Modulation of [My]-opioid receptor signal transduction and endocytosis by ADP-ribosylation factor proteins

Magdeburg, Univ., Fak. für Naturwiss., Diss., 2010

apoptosis-regulator c-FLIP : functional role in urothelial carcinoma and autoimmunity and identification of novel CD95 DISC-interacting proteins

Magdeburg, Univ., Fak. für Naturwiss., Diss., 2013

Interactions of the adaptor proteins AP2 and 14-3-3 with the presynaptic scaffolding protein Bassoon

Magdeburg, Univ., Fak. für Naturwiss., Diss., 2015

Untersuchung des humanen Proteins NF110 als Bindungspartner viraler RNAs

In der vorliegenden Masterarbeit wurde das Protein nuclear factor 110 (NF110) in vitro rückgefaltet, chromatographisch gereinigt, durch den Zusatz von L-Arginin stabilisiert und seine Affinität gegenüber RNAs untersucht. Das Ausgangsmaterial wurde mittels Dialyse zurückgefaltet und über drei aufeinander folgende Chromatographieschritte gereinigt: mittels Kationenaustauschchromatographie (capture) zur Entfernung von Fremdproteinen, Größenausschlusschromatographie (intermediate) zur Trennung des monomeren Proteins von höher-oligomeren Spezies und der Affinitätschromatographie (polishing). Diese Proteinlösung wurde unter Zusatz von 500 mM L-Arginin gelagert um eine Proteinaggregation zu unterbinden. Durch Streulichtmessungen und eine analytische Ultrazentrifugation konnte erfolgreich nachgewiesen werden, dass NF110 erst in Puffern mit mindestens 200 mM L-Arginin stabil vorliegt. Im Rahmen der vorliegenden Arbeit konnte erfolgreich bestimmt werden, dass NF110 zwischen doppelsträngiger RNA und DNA diskriminiert, während es gegenüber einzel- bzw. doppelsträngiger RNA ähnlich affin ist. Dies stellt einen großen Unterschied zu NF90 dar. Diese C-terminal verkürzte Variante interagiert mit einzelsträngiger RNA in sehr geringerem Umfang als mit doppelsträngiger RNA (Schmidt, 2015). Daher kann vermutet werden, dass der C-Terminus, insbesondere das dort befindliche GQSY-Motiv, für die Spezifität gegenüber den RNAs verantwortlich ist. Ein weiterer Fokus der Arbeit lag auf dem Einfluss von L-Arginin bei der RNA-Bindung. Mit Hilfe einer linearen freien Enthalpiebeziehung (LFER) konnte so eine Dissoziationskonstante für nicht aggregierendes NF110 ohne L-Arginin ermittelt werden. Außerdem wurde ersichtlich, dass dieser Stabilisator zwar die Aggregation hemmt, aber nur marginal die RNA-Bindung des Proteins beeinflusst

Patterns of snake evolution suggested by their proteins : a contribution in celebration of the distinguished scholarship of Robert F. Inger on the occasion of his sixty-fifth birthday / Herbert C. Dessauer, John E. Cadle, Robin Lawson.

n.s. no.34(1987)

Immunocytochemical localisation of microtubule-associated proteins 1b and 2 in the developing rat spinal cord.

