Karyology,pollen and seed morphology,and distribution of eight endangered species in the Veneto region (Northern Italy)

ABSTRACT

Eight endangered species of the Veneto region were examined from the karyological and micromorphological points of view. Their geographical distribution, the exsiccata of Herbarium Venetum (HV-PAD) and conservation problems were also considered. These species are: Cortusa matthioli L., a taxon sporadically distributed in Veneto with 2n=24 chromosomes; Calianthemum kernerianum Freyn ex Kerner, only known on Mt. Baldo (Verona) and with 2n=4x=32; Scrophularia vernalis L. with 2n=28; Kosteletzkya pentacarpos (L.) Ledeb., an entity with no regional conservation regulations and with 2n=34; Athamanta vestina A.Kerner, endemic to Italy and with 2n=22; Hottonia palustris L. with 2n=20 and in danger because of the destruction of its habitat; Sagittaria sagttifolia L. with 2n=22; Trapa natans L., a protected species in Veneto with 2n=36 and 2n=48.     

Immunization with extracellular vesicles excreted by Toxoplasma gondii confers protection in murine infection,activating cellular and humoral responses

The study aim was to analyze whether microvesicles and exosomes, named extracellular vesicles (EVs), purified from Toxoplasma gondii are able to stimulate the protective immunity of experimental mice when administered, as challenge, a highly virulent strain. EVs excreted from T. gondii tachyzoites (RH strain) were purified by chromatography and used for immunization assays in inbred mouse groups (EV-IM). Chronic infected (CHR) and naive (NI) mice were used as control groups, since the immune response is well known. After immunizations, experimental groups were challenged with 100 tachyzoites. Next, parasitemias were determined by real-time PCR (qPCR), and survival levels were evaluated daily. The humoral response was analyzed by detection of IgM, IgG, IgG1 and IgG2a, and opsonization experiments. The cellular response was evaluated in situ by immunohistochemistry on IFN-γ, IL-10, TNF-α and IL-17 expression in cells of five organs (brain, heart, liver, spleen and skeletal muscles). EV immunization reduced parasitemia and increased the survival index in two mouse lineages (A/Sn and BALB/c) infected with a lethal T. gondii strain. EV-IM mice had higher IgG1 levels than IgM or IgG2a. IgGs purified from sera of EV-IM mice were able to opsonize tachyzoites (RH strain), and mice that received these parasites had lower parasitemias, and mortality was delayed 48 h, compared with the same results from those receiving parasites opsonized with IgG purified from NI mice. Brain and spleen cells from EV-IM mice more highly expressed IFN-γ, IL-10 and TNF-α. In conclusion, EV-immunization was capable of inducing immune protection, eliciting high production of IgG1, IFN-γ, IL-10 and TNF-α.     

Absolute quantitation of endogenous proteins with precision and accuracy using a capillary Western system

Precise and accurate quantification of protein expression levels in a complex biological setting is challenging. Here, we describe a method for absolute quantitation of endogenous proteins in cell lysates using an automated capillary immunoassay system, the size-based Simple Western system (recently developed by ProteinSimple). The method was able to accurately measure the absolute amounts of target proteins at picogram or sub-picogram levels per nanogram of cell lysates. The measurements were independent of the cell matrix or the cell lysis buffer and were not affected by different antibody affinities for their specific epitopes. We then applied this method to quantitate absolute levels of expression of protein kinase C (PKC) isoforms in LNCaP and U937 cells, two cell lines used extensively for probing the downstream biological responses to PKC targeted ligands. Our absolute quantitation confirmed the predominance of PKCδ in both cells, supporting the important functional role of this PKC isoform in these cell lines. The method described here provides an approach to accurately quantitate levels of protein expression and correlate protein level with function. In addition to enhanced accuracy relative to conventional Western analysis, it circumvents the distortions inherent in comparison with signal intensities from different antibodies with different affinities.     

