Oleg Gusev

A mitochondrial superoxide theory for oxidative stress diseases and aging.

Fridovich identified CuZnSOD in 1969 and manganese superoxide dismutase (MnSOD) in 1973, and proposed ”the Superoxide Theory,” which postulates that superoxide (O2•−) is the origin of most reactive oxygen species (ROS) and that it undergoes a chain reaction in a cell, playing a central role in the ROS producing system. Increased oxidative stress on an organism causes damage to cells, the smallest constituent unit of an organism, which can lead to the onset of a variety of chronic diseases, such as Alzheimer’s, Parkinson’s, amyotrophic lateral sclerosis and other neurological diseases caused by abnormalities in biological defenses or increased intracellular reactive oxygen levels. Oxidative stress also plays a role in aging. Antioxidant systems, including non-enzyme low-molecular-weight antioxidants (such as, vitamins A, C and E, polyphenols, glutathione, and coenzyme Q10) and antioxidant enzymes, fight against oxidants in cells. Superoxide is considered to be a major factor in oxidant toxicity, and mitochondrial MnSOD enzymes constitute an essential defense against superoxide. Mitochondria are the major source of superoxide. The reaction of superoxide generated from mitochondria with nitric oxide is faster than SOD catalyzed reaction, and produces peroxynitrite. Thus, based on research conducted after Fridovich’s seminal studies, we now propose a modified superoxide theory; i.e., superoxide is the origin of reactive oxygen and nitrogen species (RONS) and, as such, causes various redox related diseases and aging.

Identification of Anhydrobiosis-related Genes from an Expressed Sequence Tag Database in the Cryptobiotic Midge Polypedilum vanderplanki (Diptera; Chironomidae)

Some organisms are able to survive the loss of almost all their body water content, entering a latent state known as anhydrobiosis. The sleeping chironomid (Polypedilum vanderplanki) lives in the semi-arid regions of Africa, and its larvae can survive desiccation in an anhydrobiotic form during the dry season. To unveil the molecular mechanisms of this resistance to desiccation, an anhydrobiosis-related Expressed Sequence Tag (EST) database was obtained from the sequences of three cDNA libraries constructed from P. vanderplanki larvae after 0, 12, and 36 h of desiccation. The database contained 15,056 ESTs distributed into 4,807 UniGene clusters. ESTs were classified according to gene ontology categories, and putative expression patterns were deduced for all clusters on the basis of the number of clones in each library; expression patterns were confirmed by real-time PCR for selected genes. Among up-regulated genes, antioxidants, late embryogenesis abundant (LEA) proteins, and heat shock proteins (Hsps) were identified as important groups for anhydrobiosis. Genes related to trehalose metabolism and various transporters were also strongly induced by desiccation. Those results suggest that the oxidative stress response plays a central role in successful anhydrobiosis. Similarly, protein denaturation and aggregation may be prevented by marked up-regulation of Hsps and the anhydrobiosis-specific LEA proteins. A third major feature is the predicted increase in trehalose synthesis and in the expression of various transporter proteins allowing the distribution of trehalose and other solutes to all tissues.

Anhydrobiosis-Associated Nuclear DNA Damage and Repair in the Sleeping Chironomid: Linkage with Radioresistance

Anhydrobiotic chironomid larvae can withstand prolonged complete desiccation as well as other external stresses including ionizing radiation. To understand the cross-tolerance mechanism, we have analyzed the structural changes in the nuclear DNA using transmission electron microscopy and DNA comet assays in relation to anhydrobiosis and radiation. We found that dehydration causes alterations in chromatin structure and a severe fragmentation of nuclear DNA in the cells of the larvae despite successful anhydrobiosis. Furthermore, while the larvae had restored physiological activity within an hour following rehydration, nuclear DNA restoration typically took 72 to 96 h. The DNA fragmentation level and the recovery of DNA integrity in the rehydrated larvae after anhydrobiosis were similar to those of hydrated larvae irradiated with 70 Gy of high-linear energy transfer (LET) ions (4He). In contrast, low-LET radiation (gamma-rays) of the same dose caused less initial damage to the larvae, and DNA was completely repaired within within 24 h. The expression of genes encoding the DNA repair enzymes occurred upon entering anhydrobiosis and exposure to high- and low-LET radiations, indicative of DNA damage that includes double-strand breaks and their subsequent repair. The expression of antioxidant enzymes-coding genes was also elevated in the anhydrobiotic and the gamma-ray-irradiated larvae that probably functions to reduce the negative effect of reactive oxygen species upon exposure to these stresses. Indeed the mature antioxidant proteins accumulated in the dry larvae and the total activity of antioxidants increased by a 3–4 fold in association with anhydrobiosis. We conclude that one of the factors explaining the relationship between radioresistance and the ability to undergo anhydrobiosis in the sleeping chironomid could be an adaptation to desiccation-inflicted nuclear DNA damage. There were also similarities in the molecular response of the larvae to damage caused by desiccation and ionizing radiation.

