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Dealing with potential stress in species that have high husbandry requirements, such as elephants, is a challenge for zoos. The objective of the present study was to determine whether positive reinforcement training (PRT) and exposure to a novel object (NOV) for enrichment induced a salivary cortisol response indicative of activation of the hypothalamic–pituitary–adrenal (HPA) axis and which factors determine individual variation in this regard in captive African elephants. We repeatedly sampled the saliva of ten animals (three zoos) for the analysis of cortisol (SACort) before and up to 60 min (in 10–15 min intervals) after the onset of PRT (three repeats) or NOV (nine repeats), which lasted 10 min. There was considerable individual variation in SACort in response to PRT or NOV. Using mixed models, we were able to control these and to reveal that PRT was associated with high SACort before and relatively low SACort after PRT, while NOV induced a moderate SACort increase. The individual differences in SACort were related to age and sex (NOV), while the effects of zoo, handling method (free vs. protected contact) and reproductive and social status were variable. We conclude that positive affective states, such as anticipation or arousal, should be taken into account when interpreting the differences in the SACort responses between PRT and NOV. In addition, understanding the individuality of stress will support management decisions aimed at promoting captive elephant welfare.
Diadenosinpolyphosphate (ApnAs), wirken in einer Vielzahl unterschiedlicher Gewebe als extrazelluläre Signalmoleküle. Ihre asymmetrische Hydrolyse durch Enzyme auf der Zelloberfläche oder durch lösliche Enzyme im extrazellulären Milieu wurde in der Literatur bereits mehrfach beschrieben. Die molekulare Identität dieser Enzyme war jedoch nicht bekannt.
In der vorliegenden Arbeit wurden die Fähigkeit von NPP1, NPP2 und NPP3, den drei Mitgliedern der E-NPP-Familie, Diadenosinpolyphosphate zu hydrolysieren, untersucht. Dazu wurden humanes NPP1 und NPP3 aus der Ratte heterolog in CHO-Zellen exprimiert und die enzymatische Aktivität wurde anhand von Membranfraktionen analysiert. Für die Charakterisierung der katalytischen Eigenschaften von NPP2 wurde eine lösliche sekretierte Form des humanen NPP2 aus partiell aufgereinigtem Vaccinia-Viruslysat eingesetzt. Es konnte gezeigt werden, dass die heterolog exprimierten Enzyme NPP1, NPP2 und NPP3 die untersuchten Diadenosinpolyphosphate Diadenosin-5´,5´´´-P1,P3-triphosphat (Ap3A), Diadenosin-5´,5´´´-P1,P4-tetraphosphat (Ap4A) und Diadenosin-5´,5´´´-P1,P5-pentaphosphat (Ap5A), sowie das Diguanosinpolyphosphat Diguanosin-5´,5´´´-P1,P4-tetraphosphat (Gp4G) hydrolysieren.. Ein Vergleich der Hydrolyseraten zeigte, dass NPP1-2 Ap3A, Ap4A, Ap5A und Gp4G mit vergleichbarer Rate hydrolysieren. NPP3 zeigte ebenfalls keine Präferenz für eines der untersuchten Diadenosinpolyphosphate, hydrolysierte aber Gp4G im Vergleich zu Ap4A deutlich langsamer. Die Hydrolyse der Dinukleotide erfolgte asymmetrisch durch Spaltung der α,β-Pyrophosphatbindung. Als primäre Hydrolyseprodukte entstanden Nukleosid-5´-Monophosphat und der verbleibende Mononukleotidrest Npn-1. Für Ap3A als Substrat wurde für NPP1-3 ein alkalisches pH-Optimum mit maximaler Aktivität bei pH 8.5-9 (NPP1 und NPP3), bzw. pH 10 (NPP2) nachgewiesen. Die enzymatische Aktivität von NPP1-3 wurde durch EDTA inhibiert und die Km-Werte für Ap3A lagen mit 5,1 ± 3,6 µM (NPP1), 8,0 ± 0,5 µM (NPP2) und 49,5 ± 17,7 µM (NPP3) im niedrigen mikromolaren Bereich. Untersuchungen zur Hemmbarkeit der NPP-vermittelten Diadenosinpolyphosphathydrolyse (Ap4A) zeigten, dass die Enzyme NPP1, NPP2 und NPP3 nach Koapplikation verschiedener P2-Rezeptorantagonisten in unterschiedlichem Ausmaß inhibiert wurden. Cibacron Blue inhibierte kräftig alle drei Enzyme, während PPADS einen stärkeren inhibitorischen Einfluss auf die katalytsiche Aktivität von NPP1 und NPP3 als auf die katalytische Aktivität von NPP2 zeigte. Suramin inhibierte dagegen nur die NPP1 und NPP2 katalysierte Ap4AHydrolyse und hatte keinen Einfluss auf NPP3. Eine Inhibierung von NPP1-3 durch NaF wurde nicht beobachtet. Eine geringe inhibitorische Wirkung auf NPP1 wurde durch die extrazellulären Matrix-Komponenten Heparin und Heparansulfat, durch ATP und die Nukleotidanaloga, AMP-CP, AMP-CPP und ATP-γ-S beobachtet. NPP2 und NPP3 wurden durch AMP-CP nicht inhibiert. Den schwächsten inhibitorischen Einfluss zeigte ATP auf die durch NPP3-vermittelte Ap4AHydrolyse.
Die hier für NPP1-3 ermittelten katalytischen Eigenschaften zeigten Übereinstimmgen aber auch Unterschiede gegenüber früheren Daten, die für die Hydrolyse von Diadenosinpolyphosphaten auf der Oberfläche von Zellen ermittelt wurden. Insgesamt sprechen die Ergebnisse dafür, dass NPP1-3 die Hauptvertreter der Diadenosinpolyphosphat-hydrolysierenden Enzyme in Säugergewebe darstellen. Möglicherweise gibt es aber auch ApnA-hydrolysierende Enzyme, die nicht zu den bisher charakterisierten Mitgliedern der E-NPP-Familie zugehören.
In einem zweiten Teil der Arbeit wurde die Expression von NPP1-3 im Gehirn der Ratte mittels Western-Blot-Analyse (Entwicklungsstadien P1, P21 und adult) untersucht. Aufgrund der geringen Spezifität der gegen NPP1 und NPP2 zur Verfügung stehenden Antikörper, konnten jedoch keine eindeutigen Aussagen zur Expression von NPP1 und NPP2 im Gehirn getroffen werden. NPP3 konnte im Rattengehirn nachgewiesen werden. Die Expression war entwicklungsabhängig und nahm mit zunehmendem Alter der Tiere deutlich ab. Die Entwicklung spezifischer Antikörper erscheint ein lohnender Ansatz, um die zelluläre Verteilung von NPP1-3 im Nervengewebe zu bestimmen.
Biallelic pathogenic variants in CLPP, encoding mitochondrial matrix peptidase ClpP, cause a rare autosomal recessive condition, Perrault syndrome type 3 (PRLTS3). It is characterized by primary ovarian insufficiency and early sensorineural hearing loss, often associated with progressive neurological deficits. Mouse models showed that accumulations of (i) its main protein interactor, the substrate-selecting AAA+ ATPase ClpX, (ii) mitoribosomes, and (iii) mtDNA nucleoids are the main cellular consequences of ClpP absence. However, the sequence of these events and their validity in human remain unclear. Here, we studied global proteome profiles to define ClpP substrates among mitochondrial ClpX interactors, which accumulated consistently in ClpP-null mouse embryonal fibroblasts and brains. Validation work included novel ClpP-mutant patient fibroblast proteomics. ClpX co-accumulated in mitochondria with the nucleoid component POLDIP2, the mitochondrial poly(A) mRNA granule element LRPPRC, and tRNA processing factor GFM1 (in mouse, also GRSF1). Only in mouse did accumulated ClpX, GFM1, and GRSF1 appear in nuclear fractions. Mitoribosomal accumulation was minor. Consistent accumulations in murine and human fibroblasts also affected multimerizing factors not known as ClpX interactors, namely, OAT, ASS1, ACADVL, STOM, PRDX3, PC, MUT, ALDH2, PMPCB, UQCRC2, and ACADSB, but the impact on downstream metabolites was marginal. Our data demonstrate the primary impact of ClpXP on the assembly of proteins with nucleic acids and show nucleoid enlargement in human as a key consequence.
The maintenance of cellular homeostasis over time is essential to avoid the degeneration of biological systems leading to aging and disease. Several interconnected pathways are active in this kind of quality control. One of them is autophagy, the vacuolar degradation of cellular components. The absence of the sorting nexin PaATG24 (SNX4 in other organisms) has been demonstrated to result in impairments in different types of autophagy and lead to a shortened lifespan. In addition, the growth rate and the size of vacuoles are strongly reduced. Here, we report how an oleic acid diet leads to longevity of the wild type and a PaAtg24 deletion mutant (ΔPaAtg24). The lifespan extension is linked to altered membrane trafficking, which abrogates the observed autophagy defects in ΔPaAtg24 by restoring vacuole size and the proper localization of SNARE protein PaSNC1. In addition, an oleic acid diet leads to an altered use of the mitochondrial respiratory chain: complex I and II are bypassed, leading to reduced reactive oxygen species (ROS) production. Overall, our study uncovers multiple effects of an oleic acid diet, which extends the lifespan of P. anserina and provides perspectives to explain the positive nutritional effects on human aging.
The accumulation of functionally impaired mitochondria is a key event in aging. Previous works with the fungal aging model Podospora anserina demonstrated pronounced age-dependent changes of mitochondrial morphology and ultrastructure, as well as alterations of transcript and protein levels, including individual proteins of the oxidative phosphorylation (OXPHOS). The identified protein changes do not reflect the level of the whole protein complexes as they function in-vivo. In the present study, we investigated in detail the age-dependent changes of assembled mitochondrial protein complexes, using complexome profiling. We observed pronounced age-depen-dent alterations of the OXPHOS complexes, including the loss of mitochondrial respiratory supercomplexes (mtRSCs) and a reduction in the abundance of complex I and complex IV. Additionally, we identified a switch from the standard complex IV-dependent respiration to an alternative respiration during the aging of the P. anserina wild type. Interestingly, we identified proteasome components, as well as endoplasmic reticulum (ER) proteins, for which the recruitment to mitochondria appeared to be increased in the mitochondria of older cultures. Overall, our data demonstrate pronounced age-dependent alterations of the protein complexes involved in energy transduction and suggest the induction of different non-mitochondrial salvage pathways, to counteract the age-dependent mitochondrial impairments which occur during aging.
The nucleus reuniens drives hippocampal goal‑directed trajectory sequences for route planning
(2023)
Goal-directed spatial navigation requires accurate estimates of one’s position and destination, as well as careful planning of a route between them to avoid known obstacles in the environment. Despite its general importance across species, the neural circuitry supporting the ability for route planning remains largely unclear. Previous studies described that place cells in the hippocampal CA1 encode the animal's next movement direction (Wood et al., 2000; Ito et al., 2015) and upcoming navigational routes (Pfeiffer & Foster, 2013). However, it has been shown that part of the CA1 activity representing the animal’s future behaviors is not necessarily generated in the hippocampus, but is derived from the medial prefrontal cortex (PFC) via the nucleus reuniens of the thalamus (RE) (Ito et al., 2015). Notably, the importance of the PFC in navigation has been demonstrated in several studies, including the recent finding of a goal map in the orbitofrontal cortex (Basu et al., 2021). Therefore, I hypothesized that information flow from the PFC to CA1 via the RE plays a key role in route planning.
To assess the animals' route planning ability, I designed a new navigation task in which a rat has to navigate to a fixed target location from various starting positions in an arena. Furthermore, by adding an L-shaped wall in the maze and removing all light sources in the experimental room, this task forced the animals to plan a wall-avoiding route without relying on direct sensory perceptions. I confirmed that rats could learn this task successfully, memorizing the wall location and taking a smooth wall-avoidance route. To test the role of the RE, I inactivated RE neurons by expressing the inhibitory opsin SwiChR++, which resulted in a significant deficit in the animal’s route planning ability, taking a longer non-smooth path to the destination. By contrast, this manipulation did not affect navigation performance when a straight goal-directed route was available, suggesting a specific role of the RE in route planning. I further found that DREADDs-mediated inactivation of neurons in the bilateral hippocampi resulted in a similar deficit in route planning ability, implying cooperation between the RE and the hippocampus.
I finally examined the activity of hippocampal CA1 neurons with and without RE inactivation. While neurons in the hippocampus exhibited brief trajectory sequences corresponding to the animal’s subsequent goal-directed journey, I found that this goal-directed bias of trajectory events was significantly reduced by RE inactivation, likely associated with route-planning deficits in these animals.
Altogether, this dissertation demonstrates the role of the RE from both behavioral and neural coding perspectives, identifying a pivotal circuit element supporting the animal’s route-planning ability.
Fungi belonging to the Rhytismatales (Ascomycota) are parasites or endophytes of plants, some are saprophytes. Their fruiting bodies are localized in different organs of the host plants belonging to many different families of gymnosperms and angiosperms. Many species of Rhytismatales are known on species of Pinaceae, Ericaceae, and Poaceae. These fungi usually have ascomata that are more or less embedded in host tissue and open by longitudinal or radial splits. They have a more or less carbonized covering stroma, thin-walled, iodine negative asci, and ascospores usually covered by gelatinous sheaths.
In the present study, two lists of species of Rhytismatales in China are presented. One is based on literature and includes 103 species in 15 genera. The second one contains the names of the species in the present study, 57 species in 20 genera based on 90 specimens I collected in the Yunnan and Anhui province in China during July to August in 2001. 31 species in the second list are new species or new records for China, so we presently know 134 species in 22 genera of Rhytismatales for China. 28 new species of Rhytismatales are proposed, 21 species from the Yunnan province and seven from the Anhui province. Among them, three new species are proposed in three new genera, Nematococcomyces, New Genus 1, and New Genus 2, respectively. The 28 new species are Cerion sp., Coccomyces spp. 1-2, Colpoma spp. 1-2, Hypoderma spp. 1-6, Lirula sp., Lophodermella sp., Lophodermium spp. 1-5, Nematococcomyces rhododendri C.-L. Hou, M. Piepenbr. & Oberw., Neococcomyces sp., New Genus 1 sp., New Genus 2 sp., Rhytisma spp. 1-2, Soleella sp., Terriera spp. 1-2, and Therrya sp. The genus Davisomycella is proposed as a synonym of Lophodermella based on observations of the morphology, ecology, and the infected organ. The four genera Cerion, Naemacyclus, Terriera, and Therrya, and three species, Hypoderma rubi, Lophodermium uncinatum, and Naemacyclus pinastri, are reported for the first time for China. All the new taxa, the newly recorded ones, as well as six species which had not been illustrated in detail before, are carefully described and illustrated by line drawings in the present study.
The results show that species of Rhytismatales are highly diverse especially in the natural vegetation in high mountainous areas in China. Most species of Rhytismatales are conspicuously host specific. The diversity of Rhytismatales is closely related to that of the preferred hosts, which are members of Pinaceae, Ericaceae, and Cupressaceae. Based on the detailed morphological observations, the significance of different morphological characteristics for a natural classification of Rhytismatales is discussed. Genera are traditionally defined by character states of a few characteristics, namely the opening patterns of ascomata, the depth of ascomata in the host tissue, and asci and ascospore shape. Data from collections in the field, detailed morphological investigation, and molecular data show, however, that the ecology, the infected organ, the host relationship, and many other characteristics have to be combined to circumscribe natural groups.
The discussion of the systematic significance of morphological characteristics is complemented by molecular data. In the present study, partial nuclear large subunit rDNA sequences of 52 specimens representing 38 species are used to analyse phylogenetic relationships for members of Rhytismatales.
Most species of Rhytismatales are placed in a monophyletic group corresponding to the Rhytismatales in the Maximum Parsimony analysis. The delimitation of the Rhytismatales from the Helotiales is, however, difficult. Cyclaneusma minus should be transferred from the Rhytismatales to the Helotiales, and Cudonia circinans and Spathularia flavida from the Helotiales to the Rhytismatales. These tranfers have previously been proposed based on SSU rDNA analysis by other authors. New Genus 1 sp. has morphological characteristics typical for species of Rhytismatales. In the LSU rDNA analysis, however, it is more closely related to Helotiales rather than toRhytismatales. Therefore New Genus 1 sp. is placed in the Helotiales.
Tryblidiopsis pinastri is morphologically intermediate between members of Rhytismataceae and Cudoniaceae. LSU rDNA sequences in the present study show that T. pinastri is more closely related to species of Cudoniaceae. Therefore, this species is removed from the Rhytismataceae to the Cudoniaceae. The delimitation of further families could not be resolved in the present analysis.
Though many new morphological, ecological, and molecular phylogenetic findings are contributed for the first time, the systematic conclusions at generic, family, and order level can only be fragmentary in the present study. With more collections and more molecular data of the worldwide 450 known and many more unknown species of Rhytismatales at hand, a natural system combining morphological and molecular analysis can be elaborated.
Einleitung
APP und die Alzheimersche Krankheit
Das Alzheimer Amyloid Precursor Protein (APP) ist ein Typ-1 Transmembranprotein mit einem Molekulargewicht von 110-135 kDa [Selkoe et al. 1988, Weidemann et al. 1989]. Es wird in allen bisher untersuchten Geweben exprimiert und weist in mehrzelligen Organismen einen hohen Konservierungsgrad auf [Robakis et al. 1987, Rosen et al. 1989]. APP ist unter anderem Vorläufer des β-A4-Peptides (Aβ), das in extrazellulären Aggregaten (Plaques) im Zentralen Nervensystem von Alzheimer-Patienten akkumuliert [Masters et al. 1985]. Die sogenannte „Amyloid-Hypothese der Alzheimerschen Erkrankung“ besagt, dass das Aβ-Peptid eine pathologische Kaskade initiiert, die zur Bildung von amyloiden Plaques, neuronaler Funktionsstörung und letztendlich Demenz führt [Hardy 1997, Selkoe 1999].
Prozessierung des APP
Der Hauptanteil des zellulären APP wird über den (nicht pathogenen) α-Sekretase-Weg prozessiert, wobei das sekretorische APP (α-sAPP) freigesetzt wird, das beinahe der gesamten N-terminalen Ektodomäne des APP entspricht. Die α-Sekretase spaltet APP innerhalb der Aβ-Domäne und verhindert somit die Bildung des pathogenen Aβ-Peptides. Kandidaten für die Katalyse dieser Spaltung sind Proteasen der ADAM-Familie [Buxbaum et al. 1998, Hooper et al. 1997, Koike et al. 1999, Lammich et al. 1999, Loechel et al. 1998].
Das Aβ-Peptid entsteht bei der sukzessiven proteolytischen Spaltung des APP durch die sogenannten β- und γ-Sekretasen. Bei der β-Sekretase handelt es sich um die Aspartat-Protease BACE (β-site APP cleaving enzyme) [Hussain et al. 1999, Sinha et al. 1999, Vassar et al. 1999, Yan et al. 1999]. Die Identität der γ-Sekretase ist noch nicht endgültig geklärt, jedoch spielen Presenilin-1 und -2 sowie Nicastrin eine Rolle bei der γ-Spaltung des APP [de Strooper et al. 1998, 1999, Struhl et al. 2000, Wolfe et al. 1999].
Unter physiologischen Bedingungen wird ca. 30% des APP durch α-Sekretasen prozessiert, ein viel geringerer Anteil dagegen durch die β-Sekretasen. Mehr als die Hälfte des zellulären APP bleibt ungespalten [Koo 2002].
Biologische Funktionen des APP
Die Funktionen des APP lassen sich unterscheiden nach Funktionen der kurzen zytoplasmatischen Domäne und der ca. 100 kDa großen Ektodomäne (α-sAPP). Die zytoplasmatische Domäne des APP stellt eine Plattform für die Bindung verschiedener Interaktionspartner dar. In Kooperation mit den Bindungspartnern spielt APP eine Rolle in unterschiedlichsten zellulären Prozessen wie vesikulärem Transport, Zellmotilität oder Genaktivierung [Review siehe Annaert und de Strooper 2002]. Die meisten Interaktionspartner der zytoplasmatischen Domäne des APP binden an die YENPTY-Sequenz nahe des C-Terminus des APP, die auch als Signal für die Endozytose des APP dient [Perez et al. 1999].
