Microsatellite inheritance and multiple paternity in the deep-sea octopus Graneledone boreopacifica (Mollusca: Cephalopoda)

Abstract. Octopodids are a globally distributed group of marine molluscs. Despite this, our knowledge of their reproductive biology rests heavily on inference, as all phases of copulation, beginning with sperm transfer, occur within the mantle cavity. Male octopuses insert a spermatophore into the female's oviduct, which is predicted to release a sperm-filled sac that either bursts to release sperm for storage or to itself be stored in a gland in the middle of the oviduct. To test whether female octopuses use sperm from multiple males to fertilize their eggs, as may be predicted from anatomy and anecdotal accounts, we apply microsatellite analysis to a partial clutch of Graneledone boreopacifica collected at 1600-m depth to test for multiple paternity. At least two genetically distinct sires contributed sperm to the hatchlings analyzed, demonstrating for the first time multiple paternity in octopodids.     

Nested species subsets of amphibians and reptiles on Neotropical forest islands

Nested species subsets are a common pattern of community assembly characteristic of many types of fragmented landscapes and insular systems. Here we describe nested subset patterns of amphibian and reptile occupancy on 23 forest islands in north-eastern Bolivia. We used observed occupancy patterns to differentiate five distributional guilds: widespread species, rare species, poor colonizers, area-sensitive species and supertramps. Amphibian occurrences were nested along a forest island isolation gradient, and when species from each of the distribution classes were removed from subsequent analyses of nestedness, we found that dispersal-limited poor colonizers were responsible for the association between nestedness and isolation. Amphibians associated with the grassland matrix at the study site showed a nested pattern linked with area, although this pattern did not scale up to all amphibians and could not be unequivocally attributed to any of the distributional guilds we recognized. There were no strong associations between two biological characteristics, body size and relative abundance in the matrix, and the likelihood of occupancy along either forest island area or isolation gradients. The relative importance of isolation in shaping nested patterns of amphibians on these forest islands may be a result of either (1) the greater range in isolation values included in this study compared with many others; (2) the long time since isolation in this landscape, manifesting a footprint of isolation not apparent in more recently fragmented patches; (3) the relatively homogeneous grassland matrix surrounding forest islands that likely provides little refuge for animals moving among forest islands.     

Insights into tRNA-Dependent Amidotransferase Evolution and Catalysis from the Structure of the Aquifex aeolicus Enzyme

Many bacteria form Gln-tRNA^Gln and Asn-tRNA^Asn by conversion of the misacylated Glu-tRNA^Gln and Asp-tRNA^Asn species catalyzed by the GatCAB amidotransferase in the presence of ATP and an amide donor (glutamine or asparagine). Here, we report the crystal structures of GatCAB from the hyperthermophilic bacterium Aquifex aeolicus, complexed with glutamine, asparagine, aspartate, ADP, or ATP. In contrast to the Staphylococcus aureus GatCAB, the A. aeolicus enzyme formed acyl-enzyme intermediates with either glutamine or asparagine, in line with the equally facile use by the amidotransferase of these amino acids as amide donors in the transamidation reaction.A water-filled ammonia channel is open throughout the length of the A. aeolicus GatCAB from the GatA active site to the synthetase catalytic pocket in the B-subunit. A non-catalytic Zn²⁺ site in the A. aeolicus GatB stabilizes subunit contacts and the ammonia channel. Judged from sequence conservation in the known GatCAB sequences, the Zn²⁺ binding motif was likely present in the primordial GatB/E, but became lost in certain lineages (e.g., S. aureus GatB). Two divalent metal binding sites, one permanent and the other transient, are present in the catalytic pocket of the A. aeolicus GatB. The two sites enable GatCAB to first phosphorylate the misacylated tRNA substrate and then amidate the activated intermediate to form the cognate products, Gln-tRNA^Gln or Asn-tRNA^Asn.     

Characterization of the Lipid Linkage Region and Chain Length of the Cellubiuronic Acid Capsule of Streptococcus pneumoniae

