Tau, a microtubule associated protein, forms abnormal aggregates in many neurodegenerative diseases such as Alzheimer disease (AD). There is an urgent need for disease-modifying therapies of AD and related tauopathies. Inhibiting the aggregation of tau and the accumulation of neurofibrillary tangles (NFTs) could be helpful in combating tau pathology. Recent studies show that tau induced toxicity is mainly due to the presence of oligomers of tau rather than the monomers and fibrillar aggregates (Kaniyappan et al., 2017, Flach et al., 2012, Lasagna-Reeves et al., 2010). <br /> To combat the toxicity of tau oligomers we developed antibodies against the purified low-n tau oligomers (dimers to hexamers) of Tau^RDΔK, the strongly aggregating repeat domain of tau. Monoclonal antibodies were tested by various biochemical and biophysical methods for their specificity to bind to the toxic oligomers. Some antibodies show specificity to aggregates of tau while others detect all forms of tau. Antibodies 2B10 and 6H1, described as representative examples, bind to tau oligomers with high specificity as judged by dotblot, dynamic light scattering (DLS) and immunofluorescence analysis. As these antibodies are dependent on tau conformations, they appear non-specific in denaturing methods like western blotting. 2B10 and 6H1 antibodies are able to inhibit the tau aggregation up to ~90% in vitro (Tau^RDΔK, hTau^P301L), as judged by the Thioflavin S fluorescence assay which is sensitive to ß-structure. In the presence of antibodies tau protein forms only up to low-n oligomers as judged by light scattering and atomic force microscopy (AFM). The choice of the pH of the column elution buffer of the antibodies plays a key role in determining the activity of the antibodies, as antibodies eluted at low pH have a higher activity compared to the same antibodies eluted at high pH. <br /> The ability of antibodies to inhibit the aggregation of tau was tested in an N2a cell model of tau pathology which expresses the pro-aggregant tau repeat domain Tau^RDΔK. Antibodies were added to the extracellular medium, without or with protein transfection reagent (Xfect) which stimulates cellular uptake. In this assay, 2B10 antibody failed to inhibit tau aggregation (ThS signal) and failed to prevent aggregation induced apoptosis (Annexin V signal). By contrast, in the split-luciferase complementation assay the antibody 2B10, applied extracellularly, was able to prevent the dimerization/oligomerization of tau. Surprisingly this antibody has only a relatively low affinity to tau but is still very active in inhibiting tau aggregation in vitro. Antibodies added extracellularly were taken up by the cells and sorted into lysosomes. Their inhibitory effect can be explained by the fact that the internalized antibody recruits the toxic tau protein or oligomers to the lysosomes for degradation. In summary, a subset of antibodies raised against the purified low-n oligomers of Tau^RDΔK are able to inhibit tau aggregation both in vitro and in a cell model of tau pathology....

Cyclic adenosine monophosphate (cAMP) is an important second messenger which can be generated in response to signals binding to stimulatory G-protein coupled receptors. Inside the cell, protein kinase A is a well-known ...

The cancer therapy of older patients is challenging, being more complex than the therapy of younger patients. Older cancer patients show a higher toxicity risk during therapy and drug-related problems are common. In order ...

Glia is now recognized as a key element in brain metabolism. In the thalamus, astrocytes and oligodendrocytes are coupled via gap junction channels and form extensive panglial networks. Interestingly and in contrast to ...

Die Entwicklung gastroretentiver Arzneiformen ist komplex, da neben der kontrollierten Freisetzung des Wirkstoffs zusätzlich die Verlängerung der Verweilzeit im Magen gewährleistet werden muss. Hierfür werden meist hydrophile ...

Phospholipases D (PLDs) are involved in different plant stress responses, exclusively to abiotic stresses like cold, salinity, and drought. The PLD enzymes catalyse the hydrolysis of membrane phospholipids into phosphatidic acid (PA) and a free head group. PA is a membranous second-messenger molecule that is involved in various stress-dependent signal transduction pathways. Earlier, an unknown protein PLDrp1 recognised as a putative target protein downstream of the PLD α1 signalling pathway. The substantial dependence of this protein from PLD α1 could be diminished under water stress conditions, raising the question about the mechanisms involved in this observation. Interestingly, the homolog of PLDrp1 with a 60% sequence similarity At3g29075 protein was identified in A. thaliana. To characterise unknown protein, At3g29075 was the main motive of this study.<br /> This study exhibits that the At3g29075 protein is a class V glycine-rich protein, with more than 21.1% lysine residues. The C-terminal end of the unknown protein At3g29075 has no similar protein or motif in the whole protein database. The protein localisation has confirmed to the cytosol and nucleus. Dehydration stress induces the expression of At3g29075. Wild Type and mutant plants were grown and phenotypically monitored upon standard growth conditions to define the protein’s function. The overexpression lines of AT3G29075 showed early flowering compared to the wild type, and at3g29075 knockdown mutant....

