Attachment and spousal caregiving

A community sample of 362 married couples participated in a study of attachment and spousal caregiving, which combined qualitative and quantitative components. The qualitative component focused on actual experiences of caregiving, assessed by participants' semi-structured accounts of a situation involving their role as caregiver for their spouse, Attachment styles and their underlying dimensions (comfort with closeness, anxiety over relationships) were related to the type of support provided, the coping strategies used in the situation, caregivers' feelings about the quality of their care, perceived effects on the couple bond, and the emotional tone of the accounts. The quantitative component tested a theoretical model of factors predicting willingness to provide care for the spouse if he or she should become dependent in later life. Measures of attachment and caregiving styles, attachment to spouse, and anticipated burden provided reliable prediction of willingness to care. The results support the conceptualization of attachment and caregiving as interrelated features of marital bonds, and they have important implications for patterns of family caregiving.

Genetic monogamy in the absence of paternity guards: the Capricorn silvereye, Zosterops lateralis chlorocephalus, on Heron Island

We investigated the genetic mating system of a socially monogamous passerine bird, the Capricorn silvereye Zosterops lateralis chlorocephalus, on an island of the Great Barrier Reef. There were no cases of extrapair paternity (EPP) among 122 offspring from 53 broods detectable by minisatellite or microsatellite DNA fingerprinting. Behavioral observations of paired birds showed that this was not a consequence of efficacious paternity guards and that females did not engage in extrapair copulation (EPC). Frequency of intrapair copulations was also low, with only 14 cases observed during 199 hours of observations of the 11 focal pairs in the fertile periods of females, and this was consistent with anatomical features of the cloacal protuberance in males. In this population, young birds form life-time pair bonds soon after gaining independence but females are obviously not attempting EPC possibly to redress this early mate choice. This is despite the fact that they breed in high density with a synchronous start and asynchronous spread of laying in a protracted season and males do not positively exhibit mate guarding behavior when females are fertile. Our results support high fidelity of socially monogamous birds on islands and are consistent with the hypothesis that sexual selection is reduced where genetic variation in fitness is limited.

Twisting and planarization in push-pull ethylenes

As determined by X-ray crystallography, Meldrum's acid derivatives 8–19 feature dihedral angles around the central CC double bonds between 3 and 83°. Hydrogen bonds between substituents RHN and the carbonyl groups favour near-planarity. Sterically demanding substituents favour large dihedral angles and zwitterionic structures as in formula 20. AM1 calculations of the structures are in excellent agreement with the experimental X-ray data, provided a dielectric field is incorporated (?= 40). This can be ascribed to the highly polar (zwitterionic) nature of the molecules. It is further predicted that all these molecules, including those that are stabilised in a planar form by intramolecular hydrogen bonds, undergo rapid rotation about the central CC bonds at room temperature. DFT calculations incorporating a dielectric field model (PCM) are in excellent agreement with the near-perpendicular arrangement of the alkene moiety in 19.

A theoretical study of the origin of rotational barriers in push-pull ethylenes

Using the B3LYP/6-31G* ab initio method, we have studied the rotation about the C=C bonds in 15 push-pull ethylenes of the general formula (X,Y)C=C(CHO)(2) [X, Y = NH2, NHCH3, N(CH3)(2), OCH3, SCH3] in the gas phase. Two stationary points (minimum and transition state) were located for all compounds. The geometry, dipole moments, natural bond orbital atomic charges, as well as the rotational barriers were examined. The torsion angle 0 depends essentially on the presence or absence of intramolecular hydrogen bonds, and the barrier is a function of the torsion angle. (C) 2002 Elsevier Science B.V. All rights reserved.

Structure of CcmG/DsbE at 1.14 angstrom resolution: High-fidelity reducing activity in an indiscriminately oxidizing environment

CcmG is unlike other periplasmic thioredoxin (TRX)like proteins in that it has a specific reducing activity in an oxidizing environment and a high fidelity of interaction. These two unusual properties are required for its role in c-type cytochrome maturation. The crystal structure of CcmG reveals a modified TRX fold with an unusually acidic active site and a groove formed from two inserts in the fold. Deletion of one of the groove-forming inserts disrupts c-type cytochrome formation. Two unique structural features of CcmG-an acidic active site and an adjacent groove-appear to be necessary to convert an indiscriminately binding scaffold, the TRX fold, into a highly specific redox protein.

beta-strand mimicking macrocyclic amino acids: Templates for protease inhibitors with antiviral activity

