Plasma membrane display of anti-viral single chain Fv fragments confers resistance to tobacco mosaic virus

We tested the hypothesis that membrane-anchored anti-viral antibodies can confer viral resistance to transgenic plants. A heterologous expression system was developed for plasma membrane targeting of anti-viral antibodies using mammalian transmembrane domains. A tobacco mosaic virus (TMV) neutralizing single-chain Fv antibody fragment (scFv24) was targeted to the endoplasmic reticulum and integrated into the plasma membrane of tobacco cells, using mammalian signal peptides and membrane receptor transmembrane domains. The human platelet-derived growth factor receptor (PDGFR) transmembrane domain or the T-cell receptor -domain (TcR) transmembrane domain was fused to the C-terminus of TMV-specific scFv24 to target expression of scFv24 as an extracellularly facing plasma membrane protein. Western blot and ELISA analyses were carried out to confirm functional expression of the recombinant fusion proteins scFv24-PDGFR and scFv24-TcR in transgenic tobacco suspension cultures and transgenic plants. Immunofluorescence and electron microscopy showed that the TcR transmembrane domain targeted scFv24 to the tobacco plasma membrane. Bioassays of viral infection showed that transgenic tobacco plants expressing scFv24-TcR were resistant to TMV infection. These results demonstrated that membrane anchored anti-viral antibody fragments are functional, can be targeted to the plasma membrane in planta and are a novel approach for engineering disease-resistant crops.     

Ethanol metabolism in guinea pig: Ethanol oxidation and its effect on NADNADH ratios,oxygen consumption,and ketogenesis in isolated hepatocytes of fed and fasted animals

Ethanol metabolism was studied in isolated hepatocytes of fed and fasted guinea pigs. Alcohol dehydrogenase (EC 1.1.1.1) activities of fed or fasted liver cells were 2.04 and 1.88 μmol/g cells/min, respectively. Under a variety of in vitro conditions, alcohol dehydrogenase operates in fed hepatocytes at 34–74% and in fasted liver cells at 23–61% of its maximum velocity, respectively. Hepatocytes of fed animals, incubated in Krebs-Ringer bicarbonate buffer, oxidized ethanol at an average rate of 0.69 μmol/g wet weight cells/min, whereas cells of 48-h fasted animals consumed only 0.44 μmol/g/min under identical conditions. Various substrates and metabolites of intermediary metabolism significantly enhanced ethanol oxidation in fed liver cells. Maximum stimulatory effects were achieved with alanine (+138%) and pyruvate (+102%), followed in decreasing order by propionate, lactate, fructose, dihydroxyacetone, and galactose. In contrast to substrate couples such as lactate/pyruvate and glycerol/dihydroxyacetone, sorbitol with or without fructose significantly inhibited ethanol oxidation. The addition of hydrogen shuttle components such as malate, aspartate, or glutamate to fasted hepatocytes resulted in significantly higher stimulation of ethanol uptake than in fed hepatocytes. Also, the degree of inhibition of shuttle activity by n-butylmalonate was more pronounced in fasted liver cells (77% inhibition) than in fed cells (59% inhibition). These data as well as oxygen kinetic studies in intact guinea pig hepatocytes utilizing uncouplers (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, dinitrophenol), electron-transport inhibitors (rotenone, antimycin), and malate-aspartate shuttle inhibitors (aminooxyacetate, n-butylmalonate) strongly suggested that the malate-aspartate shuttle is the predominant hydrogen transport system during ethanol oxidation in guinea pig liver.Comparison of the alcohol dehydrogenase-inhibitors 4-methylpyrazole and pyrazole on ethanol oxidation demonstrated that the alcohol dehydrogenase system is quantitatively the most important alcohol-metabolizing pathway in guinea pig liver. Supporting this conclusion, it was found that the H₂O₂-forming substrate glycolate slightly increased ethanol oxidation in liver cells of control animals (+26%), but prior inhibition of catalase by 3-amino-1,2,4-triazole resulted in a significant increase (+25%) instead of a decrease in alcohol oxidation. This finding does not support a quantitatively important role of peroxidatic oxidation of ethanol by catalase in liver.Cytosolic $\text{NAD}\text{NADH}$ ratios were greatly shifted toward reduction during ethanol oxidation. These reductive shifts were even more pronounced when cells were incubated in the presence of fatty acids (octanoate, oleate) plus ethanol. Inhibitor studies with 4-methylpyrazole demonstrated that the decrease of the cytosolic $\text{NAD}\text{NADH}$ ratio during fatty acid oxidation was due to an inhibition of hydrogen transport from cytosol to mitochondria and not the result of transfer of hydrogen, generated by fatty acid oxidation, from mitochondria to cytosol. Lactate plus pyruvate formation was slightly inhibited by ethanol in fed hepatocytes but greatly accelerated in fasted cells; this latter effect was mostly the result of increased lactate formation. Such regulation may represent a hepatic mechanism of alcoholic lactic acidosis as observed in human alcoholics. The ethanol-induced decrease of the mitochondrial $\text{NAD}\text{NADH}$ ratio was prevented by addition of 4-methylpyrazole. Endogenous ketogenesis was greatly increased (+80%) by ethanol in fed liver cells. This effect of ethanol was blunted in the presence of glucose. Propionate, by competing with fatty acid oxidation, was strongly antiketogenic. This effect was alleviated by ethanol. In 48-h fasted hepatocytes, endogenous ketogenesis was enhanced by 84%. Although ethanol did not further stimulate endogenous ketogenesis under these conditions, alcohol significantly increased ketogenesis in the presence of octanoate or oleate. This stimulatory effect of ethanol was almost completely prevented by 4-methylpyrazole. These findings demonstrate that the syndrome of alcoholic ketoacidosis may be due, at least partially, to the additional stimulation of ketogenesis by or from ethanol during fatty acid oxidation in the fasting state.     

