Crystal structure and thermoelectric properties of Sr-Mo substituted CaMnO3: a combined experimental and computational study.

A combination of experimental and computational techniques has been employed to study doping effects in perovskite CaMnO3. High quality Sr-Mo co-substituted CaMnO3 ceramics were prepared by the conventional mixed oxide route. Crystallographic data from X-ray and electron diffraction showed an orthorhombic to tetragonal symmetry change on increasing the Sr content, suggesting that Sr widens the transition temperature in CaMnO3 preventing phase transformation-cracking on cooling after sintering, enabling the fabrication of high density ceramics. Atomically resolved imaging and analysis showed a random distribution of Sr in the A-site of the perovskite structure and revealed a boundary structure of 90° rotational twin boundaries across {101}orthorhombic; the latter are predominant phonon scattering sources to lower the thermal conductivity as suggested by molecular dynamics calculations. The effect of doping on the thermoelectric properties was evaluated. Increasing Sr substitution reduces the Seebeck coefficient but the power factor remains high due to improved densification by Sr substitution. Mo doping generates additional charge carriers due to the presence of Mn3+ in the Mn4+ matrix, reducing electrical resistivity. The major impact of Sr on thermoelectric behaviour is the reduction of the thermal conductivity as shown experimentally and by modelling. Strontium containing ceramics showed thermoelectric figure of merit (ZT) values higher than 0.1 at temperatures above 850 K. Ca0.7Sr0.3Mn0.96Mo0.04O3 ceramics exhibit enhanced properties with S1000K = -180 µV K-1, ρ1000K = 5 × 10-5 Ωm, k1000K = 1.8 W m-1 K-1 and ZT ≈ 0.11 at 1000 K.

Strategies for positioning fluorescent probes and crosslinkers on formyl peptide ligands.

Chemoattractant receptors represent a major subset of the G-protein coupled receptor (GPCR) family. One of the best characterized, the N-formyl peptide receptor (FPR), participates in host defense responses of neutrophils. The features of the ligand which regulate its interaction with the FPR are well-known. By manipulating these features we have developed new ligands to probe structural and mechanistic aspects of the peptide-receptor interaction. Three ligand groups have been developed: 1) ligands containing a Lys residue located in positions 2 through 7 that can be conjugated to FITC (N-formyl-Met1-Lys2-Phe3-Phe4, N-formyl-Met1-Leu2-Lys3-Phe4, N-formyl-Met1-Leu2-Phe3-Lys4, N-formyl-Met1-Leu2-Phe3-Phe4-Lys5, N-formyl-nLeu1-Leu2-Phe3-nLeu4-Tyr5-Lys6 and N-formyl-Met1-Leu2-Phe3-Phe4-Gly5-Gly6-Lys7; 2) fluorescent pentapeptide ligands (N-formyl-Met-X-Phe-Phe-Lys(FITC) where X = Leu, Ala, Val or Gly); and 3) small crosslinking ligands where the photoaffinity crosslinker 4-azidosalicylic acid (ASA) was conjugated to Lys in positions 3 and 4 and p-benzoyl-phenylalanine (Bpa) was located in position 2 in N-formyl-Met1-Bpa2-Phe3-Tyr4. The peptides were characterized according to activity and affinity in human neutrophils and cell lines transfected with FPR. All of the peptides were agonists, with parallel affinity and activity. In the first group, the peptide activity decreases as Lys is placed closer to the N-formyl group and the activity is improved by 1-3 orders of magnitude by conjugation with FITC. In the second group, the dissociation rate of the peptide from the receptor increases as position 2 is replaced by aliphatic amino acids with smaller alkyl groups. In the third group, crosslinking ligands remain biologically active, display nM affinity and covalently label the FPR.

C5aR ligand peptide 3D QSAR study performed with an applied linear conformation.

A 3D quantitative structure-activity relationship study (QSAR) of binding and activation of the human C5a receptor by peptide analogs of the C-terminal binding domain of C5a anaphylatoxin is reported. Using published C5a analog affinity and activity data, this paper seeks to elucidate the pharmacophore for the high affinity C-terminal binding domain of the C5a peptide with the molecular modeling technique of comparative molecular field analysis (CoMFA). In order to model peptides for which there was incomplete conformational data, an arbitrary linear conformation was imposed upon the highly flexible C5a analogs. The resulting models yield a crossvalidated q2 of 0.889 and 0.787, for receptor-ligand affinity and EC50 calcium release activity, respectively, suggesting these models have good predictive ability for other test peptides.

