Kontrolle des Aminozuckerstoffwechsels in Corynebacterium glutamicum

Aminozucker, wie Glucosamin oder N-Acetylglucosamin, stellen für biotechno-logische Prozesse mit Corynebacterium glutamicum eine alternativer Kohlenstoffquelle dar. Als Bestandteil von Chitin sind sie weit verbreitet, können jedoch von C. glutamicum nur sehr schlecht oder, wie im Fall von N-Acetylglucosamin, gar nicht umgesetzt werden. Diese Arbeit beschreibt die Identifikation und Charakterisierung von Proteinen des Aminozuckerkatabolismus, die Wirkungsweise des an der Expression beteiligten Regulators und die Anwendung der erzielten Ergebnisse am Beispiel von L-Lysin-Produktionsstämmen. Die Annotation des Genoms zeigte, dass die für den Katabolismus der Aminozucker notwendigen Enzyme NagA (N-Acetylglucosamin-6-phosphat-Deacetylase) und NagB (Glucosamin-6-phosphats-Deaminase) vorhanden sind. Über die Substratimporter war nichts bekannt. Durch die Aufnahmemessung radioaktiv markierter Substrate konnte der Glucose-Importer PTSGlc als Aufnahmesystem von Glucosamin ermittelt werden. PTSGlc hat zwar eine vergleichsweise geringe Affinität zu Glucosamin, der Substratimport stellt jedoch nicht den wachstumslimitierenden Schritt dar. Limitierend ist die Aktivität von NagB. Durch die Überexpression von nagB konnten die Wachstumsraten von C. glutamicum auf Glucosamin verdreifacht werden. Des Weiteren wurde der Regulator der nag-Gene identifiziert und der zugrunde liegende Regulationsmechanismus beschrieben. NanR ist ein Regulator der GntR-Familie, der neben den nag-Genen auch die Expression der unmittelbar benachbarten Gene des Sialinsäurekatabolismus (nan-Gene) reguliert. Die DNA-Bindedomäne von NanR erkennt ein spezifisches Motiv, bestehend aus zwei aufeinanderfolgenden Hexanukleotidsequenzen. Durch die Bindung von NanR im Promotor der nag- und nan-Gene wird deren Expression reprimiert. N-Acetylglucosamin-6-phosphat und N-Acetylmannosamin-6-phosohat sind Effektoren von NanR, durch die der Repressor wieder von der DNA gelöst wird. Die gemeinsame Regulation der nag- und nan-Gene durch den gleichen Regulator ist bei Bakterien eine Ausnahme. In L-Lysin Produktionsstämmen wurde die Überexpression von nagB sowie die Deletion von nanR angewandt, um Glucosamin als Substrat zur Aminosäureproduktion zu nutzen. Die erzielten Ausbeuten und Produktivitäten lagen im gleichen Bereich wie bei dem gut verwertbaren Substrat Glucose. Zur Nutzung von N-Acetylglucosamin wurde der heterologe Transporter NagE aus C. glycinophilum eingesetzt. Dabei blieb der Substratimport trotz der Überexpression limitierend. Eine effektive Nutzung von N-Acetylglucosamin konnte jedoch mit den Cosubstrat zu Glucose erzielt werden

LoopBoxes -- Evaluation of a Collaborative Accessible Digital Musical Instrument

LoopBoxes is an accessible digital musical instrument designed to create an intuitive access to loop based music making for children with special educational needs (SEN). This paper describes the evaluation of the instrument in the form of a pilot study during a music festival in Berlin, Germany, as well as a case study with children and music teachers in a SEN school setting. We created a modular system composed of three modules that afford single user as well as collaborative music making. The pilot study was evaluated using informal observation and questionnaires (n = 39), and indicated that the instrument affords music making for people with and without prior musical knowledge across all age groups and fosters collaborative musical processes. The case study was based on observation and a qualitative interview. It confirmed that the instrument meets the needs of the school settings and indicated how future versions could expand access to all students, especially those experiencing complex disabilities. In addition, out-of-the-box functionality seems to be crucial for the long-term implementation of the instrument in a school setting.Comment: 10 pages, 9 figures, to be published in the Proceedings of the International Conference on New Interfaces for Musical Expression (NIME'23