Die sekretorische Ektodomäne des APP hat eine wachstumsfördernde und neuroprotektive Wirkung. Um diese Wirkung auszuüben, bindet α-sAPP an einen bisher unbekannten Rezeptor, der auf der Zelloberfläche diverser Zelltypen wie Neuronen, Fibroblasten, Thyreozyten und Keratinozyten exprimiert wird [Review siehe Schmitz et al. 2002].
Polarer Transport des APP
In polaren MDCK Zellen wird das APP-Holoprotein fast ausschließlich zur basolateralen Zelloberfläche transportiert [Haass et al. 1994]. Es wurde gezeigt, dass dieser polare Transport des APP durch Tyrosin 653 in der zytoplasmatischen Domäne des APP beeinflusst wird. Mutation dieses Tyrosins zu Alanin führte zu partieller Fehlsortierung von ca. 50% des APP zur apikalen Plasmamembran. Die Sekretion von α-sAPP dagegen fand in MDCK-Zellen unabhängig von Tyrosin 653 basolateral statt [Haass et al. 1995].
Intrazellulärer Proteintransport durch Adaptor-Protein-Komplexe
Am intrazellulären Proteintransport sind Adaptor-Protein-Komplexe (APs) beteiligt, die bestimmte Sortierungssignale in der zytoplasmatischen Domäne von Frachtproteinen erkennen. Bis heute sind vier dieser tetrameren AP-Komplexe (AP-1 bis AP-4) bekannt, die zum Teil verschiedene Isoformen einzelner Untereinheiten aufweisen, z.B. AP-1A und AP-1B [Review: Boehm und Bonifacino 2001]. Jeder AP-Komplex spielt eine Rolle in einem bestimmten Schritt des intrazellulären Proteintransportes. Für AP-1A wird eine Funktion im anterograden und retrograden Transport zwischen Endosomen und TGN beschrieben [Review: Hinners und Tooze 2003]. AP-2 vermittelt Endozytose verschiedener Transmembranproteine von der Plasmamembran [Review: Kirchhausen 2002]. AP-3 spielt eine Rolle im Proteintransport zu Lysosomen und Lysosom-ähnlichen Organellen wie Melanosomen [Robinson und Bonifacino 2001]. AP-4 sowie AP1-B sortieren Proteine zur basolateralen Plasmamembran polarer Epithelzellen [Fölsch et al. 1999, Simmen etal. 2002].
Die Sortierungsmotive, die von Adaptor-Komplexen in der zytoplasmatischen Domäne der Fracht-Proteine gebunden werden, enthalten in den meisten Fällen entweder ein Tyrosin oder zwei Leucine. Das gesamte Motiv besteht aus jeweils vier bis zehn Aminosäuren [Review siehe Bonifacino und Traub 2003].
Ziele der Arbeit
In der vorliegenden Arbeit wurde der polare Transport des APP in Epithelzellen untersucht. Ein Ziel war es, Faktoren zu finden, die den basolateralen Transport des APP in Abhängigkeit von Tyrosin 653 vermitteln. Des weiteren sollte der Transport von APP und sAPP in verschiedenen Epithelzelllinien analysiert werden. Um ein gutes Werkzeug zur Detektion von APP zu haben, wurden GFP-APP-Fusionsproteine hergestellt und charakterisiert.
Ergebnisse und Diskussion
GFP-APP-Fusionsproteine wurden hergestellt und in MDCK-, FRT- und LLC-PK1-Zellen stabil exprimiert. Die Charakterisierung der GFP-APP-Fusionsproteine durch Immunfluoreszenzanalysen zeigte, dass die chimeren Proteine im TGN sowie in peripheren Vesikeln lokalisiert sind und mit endogenem APP stark kolokalisieren. GFPAPP war somit gut geeignet, um den intrazellulären Transport des APP zu untersuchen.
Eine Analyse der zytoplasmatischen Domäne des APP im Bereich des Tyrosin 653 zeigte, dass dieses Tyrosin und die drei folgenden Aminosäuren (YTSI) ein Konsensus-Motiv für die Bindung von tetrameren Adaptor-Protein-Komplexen darstellen.
Zu Beginn dieser Arbeit waren AP-1 bis AP-3 bereits gut charakterisiert, wohingegen für AP-4 keine Funktion bekannt war. In Kollaboration mit Simmen et al. konnte gezeigt werden, dass AP-4 den basolateralen Transport einiger Proteine vermittelt [Simmen et al. 2002]. Immunfluoreszenzanalysen lokalisierten AP-4 im TGN und peripheren Vesikeln, die unterschiedlich von AP-1A/B markierten Strukturen waren. Da kaum Kolokalisation von AP-4 und AP-1A/B zu beobachten war, ist die Lokalisation von AP-4 und AP-1B, das auch eine Rolle im basolateralen Proteintransport spielt, in unterschiedlichen Subdomänen des TGN und unterschiedlichen vesikulären Strukturen anzunehmen.
Polarer Transport des APP durch Adaptor-Protein-Komplexe
Die mögliche Funktion von AP-1 und AP-4 im Transport von APP wurde zunächst mit Hilfe von in vitro-Bindungsstudien untersucht. Dazu wurde die zytoplasmatische Domäne des APP als GST-Fusionsprotein kloniert und exprimiert. Die Frachtproteinbindenden Untereinheiten von AP-1 und AP-4 wurden unter Verwendung von radioaktiv markiertem Methionin durch in vitro-Transkription und -Translation hergestellt. In Bindungsstudien interagierten AP-1A und AP-1B mit der zytoplasmatischen Domäne des APP, nicht aber AP-4. Diese Ergebnisse deuten an, dass AP-1A und AP-1B eine Rolle im intrazellulären Transport von APP spielen könnten. AP-4 dagegen scheint nicht an diesem Prozess beteiligt zu sein.
Durch Mutation des Tyrosin 653 in APP zu Alanin (Y653A) wurde die Interaktion zwischen AP-1B und APP stark verringert, was darauf hindeutet, dass dieses Tyrosin einen Teil des Bindungsmotivs für AP-1B darstellt. Übereinstimmend damit entspricht die genaue Aminosäureabfolge des Y653TSI-Motivs den Sotierungsmotiv-Präferenzen von AP-1B [Ohno et al. 1999]. Die Interaktion von AP-1A dagegen war mit WildtypAPP und der Tyrosin-Mutante vergleichbar und scheint somit auf einem anderen Interaktions-Motiv zu basieren. AP-1A und AP-1B erkennen somit unterschiedliche Sortierungsmotive in der zytoplasmatischen Domäne des APP und kooperieren möglicherweise im intrazellulären Transport des APP. Diese Ergebnisse sind der erste Bericht über eine Interaktion von Adaptor-Protein-Komplexen mit der zytoplasmatischen Domäne des APP.
Die Rolle von AP-1B im basolateralen Transport von APP wurde genauer untersucht mit Hilfe der LLC-PK1 Zelllinie, die kein AP-1B exprimiert [Ohno et al. 1999]. In LLCPK1-Zellen werden verschiedene Proteine unpolar zur apikalen und basolateralen Membran verteilt, die in MDCK-Zellen durch Interaktion mit AP-1B basolateral transportiert werden [Fölsch et al. 1999, Sugimoto et al. 2002]. Um den Transport von APP in polaren LLC-PK1-Zellen zu untersuchen, wurde Plasmamembran-ständiges GFP-APP durch zwei unabhängige Methoden nachgewiesen: die apikale oder basolaterale Oberfläche der Zellen wurde selektiv entweder biotinyliert oder mit GFPAntikörpern markiert. Beide Methoden zeigten, dass GFP-APP in LLC-PK1-Zellen sowohl an der apikalen als auch an der basolateralen Zelloberfläche lokalisiert ist. Somit wird auch APP in diesen Zellen im Vergleich zu MDCK-Zellen anders sortiert. Dieses Ergebnis festigt die Hypothese einer Funktion von AP-1B im Transport von APP, die aufgrund der Daten der in vitro-Bindungsstudien aufgestellt wurde.
Polare Sekretion des sAPP ist unabhängig vom Transport des Holoproteins
Neben dem Transport des APP-Holoproteins war auch die polare Sekretion des sAPP Thema dieser Arbeit. Es war gezeigt worden, dass basolaterale Sekretion des sAPP in MDCK-Zellen unabhängig vom Transport des APP-Holoproteins ist [Haass et al. 1995]. Dieses Ergebnis konnte in der vorliegenden Arbeit bestätigt und auf andere Zelllinien erweitert werden. Um die korrekte Sekretion von GFP-sAPP nachzuweisen, wurde die GFP-sAPP-Sekretion zunächst in polaren MDCK-Zellen untersucht, die stabil GFP-APP exprimierten. Da GFP am N-Terminus des APP angefügt ist, trägt auch das sezernierte APP die GFP-Markierung. GFP-sAPP konnte mittels Immunpräzipitation mit GFP-spezifischen Antikörpern lediglich im basolateralen Medium nachgewiesen werden. Somit sezernieren MDCK-Zellen GFP-sAPP in gleicher Polarität wie von Haass et al. für endogenes sAPP gezeigt wurde [Haass et al. 1995].
Experimente in GFP-APP exprimierenden LLC-PK1- und FRT-Zellen zeigten, dass auch hier die polare Sekretion des GFP-sAPP und der Transport des APPHoloproteins zwei unabhängige Prozesse sind. Polare LLC-PK1-Zellen transportierten GFP-APP zur apikalen und basolateralen Plasmamembran (siehe oben). GFP-sAPP-Sekretion aus polaren LLC-PK1-Zellen dagegen fand ausschließlich basolateral statt. In FRT-Zellen wurde GFP-sAPP im Gegensatz zu MDCK- und LLCPK1-Zellen apikal sezerniert. Kolokalisation des GFP-APP mit Transferrin-Rezeptor in FRT-Zellen deutete dagegen an, dass das Holoprotein wie in MDCK-Zellen basolateral transportiert wird. Dies ist auch zu erwarten, da FRT-Zellen AP-1B exprimieren und es auch in dieser Zelllinie basolateralen Transport vermittelt [A. Gonzalez, persönlich, ASCB 2003]. Nach diesen Ergebnissen zu urteilen, finden auch in FRT und LLC-PK1-Zellen APP-Transport und sAPP-Sekretion unabhängig voneinander statt.
Basolaterale sAPP-Sekretion ist unabhängig von der Ektodomäne
In MDCK-Zellen wurde zusätzlich die Sekretion eines GFP-APP untersucht, in dem der Großteil der Ektodomäne deletiert und durch GFP ersetzt wurde, die SekretaseSchnittstellen jedoch noch vorhanden waren. Durch Immunfluoreszenzanalyse wurde zunächst nachgewiesen, dass die subzelluläre Lokalisation dieser Deletionsmutante der des endogenen APP entspricht. Die Sekretion dieses stark verkürzten sAPP erfolgte wie die des Wildtyps basolateral. Dieses Ergebnis deutet an, dass die Determinante für die basolaterale Sekretion des sAPP nicht innerhalb der Ektodomäne liegt, wie in einigen älteren Publikationen angenommen wird [Haass et al. 1995, de Strooper et al. 1995]. Neuere Ergebnisse dagegen führen die polare Sekretion des sAPP auf die basolaterale Lokalisation der α-Sekretase zurück [Capell et al. 2002], was die basolaterale Sekretion der Deletionsmutante erklären könnte.
sAPP-Bindung an polaren Zellen
Durch Interaktion mit einem bisher unbekannten Rezeptorprotein erfüllt sAPP für verschiedene Zelltypen die Funktion eines Wachstumsfaktors [Saitoh et al., 1989, Pietrzik et al., 1998, Hoffmann et al., 2000]. Da viele Wachstumsfaktor-Rezeptoren selektiv entweder an der apikalen oder basolateralen Plasmamembran von Epithelzellen lokalisiert sind, wurden Bindungsstudien mit rekombinant exprimiertem sAPP (sAPPrec) an polaren FRT und MDCK-Zellen durchgeführt. Analyse der Bindung mit einem sAPPrec-spezifischen Antikörper zeigte, dass sAPP ausschließlich an der apikalen Plasmamembran beider Zelllinien bindet. Da die Sekretion des sAPP in FRT-Zellen ebenso apikal erfolgt, ist in dieser Zelllinie eine autokrine Regulation durch sAPP vorstellbar, was auch durch vorherige Ergebnisse angedeutet wurde [Pietrzik et al. 1998]. Für MDCK-Zellen, die sAPP basolateral sezernieren und apikal binden, muss ein anderer Regulationsmechanismus vorliegen. Es könnte sich um parakrine Regulation handeln, was jedoch noch bestätigt werden muss.
Fazit: In dieser Arbeit wurde zum ersten Mal gezeigt, dass tetramere Adaptor-ProteinKomplexe eine Rolle im intrazellulären Transport von APP spielen. In diesem Zusammenhang wurde die Funktion des AP-4-Komplexes in einer Kollaboration analysiert. Es wurde gezeigt, dass AP-1A und AP-1B eine Rolle im Transport von APP spielen. Eine Funktion von AP-4 im Transport von APP ist nach den vorliegenden Ergebnissen unwahrscheinlich. Untersuchungen zur APP-Sortierung in verschiedenen Epithelzelllinien zeigten, dass die Hypothese der Unabhängigkeit von APP-Transport und sAPP-Sekretion als genereller Mechanismus angesehen werden kann. Durch Analyse der sAPP-Bindung an polaren FRT- und MDCK-Zellen wurde erstmals die polare Lokalisation des putativen sAPP-Rezeptors untersucht, was einen ersten Einblick in den Mechanismus der sAPP-vermittelten Regulation in polaren Zellen ermöglichte.
Cytochrome P450 (CYP) enzymes oxidize, peroxidize and/or reduce cholesterol, vitamins, steroids, xenobiotics and numerous pharmacological substances in an oxygen- and NADPHdependent manner. Since many CYP isozymes are also capable of metabolizing arachidonic acid to biologically active products, CYP enzymes are often described as the third pathway of arachidonic acid metabolism i.e., in addition to cyclooxygenases and lipoxygenases. CYP enzymes are predominantly expressed in the liver while others, such as members of the CYP 2J, CYP 2C and CYP 4A subfamilies, can be detected in extrahepatic tissues, particularly in the cardiovascular system. Recent data suggest that a CYP 2C enzyme(s) expressed in coronary artery endothelial cells generate epoxyeicosatrienoic acids (5,6-; 8,9-; 11,12- and 14,15-EET) which contribute to the acute control of vascular tone and the longterm regulation of vascular homeostasis.
The expression of CYP 2C in coronary artery endothelial cells is regulated by a number of stimuli, such as cyclic stretch and fluid shear stress as well as by the corticosteroid cortisol and a number of CYP substrates (nifedipine, cerivastatin and -naphthoflavone). However, the signalling pathways and the transcription factors involved in regulating the expression of the gene are unknown.
Since most of the CYP 2C enzymes are transcriptionally regulated, we were interested in identifying the CYP 2C isoform(s) expressed in porcine coronary artery endothelial cells (PCAEC) as well as determining its/their promoter sequence(s). The overall goal was to study the involvement of different transcription factor binding elements in the regulation of the CYP 2C gene(s). Porcine coronary arteries were used given the possibility of analysing the results obtained at the cellular level with alterations in vascular function. Comparison of the porcine CYP 2C and the human CYP 2C8 and 2C9 promoters was also a major goal of this study.
To identify the relevant porcine CYP 2C isoform nested RT-PCR was performed using total RNA from porcine coronary artery endothelial cells. Comparison of the sequence of the product of this reaction with the NCBI database suggested that the CYP 2C expressed in PCAEC was approximately 85% homologous with the human CYP 2C9 enzyme. To obtain the full length CYP 2C isoform 5´ rapid amplification of cDNA end (5´ RACE) was performed using a downstream reverse gene specific primer which is conserved in all of the porcine CYP 2C isoforms. The intention behind using such a primer was to amplify all the possible CYP cDNAs expressed in PCAEC. With the 5´ RACE technology it was possible not only to identify the exact isoform (CYP 2C34) expressed in PCAEC, but it was also possible to amplify 550 bp of the 5´ upstream region. This result was authenticated by comparing the protein/nucleotide sequence with other human CYP 2C genes such as CYP 2C8 and CYP 2C9 as well as different porcine CYP 2C genes (CYP 2C34, CYP 2C49). Multiple protein/nucleotide sequence alignment revealed approximately 85-90% sequence identity. An exon1-2 specific radio-labelled probe of the CYP 2C34 gene was then used to screen a porcine genomic library for positive genomic clones containing the promoter region of the CYP 2C34 gene.
For the isolation of 5´ flanking region of CYP 2C34 gene a PCR-based directional genome walking strategy was used in which the positive porcine genomic BAC clones were taken as a DNA template. Four arbitrarily designed universal walking primers and a gene-specific primer derived from the CYP 2C34 gene sequence were employed and led to the identification and isolation of 1.4 kb of the 5´ flanking region.
The 1.4 kb 5´ flanking region of CYP 2C34 gene contains multiple transcription factor binding sites including glucocorticoid-responsive element (GRE), hypoxia-responsive element (HRE), CAAT-enhancer binding protein (C/EBP), stress responsive element (STRE) consensus sequences. CYP 2C34 promoter constructs were generated and reporter gene activity (luciferase) activity was compared with that of a promoterless vector (pGL3-Basic) at first in HEK cells and then in PCAEC. After using cortisol as a positive control to demonstrate that the promoter constructs generated were functional we determined the effects of physiologically relevant stimuli i.e., hypoxia and cyclic stretch. Additional experiments with zinc sulphate were performed in a preliminary analysis of the role of Zn2+ inducible transcription factors and might be cooperative heterodimerization formation with these transcription factor with C/EBP in the regulation of CYP 2C34 expression. With all these stimuli, reporter gene activity of CYP 2C34 promoter was significantly (3-8 fold) increased over values obtained in unstimulated cells.
Analysis of the regions that are essential for the induction of promoter activity in response to the different stimuli of interest have to be performed in combination with gel shift assays, siRNA experiments as well as site-directed mutagenesis experiments. Comparison of the regulation of the CYP 2C34 gene and correlation with changes in vascular function (in isolated porcine coronary arteries) should deliver information relevant to the regulation of the CYP 2C enzyme expressed in human coronary artery endothelial cells. The recent demonstration of a clinically relevant role for CYP 2C9 in coronary heart disease underlines the importance of such a study.
The epitranscriptome embodies many new and largely unexplored functions of RNA. A major roadblock in the epitranscriptomics field is the lack of transcriptome-wide methods to detect more than a single RNA modification type at a time, identify RNA modifications in individual molecules, and estimate modification stoichiometry accurately. We address these issues with CHEUI (CH3 (methylation) Estimation Using Ionic current), a new method that concurrently detects N6-methyladenosine (m6A) and 5-methylcytidine (m5C) in individual RNA molecules from the same sample, as well as differential methylation between any two conditions. CHEUI processes observed and expected nanopore direct RNA sequencing signals with convolutional neural networks to achieve high single-molecule accuracy and outperforms other methods in detecting m6A and m5C sites and quantifying their stoichiometry. CHEUI’s unique capability to identify two modification types in the same sample reveals a non-random co-occurrence of m6A and m5C in mRNA transcripts in cell lines and tissues. CHEUI unlocks an unprecedented potential to study RNA modification configurations and discover new epitranscriptome functions.
The epitranscriptome embodies many new and largely unexplored functions of RNA. A major roadblock in the epitranscriptomics field is the lack of transcriptome-wide methods to detect more than a single RNA modification type at a time, identify RNA modifications in individual molecules, and estimate modification stoichiometry accurately. We address these issues with CHEUI (CH3 (methylation) Estimation Using Ionic current), a new method that concurrently detects N6-methyladenosine (m6A) and 5-methylcytidine (m5C) in individual RNA molecules from the same sample, as well as differential methylation between any two conditions. CHEUI processes observed and expected nanopore direct RNA sequencing signals with convolutional neural networks to achieve high single-molecule accuracy and outperforms other methods in detecting m6A and m5C sites and quantifying their stoichiometry. CHEUI’s unique capability to identify two modification types in the same sample reveals a non-random co-occurrence of m6A and m5C in mRNA transcripts in cell lines and tissues. CHEUI unlocks an unprecedented potential to study RNA modification configurations and discover new epitranscriptome functions.
