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Alpha-synuclein (alpha-syn) is a small soluble protein expressed primarily at presynaptic terminals in the central nervous system. Interest in alpha-syn has increased dramatically after the discovery of a relationship between its dysfunction and several neurodegenerative diseases, including Parkinson's disease (PD). The physiological functions of alpha-syn remain to be fully defined, although recent data suggest a role in regulating membrane stability and neuronal plasticity. Various trigger factors, either environmental or genetic, can lead to a cascade of events involving misfolding or loss of normal function of alpha-syn. In dopaminergic neurons, this may promote a vicious cycle in which elevation in cytoplasmic dopamine, oxidative stress, alpha-syn dysfunction, and disruption of vesicle function lead to dopaminergic cell loss and PD. Alpha-syn dysfunction appears to be a common feature of all forms of PD. The mechanism by which alpha-syn induces neuronal cell toxicity may invoke multiple pathways, such as aggregation or interaction with other proteins and molecules, including synphilin-1, chaperone 14-3-3 protein, and dopamine itself. This complexity has hindered the development of models to study PD. The available animal models of PD, each present distinct advantages and limits. Findings to date suggest that alpha-syn-based models represent a paradigm, which is closest to the human pathology.

Endothelial cells (EC) were extracted through a lectin-based method from bone marrow of 57 patients with active multiple myeloma (MM) and compared with their healthy quiescent counterpart, human umbilical vein EC (HUVEC). MMECs exhibit specific antigens that indicate ongoing angiogenesis and embryo vasculogenesis; solid intercellular connections, hence stability of MM neovessels; and frequent interactions with plasma cells, hence tumor dissemination. They show heterogeneous antigen expression, hence existence of subsets. Their main genetic markers are indicative of a vascular phase. They show intrinsic angiogenic ability, because they rapidly form a capillary network in vitro, and extrinsic ability, because they generate numerous new vessels in vivo. They vividly secrete growth and invasive factors for plasma cells. They signal through kinases mandatory for development of neovascularization. Ultrastructurally, they are abnormal and show metabolic activation, like tumor ECs. Thalidomide heavily interferes with their functions. Vasculogenesis and angiogenesis might contribute to the MM vascular tree and progression, in the form of growth, invasion, and dissemination. In view of the heterogeneity of the antigenic phenotype of MMECs, a mixture (or a sequence) of antiangiogenic agents coupled with thalidomide would seem plausible for the biologic management of MM.

Background: Hyaluronan, or hyaluronic acid (HA), is the major hydrodynamic nonprotein component of joint synovial fluid (SF). Its unique viscoelastic properties confer remarkable shock absorbing and lubricating abilities to SF, while its enormous macromolecular size and hydrophilicity serve to retain fluid in the joint cavity during articulation. HA restricts the entry of large plasma proteins and cells into SF but facilitates solute exchange between the synovial capillaries and cartilage and other joint tissues. In addition, HA can form a pericellular coat around cells, interact with proinflammatory mediators, and bind to cell receptors, such as cluster determinant (CD)44 and receptor for hyaluronate-mediated motility (RHAMM), where it modulates cell proliferation, migration, and gene expression. All these physicochemical and biologic properties of HA have been shown to be molecular weight (MW) dependent. Objective: Intra-articular (IA) HA therapy has been used for the treatment of knee osteoarthritis (OA) for more than 30 years. However, the mechanisms responsible for the reported beneficial clinical effects of this form of treatment remain contentious. Furthermore, there are a variety of pharmaceutic HA preparations of different MW available for the treatment of OA, but the significance of their MWs with respect to their pharmacologic activities have not been reviewed previously. The objective of the present review is to redress this deficiency. Methods: We reviewed in vitro and in vivo reports to identify those pharmacologic activities of HA that were considered relevant to the ability of this agent to relieve symptoms and protect joint tissues in OA. Where possible, reports were selected for inclusion when the pharmacologic effects of HA had been studied in relation to its MW. In many studies, only a single HA preparation had been investigated. In these instances, the experimental outcomes reported were compared with similar studies undertaken with HAs of different MWs. Results: Although in vitro studies have generally indicated that high MW-HA preparations were more biologically active than HAs of lower MW, this finding was not confirmed using animal models of OA. The discrepancy may be partly explained by the enhanced penetration of the lower MW HA preparation through the extracellular matrix of the synovium, thereby maximizing its concentration and facilitating its interaction with target synovial cells. However, there is accumulating experimental evidence to show that the binding of HAs to their cellular receptors is dependent on their molecular size; the smaller HA molecular species often elicits an opposite cellular response to that produced by the higher MW preparations. Studies using large animal models of OA have shown that HAs with MWs within the range of 0.5 × 10 6 -1.0 × 10 6 Da were generally more effective in reducing indices of synovial inflammation and restoring the rheological properties of SF (visco-induction) than HAs with MW > 2.3 × 10 6 Da. These experimental findings were consistent with light and electron microscopic studies of synovial membrane and cartilage biopsy specimens obtained from OA patients administered 5 weekly IA injections of HA of MW=0.5 × 10 6 -0.73 × 10 6 Da in which evidence of partial restoration of normal joint tissue metabolism was obtained. Conclusions: By mitigating the activities of proinflammatory mediators and pain producing neuropeptides released by activated synovial cells, HA may improve the symptoms of OA. In addition, HAs within the MW range of 0.5 × 10 6 -1.0 × 10 6 Da partially restore SF rheological properties and synovial fibroblast metabolism in animal models. These pharmacologic activities of HA could account for the reported long-term clinical benefits of this OA therapy. However, clinical evidence has yet to be described to support the animal studies that indicated that HAs with MW > 2.3 × 10 6 Da may be less effective in restoring SF rheology than HAs of half this size. Semin Arthritis Rheum 32:10-37. Copyright 2002, Elsevier Science (USA). All rights reserved.

The discovery of receptor-receptor interactions (RRIs) in the early 1980s provided evidence that G-protein-coupled receptors (GPCRs) operate not only as monomers but also as heteromers, in which integration of the incoming signals takes place already at the plasma membrane level through allosteric RRIs. These integrative mechanisms give sophisticated dynamics to the structure and function of these receptor assemblies in terms of modulation of recognition, G-protein signaling and selectivity and switching to β-arrestin signaling.The present review briefly describes the concept of direct RRI and the available data on the mechanisms of oligomer formation. Further, pharmacological data concerning the best characterized heteromers involving type A GPCRs will be analyzed to evaluate their profile as possible targets for the treatment of various diseases, in particular of impacting diseases of the CNS.GPCR heteromers have the potential to open a completely new field for pharmacology with likely a major impact in molecular medicine. Novel pharmacological strategies for the treatment of several pathologies have already been proposed. However, several challenges still exist to accurately characterize the role of the identified heteroreceptor complexes in pathology and to develop heteromer-specific ligands capable of efficiently exploiting their pharmacological features.

