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The glidobactin-like natural products (GLNPs) glidobactin A and cepafungin I have been reported to be potent proteasome inhibitors and are regarded as promising candidates for anticancer drug development. Their biosynthetic gene cluster (BGC) plu1881–1877 is present in entomopathogenic Photorhabdus laumondii but silent under standard laboratory conditions. Here we show the largest subset of GLNPs, which are produced and identified after activation of the silent BGC in the native host and following heterologous expression of the BGC in Escherichia coli. Their chemical diversity results from a relaxed substrate specificity and flexible product release in the assembly line of GLNPs. Crystal structure analysis of the yeast proteasome in complex with new GLNPs suggests that the degree of unsaturation and the length of the aliphatic tail are critical for their bioactivity. The results in this study provide the basis to engineer the BGC for the generation of new GLNPs and to optimize these natural products resulting in potential drugs for cancer therapy.
The composition of cellular membranes is extremely complex and the mechanisms underlying their homeostasis are poorly understood. Organelles within a eukaryotic cell require a non-random distribution of membrane lipids and a tight regulation of the membrane lipid composition is a prerequisite for the maintenance of specific organellar functions. Physical membrane properties such as bilayer thickness, lipid packing density and surface charge are governed by the lipid composition and change gradually from the early to the late secretory pathway. As the endoplasmic reticulum (ER) is situated at the beginning of the cells secretory pathway, it has to accept and accommodate a great variety and quantity of secretory and transmembrane proteins, which enter the ER on their way to their final cellular destination. Secretory proteins can be translocated into the lumen of the ER co- or posttanslationally and membrane proteins are being inserted and released into the ER membrane. In the oxidative milieu of the ER-lumen, supported by a variety of chaperones, proteins can fold into their native form.
If the folding capacity of the ER-lumen is exceeded, an accumulation of mis- or unfolded proteins in the lumen of the ER occurs, consequently triggering the unfolded protein response (UPR). This highly conserved program activates a wide-spread transcriptional response to restore protein folding homeostasis. In fact, 7 – 8% of all genes in the yeast Saccharomyces cerevisiae (S. cerevisiae) are regulated by the UPR. The mechanism underlying the activation of the UPR by protein folding stress has been investigated thoroughly in the last decades and many of its mechanistic details have been elucidated. Recently, it became evident that aberrant lipid compositions of the ER membrane, collectively referred to as lipid bilayer stress, are equally potent in activating the UPR. The underlying molecular mechanism of this membrane-activated UPR, however, remained unclear.
This study focuses on the UPR in S. cerevisiae and characterizes the inositol requiring enzyme 1 (Ire1) as the sole UPR sensor in S. cerevisiae. Active Ire1 forms oligomers and, collaboratively with the tRNA ligase Rlg1, splices immature mRNA of the transcription factor HAC1, which results in the synthesis of mature HAC1 mRNA and the production of the active Hac1 protein, which binds to UPR-elements in the nucleus and activates the expression of UPR target genes. Here, the combination of in vivo and in vitro experiments is being used, which is supplemented by molecular dynamics (MD) simulations performed by Roberto Covino and Gerhard Hummer (MPI for Biophysics, Frankfurt), aiming to identify the molecular mechanism of Ire1 activation by lipid bilayer stress. This study focuses on the analysis of the juxta- and transmembrane region of Ire1. Bioinformatic analyses revealed a putative ER-lumenal amphipathic helix (AH) N-terminally of and partially overlapping with the transmembrane helix (TMH). This predicted AH contains a large hydrophobic face, which inserts into the ER membrane, forcing the TMH into a tilted orientation within the membrane. The resulting unusual architecture of Ire1’s AH and TMH constitutes a unique structural element required for the activation of Ire1 by lipid bilayer stress.
To investigate the function of the AH in the physiological context, different variants of Ire1 were produced under the control of their endogenous promoter and from their endogenous locus. The functional role of the AH was tested, by disrupting its amphipathic character by the introduction of charged residues into the hydrophobic face of the AH. The role of a conserved negative residue between the TMH and the AH (E540 in S. cerevisiae) was tested by substituting it by a unipolar, polar, or positively charged residue. These variants were intensively characterized using a series of assays:
This thesis provides evidence that the AH is crucial for the function of Ire1: Mutant variants with a disrupted (F531R, V535R) or otherwise modified AH (E540A) exhibited a lower degree of oligomerization and failed to catalyze the splicing of the HAC1 mRNA as the Wildtype control. Likewise, the induction of PDI1, a target gene of the UPR, was greatly reduced in mutants with a disrupted or defective AH. These data revealed an important functional role of the AH for normal Ire1 function.
An in vitro system was established to analyze the membrane-mediated oligomerization of Ire1. This system enabled the isolated functional analysis of the AH and TMH during Ire1 activation by lipid bilayer stress. A fusion construct, coding for the maltose binding protein (MBP) from Escherichia coli (E. coli), N-terminally to the AH and TMH of Ire1 was produced. The heterologous production in E. coli, the purification and reconstitution of this minimal sensor of Ire1 in liposomes was established as part of this study. To analyze the oligomeric status of the minimal sensor in different lipid environments, continuous wave electron paramagnetic resonance (cwEPR) spectroscopic experiments were performed. These experiments revealed that the molecular packing density of the lipids had a significant influence of the oligomerization of the spin-labeled membrane sensor: increasing packing densities resulted in sensor oligomerization. The AH-disruptive F531R mutant, in which the amphipathic character of the AH was destroyed, showed no membrane-sensitive changes in its oligomerization status.
Thus, the activation of Ire1 by lipid bilayer stress is achieved by a membrane-based mechanism. According to the current model, the AH induces a local membrane compression by inserting its large hydrophobic face into the membrane. As membrane thickness and acyl chain order are interconnected, this compression simultaneously results in an increased local disordering of lipid acyl chains. Supporting MD simulations performed by Roberto Covino and Gerhard Hummer revealed that the bilayer compression is significantly more pronounced in a densely packed lipid environment, than in a lipid environment of lower lipid packing density. Hence, the energetic cost of the local compression increases with the packing density of the membrane, but is compensated for by the oligomerization of Ire1. This minimization of energetic cost induced by the membrane deformation of Ire1 forms the basis for the activation of Ire1 by lipid bilayer stress.
Whereas ribosome preparations of freshly sliced potato disks do not show appreciable activity in an in-vitro amino acid incorporation system, aging of the tissue leads to a greatly enhanced incorporation activity which reaches its maximum 24 hours after slicing. If ribosomes from freshly excised disks are provided with polyuridylic acid, their activity in the incorporation of phenylalanine is increased about 8 fold.
Moreover, an RNA-fraction can be dissociated by EDTA from ribosomes of aged potato tuber slices, which sediments at 15 —18S, has a base composition different from that of 16S — rRNA, 5S-and 4S —RNA, and is not present on ribosomes of fresh slices. Its appearance is inhibited by actinomycin D and therefore most probably dependent on transcription. This compound, purified from sucrose gradients, enhances in vitro leucine incorporation into peptide material by ribosomes of fresh potato slices.
The possibility is discussed that this fraction-among other factors-is responsible for the enhanced protein synthesis after slicing plant storage organs, and is indicative of a general derepression phenomenon in these tissues.
Chromatin, RNA Polymerase, Potato Tuber Tissue, Aging Phenomenon The synthesis of RNA by chromatin-bound RNA polymerase (E.C. 2.7.7.6.) from white potato tubers proceeds at a low rate, which is enhanced after slicing the tissue, however. Concomitantly DNA template availability as measured with saturating amounts of Escherichia coli polymerase is diminished drastically. Nearest neighbor frequency analysis proved that the RNA synthesized on chromatin of intact tubers is different from that synthesized on chromatin of sliced tissue.
The RNA polymerase of white potato tubers is dependent on all four ribonucleoside triphos phates and a divalent metal ion such as Mg2+ or Mn2+ and totally inhibited by the presence of pyrophosphate. Actinomycin D blocks the formation of the RNA product, which could be shown to be a heteropolymer by nearest neighbour frequency technique. The Km of the chromatin-bound enzyme with regard to ATP, GTP, CTP and UTP was 5.1 X10-5 M, 1.6X10-5 M, 0.9X10-5 M and 0.45 X 10-5M/1 respectively, α-amanitin inhibits the overall activity to about 50%, which indicates the presence of equal amounts of polymerase I and polymerase If.