Stoichiometry of Potassium Channel Opener Action

ABSTRACT Potassium channel openers (KCOs; e.g., P1075, pinacidil) exert their effects on excitable cells by opening ATP-sensitive potassium channels. These channels are heteromultimers composed with a 4:4 stoichiometry of an inwardly rectifying K ϩ channel subunit plus a regulatory subunit comprising the receptor sites for hypoglycemic sulfonylureas and KCOs (a sulfonylurea receptor). To elucidate stoichiometry of KCO action, we analyzed P1075 sensitivity of channels coassembled from sulfonylurea receptor isoforms with high or low P1075 affinity. Concentration activation curves for cDNA ratios of 1:1 or 1:10 resembled those for channel opening resulting from interaction with a single site, whereas models for activation requiring occupation of two, three, or four sites were incongruous. We conclude KCO-induced channel activation to be mediated by interaction with a single binding site per tetradimeric complex. Potassium channel openers (KCOs) comprise a structurally diverse group of drugs with a broad spectrum of potential therapeutic applications (e.g., hypoglycemia, hypertension, arrhythmias, angina pectoris, asthma) Recent progress resulted in cloning of K ATP channels and elucidation of their subunit composition (for review, see Three isoforms of SURs have been cloned, SUR1 and two splice products of a single gene, SUR2A and SUR2B, differing only in their C-terminal 42 to 45 amino acids Notably, diversity of SURs confers tissue-specific pharmacology, with SUR2 isoforms imparting high sensitivity to KCOs and low sensitivity to sulfonylureas, and SUR1 mediating inverse sensitivities Unraveling the mechanisms involved in control of K ATP activity by SURs is of key importance for understanding molecular pharmacology of these channels. To elucidate stoichiometry of KCO action, we analyzed P1075 sensitivity of channels coassembled from SUR1 and a chimeric construct with high KCO affinity. Concentration-activation curves resembled those expected for channel opening by interaction with a single site. This work was supported by grants from the Deutsche Forschungsgemeinschaft (to M.S. and C.S.)

Correlation between body size and fatty acid and essential amino acid composition of round goby (Neogobius melanostomus) and monkey goby (Neogobius fluviatilis) from the Rhine River (Germany)

In this study correlations between body size and muscle fatty and amino acid content of two species of goby, round goby (Neogobius melanostomus) and monkey goby (Neogobius fluviatilis) caught from river Rhine (Germany) were investigated. Among saturated fatty acids (SFAs), mono- (MUFA) and polyunsaturated fatty acids (PUFAs) only SFAs were significantly higher in round goby than monkey goby (P < 0.05). In general, the correlation between body size of both gobies and the content of most of the individual fatty acids was not significant. In monkey goby, the content of palmitic acid (C16:0) and oleic acid (C18:1 n-9) was positively correlated with weight (r = 0.43) and total length (r = -0.58), respectively, and the content of docosahexaenoic acid (DHA) increased with condition factor (r = 0.50). The content of threonine, arginine, valine, phenylalanine and isoleucine in monkey goby was higher than those of round goby (P < 0.05). In round goby the three essential amino acids arginine, valine and leucine were positively (P < 0.05) correlated with body length, which indicates that longer round gobies are of higher nutritional value

Femtogram Mass Measurement of Airborne Engineered Nanoparticles using Silicon Nanopillar Resonators