The epitranscriptome embodies many new and largely unexplored functions of RNA. A major roadblock in the epitranscriptomics field is the lack of transcriptome-wide methods to detect more than a single RNA modification type at a time, identify RNA modifications in individual molecules, and estimate modification stoichiometry accurately. We address these issues with CHEUI (CH3 (methylation) Estimation Using Ionic current), a new method that concurrently detects N6-methyladenosine (m6A) and 5-methylcytidine (m5C) in individual RNA molecules from the same sample, as well as differential methylation between any two conditions. CHEUI processes observed and expected nanopore direct RNA sequencing signals with convolutional neural networks to achieve high single-molecule accuracy and outperforms other methods in detecting m6A and m5C sites and quantifying their stoichiometry. CHEUI’s unique capability to identify two modification types in the same sample reveals a non-random co-occurrence of m6A and m5C in mRNA transcripts in cell lines and tissues. CHEUI unlocks an unprecedented potential to study RNA modification configurations and discover new epitranscriptome functions.
The epitranscriptome embodies many new and largely unexplored functions of RNA. A major roadblock in the epitranscriptomics field is the lack of transcriptome-wide methods to detect more than a single RNA modification type at a time, identify RNA modifications in individual molecules, and estimate modification stoichiometry accurately. We address these issues with CHEUI (CH3 (methylation) Estimation Using Ionic current), a new method that concurrently detects N6-methyladenosine (m6A) and 5-methylcytidine (m5C) in individual RNA molecules from the same sample, as well as differential methylation between any two conditions. CHEUI processes observed and expected nanopore direct RNA sequencing signals with convolutional neural networks to achieve high single-molecule accuracy and outperforms other methods in detecting m6A and m5C sites and quantifying their stoichiometry. CHEUI’s unique capability to identify two modification types in the same sample reveals a non-random co-occurrence of m6A and m5C in mRNA transcripts in cell lines and tissues. CHEUI unlocks an unprecedented potential to study RNA modification configurations and discover new epitranscriptome functions.
The epitranscriptome embodies many new and largely unexplored functions of RNA. A major roadblock in the epitranscriptomics field is the lack of transcriptome-wide methods to detect more than a single RNA modification type at a time, identify RNA modifications in individual molecules, and estimate modification stoichiometry accurately. We address these issues with CHEUI (CH3 (methylation) Estimation Using Ionic current), a new method that concurrently detects N6-methyladenosine (m6A) and 5-methylcytidine (m5C) in individual RNA molecules from the same sample, as well as differential methylation between any two conditions. CHEUI processes observed and expected nanopore direct RNA sequencing signals with convolutional neural networks to achieve high single-molecule accuracy and outperforms other methods in detecting m6A and m5C sites and quantifying their stoichiometry. CHEUI’s unique capability to identify two modification types in the same sample reveals a non-random co-occurrence of m6A and m5C in mRNA transcripts in cell lines and tissues. CHEUI unlocks an unprecedented potential to study RNA modification configurations and discover new epitranscriptome functions.
The epitranscriptome embodies many new and largely unexplored functions of RNA. A major roadblock in the epitranscriptomics field is the lack of transcriptome-wide methods to detect more than a single RNA modification type at a time, identify RNA modifications in individual molecules, and estimate modification stoichiometry accurately. We address these issues with CHEUI (CH3 (methylation) Estimation Using Ionic current), a new method that concurrently detects N6-methyladenosine (m6A) and 5-methylcytidine (m5C) in individual RNA molecules from the same sample, as well as differential methylation between any two conditions, using signals from nanopore direct RNA sequencing. CHEUI processes observed and expected signals with convolutional neural networks to achieve high single-molecule accuracy and outperform other methods in detecting m6A and m5C sites and quantifying their stoichiometry. CHEUI’s unique capability to identify two modification types in the same sample reveals a non-random co-occurrence of m6A and m5C in mRNA transcripts in cell lines and tissues. CHEUI unlocks an unprecedented potential to study RNA modification configurations and discover new epitranscriptome functions.
The epitranscriptome embodies many new and largely unexplored functions of RNA. A significant roadblock hindering progress in epitranscriptomics is the identification of more than one modification in individual transcript molecules. We address this with CHEUI (CH3 (methylation) Estimation Using Ionic current). CHEUI predicts N6-methyladenosine (m6A) and 5-methylcytidine (m5C) in individual molecules from the same sample, the stoichiometry at transcript reference sites, and differential methylation between any two conditions. CHEUI processes observed and expected nanopore direct RNA sequencing signals to achieve high single-molecule, transcript-site, and stoichiometry accuracies in multiple tests using synthetic RNA standards and cell line data. CHEUI’s capability to identify two modification types in the same sample reveals a co-occurrence of m6A and m5C in individual mRNAs in cell line and tissue transcriptomes. CHEUI provides new avenues to discover and study the function of the epitranscriptome.
Mutations in the clk-1 gene result in slower development and increased life span in Caenorhabditis elegans. The Saccharomyces cerevisiae homologue COQ7/CAT5 is essential for several metabolic pathways including ubiquinone biosynthesis, respiration, and gluconeogenic gene activation. We show here that Coq7p/Cat5p is a mitochondrial inner membrane protein directly involved in ubiquinone biosynthesis, and that the defect in gluconeogenic gene activation in coq7/cat5 null mutants is a general consequence of a defect in respiration. These results obtained in the yeast model suggest that the effects on development and life span in C. elegans clk-1 mutants may relate to changes in the amount of ubiquinone, an essential electron transport component and a lipid soluble antioxidant.
Taphonomy and palaeoecology of Laetoli as well as Makuyuni, Arusha region in northern Tanzania
(2004)
This thesis is the result of the Hominid Corridor research Project in Tanzania since 1993 to 1995 that include Pliocene and Pleistocene localities. The localities under study include Laetoli and Manyara area in Arusha Region, northern Tanzania. The thesis has the following specific objectives: firstly, to identify taxa recovered from the studied assemblages; secondly, to underpin taphonomic history of the assemblages under study; thirdly, to elucidate further palaeoecological reconstruction of the assemblages; and finally, to examine surface fossil fauna modifications including agents of modifications either hominids or carnivores.
The Upper Laetolil Beds are dated at 3.5 million years ago (Ma) and the Ndolanya Beds are bracketed in age between 3.5 and 2.41 Ma. The Naibadad Beds, also from Laetoli area, are date to be between 2.2 to 2.1 Ma. The Naibadad Beds are correlated with the base of Bed I at Olduvai Gorge. There are so far no absolute dates for Manyara assemblages. Based on biostratigraphic correlation, the younger overlying unit, the Upper Manyara Beds are estimated to belong to Later Pleistocene and the Lower Manyara Beds are estimated to belong to Early Pleistocene. The Upper Manyara Beds are correlated to the age of Bed III at Olduvai Gorge, while the Lower Manyara Beds are interpreted to span the same contemporaneity with the upper part of Bed II at Olduvai Gorge.
At Laetoli localities, terrestrial mammals while localities from Manyara besides terrestrial mammals dominate fauna; they include aquatic species such as fish, crocodiles and hippopotamus. The main families recovered from Upper Laetolil Beds complement those already recovered from former research works by other workers. This is also true for the younger overlying stratigraphic horizon, the Upper Ndolanya Beds. Thus, mammalian families recovered from Upper Laetolil Beds include Bovidae, Carnivora, Elephantidae, Equidae, Lagomorpha, Suidae, Rodentia, Hominoidea and Rhenocerotidae. Remains of an invertebrate, Gastropoda were also recovered. For Upper Ndolanya Beds include almost the same families recovered from Upper Laetolil Beds, but based on former recovery of fossil fauna, these Beds outnumber greatly the Upper Laetolil Beds in bovid composition by 20 per cent. Such a change in species composition is noticed also from South African localities and East African localities such as the East Turkana. This is interpreted to be due to climatic change drier environments that included species adapted to such palaeoclimates.
For the first time, our team has been able to retrieve specimens identifiable to taxa, a pattern that not possible from previous workers who claimed to have recovered too sparse specimens to be identifiable to any taxon.
The Upper Manyara Beds as well as Lower Manyara taxonomic composition include aquatic species besides the large terrestrial mammalian fauna retrieved from there. In due regard, the former horizon is attributed to have affinity with Olduvai Bed III components and the latter, older horizon, is attributed to have affinity with upper parts of Bed II times at Olduvai Gorge. The Lower Manyara Beds can be said to have, in relative terms, affinity to species recovered from site RC 11 of the Chiwondo Beds, Malema region in northern Malawi, although the former site may be equable to the terminal age of the latter locality.
Fossil hominid remains; attributable to genus Homo and possibly species Homo erectus have been recovered from two localities, Mk 2 and Mk, along Lower Manyara Beds. On the other hand, stone tools, identified to belong to the Acheulian industrial technocomplex, were recovered from site Mk 4.
All of fossil fauna from Laetoli sites were mostly exfoliated and there shows to be little effect in terms of hydrodynamic sorting of the fossil bones. However, intense carnivore activity is witnessed due to the almost one to one ratio of proximal to distal ends. This is also true for the Lower Manyara Beds locality. Through examination of surface modifications of the fossil fauna, it has been established that there was carnivore consumption of ungulates. There is no evidence of hominid involvement that has to be testified by stone tools.
Interest is an important factor for successful learning that has been the subject of intensive research for decades. Although interest in nature is of great importance for environmental education, to date there is no valid and reliable measurement tool. Therefore, the purpose of this study was to develop and test a scale for interest in nature, the Nature Interest Scale (NIS). In study 1, nine items were selected based on the three dimensions of the psychological interest construct to represent interest in nature. The factor structure of this new measurement instrument, was tested using confirmatory factor analyses. The results show that the instrument represents the three dimensions of the interest construct well. In study 2 the validity (discriminant and convergent validity) as well as the reliability (internal consistency, composite reliability, test-retest reliability) of the NIS were demonstrated. In study 3, the applicability of the NIS was tested with a different target group, students with learning disabilities. The results of this factor analysis also confirm the factor structure of the scale. Thus, this study provides a valid and reliable measurement tool for individual interest in nature that can be used for future research.
Tree-related microhabitats (TreMs) describe the microhabitats that a tree can provide for a multitude of other taxonomic groups and have been proposed as an important indicator for forest biodiversity (Asbeck et al., 2021). So far, the focus of TreM studies has been on temperate forests, although many trees in the tropics harbour exceptionally high numbers of TreMs. In this study, TreMs in the lowland tropical forests of the Choco (Ecuador) and in the mountain tropical forests of Mount Kilimanjaro (Tanzania) were surveyed. Our results extend the existing typology of TreMs of Larrieu et al. (2018) to include tropical forests and enabled a comparison of the relative recordings and diversity of TreMs between tropical and temperate forests. A new TreM form, Root formations, and three new TreM groups, concavities build by fruits or leaves, dendrotelms, and root formations, were established. In total, 15 new TreM types in five different TreM groups were specified. The relative recordings of most TreMs were similar between tropical and temperate forests. However, ivy and lianas, and ferns were more common in the lowland rainforest than in temperate forests, and bark microsoil, limb breakage, and foliose and fruticose lichens in tropical montane forest than in lowland rainforest. Mountain tropical forests hosted the highest diversity for common and dominant TreM types, and lowland tropical forest the highest diversity for rare TreMs. Our extended typology of tree-related microhabitats can support studies of forest-dwelling biodiversity in tropical forests. Specifically, given the ongoing threat to tropical forests, TreMs can serve as an additional tool allowing rapid assessments of biodiversity in these hyperdiverse ecosystems.
Die Transkription vieler Gene wird über den Acetylierungsgrad der Histone reguliert. Entsprechend erweiterte die Entdeckung von Histondeacetylase-Inhibitoren das Verständnis um Transkriptions-Repressoren und ihre Rolle in der Pathogenese beträchtlich. Zur Zeit stehen die Modifikationen der Histondeacetylasen (HDACs) sowie die biologischen Rollen der verschiedenen HDAC-Isoenzyme im Zentrum intensiver Forschungsarbeiten.
In der vorliegenden Arbeit wurde anhand verschiedener Zelllinien und mit murinem Primärmaterial nachgewiesen, dass das gut verträgliche Antiepileptikum Valproinsäure (VPA) ein potenter HDAC-Inhibitor ist. Dies zeigt sich daran, dass VPA in vivo die durch HDACs vermittelte transkriptionelle Repression aufhebt und zur Akkumulation hyperacetylierter Histone führt. In vitro Enzymassays weisen darauf hin, dass VPA selbst und nicht ein hypothetischer Metabolit die Histondeacetylasen hemmt. Darüber hinaus wurde mit Bindungs- und Kompetitionsstudien festgestellt, dass eine Interaktion von VPA mit dem katalytischen Zentrum der HDACs stattfindet.
Weitere Analysen zeigten, dass VPA bevorzugt Klasse I HDACs hemmt. Durch dieses Merkmal einer erhöhten Spezifität bei gleichzeitig guter Bioverfügbarkeit definiert VPA eine neue Klasse von HDAC-Inhibitoren. Hieraus ergeben sich Hinweise auf strukturelle Anforderungen, die ein HDAC-Inhibitor erfüllen muß, um spezifischer und weniger toxisch als konventionelle Chemotherapeutika zu wirken. Außerdem eröffnete das neu entdeckte pharmakologische Wirkungsspektrum von VPA auf HDACs Erkenntnisse um zusätzliche therapeutische Einsatzmöglichkeiten dieses etablierten Arzneimittels. Bereits jetzt wird VPA in klinischen Studien an Patienten mit Krebs verabreicht.
HDAC-Inhibitoren gelten als potentielle Medikamente für die Therapie maligner Neoplasien. Deshalb besteht großes Interesse an den molekularen Mechanismen, mit denen Substanzen dieser Wirkstoffklasse das Wachstum transformierter Zellen in vitro und in vivo hemmen. In den humanen Melanomzelllinien SK-Mel-37 und Mz-Mel-19 bewirken klinisch relevante VPA-Dosen eine zeit- und dosisabhängige Akkumulation von Zellzyklusinhibitoren und hyperacetylierten Histonen, morphologische Veränderungen und eine verringerte Proliferationsrate. Die verminderte Proliferation wird von einem veränderten Zellzyklusprofil und Apoptose unter Beteiligung sowohl der extrinsisch als auch der intrinsisch bedingten Caspase-Kaskade begleitet. Dies manifestiert sich in der Spaltung der Caspasen 3, 8 und 9, einer Schädigung der Mitochondrien, der apoptotischen PARP-Spaltung, einem Abbau der genomischen DNA und einer Inaktivierung des GFP-Proteins.
Diese Analysen in Melanomzellen sprechen dafür, dass die weitgehend selektive Wirkung von VPA auf Klasse I HDACs der Mechanismus ist, mit dem diese Substanz das Wachstum bestimmter Tumorzellen hemmt. Durch Genexpressions-Analysen konnten außerdem neue Modelle zum Einfluss von VPA auf solide Tumoren postuliert werden. Darüber hinaus wurde festgestellt, dass die Expression und Induzierbarkeit der Zellzyklusregulatoren p21WAF/CIP1 und p27Kip1 und des latent cytoplasmatischen Transkriptionsfaktors Stat1 Biomarker für die Sensitivität von Melanomzellen gegenüber HDAC-Inhibitoren sind. Im Einklang hiermit wird die proapoptotische Wirkung von VPA durch das Cytokin Interferon α und den S-Phase-Inhibitor Hydroxyharnstoff deutlich gesteigert. Diese Ergebnisse sprechen für den Einsatz von VPA in tierexperimentellen und klinischen Studien.
Aufgrund der Schlüsselrolle der HDACs für die physiologische und aberrante Genexpression ist es wichtig, die Mechanismen ihrer Regulation zu kennen. In der vorliegenden Arbeit wurde anhand zahlreicher kultivierter Zelllinien und mittels eines Mausmodells gezeigt, dass therapeutisch einsetzbare VPA-Dosen neben der Hemmung enzymatischer Aktivität auch zu einer isoenzymspezifischen Verringerung der Klasse I Histondeacetylase HDAC2 führen. Als Ursache hierfür konnten eine verstärkte Poly-Ubiquitinylierung und ein proteasomaler Abbau ermittelt werden. Gleichzeitig wurden die Beteiligung etlicher Proteasen und eine veränderte Synthese oder Prozessierung der HDAC2-mRNA als Mechanismen ausgeschlossen.
Expressionsanalysen identifizierten die E2 Ubiquitinkonjugase Ubc8 als von HDAC-Inhibitoren induziertes Gen. Mittels transienter Überexpression („Gain-of-Function“) und siRNA-Experimenten („Loss-of-Function“) konnte dieses Gen als limitierender Faktor des HDAC2-Umsatzes in vivo erkannt werden. Weiterhin wurde gezeigt, dass die E3 Ubiquitinligase RLIM spezifisch mit HDAC2 interagiert. Die Expression von RLIM beziehungsweise seine enzymatische Funktion beeinflusst die HDAC2-Konzentration in vivo. Hierbei kann VPA klar von dem HDACInhibitor Trichostatin A (TSA) abgegrenzt werden. Dieser hemmt ein breites Spektrum an HDACs und induziert Ubc8, führt aber gleichzeitig zu einem proteasomal vermittelten Abbau des RLIM-Proteins. Analysen mit überexprimiertem RLIM zeigten, dass TSA aufgrund dieses Mechanismus nicht in der Lage ist, den Abbau von HDAC2 zu induzieren. Somit ist im Rahmen dieser Arbeit die Ubiquitinylierungs-Maschinerie für HDAC2 charakterisiert worden. Hierdurch sind neue Aspekte zum Zusammenspiel zwischen dem Ubiquitin-Proteasom-System und der Transkriptionsrepression nachgewiesen worden.
Isoenzymspezifische HDAC-Inhibitoren können zur Aufklärung der Funktion einzelner Histondeacetylasen beitragen, insbesondere wenn Knock-Out-Studien zu aufwendig oder aufgrund embryonaler Letalität nicht durchführbar sind. Die Wichtigkeit dieser Analysen wird gerade bei HDAC2 deutlich, da diese Histondeacetylase in vielen soliden und hämatologischen Tumoren überexprimiert ist, und ihre Deregulation möglicherweise zur Krebsentstehung beiträgt. Die in der vorliegenden Arbeit identifizierte Regulation dieses HDAC-Isoenzyms könnte Hinweise auf den Ablauf eines malignen Transformationsprozesses geben. Darüber hinaus zeigt der nachgewiesene Regulationsmechanismus Erfordernisse und potentielle Zielstrukturen einer pharmakologischen Intervention auf. Schließlich könnten die Selektivität von VPA für Klasse I HDACs zusammen mit der Spezifität für HDAC2 die Gründe für die geringen Nebenwirkungen der VPA-Behandlung bei gleichzeitigem Auftreten antitumoraler Effekte sein.
In plants, a family of more than 20 heat stress transcription factors (Hsf) controls the expression of heat stress (hs) genes. There is increasing evidence for the functional diversification between individual members of the Hsf family fulfilling distinct roles in response to various environmental stress conditions and developmental signals. In response to hs, accumulation of both heat stress proteins (Hsp) and Hsfs is induced. In tomato, the physical interaction between the constitutively expressed HsfA1 and the hs-inducible HsfA2 results in synergistic transcriptional activation (superactivation) of hs gene expression. Here, we show that the interaction is strikingly specific and not observed with other class A Hsfs. Hetero-oligomerization of the two-component Hsfs is preferred to homo-oligomerization, and each Hsf in the HsfA1/HsfA2 hetero-oligomeric complex has its characteristic contribution to its function as superactivator. Distinct regions of the oligomerization domain are responsible for specific homo- and hetero-oligomeric interactions leading to the formation of hexameric complexes. The results are summarized in a model of assembly and function of HsfA1/A2 superactivator complexes in hs gene regulation.