The carotid body may undergo different structural changes during perinatal development, aging, or in response to environmental stimuli. In the previous literature, morphometric approaches to evaluate these changes have considered quantitative first order parameters, such as volumes or densities, while changes in spatial disposition and/or complexity of structural components have not yet been considered. In the present study, different strategies for addressing morphological complexity of carotid body, apart from the overall amount of each tissue component, were evaluated and compared. In particular, we considered the spatial distribution of connective tissue in the carotid bodies of young control subjects, young opiate-related deaths and aged subjects, through analysis of dispersion (Morisita’s index), gray level co-occurrence matrix (entropy, angular second moment, variance, correlation), and fractal analysis (fractal dimension, lacunarity). Opiate-related deaths and aged subjects showed a comparable increase in connective tissue with respect to young controls. However, the Morisita’s index (p

A new in vitro model system, adding advection and shear stress associated with a flowing medium, is proposed for the investigation of nanoparticles uptake and toxicity towards endothelial cells, since these processes are normally present when nanoparticles formulations are intravenously administered. In this model system, mechanical forces normally present in vivo, such as advection and shear stress were applied and carefully controlled by growing human umbilical vein endothelial cells inside a microfluidic device and continuously infusing gold nanoparticle (Au NPs) solution in the device. The tests performed in the microfluidic device were also run in multiwells, where no flow is present, so as to compare the two model systems and evaluate if gold nanoparticles toxicity differs under static and flow culture conditions. Full characterization of Au NPs in water and in culture medium was accomplished by standard methods. Two-photon fluorescence correlation spectroscopy was also employed to map the flow speed of Au NPs in the microfluidic device and characterize Au NPs before and after interactions with the cells. Au NPs uptake in both in vitro systems was investigated through electron and fluorescence microscopy and ICP-AES, and NPs toxicity measured through standard bio-analytical tests. Comparison between experiments run in multiwells and in microfluidic device plays a pivotal role for the investigation of nanoparticle-cell interaction and toxicity assessment: our work showed that administration of equal concentrations of Au NPs under flow conditions resulted in a reduced sedimentation of nanoparticle aggregates onto the cells and lower cytotoxicity with respect to experiments run in ordinary static conditions (multiwells).

The current view on the organization of the central nervous system (CNS) is basically anchored to the paradigm describing the brain as formed by networks of neurons interconnected by synapses. Synaptic contacts are a fundamental characteristic for describing CNS operations, but increasing evidence accumulated in the last 30 years pointed to a refinement of this view. A possible overcoming of the classical "neuroscience paradigm" will be here outlined, based on the following hypotheses: (1) the basic morpho-functional unit in the brain is a compartment of tissue (functional module) where different resident cells (not only neurons) work as an integrated unit; (2) in these complex networks, a spectrum of intercellular communication processes is exploited, that can be classified according to a dichotomous criterion: wiring transmission (occurring through physically delimited channels) and volume transmission (exploiting diffusion in the extracellular space); (3) the connections between cells can themselves be described as a network, leading to an information processing occurring at different levels from cell network down to molecular level; (4) recent evidence of the existence of specialized structures (microvesicles and tunneling nanotubes) for intercellular exchange of materials, could allow a further type of polymorphism of the CNS networks based on at least transient changes in cell phenotype. When compared to the classical paradigm, the proposed scheme of cellular organization could allow a strong increase of the degrees of freedom available to the whole system and then of its plasticity. Furthermore, long range coordination and correlation can be more easily accommodated within this framework.

The central nervous system (CNS) has been proposed to be formed by functional modules (FMs) that are structurally organized as “Russian dolls”, which form transient assemblies, according to the different integrative tasks that networks within the CNS have to carry out. Integration is achieved by means of communication processes among and within FMs. Two major types of communication processes occur in the CNS, wiring transmission (WT) and volume transmission (VT). WT involves classical synaptic transmission via diffusion of a neurotransmitter across the synaptic cleft, while VT is transmission via a neuroactive substance that is carried by the extracellular and cerebrospinal fluids to distant targets. Recently, evidence has been obtained for the existence of tunneling nanotubes that mediate WT and microvesicles that mediate VT, allowing horizontal transfer of receptors, RNAs, and micro-RNAs. The recognition and decoding process at the target level involves receptor heteromers (receptor mosaics) that are generated by direct receptor–receptor interactions, as an emergent property of this system. Receptor mosaics allow the integration of incoming information at the plasma membrane level. These new aspects of the structural and functional organization of the CNS open up a new field of investigation in the physiology and pathology of the CNS.

Apoptosis represents the key mechanism for the removal of surplus, damaged, or aged cells, and deregulated apoptosis has been implicated in the etiology of diverse pathologies. There are two main pathways which are known to initiate apoptosis: the death receptor-dependent (extrinsic) pathway and the mitochondrial-dependent (intrinsic) pathway. In the intrinsic pathway, as a response to diverse signals from the cellular environment, a permeabilization of the mitochondrial outer membrane occurs, followed by the release of cytochrome c and the activation of the effector caspases, which leads to cell death. Recently, increased attention has been paid to the possible role of the protein neuroglobin, in the regulation of the apoptotic process, and data have been provided, demonstrating the ability of the protein to inhibit the intrinsic pathway of apoptosis by interacting with mitochondrial proteins. The molecular details of these interactions, however, remain largely undefined. In the present study, well recognized bioinformatics methods were applied to predict the possible interaction interfaces which the protein can exploit to interact with relevant proteins of the mitochondrial-dependent pathway of apoptosis. In the search for therapeutic approaches based on the modulation of apoptosis, such a computational prediction could represent a first, guiding step, for the design of strategies aimed at modulating these interactions, and tune the apoptotic process.

G protein-coupled receptors (GPCRs) oligomerization has emerged as a vital characteristic of receptor structure. Substantial experimental evidence supports the existence of GPCR-GPCR interactions in a coordinated and cooperative manner. However, despite the current development of experimental techniques for large-scale detection of GPCR heteromers, in order to understand their connectivity it is necessary to develop novel tools to study the global heteroreceptor networks. To provide insight into the overall topology of the GPCR heteromers and identify key players, a collective interaction network was constructed. Experimental interaction data for each of the individual human GPCR protomers was obtained manually from the STRING and SCOPUS databases. The interaction data were used to build and analyze the network using Cytoscape software. The network was treated as undirected throughout the study. It is comprised of 156 nodes, 260 edges and has a scale-free topology. Connectivity analysis reveals a significant dominance of intrafamily versus interfamily connections. Most of the receptors within the network are linked to each other by a small number of edges. DRD2, OPRM, ADRB2, AA2AR, AA1R, OPRK, OPRD and GHSR are identified as hubs. In a network representation 10 modules/clusters also appear as a highly interconnected group of nodes. Information on this GPCR network can improve our understanding of molecular integration. GPCR-HetNet has been implemented in Java and is freely available at http://www.iiia.csic.es/~ismel/GPCR-Nets/index.html. © 2014 by the authors; licensee MDPI, Basel, Switzerland., This work has been supported by the Swedish Royal Academy of Sciences (Stiftelsen B. von Beskows Fond and Stiftelsen Hierta-Retzius stipendiefond) and Karolinska Institutets Forskningsstiftelser 2011 and 2012 to D.O.B.-E., by grants from the Swedish Medical Research Council (04X-715), Telethon TV3’s La Marató Foundation 2008 and Hjärnfonden to K.F., D.O.B.-E., I.B. and W.R.-F. belong to the “Academia de Biólogos Cubanos” group. Feliciano Calvo and Carmelo Million are acknowledged for their support during the GPCR heterodimer list preparation.

Microfluidic, the technology that manipulates small amount of fluids in microscale complex devices, has undergone a remarkable development during the last decade, by targeting a significant range of applications, including biological tests and single-cell analysis, and by displaying many advantages such as reduced reagent consumption, decreased costs and faster analysis. Furthermore, the introduction of microfluidic tools has revolutionized the study of vascular functions, because the controlled three-dimensional environment and the continuous perfusion provided by the microdevice allow simulating the physiological characteristics of the circulatory system. Researchers interested in the study of vascular physiology, however, are often hampered by the difficulty in handling reduced number of cells after growth in these devices. This work shows how to apply different protocols commonly used in biology, such as the immunofluorescence technique, to cells grown in reversibly-bound microfluidic devices, obtaining results comparable to those retrieved under static conditions in multiwells. In this way, we are able to combine the advantages of microfluidic, i.e., application of continuous flow and shear stress, with classical protocols for the study of endothelial cells.

