The defensin–lipid interaction: Insights on the binding states of the human antimicrobial peptide HNP-1 to model bacterial membranes

Antimicrobial peptides are an important component of innate immunity and have generated considerable interest as a new potential class of natural antibiotics. The biological activity of antimicrobial peptides is strongly influenced by peptide–membrane interactions. Human Neutrophil Peptide 1 (HNP-1) is a 30 aminoacid peptide, belonging to the class of α-defensins. Many biophysical studies have been performed on this peptide to define its mechanism of action. Combining spectroscopic and thermodynamic analysis, insights on the interaction of the α-defensin with POPE:POPG:CL negative charged bilayers are given. The binding states of the peptide below and above the threshold concentration have been analyzed showing that the interaction with lipid bilayers is dependent by peptide concentration. These novel results that indicate how affinity and biological activities of natural antibiotics are depending by their concentration, might open new way of investigation of the antimicrobial mode of action.     

Diversity and expression of P450 genes from Dendroctonus valens LeConte (Curculionidae: Scolytinae) in response to different kairomones

Bark beetles (Curculionidae: Scolytinae) are major cause of woody plants death in the world. They colonize the stem and other parts of trees recognizing host-produced specific compounds (kairomones) and insect pheromones. Bark beetle's antennae and alimentary canal participate in the host selection identifying chemical compounds produced by trees and insects, and also in the metabolism and detoxification of these compounds. The red turpentine beetle (RTB), Dendroctonus valens LeConte, is an unaggressive species that colonize > 40 pine species (Pinaceae) in North and Central America. Several studies suggest that bark beetle cytochrome P450 enzymes are involved in monoterpene oxidation. In this study we identified by means of PCR, cloning, sequencing, and bioinformatic analysis, eleven full-length genes: five CYP4, four CYP6, and two CYP9 in the antennae and gut region of RTB, after stimulation with vapors of monoterpenes: (±)-α-pinene, (R)-(+)-α-pinene, (S)-(?)-β-pinene, (S)-(?)-α-pinene and (+)-3-carene; pine trees volatiles used by RTB as kairomones. The recovered cDNA of these genes vary from 1.5 kb to 1.8 kb and the open frame encodes from 496 to 562 amino acid proteins. The bioinformatic analysis suggests that the majority of P450 proteins encoded by these genes are membrane anchored in the endoplasmic reticulum. RT-qPCR assays showed differential expression of all CYP genes between male and female. The gene expression was dependent of monoterpenes and exposure time, with some of them sex, antennae and gut region specific. Significant differences among monoterpenes, gut region, antennae and exposure time were found. Our results suggest that some of these genes may be involved in the detoxification process of these compounds during tree colonization.     

New structure–activity relationship studies in a series of N,N-bis(cyclohexanol)amine aryl esters as potent reversers of P-glycoprotein-mediated multidrug resistance (MDR)

As a continuation of previous research on a new series of potent and efficacious P-gp-dependent multidrug resistant (MDR) reversers with a N,N-bis(cyclohexanol)amine scaffold, we have designed and synthesized several analogs by modulation of the two aromatic moieties linked through ester functions to the N,N-bis(cyclohexanol)amine, aiming to optimize activity and to extend structure–activity relationships (SAR) within the series. This scaffold, when esterified with two different aromatic carboxylic acids, gives origin to four geometric isomers (cis/trans, trans/trans, cis/cis and trans/cis).The new compounds were tested on doxorubicin-resistant erythroleukemia K562 cells (K562/DOX) in the pirarubicin uptake assay. Most of them resulted in being potent modulators of the extrusion pump P-gp, showing potency values ([I]_0.5) in the submicromolar and nanomolar range. Of these, compounds 2b, 2c, 3d, 5a–d and 6d, showed excellent efficacy with a α_max close to 1. Selected compounds (2d, 3a, 3b, 5a–d) were further studied to evaluate their doxorubicin cytotoxicity potentiation (RF) on doxorubicin-resistant erythroleukemia K562 cells and were found able to enhance significantly doxorubicin cytotoxicity on K562/DOX cells.The results of both pirarubicin uptake and the cytotoxicity assay, indicate that the new compounds of the series are potent P-gp-mediated MDR reversers. They present a structure with a mix of flexible and rigid moieties, a property that seems critical to allow the molecules to choose the most productive of the several binding modes possible in the transporter recognition site.In particular, compounds 5c and 5d, similar to the already reported analogous isomers 1c and 1d,²⁹ are potent and efficacious modulators of P-gp-dependent MDR and may be promising leads for the development of MDR-reversal drugs.     

Studies on the ectomycorrhizal community in a declining Quercus suber L. stand

This survey was carried out in a Quercus suber L. stand with many trees affected by the disease “oak decline”. Its aim was to obtain information about both the belowground ectomycorrhizal fungal community in a declining Q. suber stand as a whole, and the ectomycorrhizal fungal community of individual tree (EFT) detected in healthy and diseased plants. To this end, we first categorized the trees into four different decline classes (one for healthy plants and three for diseased plants) and then, by using morphological and molecular tools, we identified the ectomycorrhizas isolated from samples collected near the trees with different declining classes. The ectomycorrhizal community as a whole was seen to be composed of numerous ectomycorrhizal fungal species, only some of which appeared to be dominant (Cenococcum geophilum, Lactarius chrysorrheus, and some species of Tomentella genus), while most occurred sporadically. Results show that all root tips observed are mycorrhized and that decline class does not influence the number of ectomycorrhizal root tips found in the EFTs, thus oak decline does not impact the investment in ectomycorrhizal symbiosis. However, some statistical differences can be observed in the values of evenness and taxonomic distinctness in the EFT associated with trees with different states of health. Finally, both the analysis of similarity test and the ordination technique highlight a compositional difference between the EFT associated with trees in different health conditions, but also suggest that other factors may play a role in causing these differences.     

