Merging ancient and modern DNA: extinct seabird taxon rediscovered in the North Tasman Sea

Ancient DNA has revolutionized the way in which evolutionary biologists research both extinct and extant taxa, from the inference of evolutionary history to the resolution of taxonomy. Here, we present, to our knowledge, the first study to report the rediscovery of an ‘extinct’ avian taxon, the Tasman booby (Sula tasmani), using classical palaeontological data combined with ancient and modern DNA data. Contrary to earlier work, we show an overlap in size between fossil and modern birds in the North Tasman Sea (classified currently as S. tasmani and Sula dactylatra fullagari, respectively). In addition, we show that Holocene fossil birds have mitochondrial control region sequences that are identical to those found in modern birds. These results indicate that the Tasman booby is not an extinct taxon: S. dactylatra fullagari O''Brien & Davies, 1990 is therefore a junior synonym of Sula tasmani van Tets, Meredith, Fullagar & Davidson, 1988 and all North Tasman Sea boobies should be known as S. d. tasmani. In addition to reporting the rediscovery of an extinct avian taxon, our study highlights the need for researchers to be cognizant of multidisciplinary approaches to understanding taxonomy and past biodiversity.     

Extracellular calcium does not contribute to cryopreservation-induced cytotoxicity

Summary The possible role of extracellular calcium ([Ca⁺²]e) in cryopreservation-induced cytotoxicity was tested using Madin-Darby canine kidney (MDCK) cells and a fluorescent multiple endpoint assay. MDCK cells maintained in 2 mM [Ca⁺²]e and treated with the calcium ionophore, ionomycin, increased their intracellular calcium ([Ca⁺²]i) as revealed by the calcium indicator dye, Fluo3 and the bottom-reading spectrofluorometer, CytoFluor 2300. The addition of 10 mM [ethylene bis (oxyethylenenitrilo)]-tetraacetic acid (EGTA) to the extracellular medium before treatment with ionomycin blocked this ionomycin-dependent increase in [Ca⁺²]i. A number of site and activity-specific fluorescent probes were surveyed to determine which indicator dye might best reveal the ionomycin-induced cytotoxic events during this increase in [Ca⁺²]i. Although most dyes changed their emission profiles in response to calcium, neutral red was found to best reflect the loss of [Ca⁺²]i homeostasis. The NR50 for a 15-min exposure to ionomycin in the presence of 2 mM [Ca⁺²]e was approximately 2μM ionomycin, but ionomycin had little apparent effect on neutral red retention when 10 mM EGTA was added to the extracellular medium. Thus it was clear that an increase in [Ca⁺²]i could be cytotoxic to MDCK cells and that neutral red could monitor this cytotoxic episode. To test if [Ca⁺²]e was similarly cytotoxic during cryopreservation, MDCK cells were subjected to cryopreservation in the presence of dimethylsulfoxide (DMSO). In contrast to previous studies, plasma membrane integrity, not lysosomal function, seemed to best correlate with cell survival subsequent to cryopreservation. In addition, decreasing [Ca⁺²]e had no discernable effect on the retention of plasma membrane indicator dyes, neutral red, or cell survival. It is concluded that a) plasma membrane indicator dyes, not neutral red, might be better indicators of cytotoxicity occurring during cryopreservation; b) DMSO might be toxic to lysosomes during cryopreservation of cultured cells; and c) although [Ca⁺²]e can contribute to cytotoxicity, the presence of [Ca⁺²]e might not influence cryopreservation-induced cytotoxicity.     

Development in Salmo trutta at different temperatures, with a quantitative scoring method for intraspecific comparisons

The period from fertilization to the end of yolk resorption in the brown trout Salmo trutta was described as a series of 40 successive steps, each describing the maturation of multiple characters during an interval of development. An associated quantitative scoring system was devised, whereby individual embryos were assigned points ranging from 1 to 1000, on the basis of the assessment of a wide range of developmental features. Developmental score increased linearly with embryonic age at all temperatures studied (2, 6 and 10° C), indicating that the relative durations of successive intervals of development were maintained. At lower temperatures, the beginning of heartbeat, trunk muscle contractions, bile production, pectoral fin movements and hatching were all delayed relative to the rest of development. Therefore these features have been excluded from the scoring system. Maternal migratory type did not affect early developmental patterns.     

Structural characterisation of the exopolysaccharide produced by Streptococcus thermophilus EU20

Streptococcus thermophilus EU20 when grown on skimmed milk secretes a high-molecular-weight exopolysaccharide that is composed of glucose, galactose and rhamnose in a molar ratio of 2:3:2. Using chemical techniques and 1D and 2D-NMR spectroscopy (1H and 13C) the polysaccharide has been shown to possess a heptasaccharide repeating unit having the following structure: [chemical structure: see text]. Treatment of the polysaccharide with mild acid (0.5 M TFA, 100 degrees C for 1 h) liberates two oligosaccharides; the components correspond to the repeating unit and a hexasaccharide equivalent to the repeating unit minus the terminal alpha-L-Rhap.     