New amino acids are reported in which component macrocycles are constrained to mimic tripeptides locked in a beta-strand conformation. The novel amino acids involve macrocycles functionalized with both an N- and a C-terminus enabling addition of appendages at either end to modify receptor affinity, selectivity, or membrane permeability. We show that the cycles herein are effective templates within inhibitors of HIV-1 protease. Eleven compounds originating from such bifunctionalized cyclic templates are potent inhibitors of HIV-1 protease (Ki 0.3-50 nM; pH 6.5, I = 0.1 M). Unlike normal peptides comprising amino acids, five of these macrocycle-containing compounds are potent antiviral agents with sub-micromolar potencies (IC50 170-900 nM) against HIV-1 replication in human MT2 cells. The most active antiviral agents are the most lipophilic, with calculated values of LogD(6.5) greater than or equal to 4. All molecules have a conformationally constrained 17-membered macrocyclic ring that has been shown to structurally mimic a tripeptide segment (Xaa)-(Val/Ile)-(Phe/Tyr) of a peptide substrate in the extended conformation. The presence of two trans amide bonds and a para-substituted aromatic ring prevents intramolecular hydrogen bonds and fixes the macrocycle in the extended conformation. Similarly constrained macrocycles may be useful templates for the creation of inhibitors for the many other proteins and proteases that recognize peptide beta-strands.

A comparative study of carbon gasification with O-2 and CO2 by density functional theory calculations

A comparative study of carbon gasification with O-2 and CO2 was conducted by using density functional theory calculations. It was found that the activation energy and the number of active sites in carbon gasification reactions are significantly affected by both the capacity and manner of gas chemisorption. O-2 has a strong adsorption capacity and the dissociative chemisorption of O-2 is thermodynamically favorable on either bare carbon surface or even isolated edge sites. As a result, a large number of semiquinone and o-quinone oxygen can be formed indicating a significant increase in the number of active sites. Moreover, the weaker o-quinone C-C bonds can also drive the reaction forward at (ca. 30%) lower activation energy. Epoxy oxygen forms under relatively high O-2 pressure, and it can only increase the number of active sites, not further reduce the activation energy. CO2 has a lower adsorption capacity. Dissociative chemisorption of CO2 can only occur on two consecutive edge sites and o-quinone oxygen formed from CO2 chemisorption is negligible, let alone epoxy oxygen. Therefore, CO2-carbon reaction needs (ca 30%) higher activation energy. Furthermore, the effective active sites are also reduced by the manner Of CO2 chemisorption. A combination of the higher activation energy and the fewer active sites leads to the much lower reaction rate Of CO2-carbon.

Conformationally constrained macrocycles that mimic tripeptide beta-strands in water and aprotic solvents

The beta-strand conformation is unknown for short peptides in aqueous solution, yet it is a fundamental building block in proteins and the crucial recognition motif for proteolytic enzymes that enable formation and turnover of all proteins. To create a generalized scaffold as a peptidomimetic that is preorganized in a beta-strand, we individually synthesized a series of 15-22-membered macrocyclic analogues of tripeptides and analyzed their structures. Each cycle is highly constrained by two trans amide bonds and a planar aromatic ring with a short nonpeptidic linker between them. A measure of this ring strain is the restricted rotation of the component tyrosinyl aromatic ring (DeltaG(rot) 76.7 kJ mol(-1) (16-membered ring), 46.1 kJ mol(-1) (17-membered ring)) evidenced by variable temperature proton NMR spectra (DMF-d(7), 200-400 K). Unusually large amide coupling constants ((3)J(NH-CHalpha) 9-10 Hz) corresponding to large dihedral angles were detected in both protic and aprotic solvents for these macrocycles, consistent with a high degree of structure in solution. The temperature dependence of all amide NH chemical shifts (Deltadelta/T7-12 ppb/deg) precluded the presence of transannular hydrogen bonds that define alternative turn structures. Whereas similar sized conventional cyclic peptides usually exist in solution as an equilibrium mixture of multiple conformers, these macrocycles adopt a well-defined beta-strand structure even in water as revealed by 2-D NMR spectral data and by a structure calculation for the smallest (15-membered) and most constrained macrocycle. Macrocycles that are sufficiently constrained to exclusively adopt a beta-strand-mimicking structure in water may be useful pre-organized and generic templates for the design of compounds that interfere with beta-strand recognition in biology.

Secondary structure of the third extracellular loop responsible for ligand selectivity of a mammalian gonadotropin-releasing hormone receptor

