Eukaryotic translation elongation factor 1A (eEF1A) domain I from S. cerevisiae is required but not sufficient for inter-species complementation

Ethanolamine phosphoglycerol (EPG) is a protein modification attached exclusively to eukaryotic elongation factor 1A (eEF1A). In mammals and plants, EPG is linked to conserved glutamate residues located in eEF1A domains II and III, whereas in the unicellular eukaryote Trypanosoma brucei, only domain III is modified by a single EPG. A biosynthetic precursor of EPG and structural requirements for EPG attachment to T. brucei eEF1A have been reported, but nothing is known about the EPG modifying enzyme(s). By expressing human eEF1A in T. brucei, we now show that EPG attachment to eEF1A is evolutionarily conserved between T. brucei and Homo sapiens. In contrast, S. cerevisiae eEF1A, which has been shown to lack EPG is not modified in T. brucei. Furthermore, we show that eEF1A cannot functionally complement across species when using T. brucei and S. cerevisiae as model organisms. However, functional complementation in yeast can be obtained using eEF1A chimera containing domains II or III from other species. In contrast, yeast domain I is strictly required for functional complementation in S. cerevisiae.

EFFECTS OF PRENATAL DEXAMETHASONE ON HIPOPCAMPAL SEROTONIN 1A RECEPTORS IN ADULT MALE RATS

The main activation route for the stress response is the hypothalamo-pituitaryadrenal axis (HPA) and the sympatho-adrenomedullary system. The HPA axis is a neuroendocrine feedback loop mediated by an array of tissue specific hormones, receptors and neurotransmitters that regulate glucocorticoid (GC) release. GCs are steroidal hormones produced by the adrenal glands and are key players in a negativefeedback loop controlling HPA activity. They influence the HPA axis through glucocorticoid receptors in the hypothalamus and pituitary and through both glucocorticoid (GR) and mineralcorticoid receptors (MR) that are co-localized in the hippocampus. Repeated or chronic stress exerts a negative influence on these HPA axis regulatory sites and contributes to potentially pathological conditions, especially during early development. For example, chronic stress promotes increased maternal adrenal gland secretion of glucocortiocoid, leading to abnormally high concentrations of GC inthe fetal environment. The timing and maturation of the HPA axis relative to birth is highly species specific and is closely linked to landmarks in fetal development. In rats this development of the HPA axis takes place in utero and continues even shortly after birth. It is likely that the maternal endocrine environment will affect fetal development during this critical time point and may alter the overall set point for the expression ofgenes and their protein products that mediate fetal HPA axis function. Dexamethasone (DEX) is a synthetic glucocorticoid (sGC) and is a consensus treatment in preterm pregnancies used to expedite fetal lung development. However it has been shown that DEX causes long term physiological and behavioral disorders in prenatally-exposed laboratory animals. Previous studies have also shown that it alters the MR: GR receptor ratio in the hippocampus. Taking into consideration corticosteroid regulation of serotonin receptors, especially 5HT1A receptors and their putative interaction with glucocorticoid receptors in the hippocampus, we hypothesized that prenatal DEX exposure would lead to changes in the expression and function of 5HT1A receptors in the hippocampus. We administered DEX to rat dams during the last trimester of gestation and investigated the changes in these receptors in the adult rat offspring. Radioligand receptor binding assays were used to study hippocampal 5HT1A receptor binding affinity and number. Our results demonstrate that hippocampal 5HT1A receptors are increased in the DEX animalscompared with controls by 36%, with no change in binding affinity. The efficiency of ligand-induced receptor signal transduction via G-protein activation was also studied using [35S]GTPγS incorporation assay. Using this technique, we showed that there was no significant difference in the maximum ligand mediated stimulation (Emax) of 5HT1Areceptors between control and dex exposed animals. However, the intracellular signalling efficiency of hippocampal 5HT1A receptors was diminished, since a significant increase in EC50 values was obtained with the dex exposed group showing a value 51% higherEC50 than controls. Taken together these data illustrate a considerable change in the 5HT1A component of the serotonergic system following prenatal DEX exposure.

