‘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.

     

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.     

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.     

Immunoelectron microscopic studies on the localization of peptidoglycan peptide subunit pentapeptides in bacterial cell walls

The peptide subunit pentapeptide H-L-Ala-D-Glu(L-Lys-D-Ala-D-Ala-OH)-NH₂ of peptidoglycan was localized in the cell walls of several Gram-positive bacteria employing the indirect immunoferritin technique. Specific antibodies to the D-alanyl-D-alanine moiety of non-crosslinked peptide subunit pentapeptide were raised in rabbits by immunization with synthetic immunogen albumin-(CH₂CO-Gly-L-Ala-L-Ala-D-Ala-D-Ala-OH)₃₉. Specificity of these antibodies for the peptide subunit pentapeptide and not for the peptide subunit tetrapeptide was corroborated in a Farr-type radio-active hapten binding assay. Specificity of labelling with ferritin was established by immunoelectron microscopic controls, and by the excellent correlation between specific labelling of cells with ferritin and the particular peptidoglycan primary structure of bacterial strains investigated. Cells of Lactobacillus gasseri, Streptococcus pyogenes and Staphylococcus aureus revealing non-crosslinked peptide subunit pentapeptides in their peptidoglycans could specifically be labelled. Lactobacillus acidophilus and Bacillus subtilis, on the contrary, missing such pentapeptides, failed in labelling.The implication of this method to possibly localize the points of attack of penicillin or cycloserine is discussed.Abbreviations used meso-A₂pm meso-diaminopimelic acid - DSM Deutsche Sammlung für Mikroorganismen, Göttingen, FRG This paper is dedicated to Professor Gerhart Drews on the occasion of his 60th birthday     

Ultrastruktur und Bildungsweise penialer Hartstrukturen bei freilebenden Plathelminthen

Zusammenfassung Ultrastruktur und Differenzierung von Penisstiletten bzw. Stilettnadeln wurden an Vertretern verschiedener Taxa freilebender Plathelminthen untersucht. Die Ausdifferenzierung der Hartstrukturen erfolgt auf unterschiedliche Weise, jedoch stets intrazellulär. Die Stilettnadeln von Philocelis cellata (Acoela) bestehen aus Mikrofibrillen und werden sukzessiv gebildet, mit der Spitze beginnend und basalwärts fortschreitend. Eine ebenfalls sukzessive Bildungsweise zeigen die Stilettapparaturen des Taxons Paromalostomum (Macrostomida); doch bestehen die Hartstrukturen hier aus Mikrotubuli mit angelagertem elektronendichtem Material und nicht aus Mikrofibrillen. Die sukzessive Ausdifferenzierung des Stiletts von Ciliopharyngiella intermedia erfolgt ähnlich wie bei den Hartstrukturen bestimmter Proseriaten mit der Anlage von Mikrofibrillen, die mit elektronendichtem Material verkleidet werden, jedoch weist C. intermedia zusätzlich eine innere und äußere Glättungsschicht auf. Dagegen erfolgt die Stilettbildung bei Adenorhynchus balticus (Typhloplanoida), Marirhynchus longasaeta (Kalyptorhynchia) und Provortex psammophilus und P. tubiferus (Dalyellioida) simultan und ohne Anlage einer Rohform mit einem fibrillären oder tubulären Gerüst. Strukturell sehr ähnlich wie bei A. balticus und M. longasaeta, jedoch sukzessiv erfolgt die Bildung der langen Stilette bei verschiedenen Species des Taxons Promesostoma (Typhloplanoida).Die bisher bekannten feinstrukturellen Organisationsmerkmale und die verschiedenen Bildungsmodi von penialen Hartstrukturen werden unter phylogenetischen Gesichtspunkten diskutiert.

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Ultrastructure and differentiation of penial hard structures in free-living plathelminths

Summary Ultrastructure and differentiation of penis stylets and stylet needles have been investigated in several representatives of different groups of the free-living plathelminths. The formation of the hard structures occurs in different ways, but always intracellularly. The stylet needles of Philocelis cellata (Acoela) consist of microfibrils and are formed successively, beginning with the distal tip. The species of the taxon Paromalostomum (Macrostomida) have stylet apparatuses which also show a successive formation mode; however, the hard elements are built of microtubules and enveloped by electron-dense material. The successive differentiation of the stylet of Ciliopharyngiella intermedia occurs similarly to the hard structure formation of certain proseriates by building a framework of microfibrils which becomes enveloped by electron-dense material; in addition to this rough-form, in C. intermedia an intracellular smooth layer is formed on the inner and outer side of the stylet. In contrast, the stylet formation of Adenorhynchus balticus (Typhloplanoida), Marirhynchus longasaeta (Kalypthorhynchia), Provortex psammophilus and P. tubiferus (Dalyellioida) occurs synchronously and without a roughform, containing a fibrillar or tubular framework. The stylets in several species of the taxon Promesostoma (Typhloplanoida) are built in a way very similar to that in A. balticus and M. longasaeta, but successively.The fine structural properties known at present and the various formation modes of penial hard structures are discussed from the phylogenetic aspect.

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Abkürzungen ae Atriumepithelzelle - ag Atrium genitale - am Atriummuskulatur - ba Bakterium - bl Basallamina oder vergleichbare Interzellularsubstanz - bm Bulbusmuskulatur - bz Stilettbildungszelle - cw Cilienwurzel - de Ductus ejaculatorius - dr Drüsenrohr - dz Ductuszelle - fz Füllzelle - hd Hemidesmosom - hz Hüllzelle - k Kern einer Stilett- bzw. Nadelbildungszelle - m entstehende Muskulatur - ma Macula adhaerens - mc Muskelzylinder - mf Mikrofibrillen - mv Mikrovilli - n Nerv - nz Nadelbildungszelle - pm äußere Penismuskulatur - rm Ringmuskulatur - s Stilett - sd Septatdesmosom - sg Sekretgangzelle - sgz Sekretgangzelle - skg Spermakornsekretgang - skr Spermakornsekretrohr - sm stilettumhüllende Muskulatur - sn Stilettnadel - sna Stilettnaht - sp Spermium - sz Sekretzelle - vg Vesicula granulorum - vgm Muskulatur der Vesicula granulorum - za Zonula adhaerens     

Tactile localisation: the function of active antennal movements in the crayfishCherax destructor

Video recordings and single frame analysis were used to study the function of the second antennae of crayfish (Cherax destructor) as a sensory system in freely behaving animals. Walking crayfish move their antennae back and forth through horizontal angles of 100 degrees and more, relative to the body long axis. At rest, animals tend to hold their antennae at angular positions between 20 and 50 degrees. Movements of the two antennae are largely independent of each other. Before and during a turn of the body the ipsilateral antenna is moved into the direction of the turn. Solid objects are explored by repeatedly moving the antennae towards and across them. Both seeing and blinded crayfish can locate stationary objects following antennal contact. On antennal contact with a small novel object, a moving animal withdraws its antenna and attacks the object. When the antenna of a blinded crayfish is lightly touched with a brush the animal turns and attacks the point of stimulation. The direction taken and the distance covered during an attack can be correlated with: the angle at which the antenna is held at the moment of contact and the distance along the antennal flagellum at which the stimulus is applied. From behavioural evidence we conclude that crayfish use information about the angular position of their antennae and about the position of stimulated mechanoreceptors along the antennal flagellum to locate objects in their environment. We suggest ways in which an active tactile system-like the crayfish's antennae--could supply animals with information about the three-dimensional layout of their environment.