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Biomedical subjects

E Benedetti

Publications and source records attributed to E Benedetti.

At least 235 records · Page 13Linked to original sources
PubMedDate not supplied

Conformational characterization of peptides rich in the cycloaliphatic C alpha,alpha-disubstituted glycine 1-aminocyclononane-1-carboxylic acid.

A series of N- and C-protected, monodispersed homo-oligopeptides (to the pentamer level) from the cycloaliphatic C alpha,alpha-dialkylated glycine 1-aminocyclononane-1-carboxylic acid (Ac9c) and two Ala/Ac9c tripeptides have been synthesized by solution methods and fully characterized. The conformational preferences of all the model peptides were determined in deuterochloroform solution by FT-IR absorption and 1H-NMR. The molecular structures of the amino acid derivatives mCIAc-Ac9c-OH and Z-Ac9c-OtBu, the dipeptide pBrBz-(Ac9c)2-OtBu, the tetrapeptide Z-(Ac9c)4-OtBu, and the pentapeptide Z-(Ac9c)5-OtBu were determined in the crystal state by X-ray diffraction. Based on this information, the average geometry and the preferred conformation for the cyclononyl moiety of the Ac9c residue have been assessed. The backbone conformational data are strongly in favour of the conclusion that the Ac9c residue is a strong beta-turn and helix former. A comparison with the structural propensity of alpha-aminoisobutyric acid, the prototype of C alpha,alpha-dialkylated glycines, and the other extensively investigated members of the family of 1-aminocycloalkane-1-carboxylic acids (Acnc, with n = 3-8) is made and the implications for the use of the Ac9c residue in conformationally constrained analogues of bioactive peptides are briefly examined.

Amino Acids, Cyclic↗
PubMedDate not supplied

Crystal structure of the W35A mutant thioredoxin h from Chlamydomonas reinhardtii: the substitution of the conserved active site Trp leads to modifications in the environment of the two catalytic cysteines.

The conformational analysis of W35A thioredoxin h from the eukaryotic green alga Chlamydomonas reinhardtii in the solid state has been carried out by x-ray diffraction, with the aim to clarify the role of Trp in the catalysis. Comparative analysis of W35A mutant with wild-type (WT) thioredoxin shows that, even if the structural motif of thioredoxin is not perturbed, the substitution of Trp35 by an Ala leads to significant changes in protein conformation near the active site. This rearrangement increases its solvent exposure and explains the change of the pKa values of the catalytic cysteines. The substitution of the Trp residue also influences the crystal packing as well as the recognition ability of thioredoxin. The solid state analysis suggests that the Trp residue has a structural function both to force the active site in the bioactive conformation, and to mediate the protein-protein recognition.

Amino Acid Substitution↗
PubMedDate not supplied

Conformational behaviour of a cyclolinopeptide A analogue: two-dimensional NMR study of cyclo(Pro1-Pro-Phe-Phe-Ac6c-Ile-ala-Val8).

The cyclic octapeptide cyclo[-Pro1-Pro-Phe-Phe-Ac6c-Ile-ala-Val8-] [C8-Ac6c], containing the Pro1-Pro-Phe-Phe sequence, followed by a bulky helicogenic C alpha,alpha-dialkylated glycine residue Ac6c [1-aminocyclohexane-1-carboxylic acid), and a D-Ala residue at position 7 has been synthesized. This cyclic peptide is a deletion analogue of the naturally occurring cyclic nonapeptide cyclolinopeptide A (CLA). It has been designed with the aim of studying the role that the Ac6c and D-Ala residues play on the conformational behaviour of the whole molecule and their influence on the conformation of the Pro1-Pro-Phe-Phe sequence when compared with cyclolinopeptide A. C8-Ac6c has been investigated in chloroform and acetonitrile solutions by 2D NMR techniques. Only one set of sharp signals is observed in both solvents. This evidence strongly supports the hypothesis that only one conformational state exists in the chosen solvents. The interpretation of the experimental data points to the existence for C8-Ac6c of a very rigid structure stabilized by intramolecular hydrogen bonds. The measured NOE effects allow the calculation of internuclear distances, which have been used as restraints in molecular dynamic calculations. The proposed conformation of the molecule shows that the Pro-Pro-Phe segment retains the conformation observed in natural CLA both in solution and in the solid state and that the Ac6c residue indeed reinforces the ring rigidity not permitting the formation of any appropriate cavity in which inorganic cations could be complexed.

Amino Acid Sequence↗
PubMedDate not supplied

Sweet and bitter taste: structure and conformations of two aspartame dipeptide analogues.

