Antarctic ice sheet discharge driven by atmosphere-ocean feedbacks at the Last Glacial Termination.

Reconstructing the dynamic response of the Antarctic ice sheets to warming during the Last Glacial Termination (LGT; 18,000-11,650 yrs ago) allows us to disentangle ice-climate feedbacks that are key to improving future projections. Whilst the sequence of events during this period is reasonably well-known, relatively poor chronological control has precluded precise alignment of ice, atmospheric and marine records, making it difficult to assess relationships between Antarctic ice-sheet (AIS) dynamics, climate change and sea level. Here we present results from a highly-resolved 'horizontal ice core' from the Weddell Sea Embayment, which records millennial-scale AIS dynamics across this extensive region. Counterintuitively, we find AIS mass-loss across the full duration of the Antarctic Cold Reversal (ACR; 14,600-12,700 yrs ago), with stabilisation during the subsequent millennia of atmospheric warming. Earth-system and ice-sheet modelling suggests these contrasting trends were likely Antarctic-wide, sustained by feedbacks amplified by the delivery of Circumpolar Deep Water onto the continental shelf. Given the anti-phase relationship between inter-hemispheric climate trends across the LGT our findings demonstrate that Southern Ocean-AIS feedbacks were controlled by global atmospheric teleconnections. With increasing stratification of the Southern Ocean and intensification of mid-latitude westerly winds today, such teleconnections could amplify AIS mass loss and accelerate global sea-level rise.

What caused extinction of the Pleistocene megafauna of Sahul?

During the Pleistocene, Australia and New Guinea supported a rich assemblage of large vertebrates. Why these animals disappeared has been debated for more than a century and remains controversial. Previous synthetic reviews of this problem have typically focused heavily on particular types of evidence, such as the dating of extinction and human arrival, and have frequently ignored uncertainties and biases that can lead to misinterpretation of this evidence. Here, we review diverse evidence bearing on this issue and conclude that, although many knowledge gaps remain, multiple independent lines of evidence point to direct human impact as the most likely cause of extinction.

Geochronology of cave deposits at Liang Bua and of adjacent river terraces in the Wae Racang valley, western Flores, Indonesia: a synthesis of age estimates for the type locality of Homo floresiensis.

A robust timeframe for the extant cave deposits at Liang Bua, and for the river terraces in the adjoining Wae Racang valley, is essential to constrain the period of existence and time of extinction of Homo floresiensis and other biota that have been excavated at this hominin type locality. Reliable age control is also required for the variety of artifacts excavated from these deposits, and to assist in environmental reconstructions for this river valley and for the region more broadly. In this paper, we summarize the available geochronological information for Liang Bua and its immediate environs, obtained using seven numerical-age methods: radiocarbon, thermoluminescence, optically- and infrared-stimulated luminescence (collectively known as optical dating), uranium-series, electron spin resonance, and coupled electron spin resonance/uranium-series. We synthesize the large number of numerical age determinations reported previously and present additional age estimates germane to questions of hominin evolution and extinction.

Heterotrophic fixation of CO(2) in soil.

The occurrence of heterotrophic CO(2) fixation by soil microorganisms was tested in several mineral soils differing in pH and two artificial soils (a mixture of silica sand, alfalfa powder, and nutrient medium inoculated with a soil suspension). Soils were incubated at ambient ( approximately 0.05 vol%) and elevated ( approximately 5 vol%) CO(2) concentrations under aerobic conditions for up to 21 days. CO(2) fixation was detected using either a technique for determining the natural abundance of (13)C or by measuring the distribution of labeled (14)C-CO(2) in soil and bacteria. The effects of elevated CO(2) on microbial biomass (direct counts, chloroform fumigation extraction method), composition of microbial community (phospholipid fatty acids), microbial activity (respiration, dehydrogenase activity), and turnover rate were also measured. Heterotrophic CO(2) fixation was proven in all soils under study, being higher in neutral soils. The main portion of the fixed CO(2) (98-99%) was found in extracellular metabolites while only approximately 1% CO(2) was incorporated into microbial cells. High CO(2) concentration always induced an increase in microbial activity, changes in the composition of the microbial community, and a decrease in microbial turnover. The results suggest that heterotrophic CO(2) fixation could be a widespread process in soils.

Monomeric and multimeric blockers of selectins: comparison of in vitro and in vivo activity.

