The rational search for selective anticancer derivatives of the peptide Trichogin GA IV: a multi-technique biophysical approach

Peptaibols are peculiar peptides produced by fungi as weapons against other microorganisms. Previous studies showed that peptaibols are promising peptide-based drugs because they act against cell membranes rather than a specific target, thus lowering the possibility of the onset of multi-drug resistance, and they possess non-coded alpha-amino acid residues that confer proteolytic resistance. Trichogin GA IV (TG) is a short peptaibol displaying antimicrobial and cytotoxic activity. In the present work, we studied thirteen TG analogues, adopting a multidisciplinary approach. We showed that the cytotoxicity is tuneable by single amino-acids substitutions. Many analogues maintain the same level of non-selective cytotoxicity of TG and three analogues are completely non-toxic. Two promising lead compounds, characterized by the introduction of a positively charged unnatural amino-acid in the hydrophobic face of the helix, selectively kill T67 cancer cells without affecting healthy cells. To explain the determinants of the cytotoxicity, we investigated the structural parameters of the peptides, their cell-binding properties, cell localization, and dynamics in the membrane, as well as the cell membrane composition. We show that, while cytotoxicity is governed by the fine balance between the amphipathicity and hydrophobicity, the selectivity depends also on the expression of negatively charged phospholipids on the cell surface

Endogenous erythropoietin as part of the cytokine network in the pathogenesis of experimental autoimmune encephalomyelitis

Erythropoietin (EPO) is of great interest as a therapy for many of the central nervous system (CNS) diseases and its administration is protective in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Endogenous EPO is induced by hypoxic/ischemic injury, but little is known about its expression in other CNS diseases. We report here that EPO expression in the spinal cord is induced in mouse models of chronic or relapsing-remitting EAE, and is prominently localized to motoneurons. We found a parallel increase of hypoxia-inducible transcription factor (HIF)-1 alpha, but not HIF-2 alpha, at the mRNA level, suggesting a possible role of non-hypoxic factors in EPO induction. EPO mRNA in the spinal cord was co-expressed with interferon (IFN)-gamma and tumor necrosis factor (TNF), and these cytokines inhibited EPO production in vitro in both neuronal and glial cells. Given the known inhibitory effect of EPO on neuroinflammation, our study indicates that EPO should be viewed as part of the inflammatory/anti-inflammatory network in MS

Quantification and characterization of additives, plasticizers, and small microplastics (5-100 μm) in highway stormwater runoff

Highway stormwater (HSW) runoff is a significant pathway for transferring microplastics from land-based sources to the other surrounding environmental compartments. Small microplastics (SMPs, 5-100 μm), additives, plasticizers, natural, and nonplastic synthetic fibers, together with other components of micro-litter (APFs), were assessed in HSW samples via Micro-FTIR; oleo-extraction and purification procedures previously developed were optimized to accomplish this goal. The distribution of SMPs and APFs observed in distinct HSW runoff varied significantly since rainfall events may play a crucial role in the concentration and distribution of these pollutants. The SMPs' abundance varied from 11932 ± 151 to 18966 ± 191 SMPs/L. The dominating polymers were vinyl ester (VE), polyamide 6 (PA6), fluorocarbon, and polyester (PES). The APFs' concentrations ranged from 12825 ± 157 to 96425 ± 430 APFs/L. Most APFs originated from vehicle and tire wear (e.g., Dioctyl adipate or 5-Methyl-1H-benzotriazole). Other sources of these pollutants might be pipes, highway signs, packaging from garbage debris, road marking paints, atmospheric deposition, and other inputs. Assessing SMPs in HSW runoff can help evaluating the potential threat they may represent to receiving water bodies and air compartments. Besides, APFs in HSW runoff may be efficient proxies of macro- and microplastic pollution

