Proteomic analysis of 4-hydroxy-2-nonenal-modified proteins in G93A-SOD1 transgenic mice--a model of familial amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is an age-related, fatal motor neuron degenerative disease occurring both sporadically (sALS) and heritably (fALS), with inherited cases accounting for approximately 10% of diagnoses. Although multiple mechanisms likely contribute to the pathogenesis of motor neuron injury in ALS, recent advances suggest that oxidative stress may play a significant role in the amplification, and possibly the initiation, of the disease. Lipid peroxidation is one of the several outcomes of oxidative stress. Since the central nervous system (CNS) is enriched with polyunsaturated fatty acids, it is particularly vulnerable to membrane-associated oxidative stress. Peroxidation of cellular membrane lipids or circulating lipoprotein molecules generates highly reactive aldehydes, among which is 4-hydroxy-2-nonenal (HNE). HNE levels are increased in spinal cord motor neurons of ALS patients, indicating that lipid peroxidation is associated with the motor neuron degeneration in ALS. In the present study, we used a parallel proteomic approach to identify HNE-modified proteins in the spinal cord tissue of a model of fALS, G93A-SOD1 transgenic mice, in comparison to the nontransgenic mice. We found three significantly HNE-modified proteins in the spinal cord of G93A-SOD1 transgenic mice: dihydropyrimidinase-related protein 2 (DRP-2), heat-shock protein 70 (Hsp70), and possibly alpha-enolase. These results support the role of oxidative stress as a major mechanism in the pathogenesis of ALS. Structural alteration and activity decline of functional proteins may consistently contribute to the neurodegeneration process in ALS.     

Reactive oxygen species in choline deficiency induced carcinogenesis and nitrone inhibition

Reactive oxygen species and free radical processes have been considered important in cancer development for many years. Much research demonstrates that the choline-deficiency induced hepatocarcinogenesis model prominently involves reactive oxygen species. We present a summary of results obtained in our original studies of this model over the last 4 years. We have shown that -phenyl-tert-butyl nitrone (PBN) and some of its hydroxylated derivatives (the 4- and 3-hydroxylated compounds) prevent hepatocarcinogenesis in this model. Mechanistic studies have demonstrated that isolated mitochondria from the livers of rats fed the choline-deficiency defined amino acid diet produce significantly much more H₂O₂ per NADH reducing equivalents oxidized. Based on these observations, we postulate that H₂O₂ is a primary carcinogenic factor in this model. Based on studies of the action of PBN on isolated mitochondria, we postulate that the inhibiting action of PBN involves suppression of H₂O₂ production of mitochondria and generally decreasing the oxidative stress within the preneoplastic lesions. The net effect of the activity of the nitrone compounds appears to be due to their ability to shift the apoptosis/neoplastic tendency balance toward apoptosis of the cells within the preneoplastic lesions. This is considered to be the primary reason the size of the preneoplastic lesions are significantly decreased and why the nitrones are potent anti-carcinogenic agents in this model.     

Reactive oxygen species and protein oxidation in aging: a look back, a look ahead.

The existence of free radicals, as chemical entities, was inferred 100 years ago but not universally accepted for some 30-40 years. The existence and importance of free radicals in biological systems was not recognized until the mid 1950s, by a small number of visionary scientists who can be credited with founding the field of reactive oxygen biochemistry. For most of the remaining 20th century, reactive oxygen species (ROS) were considered a type of biochemical "rusting agent" that caused stochastic tissue damage and disease. As we enter the 21st century, reactive oxygen biochemistry is maturing as a discipline and establishing its importance among the biomedical sciences. It is now recognized that virtually every disease state involves some degree of oxidative stress. Moreover, we are now beginning to recognize that ROS are produced in a well-regulated manner to help maintain homeostasis on the cellular level in normal, healthy tissue. This review summarizes the history of reactive oxygen biochemistry, outlining major paradigm shifts that the field has undergone and continues to experience. The contributions of Earl Stadtman to the recent history of the field (1980-present) are especially highlighted. The role of ROS in signal transduction is presented in some detail as central to the latest paradigm shift. Emerging technologies, particularly proteomic technologies, are discussed that will facilitate further evolution in the field of reactive oxygen biochemistry.     

Buccal cells as a source of DNA for comparative animal genomic analysis

The source of DNA of adequate quality and quantity is an important consideration in genome analysis. In many animal and livestock species, easy access to DNA will facilitate the rapid and reliable genotyping of a large number of individual individuals. Here, we describe the use, for the first time, of buccal cells from non-human mammalian species as a source of DNA template for PCR and restriction analysis. The buccal cells from the pig, cow and human, were used to amplify PCR fragments that were scanned SNPs and for comparative genome analysis. The work indicates that buccal cells are also adequate sources of DNA for genome analysis of animals that have been identified as priorities in comparative genomics.     

