Screening of l‐histidine‐based ligands to modify monolithic supports and selectively purify the supercoiled plasmid DNA isoform

The growing demand of pharmaceutical‐grade plasmid DNA (pDNA) suitable for biotherapeutic applications fostered the development of new purification strategies. The surface plasmon resonance technique was employed for a fast binding screening of l ‐histidine and its derivatives, 1‐benzyl‐l ‐histidine and 1‐methyl‐l ‐histidine, as potential ligands for the biorecognition of three plasmids with different sizes (6.05, 8.70, and 14 kbp). The binding analysis was performed with different isoforms of each plasmid (supercoiled, open circular, and linear) separately. The results revealed that the overall affinity of plasmids to l ‐histidine and its derivatives was high (K_D > 10⁻⁸ M), and the highest affinity was found for human papillomavirus 16 E6/E7 (K_D = 1.1 × 10⁻¹⁰ M and K_D = 3.34 × 10⁻¹⁰ M for open circular and linear plasmid isoforms, respectively). l ‐Histidine and 1‐benzyl‐l ‐histidine were immobilized on monolithic matrices. Chromatographic studies of l ‐histidine and 1‐benzyl‐l ‐histidine monoliths were also performed with the aforementioned samples. In general, the supercoiled isoform had strong interactions with both supports. The separation of plasmid isoforms was achieved by decreasing the ammonium sulfate concentration in the eluent, in both supports, but a lower salt concentration was required in the 1‐benzyl‐l ‐histidine monolith because of stronger interactions promoted with pDNA. The efficiency of plasmid isoforms separation remained unchanged with flow rate variations. The binding capacity for pDNA achieved with the l ‐histidine monolith was 29‐fold higher than that obtained with conventional l ‐histidine agarose. Overall, the combination of either l ‐histidine or its derivatives with monolithic supports can be a promising strategy to purify the supercoiled isoform from different plasmids with suitable purity degree for pharmaceutical applications. Copyright © 2015 John Wiley & Sons, Ltd.     

Marked inhibition of Na+, K+ - ATPase activity and the respiratory chain by phytanic acid in cerebellum from young rats: possible underlying mechanisms of cerebellar ataxia in Refsum disease

Refsum disease is an autosomal recessive disorder of peroxisomal metabolism biochemically characterized by highly elevated concentrations of phytanic acid (Phyt) in a variety of tissues including the cerebellum. Reduction of plasma Phyt levels by dietary restriction intake ameliorates ataxia, a common clinical manifestation of this disorder, suggesting a neurotoxic role for this branched-chain fatty acid. Therefore, considering that the underlying mechanisms of cerebellum damage in Refsum disease are poorly known, in the present study we tested the effects of Phyt on important parameters of bioenergetics, such as the activities of the respiratory chain complexes I to IV, creatine kinase and Na⁺, K⁺- ATPase in cerebellum preparations from young rats. The activities of complexes I, II, I–III and II–III and Na⁺, K⁺- ATPase were markedly inhibited (65–85 %) in a dose-dependent manner by Phyt. In contrast, creatine kinase and complex IV activities were not altered by this fatty acid. Therefore, it is presumed that impairment of the electron flow through the respiratory chain and inhibition of Na⁺, K⁺- ATPase that is crucial for synaptic function may be involved in the pathophysiology of the cerebellar abnormalities manifested as ataxia in Refsum disease and in other peroxisomal disorders in which brain Phyt accumulates.     

Lepidoderma cristatosporum, a new species of myxomycete from Australia

A new species of myxomycete, Lepidoderma cristatosporum, is described from Australia. This species is represented by material collected on subantarctic Macquarie Island. Photographs of fruiting bodies and morphological features as observed under light microscopy are provided, along with micrographs of spore ornamentation as observed by scanning electron microscopy. In addition, the new species is compared with other taxa belonging to the genera Lepidoderma and Diderma, especially with the type material of Lepidoderma crustaceum, a very similar species, and Diderma ochraceum and D. sauteri, two species also muscicolous, for which microphotographs are provided.     

Growth patterns of common bean cultivars affect the ‘B’ value required to quantify biological N2 fixation using the 15N natural abundance technique

Plant and Soil - While soil organic matter 13C isotope helped evaluate vegetation-related change in soil organic carbon (SOC), less is understood about δ13CSOC and SOC in reforested...     

Combining GAL4 GFP enhancer trap with split luciferase to measure spatiotemporal promoter activity in Arabidopsis

In multicellular organisms different types of tissues have distinct gene expression profiles associated with specific function or structure of the cell. Quantification of gene expression in whole organs or whole organisms can give misleading information about levels or dynamics of expression in specific cell types. Tissue‐ or cell‐specific analysis of gene expression has potential to enhance our understanding of gene regulation and interactions of cell signalling networks. The Arabidopsis circadian oscillator is a gene network which orchestrates rhythmic expression across the day/night cycle. There is heterogeneity between cell and tissue types of the composition and behaviour of the oscillator. In order to better understand the spatial and temporal patterns of gene expression, flexible tools are required. By combining a Gateway®‐compatible split luciferase construct with a GAL4 GFP enhancer trap system, we describe a tissue‐specific split luciferase assay for non‐invasive detection of spatiotemporal gene expression in Arabidopsis. We demonstrate the utility of this enhancer trap‐compatible split luciferase assay (ETSLA) system to investigate tissue‐specific dynamics of circadian gene expression. We confirm spatial heterogeneity of circadian gene expression in Arabidopsis leaves and describe the resources available to investigate any gene of interest.