Untersuchungen über die Inaktivierung und Radikalbildung an suspendiertem Trypsin nach UV-Bestrahlung

Zusammenfassung Es wird eine Methode zur UV-Bestrahlung von Enzymsuspensionen in Alkohol angegeben. Durch Eintauchen der UV-Lampe in die homogene Suspension läßt sich auf einfache Weise die vom Enzym absorbierte Energie bestimmen. Die Inaktivierung wird in Abhängigkeit von der Bestrahlungsdauer gemessen und mittels der ESR-Spektroskopie die Radikalbildung untersucht. Die ESR-Spektren zeigen, daß eine sauerstoff- und wasserfreie Äthanolsuspension vakuumähnliche Bestrahlungsbedingungen liefert. Man erhält ein Radikal am-Kohlenstoff und ein Schwefelradikal vom Typ RS ·. Es wurden die Quantenausbeuten für die Inaktivierung _i und für die Radikalbildung _r ermittelt. Für Trypsin finden wir nach Bestrahlung mit UV-Licht der Wellenlänge 254 nm _i=2,4·10_–2 und _r =1,7·10_–3. Daraus ergibt sich für die Anzahl der Radikale pro inaktiviertem Molekül ein Wert von 0,07, der nahelegt, daß zwischen der gemessenen Inaktivierung und den noch vorhandenen Radikalen kein direkter Zusammenhang besteht. Untersuchungen mit einem kontinuierlichen UV-Spektrum ergaben dieselbe Radikalzahl pro inaktiviertem Molekül, wobei jedoch die Schwefelradikalausbeute geringer ist als nach Bestrahlung mit der Linie 254 nm. Bestrahlung mit Wellenlängen > 300 nm bewirkte eine teilweise Löschung der durch die Linie 254 nm erzeugten Radikale.

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Studies on inactivation and radical formation after UV-irradiation of trypsin in suspension

Summary A method for UV irradiation of suspensions of enzymes in alcohol has been described. The UV lamp was dipped into the suspension which was stirred during irradiation in order to provide a homogeneous exposure of the enzymes and to facilitate the determination of the energy absorbed in the enzymes. The inactivation has been studied as a function of exposure time, radical formations being analysed by way of EPR-spectroscopy. The EPR spectra indicated that oxygen- and waterfree suspensions in ethanol provide vacuumlike conditions for UV irradiation. A radical at the C position and a sulfur radical of the RS · type were observed. Quantum yields for the inactivation ( _i) and for radical formation ( _r) were obtained. UV irradiation of trypsin at 254 nm yielded _i=2,4·10^–2 and _r=1,7·10^–3. As a result the value of 0.07 free radicals per inactivated molecule leads to the suggestion that there is no direct relation between the inactivation and the observed radical formation. The same number of free radicals per inactivated molecule was obtained after irradiation with a continuous UV spectrum, the yield of sulfur radicals however was lower than in the 254 nm investigation. Irradiation with > 300 nm partially quenches the radicals produced at 254 nm.

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Abschließend möchte ich des im Februar 1969 verstorbenen Prof. Dr. Kurt Sommermeyer gedenken, dem ich für die Anregung zu diesem Thema und für wertvolle Diskussionen zu danken habe. Dem Deutschen Akademischen Austauschdienst danke ich für das Stipendium, das mir die Durchführung der Arbeit im Radiologischen Institut ermöglicht hat.     

Reproductive anatomy, manipulation of ovarian activity and non-surgical embryo recovery in suni (Neotragus moschatus zuluensis)

Marked disparity in the uterine horn dimensions and relative degrees of caruncle development in suni suggested that exclusive or predominant dextral implantation occurs in association with bilateral ovulatory activity. Daily urinary measurements of pregnanediol-3 alpha-glucuronide revealed an oestrous cycle of approximately 21 days in length. Ovarian activity was controlled for synchronization of oestrus by using progestagen-impregnated intravaginal sponges and multiple ovulations were induced by using exogenous gonadotrophin therapy. An effective transcervical uterine catheterization technique was developed for the non-surgical collection of embryos. The efficiency of embryo recovery performed 5 days after sponge removal was 50.0%.     

