DESIGN AND SYNTHESIS OF NOVEL SYNTHETIC ANTIOXIDANTS FOR THE TREATMENT OF OXIDATIVE STRESS RELATED DISEASES

Free radicals play an important role in many physiological processes that occur in the human body such as cellular defense responses to infectious agents and a variety of cellular signaling pathways. While at low concentrations free radicals are involved in many significant metabolic reactions, high levels of free radicals can have deleterious effects on biomolecules like proteins, lipids, and DNA. Many physiological disorders such as diabetes, ageing, neurodegenerative diseases, and ischemia-reperfusion (I/R) injury are associated with oxidative stress.1 In particular, the deleterious effects caused by I/R injury developed during organ transplantation, cardiac infarct, and stroke have become the main cause of death in the United States and Europe.1,2 In this context, we synthesized and characterized a series of novel indole-amino acid conjugates as potential antioxidants for I/R injury. The synthesis of indole-phenol conjugate compounds is also discussed. Phenolic derivatives such as caffeic acid, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), resveratrol, and its analogues are known for their significant antioxidative properties. A series of resveratrol analogues have been designed and synthesized as potential antioxidants. The radical scavenging mechanisms for potential antioxidants and assays for the in vitro evaluation of antioxidant activities are also discussed.

Role of acetyl-phosphate in activation of the Rrp2-RpoN-RpoS pathway in Borrelia burgdorferi.

Borrelia burgdorferi, the Lyme disease spirochete, dramatically alters its transcriptome and proteome as it cycles between the arthropod vector and mammalian host. During this enzootic cycle, a novel regulatory network, the Rrp2-RpoN-RpoS pathway (also known as the σ(54)-σ(S) sigma factor cascade), plays a central role in modulating the differential expression of more than 10% of all B. burgdorferi genes, including the major virulence genes ospA and ospC. However, the mechanism(s) by which the upstream activator and response regulator Rrp2 is activated remains unclear. Here, we show that none of the histidine kinases present in the B. burgdorferi genome are required for the activation of Rrp2. Instead, we present biochemical and genetic evidence that supports the hypothesis that activation of the Rrp2-RpoN-RpoS pathway occurs via the small, high-energy, phosphoryl-donor acetyl phosphate (acetyl∼P), the intermediate of the Ack-Pta (acetate kinase-phosphate acetyltransferase) pathway that converts acetate to acetyl-CoA. Supplementation of the growth medium with acetate induced activation of the Rrp2-RpoN-RpoS pathway in a dose-dependent manner. Conversely, the overexpression of Pta virtually abolished acetate-induced activation of this pathway, suggesting that acetate works through acetyl∼P. Overexpression of Pta also greatly inhibited temperature and cell density-induced activation of RpoS and OspC, suggesting that these environmental cues affect the Rrp2-RpoN-RpoS pathway by influencing acetyl∼P. Finally, overexpression of Pta partially reduced infectivity of B. burgdorferi in mice. Taken together, these findings suggest that acetyl∼P is one of the key activating molecule for the activation of the Rrp2-RpoN-RpoS pathway and support the emerging concept that acetyl∼P can serve as a global signal in bacterial pathogenesis.

Elemental and isotope compositions of lipids, kerogens and asphaltenes from samples of DSDP Hole 41-368

The thermal effects of three (one major and two minor) Miocene diabase intrusions on Cretaceous black shales from DSDP site 41-368 have been analyzed. A concentration gradient was observed, especially for the hydrocarbons, decreasing towards the major intrusion and between the three sills. The thermally-altered samples in the proximity of and between the sills contained elemental sulfur and an excess of thermally-derived pristane over phytane. whereas, the unaltered sediments contained no elemental sulfur, and more phytane than pristane. A maximum yield of the extractable hydrocarbons was observed at a depth of 7 m below the major sill. Two classes of molecular markers were present in this bitumen suite. The first was sesqui-, di- and triterpenoids and steranes. which could be correlated with both terrigenous and autochthonous sources. They were geologically mature and showed no significant changes due to the thermal stress. The second class was found in the altered samples, which contained only polynuclear aromatic hydrocarbons with low alkyl substitution and sulfur and oxygen heterocyclic aromatic compounds. These compounds were derived from pyrolytic reactions during the thermal event. Kerogen was isolated from all of these samples, but only traces of humic substances were present. The H/C, N/C, d13C, d34S and dD all exhibit the expected effects of thermal stress. The kerogen becomes more aromatized and richer in 13C, 34S and D in the proximity of and between the sills. Maturation trends were also measured by the vitrinite reflectance and electron spin resonance, where the thermal stress could be correlated with an elevated country rock temperature and an increased degree of aromaticity. The effects of in situ thermal stress on the organic-rich shales resulted in the generation and expulsion of petroliferous material from the vicinity of the sills.

