“Don’t blame the shopkeeper!!” Food, drink and confectionery advertising and British Government market controls during the Second World War

A novel series of \u3b2-lactam derivatives that was designed and synthesized to target RGD-binding and leukocyte integrins is reported. The compound library was evaluated by investigating the effects on integrin-mediated cell adhesion and cell signaling in cell lines expressing \u3b1v\u3b23, \u3b1v\u3b25, \u3b1v\u3b26, \u3b15\u3b21, \u3b1IIb\u3b23, \u3b14\u3b21, and \u3b1L\u3b22 integrins. SAR analysis of the new series of azetidinones enabled the recognition of structural elements associated with integrin selectivity. We obtained selective and potent agonists that could induce cell adhesion and promote cell signaling mediated by \u3b1v\u3b23, \u3b1v\u3b25, \u3b15\u3b21, or \u3b14\u3b21 integrin, and antagonists for the integrins \u3b1v\u3b23 and \u3b15\u3b21, as well as \u3b14\u3b21 and \u3b1L\u3b22, preventing the effects elicited by the respective endogenous agonists

Leukocyte Integrin Antagonists as a Novel Option to Treat Dry Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a complex multifactorial degenerative disease that leads to irreversible blindness. AMD affects the macula, the central part of the retina responsible for sharp central vision. Retinal pigment epithelium (RPE) is the main cellular type affected in dry AMD. RPE cells form a monolayer between the choroid and the neuroretina and are in close functional relationship with photoreceptors; moreover, RPE cells are part of the blood retina barrier that is disrupted in ocular diseases such as AMD. During ocular inflammation lymphocytes and macrophages are recruited, contact RPE and produce pro-inflammatory cytokines, which play an important role in AMD pathogenesis. The interaction between RPE and immune cells is mediated by leukocyte integrins, heterodimeric transmembrane receptors, and adhesion molecules, including VCAM-1 and ICAM-1. Within this frame, this study aimed to characterize RPE-leukocytes interaction and to investigate any potentially beneficial effects induced by integrin antagonists (DS-70, MN27 and SR714), developed in previous studies. ARPE-19 cells were co-cultured for different incubation times with Jurkat cells and apoptosis and necrosis levels were analyzed by flow cytometry. Moreover, we measured the mRNA levels of the pro-inflammatory cytokine IL-1\u3b2 and the expression of adhesion molecules VCAM-1 and ICAM-1. We found that RPE-lymphocyte interaction increased apoptosis and necrosis levels in RPE cells and the expression of IL-1\u3b2. This interaction was mediated by the binding of \u3b14\u3b21 and \u3b1L\u3b22 integrins to VCAM-1 and ICAM-1, respectively. The blockade of RPE-lymphocyte interaction with blocking antibodies highlighted the pivotal role played by integrins. Therefore, \u3b14\u3b21 and \u3b1L\u3b22 integrin antagonists were employed to disrupt RPE-lymphocyte crosstalk. Small molecule integrin antagonists proved to be effective in reducing RPE cell death and expression of IL-1\u3b2, demonstrating that integrin antagonists could protect RPE cells from detrimental effects induced by the interaction with immune cells recruited to the retina. Overall, the leukocyte integrin antagonists employed in the present study may represent a novel opportunity to develop new drugs to fight dry AMD

Eosinophil as a cellular target of the ocular anti-allergic action of mapracorat, a novel selective glucocorticoid receptor agonist

Abstract: Purpose: Glucocorticoids can either suppress gene transcription (transrepression) or activate it (transactivation). This latter process may contribute to certain side effects caused by these agents. Mapracorat (also known as BOL-303242-X or ZK 245186) is a novel selective glucocorticoid receptor agonist that maintains a beneficial anti-inflammatory activity but seems to be less effective in transactivation, resulting in a lower potential for side effects; it has been proposed for the topical treatment of inflammatory skin disorders. This study assessed the anti-allergic activity of mapracorat at the ocular level and whether eosinophils and mast cells are targets of its action