Osteocalcin (OCN) is a small noncollagenous protein mainly produced by osteoblasts and is highly represented in bones of most vertebrates. Human OCN contains up to three gamma-carboxyglutamic acid (Gla-OCN) residues at positions 17, 21 and 24 which are thought to increase calcium binding strength, improving mechanical properties of the bone matrix. Recent studies revealed that OCN exerts also important endocrine functions, affecting energy metabolism and male fertility. The latter effect seems to be mediated by the uncarboxylated form of OCN (Glu-OCN). We employed human and mouse OCN as models of fully carboxylated and uncarboxylated OCN forms to investigate, by the use of circular dichroism and molecular dynamics simulations, the respective conformational properties and Ca2+ affinity. Ca2+ binding was found to trigger a similar conformational transition in both Glu-OCN and Gla-OCN, from a disordered structure to a more compact/stable form. Notably, gamma-carboxylation increases the affinity of OCN for Ca2+ by > 30 fold suggesting that, in physiological conditions, Gla-OCN is essentially Ca2+-bound, whereas Glu-OCN circulates mainly in the Ca2+-free form.

Background . Cell populations present in human semilunar valves have not been investigated thoroughly. The aim of this study was to characterize the cell phenotypes in pulmonary valve leaflets (PVL) in comparison with aortic (AVL) valve leaflets. Methods . AVL and PVL were dissected from hearts (n = 4) harvested from transplanted patients. Leaflets were processed for immunocytochemistry analysis and Western blotting procedures using a panel of monoclonal antibodies specific for cytoskeletal/contractile antigens. Results . The fibrosa and the ventricularis layers of AVL had a higher cellularity than PVL. In PVL and AVL most cells were reactive for vimentin and nonmuscle (NM) myosin, though vimentin-positive cells were more abundant in AVL than in PVL. Sparse cells positive to antismooth muscle (SM) α-actin, calponin, and anti-SM myosin antibodies were found only at the outer edge of fibrosa. In Western blotting, AVL and PVL extracts were shown to be equally reactive for vimentin, SM α-actin, and NM myosin, whereas both valves were negative for SM myosin and SM22. Conclusions . Three distinct cell phenotypes have been identified in both valves: fibroblasts, myofibroblasts, and fetal-type SM cells whose distribution is specifically related to the valve layers. Although PVL and AVL cell populations differ quantitatively, some minor qualitative differences exist for vimentin and NM myosin distribution. These data are essential for studies aimed at repopulating valve scaffolds by using tissue engineering technology.

This work aims at presenting some hypotheses about the potential neurobiological substrate of imagery and imagination. For the present purposes, we will define imagery as the production of mental images associated with previous percepts, and imagination as the faculty of forming mental images of a novel character relating to something that has never been actually experienced by the subject but at a great extent emerges from his inner world. The two processes appear intimately related and imagery can arguably be considered as one of the main components of imagination. In this proposal, we argue that exaptation and redeployment, two basic concepts capturing important aspects of the evolution of biological structures and functions (Anderson, 2007), could also be useful in explaining imagery and imagination. As far as imagery is concerned it is proposed that neural structures originally implicated in performing certain functions, e.g., motor actions, can be reused for the imagery of the virtual execution of that function. As far as imagination is concerned we speculate that it can be the result of a "tinkering" that combines and modifies stored perceptual information and concepts leading to the creation of novel "mental objects" that are shaped by the subject peculiar inner world. Hence it is related to his self-awareness. The neurobiological substrate of the tinkering process could be found in a hierarchical model of the brain characterized by a multiplicity of functional modules (FMs) that can be assembled according to different spatial and temporal scales. Thus, it is surmised that a possible mechanism for the emergence of imagination could be represented by modulatory mechanisms controlling the perviousness of "modifiers" along the communication channels within and between FMs leading to their dynamically reassembling into novel configurations.

Conformational Protein Diseases (CPDs) comprise over forty clinically and pathologically diverse disorders in which specific altered proteins accumulate in cells or tissues of the body. The most studied are Alzheimerβ's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, prion diseases, inclusion body myopathy, and the systemic amyloidoses. They are characterised by three dimensional conformational alterations, which are often rich in β- structure. Proteins in this non-native conformation are highly stable, resistant to degradation, and have an enhanced tendency to aggregate with like protein molecules. The misfolded proteins can impart their anomalous properties to soluble, monomeric proteins with the same amino acid sequence by a process that has been likened to seeded crystallization. However, these potentially pathogenic proteins also have important physiological actions, which have not completely characterized. This opens up the question of what process transforms physiological actions into pathological actions and most intriguing, is why potentially dangerous proteins have been maintained during evolution and are present from yeasts to humans. In the present paper, we introduce the concept of mis-exaptation and of mis-tinkering since they may help in clarifying some of the double edged sword aspects of these proteins. Against this background an original interpretative paradigm for CPDs will be given in the frame of the previously proposed Red Queen Theory of Aging.

Pharyngo-oesophageal function was investigated in 12 patients with Zenker's diverticulum before and after cricopharyngeal myotomy with or without diverticulectomy using low-compliance, high-frequency oesophageal manometry. Nine healthy volunteers served as controls. The amount of muscle and connective tissue in the cricopharyngeal muscle was also measured by computerized morphometry and compared with findings in cadavers with no history of dysphagia. Preoperative manometry in patients with Zenker's diverticulum showed an incomplete relaxation of the upper oesophageal sphincter (UOS) (the residual UOS pressure at swallowing was 7.9 (range 1-20)mmHg in patients versus 0.2 (-12.5-14) mmHg in controls; P < 0.001) and increased pharyngeal intrabolus pressure (21 (range 0-52) versus 9 (range 0-16) mmHg; P < 0.01), with no pharyngo-oesophageal coordination abnormalities. Both parameters significantly decreased after myotomy. Patients with Zenker's diverticulum had significantly fewer muscle fibres in the cricopharyngeus and the muscle:connective tissue ratio was significantly lower (0.94 (range 0.8-1.4) in patients versus 1.5 (1.4-3.6) in controls; P < 0.05). This study supports the theory that Zenker's diverticulum is caused by an increased intrapharyngeal pressure at swallowing due to incomplete cricopharyngeal muscle relaxation resulting from localized sclerosis.

"Integration" is a key term in describing how nervous system can perform high level functions. A first condition to have "integration" is obviously the presence of efficient "communication processes" among the parts that have to be combined into the harmonious whole. In this respect, two types of communication processes, called wiring transmission (WT) and volume transmission (VT), respectively, were found to play a major role in the nervous system, allowing the exchange of signals not only between neurons, but rather among all cell types present in the central nervous system (CNS). A second fundamental aspect of a communication process is obviously the recognition/decoding process at target level. As far as this point is concerned, increasing evidence emphasizes the importance of supramolecular complexes of receptors (the so called receptor mosaics) generated by direct receptor-receptor interactions. Their assemblage would allow a first integration of the incoming information already at the plasma membrane level. Recently, evidence of two new subtypes of WT and VT has been obtained, namely the tunnelling nanotubes mediated WT and the microvesicle (in particular exosomes) mediated VT allowing the horizontal transfer of bioactive molecules, including receptors, RNAs and micro-RNAs. The physiological and pathological implications of these types of communication have opened up a new field that is largely still unexplored. In fact, likely unsuspected integrative actions of the nervous system could occur. In this context, a holistic approach to the brain-body complex as an indissoluble system has been proposed. Thus, the hypothesis has been introduced on the existence of a brain-body integrative structure formed by the "area postrema/nucleus tractus solitarius" (AP/NTS) and the "anteroventral third ventricle region/basal hypothalamus with the median eminence" (AV3V-BH). These highly interconnected regions operate as specialized interfaces between the brain and the body integrating brain-borne and body-borne neural and humoral signals.

Humans share the common intuition of a self that has access to an inner 'theater of mind' (Baars, 2003). The problem is how this internal theater is formed. Moving from Cook's view (Cook, 2008), we propose that the 'sentience' present in single excitable cells is integrated into units of neurons and glial cells transiently assembled into "functional modules" (FMs) organized as systems of encased networks (from cell networks to molecular networks). In line with Hebb's proposal of 'cell assemblies', FMs can be linked to form higher-order mosaics by means of reverberating circuits. Brain-level subjective awareness results from the binding phenomenon that coordinates several FM mosaics. Thus, consciousness may be thought as the global result of integrative processes taking place at different levels of miniaturization in plastic mosaics. On the basis of these neurobiological data and speculations and of the evidence of 'mirror neurons' the 'Hall of Mirrors' is proposed as a significant metaphor of consciousness. This article is part of a Special Issue entitled: Brain Integration.

