1,721,392 research outputs found
Spore germination in the fungus Syncephalastrum racemosum
Get PDFThe asexual part of the life cycle of the fungus Syncephalastrum racemosum was investigated using light microscopy, transmission and scanning electron microscopy. The structure and organisation of the vegetative mycelium and spore producing hyphae was studied in detail. Freeze-fracturing, surface replica, metal shadowing and thin sectioning techniques were used to investigate the ultrastrueture of dormant and germinated spore walls subjected to various sequential chemical treatments. The spore wall had a highly complex organisation, consisting of four, major ultrastructurally definable layers. (1) A surface layer consisting of a cross-patched network of rodlets, having a 25nm periodicity. (2) A plate-like layer covered by amorphous material.(3) A layer of thick 17»5nm diameter microfibrils. (4) An innermost layer of thin 8nm diameter microfibrils. The emergent germ tube has a smooth outer surface and an inner layer of thin microfibrils. The breakpoint between spore and germ tube is clearly delimited at various levels within the spore wall. Tentative identification of the wall layers showed: (1) the rodlet layer was mainly protein; (2) the thick microfibrils were probably a β-glucan; (3) the thin microfibrils were chitin. Freeze-fracturing of dormant and germinated spores revealed differences in plasmamembrane ultrastructure. Large particles (34nm) with complimentary large depressions (33nm) were present only in fractures of the plasmamembrane of dormant spores. During germination there is an increase in the numbers of small particles (8.4nm) in the membrane. The physiology of spore germination was studied in shake-flask and batch fermenter cultures. In the presence of glucose, spore swelling preceded outgrowth of germ tubes. Glucose was essential for initiation of these events. Swelling ceased quickly when glucose was removed. Analysis of size distributions within spore populations indicated that the larger spores germinated first. A self-inhibitor of germination was extracted from culture and spore washings, and was identified as nonanoic acid
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Novel Trypanosomatid-Bacterium Association: Evolution of Endosymbiosis in Action
Get PDFUnlabelledWe describe a novel symbiotic association between a kinetoplastid protist, Novymonas esmeraldas gen. nov., sp. nov., and an intracytoplasmic bacterium, "Candidatus Pandoraea novymonadis" sp. nov., discovered as a result of a broad-scale survey of insect trypanosomatid biodiversity in Ecuador. We characterize this association by describing the morphology of both organisms, as well as their interactions, and by establishing their phylogenetic affinities. Importantly, neither partner is closely related to other known organisms previously implicated in eukaryote-bacterial symbiosis. This symbiotic association seems to be relatively recent, as the host does not exert a stringent control over the number of bacteria harbored in its cytoplasm. We argue that this unique relationship may represent a suitable model for studying the initial stages of establishment of endosymbiosis between a single-cellular eukaryote and a prokaryote. Based on phylogenetic analyses, Novymonas could be considered a proxy for the insect-only ancestor of the dixenous genus Leishmania and shed light on the origin of the two-host life cycle within the subfamily Leishmaniinae.ImportanceThe parasitic trypanosomatid protist Novymonas esmeraldas gen. nov., sp. nov. entered into endosymbiosis with the bacterium "Ca. Pandoraea novymonadis" sp. nov. This novel and rather unstable interaction shows several signs of relatively recent establishment, qualifying it as a potentially unique transient stage in the increasingly complex range of eukaryotic-prokaryotic relationships
Characterization of CAP5.5 and CAP5.5V, atypical calpains in Trypanosoma brucei
Get PDFCalpains are a group of calcium-dependent cysteine proteases with roles ranging from cytoskeletal remodelling to signal transduction. In Trypanosoma brucei, the causal agent of African sleeping sickness, an unusually large number of calpain- like proteins exist, whose functions remain unknown. Recently, the procyclic form- specific cytoskeleton associated protein 5.5 (CAP5.5) and its bloodstream-specific paralogous variant, CAP5.5V were discovered to be essential for the proper morphogenesis of the parasite. However, precise functional roles of these proteins remain unknown. In this MSc thesis, I employed a two-pronged approach combining cell biology studies of CAP5.5 and CAP5.5V in T. brucei with in vitro assays of recombinant calpains expressed in Escherichia coli to further characterize CAP5.5 and CAP5.5V. To resolve whether the function of CAP5.5 and CAP5.5V is dependent upon proteolytic activity, I expressed and purified recombinant CAP5.5V’s catalytic domain. I demonstrated that this domain did not have any detectable autolytic activity or activity against universal protease substrates such as casein. Ectopic expression of CAP5.5::YFP::Ty in procyclic trypanosomes led to nuclear mis-positioning among other morphological defects. Additionally, I demonstrated a cell cycle dependency on incorporation of CAP5.5::YFP::Ty into the cytoskeleton and corroborate previous data that suggest that CAP5.5 is built in from the dynamic, posterior end of the cell. Investigation of the molecular determinants in CAP5.5’s sequence such as motifs or domains suggested that CAP5.5’s domain IV may be the domain mediating association with the sub-pellicular cytoskeleton
Differential Binding of Mitochondrial Transcripts by MRB8170 and MRB4160 Regulates Distinct Editing Fates of Mitochondrial mRNA in Trypanosomes
