Human Land use Threatens Endemic Wetland Species: The Case of Chorthippus lacustris (La Greca and Messina 1975) (Orthoptera: Acrididae) in Epirus, Greece

Chorthippus lacustris is an endemic grasshopper (Orthoptera) species in Epirus, Greece. Its population status, habitat characteristics, and relation to historical and current human land use are investigated. The species has a restricted and fragmented distribution pattern. Five locations, four within Pamvotida Lake basin and one in Lake Paramythia, cover a total of 0.12 km². It is strongly dependent on wet grasslands, flooded on a seasonal basis. The greatest population density is recorded in the site with the greatest diversity of dominant plant species. Ch. lacustris is estimated to have lost 85–99% of its habitat during the last 50 years due to wetland drainage. The main threat to the species survival is further habitat loss by urbanisation around Pamvotida Lake and by land conversion to agriculture in Paramythia Lake, even though both sites belong to the Natura 2000 network. The species status is Critically Endangered and it should be listed in Annex II of the Habitat Directive (92/43/EEC) as a priority species for conservation. Restoring wet grasslands, protecting them from further urbanisation and drainage, and monitoring species population are the main measures proposed for its conservation.     

Investigating the Structure and Dynamics of the PIK3CA Wild-Type and H1047R Oncogenic Mutant

The PIK3CA gene is one of the most frequently mutated oncogenes in human cancers. It encodes p110α, the catalytic subunit of phosphatidylinositol 3-kinase alpha (PI3Kα), which activates signaling cascades leading to cell proliferation, survival, and cell growth. The most frequent mutation in PIK3CA is H1047R, which results in enzymatic overactivation. Understanding how the H1047R mutation causes the enhanced activity of the protein in atomic detail is central to developing mutant-specific therapeutics for cancer. To this end, Surface Plasmon Resonance (SPR) experiments and Molecular Dynamics (MD) simulations were carried out for both wild-type (WT) and H1047R mutant proteins. An expanded positive charge distribution on the membrane binding regions of the mutant with respect to the WT protein is observed through MD simulations, which justifies the increased ability of the mutated protein variant to bind to membranes rich in anionic lipids in our SPR experiments. Our results further support an auto-inhibitory role of the C-terminal tail in the WT protein, which is abolished in the mutant protein due to loss of crucial intermolecular interactions. Moreover, Functional Mode Analysis reveals that the H1047R mutation alters the twisting motion of the N-lobe of the kinase domain with respect to the C-lobe and shifts the position of the conserved P-loop residues in the vicinity of the active site. These findings demonstrate the dynamical and structural differences of the two proteins in atomic detail and propose a mechanism of overactivation for the mutant protein. The results may be further utilized for the design of mutant-specific PI3Kα inhibitors that exploit the altered mutant conformation.     

Urotensin-II System in Genetic Control of Blood Pressure and Renal Function

Urotensin-II controls ion/water homeostasis in fish and vascular tone in rodents. We hypothesised that common genetic variants in urotensin-II pathway genes are associated with human blood pressure or renal function. We performed family-based analysis of association between blood pressure, glomerular filtration and genes of the urotensin-II pathway (urotensin-II, urotensin-II related peptide, urotensin-II receptor) saturated with 28 tagging single nucleotide polymorphisms in 2024 individuals from 520 families; followed by an independent replication in 420 families and 7545 unrelated subjects. The expression studies of the urotensin-II pathway were carried out in 97 human kidneys. Phylogenetic evolutionary analysis was conducted in 17 vertebrate species. One single nucleotide polymorphism (rs531485 in urotensin-II gene) was associated with adjusted estimated glomerular filtration rate in the discovery cohort (p = 0.0005). It showed no association with estimated glomerular filtration rate in the combined replication resource of 8724 subjects from 6 populations. Expression of urotensin-II and its receptor showed strong linear correlation (r = 0.86, p<0.0001). There was no difference in renal expression of urotensin-II system between hypertensive and normotensive subjects. Evolutionary analysis revealed accumulation of mutations in urotensin-II since the divergence of primates and weaker conservation of urotensin-II receptor in primates than in lower vertebrates. Our data suggest that urotensin-II system genes are unlikely to play a major role in genetic control of human blood pressure or renal function. The signatures of evolutionary forces acting on urotensin-II system indicate that it may have evolved towards loss of function since the divergence of primates.     