Stocking efficiency and the effects of diet preconditioning on the post-release adaptation of hatchery-reared juveniles of Atlantic salmon (Salmo salar L.) in an Atlantic temperate stream

This study explores the stocking efficiency of Atlantic salmon juveniles in a 4th order stream located at the southern limit of its Eastern Atlantic distribution. Moreover, with the idea of implementing new protocols and methods to improve ??traditional?? hatchery practices and rear a more ??wild?? like fish, the study examines the response of this species to natural diet preconditioning prior to release. The field study indicated that most juveniles disappeared from the stocked reaches within two months of their release, resulting in a recapture rate after a year of 0.0?C1.1?%. There were no differences in stocked juvenile??s density along the stream during the first months following stocking. The preconditioning experiments showed that juveniles of Atlantic salmon suffered a significant decrease in their condition factor when diet was changed from artificial pellets to live preys, either if the change occurred before (i.e., in a manipulative experiment) or after their release in the stream. Although weight, length, condition factor and specific growth rates reached by juveniles at the end of the rearing period were higher in individuals fed on pellets than in those fed on macroinvertebrates, differences disappeared only a week after their release in the stream. These results may have implications regarding the stocking success when releasing hatchery-reared Atlantic salmons in suboptimal environments. In particular, the study indicates that stocked salmon adaptation in the selected stream may not be improved by short conditioning periods to natural diet prior to their release, while we discuss potential explanations for the observed results.     

Malvidin,a red wine polyphenol,modulates mammalian myocardial and coronary performance and protects the heart against ischemia/reperfusion injury

A moderate red wine consumption and a colored fruit-rich diet protect the cardiovascular system, thanks to the presence of several polyphenols. Malvidin-3-0-glucoside (malvidin), an anthocyanidine belonging to polyphenols, is highly present in red grape skin and red wine. Its biological activity is poorly characterized, although a role in tumor cell inhibition has been found. To analyze whether and to which extent, like other food-derived polyphenols, malvidin affects the cardiovascular function, in this study, we have performed a quantitative analysis by high-performance liquid chromatography of polyphenolic content of red grape skins extract, showing that it contains a high malvidin amount (63.93±12.50 mg/g of fresh grape skin). By using the isolated and Langendorff perfused rat heart, we found that the increasing doses (1–1000 ng/ml) of the extract induced positive inotropic and negative lusitropic effects associated with coronary dilation. On the same cardiac preparations, we observed that malvidin (10^?10–10^?6 mol/L) elicited negative inotropism and lusitropism and coronary dilation. Analysis of mechanism of action revealed that malvidin-dependent cardiac effects require the activation of the phosphatidylinositol 3-kinase (PI3K)/nitric oxide (NO)/cGMP/PKG pathway and are associated with increased intracellular cGMP and the phosphorylation of endothelial NO synthase (eNOS), PI3K–AKT, ERK1/2, and GSK-3β. AKT and eNOS phosphorylation was confirmed in human umbilical vein endothelial cell. We also found that malvidin act as a postconditioning agent, being able to elicit cardioprotection against ischemia/reperfusion damages. Our results show the cardioactivity of polyphenols-rich red grape extracts and indicate malvidin as a new cardioprotective principle. This is of relevance not only for a better clarification of the beneficial cardiovascular effects of food-derived polyphenols but also for nutraceutical research.     

Somatic meiosis in the life history of Bonnemaisonia asparagoides and Bonnemaisonia clavata (Bonnemaisoniales,Rhodophyta) from the Iberian peninsula

Carpospores isolated from Bonnemaisonia asparagoides and Bonnemaisonia clavata (Bonnemaisoniaceae, Rhodophyta) and grown in culture developed into their respective ‘Hymenoclonium’ prostrate phases. In both species, young gametophytes were initiated directly on the prostrate phase from tetrasporangia-like protuberances. Comparison of the relative fluorescence area (rfa) of nuclear DNA over the sequence of life-history stages indicated that the lowest ploidy levels (1–2C) occurred in the gametophytes, whereas the lowest ploidy levels in the prostrate phases were 2–4C. Rfa data demonstrated that meiosis occurred in the tetrasporangia-like protuberances where 1C values were recorded. The present observations establish that B. asparagoides and B. clavata have a heteromorphic diplohaplontic life history, which involves a haploid gametophyte produced directly on a diploid prostate phase after somatic meiosis. We conclude that the life history of these taxa corresponds to the Lemanea-type. This indicates that the life history of several Bonnemaisoniales with a ‘Hymenoclonium’ phase but lacking tetrasporangia requires re-investigation.     