The synthesis and X-ray diffraction analysis of two dipeptide taste ligands have been carried out as part of our study of the molecular basis of taste. The compounds L-aspartyl-D-alpha-methylphenylalanine methyl ester [L-Asp-D-(alpha Me)Phe-OMe] and L-aspartyl-D-alanyl-2,2,5, 5-tetramethylcyclopentanyl ester [L-Asp-D-Ala-OTMCP] elicit bitter and sweet taste, respectively. The C-terminal residues of the two analogues adopt distinctly different conformations in the solid state. The aspartyl moiety assumes the same conformation found in other dipeptide taste ligands with the side-chain carboxylate and the amino groups forming a zwitterionic ring with a conformation defined by psi, chi 1 = 157.7 degrees, -61.5 degrees for L-Asp-D-Ala-OTMCP and 151.0 degrees, -68.8 degrees for L-Asp-D-(alpha Me)Phe-OMe. In the second residue, a left-handed helical conformation is observed for the (alpha Me)Phe residue of L-Asp-D-(alpha Me)Phe-OMe with phi 2 = 49.0 degrees and psi 2 = 47.9 degrees, while the Ala residue of L-Asp-D-Ala-OTMCP adopts a semi-extended conformation characterized by dihedral angles phi 2 = 62.8 degrees and psi 2 = -139.9 degrees. The solid-state structure of the bitter L-Asp-D-(alpha Me)Phe-OMe is extended: while the crystal structure of the sweet L-Asp-D-OTMCP roughly adopts the typical L-shaped structure shown by other sweeteners. The data of L-Asp-D-(alpha Me)Phe-OMe are compared with those of its diastereoisomer L-Asp-L-(alpha Me)Phe-OMe. Conformational analysis of the two taste ligands in solution by NMR and computer simulations agrees well with our model for sweet and bitter tastes.

Aspartame↗
PubMedDate not supplied

Inversion of 3(10)-helix screw sense in a (D-alpha Me)Leu homo-tetrapeptide induced by a guest D-(alpha Me)Val residue.

The terminally blocked tetrapeptide pBrBz-[D-(alpha Me)Leu]2-D-(alpha Me)Val-D-(alpha Me)Leu-OfBu is folded in the crystal state in a left-handed 3(10)-helical structure stabilized by two consecutive 1<--4 C = O...H-N intramolecular H-bonds, as determined by X-ray diffraction analysis. A CD study strongly supports the view that this conformation is also that largely prevailing in MeOH solution. A comparison with the published conformation of pBrBz-[D-(alpha Me)Leu]4-OfBu indicates that incorporation of a single internal beta-branched (alpha Me)Val guest residue into the host homo-tetrapeptide from the gamma-branched (alpha Me)Leu residue is responsible for a dramatic structural perturbation, i.e. an inversion of the 3(10) screw sense from right to left-handed.

Amino Acid Sequence↗
PubMedDate not supplied

Preferred conformation of peptides rich in Ac8c, a medium-ring alicyclic C (alpha,alpha)-disubstituted glycine.

A complete series of terminally blocked, monodispersed homo-oligopeptides (to the pentamer level) from the sterically demanding, medium-ring alicyclic C (alpha,alpha)-disubstituted glycine 1-aminocyclooctane-1-carboxylic acid (Ac8c), and two Ala/Ac8c tripeptides, were synthesized by solution methods and fully characterized. The preferred conformation of all the oligopeptides was determined in deuterochloroform solution by IR absorption and 1H-NMR. The molecular structures of the amino acid derivative Z-Ac8c-OH, the dipeptide pBrBz-(Ac8c)2-OH and the tripeptide pBrBz-(Ac8c)3-OtBu were assessed in the crystal state by X-ray diffraction. Conformational energy computations were performed on the monopeptide Ac-Ac8c-NHMe. Taken together, the results obtained strongly support the view that the Ac8c residue is an effective beta-turn and helix former. A comparison is also made with the conformational preferences of alpha-aminoisobutyric acid, the prototype of C (alpha,alpha)-disubstituted glycines, and of the other members of the family of 1-aminocycloalkane-1-carboxylic acids (Acnc with n = 3, 5-7) investigated so far. The implications for the use of the Ac8c residue in peptide conformational design are considered.

Amino Acids↗
PubMedDate not supplied

NMR studies of prebiotic polypertides.

Several polypeptides prepared by means of pyrocondensation have been the subject of structural investigations. Attention has been focused on the constitutional characterization of homo-and co-polymers containing Asp and Glu residues, whose role is essential for the formation of the so-called proteinoids. Contrary to the literature data based on chemical degradation, nmr studies show conclusively that in thermal poly-aspartic acid only beta-peptide linkages are present. This result casts serious doubt on the role thermal condensation might have played in prebiotic polypeptide syntheses.

Aspartic Acid↗
PubMedDate not supplied

An assessment of P-glycoprotein expression and activity in peripheral blood lymphocytes of transplant candidates.

