Stress response in medically important Mucorales

Mucorales are saprobes, ubiquitously distributed and able to infect a heterogeneous population of human hosts. The fungi require robust stress responses to survive in human host. We tested the growth of Mucorales in the presence of different abiotic stress. Eight pathogenic species of Mucorales, including Rhizopus arrhizus, Rhizopus microsporus, Rhizomucor pusillus, Apophysomyces elegans, Licthemia corymbifera, Cunninghamella bertholletiae, Syncephalastrum racemosum and Mucor racemosus, were exposed to different stress inducers: osmotic (sodium chloride and d ‐sorbitol), oxidative (hydrogen peroxide and menadione), pH, cell wall and metal ions (Cu, Zn, Fe and Mg). Wide variation in stress responses was noted: R. arrhizus showed maximum resistance to both osmotic and oxidative stresses, whereas R. pusillus and M. indicus were relatively sensitive. Rhizopus arrhizus and R. microsporus showed maximum resistance to alkaline pH, whereas C. bertholletiae, L. corymbifera, M. racemosus and A. elegans were resistant to acidic pH. Maximum tolerance was noted in R. microsporus to Cu, R. microsporus and R. arrhizus to Fe and C. bertholletiae to Zn. In contrast, L. corymbifera, A. elegans and M. indicus were sensitive to Cu, Zn and Fe respectively. In conclusion, R. arrhizus showed high stress tolerance in comparison to other species of Mucorales, and this could be the possible reason for high pathogenic potential of this fungi.     

Induction of chromosomal instability by chronic oxidative stress

Earlier studies using GM10115 cells analyzed the capability of different DNA-damaging agents to induce genomic instability and found that acute oxidative stress was relatively inefficient at eliciting a persistent destabilization of chromosomes. To determine whether this situation would change under chronic exposure conditions, the human-hamster hybrid line GM10115 was cultured under conditions of oxidative stress. Chronic treatments consisted of 1-hour incubations using a range of hydrogen peroxide (25-200 microM) or glucose oxidase (GO; 5-50 mU/ml) concentrations that were administered once daily over 10 to 30 consecutive days. The toxicity of chronic treatments was modest (- one log kill) and consistent with the low yield of first division aberrations (<5%). However, analysis of over 180 clones and 36,000 metaphases indicated that chronic oxidative stress led to a high incidence of chromosomal instability. Treatment of cells with 100 and 200 microM hydrogen peroxide or 50 mU/ml GO was found to elicit chromosomal instability in 11%, 22%, and 19% of the clones analyzed, respectively. In contrast, control clones isolated after mock treatment did not show signs of chromosomal destabilization. These data suggest that chronic oxidative stress constitutes a biochemical mechanism capable of disrupting the genomic integrity of cells.     

Tumor resistance to oxidative stress: Association with ras oncogene expression and reversal by lovastatin,an inhibitor of p21ras isoprenylation

The ras oncogene family has been implicated in tumor resistance to ionizing radiotherapy. Using the gene-transfer model, we show here that ras expression may also affect cell responses to chemical inducers of oxidative stress. Studies involving human osteosarcoma subclones, which vary in their levels of EJras expression, revealed a tight correlation between the amounts of ras-encoded mRNA and p21 produced, and the degree of resistance to doxorubicin or hydrogen peroxide. Differences in response could not be explained by increased activity of anti-oxidant enzymes such as superoxide dismutase, glutathione reductase, glutathione S-transferase or glutathione peroxidase. Moreover, there were no significant differences in glutathione levels. Although the resistant cells had elevated levels of gamma-glutamyl-transferase mRNA indicative of an increased rate of glutathione turnover, this elevation was not specific for ras-transfected cell lines. Lovastatin, an inhibitor of protein isoprenylation critical for p21 ras membrane association and function, restored the sensitivity of ras-transformed cells to doxorubicin and hydrogen peroxide. The data indicate that pharmacological agents affecting ras expression may enhance responses of some human tumors to free-radical-mediated chemotherapies. © 1995 Wiley-Liss, Inc.     