Molecular View on the iRGD Peptide Binding Mechanism: Implications for Integrin Activity and Selectivity Profiles

Receptor-selective peptides are widely used as smart carriers for specific tumor-targeted delivery. A remarkable example is the cyclic nonapeptide iRGD (CRGDKPGDC, 1) that couples intrinsic cytotoxic effects with striking tumor-homing properties. These peculiar features are based on a rather complex multistep mechanism of action, where the primary event is the recognition of RGD integrins. Despite the high number of preclinical studies and the recent success of a phase I trial for the treatment of pancreatic ductal adenocarcinoma (PDAC), there is little information available about the iRGD three-dimensional (3D) structure and integrin binding properties. Here, we re-evaluate the peptide's affinity for cancer-related integrins including not only the previously known targets alpha v beta 3 and alpha v beta 5 but also the alpha v beta 6 isoform, which is known to drive cell growth, migration, and invasion in many malignancies including PDAC. Furthermore, we use parallel tempering in the well-tempered ensemble (PT-WTE) metadynamics simulations to characterize the in-solution conformation of iRGD and extensive molecular dynamics calculations to fully investigate its binding mechanism to integrin partners. Finally, we provide clues for fine-tuning the peptide's potency and selectivity profile, which, in turn, may further improve its tumor-homing properties

Monitoring opioid receptor dimerization in living cells by bioluminescence resonance energy transfer (BRET)

Bioluminescence resonance energy transfer (BRET) is a natural phenomenon that has been successfully applied for the study of protein–protein interactions, including opioid receptor oligomers. The discovery of opioid receptor homomers and heteromers has brought to the fi nding of new functions and new way of signaling and traffi cking; therefore, opioid receptor oligomers may be considered as novel drug targets. Fusing receptors of interest with Renilla luciferase and with a fl uorescent protein (such as EYFP), it is possible to study opioid receptor dimerization using BRET

Expression of the repressor element-1 silencing transcription factor (REST) is regulated by IGF-I and PKC in human neuroblastoma cells