Comparative genome sequencing reveals genomic signature of extreme desiccation tolerance in the anhydrobiotic midge

Anhydrobiosis represents an extreme example of tolerance adaptation to water loss, where an organism can survive in an ametabolic state until water returns. Here we report the first comparative analysis examining the genomic background of extreme desiccation tolerance, which is exclusively found in larvae of the only anhydrobiotic insect, Polypedilum vanderplanki. We compare the genomes of P. vanderplanki and a congeneric desiccation-sensitive midge P. nubifer. We determine that the genome of the anhydrobiotic species specifically contains clusters of multi-copy genes with products that act as molecular shields. In addition, the genome possesses several groups of genes with high similarity to known protective proteins. However, these genes are located in distinct paralogous clusters in the genome apart from the classical orthologues of the corresponding genes shared by both chironomids and other insects. The transcripts of these clustered paralogues contribute to a large majority of the mRNA pool in the desiccating larvae and most likely define successful anhydrobiosis. Comparison of expression patterns of orthologues between two chironomid species provides evidence for the existence of desiccation-specific gene expression systems in P. vanderplanki.

Roles of mitochondria-generated reactive oxygen species on X-ray-induced apoptosis in a human hepatocellular carcinoma cell line, HLE

Abstract HLE, a human hepatocellular carcinoma cell line was transiently transfected with normal human MnSOD and MnSOD without a mitochondrial targeting signal (MTS). Mitochondrial reactive oxygen species (ROS), lipid peroxidation and apoptosis were examined as a function of time following 18.8 Gy X-ray irradiation. Our results showed that the level of mitochondrial ROS increased and reached a maximum level 2 hours after X-ray irradiation. Authentic MnSOD, but not MnSOD lacking MTS, protected against mitochondrial ROS, lipid peroxidation and apoptosis. In addition, the levels of mitochondrial ROS were consistently found to always correlate with the levels of authentic MnSOD in mitochondria. These results suggest that only when MnSOD is located in mitochondria is it efficient in protecting against cellular injuries by X-ray irradiation and that mitochondria are the critical sites of X-ray-induced cellular oxidative injuries.

Gene Expression Profiling of H9c2 Myoblast Differentiation towards a Cardiac-Like Phenotype

H9c2 myoblasts are a cell model used as an alternative for cardiomyocytes. H9c2 cells have the ability to differentiate towards a cardiac phenotype when the media serum is reduced in the presence of all-trans-retinoic acid (RA), creating multinucleated cells with low proliferative capacity. In the present study, we performed for the first time a transcriptional analysis of the H9c2 cell line in two differentiation states, i.e. embryonic cells and differentiated cardiac-like cells. The results show that RA-induced H9c2 differentiation increased the expression of genes encoding for cardiac sarcomeric proteins such as troponin T, or calcium transporters and associated machinery, including SERCA2, ryanodine receptor and phospholamban as well as genes associated with mitochondrial energy production including respiratory chain complexes subunits, mitochondrial creatine kinase, carnitine palmitoyltransferase I and uncoupling proteins. Undifferentiated myoblasts showed increased gene expression of pro-survival proteins such as Bcl-2 as well as cell cycle-regulating proteins. The results indicate that the differentiation of H9c2 cells lead to an increase of transcripts and protein levels involved in calcium handling, glycolytic and mitochondrial metabolism, confirming that H9c2 cell differentiation induced by RA towards a more cardiac-like phenotype involves remodeled mitochondrial function. PI3K, PDK1 and p-CREB also appear to be involved on H9c2 differentiation. Furthermore, complex analysis of differently expressed transcripts revealed significant up-regulation of gene expression related to cardiac muscle contraction, dilated cardiomyopathy and other pathways specific for the cardiac tissue. Metabolic and gene expression remodeling impacts cell responses to different stimuli and determine how these cells are used for biochemical assays.

Genome-wide expression analysis of reactive oxygen species gene network in Mizuna plants grown in long-term spaceflight

