Presymbiotic growth and sporal morphology are affected in the arbuscular mycorrhizal fungus Gigaspora margarita cured of its endobacteria

Some arbuscular mycorrhizal fungi contain endocellular bacteria. In Gigaspora margarita BEG 34, a homogenous population of beta-Proteobacteria is hosted inside the fungal spore. The bacteria, named Candidatus Glomeribacter gigasporarum, are vertically transmitted through fungal spore generations. Here we report how a protocol based on repeated passages through single-spore inocula caused dilution of the initial bacterial population eventually leading to cured spores. Spores of this line had a distinct phenotype regarding cytoplasm organization, vacuole morphology, cell wall organization, lipid bodies and pigment granules. The absence of bacteria severely affected presymbiotic fungal growth such as hyphal elongation and branching after root exudate treatment, suggesting that Ca. Glomeribacter gigasporarum is important for optimal development of its fungal host. Under laboratory conditions, the cured fungus could be propagated, i.e. could form mycorrhizae and sporulate, and can therefore be considered as a stable variant of the wild type. The results demonstrated that - at least for the G. margarita BEG 34 isolate - the absence of endobacteria affects the spore phenotype of the fungal host, and causes delays in the growth of germinating mycelium, possibly affecting its ecological fitness. This cured line is the first manipulated and stable isolate of an arbuscular mycorrhizal fungus.     

New species of Moravecia (Nematoda: Dracunculoidea) from body cavity of marine perciform fish Percophis brasiliensis in Argentina

A new species of dracunculoid nematode, Moravecia argentinensis n. sp. (Guyanemidae), is described from the blood vessels and body cavity of Brazilian flathead, Percophis brasiliensis Quoy et Gaimard (Perciformes: Percophidae), from off the coast of Argentina. The new species differs from the only other congeneric species, Moravecia australiensis Ribu et Lester, 2004, mainly in the number (14) of cephalic papillae, the length ratio of the muscular and glandular portions of esophagus (1:1-2), more numerous (3-5 pairs) genital papillae in the male, and the presence of a sclerotized copulatory plate. This is the second known species of Moravecia Ribu et Lester, 2004 and the first species of this genus reported from South American waters.     

Effects of ACTH and 3′,5′-cyclic purine nucleotides on the morphology and metabolism of normal adult human adrenocortical cells in primary tissue culture

Summary Stereological studies showed that treatment of normal adult human adrenocortical cells in primary culture with ACTH or cyclic-AMP for 2 days results in similar increases in the volume of cells, of the mitochondrial and membrane space compartments and of the surface area of the smooth endoplasmic reticulum and mitochondrial cristae, and decrease in the lipid content of the cells. These changes were more marked after 8 days of treatment. Treatment for 2 days with cyclic-GMP had no striking effects on cell ultrastructure, whereas an 8-day treatment led to ultrastructural changes similar to those obtained after 2 days of ACTH-or cyclic-AMP-treatment. A discrete population of untreated cortical cells maintained a slow proliferation that was not effected by exposure to cyclic-GMP, but was significantly increased in cultures treated with ACTH or cyclic-AMP. Radioimmunological studies showed that untreated cortical cells kept secreting progesterone and cortisol and that ACTH, but neither cyclic nucleotide, increased the secretion rate per cell of both hormones. These results assign a major role to cyclic-AMP and a minor one to cyclic-GMP in the mediation of the differentiation-promoting and trophic effects, but not in the steroidogenic effects of ACTH on the human adrenal cortex.The authors wish to thank Miss A. Coi and Mr. G. Gottardo for their technical assistance. These investigations were partly supported by a contract with CNR-Italy (CT 74.00226/115.3439)     

Unraveling the organization of the internal nuclear matrix: RNA-dependent anchoring of NuMA to a lamin scaffold

Using quantitative immunoelectron microscopy we show here that when the nuclear matrix is isolated from rat hepatocytes in the presence of an inhibitor of RNase activity both lamins and the nuclear mitotic apparatus protein (NuMA) preferentially localize within the electron-dense domains of the internal nuclear matrix (INM). After RNA digestion NuMA undergoes a sharp depletion, while labeling by an antibody against lamins A and C within the electron-transparent regions increases, suggesting that a subset of lamin epitopes is masked by the interaction with RNA. We were able to explain this result by visualizing for the first time a thin web of lamin protofibrils which connects the electron-dense regions. Confirmation of these changes has been obtained by immunoblot analysis and confocal microscopy. As RNA digestion results both in the release of NuMA and in the collapse of the INM, we propose that a fraction of nuclear RNA brings about the association of NuMA islands with a lamin scaffold and that this interaction is required to maintain the latter in a state of high molecular dispersion.     