Genome‐guided investigation of plant natural product biosynthesis

The medicinal plant Madagascar periwinkle, Catharanthus roseus (L.) G. Don, produces hundreds of biologically active monoterpene‐derived indole alkaloid (MIA) metabolites and is the sole source of the potent, expensive anti‐cancer compounds vinblastine and vincristine. Access to a genome sequence would enable insights into the biochemistry, control, and evolution of genes responsible for MIA biosynthesis. However, generation of a near‐complete, scaffolded genome is prohibitive to small research communities due to the expense, time, and expertise required. In this study, we generated a genome assembly for C. roseus that provides a near‐comprehensive representation of the genic space that revealed the genomic context of key points within the MIA biosynthetic pathway including physically clustered genes, tandem gene duplication, expression sub‐functionalization, and putative neo‐functionalization. The genome sequence also facilitated high resolution co‐expression analyses that revealed three distinct clusters of co‐expression within the components of the MIA pathway. Coordinated biosynthesis of precursors and intermediates throughout the pathway appear to be a feature of vinblastine/vincristine biosynthesis. The C. roseus genome also revealed localization of enzyme‐rich genic regions and transporters near known biosynthetic enzymes, highlighting how even a draft genome sequence can empower the study of high‐value specialized metabolites.     

Pattern of integration of conflicting stimuli by the crab Heterozius rotundifrons

When an animal receives several stimuli that could evoke conflicting behaviours they can either show a response to just one stimulus or show a gradation of responses to the multiple stimuli. It has been proposed [Hazlett BA. 2000. Responses to single and multiple sources of chemical cues by New Zealand crustaceans. Mar Freshw Behav Physiol. 34:1–20] that a graded response will be seen if the animals have an alternative response to one behavioural category of stimuli (the alternative-behaviour-gradual-change rule). The crab Heterozius rotundifrons has two alternative patterns of predation avoidance, either assumption of a rigid limb-extended posture for several minutes or a short period of limb extension and then rapid locomotion. We presented crabs with full strength input in one category (food stimulus) and one of seven intensities of input in a conflicting category (either chemical or visual cues associated with increased predation risk). In both cases, as the intensity of the danger signal gradually increased, there was a gradual increase in the duration of the limb-extended posture.     

The discovery of potent and long-acting oral factor Xa inhibitors with tetrahydroisoquinoline and benzazepine P4 motifs

The discovery and evaluation of potent and long-acting oral sulfonamidopyrrolidin-2-one factor Xa inhibitors with tetrahydroisoquinoline and benzazepine P4 motifs are described. Unexpected selectivity issues versus tissue plasminogen activator in the former series were addressed in the later, delivering a robust candidate for progression towards clinical studies.     

The effect of a single application of cow urine on annual N2 fixation under varying simulated grazing intensity, as measured by four 15N isotope techniques

The effects of dairy cow urine and defoliation severity on biological nitrogen fixation and pasture production of a mixed ryegrass-white clover sward were investigated over 12 months using mowing for defoliation. A single application of urine (equivalent to 746 kg N ha^–1), was applied in late spring to plots immediately after light and moderately-severe defoliation (35 mm and 85 mm cutting heights, respectively) treatments were imposed. Estimates of percentage clover N derived from N₂ fixation (%Ndfa) were compared by labelling the soil with ¹⁵N either by applying a low rate of ¹⁵N-labelled ammonium sulphate, immobilising ¹⁵N in soil organic matter, adding ¹⁵N to applied urine, or by utilising the small differences in natural abundance of ¹⁵N in soil. Urine application increased annual grass production by 85%, but had little effect on annual clover production. However, urine caused a marked decline in %Ndfa (using an average of all ¹⁵N methods) from 84% to a low of 22% by 108 days, with recovery to control levels taking almost a year. As a result, total N fixed (in above ground clover herbage) was reduced from 232 to 145 kg N ha^–1 yr^–1. Moderately–severe defoliation had no immediate effect on N₂ fixation, but after 108 days the %Ndfa was consistently higher than light defoliation during summer and autumn, and increased by up to 18%, coinciding with an increase in growth of weeds and summer-grass species. Annual N₂ fixation was 218 kg N ha^–1 yr^–1 under moderately-severe defoliation compared to 160 kg N ha^–1 yr^–1 under light defoliation. Estimates of %Ndfa were generally similar when ¹⁵N-labelled or immobilised ¹⁵N were used to label soil regardless of urine and defoliation severity. The natural abundance technique gave highly variable estimates of %Ndfa (–56 to 24%) during the first 23 days after urine application but, thereafter, estimates of %Ndfa were similar to those using ¹⁵N-labelling methods. In contrast, in urine treated plots the use of ¹⁵N-labelled urine gave estimates of %Ndfa that were 20–30% below values calculated using conventional ¹⁵N-labelling during the first 161 days. These differences were probably due to differences in the rooting depth between ryegrass and white clover in conjunction with treatment differences in ¹⁵N distribution with depth. This study shows that urine has a prolonged effect on reducing N₂ fixation in pasture. In addition, defoliation severity is a potential pasture management tool for strategically enhancing N₂ fixation.     

