‘Mild mitochondrial uncoupling’ induced protection against neuronal excitotoxicity requires AMPK activity

The preconditioning response conferred by a mild uncoupling of the mitochondrial membrane potential (Δψ_m) has been attributed to altered reactive oxygen species (ROS) production and mitochondrial Ca^2 + uptake within the cells. Here we have explored if altered cellular energetics in response to a mild mitochondrial uncoupling stimulus may also contribute to the protection. The addition of 100 nM FCCP for 30 min to cerebellar granule neurons (CGNs) induced a transient depolarization of the Δψ_m, that was sufficient to significantly reduce CGN vulnerability to the excitotoxic stimulus, glutamate. On investigation, the mild mitochondrial ‘uncoupling’ stimulus resulted in a significant increase in the plasma membrane levels of the glucose transporter isoform 3, with a hyperpolarisation of Δψ_m and increased cellular ATP levels also evident following the washout of FCCP. Furthermore, the phosphorylation state of AMP-activated protein kinase (AMPK) (Thr 172) was increased within 5 min of the uncoupling stimulus and elevated up to 1 h after washout. Significantly, the physiological changes and protection evident after the mild uncoupling stimulus were lost in CGNs when AMPK activity was inhibited. This study identifies an additional mechanism through which protection is mediated upon mild mitochondrial uncoupling: it implicates increased AMPK signalling and an adaptive shift in energy metabolism as mediators of the preconditioning response associated with FCCP-induced mild mitochondrial uncoupling.     

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.

     

Variation in feeding behaviour of Nilaparvata lugens on resistant and susceptible rice varieties

The feeding behaviour of Nilaparavata lugens was monitored on three rice varieties showing different levels of resistance in the Philippines, using a video-assisted observation method. N. lugens made more frequent, shorter probes on the moderately resistant IR46 and resistant IR62 rice varieties than on the susceptible IR22. Honeydew production was significantly lower on the resistant varieties though insect weight gains in 24 h were similar on IR46 and IR22, both being significantly greater than on the highly resistant variety.Population development, growth index and damage ratings were low on IR62 indicating antibiosis and/or non preference. When IR46 plants were infested as seedlings population increase, growth index and damage ratings were similar to those on the susceptible IR22. When infested at a later stage of plant growth the damage rating showed a moderate level of resistance though some population development was maintained, indicating antibiosis and tolerance. N. lugens started probing less frequently after surface exploration on both resistant varieties than on IR22 suggesting the presence of a resistance factor associated with the surface waxes of these varieties.

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Résumé Le comportement alimentaire de Nilaparvata lugens sur variétés de riz, sensible (IR22), partiellement résistante (IR46) et fortement résistante (IR62), a été contrôlé avec une méthode associant la vidéo à l'observation. N. lugens faisait des piqûres plus fréquentes et plus brèves sur IR46 et IR62, que sur la variété sensible. La production de miellat était significativement plus faible sur les variétés résistantes, bien que les gains de poids des insectes aient été les mêmes en 24 h sur IR46 et IR22, les deux étant significativement supérieurs à celui sur IR62.La croissance de la population, l'indice de croissance et le taux de dégâts étaient tous plus faibles sur IR62, ce qui révèle une antibiose et/ou une absence de préférence. Quand la contamination des IR46 a au lieu au stade semis, la croissance de population, l'indice de croissance et le taux de dégâts étaient semblables à ceux de la variété sensible IR22. Quand la contamination avait lieu à un stade ultérieur, le laux de dégâts révélait un niveau modéré de résistance bien qu'une certaine croissance de population se soit maintenue, ce qui révèle antibiose et tolérance.Après exploration de la surface des feuilles des deux variétés résistantes, N. lugens sondait moins fréquemment que sur IR22, ce qui laisse présumer un facteur de résistance associé aux cires superficielles de ces variétés.

     

Triggered efflux of protoberberine alkaloids from cell suspension cultures ofThalictrum rugosum

Cell cultures ofThalictrum rugosum released their protoberberine alkaloids into the medium, when cells were transferred to fresh medium lacking phosphate. The nutritional factors required and the impact of the cells' physiological state for the alkaloid excretion were analyzed. Cell cultures, having released their alkaloids into the medium, continued to grow when the alkaloid containing medium was replaced by fresh growth medium.     

Noninvasive determination of ulnar stiffness from mechanical response--in vivo comparison of stiffness and bone mineral content in humans