The Arp2/3 complex nucleates and cross-links actin filaments at the leading edge of motile cells, and its activity is stimulated by C-terminal regions of WASP/Scar proteins, called VCA domains. VCA domains contain a verprolin homology sequence (V) that binds monomeric actin and central (C) and acidic sequences (A) that bind the Arp2/3 complex. Here we show that the C domain binds to monomeric actin with higher affinity (K(d) = 10 microm) than to the Arp2/3 complex (K(d) > 200 microm). Nuclear magnetic resonance spectroscopy reveals that actin binds to the N-terminal half of the C domain and that both the V and C domains can bind actin independently and simultaneously, indicating that they interact with different sites. Mutation of conserved hydrophobic residues in the actin-binding interface of the C domain disrupts activation of the Arp2/3 complex but does not alter affinity for the complex. By chemical cross-linking the C domain interacts with the p40 subunit of the Arp2/3 complex and, by fluorescence polarization anisotropy, the binding of actin and the Arp2/3 complex are mutually exclusive. Our results indicate that both actin and Arp2/3 binding are important for C domain function but that the C domain does not form a static bridge between the two. We propose a model for activation of the Arp2/3 complex in which the C domain first primes the complex by inducing a necessary conformational change and then initiates nucleus assembly by bringing an actin monomer into proximity of the primed complex.
Gram‐negative bacteria are intrinsically resistant against cytotoxic substances by means of their outer membrane and a network of multidrug efflux systems, acting in synergy. Efflux pumps from various superfamilies with broad substrate preferences sequester and pump drugs across the inner membrane to supply the highly polyspecific and powerful tripartite resistance–nodulation–cell division (RND) efflux pumps with compounds to be extruded across the outer membrane barrier. In Escherichia coli, the tripartite efflux system AcrAB–TolC is the archetype RND multiple drug efflux pump complex. The homotrimeric inner membrane component acriflavine resistance B (AcrB) is the drug specificity and energy transduction center for the drug/proton antiport process. Drugs are bound and expelled via a cycle of mainly three consecutive states in every protomer, constituting a flexible alternating access channel system. This review recapitulates the molecular basis of drug and inhibitor binding, including mechanistic insights into drug efflux by AcrB. It also summarizes 17 years of mutational analysis of the gene acrB, reporting the effect of every substitution on the ability of E. coli to confer resistance toward antibiotics (http://goethe.link/AcrBsubstitutions). We emphasize the functional robustness of AcrB toward single‐site substitutions and highlight regions that are more sensitive to perturbation.
Echolocation allows bats to orientate in darkness without using visual information. Bats emit spatially directed high frequency calls and infer spatial information from echoes coming from call reflections in objects (Simmons 2012; Moss and Surlykke 2001, 2010). The echoes provide momentary snapshots, which have to be integrated to create an acoustic image of the surroundings. The spatial resolution of the computed image increases with the quantity of received echoes. Thus, a high call rate is required for a detailed representation of the surroundings.
One important parameter that the bats extract from the echoes is an object’s distance. The distance is inferred from the echo delay, which represents the duration between call emission and echo arrival (Kössl et al. 2014). The echo delay decreases with decreasing distance and delay-tuned neurons have been characterized in the ascending auditory pathway, which runs from the inferior colliculus (Wenstrup et al. 2012; Macías et al. 2016; Wenstrup and Portfors 2011; Dear and Suga 1995) to the auditory cortex (Hagemann et al. 2010; Suga and O'Neill 1979; O'Neill and Suga 1982).
Electrophysiological studies usually characterize neuronal processing by using artificial and simplified versions of the echolocation signals as stimuli (Hagemann et al. 2010; Hagemann et al. 2011; Hechavarría and Kössl 2014; Hechavarría et al. 2013). The high controllability of artificial stimuli simplifies the inference of the neuronal mechanisms underlying distance processing. But, it remains largely unexplored how the neurons process delay information from echolocation sequences. The main purpose of the thesis is to investigate how natural echolocation sequences are processed in the brain of the bat Carollia perspicillata. Bats actively control the sensory information that it gathers during echolocation. This allows experimenters to easily identify and record the acoustic stimuli that are behaviorally relevant for orientation. For recording echolocation sequences, a bat was placed in the mass of a swinging pendulum (Kobler et al. 1985; Beetz et al. 2016b). During the swing the bat emitted echolocation calls that were reflected in surrounding objects. An ultrasound sensitive microphone traveling with the bat and positioned above the bat’s head recorded the echolocation sequence. The echolocation sequence carried delay information of an approach flight and was used as stimulus for neuronal recordings from the auditory cortex and inferior colliculus of the bats.
Presentation of high stimulus rates to other species, such as rats, guinea pigs, suppresses cortical neuron activity (Wehr and Zador 2005; Creutzfeldt et al. 1980). Therefore, I tested if neurons of bats are suppressed when they are stimulated with high acoustic rates represented in echolocation sequences (sequence situation). Additionally, the bats were stimulated with randomized call echo elements of the sequence and an interstimulus time interval of 400 ms (element situation). To quantify neuronal suppression induced by the sequence, I compared the response pattern to the sequence situation with the concatenated response patterns to the element situation. Surprisingly, although the bats should be adapted for processing high acoustic rates, their cortical neurons are vastly suppressed in the sequence situation (Beetz et al. 2016b). However, instead of being completely suppressed during the sequence situation, the neurons partially recover from suppression at a unit specific call echo element. Multi-electrode recordings from the cortex allow assessment of the representation of echo delays along the cortical surface. At the cortical level, delay-tuned neurons are topographically organized. Cortical suppression improves sharpness of neuronal tuning and decreases the blurriness of the topographic map. With neuronal recordings from the inferior colliculus, I tested whether the echolocation sequence also induced neuronal suppression at subcortical level. The sequence induced suppression was weaker in the inferior colliculus than in the cortex. The collicular response makes the neurons able to track the acoustic events in the echolocation sequence. Collicular suppression mainly improves the signal-to-noise ratio. In conclusion, the results demonstrate that cortical suppression is not necessarily a shortcoming for temporal processing of rapidly occurring stimuli as it has previously been interpreted.
Natural environments are usually composed of multiple objects. Thus, each echolocation call reflects off multiple objects resulting in multiple echoes following the calls. At present, it is largely unexplored how neurons process echolocation sequences containing echo information from more than one object (multi-object sequences). Therefore, I stimulated bats with a multi-object sequence which contained echo information from three objects. The objects were different distances away from each other. I tested the influence of each object on the neuronal tuning by stimulating the bats with different sequences created from filtering object specific echoes from the multi-object sequence. The cortex most reliably processes echo information from the nearest object whereas echo information from distant objects is not processed due to neuronal suppression. Collicular neurons process less selectively echo information from certain objects and respond to each echo.
For proper echolocation, bats have to distinguish between own biosonar signals and the signals coming from conspecifics. This can be quite challenging when many bats echolocate adjacent to each other. In behavioral experiments, the echolocation performance of C. perspicillata was tested in the presence of potentially interfering sounds. In the presence of acoustic noise, the bats increase the sensory acquisition rate which may increase the update rate of sensory processing. Neuronal recordings from the auditory cortex and inferior colliculus could strengthen the hypothesis. Although there were signs of acoustic interference or jamming at neuronal level, the neurons were not completely suppressed and responded to the rest of the echolocation sequence.
Vocal communication is essential to coordinate social interactions in mammals and it requires a fine discrimination of communication sounds. Auditory neurons can exhibit selectivity for specific calls, but how it is affected by preceding sounds is still debated. We tackled this using ethologically relevant vocalizations in a highly vocal mammalian species: Seba’s short-tailed bat. We show that cortical neurons present several degrees of selectivity for echolocation and distress calls. Embedding vocalizations within natural acoustic streams leads to stimulus-specific suppression of neuronal responses that changes sound selectivity in disparate manners: increases in neurons with poor discriminability in silence and decreases in neurons selective in silent settings. A computational model indicates that the observed effects arise from two forms of adaptation: presynaptic frequency specific adaptation acting in cortical inputs and stimulus unspecific postsynaptic adaptation. These results shed light into how acoustic context modulates natural sound discriminability in the mammalian cortex.