The extracellular loop 3 (ECL3) of the mammalian gonadotropin-releasing hormone receptor (GnRH-R) contains an acidic amino acid (Glu(301) in the mouse GnRH-R,) that confers agonist selectivity for Are in mammalian GnRH. It is proposed that a specific conformation of ECL3 is necessary to orientate the carboxyl side chain of the acidic residue for interaction with Arg(8) of GnRH, which is supported by decreased affinity for Arg(8) GnRH but not Gln(8) GnRH when an adjacent Pro is mutated to Ala. To probe the structural contribution of the loop domain to the proposed presentation of the carboxyl side chain, we synthesized a model peptide (CGPEMLNRVSEPGC) representing residues 293-302 of mouse ECL3, where Cys and Gly residues are added symmetrically at the N and C termini, respectively, allowing the introduction of a disulfide bridge to simulate the distances at which the ECL3 is tethered to the transmembrane domains 6 and 7 of the receptor. The ability of the ECL3 peptide to bind GnRH with low affinity was demonstrated by its inhibition of GnRH stimulation of inositol phosphate production in cells expressing the GnRH-R. The CD bands of the ECL3 peptides exhibited a superposition of predominantly unordered structure and partial contributions from beta-sheet structure. Likewise, the analysis of the amide I and amide III bands from micro-Raman and FT Raman experiments revealed mainly unordered conformations of the cyclic and of the linear peptide. NMR data demonstrated the presence of a beta-hairpin among an ensemble of largely disordered structures in the cyclic peptide. The location of the turn linking the two strands of the hairpin was assigned to the three central residues L-296, N-297, and R-298. A small population of structured species among an ensemble of predominantly random coil conformation suggests that the unliganded receptor represents a variety of structural conformers, some of which have the potential to make contacts with the ligand. We propose a mechanism of receptor activation whereby binding of the agonist to the inactive receptor state induces and stabilizes a particular structural state of the loop domain, leading to further conformational rearrangements across the transmembrane domain and signal propagating interaction with G proteins. Interaction of the Glu(301) of the receptor with Arg(8) of GnRH induces a folded configuration of the ligand. Our proposal thus suggests that conformational changes of both ligand and receptor result from this interaction.

Prolonged retention after aggregation into secretory granules of human R183H-growth hormone (GH), a mutant that causes autosomal dominant GH deficiency type II

Human R183H-GH causes autosomal dominant GH deficiency type II. Because we show here that the mutant hormone is fully bioactive, we have sought to locate an impairment in its progress through the secretory pathway as assessed by pulse chase experiments. Newly synthesized wild-type and R183H-GH were stable when expressed transiently in AtT20 cells, and both formed equivalent amounts of Lubrol-insoluble aggregates within 40 min after synthesis. There was no evidence for intermolecular disulfide bond formation in aggregates of wild-type hormone or the R183H mutant. Both wildtype and R183H-GH were packaged into secretory granules, assessed by the ability of 1 mm BaCl2 to stimulate release and by immunocytochemistry. The mutant differed from wildtype hormone in its retention in the cells after packaging into secretory granules; 50% more R183H-GH than wild-type aggregates were retained in AtT20 cells 120 min after synthesis, and stimulated release of R183H-GH or a mixture of R183H-GH and wild-type that had been retained in the cell was reduced. The longer retention of R183H-GH aggregates indicates that a single point mutation in a protein contained in secretory granules affects the rate of secretory granule release.

Anaerobic ammonium oxidation by marine and freshwater planctomycete-like bacteria

Recently, two fresh water species, 'Candidatus Brocadia anammoxidans' and 'Candidatus Kuenenia stuttgartiensis', and one marine species, 'Candidatus Scalindua sorokinii', of planctomycete anammox bacteria have been identified. 'Candidatus Scalindua sorokinii' was discovered in the Black Sea, and contributed substantially to the loss of fixed nitrogen. All three species contain a unique organelle-the anammoxosome-in their cytoplasm. The anammoxosome contains the hydrazine/hydroxylamine oxidoreductase enzyme, and is thus the site of anammox catabolism. The anammoxosome is surrounded by a very dense membrane composed almost exclusively of linearly concatenated cyclobutane-containing lipids. These so-called 'ladderanes' are connected to the glycerol moiety via both ester and ether bonds. In natural and man-made ecosystems, anammox bacteria can cooperate with aerobic ammonium-oxidising bacteria, which protect them from harmful oxygen, and provide the necessary nitrite. The cooperation of these two groups of ammonium-oxidising bacteria is the microbial basis for a sustainable one reactor system, CANON (completely autotrophic nitrogen-removal over nitrite) to remove ammonia from high strength wastewater.

Linearization of a naturally occurring circular protein maintains structure but eliminates hemolytic activity

Cyclotides are a recently discovered family of disulfide rich proteins from plants that contain a circular protein backbone. They are exceptionally stable, as exemplified by their use in native medicine of the prototypic cyclotide kalata B1. The peptide retains uterotonic activity after the plant from which it is derived is boiled to make a medicinal tea. The circular backbone is thought to be in part responsible for the stability of the cyclotides, and to investigate its role in determining structure and biological activity, an acyclic derivative, des-(24-28)-kalata B1, was chemically synthesized and purified. This derivative has five residues removed from the 29-amino acid circular backbone of kalata B1 in a loop region corresponding to a processing site in the biosynthetic precursor protein. Two-dimensional NMR spectra of the peptide were recorded, assigned, and used to identify a series of distance, angle, and hydrogen bonding restraints. These were in turn used to determine a representative family of solution structures. Of particular interest was a determination of the structural similarities and differences between des-(2428)-kalata B1 and native kalata B1. Although the overall three-dimensional fold remains very similar to that of the native circular protein, removal of residues 24-28 of kalata B1 causes disruption of some structural features that are important to the overall stability. Furthermore, loss of hemolytic activity is associated with backbone truncation and linearization.