Catalase-like crystals in parathyroid gland cells of Rana temporaria L.

Summary Crystalline inclusions in parathyroid gland cell nuclei of Rana temporaria were studied by electron microscopy using a specimen tilting stage. Images were analysed by optical diffraction. Results were compared with X-ray and electron microscopic data of trigonal bovine liver catalase to which a striking resemblance of the inclusions was found.We are grateful to Professor R. Mosebach (Giessen) for discussions, to the Deutsche Forschungsgemeinschaft for a grant (La 229/4) and instruments and to Messrs. Spindler & Hoyer, Göttingen and Messrs. Rank Precision Instruments, Nürnberg for putting apparatus at our disposal and performing diffraction photographs.     

Seed dispersal by ants in the semi-arid Caatinga of North-East Brazil

BACKGROUND AND AIMS: Myrmecochory is a conspicuous feature of several sclerophyll ecosystems around the world but it has received little attention in the semi-arid areas of South America. This study addresses the importance of seed dispersal by ants in a 2500-km(2) area of the Caatinga ecosystem (north-east Brazil) and investigates ant-derived benefits to the plant through myrmecochory. METHODS: Seed manipulation and dispersal by ants was investigated during a 3-year period in the Xingó region. Both plant and ant assemblages involved in seed dispersal were described and ant behaviour was characterized. True myrmecochorous seeds of seven Euphorbiaceae species (i.e. elaiosome-bearing seeds) were used in experiments designed to: (1) quantify the rates of seed cleaning/removal and the influence of both seed size and elaiosome presence on seed removal; (2) identify the fate of seeds dispersed by ants; and (3) document the benefits of seed dispersal by ants in terms of seed germination and seedling growth. KEY RESULTS: Seed dispersal by ants involved one-quarter of the woody flora inhabiting the Xingó region, but true myrmecochory was restricted to 12.8 % of the woody plant species. Myrmecochorous seeds manipulated by ants faced high levels of seed removal (38-84 %) and 83 % of removed seeds were discarded on ant nests. Moreover, seed removal positively correlated with the presence of elaiosome, and elaiosome removal increased germination success by at least 30 %. Finally, some Euphorbiaceae species presented both increased germination and seedling growth on ant-nest soils. CONCLUSIONS: Myrmecochory is a relevant seed dispersal mode in the Caatinga ecosystem, and is particularly frequent among Euphorbiaceae trees and shrubs. The fact that seeds reach micro-sites suitable for establishment (ant nests) supports the directed dispersal hypothesis as a possible force favouring myrmecochory in this ecosystem. Ecosystems with a high frequency of myrmecochorous plants appear not to be restricted to regions of nutrient-impoverished soil or to fire-prone regions.     

Growth of Trifolium alpinum: Effects of soil properties, symbionts and pathogens

The lowland cultivation of Trifolium alpinum, a clover species found on acid soils in the Alps and suitable for the restoration of erosion areas at high altitudes, failed repeatedly in previous experiments. Three experiments were carried out in a controlled environment to elucidate the reasons for the failure and to develop possible cultivation strategies. In experiment I, T. alpinum was grown in an autochthonous soil from the Alps (high elevation) and in two allochthonous soils, a grassland soil from the Hercynian mountains (medium elevation), and an arable soil (low elevation), in which the seed propagation of T. alpinum had failed previously. The two allochthonous soils had lower contents of soil organic C and ergosterol, an indicator for fungal biomass, than the autochthonous high-elevation soil, but higher levels of exchangeable Ca and extractable P. Plants grown in the allochthonous soils achieved higher biomass and total N amounts per plant than those from the high elevation soil if inoculated with this autochthonous material to establish rhizobial infection. In the allochthonous high elevation soil, the growth of T. alpinum was P-limited as shown in experiment II. In experiment I, plants grown in the low elevation soil had a lower biomass and smaller number of active leaves at 120 days after emergence than those grown on the medium elevation soil. This difference can be explained by strong colonization with the phytophagous nematode Pratylenchus sp., as demonstrated in experiment III by comparing plant growth either in untreated or in autoclaved low-elevation soil. Successful propagation of T. alpinum at low elevation may be achieved through suitable inoculation with autochthonous soil biota, especially Rhizobia, and avoidance of soils infested by Pratylenchus species by choosing sites with acidic soil and ensuring adequate P-availability.     

