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%.     

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.     

Delivery and processing of exogenous double-stranded DNA in mouse CD34 + hematopoietic progenitor cells and their cell cycle changes upon combined treatment with cyclophosphamide and double-stranded DNA

We previously reported that fragments of exogenous double-stranded DNA can be internalized by mouse bone marrow cells without any transfection. Our present analysis shows that only 2% of bone marrow cells take up the fragments of extracellular exogenous DNA. Of these, ~ 45% of the cells correspond to CD34 + hematopoietic stem cells. Taking into account that CD34 + stem cells constituted 2.5% of the total cell population in the bone marrow samples analyzed, these data indicate that as much as 40% of CD34 + cells readily internalize fragments of extracellular exogenous DNA. This suggests that internalization of fragmented dsDNA is a general feature of poorly differentiated cells, in particular CD34 + bone marrow cells.     

In situ proteolysis for protein crystallization and structure determination

We tested the general applicability of in situ proteolysis to form protein crystals suitable for structure determination by adding a protease (chymotrypsin or trypsin) digestion step to crystallization trials of 55 bacterial and 14 human proteins that had proven recalcitrant to our best efforts at crystallization or structure determination. This is a work in progress; so far we determined structures of 9 bacterial proteins and the human aminoimidazole ribonucleotide synthetase (AIRS) domain.     

Recombinant Destabilase from Hirudo medicinalis Is Able to Dissolve Human Blood Clots In Vitro

Leeches are amazing animals that can be classified as conditionally poisonous animals since the salivary cocktail they produce is injected directly into the victim, and its components have strictly defined biological purposes, such as preventing blood clot formation. Thrombolytic drugs are mainly aimed at treating newly formed blood clots. Aged clots are stabilized by a large number of isopeptide bonds that prevent the action of thrombolytics. These bonds are destroyed by destabilase, an enzyme of the leech’s salivary glands. Here, we conducted a pilot study to evaluate the feasibility and effectiveness of the use of destabilase in relation to blood clots formed during real pathological processes. We evaluated the isopeptidase activity of destabilase during the formation of a stabilized fibrin clot. We showed that destabilase does not affect the internal and external coagulation cascades. We calculated the dose–response curve and tested the ability of destabilase to destroy isopeptide bonds in natural blood clots. The effect of aged and fresh clots dissolving ability after treatment with destabilase coincided with the morphological characteristics of clots during surgery. Thus, recombinant destabilase can be considered as a potential drug for the treatment of aged clots, which are difficult to treat with known thrombolytics.