NMR studies of mannitol-terminating oligosaccharides derived by reductive alkaline hydrolysis from brain glycoproteins

Interest in the characterisation of O-mannosyl glycan structures has been stimulated following the identification of mannitol-terminating oligosaccharides among the chains released from mammalian proteins in nervous and muscle tissues, and by the discovery of a putative human O-mannosyl transferase. Several mass spectrometry methods have been applied to structure elucidation particularly when low amounts of oligosaccharide are available for analysis. However, when sufficient amounts are available, a combination of through-bond homo- and heteronuclear, and of through-space homonuclear NMR experiments permit the complete identification of these oligosaccharide sequences. We describe here the assignment of 1H and 13C NMR chemical shifts from such experiments for four mannitol-terminating oligosaccharide alditols, GlcNAcbeta-(1-->2)Manol, Galbeta-(1-->4)GlcNAcbeta-(1-->2)Manol, Galbeta-(1-->4)[Fucalpha-(1-->3)]GlcNAcbeta-(1-->2)Manol and NeuAcalpha-(2-->3)Galbeta-(1-->4)GlcNAcbeta-(1-->2)Manol, that were released from brain glycopeptides by alkaline borohydride treatment.     

PathoGene: a pathogen coding sequence discovery and analysis resource

PathoGene is a web-based resource that streamlines the process of predicting genes in microorganisms and designs PCR primers for amplification to facilitate sequence analysis and experimentation. PathoGene currently supports primer design for every complete microbial, viral, and fungal genome as cataloged in GenBank by the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/). The resulting primers can then be subjected to a stand-alone Basic Local Alignment Search Tool (BLAST) system called PathoBLAST in which the predicted PCR product and/or primers can be compared against the genome of interest or a similar genome to find related genes or estimate primer quality.     

Protein flexibility and intrinsic disorder

Comparisons were made among four categories of protein flexibility: (1) low-B-factor ordered regions, (2) high-B-factor ordered regions, (3) short disordered regions, and (4) long disordered regions. Amino acid compositions of the four categories were found to be significantly different from each other, with high-B-factor ordered and short disordered regions being the most similar pair. The high-B-factor (flexible) ordered regions are characterized by a higher average flexibility index, higher average hydrophilicity, higher average absolute net charge, and higher total charge than disordered regions. The low-B-factor regions are significantly enriched in hydrophobic residues and depleted in the total number of charged residues compared to the other three categories. We examined the predictability of the high-B-factor regions and developed a predictor that discriminates between regions of low and high B-factors. This predictor achieved an accuracy of 70% and a correlation of 0.43 with experimental data, outperforming the 64% accuracy and 0.32 correlation of predictors based solely on flexibility indices. To further clarify the differences between short disordered regions and ordered regions, a predictor of short disordered regions was developed. Its relatively high accuracy of 81% indicates considerable differences between ordered and disordered regions. The distinctive amino acid biases of high-B-factor ordered regions, short disordered regions, and long disordered regions indicate that the sequence determinants for these flexibility categories differ from one another, whereas the significantly-greater-than-chance predictability of these categories from sequence suggest that flexible ordered regions, short disorder, and long disorder are, to a significant degree, encoded at the primary structure level.     

Freeze/thaw stress in<Emphasis Type="Italic"> Ceanothus</Emphasis> of southern California chaparral

Freeze/thaw stress was examined in chaparral shrubs of the genus Ceanothus to determine the interactive effects of freezing and drought and to consider which is the more vulnerable component, the living leaves (symplast) or the non-living water transport system (apoplast). We hypothesized that where Ceanothus species co-occurred, the more inland species C. crassifolius would be more tolerant of low temperatures than the coastal species C. spinosus, both in terms of leaf survival (LT(50), or the temperature at which there is 50% loss of function or viability) and in terms of resistance to freezing-induced embolism (measurements of percent loss hydraulic conductivity due to embolism following freeze/thaw). Cooling experiments on 2 m long winter-acclimated shoots resulted in LT(50) values of about -10 degrees C for C. spinosus versus -18 degrees C for C. crassifolius. Freeze-thaw cycles resulted in no change in embolism when the plants were well hydrated (-0.7 to -2.0 MPa). However, when plants were dehydrated to -5.0 MPa, C. spinosus became 96% embolized with freeze/thaw, versus only 61% embolism for C. crassifolius. Stems of C. crassifolius became 90% and 97% embolized at -6.6 and -8.0 MPa, respectively, meaning that even in this species, stems could be more vulnerable than leaves under conditions of extreme water stress combined with freeze/thaw events. The dominance of C. crassifolius at colder sites and the restriction of C. spinosus to warmer sites are consistent with both the relative tolerance of their symplasts to low temperatures and the relative tolerance of their apoplasts to freeze events in combination with drought stress.     

NewLeaf Plus® Russet Burbank potatoes: replicase-mediated resistance to potato leafroll virus

Potato leafroll poleovirus and the Colorado potato beetle (Leptinotarsa decemlineata (Say)) are major pests of potato in the USA. The US Department of Agriculture estimates that over 50% of annual insecticide use on potato is applied to control the Colorado potato beetle and aphids that transmit potato leafroll virus (PLRV). To address this issue, Russet Burbank potatoes have been genetically modified for a high level of resistance to infection and the resulting disease symptoms caused by PLRV and to feeding damage caused by the Colorado potato beetle. This resistance was achieved by the expression of the unmodified full-length replicase gene of PLRV and the cry3A insect control protein gene from Bacillus thuringiensis var. tenebrionis. Plant expression constructs containing various modifications of the PLRV replicase gene were produced during the development of this product. The genes in these constructs were a full-length unmodified replicase (open reading frame 2a/2b), an antisense orientation of the full-length cDNA, an open reading frame 1 translation of the full-length gene, and a gene truncation containing the 3 sense coding portion of the replicase gene. Growth chamber experiments demonstrated that transformation of plants with the full-length and 3 sense coding constructs substantially protected these potato plants from infection and disease symptoms caused by PLRV. The Russet Burbank potato expressing the full-length PLV replicase gene and the cry3A gene is a new potato product from NatureMark called NewLeaf Plus®.     

Dysgonomonas mossii sp. nov., from human sources

Phenotypic and phylogenetic studies were performed on seven unidentified gram-negative, facultatively anaerobic, coccobacillus-shaped organisms isolated from human clinical specimens. Comparative 16S rRNA gene sequencing demonstrated that four of the strains corresponded to Dysgonomonas capnocytophagoides whereas the remaining three isolates represent a new sub-line within the genus Dysgonomonas, displaying greater than 5% sequence divergence with Dysgonomonas capnocytophagoides and Dysgonomonas gadei. The three novel isolates were readily distinguished from D.capnocytophagoides and D. gadei by biochemical tests. The DNA base composition of the novel species was consistent with its assignment to the genus Dysgonomonas. Based on phylogenetic and phenotypic evidence it is proposed that the unknown species, be classified as Dysgonomonas mossii sp. nov. The type strain of Dysgonomonas mossii is CCUG 43457T (= CIP 107079T).