Fixation traps formyl peptide receptors in high and low affinity forms that can be regulated by GTP[S] in the absence of ligand.

The formyl peptide receptor on human neutrophils recognizes bacterial, N-formylated peptides and initiates a cascade of intracellular signals via a pertussis toxin sensitive Gi protein. We used fluorescence techniques to investigate the interactions of ligand (L), receptor (R), and G proteins (G), the ternary complex, in both live and fixed human neutrophils. By lightly fixing permeabilized neutrophils with a procedure that retained ligand binding, we were able to "capture' R and G in different configurations in the absence of ligand. Fixed receptors were trapped in a high affinity form (attributed to LRG) that could not be rapidly converted to low affinity by the addition of GTP[S]. Adding saturating nucleotide prior to fixation trapped receptors in a low affinity form (attributed to LR). The low affinity receptors retained the sensitivity of the native receptors to the presence of NA+. The distribution between high and low affinity receptors was modulated by GTP[S] in a dose dependent manner. The ability to redistribute low and high affinity receptor forms prior to fixation was unique to GTP[S], as compared to other non-activating nucleotides, suggesting that GTP[S] can regulate the distribution between R and RG. We suggest that precoupled receptors that give rise to high affinity ligand binding are likely to exist in native membranes in human neutrophils.

A high potency nonformylated peptide agonist for the phagocyte N-formylpeptide chemotactic receptor.

Analysis of synthetic tri- and tetrapeptides has previously indicated that N-formylation is required for high biological activity when they react with the phagocyte N-formylpeptide receptor, suggesting that the natural ligand for the receptor is from bacterial and/or mitochondrial sources. To explore this requirement further, we synthesized the pentapeptide methionyl-norleucyl-leucyl-phenylalanyl-phenylalanine (MNleLFF) and studied the effects of different NH2-terminal modifications on its activity. N-formyl-MNleLFF induced transient alterations of [Ca2+]i and superoxide production in human neutrophils with 10- and 100-fold greater potency, respectively, than the proto-type N-formylpeptide, N-formylmethionyl-leucyl-phenylalanine (fMLF). Surprisingly, N-acetyl-MNleLFF was a potent as N-formyl-MNleLFF. Moreover, the unacylated counterpart H-MNleLFF was also highly active, having an EC50 for calcium mobilization of 10 nM, and for respiratory burst activation of 100 nM. All three pentapeptides could completely desensitize calcium transients elicited by stimulation of neutrophils with fMLF, whereas the neutrophil chemoattractants C5a and interleukin 8 only weakly affected fMLF-induced transients, suggesting that they activate neutrophils via the same receptor as fMLF. Finally, all three pentapeptides activated the recombinant human N-formylpeptide receptor expressed in frog oocytes, but did not effectively activate related phagocyte receptors. These data broaden the potential sources of natural ligands for the N-formyl-peptide receptor from N-formylated bacterial and mitochondrial products to other nonformylated endogenous peptides.

The effect of perioperative blood transfusion on survival in head and neck cancer.

This Head and Neck Intergroup analysis was undertaken to evaluate further previously reported observations linking blood transfusions, which were given to patients with head and neck cancer, to a worse prognosis. This study population represents those patients registered to the Head and Neck Intergroup Trial 0034 for previously untreated resectable squamous cell carcinoma. Additional transfusion data were obtained by one of us (D.E.S.) on 217 patients and added to the Head and Neck Intergroup data set, providing an opportunity for assessing the impact of survival by other variables. The study group was separated using 13 variables. Analysis demonstrated that transfusion did not significantly decrease the locoregional control (P = .60). Multivariate analysis indicated that T stage (P = .015), N stage (P = .004), treatment received (P = .004), and Karnofsky Performance Scale (P = .031) were the only factors that did significantly influence survival. This multivariate analysis controlling for these variables demonstrated no significant effect on survival for those patients receiving transfusion during surgery (P = .55) or after surgery (P = .39). This study of 217 patients, controlled for other variables, does not demonstrate any significant negative relation between blood transfusions and either locoregional control or survival.

Structure-function analysis of signal and growth inhibition by carboxyamido-triazole, CAI.