Establishing management programs to preserve the benthic communities along the NW Pacific and the Arctic Ocean (AO) requires a deep understanding of the composition of communities and their responses to environmental stressors. In this study, we thus examine patterns of benthic community composition and patterns of species richness along the NW Pacific and Arctic Seas and investigate the most important environmental drivers of those patterns. Overall we found a trend of decreasing species richness toward higher latitudes and deeper waters, peaking in coastal waters of the eastern Philippines. The most dominant taxa along the entire study area were Arthropoda, Mollusca, Cnidaria, Echinodermata, and Annelida. We found that depth, not temperature, was the main driver of community composition along the NW Pacific and neighboring Arctic Seas. Depth has been previously suggested as a factor driving species distribution in benthic fauna. Following depth, the most influential environmental drivers of community composition along the NW Pacific and the Arctic Ocean were silicate, light, and currents. For example, silicate in Hexactinellida, Holothuroidea, and Ophiuroidea; and light in Cephalopoda and Gymnolaemata had the highest correlations with community composition. In this study, based on a combination of new samples and open-access data, we show that different benthic communities might respond differently to future climatic changes based on their taxon-specific biological, physiological, and ecological characteristics. International conservation efforts and habitat preservation should take an adaptive approach and apply measures that take the differences among benthic communities in responding to future climate change into account. This facilitates implementing appropriate conservation management strategies and sustainable utilization of the NW Pacific and Arctic marine ecosystems.
Insects with aquatic life stages can transfer sediment and water pollutants to terrestrial ecosystems, which has been described for metals, polyaromatic hydrocarbons, and polychlorinated chemicals. However, knowledge of the transfer of aquatic micropollutants released by wastewater treatment plants is scarce despite some preliminary studies on their occurrence in riparian spiders. In our study, we address a major analytical gap focusing on the transfer of the micropollutant carbamazepine from the larvae to the adult midges of Chironomus riparius using an optimized QuEChERS extraction method and HPLC–MS/MS applicable to both life stages down to the level of about three individuals. We show that the uptake of carbamazepine by larvae is concentration-dependent and reduces the emergence rate. Importantly, the body burden remained constant in adult midges. Using this information, we estimated the daily exposure of insectivorous tree swallows as terrestrial predators to carbamazepine using the energy demand of the predator and the energy content of the prey. Assuming environmentally relevant water concentrations of about 1 μg/L, the daily dose per kilogram of body weight for tree swallows was estimated to be 0.5 μg/kg/day. At places of high water contamination of 10 μg/L, the exposure may reach 5 μg/kg/day for this micropollutant of medium polarity. Considering body burden changes upon metamorphosis, this study fills the missing link between aquatic contamination and exposure in terrestrial habitats showing that wastewater pollutants can impact birds’ life. Clearly, further analytical methods for biota analysis in both habitats are urgently required to improve risk assessment.
Bird-mediated seed dispersal is crucial for the regeneration and viability of ecosystems, often resulting in complex mutualistic species networks. Yet, how this mutualism drives the evolution of seed dispersing birds is still poorly understood. In the present study we combine whole genome re-sequencing analyses and morphometric data to assess the evolutionary processes that shaped the diversification of the Eurasian nutcracker (Nucifraga), a seed disperser known for its mutualism with pines (Pinus). Our results show that the divergence and phylogeographic patterns of nutcrackers resemble those of other non-mutualistic passerine birds and suggest that their early diversification was shaped by similar biogeographic and climatic processes. The limited variation in foraging traits indicates that local adaptation to pines likely played a minor role. Our study shows that close mutualistic relationships between bird and plant species might not necessarily act as a primary driver of evolution and diversification in resource-specialized birds.
The majority of bacterial membrane-bound NiFe-hydrogenases and formate dehydrogenases have homologous membrane-integral cytochrome b subunits. The prototypic NiFe-hydrogenase of Wolinella succinogenes (HydABC complex) catalyzes H2 oxidation by menaquinone during anaerobic respiration and contains a membrane-integral cytochrome b subunit (HydC) that carries the menaquinone reduction site. Using the crystal structure of the homologous FdnI subunit of Escherichia coli formate dehydrogenase-N as a model, the HydC protein was modified to examine residues thought to be involved in menaquinone binding. Variant HydABC complexes were produced in W. succinogenes, and several conserved HydC residues were identified that are essential for growth with H2 as electron donor and for quinone reduction by H2. Modification of HydC with a C-terminal Strep-tag II enabled one-step purification of the HydABC complex by Strep-Tactin affinity chromatography. The tagged HydC, separated from HydAB by isoelectric focusing, was shown to contain 1.9 mol of heme b/mol of HydC demonstrating that HydC ligates both heme b groups. The four histidine residues predicted as axial heme b ligands were individually replaced by alanine in Strep-tagged HydC. Replacement of either histidine ligand of the heme b group proximal to HydAB led to HydABC preparations that contained only one heme b group. This remaining heme b could be completely reduced by quinone supporting the view that the menaquinone reduction site is located near the distal heme b group. The results indicate that both heme b groups are involved in electron transport and that the architecture of the menaquinone reduction site near the cytoplasmic side of the membrane is similar to that proposed for E. coli FdnI.
In Archaea, bacteria, and eukarya, ATP provides metabolic energy for energy-dependent processes. It is synthesized by enzymes known as A-type or F-type ATP synthase, which are the smallest rotatory engines in nature (Yoshida, M., Muneyuki, E., and Hisabori, T. (2001) Nat. Rev. Mol. Cell. Biol. 2, 669-677; Imamura, H., Nakano, M., Noji, H., Muneyuki, E., Ohkuma, S., Yoshida, M., and Yokoyama, K. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 2312-2315). Here, we report the first projected structure of an intact A(1)A(0) ATP synthase from Methanococcus jannaschii as determined by electron microscopy and single particle analysis at a resolution of 1.8 nm. The enzyme with an overall length of 25.9 nm is organized in an A(1) headpiece (9.4 x 11.5 nm) and a membrane domain, A(0) (6.4 x 10.6 nm), which are linked by a central stalk with a length of approximately 8 nm. A part of the central stalk is surrounded by a horizontal-situated rodlike structure ("collar"), which interacts with a peripheral stalk extending from the A(0) domain up to the top of the A(1) portion, and a second structure connecting the collar structure with A(1). Superposition of the three-dimensional reconstruction and the solution structure of the A(1) complex from Methanosarcina mazei Gö1 have allowed the projections to be interpreted as the A(1) headpiece, a central and the peripheral stalk, and the integral A(0) domain. Finally, the structural organization of the A(1)A(0) complex is discussed in terms of the structural relationship to the related motors, F(1)F(0) ATP synthase and V(1)V(0) ATPases.
Unlike other eukaryotes, plants possess a complex family of heat stress transcription factors (Hsfs) with usually more than 20 members. Among them, Hsfs A4 and A5 form a group distinguished from other Hsfs by structural features of their oligomerization domains and by a number of conserved signature sequences. We show that A4 Hsfs are potent activators of heat stress gene expression, whereas A5 Hsfs act as specific repressors of HsfA4 activity. The oligomerization domain of HsfA5 alone is necessary and sufficient to exert this effect. Due to the high specificity of the oligomerization domains, other class A Hsfs are not affected. Pull-down assay and yeast two-hybrid interaction tests demonstrate that the tendency to form HsfA4/A5 heterooligomers is stronger than the formation of homooligomers. The specificity of interaction between Hsfs A4 and A5 was confirmed by bimolecular fluorescence complementation experiments. The major role of the representatives of the HsfA4/A5 group, which are not involved in the conventional heat stress response, may reside in cell type-specific functions connected with the control of cell death triggered by pathogen infection and/or reactive oxygen species.
EF-P and its paralog EfpL (YeiP) differentially control translation of proline containing sequences
(2024)
Polyproline sequences (XPPX) stall ribosomes, thus being deleterious for all living organisms. In bacteria, translation elongation factor P (EF-P) plays a crucial role in overcoming such arrests. 12% of eubacteria possess an EF-P paralog – YeiP (EfpL) of unknown function. Here, we functionally and structurally characterize EfpL from Escherichia coli and demonstrate its yet unrecognized role in the translational stress response. Through ribosome profiling, we analyzed the EfpL arrest motif spectrum and discovered additional stalls beyond the canonical XPPX motifs at single-proline sequences (XPX), that both EF-P and EfpL can resolve. Notably, the two factors can also induce pauses. We further report that, contrary to the housekeeping EF-P, EfpL can sense the metabolic state of the cell, via lysine acylation. Together, our work uncovers a new player in ribosome rescue at proline-containing sequences, and provides evidence that co-occurrence of EF-P and EfpL is an evolutionary driver for higher bacterial growth rates.
Mitochondria and chloroplasts are of endosymbiotic origin. Their integration into cells entailed the development of protein translocons, partially by recycling bacterial proteins. We demonstrate the evolutionary conservation of the translocon component Tic22 between cyanobacteria and chloroplasts. Tic22 in Anabaena sp. PCC 7120 is essential. The protein is localized in the thylakoids and in the periplasm and can be functionally replaced by a plant orthologue. Tic22 physically interacts with the outer envelope biogenesis factor Omp85 in vitro and in vivo, the latter exemplified by immunoprecipitation after chemical cross-linking. The physical interaction together with the phenotype of a tic22 mutant comparable with the one of the omp85 mutant indicates a concerted function of both proteins. The three-dimensional structure allows the definition of conserved hydrophobic pockets comparable with those of ClpS or BamB. The results presented suggest a function of Tic22 in outer membrane biogenesis.
Background: Although Tic22 is involved in protein import into chloroplasts, the function in cyanobacteria is unknown.
Results: Cyanobacterial Tic22 is required for OM biogenesis, shares structural features with chaperones, and can be substituted by plant Tic22.
Conclusion: Tic22, involved in outer membrane biogenesis, is functionally conserved in cyanobacteria and plants.
Significance: The findings are important for the understanding of periplasmic protein transport.
Proteins of the Omp85 family are conserved in all kingdoms of life. They mediate protein transport across or protein insertion into membranes and reside in the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Omp85 proteins contain a C-terminal transmembrane β-barrel and a soluble N terminus with a varying number of polypeptide-transport-associated or POTRA domains. Here we investigate Omp85 from the cyanobacterium Anabaena sp. PCC 7120. The crystallographic three-dimensional structure of the N-terminal region shows three POTRA domains, here named P1 to P3 from the N terminus. Molecular dynamics simulations revealed a hinge between P1 and P2 but in contrast show that P2 and P3 are fixed in orientation. The P2-P3 arrangement is identical as seen for the POTRA domains from proteobacterial FhaC, suggesting this orientation is a conserved feature. Furthermore, we define interfaces for protein-protein interaction in P1 and P2. P3 possesses an extended loop unique to cyanobacteria and plantae, which influences pore properties as shown by deletion. It now becomes clear how variations in structure of individual POTRA domains, as well as the different number of POTRA domains with both rigid and flexible connections make the N termini of Omp85 proteins versatile adaptors for a plentitude of functions.
Precursor protein translocation across the outer chloroplast membrane depends on the action of the Toc complex, containing GTPases as recognizing receptor components. The G domains of the GTPases are known to dimerize. In the dimeric conformation an arginine contacts the phosphate moieties of bound nucleotide in trans. Kinetic studies suggested that the arginine in itself does not act as an arginine finger of a reciprocal GTPase-activating protein (GAP). Here we investigate the specific function of the residue in two GTPase homologues. Arginine to alanine replacement variants have significantly reduced affinities for dimerization compared with wild-type GTPases. The amino acid exchange does not impact on the overall fold and nucleotide binding, as seen in the monomeric x-ray crystallographic structure of the Arabidopsis Toc33 arginine-alanine replacement variant at 2.0A. We probed the catalytic center with the transition state analogue GDP/AlF(x) using NMR and analytical ultracentrifugation. AlF(x) binding depends on the arginine, suggesting the residue can play a role in catalysis despite the non-GAP nature of the homodimer. Two non-exclusive functional models are discussed: 1) the coGAP hypothesis, in which an additional factor activates the GTPase in homodimeric form; and 2) the switch hypothesis, in which a protein, presumably the large Toc159 GTPase, exchanges with one of the homodimeric subunits, leading to activation.
In high light, the antenna system in oxygenic photosynthetic organisms switches to a photoprotective mode, dissipating excess energy in a process called non-photochemical quenching (NPQ). Diatoms exhibit very efficient NPQ, accompanied by a xanthophyll cycle in which diadinoxanthin is de-epoxidized into diatoxanthin. Diatoms accumulate pigments from this cycle in high light, and exhibit faster and more pronounced NPQ. The mechanisms underlying NPQ in diatoms remain unclear, but it can be mimicked by aggregation of their isolated light-harvesting complexes, FCP (fucoxanthin chlorophyll-a/c protein). We assess this model system by resonance Raman measurements of two peripheral FCPs, trimeric FCPa and nonameric FCPb, isolated from high- and low-light-adapted cells (LL, HL). Quenching is associated with a reorganisation of these proteins, affecting the conformation of their bound carotenoids, and in a manner which is highly dependent on the protein considered. FCPa from LL diatoms exhibits significant changes in diadinoxanthin structure, together with a smaller conformational change of at least one fucoxanthin. For these LL-FCPa, quenching is associated with consecutive events, displaying distinct spectral signatures, and its amplitude correlates with the planarity of the diadinoxanthin structure. HL-FCPa aggregation is associated with a change in planarity of a 515-nm-absorbing fucoxanthin, and, to a lesser extent, of diadinoxanthin. Finally, in FCPb, a blue-absorbing fucoxanthin is primarily affected. FCPs thus possess a plastic structure, undergoing several conformational changes upon aggregation, dependent upon their precise composition and structure. NPQ in diatoms may therefore arise from a combination of structural changes, dependent on the environment the cells are adapted to.
Abstract
Natural plant populations often harbour substantial heritable variation in DNA methylation. However, a thorough understanding of the genetic and environmental drivers of this epigenetic variation requires large-scale and high-resolution data, which currently exist only for a few model species. Here, we studied 207 lines of the annual weed Thlaspi arvense (field pennycress), collected across a large latitudinal gradient in Europe and propagated in a common environment. By screening for variation in DNA sequence and DNA methylation using whole-genome (bisulfite) sequencing, we found significant epigenetic population structure across Europe. Average levels of DNA methylation were strongly context-dependent, with highest DNA methylation in CG context, particularly in transposable elements and in intergenic regions. Residual DNA methylation variation within all contexts was associated with genetic variants, which often co-localized with annotated methylation machinery genes but also with new candidates. Variation in DNA methylation was also significantly associated with climate of origin, with methylation levels being lower in colder regions and in more variable climates. Finally, we used variance decomposition to assess genetic versus environmental associations with differentially methylated regions (DMRs). We found that while genetic variation was generally the strongest predictor of DMRs, the strength of environmental associations increased from CG to CHG and CHH, with climate-of-origin as the strongest predictor in about one third of the CHH DMRs. In summary, our data show that natural epigenetic variation in Thlaspi arvense is significantly associated with both DNA sequence and environment of origin, and that the relative importance of the two factors strongly depends on the sequence context of DNA methylation. T. arvense is an emerging biofuel and winter cover crop; our results may hence be relevant for breeding efforts and agricultural practices in the context of rapidly changing environmental conditions.
Author summary
Variation within species is an important level of biodiversity, and it is key for future adaptation. Besides variation in DNA sequence, plants also harbour heritable variation in DNA methylation, and we want to understand the evolutionary significance of this epigenetic variation, in particular how much of it is under genetic control, and how much is associated with the environment. We addressed these questions in a high-resolution molecular analysis of 207 lines of the common plant field pennycress (Thlaspi arvense), which we collected across Europe, propagated under standardized conditions, and sequenced for their genetic and epigenetic variation. We found large geographic variation in DNA methylation, associated with both DNA sequence and climate of origin. Genetic variation was generally the stronger predictor of DNA methylation variation, but the strength of environmental association varied between different sequence contexts. Climate-of-origin was the strongest predictor in about one third of the differentially methylated regions in the CHH context, which suggests that epigenetic variation may play a role in the short-term climate adaptation of pennycress. As pennycress is currently being domesticated as a new biofuel and winter cover crop, our results may be relevant also for agriculture, particularly in changing environments.
Biotechnological processes offer better production conditions for a wide variety of goods of industrial interest. The production of aromatic compounds, for example, involves molecules of great value for cosmetic, plastic, agrochemical and pharmaceutic industries. However, the yield of such processes frequently prevents a proper implementtation that would allow the replacement of traditional production processes.
Numerous rational engineering approaches have been attempted to enhance metabolic pathways associated with desired products. Unfortunately, genetic modifications and heterologous pathway expression often lead to a higher metabolic burden on the producing organisms, ultimately leading to reduced production levels and fitness.
This project utilised adaptive laboratory evolution to better understand the development of synthetic cooperative consortia, using S. cerevisiae as a model organism. Specifically, a synthetic cooperative consortium was developed around the exchange of lysine and tyrosine, which was subjected to adaptive laboratory evolution aiming to induce mutations that would improve the system’s fitness either by enhanced production or upgraded stress resistance. Consequently, the mutant strains isolated after the evolution rounds were sequenced to identify relevant variations that could be related to the growth and production phenotypes observed.
The insights derived from this project are expected to contribute to further developing synthetic cooperative consortia with utilitarian purposes.
Background: Genome sequencing of all known eukaryotes on Earth promises unprecedented advances in biological sciences and in biodiversity-related applied fields such as environmental management and natural product research. Advances in long-read DNA sequencing make it feasible to generate high-quality genomes for many non–genetic model species. However, long-read sequencing today relies on sizable quantities of high-quality, high molecular weight DNA, which is mostly obtained from fresh tissues. This is a challenge for biodiversity genomics of most metazoan species, which are tiny and need to be preserved immediately after collection. Here we present de novo genomes of 2 species of submillimeter Collembola. For each, we prepared the sequencing library from high molecular weight DNA extracted from a single specimen and using a novel ultra-low input protocol from Pacific Biosciences. This protocol requires a DNA input of only 5 ng, permitted by a whole-genome amplification step.
Results: The 2 assembled genomes have N50 values >5.5 and 8.5 Mb, respectively, and both contain ∼96% of BUSCO genes. Thus, they are highly contiguous and complete. The genomes are supported by an integrative taxonomy approach including placement in a genome-based phylogeny of Collembola and designation of a neotype for 1 of the species. Higher heterozygosity values are recorded in the more mobile species. Both species are devoid of the biosynthetic pathway for β-lactam antibiotics known in several Collembola, confirming the tight correlation of antibiotic synthesis with the species way of life.
Conclusions: It is now possible to generate high-quality genomes from single specimens of minute, field-preserved metazoans, exceeding the minimum contig N50 (1 Mb) required by the Earth BioGenome Project.
Abstract
Natural plant populations often harbour substantial heritable variation in DNA methylation. However, a thorough understanding of the genetic and environmental drivers of this epigenetic variation requires large-scale and high-resolution data, which currently exist only for a few model species. Here, we studied 207 lines of the annual weed Thlaspi arvense (field pennycress), collected across a large latitudinal gradient in Europe and propagated in a common environment. By screening for variation in DNA sequence and DNA methylation using whole-genome (bisulfite) sequencing, we found significant epigenetic population structure across Europe. Average levels of DNA methylation were strongly context-dependent, with highest DNA methylation in CG context, particularly in transposable elements and in intergenic regions. Residual DNA methylation variation within all contexts was associated with genetic variants, which often co-localized with annotated methylation machinery genes but also with new candidates. Variation in DNA methylation was also significantly associated with climate of origin, with methylation levels being higher in warmer regions and lower in more variable climates. Finally, we used variance decomposition to assess genetic versus environmental associations with differentially methylation regions (DMRs). We found that while genetic variation was generally the strongest predictor of DMRs, the strength of environmental associations increased from CG to CHG and CHH, with climate-of-origin as the strongest predictor in about one third of the CHH DMRs. In summary, our data show that natural epigenetic variation in Thlaspi arvense is significantly associated with both DNA sequence and environment of origin, and that the relative importance of the two factors strongly depends on the sequence context of DNA methylation. T. arvense is an emerging biofuel and winter cover crop; our results may hence be relevant for breeding efforts and agricultural practices in the context of rapidly changing environmental conditions.
