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Biomedical subjects

F Fanelli

Publications and source records attributed to F Fanelli.

At least 19 recordsLinked to original sources
PubMed2006

A functional transmembrane complex: the luteinizing hormone receptor with bound ligand and G protein.

The luteinizing hormone receptor (LHR) is one of eight members in a cluster of the rhodopsin family of the large G protein-coupled receptor (GPCR) superfamily that contains some 800-900 genes in the human genome. LHR, along with its paralogons, follicle stimulating hormone receptor (FSHR) and thyroid stimulating hormone receptor, form one of the three classes in this cluster; the two other classes contain the relaxin-binding GPCRs and orphan GPCRs. These GPCRs are characterized by a relatively large ectodomain (ECD) containing leucine-rich-repeats (LRRs); in the class of glycoprotein hormone receptors, the LRR region is capped by N-terminal and C-terminal cysteine-rich regions. Binding of human chorionic gonadotropin (hCG) or luteinizing hormone to the LHR-ECD triggers a conformational change of the transmembrane region of the receptor facilitating binding and activation of Gs, followed by effector enzyme activation and subsequent intracellular signaling. Viewing LHR as a transmembrane anchoring protein that sequentially binds hCG and Gs to give the hCG-LHR-Gs complex, numerous interactions and conformational changes must be considered. There is, unfortunately, a paucity of structural data on LHR, but crystal structures exist for hCG, the homologous FSH-FSHR-ECD (N-terminal fragment) complex, rhodopsin (in the inactive state), an active form of Galphas (transducin), and the betagamma heterodimer. Using a combined experimental (site-directed mutagenesis followed by characterization in transfected cells) and computational (homology modeling and molecular dynamics simulations) approach, good working models are being developed for the protein-protein interaction faces and, in some cases, the ensuing conformational changes induced by complex formation. hCG binding to the LHR-ECD appears to involve several LRRs; LHR activation can be described in terms of disrupting a network of H-bonds in the cytosolic halves of helices 1-3, 6, and 7; and binding of LHR to Gs involves, in large part, intracellular loop 2 binding, presumably to Gsalpha at its C-terminus. Major gaps exist in our understanding at the molecular level of the six-polypeptide chain complex, hCG-LHR-Gs, but considerable progress has been made in the past few years.

Amino Acid Sequence↗
PubMed2006

Dimerization of the lutropin receptor: insights from computational modeling.

A computational approach based upon rigid-body docking, ad hoc filtering, and cluster analysis has been carried out to predict likely interfaces in LHR homodimers. Quaternary structure predictions emphasize the role of helices 4, 5 and 6, with prominence to helix 4, in mediating inter-monomer interactions. Intermolecular interactions essentially involve the transmembrane domains rather than the hydrophilic loops and do not implicate disulfide bridges.Collectively, molecular dynamics simulations on the isolated receptor and computational modeling of LHR homodimerization suggest that mutation-induced LHR activation favors H4-H4 contacts involving the highly conserved W491 from both the receptors monomers.

Amino Acid Sequence↗
PubMed2006

Quaternary structure predictions of transmembrane proteins starting from the monomer: a docking-based approach.

BACKGROUND: We introduce a computational protocol for effective predictions of the supramolecular organization of integral transmembrane proteins, starting from the monomer. Despite the demonstrated constitutive and functional importance of supramolecular assemblies of transmembrane subunits or proteins, effective tools for structure predictions of such assemblies are still lacking. Our computational approach consists in rigid-body docking samplings, starting from the docking of two identical copies of a given monomer. Each docking run is followed by membrane topology filtering and cluster analysis. Prediction of the native oligomer is therefore accomplished by a number of progressive growing steps, each made of one docking run, filtering and cluster analysis. With this approach, knowledge about the oligomerization status of the protein is required neither for improving sampling nor for the filtering step. Furthermore, there are no size-limitations in the systems under study, which are not limited to the transmembrane domains but include also the water-soluble portions. RESULTS: Benchmarks of the approach were done on ten homo-oligomeric membrane proteins with known quaternary structure. For all these systems, predictions led to native-like quaternary structures, i.e. with Calpha-RMSDs lower than 2.5 A from the native oligomer, regardless of the resolution of the structural models. CONCLUSION: Collectively, the results of this study emphasize the effectiveness of the prediction protocol that will be extensively challenged in quaternary structure predictions of other integral membrane proteins.