The processive reaction mechanisms of β-glycosyl-polymerases are poorly understood. The cellubiuronan synthase of Streptococcus pneumoniae catalyzes the synthesis of the type 3 capsular polysaccharide through the alternate additions of β-1,3-Glc and β-1,4-GlcUA. The processive multistep reaction involves the sequential binding of two nucleotide sugar donors in coordination with the extension of a polysaccharide chain associated with the carbohydrate acceptor recognition site. Degradation analysis using cellubiuronan-specific depolymerase demonstrated that the oligosaccharide-lipid and polysaccharide-lipid products synthesized in vitro with recombinant cellubiuronan synthase had a similar oligosaccharyl-lipid at their reducing termini, providing definitive evidence for a precursor-product relationship and also confirming that growth occurred at the nonreducing end following initiation on phosphatidylglycerol. The presence of a lipid marker at the reducing end allowed the quantitative determination of cellubiuronic acid polysaccharide chain lengths. As the UDP-GlcUA concentration was increased from 1 to 11.5 μm, the level of synthase in the transitory processive state decreased, with the predominant oligosaccharide-lipid product containing 3 uronic acid residues, whereas the proportion of synthase in the fully processive state increased and the polysaccharide chain length increased from 320 to 6700 monosaccharide units. In conjunction with other kinetic data, these results suggest that the formation of a complex between a tetrauronosyl oligomer and the carbohydrate acceptor recognition site plays a central role in coordinating the repetitive interaction of the synthase with the nucleotide sugar donors and modulating the chain length of cellubiuronan polysaccharide.Cellubiuronic acid, the capsular polysaccharide of type 3 Streptococcus pneumoniae, is composed of the repeating disaccharide cellobiuronic acid (-3)-β-d-GlcUA²-(1,4)-β-d-Glc-(1-) (1) and is synthesized by a processive mechanism similar to that for cellulose, chitin, hyaluronic acid, and other related β-glycans (2). This group of polysaccharides is synthesized by inverting GT-2A polymerases that are located in the plasma membrane with their active sites on the cytoplasmic face, and following chain initiation, the synthases are thought to be involved in the extrusion of the nascent chains to the external membrane face (3–8). The overall processive elaboration of these polysaccharides remains poorly understood at the molecular level. In particular, there is relatively little information concerning the initiation process, the facilitation of chain extrusion, the mechanism of translocation, and the regulation of the final chain length during the assembly of these polymers. Recent investigations in this laboratory have begun to unravel some of the details of both the early and later stages of the biosynthesis of cellubiuronan.Unlike most S. pneumoniae capsules, whose elaboration requires multiple glycosyltransferases, a polymerase, and an additional transport system (9), the assembly and transport of cellubiuronic acid in type 3 strains is carried out by the single enzyme cellubiuronan synthase (Cps3S) (3, 10, 11). Studies of the synthase in S. pneumoniae and recombinant Escherichia coli membranes have shown that assembly of the polysaccharide involves two distinct kinetic phases: 1) a transitory processive state wherein the chain is thought to be initiated by the formation of an oligosaccharide-lipid that is loosely associated with the synthase, and 2) a fully processive state in which the polysaccharide is tightly bound to the carbohydrate substrate recognition site, except for a brief period during the translocation stage of each catalytic cycle (5, 12). Each catalytic cycle in the extrusion mode provides for chain extension by the addition of a repeating disaccharide and requires the alternate association of the synthase with UDP-Glc and UDP-GlcUA, the formation of the glycosidic linkages of the respective sugars, and the release, translocation, and reattachment of the elongating chain at the synthase carbohydrate recognition site. Transition from the transitory mode to the fully processive extrusion mode correlates with the attainment of a threshold-length oligosaccharide of ∼8 sugars (12). Nod factor chito-oligosaccharides from rhizobia are synthesized by a related group of synthases that apparently are not capable of organizing into an extrusion mode (13). Significantly, the maximum length of any reported Nod-factor oligosaccharide is 6 sugars (14).Based on β-glucosidase sensitivity of singly added [¹⁴C]Glc to the terminal end of high molecular weight cellubiuronan, it was deduced that the polysaccharide grows by repetitive β-1,3-Glc and β-1,4-GlcUA additions to the nonreducing terminus (2). Oligosaccharide-lipid assembly is thought to initiate on phosphatidylglycerol (15). To date, however, there has been no quantitative demonstration of polysaccharide-lipid conjugate, and the similarity of the distal sugar-lipid linkages in the polysaccharide- and oligosaccharide-lipid products has not been verified.Cellubiuronan depolymerase is a Bacillus circulans β-endoglucuronidase that specifically cleaves cellubiuronic acid chains at GlcUA-β1,4-Glc linkages (16, 17). The polysaccharide is successively cleaved at random internal linkages, which upon completion of hydrolysis yields a series of oligosaccharide end products containing 1–4 Glc-β1,3-GlcUA disaccharide units, with the most abundant oligomer being a tetrasaccharide. The high degree of specificity of this depolymerase has provided a sensitive analytical tool to further characterize cellubiuronan oligosaccharide- and polysaccharide-lipids, and even more importantly, it has provided a means for determining the chain length of these polysaccharides. Both in vivo (18) and in vitro studies (12) indicate that the processivity of cellubiuronan synthase is modulated by the concentration of UDP-GlcUA. However, lacking well defined cellubiuronan polysaccharide size standards, there has been no way to clearly establish the actual length of the polymer synthesized under different reaction conditions. The methodology described in this article has allowed a clear demonstration of the relationship between the polysaccharide size and the UDP-GlcUA substrate concentration and in turn has led to the development of a kinetic model (38), which provides both for UDP-sugar modulation of polysaccharide chain length and for the assembly by a single-site synthase of a polysaccharide composed of a repeating heterodisaccharide.     

Membrane-targeted strategies for modulating APP and Aβ-mediated toxicity

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by numerous pathological features including the accumulation of neurotoxic amyloid-β (Aβ) peptide. There is currently no effective therapy for AD, but the development of therapeutic strategies that target the cell membrane is gaining increased interest. The amyloid precursor protein (APP) from which Aβ is formed is a membrane-bound protein, and Aβ production and toxicity are both membrane mediated events. This review describes the critical role of cell membranes in AD with particular emphasis on how the composition and structure of the membrane and its specialized regions may influence toxic or benign Aβ/APP pathways in AD. The putative role of copper (Cu) in AD is also discussed, and we highlight how targeting the cell membrane with Cu complexes has therapeutic potential in AD.     

Protein function annotation by homology-based inference

With many genomes now sequenced, computational annotation methods to characterize genes and proteins from their sequence are increasingly important. The BioSapiens Network has developed tools to address all stages of this process, and here we review progress in the automated prediction of protein function based on protein sequence and structure.