Polymer based biosensor for rapid electrochemical detection of virus infection of human cells

The demand in the field of medical diagnostics for simple, cost efficient and disposable devices is growing. Here, we present a label free, all-polymer electrochemical biosensor for detection of acute viral disease. The dynamics of a viral infection in human cell culture was investigated in a micro fluidic system on conductive polymer PEDOT:TsO microelectrodes by electrochemical impedance spectroscopy and video time lapse microscopy. Employing this sensitive, real time electrochemical technique, we could measure the immediate cell response to cytomegalovirus, and detect an infection within 3h, which is several hours before the cytopathic effect is apparent with conventional imaging techniques. Atomic force microscopy and scanning ion conductance microscopy imaging consolidate the electrochemical measurements by demonstrating early virus induced changes in cell morphology of apparent programmed cell death.     

Cu(II) mediates kinetically distinct, non-amyloidogenic aggregation of amyloid-beta peptides

Cu(II) ions are implicated in the pathogenesis of Alzheimer disease by influencing the aggregation of the amyloid-β (Aβ) peptide. Elucidating the underlying Cu(II)-induced Aβ aggregation is paramount for understanding the role of Cu(II) in the pathology of Alzheimer disease. The aim of this study was to characterize the qualitative and quantitative influence of Cu(II) on the extracellular aggregation mechanism and aggregate morphology of Aβ(1-40) using spectroscopic, microelectrophoretic, mass spectrometric, and ultrastructural techniques. We found that the Cu(II):Aβ ratio in solution has a major influence on (i) the aggregation kinetics/mechanism of Aβ, because three different kinetic scenarios were observed depending on the Cu(II):Aβ ratio, (ii) the metal:peptide stoichiometry in the aggregates, which increased to 1.4 at supra-equimolar Cu(II):Aβ ratio; and (iii) the morphology of the aggregates, which shifted from fibrillar to non-fibrillar at increasing Cu(II):Aβ ratios. We observed dynamic morphological changes of the aggregates, and that the formation of spherical aggregates appeared to be a common morphological end point independent on the Cu(II) concentration. Experiments with Aβ(1-42) were compatible with the conclusions for Aβ(1-40) even though the low solubility of Aβ(1-42) precluded examination under the same conditions as for the Aβ(1-40). Experiments with Aβ(1-16) and Aβ(1-28) showed that other parts than the Cu(II)-binding His residues were important for Cu(II)-induced Aβ aggregation. Based on this study we propose three mechanistic models for the Cu(II)-induced aggregation of Aβ(1-40) depending on the Cu(II):Aβ ratio, and identify key reaction steps that may be feasible targets for preventing Cu(II)-associated aggregation or toxicity in Alzheimer disease.     

Production of Polyclonal Antibodies to the Recombinant Potato virus M (PVM) Non‐structural Triple Gene Block Protein 1 and Coat Protein

The genes encoding the coat protein (CP) and triple gene block protein 1 (TGBp1) of Potato virus M (PVM) were cloned into expression vector pET‐45b(+) (N‐terminal 6xHis tag) and expressed in E. coli Rosetta gami‐2(DE3). The purified recombinant antigens were used for raising polyclonal antibodies. The antibodies against recombinant CP were successfully used in Western blot analysis, plate‐trapped ELISA and DAS‐ELISA as a coating for PVM detection in infected potato leaf samples. The antibodies against recombinant non‐structural protein detected the TGBp1 only in Western blot analysis. This is the first report of the production of polyclonal antibodies against recombinant coat protein and TGBp1 of PVM and their use for detecting the virus.     

Entomopathogenic fungi from Argentina for the control of Schistocerca cancellata (Orthoptera: Acrididae) nymphs: fungal pathogenicity and enzyme activity

The South American locust Schistocerca cancellata (Serville) was the most serious agricultural pest in Argentina during the first half of the last century and remains as a threat when preventive control measures are relaxed in the outbreak area. In this study, we analysed in the laboratory, the effectiveness of 26 fungal strains (isolated from both insects and soil collected in Argentina) for S. cancellata control and determined the relationship between the chitinase, protease and lipase levels in these fungi and their insecticidal activities. We observed that Beauveria bassiana (isolate LPSC 1067) caused the highest mortality (90±1.03%), the highest values of chitinolytic, proteolytic and lipolytic activity were 6.13±0.05, 2.56±0.11 and 2.33±0.47, respectively, and the lowest median lethal time was 5.96 days. This is the first time that a wide variability in chitinase, protease and lipase activity as well as in virulence has been reported in a representative sample of different entomopathogenic fungal strains from Argentina.