Clinical and molecular characterization of the potential CF disease modifier syntaxin 1A

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator gene (CFTR). Disease severity in CF varies greatly, and sibling studies strongly indicate that genes other than CFTR modify disease outcome. Syntaxin 1A (STX1A) has been reported as a negative regulator of CFTR and other ion channels. We hypothesized that STX1A variants act as a CF modifier by influencing the remaining function of mutated CFTR. We identified STX1A variants by genomic resequencing patients from the Bernese CF Patient Data Registry and applied linear mixed model analysis to establish genotype-phenotype correlations, revealing STX1A rs4363087 (c.467-38A>G) to significantly influence lung function. The same STX1A risk allele was recognized in the European CF Twin and Sibling Study (P=0.0027), demonstrating that the genotype-phenotype association of STX1A to CF disease severity is robust enough to allow replication in two independent CF populations. rs4363087 is in linkage disequilibrium to the exonic variant rs2228607 (c.204C>T). Considering that neither rs4363087 nor rs2228607 changes the amino-acid sequence of STX1A, we investigated their effects on mRNA level. We show that rs2228607 reinforces aberrant splicing of STX1A mRNA, leading to nonsense-mediated mRNA decay. In conclusion, we demonstrate the clinical relevance of STX1A variants in CF, and evidence the functional relevance of STX1A variant rs2228607 at molecular level. Our findings show that genes interacting with CFTR can modify CF disease progression.European Journal of Human Genetics advance online publication, 10 April 2013; doi:10.1038/ejhg.2013.57.

Solution structure and interaction of cupiennin 1a, a spider venom peptide, with phospholipid bilayers

The solution structure of cupiennin 1a, a 35 residue, basic antibacterial peptide isolated from the venom of the spider Cupiennius salei, has been determined by nuclear magnetic resonance (NMR) spectroscopy. The peptide was found to adopt a helix−hinge−helix structure in a membrane mimicking solvent. The hinge may play a role in allowing the amphipathic N-terminal helix and polar C-terminal helix to orient independently upon membrane binding, in order to achieve maximal antibacterial efficacy. Solid-state 31P and 2H NMR was used to further study the effects of cupiennin 1a on the dynamic properties of lipid membranes, using zwitterionic chain deuterated dimyristoylphosphatidylcholine (d54-DMPC) and anionic dimyristoylphosphatidylglycerol (DMPG) multilamellar vesicles. In d54-DMPC alone, cupiennin 1a caused a decrease in the 31P chemical shift anisotropy, indicating some interaction with the lipid head groups, and a decrease in order over the entire acyl chain. In contrast, for the mixed (d54-DMPC/DMPG) lipid system cupiennin 1a appeared to induce lateral separation of the two lipids as evidenced by the 31P spectra, in which the peptide preferentially interacted with DMPG. Little effect was observed on the deuterated acyl chain order parameters in the d54-DMPC/DMPG model membranes. Furthermore, 31P NMR relaxation measurements confirmed a differential effect on the lipid motions depending upon the membrane composition. Therefore, subtle differences are likely in the mechanism by which cupiennin 1a causes membrane lysis in either prokaryotic or eukaryotic cells, and may explain the specific spectrum of activity.

Cupiennin 1a, an antimicrobial peptide from the venom of the neotropical wandering spider Cupiennius salei, also inhibits the formation of nitric oxide by neuronal nitric oxide synthase

Cupiennin 1a (GFGALFKFLAKKVAKTVAKQAAKQGAKYVVNKQME-NH2) is a potent venom component of the spider Cupiennius salei. Cupiennin 1a shows multifaceted activity. In addition to known antimicrobial and cytolytic properties, cupiennin 1a inhibits the formation of nitric oxide by neuronal nitric oxide synthase at an IC50 concentration of 1.3 +/- 0.3 microM. This is the first report of neuronal nitric oxide synthase inhibition by a component of a spider venom. The mechanism by which cupiennin 1a inhibits neuronal nitric oxide synthase involves complexation with the regulatory protein calcium calmodulin. This is demonstrated by chemical shift changes that occur in the heteronuclear single quantum coherence spectrum of 15N-labelled calcium calmodulin upon addition of cupiennin 1a. The NMR data indicate strong binding within a complex of 1 : 1 stoichiometry.

Phosphatidylethanolamine is the precursor of the ethanolamine phosphoglycerol moiety bound to eukaryotic elongation factor 1A

In addition to its conventional role during protein synthesis, eukaryotic elongation factor 1A is involved in other cellular processes. Several regions of interaction between eukaryotic elongation factor 1A and the translational apparatus or the cytoskeleton have been identified, yet the roles of the different post-translational modifications of eukaryotic elongation factor 1A are completely unknown. One amino acid modification, which so far has only been found in eukaryotic elongation factor 1A, consists of ethanolamine-phosphoglycerol attached to two glutamate residues that are conserved between mammals and plants. We now report that ethanolamine-phosphoglycerol is also present in eukaryotic elongation factor 1A of the protozoan parasite Trypanosoma brucei, indicating that this unique protein modification is of ancient origin. In addition, using RNA-mediated gene silencing against enzymes of the Kennedy pathway, we demonstrate that phosphatidylethanolamine is a direct precursor of the ethanolamine-phosphoglycerol moiety. Down-regulation of the expression of ethanolamine kinase and ethanolamine-phosphate cytidylyltransferase results in inhibition of phosphatidylethanolamine synthesis in T. brucei procyclic forms and, concomitantly, in a block in glycosylphosphatidylinositol attachment to procyclins and ethanolamine-phosphoglycerol modification of eukaryotic elongation factor 1A.

