Multi-isotope analysis reveals that feasts in the Stonehenge environs and across Wessex drew people and animals from throughout Britain.

The great henge complexes of southern Britain are iconic monuments of the third millennium BCE, representing great feats of engineering and labor mobilization that hosted feasting events on a previously unparalleled scale. The scale of movement and the catchments that the complexes served, however, have thus far eluded understanding. Presenting the largest five-isotope system archeological dataset (87Sr/86Sr, δ34S, δ18O, δ13C, and δ15N) yet fully published, we analyze 131 pigs, the prime feasting animals, from four Late Neolithic (approximately 2800 to 2400 BCE) complexes to explore the networks that the feasts served. Because archeological evidence excludes continental contact, sources are considered only in the context of the British Isles. This analysis reveals wide-ranging origins across Britain, with few pigs raised locally. This finding demonstrates great investment of effort in transporting pigs raised elsewhere over vast distances to supply feasts and evidences the very first phase of pan-British connectivity.

Evaluation of maxillary arterial blood flow in anesthetized cats with the mouth closed and open.

The mouth-gag is a common tool used in veterinary medicine during oral and transoral procedures in cats but its use has recently been associated with the development of blindness. The goal of this study was to investigate whether maximal opening of the mouth affects maxillary artery blood flow in six anesthetized cats. To assess blood flow, the electroretinogram (ERG), brainstem auditory evoked response (BAER) and magnetic resonance angiography (MRA) were evaluated qualitatively with the mouth closed and open. During dynamic computer tomography (CT) examinations, detection of contrast medium in the maxillary artery was quantified by measuring the Hounsfield units (HUs). The peak HU, time to peak and mean HU were determined. Changes ⩾10% of these parameters were considered indicative of altered blood flow. ERG and BAER were normal with the mouth closed in all cats, but was abnormal with the mouth opened maximally in two cats and one cat, respectively. During MRA, blood flow was undetected in either maxillary artery in one cat and reduced in the right maxillary artery in two cats, when the mouth was open. During CT, the peak HU decreased ⩾10% in three cats, the time to peak was ⩾10% longer in two cats, and the mean HU was ⩾10% lower in one cat when the mouth was open. No cat developed apparent blindness or deafness. Maximal opening of the mouth caused alterations in several indicators of blood flow in some individual cats.

Heterodimeric structure of superoxide dismutase in complex with its metallochaperone.

The copper chaperone for superoxide dismutase (CCS) activates the eukaryotic antioxidant enzyme copper, zinc superoxide dismutase (SOD1). The 2.9 A resolution structure of yeast SOD1 complexed with yeast CCS (yCCS) reveals that SOD1 interacts with its metallochaperone to form a complex comprising one monomer of each protein. The heterodimer interface is remarkably similar to the SOD1 and yCCS homodimer interfaces. Striking conformational rearrangements are observed in both the chaperone and target enzyme upon complex formation, and the functionally essential C-terminal domain of yCCS is well positioned to play a key role in the metal ion transfer mechanism. This domain is linked to SOD1 by an intermolecular disulfide bond that may facilitate or regulate copper delivery.

Heterodimer formation between superoxide dismutase and its copper chaperone.

Copper, zinc superoxide dismutase (SOD1) is activated in vivo by the copper chaperone for superoxide dismutase (CCS). The molecular mechanisms by which CCS recognizes and docks with SOD1 for metal ion insertion are not well understood. Two models for the oligomerization state during copper transfer have been proposed: a heterodimer comprising one monomer of CCS and one monomer of SOD1 and a dimer of dimers involving interactions between the two homodimers. We have investigated protein-protein complex formation between copper-loaded and apo yeast CCS (yCCS) and yeast SOD1 for both wild-type SOD1 (wtSOD1) and a mutant SOD1 in which copper ligand His 48 has been replaced with phenylalanine (H48F-SOD1). According to gel filtration chromatography, dynamic light scattering, analytical ultracentrifugation, and chemical cross-linking experiments, yCCS and this mutant SOD1 form a complex with the correct molecular mass for a heterodimer. No higher order oligomers were detected. Heterodimer formation is facilitated by the presence of zinc but does not depend on copper loading of yCCS. The complex formed with H48F-SOD1 is more stable than that formed with wtSOD1, suggesting that the latter is a more transient species. Notably, heterodimer formation between copper-loaded yCCS and wtSOD1 is accompanied by SOD1 activation only in the presence of zinc. These findings, taken together with structural, biochemical, and genetic studies, strongly suggest that in vivo copper loading of yeast SOD1 occurs via a heterodimeric intermediate.

Structural basis for copper transfer by the metallochaperone for the Menkes/Wilson disease proteins.