The repressor element 1-silencing transcription factor (REST) was first identified as a protein that binds to a 21-bp DNA sequence element (known as repressor element 1 (RE1)) resulting in transcriptional repression of the neural-specific genes [Chong et al., 1995; Schoenherr and Anderson, 1995]. The original proposed role for REST was that of a factor responsible for restricting neuronal gene expression to the nervous system by silencing expression of these genes in non-neuronal cells. Although it was initially thought to repress neuronal genes in non-neuronal cells, the role of REST is complex and tissue dependent. In this study I investigated any role played by REST in the induction and patterning of differentiation of SH-SY5Y human neuroblastoma cells exposed to IGF-I. and phorbol 12- myristate 13-acetate (PMA) To down-regulate REST expression we developed an antisense (AS) strategy based on the use of phosphorothioate oligonucleotides (ODNs). In order to evaluate REST mRNA levels, we developed a real-time PCR technique and REST protein levels were evaluated by western blotting. Results showed that nuclear REST is increased in SH-SY5Y neuroblastoma cells cultured in SFM and exposed to IGF-I for 2-days and it then declines in 5-day-treated cells concomitant with a progressive neurite extension. Also the phorbol ester PMA was able to increase nuclear REST levels after 3-days treatment concomitant to neuronal differentiation of neuroblastoma cells, whereas, at later stages, it is down-regulated. Supporting these data, the exposure to PKC inhibitors (GF10923X and Gö6976) and PMA (16nM) reverted the effects observed with PMA alone. REST levels were related to morphological differentiation, expression of growth coneassociated protein 43 (GAP-43; a gene not regulated by REST) and of synapsin I and βIII tubulin (genes regulated by REST), proteins involved in the early stage of neuronal development. We observed that differentiation of SH-SY5Y cells by IGF-I and PMA was accompanied by a significant increase of these neuronal markers, an effect that was concomitant with REST decrease. In order to relate the decreased REST expression with a progressive neurite extension, I investigated any possible involvement of the ubiquitin–proteasome system (UPS), a multienzymatic pathway which degrades polyubiquinated soluble cytoplasmic proteins [Pickart and Cohen, 2004]. For this purpose, SH-SY5Y cells are concomitantly exposed to PMA and the proteasome inhibitor MG132. In SH-SY5Y exposed to PMA and MG 132, we observed an inverse pattern of expression of synapsin I and β- tubulin III, two neuronal differentiation markers regulated by REST. Their cytoplasmic levels are reduced when compared to cells exposed to PMA alone, as a consequence of the increase of REST expression by proteasome inhibitor. The majority of proteasome substrates identified to date are marked for degradation by polyubiquitinylation; however, exceptions to this principle, are well documented [Hoyt and Coffino, 2004]. Interestingly, REST degradation seems to be completely ubiquitin-independent. The expression pattern of REST could be consistent with the theory that, during early neuronal differentiation induced by IGF-I and PKC, it may help to repress the expression of several genes not yet required by the differentiation program and then it declines later. Interestingly, the observation that REST expression is progressively reduced in parallel with cell proliferation seems to indicate that the role of this transcription factor could also be related to cell survival or to counteract apotosis events [Lawinger et al., 2000] although, as shown by AS-ODN experiments, it does not seem to be directly involved in cell proliferation. Therefore, the decline of REST expression is a comparatively later event during maturation of neuroroblasts in vitro. Thus, we propose that REST is regulated by growth factors, like IGF-I, and PKC activators in a time-dependent manner: it is elevated during early steps of neural induction and could contribute to down-regulate genes not yet required by the differentiation program while it declines later for the acquisition of neural phenotypes, concomitantly with a progressive neurite extension. This later decline is regulated by the proteasome system activation in an ubiquitin-indipendent way and adds more evidences to the hypothesis that REST down-regulation contributes to differentiation and arrest of proliferation of neuroblastoma cells. Finally, the glycosylation pattern of the REST protein was analysed, moving from the observation that the molecular weight calculated on REST sequence is about 116 kDa but using western blotting this transcription factor appears to have distinct apparent molecular weight (see Table 1.1): this difference could be explained by post-translational modifications of the proteins, like glycosylation. In fact recently, several studies underlined the importance of O-glycosylation in modulating transcriptional silencing, protein phosphorylation, protein degradation by proteasome and protein–protein interactions [Julenius et al., 2005; Zachara and Hart, 2006]. Deglycosilating analysis showed that REST protein in SH-SY5Y and HEK293 cells is Oglycosylated and not N-glycosylated. Moreover, using several combination of deglycosilating enzymes it is possible to hypothesize the presence of Gal-β(1-3)-GalNAc residues on the endogenous REST, while β(1-4)-linked galactose residues may be present on recombinant REST protein expressed in HEK293 cells. However, the O-glycosylation process produces an immense multiplicity of chemical structures and monosaccharides must be sequentially hydrolyzed by a series of exoglycosidase. Further experiments are needed to characterize all the post-translational modification of the transcription factor REST

Mapracorat, a novel non-steroidal selective glucocorticoid receptor agonist for the treatment of allergic conjunctivitis

