Surgical treatment of pericardial cyst through median sternotomy.

Pericardial cysts are an uncommon benign congenital anomaly in the middle mediastinum. They are thought to result from failure of fusion of one of the mesenchymal lacunae that form the pericardial sac. The authors present the case of a 77-year-old-man with a large pericardial cyst, treated by surgical resection trough a median sternotomy. They analyze the different diagnostic alternatives and the various management options in this pathology. In the reported case the authors used a surgical resection trough a median sternotomy, to facilitate the exposure of all of the cyst, extending around the great vessels area, and on the other side of the chest.

Structural consequences of metal complexation of cyclo[Pro-Phe-Phe-Ala-Xaa]2 decapeptides.

The conformational features of both free and Ca2+-complexed cyclo[Pro-Phe-Phe-Ala-Xaa]2 (with Xaa= Glu(OtBu), Lys(CIZ), Leu, and Ala) in solution have been determined by NMR spectroscopy and extensive distance-geometry calculations. The decapeptides are conformationally homogeneous in solution and show common structural features in their free and complexed forms. The structures of the free form contain only trans peptide bonds and are topologically similar to the structure of gramicidin-S, folded up in two antiparallel extended structures, stabilized by interstrand hydrogen bonds, and closed at both ends by two beta-turns. In contrast, the Ca2+-complexed peptides present two cis peptide bonds and are generally similar to those observed for the metal-complexed forms of antamanide and related analogues, folded into a saddle shape with two beta-turns. The Glu(OtBu)-, Leu-, and Lys(ClZ)-containing peptides examined here maintain the biological activity of the cyclolinopeptide A in their ability to competitively inhibit cholate uptake. The natural antamanide and cyclolinopeptide A are both able to inhibit the uptake of bile salts into hepatocytes. They share the same postulated active sequence Pro-Phe-Phe. Based on our structural results, we conclude that the ability to adopt a global conformation, characterized by a clear amphipathic separation of hydrophobic and hydrophilic surfaces, is an important feature for the functioning of this class of peptides.

Solution structure of a sweet protein: NMR study of MNEI, a single chain monellin.

The sweet protein MNEI is a construct of 96 amino acid residues engineered by linking, with a Gly-Phe dipeptide, chains B and A of monellin, a sweet protein isolated from Discoreophyllum cuminsii. Here, the solution structure of MNEI was determined on the basis of 1169 nuclear Overhauser enhancement derived distance restraints and 184 dihedral angle restraints obtained from direct measurement of three-bond spin coupling constants. The identification of hydrogen bonded NH groups was obtained by a combination of H/(2)H exchange data and NH resonance temperature coefficients derived from a series of HSQC spectra in the temperature range 278-328 K. The good resolution of the structure is reflected by the Z-score of the quality checking program in WHAT IF (-0.61). The topology of MNEI, like that of natural monellin and of SCM, another single-chain monellin, is typical of the cystatin superfamily: an alpha-helix cradled into the concave side of a five-strand anti-parallel beta-sheet. The high resolution (14 restraints/residue) 3D structure of MNEI shows close similarity to the crystal structures of natural monellin and of SCM but differs from the solution structure of SCM. The structures of SCM in the crystal and in solution differ in some of the secondary structure elements, but most of all in the relative arrangement of the elements: the four main beta-strands that surround the helix in the crystal structure of SCM, are displaced far from the helix in the solution structure of SCM. These differences were attributed to the fact that SCM is a monomer in solution and a dimer in the crystal. This result is at variance with the observation that our solution structure, like that of SCM, corresponds to a monomeric state of the protein, as demonstrated by the insensitivity of HSQC spectra to extreme dilution (down to 20 microM). On the basis of the solution structure of MNEI it is possible to propose that the main glucophores are hosted on loop L34, whereas the N-terminal and C-terminal regions host two other important interaction regions, centered around segments 6-9 and 94-96.

Pain peptides. Solution structure of orphanin FQ2.

Orphanin FQ2 (OFQ2) is a novel heptadecapeptide generated from prepronociceptin (PPNOC), the same precursor of nociceptin/orphanin FQ and nocistatin. OFQ2 is a potent analgesic when administered both supraspinally and spinally. In order to clarify the structural relationship with all peptides generated from PPNOC, we have undertaken the conformational study of OFQ2 in water and in structure-promoting solvent media. Nuclear magnetic resonance data and theoretical calculations are consistent with a well defined helical structure from Met(5) to Ser(16). The uniform distribution of hydrophobic residues along the helix suggests that OFQ2 may interact with the transmembrane helices of a receptor akin to those of nociceptin and opioids.