氧化应激环境下大肠癌细胞VEGF转录活性的变化

目的 研究氧化应激对结肠癌细胞VEGF基因转录的影响及其在肿瘤进展中的潜在作用.方法 使用J2O2模拟大肠腔内氧化应激环境,通过不同浓度H2O2或相同浓度H2O2不同时间刺激人结肠癌细胞HT29,使癌细胞处于氧化应激状态,应用核连缀分析(Run-on assay)、northern blot、ELISA来观察VEGF基因转录活性,RNA以及蛋白质的表达水平,并进一步分析刺激强度、刺激时间与VEGF基因转录活性的关系.结果 核连缀分析显示,H2O2模拟的氧化应激环境明显增加大肠癌细胞HT-29 VEGF基因的转录活性.HT29细胞VEGF mRNA的转录在一定范围内(H2O2浓度不大于10-5 mol/L时)与氧化物刺激的强度具有时间依赖和浓度依赖关系.暴露于氧化物H2O2中的大肠癌细胞HT29 VEGF RNA转录以及蛋白质分泌增加.结论 H2O2模拟的氧化应激环境促进结肠癌细胞VEGF基因的转录活性,在大肠肿瘤的进展中具有潜在的促进作用.     

Clinical characteristics,rapid identification,molecular epidemiology and antifungal susceptibilities of Talaromyces marneffei infections in Shenzhen,China

Although case series of talaromycosis have been reported in China, their detailed clinical and microbiological characteristics have never been systematically profiled. In this study, we report the clinical characteristics, molecular epidemiology, rapid identification and antifungal susceptibilities of talaromycosis in The University of Hong Kong‐Shenzhen Hospital in Shenzhen. Seven cases of talaromycosis were observed since commencement of hospital service in 2012. Three patients were local Shenzhen residents, whereas the other four were immigrants from other parts of China. Two patients were HIV‐negative, but with underlying diseases requiring immunosuppressive therapy. Two of the seven patients succumbed. All the seven isolates were successfully identified as T. marneffei by MALDI–TOF MS using Bruker database expanded with in‐house generated T. marneffei mass spectra. MLST showed that the seven strains belonged to six different, novel sequences types. Phylogenetic analyses of the concatenated five‐locus sequence revealed that the seven strains were scattered amongst other T. marneffei strains. The MICs of itraconazole, isavuconazole, posaconazole and voriconazole against the seven clinical isolates were low but MICs of anidulafungin were high. Underlying diseases other than HIV infection are increasingly important risk factors of talaromycosis. MALDI–TOF MS is useful for rapid identification. Highly diverse T. marneffei sequence types were observed.     

Development and evaluation of a real‐time polymerase chain reaction assay for the rapid detection of Talaromyces marneffei MP1 gene in human plasma

Penicilliosis caused by Talaromyces marneffei is a common AIDS‐defining illness in South and Southeast Asia. Diagnosis is based on culture which can take up to 14 days for identification, leading to treatment delay and increased mortality. We developed a TaqMan real‐time PCR assay targeting the MP1 gene encoding an abundant cell wall protein specific to T. marneffei. The assay's performance was evaluated in MP1‐containing plasmids, clinical isolates, and plasma from HIV‐infected patients with and without penicilliosis. The assay consistently detected 10 copies of MP1‐containing plasmids per reaction and 100 T. marneffei yeast cells per millilitre plasma. There were no amplification with seven other Penicillium species and six other HIV‐associated fungal pathogens tested. The assay was evaluated in 70 patients with AIDS: 50 patients with culture‐confirmed penicilliosis and 20 patients with opportunistic infections other than penicilliosis. The diagnostic sensitivity was 70.4% (19/27, 95% CI: 51.5–84.1%) and 52.2% (12/23, 95% CI: 33.0–70.8%) in plasma samples collected prior to and within 48 h of antifungal therapy respectively. The diagnostic specificity was 100% (20/20, 95% CI: 83.9–100%). This assay provides a useful tool for the rapid diagnosis of T. marneffei infection and has the potential to improve the management of patients with penicilliosis.     

Onychomycosis in Tehran: mycological study of 504 patients

Onychomycosis is a common nail disorder resulting from the invasion of the nail plate by a dermatophyte, yeast or mould species and gives rise to some diverse clinical presentations. The purpose of the present study was to isolate and identify the causative fungi of onychomycosis in the population of Tehran, Iran. Nail samples from 504 patients with prediagnosis of onychomycosis during 2005 were examined both by direct microscopical observation of fungal elements in KOH preparations and in culture for the identification of the causative agent. All samples were inoculated on (i) Sabouraud dextrose agar (SDA, Merck), (ii) SDA with 5% chloramphenicol and cycloheximide in duplicate for dermatophyte and (iii) SDA with 5% chloramphenicol in triplicate for mould isolation. The criteria for the diagnosis of onychomycosis caused by non‐dermatophytic moulds were based on microscopical observation of fungal elements, growth of the same mould in all triplicate culture and no growth of a dermatophyte or yeast in all the cultures. Of 504 cases examined, 216 (42.8%) were mycologically proven cases of onychomycosis (144 fingernails, 72 toenails). Among the positive results, dermatophytes were diagnosed in 46 (21.3%), yeasts in 129 (59.7%) and non‐dermatophytic moulds in 41 (19%). Trichophyton mentagrophytes was the most common causative agent (n = 22), followed by Trichophyton rubrum (n = 13), Candida albicans (n = 42), Candida spp. (n = 56) and Aspergillus spp. (n = 21). Nearly half of the clinically suspected fungal nail infections are onychomycosis and yeast is responsible for most of the infections in Iran.     

