Cold-stress response during the stationary-growth phase of Antarctic and temperate-climate Penicillium strains.

Cold-induced oxidative stress during the aging of three Penicillium strains (two Antarctic and one from a temperate region) in stationary culture was documented and demonstrated a significant increase in the protein carbonyl content, the accumulation of glycogen and trehalose, and an increase in the activities of antioxidant enzymes (superoxide dismutase and catalase). The cell response to a temperature downshift depends on the degree of stress and the temperature characteristics of the strains. Our data give further support for the role of oxidative stress in the aging of fungi in stationary cultures. Comparing the present results for the stationary growth phase with our previous results for the exponential growth phase was informative concerning the relationship between the cold-stress response and age-related changes in the tested strains. Unlike the young cells, stationary-phase cultures demonstrated a more pronounced level of oxidative damage, as well as decreased antioxidant defence.

Production, purification, and characterization of a novel cold-active superoxide dismutase from the Antarctic strain Aspergillus glaucus 363.

The Antarctic fungal strain Aspergillus glaucus 363 produces cold-active (CA) Cu/Zn-superoxide dismutase (SOD). The strain contains at least one gene encoding Cu/Zn-SOD that exhibited high homology with the corresponding gene of other Aspergillus species. To our knowledge, this is the first nucleotide sequence of a CA Cu/Zn-SOD gene in fungi. An effective laboratory technology for A. glaucus SOD production in 3 L bioreactors was developed on the basis of transient cold-shock treatment. The temperature downshift to 10 °C caused 1.4-fold increase of specific SOD activity compared to unstressed culture. Maximum enzyme productivity was 64 × 10(3) U kg(-1) h(-1). Two SOD isoenzymes (Cu/Zn-SODI and Cu/Zn-SODII) were purified to electrophoretic homogeneity. The specific activity of the major isoenzyme, Cu/Zn-SODII, after Q-Sepharose chromatography was 4000 U mg(-1). The molecular mass of SODI (38 159 Da) and of SODII (15 835 Da) was determined by electrospray quadropole time-of-flight (ESI-Q-TOF) mass spectrometry and dynamic light scattering (DLS). The presence of Cu and Zn were confirmed by inductively coupled plasma mass spectrometry (ICP-MS). The N-terminal amino acid sequence of Cu/Zn-SODII revealed a high degree of structural homology with Cu/Zn-SOD from other fungi, including Aspergillus species.

Temperature-stress tolerance of the fungal strain Aspergillus niger 26: physiological and ultrastructural changes.

The study focuses on the morphological and physiological cell responses to oxidative stress induced by high temperature treatment in the industrially relevant fungus Aspergillus niger 26. Temperatures above 30 °C lead to growth suppression and changes in morphological characteristics: decrease in the size of hyphal elements and increase in "active length" by switching from slightly branched long filaments to a multitude of branched forms containing active cytoplasm. Transmission electron microscopy of fungal cultures heated at 40 °C demonstrated abnormal wavy septation with reduced amount of chitin (as shown by WGA-gold labelling), intrahyphal hyphae development, disintegration of mitochondria and extensive autolysis. Temperature-dependent decrease in the total intracellular protein content and a sharp increase (six to tenfold) in oxidatively damaged proteins were also demonstrated. Elevated temperatures caused a two and threefold increase in catalase and superoxide dismutase activities, respectively.

Temperature downshift induces antioxidant response in fungi isolated from Antarctica.

Although investigators have been studying the cold-shock response in a variety of organisms for the last two decades or more, comparatively little is known about the difference between antioxidant cell response to cold stress in Antarctic and temperate microorganisms. The change of environmental temperature, which is one of the most common stresses, could be crucial for their use in the biotechnological industry and in ecological research. We compared the effect of short-term temperature downshift on antioxidant cell response in Antarctic and temperate fungi belonging to the genus Penicillium. Our study showed that downshift from an optimal temperature to 15 degrees or 6 degrees C led to a cell response typical of oxidative stress: significant reduction of biomass production; increase in the levels of oxidative damaged proteins and accumulation of storage carbohydrates (glycogen and trehalose) in comparison to growth at optimal temperature. Cell response against cold stress includes also increase in the activities of SOD and CAT, which are key enzymes for directly scavenging reactive oxygen species. This response is more species-dependent than dependent on the degree of cold-shock. Antarctic psychrotolerant strain Penicillium olsonii p14 that is adapted to life in extremely cold conditions demonstrated enhanced tolerance to temperature downshift in comparison with both mesophilic strains (Antarctic Penicillium waksmanii m12 and temperate Penicillium sp. t35).

Heat-shock-induced oxidative stress and antioxidant response in Aspergillus niger 26.

