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F Guarnieri

Publications and source records attributed to F Guarnieri.

10 recordsLinked to original sources
PubMed1997

A surface on the G protein beta-subunit involved in interactions with adenylyl cyclases.

Receptor activation of heterotrimeric G proteins dissociates G alpha from the G betagamma complex, allowing both to regulate effectors. Little is known about the effector-interaction regions of G betagamma. We had used molecular modeling to dock a peptide encoding the region of residues 956-982 of adenylyl cyclase (AC) 2 onto Gbeta to identify residues on Gbeta that may interact with effectors. Based on predictions from the model, we synthesized peptides encoding sequences of residues 86-105 (Gbeta 86-105) and 115-135 (Gbeta 115-135) from Gbeta. The Gbeta 86-105 peptide inhibited G betagamma stimulation of AC2 and blocked G betagamma inhibition of AC1 and by itself inhibited calmodulin-stimulated AC1, thus displaying partial agonist activity. Substitution of Met-101 with Asn in this peptide resulted in the loss of both the inhibitory and partial agonist activities. Most activities of the Gbeta 115-135 peptide were similar to those of Gbeta 86-105 but Gbeta 115-135 was less efficacious in blocking G betagamma inhibition of AC1. Substitution of Tyr-124 with Val in the Gbeta 115-135 peptide diminished all of its activities. These results identify the region encoded by amino acids 84-143 of Gbeta as a surface that is involved in transmitting signals to effectors.

Adenylyl Cyclases↗
PubMed1996

Making DNA add.

Recent studies have demonstrated the feasibility of using DNA-based experiments to compute solutions to combinatorial problems. However, a prerequisite for designing a computer useful in a wide range of applications is the ability to perform mathematical calculations. The development of a DNA-based algorithm for addition is presented. The DNA representation of two nonnegative binary numbers is presented in a form permitting a chain of primer extension reactions to carry out the addition operation. To demonstrate the feasibility of this algorithm, a simple example was executed biochemically.

Algorithms↗
PubMed1996

A refined model of the thyrotropin-releasing hormone (TRH) receptor binding pocket. Experimental analysis and energy minimization of the complex between TRH and TRH receptor.

Seven transmembrane (TM) spanning, G protein-coupled receptors (GPCRs) appear to bind large glycoprotein hormones predominantly within their extracellular domains, small nonpeptidic ligands within the TM helical bundle, and peptide ligands within the extracellular domains and TM bundle. The tripeptide thyrotropin-releasing hormone (TRH, pyroGlu-His-ProNH2) may bind entirely within the TM bundle of the TRH receptor (TRH-R). We have previously demonstrated direct binding contacts between the pyroGlu of TRH and two residues in TM helix 3 (TM-3) of TRH-R and proposed a model of the binding pocket of TRH-R [Perlman, J. H., Laakkonen, L., Osman, R., & Gershengorn, M. C. (1994) J. Biol. Chem. 269, 23383-23386]. Here, we provide evidence for two additional direct interactions between TRH and TRH-R. One interaction is between the aromatic ring of Tyr 282 of TM-6 and His of TRH. This is based on a large increase in the half-maximally effective concentration (EC50) of TRH for stimulation of inositol phosphate formation by Y282A TRH-R and a loss of selectivity of this mutant receptor for TRH analogs substituted at His. We provide evidence for another interaction between Arg 306 of TM-7 and the terminal carboxamide of TRH. Using four direct interactions as anchors, a refined model of the TRH-R binding pocket was constructed using geometry optimization through energy minimization. A novel method for modeling GPCRs based on Monte Carlo and stochastic dynamics simulations is presented in the accompanying paper [Laakkonen, L. J., Guarnieri, F., Perlman, J. H., Gershengorn, M. C., & Osman, R. (1996) Biochemistry 35, 7651-7663].

Amino Acid Sequence↗
PubMed1996

A refined model of the thyrotropin-releasing hormone (TRH) receptor binding pocket. Novel mixed mode Monte Carlo/stochastic dynamics simulations of the complex between TRH and TRH receptor.