Solution structure of nocistatin, a new peptide analgesic.

Nocistatin, a new heptadecapeptide encoded in the bPNP-3 gene, has a powerful biological activity connected with the mechanisms of pain transmission. It does not bind to the opioid receptors but to another brain receptor with high affinity. In order to substantiate these novel biological data with a structural basis, we have undertaken a conformational study in solution. Proton nmr data in helicogenic solvents are consistent with a well-defined helical structure that is consistent with the nmr parameters of the C-terminal octapeptide, a shorter fragment that retains allodynia-blocking activity.

Solution structure of human beta-endorphin in helicogenic solvents: an NMR study.

Beta-endorphin is the largest natural opioid peptide. The knowledge of its bioactive conformation might be very important for the indirect mapping of the active site of opioid receptors. We have studied beta-endorphin in a variety of solution conditions with the goal of testing the intrinsic tendency of its sequence to assume a regular fold. We ran NMR experiments in water, dimethylsulfoxide and aqueous mixtures of methanol, ethylene glycol, trifluoroethanol, hexafluoracetone trihydrate and dimethylsulfoxide. The solvent in which the peptide is more ordered is the hexafluoracetone trihydrate/water mixture. The helical structure detected for beta-endorphin in this mixture at 300 K extends for the greater part of its address domain, hinting at a possible mechanism of interaction with opioid receptors: a two-point attachment involving an interaction of the helical part of the address domain (PLVTLFKNAIIKNAY) with one of the transmembrane helices and a classical interaction of the message domain (YGGF) with the receptor subsite common to all opioid receptors.

A new cacospongionolide derivative from the sponge Fasciospongia cavernosa.

Cacospongionolide F (4a), a new bioactive cacospongionolide-related sesterterpene, has been isolated from the Northern Adriatic sponge Fasciospongia cavernosa. The structure was proposed on the basis of spectroscopic data and chemical transformations. The absolute configuration was established using the modified Mosher's method. A molecular mechanics study of the dehydrodecalin ring explained the observed differences in dynamic behavior between cacospongionolide F and mamanuthaquinone, a related compound. Antimicrobial activity, brine shrimp and fish lethalities of this new compound are reported.

Solution structure of dynorphin A (1-17): a NMR study in a cryoprotective solvent mixture at 278 K.

Dynorphin A, the endogenous agonist for the kappa opioid receptor, has been studied by NMR spectroscopy in methanol, acetonitrile, DMSO and in mixtures of hexafluoroacetone/water and DMSO/water. NMR data in the DMSO/water cryomixture at 278 K are consistent with a conformer in which the N-terminal part, like the corresponding message domain of enkephalins, is poorly ordered, whereas the C-terminal part is folded in a loop centred around Pro10. The folded structure of the C-terminal part (address moiety) may shed light on the role of the essential residues Arg7, Lys11 and Lys13.

Ion-binding and pharmacological properties of Tyr6 and Tyr9 antamanide analogs.

In order to investigate the antiproliferative properties of antamanide, we have synthesized and studied two antamanide analogs where the phenylalanine residue in positions 6 or 9 is substituted by tyrosine, their corresponding linear forms and the cyclic and linear des Phe5,Phe6-Tyr9-analogs. Antamanide and its biologically active synthetic analogs are able to form highly stable complexes with metal ions, particularly Na+, K+ and Ca2+. We studied the ion-binding properties of the Tyr-antamanide analogs by CD and Tb3+ -mediated fluorescence in acetonitrile. In this medium the far-and near-UV CD spectra of the neat Tyr6-antamanide analog are very similar to that of the parent cyclic decapeptide. Substantial differences occur on the contrary in the CD spectra of the neat Tyr9-antamanide, particularly in the regions at 220 nm and 270-290 nm. In acetonitrile, as already found for antamanide, the interaction with the above-mentioned metal ions always produces evident changes in the far- and near-UV CD spectra of both analogs. On the contrary, the CD spectra of the linear deca- and octa- and of the cyclic octa-analogs are affected by the presence of metal ions only in the near-UV region. In the same solvent the Tb3+ -mediated fluorescence spectra of all the synthetic peptides are remarkably affected by the addition of ions. On the basis of the spectral total changes, by using either or both the spectroscopic techniques, it has been possible to determine the ion binding constants for all the linear and cyclic Tyr-antamanide analogs and to compare them with that of the parent peptide. The antitoxic and antiproliferative activities of these antamanide analogs have been tentatively correlated to their ion-binding properties. A preliminary account of this work was given in (1).