Author Summary: Variation within species is an important level of biodiversity, and it is key for future adaptation. Besides variation in DNA sequence, plants also harbour heritable variation in DNA methylation, and we want to understand the evolutionary significance of this epigenetic variation, in particular how much of it is under genetic control, and how much is associated with the environment. We addressed these questions in a high-resolution molecular analysis of 207 lines of the common plant field pennycress (Thlaspi arvense), which we collected across Europe, propagated under standardized conditions, and sequenced for their genetic and epigenetic variation. We found large geographic variation in DNA methylation, associated with both DNA sequence and climate of origin. Genetic variation was generally the stronger predictor of DNA methylation variation, but the strength of environmental association varied between different sequence contexts. Climate-of-origin was the strongest predictor in about one third of the differentially methylated regions in the CHH context, which suggests that epigenetic variation may play a role in the short-term climate adaptation of pennycress. As pennycress is currently being domesticated as a new biofuel and winter cover crop, our results may be relevant also for agriculture, particularly in changing environments.
Meliolales (black mildews) is an order of plant parasitic ascomycetous fungi in the tropics and subtropics. They are frequently overgrown and parasitized by other fungi, known as hyperparasites. During the last few years, species of hyperparasitic fungi on Meliolales have been collected in Benin and Panama. A new species of Paranectria and seven new reports of hyperparasites of different systematic groups are presented here with detailed descriptions and illustrations, together with new data concerning fungal hosts and host plants. The new species is called Paranectria longiappendiculata, characterized by exceptionally long appendages carried by the ascospores. New records for Benin and Panama are Calloriopsis herpotricha, Dimerosporiella cephalosporii, Isthmospora glabra, Isthmospora trichophila, Malacaria meliolicola, Paranectriella hemileiae, and Paranectriella minuta. Calloriopsis herpotricha is recorded for Africa and D. cephalosporii and P. hemileiae for America for the first time, suggesting an apparently pantropical distribution. Findings show a blatant lack of investigation on hyperparasitic fungi in the tropics. The phylogenetic positions of three of these newly reported species, C. herpotricha, D. cephalosporii, and P. minuta, are shown based on the analysis of internal transcribed spacer (ITS), large subunit (LSU), and small subunit (SSU) rDNA sequences. These sequences were generated in the context of the present study for the first time.
Earliella scabrosa is a pantropical species of Polyporales (Basidiomycota) and well-studied concerning its morphology and taxonomy. However, its pantropical intraspecific genetic diversity and population differentiation is unknown. We initiated this study to better understand the genetic variation within E. scabrosa and to test if cryptic species are present. Sequences of three DNA regions, the nuclear ribosomal internal transcribed spacer (ITS), the large subunit ribosomal DNA (LSU), and the translation elongation factor (EF1α) were analysed for 66 samples from 15 geographical locations. We found a high level of genetic diversity (haplotype diversity, Hd = 0.88) and low nucleotide diversity (π = 0.006) across the known geographical range of E. scabrosa based on ITS sequences. The analysis of molecular variance (AMOVA) indicates that the genetic variability is mainly found among geographical populations. The results of Mantel tests confirmed that the genetic distance among populations of E. scabrosa is positively correlated with the geographical distance, which indicates that geographical isolation is an important factor for the observed genetic differentiation. Based on phylogenetic analyses of combined dataset ITS-LSU-EF1α, the low intraspecific divergences (0–0.3%), and the Automated Barcode Gap Discovery (ABGD) analysis, E. scabrosa can be considered as a single species with five different geographical populations. Each population might be in the process of allopatric divergence and in the long-term they may evolve and become distinct species.
Hyperparasitic fungi on black mildews (Meliolales, Ascomycota) : hidden diversity in the tropics
(2023)
Meliolales (Sordariomycetes, Ascomycota) is a group of obligate plant parasitic microfungi mainly distributed in the tropics and subtropics. Meliolalean fungi are commonly known as “black mildews”, as they form black, superficial hyphae on the surface of vegetative and reproductive organs of vascular plants. They are considered biotrophic parasites, and the infections caused by black mildews can lead to a decrease in the photosynthetic activity of plants, as well as to an increase in the temperature and respiration rate of their leaves.
Meliolales are frequently parasitized by hyperparasitic fungi, i.e., parasitic fungi that have parasitic hosts. These hyperparasites are all Ascomycota and belong mainly to the Dothideomycetes and Sordariomycetes. Although hyperparasites represent a megadiverse group, species were only described by morphology until 1980, and the systematic position of more than 60 % of known species is still unclear. In addition, there are no DNA reference sequences available in public databases for any of the species of hyperparasites of Meliolales, and no ecological studies have been done up to now.
Before this study, no exact number of hyperparasitic fungi growing on colonies of black mildews existed. Here, we present a checklist including 189 species of fungi known to be hyperparasitic on Meliolales, but the number of existing species is likely to be even higher. The elaboration of this species checklist laid the foundations for this investigation, as it helped to understand the present state of knowledge of hyperparasitic fungi on Meliolales worldwide.
For the present study, fresh specimens of leaves infected with colonies of Meliolales and hyperparasites were opportunistically collected at 32 collection sites in Western Panama and Benin, West Africa, in 2020 and 2022, respectively. In total, 100 samples of plant specimens infected with black mildews were collected, of which 58 samples were parasitized by hyperparasitic fungi. 31 species and morphospecies of hyperparasitic fungi were identified. In addition, 35 historical specimens, including 12 type specimens, were examined for the present work.
DNA of hyperparasitic fungi was isolated directly from conidia, synnemata, apothecia, perithecia or pseudothecia of fresh and dried specimens. The main challenges faced by scientists in doing molecular studies of hyperparasitic fungi are related to the fact that the hyperparasitic fungi are intermingled with tissues of the meliolalean hosts and other organisms present in a given sample. This makes the isolation of DNA exclusively from the hyperparasite difficult. Moreover, hyperparasitic fungi on Meliolales are biotrophs and cannot be grown axenically. The hosts themselves are also biotrophic, further complicating DNA isolation from either partner. These factors have contributed to a lack of reference sequences in public databases. After more than 100 attempts, DNA of 20 specimens of hyperparasitic fungi, representing seven species, has been isolated in the context of the present investigation. Three partial nuclear gene regions were amplified and sequenced: nrLSU, nrSSU and nrITS. The datasets were assembled for phylogenetic analyses applying Maximum Likelihood (ML) and Bayesian inference (BI) methods. DNA sequences of hyperparasitic fungi on Meliolales were generated for the first time in the context of the present investigation.
Hyperparasitic fungi on Meliolales do not represent a single systematic group, but a polyphyletic ecological guild of fungi. Because of this huge diversity, only the systematics of species of perithecioid hyperparasites, as well as of the species of the genera Atractilina and Spiropes known to be hyperparasitic on black mildews was discussed in this thesis, as they represented the most common groups of fungi found in Benin and Panama. The results indicated, for example, the systematic position of Dimerosporiella cephalosporii and Paranectriella minuta in the Sordariomycetes and Dothideomycetes, respectively. In addition, the first record of a hyperparasitic fungus of black mildews in the Lecanoromycetes, namely Calloriopsis herpotricha, is reported here. The systematics of Atractilina parasitica and of some species of Spiropes is also discussed here.
In the context of the present investigation, four species new to science were described. They are presented with detailed descriptions, photos and scientific illustrations. Taxonomic studies of this thesis also generated seven new synonyms, nine new records for Benin, seven for Panama, one for Africa and two for mainland America, as well as the confirmation of one anamorph-teleomorph connection by molecular sequence data.
The ecology of hyperparasitic fungi on Meliolales is complex and far from being completely understood. The hypothesis of host specificity between hyperparasitic fungi, their meliolalean hosts and their plant hosts was tested for the first time, through a tritrophic network analysis. Results indicate that hyperparasites of Meliolales are generalists concerning genera of Meliolales, but apparently specialists at the level of order. In addition, hyperparasitic fungi tend to be found alongside their meliolalean hosts, suggesting a pantropical distribution.
Background: Nitric oxide synthase 1 adaptor protein (NOS1AP; previously named CAPON) is linked to the glutamatergic postsynaptic density through interaction with neuronal nitric oxide synthase (nNOS). NOS1AP and its interaction with nNOS have been associated with several mental disorders. Despite the high levels of NOS1AP expression in the hippocampus and the relevance of this brain region in glutamatergic signalling as well as mental disorders, a potential role of hippocampal NOS1AP in the pathophysiology of these disorders has not been investigated yet.
Methods: To uncover the function of NOS1AP in hippocampus, we made use of recombinant adeno-associated viruses to overexpress murine full-length NOS1AP or the NOS1AP carboxyterminus in the hippocampus of mice. We investigated these mice for changes in gene expression, neuronal morphology, and relevant behavioural phenotypes.
Findings: We found that hippocampal overexpression of NOS1AP markedly increased the interaction of nNOS with PSD-95, reduced dendritic spine density, and changed dendritic spine morphology at CA1 synapses. At the behavioural level, we observed an impairment in social memory and decreased spatial working memory capacity.
Interpretation: Our data provide a mechanistic explanation for a highly selective and specific contribution of hippocampal NOS1AP and its interaction with the glutamatergic postsynaptic density to cross-disorder pathophysiology. Our findings allude to therapeutic relevance due to the druggability of this molecule.
Understanding the underlying mechanisms that link psychopathology and physical comorbidities in schizophrenia is crucial since decreased physical fitness and overweight pose major risk factors for cardio-vascular diseases and decrease the patients’ life expectancies. We hypothesize that altered reward anticipation plays an important role in this. We implemented the Monetary Incentive Delay task in a MR scanner and a fitness test battery to compare schizophrenia patients (SZ, n = 43) with sex- and age-matched healthy controls (HC, n = 36) as to reward processing and their physical fitness. We found differences in reward anticipation between SZs and HCs, whereby increased activity in HCs positively correlated with overall physical condition and negatively correlated with psychopathology. On the other handy, SZs revealed stronger activity in the posterior cingulate cortex and in cerebellar regions during reward anticipation, which could be linked to decreased overall physical fitness. These findings demonstrate that a dysregulated reward system is not only responsible for the symptomatology of schizophrenia, but might also be involved in physical comorbidities which could pave the way for future lifestyle therapy interventions.
Substantial progress in the field of neuroscience has been made from anaesthetized preparations. Ketamine is one of the most used drugs in electrophysiology studies, but how ketamine affects neuronal responses is poorly understood. Here, we used in vivo electrophysiology and computational modelling to study how the auditory cortex of bats responds to vocalisations under anaesthesia and in wakefulness. In wakefulness, acoustic context increases neuronal discrimination of natural sounds. Neuron models predicted that ketamine affects the contextual discrimination of sounds regardless of the type of context heard by the animals (echolocation or communication sounds). However, empirical evidence showed that the predicted effect of ketamine occurs only if the acoustic context consists of low-pitched sounds (e.g., communication calls in bats). Using the empirical data, we updated the naïve models to show that differential effects of ketamine on cortical responses can be mediated by unbalanced changes in the firing rate of feedforward inputs to cortex, and changes in the depression of thalamo-cortical synaptic receptors. Combined, our findings obtained in vivo and in silico reveal the effects and mechanisms by which ketamine affects cortical responses to vocalisations.
Discrepancies between knockdown and knockout animal model phenotypes have long stood as a perplexing phenomenon. Several mechanisms explaining such observations have been proposed, namely the toxicity or the off-target effects of the knockdown reagents, as well as, in certain cases, genetic robustness – an organism's ability to maintain its phenotype despite genetic perturbations. In addition to these explanations, transcriptional adaptation (TA), a phenomenon defined as an event whereby a mutation in one gene leads to transcriptional upregulation or downregulation of another, adapting, gene or genes expression, has been recently proposed as an alternative explanation for the conflicting knockdown and knockout phenotype paradox.
Since its discovery in 2015, TA's precise mechanism remains a subject of ongoing research. Majority of evidence suggests that mutant mRNA degradation plays a central in TA. Epigenetic remodeling is also thought to play a role, as evidenced by an increase in active histone marks at the transcription start sites of the adapting genes. Whether mRNA degradation is indeed the key player in TA remains debated. Furthermore, it is still unknown how exactly TA develops, what adapting genes it targets, and whether genomic mutations that render mutant mRNA sensitive to degradation are required for TA to occur.
Throughout the experiments described in this Dissertation, I have designed an inducible TA system where TA can be triggered on demand and its effects on the cell’s transcriptome followed through time. I have demonstrated that degradation-prone transgenes, once induced and expressed, can be efficiently degraded, resulting in the protein loss-independent upregulation of adapting genes via TA. Adapting genes with higher degree of sequence similarity become upregulated faster than genes with lower degree of sequence similarity. Further functionality of this approach to study TA is limited by the leakiness of the inducible gene expression system; however, constitutively expressed degradation-prone transgenes were used to demonstrate TA in human cells.
In addition, I have developed an approach to target wild-type cytoplasmic mRNAs without altering the cell’s genome and reported a TA-like phenomenon, which manifested as adapting gene upregulation not relying on mutations in other genes. Cytoplasmic mRNA cleavage with CRISPR-Cas13d triggered a TA-like response in three different gene models: Actg1 knockdown, Ctnna1 knockdown, and Nckap1 knockdown. After comparing two different modes of triggering TA, CRISPR-Cas9 knockout versus CRISPR-Cas13d knockdown, I reported little overlap between the dysregulated genes and suggested that diverse mRNA degradation modes led to distinct TA responses. In addition, the transcriptional increase of Actg2 caused by CRISPR-Cas13d-mediated Actg1 mRNA cleavage did not require chromatin accessibility changes.
Experiments and genetic tools described in this dissertation investigated how TA develops from its earliest onset, how it affects the global transcriptome of the cell, as well as provided compelling evidence for an mRNA degradation-central TA mechanism. I have created tools to study both direct and indirect TA gene targets and unveiled important insights into the temporal dynamics of TA. Genes with higher sequence similarity were found to be upregulated more rapidly than those with lower similarity. Furthermore, it was revealed that the epigenetic properties of TA responses vary depending on the triggering mechanism. Cas13d-mediated degradation of wild-type mRNAs led to immediate transcriptional enhancement independent of epigenetic changes, which stood in contrast to previously measured alterations in chromatin accessibility in CRISPR-Cas9 mutants. This research has thus significantly advanced our knowledge of TA and provided valuable tools and findings that contribute to the broader understanding of gene expression regulation in response to mRNA degradation.
RNA-binding proteins (RBPs) control every RNA metabolic process by multiple protein-RNA and protein-protein interactions. Their roles have largely been analyzed by crude mutations, which abrogate multiple functions at once and likely impact the structural integrity of the large ribonucleoprotein particles (RNPs) these proteins function in. Using UV-induced RNA-protein crosslinking of entire cells, protein complex purification and mass spectrometric analysis, we identified >100 in vivo RNA crosslinks in 16 nuclear mRNP components in Saccharomyces cerevisiae. For functional analysis, we chose Npl3, which displayed crosslinks in its two RNA recognition motifs (RRMs) and in the connecting flexible linker region. Both RRM domains and the linker uniquely contribute to RNA recognition as revealed by NMR and structural analyses. Interestingly, mutations in these regions cause different phenotypes, indicating distinct functions of the different RNA-binding domains. Notably, an npl3-Linker mutation strongly impairs recruitment of several mRNP components to chromatin and incorporation of other mRNP components into nuclear mRNPs, establishing a so far unknown function of Npl3 in nuclear mRNP assembly. Taken together, our integrative analysis uncovers a specific function of the RNA-binding activity of the nuclear mRNP component Npl3. This approach can be readily applied to RBPs in any RNA metabolic process.
RNA-binding proteins (RBPs) control every RNA metabolic process by multiple protein-RNA and protein-protein interactions. Their roles have largely been analyzed by crude mutations, which abrogate multiple functions at once and likely impact the structural integrity of the large messenger ribonucleoprotein particle (mRNP) assemblies, these proteins often function in. Using UV-induced RNA-protein crosslinking and subsequent mass spectrometric analysis, we first identified more than 100 in vivo RNA crosslinks in 16 nuclear mRNP components in S. cerevisiae. For functional analysis, we chose Npl3, for which we determined crosslinks in its two RNA recognition motifs (RRM) and in the flexible linker region connecting the two. Using NMR and structural analyses, we show that both RRM domains and the linker uniquely contribute to RNA recognition. Interestingly, mutations in these regions cause different phenotypes, indicating distinct functions of the different RNA-binding domains of Npl3. Notably, the npl3-Linker mutation strongly impairs recruitment of several mRNP components to chromatin and incorporation of further mRNP components into nuclear mRNPs, establishing a function of Npl3 in nuclear mRNP assembly. Taken together, we determined the specific function of the RNA-binding activity of the nuclear mRNP component Npl3, an approach that can be applied to many RBPs in any RNA metabolic process.
Zinc finger (ZnF) domains appear in a pool of structural contexts and despite their small size achieve varying target specificities, covering single-stranded and double-stranded DNA and RNA as well as proteins. Combined with other RNA-binding domains, ZnFs enhance affinity and specificity of RNA-binding proteins (RBPs). The ZnF-containing immunoregulatory RBP Roquin initiates mRNA decay, thereby controlling the adaptive immune system. Its unique ROQ domain shape-specifically recognizes stem-looped cis-elements in mRNA 3’-untranslated regions (UTR). The N-terminus of Roquin contains a RING domain for protein-protein interactions and a ZnF, which was suggested to play an essential role in RNA decay by Roquin. The ZnF domain boundaries, its RNA motif preference and its interplay with the ROQ domain have remained elusive, also driven by the lack of high-resolution data of the challenging protein. We provide the solution structure of the Roquin-1 ZnF and use an RBNS-NMR pipeline to show that the ZnF recognizes AU-rich elements (ARE). We systematically refine the contributions of adenines in a poly(U)-background to specific complex formation. With the simultaneous binding of ROQ and ZnF to a natural target transcript of Roquin, our study for the first time suggests how Roquin integrates RNA shape and sequence specificity through the ROQ-ZnF tandem.
Highlights
• Protocol for extracting and analyzing pollen grains from fossil insects
• Individual fossil grains can be analyzed using a combined approach
• Simple and fast TEM embedding and sectioning protocol
• Protocol enables a taxonomic assignment of pollen
Summary
This protocol explains how to extract pollen from fossil insects with subsequent descriptions of pollen treatment. We also describe how to document morphological and ultrastructural features with light-microscopy and electron microscopy. It enables a taxonomic assignment of pollen that can be used to interpret flower-insect interactions, foraging and feeding behavior of insects, and the paleoenvironment. The protocol is limited by the state of the fossil, the presence/absence of pollen on fossil specimens, and the availability of extant pollen for comparison.
Highlights
• Seed size mediates seedling recruitment in tropical forests and pastures.
• Large-seeded species recruited better than small-seeded species in the forest.
• Recruitment of large-seeded species in pastures was limited by surface temperature.
• Large-seeded species should be protected against drought in regenerating pastures.
Abstract
Seedling recruitment is a key process of plant regeneration that often depends on plant functional traits, such as seed size. To optimize forest restoration efforts, we need to better understand how seedling recruitment of different seed sizes varies along environmental gradients with strong variation in abiotic and biotic factors. To understand these interacting effects, we conducted a sowing experiment with different-sized seeds in forests and pastures in the tropical mountains of southern Ecuador. We quantified seedling recruitment in relation to temperature, soil moisture and biotic pressures. We sowed seeds of five tree species of varying seed size at three elevations (1000, 2000 and 3000 m a.s.l.) in primary forest and pastures. We tested (1) how habitat type influences the recruitment of seedlings belonging to three small- and two large-seeded species, and (2) how abiotic and biotic factors limit seedling recruitment of species with different seed sizes. We found that seedlings of the two large-seeded species recruited better than seedlings of the three small-seeded species, but only in the forest habitat. Seedling recruitment of large seeds was primarily limited by high surface temperature, which explains lower recruitment of large seeds in pastures compared to forests. Our study shows that seed size can be a key trait mediating variability in seedling recruitment in tropical ecosystems. We conclude that restoration measures should aim to mitigate extreme temperatures in tropical pastures to aid the natural regeneration of large-seeded tree species.