The straightforward anatomical organisation of the developing and mature rat spinal cord was used to determine and interpret the time of appearance and expression patterns of microtubule-associated proteins (MAP) 1b and 2. Immunoblots revealed the presence of MAP1b and 2 in the early embryonic rat spinal cord and confirmed the specificity of the used anti-MAP mouse monoclonal antibodies. The immunocytochemical data demonstrated a rostral-to-caudal and ventral-to-dorsal gradient in the expression of MAP1b/2 within the developing spinal cord. In the matrix layer, MAP1b was found in a distinct radial pattern distributed between the membrana limitans interna and externa between embryonal day (E)12 and E15. Immunostaining for vimentin revealed that this MAP1b pattern was morphologically and topographically different from the radial glial pattern which was present in the matrix layer between E13 and E19. The ventral-to-dorsal developmental gradient of the MAP1b staining in the spinal cord matrix layer indicates a close involvement of MAP1b either in the organisation of the microtubules in the cytoplasmatic extensions of the proliferating neuroblasts or neuroblast mitosis. MAP2 could not be detected in the developing matrix layer. In the mantle and marginal layer, MAP1b was abundantly present between E12 and postnatal day (P)0. After birth, the staining intensity for MAP1b gradually decreased in both layers towards a faint appearance at maturity. The distribution patterns suggest an involvement of MAP1b in the maturation of the motor neurons, the contralaterally and ipsilaterally projecting axons and the ascending and descending long axons of the rat spinal cord. MAP2 was present in the spinal cord grey matter between E12 and maturity, which reflects a role for MAP2 in the development as well as in the maintenance of microtubules. The present description of the expression patterns of MAP1b and 2 in the developing spinal cord suggests important roles of the two proteins in various morphogenetic events. The findings may serve as the basis for future studies on the function of MAP1b and 2 in the development of the central nervous system.

Bioluminescent sensor proteins for point-of-care therapeutic drug monitoring.

For many drugs, finding the balance between efficacy and toxicity requires monitoring their concentrations in the patient's blood. Quantifying drug levels at the bedside or at home would have advantages in terms of therapeutic outcome and convenience, but current techniques require the setting of a diagnostic laboratory. We have developed semisynthetic bioluminescent sensors that permit precise measurements of drug concentrations in patient samples by spotting minimal volumes on paper and recording the signal using a simple point-and-shoot camera. Our sensors have a modular design consisting of a protein-based and a synthetic part and can be engineered to selectively recognize a wide range of drugs, including immunosuppressants, antiepileptics, anticancer agents and antiarrhythmics. This low-cost point-of-care method could make therapies safer, increase the convenience of doctors and patients and make therapeutic drug monitoring available in regions with poor infrastructure.

Genome of an arbuscular mycorrhizal fungus provides insight into the oldest plant symbiosis.

The mutualistic symbiosis involving Glomeromycota, a distinctive phylum of early diverging Fungi, is widely hypothesized to have promoted the evolution of land plants during the middle Paleozoic. These arbuscular mycorrhizal fungi (AMF) perform vital functions in the phosphorus cycle that are fundamental to sustainable crop plant productivity. The unusual biological features of AMF have long fascinated evolutionary biologists. The coenocytic hyphae host a community of hundreds of nuclei and reproduce clonally through large multinucleated spores. It has been suggested that the AMF maintain a stable assemblage of several different genomes during the life cycle, but this genomic organization has been questioned. Here we introduce the 153-Mb haploid genome of Rhizophagus irregularis and its repertoire of 28,232 genes. The observed low level of genome polymorphism (0.43 SNP per kb) is not consistent with the occurrence of multiple, highly diverged genomes. The expansion of mating-related genes suggests the existence of cryptic sex-related processes. A comparison of gene categories confirms that R. irregularis is close to the Mucoromycotina. The AMF obligate biotrophy is not explained by genome erosion or any related loss of metabolic complexity in central metabolism, but is marked by a lack of genes encoding plant cell wall-degrading enzymes and of genes involved in toxin and thiamine synthesis. A battery of mycorrhiza-induced secreted proteins is expressed in symbiotic tissues. The present comprehensive repertoire of R. irregularis genes provides a basis for future research on symbiosis-related mechanisms in Glomeromycota.

Expression of SCG10 and stathmin proteins in the rat olfactory system during development and axonal regeneration.

