A detailed study of the periodate oxidation of sialic acids in glycoproteins

Periodate oxidation of terminalN-acetyl- andN-glycoloylneuraminic acid residues in the mucins from edible bird nest substance and pig submandibular gland, respectively, can be carried out under conditions which exclusively give rise to the formation of the C-7 analogues of these sialic acids. In contrast, the C-8 compounds can be obtained in a maximum yield of about 40%. Under identical conditions,N-glycoloylneuraminic acid is oxidized about 1.5 times faster than theN-acetylated derivative. After release of the sialic acids by acid hydrolysis, the characterization of the oxidation products was carried out by TLC, by GLC and GLC-MS of the corresponding pertrimethylsilyl derivatives, and by 500-MHz¹H-NMR spectroscopy. In addition, molar response factors for GLC analysis and extinction coefficients in the orcinol/Fe³⁺/HCl assay were determined.     

Patterns of divergence during evolution of alpha 1-proteinase inhibitors in mammals

alpha 1-Proteinase inhibitor (alpha 1-PI), a member of the serine proteinase inhibitor superfamily, has a primary role in controlling neutrophil elastase activity within the mammalian circulation. Several studies have indicated that the reactive center region of alpha 1-PI, the amino acid sequence of which is critical to recognition of and binding to target proteinases, is highly divergent within and among species. This appears to be a consequence of accelerated rates of evolution that may have been driven by positive Darwinian selection. In order to examine this and other features of alpha 1-PI evolution in more detail, we have isolated and sequenced cDNAs representing alpha 1- PI mRNAs of the mouse species Mus saxicola and Mus minutoides and have compared these with a number of other mammalian alpha 1-PI mRNAs. Relative to other mammalian mRNAs, the extent of nonsynonymous substitution is generally high throughout the alpha 1-PI mRNA molecule, indicating greater overall rates of amino acid substitution. Within and among mouse species, the 5'-half of the mRNA, but not the 3'-half, has been homogenized by concerted evolution. Finally, the reactive center is under diversifying or positive Darwinian selection in murid rodents (rats, mice) and guinea pigs yet is under purifying selection in primates and artiodactyls. The significance of these findings to alpha 1-PI function and the possible selective forces driving evolution of serpins in general are discussed.      

Seasonal Sexual Segregation by Monomorphic Sooty Shearwaters Puffinus griseus Reflects Different Reproductive Roles during the Pre-Laying Period

Tracking technology has revolutionized knowledge of seabird movements; yet, few studies have examined sex differences in distribution and behavior of small to medium-sized, sexually-monomorphic seabirds. Application of bird-borne geolocation-immersion loggers revealed seasonal segregation in the sexually-monomorphic Sooty Shearwater Puffinus griseus, mainly in the pre-laying period, when there were clear differences in reproductive roles. Shearwaters first returned to the Falkland Islands on 27 Sept±8 d; males, on average, 8 d earlier than females. Prior to egg-laying, distribution at sea, colony attendance and behaviour depended on sex. Males foraged locally over the southern Patagonian Shelf and Burdwood Bank, spending mainly single days at sea and intervening nights in the burrow. Females, who flew for more of the day during this time, foraged in more distant areas of the northern Patagonian Shelf and Argentine Basin that were deeper, warmer and relatively more productive. Attendance of females at the colony was also more variable than that of males and, overall, males were present for significantly more of the pre-laying period (38 vs. 19% of time). Sex differences were reduced following egg-laying, with males and females using similar foraging areas and making trips of similar mean duration in incubation (7.6±2.7 d) and chick-rearing (1.4±1.3 d). Congruence continued into the non-breeding period, with both sexes showing similar patterns of activity and areas of occupancy in the NW Atlantic. Thus, seasonal changes in reproductive roles influenced patterns of sexual segregation; this occurred only early in the season, when male Sooty Shearwaters foraged locally, returning regularly to the colony to defend (or maintain) the burrow or the mate, while females concentrated on building resources for egg development in distant and relatively more productive waters.     

Zebrafish AID is capable of deaminating methylated deoxycytidines

Activation-induced cytidine deaminase (AID) deaminates deoxycytidine (dC) to deoxyuracil (dU) at immunoglobulin loci in B lymphocytes to mediate secondary antibody diversification. Recently, AID has been proposed to also mediate epigenetic reprogramming by demethylating methylated cytidines (mC) possibly through deamination. AID overexpression in zebrafish embryos was shown to promote genome demethylation through G:T lesions, implicating a deamination-dependent mechanism. We and others have previously shown that mC is a poor substrate for human AID. Here, we examined the ability of bony fish AID to deaminate mC. We report that zebrafish AID was unique among all orthologs in that it efficiently deaminates mC. Analysis of domain-swapped and mutant AID revealed that mC specificity is independent of the overall high-catalytic efficiency of zebrafish AID. Structural modeling with or without bound DNA suggests that efficient deamination of mC by zebrafish AID is likely not due to a larger catalytic pocket allowing for better fit of mC, but rather because of subtle differences in the flexibility of its structure.     