RBFOX1 is a highly pleiotropic gene that contributes to several psychiatric and neurodevelopmental disorders. Both rare and common variants in RBFOX1 have been associated with several psychiatric conditions, but the mechanisms underlying the pleiotropic effects of RBFOX1 are not yet understood. Here we found that, in zebrafish, rbfox1 is expressed in spinal cord, mid- and hindbrain during developmental stages. In adults, expression is restricted to specific areas of the brain, including telencephalic and diencephalic regions with an important role in receiving and processing sensory information and in directing behaviour. To investigate the effect of rbfox1 deficiency on behaviour, we used rbfox1sa15940, a rbfox1 loss-of-function line. We found that rbfox1sa15940 mutants present hyperactivity, thigmotaxis, decreased freezing behaviour and altered social behaviour. We repeated these behavioural tests in a second rbfox1 loss-of-function line with a different genetic background, rbfox1del19, and found that rbfox1 deficiency affects behaviour similarly in this line, although there were some differences. rbfox1del19 mutants present similar thigmotaxis, but stronger alterations in social behaviour and lower levels of hyperactivity than rbfox1sa15940 fish. Taken together, these results suggest that rbfox1 deficiency leads to multiple behavioural changes in zebrafish that might be modulated by environmental, epigenetic and genetic background effects, and that resemble phenotypic alterations present in Rbfox1-deficient mice and in patients with different psychiatric conditions. Our study thus highlights the evolutionary conservation of rbfox1 function in behaviour and paves the way to further investigate the mechanisms underlying rbfox1 pleiotropy on the onset of neurodevelopmental and psychiatric disorders.
RBFOX1 is a highly pleiotropic gene that contributes to several psychiatric and neurodevelopmental disorders. Both rare and common variants in RBFOX1 have been associated with several psychiatric conditions, but the mechanisms underlying the pleiotropic effects of RBFOX1 are not yet understood. Here we found that, in zebrafish, rbfox1 is expressed in spinal cord, mid- and hindbrain during developmental stages. In adults, expression is restricted to specific areas of the brain, including telencephalic and diencephalic regions with an important role in receiving and processing sensory information and in directing behaviour. To investigate the effect of rbfox1 deficiency on behaviour, we used rbfox1sa15940, a rbfox1 loss-of-function line. We found that rbfox1sa15940 mutants present hyperactivity, thigmotaxis, decreased freezing behaviour and altered social behaviour. We repeated these behavioural tests in a second rbfox1 loss-of-function line with a different genetic background, rbfox1del19, and found that rbfox1 deficiency affects behaviour similarly in this line, although there were some differences. rbfox1del19 mutants present similar thigmotaxis, but stronger alterations in social behaviour and lower levels of hyperactivity than rbfox1sa15940 fish. Taken together, these results suggest that rbfox1 deficiency leads to multiple behavioural changes in zebrafish that might be modulated by environmental, epigenetic and genetic background effects, and that resemble phenotypic alterations present in Rbfox1-deficient mice and in patients with different psychiatric conditions. Our study thus highlights the evolutionary conservation of rbfox1 function in behaviour and paves the way to further investigate the mechanisms underlying rbfox1 pleiotropy on the onset of neurodevelopmental and psychiatric disorders.
Parallel multisite recordings in the visual cortex of trained monkeys revealed that the responses of spatially distributed neurons to natural scenes are ordered in sequences. The rank order of these sequences is stimulus-specific and maintained even if the absolute timing of the responses is modified by manipulating stimulus parameters. The stimulus specificity of these sequences was highest when they were evoked by natural stimuli and deteriorated for stimulus versions in which certain statistical regularities were removed. This suggests that the response sequences result from a matching operation between sensory evidence and priors stored in the cortical network. Decoders trained on sequence order performed as well as decoders trained on rate vectors but the former could decode stimulus identity from considerably shorter response intervals than the latter. A simulated recurrent network reproduced similarly structured stimulus-specific response sequences, particularly once it was familiarized with the stimuli through non-supervised Hebbian learning. We propose that recurrent processing transforms signals from stationary visual scenes into sequential responses whose rank order is the result of a Bayesian matching operation. If this temporal code were used by the visual system it would allow for ultrafast processing of visual scenes.
Folding of G-protein coupled receptors (GPCRs) according to the two-stage model (Popot, J. L., and Engelman, D. M. (1990) Biochemistry 29, 4031–4037) is postulated to proceed in 2 steps: partitioning of the polypeptide into the membrane followed by diffusion until native contacts are formed. Herein we investigate conformational preferences of fragments of the yeast Ste2p receptor using NMR. Constructs comprising the first, the first two, and the first three transmembrane (TM) segments, as well as a construct comprising TM1–TM2 covalently linked to TM7 were examined. We observed that the isolated TM1 does not form a stable helix nor does it integrate well into the micelle. TM1 is significantly stabilized upon interaction with TM2, forming a helical hairpin reported previously (Neumoin, A., Cohen, L. S., Arshava, B., Tantry, S., Becker, J. M., Zerbe, O., and Naider, F. (2009) Biophys. J. 96, 3187–3196), and in this case the protein integrates into the hydrophobic interior of the micelle. TM123 displays a strong tendency to oligomerize, but hydrogen exchange data reveal that the center of TM3 is solvent exposed. In all GPCRs so-far structurally characterized TM7 forms many contacts with TM1 and TM2. In our study TM127 integrates well into the hydrophobic environment, but TM7 does not stably pack against the remaining helices. Topology mapping in microsomal membranes also indicates that TM1 does not integrate in a membrane-spanning fashion, but that TM12, TM123, and TM127 adopt predominantly native-like topologies. The data from our study would be consistent with the retention of individual helices of incompletely synthesized GPCRs in the vicinity of the translocon until the complete receptor is released into the membrane interior.
Lantibiotics are peptide-derived antibiotics that inhibit the growth of Gram-positive bacteria via interactions with lipid II and lipid II-dependent pore formation in the bacterial membrane. Due to their general mode of action the Gram-positive producer strains need to express immunity proteins (LanI proteins) for protection against their own lantibiotics. Little is known about the immunity mechanism protecting the producer strain against its own lantibiotic on the molecular level. So far, no structures have been reported for any LanI protein. We solved the structure of SpaI, a LanI protein from the subtilin producing strain Bacillus subtilis ATCC 6633. SpaI is a 16.8-kDa lipoprotein that is attached to the outside of the cytoplasmic membrane via a covalent diacylglycerol anchor. SpaI together with the ABC transporter SpaFEG protects the B. subtilis membrane from subtilin insertion. The solution-NMR structure of a 15-kDa biologically active C-terminal fragment reveals a novel fold. We also demonstrate that the first 20 N-terminal amino acids not present in this C-terminal fragment are unstructured in solution and are required for interactions with lipid membranes. Additionally, growth tests reveal that these 20 N-terminal residues are important for the immunity mediated by SpaI but most likely are not part of a possible subtilin binding site. Our findings are the first step on the way of understanding the immunity mechanism of B. subtilis in particular and of other lantibiotic producing strains in general.
The solution structure of the lantibiotic immunity protein NisI and its interactions with nisin
(2015)
Many Gram-positive bacteria produce lantibiotics, genetically encoded and posttranslationally modified peptide antibiotics, which inhibit the growth of other Gram-positive bacteria. To protect themselves against their own lantibiotics these bacteria express a variety of immunity proteins including the LanI lipoproteins. The structural and mechanistic basis for LanI-mediated lantibiotic immunity is not yet understood. Lactococcus lactis produces the lantibiotic nisin, which is widely used as a food preservative. Its LanI protein NisI provides immunity against nisin but not against structurally very similar lantibiotics from other species such as subtilin from Bacillus subtilis. To understand the structural basis for LanI-mediated immunity and their specificity we investigated the structure of NisI. We found that NisI is a two-domain protein. Surprisingly, each of the two NisI domains has the same structure as the LanI protein from B. subtilis, SpaI, despite the lack of significant sequence homology. The two NisI domains and SpaI differ strongly in their surface properties and function. Additionally, SpaI-mediated lantibiotic immunity depends on the presence of a basic unstructured N-terminal region that tethers SpaI to the membrane. Such a region is absent from NisI. Instead, the N-terminal domain of NisI interacts with membranes but not with nisin. In contrast, the C-terminal domain specifically binds nisin and modulates the membrane affinity of the N-terminal domain. Thus, our results reveal an unexpected structural relationship between NisI and SpaI and shed light on the structural basis for LanI mediated lantibiotic immunity.
Previous studies towards reduced oxygen availability have mostly focused on changes in total mRNA expression, neglecting underlying transcriptional and post-transcriptional events. Therefore, we generated a comprehensive overview of hypoxia-induced changes in total mRNA expression, global de novo transcription, and mRNA stability in monocytic THP-1 cells. Since hypoxic episodes often persist for prolonged periods, we further compared the adaptation to acute and chronic hypoxia. While total mRNA changes correlated well with enhanced transcription during short-term hypoxia, mRNA destabilization gained importance under chronic conditions. Reduced mRNA stability not only added to a compensatory attenuation of immune responses, but also, most notably, to the reduction in nuclear-encoded mRNAs associated with various mitochondrial functions. These changes may prevent the futile production of new mitochondria under conditions where mitochondria cannot exert their full metabolic function and are indeed actively removed by mitophagy. The post-transcriptional mode of regulation might further allow for the rapid recovery of mitochondrial capacities upon reoxygenation. Our results provide a comprehensive resource of functional mRNA expression dynamics and underlying transcriptional and post-transcriptional regulatory principles during the adaptation to hypoxia. Furthermore, we uncover that RNA stability regulation controls mitochondrial functions in the context of hypoxia.
Background: The causative agent of Chagas disease, Trypanosoma cruzi, and its nonpathogenic relative, Trypanosoma rangeli, are transmitted by haematophagous triatomines and undergo a crucial ontogenetic phase in the insect’s intestine. In the process, the parasites interfere with the host immune system as well as the microbiome present in the digestive tract potentially establishing an environment advantageous for development. However, the coherent interactions between host, pathogen and microbiota have not yet been elucidated in detail. We applied a metagenome shotgun sequencing approach to study the alterations in the microbiota of Rhodnius prolixus, a major vector of Chagas disease, after exposure to T. cruzi and T. rangeli focusing also on the functional capacities present in the intestinal microbiome of the insect.
Results: The intestinal microbiota of R. prolixus was dominated by the bacterial orders Enterobacterales, Corynebacteriales, Lactobacillales, Clostridiales and Chlamydiales, whereas the latter conceivably originated from the blood used for pathogen exposure. The anterior and posterior midgut samples of the exposed insects showed a reduced overall number of organisms compared to the control group. However, we also found enriched bacterial groups after exposure to T. cruzi as well as T rangeli. While the relative abundance of Enterobacterales and Corynebacteriales decreased considerably, the Lactobacillales, mainly composed of the genus Enterococcus, developed as the most abundant taxonomic group. This applies in particular to vectors challenged with T. rangeli and at early timepoints after exposure to vectors challenged with T. cruzi. Furthermore, we were able to reconstruct four metagenome-assembled genomes from the intestinal samples and elucidate their unique metabolic functionalities within the triatomine microbiome, including the genome of a recently described insect symbiont, Candidatus Symbiopectobacterium, and the secondary metabolites producing bacteria Kocuria spp.
Conclusions: Our results facilitate a deeper understanding of the processes that take place in the intestinal tract of triatomine vectors during colonisation by trypanosomal parasites and highlight the influential aspects of pathogen-microbiota interactions. In particular, the mostly unexplored metabolic capacities of the insect vector’s microbiome are clearer, underlining its role in the transmission of Chagas disease.
Understanding how species relate mechanistically to their environment via traits is a central goal in ecology. Many macroecological rules were found for macroorganisms, however, whether they can explain microorganismal macroecological patterns still requires investigation. Further, whether macroecological rules are also applicable in microclimates is largely unexplored. Here we use fruit body-forming fungi to understand both aspects better. A recent study showed first evidence for the thermal-melanism hypothesis (Bogert’s rule) in fruit body-forming fungi and relied on a continental spatial scale with large grid size. At large spatial extent and grid sizes, other factors like dispersal limitation or local microclimatic variability might influence observed patterns besides the rule of interest. Therefore, we test fungal assemblage fruit body color lightness along a local elevational gradient (mean annual temperature gradient of 7°C) while considering the vegetation cover as a proxy for local variability in microclimate. Using multivariate linear modeling, we found that fungal fruiting assemblages are significantly darker at lower mean annual temperatures supporting the thermal-melanism hypothesis. Further, we found a non-significant trend of assemblage color lightness with vegetation cover. Our results support Bogert’s rule for microorganisms with macroclimate, which was also found for macroorganisms.
Out-of-school laboratories, also called student labs, are an advantageous opportunity to teach biological subjects. Particularly in the case of complex fields such as neurobiology, student labs offer the opportunity to learn about difficult topics in a practical way. Due to numerous advantages, digital student labs are becoming increasingly popular nowadays. In this study, we investigated the effect of an electrophysiological setup for a virtual experiment with and without hands-on elements on participant motivation and technology acceptance. For this purpose, 235 students were questioned during a student laboratory day. The surveyed students showed high motivation and technology acceptance for the virtual experiment. In the comparison, the electrophysiological setup with hands-on elements performs better in the intrinsic components than the setup without hands-on elements: Thus, the hands-on approach is rated as more interesting and the perceived enjoyment scores higher. Nevertheless, both experimental groups show high values, so that the results of the study support the positive influence of digital laboratory as well as a positive influence of hands-on elements.
Determining the structure and mechanisms of all individual functional modules of cells at high molecular detail has often been seen as equal to understanding how cells work. Recent technical advances have led to a flush of high-resolution structures of various macromolecular machines, but despite this wealth of detailed information, our understanding of cellular function remains incomplete. Here, we discuss present-day limitations of structural biology and highlight novel technologies that may enable us to analyze molecular functions directly inside cells. We predict that the progression toward structural cell biology will involve a shift toward conceptualizing a 4D virtual reality of cells using digital twins. These will capture cellular segments in a highly enriched molecular detail, include dynamic changes, and facilitate simulations of molecular processes, leading to novel and experimentally testable predictions. Transferring biological questions into algorithms that learn from the existing wealth of data and explore novel solutions may ultimately unveil how cells work.
Highlights
• PUR, PVC and PLA microplastics affect life-history parameters of Daphnia magna.
• Natural kaolin particles are less toxic than microplastics.
• Microplastic toxicity is material-specific, e.g. PVC is most toxic on reproduction.
• In case of PVC, plastic chemicals are the main driver of microplastic toxicity.
• PLA bioplastics are similarly toxic as conventional plastics.
Abstract
Given the ubiquitous presence of microplastics in aquatic environments, an evaluation of their toxicity is essential. Microplastics are a heterogeneous set of materials that differ not only in particle properties, like size and shape, but also in chemical composition, including polymers, additives and side products. Thus far, it remains unknown whether the plastic chemicals or the particle itself are the driving factor for microplastic toxicity. To address this question, we exposed Daphnia magna for 21 days to irregular polyvinyl chloride (PVC), polyurethane (PUR) and polylactic acid (PLA) microplastics as well as to natural kaolin particles in high concentrations (10, 50, 100, 500 mg/L, ≤ 59 μm) and different exposure scenarios, including microplastics and microplastics without extractable chemicals as well as the extracted and migrating chemicals alone. All three microplastic types negatively affected the life-history of D. magna. However, this toxicity depended on the endpoint and the material. While PVC had the largest effect on reproduction, PLA reduced survival most effectively. The latter indicates that bio-based and biodegradable plastics can be as toxic as their conventional counterparts. The natural particle kaolin was less toxic than microplastics when comparing numerical concentrations. Importantly, the contribution of plastic chemicals to the toxicity was also plastic type-specific. While we can attribute effects of PVC to the chemicals used in the material, effects of PUR and PLA plastics were induced by the mere particle. Our study demonstrates that plastic chemicals can drive microplastic toxicity. This highlights the importance of considering the individual chemical composition of plastics when assessing their environmental risks. Our results suggest that less studied polymer types, like PVC and PUR, as well as bioplastics are of particular toxicological relevance and should get a higher priority in ecotoxicological studies.
Nisin-producing Lactococcus lactis strains show a high degree of resistance to the action of nisin, which is based upon expression of the self-protection (immunity) genes nisI, nisF, nisE, and nisG. Different combinations of nisin immunity genes were integrated into the chromosome of a nisin-sensitive Bacillus subtilis host strain under the control of an inducible promoter. For the recipient strain, the highest level of acquired nisin tolerance was achieved after coordinated expression of all four nisin immunity genes. But either the lipoprotein NisI or the ABC transporter-homologous system NisFEG, respectively, were also able to protect the Bacillus host cells. The acquired immunity was specific to nisin and provided no tolerance to subtilin, a closely related lantibiotic. Quantitative in vivo peptide release assays demonstrated that NisFEG diminished the quantity of cell-associated nisin, providing evidence that one role of NisFEG is to transport nisin from the membrane into the extracellular space. NisI solubilized from B. subtilis membrane vesicles and recombinant hexahistidine-tagged NisI from Escherichia coli interacted specifically with nisin and not with subtilin. This suggests a function of NisI as a nisin-intercepting protein.
Highlights
• Three ecological groups were identified based on distributional patterns.
• Old assessments were confirmed with the latest occurrence data.
• For each group, we derived different population trends in times of global change.
• Global change elevates importance of vector-borne diseases.
• Our results serve as base for effective Simuliidae monitoring.
Abstract
The black fly genus Simulium includes medically and ecologically important species, characterized by a wide variation of ecological niches largely determining their distributional patterns. In a rapidly changing environment, species-specific niche characteristics determine whether a species benefits or not. With aquatic egg, larval and pupal stages followed by a terrestrial adult phase, their spatial arrangements depend upon the interplay of aquatic conditions and climatic-landscape parameters in the terrestrial realm. The aim of this study was to enhance the understanding of the distributional patterns among Simulium species and their ecological drivers. In an ecological niche modelling approach, we focused on 12 common black fly species with different ecological requirements. Our modelling was based on available distribution data along with five stream variables describing the climatic, land-cover, and topographic conditions of river catchments. The modelled freshwater habitat suitability was spatially interpolated to derive an estimate of the adult black flies' probability of occurrence. Based on similarities in the spatial patterns of modelled habitat suitability we were able to identify three biogeographical groups, which allows us to confirm old assessments with current occurrence data: (A) montane species, (B) broad range species and (C) lowland species. The five veterinary and human medical relevant species Simulium equinum, S. erythrocephalum, S. lineatum, S. ornatum and S. reptans are mainly classified in the lowland species group. In the course of climatic changes, it is expected that biocoenosis will slightly shift towards upstream regions, so that the lowland group will presumably emerge as the winner. This is mainly explained by wider ecological niches, including a higher temperature tolerance and tolerance to various pollutants. In conclusion, these findings have significant implications for human and animal health. As exposure to relevant Simulium species increases, it becomes imperative to remain vigilant, particularly in investigating the potential transmission of pathogens.
Seit Jahrzehnten finden Kunststoffe aufgrund ihrer vorteilhaften Materialeigenschaften wie z. B. Formbarkeit und im Vergleich zu Glas oder Metall geringe Kosten und leichtes Gewicht, vermehrt Anwendung in allen Bereichen des täglichen Lebens. Einhergehend gelangen Kunststoffe zunehmend in die Umwelt, und reichern sich dort an. Besondere Aufmerksamkeit erfahren Partikel im Größenbereich von 1-1000 µm, sogenanntes Mikroplastik (MP), welches entweder direkt eingetragen wird oder in der Umwelt durch Fragmentierung größerer Plastikteile entsteht. Lange Zeit fokussierte sich die MP Forschung vorrangig auf aquatische Ökosysteme, obwohl Schätzungen davon ausgehen, dass die Kunststoffeinträge in terrestrischen Ökosystemen um ein Vielfaches höher sind. Besonders relevante Eintragspfade sind neben der unsachgemäßen Entsorgung von Abfällen, die landwirtschaftliche Klärschlamm- und Kompostdüngung und der zunehmende Einsatz von Mulchfolien, sowie der im Straßenverkehr generierte Reifenabrieb.