Adaptation of human leukemia HL-60 cells to hydrogen peroxide as oxidative stress

The growth inhibitory effect on human leukemia HL-60 cells of hydrogen peroxide as an oxidative stress and the possibility of adaptation of the cells to this stress were examined. When HL-60 cells were treated with various concentrations of hydrogen peroxide, concentrations of 5-200 microM were found to be sublethal. When HL-60 cells were repeatedly exposed to 50 and 100 microM hydrogen peroxide, they began to grow stably in the medium with the drug after a few months, indicating that they had become adapted to the drug. Sublines HP50-2 and HP100-1 cloned from these cultures were approximately 40-fold and 340-fold more resistant, respectively, than the parent cells. These resistant sublines tended to form cell aggregates in stationary culture, suggesting an alteration in their surface membrane. The HP50-2 and HP100-1 lines will be useful in the studies of many cellular mechanisms associated with reactive oxygens.     

ABT-737, a BH3 mimetic, induces glutathione depletion and oxidative stress

Purpose

This study assessed the role of oxidative stress and loss of glutathione in ABT-737-induced apoptosis.

Methods

Jurkat human acute lymphocytic leukemia cells and HeLa cells transfected with a tet-regulated Bcl-2 expression system were treated with ABT-737 or its less active stereoisomer. GSH concentrations, intracellular reactive oxygen species (ROS), caspase activation and apoptotic DNA fragmentation were measured.

Results

ABT-737 induced oxidative stress through decreased GSH and increased intracellular hydrogen peroxide and superoxide levels. Apoptotic DNA fragmentation and caspase activation were the consequences of this oxidative stress. Combining ABT-737 with ROS-inducing agents such as adaphostin or etoposide enhanced cell death.

Conclusions

These results demonstrate that inhibition of Bcl-2 causes a loss of GSH, an increase in ROS, caspase activation and subsequent apoptosis. Clinically, redox alterations as a consequence of Bcl-2 inhibition by ABT-737 should be considered in devising combination therapies with this novel agent or its derivatives.     

Chemopreventive action of dexamethasone and α-tocopherol in oxidative stressed cells

Introduction: Recent research indicates a close connection of inflammation and cancer as presumed by Virchow in 1893. The growing understanding of cellular signalling and regulatory pathways reveals multiple links between inflammation and cancer. This study was designed to evaluate the influence of the anti-inflammatory drug dexamethasone and the antioxidant α-tocopherol on oxidative induced DNA damage, a major factor in the development of malignancies. Material and methods: Miniorgan cultures (MOC) of fresh biopsied human nasal mucosa were used to keep cells in their microenvironment and thus to mimic in vivo conditions. MOC were pretreated with dexamethasone and α-tocopherol in different concentrations on 1 or on 5 days before oxidative DNA damage was introduced by hydrogen peroxide. The effect of these substances on DNA damage was evaluated using the alkaline single cell microgel electrophoresis (Comet Assay). Results: Dexamethasone induced slight, but considerable DNA fragmentation by itself. It effectively protected cells from hydrogen peroxide induced DNA damage, leading to a maximum decrease of about 45% when preincubated on 5 days at 20 μM. α-Tocopherol most effectively reduced oxidative DNA fragmentation by about 38% when MOC were pretreated 5 days at 20 μM. Discussion: Our experimental data clearly shows the DNA protective action of dexamethasone and α-tocopherol with regard to oxidatively induced DNA damage, a major pathogenetic factor that inflammation and cancer have in common.     