Grenadier abundance examined at varying spatial scales in deep waters off Newfoundland, Canada, with special focus on the influence of corals

There is a growing body of research examining the effects of corals on fish communities, species abundances, and biodiversity. Yet, few studies have quantitatively examined what factors are influencing the distribution of individual fish species. In general, many researchers believe they know what influences the distribution of grenadiers on large spatial scales, but numerous studies have shown the distributions of organisms are often determined by various factors that change in relative importance when viewed at differing scales. Our study used video collected from three deep canyons off Newfoundland, Canada (North west Atlantic) to examine how the factors apparently influencing the distribution of four grenadiers (Macrouridae: Coryphaenoides rupestris, Coryphaenoides carapinus, Nezumia bairdii, and Macrourus berglax) change when assessed at varying spatial scales. We paid special attention to the influence of deep-water corals found in the study area (large gorgonians/antipatharians, small gorgonians, sea pens, soft corals, and cup corals). The factors that influenced grenadier presence and/or abundance (and the magnitude of this effect) varied as different sampling resolutions were examined. We found C. rupestris abundance was positively related to cup coral abundance in transects longer than 10 m, likely as a result of similar habitat preferences between both taxa. When significant relationships between depth and C. rupestris presence and/or abundance were found, they were always negative. Depth was a significant predictor of C. carapinus abundance in transects longer than 10 m. Very few predictors of M. berglax abundance or presence could be found. Depth and the number of small gorgonians were consistent predictors of N. bairdii abundance.     

Tyrosine phosphorylation of Munc18‐1 inhibits synaptic transmission by preventing SNARE assembly

Tyrosine kinases are important regulators of synaptic strength. Here, we describe a key component of the synaptic vesicle release machinery, Munc18‐1, as a phosphorylation target for neuronal Src family kinases (SFKs). Phosphomimetic Y473D mutation of a SFK phosphorylation site previously identified by brain phospho‐proteomics abolished the stimulatory effect of Munc18‐1 on SNARE complex formation (“SNARE‐templating”) and membrane fusion in vitro. Furthermore, priming but not docking of synaptic vesicles was disrupted in hippocampal munc18‐1‐null neurons expressing Munc18‐1_Y473D. Synaptic transmission was temporarily restored by high‐frequency stimulation, as well as by a Munc18‐1 mutation that results in helix 12 extension, a critical conformational step in vesicle priming. On the other hand, expression of non‐phosphorylatable Munc18‐1 supported normal synaptic transmission. We propose that SFK‐dependent Munc18‐1 phosphorylation may constitute a potent, previously unknown mechanism to shut down synaptic transmission, via direct occlusion of a Synaptobrevin/VAMP2 binding groove and subsequent hindrance of conformational changes in domain 3a responsible for vesicle priming. This would strongly interfere with the essential post‐docking SNARE‐templating role of Munc18‐1, resulting in a largely abolished pool of releasable synaptic vesicles.     

Softening the agricultural matrix: a novel agri‐environment scheme that balances habitat restoration and livestock grazing

The loss and degradation of woody vegetation in the agricultural matrix represents a key threat to biodiversity. Strategies for habitat restoration in these landscapes should maximize the biodiversity benefit for each dollar spent in order to achieve the greatest conservation outcomes with scarce funding. To be effective at scale, such strategies also need to account for the opportunity cost of restoration to the farmer. Here, we critique the Whole‐of‐Paddock Rehabilitation program, a novel agri‐environment scheme which seeks to provide a cost‐effective strategy for balancing habitat restoration and livestock grazing. The scheme involves the revegetation of large (minimum 10 ha) fields, designed to maximize biodiversity benefits and minimize costs while allowing for continued agricultural production. The objectives and design of the scheme are outlined, biodiversity and production benefits are discussed, and we contrast its cost‐effectiveness with alternative habitat restoration strategies. Our analysis indicates that this scheme achieves greater restoration outcomes at approximately half the cost of windbreak‐style plantings, the prevailing planting configuration in southeastern Australia, largely due to a focus on larger fields, and the avoidance of fencing costs through the use of existing farm configuration and infrastructure. This emphasis on cost‐effectiveness, the offsetting of opportunity costs through incentive payments, and the use of a planting design that seeks to maximize biodiversity benefits while achieving production benefits to the farmer, has the potential to achieve conservation in productive parts of the agricultural landscape that have traditionally been “off limits” to conservation.     

Differences in the enzymatic efficiency of human and bony fish AID are mediated by a single residue in the C terminus modulating single-stranded DNA binding

Activation-induced cytidine deaminase (AID) mediates antibody diversification by deaminating deoxycytidines to deoxyuridine within immunoglobulin genes. However, it also generates genome-wide DNA lesions, leading to transformation. Though the biochemical properties of AID have been described, its 3-dimensional structure has not been determined. Hence, to investigate the relationship between the primary structure and biochemical characteristics of AID, we compared the properties of human and bony fish AID, since these are most divergent in amino acid sequence. We show that AIDs of various species have different catalytic rates that are thermosensitive and optimal at native physiological temperatures. Zebrafish AID is severalfold more catalytically robust than human AID, while catfish AID is least active. This disparity is mediated by a single amino acid difference in the C terminus. Using functional assays supported by models of AID core and surface structure, we show that this residue modulates activity by affecting ssDNA binding. Furthermore, the cold-adapted catalytic rates of fish AID result from increased ssDNA binding affinity at lower temperatures. Our work suggests that AID may generate DNA damage with variable efficiencies in different organisms, identifies residues critical in regulating AID activity, and provides insights into the evolution of the APOBEC family of enzymes.