Caveolin‐1 down‐regulation is required for Wnt5a‐Frizzled 2 signalling in Ha‐RasV12‐induced cell transformation

Caveolin‐1 (Cav1) is down‐regulated during MK4 (MDCK cells harbouring inducible Ha‐Ras^V12 gene) transformation by Ha‐Ras^V12. Cav1 overexpression abrogates the Ha‐Ras^V12‐driven transformation of MK4 cells; however, the targeted down‐regulation of Cav1 is not sufficient to mimic this transformation. Cav1‐silenced cells, including MK4/shCav1 cells and MDCK/shCav1 cells, showed an increased cell area and discontinuous junction‐related proteins staining. Cellular and mechanical transformations were completed when MDCK/shCav1 cells were treated with medium conditioned by MK4 cells treated with IPTG (MK4+I‐CM) but not with medium conditioned by MK4 cells. Nanoparticle tracking analysis showed that Ha‐Ras^V12‐inducing MK4 cells increased exosome‐like microvesicles release compared with their normal counterparts. The cellular and mechanical transformation activities of MK4+I‐CM were abolished after heat treatment and exosome depletion and were copied by exosomes derived from MK4+I‐CM (MK4+I‐EXs). Wnt5a, a downstream product of Ha‐Ras^V12, was markedly secreted by MK4+I‐CM and MK4+I‐EXs. Suppression of Wnt5a expression and secretion using the porcupine inhibitor C59 or Wnt5a siRNA inhibited the Ha‐Ras^V12‐ and MK4+I‐CM‐induced transformation of MK4 cells and MDCK/shCav1 cells, respectively. Cav1 down‐regulation, either by Ha‐Ras^V12 or targeted shRNA, increased frizzled‐2 (Fzd2) protein levels without affecting its mRNA levels, suggesting a novel role of Cav1 in negatively regulating Fzd2 expression. Additionally, silencing Cav1 facilitated the internalization of MK4+I‐EXs in MDCK cells. These data suggest that Cav1‐dependent repression of Fzd2 and exosome uptake is potentially relevant to its antitransformation activity, which hinders the activation of Ha‐Ras^V12‐Wnt5a‐Stat3 pathway. Altogether, these results suggest that both decreasing Cav1 and increasing exosomal Wnt5a must be implemented during Ha‐Ras^V12‐driven cell transformation.     

Stochastic Models and Numerical Algorithms for a Class of Regulatory Gene Networks

Regulatory gene networks contain generic modules, like those involving feedback loops, which are essential for the regulation of many biological functions (Guido et al. in Nature 439:856–860, 2006). We consider a class of self-regulated genes which are the building blocks of many regulatory gene networks, and study the steady-state distribution of the associated Gillespie algorithm by providing efficient numerical algorithms. We also study a regulatory gene network of interest in gene therapy, using mean-field models with time delays. Convergence of the related time-nonhomogeneous Markov chain is established for a class of linear catalytic networks with feedback loops.     

Long‐term regeneration of a tropical plant community after sand mining

Sandy coastal plant communities in tropical regions have been historically under strong anthropic pressure. In Brazil, these systems shelter communities with highly plastic plant species. However, the potential of these systems to regenerate without human assistance after disturbances has hardly been examined. We determined the natural regeneration of a coastal sandy plain vegetation (restinga) in Brazil, 16 years after the end of sand removal. We inventoried 38 plots: 20 within a sand‐mined site and 18 in an adjacent undisturbed site. We expected lower diversity values in the sand‐mined site compared to the undisturbed site, but similar species composition between the two sites due to the spatial proximity of the two sites and the high plasticity of restinga species. Species were ranked using abundance and importance value index in both sites, and comparisons were performed using Rényi entropy profiles, rarefaction curves, principal component analysis, and redundancy analysis. Species composition and dominant species differed markedly between the two sites. Bromeliads and Clusia hilariana, well‐known nurse plants, dominated the undisturbed site but were almost absent in the regenerating site. Species richness did not differ between both sites, but diversity was higher in the undisturbed site. Within‐site composition differences in the mined area were associated with field characteristics. Interestingly, species classified as subordinate or rare in the undisturbed site became dominants in the regenerating site. These newer dominants in the sand‐mined site are not those known as nurse plants in other restingas, thus yielding strong implications for restoration.     

