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Nach Bestrahlung eines kleinen Bezirkes der Froschschwimmhaut mit verschiedenen ultravioletten Wellenlängen (λ=254 mµ, 280 mµ, 297 mµ, 313 mµ, 366 mµ) wurde Stase in den Kapillaren beobachtet. Die Wirksamkeit der verschiedenen Wellenlängen ist dabei unterschiedlich. Messungen der Durchlässigkeit der Schwimmhaut wurden durchgeführt und erlaubten Schlüsse auf die in das Kapillargebiet hingelangende Strahlung. Während der Bestrahlungen konnten Durchlässigkeits-Veränderungen der Schwimmhaut beobachtet und gemessen werden. Die Ergebnisse werden diskutiert. Histologische Untersuchungen erweiterten das Bild über die Veränderungen im Gewebe durch Strahleneinwirkung.
1. Die Lebensdauer und Entwicklungsfähigkeit unbesamter Seeigeleier in Coffeinlösungen (1 : 250 bis 1 : 2000) ist gegenüber der in normalem Seewasser deutlich verlängert.
2. Das Optimum der Lebensverlängerung liegt etwa bei einer Konzentration von 1 : 1000 bis 1 : 1250.
3. Der Zellkern kann sich unter Coffeineinfluß „aufblähen“. und zwar bis zum 3-fachen seines normalen Umfanges.
4. Das Coffein ruft bei gleichbleibender Zellgröße eine Herabsetzung der Viskosität der Zelloberfläche hervor und ermöglicht bei gallertlosen Eiern, die sich berühren, ein Aneinanderlegen und Abplatten der Eier bis zu Reihen-Eiern und Pflasterbildungen. Derartige Eier können sich nach Zurückbringen in Seewasser und Besamung noch am 4. und 5. Tage zu Plutei aller Normalitäts-Stufen entwickeln.
5. Aus den Reiheneiern können durch Verschmelzen braun gefärbte Riesen-Eier hervorgehen, die nicht mehr entwicklungsfähig, aber gegen Zerfall sehr widerstandsfähig sind.
Epithelial cells enable essential physiological functions, including absorption, morphogenesis, secretion, and transport. To execute these functions, epithelial cells often form three-dimensional shapes that include curved sheets of cells surrounding a pressurized fluid-filled lumen. These three-dimensional tissues (called domes) are essential for organ function, but when they are not working properly, developmental defects, inflammation, and cancer can ensue. Recently, it has been shown that the cells that form domes show active superelasticity on micropatterned plates.
We show here that the immortalized renal proximal tubule epithelial cell line, LLC-PK1, stereotypically forms tubules in 10 days. Tubule formation takes place in 4 stages. When cells are plated on a culture dish, they form a monolayer on the 1st day; on the 3rd day, three-dimensional structures are formed, called domes; and after the 4.5th day, these domes start fusing to begin the transition stage and transit to the tubule stage. At the end of the 10th day, differentiated, elongated, and matured tubes form (Figure 3.1). Therefore, tubule formation is a self-organized, stereotypic morphogenetic program under long-term, unperturbed tissue culture conditions.
We propose that tubulogenesis is a two-step process in proximal tubules by doming and wrapping. The process begins with dome formation, and as the cell layers come together in the transition stage at the edge of the dome, this leads to the formation of the lumen of the eventual tubule. We also found that F-actin provides the mechanical strength during the formation of these three-dimensional structures during tubule formation. To better understand this 4-step process on a molecular level, we performed proteomics of tubule formation to identify the different proteins that play a significant role in proximal tubule development. Importantly, we identified proximal tubule markers like synaptopondin, angiotensin 1-10, collectrin, polycystin 1, and polycystin 2. These proteins play an important role in renal tube formation and differentiation.
Cell division is carried out by highly conserved cyclin-CDK complexes, which phosphorylate various cellular components. Cyclin-CDKs act differently depending on the cell cycle phase and work cooperatively to create DNA replication and cytokinesis. Therefore, we identified that cyclin-B1, marker of proliferation Ki-67, the RAD51 recombinase, and proliferating cell nuclear antigen (PNCA) are upregulated in the monolayer stage, and the expression decreases as tubule formation takes place. The proximal tubule reabsorbs 60-65% of the glomerulus filtrate. Therefore, it requires a lot of energy generated by using the fatty acid oxidation (FAO) pathway. In our model, we found FAO expression is higher than that of the other metabolic pathways.