Lipids in ocean particulate matter and bottom sediments

Lipid contents both in particulate matter and bottom sediments decreases with passage from the shelf toward the open ocean. Lipid concentration in particulate matter collected by a separator (Ls) decreases by a factor of 7 (from 7.05 to 0.95 % of dry matter), while in particulate matter collected on filters (Lf) it decreases by a factor of 13 (from 78 to 6 µg/l) in the vicinity of the Limpopo River and by a factor of 6 (from 74 to 13 µg/l) in the vicinity of the Zambezi River. Concentration of Lf also decreased with depth. In the upper sediment layers lipid concentration was 0.0028-0.039% of dry matter; all mud samples were richer in lipids, than sand samples. During sedimentogenesis there is an increase in proportion of lipids relative to other classes of organic matter, proportion of low-polarity compounds increases among the lipids, and proportion of hydrocarbons rises among these compounds. Sediments inherit composition of particulate matter to the greatest degree in the vicinity of river mouths.

Lipid composition of particulate matter measured on water bottle samples during POLARSTERN cruise ARK-XI/1

The lipid composition of particulate matter in oceanic environments can provide informations on the nature and origin of the organic matter as well as on their transformation processes. Molecular characteristics for lipids in the Arctic environment have been used as indicators of the sources and transformation of organic particulate matter (Smith et al., 1997; Fahl and Stein, 1997, 1999). However, the features of the lipid composition of particulate matter in the Arctic with its high seasonality of ice Cover and primary productivity has been studied insufficiently. Lipids are one of the most important compounds of organic matter. On the one hand, the composition of lipids is a result of the variability of biological sources (phyto- and zooplankton, higher plants, bacteria etc.). On the other hand, the lipid composition of particulate matter is undergone significant alteration during vertical transport. The organic matter balance in the Arctic marginal seas, such as the Kara and Laptev seas, is characterized by the significant supply of dissolved and particulate material by the major Eurasian rivers - Ob, Yenisei and Lena (Cauwet and Sidorov, 1996; Gordeev et al., 1996, Martin et al., 1993). In relation to the world's ocean the primary productivity values are lower in the Arctic seas due to the ice-cover. However local increased values of primary productivity can be connected with the melting processes inducing increased phytoplankton growth near ice-edge (Nelson et al., 1989; Fahl and Stein, 1997) and enhanced river supply of nutrients, These features can influence the proportion of allochtonous and autochtonous components of the organic matter in the Arctic marginal seas (Fahl and Stein, 1997; Stein and Fahl, 1999). Furthermore, increased lipid contents in aquatic environments were found near density discontinuities (Parish et al., 1988). Although being less informative than lipid studies on the molecular level the character of lipid composition analysis on the group could also be used for studying of particulate organic matter and its transformation in sedimentation processes in the Arctic. In this paper the investigation of the characteristics of lipid composition performed by Alexandrova and Shevchenko (1997) in Arctic seas was continued.

Composition of particulate lipids in the intermediate zone between the Kara and Laptev Seas

Chemical group composition of particulate lipids from the intermediate zone between the Kara and Laptev Seas is studied by thin-layer chromatography with flame ionization detection (IATROSCAN TH-10). Hydrocarbons and complex polar lipids similar to those found in the previously studied southeastern area of the Kara Sea are basic components of particulate lipids. High content of triglycerides in the upper layers of the water mass north of the Severnaya Zemlya Islands is a characteristic feature of group composition of particulate lipids. Distribution of triglycerides correlates with localization of the ice cover boundary and complies with process of phytoplankton blooming in the ice edge zone. Distribution of lipid concentration depends on water stratification in the intermediate zone between the Kara and Laptev Seas.

Concentrations of particulate organic carbon in the Kara Sea and in the Obskaya Guba (Ob River estuary) in September 1993

Contents and distribution of particulate lipids were studied by thin-layer chromatography technique with flame ionization detection (Iatroscan TH-10) along the transect from the Ob River towards the Kara Sea. Lipid contents range from 18.4 to 266 µg/l with, average 84.97 µg/l, which comprises from 4.06 to 58.32 % of total particulate organic matter. Principal constituents of particulate lipids are hydrocarbons (32.14 % of total lipids on the average), polar compounds (29.85 %), wax and sterol esters (13.04 %), and mono- and diglycerides (12.52 %). Secondary components are presented by fatty acid esters (5.14 %), free fatty acids (4.56 %), triglycerides (2.32 %), and sterols (1.04 %). Specific composition of particulate lipids along the Ob River - Kara Sea transect is formed under strong impact of river run-off. Particulate lipid composition reflects differences between processes of organic matter transformation in estuarine and marine parts of the transect, as well as peculiarities of species composition of Arctic living organisms.