The potency of the oligosaccharides SiaLe(x), SiaLe(a), HSO(3)Le(x), and HSO(3)Le(a), their conjugates with polyacrylamide (PAA, 40 kD), and other monomeric and polymeric selectin inhibitors has been compared with that of the polysaccharide fucoidan. The following assay systems were used: 1) a 96-well assay based either on the use of recombinant E-, P-, and L-selectins or an analogous assay with natural P-selectin isolated from human platelets; 2) a platelet-based P-selectin cell assay; and 3) a rat model of peritoneal inflammation. IC(50) values for the neoglycoconjugate SiaLe(a)-PAA were 6, 40, and 85 microM for recombinant E-, P-, and L-selectins, respectively; all monomeric inhibitors were about two orders of magnitude weaker. PAA-conjugates, containing as a ligand tyrosine-O-sulfate (sTyr) in addition to one of the sialylated oligosaccharides, were the most potent synthetic blockers in vitro. Compared with fucoidan, the most potent known P- and L-selectin blocker, the bi-ligand glycoconjugate HSO(3)Le(a)-PAA-sTyr displayed similar inhibitory activity in vitro towards L-selectin and about ten times lower activity towards P-selectin. All of the tested synthetic polymers displayed a similar ability to inhibit neutrophil extravasation in the peritonitis model (in vivo) at 10 mg/kg. The data provide evidence that monomeric SiaLe(x) is considerably more effective as a selectin blocker in vivo than in vitro, whereas the opposite is true for fucoidan and the bi-ligand neoglycoconjugate HSO(3)Le(a)-PAA-sTyr.

Archaeology and age of a new hominin from Flores in eastern Indonesia.

Excavations at Liang Bua, a large limestone cave on the island of Flores in eastern Indonesia, have yielded evidence for a population of tiny hominins, sufficiently distinct anatomically to be assigned to a new species, Homo floresiensis. The finds comprise the cranial and some post-cranial remains of one individual, as well as a premolar from another individual in older deposits. Here we describe their context, implications and the remaining archaeological uncertainties. Dating by radiocarbon (14C), luminescence, uranium-series and electron spin resonance (ESR) methods indicates that H. floresiensis existed from before 38,000 years ago (kyr) until at least 18 kyr. Associated deposits contain stone artefacts and animal remains, including Komodo dragon and an endemic, dwarfed species of Stegodon. H. floresiensis originated from an early dispersal of Homo erectus (including specimens referred to as Homo ergaster and Homo georgicus) that reached Flores, and then survived on this island refuge until relatively recently. It overlapped significantly in time with Homo sapiens in the region, but we do not know if or how the two species interacted.

Single-nucleotide polymorphism alleles in the insulin receptor gene are associated with typical migraine.

We have identified a migraine locus on chromosome 19p13.3/2 using linkage and association analysis. We isolated 48 single-nucleotide polymorphisms within the locus, of which we genotyped 24 in a Caucasian population comprising 827 unrelated cases and 765 controls. Five single-nucleotide polymorphisms within the insulin receptor gene showed significant association with migraine. This association was independently replicated in a case-control population collected separately. We used experiments with insulin receptor RNA and protein to investigate functionality for the migraine-associated single-nucleotide polymorphisms. We suggest possible functions for the insulin receptor in migraine pathogenesis.

Identification of human complement Factor H as a ligand for L-selectin.

The selectin family of adhesion molecules (E-, P- and L-selectins) is involved in leukocyte recruitment to sites of inflammation and tissue damage. Recently it has been shown that L-selectin is involved not only in leukocyte tethering and rolling, but also plays an important role in leukocyte activation. For example, glycosylation-dependent cell-adhesion molecule 1 (GlyCAM-1), a known ligand for L-selectin, has been shown to enhance beta2-integrin function. GlyCAM-1 is a secreted protein and is present in mouse serum at a concentration of approx. 1.5 microg/ml. There is no obvious GlyCAM-1 homologue in man and, to date, L-selectin ligand(s) from human serum have not been characterized. Therefore we have used L-selectin affinity chromatography, followed by ion-exchange chromatography, to isolate specific ligand(s) for L-selectin. Using this procedure, we have isolated three major glycoproteins of apparent molecular masses 170 kDa, 70kDa and 50 kDa. The 170 kDa protein band was digested with trypsin and peptides were analysed by delayed extraction matrix-assisted laser desorption ionization MS and protein database searching. The 170 kDa protein was identified as the human complement protein Factor H. Human Factor H, isolated by a different method, was shown to bind specifically to L-selectin in the presence of CaCl2, and binding was inhibited by anti-L-selectin antibodies, fucoidan and lipopolysaccharide. Only a part of the purified Factor H preparation bound to immobilized L-selectin. The interaction of Factor H with leukocyte L-selectin was shown to induce the secretion of tumour necrosis factor-alpha (TNF-alpha). Pretreatment of Factor H with sialidase reduced both the binding of L-selectin to Factor H and the Factor H-induced L-selectin-mediated TNF-alpha secretion by leukocytes. Taken together, these results demonstrate that a post-translationally modified form of human plasma Factor H is a potential physiological ligand for L-selectin.