Synthesis and structural characterization of soluble neuromelanin analogs provides important clues to its biosynthesis

Elucidating the structure and biosynthesis of neuromelanin (NM) would be an important step towards understanding its putative role in the pathogenesis of Parkinson’s disease. A useful complement to studies aimed at unraveling the origin and properties of this essentially insoluble natural substance is the preparation of synthetic derivatives that resemble NM. With this aim in mind, water-soluble conjugates between dopamine-derived melanin and bovine serum albumin (BSA) were synthesized. Melanin–BSA adducts were prepared with both eumelanic oligomers obtained through the oxidative polymerization of dopamine and pheomelanic oligomers obtained under the same conditions from dopamine and cysteine. Iron ions were added during the synthesis to understand the interaction between the pigment and this metal ion, as the NM in neurons in several human brain regions contains significant amounts of iron. The structures of the conjugates were analyzed by ¹H NMR spectroscopy and controlled proteolysis/MS experiments. The binding of iron(III) ions was evaluated by ICP analysis and EPR spectroscopy. The EPR signal from bound iron(III) indicated high-spin octahedral sites and, as also seen for NM, the signal is coupled to a signal from a radical associated with the melanic components of the conjugates. However, the intensity of the EPR signal from iron suggested a reduced fraction of the total iron, indicating that most of the iron is strongly coupled in clusters within the matrix. The amount of paramagnetic, mononuclear iron(III) was greater in the pheomelanin–BSA conjugates, suggesting that iron clustering is reduced in the sulfur-containing pigment. Thus, the melanin–BSA conjugates appear to be good models for the natural pigment.     

A proteomics approach to investigate the process of Zn hyperaccumulation in Noccaea caerulescens (J & C. Presl) F.K. Meyer

Zinc (Zn) is an essential trace element in all living organisms, but is toxic in excess. Several plant species are able to accumulate Zn at extraordinarily high concentrations in the leaf epidermis without showing any toxicity symptoms. However, the molecular mechanisms of this phenomenon are still poorly understood. A state‐of‐the‐art quantitative 2D liquid chromatography/tandem mass spectrometry (2D‐LC‐MS/MS) proteomics approach was used to investigate the abundance of proteins involved in Zn hyperaccumulation in leaf epidermal and mesophyll tissues of Noccaea caerulescens. Furthermore, the Zn speciation in planta was analyzed by a size‐exclusion chromatography/inductively coupled plasma mass spectrometer (SEC‐ICP‐MS) method, in order to identify the Zn‐binding ligands and mechanisms responsible for Zn hyperaccumulation. Epidermal cells have an increased capability to cope with the oxidative stress that results from excess Zn, as indicated by a higher abundance of glutathione S‐transferase proteins. A Zn importer of the ZIP family was more abundant in the epidermal tissue than in the mesophyll tissue, but the vacuolar Zn transporter MTP1 was equally distributed. Almost all of the Zn located in the mesophyll was stored as Zn–nicotianamine complexes. In contrast, a much lower proportion of the Zn was found as Zn–nicotianamine complexes in the epidermis. However, these cells have higher concentrations of malate and citrate, and these organic acids are probably responsible for complexation of most epidermal Zn. Here we provide evidence for a cell type‐specific adaptation to excess Zn conditions and an increased ability to transport Zn into the epidermal vacuoles.     

Loss of TRAIL-Receptors Is a Recurrent Feature in Pancreatic Cancer and Determines the Prognosis of Patients with No Nodal Metastasis after Surgery

Introduction

Agonistic antibodies targeting TRAIL-receptors 1 and 2 (TRAIL-R1 and TRAIL-R2) are being developed as a novel therapeutic approach in cancer therapy including pancreatic cancer. However, the cellular distribution of these receptors in primary pancreatic cancer samples has not been sufficiently investigated and no study has yet addressed the issue of their prognostic significance in this tumor entity.

Aims and Methods

Applying tissue microarray (TMA) analysis, we performed an immunohistochemical assessment of TRAIL-receptors in surgical samples from 84 consecutive patients affected by pancreatic adenocarcinoma and in 26 additional selected specimens from patients with no lymph nodes metastasis at the time of surgery. The prognostic significance of membrane staining and staining intensity for TRAIL-receptors was evaluated.

Results

The fraction of pancreatic cancer samples with positive membrane staining for TRAIL-R1 and TRAIL-R2 was lower than that of cells from surrounding non-tumor tissues (TRAIL-R1: p<0.001, TRAIL-R2: p = 0.006). In addition, subgroup analyses showed that loss of membrane staining for TRAIL-R2 was associated with poorer prognosis in patients without nodal metastases (multivariate Cox regression analysis, Hazard Ratio: 0.44 [95% confidence interval: 0.22−0.87]; p = 0.019). In contrast, analysis of decoy receptors TRAIL-R3 and -R4 in tumor samples showed an exclusively cytoplasmatic staining pattern and no prognostic relevance.

Conclusion

This is a first report on the prognostic significance of TRAIL-receptors expression in pancreatic cancer showing that TRAIL-R2 might represent a prognostic marker for patients with early stage disease. In addition, our data suggest that loss of membrane-bound TRAIL-receptors could represent a molecular mechanism for therapeutic failure upon administration of TRAIL-receptors-targeting antibodies in pancreatic cancer. This hypothesis should be evaluated in future clinical trials.