Diverging patterns of mitochondrial and nuclear DNA diversity in subarctic black spruce: imprint of a founder effect associated with postglacial colonization

High-latitude ecotonal populations at the species margins may exhibit altered patterns of genetic diversity, resulting from more or less recent founder events and from bottleneck effects in response to climate oscillations. Patterns of genetic diversity were investigated in nine populations of the conifer black spruce (Picea mariana [Mill.] BSP.) in northwestern Québec, Canada, using seed-dispersed mitochondrial (mt) DNA and nuclear (nc) DNA. mtDNA diversity (mitotypes) was assessed at three loci, and ncDNA diversity was estimated for nine expressed sequence tag polymorphism (ESTP) loci. Sampling included populations from the boreal forest and the southern and northern subzones of the subarctic forest-tundra, a fire-born ecotone. For ncDNA, populations from all three vegetation zones were highly diverse with little population differentiation (thetaN = 0.014); even the northernmost populations showed no loss of rare alleles. Patterns of mitotype diversity were strikingly different: within-population diversity and population differentiation were high for boreal forest populations [expected heterozygosity per locus (HE) = 0.58 and thetaM = 0.529], but all subarctic populations were fixed for a single mitotype (HE = 0). This lack of variation suggests a founder event caused by long-distance seed establishment during postglacial colonization, consistent with palaeoecological data. The estimated movement of seeds alone (effective number of migrants per generation, NmM < 2) was much restricted compared to that estimated from nuclear variants, which including pollen movement (NmN > 17). This could account for the conservation of a founder imprint in the mtDNA of subarctic black spruce. After reduction, presumably in the early Holocene, the diversity in ncDNA would have been replenished rapidly by pollen-mediated gene flow, and maintained subsequently through vegetative layering during the current cooler period covering the last 3000 years.     

A single Na+-Pi cotransporter in Toxoplasma plays key roles in phosphate import and control of parasite osmoregulation

Inorganic ions such as phosphate, are essential nutrients required for a broad spectrum of cellular functions and regulation. During infection, pathogens must obtain inorganic phosphate (P_i) from the host. Despite the essentiality of phosphate for all forms of life, how the intracellular parasite Toxoplasma gondii acquires P_i from the host cell is still unknown. In this study, we demonstrated that Toxoplasma actively internalizes exogenous P_i by exploiting a gradient of Na⁺ ions to drive P_i uptake across the plasma membrane. The Na⁺-dependent phosphate transport mechanism is electrogenic and functionally coupled to a cipargarmin sensitive Na⁺-H⁺-ATPase. Toxoplasma expresses one transmembrane P_i transporter harboring PHO4 binding domains that typify the PiT Family. This transporter named TgPiT, localizes to the plasma membrane, the inward buds of the endosomal organelles termed VAC, and many cytoplasmic vesicles. Upon P_i limitation in the medium, TgPiT is more abundant at the plasma membrane. We genetically ablated the PiT gene, and ΔTgPiT parasites are impaired in importing P_i and synthesizing polyphosphates. Interestingly, ΔTgPiT parasites accumulate 4-times more acidocalcisomes, storage organelles for phosphate molecules, as compared to parental parasites. In addition, these mutants have a reduced cell volume, enlarged VAC organelles, defects in calcium storage and a slightly alkaline pH. Overall, these mutants exhibit severe growth defects and have reduced acute virulence in mice. In survival mode, ΔTgPiT parasites upregulate several genes, including those encoding enzymes that cleave or transfer phosphate groups from phosphometabolites, transporters and ions exchangers localized to VAC or acidocalcisomes. Taken together, these findings point to a critical role of TgPiT for P_i supply for Toxoplasma and also for protection against osmotic stresses.     

