Evidence for rolling circle replication of tandem genes in Drosophila

Extrachromosomal circular DNA (eccDNA) is one characteristic of the plasticity of the eukaryotic genome. It is found in various organisms and contains sequences derived primarily from repetitive chromosomal DNA. Using 2D gel electrophoresis, we have previously detected eccDNA composed of chromosomal tandem repeats throughout the life cycle of Drosophila. Here, we report for the first time evidence suggesting the occurrence of rolling circle replication of eccDNA in Drosophila. We show, on 2D gels, specific structures that can be enriched by benzoylated naphthoylated DEAE-cellulose chromatography and were identified in other systems as rolling circle intermediates (RCIs). These RCIs are homologous to histone genes, Stellate and Suppressor of Stellate, which are all organized in the chromosomes as tandem repeats. RCIs are detected throughout the life cycle of Drosophila and in cultured fly cells. These structures are found regardless of the expression of the replicated gene or of its chromosomal copy number.     

Botrytis cinerea BcNma is involved in apoptotic cell death but not in stress adaptation

Apoptotic-like programmed cell death (PCD) occurs naturally in fungi during development and might also be induced by external conditions. Candidate apoptotic genes have been characterized in several model fungal species but not in plant pathogenic fungi. Here we report on the isolation and characterization of BcNMA, an orthologue of the human pro-apoptotic gene HtrA2 from the plant pathogen Botrytis cinerea. The predicted BcNma protein shows high homology to the previously characterized Nma111p from Saccharomyces cerevisiae and despite some structural differences it complemented the function of Nma111p in Δnma111 mutant strains. BcNMA-over-expression and mutant strains had enhanced or reduced appearance of apoptotic markers, respectively. However there was no difference in growth response of the wild type and BcNMA-transgenic strains to application of various stresses, and the effect on pathogenicity was marginal in both the over-expression and mutant strains. When considered together these results suggest that although BcNma has a pro-apoptotic activity, it is not a major regulator of apoptosis. The protein probably has additional roles that are unrelated to apoptosis, which lead to the pleotrophic phenotype of the transgenic strains and lack of a clear effect on stress adaptation and pathogenicity.     

A Ca2+-activated ATPase specifically released by Ca2+ shock from Paramecium tetraurelia

Deciliation of Paramecium tetraurelia by a Ca²⁺ shock procedure releases a discrete set of proteins which represent about 1% of the total cell protein. Marker enzymes for cytoplasm (hexokinase), endoplasmic reticulum (glucose-6-phosphatase), peroxisomes (catalase), and lysosomes (acid phosphatase) were not released by this treatment. Among the proteins selectively released is a Ca²⁺-dependent ATPase. This enzyme has a broad substrate specificity which includes GTP, ATP, and UTP, and it can be activated by Ca²⁺, Sr²⁺, or Ba²⁺, but not by Mg²⁺ or by monovalent cations. The crude enzyme has a specific activity of 2–3 μmol/min per mg; the optimal pH for activity is 7.5. ATPase, GTPase, and UTPase all reside in the same protein, which is inhibited by ruthenium red, is irreversibly denatured at 50°C, and which has a sedimentation coefficient of 8–10 S. This enzyme is compared with other surface-derived ATPases of ciliated protozoans, and its possible roles are discussed.     

Function from within: Autophagy induction by HPSE/heparanase—new possibilities for intervention

HPSE (heparanase) is the predominant enzyme in mammals capable of cleaving heparan sulfate, an activity highly implicated in cellular invasion and tumor metastasis. HPSE expression is induced in many types of cancer and increased HPSE levels are most often associated with increased tumor metastasis and reduced patient survival post operation. In addition, HPSE induction is associated with progression of the primary tumors but the mechanism(s) underlying tumor expansion by HPSE have not been sufficiently resolved. Our results establish a role for heparanase in modulating autophagy in normal and malignant cells, thereby conferring growth advantages as well as resistance to chemotherapy.     