Evidence is accumulating that calcium homeostasis and calcium-regulated events may be selectively important in generation and maintenance of the malignant phenotype. CAI, a carboxyamido-triazole with a halogenated benzophenone tail, is a novel inhibitor of receptor-operated calcium influx and arachidonic acid release which inhibits malignant proliferation, invasion, and metastasis. The focus of this investigation was structural analysis of CAI and to determine if the inhibition of calcium influx and arachidonic acid release by CAI and its antiproliferative activity were mediated through the same chemical domains. Four families of molecular modifications of the CAI parent were synthesized: (I) modification or substitution of the triazole ring; (II) removal of the substituted benzophenone tail; (III) dehalogenation or partial truncation of the benzophenone moiety; and (IV) removal of the triazole and altered substitutions of the benzophenone tail. Compounds were tested for the inhibition of calcium influx and arachidonic acid release and inhibition of proliferation and colony formation in soft agar using the malignant CHO line transfected with the m5 muscarinic receptor and the A2058 human melanoma cell line. Only CAI and Group I compounds inhibited stimulated calcium influx, arachidonic acid release, and proliferation. Linear regression analysis of the relationship of the 50% inhibitory concentration values for all compounds in inhibition of calcium influx and arachidonate release was statistically significant (r2 = 0.993). Similarly, a linear relationship was demonstrated between inhibition of calcium influx and inhibition of tumor cell proliferation (r2 = 0.971). Groups II-IV had minimal or no signal or growth inhibitory activity. This investigation provides the first evidence for a coordinate link between calcium influx, calcium-mediated arachidonic acid release, and malignant proliferation and metastasis and constitutes the initial analysis of structurally important domains of the CAI molecule.

Multiparameter flow cytometric analysis of a pH sensitive formyl peptide with application to receptor structure and processing kinetics.

Environmentally sensitive molecules have many potential cellular applications. We have investigated the utility of a pH sensitive ligand for the formyl peptide receptor, CHO-Met-Leu-Phe-Phe-Lys (SNAFL)-OH (SNAFL-seminaphtho-fluorescein), because in previous studies (Fay et al.: Biochemistry 30:5066-5075, 1991) protonation has been used to explain the quenching when the fluoresceinated formyl pentapeptide ligand binds to this receptor. Moreover, acidification in intracellular compartments is a general mechanism occurring in cells during processing of ligand-receptor complexes. Because the protonated form of SNAFL is excited at 488 nm with emission at 530 nm and the unprotonated form is excited at 568 nm with emission at 650 nm, the ratio of protonated and unprotonated forms can be examined by multiparameter flow cytometry. We found that the receptor-bound ligand is sensitive to both the extracellular and intracellular pH. There is a small increase in the pKa of the ligand upon binding to the receptor consistent with protonation in the binding pocket. Once internalized, spectral changes in the probe consistent with acidification and ligand dissociation from the receptor are observed.

Analysis of the active conformation of angiotensin II: a comparison of AII and non-peptide AII antagonists.

Several molecular models of angiotensin II (AII) were constructed using information derived primarily from conformationally constrained AII analogues. In order to refine the original models, AII was compared using computer-aided molecular graphics with several of the recently reported non-peptide AII antagonists. As a first approximation, the imidazole, phenyl ring and acidic moiety (i.e., carboxyl, tetrazole) of the non-peptide AII antagonists were overlapped with the corresponding features of the peptide. AII and Dup 753 were overlapped using Multifit (SYBYL) and then both were optimized individually and refit; this procedure was repeated three times to generate a working model of AII. The less-potent non-peptide AII antagonists S 8308 and Exp 6155 could also be overlaid with the model of AII, albeit not as well as DuP 753. The working model of AII, which has a twisted U-shape, places the imidazole of His6 and the terminal carboxylate approximately 8.7 A apart.

The significance of functional receptor heterogeneity in the biological responses of the rabbit neutrophil to stimulation by chemotactic formyl peptides.

The characteristics of binding to the chemotactic receptors on rabbit peritoneal neutrophils were examined for seven formyl peptide analogues. These receptor-binding characteristics were compared with the abilities of the analogues to induce the biological responses of degranulation and chemotaxis. Five of the analogues showed distinct functional heterogeneity in their receptor-binding patterns, whereas the two most potent compounds displayed homogeneous binding patterns. The relative potencies of the formyl peptide analogues for stimulation of degranulation correlated well with their relative potencies for high-affinity, but not low-affinity, binding. The biphasic patterns for stimulation of chemotactic migration were similar for the less potent analogues, and their potencies paralleled those for both degranulation and receptor binding. In contrast, the most potent analogues induced a greater maximal extent of chemotactic migration than the other compounds, but displayed a lower than expected potency (i.e. they required higher than expected concentrations). These anomalies in the patterns of the chemotactic response cannot be reconciled with a simple receptor model comprising two independent classes of receptors. Instead, a model comprising interconvertible states of different affinities is proposed. The state of higher affinity appears to play a central role in initiation of both degranulation and chemotaxis. The more potent formyl peptide analogues are thought to stabilize an activated, higher-affinity, state of the receptor; this can explain their greater efficacy in stimulating chemotaxis. The proposed model may also be applicable to other receptors that are coupled by a guanine-nucleotide-binding regulatory protein to their associated effector.