Characterization of E-selectin-binding epitopes expressed by skin-homing T cells.

The glycoprotein counter-receptors for E-selectin borne on skin-homing T cells are poorly defined. In this study we have used flow cytometry to investigate the surface expression of potential carbohydrate ligands for E-selectin on HUT78, a skin-homing cutaneous T-cell lymphoma. These cells possessed high surface expression of the KM-93 epitope but not HECA 452 or CSLEX1 epitopes. The KM-93 antibody also blocked the binding of HUT78 cells to E-selectin. All these antibodies are reported to recognize sialyl Lewis X (sLex)-like molecules. Using an E-selectin affinity matrix, the main glycoprotein isolated from HUT78 cells was a molecular species of 90 000 MW. Other minor species of molecular weights 40 000, 60 000, 100 000, 120 000 and 200 000 were also identified as potential counter-receptors for E-selectin. Four of the purified counter-receptors (90 000, 100 000, 120 000 and 200 000 MW) stained positive with the KM-93 antibody. Immunoblot analysis of these purified glycoproteins established the identity of the 90 000 MW glycoprotein as l-selectin. Furthermore, an anti-l-selectin antibody inhibited the binding of HUT78 cells to E-selectin, probably by steric inhibition of the carbohydrate ligand for E-selectin that is borne on the C-type lectin domain of l-selectin. These results suggest that a carbohydrate epitope on l-selectin may act as a ligand for E-selectin on skin-homing T cells.

Acceptor specificity of the human leukocyte alpha3 fucosyltransferase: role of FucT-VII in the generation of selectin ligands.

The alpha3 fucosyltransferase, FucT-VII, is one of the key glycosyltransferases involved in the biosynthesis of the sialyl Lewis X (sLex) antigen on human leukocytes. The sialyl Lewis X antigen (NeuAcalpha(2-3)Galbeta(1-4)[Fucalpha(1-3)]GlcNAc-R) is an essential component of the recruitment of leukocytes to sites of inflammation, mediating the primary interaction between circulating leukocytes and activated endothelium. In order to characterize the enzymatic properties of the leukocyte alpha3 fucosyltransferase FucT-VII, the enzyme has been expressed in Trichoplusia ni insect cells. The enzyme is capable of synthesizing both sLexand sialyl-dimeric-Lexstructures in vitro , from 3'-sialyl-lacNAc and VIM-2 structures, respectively, with only low levels of fucose transfer observed to neutral or 3'-sulfated acceptors. Studies using fucosylated NeuAcalpha(2-3)-(Galbeta(1-4)GlcNAc)3-Me acceptors demonstrate that FucT-VII is able to synthesize both di-fucosylated and tri-fucosylated structures from mono-fucosylated precursors, but preferentially fucosylates the distal GlcNAc within a polylactosamine chain. Furthermore, the rate of fucosylation of the internal GlcNAc residues is reduced once fucose has been added to the distal GlcNAc. These results indicate that FucT-VII is capable of generating complex selectin ligands, in vitro , however the order of fucose addition to the lactosamine chain affects the rate of selectin ligand synthesis.

Scintillation proximity assay for E-, P-, and L-selectin utilizing polyacrylamide-based neoglycoconjugates as ligands.

In this study, a novel scintillation proximity assay (SPA) that uses radiolabeled soluble neoglycoconjugates as synthetic alternatives to the natural E-, P-, and L-selectin counterligands was developed. The neoglycoconjugates contained sialyl LewisX or sialyl LewisA attached via a three-carbon spacer to a poly[N-(hydroxyethyl)acrylamide] backbone, thus presenting the carbohydrates in a multivalent form. Selectin-ZZ fusion proteins were immobilized on anti-rabbit IgG-coated SPA beads via a rabbit IgG bridge. The neoglycoconjugate ligands bound to all three bead-immobilized selectins, with the highest binding levels apparent with E-selectin. Saturation binding studies with E-selectin revealed a complex interaction indicative of two or more binding affinities. The response to carbohydrate inhibitors was comparable in E-selectin assays that used either the neoglycoconjugates or the tritium-labeled HL60 cells as selectin counterligands. The incorporation of tyrosine sulfate groups into the backbone of the neoglycoconjugate resulted in enhanced binding avidity to both P- and L-selectin, indicating that the sulfate-containing neoglycoconjugates are viable synthetic mimics of the natural P- and L-selectin counterligands. The use of these radiolabeled neoglycoconjugates in conjunction with SPA results in a format ideally suited for the high-throughput screening for selectin antagonists. Furthermore, this approach can potentially be used to measure other low-avidity lectin-carbohydrate interactions.