Endothelial cell (EC) differentiation on basement membrane matrix is a highly specific process, which recapitulates many steps in blood vessel formation, including cell adhesion, migration, alignment, protease secretion and tubule formation. For this reason, it is presently considered as a reliable in vitro tool to identify factors with potential anti- or proangiogenic properties. Examples include pharmacological trials, as well as studies focused on the identification of endogenous factors involved in the modulation of the angiogenic process. The EC tube formation on basement membrane matrix is generally used as a first screen that is followed by additional in vitro and ex vivo or in vivo assays. Being quite easy to perform and not focused on a single specific step of the angiogenic process, as a first screen assay, it has many advantages over other in vitro tests. Moreover, the results provided by the tube formation on basement membrane assay can be accurately quantified by morphometric techniques and/or image analysis. Methodological issues concerning the assay and the quantitative evaluation of the results it provides are the focus of this chapter.

Conformational protein diseases of the human central nervous system represent a subject that has crucial theoretical and medical implications. They include several important neurodegenerative diseases, such as Alzheimer's, Parkinson's, Huntington's and Creutzfeldt-Jacob's diseases, amyotrophic lateral sclerosis, and the tauopathies. They occur when soluble proteins undergo conformational rearrangements becoming capable of aggregate into β-sheets conformations leading to the production of insoluble complexes known as amyloid deposits, that accumulate and lead to neurons and glial cells death. Theoretical and experimental evidence indicates that a key role in the conformational changes leading to amyloid formation is played by short sequence stretches within a given protein. Thus, the identification of protein regions potentially involved in aggregate formation and the characterization of their properties are relevant questions in the study of conformational proteins diseases. To address these questions, bioinformatics methods might provide an important contribution, suggesting possible mechanisms of protein aggregation, and focusing and orienting the experimental work. Thus, in the first part of the present review bioinformatics methods specifically attempting to predict aggregation-prone regions in proteins will be briefly described. Furthermore, the results provided by the combined use of some of them to analyze a set of particularly important proteins involved in human degenerative diseases will be discussed.

It is proposed that the moonlighting concept can be applied to G protein coupled receptors (GPCRs) as, obviously, they can carry out different types of functions. The same motifs in, for example, the third intracellular loop, can moonlight by switching between receptor-receptor interactions and interactions with signaling proteins such as G proteins or calmodulin. A "guide-and-clasp" manner of receptor-receptor interactions has been proposed where the "adhesive guides" may be the triplet homologies. As an example, the triplets AAR (or RAA) and AAE (or EAA) homologies in A(2A) R-D2 R heteromers may guide-and-clasp binding not only of the two protomers but also of calmodulin and G(i) . A beautiful moonlighting phenomenon in the A(2A) R-D2 R heteromer is that the positively charged D2 R N-terminal third intracellular loop epitope (VLRRRRKRVN) may switch between bindings to the negatively charged A(2A) R epitope (SAQEpSQGNT), localized in the medium segment of the C terminus of the A2A receptor to several negative epitopes of calmodulin. Furthermore, overlapping motifs may favor moonlighting to G(i/o) via inter alia electrostatic interaction between triplets AAR(in D2 R third intracellular loop) and AAE (G(i/alpha1) ) (and/or their symmetric variants) contributing to guide-and-clasp D2 R-G(i) interactions Thus, moonlighting in GPCR heteromers can take place via allosteric receptor-receptor interactions and is also described in D1 R-D2 R, D2 R-5-HT2 R,and A1 R-P2Y1 heteromers. Allosteric receptor-receptor interactions in GPCR-receptor tyrosine kinases (RTKs) heteromers and postulated ion channel receptor-RTK heteromers-like, for example, AMPA-NMDA-TrkB heteromers may lead to moonlighting of the participating GPCR and RTK protomers altering, for example, the pattern of the five major signaling pathways of the RTKs favoring MAPK and/or mTOR signaling with high relevance for neurodegenerative processes and depression induced atrophy of neurons. Moonlighting may also develop in the intracellular loops and C-terminal of the GPCRs as a result of dynamic allosteric interactions between different types of G proteins and other receptor interacting proteins in these domains of the receptor.

Urotensin-II (U-II) is an endogenous peptide recently characterized as a "nonclassic" pro-angiogenic cytokine. In fact, human vascular endothelial cells express the U-II receptor and exhibit a strong in vitro angiogenic response to the peptide, which was specifically triggered by the binding of U-II to its receptor and involved the activation of ERK1/2 and PI3K/Akt signaling pathways. Moreover, available studies, designed to investigate the pro-angiogenic effect quite shortly following U-II stimulation, suggested that the angiogenic action of the peptide was direct and not associated with an increased expression of vascular endothelial growth factor (VEGF) and/or its receptors. In the present study, the expression of three pro-angiogenic factors, namely VEGF, endothelin-1, and adrenomedullin, was studied in human umbilical vein endothelial cells (HUVEC) for longer times of U-II stimulation. RT-PCR and Western blot indicated that in HUVEC, exposed for at least 24h to U-II, the expression of the three angiogenic molecules was significantly increased at both mRNA and protein level, opening the possibility that U-II, not only could exert a direct stimulation of an angiogenic phenotype in endothelial cells quite shortly following exposure to the peptide, but could also further enhance the process indirectly by inducing in the cells a delayed production of other pro-angiogenic factors. Interestingly, a preliminary analysis of the time course of the in vitro capillary-like pattern formation was consistent with this view, suggesting a two phase temporal dynamics of the process.

This work investigated the in vitro and in vivo anti-angiogenic activity of some pyrroloazaflavones, exactly 2-phenyl-1H-pyrrolo[2,3-h]quinolin-4(7H)ones, with vinblastine as reference compound. Growth inhibitory activity, migration, and capillary-like structures formation were determined in human umbilical vein endothelial cell cultures, and Matrigel plug assay was carried out to evaluate in vivo effects on angiogenesis. Collectively, our results indicate that some pyrroloazaflavone derivatives, at non-cytotoxic concentrations and like vinblastine are able: (i) to exert in vitro anti-angiogenic activity and (ii) to counteract in vitro and in vivo the pro-angiogenic effects of fibroblast growth factor-2 (FGF-2).

Computational systems are useful in neuroscience in many ways. For instance, they may be used to construct maps of brain structure and activation, or to describe brain processes mathematically. Furthermore, they inspired a powerful theory of brain function, in which the brain is viewed as a system characterized by intrinsic computational activities or as a "computational information processor. "Although many neuroscientists believe that neural systems really perform computations, some are more cautious about computationalism or reject it. Thus, does the brain really compute? Answering this question requires getting clear on a definition of computation that is able to draw a line between physical systems that compute and systems that do not, so that we can discern on which side of the line the brain (or parts of it) could fall. In order to shed some light on the role of computational processes in brain function, available neurobiological data will be summarized from the standpoint of a recently proposed taxonomy of notions of computation, with the aim of identifying which brain processes can be considered computational. The emerging picture shows the brain as a very peculiar system, in which genuine computational features act in concert with noncomputational dynamical processes, leading to continuous self-organization and remodeling under the action of external stimuli from the environment and from the rest of the organism.