BackgroundSpaceflight environment have been shown to generate reactive oxygen species (ROS) and induce oxidative stress in plants, but little is known about the gene expression of the ROS gene network in plants grown in long-term spaceflight. The molecular response and adaptation to the spaceflight environment of Mizuna plants harvested after 27 days of cultivation onboard the International Space Station (ISS) were measured using genome-wide mRNA expression analysis (mRNA-Seq).ResultsTotal reads of transcripts from the Mizuna grown in the ISS as well as on the ground by mRNA-Seq showed 8,258 and 14,170 transcripts up-regulated and down-regulated, respectively, in the space-grown Mizuna when compared with those from the ground-grown Mizuna. A total of 20 in 32 ROS oxidative marker genes were up-regulated, including high expression of four hallmarks, and preferentially expressed genes associated with ROS-scavenging including thioredoxin, glutaredoxin, and alternative oxidase genes. In the transcription factors of the ROS gene network, MEKK1-MKK4-MPK3, OXI1-MKK4-MPK3, and OXI1-MPK3 of MAP cascades, induction of WRKY22 by MEKK1-MKK4-MPK3 cascade, induction of WRKY25 and repression of Zat7 by Zat12 were suggested. RbohD and RbohF genes were up-regulated preferentially in NADPH oxidase genes, which produce ROS.ConclusionsThis large-scale transcriptome analysis revealed that the spaceflight environment induced oxidative stress and the ROS gene network activation in the space-grown Mizuna. Among transcripts altered in expression by space conditions, some were common genes response to abiotic and biotic stress. Furthermore, certain genes were exclusively up-regulated in Mizuna grown on the ISS. Surprisingly, Mizuna grew in space normally, as well as on the ground, demonstrating that plants can acclimate to long-term exposure in the spaceflight environment by reprogramming the expression of the ROS gene network.

Diversity of the expression profiles of late embryogenesis abundant (LEA) protein encoding genes in the anhydrobiotic midge Polypedilum vanderplanki

AbstractMain conclusionIn the anhydrobiotic midgePolypedilum vanderplanki, LEA family proteins are likely to play distinct temporal and spatial roles in the larvae throughout the process of desiccation and rehydration. The larvae of the anhydrobiotic midge, P. vanderplanki, which can tolerate almost complete desiccation, accumulate late embryogenesis abundant (LEA) proteins in response to drying. Using complete genome data of the midge, we have identified 27 PvLea1-like genes based on the similarity to previously characterized PvLea1 gene belonging to group 3 LEA proteins. Generally, group 3 LEA proteins are characterized by several repetitions of an 11-mer motif. However, some PvLea genes lack the canonical motif in their sequences. We performed the detailed characterization of all 27 PvLea genes in terms of biochemical and biophysical properties and conserved motifs. The motif analysis among their amino acid sequences revealed that all 27 PvLEA proteins have at least one of two types of motifs (motif 1: G AKDTTKEKLGE AKDATAEKLG or motif 2: KD ILExAKDKLxD AKDAVKEKL), indicating the presence of at least two repeated 11-mer LEA motifs. Most of PvLEA proteins were localized to the cytosol. We also performed quantitative real-time PCR of all 27 PvLea genes in detail during the process of desiccation and rehydration. The expression of these genes was upregulated at the beginning of dehydration, the latter phase of the desiccation process and on rehydration process. These data suggested that each LEA protein is likely to play distinct temporal and spatial roles in the larvae throughout the process of desiccation and rehydration.

MnSOD downregulation induced by extremely low 0.1 mGy single and fractionated X-rays and microgravity treatment in human neuroblastoma cell line, NB-1

A human neuroblastoma cell line, NB-1, was treated with 24 h of microgravity simulation by clinostat, or irradiated with extremely small X-ray doses of 0.1 or 1.0 mGy using single and 10 times fractionation regimes with 1 and 2 h time-intervals. A quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) examination was performed for apoptosis related factors (BAX, CYTC, APAF1, VDAC1–3, CASP3, CASP8, CASP9 P53, AIF, ANT1 and 2, BCL2, MnSOD, autophagy related BECN and necrosis related CYP-40. The qRT-PCR results revealed that microgravity did not result in significant changes except for a upregulation of proapoptotic VDAC2, and downregulations of proapoptotic CASP9 and antiapoptotic MnSOD. After 0.1 mGy fractionation irradiation, there was increased expression of proapoptotic APAF1 and downregulation of proapoptotic CYTC, VDAC2, VDAC3, CASP8, AIF, ANT1, and ANT2, as well as an increase in expression of antiapoptotic BCL2. There was also a decrease in MnSOD expression with 0.1 mGy fractionation irradiation. These results suggest that microgravity and low-dose radiation may decrease apoptosis but may potentially increase oxidative stress.

Study of the effects of the outer space environment on dormant forms of microorganisms, fungi and plants in the 'Expose-R' experiment

Investigations of the effects of solar radiation combined with the spaceflight factors on biological objects were performed in the «EXPOSE-R» experimenton the outersurface ofISS. After more than 1 year of outerspaceexposure,thesporesofmicroorganismsandfungi,aswellastwospeciesofplantseedswereanalysed for viability and the set of biological properties. The experiment provided evidence that not only bacterial and fungalsporesbutalsodormantformsofplantshadthecapabilitytosurvivealong-termexposuretoouterspace. Received 11 August 2014, accepted 6 November 2014