Für eine Abschätzung und Bewertung der MP-Belastung in Böden sind analytische Messungen von MP in Umweltproben essenziell, derzeit jedoch kaum existent, da MP im Boden partikulär und heterogen verteilt vorliegt und deshalb nur schwierig zu detektieren ist. Die für viele Analyseverfahren notwendige Isolation der Kunststoffpartikel, sowie die für repräsentative Messungen erforderliche Aufbereitung großer Probenvolumina stellen besondere analytische Herausforderungen mit großem Kosten- und Zeitaufwand dar. Chromatografische Verfahren finden wenig Anwendung, bieten aber vorteilhafte Voraussetzungen als Screeningverfahren für die Untersuchung von Böden, da sie nicht zwangsweise eine Partikelisolation verlangen, und zudem als Ergebnis einen Massegehalt liefern.
Diese Dissertation zeigt drei Anwendungen Chromatografie basierter Analyseverfahren zur Charakterisierung von MP im Boden. Erstmalig wurde die Thermo-Extraktion-Desorption-Gaschromatografie-Massenspektrometrie (TED-GC/MS) für die Analytik von Reifenabrieb in realen Umweltproben angewandt bei minimaler Probenaufbereitung. Dafür wurde ein Straßenrandboden umfangreich beprobt und analysiert, und es konnte neben der Eignung der analytischen Methode auch eine repräsentative Probenahmestrategie und räumliche Verteilungsmuster von Reifenabrieb im Boden demonstriert werden.
Der zweite Forschungsschwerpunkt lag auf der Methodenentwicklung und validierung eines neuartigen chemischen Extraktionsverfahrens für die Bestimmung von Polyestern in Bodenproben. Das Verfahren basiert auf der hydrolytischen Spaltung von Polyestern in ihre Monomere, deren flüssigchromatografische Abtrennung von Matrixbestandteilen und der Detektion mittels UV-Absorption. Das Verfahren verlangt neben der Extraktion keine weiteren Probenaufbereitungsschritte, ist für unterschiedliche Umweltmatrizes geeignet und ist damit z. B. prädestiniert für den Nachweis von Polyesterfasern auf gedüngten landwirtschaftlichen Flächen.
MP ist nicht nur aufgrund seiner Persistenz problematisch, sondern auch, weil es hydrophobe organische Schadstoffe aus dem Umweltmedium anreichern und transportieren kann. Maßgeblich für das Sorptionsverhalten sind die Materialeigenschaften des zugrunde liegenden Kunststoffes, welche Änderungen durch Alterungsprozessen unterliegen. Der Zusammenhang zwischen Materialalterung und Sorptionsverhalten wurde in früheren Studien kontrovers diskutiert und ist der dritte Teil dieser Arbeit. In einem Sorptionsexperiment konnte mittels Headspace-Gaschromatografie mit Flammenionisations-Detektion die Aufnahme von Aromaten an den Kunststoffen Polypropylen und Polystyrol quantifiziert werden. Die Kunststoffe wurden materialwissenschaftlich charakterisiert, teilweise künstlich gealtert und die daraus resultierende Änderungen der Materialeigenschaften sowie einhergehenden Änderungen des Sorptionsverhaltens erfasst. Dadurch war es möglich den Einfluss einzelner Materialeigenschaften auf das Sorptionsverhalten zu bewerten, Rückschlüsse auf zugrunde liegende Sorptionsmechanismen zu treffen und zu zeigen, dass in vorliegendem Experiment die Polymeralterung bei MP nicht zu einer erhöhten Schadstoffsorption führte.
Trait-dependent effects of biotic and abiotic filters on plant regeneration in Southern Ecuador
(2024)
Tropical forests have always fascinated scientists due to their unique biodiversity. However, our understanding of ecological processes shaping the complexity of tropical rainforests is still relatively poor. Plant regeneration is one of the processes that remain understudied in the tropics although this is a key process defining the structure, diversity and assembly of tropical plant communities. In my dissertation, I combine experimental, observational and trait-based approaches to identify processes shaping the assembly of seedling communities and compare associations between environmental conditions and plant traits across plant life stages. By working along a steep environmental gradient in the tropical mountains of Southern Ecuador, I was able to investigate how processes of plant regeneration vary in response to biotic and abiotic factors in tropical montane forests.
My dissertation comprises three complementary chapters, each addressing an individual research question. First, I studied how trait composition in plant communities varies in relation to the broad- and local-scale environmental conditions and across the plant life cycle. I measured key traits reflecting different ecological strategies of plants that correspond to three stages of the plant life cycle (i.e., adult trees, seed rain and recruiting seedlings). I worked on 81 subplots along an elevational gradient covering a large climatic gradient at three different elevations (1000, 2000 and 3000 m a.s.l.). In addition, I measured soil and light conditions at the local spatial scale within each subplot. My findings show that the trait composition of leaves, seeds and seedlings changed similarly across the elevational gradient, but that the different life stages responded differently to the local gradients in soil nutrients and light availability. Consequently, my findings highlight that trait-environment associations in plant communities differ between large and small spatial scales and across plant life stages.
Second, I investigated how seed size affects seedling recruitment in natural forests and in pastures in relation to abiotic and biotic factors. I set up a seed sowing experiment in both habitat types and sowed over 8,000 seeds belonging to seven tree species differing in seed size. I found that large-seeded species had higher proportions of recruitment in the forests compared to small-seeded species. However, small-seeded species tended to recruit better in pastures compared to large-seeded species. I showed that high surface temperature was the main driver of differences in seedling recruitment between habitats, because it limited seedling recruitment of large-seeded species. The results from this experiment show that pasture restoration requires seed addition of large-seeded species and active protection of recruiting seedlings in order to mitigate harmful conditions associated with high temperatures in deforested areas.
Third, I examined the associations between seedling beta-diversity and different abiotic and biotic factors between and within elevations. I applied beta-diversity partitioning to obtain two components of beta-diversity: species turnover and species richness differences. I associated these components of beta-diversity with biotic pressures by herbivores and fungal pathogens and environmental heterogeneity in light and soil conditions. I found that species turnover in seedling communities was positively associated with the dissimilarity in biotic pressures within elevations and with environmental heterogeneity between elevations. Further, I found that species richness differences increased primarily with increasing environmental heterogeneity within elevations. My findings show that the associations between beta-diversity of seedling communities and abiotic and biotic factors are scale-dependent, most likely due to differences in species sorting in response to biotic pressures and species coexistence in response to environmental heterogeneity.
My dissertation reveals that studying processes of community assembly at different plant life stages and spatial scales can yield new insights into patterns and processes of plant regeneration in tropical forests. I investigated how community assembly processes are governed by abiotic and biotic filtering across and within elevations. I also experimentally explored how the process of seedling recruitment depends on seed size-dependent interactions, and verified how these effects are associated with abiotic and biotic filtering. Identifying such processes is crucial to inform predictive models of environmental change on plant regeneration and successful forest restoration. Further exploration of plant functional traits and their associations with local-scale environmental conditions could effectively support local conservation efforts needed to enhance forest cover in the future and halt the accelerating loss of biodiversity.
Calcium (Ca2+) elevation is an essential secondary messenger in many cellular processes, including disease progression and adaptation to external stimuli, e.g., gravitational load. Therefore, mapping and quantifying Ca2+ signaling with a high spatiotemporal resolution is a key challenge. However, particularly on microgravity platforms, experiment time is limited, allowing only a small number of replicates. Furthermore, experiment hardware is exposed to changes in gravity levels, causing experimental artifacts unless appropriately controlled. We introduce a new experimental setup based on the fluorescent Ca2+ reporter CaMPARI2, onboard LED arrays, and subsequent microscopic analysis on the ground. This setup allows for higher throughput and accuracy due to its retrograde nature. The excellent performance of CaMPARI2 was demonstrated with human chondrocytes during the 75th ESA parabolic flight campaign. CaMPARI2 revealed a strong Ca2+ response triggered by histamine but was not affected by the alternating gravitational load of a parabolic flight.
In Saccharomyces cerevisiae, the NDI1 gene encodes a mitochondrial NADH dehydrogenase, the catalytic side of which projects to the matrix side of the inner mitochondrial membrane. In addition to this NADH dehydrogenase, S. cerevisiae exhibits another mitochondrial NADH-dehydrogenase activity, which oxidizes NADH at the cytosolic side of the inner membrane. To investigate whether open reading frames YMR145c/NDE1 and YDL 085w/NDE2, which exhibit sequence similarity with NDI1, encode the latter enzyme, NADH-dependent mitochondrial respiration was assayed in wild-type S. cerevisiae and nde deletion mutants. Mitochondria were isolated from aerobic, glucose-limited chemostat cultures grown at a dilution rate (D) of 0. 10 h-1, in which reoxidation of cytosolic NADH by wild-type cells occurred exclusively by respiration. Compared with the wild type, rates of mitochondrial NADH oxidation were about 3-fold reduced in an nde1Delta mutant and unaffected in an nde2Delta mutant. NADH-dependent mitochondrial respiration was completely abolished in an nde1Delta nde2Delta double mutant. Mitochondrial respiration of substrates other than NADH was not affected in nde mutants. In shake flasks, an nde1Delta nde2Delta mutant exhibited reduced specific growth rates on ethanol and galactose but not on glucose. Glucose metabolism in aerobic, glucose-limited chemostat cultures (D = 0.10 h-1) of an nde1Delta nde2Delta mutant was essentially respiratory. Apparently, under these conditions alternative systems for reoxidation of cytosolic NADH could replace the role of Nde1p and Nde2p in S. cerevisiae.
In the diazotroph Klebsiella pneumoniae the flavoprotein NifL inhibits the activity of the nif-specific transcriptional activator NifA in response to molecular oxygen and combined nitrogen. Sequestration of reduced NifL to the cytoplasmic membrane under anaerobic and nitrogen-limited conditions impairs inhibition of cytoplasmic NifA by NifL. To analyze whether NifL is reduced by electrons directly derived from the reduced menaquinone pool, we studied NifL reduction using artificial membrane systems containing purified components of the anaerobic respiratory chain of Wolinella succinogenes. In this in vitro assay using proteoliposomes containing purified formate dehydrogenase and purified menaquinone (MK6) or 8-methylmenaquinone (MMK6) from W. succinogenes, reduction of purified NifL was achieved by formate oxidation. Furthermore, the respective reduction rates, which were determined using equal amounts of NifL, have been shown to be directly dependent on the concentration of both formate dehydrogenase and menaquinones incorporated into the proteoliposomes, demonstrating a direct electron transfer from menaquinone to NifL. When purified hydrogenase and MK6 from W. succinogenes were inserted into the proteoliposomes, NifL was reduced with nearly the same rate by hydrogen oxidation. In both cases reduced NifL was found to be highly associated to the proteoliposomes, which is in accordance with our previous findings in vivo. On the bases of these experiments, we propose that the redox state of the menaquinone pool is the redox signal for nif regulation in K. pneumoniae by directly transferring electrons onto NifL under anaerobic conditions.
Arsenic trioxide is a toxic metalloid and carcinogen that is also used as an anticancer drug, and for this reason it is important to identify the routes of arsenite uptake by cells. In this study the ability of hexose transporters to facilitate arsenic trioxide uptake in Saccharomyces cerevisiae was examined. In the absence of glucose, strains with disruption of the arsenite efflux gene ACR3 accumulated high levels of (73)As(OH)(3). The addition of glucose inhibited uptake by approximately 80%. Disruption of FPS1, the aquaglyceroporin gene, reduced glucose-independent uptake by only about 25%, and the residual uptake was nearly completely inhibited by hexoses, including glucose, galactose, mannose, and fructose but not pentoses or disaccharides. A strain lacking FPS1, ACR3, and all genes for hexose permeases except for HXT3, HXT6, HXT7, and GAL2 exhibited hexose-inhibitable (73)As(OH)(3) uptake, whereas a strain lacking all 18 hexose transport-related genes (HXT1 to HXT17 and GAL2), FPS1 and ACR3, exhibited <10% of wild type (73)As(OH)(3) transport. When HXT1, HXT3, HXT4, HXT5, HXT7, or HXT9 was individually expressed in that strain, hexose-inhibitable (73)As(OH)(3) uptake was restored. In addition, the transport of [(14)C]glucose was inhibited by As(OH)(3). These results clearly demonstrate that hexose permeases catalyze the majority of the transport of the trivalent metalloid arsenic trioxide.
Secretins are a family of large bacterial outer membrane protein complexes mediating the transport of complex structures, such as type IV pili, DNA and filamentous phage, or various proteins, such as extracellular enzymes and pathogenicity determinants. PilQ of the thermophilic bacterium Thermus thermophilus HB27 is a member of the secretin family required for natural transformation. Here we report the isolation, structural, and functional analyses of a unique PilQ from T. thermophilus. Native PAGE, gel filtration chromatography, and electrophoretic mobility shift analyses indicated that PilQ forms a macromolecular homopolymeric complex that binds dsDNA. Electron microscopy showed that the PilQ complex is 15 nm wide and 34 nm long and consists of an extraordinary stable "cone" and "cup" structure and five ring structures with a large central channel. Moreover, the electron microscopic images together with secondary structure analyses combined with structural data of type II protein secretion system and type III protein secretion system secretins suggest that the individual rings are formed by conserved domains of alternating α-helices and β-sheets. The unprecedented length of the PilQ complex correlated well with the distance between the inner and outer membrane of T. thermophilus. Indeed, PilQ was found immunologically in both membranes, indicating that the PilQ complex spans the entire cell periphery of T. thermophilus. This is consistent with the hypothesis that PilQ accommodates a PilA4 comprising pseudopilus mediating DNA transport across the outer membrane and periplasmic space in a single-step process.
DNA translocators of natural transformation systems are complex systems critical for the uptake of free DNA and provide a powerful mechanism for adaptation to changing environmental conditions. In natural transformation machineries, outer membrane secretins are suggested to form a multimeric pore for the uptake of external DNA. Recently, we reported on a novel structure of the DNA translocator secretin complex, PilQ, in Thermus thermophilus HB27 comprising a stable cone and cup structure and six ring structures with a large central channel. Here, we report on structural and functional analyses of a set of N-terminal PilQ deletion derivatives in T. thermophilus HB27. We identified 136 N-terminal residues exhibiting an unusual ααβαββα fold as a ring-building domain. Deletion of this domain had a dramatic effect on twitching motility, adhesion, and piliation but did not abolish natural transformation. These findings provide clear evidence that the pilus structures of T. thermophilus are not essential for natural transformation. The truncated complex was not affected in inner and outer membrane association, indicating that the 136 N-terminal residues are not essential for membrane targeting. Analyses of complex formation of the truncated PilQ monomers revealed that the region downstream of residue 136 is required for multimerization, and the region downstream of residue 207 is essential for monomer stability. Possible implications of our findings for the mechanism of DNA uptake are discussed.
Glucokinase (GK) is a key enzyme of glucose metabolism in liver and pancreatic beta-cells, and small molecule activators of GK (GKAs) are under evaluation for the treatment of type 2 diabetes. In liver, GK activity is controlled by the GK regulatory protein (GKRP), which forms an inhibitory complex with the enzyme. Here, we performed isothermal titration calorimetry and surface plasmon resonance experiments to characterize GK-GKRP binding and to study the influence that physiological and pharmacological effectors of GK have on the protein-protein interaction. In the presence of fructose-6-phosphate, GK-GKRP complex formation displayed a strong entropic driving force opposed by a large positive enthalpy; a negative change in heat capacity was observed (Kd = 45 nm, DeltaH = 15.6 kcal/mol, TDeltaS = 25.7 kcal/mol, DeltaCp = -354 cal mol(-1) K(-1)). With k(off) = 1.3 x 10(-2) s(-1), the complex dissociated quickly. The thermodynamic profile suggested a largely hydrophobic interaction. In addition, effects of pH and buffer demonstrated the coupled uptake of one proton and indicated an ionic contribution to binding. Glucose decreased the binding affinity between GK and GKRP. This decrease was potentiated by an ATP analogue. Prototypical GKAs of the amino-heteroaryl-amide type bound to GK in a glucose-dependent manner and impaired the association of GK with GKRP. This mechanism might contribute to the antidiabetic effects of GKAs.
The traffic AAA-ATPase PilF is essential for pilus biogenesis and natural transformation of Thermus thermophilus HB27. Recently, we showed that PilF forms hexameric complexes containing six zinc atoms coordinated by conserved tetracysteine motifs. Here we report that zinc binding is essential for complex stability. However, zinc binding is neither required for pilus biogenesis nor natural transformation. A number of the mutants did not exhibit any pili during growth at 64 °C but still were transformable. This leads to the conclusion that type 4 pili and the DNA translocator are distinct systems. At lower growth temperatures (55 °C) the zinc-depleted multiple cysteine mutants were hyperpiliated but defective in pilus-mediated twitching motility. This provides evidence that zinc binding is essential for the role of PilF in pilus dynamics. Moreover, we found that zinc binding is essential for complex stability but dispensable for ATPase activity. In contrast to many polymerization ATPases from mesophilic bacteria, ATP binding is not required for PilF complex formation; however, it significantly increases complex stability. These data suggest that zinc and ATP binding increase complex stability that is important for functionality of PilF under extreme environmental conditions.
Background: In the face of ongoing climate warming, vector-borne diseases are expected to increase in Europe, including tick-borne diseases (TBD). The most abundant tick-borne diseases in Germany are Tick-Borne Encephalitis (TBE) and Lyme Borreliosis (LB), with Ixodes ricinus as the main vector.
Methods: In this study, we display and compare the spatial and temporal patterns of reported cases of human TBE and LB in relation to some associated factors. The comparison may help with the interpretation of observed spatial and temporal patterns.
Results: The spatial patterns of reported TBE cases show a clear and consistent pattern over the years, with many cases in the south and only few and isolated cases in the north of Germany. The identification of spatial patterns of LB disease cases is more difficult due to the different reporting practices in the individual federal states. Temporal patterns strongly fluctuate between years, and are relatively synchronized between both diseases, suggesting common driving factors. Based on our results we found no evidence that weather conditions affect the prevalence of both diseases. Both diseases show a gender bias with LB bing more commonly diagnosed in females, contrary to TBE being more commonly diagnosed in males.
Conclusion: For a further investigation of of the underlying driving factors and their interrelations, longer time series as well as standardised reporting and surveillance system would be required.
Unter den weltweit in ständigem Gebrauch befindlichen Chemikalien befinden sich nicht nur Verbindungen mit akuter toxischer Wirkung, sondern auch solche mit Wirkung auf das endokrine System. Eine große Rolle spielt hier vor allem die Störung der Geschlechtsdifferenzierung und der Reproduktion, ausgelöst durch natürliche oder synthetische Chemikalien mit endokrinem Potential, sogenannte endokrine Disruptoren (ED). Diese Chemikalien können über unterschiedliche Eintragspfade in die Umwelt gelangen. Seit Mitte des 20. Jahrhunderts werden mehr und mehr Fälle bekannt, in denen anthropogene Chemikalien die Pflanzen- und Tierwelt belasten, darunter zahlreiche Befunde zu Störungen des Hormonsystems von Mensch und Tier.
Im Rahmen der Gefahren- und Risikobewertung steht bereits eine Vielzahl harmonisierter Prüfrichtlinien für die Identifizierung und Evaluierung der Effekte von (potentiellen) ED zur Verfügung. Um die Gesamtheit aller potentiellen Interaktionen von ED mit dem Hormonsystem detektieren zu können, ist die In-vivo-Untersuchung an Vertebraten in der Chemikalienregistrierung bisher unabdingbar. Bei der Untersuchung endokriner Potentiale in höheren Vertebraten spielen vor allem nager- und vogelbasierte Testsysteme eine wichtige Rolle. Diese bergen jedoch einen hohen zeitlichen, personellen und finanziellen Aufwand und erfordern eine massive Zahl an Versuchstieren, die für diese Tests benötigt werden. Darüber hinaus beinhalten Tierversuche eine Vielzahl von Problemen einschließlich ethischer Bedenken, die sich als Konsequenz der Tierhaltung unter Versuchsbedingungen ergeben. Ein sehr interessanter und vielversprechender Ansatz zur Reduktion von Tierversuchen ist die Entwicklung eines standardisierten Verfahrens für die Untersuchung potentieller ED in Vogelembryonen. Auf Vogelembryonen basierende In-ovo-Modelle stellen einen Mittelweg zwischen In-vitro- und In-vivo-Testsystemen dar. Mit dem Vogeleitest wird der sich entwickelnde Embryo, das für ED sensitivste Entwicklungsstadium im Leben eines Organismus, berücksichtigt.
