Isomeric lipid signatures reveal compartmentalized fatty acid metabolism in cancer

The cellular energy and biomass demands of cancer drive a complex dynamic between uptake of extracellular FAs and their de novo synthesis. Given that oxidation of de novo synthesized FAs for energy would result in net-energy loss, there is an implication that FAs from these two sources must have distinct metabolic fates; however, hitherto, all FAs have been considered part of a common pool. To probe potential metabolic partitioning of cellular FAs, cancer cells were supplemented with stable isotope-labeled FAs. Structural analysis of the resulting glycerophospholipids revealed that labeled FAs from uptake were largely incorporated to canonical (sn-) positions on the glycerol backbone. Surprisingly, labeled FA uptake also disrupted canonical isomer patterns of the unlabeled lipidome and induced repartitioning of n-3 and n-6 PUFAs into glycerophospholipid classes. These structural changes support the existence of differences in the metabolic fates of FAs derived from uptake or de novo sources and demonstrate unique signaling and remodeling behaviors usually hidden from conventional lipidomics.     

α-MSH Stimulates Glucose Uptake in Mouse Muscle and Phosphorylates Rab-GTPase-Activating Protein TBC1D1 Independently of AMPK

The melanocortin system includes five G-protein coupled receptors (family A) defined as MC1R-MC5R, which are stimulated by endogenous agonists derived from proopiomelanocortin (POMC). The melanocortin system has been intensely studied for its central actions in body weight and energy expenditure regulation, which are mainly mediated by MC4R. The pituitary gland is the source of various POMC-derived hormones released to the circulation, which raises the possibility that there may be actions of the melanocortins on peripheral energy homeostasis. In this study, we examined the molecular signaling pathway involved in α-MSH-stimulated glucose uptake in differentiated L6 myotubes and mouse muscle explants. In order to examine the involvement of AMPK, we investigate α-MSH stimulation in both wild type and AMPK deficient mice. We found that α-MSH significantly induces phosphorylation of TBC1 domain (TBC1D) family member 1 (S237 and T596), which is independent of upstream PKA and AMPK. We find no evidence to support that α-MSH-stimulated glucose uptake involves TBC1D4 phosphorylation (T642 and S704) or GLUT4 translocation.     

The ability of the biological control agent Bacillus subtilis, strain BB, to colonise vegetable brassicas endophytically following seed inoculation

The ability of Bacillus subtilis, strain BB, to colonise cabbage seedlings endophytically was examined following seed inoculation. Strain BB was recovered from different plant parts including leaves (cotyledons), stem (hypocotyl) and roots. While high bacterial populations persisted in the roots and lower stem, they were lower in the upper stem and leaves through time. In addition to cabbage, strain BB colonised endophytically the roots of 5 other vegetable brassicas. Fatty acid methyl ester (FAME) and PCR fingerprinting analysis confirmed the reliability of the detection method. Studies conducted with transmission electron microscope (TEM) showed that BB mainly colonised intercellular spaces of cortical tissues including intercellular spaces close to the conducting elements of roots and stem of cabbage seedlings. Gold labelling was specifically associated with BB and the fibrillar material filling the intercellular spaces where bacterial cells were found.     

Cell surface T3 expression requires the presence of both alpha- and beta-chains of the T cell receptor

We have identified a surface T3- Jurkat variant which has a defective alpha-chain but which possesses an intact beta-chain. The transfection of a functional mouse alpha-chain into this human T cell induces the expression of surface T3 molecules associated with mouse alpha-human beta-heterodimers detected by anticlonotypic antibodies. Treatment of the transfectant with anti-T3, anti-mouse Ti-alpha, or anti-human Ti-beta antibodies clears all Ti-T3 complexes from the surface. These results demonstrate that functional alpha- and beta-chains are both required for expression of T3 on the cell membrane, and that the Ti heterodimers present and associated with T3 on Jurkat cells involve only alpha- and beta-chains.     

Three-dimensional reconstruction of a human metaphase chromosome from electron micrographs

A complete human metaphase chromosome has been reconstructed from a series of electron microscopical projections obtained by tilting the specimen stage at 3 degree intervals from –60 to +60 degrees. The reconstructed structure is about 3.0 m long, 1.6 m wide, and 0.8 m thick. The mass distribution was fairly homogeneous within the chromatids and neither a hollow nor a dense core was observed. The distribution and course of fibers observed are most consistent with a looping model of chromosome structure.     

