New World Simian Foamy Virus Infections In Vivo and In Vitro

Foamy viruses (FV) are complex retroviruses that naturally infect all nonhuman primates (NHP) studied to date. Zoonotic transmission of Old World NHP simian foamy viruses (SFV) has been documented, leading to nonpathogenic persistent infections. To date, there have been no reports concerning zoonotic transmission of New World monkey (NWM) SFV to humans and resulting infection. In this study, we developed a Western blot assay to detect antibodies to NWM SFV, a nested PCR assay to detect NWM SFV DNA, and a β-galactosidase-containing indicator cell line to assay replication of NWM SFV. Using these tools, we analyzed the plasma and blood of 116 primatologists, of whom 69 had reported exposures to NWM. While 8 of the primatologists tested were seropositive for SFV from a NWM, the spider monkey, none had detectable levels of viral DNA in their blood. We found that SFV isolated from three different species of NWM replicated in some, but not all, human cell lines. From our data, we conclude that while humans exposed to NWM SFV produce antibodies, there is no evidence for long-term viral persistence.     

Genetic structure offers insights into the evolution of migration and the taxonomy of the Barred Long‐tailed Cuckoo Cercococcyx montanus species complex

Barred Long‐tailed Cuckoo (Cercococcyx montanus) currently comprises two morphologically distinct subspecies, one resident in the Albertine Rift (montanus) and one in east and southeast Africa (patulus) in which there are migrations that are poorly understood. Based on nuclear and mitochondrial DNA sequences, we find that two specimens collected in relatively low‐elevation forest in the Albertine Rift were correctly identified from plumage as the migratory subspecies whose closest known breeding area is > 800 km to the east. We discuss ways in which this unique migratory pattern could have evolved and argue that migration was gained and then lost in the C. montanus complex. Based on consistent morphological and genetic differences, we suggest that Barred Long‐tailed Cuckoo is best treated as two species, one of which (C. montanus) is a non‐migratory Albertine Rift endemic.     

A new interpretation of the bee fossil Melitta willardi Cockerell (Hymenoptera,Melittidae) based on geometric morphometrics of the wing

Although bees are one of the major lineages of pollinators and are today quite diverse, few well-preserved fossils are available from which to establish the tempo of their diversification/extinction since the Early Cretaceous. Here we present a reassessment of the taxonomic affinities of Melitta willardi Cockerell 1909, preserved as a compression fossil from the Florissant shales of Colorado, USA. Based on geometric morphometric wing shape analyses M. willardi cannot be confidently assigned to the genus Melitta Kirby (Anthophila, Melittidae). Instead, the species exhibits phenotypic affinity with the subfamily Andreninae (Anthophila, Andrenidae), but does not appear to belong to any of the known genera therein. Accordingly, we describe a new genus, Andrenopteryx gen. n., based on wing shape as well as additional morphological features and to accommodate M. willardi. The new combination Andrenopteryx willardi (Cockerell) is established.     

Potent second generation vinyl sulfonamide inhibitors of the trypanosomal cysteine protease cruzain

A new family of potent N-alkoxyvinylsulfonamide inhibitors of cruzain have been developed. Inhibitor 13 has a second order inactivation rate constant of 6,480,000s(-1)M(-1) versus cruzain, and is also highly effective against Trypanosoma cruzi trypomastigotes in a tissue culture assay.     

A Contiguous Compartment Functions as Endoplasmic Reticulum and Endosome/Lysosome in Giardia lamblia

