Selection of transgenic <Emphasis Type="Italic">Petunia</Emphasis> plants using the green fluorescent protein (GFP)

Selectable marker genes are needed for efficient transformation of plants. The present study focused on testing the applicability of green fluorescent protein (GFP) for selecting transgenic Petunia hybrida plants without applying antibiotics or herbicides. Based on a transient gene expression assay, the efficiency of two gfp genes, mGFP-4 and smRS-GFP, was compared. Two days after infiltration of Agrobacterium tumefaciens, GFP expression was recorded in leaf epidermal cells. The intensity of smRS-GFP fluorescence was higher than that of mGFP-4 and easier to distinguish from other unspecific fluorescent signals in Petunia. Transformations using the pMen65smRS-GFP vector, which contained the neomycin phosphotransferase II (nptII) gene, resulted in callus and shoots that visually and clearly expressed detectable GFP levels; in addition, this vector made it possible to exclusively select transformed plants using GFP. The transformation efficiencies achieved by using GFP selection versus combined kanamycin and GFP selection (nptII+GFP) were compared in four Petunia genotypes with a transformation experiment with four replications. In three out of four Petunia cultivars a higher transformation frequency was achieved by using nptII+GFP selection. Southern blot hybridisation revealed single and multiple integrations of smRS-GFP in Petunia. Single copy plants showed intensive expression in all parts of the plants, whereas a higher copy number led to only weak or partial expression of smRS-GFP allowing the visual selection of single copy events. Thus, it is possible to select transgenic Petunia plants based on their GFP expressions without applying antibiotics or herbicides.     

Schizosaccharomyces pombe Ofd2 is a nuclear 2-oxoglutarate and iron dependent dioxygenase interacting with histones

2-Oxoglutarate (2OG) dependent dioxygenases are ubiquitous iron containing enzymes that couple substrate oxidation to the conversion of 2OG to succinate and carbon dioxide. They participate in a wide range of biological processes including collagen biosynthesis, fatty acid metabolism, hypoxic sensing and demethylation of nucleic acids and histones. Although substantial progress has been made in elucidating their function, the role of many 2OG dioxygenases remains enigmatic. Here we have studied the 2OG and iron (Fe(II)) dependent dioxygenase Ofd2 in Schizosaccharomyces pombe, a member of the AlkB subfamily of dioxygenases. We show that decarboxylation of 2OG by recombinant Ofd2 is dependent on Fe(II) and a histidine residue predicted to be involved in Fe(II) coordination. The decarboxylase activity of Ofd2 is stimulated by histones, and H2A has the strongest effect. Ofd2 interacts with all four core histones, however, only very weakly with H4. Our results define a new subclass of AlkB proteins interacting with histones, which also might comprise some of the human AlkB homologs with unknown function.     

Genetic variation of an odorant receptor OR7D4 and sensory perception of cooked meat containing androstenone

Although odour perception impacts food preferences, the effect of genotypic variation of odorant receptors (ORs) on the sensory perception of food is unclear. Human OR7D4 responds to androstenone, and genotypic variation in OR7D4 predicts variation in the perception of androstenone. Since androstenone is naturally present in meat derived from male pigs, we asked whether OR7D4 genotype correlates with either the ability to detect androstenone or the evaluation of cooked pork tainted with varying levels of androstenone within the naturally-occurring range. Consistent with previous findings, subjects with two copies of the functional OR7D4 RT variant were more sensitive to androstenone than subjects carrying a non-functional OR7D4 WM variant. When pork containing varying levels of androstenone was cooked and tested by sniffing and tasting, subjects with two copies of the RT variant tended to rate the androstenone-containing meat as less favourable than subjects carrying the WM variant. Our data is consistent with the idea that OR7D4 genotype predicts the sensory perception of meat containing androstenone and that genetic variation in an odorant receptor can alter food preferences.     

Leupeptin enhances cell surface localization of fibroblast growth factor receptor 1 in adult sensory neurons by increased recycling

Fibroblast growth factors (FGFs) act as trophic factors during development and regeneration of the nervous system. FGFs mediate their responses by activation of four types of FGF receptors (FGFR1-4). FGFR1 is expressed in adult sensory neurons of dorsal root ganglia (DRG), and overexpression of FGFR1 enhances FGF-2-induced elongative axon growth in vitro. Ligand-induced activation of FGFR1 is followed by endocytosis and rapid lysosomal degradation. We previously reported that the lysosomal inhibitor leupeptin prevents degradation of FGFR1 and promotes FGF-2-induced elongative axon growth of DRG neurons overexpressing FGFR1. Therefore, we analyzed the effects of leupeptin on intracellular sorting of FGFR1 in PC12 pheochromocytoma cells and DRG neurons. Leupeptin increased colocalization of FGFR1 with lysosomes. Furthermore, leupeptin enhanced the cell surface localization of FGFR1 by increased receptor recycling and this effect was abolished by the recycling inhibitor monensin. In addition, a lysine mutant of FGFR1, which is preferentially recycled back to the cell surface, promoted elongative axon growth of DRG neurons similar to leupeptin. In contrast, the lysosomal inhibitor bafilomycin had no effect on surface localization of FGFR1, inhibited axon growth of DRG neurons and abolished the effects of leupeptin on receptor recycling. Together, our results strongly imply that increased recycling of FGFR1 promotes axon elongation, but not axonal branching, of adult DRG neurons in vitro.     