Acinetobacter baumannii virulence is mediated by the concerted action of three phospholipases D
(2015)
Acinetobacter baumannii causes a broad range of opportunistic infections in humans. Its success as an emerging pathogen is due to a combination of increasing antibiotic resistance, environmental persistence and adaptation to the human host. To date very little is known about the molecular basis of the latter. Here we demonstrate that A. baumannii can use phosphatidylcholine, an integral part of human cell membranes, as sole carbon and energy source. We report on the identification of three phospholipases belonging to the PLD superfamily. PLD1 and PLD2 appear restricted to the bacteria and display the general features of bacterial phospholipases D. They possess two PLDc_2 PFAM domains each encompassing the HxKx4Dx6GS/GGxN (HKD) motif necessary for forming the catalytic core. The third candidate, PLD3, is found in bacteria as well as in eukaryotes and harbours only one PLDc_2 PFAM domain and one conserved HKD motif, which however do not overlap. Employing a markerless mutagenesis system for A. baumannii ATCC 19606T, we generated a full set of PLD knock-out mutants. Galleria mellonella infection studies as well as invasion experiments using A549 human lung epithelial cells revealed that the three PLDs act in a concerted manner as virulence factors and are playing an important role in host cell invasion.
Simple Summary: Tooth roots are increasingly applied for bone reconstruction before implant placement. Growth factors stored in the dentin are assumed to enhance bone regeneration, however, the evidence is low. To this aim, collagen membranes were coated with dentin lysates obtained from extracted porcine teeth or remain untreated. The collagen membranes were tested for their capacity to stimulate bone formation in rat calvarial bone defects. After four weeks of healing, micro-computed tomography and histological analyses revealed that dentin lysates coating had no significant impact on the rather strong bone regeneration reaching a nearly complete defect closure even in untreated defects. It can thus be concluded that dentin lysates do not hinder bone regeneration. Conclusions concerning a possible stimulation of bone regeneration by dentin lysates should not be drawn.
Abstract: Autogenous tooth roots are increasingly applied as a grafting material in alveolar bone augmentation. Since tooth roots undergo creeping substitution similar to bone grafts, it can be hypothesized that osteoclasts release the growth factors stored in the dentin thereby influencing bone formation. To test this hypothesis, collagen membranes were either soaked in acid dentin lysates (ADL) from extracted porcine teeth or serum–free medium followed by lyophilization. Thereafter, these membranes covered standardized 5-mm-diameter critical-size defects in calvarial bone on rats. After four weeks of healing, micro-computed tomography and histological analyses using undecalcified thin ground sections were performed. Micro-computed tomography of the inner 4.5 mm calvaria defects revealed a median bone defect coverage of 91% (CI: 87–95) in the ADL group and 94% (CI: 65–100) in the control group, without significant differences between the groups (intergroup p > 0.05). Furthermore, bone volume (BV) was similar between ADL group (5.7 mm3, CI: 3.4–7.1) and control group (5.7 mm3, CI: 2.9–9.7). Histomorphometry of the defect area confirmed these findings with bone area values amounting to 2.1 mm2 (CI: 1.2–2.6) in the ADL group and 2.0 mm2 (CI: 1.1–3.0) in the control group. Together, these data suggest that acid dentin lysate lyophilized onto collagen membranes failed to modulate the robust bone formation when placed onto calvarial defects.
Niemann-Pick type C (NPC) disease, a lysosomal storage disorder caused by defective NPC1/NPC2 function, results in the accumulation of cholesterol and glycosphingolipids in lysosomes of affected organs, such as liver and brain. Moreover, increase of mitochondrial cholesterol (mchol) content and impaired mitochondrial function and GSH depletion contribute to NPC disease. However, the underlying mechanism of mchol accumulation in NPC disease remains unknown. As STARD1 is crucial in intramitochondrial cholesterol trafficking and acid ceramidase (ACDase) has been shown to regulate STARD1, we explored the functional relationship between ACDase and STARD1 in NPC disease. Liver and brain of Npc1−/− mice presented a significant increase in mchol levels and STARD1 expression. U18666A, an amphiphilic sterol that inhibits lysosomal cholesterol efflux, increased mchol levels in hepatocytes from Stard1f/f mice but not Stard1ΔHep mice. We dissociate the induction of STARD1 expression from endoplasmic reticulum stress, and establish an inverse relationship between ACDase and STARD1 expression and LRH-1 levels. Hepatocytes from Npc1+/+ mice treated with U18666A exhibited increased mchol accumulation, STARD1 upregulation and decreased ACDase expression, effects that were reversed by cholesterol extraction with 2-hydroxypropyl-β-cyclodextrin. Moreover, transfection of fibroblasts from NPC patients with ACDase, decreased STARD1 expression and mchol accumulation, resulting in increased mitochondrial GSH levels, improved mitochondrial functional performance, decreased oxidative stress and protected NPC fibroblasts against oxidative stress-mediated cell death. Our results demonstrate a cholesterol-dependent inverse relationship between ACDase and STARD1 and provide a novel approach to target the accumulation of cholesterol in mitochondria in NPC disease.
Neuronal nAChRs are a diverse family of pentameric ion channels with wide distribution throughout cells of the nervous and immune systems. However, the role of specific subtypes in normal and pathological states remains poorly understood due to the lack of selective probes. Here, we used a binding assay based on acetylcholine-binding protein (AChBP), a homolog of the nicotinic acetylcholine ligand-binding domain, to discover a novel alpha-conotoxin (alpha-TxIA) in the venom of Conus textile. alpha-TxIA bound with high affinity to AChBPs from different species and selectively targeted the alpha3beta2 nAChR subtype. A co-crystal structure of Ac-AChBP with the enhanced potency analog TxIA(A10L), revealed a 20° backbone tilt compared to other AChBP–conotoxin complexes. This reorientation was coordinated by a key salt bridge formed between Arg5 (TxIA) and Asp195 (Ac-AChBP). Mutagenesis studies, biochemical assays and electrophysiological recordings directly correlated the interactions observed in the co-crystal structure to binding affinity at AChBP and different nAChR subtypes. Together, these results establish a new pharmacophore for the design of novel subtype-selective ligands with therapeutic potential in nAChR-related diseases.
Acetylcholine (ACh) is the major excitatory neurotransmitter in the insect central nervous system (CNS). However, besides the neuronal expression of ACh receptors (AChR), the existence of non-neuronal AChR in honeybees is plausible. The cholinergic system is a popular target of insecticides because the pharmacology of insect nicotinic acetylcholine receptors (nAChRs) differs substantially from their vertebrate counterparts. Neonicotinoids are agonists of the nAChR and are largely used in crop protection. In contrast to their relatively high safety for humans and livestock, neonicotinoids pose a threat to pollinating insects such as bees. In addition to its effects on behavior, it becomes increasingly evident that neonicotinoids affect developmental processes in bees that appear to be independent of neuronal AChRs. Brood food (royal jelly, worker jelly, or drone jelly) produced in the hypopharyngeal glands of nurse bees contains millimolar concentrations of ACh, which is required for proper larval development. Neonicotinoids reduce the secreted ACh-content in brood food, reduce hypopharyngeal gland size, and lead to developmental impairments within the colony. We assume that potential hazards of neonicotinoids on pollinating bees occur neuronally causing behavioral impairments on adult individuals, and non-neuronally causing developmental disturbances as well as destroying gland functioning.