P-selectin binds to bacterial lipopolysaccharide.

Multiple organ failure associated with disseminated intravascular coagulation is a frequent complication in septic shock patients. Accumulation of platelets and neutrophils in the organs contributes to the manifestation of lipopolysaccharide (LPS)-induced organ failure. Although a direct interaction between LPS and platelets is well documented, the nature of the surface receptor for LPS on platelets is unknown. In this article we show that P-selectin is a receptor for LPS. The binding of LPS to P-selectin is independent of Ca2+, and is blocked by antibodies to P-selectin, lipid A and fucoidan. Platelets pre-treated with thrombin showed fourfold higher binding of fluorescein isothiocyanate (FITC)-conjugated LPS compared to untreated platelets and the binding of FITC-conjugated LPS to platelets was blocked in the presence of anti-P-selectin antibodies. It is likely that the binding of LPS via P-selectin on activated platelets or epithelium could have a significant role in the pathophysiology of organ failure in septic shock.

Potential pro-inflammatory effects of soluble E-selectin upon neutrophil function.

Appropriate recruitment of neutrophils to sites of infection or tissue injury is a key event in the inflammatory response. A number of studies have shown the critical role of selectins in tethering and rolling of neutrophils on vascular endothelium, as well as a more complex regulatory role, since they have the potential to alter leukocyte recruitment by triggering beta2 integrin-mediated adhesion. In this study, we report that in contrast to patients "at risk" of developing acute respiratory disease syndrome (ARDS), elevated plasma levels of soluble E-selectin are found in patients with established disease. Since neutrophil granulocytes are implicated in ARDS pathogenesis, we have investigated the possibility of a link between elevated soluble plasma E-selectin levels and disease progression by examining the effects of soluble recombinant E-selectin (E-zz) upon neutrophil function. In this paper, we describe the novel finding that exposure of neutrophils to E-zz potentiates a number of neutrophil functions which may act to drive inflammatory processes. Although neutrophil deformability, an important parameter determining retention within the lung microvasculature, was not affected by E-zz, neutrophil polarization was observed. In addition, neutrophil beta2 integrin-mediated adhesion was found to be augmented by E-zz without alteration in levels of surface expression of alphaMbeta2 or the "activation" reporter epitope defined by monoclonal antibody 24. Concomitantly with increased beta2 integrin-mediated adhesion, we observed an inhibition of formyl-Met-Leu-Phe-directed chemotaxis. Together with an augmentation of neutrophil reactive oxidant species production and release of superoxide anions, these data raise the possibility that soluble E-selectin exerts pro-inflammatory effects upon neutrophil function at sites of inflammation, thereby exacerbating disease processes.

Measurement of alpha(1-3) fucosyltransferase activity using scintillation proximity.

The alpha 3 fucosyltransferases are a family of glycosyltransferases involved in the addition of fucose onto glycoproteins and glycolipids. One of the best defined roles for the alpha 3 fucosyltransferases is in the biosynthesis of the carbohydrate antigen sialyl Lewis X, the minimal ligand for the selectin family of adhesion molecules. We describe here the development of a single-step assay for the measurement of alpha 3 fucosyltransferase activity based on the principle of scintillation proximity. The fucosyltransferase catalyses the transfer of [3H]fucose, from GDP-[3H]fucose, onto the sugar chains of a glycoprotein acceptor noncovalently bound to a scintillant-impregnated microsphere (SPA bead). The resultant signal can be used as a measure of enzyme activity. Due to the nature of this assay no steps are required to separate unused substrate from product. Kinetic data from the assay compare favorably with those obtained from assays currently used for the alpha 3 fucosyltransferases. This SPA-based assay appears generic for the alpha 3 fucosyltransferases and readily adaptable for other glycosyltransferases. The particular advantage of the assay is anticipated to be found in the simple, routine testing of a large number of samples.

L-selectin: a novel receptor for lipopolysaccharide and its potential role in bacterial sepsis.