An immunogold evaluation of cortical actin reorganization in pubertal Sertoli cells in vitro after PMA treatment

The aim of the present study was to investigate stress fibers and cortical actin reorganization in pubertal Sertoli cells in vitro after PMA treatment. Actin was studied by means of immunogold labeling, using the 'Progressive Lowering of Temperature' technique (PLT). Actin rearrangement was evaluated by a quantitative analysis of the gold label distribution. Eight hours after addition of 10(-7) M PMA, rearrangement of cortical actin was minimal, but stress fiber perturbation was significant as shown by immunogold labeling distribution measurements. PMA-mediated F-actin reorganization and redistribution in non-neoplastic cells is discussed, since these phenomena have been closely linked with cell transformation.     

Molecular basis for the binding of competitive inhibitors of maize polyamine oxidase

Maize polyamine oxidase (MPAO), the only member of the polyamine oxidase (PAO) family whose three-dimensional structure is known, is characterized by a 30 A long U-shaped catalytic tunnel located between the substrate binding domain and the FAD. To shed light on the MPAO ligand binding mode, we studied the inhibition properties of linear diamines, agmatine, prenylagmatine (G3), G3 analogues, and guazatine, and analyzed the structural determinants of their biological activity. Linear diamines competitively inhibited MPAO, with the inhibitory activity increasing as a function of the number of methylene groups. With regard to the guanidino competitive inhibitors, including agmatine, G3, and G3 analogues, the presence of a hydrophobic substituent constitutes the principal factor influencing MPAO inhibition, as the addition of a hydrophobic substituent to the guanidino group of both G3 and G3 analogues greatly increases the inhibitory activity. Moreover, results obtained by a molecular modeling procedure indicated that in their preferred orientation, G3 analogues point the ammonium group toward the narrow entrance of the tunnel, while the terminal hydrophobic group is located within the large entrance. The high binding affinity for MPAO exhibited by G3 and G3 analogues bearing a prenyl group as a substituent on the guanidino moiety is in agreement with the observation that the prenyl group binds in a well-defined hydrophobic pocket, mainly formed by aromatic residues. Finally, docking simulations performed with the charged and uncharged forms of MPAO inhibitors indicate that the stereoelectronic properties of the MPAO active site are consistent with the binding of inhibitors in the protonated form.     

Hyaluronan and its binding proteins in the epithelium and intraluminal fluid of the bovine oviduct

Hyaluronan (HA) is involved in several important steps of sperm storage and of fertilization. This study investigates the presence and concentration of HA in oviductal fluid (ODF), together with the localization of HA and the presence of hyaluronan-binding proteins (HABPs) in the oviductal epithelium of normally cycling dairy heifers and cows. The concentration and amount of HA in ODF, collected over the course of several oestrous cycles via catheters placed in the isthmic and ampullar tubal segments, were measured using an ELISA. The concentration and amount of HA in ODF did not vary significantly between these anatomical regions, nor between the stages of the oestrous cycle (p > 0.05), although the amount of HA seemed to peak during oestrous. The most HA per day (2.9 +/- 0.64 microg, least square mean +/- SEM) was produced on the day of ovulation, whereas the lowest amount (1.25 +/- 0.68 microg) was produced 4 days before ovulation. To investigate the localization of HA, tissue samples were retrieved at well-defined stages of the oestrous cycle and from corresponding regions of the oviduct. Sections and protein extracts from the tissue samples were studied histochemically using biotinylated HABP and immunoblotted with fluorescein isothiocyanate (FITC)-HA, respectively. Presence of HA labelling in the oviductal epithelium was restricted to the sperm reservoir, a localization that seemed to be cycle-independent. The immunoblotting of samples from the lining epithelium revealed seven bands of HABPs. We confirm that the bovine oviduct produces HA and its binding proteins, and that HA is mainly localized to the epithelium of the sperm reservoir.     

Hexapeptide Tandem Repeats Dictate the Formation of Silkmoth Chorion,a Natural Protective Amyloid

Silkmoth chorion is a fibrous structure composed mainly of two major protein classes, families A and B. Both families of silkmoth chorion proteins present a highly conserved, in sequence and in length, central domain, consisting of Gly-rich tandem hexapeptide repetitive segments, flanked by two more variable N-terminal and C-terminal arms. Primary studies identified silkmoth chorion as a functional protective amyloid by unveiling the amyloidogenic properties of the central domain of both protein families. In this work, we attempt to detect the principal source of amyloidogenicity of the central domain by focusing on the role of the tandem hexapeptide sequence repeats. Concurrently, we discuss a possible mechanism for the self-assembly of class A protofilaments, suggesting that the aggregation-prone hexapeptide building blocks may fold into a triangle-shaped β-helical structure.     