The dynamic evolution of organelle compartmentalization in eukaryotes and how strictly compartmentalization is maintained are matters of ongoing debate. While the endoplasmic reticulum (ER) is classically envisioned as the site of protein cotranslational translocation, it has recently been proposed to have pluripotent functions. Using transfected reporter constructs, organelle-specific markers, and functional enzyme assays, we now show that in an early-diverging protozoan, Giardia lamblia, endocytosis and subsequent degradation of exogenous proteins occur in the ER or in an adjacent and communicating compartment. The Giardia endomembrane system is simple compared to those of typical eukaryotes. It lacks peroxisomes, a classical Golgi apparatus, and canonical lysosomes. Giardia orthologues of mammalian lysosomal proteases function within an ER-like tubulovesicular compartment, which itself can dynamically communicate with clathrin-containing vacuoles at the periphery of the cell to receive endocytosed proteins. These primitive characteristics support Giardia''s proposed early branching and could serve as a model to study the compartmentalization of endocytic and lysosomal functions into organelles distinct from the ER. This system also may have functional similarity to the retrograde transport of toxins and major histocompatibility complex class I function in the ER of mammals.A key event in the evolution of eukaryotic cells was the compartmentalization of cellular functions into distinct organelles responsible for protein synthesis, sorting, secretion, endocytosis, and degradation (38). However, it is clear from ultrastructural and biochemical analysis of many eukaryotic cells that these functionally distinct compartments often share common aspects of biogenesis and function and, in some cases, a common tubulovesicular network (TVN) (2). For example, one current debate concerns a putative role for the endoplasmic reticulum (ER) in phagocytosis (11, 42). Gagnon et al. (11) proposed that the ER was involved in direct uptake of material from the extracellular environment via fusion with the plasma membrane. This hypothesis was based on the presence of ER markers at the initial stage of phagosome formation in mammalian macrophages. Touret et al. (42), however, found no evidence for direct ER-plasma membrane communication in either macrophages or dendritic cells. Nevertheless, the concept of pluripotent functions for the ER was left unresolved, and these studies underscore the potential for an ER function in phagocytosis or endocytosis, particularly in regard to antigen processing for major histocompatibility complex (MHC) class I presentation. Furthermore, there are intriguing examples of exogenous toxins and viruses entering mammalian cells via the ER (19, 36). Because cellular compartmentalization is a defining eukaryotic trait, clues from early-diverging eukaryotic cells could provide valuable insights into the way in which compartmentalization and discrete organelle functions evolved.Giardia evolutionary status continues to be a matter of debate. Either Giardia lamblia is one of the earliest branches of the eukaryotic tree, with an estimated point of divergence of 1.7 to 2.1 billion years (3, 16, 17, 35, 38), or it is a very simple cell that has lost endomembrane complexity and classic organelle morphology by evolutionary reduction (6). Giardia has a simple life cycle that includes a replicating trophozoite stage and, under certain environmental conditions, an infectious, environmentally resistant cyst. The cyst form allows the replicative trophozoite to persist under conditions of desiccation outside the host and in harsh chemical environments like the host stomach. Following passage through the acidic stomach into the alkaline duodenum, the trophozoite form excysts and resides in the upper small intestine of its vertebrate host, where it opportunistically scavenges nutrients by uncharacterized endocytic pathways.The endomembrane system of the vegetative trophozoite form of Giardia lacks complexity compared to typical eukaryotic cells. In many extant eukaryotic cells, the majority of secretory and organelle-resident proteins are delivered by cotranslational translocation to the ER lumen, prior to maturation and subsequent targeting to the Golgi apparatus (2). In Giardia, no morphological equivalent of the classic mammalian-cell Golgi apparatus has been identified, and the transient secretory pathway for cyst wall synthesis is induced only under specific conditions (13, 14, 34). Giardia does contain two nuclei, a glycogen-rich cytoplasm, acidified peripheral vacuoles (PVs), and a labyrinthine TVN, segments of which are decorated with ribosomes, consistent with rough ER (5, 21, 22). Giardia also contains a multigene family of cysteine endoproteases that are orthologous to the cathepsin L and cathepsin B found in lysosomes of higher organisms and are therefore useful markers of cell compartments where protein degradation takes place (29, 46; Giardia genome project [http://www.giardiadb.org]). Due to the limited availability of established ER/endocytic subcompartment markers and the lack of classical genetic techniques, the discrete endocytic pathway of Giardia has not been fully characterized. Despite preliminary reports, RNA interference has not been established as a reliable and consistent genetic approach (43). In spite of these limitations, we were able to use reporter gene constructs, organelle-specific markers, and functional protease cytochemistry to show that endocytosis and degradation of exogenous proteins takes place in the ER-like TVN. Such primitive characteristics of Giardia''s endomembrane system support Giardia''s proposed early branching and could be used as an analogous model to study the compartmentalization of endocytic/lysosomal functions into organelles distinct from the ER. Alternatively, Giardia may have had a more complex and “modern” endocytic system that has been lost by evolutionary reduction (6). These observations also have important implications for recent theories of pluripotent mammalian ER functions, including its roles in phagocytosis, entry of toxins and viruses, and MHC class I function (7, 19, 36).     

