Biogenesis, structure, and immune-suppressive effects of virus-like particles of a Drosophila parasitoid, Leptopilina victoriae

Drosophila melanogaster larvae are attacked by virulent strains of parasitoid wasps. Females of Leptopilina heterotoma produce virus-like particles (VLPs) that efficiently destroy lamellocytes, a major larval immune effector cell type. We report here that L. victoriae, a closely related wasp species, also produces VLPs that trigger immune suppression responses in fly hosts. We compare the ability of immune suppression of the two parasitoids using a mutant host strain hopscotch^{Tumorous-lethal} (hop^Tum-l). hop^Tum-l larvae have two defects of hematopoietic origin: overproliferation of hemocytes and constitutive encapsulation of self-tissue by lamellocytes. The encapsulation phenotype is suppressed weakly by L. victoriae and strongly by L. heterotoma. In vitro studies on hop^Tum-l lamellocytes show that VLP-containing fluid from either wasp species induces lamellocyte lysis, but with different kinetics.Previously undocumented precursors of L. victoriae VLPs are synthesized in the long gland and are first visible within canals connecting secretory cells to the long gland lumen. VLP assembly occurs in the lumen. VLPs show multiple electron-dense projections surrounding a central core. Maturing particles appear segmented, singly or in arrays, embedded in the reservoir matrix. In sections, mature particles are pentagonal or hexagonal; the polygon vertices extending into spikes. Our results suggest that L. victoriae is likely to promote immune suppression by an active mechanism that is mediated by VLPs, similar to that used by L. heterotoma.     

Alterations in heart looping induced by overexpression of the tight junction protein Claudin-1 are dependent on its C-terminal cytoplasmic tail

In vertebrates, the positioning of the internal organs relative to the midline is asymmetric and evolutionarily conserved. A number of molecules have been shown to play critical roles in left-right patterning. Using representational difference analysis to identify genes that are differentially expressed on the left and right sides of the chick embryo, we cloned chick Claudin-1, an integral component of epithelial tight junctions. Here, we demonstrate that retroviral overexpression of Claudin-1, but not Claudin-3, on the right side of the chick embryo between HH stages 4 and 7 randomizes the direction of heart looping. This effect was not observed when Claudin-1 was overexpressed on the left side of the embryo. A small, but reproducible, induction of Nodal expression in the perinodal region on the right side of the embryo was noted in embryos that were injected with Claudin-1 retroviral particles on their right sides. However, no changes in Lefty,Pitx2 or cSnR expression were observed. In addition, Flectin expression remained higher in the left dorsal mesocardial folds of embryos with leftwardly looped hearts resulting from Claudin-1 overexpression on the right side of the embryo. We demonstrated that Claudin-1's C-terminal cytoplasmic tail is essential for this effect: mutation of a PKC phosphorylation site in the Claudin-1 C-terminal cytoplasmic domain at threonine-206 eliminates Claudin-1's ability to randomize the direction of heart looping. Taken together, our data provide evidence that appropriate expression of the tight junction protein Claudin-1 is required for normal heart looping and suggest that phosphorylation of its cytoplasmic tail is responsible for mediating this function.     

K+ channels involved in contractility of rabbit small intestine

Most excitable cells, including gastrointestinal smooth muscle cells, express several types of K+ channels. The aim of this study was to examine the types of K' channels involved in the contractility of longitudinal smooth muscle of rabbit small intestine in vitro. Spontaneous contractions and KCl-stimulated contractions were reduced by atropine, phentolamine, propranolol, suramin, tetrodotoxin and indomethacin. The amplitude and tone of spontaneous contractions were increased by apamin, charybdotoxin, iberiotoxin, E4031, tetraetylammonium (TEA) and BaCl2. The frequency of contractions was reduced in the presence of apamin and TEA and increased by charybdotoxin. It was found that 4-aminopyridine increased the tone of spontaneous contractions and reduced the amplitude and frequency of contractions. Glibenclamide did not modify the amplitude, frequency or tone of contractions. KCl-stimulated contractions were increased by E4031, were not modified by apamin, glibenclamide, NS1619 or diazoxide, and were reduced by charybdotoxin, TEA, 4-aminopyridine or BaCl2. These results suggest that both Ca2+-activated K+ channels of small and high conductance, and HERG K+ channels and inward rectifier K+ channels participate in spontaneous contractions of small intestine. On the other hand, voltage-dependent K+ channels, HERG K+ channels, inward rectifier K+ channels and high conductance Ca2+-activated K+ channels are involved in KCl-stimulated contractions.     

