The elongation of amylose and amylopectin chains in isolated starch granules

The aim of this work was to investigate the conditions required for amylose synthesis in starch granules. Although the major granule-bound isoform of starch synthase - GBSSI - catalyses the synthesis of amylose in vivo, ¹⁴C from ADP[¹⁴C]glucose was incorporated primarily into a specific subset of amylopectin chains when supplied to starch granules isolated from pea (Pisum sativum L.) embryos and potato (Solanum tuberosum L.) tubers. Incubation of granules with soluble extracts of these organs revealed that the extracts contained compounds that increased the incorporation of ¹⁴C into amylose. These compounds were rendered inactive by treatment of the extracts with α-glucosidase, suggesting that they were malto-oligosaccharides. Consistent with this idea, provision of pure malto-oligosaccharides to isolated granules resulted in a dramatic shift in the pattern of incorporation of ¹⁴C, from amylopectin chains to amylose molecules. Comparison of the pattern of incorporation in granules from wild-type peas and lam mutant peas which lack GBSSI showed that this effect of malto-oligosaccharides was specifically on GBSSI. The significance of these results for understanding of the synthesis of amylose and amylopectin in storage organs is discussed.     

No Evidence‐Based Restoration Without a Sound Evidence Base: A Reply to Guldemond et al.

Evidence‐based practice is not possible without an evidence base. Guldemond et al. confuse our attempt at assessing the status of the evidence base of restoration programs in South Africa with attempting to assess whether restoration is evidence‐based. While we fully agree with them that there is a need to assess whether practitioners use evidence in their decision‐making, we assert that use of evidence is the last step in the evidence‐based approach. It is preceded by the generation (and documentation) of evidence through baseline condition assessment, proper goal setting, sound monitoring of the impacts of the chosen intervention as well as effective dissemination of resulting evidence. To answer the question whether restoration is evidence‐based would require the assessment of all stages from generation to use. We chose to start at the beginning, a logical place to start.     

Recreating a functional ancestral archosaur visual pigment

The ancestors of the archosaurs, a major branch of the diapsid reptiles, originated more than 240 MYA near the dawn of the Triassic Period. We used maximum likelihood phylogenetic ancestral reconstruction methods and explored different models of evolution for inferring the amino acid sequence of a putative ancestral archosaur visual pigment. Three different types of maximum likelihood models were used: nucleotide-based, amino acid-based, and codon-based models. Where possible, within each type of model, likelihood ratio tests were used to determine which model best fit the data. Ancestral reconstructions of the ancestral archosaur node using the best-fitting models of each type were found to be in agreement, except for three amino acid residues at which one reconstruction differed from the other two. To determine if these ancestral pigments would be functionally active, the corresponding genes were chemically synthesized and then expressed in a mammalian cell line in tissue culture. The expressed artificial genes were all found to bind to 11-cis-retinal to yield stable photoactive pigments with lambda(max) values of about 508 nm, which is slightly redshifted relative to that of extant vertebrate pigments. The ancestral archosaur pigments also activated the retinal G protein transducin, as measured in a fluorescence assay. Our results show that ancestral genes from ancient organisms can be reconstructed de novo and tested for function using a combination of phylogenetic and biochemical methods.     

UV resistance of Bacillus anthracis spores revisited: validation of Bacillus subtilis spores as UV surrogates for spores of B. anthracis Sterne

Recent bioterrorism concerns have prompted renewed efforts towards understanding the biology of bacterial spore resistance to radiation with a special emphasis on the spores of Bacillus anthracis. A review of the literature revealed that B. anthracis Sterne spores may be three to four times more resistant to 254-nm-wavelength UV than are spores of commonly used indicator strains of Bacillus subtilis. To test this notion, B. anthracis Sterne spores were purified and their UV inactivation kinetics were determined in parallel with those of the spores of two indicator strains of B. subtilis, strains WN624 and ATCC 6633. When prepared and assayed under identical conditions, the spores of all three strains exhibited essentially identical UV inactivation kinetics. The data indicate that standard UV treatments that are effective against B. subtilis spores are likely also sufficient to inactivate B. anthracis spores and that the spores of standard B. subtilis strains could reliably be used as a biodosimetry model for the UV inactivation of B. anthracis spores.     