Ebola and Marburg viruses: pathogenesis and development of countermeasures

Ebola and Marburg viruses, family Filoviridae, are among the best known examples of emerging and re-emerging pathogens. Although outbreaks have been sporadic and geographically restricted to areas of Central Africa, the hemorrhagic fevers caused by these viruses are remarkably severe and are associated with high case fatality rates often exceeding 80 percent. In addition to humans, these viruses have decimated populations of wild apes in Central Africa. Currently, there are no vaccines or effective therapies available for human use. Progress in understanding the geneses of the pathophysiological changes that make filoviral infections of humans so destructive has been slow, primarily because these viruses require special containment for safe research. However, an increasing understanding of the molecular mechanisms of filoviral pathogenesis, facilitated by the development of new tools to elucidate critical regulatory elements in the viral life cycle, is providing new targets that can be exploited for therapeutic interventions. In addition, substantial progress has been made in developing recombinant vaccines against these viruses.     

Induction of Akt phosphorylation in rat primary astrocytes by H2O2 occurs upstream of phosphatidylinositol 3-kinase: no evidence for oxidative inhibition of PTEN

Phosphorylation of the serine/threonine kinase Akt has previously been shown to be increased by treatment of cells with H2O2; the target of H2O2 has not been clearly identified. Here we show that treatment of rat primary astrocytes with H2O2 resulted in increased Akt phosphorylation that was blocked by wortmannin. The thiol-reducing agent N-acetylcysteine had only a slight inhibitory effect. Treatment with rotenone or antimycin A also resulted in increased wortmannin-sensitive Akt phosphorylation, probably by increasing intracellular H2O2 generation by blocking mitochondrial electron transport. Addition of phosphatidylinositol 3,4-bisphosphate to cells also resulted in an increase in Akt phosphorylation. This increase was additive to that induced by H2O2 and was also blocked by wortmannin. These results suggest that activation of Akt by H2O2 occurs upstream of phosphatidylinositol 3-kinase (PI 3-K) activity in astrocytes. The data indicate that major oxidative effects do not occur at the level of the PI 3-K-antagonizing phosphatase PTEN.     

Measurement of 3-nitrotyrosine and 5-nitro-gamma-tocopherol by high-performance liquid chromatography with electrochemical detection

Nitric oxide (NO) is a lipophilic gaseous molecule synthesized by the enzymatic oxidation of L-arginine. During periods of inflammation, phagocytic cells generate copious quantities of NO and other reactive oxygen species. The combination of NO with other reactive oxygen species promotes nitration of ambient biomolecules, including protein tyrosine residues and membrane-localized gamma-tocopherol. The oxidative chemistry of NO and derived redox congeners is reviewed. Techniques are described for the determination of 3-nitro-tyrosine and 5-nitro-gamma-tocopherol in biological samples using high-performance liquid chromatography with electrochemical detection.     

River network saturation concept: factors influencing the balance of biogeochemical supply and demand of river networks

River networks modify material transfer from land to ocean. Understanding the factors regulating this function for different gaseous, dissolved, and particulate constituents is critical to quantify the local and global effects of climate and land use change. We propose the River Network Saturation (RNS) concept as a generalization of how river network regulation of material fluxes declines with increasing flows due to imbalances between supply and demand at network scales. River networks have a tendency to become saturated (supply???demand) under higher flow conditions because supplies increase faster than sink processes. However, the flow thresholds under which saturation occurs depends on a variety of factors, including the inherent process rate for a given constituent and the abundance of lentic waters such as lakes, ponds, reservoirs, and fluvial wetlands within the river network. As supply increases, saturation at network scales is initially limited by previously unmet demand in downstream aquatic ecosystems. The RNS concept describes a general tendency of river network function that can be used to compare the fate of different constituents among river networks. New approaches using nested in situ high-frequency sensors and spatially extensive synoptic techniques offer the potential to test the RNS concept in different settings. Better understanding of when and where river networks saturate for different constituents will allow for the extrapolation of aquatic function to broader spatial scales and therefore provide information on the influence of river function on continental element cycles and help identify policy priorities.     

Multiple-step,one-pot synthesis of 2-substituted-3-phosphono-1-thia-4-aza-2-cyclohexene-5-carboxylates and their corresponding ethyl esters

The multiple-step, one-pot procedure for a series of 2-substituted-3-phosphono-1-thia-4-aza-2-cyclohexene-5-carboxylates, analogues of the natural, sulfur amino acid metabolite lanthionine ketimine (LK), its 5-ethyl ester (LKE) and 2-substituted LKEs is described. Initiating the synthesis with the Michaelis-Arbuzov preparation of α-ketophosphonates allows for a wide range of functional variation at the 2-position of the products. Nine new compounds were synthesized with overall yields range from 40 to 62%. In addition, the newly prepared 2-isopropyl-LK-P, 2-n-hexyl-LKE-P and 2-ethyl-LKE were shown to stimulate autophagy in cultured cells better than that of the parent compound, LKE.