Somatostatin coupling to adenylyl cyclase activity in the mouse retina

The peptide somatostatin-14 (SRIF) acts in the mammalian retina through its distinct receptors (sst1-5). Scarce information is available on SRIF function in the retina, including the elucidation of transduction pathways mediating SRIF action. We have investigated SRIF and SRIF receptor modulation of adenylyl cyclase (AC) activity in both wild type (WT) retinas and sst1 or sst2 knock-out (KO) retinas which are known to over-express sst2 or sst1 receptors, respectively. In WT retinas, application of SRIF compounds does not affect forskolin-stimulated AC activity. In contrast, activation of sst1 or sst2 receptors inhibits AC in the presence of sst2 or sst1 receptor antagonists, respectively. Results from sst1 KO retinas demonstrate that either SRIF or octreotide, pertussis toxin-dependently inhibit AC activity. In contrast, in sst2 KO retinas, neither SRIF nor CH-275, an sst1 receptor agonist, are found to influence AC activity. As revealed by immunoblotting experiments, in sst1 KO retinas, levels of Goα proteins are 60% higher than in WT retinas and this increase in Goα protein levels is concomitant with an increase in sst2A receptor expression. We conclude that interactions between sst1 and sst2 receptors may prevent SRIF effects on AC activity. In addition, we suggest that the density of sst2 receptors and/or Goα proteins may represent the rate-limiting factor for the sst2 receptor-mediated inhibition of AC.L'articolo è disponibile sul sito dell'editore http://www.springerlink.com

Interactions of GFAP with ceftriaxone and phenytoin: SRCD and molecular docking and dynamic simulation

Abstract Background GFAP is the major intermediate filament protein in mature astrocytes. Its increased expression and aggregation was firstly associated to Alexander's disease, and successively in different neurological diseases including scrapie, Alzheimer's and Creutzfeld–Jacob diseases. Recently, ceftriaxone a multi-potent β-lactam antibiotic able to overcome the blood–brain barrier, successfully eliminated the cellular toxic effects of misfolded mutated GFAP, similarly to phenytoin sodium, in a cellular model of Alexander's disease and inhibited α-synuclein aggregation protecting PC12 cells from the exposure to 6-hydroxydopamine. Methods In this study, synchrotron radiation circular dichroism spectroscopy has been used to obtain structural information about the GFAP-ceftriaxone (phenytoin) interactions, while computational methods allowed the identification of the relevant putative binding site of either ceftriaxone or phenytoin on the dimer structure of GFAP, permitting to rationalize the spectroscopic experimental results. Results We found that GFAP exhibited enhanced stability upon the addition of two equivalents of each ligands with ceftriaxone imparting a more spontaneous interactions and a more ordered complex system than phenytoin. Conclusions SRCD data and MD models indicate a stronger protective effect of ceftriaxone in neurological disorders characterized by an increased production and polymerization of GFAP. General significance This result, in addition to our previous works in which we documented that ceftriaxone interacts with α-synuclein inhibiting its pathological aggregation and that a cyclical treatment with this molecule in a patient with adult-onset Alexander's disease halted, and partly reversed, the progression of neurodegeneration, suggests the possibility of a chaperone-like effect of ceftriaxone on protein involved in specific neurodegenerative diseases

Conformationally Constrained Peptides with High Affinity to the Vascular Endothelial Growth Factor

The design of efficient vascular endothelial growth factor (VEGF) inhibitors is a high-priority research area aimed at the treatment of pathological angiogenesis. Among other compounds, v114* has been identified as a potent VEGF-binding peptide. In order to improve the affinity to VEGF, we built a conformational constrain in its structure. To this aim, C-alpha-tetrasubstituted amino acid Aib was introduced into the N-terminal tail, peptide loop, or C-terminal helix. NMR studies confirmed the stabilization of the helical conformation in proximity to the Aib residue. We found that the induction of the N-terminal helical structure or stabilization of the C-terminal helix can noticeably increase the peptide affinity to the VEGF. These peptides efficiently inhibited VEGF-stimulated cell proliferation as well. The insertion of the non-proteinogenic Aib residue significantly enhanced the stability of the peptides in the vitreous environment. Thus, these Aib-containing peptides are promising candidates for the design of VEGF inhibitors with improved properties.Peer reviewe

