Analysis of Intracellular Nucleotides by Capillary Electrophoresis–Mass Spectrometry

A pilot study using capillary electrophoresis with mass spectrometry for the analysis of nucleotides in human erythrocytes is presented. Erythrocytes were incubated with 5-amino-4-imidazolecarboxamide riboside in order to mimic situation in defect of purine metabolism—AICA-ribosiduria. Characteristic AICA-ribotides together with normal nucleotides were separated by capillary electrophoresis in acetate buffer (20 mmol/L, pH 4.4) and identified on line by mass spectrometry.     

Nucleoside Phosphonates. Development of Synthetic Methods and Reagents

Abstract

In this paper a short account of our recent research concerning development of new synthetic methods and new reagents for the preparation of DNA and RNA fragments and their analogues is given.

     

Stereochemistry of Internucleotide Bond Formation by the H-Phosphonate Method. 1. Synthesis and 31P Nmr Analysis of 16 Diribonulceoside (3′-5′)-H-Phosphonates and the Corresponding Phosphorothioates

Sixteen diribonucleoside (3′-5′)-H-phosphonates were synthesized via condensation of the protected ribonucleoside 3′-H-phosphonates with nucleosides, and the influence of a nucleoside sequence on the observed stereoselectivity was analyzed. ₃₁P NMR spectroscopy was used to evaluate a relationship between chemical shift and absolute configuration at the phosphorous center of the H-phosphonate diesters as well as of the corresponding phosphorothioate diesters. Although for the most cases such correlation was found, there was however several exceptions to the rule where the relative positions of resonances arising from R _P and S _P diastereomers were reversed.     

Chlamydia pneumoniae infection acts as an endothelial stressor with the potential to initiate the earliest heat shock protein 60-dependent inflammatory stage of atherosclerosis

We identified increased expression and redistribution of the intracellular protein 60-kDa human heat shock protein (hHSP60) (HSPD1) to the cell surface in human endothelial cells subjected to classical atherosclerosis risk factors and subsequent immunologic cross-reactivity against this highly conserved molecule, as key events occurring early in the process of atherosclerosis. The present study aimed at investigating the role of infectious pathogens as stress factors for vascular endothelial cells and, as such, contributors to early atherosclerotic lesion formation. Using primary donor-matched arterial and venous human endothelial cells, we show that infection with Chlamydia pneumoniae leads to marked upregulation and surface expression of hHSP60 and adhesion molecules. Moreover, we provide evidence for an increased susceptibility of arterial endothelial cells for redistribution of hHSP60 to the cellular membrane in response to C. pneumoniae infection as compared to autologous venous endothelial cells. We also show that oxidative stress has a central role to play in endothelial cell activation in response to chlamydial infection. These data provide evidence for a role of C. pneumoniae as a potent primary endothelial stressor for arterial endothelial cells leading to enrichment of hHSP60 on the cellular membrane and, as such, a potential initiator of atherosclerosis.     

Tidal marsh plant responses to elevated CO2, nitrogen fertilization,and sea level rise

Elevated CO₂ and nitrogen (N) addition directly affect plant productivity and the mechanisms that allow tidal marshes to maintain a constant elevation relative to sea level, but it remains unknown how these global change drivers modify marsh plant response to sea level rise. Here we manipulated factorial combinations of CO₂ concentration (two levels), N availability (two levels) and relative sea level (six levels) using in situ mesocosms containing a tidal marsh community composed of a sedge, Schoenoplectus americanus, and a grass, Spartina patens. Our objective is to determine, if elevated CO₂ and N alter the growth and persistence of these plants in coastal ecosystems facing rising sea levels. After two growing seasons, we found that N addition enhanced plant growth particularly at sea levels where plants were most stressed by flooding (114% stimulation in the + 10 cm treatment), and N effects were generally larger in combination with elevated CO₂ (288% stimulation). N fertilization shifted the optimal productivity of S. patens to a higher sea level, but did not confer S. patens an enhanced ability to tolerate sea level rise. S. americanus responded strongly to N only in the higher sea level treatments that excluded S. patens. Interestingly, addition of N, which has been suggested to accelerate marsh loss, may afford some marsh plants, such as the widespread sedge, S. americanus, the enhanced ability to tolerate inundation. However, if chronic N pollution reduces the availability of propagules of S. americanus or other flood‐tolerant species on the landscape scale, this shift in species dominance could render tidal marshes more susceptible to marsh collapse.     

Predicting shifts in parasite distribution with climate change: a multitrophic level approach

Climate change likely will lead to increasingly favourable environmental conditions for many parasites. However, predictions regarding parasitism's impacts often fail to account for the likely variability in host distribution and how this may alter parasite occurrence. Here, we investigate potential distributional shifts in the meningeal worm, Parelaphostrongylosis tenuis, a protostrongylid nematode commonly found in white‐tailed deer in North America, whose life cycle also involves a free‐living stage and a gastropod intermediate host. We modelled the distribution of the hosts and free‐living larva as a complete assemblage to assess whether a complex trophic system will lead to an overall increase in parasite distribution with climate change, or whether divergent environmental niches may promote an ecological mismatch. Using an ensemble approach to climate modelling under two different carbon emission scenarios, we show that whereas the overall trend is for an increase in niche breadth for each species, mismatches arise in habitat suitability of the free‐living larva vs. the definitive and intermediate hosts. By incorporating these projected mismatches into a combined model, we project a shift in parasite distribution accounting for all steps in the transmission cycle, and identify that overall habitat suitability of the parasite will decline in the Great Plains and southeastern USA, but will increase in the Boreal Forest ecoregion, particularly in Alberta. These results have important implications for wildlife conservation and management due to the known pathogenicity of parelaphostrongylosis to alternate hosts including moose, caribou and elk. Our results suggest that disease risk forecasts which fail to consider biotic interactions may be overly simplistic, and that accounting for each of the parasite's life stages is key to refining predicted responses to climate change.     

Aquatic Ecosystem Responses to Rapid Recovery from Extreme Acidification and Metal Contamination in Lakes Near Wawa, Ontario

In the region northeast of Wawa, Ontario (Canada), many circumneutral lakes downwind of a nearby iron-sintering plant were strongly acidified (pH 3–4) in response to the emissions of large amounts of sulfur dioxide from 1939–1998. Following closure of the plant in 1998, lakewater pH has returned to circumneutral conditions due to the high buffering capacity of the local geological substrate. Prior paleolimnological analyses of dated sediment cores have detected some biological recovery among algal communities (diatoms and chrysophytes), although they have not returned to their pre-impact assemblages. Here we take a broader ecosystem approach, and build upon the algal analyses by examining cladoceran sedimentary assemblages, and spectrally-inferred chlorophyll a and dissolved organic carbon (DOC) from the same dated sediment cores. Similar to the algal communities, recent cladoceran sedimentary assemblages from three impacted lakes remain in an altered state relative to the pre-impact period (for example, increased relative abundances of Chydorus brevilabris and reduced cladoceran density in sediments). However, trends in the spectrally-inferred chlorophyll a and DOC were mixed, with long-term decreases in the study lake closest to the plant and long-term increases within the other lakes. Collectively, the multi-proxy paleolimnological analyses of these markedly acidified lakes demonstrate the delayed biological recovery from acidification (and differences in timing) across multiple trophic levels, despite the near-elimination of acid deposition almost a decade previously, which led to a striking recovery in lakewater pH and increased food availability.