One of the key functions of blood vessels is to transport nutrients and oxygen to distant tissues and organs in the body. When blood supply is insufficient, new vessels form to meet the metabolic tissue demands and to re-establish cellular homeostasis. Expansion of the vascular network through sprouting angiogenesis requires the specification of ECs into leading (sprouting) tip and following (non-sprouting) stalk cells. Attracted by guidance cues tip cells dynamically extend and retract filopodia to navigate the nascent vessel sprout, whereas trailing stalk cells proliferate to form the extending vascular tube. All of these processes are under the control of environmental signals (e.g. hypoxia, metabolism) and numerous cytokines and peptide growth factors. The Dll4/Notch pathway coordinates several critical steps of angiogenic blood vessel growth. Even subtle alterations in Notch activity can profoundly influence endothelial cell behavior and blood vessel formation, yet little is known about the intrinsic regulation and dynamics of Notch signaling in endothelial cells. In addition, it remains an open question, how different growth factor signals impinging on sprouting ECs are coordinated with local environmental cues originating from nutrient-deprived, hypoxic tissue to achieve a balanced endothelial cell response. Acetylation of lysines is a critical posttranslational modification of histones, which acts as an important regulatory mechanism to control chromatin structure and gene transcription. In addition to histones, several non-histone proteins are targeted for acetylation reversible acetylation is emerging as a fundamental regulatory mechanism to control protein function, interaction and stability. Previous studies from our group identified the NAD+-dependent deacetylase SIRT1 as a key regulator of blood vessel growth controlling endothelial angiogenic responses. These studies revealed that SIRT1 is highly expressed in the vascular endothelium during blood vessel development, where it controls the angiogenic activity of endothelial cells. Moreover, in this work SIRT1 has been shown to control the activity of key regulators of cardiovascular homeostasis such as eNOS, Foxo1 and p53. The present study describes that SIRT1 antagonizes Notch signaling by deacetylating the Notch intracellular domain (NICD). We showed that loss of SIRT1 enhances DLL4-induced endothelial Notch responses as assessed by different luciferase responsive elements as well as transcriptional analysis of Notch endogenous target genes activation. Conversely, SIRT1 gain of function by overexpression of pharmacological activation decreases induction of Notch targets in response to DLL4 stimulation. We also showed that the NICD can be directly acetylated by PC AF and p300 and that SIRT1 promotes deacetylation of NICD. We have identified 14 lysines that are targeted for acetylation and their mutation abolishes the effects of SIRT1 of Notch responses. Furthermore, over-expression or activation of SIRT1 significantly reduces the levels of NICD protein. Moreover, SIRT1-mediated NICD degradation can be reversed by blockade of the proteasome suggesting a mechanism resulting from ubiquitin-mediated proteolysis. Indeed, we have shown that SIRT1 knockdown or pharmacological inhibition decreased NICD ubiquitination. We propose a novel molecular mechanism of modulation of the amplitude and duration of Notch responses in which acetylation increases NICD stability and therefore permanence at the promoters, while SIRT1, by inducing NICD degradation through its deacetylation, shortens Notch responses. In order to evaluate the physiological relevance of our findings we used different models in which the Notch functions during blood vessel formation have been extensively characterized. First, retinal angiogenesis in mice lacking SIRT1 activity shows decreased branching and reduced endothelial proliferation, similar to what happens after Notch gain of function mutations. ECs from these mice exhibit increased expression of Notch target genes. Second, these results were reproducible during intersomitic vessel growth in sirt1-deficient zebrafish. In both models, the defects could be partially rescued by inhibition of Notch activation. Third, we used an in vitro model of vessel sprouting from differentiating embryonic bodies in response to VEGF in a collagen matrix. Our results showed that Sirt1-deficient cells shows impaired sprouting which correlated with increased NICD levels. In addition, when in competition with wild-type cells in this assay, Sirt1-deficient cells are more prone to occupy the stalk cell position. Taken together, our study identifies reversible acetylation of NICD as a novel molecular mechanism to adapt the dynamics of Notch signaling and suggest that SIRT1 acts as a rheostat to fine-tune endothelial Notch responses. The NAD+-dependent feature of SIRT1 activity possibly links endothelial Notch responses to environmental cues and metabolic changes during nutrient deprivation in ischemic environments or upon other cellular stresses.
The enzyme acetyl-CoA carboxylase (ACC) plays a crucial role in fatty acid metabolism. In recent years, ACC has been recognized as a promising drug target for treating different diseases. However, the role of ACC in vascular endothelial cells (ECs) has been neglected so far. To characterize the role of ACC, we used the ACC inhibitor, soraphen A, as a chemical tool, and also a gene silencing approach. We found that ACC1 was the predominant isoform in human umbilical vein ECs as well as in human microvascular ECs and that soraphen A reduced the levels of malonyl-CoA. We revealed that ACC inhibition shifted the lipid composition of EC membranes. Accordingly, membrane fluidity, filopodia formation, and migratory capacity were reduced. The antimigratory action of soraphen A depended on an increase in the cellular proportion of PUFAs and, most importantly, on a decreased level of phosphatidylglycerol. Our study provides a causal link between ACC, membrane lipid composition, and cell migration in ECs. Soraphen A represents a useful chemical tool to investigate the role of fatty acid metabolism in ECs and ACC inhibition offers a new and valuable therapeutic perspective for the treatment of EC migration-related diseases.
The enzyme acetyl-CoA carboxylase (ACC) plays a fundamental role in the fatty acid metabolism. It regulates the first and rate limiting step in the biosynthesis of fatty acids by catalyzing the carboxylation of acetyl-CoA to malonyl-CoA and exists as two different isoforms, ACC1 and ACC2. In the last few years, ACC has been reported as an attractive drug target for treating different diseases, such as insulin resistance, hepatic steatosis, dyslipidemia, obesity, metabolic syndrome and nonalcoholic fatty liver disease. An altered fatty acid metabolism is also associated with cancer cell proliferation. In general, the inhibition of ACC provides two possibilities to regulate the fatty acid metabolism: It blocks the de novo lipogenesis in lipogenic tissues and stimulates the mitochondrial fatty acid β-oxidation. Surprisingly, the role of ACC in human vascular endothelial cells has been neglected so far. This work aimed to investigate the role of the ACC/fatty acid metabolism in regulating important endothelial cell functions like proliferation, migration and tube formation.
To investigate the function of ACC, the ACC-inhibitor soraphen A as well as an siRNA-based approach were used. This study revealed that ACC1 is the predominant isoform both in human umbilical vein endothelial cells (HUVECs) and in human dermal microvascular endothelial cells (HMECs). Inhibition of ACC via soraphen A resulted in decreased levels of malonyl-CoA and shifted the lipid composition of endothelial cell membranes. Consequently, membrane fluidity, filopodia formation and the migratory capacity were attenuated. Increasing amounts of longer acyl chains within the phospholipid subgroup phosphatidylcholine (PC) were suggested to overcompensate the shift towards shorter acyl chains within phosphatidylglycerol (PG), which resulted in a dominating effect on regulating the membrane fluidity. Most importantly, this work provided a link between changes in the phospholipid composition and altered endothelial cell migration. The antimigratory effect of soraphen A was linked to a reduced amount of PG and to an increased amount of polyunsaturated fatty acids (PUFAs) within the phospholipid cell membrane. This link was unknown in the literature so far. Interestingly, a reduced filopodia formation was observed upon ACC inhibition via soraphen A, which presumably caused the impaired migratory capacity.
This work revealed a relationship between ACC/fatty acid metabolism, membrane lipid composition and endothelial cell migration. The natural compound soraphen A emerged as a valuable chemical tool to analyze the role of ACC/fatty acid metabolism in regulating important endothelial cell functions. Furthermore, regulating endothelial cell migration via ACC inhibition promises beneficial therapeutic perspectives for the treatment of cell migration-related disorders, such as ischemia reperfusion injury, diabetic angiopathy, macular degeneration, rheumatoid arthritis, wound healing defects and cancer.