Effects of CO2 anaesthesia on central nervous system activity in swine

The objective of the study was to examine the changes in central nervous system (CNS) activity and physical behaviour during induction and awakening from CO2 anaesthesia. Two studies, each using pigs immersed into 90% CO2 gas for a period of 60 s were performed. In study 1, we monitored middle latency auditory evoked potentials (changes in latencies, amplitudes and a depth of anaesthesia index), electroencephalographic parameters (delta, theta, alpha and beta electroencephalographic power and 95% spectral edge frequency) and heart rate; and in study 2, we monitored body movements and arterial and venous partial pressure of CO2 and O2. No behavioural signs of distress were observed during the early part of the induction. The swine exhibited muscular activity from 13-30 s after induction-start as well as during awakening from anaesthesia, possibly because of a transitory weaker suppression of the brain stem than of the cortex. The CNS and blood gas parameters started to change from the very start of induction. The CNS suppression lasted only approximately one minute after the end of the induction period. The two studies indicated a good temporal relationship between changes in amplitude, depth of anaesthesia index, spectral edge frequency, and arterial PCO2 during the induction period.     

Chemometric exploration of an amperometric biosensor array for fast determination of wastewater quality

Four wastewater samples of different treatment qualities; untreated, alarm, alert and normal, from a Swedish chemi-thermo-mechanical pulp mill and pure water were investigated using an amperometric bio-electronic tongue in a batch cell. The aim was to explore enzymatically modified screen-printed amperometric sensors for the discrimination of wastewater quality and to counteract the inherent drift. Seven out of eight platinum electrodes on the array were modified with four different enzymes; tyrosinase, horseradish peroxidase, acetyl cholinesterase and butyryl cholinesterase. At a constant potential the current intensity on each sensor was measured for 200s, 100s before injection and 100s after injection of the sample. The dynamic biosensor response curves from the eight sensors were used for principal component analysis (PCA). A simple baseline and sensitivity correction equivalent to multiplicative drift correction (MDC), using steady state intensities of reference sample (catechol) recordings, was employed. A clear pattern emerged in perfect agreement with prior knowledge of the samples explaining 97% of the variation in the data by two principal components (PCs). The first PC described the treatment quality of the samples and the second PC described the difference between treated and untreated samples. Horseradish peroxidase and pure platinum sensors were found to be the determinant sensors, while the rest did not contribute much to the discrimination. The wastewater samples were characterized by the chemical oxygen demand (COD), biological oxygen demand (BOD), total organic carbon (TOC), inhibition of nitrification, inhibition of respiration and toxicity towards Vibrio fischeri using Microtox, the freshwater alga Pseudokirchneriella subcapita and the freshwater crustacean Daphnia magna.     

Glycolipids and the structure of chlorosomes in green bacteria

1. The cellular content of galactolipids in Chlorobium and Chloroflexus is not related to bacteriochlorophyll content nor to the total amount of chlorosome material in the cells.
2. Chlorosomes of both bacteria were agglutinated by Ricinus lectin and the agglutination was increased after treatment of the chlorosomes with trypsin.
3. When cell free preparations of both bacteria were treated with trypsin prior to centrifugation on sucrose gradients, the resulting chlorosome fractions were less contaminated with material derived from the cytoplasmic membrane than when trypsin was not employed.

     

The effect of pH on silver staining of nucleolus organizer regions

The effect of pH on silver staining of the nucleolus organizer regions (NORs) of human chromosomes has been investigated between pH 6.5 and 12.0. Nonvolatile mixtures of ethanolamine and ethanolammonium nitrate replaced the ammonia of standard procedures. The optimal NOR staining obtained at pH 3.5 by the silver staining procedure of Howell and Black served as a standard; this procedure stained all ten NORs in 90% of mitoses. Similar NOR staining was found in 75% of mitoses stained at pH 11.7 or 11.8, but only in 10-15% of mitoses stained between pH 11.6 and 10.0. Between pH 10.0 and 9.0 NOR staining was incomplete, and between pH 8.5 and 6.5 there was no NOR staining.     

Improved Postharvest Quality of Inflorescences of <Emphasis Type="Italic">fbp1::etr1-1</Emphasis> Transgenic <Emphasis Type="Italic">Burrageara</Emphasis> ‘Stefan Isler Lava Flow’

Flowers of the complex orchid hybrid Burrageara ‘Stefan Isler Lava Flow’ had been shown previously to react sensitively to ethylene. Via Agrobacterium tumefaciens, the mutant ethylene receptor ETHYLENE RESPONSE 1 (etr1-1) from Arabidopsis thaliana under the control of the flower-specific promoter FLOWER BINDING PROTEIN 1 (fbp1) from Petunia hybrida was transferred to Burrageara. One single-copy event was analyzed in this study aiming to investigate the expression of the fbp1::etr1-1 transgene in different plant and flower organs by quantitative RT-PCR and the reaction of flowers and inflorescences to ethylene. It was shown that the heterologous promoter led to high expression levels in the perianth of the orchid flowers compared to low levels in leaves and roots. The expression shift to the first whorl (sepals) described here corresponds to extended expression of endogenous B class MADS box homeotic genes in orchids in general. The transgenic plants grew and developed similar to the wild-type plants, except for slightly faster rooting in vitro and smaller flowers. Flower longevity was improved by 7 days in 10 ppm ethylene. Moreover, bud drop starting at day 5 of incubation of inflorescences in 10 ppm ethylene in the wild-type was efficiently prevented for at least 19 days in the fbp1::etr1-1 transgenic plants. The function of the tissue-specific promoter fbp1 and the mutant receptor etr1-1 was shown for the first time in a monocotyledonous plant.