An approach referred to as Mechanical Response Tissue Analysis (MRTA) has been developed for the noninvasive determination of mechanical properties of the constituents of the intact limb. Of specific interest in the present study is the bending stiffness of the ulna. The point mechanical impedance properties in the low frequency regime, between 60 and 1,600 Hz are used. The procedure requires a proper design of the probe for good contact of the skin at midshaft and proper support of the proximal and distal ends of the forearm to obtain an approximation to "simple support" of the ulna. A seven-parameter model for the mechanical response is then valid, which includes the first mode of anterior-posterior beam bending of the ulna, the damping and spring effect of the soft tissue between probe and bone, and the damping of musculature. A dynamic analyzer (HP3562A) provides in seconds the impedance curve and the pole-zero curve fit. The physical parameters are obtained from a closed-form solution in terms of the curve-fit parameters. The procedure is automated and is robust and analytically reliable at about the five percent level. Some 80 human subjects have been evaluated by this mechanical response system and by the Norland single photon absorptiometer, providing for the first time in vivo, a comparison of elastic bending stiffness (ulna) and bone mineral content (radius). Three functional parameters of potential clinical value are the cross-sectional bending stiffness EI, the axial load capability Pcr (Euler buckling load) and the bone "sufficiency" S, defined as the ratio of Pcr to body weight. The correlation between EI and bone mineral (r = 0.81) is only slightly less than previous in vitro results with both measurements on the same bone (r = 0.89). When sufficiency is taken into consideration, the correlation of Pcr and bone mineral content is improved (r = 0.89). An implication is that "quality" of bone is a factor which is not indicated by bone mineral content but which is indicated by stiffness. Bone mineral is necessary for proper stiffness but not sufficient. Therefore mechanical measurement should provide a new dimension to be used toward a better understanding of the factors related to bone health and disease.     

Single-strand conformation polymorphism (SSCP) analysis of exon 11 of the CFTR gene reliably detects more than one third of non-ΔF508 mutations in German cystic fibrosis patients

Summary In Central Europe, the F508 deletion accounts for approximately 75% of mutations in the cystic fibrosis transmembrane conductance regulator gene causing cystic fibrosis. The remainder comprise a large number of individually infrequent mutations whose detection requires a disproportionately large effort. However, a sizeable proportion of non-F508 mutations have been found to cluster within exon 11. We have taken advantage of this clustering to detect a total of five previously described point mutations present on 26/72 (36%) non-F508 chromosomes by polymerase chain reaction/direct sequencing of exon 11. These exon 11 mutations were then subjected to single-strand conformation polymorphism (SSCP) analysis, which was shown (i) to discriminate reliably between mutant and wildtype alleles and (ii) to generate reproducible mutation-specific band patterns. This analysis thus represents the first attempt to assess SSCP analysis retrospectively, and serves to illustrate the potential of this screening technique in diagnostic medicine.     

Molecular genetic analysis of 67 patients with duchenne/becker muscular dystrophy

Summary A total of 56 Duchenne muscular dystrophy (DMD) patients and 11 Becker muscular dystrophy (BMD) patients was analyzed by extended multiplex amplification of the DMD/BMD gene; deletions were found in 60% of these patients. The data obtained were used to test the frameshift hypothesis and to compare the distribution of familial versus isolated cases. A significant correlation was found between deletions and isolated cases. Additional experiments were performed in order to determine the deletion breakpoints more precisely. These data are a prerequisite for carrier analysis in the respective families by detection or exclusion of aberrant cDNA fragments derived from ectopic lymphocyte RNA. This diagnostic technique is illustrated by 5 examples.     

Isolation of cDNA clones for fourteen nuclear-encoded thylakoid membrane proteins

Summary Spinach cDNA libraries, made from polyadenylated seedling RNA, have been constructed in pBR322 and the expression vector gt11. Recombinant plasmids or phage for 14 intrinsic and peripheral thylakoid membrane proteins and one stromal protein have been identified. They encode components containing antigenic determinants against the lysine-rich 34 kd, the 23 kd and 16 kd proteins all associated with the water-splitting apparatus of the photosystem II reaction center, the ATP synthase subunits gamma, delta and CF_o-II, the Rieske Fe/S protein of the cytochrome b/f complex, subunits 2, 3, 5 and 6 of the photosystem I reaction center, plastocyanin, ferredoxin oxidoreductase, chlorophyll a/b-binding apoproteins of the lightharvesting complex associated with photosystem II, and the small subunit of the stromal enzyme ribulose bisphosphate corboxylase/oxygenase. The cDNA inserts lack complementarity to plastid DNA but hybridize to restricted nuclear DNA as well as to discrete poly A⁺-mRNA species. The precursor products obtained after translation of hybrid selected RNA fractions in a wheat germ assay are imported and processed by isolated unbroken spinach chloroplasts. The imported components comigrate with the respective authentic proteins.     

Molecular cloning of a pea mRNA encoding an early light induced,nuclear coded chloroplast protein

Summary cDNA clones were isolated for a chloroplast protein, the mRNA of which is induced to maximum levels within 2–4 h after onset of illumination in five day old, etiolated pea seedlings. The cDNA library was constructed from poly(A)⁺-mRNA which was isolated from 4 h illuminated seedlings. The extremely short induction period of the early light induced protein(ELIP)-mRNA established the basis of our screening procedure. Colony hybridization experiments were performed with³²P-labelled cDNA probes, synthesized from RNA of seedlings which had been exposed to different programs of illumination. Plasmid DNAs were isolated from colonies showing strong hybridization signals exclusively with cDNA corresponding to the 4 h-mRNA. Hybrid released translation of preselected plasmids p 17/C2 and p17/C4 revealed a peptide of M_r 24 000. After posttranslational importin vitro, the processed product of M_r 17 000 appears in the chloroplast. Using these clones, the expression of the ELIP-mRNA was investigated by DOT-hybridization. The ELIP-mRNA reaches maximum levels within 2–4 hours after onset of illumination. Our results correspond precisely to thein vivo characteristics and indicate positive identification of the sought clones.