Conformational dynamics of the formyl peptide receptor: a prototype for studies of receptor dynamics and binding pocket structure.

We have used spectrofluorometric and flow cytometric techniques to examine the interactions of formyl peptide ligands (L) with their cell surface receptors (R). Kinetic studies suggest that L binds to R at a diffusion limited rate and that R undergoes rapid transitions involving three states (LR, LRG, the ternary complex of L and R with the G protein, and a desensitized receptor "LRX" which forms within seconds) prior to internalization. A spectroscopic analysis of the interaction between L and R show that the binding pocket of R is large enough to contain no more than 6 amino acids and that a fluorescein-labelled pentapeptide is quenched upon binding to R. We hypothesize that histidine 90 (putatively located in the extracellular loop connecting the second and third transmembrane domains) protonates L and quenches the probe. New technology will extend the analysis of structure and dynamics to low affinity peptide receptors of living biological systems. Such technology will have implications in the design of peptidomimetic ligand and drug molecules.

N alpha-formylated and tert-butyloxycarbonylated Phe-(Leu-Phe)n and (Leu-Phe)n peptides as agonists and antagonists of the chemotactic formylpeptide receptor of the rabbit peritoneal neutrophil.

The various diastereomers of the N alpha-formylated(CHO) and tert-butyloxycarbonylated (t-Boc) Phe-(Leu-Phe)n and (Leu-Phe)n methyl esters, where n = 1-2 and 1-3, respectively, have been newly synthesized and their physical properties described. The CHO-blocked peptides are all able to release beta-glucosaminidase from rabbit peritoneal neutrophils in a concentration-dependent manner. There is a strong effect of primary structure and of chirality on their biology activity; lengthening the peptide chain distinctly increases activity in each series and within a series the activity decreases in the order: all-L greater than D-L much greater than all-D. Of the t-Boc protected synthetic precursors, the all-L isomers have definite but weak agonist activity; the agonist activity of the other isomers is equivocal or not detectable. All the t-Boc peptides, however, are capable of acting as weak, specific antagonists. There is a dependence of antagonist activity on primary structure, but this is variable and contingent on the nature of the peptide. Similarly, an effect of chirality on antagonist activity, although present, also depends on the structure of the peptide. In the one instance directly tested, t-Boc-L-Phe-(D-Leu-L-Phe)2-OMe (OMe, methoxy) was found to be distinctly less active than the corresponding free acid.

Fluorescence analysis of the size of a binding pocket of a peptide receptor at natural abundance.

We have studied the topography of interaction of a family of fluorescent formyl peptides containing four (CHO-Met-Leu-Phe-Lys-fluorescein), five (CHO-Met-Leu-Phe-Phe-Lys- fluorescein), and six (CHO-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein and CHO-Met-Leu-Phe-Phe-Phe-Lys- fluorescein) amino acids with their receptor using spectroscopic methods adapted to small sample volumes. Only the fluorescent peptides containing four and five amino acids were quenched upon binding to the receptor, indicating physical contact of the chromophore with the receptor. In contrast, only the hexapeptides were accessible to antibodies to fluorescein. Taken together, these results suggest that the carboxy terminus of the tetrapeptide or the pentapeptide is protected in the receptor binding pocket while the fluorescein on the carboxy terminus of either hexapeptide is exposed and recognized by the antibody to fluorescein. These results indicate that the binding pocket accommodates at least five but no more than six amino acids.

Conformationally restricted formyl methionyl tripeptide chemoattractants: a three-dimensional structure-activity study of analogs incorporating a C alpha,alpha-dialkylated glycine at position 2.

The conformationally restricted CHO-L-Met-Xxx-L-Phe-OY (where Xxx = Aib, Ac3c, Ac5c, Ac6c, and Ac7c; Y = H, Me) tripeptides, analogs of the chemoattractant CHO-L-Met-L-Leu-L-Phe-OH, have been synthesized in solution by classical methods and fully characterized. Compounds were compared to determine the combined effect of backbone conformational preferences and side-chain bulkiness on the relation of three-dimensional structure to biological activity. Each peptide was tested for its ability to induce granule enzyme secretion from rabbit peritoneal polymorphonuclear leukocytes. In parallel, a conformational analysis on the CHO-blocked peptide and their tertbutyloxycarbonylated synthetic precursors was performed in the crystal state and in solution using X-ray diffraction, infrared absorption, and 1H nuclear magnetic resonance. The biological and conformational data are discussed in relation to the proposed model of the chemotactic peptide receptor of rabbit neutrophils.