Characterization of reproduction-specific genes in a marine bivalve mollusc: influence of maturation stage and sex on mRNA expression

Numerous studies have investigated the reproduction mechanisms in mollusc species at a biochemical and physiological level; few have described these mechanisms at a molecular level, despite great commercial interest in several mollusc species. We investigated genes involved in gonad maturation of the marine scallop Argopecten purpuratus. A cDNA library was made from gonad tissue. After sequence analysis, 418 unique genes were characterized, of these, about 80% were of unknown function. Among the identified sequences, we analyzed the mRNA expression by real-time PCR of 7 genes involved in reproduction mechanisms, either directly: testis-specific serine/threonine-protein kinase (TSSK), vitellogenin (Vg), and spermatogenesis and centriole associated 1 (SCA) or indirectly: calcineurin A (CNA), centrin, RNA-specific adenosine deaminase (ADAR), and cytidine deaminase (CDA). The real-time PCR analyses were conducted on different tissues of mature and immature scallops (testis, ovary, immature gonad, gill, digestive gland and mantle). The genes studied, presented (1) a strong tissue-dependent expression pattern (higher expression in gonad tissues than in all other tissues) and (2) a sex- and maturation-specific expression pattern (except centrin). This is the first time that the expression of specific genes involved in reproduction mechanisms in a marine mollusc has been described at the molecular level.     

Glucose-induced Ubiquitylation and Endocytosis of the Yeast Jen1 Transporter: ROLE OF LYSINE 63-LINKED UBIQUITIN CHAINS*

Protein ubiquitylation is essential for many events linked to intracellular protein trafficking. Despite the significance of this process, the molecular mechanisms that govern the regulation of ubiquitylation remain largely unknown. Plasma membrane transporters are subjected to tightly regulated endocytosis, and ubiquitylation is a key signal at several stages of the endocytic pathway. The yeast monocarboxylate transporter Jen1 displays glucose-regulated endocytosis. We show here that casein kinase 1-dependent phosphorylation and HECT-ubiquitin ligase Rsp5-dependent ubiquitylation are required for Jen1 endocytosis. Ubiquitylation and endocytosis of Jen1 are induced within minutes in response to glucose addition. Jen1 is modified at the cell surface by oligo-ubiquitylation with ubiquitin-Lys⁶³ linked chain(s), and Jen1-Lys³³⁸ is one of the target residues. Ubiquitin-Lys⁶³-linked chain(s) are also required directly or indirectly to sort Jen1 into multivesicular bodies. Jen1 is one of the few examples for which ubiquitin-Lys⁶³-linked chain(s) was shown to be required for correct trafficking at two stages of endocytosis: endocytic internalization and sorting at multivesicular bodies.Ubiquitylation is one of the most prevalent protein post-translational modifications in eukaryotes. In addition to its role in promoting proteasomal degradation of target proteins, ubiquitylation has been shown to regulate multiple processes, including DNA repair, signaling, and intracellular trafficking. Ubiquitylation serves as a key signal mediating the internalization of plasma membrane receptors and transporters, followed by their intracellular transport and subsequent recycling or lysosomal/vacuolar degradation (1, 2). In Saccharomyces cerevisiae, transporters usually display both constitutive and accelerated endocytosis regulated by factors such as excess substrate, changes in nutrient availability, and stress conditions. Ubiquitylation of these cell surface proteins acts as a signal triggering their internalization (1). A single essential E3⁴ ubiquitin ligase, Rsp5, has been implicated in the internalization of most, if not all, endocytosed proteins (3). Rsp5 is the unique member in S. cerevisiae of the HECT (homologous to E6AP COOH terminus)-ubiquitin ligases of the Nedd4/Rsp5 family (4). In a few cases, Rsp5-dependent cell surface ubiquitylation was shown to involve PY-containing adapters that bind to Rsp5 (5–7). Rsp5-mediated ubiquitylation is also required for sorting into multivesicular bodies (MVBs) of endosomal membrane proteins that come from either the plasma membrane (through endocytosis) or the Golgi (through vacuolar protein sorting (VPS) pathway) (8). Although much progress has been made in elucidating the mechanistic basis of various steps in protein trafficking, the precise requirement for a specific type and length of Ub chains at various stages of the endocytic pathway remains to be addressed.The ubiquitin profile needed for proper internalization has been established for some yeast membrane proteins (1). The α-factor receptor Ste2 was described as undergoing monoubiquitylation on several lysines (multimonoubiquitylation). The a-factor receptor, Ste3p; the general transporter of amino acids, Gap1; the zinc transporter, Ztr1; and the uracil transporter, Fur4, have been shown to be modified by short chains of two to three ubiquitins, each attached to one, two, or more target lysine residues (oligo-ubiquitylation). Among them, Fur4 and Gap1 were the only transporters demonstrated to undergo plasma membrane oligo-ubiquitylation with ubiquitin residues linked via ubiquitin-Lys⁶³ (9, 10). In addition, the two siderophore transporters Arn1 and Sit1 were also shown to undergo Lys⁶³-linked cell surface ubiquitylation (11, 12). Whether these four transporters are representative of a larger class of plasma membrane substrates remains to be determined. Little is known about the type of ubiquitylation involved and/or required for sorting to MVBs. Some MVB cargoes appear to undergo monoubiquitylation (8), whereas Sna3, an MVB cargo of unknown function, undergoes Lys⁶³-linked ubiquitylation (13). Lys⁶³-linked ubiquitin chains were also recently reported to be required, directly or indirectly, for MVB sorting of the siderophore transporter, Sit1, when trafficking through the VPS pathway in the absence of its external substrate (11). In agreement with the possibility that additional membrane-bound proteins might undergo Lys⁶³-linked ubiquitylation, a proteomic study aiming to uncover ubiquitylated yeast proteins showed that Lys⁶³-ubiquitin chains are far more abundant than previously thought (14).The transport of monocarboxylates, such as lactate and pyruvate, as well as ketone bodies across the plasma membrane is essential for the metabolism of cells of various organisms. A family of monocarboxylate transporters has been reported that includes mainly mammalian members (15). In S. cerevisiae, two monocarboxylate-proton symporters have been described, Jen1 and Ady2 (16, 17). These transporters exhibit differences in their mechanisms of regulation and specificity. Jen1 is a lactate-pyruvate-acetate-propionate transporter induced in lactic or pyruvic acid-grown cells (18). Ady2, which accepts acetate, propionate, or formate, is present in cells grown in non-fermentable carbon sources (19). Jen1 has unique regulatory characteristics and has been extensively studied. It was the first secondary porter of S. cerevisiae characterized by heterologous expression in Pichia pastoris at both the cell and the membrane vesicle levels (20). The addition of glucose to lactic acid-grown cells very rapidly triggers loss of Jen1 activity and repression of JEN1 gene expression (21, 22). Newly synthesized Jen1-GFP fusion protein is sorted to the plasma membrane in an active and stable form, and loss of Jen1-GFP activity upon glucose addition is the result of its endocytosis followed by vacuolar degradation (23). Data from large scale analyses based on mass spectrometry approaches led to the detection of two sites of ubiquitylation for Jen1, one located in the N terminus of the protein and the second in the central loop (14), and several sites of phosphorylation in the N terminus, central loop, and C terminus of the protein (14, 24). In the present study, we aimed at further characterizing the internalization step of endocytosis of the transporter Jen1 and the potential role of the phosphorylation and ubiquitylation events required for its correct endocytic trafficking.     