Acclimatisation
(1876)
Mitochondial NADH:ubiquinone oxidoreductase (complex I) the largest multiprotein enzyme of the respiratory chain, catalyses the transfer of two electrons from NADH to ubiquinone, coupled to the translocation of four protons across the membrane. In addition to the 14 strictly conserved central subunits it contains a variable number of accessory subunits. At present, the best characterized enzyme is complex I from bovine heart with a molecular mass of about 980 kDa and 32 accessory proteins. In this study, the subunit composition of mitochondrial complex I from the aerobic yeast Y. lipolytica has been analysed by a combination of proteomic and genomic approaches. The sequences of 37 complex I subunits were identified. The sum of their individual molecular masses (about 930 kDa) was consistent with the native molecular weight of approximately 900 kDa for Y. lipolytica complex I obtained by BN-PAGE. A genomic analysis with Y. lipolytica and other eukaryotic databases to search for homologues of complex I subunits revealed 31 conserved proteins among the examined species. A novel protein named “X” was found in purified Y. lipolytica complex I by MALDI-MS. This protein exhibits homology to the thiosulfate sulfurtransferase enzyme referred to as rhodanese. The finding of a rhodanese-like protein in isolated complex I of Y. lipolytica allows to assume a special regulatory mechanism of complex I activity through control of the status of its iron-sulfur clusters. The second part of this study was aimed at investigating the possible role of one of these extra subunits, 39 kDa (NUEM) subunit which is related to the SDRs-enzyme family. The members of this family function in different redox and isomerization reactions and contain a conserved NAD(P)H-binding site. It was proposed that the 39 kDa subunit may be involved in a biosynthetic pathway, but the role of this subunit in complex I is unknown. In contrast to the situation in N. crassa, deletion of the 39 kDa encoding gene in Y. lipolytica led to the absence of fully assembled complex I. This result might indicate a different pathway of complex I assembly in both organisms. Several site-directed mutations were generated in the nucleotide binding motif. These had either no effect on enzyme activity and NADPH binding, or prevented complex I assembly. Mutations of arginine-65 that is located at the end of the second b-strand and responsible for selective interaction with the 2’-phosphate group of NADPH retained complex I activity in mitochondrial membranes but the affinity for the cofactor was markedly decreased. Purification of complex I from mutants resulted in decrease or loss of ubiquinone reductase activity. It is very likely that replacement of R65 not only led to a decrease in affinity for NADPH but also caused instability of the enzyme due to steric changes in the 39 kDa subunit. These data indicate that NADPH bound to the 39 kDa subunit (NUEM) is not essential for complex I activity, but probably involved in complex I assembly in Y. lipolytica.
Peracarid data were collected in the Southern Ocean and South Atlantic Ocean. Sampling was performed during nine different expeditions on board of RRS James Clark Ross and RV Polarstern, using epibenthic sledges (EBS) at depth ranging between 160–6348 m at 109 locations. The correlation between environmental variables and peracarid abundance was investigated. Abundance data comprise a total of 128570 peracarids (52366 were amphipods, 28516 were cumaceans, 36142 isopods, 5676 mysidaceans and 5870 were tanaidaceans). The presented data are useful to investigate the composition and abundance patterns of peracarid orders at a wide depth range and spatial scale in the Southern Ocean. They can also be reused to compare their abundance with that of other taxa in broader ecological surveys.
The association of Schlen k’s hydrocarbon was studied by means of osmometric and magnetic measurements. The mixed chain-ring-association can be explained satisfactorily assuming that two different dimers and four monomer species participate in the equilibria, including a monomeric diamagnetic ring. The equilibria existing between the different species are discussed. For the equilibria between the monomer and dimer species, which can be detected in solutions of normal viscosity by means of ESR-measurements, the unexpected values of ΔH=0 for the enthalpie of association and ΔS= +19.7 e. u. for the entropie of association were found.
Aim: The identification of the mechanisms determining spatial variation in biological diversity along elevational gradients is a central objective in ecology and biogeography. Here, we disentangle the direct and indirect effects of abiotic drivers (climatic conditions, and land use) and biotic drivers (vegetation structure and food resources) on functional diversity and composition of bird and bat assemblages along a tropical elevational gradient. Location: Southern slopes of Mt. Kilimanjaro, Tanzania, East Africa. Methods: We counted birds and recorded bat sonotypes on 58 plots distributed in near-natural and anthropogenically modified habitats from 700 to 4,600 m above sea level. For the recorded taxa, we compiled functional traits related to movement, foraging and body size from museum specimens and databases. Further, we recorded mean annual temperature, precipitation, vegetation complexity as well as the number of fruits, flowers, and insect biomass as measures of resource availability on each study site. Results: Using path analyses, we found similar responses of bird and bat functional diversity to the variation in abiotic and biotic drivers along the elevational gradient. In contrast, the functional composition of both taxa showed distinct responses to abiotic and biotic drivers. For both groups, direct temperature effects were most important, followed by resource availability, precipitation and vegetation complexity. Main Conclusions: Our findings indicate that physiological and metabolic constraints imposed by temperature and resource availability determine the functional diversity of bird and bat assemblages, whereas the composition of individual functional traits is driven by taxon-specific processes. Our study illustrates that distinct filtering mechanisms can result in similar patterns of functional diversity along broad environmental gradients. Such differences need to be taken into account when it comes to conserving the functional diversity of flying vertebrates on tropical mountains.
During CNS development and adult neurogenesis, immature neurons travel from the germinal zones towards their final destination using cellular substrates for their migration. Classically, radial glia and neuronal axons have been shown to act as physical scaffolds to support neuroblast locomotion in processes known as gliophilic and neurophilic migration, respectively (Hatten, 1999; Marin and Rubenstein, 2003; Rakic, 2003). In adulthood, long distance neuronal migration occurs in a glial-independent manner since radial glia cells differentiate into astrocytes after birth. A series of studies highlight a novel mode of neuronal migration that uses blood vessels as scaffolds, the so-called vasophilic migration. This migration mode allows neuroblast navigation in physiological and also pathological conditions, such as neuronal precursor migration after ischemic stroke or cerebral invasion of glioma tumor cells. Here we review the current knowledge about how vessels pave the path for migrating neurons and how trophic factors derived by glio-vascular structures guide neuronal migration both during physiological as well as pathological processes
Diploid transgenic organisms are either hemi- or homozygous. Genetic assays are, therefore, required to identify the genotype. Our AGameOfClones vector concept uses two clearly distinguishable transformation markers embedded in interweaved, but incompatible Lox site pairs. Cre-mediated recombination leads to hemizygous individuals that carry only one marker. In the following generation, heterozygous descendants are identified by the presence of both markers and produce homozygous progeny that are selected by the lack of one marker. We prove our concept in Tribolium castaneum by systematically creating multiple functional homozygous transgenic lines suitable for long-term fluorescence live imaging. Our approach saves resources and simplifies transgenic organism handling. Since the concept relies on the universal Cre-Lox system, it is expected to work in all diploid model organisms, for example, insects, zebrafish, rodents and plants. With appropriate adaptions, it can be used in knock-out assays to preselect homozygous individuals and thus minimize the number of wasted animals.
The RNA cleaving catalyst tris(2-aminobenzimidazole) when attached to the 5’ terminus of oligonucleotides cuts complementary RNA strands in a highly site-specific manner. Conjugation was previously achieved by the acylation of an amino linker by an active ester of the catalyst. However, this procedure was low yielding and not reliable. Here, a phosphoramidite building block is described that can be coupled to oligonucleotides by manual solid phase synthesis in total yields around 85%. Based on this chemistry, we have now studied the impact of LNA (locked nucleic acids) nucleotides on the rates and the site-specificities of RNA cleaving conjugates. The highest reaction rates and the most precise cuts can be expected when the catalyst is attached to a strong 5’ closing base pair and when the oligonucleotide contains several LNA units that are equally distributed in the strand. However, when placed in the 5’ position, LNA building blocks tend to diminish the specificity of RNA cleavage.
Non-alcoholic steatohepatitis (NASH) - a hepatic manifestation of the metabolic syndrome - is a multifactorial disease with alarming global prevalence. It involves steatosis, inflammation and fibrosis in the liver, thus demanding multiple modes of action for robust therapeutic efficacy. Aiming to fuse complementary validated anti-NASH strategies in a single molecule, we have designed and systematically optimized a scaffold for triple activation of farnesoid X receptor (FXR), peroxisome proliferator-activated receptor (PPAR) α and PPARδ. Pilot profiling of the resulting triple modulator demonstrated target engagement in native cellular settings and in mice, rendering it a suitable tool to probe the triple modulator concept in vivo. In DIO NASH in mice, the triple agonist counteracted hepatic inflammation and reversed hepatic fibrosis highlighting the potential of designed polypharmacology in NASH.
A toolbox for the generation of chemical probes for Baculovirus IAP Repeat containing proteins
(2022)
E3 ligases constitute a large and diverse family of proteins that play a central role in regulating protein homeostasis by recruiting substrate proteins via recruitment domains to the proteasomal degradation machinery. Small molecules can either inhibit, modulate or hijack E3 function. The latter class of small molecules led to the development of selective protein degraders, such as PROTACs (PROteolysis TArgeting Chimeras), that recruit protein targets to the ubiquitin system leading to a new class of pharmacologically active drugs and to new therapeutic options. Recent efforts have focused on the E3 family of Baculovirus IAP Repeat (BIR) domains that comprise a structurally conserved but diverse 70 amino acid long protein interaction domain. In the human proteome, 16 BIR domains have been identified, among them promising drug targets such as the Inhibitors of Apoptosis (IAP) family, that typically contain three BIR domains (BIR1, BIR2, and BIR3). To date, this target area lacks assay tools that would allow comprehensive evaluation of inhibitor selectivity. As a consequence, the selectivity of current BIR domain targeting inhibitors is unknown. To this end, we developed assays that allow determination of inhibitor selectivity in vitro as well as in cellulo. Using this toolbox, we have characterized available BIR domain inhibitors. The characterized chemical starting points and selectivity data will be the basis for the generation of new chemical probes for IAP proteins with well-characterized mode of action and provide the basis for future drug discovery efforts and the development of PROTACs and molecular glues.