We found expression of an intricate protein network in mitochondria, which we interpret as a sign of mitochondrial homeostasis being vital for the FAO pathway to work. Furthermore, we also identified different types of transporters at each stage of proximal tubule formation, and we could recognize different cytoskeletal components playing a significant role in each stage of proximal tubule formation, for instance, at the monolayer stage, vimentin expression is high, and its expression is reduced as tubules form. Hence, this 2D system, at this step of characterization, seems suitable to use to study differential transport protein expression and how this might relate to physiological functions and syndromes.
Next, we inhibited different transporters using specific inhibitors and analyzed the effect on dome and tubule formation. We identified that Na+/K+ ATPase and vacuolar H+ ATPase play a significant role in the process of epithelial dynamics. Digoxin (a Na+/K+ ATPase inhibitor) treatment inhibits dome and tubule formation. Bafilomycin (a v-ATPase inhibitor) treatment demonstrated a delay in dome and tube formation. Therefore, this study shows that this 2D proximal tubule novel system can be used for screening of pharmacological leads in the context of specific aspects of kidney physiology.
Despite the recent success in growing kidney organoids, they are not well suited to investigate various pathophysiological conditions in vitro for several reasons: They grow in 3D and form a tissue that later needs to be dissected/cleared and stained to investigate pathophysiological changes. Moreover, organoids require complex and expensive protocols for generation and are challenging to use in screening approaches. Therefore, we set out to demonstrate feasibility for our 2D system using normal renal epithelial cells, which are the origin of various pathological conditions, to study pathophysiological conditions.
Due to their sessile nature, plants are constantly exposed to an everchanging environment. When these changes exceed certain limits, they can significantly impact plant growth and development, which, in case of crop plants, has consequences on food security. Exposure to high temperatures causes heat stress (HS), one of the most devastating stresses that plants can face. The survival and recovery from HS are dependent on the activation of the HS response (HSR), a collection of molecular mechanisms conferring HS tolerance by maintaining the cellular homeostasis. Stress responses follow a strictly orchestrated network of signal perception and -transduction, ultimately resulting in an adaptive cellular output. Thereby, the massive reshaping of the transcriptome plays a major part, in which heat stress transcription factors (HSFs) play the key role by inducing the expression of HS-responsive genes, including heat shock proteins and other transcription factors. Additionally, alternative splicing (AS), the selective usage of splice sites, contributes to the rapid adjustment of the transcriptome landscape by producing different mRNA variants from a single gene. Consequently, this results in the reduction of translatable transcripts by nonsense-mediated mRNA-decay or nuclear retention, but also enhances the proteome diversity by allowing the synthesis of protein isoforms with distinct functions. AS thereby modulates the activity of important regulatory factors like HSFA2 in Solanum lycopersicum (tomato). HSFA2 is the key factor of acquired thermotolerance (ATT), which enables the ability to survive a potentially lethal HS through pre-exposure to a preceding mild HS. Temperature-dependent AS leads to the synthesis of two HSFA2 protein variants, whereby inhibition of splicing ensures the synthesis of the stable isoform HSFA2-I that is required for ATT.
Transcriptome analysis of several plant species exposed to HS has highlighted the strong impact of high temperatures on the regulation of pre-mRNA splicing. Despite its importance, little is known about the molecular basis of the AS regulation in plants. Particularly for an economically important crop like tomato, understanding the regulation of HS-sensitive AS will contribute to the description of such an important regulatory mechanism but also might offer new insights for increasing HS resilience. Serine/arginine-rich proteins (SR proteins) are central regulators of constitutive and AS by modulating the splice site selection by the spliceosome. This study describes two members of the RS2Z subfamily of SR proteins in tomato, namely RS2Z35 and RS2Z36, which act as core regulators of AS under HS and consequently as central factors for thermotolerance. This study investigates the interaction of the two RS2Z proteins with the HSFA2 pre-mRNA and provides evidence for their function as splicing repressors in this particular AS event. Thereby, RS2Z proteins play an important role in the HSR by modulating the AS of the key factor of the ATT. Furthermore, based on global transcriptome analysis of knockout mutants of single or both RS2Z genes, it is demonstrated that RS2Z proteins are involved in the splicing of pre-mRNAs of almost 2000 genes. Moreover, RS2Z proteins act as splicing regulators and take part in a large portion of HS-induced AS events, thus playing a broader role in AS regulation. Furthermore, the HS-induced RS2Z36 is involved in basal thermotolerance (BTT), highlighting its importance for the basic HS resilience capacity of tomato. In addition, RNA sequencing demonstrates that RS2Z proteins–especially RS2Z36–regulate the expression of proteins involved in plant immunity. The study thereby provides experimental evidence for the important and essential role of SR proteins for plant thermotolerance and suggests the existence of RS2Z-mediated crossroads of different stress responses.