Impacts of groundwater discharge at Myora Springs (North Stradbroke Island, Australia) on the phenolic metabolism of eelgrass, Zostera muelleri, and grazing by the juvenile rabbitfish, Siganus fuscescens

Myora Springs is one of many groundwater discharge sites on North Stradbroke Island (Queensland, Australia). Here spring waters emerge from wetland forests to join Moreton Bay, mixing with seawater over seagrass meadows dominated by eelgrass, Zostera muelleri. We sought to determine how low pH / high CO2 conditions near the spring affect these plants and their interactions with the black rabbitfish (Siganus fuscescens), a co-occurring grazer. In paired-choice feeding trials S. fuscescens preferentially consumed Z. muelleri shoots collected nearest to Myora Springs. Proximity to the spring did not significantly alter the carbon and nitrogen contents of seagrass tissues but did result in the extraordinary loss of soluble phenolics, including Folin-reactive phenolics, condensed tannins, and phenolic acids by ?87%. Conversely, seagrass lignin contents were, in this and related experiments, unaffected or increased, suggesting a shift in secondary metabolism away from the production of soluble, but not insoluble, (poly)phenolics. We suggest that groundwater discharge sites such as Myora Springs, and other sites characterized by low pH, are likely to be popular feeding grounds for seagrass grazers seeking to reduce their exposure to soluble phenolics.

Production of biodiesel from animal fat using supercritical ethanol

Biodiesel is currently produced from a catalytic transesterification reaction of various types of edible and non-edible oil with methanol. The use of waste animal tallow instead of edible oils opens a route to recycle this waste. This material has the advantage of lower costs but the problem of high content of free fatty acids, becoming necessary a pre-esterification reaction that increases the cost of the catalytic process. The production of biodiesel using supercritical alcohols is appropriate for materials with high acidity and water content, therefore the use of this process with animal fat is a promising alternative. Ethanol has been used because it can be produced from biomass via fermentation resulting in a complete renewable biodiesel, instead of methanol that derives from fossil feedstocks. Two different processes have been studied: first, the direct transesterification of animal fat using supercritical ethanol and second a two-step process where the first step is a hydrolysis of the animal fat and the second step is the esterification of the resulting fatty acids. The temperature, the molar ratio ethanol:fat and the time have been modified in the different reactions to study the effect in the final conversion and the degradation of the unsaturated fatty acid esters, main inconvenient of these high temperature and pressure processes.

Localized Changes in Peroxidase Activity Accompany Hydrogen Peroxide Generation during the Development of a Nonhost Hypersensitive Reaction in Lettuce1

Peroxidase activity was characterized in lettuce (Lactuca sativa L.) leaf tissue. Changes in the activity and distribution of the enzyme were examined during the development of a nonhost hypersensitive reaction (HR) induced by Pseudomonas syringae (P. s.) pv phaseolicola and in response to an hrp mutant of the bacterium. Assays of activity in tissue extracts revealed pH optima of 4.5, 6.0, 5.5 to 6.0, and 6.0 to 6.5 for the substrates tetramethylbenzidine, guaiacol, caffeic acid, and chlorogenic acid, respectively. Inoculation with water or with wild-type or hrp mutant strains of P. s. pv phaseolicola caused an initial decline in total peroxidase activity; subsequent increases depended on the hydrogen donor used in the assay. Guaiacol peroxidase recovered more rapidly in tissues undergoing the HR, whereas changes in tetramethylbenzidine peroxidase were generally similar in the two interactions. In contrast, increases in chlorogenic acid peroxidase were significantly higher in tissues inoculated with the hrp mutant. During the HR, increased levels of Mn2+/2,4-dichlorophenol-stimulated NADH and NADPH oxidase activities, characteristic of certain peroxidases, were found in intercellular fluids and closely matched the accumulation of H2O2 in the apoplast. Histochemical analysis of peroxidase distribution by electron microscopy revealed a striking, highly localized increase in activity within the endomembrane system and cell wall at the sites of bacterial attachment. However, no clear differences in peroxidase location were observed in tissue challenged by the wild-type strain or the hrp mutant. Our results highlight the significance of the subcellular control of oxidative reactions leading to the generation of reactive oxygen species, cell wall alterations, and the HR.