Enterobacter radicincitans sp. nov., a plant growth promoting species of the family Enterobacteriaceae

A plant growth promoting bacterial isolate (D5/23T) from the phyllosphere of winter wheat, able to fix atmospheric nitrogen and to produce auxines and cytokinins was investigated in a polyphasic taxonomy approach. Phylogenetic analyses using the 16S rRNA gene sequence of the strain clearly indicated that the strain belonged to the family Enterobacteriaceae, most closely related to Enterobacter cloacae with 99.0% and Enterobacter dissolvens with 98.5% sequence similarity. Phylogenetic analysis derived from the sequence of the rpoB gene showed the highest sequence similarities to Enterobacter cowanii (93.0%) but supported the distinct position of strain D5/23T. The isolate produced a fatty acid pattern typical for members of the family Enterobacteriaceae. On the basis of the phylogenetic analyses, DNA-DNA hybridizations, and the unique physiological and biochemical characteristics, we propose that strain D5/23T represents a new species of the genus Enterobacter for which we propose the name Enterobacter radicincitans sp. nov.     

Superresolution imaging of biological nanostructures by spectral precision distance microscopy

For the improved understanding of biological systems on the nanoscale, it is necessary to enhance the resolution of light microscopy in the visible wavelength range beyond the limits of conventional epifluorescence microscopy (optical resolution of about 200 nm laterally, 600 nm axially). Recently, various far-field methods have been developed allowing a substantial increase of resolution ("superresolution microscopy", or "lightoptical nanoscopy"). This opens an avenue to 'nano-image' intact and even living cells, as well as other biostructures like viruses, down to the molecular detail. Thus, it is possible to combine light optical spatial nanoscale information with ultrastructure analyses and the molecular interaction information provided by molecular cell biology. In this review, we describe the principles of spectrally assigned localization microscopy (SALM) of biological nanostructures, focusing on a special SALM approach, spectral precision distance/position determination microscopy (SPDM) with physically modified fluorochromes (SPDM(Phymod) . Generally, this SPDM method is based on high-precision localization of fluorescent molecules, which can be discriminated using reversibly bleached states of the fluorophores for their optical isolation. A variety of application examples is presented, ranging from superresolution microscopy of membrane and cytoplasmic protein distribution to dual-color SPDM of nuclear proteins. At present, we can achieve an optical resolution of cellular structures down to the 20-nm range, with best values around 5 nm (～1/100 of the exciting wavelength).     

State-of-the art data normalization methods improve NMR-based metabolomic analysis

Extracting biomedical information from large metabolomic datasets by multivariate data analysis is of considerable complexity. Common challenges include among others screening for differentially produced metabolites, estimation of fold changes, and sample classification. Prior to these analysis steps, it is important to minimize contributions from unwanted biases and experimental variance. This is the goal of data preprocessing. In this work, different data normalization methods were compared systematically employing two different datasets generated by means of nuclear magnetic resonance (NMR) spectroscopy. To this end, two different types of normalization methods were used, one aiming to remove unwanted sample-to-sample variation while the other adjusts the variance of the different metabolites by variable scaling and variance stabilization methods. The impact of all methods tested on sample classification was evaluated on urinary NMR fingerprints obtained from healthy volunteers and patients suffering from autosomal polycystic kidney disease (ADPKD). Performance in terms of screening for differentially produced metabolites was investigated on a dataset following a Latin-square design, where varied amounts of 8 different metabolites were spiked into a human urine matrix while keeping the total spike-in amount constant. In addition, specific tests were conducted to systematically investigate the influence of the different preprocessing methods on the structure of the analyzed data. In conclusion, preprocessing methods originally developed for DNA microarray analysis, in particular, Quantile and Cubic-Spline Normalization, performed best in reducing bias, accurately detecting fold changes, and classifying samples.

     

Sleep-related obstructive disordered breathing in cleft palate patients after palatoplasty

Sleep-disordered breathing is frequently associated with children presenting congenital midface defects. Because of structural and functional anomalies in the upper airway, children with cleft palate, especially after surgery, may carry a higher risk of developing sleep-disordered breathing. However, the presence of such sleep-disordered breathing in older cleft palate children has not been emphasized. The aim of this comparative overnight cardiorespiratory sleep study was to evaluate cleft palate patients according to sleep-disordered breathing. A group of 43 cleft palate children (17 girls and 26 boys; mean age, 12.1 +/- 3.8 years) was compared with a control group of 20 randomly selected, noncleft children matched for age, sex, and body mass index. None of the patients suffered from manifest sleep-disordered breathing. Cleft palate patients had a statistically significantly higher respiratory disturbance index and snoring index, but no increased apnea index. The data suggest that cleft palate patients having undergone primary closure of the palate demonstrate microsymptoms of nocturnal upper airway obstruction.