Amino Acid Sequence↗
PubMed2006

Stent graft in TIPS: technical and procedural aspects.

Transjugular intrahepatic portosystemic shunt (TIPS) is a nonoperative therapeutic option for the management of portal hypertension, variceal bleeding, recurrent ascites, Budd-Chiari syndrome. In view of the many issues surrounding the use of TIPS, in 1994 the US National Digestive Diseases Advisory Board convened a scientific conference to review the current data available and to establish the indications and controindications for this procedure. However there are still unsolved problems especially short primary patency of the shunt due to intimal hyperplasia, which causes a reduction of the shunt lumen thus favoring a return of the portal hypertension with recurrent variceal bleeding. Several study were performed in the last years to evaluate the efficacy of covered stent in order to reduce shunt disfunction secondary to intimal hyperplasia. PTFE seems to be more efficient in the prevention of restenoses. In our experience more then 100 patients were treated with the Viatorr stent-graft. After a follow-up ranging from 1 to 50 months we reported a 1- year primary patency rate of 83.8%. However the use of the stentgraft is correlate with a high rate of hepatic hencefalopathy (46.6 %). In case of hepatic hencefalopathy refractory to the conventional medical therapy, TIPS reduction should be performed.

Ascites↗
PubMed2006

Multidetector-row CT angiography of renal artery stenosis in 50 consecutive patients: prospective interobserver comparison with DSA.

PURPOSE: The purpose of this study was to establish the diagnostic value of multidetector-row computed tomography (MDCT) angiography compared with digital subtraction angiography (DSA) for detection and quantification of both main and accessory renal artery stenosis in patients with secondary hypertension. MATERIALS AND METHODS: Fifty consecutive patients scheduled for DSA were considered candidates for MDCT angiography. In all patients, MDCT angiography of the abdominal aorta was performed before DSA. For the purpose of interpretation, the arteries were separately interpreted either with DSA or MDCT angiography in order to provide qualitative and quantitative information. For qualitative evaluation, one experienced reader graded the opacification of renal arteries as excellent, good or poor; for quantitative evaluation, MDCT and DSA were independently evaluated for the number of renal arteries and the presence, location and degree of stenosis in random order by three readers. On the basis of consensus readings, calculations of sensitivity, specificity, accuracy, positive predictive value (PPV) and negative predictive value (NPV) for detection of degree of stenosis were made by using DSA findings as the standard of reference. Interobserver variability was also assessed. RESULTS: With regard to qualitative analysis, arterial enhancement was considered excellent in 39 patients and good in 11. For quantitative analysis, 73 arteries were classified as normal with DSA. Although 72 of these were also classified as normal with CT angiography, one was overestimated by one grade; at DSA, 16 arteries were classified as moderately stenotic; in two arteries, there was an overestimation of one grade. Perfect correlation was achieved for the diagnosis of occlusion. In two patients, all three readers detected multiple severe stenoses on both modalities, with a "string-of-beads" appearance typical of fibromuscular dysplasia. Accessory arteries were correctly identified as such by all three readers on either DSA or MDCT. Levels of sensitivity, specificity and accuracy regarding degree of stenosis were 100%, 98.6% and 96.9%, respectively, with PPV and NPV of 97.6% and 100%, respectively. When we considered significant arterial stenosis (50%-100% luminal narrowing), sensitivity, specificity and accuracy were 100%, 97.3% and 97.8%, respectively, with a PPV and NPV of 98.2% and 97.8%, respectively. For all observers, interobserver agreement was almost perfect (k=0.81-1) for both MDCT and DSA, with a k value between 0.82 and 0.95. CONCLUSIONS: MDCT angiography is very accurate and robust, even for the assessment of renal artery stenosis, and has the potential to become a viable substitute, in most cases, for diagnostic catheter-based DSA.

Adult↗
PubMed2005

Phase II study of sequential hormonal therapy with anastrozole/exemestane in advanced and metastatic breast cancer.