Epitope map for a growth hormone receptor agonist monoclonal antibody, MAb 263

Monoclonal antibody (MAb) 263 is a widely used monoclonal antibody that recognizes the extracellular domain (ECD) of the GH receptor. It has been shown to act as a GH agonist both in vitro and in vivo, and we report here that it must be divalent to exert its effect on the full-length receptor. To understand the mechanism of its agonist action, we have determined the precise epitope for this antibody using a novel random PCR mutagenesis approach together with expression screening in yeast. A library of 5200 clones of rabbit GH receptor ECD mutants were screened both with MAb 263 and with an anticarboxy-tag antibody to verify complete ECD expression. Sequencing for clones that expressed complete ECD but were not MAb 263 positive identified 20 epitope residues distributed in a discontinuous manner throughout the ECD. The major part of the epitope, as revealed after mapping onto the crystal structure model of the ECD molecule, was located on the side and upper portion of domain 1, particularly within the D - E strand disulfide loop 79 - 96. Molecular dynamics docking of an antibody of the same isotype as MAb 263 was used to dock the bivalent antibody to the 1528-Angstrom(2) epitope and to visualize the likely consequences of MAb binding. The minimized model enables the antibody to grasp two receptors in a pincer-like movement from opposite sides, facilitating alignment of the receptor dimerization domains in a manner similar to, but not identical with, GH.

Synthesis and characterization of delta-atracotoxin-ar1a, the lethal neurotoxin from venom of the Sydney funnel-web spider (Atrax robustus)

delta-Atracotoxin-Ar1a (delta-ACTX-Ar1a) is the major polypeptide neurotoxin isolated from the venom of the male Sydney funnel-web spider, Atrax robustus. This neurotoxin targets both insect and mammalian voltage-gated sodium channels, where it competes with scorpion alpha-toxins for neurotoxin receptor site-3 to slow sodium-channel inactivation. Progress in characterizing the structure and mechanism of action of this toxin has been hampered by the limited supply of pure toxin from natural sources. In this paper, we describe the first successful chemical synthesis and oxidative refolding of the four-disulfide bond containing delta-ACTX-Ar1a. This synthesis involved solid-phase Boc chemistry using double coupling, followed by oxidative folding of purified peptide using a buffer of 2 M GdnHCl and glutathione/glutathiol in a 1:1 mixture of 2-propanol (pH 8.5). Successful oxidation and refolding was confirmed using both chemical and pharmacological characterization. Ion spray mass spectrometry was employed to confirm the molecular weight. H-1 NMR analysis showed identical chemical shifts for native and synthetic toxins, indicating that the synthetic toxin adopts the native fold. Pharmacological studies employing whole-cell patch clamp recordings from rat dorsal root ganglion neurons confirmed that synthetic delta-ACTX-Ar1a produced a slowing of the sodium current inactivation and hyperpolarizing shifts in the voltage-dependence of activation and inactivation similar to native toxin. Under current clamp conditions, we show for the first time that delta-ACTX-Ar1a produces spontaneous repetitive plateau potentials underlying the clinical symptoms seen during envenomation. This successful oxidative refolding of synthetic delta-ACTX-Ar1a paves the way for future structure-activity studies to determine the toxin pharmacophore.

New structure formation on gamma-irradiation of poly(chlorotrifluoroethylene)

The radiation chemistry of PCTFE at different temperatures has been studied. The polymer was irradiated under vacuum to absorbed doses of up to 1500 kGy. Three irradiation temperatures were chosen. These included ambient temperature, a temperature well above the T, and a temperature above the crystalline melting temperature. These were 298, 423 and 493 K, respectively. The formation of new structures was identified by solid-state FTIR and F-19 NMR. No branching was observed below the melting temperature, but branches were observed above the melting temperature. G-values for chain-end formation were 1.5 and 2.4 at room temperature and 423 K, respectively and the G-value for the formation of double bonds was found to be < 0.1. For the irradiations at 493 K, the G-values for the formation of chain ends, double bonds and branching points were 3.6, 0.2 and 0.5, respectively. The presence of long-chain branches within the polymer structure could not be proven for radiolysis at 493 K, but scission predominates and network formation does not occur upon irradiation. DSC studies of the polymers irradiated at ambient temperature were consistent with chain scission leading to an increase in the percentage crystallinity, as observed for other fluoropolymers. (C) 2003 Elsevier Science Ltd. All rights reserved.