Bovine mammary gland UDP-GalNAc:GlcNAcbeta-R beta1-->4-N- acetylgalactosaminyltransferase is glycoprotein hormone nonspecific and shows interaction with alpha-lactalbumin

We have identified a novel N -acetylgalactosaminyltransferase activity in lactating bovine mammary gland membranes. Acceptor specificity studies and analysis of products obtained in vitro by 400 MHz1H-NMR spectroscopy revealed that the enzyme catalyses the transfer of N - acetylgalactosamine (GalNAc) from UDP-GalNAc to acceptor substrates carrying a terminal, beta-linked N -acetylglucosamine (GlcNAc) residue and establishes a beta1-->4-linkage forming a GalNAcbeta1-->4GlcNAc ( N, N '-diacetyllactosediamine, lacdiNAc) unit. Therefore, the enzyme can be identified as a UDP-GalNAc:GlcNAcbeta-R beta1-->4-N- acetylgalactosaminyltransferase (beta4-GalNAcT). This enzyme resembles invertebrate beta4-GalNAcT as well as mammalian beta4- galactosyltransferase (beta4-GalT) in acceptor specificity. It can, however, be clearly distinguished from the pituitary hormone-specific beta4-GalNAcT by its incapability of acting with an elevated activity on a glycoprotein substrate carrying a hormone-specific peptide motif. Furthermore, the GalNAcT activity appeared not to be due to a promiscuous action of a beta4-GalT as could be demonstrated by comparing the beta4-GalNAcT and beta4-GalT activities of the mammary gland, bovine colostrum, and purified beta4-GalT, by competition studies with UDP-GalNAc and UDP-Gal, and by use of an anti-beta4-GalT polyclonal inhibiting antibody. Interestingly, under conditions where mammalian beta4-GalT forms with alpha-lactalbumin (alpha-LA) the lactose synthase complex, the mammary gland beta4-GalNAcT was similarly induced by alpha-LA to act on Glc with an increased efficiency yielding the lactose analog GalNAcbeta1-->4Glc. This enzyme thus forms the second example of a mammalian glycosyltransferase the specificity of which can be modified by this milk protein. It is proposed that the mammary gland beta4-GalNAcT functions in the synthesis of lacdiNAc- based, complex-type glycans frequently occurring on bovine milk glycoproteins. The action of this enzyme is to be considered when aiming at the production of properly glycosylated protein biopharmaceuticals in the milk of transgenic dairy animals.      

Unusual filamentous virus-like particles in symptomless blackberry associated with severe leaf curling in the virus indicator Rubus macraei, and anomalous data using a degenerate badnavirus primer in PCR of Rubus species

Electron microscopy of ultrathin sections of leaves of symptomless Himalaya Giant blackberry and of the virus indicator species, Rubus macraei, showing severe leaf curl symptoms following graft inoculation with scions from this blackberry, detected highly flexuous virus‐like particles with an unusual ‘beaded’ structure. Such particles were restricted to a few vascular cells and were distinct from P‐protein common in some such cells. This virus, provisionally named Hawaiian rubus leaf curl virus (HRLCV), symptomlessly infected a wide range of Rubus species and cultivars. Badnavirus‐like bacilliform particles were observed in some cells of a single R. macraei plant showing leaf curl symptoms following graft inoculation with the causal agent of this disease symptom from Himalaya Giant blackberry after passage through red raspberry, but not in any other material. PCR with primer sets for the badnaviruses Rubus yellow net virus and Gooseberry veinbanding associated virus, showed that no Rubus sources studied contained these viruses. However, using a sequence‐specific primer set designed from the sequence of the product generated with a badnavirus degenerate primer set, a specific product was amplified from healthy plants of all of 16 raspberry cultivars and two Rubus species, but not from 16 blackberry cultivars (including cv. Himalaya Giant). All of these sources were free from viruses known to occur in Rubus. Sequence analysis of this product showed no homology with any known badnavirus, or with any other published sequences. It seems most likely therefore that a region of the raspberry genome has been amplified using the degenerate badnavirus primer set and that it is absent from the blackberry genome.