The concept of intra-membrane receptor–receptor interactions (RRIs) between different types of G protein-coupled receptors (GPCRs) and evidence for their existence was introduced by Agnati and Fuxe in 1980/81 through the biochemical analysis of the effects of neuropeptides on the binding characteristics of monoamine receptors in membrane preparations from discrete brain regions and functional studies of the interactions between neuropeptides and monoamines in the control of specific functions such as motor control and arterial blood pressure control in animal models.Whether GPCRs can form high-order structures is still a topic of an intense debate. Increasing evidence, however, suggests that the hypothesis of the existence of high-order receptor oligomers is correct. A fundamental consequence of the view describing GPCRs as interacting structures, with the likely formation at the plasma membrane of receptor aggregates of multiple receptors (Receptor Mosaics) is that it is no longer possible to describe signal transduction simply as the result of the binding of the chemical signal to its receptor, but rather as the result of a filtering/integration of chemical signals by the Receptor Mosaics (RMs) and membrane-associated proteins. Thus, in parallel with experimental research, significant efforts were spent in bioinformatics and mathematical modelling. We review here the main approaches that have been used to assess the interaction interfaces allowing the assembly of GPCRs and to shed some light on the integrative functions emerging from the complex behaviour of these RMs. Particular attention was paid to the RMs generated by adenosine A2A, dopamine D2, cannabinoid CB1, and metabotropic glutamate mGlu5 receptors (A2A, D2, CB1 and mGlu5, respectively), and a possible approach to model the interplay between the D2–A2A–CB1 and D2–A2A–mGlu5 trimers is proposed. This article is part of a Special Issue entitled: “Adenosine Receptors”.

Thymosin-beta 4 (T beta 4) is a G-actin sequestering peptide involved in regeneration and remodeling of injured tissues. In this work, we have designed and synthesized three peptide sequences containing the N-terminus (TYB4-n), the central part (TYB4-i) or the C-terminus (TYB4-c) of T beta 4. All fragments are overlapping on the main central binding actin site. After a structural characterization, we have evaluated in vitro and in vivo their pro-angiogenic effects. The results of this study have shown that: (i) each fragment reproduces the native conformation; (ii) T beta 4-derived peptides exert both in vitro and in vivo pro-angiogenic effects; (iii) their in vitro effect seem to be related to the activation of several signaling pathways and is positively modulated by the N-terminus of T beta 4. (C) 2011 Elsevier Inc. All rights reserved.

To create computerized three-dimensional models of the crural fascia and of the superficial layer of the thoracolumbar fascia. Serial sections of these two fasciae, stained with Azan-Mallory, van Gieson and anti-S100 antibody stains, were recorded. The resulting images were merged (Image Zone 5.0 software) and aligned (MatLab Image Processing Toolkit). Color thresholding was applied to identify the structures of interest. 3D models were obtained with Tcl/Tk scripts and Paraview 3.2.1 software. From these models, the morphometric features of these fasciae were evaluated with ImageJ. In the crural fascia, collagen fibers represent less than 20% of the total volume, arranged in three distinct sub-layers (mean thickness, 115 μm), separated by a layer of loose connective tissue (mean thickness, 43 μm). Inside a single sub-layer, all the fibers are parallel, whereas the angle between the fibers of adjacent layers is about 78°. Elastic fibers are less than 1%. Nervous fibers are mostly concentrated in the middle layer. The superficial layer of the thoracolumbar fascia is also formed of three thinner sub-layers, but only the superficial one is similar to the crural fascia sub-layers, the intermediate one is similar to a flat tendon, and the deep one is formed of loose connective tissue. Only the superficial sub-layer has rich innervation and a few elastic fibers. Computerized three-dimensional models provide a detailed representation of the fascial structure, for better understanding of the interactions among the different components. This is a fundamental step in understanding the mechanical behavior of the fasciae and their role in pathology.

In this study, the extent of angiogenesis, evaluated as microvascular density, and the immunoreactivity of tumor cells to erythropoietin (Epo) and of endothelial cells to Epo receptor (EpoR) have been correlated in human glioma specimens, and the effect of anti-Epo antibody on glioma-induced angiogenesis in vivo in the chick embryo chorioallantoic membrane (CAM) has been investigated. Results show that: (1) Epo/EpoR expression correlates with angiogenesis, (2) in the CAM assay, tumor bioptic specimens induce a strong angiogenic response, comparable to that induced by VEGF, and (3) an anti-Epo antibody co-administered with tumor bioptic specimens significantly inhibits the angiogenic response. These findings suggest the presence of a loop in the Epo/EpoR system, i.e. Epo is secreted by glioma tumor cells and it affects glioma vascular endothelial cells via its receptor and promotes angiogenesis in a paracrine manner. Moreover, as demonstrated by in vivo experiments, Epo is responsible for the strong angiogenic response induced by human glioma bioptic specimens, because an anti-Epo antibody is able to significantly inhibit this response.

It has been estimated that at least 50% of the drugs available on the market act on G-protein coupled receptors (GPCRs) and most of these are basically or agonists or antagonists of this type of receptors. Herein, we propose new putative targets for drug development based on recent data on GPCR allosterism and on the existence of receptor mosaics (RMs). The main target for drug development is still GPCRs, but the focus is not the orthosteric binding pocket. According to the mosaic model of the plasma membrane, we mainly discuss the possibility of indirect modulatory pharmacological actions on expression/function of GPCRs. In particular, the following two new targets will be analyzed: a) The possibility of pharmacological interventions on the roamer-type of volume transmission (VT), which allow the intercellular transfer of set of signal molecules such as GPCRs, tetraspanins and ribonucleic acids. Thus, there is the possibility of pharmacological interventions on the decoding capabilities of neurons and/or glial cells by means of an action on composition and release of micro-vesicles. b) The possibility of pharmacological interventions on epigenetic mechanisms by taking into account their inter-relationships with GPCRs. As a matter of fact, there are epigenetic changes that are characteristic of periods of developmental plasticity that could provide a target for therapeutic intervention in the event of brain damage. We believe that almost all the biochemical knowledge presently available on GPCRs can be used in the development of these new pharmacological approaches.

While the G protein-coupled receptor (GPCR) oligomerization has been questioned during the last fifteen years, the existence of a multi-receptor complex involving direct receptor–receptor interactions, called receptor oligomers, begins to be widely accepted. Eventually, it has been postulated that oligomers constitute a distinct functional form of the GPCRs with essential receptorial features. Also, it has been proven, under certain circumstances, that the GPCR oligomerization phenomenon is crucial for the receptor biosynthesis, maturation, trafficking, plasma membrane diffusion, and pharmacology and signalling. Adenosine receptors are GPCRs that mediate the physiological functions of adenosine and indeed these receptors do also oligomerize. Accordingly, adenosine receptor oligomers may improve the molecular mechanism by which extracellular adenosine signals are transferred to the G proteins in the process of receptor transduction. Importantly, these adenosine receptor-containing oligomers may allow not only the control of the adenosinergic function but also the fine-tuning modulation of other neurotransmitter systems (i.e. dopaminergic and glutamatergic transmission). Overall, we underscore here recent significant developments based on adenosine receptor oligomerization that are essential for acquiring a better understanding of neurotransmission in the central nervous system under normal and pathological conditions. This article is part of a Special Issue entitled: “Adenosine Receptors”.

