Substrate specificity of cerebral GDP-fucose: glycoprotein fucosyltransferase

Solubilized sheep brain fucosyltransferase was shown to transfer fucose from GDP-fucose onto glycoprotein and glycopeptide acceptors, such as asialofetuin, asialotransferrin, their glycopeptides and glycopeptides from ovalbumin, but not on to monosaccharides and disaccharides such as galactose, N-acetylglucosamine and lactose. Competition studies between asialofetuin and glycopeptide V from ovalbumin provided evidence that both substrates compete for a common enzyme active site. The position of the fucosyl linkage was then investigated. Endo-beta-N-glucosaminidase D digestion of fucosylated and acetylated glycopeptide V showed that fucose is not linked to asparagine-linked N-acetylglucosamine. Hydrazinolysis and nitrous acid deamination performed on asialofetuin and glycopeptide V proved that fucose is not linked to external galactose or N-acetylglucosamine either. Thus we assume that fucose is linked to the oligomannochitobiosyl core of the glycan, and probably to the second N-acetylglucosamine.     

Quantifying genotyping errors in noninvasive population genetics

The use of noninvasively collected samples greatly expands the range of ecological issues that may be investigated through population genetics. Furthermore, the difficulty of obtaining reliable genotypes with samples containing low quantities of amplifiable DNA may be overcome by designing optimal genotyping schemes. Such protocols are mainly determined by the rates of genotyping errors caused by false alleles and allelic dropouts. These errors may not be avoided through laboratory procedure and hence must be quantified. However, the definition of genotyping error rates remains elusive and various estimation methods have been reported in the literature. In this paper we proposed accurate codification for the frequencies of false alleles and allelic dropouts. We then reviewed other estimation methods employed in hair- or faeces-based population genetics studies and modelled the bias associated with erroneous methods. It is emphasized that error rates may be substantially underestimated when using an erroneous approach. Genotyping error rates may be important determinants of the outcome of noninvasive studies and hence should be carefully computed and reported.     

Sexual isolation with and without ecological isolation in marine isopods Jaera albifrons and J. praehirsuta

Sexual barriers associated with mate choice are often found to be associated with some level of ecological isolation between species. The independence and relative strength of sexual isolation are thus difficult to assess. Here, we take advantage of a pair of marine isopod species (Jaera albifrons and J. praehirsuta) that show sexual isolation and coexist in populations where they share the same microhabitat or not (i.e. without or with ecological isolation). We estimated the strength of sexual isolation between J. albifrons and J. praehirsuta using no‐choice trials and a multiple‐choice experimental population. We found that sexual isolation is strong in both the presence and the absence of ecological isolation, but that it is asymmetric and fails to prevent interspecific gene flow entirely. First‐generation intrinsic post‐zygotic barriers were low, and there was no sexual isolation within J. praehirsuta across habitats. The J. albifrons/J. praehirsuta species pair thus provides an example where the role of sexual isolation as a barrier to gene flow (a) does not depend upon current ecological isolation, (b) seems to have evolved independently of local ecological conditions, but (c) is insufficient to complete speciation entirely on its own.     

Description of microsatellite markers and genotyping performances using feathers and buccal swabs for the Ivory gull (Pagophila eburnea)

We report 22 new polymorphic microsatellites for the Ivory gull (Pagophila eburnea), and we describe how they can be efficiently co-amplified using multiplexed polymerase chain reactions. In addition, we report DNA concentration, amplification success, rates of genotyping errors and the number of genotyping repetitions required to obtain reliable data with three types of noninvasive or nondestructive samples: shed feathers collected in colonies, feathers plucked from living individuals and buccal swabs. In two populations from Greenland (n=21) and Russia (Severnaya Zemlya Archipelago, n=21), the number of alleles per locus varied between 2 and 17, and expected heterozygosity per population ranged from 0.18 to 0.92. Twenty of the markers conformed to Hardy-Weinberg and linkage equilibrium expectations. Most markers were easily amplified and highly reliable when analysed from buccal swabs and plucked feathers, showing that buccal swabbing is a very efficient approach allowing good quality DNA retrieval. Although DNA amplification success using single shed feathers was generally high, the genotypes obtained from this type of samples were prone to error and thus need to be amplified several times. The set of microsatellite markers described here together with multiplex amplification conditions and genotyping error rates will be useful for population genetic studies of the Ivory gull.     

MicroRNA expression profiling in neurogenesis of adipose tissue-derived stem cells

Adipose tissue-derived stem cells (ADSCs) are one population of adult stem cells that can self renew and differentiate into multiple lineages. Because of advantages in method and quantity of acquisition, ADSCs are gaining attention as an alternative source of bone marrow mesenchymal stem cells. In this study, we performed microRNA profiling of undifferentiated and of neurally-differentiated ADSCs to identify the responsible microRNAs in neurogenesis using this type of stem cell. MicroRNAs from four different donors were analysed by microarray. Compared to the undifferentiation control, we identified 39–101 microRNAs with more than two-fold higher expression and 3–9 microRNAs with two-fold lower expression. The identified microRNAs were further analysed in terms of gene ontology (GO) in relation with neurogenesis, based on their target mRNAs predicted by computational analysis. This study revealed the specific microRNAs involved in neurogenesis via microRNA microarray, and may provide the basic information for genetic induction of adult stem cell differentiation using microRNAs.     

RIG-I/MDA5/MAVS are required to signal a protective IFN response in rotavirus-infected intestinal epithelium

Rotavirus is a dsRNA virus that infects epithelial cells that line the surface of the small intestine. It causes severe diarrheal illness in children and ～500,000 deaths per year worldwide. We studied the mechanisms by which intestinal epithelial cells (IECs) sense rotavirus infection and signal IFN-β production, and investigated the importance of IFN-β production by IECs for controlling rotavirus production by intestinal epithelium and virus excretion in the feces. In contrast with most RNA viruses, which interact with either retinoic acid-inducible gene I (RIG-I) or melanoma differentiation-associated gene 5 (MDA5) inside cells, rotavirus was sensed by both RIG-I and MDA5, alone and in combination. Rotavirus did not signal IFN-β through either of the dsRNA sensors TLR3 or dsRNA-activated protein kinase (PKR). Silencing RIG-I or MDA5, or their common adaptor protein mitochondrial antiviral signaling protein (MAVS), significantly decreased IFN-β production and increased rotavirus titers in infected IECs. Overexpression of laboratory of genetics and physiology 2, a RIG-I-like receptor that interacts with viral RNA but lacks the caspase activation and recruitment domains required for signaling through MAVS, significantly decreased IFN-β production and increased rotavirus titers in infected IECs. Rotavirus-infected mice lacking MAVS, but not those lacking TLR3, TRIF, or PKR, produced significantly less IFN-β and increased amounts of virus in the intestinal epithelium, and shed increased quantities of virus in the feces. We conclude that RIG-I or MDA5 signaling through MAVS is required for the activation of IFN-β production by rotavirus-infected IECs and has a functionally important role in determining the magnitude of rotavirus replication in the intestinal epithelium.