Auf Grund vergleichender Prüfungen im Riesengebirge, im Schwarzwald und im Allgäu zwischen 1250 und 2220 m Meereshöhe wird nachgewiesen, daß die bisher allein bekannt gewordene Beziehung zwischen Lichtfeld und Chlorophyllgehalt der Art, daß der Farbstoffgehalt mit der Lichtintensität bis zur ökologisch maximalen Strahlung ansteigt, nur für die Angehörigen des photostabilen Reaktionstypus gilt.
Neben diesem ist selbst in frei-sonnigen Pflanzenvereinen alpiner Matten und Felsfluren ein photolabiler Typus weit verbreitet, dessen maximal bestrahlte Sonnenblätter im Verlauf des Sommers im Vergleich zu gleich alten Schattenblättern mäßige bis starke Depressionen des (flächenrelativen) Chlorophyllwertes aufweisen.
Der extrem photostabile ist mit dem extrem photolabilen Reaktionstypus durch alle Übergänge verbunden. Allein die Abstufungen der Resistenz können weder auf das Vorleben in bestimmtem Strahlungsklima bzw. bestimmter Höhenlage, noch auf die Zugehörigkeit zu einem bestimmten ökologischen Verbreitungstypus zurückgeführt werden. Photostabile Tieflandpflanzen wie Silene inflata und Anthyllis vulneraria erweisen sich auch in 2220 m Höhe als photostabil, während viele Alpenpflanzen auch in ihrem natürlichen Verbreitungsgebiet ausgesprochen photolabil angetroffen werden.
In synchronen Vergleichsversuchen aus 1415 m Höhe werden an photolabilen Stauden die Zeitkurven der Veränderungen des Chlorophyllwertes beim Übergang aus diffusem Licht in die Gesamtstrahlung und umgekehrt verfolgt. Während im ersten Fall im Verlauf einer Schönwetterperiode die (bisherigen) Scha-Blätter rasch photochemische Chlorophyllzerstörungen erleiden, erfahren die (bisherigen) So-Blätter im diffusen Licht zur gleichen Zeit eine Zunahme des Chlorophylls.
Es wird die Brauchbarkeit der Wärmeleitfähigkeitsmessung zur Ermittlung des Grundumsatzes geprüft.
Durch Diffusion des Meßgases zu den Hitzdrähten, eine Anordnung, die in der industriellen Meßtechnik häufig Anwendung findet, wird erreicht, daß die Messung weitgehend von der linearen Strömungsgeschwindigkeit unabhängig wird. Die Empfindlichkeit der Sauerstoffbestimmung wird gesteigert durch Erhöhung der Hitzdrahttemperatur. Eine Anordnung zur Bestimmung des Grundumsatzes am Menschen wird beschrieben, die es gestattet, im „offenen System" laufende Messungen durchzuführen. Die laufende Messung“ wird den integrierenden Methoden gegenübergestellt.
Bestrahlung mit UV und UR einschließlich sichtbaren Lichts bedingt bei weißen Mäusen und Hühnern eine Senkung im Gasstoffwechsel von durchschnittlich 25% und im Dauerversuch eine deutliche Herabsetzung des Grundstoffwechsels, während Lichtmangel bei weißen Mäusen den Stoffwechsel erhöht.
Sichtbares Licht, UV oder UR allein bewirken keinen Stoffwechselabfall.
Für die Stoffwechselerniedrigung nach Bestrahlung ist eine bestimmte Kombination von UV und UR notwendig.
Die Wirkung der Bestrahlung beruht mit großer Wahrscheinlichkeit auf einer Senkung des Tonus des Sympathicus und einer Verschiebung der vegetativen Regulationslage zur Vagotonie.
Eigenbestrahlungsversuche (G.) zeigten eine gleiche Senkung des durch Hyperthyreose erhöhten Grundstoffwechsels.
Motivation Expert curation to differentiate between functionally diverged homologs and those that may still share a similar function routinely relies on the visual interpretation of domain architecture changes. However, the size of contemporary data sets integrating homologs from hundreds to thousands of species calls for alternate solutions. Scoring schemes to evaluate domain architecture similarities can help to automatize this procedure, in principle. But existing schemes are often too simplistic in the similarity assessment, many require an a-priori resolution of overlapping domain annotations, and those that allow overlaps to extend the set of annotations sources cannot account for redundant annotations. As a consequence, the gap between the automated similarity scoring and the similarity assessment based on visual architecture comparison is still too wide to make the integration of both approaches meaningful.