Morphological techniques (histology and electron microscopy), as well as immunofluorescence assays, were applied to the study of the localization and smooth muscle cell (SMC) composition of atherosclerotic lesions in Watanabe heritable hyperlipidemic (WHHL) rabbits during a 4.5-month period. Vascular segments from different arteries (carotid, coronary, and iliac arteries) or from the same vessel at different levels (aorta) of animals at days 7, 15, 30, 40, 60, 90, and 135 showed that the atherosclerotic lesion first became visible at the level of the aortic arch in 60-day-old WHHL animals. Histological examination of serial cryosections from this vascular region indicated that the vascular lesion arose from a cavity in the media layer, located anatomically at the level of the juncture of the ligamentum arteriosum with the aortic arch. This aortic arch cavity is formed during the postnatal closure of the ductus arteriosus and is characterized by the presence of a thickened intima, which was absent in the other vascular regions examined. Immunofluorescence comparison of normal and atherosclerotic tissues from the aortic arch cavity wall with the use of monoclonal antibodies specific for smooth muscle and nonmuscle myosin isoforms revealed the existence of distinct SMC populations. SMCs in the thickened intima showed a myosin isoform pattern peculiar to cells with a degree of maturation intermediate between the fully differentiated and the developing (fetal) aortic SMCs. By contrast, SMCs present in atherosclerotic lesions displayed a predominant fetal-type pattern of myosin isoform expression. The achievement of this myosin isoform content seems to be correlated with the accumulation of lipids in the intima. In the media subjacent to the intimal thickening or atherosclerotic lesion, SMCs primarily displayed an intermediate degree of maturation. In older WHHL animals and at this aortic level, the SMC composition of the atherosclerotic lesion did not change, whereas in the subjacent media, the cells of intermediate type almost disappeared. In the vascular regions in which the atherosclerotic lesion appeared at later stages, such as near the aortic bifurcation, the distribution of fetal and intermediate cell types in the atherosclerotic wall was similar to that taken at the aortic arch level. These results indicate that there is 1) a preferential anatomic site from which atherogenesis initiates in WHHL rabbits; 2) a time correlation between the accumulation of lipids in the wall and the phenotypic change of SMCs toward a poorly differentiated cell type; and 3) the tendency for SMCs to follow the same differentiation pattern in early atherosclerotic lesions, irrespective of the site and time at which they develop.

Based on indications of direct physical interactions between neuropeptide and monoamine receptors in the early 1980s, the term receptor-receptor interactions was introduced and later on the term receptor heteromerization in the early 1990s. Allosteric mechanisms allow an integrative activity to emerge either intramolecularly in G protein-coupled receptor (GPCR) monomers or intermolecularly via receptor-receptor interactions in GPCR homodimers, heterodimers, and receptor mosaics. Stable heteromers of Class A receptors may be formed that involve strong high energy arginine-phosphate electrostatic interactions. These receptor-receptor interactions markedly increase the repertoire of GPCR recognition, signaling and trafficking in which the minimal signaling unit in the GPCR homomers appears to be one receptor and one G protein. GPCR homomers and GPCR assemblies are not isolated but also directly interact with other proteins to form horizontal molecular networks at the plasma membrane.

Intermedin (IMD) is a recently discovered peptide closely related to adrenomedullin. Its principal physiological activity is its role in the regulation of the cardiovascular system, where it exerts a potent hypotensive effect. In addition, data were recently provided showing that this peptide is able to exert a clearcut pro-angiogenic effect both in vitro and in vivo. IMD acts through the non-selective interaction with receptor complexes formed by the dimerization of calcitonin-like receptor (CLR) with the receptor activity-modifying proteins RAMP1, 2 or 3. Thus, in the present study, the role of CLR/RAMP complexes in mediating the pro-angiogenic effect induced by IMD on human umbilical vein endothelial cells (HUVECs) cultured on Matrigel was examined. Real-time PCR demonstrated the expression of IMD, CLR/RAMP1 and CLR/RAMP2 (but not CLR/RAMP3) mRNA in HUVECs. IMD exerted a significant in vitro angiogenic action, specifically triggered by the binding of the peptide to CLR/RAMP complexes. Both CLR/RAMP1 and CLR/RAMP2 appeared to mediate the pro-angiogenic effect, which was associated with a significant increase of vascular endothelial growth factor (VEGF) mRNA expression 18 h following IMD administration, indicating that the observed pro-angiogenic effects are related, at least in part, to an increased synthesis of this growth factor promoted by the peptide. Western blot analysis, however, showed a significant increase of VEGF receptor-2 phosphorylation as early as 5 min following IMD administration, indicating that IMD induces a pro-angiogenic response in human vascular endothelial cells not only via CLR/RAMP-induced release of VEGF, but also during signal initiation and propagation by transactivating the VEGF receptor-2 machinery.

The proposal on the existence of two main modes of intercellular communication in the central nervous system (CNS) was introduced in 1986 and called wiring transmission (WT) and volume transmission (VT). The major criterion for this classification was the different characteristics of the communication channel with physical boundaries well delimited in the case of WT (axons and their synapses; gap junctions) but not in the case of VT (the extracellular fluid filled tortuous channels of the extracellular space and the cerebrospinal fluid filled ventricular space and sub-arachnoidal space). The basic dichotomic classification of intercellular communication in the brain is still considered valid, but recent evidence on the existence of unsuspected specialized structures for intercellular communication, such as microvesicles (exosomes and shedding vesicles) and tunnelling nanotubes, calls for a refinement of the original classification model. The proposed updating is based on criteria which are deduced not only from these new findings but also from concepts offered by informatics to classify the communication networks in the CNS. These criteria allowed the identification also of new sub-classes of WT and VT, namely the "tunnelling nanotube type of WT" and the "Roamer type of VT." In this novel type of VT microvesicles are safe vesicular carriers for targeted intercellular communication of proteins, mtDNA and RNA in the CNS flowing in the extracellular fluid along energy gradients to reach target cells. In the tunnelling nanotubes proteins, mtDNA and RNA can migrate as well as entire organelles such as mitochondria. Although the existence and the role of these new types of intercellular communication in the CNS are still a matter of investigation and remain to be fully demonstrated, the potential importance of these novel types of WT and VT for brain function in health and disease is discussed.

Angiogenesis, the process through which new blood vessels arise from pre-existing ones, is regulated by numerous “classic” factors and other “nonclassic” regulators of angiogenesis. Among these latter urotensin-II is a cyclic 11-amino acid (human) or 15-amino acid (rodent) peptide, originally isolated from the fish urophysis, which exerts a potent systemic vasoconstrictor and hypertensive effect. This review article summarizes the literature data concerning the involvement of urotensin-II in angiogenesis.

Human vascular endothelial cells express the urotensin-II (U-II) receptor and exhibit a strong in vitro angiogenic response to the peptide. Thus, in the present study an in vitro model, based on human umbilical vein endothelial cells (HUVEC) cultured on Matrigel, was used to characterize more in detail the signaling pathways that control the pro-angiogenic action of U-II. The activation of the U-II receptor (UT) was associated with an increase of intracellular calcium concentration. Both calcium rise and pro-angiogenic effect of the peptide can be blocked by U73122, a selective inhibitor of phospholipase-C, indicating that the signal transduction from UT mainly involves the phospholipase-C/IP 3 pathway. As far as the downstream signaling pathways are concerned, western blot analyses and experiments with specific inhibitors indicated that the U-II-induced self-organization of the cells into capillary-like structures was PKC dependent and involved the activation of the ERK1/2, but not p38-MAPK, transduction pathway. Interestingly, the pharmacological inhibition of PI3K (obtained with LY294002), hindered the capacity of U-II to induce a proangiogenic effect on HUVEC, suggesting that PI3K-dependent pathways also play a role in regulating the process.

The effects of low and high concentrations of cocaine have been studied in vitro on the trafficking of plasma membrane A(2A) and D(2) immunoreactivities in previously characterized A(2A)-D(2) CHO cell lines. Receptor double immunofluorescence staining was performed with D(2) and A(2A) antibodies, planar lipid rafts immunolabeling with biotinylated cholera toxin subunit B and membrane invaginations with an anti-caveolin-1 antibody. A computer-assisted image analysis demonstrated a substantial and highly significant rise of membrane-associated D(2) immunoreactivity (IR) after 8 h of exposure to a low concentration of cocaine (150 nM). At this low concentration of cocaine, there was also an increase of membrane associated A(2A) immunoreactivity but smaller and less significant. However, this increase became considerably larger and highly significant at 150 microM at which concentration the rise of D(2) immunoreactivity had begun to disappear. It may be suggested that an allosteric action of cocaine at 150 nM on the D(2) receptors may primarily increase the insertion of D(2) monomers, homomers and also of a subpopulation of A(2A)-D(2) heteromers from the cytoplasm into the plasma membrane due to the conformational change induced by cocaine in the D(2) receptor. The planar lipid rafts and the caveolae are only affected by the higher concentrations of cocaine. It is proposed that changes in D(2) and A(2A)-D(2) trafficking induced by allosteric actions of cocaine at D(2) receptors may contribute to the alterations of D(2) signaling found in cocaine abusers.