Prey stage preference and functional response of Euseius hibisci to Tetranychus urticae (Acari: Phytoseiidae, Tetranychidae)

The aims of this study were: (a) determine the prey stage preference of female Euseius hibisci (Chant) (Phytoseiidae) at constant densities of different stages of Tetranychus urticae Koch (Tetranychidae), (b) assess the functional response of the predator females to the varying densities of eggs, larvae, or protonymphs of T. urticae, and (c) estimate the functional response of E. hibisci when pollen of Ligustrum ovalifolium was present as well. We conducted experiments on excised pieces of strawberry leaf arenas (Fragaria ananassa) under laboratory conditions of 25 ± 2 °C, 60 ± 5% RH and 12 h photophase. Our results indicated that the predator consumed significantly more prey eggs than other prey stages. Consumption of prey deutonymphs and adults was so low that they were excluded from the non-choice functional response experiments. The functional response on all food items was of type II. The two parameters of the functional response were estimated for each prey type by means of the adjusted non-linear regression model. The highest estimated value a (instantaneous rate of discovery) and the lowest value of T_h (handling time, including digestion) were found for the predator feeding on prey eggs, and a was lowest and T_h highest when fed protonymphs. Using the jack-knife method, the values for the functional response parameters were estimated. The values of a and T_h produced by the model were similar among all prey types except for the eggs, which were different. Using pollen simultaneously with prey larvae decreased the consumption of the latter over the full range of prey densities The suitability of this predator for biological control of T. urticae on strawberry is discussed.     

Lipophosphoglycans from Leishmania amazonensis Strains Display Immunomodulatory Properties via TLR4 and Do Not Affect Sand Fly Infection

The immunomodulatory properties of lipophosphoglycans (LPG) from New World species of Leishmania have been assessed in Leishmania infantum and Leishmania braziliensis, the causative agents of visceral and cutaneous leishmaniasis, respectively. This glycoconjugate is highly polymorphic among species with variation in sugars that branch off the conserved Gal(β1,4)Man(α1)-PO₄ backbone of repeat units. Here, the immunomodulatory activity of LPGs from Leishmania amazonensis, the causative agent of diffuse cutaneous leishmaniasis, was evaluated in two strains from Brazil. One strain (PH8) was originally isolated from the sand fly and the other (Josefa) was isolated from a human case. The ability of purified LPGs from both strains was investigated during in vitro interaction with peritoneal murine macrophages and CHO cells and in vivo infection with Lutzomyia migonei. In peritoneal murine macrophages, the LPGs from both strains activated TLR4. Both LPGs equally activate MAPKs and the NF-κB inhibitor p-IκBα, but were not able to translocate NF-κB. In vivo experiments with sand flies showed that both stains were able to sustain infection in L. migonei. A preliminary biochemical analysis indicates intraspecies variation in the LPG sugar moieties. However, they did not result in different activation profiles of the innate immune system. Also those polymorphisms did not affect infectivity to the sand fly.     

Binding of sulphonated indigo derivatives to RepA-WH1 inhibits DNA-induced protein amyloidogenesis

The quest for inducers and inhibitors of protein amyloidogenesis is of utmost interest, since they are key tools to understand the molecular bases of proteinopathies such as Alzheimer, Parkinson, Huntington and Creutzfeldt–Jakob diseases. It is also expected that such molecules could lead to valid therapeutic agents. In common with the mammalian prion protein (PrP), the N-terminal Winged-Helix (WH1) domain of the pPS10 plasmid replication protein (RepA) assembles in vitro into a variety of amyloid nanostructures upon binding to different specific dsDNA sequences. Here we show that di- (S2) and tetra-sulphonated (S4) derivatives of indigo stain dock at the DNA recognition interface in the RepA-WH1 dimer. They compete binding of RepA to its natural target dsDNA repeats, found at the repA operator and at the origin of replication of the plasmid. Calorimetry points to the existence of a major site, with micromolar affinity, for S4-indigo in RepA-WH1 dimers. As revealed by electron microscopy, in the presence of inducer dsDNA, both S2/S4 stains inhibit the assembly of RepA-WH1 into fibres. These results validate the concept that DNA can promote protein assembly into amyloids and reveal that the binding sites of effector molecules can be targeted to inhibit amyloidogenesis.     

Pentacycloundecane lactam vs lactone norstatine type protease HIV inhibitors: binding energy calculations and DFT study

Background

Novel pentacycloundecane (PCU)-lactone-CO-EAIS peptide inhibitors were designed, synthesized, and evaluated against wild-type C-South African (C-SA) HIV-1 protease. Three compounds are reported herein, two of which displayed IC₅₀ values of less than 1.00 μM. A comparative MM-PB(GB)SA binding free energy of solvation values of PCU-lactam and lactone models and their enantiomers as well as the PCU-lactam-NH-EAIS and lactone-CO-EAIS peptide inhibitors and their corresponding diastereomers complexed with South African HIV protease (C-SA) was performed. This will enable us to rationalize the considerable difference between inhibitory concentration (IC₅₀) of PCU-lactam-NH-EAIS and PCU-lactone-CO-EAIS peptides.