Results Here, we present FAS, a scoring system for the comparison of multi-layered feature architectures integrating information from a broad spectrum of annotation sources. Feature architectures are represented as directed acyclic graphs, and redundancies are resolved in the course of comparison using a score maximization algorithm. A benchmark using more than 10,000 human-yeast ortholog pairs reveals that FAS consistently outperforms existing scoring schemes. Using three examples, we show how automated architecture similarity assessments can be routinely applied in the benchmarking of orthology assignment software, in the identification of functionally diverged orthologs, and in the identification of entries in protein collections that most likely stem from a faulty gene prediction.
Pectin-rich residues are considered as promising feedstocks for sustainable production of platform chemicals. Enzymatic hydrolysis of extracted sugar beet press pulp (SBPP) releases the main constituent of pectin, d-galacturonic acid (d-GalA). Using engineered Saccharomyces cerevisiae, d-GalA is then reduced to l-galactonate (l-GalOA) with sorbitol as co-substrate. The current work addresses the combination of enzymatic hydrolysis of pectin in SBPP with a consecutive optimized biotransformation of the released d-GalA to l-GalOA in simple batch processes in stirred-tank bioreactors. Process conditions were first identified with synthetic media, where a product concentration of 9.9 g L-1 L-GalOA was obtained with a product selectivity of 99% (L-GalOA D-GalA-1) at pH 5 with 4% (w/v) sorbitol within 48 h. A very similar batch process performance with a product selectivity of 97% was achieved with potassium citrate buffered SBPP hydrolysate, demonstrating for the first time direct production of L-GalOA from hydrolyzed biomass using engineered S. cerevisiae. Combining the hydrolysis process of extracted SBPP and the biotransformation process with engineered S. cerevisiae paves the way towards repurposing pectin-rich residues as substrates for value-added chemicals.
The interaction between the Heat Shock Proteins 70 and 40 is at the core of the ATPase regulation of the chaperone machinery that maintains protein homeostasis. However, the structural details of this fundamental interaction are still elusive and contrasting models have been proposed for the transient Hsp70/Hsp40 complexes. Here we combine molecular simulations based on both coarsegrained and atomistic models with co-evolutionary sequence analysis to shed light on this problem by focusing on the bacterial DnaK/DnaJ system. The integration of these complementary approaches resulted into a novel structural model that rationalizes previous experimental observations. We identify an evolutionary-conserved interaction surface formed by helix II of the DnaJ J-domain and a groove on lobe IIA of the DnaK nucleotide binding domain, involving the inter-domain linker.
The basidiomycete smut fungi are predominantly plant parasitic, causing severe losses in some crops. Most species feature a saprotrophic haploid yeast stage, and several smut fungi are only known from this stage, with some isolated from habitats without suitable hosts, e.g. from Antarctica. Thus, these species are generally believed to be apathogenic, but recent findings that some of these might have a plant pathogenic sexual counterpart, casts doubts on the validity of this hypothesis. Here, four Pseudozyma genomes were re-annotated and compared to published smut pathogens and the well-characterised effector gene Pep1 from these species was checked for its ability to complement a Pep1 deletion strain of Ustilago maydis. It was found that 113 high-confidence putative effector proteins were conserved among smut and Pseudozyma genomes. Among these were several validated effector proteins, including Pep1. By genetic complementation we show that Pep1 homologs from the supposedly apathogenic yeasts restore virulence in Pep1-deficient mutants Ustilago maydis. Thus, it is concluded that Pseudozyma species have retained a suite of effectors. This hints at the possibility that Pseudozyma species have kept an unknown plant pathogenic stage for sexual recombination or that these effectors have positive effects when colonising plant surfaces.