N-terminal aromatic residues closely impact the cytolytic activity of cupiennin 1a, a major spider venom peptide

Cupiennins are small cationic a-helical peptides from the venom of the ctenid spider Cupiennius salei which are characterized by high bactericidal as well as hemolytic activities. To gain insight into the determinants responsible for the broad cytolytic activities, two analogues of cupiennin 1a with different N-terminal hydrophobicities were designed. The insecticidal, bactericidal and hemolytic activities of these analogues were assayed and compared to the native peptide. Specifically, substitution of two N-terminal Phe residues by Ala results in less pronounced insecticidal and cytolytic activity, whereas a substitution by Lys reduces strongly its bactericidal activity and completely diminishes its hemolytic activity up to very high tested concentrations. Biophysical analyses of peptide/bilayer membrane interactions point to distinct interactions of the analogues with lipid bilayers, and dependence upon membrane surface charge. Indeed, we find that lower hemolytic activity was correlated with less surface association of the analogues. In contrast, our data indicate that the reduced bactericidal activity of the two cupiennin 1a analogues likely correspond to greater bilayer-surface localization of the peptides. Overall, ultimate insertion and destruction of the host cell membrane is highly dependent on the presence of Phe-2 and Phe-6 (Cu 1a) or Leu-6 (Cu 2a) in the N-terminal sequences of native cupiennins.

Responses to rapid warming at Termination 1a at Gerzensee (Central Europe): Primary succession, albedo, soils, lake development, and ecological interactions

The transition from the Oldest Dryas to the Bølling around 14,685 cal yr BP was a period of extremely rapid climatic warming. From a single core of lake marl taken at Gerzensee (Switzerland) we studied the transition in stable isotopes of oxygen and carbon on bulk sediment and charophyte remains, as well as on monospecific samples of ostracods, after Pisidium a; in addition pollen, chironomids, and Cladocera were analyzed. The δ18O record serves as an estimate of mean air temperature, and by correlation to the one from NGRIP in Greenland it provides a timescale. The timing of responses: The statistically significant zone boundaries of the biostratigraphies are telescoped at the rapid increase of about 3‰ in δ18O at the onset of Bølling. Biotic responses may have occurred within sampling resolution (8 to 16 years), although younger zone boundaries are less synchronous. Gradual and longer-lasting responses include complex processes such as primary or secular succession. During the late-glacial interstadial of Bølling and Allerød, two stronger and two weaker cool phases were found. Biological processes involved in the responses occurred on levels of individuals (e.g. pollen productivity), of populations (increases or decreases, immigration, or extinction), and on the ecosystem level (species interactions such as facilitation or competition). Abiotic and biotic interactions include pedogenesis, nitrogen-fixation, nutrient cycling, catchment hydrology, water chemistry of the lake and albedo (controlled by the transition from tundra to forest). For the Swiss Plateau this major change in vegetation induced a change in the mammal fauna, which in turn led to changes in the tool-making by Paleolithic people.

Die Handschriften der Stadt- und Universitätsbibliothek Frankfurt am Main : (Fortsetzung von Teil 1A)

beschr. von Ernst Róth und Leo Prijs

Na 1 Nachlass Max Horkheimer, 661 - Berichte über die Arbeit des Instituts für Sozialforschung, "Research-Project on Anti-Semitism" u.a. (p. IX 78-IX 92.1a)