Hormonal therapy is the preferred systemic treatment for recurrent or metastatic, post-menopausal hormone-receptor-positive breast cancer. Previous studies have shown that there is no cross-resistance between exemestane and reversible aromatase inhibitors. Exposure to hormonal therapy does not hamper later response to chemotherapy. Patients with locally advanced or metastatic, hormonal receptor positive or unknown, breast cancer were treated with oral anastrozole, until disease progression, followed by oral exemestane until new evidence of disease progression. The primary end point of the study was clinical benefit, defined as the sum of complete responses (CR), partial responses (PR) and > 24 weeks stable disease (SD). In all, 100 patients were enrolled in the study. Anastrozole produced eight CR and 19 PR for an overall response rate of 27% (95% CI: 18.6-36.8%). An additional 46 patients had long-term (> 24 weeks) SD for an overall clinical benefit of 73% (95% CI: 63.2-81.4). Median time to progression (TTP) was 11 months (95% CI: 10-12). A total of 50 patients were evaluated for the second-line treatment: exemestane produced one CR and three PR; 25 patients had SD which lasted > or = 6 months in 18 patients. Median TTP was 5 months. Toxicity of treatment was low. Our study confirms that treatment with sequential hormonal agents can extend the period of time during which endocrine therapy can be used, thereby deferring the decision to use chemotherapy.

Administration, Oral↗
PubMed2002

Interventional radiology techniques in the treatment of complications due to videolaparoscopic cholecystectomy.

INTRODUCTION: The development of videolaparoscopic cholecystectomy (VLC) has represented an important achievement in the search for minimally invasive surgical procedures, and especially in the surgical management of such a common and costly disease as cholelithiasis. However, the literature shows that, while VLC carries a similar mortality rate to open surgery, it has a greater incidence (2-5 times) of iatrogenic injury to the biliary tract and hepatic hilum [3, 5, 10, 25, 28]; this incidence further increases in cases of so-called "hard cholecystis" [10, 13, 25, 28, 30]. An equally minimally invasive technique is therefore needed to treat these lesions; this technique should be effective and safe, allow for shorter hospital stays and lower costs, and be made available at all the centres where VLC is performed. MATERIAL AND METHODS: We evaluated 60 patients (28 women and 22 men, age range 41-71) with 24 peritoneal collections (14 bilomas, 10 abscesses), 35 biliary stenosis (clips, chronic-developing lesions), and 2 vascular lesions as direct iatrogenic post-LC injuries. The most frequent clinical symptoms were jaundice, pain, sepsis, abdominal distension, and abdominal colic due to retained common bile duct stones. We carried out 60 percutaneous biliary reconstruction procedures, 44 biliary drainages (4 internal, 4 external), 6 combined radiological-endoscopic approaches with the Rendez-Vous technique, and 2 embolisations. RESULTS: All procedures had 100% technical success; the biliary reconstruction procedures had a 2% morbidity rate (sepsis, cholangitis). The clinical emergencies (choleperitoneum, haemoperitoneum, severe jaundice) resolved in 72 hours for 55 of the 60 patients (92%). The combined percutaneous-endoscopic procedures with the biliary Rendez-Vous technique were successfully performed in 5 out of 6 cases, allowing reconstruction of bile duct continuity and remission of symptoms, without the need to resort to surgical anastomosis. At a recent follow-up of the patients who had been managed only percutaneously, one showed patency of the bile ducts and had the stent removed after 18 months, and two showed optimal functioning of the stent that was still in place after 5 and 8 months respectively; two patients were lost to follow-up. Primary biliary patency at 60 months from biliary reconstruction was 75%. Maximum and immediate dilatation was obtained at the first attempt in most cases. Reoperation was avoided in 59 out of 60 patients. Only one patient with full transection of the common bile duct was reoperated on after insertion of a biliary drainage catheter. The overall length of hospital stays was between 1 and 4 weeks. CONCLUSIONS: Interventional radiology carried out according to indications seems to be the most natural way to treat the complications of VLC. It spares the patients, who opted for a minimally invasive technique like VLC, the need to undergo open surgery, and allows for shorter hospital stays and more efficient cost management for the hospital.