Evaluation of the efficacy of two mouthrinses formulated for the relief of xerostomia of diverse origin in adult subjects

doi: 10.1111/j.1741‐2358.2012.00626.x Evaluation of the efficacy of two mouthrinses formulated for the relief of xerostomia of diverse origin in adult subjects Objective: To evaluate the efficacy of two new mouthrinses in the reduction of xerostomía‐associated symptomatology. Background: Xerostomia is a common chronic health condition that affects a great number of adults and significantly deteriorates quality of life, such that treatment is necessary. Materials and methods: Sixty‐seven adult subjects of both sexes presenting xerostomia of diverse origin were selected. Mouthrinses were tested using a double‐blind, randomized, cross‐over clinical trial with an intervining wash out period. Results: The 100% of subjects presented sensation of dry mouth, and 86% stated sensation of thick saliva. Burning tongue sensation, need to drink liquids to swallow and the sensation of swallowing difficulty were recorded in more than 50% of the patients. The most frequent pathologies in the sample were depression, arthritis, and arterial hypertension. Results of the clinical tests showed that mouthrinse 1 relieves sensation of dry mouth, need to drink liquids, and swallowing difficulty. In contrast, mouthrinse 2 relieves only latter two symptoms. Both rinses were more effective in relieving xerostomía‐associated symptomatology in patients taking 3 or more medicines simultaneously. Conclusion: Both mouthrinses were effective in relieving various xerostomia symptoms, could be distributed at a low cost, thereby improving the quality of life of population affected.     

Non-invasive prenatal diagnosis of single-gene disorders from maternal blood

Prenatal diagnosis (PD) is available for pregnancies at risk of monogenic disorders. However, PD requires the use of invasive obstetric techniques for fetal-sample collection and therefore, involves a risk of fetal loss. Circulating fetal DNA in the maternal bloodstream is being used to perform non-invasive prenatal diagnosis (NIPD). NIPD is a challenging discipline because of the biological features of the maternal blood sample. Maternal blood is an unequal mixture of small (and fragmented) amounts of fetal DNA within a wide background of maternal DNA. For this reason, initial NIPD studies have been based on the analysis of specific paternally inherited fetal tracts not present in the maternal genome so as to ensure their fetal origin. Following this strategy, different NIPD studies have been carried out, such as fetal-sex assessment for pregnancies at risk of X-linked disorders, RhD determination, and analysis of single-gene disorders with a paternal origin. The study of the paternal mutation can be used for fetal diagnosis of dominant disorders or to more accurately assess the risk of an affected child in case of recessive diseases. Huntington's disease, cystic fibrosis, or achondroplasia are some examples of diseases studied using NIPD. New technologies are opening NIPD to the analysis of maternally inherited fetal tracts. NIPD of trisomy 21 is the latest study derived from the use of next-generation sequencing (NGS).     

Galactosylated Fucose Epitopes in Nematodes: INCREASED EXPRESSION IN A CAENORHABDITIS MUTANT ASSOCIATED WITH ALTERED LECTIN SENSITIVITY AND OCCURRENCE IN PARASITIC SPECIES

The modification of α1,6-linked fucose residues attached to the proximal (reducing-terminal) core N-acetylglucosamine residue of N-glycans by β1,4-linked galactose ("GalFuc" epitope) is a feature of a number of invertebrate species including the model nematode Caenorhabditis elegans. A pre-requisite for both core α1,6-fucosylation and β1,4-galactosylation is the presence of a nonreducing terminal N-acetylglucosamine; however, this residue is normally absent from the final glycan structure in invertebrates due to the action of specific hexosaminidases. Previously, we have identified two hexosaminidases (HEX-2 and HEX-3) in C. elegans, which process N-glycans. In the present study, we have prepared a hex-2;hex-3 double mutant, which possesses a radically altered N-glycomic profile. Whereas in the double mutant core α1,3-fucosylation of the proximal N-acetylglucosamine was abolished, the degree of galactosylation of core α1,6-fucose increased, and a novel Galα1,2Fucα1,3 moiety attached to the distal core N-acetylglucosamine residue was detected. Both galactosylated fucose moieties were also found in two parasitic nematodes, Ascaris suum and Oesophagostomum dentatum. As core modifications of N-glycans are known targets for fungal nematotoxic lectins, the sensitivity of the C. elegans double hexosaminidase mutant was assessed. Although this mutant displayed hypersensitivity to the GalFuc-binding lectin CGL2 and the N-acetylglucosamine-binding lectin XCL, the mutant was resistant to CCL2, which binds core α1,3-fucose. Thus, the use of C. elegans mutants aids the identification of novel N-glycan modifications and the definition of in vivo specificities of nematotoxic lectins with potential as anthelmintic agents.     