The activation of leukocytes by bacterial cell wall lipopolysaccharide (LPS) contributes to the pathogenesis of septic shock. It is well established that, in the presence of plasma LPS-binding protein (LBP), LPS binds with high affinity to CD14. The binding of LPS to CD14 has been associated with the activation of cells, although available evidence indicates that CD14 itself does not transduce intracellular signalling. The physiological function of this interaction is to promote host defense mechanisms of cells to combat the infection and clear LPS from the circulation. At higher concentrations of LPS, however, the activation of cells can take place in the absence of LBP and CD14, presumably through a distinct low-affinity signalling LPS receptor. On the evidence published by us and others, we propose that in neutrophils, and possibly other leukocytes, L-selectin can act as a low-affinity LPS receptor.

L-Selectin--a signalling receptor for lipopolysaccharide.

The activation of leukocytes by bacterial cell-wall lipopolysaccharide contributes to the pathogenesis of septic shock. We propose that in neutrophils, and possibly other leukocytes, L-selectin can act as a low-affinity lipopolysaccharide receptor. Inhibitors of L-selectin may therefore be of therapeutic value in treating this life-threatening condition.

Lewis X biosynthesis in Helicobacter pylori. Molecular cloning of an alpha(1,3)-fucosyltransferase gene.

The lipopolysaccharide of certain strains of Helicobacter pylori was recently shown to contain the Lewis X (Lex) trisaccharide (Galbeta-1, 4-(Fucalpha(1,3))-GlcNAc). Lex is an oncofetal antigen which appears on human gastric epithelium, and its mimicry by carbohydrate structures on the surface of H. pylori may play an important part in the interaction of this pathogen with its host. Potential roles for bacterial Lex in mucosal adhesion, immune evasion, and autoantibody induction have been proposed (Moran, A. P., Prendergast, M. M., and Appelmelk, B. J. (1996) FEMS Immunol. Med. Microbiol. 16, 105-115). In mammals, the final step of Lex biosynthesis is the alpha(1,3)-fucosylation of GlcNAc in a terminal Galbeta(1-->4)GlcNAc unit, and a corresponding GDP-fucose:N-acetylglucosaminyl alpha(1,3) fucosyltransferase (alpha(1,3)-Fuc-T) activity was recently discovered in H. pylori extracts. We used part of a human alpha(1, 3)-Fuc-T amino acid sequence to search an H. pylori genomic data base for related sequences. Using a probe based upon weakly matching data base sequences, we retrieved clones from a plasmid library of H. pylori DNA. DNA sequence analysis of the library clones revealed a gene which we have named fucT, encoding a protein with localized homology to the human alpha(1,3)-Fuc-Ts. We have demonstrated that fucT encodes an active Fuc-T enzyme by expressing the gene in Escherichia coli. The recombinant enzyme shows a strong preference for type 2 (e.g. LacNAc) over type 1 (e.g. lacto-N-biose) acceptors in vitro. Certain residues in a short segment of the H. pylori protein are completely conserved throughout the alpha(1,3)-Fuc-T family, defining an alpha(1,3)-Fuc-T motif which may be of use in identifying new fucosyltransferase genes.

Chemical modification of an alpha 3-fucosyltransferase; definition of amino acid residues essential for enzyme activity.

The biosynthesis of the carbohydrate antigen sialyl Lewis X (sLe(x)) is dependent on the activity of an alpha 3-fucosyltransferase (EC 2.4.1.152, GDP-fucose:Gal beta (1-4)GlcNAc-R alpha (1-3)fucosyltransferase). This enzyme catalyses the transfer of fucose from GDP-beta-fucose to the 3-OH of N-acetylglucosamine present in lactosamine acceptors. In this report, we have investigated the amino acids essential for the activity of a recombinant alpha 3-fucosyltransferase (FucT-VI) through chemical modification of the enzyme with group-selective reagents. FucT-VI activity was found to be particularly sensitive to the histidine-selective reagent diethylpyrocarbonate and the cysteine reagent N-ethylmaleimide, with IC50 values of less than 200 microM. Reagents selective for arginine and lysine had no effect on enzyme activity. The inclusion of GDP-beta-fucose during preincubation with NEM reduces the rate of inactivation whereas inclusion of an acceptor saccharide for the enzyme, Gal beta (1-4)GlcNAc, had no effect. No protective effect with either GDP-beta-fucose or Gal beta (1-4)GlcNAc was observed on treatment of the enzyme with diethylpyrocarbonate. These data suggest that in addition to an NEM-reactive cysteine in, or adjacent to, the substrate-binding site of the enzyme, FucT-VI possesses histidine residue(s) that are essential for enzyme activity.