Working memory and conscious perception are thought to share similar brain mechanisms, yet recent reports of non-conscious working memory challenge this view. Combining visual masking with magnetoencephalography, we investigate the reality of non-conscious working memory and dissect its neural mechanisms. In a spatial delayed-response task, participants reported the location of a subjectively unseen target above chance-level after several seconds. Conscious perception and conscious working memory were characterized by similar signatures: a sustained desynchronization in the alpha/beta band over frontal cortex, and a decodable representation of target location in posterior sensors. During non-conscious working memory, such activity vanished. Our findings contradict models that identify working memory with sustained neural firing, but are compatible with recent proposals of ‘activity-silent’ working memory. We present a theoretical framework and simulations showing how slowly decaying synaptic changes allow cell assemblies to go dormant during the delay, yet be retrieved above chance-level after several seconds.
The Neotropical ambrosia beetle genus Camptocerus Dejean was revised. Monophyly of the genus was tested using 66 morphological characters in a cladistic analysis. Camptocerus was recovered as monophyletic and 31 species were recognized. Six new synonyms were discovered: C. auricomus Blandford 1896 (= C. striatulus Hagedorn 1905), C. inoblitus (Schedl) 1939 (= C. morio (Schedl) 1952), C. niger (Fabricius) 1801 (= C. tectus Eggers 1943), C. opacicollis (Eggers) 1929 (= C. infidelis Wood 1969; = C. uniseriatus Schedl 1972), C. suturalis (Fabricius) 1801 (= C. cinctus Chapuis 1869). Two species were removed from synonymy: C. charpentierae Schedl and C. hirtipennis Schedl. Twelve new species of Camptocerus were described: C. coccoformus (Brazil, Ecuador), C. distinctus (Ecuador), C. doleae (Ecuador), C. igniculus (Brazil), C. mallopterus (Ecuador), C. noel (widely distributed across Amazonia), C. petrovi (Ecuador), C. pilifrons (Ecuador), C. pseudoangustior (widely distributed across Amazonia), C. satyrus (Brazil), C. unicornus (Brazil) and C. zucca (Ecuador). Lectotypes are here designated for the following species: Camptocerus auricomus Blandford, Camptocerus squammiger Chapuis, Hylesinus gibbus Fabricius, Hylesinus suturalis Fabricius, Hylesinus fasciatus Fabricius. A key, diagnosis, distribution, host records and images were provided for each species.
Demographic change is supposed to be the most important indirect driver for changing biodiversity. In this article, a systematic review of 148 studies was conducted to examine the scientific evidence for this relationship and to identify potential gaps in research. We explored the spatial distribution of studies, the categories addressed with respect to biodiversity and demographic change, and the ways in which their relationships were conceptualised (spatially and temporally) and valued. The majority of studies were carried out in Africa, Europe and North America. Our analysis confirms the trend that demographic phenomena were mostly found to negatively influence biodiversity. However, a considerable number of studies also point towards impacts that were context dependent, either positive or negative under certain circumstances. In addition to that we identified significant gaps in research. In particular, there is a lack of addressing (1) other demographic aspects such as population decline, age structure or gender differences, (2) spatial variability of, e.g. human population growth, (3) long-term effects of demographic processes, and (4) the context dependency (e.g. regulations/law enforcement, type of human activities, and choice of scale or proxy). We conclude there is evidence that the relationship between biodiversity and demographic change is much more complex than expected and so far represented in research. Thus, we call for a social–ecological biodiversity research that particularly focusses on the functional relation between biodiversity and human activities, namely the different types, context, and interdependent dynamics (spatial and temporal) of this complex relation.
Invasive plants represent a significant financial burden for land managers and also have the potential to severely degrade ecosystems. Arthropods interact strongly with plants, relying on them for food, shelter, and as nurseries for their young. For these reasons, the impacts of plant invasions are likely strongly reflected by arthropod community dynamics including diversity and abundances. A systematic review was conducted to ascertain the state of the literature with respect to plant invaders and their associated arthropod communities. We found that the majority of studies did not biogeographically contrast arthropod community dynamics from both the home and away ranges and that studies were typically narrow in scope, focusing only on the herbivore feeding guild, rather than assessing two or more trophic levels. Importantly, relative arthropod richness was significantly reduced on invasive plant species. Phylogenetic differences between the invasive and local plant community as well as the plant functional group impact arthropod diversity patterns. A framework highlighting some interaction mechanisms between multiple arthropod trophic levels and native and invasive plants is discussed and future research directions relating to these interactions and the findings herein are proposed.
Many cellular processes are regulated via pH, and maintaining the pH of different organelles is crucial for cell survival. A pH-sensitive GFP variant, the so-called pHluorin, has proven to be a valuable tool to study the pH of the cytosol, mitochondria and other organelles in vivo. We found that the fluorescence intensity of Endoplasmic Reticulum (ER)-targeted pHluorin in the yeast Saccharomyces cerevisiae was very low and barely showed pH sensitivity, probably due to misfolding in the oxidative environment of the ER. We therefore developed a superfolder variant of pHluorin which enabled us to monitor pH changes in the ER and the cytosol of S. cerevisiae in vivo. The superfolder pHluorin variant is likely to be functional in cells of different organisms as well as in additional compartments that originate from the secretory pathway like the Golgi apparatus and pre-vacuolar compartments, and therefore has a broad range of possible future applications.
Non-coding variations located within regulatory elements may alter gene expression by modifying Transcription Factor (TF) binding sites and thereby lead to functional consequences like various traits or diseases. To understand these molecular mechanisms, different TF models are being used to assess the effect of DNA sequence variations, such as Single Nucleotide Polymorphisms (SNPs). However, few statistical approaches exist to compute statistical significance of results but they often are slow for large sets of SNPs, such as data obtained from a genome-wide association study (GWAS) or allele-specific analysis of chromatin data.
Results We investigate the distribution of maximal differential TF binding scores for general computational models that assess TF binding. We find that a modified Laplace distribution can adequately approximate the empirical distributions. A benchmark on in vitro and in vivo data sets showed that our new approach improves on an existing method in terms of performance and speed. In applications on large sets of eQTL and GWAS SNPs we could illustrate the usefulness of the novel statistic to highlight cell type specific regulators and TF target genes.
Conclusions Our approach allows the evaluation of DNA changes that induce differential TF binding in a fast and accurate manner, permitting computations on large mutation data sets. An implementation of the novel approach is freely available at https://github.com/SchulzLab/SNEEP.
A state record for the Oconee scorpionfly, Panorpa oconee Byers (Mecoptera: Panorpidae), in Florida
(2010)
I provide the first state record for the Oconee scorpionfly, Panorpa oconee Byers, from Putnam County, Florida. This is the southernmost record for P. oconee, extends its range 321 km south of its known distribution and, if valid, adds a seventh described species of panorpid, and twelfth mecopteran, indigenous to Florida.
Background: Mitochondrial DNA sequencing increasingly results in the recognition of genetically divergent, but morphologically cryptic lineages. Species delimitation approaches that rely on multiple lines of evidence in areas of co-occurrence are particularly powerful to infer their specific status. We investigated the species boundaries of two cryptic lineages of the land snail genus Trochulus in a contact zone, using mitochondrial and nuclear DNA marker as well as shell morphometrics.
Results: Both mitochondrial lineages have a distinct geographical distribution with a small zone of co-occurrence. In the same area, we detected two nuclear genotype clusters, each being highly significantly associated to one mitochondrial lineage. This association however had exceptions: a small number of individuals in the contact zone showed intermediate genotypes (4%) or cytonuclear disequilibrium (12%). Both mitochondrial lineage and nuclear cluster were statistically significant predictors for the shell shape indicating morphological divergence. Nevertheless, the lineage morphospaces largely overlapped (low posterior classification success rate of 69% and 78%, respectively): the two lineages are truly cryptic.