Cells deficient in oxidative DNA damage repair genes Myh and Ogg1 are sensitive to oxidants with increased G2/M arrest and multinucleation

Oxidative stress generated from endogenous and exogenous sourcescauses oxidative DNA damage. The most frequent mutagenic baselesion 7,8-dihydro-8-oxoguanine and the resulting mismatchedadenine are removed by OGG1 and MYH in mammals. Deficienciesin human MYH or mouse MYH and OGG1 result in tumor predispositionbut the underlying molecular mechanism is not fully understood.To facilitate the study of the roles of MYH and OGG1 in theprotection against oxidative stress, we generated mouse embryonicfibroblast cell lines deficient in these genes. Myh and Ogg1double knockout cells were more sensitive than wild type tooxidants (hydrogen peroxide and t-butyl hydroperoxide), butnot to cis-platinum or -irradiations. The low dosage oxidativestress resulted in more reduction of S phase and increase ofG₂/M phase in Myh^–/–Ogg1^–/– cells thanin wild-type cells, but a similar level of cell death in bothcells. The oxidants also induced more multinucleated cells inMyh^–/–Ogg1^–/– cells than in wild-type,accompanied by centrosome amplification and multipolar spindleformation. Thus, under oxidative stress, Myh and Ogg1 are likelyrequired for normal cell-cycle progression and nuclear division,suggesting multiple roles of Myh and Ogg1 in the maintenanceof genome stability and tumor prevention. Abbreviations: H₂O₂, hydrogen peroxide; MEF, mouse embryo fibroblast; TBH, t-butyl hydroperoxide Received September 26, 2007; revised January 17, 2008; accepted January 26, 2008.     

Endoplasmic Reticulum Stress and Unfolded Protein Response in Atm-Deficient Thymocytes and Thymic Lymphoma Cells Are Attributable to Oxidative Stress

Both oxidative stress and endoplasmic reticulum (ER) stress have been implicated in carcinogenesis. It is well documented that cells deficient in the ataxia-telangiectasia mutated (ATM) gene undergo oxidative stress, which is critically involved in thymic lymphomagenesis in Atm-/- mice. Here we demonstrate that undifferentiated Atm-/- thymocytes show signs of ER stress and of the unfolded protein response (UPR). Using two-dimensional (2-D) gel electrophoresis and mass spectrometry (MS) analysis, we identified 22 differentially expressed proteins, including the ER stress marker glucose-regulated protein 78 (GRP78), in Atm-/- thymocytes and in Atm-/- thymic lymphoma cells relative to Atm+/+ thymocytes. The phosphorylated α subunit of eukaryotic translation initiation factor 2 (p-eIF2α), a UPR marker, was also increased in Atm-/- thymocytes. Cells of the ATL-1 line, which were derived from an Atm-/- mouse thymic lymphoma, were more sensitive to the ER stress inducer tunicamycin than were Atm+/+ thymic leukemia ASL-1 cells. Notably, treatment with hydrogen peroxide duplicated the effects of ATM deficiency in cultured thymocytes, and treatment with the novel cell-permeable thiol antioxidant N-acetylcysteine amide (AD4) reduced elevated p-eIF2α levels in thymocytes of Atm-/- mice. Thus, we propose that ER stress and the UPR are secondary to oxidative stress in Atm-/- thymocytes.     

MnSOD Val-9Ala Genotype, Pro- and Anti-oxidant Environmental Modifiers, and Breast Cancer Among Women on Long Island, New York

Excessive oxidative stress may induce and promote breast carcinogenesis. Manganese superoxide dismutase (MnSOD) is critical to management of oxidative stress by catalyzing the formation of hydrogen peroxide from two superoxide anions. To examine the relationship between MnSOD Val-9Ala polymorphism, breast cancer and potential modifiers, we analyzed data from a large population-based case-control study. Study participants completed an in-home interviewer-administered questionnaire, and self-completed a Block food frequency questionnaire. Age-adjusted unconditional logistic models included 1034 cases and 1084 controls. As compared with Val/Val genotype, we found no association between MnSOD Ala/Val (OR = 0.98, 95% CI: 0.79–1.21) and Ala/Ala (OR = 1.00, 95% CI: 0.79–1.28) genotypes and breast cancer. Results did not differ by menopausal status, stage of tumor, or estrogen and progesterone receptor status. No discernable patterns of interaction were found between this MnSOD variant and anti-oxidative exposures, including fruit and vegetable intake or NSAID use, or pro-oxidant exposures, including smoking and alcohol. This study provides little evidence that variation in Val-9Ala polymorphism of MnSOD alone or through substantial interaction with key exposures believed to be pro- or anti-oxidant properties influences breast cancer risk.     