The membrane-associated protein SCG10 is expressed specifically by neuronal cells. Recent experiments have suggested that it promotes neurite outgrowth by increasing microtubule dynamics in growth cones. SCG10 is related to the ubiquitous but neuron-enriched cytosolic protein stathmin. To better understand the role played by SCG10 and stathmin in vivo, we have analyzed the expression and localization of these proteins in both the olfactory epithelium and the olfactory bulb in developing and adult rats, as well as in adult bulbectomized rats. The olfactory epithelium is exceptional in that olfactory receptor neurons constantly regenerate and reinnervate the olfactory bulb throughout animal life-span. SCG10 and stathmin expression in the olfactory receptor neurons was found to be regulated during embryonic and postnatal development and to correlate with neuronal maturation. Whereas SCG10 expression was restricted to immature olfactory receptor neurons (GAP-43-positive, olfactory marker protein-negative), stathmin was also expressed by the basal cells. In the olfactory bulb of postnatal and adult rats, a moderate to strong SCG10 immunoreactivity was present in the olfactory nerve layer, whereas no labeling was detected in the glomerular layer. Olfactory glomeruli also showed no apparent immunoreactivity for several cytoskeletal proteins such as tubulin and microtubule-associated proteins. In unilaterally bulbectomized rats, SCG10 and stathmin were seen to be up-regulated in the regenerating olfactory epithelium at postsurgery stages corresponding to olfactory axon regeneration. Our data strongly suggest that, in vivo, both SCG10 and stathmin may play a role in axonal outgrowth during ontogenesis as well as during axonal regeneration.

Pupal cocoons affect sanitary brood care and limit fungal infections in ant colonies.

BACKGROUND: The brood of ants and other social insects is highly susceptible to pathogens, particularly those that penetrate the soft larval and pupal cuticle. We here test whether the presence of a pupal cocoon, which occurs in some ant species but not in others, affects the sanitary brood care and fungal infection patterns after exposure to the entomopathogenic fungus Metarhizium brunneum. We use a) a comparative approach analysing four species with either naked or cocooned pupae and b) a within-species analysis of a single ant species, in which both pupal types co-exist in the same colony. RESULTS: We found that the presence of a cocoon did not compromise fungal pathogen detection by the ants and that species with cocooned pupae increased brood grooming after pathogen exposure. All tested ant species further removed brood from their nests, which was predominantly expressed towards larvae and naked pupae treated with the live fungal pathogen. In contrast, cocooned pupae exposed to live fungus were not removed at higher rates than cocooned pupae exposed to dead fungus or a sham control. Consistent with this, exposure to the live fungus caused high numbers of infections and fungal outgrowth in larvae and naked pupae, but not in cocooned pupae. Moreover, the ants consistently removed the brood prior to fungal outgrowth, ensuring a clean brood chamber. CONCLUSION: Our study suggests that the pupal cocoon has a protective effect against fungal infection, causing an adaptive change in sanitary behaviours by the ants. It further demonstrates that brood removal-originally described for honeybees as "hygienic behaviour"-is a widespread sanitary behaviour in ants, which likely has important implications on disease dynamics in social insect colonies.

Analysis of toxoplasma gondii proteins after Triton X-114 solubilization and hidropholic chromotograhy

The distribution of the surface proteins of toxoplasma gondii radiodinated were studied using the phase separation technique and ability of binding in the phenyl-Sepharose column. Eight polypeptides with Mr 22 to 180 distributed exclusively in the detergent rich-phase, while six polypeptides with mol. wt. 15,000 to 76,000 distributed exclusively in the detergent poor-phase. Twopolypeptides with 15,000 and 70,000 distributed on both phase. All the polypeptides present in the detergent rich-phase binding in the phenyl-Sepharose column, and can be isolated in two peak according with their relative hydrophobicities.two polypeptides hydrophobic with Mr 60 and 66 recognized by human serum were isolated by the association of the two technique. Our result showed that the surface proteins of t. gondii present different degrees of hydrophobicity and that the use of hydrophobic interaction chromatography after Triton X-114 extraction may be an important isolation method of membrane proteins.