Cigarette smoke extract induces oxidative stress and apoptosis in human lung fibroblasts

Cigarette smoke is a mixture of chemicals having direct and/or indirect toxic effects on different lung cells. We investigated the effect of cigarette smoke on human lung fibroblasts (HFL-1) oxidation and apoptosis. Cells were exposed to various concentrations (1, 5, and 10%) of cigarette smoke extract (CSE) for 3 h, and oxidative stress and apoptosis were assessed by fluorescence-activated cell sorting and confocal laser fluorescence microscopy. Both oxidative stress and apoptosis exhibited a dose-response relationship with CSE concentrations. Lung fibroblasts also showed marked DNA fragmentation at the Comet assay after exposure to 10% CSE. Coincubation of HLF-1 cells with N-acetylcysteine (1 mM) during CSE exposure significantly reduced oxidative stress, apoptosis, and DNA fragmentation, whereas preincubation (3 h) with the glutathione-depleting agent buthionine sulfoximine (125 microM) produced a significant increase of oxidative stress. Cigarette smoke is a potent source of oxidative stress, DNA damage, and apoptosis for HFL-1 cells, and we speculate that this could contribute to the development of pulmonary emphysema in the lungs of smokers.     

Angiogenesis and nerve regeneration in a model of human skin equivalent transplant

The angiogenesis and reinnervation were studied in a porcine model of human skin equivalent (SE) graft and the relationship between the two processes was investigated. Confocal laser scanning microscopy was used to monitor, during the healing process, the pattern of vascularization and reinnervation at different time points. The SE was obtained by co-culturing fibroblasts and keratinocytes on a collagen-glycosaminoglycan-chitosan biopolymer and grafted on dorsal wounds generated by full-thickness resection in 25/30 Kg Large white pigs. Frozen sections were obtained from biopsies performed in autograft and xenograft, then were immunolabeled by using the endothelial marker lectin Lactifolia and with the neuronal marker gene product PGP9.5. Cajal staining was also used to visualize the nerve fibers. The results show that the vascularization precedes the innervation process. These data are consistent with the view that the development of nervous tissue is driven by nutritional and trophic factors provided by the vascular system. The arborization of the two systems observed during the third week from the graft might play a key role in maintaining the healing process and the graft survival.     

Co-Transplantation of Endothelial Progenitor Cells and Pancreatic Islets to Induce Long-Lasting Normoglycemia in Streptozotocin-Treated Diabetic Rats

Graft vascularization is a crucial step to obtain stable normoglycemia in pancreatic islet transplantation. Endothelial progenitor cells (EPCs) contribute to neoangiogenesis and to the revascularization process during ischaemic events and play a key role in the response to pancreatic islet injury. In this work we co-transplanted EPCs and islets in the portal vein of chemically-induced diabetic rats to restore islet vascularization and to improve graft survival. Syngenic islets were transplanted, either alone or with EPCs derived from green fluorescent protein (GFP) transgenic rats, into the portal vein of streptozotocin-induced diabetic rats. Blood glucose levels were monitored and intraperitoneal glucose tolerance tests were performed. Real time-PCR was carried out to evaluate the gene expression of angiogenic factors. Diabetic-induced rats showed long-lasting (6 months) normoglycemia upon co-transplantation of syngenic islets and EPCs. After 3–5 days from transplantation, hyperglycaemic levels dropped to normal values and lasted unmodified as long as they were checked. Further, glucose tolerance tests revealed the animals'' ability to produce insulin on-demand as indexed by a prompt response in blood glucose clearance. Graft neovascularization was evaluated by immunohistochemistry: for the first time the measure of endothelial thickness revealed a donor-EPC-related neovascularization supporting viable islets up to six months after transplant. Our results highlight the importance of a newly formed viable vascular network together with pancreatic islets to provide de novo adequate supply in order to obtain enduring normoglycemia and prevent diabetes-related long-term health hazards.     

A molecular phylogenetic study of the freshwater talitrid amphipod Orchestia cavimana (Crustacea).

In this paper we performed a molecular phylogenetic study of Orchestia cavimana, the sole talitrid amphipod inhabiting beaches of European freshwater lakes and rivers. For that purpose, we have PCR amplified and sequenced regions of the mitochondrial cytochrome oxidase subunit I (COI) gene, basing our analysis on both nucleotide and amino acid sequences and considering also structural classes of the COI enzyme. Phylogenetic analyses were conducted by neighbour-joining (NJ) and maximum-parsimony (MP) methods comparing homologous sequences of talitrids and other Crustacea. In both NJ and MP trees, O. cavimana shows a basal placement with respect to other talitrid amphipods.     

