Uniformity of organellar DNA in Aldrovanda vesiculosa, an endangered aquatic carnivorous species, distributed across four continents

Organellar DNA from the widely distributed but rare and critically endangered aquatic carnivorous plant Aldrovanda vesiculosa (Droseraceae) was examined. Six chloroplast intergenic regions (3700 nt in total) were sequenced before analyzing the Southern-RFLP (Restriction Fragment Length Polymorphism) of 2 mt gene flanking regions. Only two different chloroplast haplotypes among 15 A. vesiculosa accessions from Africa, Australia, Europe, and Japan were found, generally distinguishing European and non-European plants, with two exceptions. Genetic variation observed in A. vesiculosa appears to be even lower than in other aquatic species with a similar world-wide distribution. A recent bottleneck followed by long-distance dispersal by water birds or low mutation rates could be responsible for the observed genetic uniformity. Estimation of genetic distances based on six chloroplast intergenic regions led to the conclusion that the chloroplast genome of A. vesiculosa matches more closely to that of Drosera regia than Dionaea muscipula, a sister genus sharing snapping traps. The inconsistency between genetic distance estimates based on nuclear and cytoplasmic markers may reflect a chloroplast capture. In A. vesiculosa, a four amino acids substitution (TGWS) in the amino acid sequence of ATP synthase alpha subunit (ATP1), highly conserved mitochondrial protein, was discovered, unique among all organisms based on current knowledge.     

Toxoplasma gondii grown in human cells uses GalNAc-containing glycosylphosphatidylinositol precursors to anchor surface antigens while the immunogenic Glc-GalNAc-containing precursors remain free at the parasite cell surface

Toxoplasma gondii is a ubiquitous parasite that infects nearly all warm-blooded animals. Developmental switching in T. gondii, from the virulent tachyzoite to the relatively quiescent bradyzoite stage, is responsible for the disease propagation after alteration of the immune status of the carrier. The redifferentiation event is characterized by an over expression of a tachyzoite specific set of glycosylphosphatidylinositol anchored surface antigens and free GPIs. T. gondii grown in animal cells uses two glycosylphosphatidylinositol precursors to anchor the parasite surface proteins. The first form has an N-acetylgalactosamine residue bound to a conserved three-mannosyl core glycan, while the second structure contains an additional terminal glucose linked to the N-acetylgalactosamine side branch. Sera from persons infected with T. gondii reacted only with the glucose-N-acetylgalactosamine-containing structure. Here we report that T. gondii cultured in human cells uses predominantly the N-acetylgalactosamine-containing structure to anchor the parasite surface antigens. On the other hand, glycosylphosphatidylinositol structures having an additional terminal glucose are found exclusively on the parasite cell surface as free glycolipids participating in the production of cytokines that are implicated in the pathogenesis of T. gondii. We also provide evidence that such free glycosylphosphatidylinositols are restricted mainly to the lipid microdomains in the parasite cell surface membrane and mostly associated with proteins involved in the parasite motility as well as invasion of the host cell.     

Inhibitors of glycosyl-phosphatidylinositol anchor biosynthesis

Glycosyl-phosphatidylinositol (GPI) is a complex glycolipid structure that acts as a membrane anchor for many cell-surface proteins of eukaryotes. GPI-anchored proteins are particularly abundant in protozoa such as Trypanosoma brucei, Leishmania major, Plasmodium falciparum and Toxoplasma gondii, and represent the major carbohydrate modification of many cell-surface parasite proteins. Although the GPI core glycan is conserved in all organisms, many differences in additional modifications to GPI structures and biosynthetic pathways have been reported. Therefore, the characteristics of GPI biosynthesis are currently being explored for the development of parasite-specific inhibitors. In vitro and in vivo studies using sugars and substrate analogues as well as natural compounds have shown that it is possible to interfere with GPI biosynthesis at different steps in a species-specific manner. Here we review the recent and promising progress in the field of GPI inhibition.     

Plasmodium falciparum: glycosylation status of Plasmodium falciparum circumsporozoite protein expressed in the baculovirus system

We expressed the main surface antigen of Plasmodium falciparum sporozoites, the circumsporozoite protein (CSP), in High Five (Trichoplusia ni) insect cells using the baculovirus system. Significant amounts of the recombinant protein could be obtained, as judged by SDS-PAGE, Western blot, and immunofluorescence analysis. The cellular localization for recombinant CSP was determined by immunofluorescence. The high fluorescence signal of the permeabilized cells, relative to that of fixed nonpermeabilized cells, revealed a clear intracellular localization of this surface antigen. Analysis of possible posttranslational modifications of CSP showed that this recombinant protein is only N-glycosylated in the baculovirus system. Although DNA-sequence analysis revealed a GPI-cleavage/attachment site, no GPI anchor could be demonstrated. These analyses show that the glycosylation status of this recombinant protein may not reflect its native form in P. falciparum. The impact of these findings on vaccine development will be discussed.     