Covalent Graft of Lipopeptides and Peptide Dendrimers to Cellulose Fibers

Introduction: Bacterial proliferation in health environments may lead to the development of specific pathologies, but can be highly dangerous under particular conditions, such as during chemotherapy. To limit the spread of infections, it is helpful to use gauzes and clothing containing antibacterial agents. As cotton tissues are widespread in health care environments, in this contribution we report the preparation of cellulose fibers characterized by the covalent attachment of lipopeptides as possible antimicrobial agents. Aim: To covalently link peptides to cotton samples and characterize them. Peptides are expected to preserve the features of the fabrics even after repeated washing and use. Peptides are well tolerated by the human body and do not induce resistance in bacteria. Materials and Methods: A commercially available cotton tissue (specific weight of 150 g/m2, 30 Tex yarn fineness, fabric density of 270/230 threads/10 cm in the warp and weft) was washed with alkali and bleached and died. A piece of this tissue was accurately weighed, washed with methanol (MeOH) and N,N-dimethylformamide (DMF), and air-dried. Upon incubation with epibromohydrin, followed by treatment with Fmoc-NH-CH2CH2-NH2 and Fmoc removal, the peptides were synthesized by incorporating one amino acid at a time, beginning with the formation of an amide bond with the free NH2 of 1,2\u2013diaminoethane. We also linked to the fibers a few peptide dendrimers, because the mechanism of action of these peptides often requires the formation of clusters. We prepared and characterized seven peptide-cotton samples. Results: The new peptide-cotton conjugates were characterized by means of FT-IR spectroscopy and X-ray Photoelectron Spectroscopy (XPS). This latter technique allows for discriminating among different amino acids and thus different peptide-cotton samples. Some samples maintain a pretty good whiteness degree even after peptide functionalization. Interestingly, these samples also display encouraging activities against a Gram positive strain. Conclusions: Potentially antimicrobial lipopeptides can be covalently linked to cotton fabrics, step-by-step. It is also possible to build on the cotton Lys-based dendrimers. XPS is a useful technique to discriminate among different types of nitrogen. Two samples displaying some antibacterial potency did also preserve their whiteness index

Biological activity of somatostatin receptors in GC rat tumour somatotrophs: evidence with sst1-sst5 receptor-selective nonpeptidyl agonists

The physiological actions of somatostatin-14 (SRIF) receptor subtypes (sst1-sst5), which are endogenously expressed in GC cells, have not yet been elucidated, although there is evidence that sst2 receptors are negatively coupled to cytosolic free Ca2+ concentration ([Ca2+]i) and cAMP accumulation. In addition, both sst1 and sst2 receptors are negatively coupled to growth hormone (GH) secretion in GC cells. Here we report on studies concerning the expression, the pharmacology and the functional role of native SRIF receptors in GC cells with the use of five nonpeptidyl agonists, highly selective for each of the SRIF receptors. Radioligand binding studies show that sst2 and sst5 receptors are present at different relative densities, while the presence of sst3 and sst4 receptors appears to be negligible. The absence of sst1 receptor binding was unexpected in view of sst1 receptor functional effects on GH secretion. This suggests very efficient receptor-effector coupling of a low density population of sst1 receptors. Functionally, only sst2 receptors are coupled to the inhibition of [Ca2+]i and cAMP accumulation and the selective activation of sst5 receptors facilitates the stimulation of adenylyl cyclase activity through Gi/o proteins. This effect was not observed when sst2 and sst5 receptors were simultaneously activated, suggesting that there is a functional interaction between sst2 and sst5 receptors. In addition, sst1, sst2 and sst5 receptor activation inhibits GH release, further indicating that SRIF can modulate GH secretion in GC cells through mechanisms both dependent and independent on [Ca2+]i and cAMP-dependent pathways. The present data suggest SRIF-mediated functional effects in GC cells to be very diverse and provide compelling arguments to propose that multiple native SRIF receptors expressed in the same cells are not simply redundant, but contribute to marked signalling diversity.L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com