To extend the knowledge about the relationship between heat shock and oxidative stress in lower eukaryotes, the filamentous fungus Aspergillus niger 26 was chosen as a model system. Here, the response of A. niger cells to heat shock is reported. The temperature treatment significantly increased the levels of reactive oxygen species, superoxide anions (O2), and hydrogen peroxide and the rate of cyanide-resistant respiration as a marker of oxidative stress. Enhanced reactive oxygen species generation coincided with an increase in the content of oxidative damaged protein and in the accumulation of the storage carbohydrates trehalose and glycogen. Thermal survival of the A. niger cells corresponded to a significant increase in the levels of the antioxidant enzymes superoxide dismutase and catalase for all variants. These observations suggest that heat and oxidative stress have a common cellular effect.

Cell response of Antarctic and temperate strains of Penicillium spp. to different growth temperature.

The effect of growth temperature (10, 15, 20, 25, and 30 degrees C) on the cell response was compared between two Antarctic Penicillium sp. strains (Penicillium sp. p14 and Penicillium sp. m12) and a European temperate strain, Penicillium sp. t35. According to the temperature profiles, Penicillium sp. p14 was identified as psychrophilic, while Penicillium sp. m12 and Penicillium sp. t35 as mesophilic fungi, respectively. The results demonstrated that the growth at low temperature does clearly induce oxidative stress events in all strains tested. Decreases in growth temperature below the optimal coincided with markedly enhanced protein carbonyl content, an indicator of oxidatively damaged proteins. Also, the cellular response to growth temperature in terms of reserve carbohydrate was determined. In the mesophilic strains there was essentially no enhancement of glycogen content. This was in contrast to the psychrophilic Penicillium sp. p14, which gradually accumulated glycogen in response to cold (10 degrees C) during the exponential phase. In addition, elevated endogenous levels of trehalose upon low-temperature stress were exhibited by all model microorganisms. Compared with temperate mesophilic Penicillium sp. t35, Antarctic strains (psychrophilic Penicillium sp. p14 and mesophilic Penicillium sp. m12) demonstrated a marked rise in activities of protective enzymes such as superoxide dismutase and catalase at decreasing temperatures. The results suggested that low-temperature resistance is partially associated with enhanced scavenging systems.

Oxidative stress response of filamentous fungi induced by hydrogen peroxide and paraquat.

Although, oxidative stress response, which protects organisms from deleterious effects of reactive oxygen species (ROS), has been extensively studied in pro- and eukaryotes, the information about filamentous fungi is fragmentary. We investigated the effect of two ROS-generating agents (paraquat, PQ, and H2O2) on cellular growth and antioxidant enzyme induction in 12 fungal species. Our results indicate that exposure of fungal spores or mycelia to PQ and H2O2 promoted oxidative stress, as evidenced by remarkable inhibition of spore germination and biomass production; stimulation of cyanide-resistant respiration; accumulation of oxidative modified proteins. Cell responses against both superoxide and peroxide stresses include enhanced expression of superoxide dismutase (SOD) and catalase, however, the extent was different: treatment with PQ increased mainly SOD, whereas exogenous H2O2 led to enhanced catalase. We also found that G6PD has a role in the mechanism of protection against superoxide and peroxide stresses. The activation of antioxidant enzyme defence was blocked by the translation inhibitor, cycloheximide, suggesting that there was de novo enzyme synthesis.

A novel glycosylated Cu/Zn-containing superoxide dismutase: production and potential therapeutic effect.

The fungal strain Humicola lutea 103 produces a naturally glycosylated Cu/Zn SOD. To improve its yield, the effect of an increased concentration of dissolved oxygen (DO) on growth and enzyme biosynthesis by the producer, cultivated in a 3 l bioreactor, was examined. Exposure to a 20% DO level caused a 1.7-fold increase of SOD activity compared to the DO-uncontrolled culture. Maximum enzyme productivity of SOD was approximately 300 x 10(3) U (kg wet biomass)(-1). The novel enzyme was purified to electrophoretic homogeneity. The presence of Cu and Zn were confirmed by atomic absorption spectrometry. The molecular mass of H. lutea Cu/Zn SOD was calculated to be 31870 Da for the whole molecule and 15936 Da for the structural subunits. The N-terminal sequence revealed a high degree of structural homology with Cu/Zn SOD from other prokaryotic and eukaryotic sources. H. lutea Cu/Zn SOD was used in an in vivo model for the demonstration of its protective effect against myeloid Graffi tumour in hamsters. Comparative studies revealed that the enzyme (i) elongated the latent time for tumour appearance, (ii) inhibited tumour growth in the early stage of tumour progression (73-75% at day 10) and (iii) increased the mean survival time of Graffi-tumour-bearing hamsters. Moreover, the fungal Cu/Zn SOD exhibited a strong protective effect on experimental influenza virus infection in mice. The survival rate increased markedly, the time of survival rose by 5.2 d and the protective index reached 86%. The H. lutea SOD protected mice from mortality more efficiently compared to the selective antiviral drug ribavirin and to commercial bovine SOD. In conclusion, our results suggest that appropriate use of the novel fungal SOD, applied as such or in combination with selective inhibitors, could outline a promising strategy for the treatment of myeloid Graffi tumour and influenza virus infection.