Das Ziel der vorliegenden Arbeit war die Entwicklung und Eignungsuntersuchung eines auf dem Embryo des Haushuhns (Gallus gallus domesticus) basierenden Testsystems für den Nachweis von ED. Das resultierende Testsystem soll als Alternativmethode zu bisher etablierten nager- und vogelbasierten Testsystemen für die Untersuchung der Effekte hormonell aktiver Substanzen auf die Geschlechtsdifferenzierung in höheren Wirbeltieren eingesetzt werden.
Die im Rahmen der vorliegenden Dissertation durchgeführten Arbeiten umfassten sowohl die Charakterisierung der Normalentwicklung des Hühnerembryos, unbeeinflusst durch ED, als auch die morphologisch-histologischen Veränderungen der Gonaden von substanzexponierten Embryonen. Für die Untersuchung substanzbedingter Effekte, welche den Schwerpunkt der vorliegenden Arbeit darstellen, wurden die Embryonen gegenüber verschiedenen (anti)estrogenen und (anti)androgenen Substanzen exponiert. Unter Einfluss der Estrogene Bisphenol A (BPA) und 17α-Ethinylestradiol (EE2) entwickelten sich die Keimdrüsen der Männchen zu Ovotestes, während Weibchen ein Ovar mit deutlich schmalerem Cortex ausbildeten. Unter Einfluss der Antiestrogene Fulvestrant und Tamoxifen blieben Effekte auf die Gonaden männlicher Embryonen aus, eine durch das potente Estrogen EE2 hervorgerufene Feminisierung männlicher Gonaden konnte durch beide Substanzen jedoch effektiv antagonisiert werden. Weibchen bilden unter Einfluss von Tamoxifen deutlich schmalere linke Gonaden mit einem missgebildeten Cortex aus. Unter Einfluss der Androgene Tributylzinn (TBT) und 17α-Methyltestosteron (MT) blieben die Effekte auf männliche Embryonen aus, während die Weibchen anatomisch virilisierte Gonaden und eine Reduktion des linken gonadalen Cortex aufwiesen. Allein die untersuchten antiandrogenen Versuchssubstanzen Cyproteronacetat (CPA), Flutamid und p,p´-Dichlorodiphenyldichloroethen (p,p´-DDE) hatten keinen Effekt auf die gonadale Geschlechtsdifferenzierung männlicher und weiblicher Hühnerembryonen.
Es konnte gezeigt werden, dass der Embryo von G. gallus domesticus einen sensitiven Organismus innerhalb des Tierreichs darstellt und hinreichend sensitiv auf eine Reihe von endokrin wirksamen und reproduktionstoxischen Chemikalien reagiert. Anatomische und histologische Änderungen der Gonaden können daher als Biomarker für die Wirkung von ED bei Vögeln nützlich sein. Die untersuchten Endpunkte beziehen sich jedoch auf apikale Effekte und liefern keine mechanistischen Informationen zu den untersuchten Substanzen. Der
Hühnereitest ist eine sinnvolle Ergänzung zur bestehenden OECD-Testbatterie und zeichnet sich besonders durch seine kostengünstige und einfache Handhabung im Labor sowie einfach durchzuführende Tests aus. Durch die vergleichsweise kurze Versuchsdauer von nur 19 Tagen ist ein schnelles Substanzscreening möglich, welches zeitlich deutliche Vorteile gegenüber den etablierten nager- und vogelbasierten Testsystemen hat. Als Alternative zu bisherigen Assays könnte der vorgeschlagene Hühnereitest dazu beitragen, im Rahmen der (öko)toxikologischen Gefährdungs- und Risikobewertung von Chemikalien künftig weniger Versuchstiere zu verwenden.
The hydrophobic thickness of membranes, which is manly defined by fatty acids, influences the packing of transmembrane domains of proteins and thus can modulate the activity of these proteins. We analyzed the dynamics of the dimerization of Glycophorin A (GpA) by molecular dynamics simulations to describe the fatty acid dependence of the transmembrane region assembly. GpA represents a well-established model for dimerization of single transmembrane helices containing a GxxxG motif in vitro and in silico. We performed simulations of the dynamics of the NMR-derived dimer as well as self-assembly simulations of monomers in membranes composed of different fatty acid chains and monitored the formed interfaces and their transitions. The observed dimeric interfaces, which also include the one known from NMR, are highly dynamic and converted into each other. The frequency of interface formation and the preferred transitions between interfaces similar to the interface observed by NMR analysis strongly depend on the fatty acid used to build the membrane. Molecular dynamic simulations after adaptation of the helix topology parameters to better represent NMR derived structures of single transmembrane helices yielded an enhanced occurrence of the interface determined by NMR in molecular dynamics simulations. Taken together we give insights into the influence of fatty acids and helix conformation on the dynamics of the transmembrane domain of GpA.
Analysis of whole cell lipid extracts of bacteria by means of ultra-performance (UP)LC-MS allows a comprehensive determination of the lipid molecular species present in the respective organism. The data allow conclusions on its metabolic potential as well as the creation of lipid profiles, which visualize the organism's response to changes in internal and external conditions. Herein, we describe: i) a fast reversed phase UPLC-ESI-MS method suitable for detection and determination of individual lipids from whole cell lipid extracts of all polarities ranging from monoacylglycerophosphoethanolamines to TGs; ii) the first overview of a wide range of lipid molecular species in vegetative Myxococcus xanthus DK1622 cells; iii) changes in their relative composition in selected mutants impaired in the biosynthesis of α-hydroxylated FAs, sphingolipids, and ether lipids; and iv) the first report of ceramide phosphoinositols in M. xanthus, a lipid species previously found only in eukaryotes.
ω-Azido fatty acids as probes to detect fatty acid biosynthesis, degradation, and modification
(2014)
FAs play a central role in the metabolism of almost all known cellular life forms. Although GC-MS is regarded as a standard method for FA analysis, other methods, such as HPLC/MS, are nowadays widespread but are rarely applied to FA analysis. Here we present azido-FAs as probes that can be used to study FA biosynthesis (elongation, desaturation) or degradation (β-oxidation) upon their uptake, activation, and metabolic conversion. These azido-FAs are readily accessible by chemical synthesis and their matization with high sensitivity by HPLC/MS, contributing a powerful tool to FA analysis, and hence, lipid analysis in general.
A first model of the three-dimensional structure of the photosynthetic reaction center of the mutant T1 (SerL 223 → Ala, ArgL 217 → His) from Rhodopseudomonas viridis, resistant toward the triazine herbicide terbutryn (2-methylthio-4-ethylamino-6-f-butylamino-5-triazine), has been developed from X-ray data measured to a resolution of 2.5 Å. The secondary quinone, QB, which in T1 binds better than in the wild type, is present in the crystals. Both substituted residues are clearly visible in the difference fourier map. The replacement of these two residues in the QB site causes only minor changes in the overall structure of the protein.
Young poplar cuttings (Populus nigra L. cv. Loenen and P. maximowiczii Henry x P. nigra L. cv. Rochester) were exposed for six weeks in open-top chambers to realistic concentrations of pollutant mixtures: 1) control; 2) SO2/NOx; 3)O3/ NOx and 4)SO2/O3/NOx. In this sequence of fumigation variants, the degree of influence of the various parameters of the nitrogen metabolism and of premature leaf drop increased very frequently compared to the control plants, P. nigra L. proving to be the more sensitive species.
The elevated Kjeldahl nitrogen content of the fumigated leaves was accompanied by either an increase in free amino acids or in total protein or, in the case of particularly large rises (SO2/O3/NOx variants), by increases in both substance groups. Proteolytic processes as a cause of the elevated content of free amino acids could be excluded to a large extent. A diminished de novo synthesis of proteins obviously led to a shift in the amino acid/protein relationship. In the younger fumigated leaves, the total concentration of free amino acids exceeded the values of the older leaves. The elevated amino acid content of the fumigated leaves was produced to a high degree by the glycolate pathway and the Krebs cycle. The increased turnover of the carbon skeletons was connected with a drastic starch degradation, especially in the older leaves.
The interaction of the amino acid and carbohydrate metabolisms is probably an important regulator in the promotion of rapid growth of young leaves in order to compensate premature leaf loss.
Membrane-embedded β-barrel proteins are found in the outer membranes (OM) of Gram-negative bacteria, mitochondria and chloroplasts. In eukaryotic cells, precursors of these proteins are synthesized in the cytosol and have to be sorted to their corresponding organelle. Currently, the signal that ensures their specific targeting to either mitochondria or chloroplasts is ill-defined. To address this issue, we studied targeting of the chloroplast β-barrel proteins Oep37 and Oep24. We found that both proteins can be integrated in vitro into isolated plant mitochondria. Furthermore, upon their expression in yeast cells Oep37 and Oep24 were exclusively located in the mitochondrial OM. Oep37 partially complemented the growth phenotype of yeast cells lacking Porin, the general metabolite transporter of this membrane. Similarly to mitochondrial β-barrel proteins, Oep37 and Oep24 expressed in yeast cells were assembled into the mitochondrial OM in a pathway dependent on the TOM and TOB complexes. Taken together, this study demonstrates that the central mitochondrial components that mediate the import of yeast β-barrel proteins can deal with precursors of chloroplast β-barrel proteins. This implies that the mitochondrial import machinery does not recognize signals that are unique to mitochondrial β-barrel proteins. Our results further suggest that dedicated targeting factors had to evolve in plant cells to prevent mis-sorting of chloroplast β-barrel proteins to mitochondria.
A non-radioactive cell-free assay was developed to quantitatively determine inhibition of plant-type phytoene desaturase by bleaching herbicides. An active desaturase was prepared from an appropriately cloned E. coli transformant. Another E. coli transformant was used to produce the required phytoene. Phytofluene and t-carotene, the products of the desaturase reaction, were either determined by HPLC or optical absorption spectra. Enzyme kinetics and inhibition data for the bleaching tetrazole herbicide WL110547 are presented as an example.
The accumulation and distribution of characteristic secondary products in the different organs of an Aloe plant (A. succotrina Lam.) were studied by high performance liquid chromatography for the first time. In the leaves of the Aloe plant, only anthrone-C-glycosyls of the 7-hydroxyaloin type and, for the first time in plant material, the free anthraquinone 7-hydroxyaloeemodin were found. In contrast to previous reports on the distribution of secondary products in Aloe plants, anthrone-C-glycosyls were also detected in flowers, bracts and the inflorescence axis of the species examined. Aloesaponol I, a tetrahydroanthracene aglycone, was only present in the underground organs and in the stem. The 2-alkylchromone-C-glucosyl aloeresin B showed no specific occurrence as it was found in every type of organ. Based on these results and the findings of recent studies on Aloe roots and flowers, a distribution scheme of polyketide types in the Aloe plant was established. It suggests a separate and independent anthranoid metabolism for underground Aloe organs and stem on the one hand, and for leaves and inflorescence organs on the other hand. In the latter structures anthranoid metabolism seems to be additionally compartmentalized as the anthranoid pro files of inflorescence organs and leaves differ in two points relevant to anthranoid biosynthe sis: firstly, the occurrence of anthrone aglycones and secondly, the individual content of corresponding anthrone-C-glucosyl diastereomers.
The anion transport protein of the human erythrocyte membrane, band 3, was solubilized and purified in solutions of the non-ionic detergent nonaethylene glycol lauryl ether and then reconstituted in spherical egg phosphatidylcholine bilayers as described earlier (U. Scheuring, K. Kollewe, W. Haase, and D. Schubert, J. Membrane Biol. 90, 123-135 (1986)). The resulting paucilamellar proteoliposom es of average diameter 70 nm were transformed into smaller vesicles by French press treatment and fractionated according to size by gel filtration. The smallest protein-containing liposomes obtained had diameters around 32 nm; still smaller vesicles were free of protein. All proteoliposome samples studied showed a rapid sulfate efflux which was sensitive to specific inhibitors of band 3-mediated anion exchange. In addition, the orientation of the transport protein in the vesicle membranes was found to be “right-side-out” in all samples. This suggests that the orientation of the protein in the vesicle membranes is dictated by the shape of the protein’s intramembrane domain and that this domain has the form of a truncated cone or pyramid.
Physiological conditions which lead to changes in total carotenoid content in tomato plantlets were identified. Carotenoid levels were found to increase after the onset of a dark period during a normal 24h cycle. This rapid initial increase is followed by a steady decrease in carotenoid content throughout the night. A decrease in the expression of several carotenogenic genes, namely pds, zds (carotenoid desaturases) and ptox (plastid terminal oxidase), was observed following the removal of the light (when carotenoid content is at its highest). An increase in gene expression was observed before the return to light for pds and zds (when carotenoid levels were at their lowest), or following the return to light for ptox. The phytoene desaturation inhibitor norflurazon leads to a decrease coloured carotenoid content and, in the light, this correlated with pds and zds gene induction. In the dark, norflurazon treatment led to only a weak decrease in carotenoid content and only a small increase in pds and zds gene expression. The striking absence of phytoene accumulation under norflurazon treatment in the dark suggests a down-regulation of carotenoid formation in darkness. However, prolonged dark conditions, or treatment with photosynthetic inhibitors, surprisingly led to higher carotenoid levels, which correlated with decreased expression of most examined genes. In addition to light, which acts in a complex way on carotenoid accumulation and gene expression, our results are best explained by a regulatory effect of carotenoid levels on the expression of several biosynthetic genes. In addition, monitoring of protein amounts for phytoene desaturase and plastid terminal oxidase (which sometimes do not correlate with gene expression) indicate an even more complex regulatory pattern.
Pheromonal synergism and inhibition in P. flammea was further studied through electrophysiological and field trapping tests. Z11-tetradecenyl acetate and Z11-hexa - decenyl acetate, each acting upon a separate type of male sensory cell, were equally effective in synergizing attraction responses to the major pheromone component, Z9-tetradecenyl acetate. Addition of Z7-dodecenyl acetate to these lures reduced captures. Male attraction specificity markedly varied with local moth density.
The role of TolC has largely been explored in proteobacteria, where it functions as a metabolite and protein exporter. In contrast, little research has been carried out on the function of cyanobacterial homologues, and as a consequence, not much is known about the mechanism of cyanobacterial antibiotic uptake and metabolite secretion in general. It has been suggested that the TolC-like homologue of the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120, termed heterocyst glycolipid deposition protein D (HgdD), is involved in both protein and lipid secretion. To describe its function in secondary metabolite secretion, we established a system to measure the uptake of antibiotics based on the fluorescent molecule ethidium bromide. We analyzed the rate of porin-dependent metabolite uptake and confirmed the functional relation between detoxification and the action of HgdD. Moreover, we identified two major facilitator superfamily proteins that are involved in this process. It appears that anaOmp85 (Alr2269) is not required for insertion or assembly of HgdD, because an alr2269 mutant does not exhibit a phenotype similar to the hgdD mutant. Thus, we could assign components of the metabolite efflux system and describe parameters of detoxification by Anabaena sp. PCC 7120.
The TolC-like protein HgdD of the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 is part of multiple three-component "AB-D" systems spanning the inner and outer membranes and is involved in secretion of various compounds, including lipids, metabolites, antibiotics, and proteins. Several components of HgdD-dependent tripartite transport systems have been identified, but the diversity of inner membrane energizing systems is still unknown. Here we identified six putative resistance-nodulation-cell division (RND) type factors. Four of them are expressed during late exponential and stationary growth phase under normal growth conditions, whereas the other two are induced upon incubation with erythromycin or ethidium bromide. The constitutively expressed RND component Alr4267 has an atypical predicted topology, and a mutant strain (I-alr4267) shows a reduction in the content of monogalactosyldiacylglycerol as well as an altered filament shape. An insertion mutant of the ethidium bromide-induced all7631 did not show any significant phenotypic alteration under the conditions tested. Mutants of the constitutively expressed all3143 and alr1656 exhibited a Fox(-) phenotype. The phenotype of the insertion mutant I-all3143 parallels that of the I-hgdD mutant with respect to antibiotic sensitivity, lipid profile, and ethidium efflux. In addition, expression of the RND genes all3143 and all3144 partially complements the capability of Escherichia coli ΔacrAB to transport ethidium. We postulate that the RND transporter All3143 and the predicted membrane fusion protein All3144, as homologs of E. coli AcrB and AcrA, respectively, are major players for antibiotic resistance in Anabaena sp. PCC 7120.
By a comparative thin layer chromatographic screening of the methanol-soluble leaf exudates from more than 400 Aloe plants (183 species), 5-hydroxyaloin A was identified in 20 species. Whilst 13 of the 20 species revealed interindividual variations concerning to the occurrence of 5-hydroxyaloin A, this anthrone-C-glucosyl was unambiguously detected in each individual of 6 Aloe species. In the leaf exudates from A. marlothii Berger 5-hydroxyaloin A was only traceable in the aloin-containing chemivars. The complete anthrone-C-glucosyl pattern of these 7 clearly characterized species has been determined additionally by qualitative and quantitative high performance liquid chromatography: The results obtained demonstrate that 5-hydroxyaloin only occurs in the more stable A-configuration (10 R, 1′S), thus being till now the only anthrone-C-glycosyl which has not been found as diastereomeric pair genuinely in plants. As well, 5-hydroxyaloin A characterizes a quantitatively significant hydroxylating pathway in biosynthesis of anthranoids. It is discussed as a chemotaxonomic marker of the genus Aloe, especially of the sections Pachydendron and Eualoe.
In haploid and diploid S. cerevisiae the dimer yield ratio TT̂/CT̂ is found to be 1.2/1 and 1.3/1, resp., at the UV (254 nm) unit dose 1 erg/mm2, the share of TT̂ and CT̂ in a UV (254 nm) lethal hit being 0.7 TT̂ and 0.6 CT̂. A general formulation of the UV lethal hit is given and discussed. The TT̂ + CT̂ yields obtained for S. cerevisiae are compared to those reported for other organisms. It is found that there obviously exists a directly proportional linear correlation between genome size and TT̂ + CT̂ yield for the UV dose range well below the stationary levels of the TT̂ and CT̂ formation kinetics.
A screening procedure is presented which allows the isolation of yeast mutants (typ tlr) with highly efficient utilization of exogenous deoxythymidine-5′-monophosphate (5′-dTMP) (>50% ). Data are given concerning the phenomenon of 5′-dTMP utilization in general: (i) The ability of S. cerevisiae to incorporate exogenous 5′-dTMP was found to already be a wild type feature of this yeast, i. e. apparently not to be due to any mutation such as typ , tup, tmp per or tum. Consequently these mutations are interpreted as amplifiers of a pre-given wild type potency. So far eight stages of 5′-dTMP utilization were detected as classified by the optimal 5′-dTMP requirement, with 5′-dTMP biosynthesis blocked, of the corresponding mutant strains isolated. All of them fit well into a mathematical series of the type “2n × 1.5” (n = 0, 1, 2, … , 11), where the product term for n = 11 represents the 5′-dTMP requirement (μg/ml) of the best 5′-dTMP utilizing wild type strain found, (ii) Amplification of the 5′-dTMP utilizing potency obviously is due to any genetically determined alteration of the yeast 5′-dTMP uptaking principle itself or of physiological processes accompanying the monophosphate’s uptake, (iii) The functioning of 5′-dTMP uptake requires acidic (≦ pH 6) conditions in the yeast cell’s outer environment, (iv) Some yeast typ and typ tlr mutants were found to exhibit a more or less pronounced sensitivity towards exogenously offered 5′dTM P. The response of a sensitive strain towards inhibitory concentrations of the nucleotide apparently is co-conditioned by the presence or absence of thymidylate biosynthesis. With 5′-dTMP biosynthesis blocked the 5′-dTMP mediated inhibition is a permanent one and finally leads to the death of a cell. With a functioning thymidylate biosynthesis, in contrast, the inhibition is only temporary, (v) Yeast typ or typ tlr strains were observed to dephosphorylate exogenous 5′-dTMP to thymidine due to a phosphatase activity which cannot be eliminated at pH 7 + 70 mм inorganic phosphate conditions in the growth medium. This 5′-dTMP cleavage obviously occurs outside the cell and does not seem to be correlated both to the monophosphate’s uptake and to the phenomenon of 5′-dTMP sensitivity. The destruction of 5′-dTMP does not disturb (5′-dTMP) DNA-specific labelling.