Kinetic β-deuterium isotope effects suggest a covalent mechanism for the protein folding enzyme peptidylprolyl cis/trans-isomerase

The cis/trans interconversion of Glt-Ala-Ala-Pro-Phe-4-nitroanilide and Glt-Ala-Gly-Pro-Phe-4-nitroanilide was studied both enzymatically and nonenzymatically by measuring kinetic β-deuterium isotope effects. The hydrogen atom at the α-carbon atom of the Xaa residue within the Xaa-Pro moiety was substituted by deuterium. In the nonenzymatic case the transition state of rotation is reflected by k_H/k_D > 1. When catalysed by 17 kDa PPIase the same bond rotation is characterized by k_H/k_D < 1. This suggests a covalent mechanism of catalysis which involves an approximately tetravalent carbon of the prolyl imidic bond for the transition state of reaction.     

Packing of the 30 nm chromatin fiber in the human metaphase chromosome

The human genetic material is packed hierarchically within the metaphase chromosome: the DNA moleculet together with histone proteins form 11 nm diameter nucleosomes, which are then ordered into the 30 nm thick chromatin fiber. Little is known about the packing of this fiber within the chromosome. We have developed a tracking algorithm with which we followed its path within a three-dimensional reconstruction of a human chromosome computed from a series of electron micrographie projections. Fiber segments were seen to form loops of 100–350 nm diameter. Our observations indicate that these loops — which themselves show no preferred orientation — are organised into regions of roughly 200 nm axial extent.     

Die Bestandsentwicklung von Kleinv?geln in Mitteleuropa: Analyse von Fangzahlen

Zusammenfassung Im Mettnau-Reit-Illmitz-Programm (MRI-Programm) der Vogelwarte Radolfzell wurden von 1974–1983 auf drei Fangstationen in Süddeutschland, Norddeutschland und Ostösterreich (Abb. 1) 184 939 Vögel von 37 Kleinvogelarten gefangen. Die Fänge erfolgten jährlich während der gesamten Wegzugperiode von Ende Juni bis Anfang November an Durchzüglern in Japannetzen unter strikter und umfassender Standardisierung aller Fang- und Arbeitsmethoden. Die Fangzahlen (Tab. 1, Abb. 2–6) werden in fünf Regressionsanalysen (Tab. 2–9) auf systematische Änderungen untersucht.Von insgesamt 496 errechenbaren Koeffizienten waren 317 oder 63,9% negativ und nur 179 oder 38,1 % positiv. Bei 34 der 37 untersuchten Arten ließen sich signifikante Trends errechnen. Sie sind für 20 oder 54 % dieser Arten ausschließlich oder überwiegend negativ. 14 Arten zeigten mindestens auf zwei Stationen negative Trends. Nur für insgesamt 10 Arten ließen sich überwiegend positive Trends errechnen. Faßt man negative Trends und Tendenzen (Vorzeichen) zusammen, so ergibt sich für 26 oder 70 % der untersuchten Arten ein negatives Bild. Dieses stark negative Gewicht ist im statistischen Sinne keine zufällige Erscheinung. Da die Bestandszunahmen die Abnahmen nicht aufwiegen konnten, zeigten auch die jährlichen Gesamtfangzahlen aller drei Stationen negative Trends. Die mittlere jährliche Abnahme betrug auf den Stationen Mettnau, Reit und Illmitz etwa 1,6 %. Die Tendenzen und Trends der einzelnen Arten stimmen auf den drei Stationen weitgehend überein. Sie lassen für ihr Zustandekommen auf weitgehend gleichförmige Ursachen bei den durch Mitteleuropa wandernden Populationen schließen.Die Fangzahlen und Literaturdaten zeigen, daß beträchtliche Teile unserer Kleinvogelwelt von Rückgangserscheinungen betroffen sind, wie wir sie von vielen Großvogelarten seit langem kennen. Bei einer Reihe von Arten sind die Abnahmen gravierend und die jährlichen Fangzahlen inzwischen so niedrig, daß sich bei ihnen mit den in den 70er Jahren konzipierten Fanganlagen eine Reihe von Fragestellungen nicht mehr untersuchen lassen. Wir können der weiteren Entwicklung der Bestände unserer derzeit noch artenreichen Kleinvogelwelt — wie der vieler Großvögel — nur mit größter Sorge entgegensehen. Die Ursachen der Rückgänge sind weitgehend unbekannt, und demzufolge mangelt es fast vollständig an geeigneten Gegenmaßnahmen. Bisherige Schutzmaßnahmen haben die Rückgänge nicht aufhalten können. Künftiger Vogelschutz wird sich folglich weitergehender Maßnahmen bedienen müssen.