Conformational studies of RGDechi peptide by natural‐abundance NMR spectroscopy

Integrins are heterodimeric cell‐surface proteins that play important roles during developmental and pathological processes. Diverse human pathologies involve integrin adhesion including thrombotic diseases, inflammation, tumour progression, fibrosis, and infectious diseases. Although in the past decade, novel integrin‐inhibitor drugs have been developed for integrin‐based medical applications, the structural determinants modulating integrin‐ligands recognition mechanisms are still poorly understood, reducing the number of integrin subtype exclusive antagonists. In this scenario, we have very recently showed, by means of chemical and biological assays, that a chimeric peptide (named RGDechi), containing a cyclic RGD motif linked to an echistatin C‐terminal fragment, is able to interact with the components of integrin family with variable affinities, the highest for α_vβ3. Here, in order to understand the mechanistic details driving the molecular recognition mechanism of α_vβ₃ by RGDechi, we have performed a detailed structural and dynamics characterization of the free peptide by natural abundance nuclear magnetic resonance (NMR) spectroscopy. Our data indicate that RGDechi presents in solution an heterogeneous conformational ensemble characterized by a more constrained and rigid pentacyclic ring and a largely unstructured acyclic region. Moreover, we propose that the molecular recognition of α_vβ₃ integrin by RGDechi occurs by a combination of conformational selection and induced fit mechanisms. Finally, our study indicates that a detailed NMR characterization, by means of natural abundance ¹⁵N and ¹³C, of a mostly unstructured bioactive peptide may provide the molecular basis to get essential structural insights into the binding mechanism to the biological partner.     

Molecular strategies to replace the structural metal site in the prokaryotic zinc finger domain

The specific arrangement of secondary elements in a local motif often totally relies on the formation of coordination bonds between metal ions and protein ligands. This is typified by the ~ 30 amino acid eukaryotic zinc finger motif in which a β-sheet and an α-helix are clustered around a zinc ion by various combinations of four ligands.     

Molecular basis of the PED/PEA15 interaction with the C-terminal fragment of phospholipase D1 revealed by NMR spectroscopy

PED/PEA15 is a small protein involved in many protein–protein interactions that modulates the function of a number of key cellular effectors involved in major cell functions, including apoptosis, proliferation and glucose metabolism. In particular, PED/PEA15 interacts with the phospholipase D (PLD) isoforms 1 and 2 increasing protein kinase C-α isoform activity and affects both insulin-stimulated glucose transport and glucose-stimulated insulin secretion.     

Physiological and Biochemical Effects of an Aqueous Extract of Lemna minor L. as a Potential Biostimulant for Maize

Journal of Plant Growth Regulation - Biostimulants are receiving increasing attention for their beneficial effects on crops, driving interest in identifying new plant extracts that could exert such...     

Assessing Archeal Indicators of Performance by RT-qPCR Methods During Anaerobic Co-digestion of Organic Wastes

Biogas is a renewable energy resource produced during the anaerobic digestion of various organic substrates. A wide community of microorganisms is involved, including methanogens. These Archaea are the biologic key to the process because they accomplish the methane-forming reaction. Despite its crucial role, the microbiome inside the digester is poorly understood. The aim of this work is to develop bioindicators of efficiency for the anaerobic process through the quantification and characterisation of the methanogens and sulphate-reducing bacteria. From a full-scale digester fed with organic wastes, 31 samples were collected. Temperature, pH, acidity, alkalinity and biogas quantity and quality were monitored over time. The methanogens were detected from the samples both in total and as belonging to different taxa units. These evaluations, by real-time quantitative PCR (RT-qPCR) methods, produced valuable results for Methanosarcina, Methanosaeta, Methanocorpusculaceae and sulphate-reducing bacteria. Methanosarcina was the most abundant family, followed by Methanocorpusculaceae and then Methanosaeta. The methanogen taxa are significantly and directly correlated with each other (p?<?0.05). Methanosaeta and Methanocorpusculaceae are present in significantly different amounts at different temperatures. While Methanosaeta levels also change when the organic load increases (t test, p?<?0.05), Methanosarcina is more tolerant, and its levels are quite constant. Methanosarcina and Methanosaeta are proposed to be bioindicators of the stability of the process (the first) and of susceptibility (the second) to detect early sufferance conditions in the digester. These methods will be useful in the control and optimisation of an eco-friendly waste-to-energy system.