synergic tumor suppression. We used a mixture of AMPK-activating phytochemical extracts including curcumin, quercetin, catechins, and resveratrol. Survival analysis was carried out in a B16F10 melanoma model fed a control diet (62.14% kcal carbohydrate, 24.65% kcal protein and 13.2% kcal fat), a control diet with multiple phytochemicals (MP), LCD (16.5, 55.2, and 28.3% kcal, respectively), LCD with multiple phytochemicals (LCDmp), a moderate-carbohydrate diet (MCD, 31.9, 62.4, and 5.7% kcal, respectively), or MCD with phytochemicals (MCDmp). Compared with the control group, MP, LCD, or MCD intervention did not produce survival benefit, but LCDmp (22.80±1.58 vs. 28.00±1.64 days, P=0.040) and MCDmp (23.80±1.08 vs. 30.13±2.29 days, P=0.008) increased the median survival time significantly. Suppression of the IGF-1R/PI3K/Akt/mTOR signaling, activation of the AMPK/SIRT1/LKB1pathway, and NF-κB suppression were the critical tumor-suppression mechanisms. In addition, SIRT1 suppressed proliferation of the B16F10 and A375SM cells under a low-glucose condition. Alterations in histone methylation within Pten and FoxO3a were observed after the MCDmp intervention. In the transgenic liver cancer model developed by hydrodynamic transfection of the HrasG12V and shp53, MCDmp and LCDmp interventions induced significant cancer-prevention effects. Microarray analysis showed that PPARα increased with decreased IL-6 and NF-κB within the hepatocytes after an MCDmp intervention. In conclusion, an isocaloric carbohydrate-restriction diet and natural AMPK-activating agents induce synergistic anticancer effects. SIRT1 acts as a tumor suppressor under a low-glucose condition.

Genetic and Biochemical Characterization of Monokaryotic Progeny Strains of Button Mushroom (Agaricus bisporus).

To promote the selection of promising monokaryotic strains of button mushroom (Agaricus bisporus) during breeding, 61 progeny strains derived from basidiospores of two different lines of dikaryotic parental strains, ASI1038 and ASI1346, were analyzed by nucleotide sequencing of the intergenic spacer I (IGS I) region in their rDNA and by extracellular enzyme assays. Nineteen different sizes of IGS I, which ranged from 1,301 to 1,348 bp, were present among twenty ASI1346-derived progeny strains, while 15 different sizes of IGS I, which ranged from 700 to 1,347 bp, were present among twenty ASI1038-derived progeny strains. Phylogenetic analysis of the IGS sequences revealed that different clades were present in both the ASI10388- and ASI1346-derived progeny strains. Plating assays of seven kinds of extracellular enzymes (ß-glucosidase, avicelase, CM-cellulase, amylase, pectinase, xylanase, and protease) also revealed apparent variation in the ability to produce extracellular enzymes among the 40 tested progeny strains from both parental A. bisporus strains. Overall, this study demonstrates that characterization of IGS I regions and extracellular enzymes is useful for the assessment of the substrate-degrading ability and heterogenicity of A. bisporus monokaryotic strains.

Characterization of Myrothecium roridum Isolated from Imported Anthurium Plant Culture Medium.

During an investigation of microorganisms and pests in plant culture media from imported anthurium pots, a fungal isolate (DUCC4002) was detected. Based on its morphological characters including colony shape on potato dextrose agar, the microstructures of spores observed by light and scanning electron microscopy and the results of phylogenetic analysis using an internal transcribed spacer rDNA sequence, the fungal isolate was identified as Myrothecium roridum. Pathogenicity testing on anthurium leaves revealed that the fungus could colonize and produce sporodochia on the inoculated leaves. This is the first report of M. roridum detected in imported plant culture medium in Korea.

Diallyl trisulfide induces apoptosis in human breast cancer cells through ROS-mediated activation of JNK and AP-1.