Study of potent cytotoxic activity of Helleborus cyclophyllus Boiss against a human adenocarcinoma cell line

Helleborus cyclophyllus Boiss is a rhizomatous plant species, with strong allelochemical properties, that has been used since ancient times for its therapeutic properties. In the present study we investigated the ability of an aqueous-soluble fraction of the methanol extract of H. cyclophyllus Boiss leaves, to induce apoptotic cell death on A549 human bronchial epithelial adenocarcinoma cells. A primary human lung fibroblasts’ cell line was used as a model of normal-healthy cells for comparison. Cell morphology was examined after appropriate staining, cytotoxic activity of the extract was determined by the MTT assay, the type of cell death was analyzed by flow cytometry, confirmation of apoptosis was evaluated with the analysis of caspase-3, PARP1 by western blotting, while the chemical composition was assessed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). H. cyclophyllus Boiss extract was selectively active on A549 cells inducing significant morphological changes, even at low concentrations. Characteristic morphological alterations included the release of vesicular formations from A549 cell membranes (ectosomes), detachment of cells from their substrate, generation of a large vesicle into the cytoplasm (thanatosome) and the formation of apoptotic bodies. The selective apoptotic action on treated cells was also confirmed by biochemical criteria. Low concentrations, however, did not affect normal cells. The phytochemical analysis of the extract revealed the presence of cardiac glucosides, bufadienolides and phytoecdysteroids. To the best of our knowledge, the above-mentioned sequences of events leading selectively cancer cells to apoptosis, has not been reported before.Electronic supplementary materialThe online version of this article (10.1007/s10616-020-00425-4) contains supplementary material, which is available to authorized users.     

The Pattern of Change in the Abundances of Specific Bacterioplankton Groups Is Consistent across Different Nutrient-Enriched Habitats in Crete

A common source of disturbance for coastal aquatic habitats is nutrient enrichment through anthropogenic activities. Although the water column bacterioplankton communities in these environments have been characterized in some cases, changes in α-diversity and/or the abundances of specific taxonomic groups across enriched habitats remain unclear. Here, we investigated the bacterial community changes at three different nutrient-enriched and adjacent undisturbed habitats along the north coast of Crete, Greece: a fish farm, a closed bay within a town with low water renewal rates, and a city port where the level of nutrient enrichment and the trophic status of the habitat were different. Even though changes in α-diversity were different at each site, we observed across the sites a common change pattern accounting for most of the community variation for five of the most abundant bacterial groups: a decrease in the abundance of the Pelagibacteraceae and SAR86 and an increase in the abundance of the Alteromonadaceae, Rhodobacteraceae, and Cryomorphaceae in the impacted sites. The abundances of the groups that increased and decreased in the impacted sites were significantly correlated (positively and negatively, respectively) with the total heterotrophic bacterial counts and the concentrations of dissolved organic carbon and/or dissolved nitrogen and chlorophyll α, indicating that the common change pattern was associated with nutrient enrichment. Our results provide an in situ indication concerning the association of specific bacterioplankton groups with nutrient enrichment. These groups could potentially be used as indicators for nutrient enrichment if the pattern is confirmed over a broader spatial and temporal scale by future studies.     

An N-terminal pro-atrial natriuretic peptide (NT-proANP) ‘aggregation-prone’ segment involved in isolated atrial amyloidosis

Isolated atrial amyloidosis (IAA) is a common localized form of amyloid deposition within the atria of the aging heart. The main constituents of amyloid fibrils are atrial natriuretic peptide (ANP) and the N-terminal part of its precursor form (NT-proANP). An ‘aggregation-prone’ heptapeptide (¹¹⁴KLRALLT¹²⁰) was located within the NT-proANP sequence. This peptide self-assembles into amyloid-like fibrils in vitro, as electron microscopy, X-ray fiber diffraction, ATR FT-IR spectroscopy and Congo red staining studies reveal. Consequently, remedies/drugs designed to inhibit the aggregation tendency of this ‘aggregation-prone’ segment of NT-proANP may assist in prevention/treatment of IAA, congestive heart failure (CHF) or atrial fibrillation (AF).