Synthesis and Antiviral Activity of 5′-O-Sulfamoyluridine Derivatives

Abstract

A series of 5′-O-[[[[(alkyl)oxy]carbonyl] amino] sulfonyl] uridines have been synthesized by reaction of cyclohexanol, palmityl alcohol, 1,2-di-O-benzoylpropanetriol and 2,3,4,6-tetra-O-benzoyl-L-glucopyranose with chlorosulfonyl isocyanate and 2,3′-O-isopropylidene-uridine. Another series of 5′-O-(N-ethyl and N-isopropylsulfamoyl) uridines have been prepared by reaction of 2′,3′-O-isopropylidene and 2′,3′-di-O-acetyluridine with N-ethylsulfamoyl and N-isopropylsulfamoyl chlorides. All compounds were tested against HSV-2, VV, SV and ASFV viruses. 2′,3′-Di-O-acetyl-5′-O-(N-ethyl and N-isopropylsulfamoyl) uridine showed significant activities against HSV-2. 5′-O-[[[[(2,3,4,6-Tetra-O-benzoyl-β-L-glucopyranosyl)oxy]carbonyl]amino] sulfonyl]-2′,3′-O-isopropylideneuridine was very active against ASFV.     

Chronic Nicotine Treatment Impacts the Regulation of Opioid and Non-opioid Peptides in the Rat Dorsal Striatum

The chronic use of nicotine, the main psychoactive ingredient of tobacco smoking, alters diverse physiological processes and consequently generates physical dependence. To understand the impact of chronic nicotine on neuropeptides, which are potential molecules associated with dependence, we conducted qualitative and quantitative neuropeptidomics on the rat dorsal striatum, an important brain region implicated in the preoccupation/craving phase of drug dependence. We used extensive LC-FT-MS/MS analyses for neuropeptide identification and LC-FT-MS in conjunction with stable isotope addition for relative quantification. The treatment with chronic nicotine for 3 months led to moderate changes in the levels of endogenous dorsal striatum peptides. Five enkephalin opioid peptides were up-regulated, although no change was observed for dynorphin peptides. Specially, nicotine altered levels of nine non-opioid peptides derived from precursors, including somatostatin and cerebellin, which potentially modulate neurotransmitter release and energy metabolism. This broad but selective impact on the multiple peptidergic systems suggests that apart from the opioid peptides, several other peptidergic systems are involved in the preoccupation/craving phase of drug dependence. Our finding permits future evaluation of the neurochemical circuits modulated by chronic nicotine exposure and provides a number of novel molecules that could serve as potential therapeutic targets for treating drug dependence.Nicotine is the main psychoactive ingredient of tobacco (1). By acting on the nicotinic acetylcholine receptors located in diverse brain areas, nicotine generates psychoactive effects such as euphoria, reduced stress, increased energy, and enhanced cognitive functions (2). Chronic nicotine use alters various aspects of neurochemical transmission and has a strong impact on diverse physiological processes (2), resulting in drug-seeking and drug-taking behaviors for normal smokers and for a considerable number of patients suffering from schizophrenia and Alzheimer disease, who use nicotine for self-medication (3, 4). The dorsal striatum (DS)¹ is one of the key brain regions that has been associated with neural regulation during chronic nicotine exposure (5). In particular, the DS is involved in habit formation during the preoccupation/craving (later) phase of nicotine dependence characterized by compulsive drug-taking (6). Behavioral changes associated with nicotine dependence have been linked to small molecule neurotransmitter systems, including the glutamate and dopamine system in the DS (7). The DS is also known to contain diverse neuropeptides, many of which are probably critical mediators of physiological processes that are associated with nicotine, such as the regulation of reinforcement and energy metabolism. However, neuropeptides have not been extensively investigated in the DS during long periods of nicotine administration.Immunoassay studies have shown that neuropeptides, including substance P, neuropeptide Y, and opioid peptides, including the enkephalins, are expressed by inhibitory neurons (8), which make up a large majority of the neurons in the DS (9). Many of these inhibitory GABAergic neurons express nicotinic cholinergic receptors (10), suggesting that nicotine administration may regulate their activity, leading to variations in the release of neuropeptides, as well as the inhibitory neurotransmitter GABA. Previous investigations of peptide regulation during chronic nicotine administration in the striatum have exclusively focused on the class of opioid peptides, which are thought to play an important role in the control of diverse physiological processes, including reward processing, nociception, and regulation of emotions (11, 12). Available studies have focused on the analysis of three opioid peptides, their precursors, or receptors as follows: met-enkephalin, dynorphin, and β-endorphin, using conventional techniques like immunoassays (13, 14). There is considerable variability in reported changes of peptide levels in the striatum during chronic nicotine administration. For example, when animals are treated with 1 mg/kg free base nicotine (daily for 14 days), met-enkephalin increased in the striatum (15). By contrast, met-enkephalin is reduced in the striatum when rats are treated with 0.3 mg/kg nicotine (three times/day for 14 days) (16). A number of factors might contribute to this observed variability, including the exact dosing, daily frequency, time span of administration, and delivery method of nicotine. Furthermore, as individual studies have each so far generally examined a single opioid peptide, there is currently little reliable information about peptide co-regulation, even for these well studied opioid peptides. In addition to these opioid peptides, the DS expresses peptides from other peptide families, which are also potential targets under the regulation of chronic nicotine treatment. So far, however, there is no information available about changes of these non-opioid peptides during chronic nicotine administration.In this study, our aim was to use a neuropeptidomics approach (17) to provide a comprehensive characterization of dorsal striatal neuropeptides after long term nicotine chronic treatment in adult rats using oral administration. The main advantage of this approach is that it allows the simultaneous monitoring of many peptides from the same brain tissue derived from a single drug protocol. We used a combination of a robust sample preparation method (18), high accuracy LC-MS analysis (19, 20), and the use of multiple synthetic internal standards (21) to compare peptide levels in the DS between chronic nicotine and control animals. Our peptidome analysis determined 14 peptides exhibiting significant changes following chronic nicotine administration. Among these peptides were members of the opioid family that had previously been associated with nicotine dependence, as well as a number of newly identified peptides, including members of the secretogranin, cholecystokinin, and somatostatin families. This greatly expands the present scope of peptide involvement in drug dependence in the dorsal striatum.     