Results

The PCU-lactam model exhibited more negative calculated binding free energies of solvation than the PCU-lactone model. The same trend was observed for the PCU-peptide inhibitors, which correspond to the experimental activities for the PCU-lactam-NH-EAIS peptide (IC₅₀ = 0.076 μM) and the PCU-lactone-CO-EAIS peptide inhibitors (IC₅₀ = 0.850 μM). Furthermore, a density functional theory (DFT) study on the natural atomic charges of the nitrogen and oxygen atoms of the three PCU-lactam, PCU-lactim and PCU-lactone models were performed using natural bond orbital (NBO) analysis. Electrostatic potential maps were also used to visualize the electron density around electron-rich regions. The asymmetry parameter (η) and quadrupole coupling constant (χ) values of the nitrogen and oxygen nuclei of the model compounds were calculated at the same level of theory. Electronic molecular properties including polarizability and electric dipole moments were also calculated and compared. The Gibbs theoretical free solvation energies of solvation (∆G_solv) were also considered.

Conclusions

A general trend is observed that the lactam species appears to have a larger negative charge distribution around the heteroatoms, larger quadrupole constant, dipole moment and better solvation energy, in comparison to the PCU-lactone model. It can be argued that these characteristics will ensure better eletronic interaction between the lactam and the receptor, corresponding to the observed HIV protease activities in terms of experimental IC₅₀ data.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-015-0115-5) contains supplementary material, which is available to authorized users.     

Activation of the lysosome-associated p61Hck isoform triggers the biogenesis of podosomes

Haematopoietic cell kinase (Hck) is a protein tyrosine kinase of the Src family specifically expressed in phagocytes as two isoforms, p59Hck and p61Hck, present at the plasma membrane and lysosomes, respectively. We report that ectopic expression of a constitutively active mutant of p61Hck (p61Hck(ca)) triggered the de novo formation of actin-rich rings at the ventral face of the cells that we characterized as bona fide podosome rosettes, structures involved in cell migration. Their formation required the adaptor domains and the kinase activity of p61Hck, the integrity of microfilament and microtubule networks and concerted action of Cdc42, Rac and Rho. Podosome rosette formation was either abolished when p61Hck(ca) was readdressed from lysosomes to the cytosol or triggered when p59Hck(ca) was relocalized to lysosomes. Lysosomal markers were present at podosome rosettes. By stimulating exocytosis of p61Hck(ca) lysosomes with a calcium ionophore, the formation of podosome rosettes was enhanced. Interestingly, we confirm that, in human macrophages, Hck and lysosomal markers were present at podosomes which were spatially reorganized as clusters, a foregoing step to form rosettes, upon expression of p61Hck(ca). We propose that lysosomes, under the control of p61Hck, are involved in the biogenesis of podosomes, a key phenomenon in the migration of phagocytes.     

Protein–Protein and Protein–Membrane Associations in the Lignin Pathway

Supramolecular organization of enzymes is proposed to orchestrate metabolic complexity and help channel intermediates in different pathways. Phenylpropanoid metabolism has to direct up to 30% of the carbon fixed by plants to the biosynthesis of lignin precursors. Effective coupling of the enzymes in the pathway thus seems to be required. Subcellular localization, mobility, protein–protein, and protein–membrane interactions of four consecutive enzymes around the main branch point leading to lignin precursors was investigated in leaf tissues of Nicotiana benthamiana and cells of Arabidopsis thaliana. CYP73A5 and CYP98A3, the two Arabidopsis cytochrome P450s (P450s) catalyzing para- and meta-hydroxylations of the phenolic ring of monolignols were found to colocalize in the endoplasmic reticulum (ER) and to form homo- and heteromers. They moved along with the fast remodeling plant ER, but their lateral diffusion on the ER surface was restricted, likely due to association with other ER proteins. The connecting soluble enzyme hydroxycinnamoyltransferase (HCT), was found partially associated with the ER. Both HCT and the 4-coumaroyl-CoA ligase relocalized closer to the membrane upon P450 expression. Fluorescence lifetime imaging microscopy supports P450 colocalization and interaction with the soluble proteins, enhanced by the expression of the partner proteins. Protein relocalization was further enhanced in tissues undergoing wound repair. CYP98A3 was the most effective in driving protein association.