The Evolutionary History of the Genus Acanthamoeba and the Identification of Eight New 18S rRNA Gene Sequence Types

ABSTRACT The 18S rRNA gene ( Rns ) phylogeny of Acanthamoeba is being investigated as a basis for improvements in the nomenclature and taxonomy of the genus. We previously analyzed Rns sequences from 18 isolates from morphological groups 2 and 3 and found that they fell into four distinct evolutionary lineages we called sequence types T1-T4. Here, we analyzed sequences from 53 isolates representing 16 species and including 35 new strains. Eight additional lineages (sequence types T5-T12) were identified. Four of the 12 sequence types included strains from more than one nominal species. Thus, sequence types could be equated with species in some cases or with complexes of closely related species in others. The largest complex, sequence type T4, which contained six closely related nominal species, included 24 of 25 keratitis isolates. Rns sequence variation was insufficient for full phylogenetic resolution of branching orders within this complex, but the mixing of species observed at terminal nodes confirmed that traditional classification of isolates has been inconsistent. One solution to this problem would be to equate sequence types and single species. Alternatively, additional molecular information will be required to reliably differentiate species within the complexes. Three sequence types of morphological group 1 species represented the earliest divergence in the history of the genus and, based on their genetic distinctiveness, are candidates for reclassification as one or more novel genera.     

Nitration of the tyrosyl radical in ribonucleotide reductase by nitrogen dioxide: a gamma radiolysis study

Nitrogen dioxide is a product of peroxynitrite homolysis and peroxidase-catalyzed oxidation of nitrite. It is of great importance in protein tyrosine nitration because most nitration pathways end with the addition of *NO2 to a one-electron-oxidized tyrosine. The rate constant of this radical addition reaction is high with free tyrosine-derived radicals. However, little is known of tyrosine radicals in proteins. In this paper, we have used *NO2 generated by gamma radiolysis to study the nitration of the R2 subunit of ribonucleotide reductase, which contains a long-lived tyrosyl radical on Tyr122. Most of the nitration occurred on Tyr122, but nonradical tyrosines were also modified. In addition, peptidic bonds close to nitrated Tyr122 could be broken. Nitration at Tyr122 was not observed with a radical-free metR2 protein. The estimated rate constant of the Tyr122 radical reaction with *NO2 was of 3 x 10(4) M(-1) s(-1), thus several orders of magnitude lower than that of a radical on free tyrosine. Nitration rate of other tyrosine residues in R2 was even lower, with an estimated value of 900 M(-1) s(-1). This study shows that protein environment can significantly reduce the reactivity of a tyrosyl radical. In ribonucleotide reductase, the catalytically active radical residue is very efficiently protected against nitrogen oxide attack and subsequent nitration.     

1-Thiazol-2-yl-N-3-methyl-1H-pyrozole-5-carboxylic acid derivatives as antitumor agents

A class of substituted 1-thiazol-2-yl-N-3-methyl-1H-pyrozole-5-carboxylic acid derivatives was found to have potent anti-proliferative activity against a broad range of tumor cell lines. A compound from this class (14) was profiled across a broad panel of hematologic and solid tumor cancer cell lines demonstrating cell cycle arrest at the G0/G1 interphase and has potent anti-proliferative activity against a distinct and select set of cancer cell types with no observed effects on normal human cells. An example is the selective inhibition of human B-cell lymphoma cell line (BJAB). Compound 14 was orally bioavailable and tolerated well in mice. Synthesis and structure activity relationships (SAR) in this series of compounds are discussed.     

Circulating microparticles carry oxidation-specific epitopes and are recognized by natural IgM antibodies

Oxidation-specific epitopes (OSEs) present on apoptotic cells and oxidized low density lipoprotein (OxLDL) represent danger-associated molecular patterns that are recognized by different arcs of innate immunity, including natural IgM antibodies. Here, we investigated whether circulating microparticles (MPs), which are small membrane vesicles released by apoptotic or activated cells, are physiological carriers of OSEs. OSEs on circulating MPs isolated from healthy donors and patients with ST-segment elevation myocardial infarction (STE-MI) were characterized by flow cytometry using a panel of OSE-specific monoclonal antibodies. We found that a subset of MPs carry OSEs on their surface, predominantly malondialdehyde (MDA) epitopes. Consistent with this, a majority of IgM antibodies bound on the surface of circulating MPs were found to have specificity for MDA-modified LDL. Moreover, we show that MPs can stimulate THP-1 (human acute monocytic leukemia cell line) and human primary monocytes to produce interleukin 8, which can be inhibited by a monoclonal IgM with specificity for MDA epitopes. Finally, we show that MDA⁺ MPs are elevated at the culprit lesion site of patients with STE-MI. Our results identify a subset of OSE⁺ MPs that are bound by OxLDL-specific IgM. These findings demonstrate a novel mechanism by which anti-OxLDL IgM antibodies could mediate protective functions in CVD.     

Could the acid–base status of Antarctic sea urchins indicate a better‐than‐expected resilience to near‐future ocean acidification?

Increasing atmospheric carbon dioxide concentration alters the chemistry of the oceans towards more acidic conditions. Polar oceans are particularly affected due to their low temperature, low carbonate content and mixing patterns, for instance upwellings. Calcifying organisms are expected to be highly impacted by the decrease in the oceans' pH and carbonate ions concentration. In particular, sea urchins, members of the phylum Echinodermata, are hypothesized to be at risk due to their high‐magnesium calcite skeleton. However, tolerance to ocean acidification in metazoans is first linked to acid–base regulation capacities of the extracellular fluids. No information on this is available to date for Antarctic echinoderms and inference from temperate and tropical studies needs support. In this study, we investigated the acid–base status of 9 species of sea urchins (3 cidaroids, 2 regular euechinoids and 4 irregular echinoids). It appears that Antarctic regular euechinoids seem equipped with similar acid–base regulation systems as tropical and temperate regular euechinoids but could rely on more passive ion transfer systems, minimizing energy requirements. Cidaroids have an acid–base status similar to that of tropical cidaroids. Therefore Antarctic cidaroids will most probably not be affected by decreasing seawater pH, the pH drop linked to ocean acidification being negligible in comparison of the naturally low pH of the coelomic fluid. Irregular echinoids might not suffer from reduced seawater pH if acidosis of the coelomic fluid pH does not occur but more data on their acid–base regulation are needed. Combining these results with the resilience of Antarctic sea urchin larvae strongly suggests that these organisms might not be the expected victims of ocean acidification. However, data on the impact of other global stressors such as temperature and of the combination of the different stressors needs to be acquired to assess the sensitivity of these organisms to global change.