The auditory midbrain (inferior colliculus, IC) plays an important role in sound processing, acting as hub for acoustic information extraction and for the implementation of fast audio-motor behaviors. IC neurons are topographically organized according to their sound frequency preference: dorsal IC regions encode low frequencies while ventral areas respond best to high frequencies, a type of sensory map defined as tonotopy. Tonotopic maps have been studied extensively using artificial stimuli (pure tones) but our knowledge of how these maps represent information about sequences of natural, spectro-temporally rich sounds is sparse. We studied this question by conducting simultaneous extracellular recordings across IC depths in awake bats (Carollia perspicillata) that listened to sequences of natural communication and echolocation sounds. The hypothesis was that information about these two types of sound streams is represented at different IC depths since they exhibit large differences in spectral composition, i.e., echolocation covers the high-frequency portion of the bat soundscape (> 45 kHz), while communication sounds are broadband and carry most power at low frequencies (20–25 kHz). Our results showed that mutual information between neuronal responses and acoustic stimuli, as well as response redundancy in pairs of neurons recorded simultaneously, increase exponentially with IC depth. The latter occurs regardless of the sound type presented to the bats (echolocation or communication). Taken together, our results indicate the existence of mutual information and redundancy maps at the midbrain level whose response cannot be predicted based on the frequency composition of natural sounds and classic neuronal tuning curves.
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.
Although new advances in neuroscience allow the study of vocal communication in awake animals, substantial progress in the processing of vocalizations has been made from brains of anaesthetized preparations. Thus, understanding how anaesthetics affect neuronal responses is of paramount importance. Here, we used electrophysiological recordings and computational modelling to study how the auditory cortex of bats responds to vocalizations under anaesthesia and in wakefulness. We found that multifunctional neurons that process echolocation and communication sounds were affected by ketamine anaesthesia in a manner that could not be predicted by known anaesthetic effects. In wakefulness, acoustic contexts (preceding echolocation or communication sequences) led to stimulus-specific suppression of lagging sounds, accentuating neuronal responses to sound transitions. However, under anaesthesia, communication contexts (but not echolocation) led to a global suppression of responses to lagging sounds. Such asymmetric effect was dependent on the frequency composition of the contexts and not on their temporal patterns. We constructed a neuron model that could replicate the data obtained in vivo. In the model, anaesthesia modulates spiking activity in a channel-specific manner, decreasing responses of cortical inputs tuned to high-frequency sounds and increasing adaptation in the respective cortical synapses. Combined, our findings obtained in vivo and in silico reveal that ketamine anaesthesia does not reduce uniformly the neurons’ responsiveness to low and high frequency sounds. This effect depends on combined mechanisms that unbalance cortical inputs and ultimately affect how auditory cortex neurons respond to natural sounds in anaesthetized preparations.
Communication sounds are ubiquitous in the animal kingdom, where they play a role in advertising physiological states and/or socio-contextual scenarios. Distress sounds, for example, are typically uttered in distressful scenarios such as agonistic interactions. Here, we report on the occurrence of superfast temporal periodicities in distress calls emitted by bats (species Carollia perspicillata). Distress vocalizations uttered by this bat species are temporally modulated at frequencies close to 1.7 kHz, that is, ∼17 times faster than modulation rates observed in human screams. Fast temporal periodicities are represented in the bats’ brain by means of frequency following responses, and temporally periodic sounds are more effective in boosting the heart rate of awake bats than their demodulated versions. Altogether, our data suggest that bats, an animal group classically regarded as ultrasonic, can exploit the low frequency portion of the soundscape during distress calling to create spectro-temporally complex, arousing sounds.
Neural oscillations are at the core of important computations in the mammalian brain. Interactions between oscillatory activities in different frequency bands, such as delta (1-4 Hz), theta (4-8 Hz), or gamma (>30 Hz), are a powerful mechanism for binding fundamentally distinct spatiotemporal scales of neural processing. Phase-amplitude coupling (PAC) is one such plausible and well-described interaction, but much is yet to be uncovered regarding how PAC dynamics contribute to sensory representations. In particular, although PAC appears to have a major role in audition, the characteristics of coupling profiles in sensory and integration (i.e. frontal) cortical areas remain obscure. Here, we address this question by studying PAC dynamics in the frontal-auditory field (FAF; an auditory area in the bat frontal cortex) and the auditory cortex (AC) of the bat Carollia perspicillata. By means of simultaneous electrophysiological recordings in frontal and auditory cortices examining local-field potentials (LFPs), we show that the amplitude of gamma-band activity couples with the phase of low-frequency LFPs in both structures. Our results demonstrate that the coupling in FAF occurs most prominently in delta/high-gamma frequencies (1-4/75-100 Hz), whereas in the AC the coupling is strongest in the theta/low-gamma (2-8/25-55 Hz) range. We argue that distinct PAC profiles may represent different mechanisms for neuronal processing in frontal and auditory cortices, and might complement oscillatory interactions for sensory processing in the frontal-auditory cortex network.