Antiviral compounds obtained from microalgae commonly used as carotenoid sources

Pressurized liquid extraction (PLE), an environmentally friendly technique, has been used to obtain antiviral compounds from microalgae commonly used as carotenoid sources: Haematococcus pluvialis and Dunaliella salina. The antiviral properties of PLE extracts (hexane, ethanol and water) were evaluated against herpes simplex virus type 1 (HSV-1) at different stages during viral infection. Pretreatment of Vero cells with 75?μg?mL^?1 of H. pluvialis ethanol extract inhibited virus infection by approximately 85%, whereas the same concentration of water and hexane extracts reduced the virus infectivity 75% and 50%, respectively. D. salina extracts were less effective than H. pluvialis extracts and presented a different behaviour since water and ethanol extracts produced a similar virus inhibition (65%). Moreover, H. pluvialis ethanol extract was also the most effective against HSV-1 intracellular replication. The antiviral activity of water PLE extracts was found to correlate with polysaccharides since the polysaccharide-rich fraction isolated from these extracts showed higher antiviral activity than the original water extracts. A gas chromatography-mass spectrometry (GC-MS) characterization of the H. pluvialis ethanol extract showed the antiviral activity of this extract could be partially related with the presence of short-chain fatty acids, although other compounds could be involved in this activity; meanwhile, in the case of D. salina ethanol extract other compounds seemed to be implied, such as: β-ionone, neophytadiene, phytol, palmitic acid and α-linolenic acid. The results demonstrate the use of PLE allows obtaining antiviral compounds from microalgae used as carotenoids sources, which gives the microalgae biomass an added value.     

Effectors of tridimensional cell morphogenesis and their evolution

One of the most challenging problems in biology resides in unraveling the molecular mechanisms, hardwired in the genome, that define and regulate the multiscale tridimensional organization of organs, tissues and individual cells. While works in cultured cells have revealed the importance of cytoskeletal networks for cell architecture, in vivo models are now required to explore how such a variety in cell shape is produced during development, in interaction with neighboring cells and tissues. The genetic analysis of epidermis development in Drosophila has provided an unbiased way to identify mechanisms remodeling the shape of epidermal cells, to form apical trichomes during terminal differentiation. Since hearing in vertebrates relies on apical cell extensions in sensory cells of the cochlea, called stereocilia, the mapping of human genes causing hereditary deafness has independently identified several factors required for this peculiar tridimensional organization. In this review, we summarized recent results obtained toward the identification of genes involved in these localized changes in cell shape and discuss their evolution throughout developmental processes and species.     

Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting

Functional analysis of genome sequences requires methods for cloning DNA of interest. However, existing methods, such as library cloning and screening, are too demanding or inefficient for high-throughput application to the wealth of genomic data being delivered by massively parallel sequencing. Here we describe direct DNA cloning based on the discovery that the full-length Rac prophage protein RecE and its partner RecT mediate highly efficient linear-linear homologous recombination mechanistically distinct from conventional recombineering mediated by Redαβ from lambda phage or truncated versions of RecET. We directly cloned all ten megasynthetase gene clusters (each 10–52 kb in length) from Photorhabdus luminescens into expression vectors and expressed two of them in a heterologous host to identify the metabolites luminmycin A and luminmide A/B. We also directly cloned cDNAs and exactly defined segments from bacterial artificial chromosomes. Direct cloning with full-length RecE expands the DNA engineering toolbox and will facilitate bioprospecting for natural products.     

Proliferating Cell Nuclear Antigen (PCNA) Interactions in Solution Studied by NMR

PCNA is an essential factor for DNA replication and repair. It forms a ring shaped structure of 86 kDa by the symmetric association of three identical protomers. The ring encircles the DNA and acts as a docking platform for other proteins, most of them containing the PCNA Interaction Protein sequence (PIP-box). We have used NMR to characterize the interactions of PCNA with several other proteins and fragments in solution. The binding of the PIP-box peptide of the cell cycle inhibitor p21 to PCNA is consistent with the crystal structure of the complex. A shorter p21 peptide binds with reduced affinity but retains most of the molecular recognition determinants. However the binding of the corresponding peptide of the tumor suppressor ING1 is extremely weak, indicating that slight deviations from the consensus PIP-box sequence dramatically reduce the affinity for PCNA, in contrast with a proposed less stringent PIP-box sequence requirement. We could not detect any binding between PCNA and the MCL-1 or the CDK2 protein, reported to interact with PCNA in biochemical assays. This suggests that they do not bind directly to PCNA, or they do but very weakly, with additional unidentified factors stabilizing the interactions in the cell. Backbone dynamics measurements show three PCNA regions with high relative flexibility, including the interdomain connector loop (IDCL) and the C-terminus, both of them involved in the interaction with the PIP-box. Our work provides the basis for high resolution studies of direct ligand binding to PCNA in solution.