Recognition of Yeast by Murine Macrophages Requires Mannan but Not Glucan

The first barrier against infection by Candida albicans involves fungal recognition and destruction by phagocytic cells of the innate immune system. It is well established that interactions between different phagocyte receptors and components of the fungal cell wall trigger phagocytosis and subsequent immune responses, but the fungal ligands mediating the initial stage of recognition have not been identified. Here, we describe a novel assay for fungal recognition and uptake by macrophages which monitors this early recognition step independently of other downstream events of phagocytosis. To analyze infection in live macrophages, we validated the neutrality of a codon-optimized red fluorescent protein (yEmRFP) biomarker in C. albicans; growth, hyphal formation, and virulence in infected mice and macrophages were unaffected by yEmRFP production. This permitted a new approach for studying phagocytosis by carrying out competition assays between red and green fluorescent yeast cells to measure the efficiency of yeast uptake by murine macrophages as a function of dimorphism or cell wall defects. These competition experiments demonstrate that, given a choice, macrophages display strong preferences for phagocytosis based on genus, species, and morphology. Candida glabrata and Saccharomyces cerevisiae are taken up by J774 macrophage cells more rapidly than C. albicans, and C. albicans yeast cells are favored over hyphal cells. Significantly, these preferences are mannan dependent. Mutations that affect mannan, but not those that affect glucan or chitin, reduce the uptake of yeast challenged with wild-type competitors by both J774 and primary murine macrophages. These results suggest that mannose side chains or mannosylated proteins are the ligands recognized by murine macrophages prior to fungal uptake.Candida albicans is an opportunistic fungus that normally resides in the human gut (26) and can cause mucosal infections. When host immune defenses are compromised or when anatomical breaches permit extreme fungal burdens, systemic and often lethal fungal colonization throughout the body can occur. In hospital-acquired bloodstream infections, the rate of mortality, hospital cost, and length of stay associated with disseminated candidiasis now outrank those associated with bacterial infections (37, 43). The most effective host barrier that limits Candida infections is microbial destruction by phagocytic cells of the innate immune system. In a healthy host, phagocytes—macrophages, neutrophils, and dendritic cells—recognize, ingest, and destroy the invading yeast by phagocytosis.The first step of a fungal infection requires the recognition of yeast by phagocytes. Despite the medical importance of this reaction, it remains poorly understood. As the interface between the yeast and its host, the fungal cell wall is crucial for recognition. The wall is a complex structure consisting of an elastic network of polysaccharides (glucans and chitin) that surrounds the plasma membrane and that in most yeast and fungi contains many different heavily mannosylated proteins (mannan) anchored to the wall in various ways (9, 27,–29). Three distinct layers that correspond to these three components can be seen by electron microscopy. The innermost layer is enriched with a small amount of chitin, the outermost layer consists of mannan, and in between these layers are flexible fibrils of β1,3-glucan. Another glucan (β1,6 linked) is relatively minor in amount but is important for maintaining wall structure because it cross-links β1,3-glucan to wall proteins and to chitin (24, 30). Yeast survival relies on the integrity of the cell wall because it shields the yeast from physical stress and osmotic shock. The wall also maintains cell shape, which is a precondition for growth and morphogenesis. The rapid switch between the yeast and hyphal forms that is essential for C. albicans virulence underscores the plasticity of the wall, whose composition, thickness, and structure vary tremendously in response to changes in the environment.Many phagocytic receptors implicated in fungal recognition have been identified. The interactions between these receptors and fungal wall components activate an array of host defense signaling pathways that promote actin cytoskeletal rearrangements and the membrane remodeling required for phagocytosis, production of toxic metabolites and hydrolytic enzymes within the phagosome that destroy the ingested yeast, and secretion of cytokines that are pro- or anti-inflammatory (for a review, see references 18, 31, and 36). These receptors are members of the C-type lectin receptor and Toll-like receptor families and include proteins that can recognize mannose, glucan, and, possibly, chitin or, possibly, multiligand combinations of these carbohydrates (for reviews, see references 22 and 49). Despite a wealth of information about the signaling cascades elicited by these host receptors, the identity of the fungal cell wall ligands that mediate the initial recognition event during host-fungal interactions remains unclear, in large part because good model systems for studying host-fungal interactions in the context of the live infective environment have been unavailable. Most current assays of fungal recognition rely on indirect readouts, for instance, virulence or cytokine production, which cannot distinguish the initial step of fungal recognition from other downstream events of phagocytosis. In addition, different experimental systems for studying fungal phagocytosis use different cell types that may display unique interactions with C. albicans and vice versa. Thus, there are conflicts in the literature about the contributions of fungal cell wall components to host recognition and phagocytosis.Here, we make use of a novel assay to help clarify discrepancies that currently exist in this field. We developed a biologically neutral red fungal fluorescent biomarker that can be stably introduced into most yeast and fungi to monitor C. albicans-host interactions during infection in live cells or animals. This permitted development of quantitative competition assays to measure uptake by macrophages of red fluorescent protein (RFP)- or green fluorescent protein (GFP)-labeled cells as a single isolated event within the complex process of phagocytosis in live cells. We apply this system to address two fundamental questions regarding fungal recognition by murine macrophages. First, do these macrophages display a preference toward yeast forms versus filamentous fungal forms, and second, how do the various fungal cell wall components contribute to this preference during the initial stage of fungal recognition? We demonstrate that, given a choice, J774 macrophages recognize and ingest yeast cells far more rapidly and efficiently than hyphal cells. Importantly, competitive fungal uptake by murine macrophages, both immortalized cell lines and primary cells, is markedly inhibited by reduction of cell wall mannan but not glucan or chitin. This points to a critical role for mannose side chains or mannosylated proteins as key fungal recognition ligands.     