BACKGROUND: P-glycoprotein (P-gp) is involved in the transport of the xenobiotic immunosuppressive agents and many cytokines, such as IL-2 and IFN-gamma. Hence, P-gp activity on peripheral blood lymphocytes (PBLs) could affect the pharmacologic response to xenobiotic immunosuppressants and immune responsiveness. The objectives of this study were to (1) determine the level of P-gp expression and activity on PBLs of kidney transplant candidates; and (2) determine whether P-gp expression correlates with P-gp activity. METHODS: We measured P-gp expression and activity on CD3(+)/CD8(+), CD3(+)/CD4(+), B lymphocytes, and NK cells of 36 kidney transplant candidates using a flow cytometric assay. P-gp activity was determined for each subpopulation of cells by the ratio of the mean Rhodamine 123 fluorescence (MFI Rh123) in the presence of verapamil divided by the MFI Rh123 in the absence of verapamil. P-gp expression was noted as the percentage of P-gp(+) cells. RESULTS: NK cells exhibited the greatest amount of P-gp activity (MFI Rh123 = 20.2 +/- 16.4) compared with other cell populations (P < .05). P-gp efflux activity was also significantly elevated in CD3(+)/CD8(+) cells (13.9 +/- 10.5) compared with B lymphocytes (4.9 +/- 2.7; P < .05) and CD4/CD3(+) cells (2.4 +/- 1.0; P < .05). P-gp expression was significantly higher in B lymphocytes (11.7 +/- 9.5) and NK cells (10.2 +/- 7.3) when compared with CD3(+)/CD8(+) cells (7.3 +/- 6.9) and CD3(+)/CD4(+) cells (6.4 +/- 3.8). P-gp expression was highly variable and did not correlate with P-gp activity (P > .05). CONCLUSIONS: CD3(+)/CD8(+) cells and NK cells, exhibited significantly increased P-gp activity compared with the other cell populations. P-gp expression is not a good correlate of P-gp activity. These findings may have important implications for the use of immunosuppressive drugs posttransplant and immune responsiveness after transplantation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMedDate not supplied

Hepatic ischemia/reperfusion injury can be modulated with thymoglobulin induction therapy.

BACKGROUND: Thymoglobulin induction therapy has been shown to ameliorate delayed graft function and possibly decrease ischemia reperfusion injury in cadaver renal transplant recipients. This controlled randomized trial was designed to assess whether thymoglobulin also protects liver transplant recipients from ischemia reperfusion injury. PATIENTS AND METHODS: Twenty-two cadaver liver transplant recipients were randomized to receive either thymoglobulin (1.5 mg/kg per dose) during the anhepatic period and two doses every other day or no thymoglobulin. No differences in recipient or donor demographics were present. Maintenance immunosupression consisted of tacrolimus (or cyclosporine) and steroids for both groups. Donor biopsies were obtained during organ procurement, cold storage, and 1 hour after revascularization. Postoperative liver function tests were monitored. Early graft function, length of stay, patient and graft survival rates, incidence of primary nonfunction, and rate of rejection were assessed. RESULTS: Patient and graft survival at 3 months was 100%. There was no incidence of primary graft nonfunction and no need for retransplantation. The incidence of acute rejection was similar between the two groups. Although donor livers randomized to thymoglobulin had less optimal preimplantation biopsies, these recipients had significant decreases in ALT at day 1 compared to the control group (P = .02), near significant decreases of total bilirubin at day 5, and shorter length of hospitalization. CONCLUSION: Thymoglobulin allowed for more compromised liver grafts to be transplanted with less clinical evidence of ischemia reperfusion injury and improved function.

Antilymphocyte Serum↗
PubMedDate not supplied

Different reconstitution of peripheral blood lymphocytes and dendritic cells in liver and kidney transplant patients.

T cells and dendritic cells are responsible for immune alloreactivity or tolerance after transplantation. In this study, we compared the levels of circulating T, B, and NK lymphocytes, as well as monocytes, plasmacytoid dendritic cells, and myeloid dendritic cells, in adult patients undergoing a liver transplant or kidney transplant. Our findings show that candidates for liver transplant had significantly lower levels of circulating T, B, and dendritic cells than candidates for kidney transplant. Nevertheless, liver transplant patients showed a greater T-cell recovery, despite the use of thymoglobulin, as compared with kidney transplant patients who were induced with Daclizumab. In four kidney transplant patients with allograft rejection we observed a dramatic drop of circulating T and dendritic cells at the time of rejection, and while myeloid dendritic cells and CD4(+) and CD8(+) cells rapidly recovered after 1 month, plasmacytoid dendritic cells and CD4(+)CD25(+) T-cell numbers remained significantly lower than in patients without rejection. Future studies will evaluate the monitoring of circulating CD4(+)CD25(+) T cells and myeloid dendritic cell:plasmacytoid dendritic cell ratio as potential biomarkers for rejection or, alternatively, for withdrawal of immune suppression.

Adult↗