Mapping the semantic space for the subjective experience of emotional responses to odors

Two studies were conducted to examine the nature of the verbal labels that describe emotional effects elicited by odors. In Study 1, a list of terms selected for their relevance to describe affective feelings induced by odors was assessed while participants were exposed to a set of odorant samples. The data were submitted to a series of exploratory factor analyses to 1) reduce the set of variables to a smaller set of summary scales and 2) get a preliminary sense of the differentiation of affective feelings elicited by odors. The goal of Study 2 was to replicate the findings of Study 1 with a larger sample of odorant samples and participants and to validate the preliminary model obtained in Study 1 by using confirmatory factor analysis. Overall, the findings point to a structure of affective responses to odors that differs from the classical taxonomies of emotion such as posited by discrete or bidimensional emotion theories. These findings suggest that the subjective affective experiences or feelings induced by odors are structured around a small group of dimensions that reflect the role of olfaction in well-being, social interaction, danger prevention, arousal or relaxation sensations, and conscious recollection of emotional memories.     

Subcutaneous graft of D1 mouse mesenchymal stem cells leads to the formation of a bone-like structure

Mesenchymal stem cells (MSC) are capable of both self-renewal and multi-lineage differentiation into mesoderm-type cells such as osteoblasts, chondrocytes, adipocytes and myocytes. Together the multipotent nature of MSCs and the facility to expand them in vitro make these cells ideal resources for regenerative medicine, particularly for bone reconstruction, and therefore research efforts focused on defining efficient protocols for directing their differentiation into the requisite lineage. Despite much progress in identifying mechanisms and factors that direct and control in vitro osteogenic differentiation of MSCs, a rapid and simple model to evaluate in vivo tissue formation is still lacking. Here, we describe the unique capacity of the murine bone marrow-derived D1 MSC cell line, which differentiates in vitro into at least three cell lineages, to form in vivo a structure resembling bone. This bone-like structure was obtained after subcutaneous grafting of D1 cells into immunocompetent mice without the need of neither an osteogenic factor nor scaffold material. These data allow us to propose this cell model as a tool for exploring in vivo the mechanisms and/or factors that govern and potentially regulate osteogenesis.     

Genetic Resistance to Scrapie Infection in Experimentally Challenged Goats

In goats, several field studies have identified coding mutations of the gene encoding the prion protein (I/M₁₄₂, N/D₁₄₆, S/D₁₄₆, R/Q₂₁₁, and Q/K₂₂₂) that are associated with a lower risk of developing classical scrapie. However, the data related to the levels of resistance to transmissible spongiform encephalopathies (TSEs) of these different PRNP gene mutations are still considered insufficient for developing large-scale genetic selection against scrapie in this species. In this study, we inoculated wild-type (WT) PRNP (I₁₄₂R₁₅₄R₂₁₁Q₂₂₂) goats and homozygous and/or heterozygous I/M₁₄₂, R/H₁₅₄, R/Q₂₁₁, and Q/K₂₂₂ goats with a goat natural scrapie isolate by either the oral or the intracerebral (i.c.) route. Our results indicate that the I/M₁₄₂ PRNP polymorphism does not provide substantial resistance to scrapie infection following intracerebral or oral inoculation. They also demonstrate that H₁₅₄, Q₂₁₁, and K₂₂₂ PRNP allele carriers are all resistant to scrapie infection following oral exposure. However, in comparison to WT animals, the H₁₅₄ and Q₂₁₁ allele carriers displayed only moderate increases in the incubation period following i.c. challenge. After i.c. challenge, heterozygous K₂₂₂ and a small proportion of homozygous K₂₂₂ goats also developed the disease, but with incubation periods that were 4 to 5 times longer than those in WT animals. These results support the contention that the K₂₂₂ goat prion protein variant provides a strong but not absolutely protective effect against classical scrapie.     

NQO1-dependent redox cycling of idebenone: effects on cellular redox potential and energy levels

Short-chain quinones are described as potent antioxidants and in the case of idebenone have already been under clinical investigation for the treatment of neuromuscular disorders. Due to their analogy to coenzyme Q10 (CoQ10), a long-chain quinone, they are widely regarded as a substitute for CoQ10. However, apart from their antioxidant function, this provides no clear rationale for their use in disorders with normal CoQ10 levels. Using recombinant NAD(P)H:quinone oxidoreductase (NQO) enzymes, we observed that contrary to CoQ10 short-chain quinones such as idebenone are good substrates for both NQO1 and NQO2. Furthermore, the reduction of short-chain quinones by NQOs enabled an antimycin A-sensitive transfer of electrons from cytosolic NAD(P)H to the mitochondrial respiratory chain in both human hepatoma cells (HepG2) and freshly isolated mouse hepatocytes. Consistent with the substrate selectivity of NQOs, both idebenone and CoQ1, but not CoQ10, partially restored cellular ATP levels under conditions of impaired complex I function. The observed cytosolic-mitochondrial shuttling of idebenone and CoQ1 was also associated with reduced lactate production by cybrid cells from mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) patients. Thus, the observed activities separate the effectiveness of short-chain quinones from the related long-chain CoQ10 and provide the rationale for the use of short-chain quinones such as idebenone for the treatment of mitochondrial disorders.