Multiple lines of evidence support an inverse association between consumption of garlic and the risk of cancer. Chemopreventive effects of garlic have been attributed to its oil-soluble sulfur ingredients, such as diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS), but their underlying molecular mechanisms remain largely unresolved. In the present study, we found that DATS showed the most potent anti-proliferative effects in human breast cancer MCF-7 cells. MCF-7 cells treated with DATS underwent apoptotic death as revealed by a progressive increase in the proportion of the sub-G0/G1 cell population and a typical pattern of annexin V/propidium iodide staining. DATS induced phosphorylation of the antiapoptotic Bcl-2 and proteolytic cleavage of poly(ADP-ribose)polymerase (PARP) in MCF-7 cells. DATS treatment activated c-Jun N-terminal kinase (JNK). DATS-induced apoptosis was blunted in MCF-7 cells treated with a specific JNK inhibitor SP600125 or transiently transfected with dominant negative JNK. DATS treatment resulted in accumulation of reactive oxygen species (ROS). DATS-induced apoptosis as well as activation of JNK was abrogated by N-acetyl-l-cysteine (NAC). Furthermore, DATS induced phosphorylation and expression of c-Jun, which were attenuated by NAC. MCF-7 cells treated with DATS also exhibited increased DNA binding activity of AP-1, which was blocked by NAC and the JNK inhibitor. Proteolytic cleavage of PARP induced by DATS was abrogated in the cells transfected with c-jun siRNA. Oral administration of 5µmol/kg DATS to female Balb/c mice inhibited the growth of human MCF-7 cell tumor xenografts. These results suggest that DATS-induced apoptosis is mediated through ROS generation and subsequent activation of JNK and AP-1.

Protective effects of magnesium lithospermate B against diabetic atherosclerosis via Nrf2-ARE-NQO1 transcriptional pathway.

Hyperglycemia-induced oxidative stress is known to play an important role in the development of several diabetic complications, including atherosclerosis. Although a number of antioxidants are available, none have been found to be suitable for regulating the oxidative stress response and enhancing antioxidative defense mechanisms. In this study, we evaluated the effects of magnesium lithospermate B (LAB) against oxidative stress. We also endeavored to identify the target molecule of LAB in vascular smooth muscle cells (VSMCs) and the underlying biochemical pathways related to diabetic atherosclerosis. Modified MTT and transwell assays showed that the increased proliferation and migration of rat aortic VSMCs in culture with high glucose was significantly inhibited by LAB. LAB also attenuated neointimal hyperplasia after balloon catheter injury in diabetic rat carotid arteries. To determine molecular targets of LAB, we studied the effects of LAB on aldose reductase (AR) activity, O-GlcNAcylation, and protein kinase C (PKC) activity in VSMCs under normoglycemic or hyperglycemic conditions and showed the improvement of major biochemical pathways by LAB. Potential involvement of the nuclear factor erythroid 2-related factor-2 (Nrf2)--antioxidant responsive element (ARE)-NAD(P)H: quinone oxidoreductase-1 (NQO1) pathway was assessed using siRNA methods. We found that LAB activates the NQO1 via the Nrf2-ARE pathway, which plays an important role in inhibition of the major molecular mechanisms that lead to vascular damage and the proliferation and migration of VSMCs. Together, these findings demonstrate that the induction of the Nrf2-ARE-NQO1 pathway by LAB could be a new therapeutic strategy to prevent diabetic atherosclerosis.

Polymorphisms of CYP1A1 and GSTM1 genes and susceptibility to oral cancer.