"Institut für Sozialforschung: Research Projects", Tabellarische Zusammenstellung (Juni 1953), Typoskript, 2 Blatt; "Institut für Sozialforschung an der Johann Wolfgang Goethe-Universität Frankfurt, Germany: Memorandum" (1953), a) Typoskript, 12 Blatt, b) Typoskript, 13 Blatt; "Projects of the Institute for Social Research" (1.7.1954), Typoskript, 5 Blatt; "Institut für Soziaforschung: Mitteilungen an die Presse" (November 1955), als Typoskript vervielfältigt, 6 Blatt; Clark: "Institut für Sozialforschung an der Johann Wolfgang Goethe-Universität in Frankfurt am Main" (etwa 1955), Typoskript, englisch, mit handschriftlichen Korrekturen, unter anderem von Max Horkheimer, 2 Blatt; "Institut für Sozialforschung" (6.3.1958), a) als Typoskript vervielfältigt, 10 Blatt, b) Typoskript, 22 Blatt, c) Kurzfassung, Typoskript, 3 Blatt, d) Teilstück, Typoskript, 1 Blatt, e) Sonderdruck, 15 Seiten; Abgeschlossene und laufende Arbeiten des Instituts. Tabellarische Zusammenstellung (1958?), Typoskript, 1 Blatt; "Laufende Studien" Tabellarische Aufstellung (25.5.1961), Typoskript, 1 Blatt; Friedrich Pollock (?): Anmerkungen zu Paul Klukes Darstellung des Instituts für Sozialforschung in dessen "Geschichte der Frankfurter Universität" (8.9.1969), Typoskript mit handschriftlichen Korrekturen, 4 Blatt; Theodor W. Adorno: Über die Arbeiten des Instituts für Sozialforschung (Datierung unklar, etwa 1941-45), Teilstück eines Entwurfs, Typoskript mit eigenhändiger Korrektur, 2 Blatt; Über die wesentliche Aufgabe des Instituts: Vereinigung geisteswissenschaftlicher und empirischer Methoden (Datierung unklar), Typoskript, 1 Blatt; "Forschungsprojekte des Instituts" (Datierung unklar, etwa 1950), Typoskript mit handschriftlichen Ergänzungen von Friedrich Pollock, 5 Blatt; "Vorträge im Institut für Sozialforschung. Einladungen und tabellarische Aufstellungen" (1953 u.a.), 5 Blatt; "Vorlesungen und Übungen im Institut für Sozialforschung, Ankündigungen aus den Jahren 1952-64, 9 Blatt; "International Institute of Social Research: Research-Project on Anti-Semitism" (1939-42) (veröffentlicht in Studies in Philosophy and Social Science Bd. IX, 1941, S. 124-143): 1. "Research Project on Anti-Semitism", a) Typoskript, 45 Blatt;

Na 1 Nachlass Max Horkheimer, 695 - "The Collapse of German Democracy and the Expansion of National Socialism", "Cultural Aspects of National Socialism" u.a. (p. IX 169.1a - IX 170.1-4)

"The Collapse of German Democracy and the Expansion of National Socialism" (1940):; 1. Darstellung des Forschungsprojekts (15.9.1940), b. Typoskript mit handschriftlichen Korrekturen, 78 Blatt; 2. "Research work on recent trends in the history of ideas (parts of the Research project on the Collapse of German Democracy would be included)". Als Memorandum zur Eröffnung zur Eröffnung einer Zweigstelle des Instituts in Los Angeles (12.12.1940): a) Typoskript, 2 Blatt, b) Teilstück, Typoskript mit handschriftlichen Korrekturen, 1 Blatt, c) Teilstück, Typoskript mit handschriftlichen Korrekturen, 1 Blatt, d) Teilstück, Typoskript, 1 Blatt, e) Teilstück, Typoskript, 1 Blatt, f) Entwurf, Typoskript mit handschriftlichen Korrekturen und Manuskript, 3 Blatt; 3. University of California, Los Angeles: 2 Briefe (Abschrift) von Max Horkheimer, o.O., 1940, 2 Briefe (Abschrift) an Max Horkheimer, 1940, 2 Blatt; A.R.L. Gurland: "Survey of Structural Changes in the German Economy, 1933 to 1939. Technological Bases and Organizational Forms of the National Socialist Economic System". Typoskript mit handschriftlichen Korrekturen unter anderem von Theodor W. Adorno, 48 Blatt (formal nicht identisch mit "Technological Trends and Economic Structure under National Socialism", Studies in Philosophy and Social Science, Bd. IX, 1941, S. 226ff.); "Cultural Aspects of National Socialism. A Research Project" (1941):; 1. Institute of Social Research: Mitteilung über das Forschungsprojekt und das 'Supplementary Statement', Typoskript, englisch, 4 Blatt; 2. Supplementary Statement to the Research Project, a) Typoskript, 14.4.1941, 63 Blatt, b) Typoskript, 12.4.1941, mit handschriftlichen Korrekturen, 35 Blatt; 3. "Cultural Aspects of National Socialism. A Research Project" (24.2.1941), a) als Typoskript vervielfältigt, 54 Blatt, b) Typoskript mit handschriftlichen Korrekturen, 34 Blatt, c) Fassung Januar 1941, Typoskript mit handschriftlichen Korrekturen, 40 Blatt; 4. Inhaltsverzeichnisse, mit handschriftlichen Korrekturen, 3 Blatt;