The available evidence for receptor-receptor interactions between adenosine A(2A), dopamine D(2), cannabinoid CB(1), and metabotropic glutamate mGlu(5) receptors (A(2A), D(2), CB(1), and mGlu(5), respectively) is revised under the "receptor mosaic" perspective. Furthermore, the concept of "hub receptor" is defined in accordance with informatics and it is tentatively illustrated in the case of the hypothesized tetramer formed by the above mentioned receptors. On the basis of some biochemical features of the four receptors and of a bioinformatics analysis, an objective deduction of their "similarity" has been obtained. To this aim the Canberra, Euclidean and Chebyshev multivariate distance metrics have been used. It is interesting to note that A(2A) and D(2) are the most different ones, while CB(1) and mGlu(5) are the most similar ones among the four receptors analyzed. Finally, by means of a bioinformatics analysis based on different approaches the possible binding sites mediating G-protein-coupled receptor (GPCR) interactions have been indicated. It is interesting to note that in some instances accordance has been found between the bioinformatics indications and the available experimental data.

Intussusceptive microvascular growth (IMG), which occurs by splitting of the existing vasculature by transluminal pillars or transendothelial bridges, has been demonstrated in several tumors such as colon and mammary carcinomas, melanoma and B-cell non-Hodgkin's lymphomas. In this study, we have correlated in human glioma the extent of angiogenesis, evaluated as microvascular density, the immunoreactivity of tumor cells to vascular endothelial growth factor (VEGF), vessel diameter and IMG to the tumor stage. Results demonstrate for the first time a relationship in human glioma progression between angiogenesis, VEGF immunoreactivity of tumor cells, vessel diameter and the number of connections of intraluminal tissue folds with the opposite vascular wall, expression of IMG and suggest that IMG could be a mechanism of compensatory vascular growth occurring in human glioma. The advantages are that (1) blood vessels are generated more rapidly; (2) it is energetically and metabolically more economic; (3) the capillaries thereby formed are less leaky.

It is well known that tumor growth is strictly dependent on neo-vessel formation inside the tumor mass and that cell adhesion is required to allow EC proliferation and migration inside the tumor. In this work, we have evaluated the in vitro and in vivo effects on angiogenesis of some peptides, originally designed to promote cell adhesion on biomaterials, containing RGD motif mediating cell adhesion via integrin receptors [RGD, GRGDSPK, and (GRGDSP)4K] or the heparin-binding sequence of human vitronectin that interacts with HSPGs [HVP(351–359)]. Cell adhesion, proliferation, migration, and capillary-like tube formation in Matrigel were determined on HUVECs, whereas the effects on in vivo angiogenesis were evaluated using the CAM assay. (GRGDSP)4K linear sequence inhibited cell adhesion, decreased cell proliferation, migration and morphogenesis in Matrigel, and induced anti-angiogenic responses on CAM at higher degree than that determined after incubation with RGD or GRGDSPK. Moreover, it counteracted both in vitro and in vivo the pro-angiogenic effects induced by the Fibroblast growth factor (FGF-2). On the other hand, HVP was not able to affect cell adhesion and appeared less effective than (GRGDSP)4K. Our data indicate that the activity of RGD-containing peptides is related to their adhesive properties, and their effects are modulated by the number of cell adhesion motifs and the aminoacidic residues next to these sequences. The anti-angiogenic properties of (GRGDSP)4K seem to depend on its interaction with integrins, whereas the effects of HVP may be partially due to an impairment of HSPGs/FGF-2. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.

Micro-vesicles can be released by different cell types and operate as 'safe containers' mediating inter-cellular communication. In this work we investigated whether cultured myoblasts could release exosomes. The reported data demonstrate, for the first time, that C2C12 myoblasts release micro-vesicles as shown by the presence of two exosome markers (Tsg101 and Alix proteins). Using real-time PCR analysis it was shown that these micro-vesicles, like other cell types, carry mtDNA. Proteomic characterization of the released micro-vesicle contents showed the presence of many proteins involved in signal transduction. The bioinformatics assessment of the Disorder Index and Aggregation Index of these proteins suggested that C2C12 micro-vesicles mainly deliver the machinery for signal transduction to target cells rather than key proteins involved in hub functions in molecular networks. The presence of IGFBP-5 in the purified micro-vesicles represents an exception, since this binding protein can play a key role in the modulation of the IGF-1 signalling pathway. In conclusion, the present findings demonstrate that skeletal muscle cells release micro-vesicles, which probably have an important role in the communication processes within skeletal muscles and between skeletal muscles and other organs. In particular, the present findings suggest possible new diagnostic approaches to skeletal muscle diseases.

The B subunit of cholera toxin, which binds specifically to GM1 ganglioside on cell surfaces, has previously been shown to modulate intracellular calcium levels and growth in several cell types. To explore a role for such changes in calcium in the growth regulatory function of cell-associated GM1 in neurons, dissociated neurons from chicken embryonic day 8 dorsal root ganglia were exposed to the B subunit. To enhance sensitivity to B subunit, some neurons were also enriched with added GM1 (100 μM) and then exposed to B subunit. Incubation of naive cultures with 1 μg/ml of the B subunit was sufficient to produce modest increases in intracellular free calcium above basal levels in a minor percentage of cells for at least 5 min, as measured by fura-2 fluorescence imaging. Pretreatment of the cells with GM1 for 48 hr increased even further the elevations in intracellular free calcium and the percentage of responding neurons observed after B subunit exposure. These in creases in intracellular calcium required the presence of external Ca2+, but were not inhibited by calcium channel blockers. Such changes in calcium were accompanied by fine alterations in morphology affecting mostly the branching of neurites and were more pronounced in the presence of GM1. However, the morphological changes did not result in altered neurofilament protein expression. Immunogold electron microscopy using anti-choleragenoid depicted extensive aggregations of immunoreactive gold particles on neuronal surfaces, which were more extensive in cells treated with GM1. The results demonstrate that cell incorporated GM1 may modulate calcium fluxes, perhaps accounting for the growth regulatory functions of GM1 in both neuronal and other cell types. © 1992 Wiley-Liss, Inc.

In situ hybridization and Northern blotting were used to study the expression of brain-derived neurotrophic factor (BDNF) mRNA in the rat brain following photochemical stroke. A focal thrombotic lesion of the sensorimotor cortex was produced by intravenously injecting the light-sensitive dye rose bengal and exposing the skull to a controlled beam of light. Four hours after the light exposure the level of BDNF mRNA was increased in the hippocampus and cortex ipsilateral and perifocal to the lesion. The stroke-induced BDNF mRNA increase was prevented by the non-competitive glutamate receptor blocker dizocilpine (MK-801). The results indicate that the activation of N-methyl-D-aspartate (NMDA)-sensitive glutamate receptors is involved in the stroke-triggered stimulation of BDNF mRNA increase.

A recently proposed approach was used to model the self-organization into capillary-like structures of human vascular endothelial cells cultured on Matrigel. The model combines a Cellular Potts Model, considering cell adhesion, cytoskeletal rearrangement and chemotaxis, and a Partial Differential Equation model describing the release and the diffusion of a chemoattractant. The results were compared with the data from real in vitro experiments to establish the capability of the model to accurately reproduce both the spontaneous self-assembly of unstimulated cells and their self-organization in the presence of the proangiogenic factor adrenomedullin. The results showed that the model can accurately reproduce the selfassembly of unstimulated cells, but it failed in reproducing the adrenomedullin-induced self-organization of the cells. The extension of the model to include cell proliferation led to a good match between simulated and experimental patterns in both cases with predicted proliferation rates in agreement with the data of cell proliferation experiments. Developmental Dynamics 238:1951–1963, 2009. © 2009 Wiley-Liss, Inc.