Forced adhesive growth of K562 leukemic cells that normally grow in suspension induces variations in membrane lipids and energy metabolism: a proton NMR study.

The mechanisms responsible for the adhesion of cells onto a material's surface and the effects that that adhesion may have on cell structure and function are fundamental questions in biomaterials research. We recently demonstrated that the erythroleukemic cell line K562, which normally grows in suspension, can be induced to grow attached to a polylysine-coated solid surface in an anchorage-dependent manner. In this study, the effects of the growth of K562 cells onto polylysine were further investigated utilizing 500 MHz 1H-NMR spectroscopy. The NMR results showed that when K562 cells are grown attached to a positively-charged polylysine surface, there are alterations in lipids and energy metabolism. In particular, there was a 31% increase in phosphatidylcholine and a 15% decrease in each of its two precursors, glycerophosphatidylcholine and choline, as well as a 20% increase in CH2 lipids and a 7% decrease in CH3 lipids in treated cells compared to the controls. These results suggest that adhesive growth can induce strong variations in membrane structure, including the membrane fluidity of K562 cells. In addition, in cells attached to polylysine there was about a 10% decrease in creatine (together with phosphocreatine), a 20% increase in gamma-glutamate, a 15% increase in beta-glutamate, and a 24% decrease in lactate. This second set of results, which is closely related to energy metabolism, indicates that not only does adhesive growth induce changes in K562 cell membrane structure, but also in the utilization of energy in these cells. The data are discussed in view of the possible role played by surface charge in affecting cell structure and function in cells that come into direct contact with charged biopolymers.

Changes in rat liver microcirculation after experimental hepatic arterial embolization: comparison of different embolic agents.

PURPOSE: To evaluate the effects of hepatic arterial embolization on hepatic microcirculation in the rat liver by using different particulate agents. MATERIALS AND METHODS: Polylactic acid microspheres, polyvinyl alcohol particles, and absorbable gelatin powder were injected into the hepatic artery of 50 rats. Saline was used as the control agent. Flow characteristics of hepatic microcirculation were qualitatively assessed on days 0 and 7 after embolization by using in vivo microscopy. Histologic specimens of the rat liver were analyzed. RESULTS: The polylactic acid (1-5 microns) injected into the hepatic artery was seen circulating through the sinusoids into the central venules. The slowing of flow observed with the injection of larger (50-200-micron) particles reflected the arterial occlusion occurring more proximally. After 7 days, all embolic agents caused vascular occlusion that led to necrosis and fibrosis. Networks of irregular, high-speed vessels that resembled arterioles and bypassed the normal sinusoids were observed. CONCLUSION: The necrotic areas observed after experimental distal occlusion of the hepatic arteries in the rats were bypassed by vessels similar to the capillarized sinusoids observed in the cirrhotic liver in humans. These vessels acted as sinusoidal shunts in the embolized territories.

Conformational sampling of bioactive conformers: a low-temperature NMR study of 15N-Leu-enkephalin.

Conformational studies of enkephalins are hampered by their high flexibility which leads to mixtures of quasi-isoenergetic conformers in solution and makes NOEs very difficult to detect in NMR spectra. In order to improve the quality of the NMR data, Leu-enkephalin was synthesized with 15N-labelled uniformly on all amide nitrogens and examined in a viscous solvent medium at low temperature. HMQC NOESY spectra of the labelled Leu-enkephalin in a DMSOd6/H2O) mixture at 275 K do show numerous NOEs, but these are not consistent with a single conformer and are only sufficient to describe the conformational state as a mixture of several conformers. Here a different approach to the structure-activity relationships of enkephalins is presented: it is possible to analyse the NMR data in terms of limiting canonical structures (i.e. beta- and gamma-turns) and finally to select only those consistent with the requirements of delta selective agonists and antagonists. This strategy results in the prediction of a family of conformers that may be useful in the design of new delta selective opioid peptides.

Structure of cyclic peptides: the crystal and solution conformation of cyclo(Phe-Phe-Aib-Leu-Pro).