PURPOSE: Oral cancer is the fifth most common form of cancer in the world and comprises 6.5% of all cancer deaths. Since one of the major risk factors for oral cancer is tobacco use, we hypothesized that polymorphic genes coding for tobacco carcinogen-metabolizing enzymes may play a role in oral cancer susceptibility. MATERIALS AND METHODS: To investigate the association between polymorphisms of the CYP1A1 and GSTM1 genes and risks for oral squamous cell carcinoma (OSCC) in the Korean population, the prevalence of the CYP1A1 Mspl and GSTM1 null polymorphisms were examined in 72 patients with histologically confirmed primary OSCC, as well as in 221 healthy control subjects. RESULTS: A significant risk increase for oral cancer was observed among subjects with the homozygous CYP1A1 (m2/m2) genotype (OR=3.8, 95% CI=1.9-7.7), but not the GSTM1 null genotype (OR=0.7, 95% CI=0.4-1.3). Risk for oral cancer was significantly increased in subjects with the homozygous CYP1A1 (m2/m2)genotype, regardless of smoking history (smokers; OR=4.4; 95% CI=1.2-16.3; non- smokers OR=4.9; 95% CI=1.9-12.5). Using the potentially most protective genotype GSTM1 (+)/CYP1A1 [(m1/m1)+ (m1/m2)] as the reference group, an increased risk for oral cancer was observed among subjects with the GSTM1 (+)/ CYP1A1 (m2/m2) (OR= 2.0, 95% CI=0.8-5.2), and GSTM1 (-)/ CYP1A1 (m2/m2) (OR=4.9, 95% CI=1.5-15.5) genotypes (p < 0.009, (chi2 trend test). CONCLUSION: Our results suggest that individuals with a genotype of CYP1A1 (m2/m2) and GSTM1 (-) are highly susceptible for OSCC and that the CYP1A1 (m2/m2) genotype is closely associated with increased risk of OSCC in Koreans.

Xanthorrhizol, a natural sesquiterpenoid from Curcuma xanthorrhiza, has an anti-metastatic potential in experimental mouse lung metastasis model.

Xanthorrhizol is a sesquiterpenoid compound isolated from the rhizome of Curcuma xanthorrhiza. In this study, the anti-metastatic activity of xanthorrhizol was evaluated by using an in vivo mouse lung metastasis model and a tumor mass formation assay. Interestingly, xanthorrhizol dramatically inhibited the formation of tumor nodules in the lung tissue and the intra-abdominal tumor mass formation. Next, to examine the mechanism of the anti-metastatic action of xanthorrhizol in the mouse lung metastasis, expression patterns of the several intracellular signaling molecules were evaluated using the lung tissues with tumor nodules. Higher expression levels of cyclooxygenase-2 (COX-2), matrix metalloproteinase-9 (MMP-9), and phosphorylated extracellular signal-regulated kinase (ERK) were observed in the metastatic group compared with control, but these were attenuated by the treatment of xanthorrhizol. In conclusion, xanthorrhizol exerts anti-metastatic activity in vivo and this effect could be highly linked to the metastasis-related multiplex signal pathway including ERK, COX-2, and MMP-9.

Polymorphisms in genes coding for enzymes metabolizing smoking-derived substances and the risk of periodontitis.

OBJECTIVES: Although the direct cause for periodontitis is oral bacterial infection, its progression depends upon genetic and environmental factors. Smoking, one of the environmental factors, is a risk factor for the development and severity of periodontitis. Therefore, individual susceptibility to periodontitis may be influenced by the polymorphisms of genes coding for enzymes metabolizing tobacco-derived substances. The object of this study is to investigate roles of genetic polymorphisms of these metabolizing enzymes in the risk for periodontitis. MATERIAL AND METHODS: We investigated three important enzymes: cytochrome P450 (CYP) 1A1, CYP2E1 and glutathione S-transferase (GST) M1, involved in the metabolic activation and detoxification of tobacco-derived substances. The prevalence of the polymorphisms of these genes was examined in 115 patients with periodontitis as well as in 126 control subjects. RESULTS: Significantly increased risk for periodontitis was observed for subjects with the polymorphic CYP1A1 m2 allele (odds ratio (OR)=2.3, 95% confidence interval (CI)=1.2-4.4). A significant risk increase for periodontitis associated with the GSTM1 allele was observed (OR=2.1, 95% CI=1.3-3.6). However, no association was observed between the CYP2E1 Pst1 polymorphism and risk for periodontitis (OR=1.3, 95% CI=0.6-2.5). CONCLUSION: These results suggest that the GSTM1 and CYP1A1 polymorphisms may play an important role in risk for periodontitis.