Multiple Residues in the Second Extracellular Loop Are Critical for M3 Muscarinic Acetylcholine Receptor Activation

Recent studies suggest that the second extracellular loop (o2 loop) of bovine rhodopsin and other class I G protein-coupled receptors (GPCRs) targeted by biogenic amine ligands folds deeply into the transmembrane receptor core where the binding of cis-retinal and biogenic amine ligands is known to occur. In the past, the potential role of the o2 loop in agonist-dependent activation of biogenic amine GPCRs has not been studied systematically. To address this issue, we used the M3 muscarinic acetylcholine receptor (M3R), a prototypic class I GPCR, as a model system. Specifically, we subjected the o2 loop of the M3R to random mutagenesis and subsequently applied a novel yeast genetic screen to identity single amino acid substitutions that interfered with M3R function. This screen led to the recovery of about 20 mutant M3Rs containing single amino acid changes in the o2 loop that were inactive in yeast. In contrast, application of the same strategy to the extracellular N-terminal domain of the M3R did not yield any single point mutations that disrupted M3R function. Pharmacological characterization of many of the recovered mutant M3Rs in mammalian cells, complemented by site-directed mutagenesis studies, indicated that the presence of several o2 loop residues is important for efficient agonist-induced M3R activation. Besides the highly conserved Cys220 residue, Gln207, Gly211, Arg213, Gly218, Ile222, Phe224, Leu225, and Pro228 were found to be of particular functional importance. In general, mutational modification of these residues had little effect on agonist binding affinities. Our findings are therefore consistent with a model in which multiple o2 loop residues are involved in stabilizing the active state of the M3R. Given the high degree of structural homology found among all biogenic amine GPCRs, our findings should be of considerable general relevance

Studio CFD dell'influenza della forma della camera di combustione di un motore ad iniezione diretta sul miscelamento tra aria ed idrogeno

L'obiettivo del presente lavoro è quello di simulare, tramite un codice fluidodinamico, l'iniezione diretta dell'idrogeno e il conseguente miscelamento con l'aria, in un motore a combustione interna di 500 cm3, e di confrontare i risultati ottenuti con un modello preesistente validato con i dati sperimentali ottenuti precedentemente al banco dal personale di laboratorio. Il modello è stato usato per verificare come la geometria della camera di combustione influenzasse il miscelamento aria-idrogeno nel cilindro. Partendo dal modello 3D del motore Diesel originale, già modellato per lavori precedenti e, sul CAD SolidWorks 2010, sono state effettuate le modifiche che hanno permesso la trasformazione del motore Diesel a motore ad idrogeno. La geometria è stata riportata nel software Gambit, con il quale è stata creata la griglia di calcolo. La mesh così ottenuta è stata quindi riportata su Fluent 14 che, mediante l'imposizione delle condizioni al contorno, delle condizioni iniziali, dei parametri di movimento della griglia e delle impostazioni del solutore, ha elaborato le soluzioni sul dominio in esame. Dopo la validazione del modello tramite il confronto con i dati preesistenti, è stata effettuata una campagna di prove al variare di alcuni elementi geometrici della bowl; è stata inoltre definita un'efficienza di miscelamento che permettesse una valutazione quantitativa della qualità del miscelamento aria-idrogeno nella camera di combustione

Integrated analysis of the Neogene–Quaternary Valdera-Volterra Basin (Northern Apennines). Evidence for composite development of hinterland basins

The Neogene and Quaternary hinterland basins of the Northern Apennine have been the subject of different tectonic interpretations. Several studies considered these basins as the result of polyphase normal faulting framed in a continuous crustal extensional regime since the middle Miocene. On the contrary, geophysical and geological studies provided evidence of the important role played by out-of-sequence thrusts and backthrusts in the evolution of these basins during a prolongated and intense period of shortening. Here we present an integrated analysis of 2D stacked seismic reflection profiles, stratigraphic and geophysical data from deep exploration wells, gravity data, and published geological and biostratigraphic data for the Valdera-Volterra basin (central Tuscany, Italy). The results support a polyphase and composite evolution of the basin, subdivided into three main phases. During the late Tortonian?Zanclean, the growth of major thrust-related anticlines controlled the evolution of the sedimentary basin. The growth of a syncline determined the creation of accommodation space for the sediments. This main compressional deformation occurred during the Messinian and ended during the Late Zanclean. NE migration of the depocentre during the Early Zanclean was identified, likely possibly due to a differential activity growth between the bordering anticlines. During the Piacenzian, an extensional phase has been recognised, superposed to the previous compressive phase. During the Latest Piacenzian?Early Pleistocene (?), a final compressional phase took place resulting in the positive inversion of the Piacenzian WSW dipping main border fault

Atypical antipsychotics and metabolic syndrome : from molecular mechanisms to clinical differences