Peroxisomes contribute to the acylcarnitine production when the carnitine shuttle is deficient

Fatty acid β-oxidation may occur in both mitochondria and peroxisomes. While peroxisomes oxidize specific carboxylic acids such as very long-chain fatty acids, branched-chain fatty acids, bile acids, and fatty dicarboxylic acids, mitochondria oxidize long-, medium-, and short-chain fatty acids. Oxidation of long-chain substrates requires the carnitine shuttle for mitochondrial access but medium-chain fatty acid oxidation is generally considered carnitine-independent. Using control and carnitine palmitoyltransferase 2 (CPT2)- and carnitine/acylcarnitine translocase (CACT)-deficient human fibroblasts, we investigated the oxidation of lauric acid (C12:0). Measurement of the acylcarnitine profile in the extracellular medium revealed significantly elevated levels of extracellular C10- and C12-carnitine in CPT2- and CACT-deficient fibroblasts. The accumulation of C12-carnitine indicates that lauric acid also uses the carnitine shuttle to access mitochondria. Moreover, the accumulation of extracellular C10-carnitine in CPT2- and CACT-deficient cells suggests an extramitochondrial pathway for the oxidation of lauric acid. Indeed, in the absence of peroxisomes C10-carnitine is not produced, proving that this intermediate is a product of peroxisomal β-oxidation. In conclusion, when the carnitine shuttle is impaired lauric acid is partly oxidized in peroxisomes. This peroxisomal oxidation could be a compensatory mechanism to metabolize straight medium- and long-chain fatty acids, especially in cases of mitochondrial fatty acid β-oxidation deficiency or overload.     