Catalytic function of PLA2G6 is impaired by mutations associated with infantile neuroaxonal dystrophy but not dystonia-parkinsonism

Background

Mutations in the PLA2G6 gene have been identified in autosomal recessive neurodegenerative diseases classified as infantile neuroaxonal dystrophy (INAD), neurodegeneration with brain iron accumulation (NBIA), and dystonia-parkinsonism. These clinical syndromes display two significantly different disease phenotypes. NBIA and INAD are very similar, involving widespread neurodegeneration that begins within the first 1–2 years of life. In contrast, patients with dystonia-parkinsonism present with a parkinsonian movement disorder beginning at 15 to 30 years of age. The PLA2G6 gene encodes the PLA2G6 enzyme, also known as group VIA calcium-independent phospholipase A₂, which has previously been shown to hydrolyze the sn-2 acyl chain of phospholipids, generating free fatty acids and lysophospholipids.

Methodology/Principal Findings

We produced purified recombinant wildtype (WT) and mutant human PLA2G6 proteins and examined their catalytic function using in vitro assays with radiolabeled lipid substrates. We find that human PLA2G6 enzyme hydrolyzes both phospholipids and lysophospholipids, releasing free fatty acids. Mutations associated with different disease phenotypes have different effects on catalytic activity. Mutations associated with INAD/NBIA cause loss of enzyme activity, with mutant proteins exhibiting less than 20% of the specific activity of WT protein in both lysophospholipase and phospholipase assays. In contrast, mutations associated with dystonia-parkinsonism do not impair catalytic activity, and two mutations produce a significant increase in specific activity for phospholipid but not lysophospholipid substrates.

Conclusions/Significance

These results indicate that different alterations in PLA2G6 function produce the different disease phenotypes of NBIA/INAD and dystonia-parkinsonism. INAD/NBIA is caused by loss of the ability of PLA2G6 to catalyze fatty acid release from phospholipids, which predicts accumulation of PLA2G6 phospholipid substrates and provides a mechanistic explanation for the accumulation of membranes in neuroaxonal spheroids previously observed in histopathological studies of INAD/NBIA. In contrast, dystonia-parkinsonism mutations do not appear to directly impair catalytic function, but may modify substrate preferences or regulatory mechanisms for PLA2G6.     

The role of the disulfide bond in the interaction of islet amyloid polypeptide with membranes

Human islet amyloid polypeptide (hIAPP) forms amyloid fibrils in pancreatic islets of patients with type 2 diabetes mellitus. It has been suggested that the N-terminal part, which contains a conserved intramolecular disulfide bond between residues 2 and 7, interacts with membranes, ultimately leading to membrane damage and β-cell death. Here, we used variants of the hIAPP_1–19 fragment and model membranes of phosphatidylcholine and phosphatidylserine (7:3, molar ratio) to examine the role of this disulfide in membrane interactions. We found that the disulfide bond has a minor effect on membrane insertion properties and peptide conformational behavior, as studied by monolayer techniques, ²H NMR, ThT-fluorescence, membrane leakage, and CD spectroscopy. The results suggest that the disulfide bond does not play a significant role in hIAPP–membrane interactions. Hence, the fact that this bond is conserved is most likely related exclusively to the biological activity of IAPP as a hormone.