Transport and dynamics of molecules dissolved in maize root cortex membranes

Translational diffusion of a fluorescent sterol probe was measured in the plasma membranes of protoplasts isolated from cortical cells of the primary root of maize seedlings. The apparent lateral diffusion coefficient was typically observed to be nearly insensitive to temperature, while the mobile fraction increased with increasing temperature. These fluorescence photobleaching recovery (FPR) measurements were compared with the electron paramagnetic resonance (EPR) spectra of the methyl ester of 13-doxyl palmitic acid in membranes of corn root tissue in situ. The complex spectra observed with this probe were analyzed as weighted sums of simpler spectra of various order parameters and rotational correlation times. The reconstituted spectra calculated from the model show that EPR also detects a mobile (less ordered, fluid) fraction, distinguished by the order parameter S=0.1 to 0.2, which becomes more abundant as temperature increases and is qualitatively comparable to the mobile fraction determined by the FPR method. The observed results on the mobile fractions and the diffusion rates for translational (FPR) as well as rotational (EPR) motions are interpreted in terms of membrane organization, thus providing information on the population and structural patterns of the coexisting domains with a special emphasis on the response of the membrane to temperature changes.This work was supported in part by grants from the Ministry of Science and Technology of the Republic of Slovenia and the International Research Program of the U.S. Department of Agriculture (USDA-JF 814-51) to M.S., and by grants from the Competitive Grants Program of the U.S. Department of Agriculture (88-37264-3807 and 90-37264-5471) to E.A.N.     

Eurotium (Aspergillus) repens metabolites and their biological activity

Eurotium repens mycelium cultivated under static conditions was used to isolate and identify metabolities—echinulin, physcion, erythroglaucin, flavoglaucin and asperentin; the filtrate of the culture yielded asperentin 8-methylether. The broadest biological activity spectrum was displayed by asperentin which had antibacterial and antifungal effects and, at a concentration of 86 ώg/ml, caused 50 % mor7 tality inArtemia saline larvae. The highest cytotoxicity towards HeLa cells was found in physcion which caused 50 % growth inhibition at a concentration of 0.1 ώg/ml.     

Interactions of Methicillin Resistant Staphylococcus aureus USA300 and Pseudomonas aeruginosa in Polymicrobial Wound Infection

Understanding the pathology resulting from Staphylococcus aureus and Pseudomonas aeruginosa polymicrobial wound infections is of great importance due to their ubiquitous nature, increasing prevalence, growing resistance to antimicrobial agents, and ability to delay healing. Methicillin-resistant S. aureus USA300 is the leading cause of community-associated bacterial infections resulting in increased morbidity and mortality. We utilized a well-established porcine partial thickness wound healing model to study the synergistic effects of USA300 and P. aeruginosa on wound healing. Wound re-epithelialization was significantly delayed by mixed-species biofilms through suppression of keratinocyte growth factor 1. Pseudomonas showed an inhibitory effect on USA300 growth in vitro while both species co-existed in cutaneous wounds in vivo. Polymicrobial wound infection in the presence of P. aeruginosa resulted in induced expression of USA300 virulence factors Panton-Valentine leukocidin and α-hemolysin. These results provide evidence for the interaction of bacterial species within mixed-species biofilms in vivo and for the first time, the contribution of virulence factors to the severity of polymicrobial wound infections.     