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The development of songbird populations in central Europe: Analysis of trapping data

Summary In the Mettnau-Reit-Illmitz-Program (MRI-Program) of the Vogelwarte Radolfzell 184 939 birds of 37 songbird species were caught in the period 1974–1983 on three trapping stations in S. and N. Germany and E. Austria (Fig. 1). Trapping of passage migrants in mist nets occurred annually from the end of June to the beginning of November, the entire autumn migratory period. All trapping and working methods were strictly and comprehensively standardized. The trapping figures (Tab. 1, Fig. 2–6) were analyzed with respect to systematic alterations to gain information about changes in the population levels of species migrating through central Europe. The investigation is based above all on five regression analyses (Tab. 2–9).The most important individual results are: out of a total of 496 coefficients which could be calculated 317 or 63.9 % were negative and only 179 or 38.1 % positive. For 34 of the 37 investigated species significant trends could be calculated. In 20 or 54 % of these species they were (exclusively or predominately) negative. 14 species showed negative trends at least at two stations. Mainly positive trends could be found in only 10 species. Taking negative trends and tendencies (negative signs) together, 26 or 70 % of the species were included. This strongly negative bias is not accidental in a statistical sense. Since the increases could not balance the declines, the annual trapping totals of all three stations also showed negative trends. The mean annual decline in the stations Mettnau, Reit and Illmitz was about 1.6 %. Trends and tendencies of the species show a high degree of agreement for the three stations. This suggests that in the species migrating through central Europe rather uniform factors control the population levels. From the most recent literature results, it appears that most of the species with declining trapping figures in the MRI-Program demonstrate decreases elsewhere.The trapping figures of the MRI-Program and data from the literature together clearly indicate that considerable parts of our small birds are affected by declines as have been known in many large bird species for long time. In a number of species the decreases have been quite dramatic and their annual trapping totals are now so low that a number of problems can no longer be studied. We should be alarmed when considering this negative development among our small birds as we must be with respect to many large bird species. The reasons for the declines are almost unkown and thus there is lack of appropriate counter-measures. Hitherto existing efforts for conservation which are briefly discussed have not been able to prevent the decline. Consequently, bird conservation in the future has to be based on more effective methods. Proposals, briefly raised, will be discussed in a forthcoming paper.

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Mit Unterstützung der Deutschen Forschungsgemeinschaft und gefördert mit Hilfe von Forschungsmitteln des Landes Niedersachsen. 15. Mitteilung aus dem MRI-Programm     

Cloning and Characterization of Yeast LEU4, One of Two Genes Responsible for α-Isopropylmalate Synthesis

By complementation of an alpha-isopropylmalate synthase-negative mutant of Saccharomyces cerevisiae (leu4 leu5), a plasmid was isolated that carried a structural gene for alpha-isopropylmalate synthase. Restriction mapping and subcloning showed that sequences sufficient for complementation of the leu4 leu5 strain were located within a 2.2-kilobase SalI-PvuII segment. Southern transfer hybridization indicated that the cloned DNA was derived intact from the yeast genome. The cloned gene was identified as LEU4 by integrative transformation that caused gene disruption at the LEU4 locus. When this transformation was performed with a LEU4fbr LEU5 strain, the resulting transformants had lost the 5',5',5'-trifluoro-D,L-leucine resistance of the recipient strain but were still Leu+. When it was performed with a LEU4 leu5 recipient, the resulting transformants were Leu-. The alpha-isopropylmalate synthase of a transformant that carried the LEU4 gene on a multicopy plasmid (in a leu5 background) was characterized biochemically. The transformant contained about 20 times as much alpha-isopropylmalate synthase as wild type. The enzyme was sensitive to inhibition by leucine and coenzyme A, was inactivated by antibody generated against alpha-isopropylmalate synthase purified from wild type and was largely confined to the mitochondria. The subunit molecular weight was 65,000-67,000. Limited proteolysis generated two fragments with molecular weights of about 45,000 and 23,000. Northern transfer hybridization showed that the transformant produced large amounts of LEU4-specific RNA with a length of about 2.1 kilonucleotides. The properties of the plasmid-encoded enzyme resemble those of a previously characterized alpha-isopropylmalate synthase that is predominant in wild-type cells. The existence in yeast of a second alpha-isopropylmalate synthase activity that depends on the presence of an intact LEU5 gene is discussed.