Temporal dynamics of plant community regeneration sources during tallgrass prairie restoration

Ecological restoration aims to augment and steer the composition and contribution of propagules for community regeneration in degraded environments. We quantified patterns in the abundance, richness, and diversity of seed and bud banks across an 11-year chronosequence of restored prairies and in prairie remnants to elucidate the degree to which the germinable seed bank, emerged seedlings, belowground buds, and emerged ramets were related to community regeneration. There were no directional patterns in the abundance, richness, or diversity of the germinable seed bank across the chronosequence. Emerged seedling abundance of sown species decreased during restoration. Richness and diversity of all emerged seedlings and non-sown emerged seedling species decreased across the chronosequence. Conversely, abundance and richness of belowground buds increased with restoration age and belowground bud diversity of sown species increased across the chronosequence. Numbers of emerged ramets also increased across the chronosequence and was driven primarily by the number of graminoid ramets. There were no temporal changes in abundance and richness of sown and non-sown emerged ramets, but diversity of sown emerged ramets increased across the chronosequence. This study demonstrates that after initial seeding, plant community structure in restored prairies increasingly reflects the composition of the bud bank.     

Anammox bacterial populations in deep marine hypersaline gradient systems

To extend the knowledge of anaerobic ammonium oxidation (anammox) habitats, bacterial communities were examined in two hypersaline sulphidic basins in Eastern Mediterranean Sea. The 2 m thick seawater–brine haloclines of the deep anoxic hypersaline basins Bannock and L’Atalante were sampled in intervals of 10 cm with increasing salinity. ¹⁵N isotope pairing incubation experiments showed the production of ²⁹N₂ and ³⁰N₂ gases in the chemoclines, ranging from 6.0 to 9.2 % salinity of the L’Atalante basin. Potential anammox rates ranged from 2.52 to 49.65 nmol N₂ L^?1 day^?1 while denitrification was a major N₂ production pathway, accounting for more than 85.5 % of total N₂ production. Anammox-related 16S rRNA genes were detected along the L’Atalante and Bannock haloclines up to 24 % salinity, and the amplification of the hydrazine synthase genes (hzsA) further confirmed the presence of anammox bacteria in Bannock. Fluorescence in situ hybridisation and sequence analysis of 16S rRNA genes identified representatives of the marine anammox genus ‘Candidatus Scalindua’ and putatively new operational taxonomic units closely affiliated to sequences retrieved in marine environments that have documented anammox activity. ‘Scalindua brodae’ like sequences constituted up to 84.4 % of the sequences retrieved from Bannock. The anammox community in L’Atalante was different than in Bannock and was stratified according to salinity increase. This study putatively extends anammox bacterial habitats to extremely saline sulphidic ecosystems.     

Isolation and characterisation of human pulmonary microvascular endothelial cells from patients with severe emphysema

Background

Loss of the pulmonary microvasculature in the pathogenesis of emphysema has been put forward as a credible alternative to the classical inflammatory cell driven proteolysis hypothesis. Mechanistic studies in this area have to date employed animal models, immortalised cell lines, primary endothelial cells isolated from large pulmonary arteries and non-pulmonary tissues and normal human pulmonary microvascular endothelial cells. Although these studies have increased our understanding of endothelial cell function, their relevance to mechanisms in emphysema is questionable. Here we report a successful technique to isolate and characterise primary cultures of pulmonary microvascular endothelial cells from individuals with severe emphysema.

Methods

A lobe of emphysematous lung tissue removed at the time of lung transplantation surgery was obtained from 14 patients with severe end-stage disease. The pleura, large airways and large blood vessels were excised and contaminating macrophages and neutrophils flushed from the peripheral lung tissue before digestion with collagenase. Endothelial cells were purified from the cell mixture via selection with CD31 and UEA-1 magnetic beads and characterised by confocal microscopy and flow cytometry.

Results

Successful isolation was achieved from 10 (71%) of 14 emphysematous lungs. Endothelial cells exhibited a classical cobblestone morphology with high expression of endothelial cell markers (CD31) and low expression of mesenchymal markers (CD90, αSMA and fibronectin). E-selectin (CD62E) was inducible in a proportion of the endothelial cells following stimulation with TNFα, confirming that these cells were of microvascular origin.

Conclusions

Emphysematous lungs removed at the time of transplantation can yield large numbers of pulmonary microvasculature endothelial cells of high purity. These cells provide a valuable research tool to investigate cellular mechanisms in the pulmonary microvasculature relevant to the pathogenesis of emphysema.