Get PDFA dozen mRNAs are edited by multiple insertions and/or deletions of uridine residues in the mitochondrion of Trypanosoma brucei Several protein complexes have been implicated in performing this type of RNA editing, including the mitochondrial RNA-binding complex 1 (MRB1). Two paralogous novel RNA-binding proteins, MRB8170 and MRB4160, are loosely associated with the core MRB1 complex. Their roles in RNA editing and effects on target mRNAs are so far not well understood. In this study, individual-nucleotide-resolution UV-cross-linking and affinity purification (iCLAP) revealed a preferential binding of both proteins to mitochondrial mRNAs, which was positively correlated with their extent of editing. Integrating additional in vivo and in vitro data, we propose that binding of MRB8170 and/or MRB4160 onto pre-mRNA marks it for the initiation of editing and that initial binding of both proteins may facilitate the recruitment of other components of the RNA editing/processing machinery to ensure efficient editing. Surprisingly, MRB8170 also binds never-edited mRNAs, suggesting that at least this paralog has an additional role outside RNA editing to shape the mitochondrial transcriptome. IMPORTANCE: Trypanosoma brucei mitochondrial mRNAs undergo maturation by RNA editing, a unique process involving decrypting open reading frames by the precise deletion and/or insertion of uridine (U) residues at specific positions on an mRNA. This process is catalyzed by multiprotein complexes, such as the RNA editing core complex, which provides the enzymatic activities needed for U insertion/deletion at a single editing site. Less well understood is how RNA editing occurs throughout an mRNA bearing multiple sites. To address this question, we mapped at single-nucleotide resolution the RNA interactions of two unique RNA-binding proteins (RBPs). These RBPs are part of the mitochondrial RNA-binding complex 1, hypothesized to mediate multiple rounds of RNA editing. Both RBPs were shown to mark mRNAs for the process in correlation with the number of editing sites on the transcript. Surprisingly, one also binds mRNAs that bypass RNA editing, indicating that it may have an additional role outside RNA editing
Mutations in the lethal ciliopathy Meckel-Gruber syndrome alter the subcellular distribution of actin-binding proteins and disrupt the actin cytoskeleton
No full textMKS3, encoding a novel trans-membrane receptor, meckelin with similarity to frizzled proteins, is mutated in Meckel–Gruber syndrome (MKS), an autosomal recessive lethal ciliopathy.Meckelin is a ciliary protein, but it also localises to the actin cytoskeleton at baso-lateral and basal cell surfaces. Filamin A (FLNa) and specific isoforms of nesprin-2 are actin-binding proteins (ABP) that interact with meckelin. Both are important for maintenance and remodelling of the actin cytoskeleton at the cell membrane and nuclear envelope. Meckelin and FLNa localised throughout ciliogenesis and in post-mitotic ciliated cells. However, a meckelin–nesprin-2 interaction was seen only during early ciliogenesis prior to the establishment of cell polarity and centriole migration; crucial steps in cilia formation. siRNA knockdown of meckelin caused loss of cilia and failure of centriole migration. This was also seen for FLNa and nesprin-2 indicating that mutation of ABPs could lead, or contribute, to a ciliopathy phenotype.In MKS patient fibroblasts, nuclear membrane architecture was distorted and cell-migration reduced; both indicative of disruption of the actin cytoskeleton. In addition, RhoA-GTPase activity was increased. Small GTPases are key regulators of actin assembly and remodelling pathways and the appearance of actin stress fibres was concurrent with this increased activity. Both FLNA and nesprin-2 were redirected to these fibres showing loss of meckelin altered distribution of ABPs.These novel findings suggest the actin cytoskeleton and actin-binding proteins play a crucial role in ciliogenesis and cilia function. This has important implications for defining the ciliopathy phenotype and determining the multi-functional role of ciliary proteins
Generation, regulation and function of morphology in Leishmania and Trypanosoma
Get PDFLittle is known about the generation of Leishmania morphology and the function of morphology in trypanosomatids, despite every species having characteristic cell shapes and undergoing changes in morphology between life cycle stages. To address this I analysed morphogenesis of the cell body and flagellum through the cell cycle of the Leishmania insect (promastigote) life cycle stage using a novel method for determining cell cycle stage from cell size and DNA content. This showed cell body morphology is generated by growth and then remodelling of cell shape around mitosis and cytokinesis. Mathematical modelling of flagellum growth indicated flagellum length continues to increase over multiple cell cycles and does not reach a defined length. I also observed little link between the cell cycle and flagellum length regulation during differentiation to the mammalian macrophage-inhabiting (amastigote) life cycle stage. Analysis of motility showed the diverse flagellar lengths of promastigote Leishmania cells bestow different swimming abilities, and the capacity of Leishmania promastigotes for highly directional swimming differs sharply from trypomastigote Trypanosoma brucei. This difference did not arise from altered flagellar beating therefore appeared to be linked to morphology. Together these indicate the mechanisms of cell body morphogenesis, flagellum length regulation, life cycle stage differentiation and the swimming abilities of the cells the morphogenetic processes generate differ significantly between Leishmania and T. brucei. These insights motivated the programming of automated micrograph analysis tools based on a new DNA staining method to support similar future morphometric analyses. This is the first comprehensive comparison of morphogenesis and function of morphology in a promastigote and a trypomastigote and, by considering these new insights in the context of existing molecular biology and the morphological diversity across many trypanosomatid species, give insight into basic Leishmania biology, the shared molecular mechanisms underlying morphogenesis and the potential functions of the diverse morphologies which are seen in different trypanosomatid species and life cycle stages
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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