Conclusions: The integrative approach using multiple lines of evidence supported the hypothesis that the investigated Trochulus lineages are reproductively isolated species. In the small contact area, however, the lineages hybridise to a limited extent. This detection of a hybrid zone adds an instance to the rare reported cases of hybridisation in land snails.
Biodiversity research heavily relies on recent and older literature, and the data contained therein. Despite great effort, large parts of the literature and the data it holds are still not available in appropriate formats needed for efficient compilation and analysis. As a part of the current funding strategy of the German Research Council (Deutsche Forschungsgemeinschaft, DFG), and resulting from an extensive dialogue with the scientific community in Germany, a "Specialised Information Service" (Fachinformationsdienst, FID) for Biodiversity Research will be established with the objective of making further segments of literature about biodiversity available in up-to-date formats. This project, starting 2017, is conducted by the University Library Johann Christian Senckenberg (Frankfurt/Main, Germany) together with the Senckenberg Gesellschaft für Naturforschung and the Text Technology Lab of the Goethe University (Frankfurt/Main).
The new Specialised Information Service for Biodiversity Research (FID Biodiversitätsforschung) comprises four core elements: (A) A text mining approach which encompasses advanced text technologies and a large body of 20th century literature; (B) the digitisation of selected German biodiversity literature; (C) a platform für Open Access journals; and (D) Acquisition of specialised print literature.
The dependence of the Escherichia coli Na+H+ antiporter A (EcNhaA) pH sensor mutant E241C on H+ and Na+ concentrations was tested using a solid supported membrane (SSM) based electrophysiological approach. Proteoliposome preparations with right side out (RSO) oriented carriers were used to investigate the passive downhill uptake mode (physiologically the reverse transport mode) at zero membrane potential. Na+ concentration gradients established with a rapid solution exchange acted as the driving force. When a Na+ concentration gradient was established at symmetrical pH, the transport activity of the E241C EcNhaA variant was similar to that of the wildtype EcNhaA, with no shift of the bell-shaped pH dependence, an increase of the KmNa at acidic pH and a decrease of the KmNa at alkaline pH, supporting the model of a competitive binding of Na+ and H+ to a common binding site.
A small-world network has been suggested to be an efficient solution for achieving both modular and global processing-a property highly desirable for brain computations. Here, we investigated functional networks of cortical neurons using correlation analysis to identify functional connectivity. To reconstruct the interaction network, we applied the Ising model based on the principle of maximum entropy. This allowed us to assess the interactions by measuring pairwise correlations and to assess the strength of coupling from the degree of synchrony. Visual responses were recorded in visual cortex of anesthetized cats, simultaneously from up to 24 neurons. First, pairwise correlations captured most of the patterns in the population´s activity and, therefore, provided a reliable basis for the reconstruction of the interaction networks. Second, and most importantly, the resulting networks had small-world properties; the average path lengths were as short as in simulated random networks, but the clustering coefficients were larger. Neurons differed considerably with respect to the number and strength of interactions, suggesting the existence of "hubs" in the network. Notably, there was no evidence for scale-free properties. These results suggest that cortical networks are optimized for the coexistence of local and global computations: feature detection and feature integration or binding.
Small RNAs have been implicated in numerous cellular processes, including effects on chromatin structure and the repression of transposons. We describe the generation of a small RNA response at DNA ends in Drosophila that is analogous to the recently reported double-strand break (DSB)-induced RNAs or Dicer- and Drosha-dependent small RNAs in Arabidopsis and vertebrates. Active transcription in the vicinity of the break amplifies this small RNA response, demonstrating that the normal messenger RNA contributes to the endogenous small interfering RNAs precursor. The double-stranded RNA precursor forms with an antisense transcript that initiates at the DNA break. Breaks are thus sites of transcription initiation, a novel aspect of the cellular DSB response. This response is specific to a double-strand break since nicked DNA structures do not trigger small RNA production. The small RNAs are generated independently of the exact end structure (blunt, 3′- or 5′-overhang), can repress homologous sequences in trans and may therefore—in addition to putative roles in repair—exert a quality control function by clearing potentially truncated messages from genes in the vicinity of the break.
The entire chemical modification repertoire of yeast ribosomal RNAs and the enzymes responsible for it have recently been identified. Nonetheless, in most cases the precise roles played by these chemical modifications in ribosome structure, function and regulation remain totally unclear. Previously, we demonstrated that yeast Rrp8 methylates m1A645 of 25S rRNA in yeast. Here, using mung bean nuclease protection assays in combination with quantitative RP-HPLC and primer extension, we report that 25S/28S rRNA of S. pombe, C. albicans and humans also contain a single m1A methylation in the helix 25.1. We characterized nucleomethylin (NML) as a human homolog of yeast Rrp8 and demonstrate that NML catalyzes the m1A1322 methylation of 28S rRNA in humans. Our in vivo structural probing of 25S rRNA, using both DMS and SHAPE, revealed that the loss of the Rrp8-catalyzed m1A modification alters the conformation of domain I of yeast 25S rRNA causing translation initiation defects detectable as halfmers formation, likely because of incompetent loading of 60S on the 43S-preinitiation complex. Quantitative proteomic analysis of the yeast Δrrp8 mutant strain using 2D-DIGE, revealed that loss of m1A645 impacts production of specific set of proteins involved in carbohydrate metabolism, translation and ribosome synthesis. In mouse, NML has been characterized as a metabolic disease-associated gene linked to obesity. Our findings in yeast also point to a role of Rrp8 in primary metabolism. In conclusion, the m1A modification is crucial for maintaining an optimal 60S conformation, which in turn is important for regulating the production of key metabolic enzymes.
The entire chemical modification repertoire of yeast ribosomal RNAs and the enzymes responsible for it have recently been identified. Nonetheless, in most cases the precise roles played by these chemical modifications in ribosome structure, function and regulation remain totally unclear. Previously, we demonstrated that yeast Rrp8 methylates m1A645 of 25S rRNA in yeast. Here, using mung bean nuclease protection assays in combination with quantitative RP-HPLC and primer extension, we report that 25S/28S rRNA of S. pombe, C. albicans and humans also contain a single m1A methylation in the helix 25.1. We characterized nucleomethylin (NML) as a human homolog of yeast Rrp8 and demonstrate that NML catalyzes the m1A1322 methylation of 28S rRNA in humans. Our in vivo structural probing of 25S rRNA, using both DMS and SHAPE, revealed that the loss of the Rrp8-catalyzed m1A modification alters the conformation of domain I of yeast 25S rRNA causing translation initiation defects detectable as halfmers formation, likely because of incompetent loading of 60S on the 43S-preinitiation complex. Quantitative proteomic analysis of the yeast Δrrp8 mutant strain using 2D-DIGE, revealed that loss of m1A645 impacts production of specific set of proteins involved in carbohydrate metabolism, translation and ribosome synthesis. In mouse, NML has been characterized as a metabolic disease-associated gene linked to obesity. Our findings in yeast also point to a role of Rrp8 in primary metabolism. In conclusion, the m1A modification is crucial for maintaining an optimal 60S conformation, which in turn is important for regulating the production of key metabolic enzymes.
Poster presentation: Introduction Dopaminergic neurons in the midbrain show a variety of firing patterns, ranging from very regular firing pacemaker cells to bursty and irregular neurons. The effects of different experimental conditions (like pharmacological treatment or genetical manipulations) on these neuronal discharge patterns may be subtle. Applying a stochastic model is a quantitative approach to reveal these changes. ...
A role of the Qв binding protein in the mechanism of cyanobacterial adaptation to light intensity?
(1986)
Growth of the unicellular blue-green alga Anacystis nidulans in media containing sublethal concentrations of DCMU-type inhibitors of photosynthetic electron transport in strong white light gave rise to shade type appearance in this organism, as characterized by an increased ratio of phycocyanin to chlorophyll and reduced ratios, both, of carotenoids to chlorophyll and of total chlorophyll to P700. Shade type in Anacystis was caused neither by phenolic inhibitors tested nor by those known to bind to the cytochrome b6/f-complex. Surprisingly enough, the molar ratio of phycocyanin to chlorophyll in artificially shade adapted Anacystis1 grown in strong white light in the presence of 10-6 м atrazine, was found to increase with temperature for a given light intensity and with light intensity for a given temperature.