Adult↗
PubMed2001

Mutational and computational analysis of the alpha(1b)-adrenergic receptor. Involvement of basic and hydrophobic residues in receptor activation and G protein coupling.

To investigate their role in receptor coupling to G(q), we mutated all basic amino acids and some conserved hydrophobic residues of the cytosolic surface of the alpha(1b)-adrenergic receptor (AR). The wild type and mutated receptors were expressed in COS-7 cells and characterized for their ligand binding properties and ability to increase inositol phosphate accumulation. The experimental results have been interpreted in the context of both an ab initio model of the alpha(1b)-AR and of a new homology model built on the recently solved crystal structure of rhodopsin. Among the twenty-three basic amino acids mutated only mutations of three, Arg(254) and Lys(258) in the third intracellular loop and Lys(291) at the cytosolic extension of helix 6, markedly impaired the receptor-mediated inositol phosphate production. Additionally, mutations of two conserved hydrophobic residues, Val(147) and Leu(151) in the second intracellular loop had significant effects on receptor function. The functional analysis of the receptor mutants in conjunction with the predictions of molecular modeling supports the hypothesis that Arg(254), Lys(258), as well as Leu(151) are directly involved in receptor-G protein interaction and/or receptor-mediated activation of the G protein. In contrast, the residues belonging to the cytosolic extensions of helices 3 and 6 play a predominant role in the activation process of the alpha(1b)-AR. These findings contribute to the delineation of the molecular determinants of the alpha(1b)-AR/G(q) interface.

Amino Acid Sequence↗
PubMed2001

Phenylpiperazinylalkylamino substituted pyridazinones as potent alpha(1) adrenoceptor antagonists.

QSAR models have been used for designing a series of compounds characterized by a N-phenylpiperazinylalkylamino moiety linked to substituted pyridazinones, which have been synthesized. Measurements of the binding affinities of the new compounds toward the alpha(1a)-, alpha(1b)-, and alpha(1d)-AR cloned subtypes as well as the 5-HT(1A) receptor have been done validating, at least in part, the estimations of the theoretical models. This study provides insight into the structure activity relationships of the alpha(1)-ARs ligands and their alpha(1)-AR/5-HT(1A) selectivity.

Adrenergic alpha-Antagonists↗
PubMed2001

Emergency surgery for retrograde extension of type B dissection after endovascular stent graft repair.

Endovascular stent graft repair of type B dissection is a new and alternative treatment to the surgical or medical therapy. This technique is not free from minor or major complications and we herein report the case of a patient who developed a retrograde dissection after endovascular stent graft placement. The emergent surgical treatment undertaken consisted of ascending aorta replacement without treating the arch in account of the presence of the endoluminal prosthesis.

Aortic Dissection↗
PubMed2001

Lutropin receptor function: insights from natural, engineered, and computer-simulated mutations.

The lutropin receptor is a member of the superfamily of G protein-coupled receptors and, in humans, binds two closely related ligands, members of the heterodimer glycoprotein hormone family. As reviewed herein, comparative analyses of natural, engineered, and computer-simulated mutations of the lutropin receptor have provided considerable insight into the molecular basis of receptor function.

Amino Acid Sequence↗
PubMed2001

Pleiotropic effects of substitutions of a highly conserved leucine in transmembrane helix III of the human lutropin/choriogonadotropin receptor with respect to constitutive activation and hormone responsiveness.

It has been shown previously that a naturally occurring mutation of the human LH/CG receptor (hLHR), which replaces L457 in helix III with arginine, results in a receptor that constitutively elevates basal cAMP but does not respond to human CG (hCG) with further cAMP production. In the present study, substitutions of L457 with several amino acids were examined. The constitutive activation of cAMP production was observed only when L457 was replaced with a positively charged residue. Although constitutive activation of the inositol phosphate pathway could not be detected when measuring inositol phosphate production, the use of a more sensitive reporter gene assay for protein kinase C activation revealed the constitutive activation of this pathway by the R- and K-substituted mutants. Therefore, L457 of the hLHR plays a key role in stabilizing the receptor in an inactive conformation. Molecular modeling shows that the insertion of R, K, or H at position 457 triggers the receptor transition toward an active state due to the proximity of an anionic amino acid, D578, in helix VI. These substitutions cause perturbations in helix III-helix VI and helix III-helix VII interactions that culminate in the opening of a solvent-accessible site in the cytosolic domains potentially involved in Gs recognition. Interestingly, L457R was completely unresponsive and the K- and H-substituted L457 hLHR mutants were significantly blunted in their cAMP responses to hCG stimulation. Cells expressing L457R were also unresponsive to hCG with regards to increased inositol phosphate production. Other substitutions of L457 were identified, though, that selectively permit the hormonal stimulation of only one of the two signaling pathways. These results suggest a pivotal role for L457 in hormone-stimulated signal transduction by the hLHR.