The Hah1 metallochaperone protein is implicated in copper delivery to the Menkes and Wilson disease proteins. Hah1 and the N-termini of its target proteins belong to a family of metal binding domains characterized by a conserved MT/HCXXC sequence motif. The crystal structure of Hah1 has been determined in the presence of Cu(I), Hg(II), and Cd(II). The 1.8 A resolution structure of CuHah1 reveals a copper ion coordinated by Cys residues from two adjacent Hah1 molecules. The CuHah1 crystal structure is the first of a copper chaperone bound to copper and provides structural support for direct metal ion exchange between conserved MT/HCXXC motifs in two domains. The structures of HgHah1 and CdHah1, determined to 1.75 A resolution, also reveal metal ion coordination by two MT/HCXXC motifs. An extended hydrogen bonding network, unique to the complex of two Hah1 molecules, stabilizes the metal binding sites and suggests specific roles for several conserved residues. Taken together, the structures provide models for intermediates in metal ion transfer and suggest a detailed molecular mechanism for protein recognition and metal ion exchange between MT/HCXXC containing domains.

Crystal structure of the second domain of the human copper chaperone for superoxide dismutase.

The human copper chaperone for superoxide dismutase (hCCS) delivers the essential copper ion cofactor to copper,zinc superoxide dismutase (SOD1), a key enzyme in antioxidant defense. Mutations in SOD1 are linked to familial amyotrophic lateral sclerosis (FALS), a fatal neurodegenerative disorder. The molecular mechanisms by which SOD1 is recognized and activated by hCCS are not understood. To better understand this biochemical pathway, we have determined the X-ray structure of the largest domain of hCCS (hCCS Domain II) to 2. 75 A resolution. The overall structure is closely related to that of its target enzyme SOD1, consisting of an eight-stranded beta-barrel and a zinc-binding site formed by two extended loops. The first of these loops provides the ligands to a bound zinc ion, and is analogous to the zinc subloop in SOD1. The second structurally resembles the SOD1 electrostatic channel loop, but lacks many of the residues important for catalysis. Like SOD1 and yCCS, hCCS forms a dimer using a highly conserved interface. In contrast to SOD1, however, the hCCS structure does not contain a copper ion bound in the catalytic site. Notably, the structure reveals a single loop proximal to the dimer interface which is unique to the CCS chaperones.

Crystal structure of the copper chaperone for superoxide dismutase.

Cellular systems for handling transition metal ions have been identified, but little is known about the structure and function of the specific trafficking proteins. The 1.8 A resolution structure of the yeast copper chaperone for superoxide dismutase (yCCS) reveals a protein composed of two domains. The N-terminal domain is very similar to the metallochaperone protein Atx1 and is likely to play a role in copper delivery and/or uptake. The second domain resembles the physiological target of yCCS, superoxide dismutase I (SOD1), in overall fold, but lacks all of the structural elements involved in catalysis. In the crystal, two SOD1-like domains interact to form a dimer. The subunit interface is remarkably similar to that in SOD1, suggesting a structural basis for target recognition by this metallochaperone.

The structure of retinal dehydrogenase type II at 2.7 A resolution: implications for retinal specificity.

Retinoic acid, a hormonally active form of vitamin A, is produced in vivo in a two step process: retinol is oxidized to retinal and retinal is oxidized to retinoic acid. Retinal dehydrogenase type II (RalDH2) catalyzes this last step in the production of retinoic acid in the early embryo, possibly producing this putative morphogen to initiate pattern formation. The enzyme is also found in the adult animal, where it is expressed in the testis, lung, and brain among other tissues. The crystal structure of retinal dehydrogenase type II cocrystallized with nicotinamide adenine dinucleotide (NAD) has been determined at 2.7 A resolution. The structure was solved by molecular replacement using the crystal structure of a mitochondrial aldehyde dehydrogenase (ALDH2) as a model. Unlike what has been described for the structures of two aldehyde dehydrogenases involved in the metabolism of acetaldehyde, the substrate access channel is not a preformed cavity into which acetaldehyde can readily diffuse. Retinal dehydrogenase appears to utilize a disordered loop in the substrate access channel to discriminate between retinaldehyde and short-chain aldehydes.

Purification, crystallization and preliminary X-ray diffraction studies of retinal dehydrogenase type II.

One enzyme which catalyzes the last step of the formation of the hormone retinoic acid from vitamin A (retinol) is retinal dehydrogenase type II (Ra1DH2). Ra1DH2, expressed in the Escherichia coli BL21(DE3) strain, was purified and crystallized using ammonium sulfate as a precipitant. These crystals belong to the space group P212121 (a = 108, b = 150, c = 168 A, alpha = beta = gamma = 90 degrees).