Previous mutational and computational studies of the thyrotropin-releasing hormone (TRH) receptor identified several residues in its binding pocket [see accompanying paper, Perlman et al. (1996) Biochemistry 35, 7643-7650]. On the basis of the initial model constructed with standard energy minimization techniques, we have conducted 15 mixed mode Monte Carlo/stochastic dynamics (MC-SD) simulations to allow for extended sampling of the conformational states of the ligand and the receptor in the complex. A simulated annealing protocol was adopted in which the complex was cooled from 600 to 310 K in segments of 30 ps of the MC-SD simulations for each change of 100 K. Analysis of the simulation results demonstrated that the mixed mode MC-SD protocol maintained the desired temperature in the constant temperature simulation segments. The elevated temperature and the repeating simulations allowed for adequate sampling of the torsional space of the complex with successful conservation of the general structure and good helicity of the receptor. For the analysis of the interaction between TRH and the binding pocket, TRH was divided into four groups consisting of pyroGlu, His, ProNH2, and the backbone. The pairwise interaction energies of the four separate portions of TRH with the corresponding residues in the receptor provide a physicochemical basis for the understanding of ligand-receptor complexes. The interaction of pyroGlu with Tyr106 shows a bimodal distribution that represents two populations: one with a H-bond and another without it. Asp195 was shown to compete with pyroGlu for the H-bond to Tyr106. Simulations in which Asp195 was interacting with Arg283, thus removing it from the vicinity of Tyr106, resulted in a stable H-bond to pyroGlu. In all simulations His showed a van der Waals attraction to Tyr282 and a weak electrostatic repulsion from Arg 306. The ProNH2 had a strong and frequent H-bonding interaction with Arg306. The backbone carbonyls show a frequent H-bonding interaction with the OH group of Tyr282 and strong, often multiple, interactions with Arg306. Three structures, which maintained these interactions simultaneously, were selected as candidates for ligand-receptor complexes. These show persistent interactions of TRH with Ile 109 and Ile 116 in HX 3 and with Tyr310 and Ser313 in HX 7, which will be tested to refine the structure of the ligand-receptor complex. The superposition of the three structures shows the extent of structural flexibility of the receptor and the ligand in the complex. The backbone of TRH inside the receptor is in an alpha-helical conformation, suggesting that the receptor, through its interaction with the ligand, provides the energy required for the conformational change in the ligand from an extended to the folded form.

Amino Acid Sequence↗
PubMed1996

Immunization with granulocyte-macrophage colony-stimulating factor-transduced, but not B7-1-transduced, lymphoma cells primes idiotype-specific T cells and generates potent systemic antitumor immunity.

Recently, genetically modified tumor cell vaccines have been described for nonhematopoietic cancers in which the relevant Ags are unknown. Several of these cell-based vaccine strategies have been shown to induce T cell-mediated systemic antitumor immunity, either by enhancing the processing and presentation of tumor Ags by host APCs or by facilitating effective Ag presentation by the tumor vaccine itself. These strategies were compared in a model B cell lymphoma, a tumor derived from APCs, which have the inherent capacity to activate Ag-specific T cells. Eradication of pre-established systemic lymphoma was achieved following immunization with lymphoma cells engineered to produce granulocyte-macrophage (GM)-CSF, and to a lesser extent cells producing IL-4, whereas vaccination with lymphoma cells transfected with the genes encoding IL-2 or B7-1 had no effect. The systemic immunity generated by GM-CSF- or IL-4-transfected lymphoma required both CD4+ and CD8+ T cells. Previous immunotherapeutic strategies for the treatment of lymphoma have focused on the generation of Ab responses targeted to the unique Ig Id as a tumor-specific Ag. Anti-idiotypic Abs were undetectable in animals vaccinated with GM-CSF-transduced lymphoma cells. In contrast, such immunization did result in the induction of Id-specific T cell responses. This is the first demonstration that T cell responses specific for a native tumor Ag are generated by GM-CSF-transduced tumor cell-based vaccination, suggesting that B cell lymphoma may be a suitable disease for genetically modified tumor vaccine strategies.