Bacterial Endosymbioses of Gutless Tube-Dwelling Worms in Nonhydrothermal Vent Habitats

Gutless tube-dwelling worms of pogonophorans (also known as frenulates) and vestimentiferans depend on primary production of endosymbiotic bacteria. The endosymbionts include thiotrophs that oxidize sulfur for autotrophic production and methanotrophs that oxidize and assimilate methane. Although most of the pogonophoran and vestimentiferan tube worms possess single thiotrophic 16S rRNA genes (16S rDNA) related to γ-proteobacteria, some pogonohorans are known to bear single methanotroph species or even dual symbionts of thiotrophs and methanotrophs. The vestimentiferan Lamellibrachia sp. L1 shows symbiotic 16S rDNA sequences of α-, β-, γ-, and ε-proteobacteria, varying among specimens, with RuBisCO form II gene (cbbM) sequences related to β-proteobacteria. An unidentified pogonophoran from the world’s deepest cold seep, 7326-m deep in the Japan Trench, hosts a symbiotic thiotroph based on 16S rDNA with the RuBisCO form I gene (cbbL). In contrast, a shallow-water pogonophoran (Oligobrachia mashikoi) in coastal Japan Sea has a methanotrophic 16S rDNA and thiotrophic cbbL, which may suggest the feature of type X methanotrophs. These observations demonstrate that pogonophoran and vestimentiferan worms have higher plasticity in bacterial symbioses than previously suspected.     

Comparative analysis of cytokines and growth factors in the conditioned media of stem cells from the pulp of deciduous,young, and old permanent tooth

ObjectivesTo compare and analyze the secretome profile of stem cells obtained from the deciduous tooth (SHEDs), young (yDPSCs), and old permanent tooth (oDPSCs).MethodsAll the stem cells were assessed for mesenchymal stem cell markers. The stem cells were differentiated into osteoblasts and chondrocytes using lineage-specific differentiation media. Conditioned media was collected from growing stem cells, and a cytometric bead array was performed to estimate secreted cytokines and growth factor levels by flow cytometry. Gene expresseion levels were assessed by real-time quantitative polymerase chain reaction.ResultsAge did not affect the mesenchymal characteristics of dental stem cells from various age groups. The secretomes of SHEDs and young yDPSCs exhibit more growth factors and lesser pro-inflammatory cytokines than oDPSCs. Osteo and chondrogenic differentiation potential were higher in SHEDs and young yDPSCs than in the oDPSCs. TLR1, TLR2, TLR3 show decreased expression levels with age and TLR5, TLR6 show increased expression with age.ConclusionThe superior regenerative potential of SHEDs and yDPSCs may be due to the higher growth factors and lower pro-inflammatory cytokine profile.     

Triphala extract negates arecoline-induced senescence in oral mucosal epithelial cells in vitro

BackgroundArecoline found in areca nut causes oral submucous fibrosis. Triphala is an Ayurvedic medicinal preparation used to improve overall physical wellness that has also been shown to improve oral health.ObjectivesTo assess the activity of Triphala extract on arecoline-induced senescence in oral mucosal epithelial cells in vitro.Materials and methodsOral mucosal epithelial cells were isolated and cultured in vitro. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to assess the viability of treated cells, while senescence was assessed by senescence-associated-β-galactosidase staining. Cell surface marker expression was analyzed by flow cytometry. Finally, real-time quantitative polymerase chain reaction was performed to examine gene expression levels.ResultsTriphala extract (5 µg/mL) reversed the cell senescence activity of arecoline, as evidenced by reduced β-galactosidase activity, increased Ki-67 marker expression, and reduced expression of senescence-related genes p16 and p21.ConclusionTriphala extract helped to reduce the pathological effects of arecoline-induced pathogenesis.Clinical relevance.Arecoline found in the areca nut causes oral pathological conditions including oral submucous fibrosis. Our results showed that Triphala counteracted the adverse effects of arecoline, in particular, negating senescence in oral mucosal epithelial cells. As a translational effect, Triphala treatment could restore normal epithelial thickness in oral submucous fibrosis, thus reducing the clinical severity of the disease. This reestablishment of oral homeostasis would help to improve oral health-related quality of life in patients with oral submucous fibrosis.     

Phosphorylation of Marburg virus matrix protein VP40 triggers assembly of nucleocapsids with the viral envelope at the plasma membrane

Marburg virus (MARV) matrix protein VP40 plays a key role in virus assembly, recruiting nucleocapsids and the surface protein GP to filopodia, the sites of viral budding. In addition, VP40 is the only MARV protein able to induce the release of filamentous virus-like particles (VLPs) indicating its function in MARV budding. Here, we demonstrated that VP40 is phosphorylated and that tyrosine residues at positions 7, 10, 13 and 19 represent major phosphorylation acceptor sites. Mutagenesis of these tyrosine residues resulted in expression of a non-phosphorylatable form of VP40 (VP40(mut) ). VP40(mut) was able to bind to cellular membranes, produce filamentous VLPs, and inhibit interferon-induced gene expression similarly to wild-type VP40. However, VP40(mut) was specifically impaired in its ability to recruit nucleocapsid structures into filopodia, and released infectious VLPs (iVLPs) had low infectivity. These results indicated that tyrosine phosphorylation of VP40 is important for triggering the recruitment of nucleocapsids to the viral envelope.