Browsing by Subject "Bindeproteine"

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Lokalisation von Pheromon-Rezeptoren und -Bindeproteinen in antennalen Sensillen von Insekten
(2007) Gohl, Thomas; Breer, Heinz
The remarkable reactivity of moth to specific pheromones is based on the extreme selectivity and sensitivity of sensory cells in the male antennae. This feature is supposed to be based on cells equipped with specific receptors. Only the sequencing of the genomes of Bombyx mori and Heliothis virescens provided the possibility to identify candidate genes of olfactory receptors in moths. Upon detailed inspection of candidate receptors it turned out that within the generally very heterogeneous group of receptor-genes a subfamily exists containing members of both species showing striking sequence homology. A conservation of the primary structure of receptors for pheromones has been postulated. For a continuing characterization in these studies different approaches were used to verify that these receptors are indeed expressed in cells of pheromone-sensitive sensilla (sensilla trichodea). By means of ?whole mount? in situ hybridization experiments the RNA of the receptor types BmOR1 and BmOR3 could be visualized in directly neighboring cells reflecting the topology of trichoid sensilla. Also some of the Heliothis receptor types (HR13, HR14, HR16) could be assigned to sensilla trichodea. In addition to the specific receptors, the pheromone binding proteins (PBPs) are expected to play an important role in the detection of hydrophobic pheromone molecules. PBPs are produced by glia-like cells surrounding the sensory neurons. In double in situ hybridization experiments it could be shown that HR13-cells are indeed surrounded by cells expressing HvirPBP1 and HvirPBP2. Analysis comparing the topology of different receptor-types showed that cells expressing HR13 can be assigned to sensilla trichodea type A, whereas HR14 and HR16 are expressed in cells of sensilla trichodea type C. This characteristic expression pattern is considered as a further indication that these candidate-receptors are indeed pheromone-receptors. The assignment of individual receptor-types to distinct sensilla-types provides the basis for investigating the functional implications of receptor-types for the registration of main or minor components of complex pheromone-blends. Further it turned out that HR13 shows coexpression with SNMP1 (sensory neuron membrane protein 1) which is considered as a ?marker?-protein for antennal sensory neurons. This is however not the case for receptor types HR14 and HR16. In search of further SNMP-types screening-experiments were carried out which led to the identification of a novel SNMP-type (SNMP2) of Heliothis virescens. Subsequent studies concerning the expression of SNMP2 showed that the topologic distribution of SNMP2-cells is comparable to SNMP1-cells, but they show a different morphology. Further experiments revealed that SNMP2 is in fact expressed in PBP-producing cells. These findings imply that the proposed putative function of SNMPs has to be reconsidered. One major goal of this study was the attempt, to identify receptor-relevant cells by visualization of mRNA via in situ hybridization but to visualize the localization of the receptor-protein via immunohistochemical approaches. Although the generation of antibodies for olfactory receptors is very difficult, it was possible to raise antibodies specific for receptor type HR13. Using these antibodies in immunohistochemical approaches allowed to also visualize HR13-receptor-protein. By means of double-staining experiments using HR13-specific antisense RNA-probes and anti-HR13 antibodies mRNA and protein were visualized in the same specific cells. Using confocal laserscanning microscopy, it was possible to document that receptor-protein was indeed located in the sensory dendrites. Further, the receptor-protein was also visualized in the axonal processes of sensory cells and the receptor-specific staining revealed that within the antennal nerve HR13-axons appear to be organized in fascicles. These HR13-immunolabeled fascicles were visible until they reach the ?sorting zone? of the antennal lobe; in contrast to mouse olfactory bulb, no receptor specific staining was visible in the antennal lobe.