A solid-state and solution conformation analyses of the cyclopentapeptide cyclo(Phe-Phe-Aib-Leu-Pro) has been carried out by X-ray diffraction and nuclear magnetic resonance techniques. The structure of the hexagonal crystals, grown from a methanol solution [a = b = 16.530(4) A, c = 21.356(9) A, space group P6(5), Z = 6], shows the presence of one intramolecular N-H ..O=C hydrogen bond with the formation of a gamma-turn (C7). The Aib3 residue, at the center of the gamma-turn, presents unexpected values of the torsion angles [phi = 70.5 degrees and psi = -73.8 degrees], which have been observed only once before for this helicogenic residue. A cis peptide bond occurs between Leu4 and Pro5; all other peptide bonds are trans. The overall conformation for the cyclopentapeptide with one cis-peptide bond on one side and an intramolecular gamma-turn on the opposite side results in an equatorial topology of the side-chains of the Phe1, Phe2 and Leu4 residues. Indeed, the Calpha-Cbeta and Cbeta-Cgamma bonds of these residues lie approximately in the mean plane of the cyclic ring system. The structure is compared with data in the literature on cyclic pentapeptides. In addition the Pro-Phe-Phe moiety shows a conformation similar to that observed in other larger cyclic bioactive peptides, which indicates a reduced number of conformations for this sequence. The solution study was carried out in three different solvent systems: chloroform, acetonitrile and methanol in the temperature interval 220-300 K. In all three solvents the room temperature spectra show that the peptide is conformationally nonhomogeneous. In acetonitrile at low temperatures it is possible to reduce the conformational equilibrium to two predominant conformers which differ for the cis-trans isomerism of the Leu4-Pro5 peptide bond.

Solution conformational analysis of sodium complexed [Gly6]- and [Gly9]-antamanide analogs.

To investigate the conformational flexibility of metal-complexed cyclodecapeptides, we synthesized and studied two antamanide analogs, in which the phenylalanine residue in position 6 or 9 of the sequence was substituted by Gly. Previous conformational studies on antamanide suggested that these backbone regions are affected by conformational variation. The NMR conformational study showed a high degree of flexibility for the two analogs. With sodium ions, on the other hand, [Gly9]-antamanide was able to form a fairly stable equimolar complex, whereas [Gly6]-antamanide showed a conformational heterogeneity, with one prevailing conformer. For the [Gly9]-antamanide analog, the whole NMR data, combined with extensive theoretical calculations, were consistent with the presence of 1) two beta-turns of type I, centered on Gly9-Phe10 and Ala4-Phe5, respectively; 2) a central cavity with a six-carbonyl oxygen cage, optimal for a Na+ hexacoordination; 3) strongly H-bonded amide protons for residues 1 and 6, both involved in the formation of the two type I beta-turns, which, however, exhibited some fluctuations during the molecular dynamics simulations. For the [Gly6]-antamanide-Na+ complex the prevailing conformer was consistent with a more open structure, with the partial solvent exposure of all the amide protons; that is, the Gly residue in position 6 increases the flexibility of this critical site more than does the Gly in position 9. These data in some way parallel the results of the cytotoxicity tests on B16-F10 transformed cells for the two analogs: [Gly9]-antamanide is cytotoxic after 48 h exposure, whereas [Gly6]-antamanide is almost inactive. On the contrary, both analogs are practically inactive in vivo against phalloidin.

Design and solution structure of a partially rigid opioid antagonist lacking the basic center--models of antagonism.

To discriminate between two general models of antagonism (participation and allosteric), an opioid antagonist lacking the basic nitrogen of tyramine was designed and characterized. Cyclo-[Tyr(Me)2-Tic-], the diketopiperazine of 2,6-dimethyltyrosyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, is a partially rigid opioid antagonist; its pA2 (5.8) is one smaller than that of N,N-bisallyl-enkephalin but it has a very high binding affinity (10 nM) and has a delta selectivity (66 with respect to the binding to mu receptors) higher than that of naltrindole. The conformational state of this diketopiperazine, studied under a variety of solvent and temperature conditions by NMR and molecular dynamics, can be described in terms of only three conformers whose relative populations vary widely with solvent. Only one of the three conformers, characterized by a 90 degree arrangement of the aromatic rings of Tyr(Me)2 and Tic similar to those of rigid agonists and of the bioactive conformation of the corresponding linear antagonist, is consistent with the antagonist activity. This finding favors the participation model among the general mechanisms proposed to explain antagonism. Due to the simple composition of the conformational mixture and to the rigidity of the molecule, it is possible to propose a quantitative explanation for the discrepancy between the very high binding affinity (10 nM) and the fairly small in mouse vas deferens value (1.5 microM).