AbstractSilicon nanopillar resonators were fabricated and used for sensing airborne engineered nanoparticles (ENPs) by monitoring resonant frequency shifts induced by the mass of trapped ENPs. Inductively coupled plasma (ICP) cryogenic dry etching and thermal oxidation were utilized in sensor fabrication. An electrostatic aerosol sampler and piezo shear actuator were used to collect the flowing ENPs and excite the nanopillars in resonant mode, respectively. By using the fundamental bending frequency, the sensor can achieve a mass sensitivity of 7.41Hz/fg, which enables its application to a nanobalance to detect airborne ENPs in the femtogram regime. A simple cleaning method of polluted nanopillars was also performed to remove the adhered ENPs, thus efficiently extend the operating life of the sensor. This developed sensor is targeted for use in airborne ENP sensing applications

Cytotoxic CD8+ T cell ablation enhances the capacity of regulatory T cells to delay viral elimination in Theiler's murine encephalomyelitis

Theiler's murine encephalomyelitis (TME) of susceptible mouse strains is a commonly used infectious animal model for multiple sclerosis. The study aim was to test the hypothesis whether cytotoxic T cell responses account for the limited impact of regulatory T cells on antiviral immunity in TME virus-induced demyelinating disease (TMEV-IDD) resistant C57BL/6 mice. TME virus-infected C57BL/6 mice were treated with (i) interleukin-2/-anti-interleukin-2-antibody-complexes to expand regulatory T cells (“Treg-expansion”), (ii) anti-CD8-antibodies to deplete cytotoxic T cells (“CD8-depletion”) or (iii) with a combination of Treg-expansion and CD8-depletion (“combined treatment”) prior to infection. Results showed that “combined treatment”, but neither sole “Treg-expansion” nor “CD8-depletion,” leads to sustained hippocampal infection and virus spread to the spinal cord in C57BL/6 mice. Prolonged infection reduces myelin basic protein expression in the spinal cord together with increased accumulation of β-amyloid precursor protein in axons, characteristic of myelin loss and axonal damage, respectively. Chronic spinal cord infection upon “combined treatment” was also associated with increased T and B cell recruitment, accumulation of CD107b+ microglia/macrophages and enhanced mRNA expression of interleukin (IL)-1α, IL-10 and tumor necrosis factor α. In conclusion, data revealed that the suppressive capacity of Treg on viral elimination is efficiently boosted by CD8-depletion, which renders C57BL/6 mice susceptible to develop chronic neuroinfection and TMEV-IDD

Transcription of sialic acid catabolism genes in Corynebacterium glutamicum is subject to catabolite repression and control by the transcriptional repressor NanR

Uhde A, Brühl N, Goldbeck O, et al. Transcription of sialic acid catabolism genes in Corynebacterium glutamicum is subject to catabolite repression and control by the transcriptional repressor NanR. J Bacteriol. 2016;198(16):2204-2218

Intact interleukin-10 receptor signaling protects from hippocampal damage elicited by experimental neurotropic virus infection of SJL mice

Abstract Theiler’s murine encephalomyelitis virus (TMEV) infection represents an experimental mouse model to study hippocampal damage induced by neurotropic viruses. IL-10 is a pleiotropic cytokine with profound anti-inflammatory properties, which critically controls immune homeostasis. In order to analyze IL-10R signaling following virus-induced polioencephalitis, SJL mice were intracerebrally infected with TMEV. RNA-based next generation sequencing revealed an up-regulation of Il10, Il10rα and further genes involved in IL-10 downstream signaling, including Jak1, Socs3 and Stat3 in the brain upon infection. Subsequent antibody-mediated blockade of IL-10R signaling led to enhanced hippocampal damage with neuronal loss and increased recruitment of CD3+ T cells, CD45R+ B cells and an up-regulation of Il1α mRNA. Increased expression of Tgfβ and Foxp3 as well as accumulation of Foxp3+ regulatory T cells and arginase-1+ macrophages/microglia was detected in the hippocampus, representing a potential compensatory mechanism following disturbed IL-10R signaling. Additionally, an increased peripheral Chi3l3 expression was found in spleens of infected mice, which may embody reactive regulatory mechanisms for prevention of excessive immunopathology. The present study highlights the importance of IL-10R signaling for immune regulation and its neuroprotective properties in the context of an acute neurotropic virus infection