Thermal regulation of FMLP receptors on human neutrophils.

Polymorphonuclear leukocytes (PMNs) from subjects diagnosed as having juvenile periodontitis (JP) have been categorized on the basis of their chemotactic (CTX) response to f-met-leu-phe (FMLP) when assayed concurrently with PMNs from periodontally healthy subjects (HP). When PMNs from JP groups demonstrating depressed CTX were assayed for lysosomal enzyme secretion (LES) in response to FMLP, there were no significant differences with respect to rate or amount. Significant differences were observed between HP and chemotactically depressed JP cells when assessed for FMLP receptor ligand binding at 23 degrees C, but not at 4 degrees C. Receptor differences observed at 23 degrees C in HP cells included an increase in amount of total binding, number of receptors, and available displaceable binding sites, compared with the chemotactically depressed JP PMNs, whereas the receptor affinities were similar. These data suggest that differences in FMLP receptor density in JP PMN that are chemotactically depressed may be related to processes that modulate receptor mobility and/or expression.

A computergraphic determination of the chemotactic peptide preferred conformation.

Replacement of leucine in the chemotactic peptide For-Met-Leu-Phe by the sterically constrained amino acids alpha-aminoisobutyric acid and aminocyclohexanecarboxylic acid affords compounds of equal or greater activity than the parent. NMR studies indicate that the parent compound is present as a beta-sheet in solution, whereas the analogues prefer a beta-turn. Application of molecular modelling would indicate that the beta-turn conformer is energetically preferable and thus suggests that it is the orientation adopted by the peptides.

Synthetic formyl-methionyl chemoattractants: a conformation-activity study of oxidized tripeptides.

The two diastereomeric sulphoxides and the sulphone derived from the formyl-methionyl tripeptide chemoattractant CHO-L-Met-L-Phe-OMe have been synthesized and fully characterized. The diastereomeric sulphoxide tripeptides have been separated at the stage of their N-tert-butyloxycarbonyl synthetic precursors. All of the oxidized sulphur derivatives induce secretion of granule enzymes with ED50s from 1-2 x 10(-9) M with no significant differences in activity among them. They are also active to the same relative extent in inducing chemotaxis. In parallel, a solution conformational analysis has been performed in solvents of widely different polarities and capabilities of H-bond formation using circular dichroism, infrared absorption and 1H nuclear magnetic resonance. No significant propensity for formation of intramolecularly (C = O...H-N) H-bonded folded forms has been detected in any of the four tripeptides. Intermolecular S = O...H-N interactions are postulated to tentatively explain the 1H nuclear magnetic resonance behavior of the Met and, particularly, Leu NH resonances of the two sulphoxide tripeptides in CDCl3 solution. The biological and conformational data agree with the recently proposed model of the chemotactic peptide receptor of rabbit neurotrophils, which involves the extended backbone of the integrity of the Met side-chain sulphide sulphur atom as a corollary point of ligand interaction.

Characteristics of binding of a potent chemotactic formyl tetrapeptide, formylmethionyl-leucyl-phenylalanyl-phenylalanine, to the receptors on rabbit neutrophils.

Binding of a potent chemotactic formyl tetrapeptide, formylmethionyl-leucyl-phenylalanyl-phenylalanine (fMet-Leu-Phe-Phe), to the formyl peptide receptors on the rabbit neutrophil was assessed by two approaches. A tritiated preparation of fMet-Leu-Phe-Phe was used for direct binding studies, whereas indirect studies comprised an assessment of the ability of the formyl tetrapeptide to competitively inhibit the binding of 35S-labeled formylmethionyl-leucyl-phenylalanine. These two approaches yielded analogous results. The formyl tetrapeptide fMet-Leu-Phe-Phe showed rapid and saturable binding to the same chemotactic receptors as the less potent formyl tripeptides with which it was compared. Its equilibrium-binding pattern, however, was different: fMet-Leu-Phe-Phe showed a homogeneous binding pattern, in contrast to the heterogeneity seen with the less potent compounds. The relative potencies for high-affinity binding of the two standard formyl tripeptides and fMet-Leu-Phe-Phe correlated well with their relative potencies for stimulating the biological response of degranulation; the relative potencies for low-affinity binding correlated less well.