Animals↗
PubMed1996

Restricted analogues provide evidence of a biologically active conformation of thyrotropin-releasing hormone.

Thyrotropin-releasing hormone (TRH) is a tripeptide (< Glu-His-Pro-NH2) that signals through a G protein-coupled receptor. TRH is a highly flexible molecule that can assume many conformations in solution. To attempt to delineate the biologically active conformation of TRH, we synthesized a pair of conformationally restricted cyclohexyl/Ala2-TRH analogues. The diastereomeric analogues use a lactam ring to restrict two of the six free torsional angles of TRH and constrain the X-Pro-NH2 peptide bond to trans. Unrestricted cyclohexyl/Ala2-TRH exhibited a 650-fold lower affinity than TRH for TRH receptor and was 430-fold less potent than TRH in stimulating inositol phosphate second messenger formation. One diastereomer exhibited higher affinity and potency than the unrestricted analogue despite the presence of the methylene bridge and fused ring, whereas the other showed lower affinity and potency. Computer simulations predicted that the positions of the cyclohexyl/Ala2 and Pro-NH2 moieties relative to < glutamate were different in the two analogues and that the conformation of the higher affinity analogue is different from that of trans-TRH in solution but is superimposable on that of trans-TRH found in a model of the TRH/TRH receptor complex. These experimental findings identify a favored relative position of < glutamate and Pro-NH2 in the more active conformation of two diastereomeric analogues of TRH and provide independent support for the model of the TRH/TRH receptor complex.

Amino Acid Sequence↗
PubMed1994

[New surgical technique for the repair of abdominal hernia with mesh].

The use of prostheses is indispensable in major anterior abdominal hernias when it is not possible to close the edges of the hernial opening without strain. The most commonly used prostheses are made from dacron, polypropylene (PPNE) and expanded polytetrafluoroethylene (EPTF) mesh. It is important to point out that, in spite of the excellent characteristics of these materials, there is no ideal prosthesis. There is a well-known increase in the risk of rejection and infection when mesh is placed in contact with the subcutis and the formation of adhesions, with the possibility of occlusions and fistulas, when it is in direct contact with the intestine. Whereas EPTF does not cause problems of adhesion, it has a greater tendency to cause rejection and infection if placed in the subcutaneous area. Moreover, there is a increased risk of displacement and it is also very costly. The opposite is true of dacron and PPNE mesh. When the mesh serves to "replace" a wall defect and it cannot be isolated by the interposition of the peritoneum or muscular fascia, it comes into direct contact with the intestine and subcutaneous tissue. The technique outlined here aims to isolate the prosthesis from the intestinal loops and subcutaneous tissue. The hernial sac itself is used for this purpose and is divided into two flaps by a cut perpendicular to the wall. The free edge of one flap is sutured to the inside of the hernial opening. The mesh is then sutured half inside and half outside the hernial opening.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1992

A new technique for indirect inguinal hernia repair.

This report concerns a preliminary study of a new technique for indirect herniorraphy, which was used in 242 patients from December 1988 through March 1991. Its main characteristics are the creation of a deep neo-inguinal ring in a more medial site, shortening of the inguinal canal by transposition of the superficial ring to the point where the inferior border of the internal oblique muscle is well represented, reinforcement of the inguinal canal by overlapping the external oblique aponeurosis in a double-breast fashion, and maintenance of the cremasteric muscle. Follow-up in our outpatient clinic was carried out at 1, 6, 12, and 24 months in 71% of our patients. There were no recurrences, except for one crural pseudo-recurrence, no mortality, and no testicular atrophy. Thirteen percent of patients had subcutaneous serous collection; 1% had hematomas; 2% temporary testicular edema; and 0.4% wound infection.

Abdominal Muscles↗