Safety assessment of coagulase-negative staphylococci used in food production
(2015) Seitter, Marion; Hertel, Christian
Coagulase-negative staphylococci (CNS) are used in starter cultures for the production of fermented meat products due to their involvement in the development of desired red color, characteristic flavor as well as ensuring stability. But also other CNS species like S. condimenti, S. piscifermentans, S. equorum and S. succinus have a potential for future use in starter cultures. The safety of fermented food products is principally proven by long-term experience as traditional methods are considered safe based on their long “history of safe use”. However, for the last mentioned species long-term experience concerning sanitary harmlessness exists only with limitations. To get an insight in safety relevant properties of food associated CNS in Chapter III-V strains of the species S. carnosus, S. condimenti and S. piscifermentans (S. carnosus-group) as well as S. equorum, S. succinus and S. xylosus (S. xylosus-group) were phenotypically and partly genotypically investigated. Based on these insights in Chapter VI a DNA microarray was developed for rapid and simultaneous detection of various safety relevant properties in CNS with future use in the food production. To increase the application potential of this microarray, additionally technological relevant properties were considered in the array design. Subsequently, the designed microarray was used for the genotypic investigation of phenotypically characterized CNS concerning the presence of safety relevant properties. In Chapter III, antibiotic resistances of 330 CNS belonging to S. carnosus- and S. xylosus-group isolated from food and starter cultures were examined. Resistances to 21 antibiotics were phenotypically determined and resistance genes blaZ, lnuA and tetK were detected in strains showing phenotypic resistances to ß-lactam antibiotics, lincomycin and tetracycline. Antibiotic resistance profiles in strains of the species S. equorum, S. succinus and S. piscifermentans are described and due to the high number of investigated strains an insight regarding the occurrence of antibiotic resistances in food associated CNS is given. In Chapter IV toxin production of food associated CNS belonging to S. carnosus- and S. xylosus-group was investigated. First, 330 strains isolated from food, starter cultures and clinical isolates have been analyzed to hemolytic activity on human and sheep blood agar plates. Secondly, the ability of 35 selected strains to produce staphylococcal enterotoxins, toxic shock syndrome toxin 1 and exfoliative toxin A has been examined by immunoblot analysis. The chapter demonstrates that CNS strains present in high numbers in fermented food cannot necessarily be regarded as safe. Thus, strains used in the production of fermented food should be analyzed with respect of their toxigenic potential to avoid negative effects on human health. Chapter V is dealing with the formation of binding proteins to extracellular matrix proteins (ECM) and the production of biogenic amines (BA) by 32 CNS of S. carnosus- and S. xylosus-group. Binding capacity of CNS to the ECM fibronectin and fibrinogen was investigated by detection of fluorescent labeled cells which were added to microtiter plates coated with ECM. The formation of six important BA was examined by HPLC using growing and resting cells. By the results of this chapter the ability of food associated CNS to develop undesired properties like the formation of binding proteins to ECM and BA was demonstrated. Thus, further research is needed concerning potential risks and the importance on human health if strains with these properties are used in the production of fermented food. In Chapter VI, the design of a polynucleotide based DNA microarray as screening tool to detect genes of potential health concern and technological relevance in food associated CNS is described. The array considered 220 genes encoding for antibiotic resistances, hemolysins, toxins, amino acid decarboxylases (involved in the formation of BA), binding proteins to ECM, lipases, proteases, stress response factors, and nitrate dissimilation. Hybridization experiments were performed using genomic DNA isolated of 32 in Chapter III-V phenotypically characterized CNS allowing the detection of e.g. antibiotic resistance genes blaZ, lnuA, and tetK. Genes coding for decarboxylases as well as fibronectin and fibrinogen binding proteins were rarely correlated with the phenotype. Toxin genes could not be detected, whereas technological relevant genes like genes coding for proteases, lipases, catalase, superoxide dismutase or genes involved in dissimilatory nitrate reduction resulted in hybridization signals. The present thesis provides data concerning safety relevant properties in food associated CNS which are important for accurate safety assessment. Comparison of the results of Chapter III-V with them of Chapter VI showed that antibiotic resistances, formation of toxins and binding proteins to ECM are more present in strains of S. xylosus- than in S. carnosus-group. In context with safety assessment of food associated CNS, the designed microarray can be used as screening tool for the detection of safety relevant combined with technologically important properties (nitrate dissimilation, control of oxidative damage by catalase, flavor formation by proteases and lipases). Summarizing, the array is able to make a contribution in enhancing the selection criteria of CNS used as starter organisms in respect to food safety as well as technologically relevant properties.