Role of Scarf and its binding target proteins in epidermal calcium homeostasis

The novel Ca2+-binding protein, Scarf (skin calmodulin-related factor) belongs to the calmodulin-like protein family and is expressed in the differentiated layers of the epidermis. To determine the roles of Scarf during stratification, we set out to identify the binding target proteins by affinity chromatography and subsequent analysis by mass spectrometry. Several binding factors, including 14-3-3s, annexins, calreticulin, ERp72 (endoplasmic reticulum protein 72), and nucleolin, were identified, and their interactions with Scarf were corroborated by co-immunoprecipitation and co-localization analyses. To further understand the functions of Scarf in epidermis in vivo, we altered the epidermal Ca2+ gradient by acute barrier disruption. The change in the expression levels of Scarf and its binding target proteins were determined by immunohistochemistry and Western blot analysis. The expression of Scarf, annexins, calreticulin, and ERp72 were up-regulated by Ca2+ gradient disruption, whereas the expression of 14-3-3s and nucleolin was reduced. Because annexins, calreticulin, and ERp72 have been implicated in Ca2+-induced cellular trafficking, including the secretion of lamellar bodies and Ca2+ homeostasis, we propose that the interaction of Scarf with these proteins might be crucial in the process of barrier restoration. On the other hand, down-regulation of 14-3-3s and nucleolin is potentially involved in the process of keratinocyte differentiation and growth inhibition. The calcium-dependent localization and up-regulation of Scarf and its binding target proteins were studied in mouse keratinocytes treated with ionomycin and during the wound-healing process. We found increased expression and nuclear presence of Scarf in the epidermis of the wound edge 4 and 7 days post-wounding, entailing the role of Scarf in barrier restoration. Our results suggest that Scarf plays a critical role as a Ca2+ sensor, potentially regulating the function of its binding target proteins in a Ca2+-dependent manner in the process of restoration of epidermal Ca2+ gradient as well as during epidermal barrier formation.     

Mechanisms of EDDHA effects on the promotion of floral induction in the long-day plant Lemna minor (L.)

EDDHA added in an optimal concentration (20.5 mumol.L-1) to a modified Pirson-Seidel nutrient solution induces flowering in some clones of the species Lemna minor, Lemna gibba and Spirodela polyrrhiza, which in the absence of EDDHA in the same nutrient solution do not flower. By adding EDDHA (20.5 mumol.L-1), floral induction under LD conditions is optimally promoted in the long-day (LD) species Lemna minor. After adding EDDHA to the nutrient solution, before floral induction and during flowering, Zn, Mn and Cu content is significantly increased in plants. Zn-EDDHA (0.86 mumol.L-1), Mn-EDDHA (1.51 mumol.L-1) and Cu-EDDHA (0.12 mumol.L-1), when used individually, greatly promote flowering under LD conditions as compared to flowering in the same nutrient solution with an equivalent quantity of Zn, Mn or Cu in the nonchelate form. If, on the other hand, Zn-EDDHA and Mn-EDDHA are added to the nutrient solution together (instead of Zn and Mn in nonchelate form), their effect on the promotion of flowering is less than in the case of their individual use. This shows that there is antagonism between Zn-EDDHA and Mn-EDDHA that is eliminated by adding EDDHA to the nutrient solution. We obtained the highest percentage of flowering plants (i.e. 74%) if we added EDDHA (20.5 mumol.L-1) to the nutrient solution containing Mn, Zn and Cu in chelate form. 74% of flowering plants actually means that flowering was achieved in all physiologically mature plants. Our results show that EDDHA promotes floral induction in Lemna minor under LD conditions, especially through chelating Zn, Mn and Cu, and, in addition, through eliminating the antagonism between Mn and Zn chelates EDDHA. Zn-EDDHA (0.86 mumol.L-1) also promote floral differentiation, especially cell division of microspore mother cells into dyads and those into microspore tetrads, which can be seen in microphotographs. When investigating possible pathways through which Mn-EDDHA, Zn-EDDHA and Cu-EDDHA promote flowering, we studied the effects of various concentrations of IAA and sucrose added to the nutrient solution as well. The results support the hypothesis that one of the possible pathways in which Mn-EDDHA promotes floral induction is through auxin oxidase, whereas Zn-EDDHA and Cu-EDDHA probably promote it through the enhancement of the photosynthesis and synthesis of sucrose.     

Elimination of bean seed-borne bacteria by thermotherapy and meristem culture

Plant Cell, Tissue and Organ Culture (PCTOC) -