DNA recombination induced by aflatoxin B1 activated by cytochrome P450 1A enzymes

Mutations in tumor suppressor genes are intricately associated with the etiology of neoplasia. Often, such mutations are followed by the second, functional alleles of tumor suppressor genes, a phenomenon known as loss of heterozygosity. Loss of heterozygosity may occur by different molecular mechanisms, including mitotic recombination, and it is conceivable that these molecular events are influenced by endogenous as well as exogenous factors. To test whether mitotic recombination is induced by certain carcinogens, we genetically engineered a Saccharomyces cerevisiae tester strain so that it metabolized two important classes of carcinogens, polycyclic aromatic hydrocarbons and heterocyclic arylamines. This was accomplished by expressing human cDNA's coding for the cytochrome P450 (CYP) enzymes CYP1A1 or CYP1A2 in combination with NADPH-CYP oxidoreductase in a strain heterozygous for two mutations in the trp5 gene. Microsomes isolated form the transformed yeast strains activated various xenobiotics to powerful mutagens that were detected in the were detected in the Ames test. Of these, the mycotoxin aflatoxin B1, when activated intracellularly in the strains containing either human CYP enzyme, significantly induced mitotic recombination. These results are discussed in light of possible mechanisms that are involved in aflatoxin B₁-mediated hepatocarcinogenesis. Similarly, benzo[a]pyrene-trans-7,8-dihydrodiol and 3-amino-1-methyl-5H-pyrido [4,3-b]indole were activated to recombinagenic products, whereas benozo[a]pyrene and 2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline were negative in this assay. Our results argue that the contstructed yeast strains may be a valuable tool for the investigation of drug-induced mitotic recombination. ©1994 Wiley-Liss, Inc.     

Cordycepin prevents oxidative stress-induced inhibition of osteogenesis

Oxidative stress is known to be involved in impairment of osteogenesis and age-related osteoporosis. Cordycepin is one of the major bioactive components of Cordyceps militaris that has been shown to exert antioxidant and anti-inflammatory activities. However, there are few reports available regarding the effects of cordycepin on osteogenesis and the underlying mechanism. In this study, we investigated the potential osteoprotective effects of cordycepin and its mechanism systematically using both in vitro model as well as in vivo mouse models. We discovered that hydrogen peroxide (H₂O₂) induced inhibition of osteogenesis which was rescued by cordycepin treatment in human bone marrow mesenchymal stem cells (BM-MSCs). Cordycepin exerted its protective effects partially by increasing or decreasing expression of osteogenic and osteoclastogenesis marker genes. Treatment with cordycepin increased Wnt-related genes'' expression whereas supplementation of Wnt pathway inhibitor reversed its protective effects. In addition, administration of cordycepin promoted osteogenic differentiation of BM-MSCs by reducing oxidative stress in both ovariectomized and aged animal models. Taken together, these results support the protective effects of cordycepin on oxidative stress induced inhibition of osteogenesis by activation of Wnt pathway.     

Induction of the heme oxygenase gene in human skin fibroblasts by hydrogen peroxide and UVA (365 nm) radiation: evidence for the involvement of the hydroxyl radical

The induction of heme oxygenase by both hydrogen peroxide and UVA (365 nm) radiation in normal human skin fibroblasts is prevented by prior treatment of cells with the specific iron chelators, o-phenanthroline or desferrioxamine. In addition, both iron chelators protected cells against the lethal effects of H2O2 treatment or UVA irradiation. We propose that the generation of the highly reactive hydroxyl radical by an iron catalyzed Fenton reaction is involved both in the induction of this stress response and, at least in part, in cell killing by the two treatments. These results are also consistent with the idea that the heme oxygenase gene is induced in response to oxidative stress and that its induction may constitute an inducible protective mechanism against oxidative damage induced by both hydrogen peroxide and UVA radiation.     

Comparative pathogenicity of opportunistic black yeasts in Aureobasidium

Aureobasidium pullulans and A. melanogenum are black‐yeast‐like surface colonisers and are commonly encountered as contaminants in the hospital. The species are able to produce melanin which play a role in protection against environmental stress and irradiation. Aureobasidium melanogenum shows higher frequency in opportunistic infections compared to A. pullulans. Comparative pathogenicity of opportunistic black yeasts between Aureobasidium pullulans and A. melanogenum to explain the observed differences in frequency in infection. Degrees of melanisation and thermotolerance were measured, and virulence of strains from different sources was examined in Galleria mellonela and murine infection models. Aureobasidium melanogenum responds with increased melanisation to temperature stress and generally survives at 37°C, A. pullulans on average scored less on these parameters. In the murine model, differences between species were not significant, but the melanised A. melanogenum group showed the highest virulence. This result was not reproducible in Galleria mellonella larvae at 25°C. The A. melanogenum black group showed higher pathogenicity in murine model, indicating that the combination of melanisation and thermotolerance rather than species affiliation is instrumental. Galleria larvae did not survive very well at 37°C, and hence, this model is judged insufficient to detect the small virulence differences observed in Aureobasidium.