Atypical antipsychotics (AAPs) are commonly prescribed medications to treat schizophre-nia, bipolar disorders and other psychotic disorders. However, they might cause metabolic syndrome (MetS) in terms of weight gain, dyslipidemia, type 2 diabetes (T2D), and high blood pressure, which are responsible for reduced life expectancy and poor adherence. Importantly, there is clear evidence that early metabolic disturbances can precede weight gain, even if the latter still remains the hallmark of AAPs use. In fact, AAPs interfere profoundly with glucose and lipid homeostasis acting mostly on hypothalamus, liver, pancreatic β-cells, adipose tissue, and skeletal muscle. Their ac-tions on hypothalamic centers via dopamine, serotonin, acetylcholine, and histamine receptors affect neuropeptides and 5′ AMP-activated protein kinase (AMPK) activity, thus producing a supra-physiological sympathetic outflow augmenting levels of glucagon and hepatic glucose production. In addition, altered insulin secretion, dyslipidemia, fat deposition in the liver and adipose tissues, and insulin resistance become aggravating factors for MetS. In clinical practice, among AAPs, olan-zapine and clozapine are associated with the highest risk of MetS, whereas quetiapine, risperidone, asenapine and amisulpride cause moderate alterations. The new AAPs such as ziprasidone, lurasi-done and the partial agonist aripiprazole seem more tolerable on the metabolic profile. However, these aspects must be considered together with the differences among AAPs in terms of their efficacy, where clozapine still remains the most effective. Intriguingly, there seems to be a correlation between AAP’s higher clinical efficacy and increase risk of metabolic alterations. Finally, a multidisciplinary approach combining psychoeducation and therapeutic drug monitoring (TDM) is proposed as a first-line strategy to avoid the MetS. In addition, pharmacological treatments are discussed as well.Publisher PDFPeer reviewe

Unraveling the Functional Significance of Unstructured Regions in G Protein-Coupled Receptors

Unstructured regions in functional proteins have gained attention in recent years due to advancements in informatics tools and biophysical methods. G protein-coupled receptors (GPCRs), a large family of cell surface receptors, contain unstructured regions in the form of the i3 loop and C-terminus. This review provides an overview of the functional significance of these regions in GPCRs. GPCRs transmit signals from the extracellular environment to the cell interior, regulating various physiological processes. The i3 loop, located between the fifth and sixth transmembrane helices, and the C-terminus, connected to the seventh transmembrane helix, are determinant of interactions with G proteins and with other intracellular partners such as arrestins. Recent studies demonstrate that the i3 loop and C-terminus play critical roles in allosterically regulating GPCR activation. They can act as autoregulators, adopting conformations that, by restricting G protein access, modulate receptor coupling specificity. The length and unstructured nature of the i3 loop and C-terminus provide unique advantages in GPCR interactions with intracellular protein partners. They act as "fishing lines", expanding the radius of interaction and enabling GPCRs to tether scaffolding proteins, thus facilitating receptor stability during cell membrane movements. Additionally, the i3 loop may be involved in domain swapping between GPCRs, generating novel receptor dimers with distinct binding and coupling characteristics. Overall, the i3 loop and C-terminus are now widely recognized as crucial elements in GPCR function and regulation. Understanding their functional roles enhances our comprehension of GPCR structure and signaling complexity and holds promise for advancements in receptor pharmacology and drug development

Mesenchymal Stem Cell in Pancreatic Islet Transplantation

Pancreatic islet transplantation is a therapeutic option for achieving physiologic regulation of plasma glucose in Type 1 diabetic patients. At the same time, mesenchymal stem cells (MSCs) have demonstrated their potential in controlling graft rejection, the most fearsome complication in organ/tissue transplantation. MSCs can interact with innate and adaptive immune system cells either through direct cell-cell contact or through their secretome including exosomes. In this review, we discuss current findings regarding the graft microenvironment of pancreatic islet recipient patients and the crucial role of MSCs operation as cell managers able to control the immune system to prevent rejection and promote endogenous repair. We also discuss how challenging stressors, such as oxidative stress and impaired vasculogenesis, may jeopardize graft outcomes. In order to face these adverse conditions, we consider either hypoxia-exposure preconditioning of MSCs or human stem cells with angiogenic potential in organoids to overcome islets' lack of vasculature. Along with the shepherding of carbon nanotubes-loaded MSCs to the transplantation site by a magnetic field, these studies look forward to exploiting MSCs stemness and their immunomodulatory properties in pancreatic islet transplantation

Enlightening G-protein-coupled receptors on the plasma membrane using super-resolution photoactivated localization microscopy

The possibility to visualize and image the arrangement of proteins within the cell at the molecular level has always been an attraction for scientists in biological research. In particular, for signalling molecules such as GPCRs (G-protein-coupled receptors), the existence of protein aggregates such as oligomers or clusters has been the topic of extensive debate. One of the reasons for this lively argument is that the molecular size is below the diffraction-limited resolution of the conventional microscopy, precluding the direct visualization of protein super-structures. On the other hand, new super-resolution microscopy techniques, such as the PALM (photoactivated localization microscopy), allow the limit of the resolution power of conventional optics to be broken and the localization of single molecules to be determined with a precision of 10-20 nm, close to their molecular size. The application of super-resolution microscopy to study the spatial and temporal organization of GPCRs has brought new insights into receptor arrangement on the plasma membrane. Furthermore, the use of this powerful microscopy technique as a quantitative tool opens up the possibility for investigating and quantifying the number of molecules in biological assemblies and determining the protein stoichiometry in signalling complexes

Identification of the factors affecting co-localization precision for quantitative multicolor localization microscopy

This work explores the potential of multi-color Photoactivated Localization Microscopy (PALM) imaging to probe sub-diffraction limit interactions between proteins with spectrally separated labels. Using a PALM setup built around a commercial microscope axially stabilized to nm-level, we determined the ultimate registration accuracy that could be achieved (10 nm) and compared the performance of three different pairs of fluorescent proteins that can be used in dual color PALM. Fusion constructs were cloned and imaged either in vitro or at the cell plasma membrane, allowing to identify a current limit to co-localization precision of approximately 30-40 nm. We identified the better performing pair and present a concluding perspective application to a co-clustering study. © 2012 Annibale et al