Adenylyl Cyclases↗
PubMed2000

Theoretical study on mutation-induced activation of the luteinizing hormone receptor.

Here, three-dimensional model building and molecular dynamics simulations of the luteinizing hormone receptor have been employed to generate hypotheses about the molecular mechanisms underlying the activation of the receptor induced by naturally occurring activating mutations. The comparative analysis of the wild-type receptor and of 16 constitutively active or inactive mutants has been instrumental in inferring the structural/dynamic features which could characterize the inactive and the active forms of the receptor. These features have been also employed for predicting the functional behavior of new receptor mutants. The results of this study might provide a structural framework to interpret the pathological effects induced by mutations of the luteinizing hormone receptor. In addition, the proposed theoretical model could be useful for engineering new mutations or ligands able to modulate receptor function.

Amino Acid Motifs↗
PubMed2000

The alpha 1a and alpha 1b-adrenergic receptor subtypes: molecular mechanisms of receptor activation and of drug action.

In this chapter we summarize some aspects of the structure-functional relationship of the alpha 1a and alpha 1b-adrenergic receptor subtypes related to the receptor activation process as well as the effect of different alpha-blockers on the constitutive activity of the receptor. Molecular modeling of the alpha 1a and alpha 1b-adrenergic receptor subtypes and computational simulation of receptor dynamics were useful to interpret the experimental findings derived from site directed mutagenesis studies.

Animals↗
PubMed2000

Theoretical investigation of substrate specificity for cytochromes P450 IA2, P450 IID6 and P450 IIIA4.

Three-dimensional models of the cytochromes P450 IA2, P450 IID6 and P450 IIIA4 were built by means of comparative modeling using the X-ray crystallographic structures of P450 CAM, P450 BM-3, P450 TERP and P450 ERYF as templates. The three cytochromes were analyzed both in their intrinsic structural features and in their interaction properties with fifty specific and non-specific substrates. Substrate/enzyme complexes were obtained by means of both automated rigid and flexible body docking. The comparative analysis of the three cytochromes and the selected substrates, in their free and bound forms, allowed for the building of semi-quantitative models of substrate specificity based on both molecular and intermolecular interaction descriptors. The results of this study provide new insights into the molecular determinants of substrate specificity for the three different eukaryotic P450 isozymes and constitute a useful tool for predicting the specificity of new compounds.

Amino Acid Sequence↗
PubMed2000

Molecular basis of ligand binding and receptor activation in the oxytocin and vasopressin receptor family.

Although it is now widely accepted that G-protein-coupled receptors exist in at least two allosteric states, inactive and active, and that the spontaneous equilibrium between the two is regulated by various events including the binding of specific agonists and antagonists, the molecular counterparts of these functionally different states are still poorly understood. In this paper, we review our current knowledge concerning the structure-function relationships of the oxytocin and vasopressin receptors, focusing in particular on the process of receptor activation. Using a combined approach of site-directed mutagenesis and molecular modelling, we investigated the molecular events leading to agonist-dependent and -independent receptor activation in the human oxytocin receptor. Our analysis allows us to propose that the active conformations of this receptor are characterised by similar rearrangements of its cytosolic regions that ultimately lead to the opening of a putative docking site for the G-protein. Furthermore, the dynamics of these motions are similar to that observed in the alpha1B-adrenergic receptor, thus suggesting that, although activated by different ligands, the process of receptor isomerization in these two receptors is regulated by the same cluster of highly conserved residues and that common molecular events are responsible for receptor activation in different G-protein-coupled receptors.

Binding Sites↗