Non-fermented Dairy Desserts with Potentially Probiotic Autochthonous Lactobacilli and Products from Peel of Jabuticaba (Myrciaria cauliflora)

Probiotics and Antimicrobial Proteins - The influence of two autochthonous lactobacilli strains with probiotic potential (Lactobacillus mucosae CNPC007 and Lactobacillus plantarum CNPC020) in...     

The presequence of fumarase is exposed to the cytosol during import into mitochondria

The majority of mitochondrial proteins can be imported into mitochondria following termination of their translation in the cytosol. Import of fumarase and several other proteins into mitochondria does not appear to occur post-translationally according to standard in vivo and in vitro assays. However, the nature of interaction between the translation and translocation apparatuses during import of these proteins is unknown. Therefore, a major question is whether the nascent chains of these proteins are exposed to the cytosol during import into mitochondria. We asked directly if the presequence of fumarase can be cleaved by externally added mitochondrial processing peptidase (MPP) during import, using an in vitro translation-translocation coupled reaction. The presequence of fumarase was cleaved by externally added MPP during import, indicating a lack of, or a loose physical connection between, the translation and translocation of this protein. Exchanging the authentic presequence of fumarase for that of the more efficient Su9-ATPase presequence reduced the exposure of fumarase precursors to externally added MPP en route to mitochondria. Therefore, exposure to cytosolic MPP is dependent on the presequence and not on the mature part of fumarase. On the other hand, following translation in the absence of mitochondria, the authentic fumarase presequence and that of Su9-ATPase become inaccessible to added MPP when attached to mature fumarase. Thus, folding of the mature portion of fumarase, which conceals the presequence, is the reason for its inability to be imported in classical post-translational assays. Another unique feature of fumarase is its distribution between the mitochondria and the cytosol. We show that in vivo the switch of the authentic presequence with that of Su9-ATPase caused more fumarase molecules to be localized to the mitochondria. A possible mechanism by which the cytosolic exposure, the targeting efficiency, and the subcellular distribution of fumarase are dictated by the presequence is discussed.     

Yeast-to-hyphal transition triggers formin-dependent Golgi localization to the growing tip in Candida albicans

Rapid and long-distance secretion of membrane components is critical for hyphal formation in filamentous fungi, but the mechanisms responsible for polarized trafficking are not well understood. Here, we demonstrate that in Candida albicans, the majority of the Golgi complex is redistributed to the distal region during hyphal formation. Randomly distributed Golgi puncta in yeast cells cluster toward the growing tip during hyphal formation, remain associated with the distal portion of the filament during its extension, and are almost absent from the cell body. This restricted Golgi localization pattern is distinct from other organelles, including the endoplasmic reticulum, vacuole and mitochondria, which remain distributed throughout the cell body and hypha. Hyphal-induced positioning of the Golgi and the maintenance of its structural integrity requires actin cytoskeleton, but not microtubules. Absence of the formin Bni1 causes a hyphal-specific dispersal of the Golgi into a haze of finely dispersed vesicles with a sedimentation density no different from that of normal Golgi. These results demonstrate the existence of a hyphal-specific, Bni1-dependent cue for Golgi integrity and positioning at the distal portion of the hyphal tip, and suggest that filamentous fungi have evolved a novel strategy for polarized secretion, involving a redistribution of the Golgi to the growing tip.