Zidovudine (AZT) is an antiretroviral drug widely used in the treatment of human immunodeficiency virus (HIV)-infected patients, whose prolonged administration was found to cause toxic lesions in cardiomyocytes in humans and experimental animals. Alterations in adrenocortical secretion were frequently observed in HIV patients, but it is not clear whether medication is involved in the production of these complications. Hence, we studied in vivo and in vitro, the effects of AZT on the rat adrenal cortex. The prolonged AZT administration (100 mg/kg per day for 4 months) did not cause overt qualitative morphological alterations of adrenocortical cells, which, however, underwent a net hypertrophy. Hypertrophy is associated with increases in the volume and surface area per cell of the mitochondrial compartment and smooth endoplasmic reticulum (where the enzymes of steroid synthesis are located), and a marked decrease in the volume of the lipid-droplet compartment (where cholesterol and its esters, the precursors of steroid hormones, are stored). AZT chronic treatment induced rises in the plasma concentrations of aldosterone and corticosterone, and in the basal and ACTH-stimulated in vitro secretion of these hormones from adrenal slices. The 24-h exposure to AZT (10(-5) M) did not significantly affect either secretory activity or proliferation and apoptotic rates of cultured rat adrenocortical cells. Taken together, these findings suggest that AZT chronic treatment enhances the growth and steroidogenic capacity of rat adrenal cortex, probably by activating the central branch of the hypothalamic-pituitary-adrenal axis. The toxic activity of AZT is thought to depend on increased production of ROS. On these grounds, it is likely that the lack of toxic effect of AZT on adrenocortical cells is due to their very elevated content in vitamin C, which prevents the deleterious effect of the AZT-induced increase in intracellular ROS production.

Urotensin-II (U-II), along its receptor UT, is widely expressed in the cardiovascular system, where it exerts regulatory actions under both physiological and pathological conditions. In the present study, human vascular endothelial cells (EC) from one arterious and three venous vascular beds were used to investigate in vitro their heterogeneity in terms of expression of U-II and UT and of angiogenic response to the peptide. Real-time PCR and immunocytochemistry demonstrated the expression of UT, as mRNA and protein, in all the EC populations investigated. U-II, on the contrary, was detectable only in EC from aorta and umbilical vein. U-II did not affect the proliferation rate of adult human EC, but induced a moderate proliferative effect on EC from human umbilical vein. When tested in the Matrigel assay, however, all EC exhibited a strong angiogenic response to the peptide, comparable to that of fibroblast growth factor-2 (FGF-2) and it was not associated to an increased expression of vascular endothelial growth factor (VEGF) and/or its receptors. The angiogenic effect of U-II was abolished by the UT antagonist palosuran. Overall, these data suggest that U-II, in addition to the well known role in the regulation of cardiovascular function, also exert a specific angiogenic activity.

The functional role of the LIM-domain protein Hic-5 was investigated in microvascular endothelial cells using a siRNA approach. Knock down of Hic-5 reduced endothelial cell spreading and impaired structural organization of the cells on basement membrane extracts. Furthermore, Hic-5 was involved in the regulation of the multifunctional protein connective tissue growth factor (CTGF, CCN2). Upon Hic-5 down-regulation, induction of CTGF by lysophosphatidic acid or colchicine was reduced. Inhibition of CTGF expression was even more pronounced in cells treated with transforming growth factor beta and inhibitors of histone deacetylases. Treatment of endothelial cells with Hic-5 siRNA reduced CTGF promoter activity. Mutation analyses of the promoter revealed transcription factors binding to the basic control element as part of the proposed Hic-5-modulated transcription complex. Further analyses showed down-regulation of Hic-5 protein upon overnight treatment with inhibitors of histone deacetylases. These data suggest that the reduced expression of Hic-5 may contribute to the anti-angiogenic effects of histone deacetylase inhibitors.

The miR-17 family of microRNAs has recently been recognized for its importance during lung development. The transgenic overexpression of the entire miR-17–92 cluster in the lung epithelium led to elevated cellular proliferation and inhibition of differentiation, while targeted deletion of miR-17–92 and miR-106b–25 clusters showed embryonic or early post-natal lethality. Herein we demonstrate that miR-17 and its paralogs, miR-20a, and miR-106b, are highly expressed during the pseudoglandular stage and identify their critical functional role during embryonic lung development. Simultaneous downregulation of these three miRNAs in explants of isolated lung epithelium altered FGF10 induced budding morphogenesis, an effect that was rescued by synthetic miR-17. E-Cadherin levels were reduced, and its distribution was altered by miR-17, miR-20a and miR-106b downregulation, while conversely, beta-catenin activity was augmented, and expression of its downstream targets, including Bmp4 as well as Fgfr2b, increased. Finally, we identified Stat3 and Mapk14 as key direct targets of miR-17, miR-20a, and miR-106b and showed that simultaneous overexpression of Stat3 and Mapk14 mimics the alteration of E-Cadherin distribution observed after miR-17, miR-20a, and miR-106b downregulation. We conclude that the mir-17 family of miRNA modulates FGF10–FGFR2b downstream signaling by specifically targeting Stat3 and Mapk14, hence regulating E-Cadherin expression, which in turn modulates epithelial bud morphogenesis in response to FGF10 signaling.

There is increasing evidence suggesting a wider biological role of erythropoietin (Epo) and Epo receptor (EpoR) not related to erythropoiesis, such as the detection of EpoR in other cells, i.e. polymorphonuclear leukocytes, megakaryocytes, endothelial, myocardial and neural cells. In this study, by using a mouse model (designated tg6) that constitutively overexpresses human Epo in an oxygen-independent manner, we have investigated mast cell and macrophage number and distribution in duodenal mucosa using immunohistochemical, morphometric and image analysis methods. The results showed that tryptase-positive mast cells and BM8-positive macrophages were more numerous in duodenal mucosa specimens of tg6 mice compared with wild-type mice. Moreover, whereas in wild-type specimens both mast cells and macrophages were generally scattered throughout the villus, in tg6 specimens they were aligned along the axis of the villus. Morphometric analysis confirms this observation, and the quantitative analysis of the spatial distribution of the cells in duodenal villi indicated that in both wild-type and tg6 groups the macrophage and mast cell distribution was characterized by significant deviations from randomness. In addition, an increased number of c-kit-positive cells have been identified in the villus axis of tg6 mice, indicating an expanded compartment of mast cell precursors in the intestinal mucosa of these animals. Finally, we have also demonstrated that in tg6 specimens the number of duodenal epithelial cells positive for Epo were significantly higher as compared to wild type. Overall, these data confirm that Epo, acting as a general stimulator of the hemopoietic compartment, is able to induce an expansion of two effectors of the immune response, mast cells and macrophages, in a specific peripheral site, the duodenal mucosa, in the tg6 mouse experimental model.

Terminal arborizations expressing nerve growth factor receptor (NGF-R) have been detected with immunohistochemistry in the reticular thalamic nucleus of neonate, adult and aged rats. Intracerebroventricular administration of nerve growth factor (NGF) resulted in a dramatic increase in NGF-R immunoreactivity throughout the lifespan. This effect was paralleled by a concomitant increase in NGF-R immunopositivity in the neurons of the basal forebrain, which was here demonstrated also in aged animals, thus indicating that the NGF-R immunoreactivity within the reticular thalamic nucleus derives in all likelihood from cholinergic neuronal cell bodies of the basal forebrain. Our results demonstrate a prominent ability of NGF to up-regulate its receptors within fibres innervating the reticular thalamic nucleus, and show that this up-regulation of NGF-R is maintained throughout the lifetime. Altogether this indicates that the reticular thalamic nucleus may represent a new, important site of action of endogenous NGF or NGF-like molecules within the brain. In view of the crucial role played by the reticular thalamic nucleus in gating thalamocortical information, the autoregulation of NGF-R in this structure may have important concomitants in both physiological and pathological conditions.