The Local Complement Activation on Vascular Bed of Patients with Systemic Sclerosis: A Hypothesis-Generating Study

ObjectiveThe role of complement system in the pathogenesis of systemic sclerosis (SSc) has been debated during the last decade but an evident implication in this disease has never been found. We carried out an explorative study on SSc patients to evaluate the expression of soluble and local C5b-9 complement complex and its relation with a complement regulator, the Membrane Cofactor Protein (MCP, CD46) on skin vascular bed as target distinctive of SSc disease. We also analyzed two polymorphic variants in the complement activation gene cluster involving the MCP region.MethodsC5b-9 plasma levels of SSc patients and healthy subjects were analyzed by ELISA assay. Archival skin biopsies of SSc patients and controls were subjected to immunofluorescence analysis to detect C5b-9 and MCP on vascular endothelial cells. The expression of MCP was validated by immunoblot analysis with specific antibody. Polymorphic variants in the MCP gene promoter were tested by a quantitative PCR technique-based allelic discrimination method.ResultsEven though circulating levels of C5b-9 did not differ between SSc and controls, C5b-9 deposition was detected in skin biopsies of SSc patients but not in healthy subjects. MCP was significantly lower in skin vessels of SSc patients than in healthy controls and was associated with the over-expression of two polymorphic variants in the MCP gene promoter, which has been related to more aggressive phenotypes in other immune-mediated diseases.ConclusionsOur results firsty document the local complement activation with an abnormal expression of MCP in skin vessels of SSc patients, suggesting that a subset of SSc patients might be exposed to more severe organ complications and clinical evolution due to abnormal local complement activation.     

A Multicenter,Randomized, Controlled Trial of Osteopathic Manipulative Treatment on Preterms

BackgroundDespite some preliminary evidence, it is still largely unknown whether osteopathic manipulative treatment improves preterm clinical outcomes.ResultsA total of 695 newborns were randomly assigned to either the study group (n= 352) or the control group (n=343). A statistical significant difference was observed between the two groups for the primary outcome (13.8 and 17.5 days for the study and control group respectively, p<0.001, effect size: 0.31). Multivariate analysis showed a reduction of the length of stay of 3.9 days (95% CI -5.5 to -2.3, p<0.001). Furthermore, there were significant reductions with treatment as compared to usual care in cost (difference between study and control group: 1,586.01€; 95% CI 1,087.18 to 6,277.28; p<0.001) but not in daily weight gain. There were no complications associated to the intervention.ConclusionsOsteopathic treatment reduced significantly the number of days of hospitalization and is cost-effective on a large cohort of preterm infants.     

Mapping of p140Cap Phosphorylation Sites: The EPLYA and EGLYA Motifs Have a Key Role in Tyrosine Phosphorylation and Csk Binding,and Are Substrates of the Abl Kinase

