Bone loss recovery in mice following microgravity with concurrent bone-compartment-specific osteocyte characteristics.

Space missions provide the opportunity to investigate the influence of gravity on the dynamic remodelling processes in bone. Mice were examined following space flight and subsequent recovery to determine the effects on bone compartment-specific microstructure and composition. The resulting bone loss following microgravity recovered only in trabecular bone, while in cortical bone the tissue mineral density was restored after only one week on Earth. Detection of TRAP-positive bone surface cells in the trabecular compartment indicated increased resorption following space flight. In cortical bone, a persistent reduced viability of osteocytes suggested an impaired sensitivity to mechanical stresses. A compartment-dependent structural recovery from microgravity-induced bone loss was shown, with a direct osteocytic contribution to persistent low bone volume in the cortical region even after a recovery period. Trabecular recovery was not accompanied by changes in osteocyte characteristics. These post-space-flight findings will contribute to the understanding of compositional changes that compromise bone quality caused by unloading, immobilisation, or disuse.

Retrograde crossing stent placement strategies at the basilar apex for the treatment of wide necked aneurysms: reconstructive and deconstructive opportunities.

Neck remodeling devices such as the Neuroform or Enterprise greatly facilitate the endovascular treatment of wide necked basilar apex aneurysms. The complex anatomy of the basilar apex affords opportunity for antegrade, multiple crossing and retrograde device placement strategies to facilitate coil embolization. A retrograde approach is possible in the presence of a posterior communicating artery large enough to allow device navigation. Our experience with a retrograde device placement strategy in three patients is reported. In two patients, device positioning extended from one P1 segment of the posterior cerebral artery to the other across the basilar apex. In one patient, device positioning extended from the P1 segment of the posterior cerebral artery across the basilar apex into the opposite superior cerebellar artery. All patients underwent reconstructive or deconstructive uncomplicated coil embolization after device placement with stable aneurysm occlusion on follow-up angiography. In appropriate anatomic situations, retrograde stent placement across the basilar apex through a posterior communicating artery may represent a preferred strategy for wide necked basilar apex aneurysms.

Nature of hydrogen transfer in soybean lipoxygenase 1: separation of primary and secondary isotope effects.

Previous measurements of the kinetics of oxidation of linoleic acid by soybean lipoxygenase 1 have indicated very large deuterium isotope effects, but have not been able to distinguish the primary isotope effect from the alpha-secondary effect. To address this question, singly deuterated linoleic acid was prepared, and enantiomerically resolved using the enzyme itself. Noncompetitive measurements of the primary deuterium isotope effect give a value of ca. 40 which is temperature-independent. The enthalpy of activation is low and isotope-independent, and there is a large isotope effect on the Arrhenius prefactor. A very large apparent secondary isotope effect (ca. 2.1) is measured with deuterium in the primary position, but a greatly reduced value (1.1) is observed with protium in the primary position. Mutagenesis of the active site leads to a significant reduction in k(cat) and perturbed isotope effects, in particular, a secondary effect of 5.6 when deuterium is in the primary position. The anomalous secondary isotope effects are shown to arise from imperfect stereoselectivity of hydrogen abstraction which, for the mutant, is attributed to a combination of inverse substrate binding and increased flexibility at the reactive carbon. After correction, a very large primary (76-84) and small secondary (1.1-1.2) kinetic isotope effects are calculated for both mutant and wild-type enzymes. The weight of the evidence is taken to favor hydrogen tunneling as the primary mechanism of hydrogen transfer.

Analysis of the conserved glycosylation site in the nicotinic acetylcholine receptor: potential roles in complex assembly.

BACKGROUND: Assembly of the functional nicotinic acetylcholine receptor (nAChR) is dependent on a series of exquisitely coordinated events including polypeptide synthesis and processing, side-chain elaboration through post-translational modifications, and subunit oligomerization. A 17-residue sequence that includes a cystine disulfide and an N-linked glycosylation site is conserved in the extracellular domain of each of the nAChR subunits, and is involved in intersubunit interactions that are critical for assembly of intact, pentameric complexes. A polypeptide representing the relevant sequence from the alpha-subunit of the nAChR (Ac-Tyr-Cys-Glu-Ile-Ile-Val-Thr-His-Phe-Pro-Phe-Asp-Gln-Gln Asn-Cys-Thr-NH2) is small enough to allow detailed structural analysis, which may provide insight into the role of glycosylation in the maturation process that leads to ion-channel assembly. We therefore investigated the effect of N-linked glycosylation on the structure of this heptadecapeptide. RESULTS: Thermodynamic analysis shows that glycosylation alters disulfide formation in the loop peptide, shifting the equilibrium in favor of the disulfide. Spectroscopic studies reveal that the cis/trans amide isomer ratio of the proline is also affected by the modification, with a resultant shift in the equilibrium in favor of the trans isomer, even though the proline is several residues removed from the glycosylation site. Two-dimensional NMR analysis of the glycopeptide does not indicate the presence of any specific interactions between the carbohydrate and the peptide. CONCLUSIONS: These studies demonstrate that glycosylation can have a significant influence on disulfide formation and proline isomerization in a local peptide sequence. As both these processes are considered slow steps in protein folding, it is evident that N-linked glycosylation has important indirect roles that influence the folding of the receptor subunit and assembly of the pentameric complex.

Conformational implications of asparagine-linked glycosylation.

The effects of cotranslational protein modification on the process of protein folding are poorly understood. Time-resolved fluorescence energy transfer has been used to assess the impact of glycosylation on the conformational dynamics of flexible oligopeptides. The peptide sequences examined are selected from glycoproteins of known three-dimensional structure. The energy transfer modulation associated with N-linked glycosylation is consistent with the glycopeptides sampling different conformational profiles in water. Results show that glycosylation causes the modified peptides to adopt a different ensemble of conformations, and for some peptides this change may lead to conformations that are more compact and better approximate the conformation of these peptides in the final folded protein. This result further implies that cotranslational glycosylation can trigger the timely formation of structural nucleation elements and thus assist in the complex process of protein folding.

Mechanism of irreversible inhibition of O2 evolution in photosystem II by Tris(hydroxymethyl)aminomethane.

The dark reaction of tris(hydroxymethyl)aminomethane (Tris) with the O2-evolving center of photosystem II (PSII) in the S1 state causes irreversible inhibition of O2 evolution. Similar inhibition is observed for several other amines: NH3, CH3NH2, (CH3)2NH, ethanolamine, and 2-amino-2-ethyl-1,3-propanediol. In PSII membranes, both depleted of the 17- and 23-kDa polypeptides and undepleted, the rate of reaction of Tris depends inversely upon the Cl- concentration. However, the rate of reaction of Tris is about 2-fold greater with PSII membranes depleted of the 17- and 23-kDa polypeptides than with undepleted PSII membranes. We have used low-temperature electron paramagnetic resonance (EPR) spectroscopy to study the effect of Tris on the oxidation state of the Mn complex in the O2-evolving center, to monitor the electron-donation reactions in Tris-treated samples, and to observe any loss of the Mn complex (forming Mn2+ ions) after Tris treatment. We find that Tris treatment causes loss of electron-donation ability from the Mn complex at the same rate as inhibition of O2 evolution and that Mn2+ ions are released. We conclude that Tris reduces the Mn complex to labile Mn2+ ions, without generating any kinetically stable, partially reduced intermediates, and that the reaction occurs at the Cl(-)-sensitive site previously characterized in studies of the reversible inhibition of O2 evolution by amines.