Solution conformation of nociceptin.

Nociceptin, a novel heptadecapeptide, interacts with ORL1 a G protein-coupled receptor whose sequence is closely related to that of the kappa opioid receptor but has no opioid activity. We have investigated the conformational preferences of Nociceptin also in comparison to Dynorphin A. The N-terminal part of Nociceptin has the same conformational preferences of the message of endogenous opioids but the C-terminal part of the sequence is more flexible than the corresponding address of Dynorphin A. [Tyr1]-Nociceptin, while retaining nociceptive activity, has also an opioid activity comparable to that of enkephalins.

Conformational analysis of three NK1 tripeptide antagonists: a proton nuclear magnetic resonance study.

Two new peptides, tailored after Ac-Thr-D-Trp(CHO)-Phe-NMeBzl (TRI), namely, Ac-Thr-D-Trp(CHO)-Phe-NMe alpha MeBzl (TRA) and Ac-Thr-D-Trp(CHO)-Oic-NMeBzl (TOI), in which Phe is replaced by (3aS, 7aS)-octahydroindole-2-carboxylic acid, proved more potent and selective NK1 antagonists. The conformational properties of all three compounds were investigated in solution by NMR spectroscopy and those of TRI analyzed in greater detail by means of systematic computer-assisted modeling. All conformers whose energy differs by less than 9 kcal/mol from the absolute minimum are different from the conformer proposed in a previous molecular modeling study by the discovers of TRI. Parallel calculations for TRA and TOI yield low-energy conformers similar to those of TRI but in a slightly different order. Comparison of the shapes of low-energy conformers of all three peptides with those of four typical rigid NK1 antagonists shows that putative bioactive conformations are indeed present in solution.

Delta-selective opioid peptides containing a single aromatic residue in the message domain: an NMR conformational analysis.

The sequence of deltorphin I, a delta-selective opioid agonist, has been systematically modified by inserting conformationally constrained C alpha, alpha disubstituted apolar residues in the third position. As expected, substitution of Phe with Ac6c, Ac5c and Ac3c yields analogues with decreasing but sizeable affinity. Surprisingly, substitution with Aib yields an analogue with almost the same binding affinity of the parent compound but with a greatly increased selectivity. This is the first case of a potent and very selective opioid peptide containing a single aromatic residue in the message domain, that is, only Tyr1. Here we report a detailed conformational analysis of [Aib3]deltorphin I and [Ac6c3]deltorphin I in DMSO at room temperature and in a DMSO/water cryomixture at low temperature, based on NMR spectroscopy and energy calculations. The peptides are highly structured in both solvents, as indicated by the exceptional finding of a nearly zero temperature coefficient of Val5 NH resonance. NMR data cannot be explained on the basis of a single structure but it was possible to interpret all NMR data on the basis of a few structural families. The conformational averaging was analysed by means of an original computer program that yields qualitative and quantitative composition of the mixture. Comparison of the preferred solution conformation with two rigid delta-selective agonists shows that the shapes of [Aib3]deltorphin I and [Ac6c3]deltorphin I are consistent with those of rigid agonists and that the message domain of opioid peptides can be defined only in conformational terms.

Solution and solid state structure of an aib-containing cyclodecapeptide inhibiting the cholate uptake in hepatocytes.

The conformational analysis of synthetic cyclodecapeptide c(Pro-Phe-phe-Aib-Leu)2 related to the cyclolinopeptide A, in the solid state and solution, has been carried out by x-ray diffraction and nmr spectroscopy. The structure of the monoclinic form obtained from methanol [a = 11.351 (5) A, b = 27.455 (2) A, c = 12.716 (8) A, beta = 99.65 (3) degrees; space group P2(1); Z = 2] shows the presence of six intramolecular NH...CO hydrogen bonds, with formation of four turns (three of type I and one of type III) and two C16 ring structures. All peptide units are trans. The solution structure, as found by nmr, indicates that, at room temperature, the peptide is conformationally homogeneous; the structure determined is perfectly symmetrical and topologically similar to that found in the solid state. The cyclodecapeptide exhibits similar biological activity to cyclolinopeptide A.