Protein phosphorylation tightly regulates specific binding of effector proteins that control many diverse biological functions of cells (e. g. signaling, migration and proliferation). p140Cap is an adaptor protein, specifically expressed in brain, testis and epithelial cells, that undergoes phosphorylation and tunes its interactions with other regulatory molecules via post-translation modification. In this work, using mass spectrometry, we found that p140Cap is in vivo phosphorylated on tyrosine (Y) within the peptide GEGLpYADPYGLLHEGR (from now on referred to as EGLYA) as well as on three serine residues. Consistently, EGLYA has the highest score of in silico prediction of p140Cap phosphorylation. To further investigate the p140Cap function, we performed site specific mutagenesis on tyrosines inserted in EGLYA and EPLYA, a second sequence with the same highest score of phosphorylation. The mutant protein, in which both EPLYA/EGLYA tyrosines were converted to phenylalanine, was no longer tyrosine phosphorylated, despite the presence of other tyrosine residues in p140Cap sequence. Moreover, this mutant lost its ability to bind the C-terminal Src kinase (Csk), previously shown to interact with p140Cap by Far Western analysis. In addition, we found that in vitro and in HEK-293 cells, the Abelson kinase is the major kinase involved in p140Cap tyrosine phosphorylation on the EPLYA and EGLYA sequences. Overall, these data represent an original attempt to in vivo characterise phosphorylated residues of p140Cap. Elucidating the function of p140Cap will provide novel insights into its biological activity not only in normal cells, but also in tumors.     

P130Cas-associated protein (p140Cap) as a new tyrosine-phosphorylated protein involved in cell spreading

Integrin-mediated cell adhesion stimulates a cascade of signaling pathways that control cell proliferation, migration, and survival, mostly through tyrosine phosphorylation of signaling molecules. p130Cas, originally identified as a major substrate of v-Src, is a scaffold molecule that interacts with several proteins and mediates multiple cellular events after cell adhesion and mitogen treatment. Here, we describe a novel p130Cas-associated protein named p140Cap (Cas-associated protein) as a new tyrosine phosphorylated molecule involved in integrin- and epidermal growth factor (EGF)-dependent signaling. By affinity chromatography of human ECV304 cell extracts on a MBP-p130Cas column followed by mass spectrometry matrix-assisted laser desorption ionization/time of flight analysis, we identified p140Cap as a protein migrating at 140 kDa. We detected its expression in human, mouse, and rat cells and in different mouse tissues. Endogenous and transfected p140Cap proteins coimmunoprecipitate with p130Cas in ECV304 and in human embryonic kidney 293 cells and associate with p130Cas through their carboxy-terminal region. By immunofluorescence analysis, we demonstrated that in ECV304 cells plated on fibronectin, the endogenous p140Cap colocalizes with p130Cas in the perinuclear region as well as in lamellipodia. In addition p140Cap codistributes with cortical actin and actin stress fibers but not with focal adhesions. We also show that p140Cap is tyrosine phosphorylated within 15 min of cell adhesion to integrin ligands. p140Cap tyrosine phosphorylation is also induced in response to EGF through an EGF receptor dependent-mechanism. Interestingly expression of p140Cap in NIH3T3 and in ECV304 cells delays the onset of cell spreading in the early phases of cell adhesion to fibronectin. Therefore, p140Cap is a novel protein associated with p130Cas and actin cytoskeletal structures. Its tyrosine phosphorylation by integrin-mediated adhesion and EGF stimulation and its involvement in cell spreading on matrix proteins suggest that p140Cap plays a role in controlling actin cytoskeleton organization in response to adhesive and growth factor signaling.     

The influence of the polar head and the hydrophobic chain on the skin penetration enhancement effect of poly(ethylene glycol) derivatives

The effect of a homologue series of nonionic surfactants, namely poly(ethylene glycol) (PEG) fatty acid esters, differing in oxyethylene (PEG 8, PEG 12, and PEG 40) and fatty acid (stearate, mono and di-laurate, and mono and di-oleate) chain lengths, on in vitro skin permeability of ketoprofen (KTP) vehicled in plasters was investigated. The drug diffusion through hairless mouse skin as well as the effect of the surfactant type and strength was studied by Franz diffusion cells and ATR-FTIR spectroscopy. The use of PEG stearate series revealed that the surfactant with the largest polar head, namely PEG 40, was ineffective in enhancing the skin permeation of KTP, independently of the plaster concentrations. The effect of the hydrophobic chain was investigated only by using the shortest oxyethylene chains. The experimental results revealed that the oxyethylene chain length of surfactants appeared to be more influent than the alkyl chain. The prediction of the absorption enhancing capability of these PEG derivatives appeared related to the vehicle other than the proper combination of the number of ethylene oxide groups and alkyl groups.