Mutants of Anaeystis with a reduced binding capacity for DCMU-type herbicides due to an amino acid exchange in the 32 kDa Qв-binding polypeptide, also called D-1 protein, were ob- served to show shade type appearance in strong light, to respond very little to changes in light intensity and to show a reduced capability to further change their appearance to shade type by binding of competitors of Ob to the 32 kDa polypeptide.
In Anaeystis a concentration of atrazine (10-7 м), ten times lower than the one causing the highest rate of shade adaptation (10-6 м), was shown to induce an optimum in cell density, which in turn resulted in an optimum in light-dependent O2 evolution. Both factors together might be responsible for the so-called greening effect observed in higher plants treated with sublethal concentrations of DCMU-type inhibitors of photosynthetic electron transport.
A risk based assessment approach for chemical mixtures from wastewater treatment plant effluents
(2022)
In this study, 56 effluent samples from 52 European wastewater treatment plants (WWTPs) were investigated for the occurrence of 499 emerging chemicals (ECs) and their associated potential risks to the environment. The two main objectives were (i) to extend our knowledge on chemicals occurring in treated wastewater, and (ii) to identify and prioritize compounds of concern based on three different risk assessment approaches for the identification of consensus mixture risk drivers of concern. Approaches include (i) PNEC and EQS-based regulatory risk quotients (RQs), (ii) species sensitivity distribution (SSD)-based hazard units (HUs) and (iii) toxic units (TUs) for three biological quality elements (BQEs) algae, crustacean, and fish.
For this purpose, solid-phase extracts were analysed with wide-scope chemical target screening via liquid chromatography high-resolution mass spectrometry (LC-HRMS), resulting in 366 detected compounds, with concentrations ranging from < 1 ng/L to > 100 µg/L. The detected chemicals were categorized with respect to critical information relevant for risk assessment and management prioritization including: (1) frequency of occurrence, (2) measured concentrations, (3) use groups, (4) persistence & bioaccumulation, and (5) modes of action. A comprehensive assessment using RQ, HU and TU indicated exceedance of risk thresholds for the majority of effluents with RQ being the most sensitive metric. In total, 299 out of the 366 compounds were identified as mixture risk contributors in one of the approaches, while 32 chemicals were established as consensus mixture risk contributors of high concern, including a high percentage (66%) of pesticides and biocides. For samples which have passed an advanced treatment using ozonation or activated carbon (AC), consistently much lower risks were estimated.
Plant-fungal interactions are essential for understanding the distribution and abundance of plants species. Recently, arbuscular mycorrhizal fungal (AMF) partners of non-indigenous invasive plants have been hypothesized to be a critical factor influencing the invasion processes. AMF are known to improve nutrient and moisture uptake, as well as disrupt parasitic and pathogenic microbes in the host plant. Such benefits may enable invaders to establish significant and persistent populations in environments previously dominated by natives. Coupling these findings with studies on invader pathogen-disrupting root exudates is not well documented in the literature describing plant invasion strategies. The interaction effects of altered AMF associations and the impact of invader root exudates would be more relevant than understanding the AMF dynamics or the phytochemistry of successful invaders in isolation, particularly given that AMF and root exudates can have a similar role in pathogen control but function quite differently. One means to achieve this goal is to assess these strategies concurrently by characterizing both the general (mostly pathogens or commensals) and AM-specific fungal colonization patterns found in field collected root samples of successful invaders, native plants growing within dense patches of invaders, and native plants growing separately from invaders. In this review I examine the emerging evidence of the ways in which AMF-plant interactions and the production of defensive root exudates provide pathways to invasive plant establishment and expansion, and conclude that interaction studies must be pursued to achieve a more comprehensive understanding of successful plant invasion.
Twenty-nine species of caddisflies in the genus Agapetus Curtis in eastern and central North America are reviewed. Twelve are described as new species: Agapetus aphallus (known only from females); Agapetus baueri, Agapetus flinti, Agapetus harrisi, Agapetus hesperus, Agapetus ibis, Agapetus kirchneri, Agapetus meridionalis, Agapetus pegram, Agapetus ruiteri, Agapetus stylifer, and Agapetus tricornutus. Agapetus rossi Denning 1941 is recognized as a junior subjective synonym of Agapetus walkeri (Betten and Mosely 1940), new synonym. A key to males is provided, and species’ distributions are mapped.
The species of the genus Lathropus Erichson are reviewed for Florida and the West Indies, excluding the Lesser Antilles. Seven species are recorded from this region, three of which are described as new: Lathropus chickcharnie Thomas, new species, Lathropus jamaicensis Thomas, new species, and Lathropus rhabdophloeoides Thomas, new species. A lectotype is designated for Lathropus vernalis Casey, and Lathropus striatus Casey is synonymized under Lathropus vernalis Casey, new synonymy. Illustrations and a key to the species of this geographgic region are provided.
Neurons collect their inputs from other neurons by sending out arborized dendritic structures. However, the relationship between the shape of dendrites and the precise organization of synaptic inputs in the neural tissue remains unclear. Inputs could be distributed in tight clusters, entirely randomly or else in a regular grid-like manner. Here, we analyze dendritic branching structures using a regularity index R, based on average nearest neighbor distances between branch and termination points, characterizing their spatial distribution. We find that the distributions of these points depend strongly on cell types, indicating possible fundamental differences in synaptic input organization. Moreover, R is independent of cell size and we find that it is only weakly correlated with other branching statistics, suggesting that it might reflect features of dendritic morphology that are not captured by commonly studied branching statistics. We then use morphological models based on optimal wiring principles to study the relation between input distributions and dendritic branching structures. Using our models, we find that branch point distributions correlate more closely with the input distributions while termination points in dendrites are generally spread out more randomly with a close to uniform distribution. We validate these model predictions with connectome data. Finally, we find that in spatial input distributions with increasing regularity, characteristic scaling relationships between branching features are altered significantly. In summary, we conclude that local statistics of input distributions and dendrite morphology depend on each other leading to potentially cell type specific branching features.
It is widely acknowledged that biodiversity change is affecting human well-being by altering the supply of Nature's Contributions to People (NCP). Nevertheless, the role of individual species in this relationship remains obscure. In this article, we present a framework that combines the cascade model from ecosystem services research with network theory from community ecology. This allows us to quantitatively link NCP demanded by people to the networks of interacting species that underpin them. We show that this “network cascade” framework can reveal the number, identity and importance of the individual species that drive NCP and of the environmental conditions that support them. This information is highly valuable in demonstrating the importance of biodiversity in supporting human well-being and can help inform the management of biodiversity in social-ecological systems.
Energy-converting hydrogenases (Ech) are ancient, membrane-bound enzymes that use reduced ferredoxin (Fd) as an electron donor to reduce protons to molecular H2. Experiments with whole cells, membranes and vesicle-fractions suggest that proton reduction is coupled to proton translocation across the cytoplasmatic membrane, but this has never been demonstrated with a purified enzyme. To this end, we produced a His-tagged Ech complex in the thermophilic and anaerobic bacterium Thermoanaerobacter kivui. The enzyme could be purified by affinity chromatography from solubilized membranes with full retention of its eight subunits, as well as full retention of physiological activities, i.e., H2-dependent Fd reduction and Fd2--dependent H2 production. We found the purified enzyme contained 34.2 ± 12.2 mol of iron/mol of protein, in accordance with seven predicted [4Fe-4S]-clusters and one [Ni-Fe]-center. The pH and temperature optima were at 7 to 8 and 66 °C, respectively. Notably, we found that the enzymatic activity was inhibited by N,N′-dicyclohexylcarbodiimide, an agent known to bind ion-translocating glutamates or aspartates buried in the cytoplasmic membrane and thereby inhibiting ion transport. To demonstrate the function of the Ech complex in ion transport, we further established a procedure to incorporate the enzyme complex into liposomes in an active state. We show the enzyme did not require Na+ for activity and did not translocate 22Na+ into the proteoliposomal lumen. In contrast, Ech activity led to the generation of a pH gradient and membrane potential across the proteoliposomal membrane, demonstrating that the Ech complex of T. kivui is a H+-translocating, H+-reducing enzyme.