**5. Acknowledgments**

We are grateful to Professor Toshiaki Umezawa from Kyoto University for his kind support and synthesis of the β-*O*-4 lignin model compound. This work was supported by the "Academic Frontier" Project for Private Universities, with a matching fund subsidy from the Ministry of Education, Culture, Sports, Science and Technology (2004–2008), Japan. The Article Processing Charges for this chapter were provided by a fund from The General Environmental Technos Co. Ltd., Osaka, Japan.

### **6. References**

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induced conditions, were both identified as laccase (Lcc1) by Q-TOF mass spectrometry. Four major, intracellular, MnP isozyme bands were detected in mycelial extracts obtained from *L. edodes* cultured under MnP-induced conditions. Of these isozyme bands, two exhibited the same mobilities as extracellular MnP isozymes, while the other two bands, MnP-i (63) and MnP-i (66), were strictly intracellular. The intracellular MnP isozymes were expressed during the initial stage of culture, either several days before, or coincident with, the expression of the extracellular MnP isozymes. Compared to intracellular MnP isozymes, the extracellular MnP isozymes maintained relatively high activities for up to 40 days of culture. While glycosidase treatment of crude enzyme solutions prior to electrophoresis had no effect on the activities of the extracellular MnP isozymes, such treatment completely inactivated the two strictly intracellular MnP isozymes, implying that the intracellular isozymes were active as glycosylated proteins. Both of the extracellular MnP isozymes detected in the dikaryon were also detected in monokaryotic progeny, suggesting that although these isozymes may be encoded by different loci, they are not under allelic control. Southern blot analysis revealed that the probe *lemnp2* region hybridized with the four of the monokaryotic strains used, all of which exhibited the same two hybridization signals that were observed in the parent dikaryon. These observations suggest that there are two copies of *lemnp2* in the *L. edodes* haploid genome. Moreover, degradation assays involving the addition of the β-*O*-4 lignin model compound in cultures under MnP- and Lcc-induced conditions suggest that, rather than laccase (Lcc1), manganese peroxidase (LeMnP2) is a

In response to the crucial role played by basidiomycetous fungi in the carbon cycle by degrading lignocelluloses, considerable effort has focused on the functional genomics related to the enzymatic systems and mechanisms involved in lignin degradation, particularly in a few model fungus species. Nevertheless, fungal succession on dead logs and leaf litter in nature show that complete degradation of lignocelluloses is a commensal and competitive process affected by numerous fungi. The assay system presented here would be practical and convenient, not only as a method of screening isozymes of value in mushroom breeding and cultivation, but also for evaluating the lignin-degrading abilities of fungi and assessing the antagonistic interactions of different strains under experimental

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**21** 

*Turkey* 

Pnar Erkekoglu

**Protection Studies by Antioxidants Using Single** 

Oxidation-reduction reactions, simply referred as "redox" reactions, describe all the chemical reactions in which atoms have their oxidation state changed. This can either be a simple redox process like the oxidation of carbon (C) to carbon dioxide (CO2) or the reduction of C by hydrogen (H) to yield methane (CH4). However, in biology redox reactions are rather complex and 'redox biology' is fundamental to aerobic life **(Peters et al., 2008; Baliga et al., 2007)**. The simplest example to give is the oxidation of glucose to CO2 and

Aerobes are constantly subject to free radicals, but modulate their actions by synthesizing antioxidants. Free radicals are atoms, molecules, or ions with one or more unpaired electrons on an open shell configuration **(Gutteridge & Halliwell, 2000)**. The simplest form is the atomic H. There are many types of free radicals in living systems, but both nitrogen (N) and oxygen (O) radicals are the main concern for the researchers of several fields as they are suspected to be the underlying factors of several conditions and diseases **(Halliwell, 2006)**. O2 toxicity was suggested to be due to the inactivation of a variety of enzymes (particularly of antioxidant enzymes) by targeting the thiol group of cysteine residues. In the last decades, molecular biology techniques established that the toxic effects of O2 are directly linked to its reactive forms, the reactive oxygen species (ROS), acting on cellular components. Oxidative stress is a serious imbalance between the generation of ROS and antioxidant protection in favor of the former, causing excessive oxidative damage **(Dröge, 2002; Halliwell, 2011)**. Oxidative stress and ROS can account for changes that may be detrimental to the cells **(Dröge, 2002)**. ROS are shown to contribute to cellular damage, apoptosis and cell death **(Dalton et al., 1999; Finkel, 1998)**. The link between O2 toxicity and many pathologies, e.g. pulmonary diseases, **(Frankl, 1991)**, and its effect on swelling of the blood–gas barrier **(Drath et al., 1981)**, retina defects **(Geller et al., 2006)**, bowel disease **(Grisham, 1994)** neurodegeneration **(Wang et al., 2006)**, cancer **(Cerutti, 1994)**, diabetes **(Seet et al., 2010)** and ageing **(Irminger-Finger, 2007)** is very well-established. Besides, in the last decade a relationship between obesity and ROS was demonstrated **(Seet et al., 2010;** 

Antioxidant is a molecule that protects a biological target against oxidative damage **(Halliwell, 2011)**. Accumulating data implicate that both low antioxidant status and genetics may contribute to the risk of several types of malignancies **(Peters et al., 2008;** 

**1. Introduction** 

**Halliwell, 2011)**.

water in photosynthesis **(Halliwell, 2006)**.

**Cell Gel Electrophoresis (Comet Assay)** 

*Hacettepe University, Faculty of Pharmacy, Department of Toxicology, Ankara* 

