Isolation,characterization and application of a species-specific repeated sequence from Haynaldia villosa

A species-specific repeated sequence, pHvNAU62, was cloned from Haynaldia villosa, a wheat relative of great importance. It strongly hybridized to H. villosa, but not to wheat. In situ hybridization localized this sequence to six of seven H. villosa chromosome pairs in telomeric or sub-telomeric regions. Southern hybridization to whea-H. villosa addition lines showed that chromosomes 1V through 6V gave strong signals in ladders while chromosome 7V escaped detection. In addition to H. villosa, several Triticeae species were identified for a high abundance of the pHvNAU62 repeated sequence, among which Thinopyrum bassarabicum and Leymus racemosus produced the strongest signals. Sequence analysis indicated that the cloned fragment was 292 bp long, being AT rich (61%), and showed 67% homology of pSc7235, a rye repeated sequence. Isochizomer analysis suggested that the present repeated sequence was heavily methylated at the cytosine of the CpG dimer in the genome of H. villosa.It was also demonstrated that pHvNAU62 is useful in tagging the introduced 6VS chromosome arm, which confers a resistance gene to wheat powdery mildew, in the segregating generations.     

Photolithotrophic cultivation of <Emphasis Type="Italic">Laminaria japonica</Emphasis> gametophyte cells in a silicone tubular membrane-aerated photobioreactor

Gametophyte cells of brown algae Laminaria japonica were employed both in a modified silicone tubular membrane-aerated photobioreactor (bubble-less cultivation mode) and a bubble-column photobioreactor (bubbling cultivation mode), to study different gas–liquid mixing modes on cell growth rate and cell physiological status. With an inoculum density of 50 mg DCW l⁻¹, in modified artificial Pacific seawater (APSW) medium at 13°C, light intensity of 60 μE m⁻² s⁻¹, light cycle of 16/8 h L/D, and aeration rate of 60 ml min⁻¹, the specific growth rates were 0.082 d⁻¹ for bubble-less mode and 0.070 d⁻¹ for bubbling mode with biomass, in the form of dry cell density, increasing 10.9 and 6.8 times, respectively, during the 36 days’ photolithotrophic cultivation. The specific oxygen evolution rate under bubble-less mode was 39.6% higher than under bubbling mode on the 18th day. The gametophyte cells grew in cell aggregates with clump sizes, at day 36, of 1.5 mm and 0.5 mm diameter under bubble-less and bubbling mode respectively and cell injury percentages of 5.1% and 21.1%, respectively. The silicone tubular membrane-aerated photobioreactor was better suited for the cultivation of fragile macroalgal gametophyte cells due to the absence of hydrodynamic shear stress caused by fluid turbulence and the presence of a bubble-less gas supply.     

Mutagenesis of a bacteriophage lytic enzyme PlyGBS significantly increases its antibacterial activity against group B streptococci

Group B streptococci (GBS) are the leading cause of neonatal meningitis and sepsis worldwide. Intrapartum antibiotic prophylaxis (IAP) is the current prevention strategy given to pregnant women with confirmed vaginal GBS colonization. Due to antibiotic resistance identified in GBS, we previously developed another strategy using a bacteriophage lytic enzyme, PlyGBS, to reduce vaginal GBS colonization. In this study, various DNA mutagenesis methods were explored to produce PlyGBS mutants with increased lytic activity against GBS. Several hyperactive mutants were identified that contain only the endopeptidase domain found in the N-terminal region of PlyGBS and represent only about one-third of the wild-type PlyGBS in length. Significantly, these mutants not only have 18–28-fold increases in specific activities compared to PlyGBS, but they also have a similar activity spectrum against several streptococcal species. One of the hyperactive mutants, PlyGBS90-1, reduced the GBS colonization from >5 logs of growth per mouse to <50 colony-forming units (cfu) 4 h post treatment (∼4-log reduction) using a single dose in a mouse vaginal model. A reduction in GBS colonization before delivery should significantly reduce neonatal GBS infection providing a safe alternative to IAP.     

Engineered xyloglucan specificity in a carbohydrate-binding module

The field of plant cell wall biology is constantly growing and consequently so is the need for more sensitive and specific probes for individual wall components. Xyloglucan is a key polysaccharide widely distributed in the plant kingdom in both structural and storage tissues that exist in both fucosylated and non-fucosylated variants. Presently, the only xyloglucan marker available is the monoclonal antibody CCRC-M1 that is specific to terminal alpha-1,2-linked fucosyl residues on xyloglucan oligo- and polysaccharides. As a viable alternative to searches for natural binding proteins or creation of new monoclonal antibodies, an approach to select xyloglucan-specific binding proteins from a combinatorial library of the carbohydrate-binding module, CBM4-2, from xylanase Xyn10A of Rhodothermus marinus is described. Using phage display technology in combination with a chemoenzymatic method to anchor xyloglucan to solid supports, the selection of xyloglucan-binding modules with no detectable residual wild-type xylan and beta-glucan-binding ability was achieved.     

Cytosolic prion protein is not toxic and protects against Bax-mediated cell death in human primary neurons

Recently, it was observed that reverse-translocated cytosolic PrP and PrP expressed in the cytosol induce rapid death in neurons (Ma, J., Wollmann, R., and Lindquist, S. (2002) Science 298, 1781-1785). In this study, we investigated whether accumulation of prion protein (PrP) in the cytosol is toxic to human neurons in primary culture. We show that in these neurons, a single PrP isoform lacking signal peptide accumulates in the cytosol of neurons treated with epoxomicin, a specific proteasome inhibitor. Therefore, endogenously expressed PrP is subject to the endoplasmic reticulum-associated degradation (ERAD) pathway and is degraded by the proteasome in human primary neurons. In contrast to its toxicity in N2a cells, reverse-translocated PrP (ERAD-PrP) is not toxic even when neurons are microinjected with cDNA constructs to overexpress either wild-type PrP or mutant PrPD178N. We found that ERAD-PrP in human neurons remains detergent-soluble and proteinase K-sensitive, in contrast to its detergent-insoluble and proteinase K-resistant state in N2a cells. Furthermore, not only is microinjection of a cDNA construct expressing CyPrP not toxic, it protects these neurons against Bax-mediated cell death. We conclude that in human neurons, ERAD-PrP is not converted naturally into a form reminiscent of scrapie PrP and that PrP located in the cytosol retains its protective function against Bax. Thus, it is unlikely that simple accumulation of PrP in the cytosol can cause neurodegeneration in prion diseases.     

The strain HEB01 of Fusarium sp., a new pathogen that infects brown soft scale

The objectives of the present study were to identify a fungal strain, HEB01, isolated from naturally infected brown soft scale, Coccus hesperidum L. (Hemiptera: Coccidae), and to determine whether it is an entomopathogenic fungus. Fungal culture, reinoculation test, morphological observations, and infection behaviors were investigated. Additionally, the fungal gene sequence of translation elongation factor 1-a (EF-1a) was obtained for molecular identification. The results showed that the fungal strain HEB01 belongs to the Fusarium incarnatum-equiseti species complex and is part of the family Nectriaceae (Hypocreales: Sordariomycetes). The inoculation test and observations of infection behaviors indicated that strain HEB01 is a pathogenic fungus and confirmed that it infects brown soft scale. Thus, the HEB01 strain of Fusarium incarnatum-equiseti is the first pathogen in the genus Fusarium to be isolated from a brown soft scale.