Glucocorticoids are used to treat chronic and severe forms of allergic conjunctivitis. Although they exert a rapid and powerful therapeutic activity, relevant side effects may limit their ocular use: increase of intraocular pressure, cataract formation and reduced resistance to infections. New glucocorticoids displaying the same potency of classical glucocorticoids but with fewer adverse effects are needed for the treatment of ocular disorders. Mapracorat (also known as ZK245186 or BOL-303242-X) is a novel non-steroidal selective glucocorticoid receptor agonist that is in the first phases of clinical evaluation (Phase II Clinical trials) for topical treatment of inflammatory skin and ocular disorders. Mapracorat binds selectively to human glucocorticoid receptor and displays powerful anti-inflammatory effects. In experimental models of ocular diseases, mapracorat reduces clinical symptoms, eosinophil recruitment, chemokines and pro-inflammatory cytokines production at ocular level, confirming that it acts preventing both the early and late phase of allergic response. Interestingly, mapracorat induces a lower increase of intraocular pressure in comparison to the classical glucocorticoid dexamethasone. Several clinical trials are ongoing to investigate the efficacy and safety of mapracorat for the treatment of several ocular diseases. Transrepressive mechanisms are thought to account for the majority of mapracorat's antiinflammatory effects; however, the induction of anti-inflammatory proteins likely involved in transactivation events may contribute to mapracorat-mediated anti-inflammatory properties and deserve to be further investigated in suitable in vivo and in vitro models. These observations may influence how novel "differential" ligands are discovered, identified and evaluated

Transcriptional activation of human mu-opioid receptor gene by insulin-like growth factor-I in neuronal cells is modulated by the transcription factor REST

The human mu-opioid receptor gene (OPRM1) promoter contains a DNA sequence binding the repressor element 1 silencing transcription factor (REST) that is implicated in transcriptional repression. We investigated whether insulin-like growth factor I (IGF-I), which affects various aspects of neuronal induction and maturation, regulates OPRM1 transcription in neuronal cells in the context of the potential influence of REST. A series of OPRM1-luciferase promoter/reporter constructs were transfected into two neuronal cell models, neuroblastoma-derived SH-SY5Y cells and PC12 cells. In the former, endogenous levels of human mu-opioid receptor (hMOPr) mRNA were evaluated by real-time PCR. IGF-I up-regulated OPRM1 transcription in: PC12 cells lacking REST, in SH-SY5Y cells transfected with constructs deficient in the REST DNA binding element, or when REST was down-regulated in retinoic acid-differentiated cells. IGF-I activates the signal transducer and activator of transcription-3 signaling pathway and this transcription factor, binding to the signal transducer and activator of transcription-1/3 DNA element located in the promoter, increases OPRM1 transcription. We propose that a reduction in REST is a critical switch enabling IGF-I to up-regulate hMOPr. These findings help clarify how hMOPr expression is regulated in neuronal cells

Role of nociceptin/orphanin FQ in thermoregulation

Nociceptin/Orphanin FQ (N/OFQ) is a 17-amino acid peptide that binds to the nociceptin receptor (NOP). N/OFQ and NOP receptors are expressed in numerous brain areas. The generation of specific agonists, antagonists and receptor-deficient mice or rats has enabled progress in elucidating the biological functions of N/OFQ. These tools have been employed to identify the biological significance of the N/OFQ system and how it interacts with other endogenous systems to regulate several body functions. The present review focuses on the role of N/OFQ in the regulation of body temperature and its relationship with energy balance. Critical evaluation of the literature data suggests that N/OFQ, acting through the NOP receptor, may cause hypothermia by influencing the complex thermoregulatory system that operates as a federation of independent thermoeffector loops to control body temperature at the hypothalamic level. Furthermore, N/OFQ counteracts hyperthermia elicited by cannabinoids or µ-opioid agonists. N/OFQ-induced hypothermia is prevented by ω-conotoxin GVIA, an N-type calcium channel blocker. Hypothermia induced by N/OFQ is considered within the framework of the complex action that this neuropeptide exerts on energy balance. Energy stores are regulated through the complex neural controls exerted on both food intake and energy expenditure. In laboratory rodents, N/OFQ stimulates consummatory behavior and decreases energy expenditure. Taken together, these studies support the idea that N/OFQ contributes to the regulation of energy balance by acting as an “anabolic” neuropeptide as it elicits effects similar to those produced in the hypothalamus by other neuropeptides such as orexins and neuropeptide