Pollination syndromes represent groups of floral phenotypes that have originated and diversified in interaction with biotic and abiotic pollen vectors. Plant trait pattern that constitute respective syndromes have been ...

Nearly 50 million people worldwide suffer from epilepsy, with 1/3 of the patients remaining without seizure control. This high number emphasizes the importance to develop new anti-epileptic drugs (AEDs) and to understand ...

Die vorliegende Arbeit befasste sich mit dem zentralen Kohlenstoff- und Energiemetabolismus von Prevotella (P.) copri, eines der relevantesten Bakterien der menschlichen Darmflora. Um zu verstehen, wie die Prädominanz dieses Organismus innerhalb der Mikrobiota zustande kommt, wurden seine grundlegenden Wachstumseigenschaften erfasst, der Zentralstoffwechsel detailliert rekonstruiert und die gewonnen Daten mit zwei weiteren zentralen Bakterien der menschlichen Intestinalflora (Bacteroides vulgatus und Parabacteroides johnsonii) verglichen. Zusätzlich wurde überprüft, ob sich P. copri aufgrund seiner metabolischen Eigenschaften zur biotechnologischen Produktion der zukunftsträchtigen Plattformchemikalie Succinat einsetzen lässt. Im Zuge von Wachstumsexperimenten mit Komplex- und Minimalmedien konnte gezeigt werden, dass P. copri mit kurzen Verdopplungszeiten hohe optische Dichten erreicht und Vitamin K, Hämin und Vitamin B12 benötigt. Außerdem lebt der Organismus strikt CO₂-abhängig, was bemerkenswerte Implikationen für die Darmflorazusammensetzung des Menschen mit sich bringt. Des Weiteren konnte anhand von bioinformatischen, transkriptionellen und enzymatischen Untersuchungen ein zentrales Stoffwechselmodell von P. copri für die Verstoffwechselung von Glucose erstellt werden. Das Bakterium baut Glucose über den EMP-Weg ab, wobei PEP und Pyruvat die zentralen Stoffwechselintermediate darstellen. PEP wird zu Fumarat verstoffwechselt, das in der anaeroben Atmungskette von P. copri als terminaler Elektronenakzeptor fungiert, während Pyruvat entweder über eine Pyruvat-Formiat-Lyase oder eine Pyruvat:Ferredoxin-Oxidoreduktase zu Acetyl-CoA umgesetzt wird. Acetyl-CoA wird letztlich zu Acetat verstoffwechselt. Die funktionelle Einheit der Atmungskette von P. copri besteht aus den Enzymen Nqr, einer „kopflosen“ NDHI-Variante und der Fumaratreduktase. Elektronen für die Reduktion von Fumarat werden über NADH und Fd_red bereitgestellt und es wird ein elektrochemischer Ionengradient mit den Kopplungsionen H⁺ und Na⁺ erzeugt. Durch Bestimmung des Trockengewichts, des Substratverbrauchs und der Produktbildung von Kulturen in unterschiedlichen Wachstumsphasen konnte außerdem der Fluss der Intermediate im zentralen Energie- und Kohlenstoffmetabolismus von P. copri rekonstruiert werden. Hier zeigte sich unter anderem, dass die CO₂-Abhängigkeit von P. copri durch das Ungleichgewicht im CO₂-Bedarf der PEP-Carboxykinase und der CO₂-Freisetzung der Pyruvat:Ferredoxin-Oxidoreduktase zustande kam, was direkt mit der gebildeten Menge an Formiat korrelierte. Zusätzlich wurden Versuche unternommen, um die Plattformchemikalie Succinat mit Hilfe von P. copri zu produzieren. Unter Ca(HCO₃)₂- und Ca(OH)₂-gepufferten Bedingungen konnten 0,5-0,7 g Succinat pro g Glucose umgesetzt werden mit einem maximalen Produkttiter von 25 g l^-1 (≈215 mM). Damit stellte sich P. copri als ein vielversprechender Kandidat für die biotechnologische Succinat-Produktion heraus....

Voltage-gated sodium channels (VGSC) are important key regulators in excitable tissue that initiate and propagate the action potential in specifically excitable tissue such as brain nerves or muscle. In order to understand ...