Expression of a C3 plant Rubisco SSU gene in regenerated C4 Flaveria plants

We report the successful transformation, via Agrobacterium tumefaciens infection, and regeneration of two species of the genus Flaveria: F. brownii and F. palmeri. We document the expression of a C₃ plant gene, an abundantly expressed ribulose 1,5-bisphosphate carboxylase/oxygenase small subunit gene isolated from petunia, in these C₄ plants. The organ-specific expression of this petunia gene in Flaveria brownii is qualitatively identical to its endogenous pattern of expression.     

LRGUK-1 Is Required for Basal Body and Manchette Function during Spermatogenesis and Male Fertility

Male infertility affects at least 5% of reproductive age males. The most common pathology is a complex presentation of decreased sperm output and abnormal sperm shape and motility referred to as oligoasthenoteratospermia (OAT). For the majority of OAT men a precise diagnosis cannot be provided. Here we demonstrate that leucine-rich repeats and guanylate kinase-domain containing isoform 1 (LRGUK-1) is required for multiple aspects of sperm assembly, including acrosome attachment, sperm head shaping and the initiation of the axoneme growth to form the core of the sperm tail. Specifically, LRGUK-1 is required for basal body attachment to the plasma membrane, the appropriate formation of the sub-distal appendages, the extension of axoneme microtubules and for microtubule movement and organisation within the manchette. Manchette dysfunction leads to abnormal sperm head shaping. Several of these functions may be achieved in association with the LRGUK-1 binding partner HOOK2. Collectively, these data establish LRGUK-1 as a major determinant of microtubule structure within the male germ line.     

Bioethanol,biohydrogen and biogas production from wheat straw in a biorefinery concept

The production of bioethanol, biohydrogen and biogas from wheat straw was investigated within a biorefinery framework. Initially, wheat straw was hydrothermally liberated to a cellulose rich fiber fraction and a hemicellulose rich liquid fraction (hydrolysate). Enzymatic hydrolysis and subsequent fermentation of cellulose yielded 0.41 g-ethanol/g-glucose, while dark fermentation of hydrolysate produced 178.0 ml-H₂/g-sugars. The effluents from both bioethanol and biohydrogen processes were further used to produce methane with the yields of 0.324 and 0.381 m³/kg volatile solids (VS)_added, respectively. Additionally, evaluation of six different wheat straw-to-biofuel production scenaria showed that either use of wheat straw for biogas production or multi-fuel production were the energetically most efficient processes compared to production of mono-fuel such as bioethanol when fermenting C6 sugars alone. Thus, multiple biofuels production from wheat straw can increase the efficiency for material and energy and can presumably be more economical process for biomass utilization.     

High-performance liquid chromatographic method for the determination of mycophenolate mofetil in human plasma

A method for the quantification of mycophenolate mofetil (MMF, CellCept) in plasma using solid-phase extraction and HPLC is described here. A solution of internal standard is added to a 0.5-ml plasma aliquot. The resulting sample is treated with water and dilute HCl and applied to a C₁₈ solid-phase extraction column. After a water wash, the MMF and internal standard are eluted with methanol-0.1 M citrate-phosphate buffer, pH 2.6 (80:20, v/v). A 20-μl aliquot of the eluate is injected onto a C₁₈ column (5 μm particle size, 150 × 4.6 mm I.D.) and eluted at ambient temperature with acetonitrile-0.05 M citrate-phosphate buffer, pH 3.6, containing 0.02 M heptanesulfonic acid (41:59, v/v). Quantification is achieved by UV detection at 254 nm. The method is reproducible, accurate and specific for MMF. Using 0.5 ml of plasma for analysis, the quantification limit is 0.400 μg/ml and the range is 0.400–20 μg/ml. Based on the stability profile of MMF in plasma, it is recommended that blood samples collected following intravenous infusion be immediately stored on ice and that plasma be prepared rapidly, immediately stored frozen at −80°C and analyzed within four months of collection.     

Using time-resolved fluorescence to measure serum venom-specific IgE and IgG

We adapted DELFIA™ (dissociation-enhanced lanthanide fluoroimmunoassay), a time resolved fluorescence method, to quantitate whole venom specific and allergenic peptide-specific IgE (sIgE), sIgG₁ and sIgG₄ in serum from people clinically allergic to Australian native ant venoms, of which the predominant cause of allergy is jack jumper ant venom (JJAV). Intra-assay CV was 6.3% and inter-assay CV was 13.7% for JJAV sIgE. DELFIA and Phadia CAP JJAV sIgE results correlated well and had similar sensitivity and specificity for the detection of JJAV sIgE against intradermal skin testing as the gold standard. DELFIA was easily adapted for detecting sIgE to a panel of other native ant venoms.