Über die Wirkung verschiedener Faktoren,insbesondere narkotisierender Substanzen auf die vitale Methylenblaufärbung bei in vitro gezüchteten Fibrocyten

Ohne Zusammenfassung     

Das Verhalten der histochemisch nachweisbaren Phosphorylaseaktivität am anoxisch ruhenden und anoxisch tätigen (ischämischen) Rattenherzmuskel

Zusammenfassung In vergleichenden Untersuchungen wird fermenthistochemisch die Phosphorylaseaktivität am anoxisch ruhenden, anoxisch tätigen und ischämischen (Herzinfarkt) Herzmuskel dargestellt. Während bei letzterem bereits nach 5 min ein völliger Verlust der Enzymaktivität zu bemerken ist, ist die Phosphorylaseaktivität am anoxisch ruhenden Herzmuskel selbst nach 20 Minuten Anoxie an alkoholfixierten Schnitten nicht, an unfixierten nur gering vermindert. Beim anoxisch tätigen Herzmuskel tritt auch ein rascher Aktivitätsverlust ein. Diese Ergebnisse werden mit bisher bekannten biochemischen und histochemischen Befunden verglichen und beobachtete Unterschiede auf eine abweichende Versuchsanordnung zurückgeführt.

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Summary A comparative enzyme-histochemical study is carried out on the phosporylase activity of anoxic inactive, anoxic active, and ischaemic (cardiac infarction) cardiac musculature. In the case of the latter the enzymatic activity ceases completely after 5 minutes, whereas in the case of the inactive anoxic cardiac musculature no decrease of the phosphorylase activity is observed after 20 min of anoxia (alcohol fixed sections); the decrease in enzymatic activity in unfixed material is almost negligible. Anoxic active cardiac musculature shows a rapid decrease in activity. These findings are compared to known biochemical and histochemical results; the obvious differences are thought to be due to different experimental conditions.

     

Detoxification of naturally occurring chromenes in larvae of the generalist herbivore Spodoptera littoralis (Noctuidae)

Five naturally occurring chromenes from the Asteraceae including the insecticidal compounds precocene II (1) and encecalin (2) were administered to last instar larvae of Spodoptera littoralis via the food or by topical contact. Metabolites formed and excreted via the frass were analysed by GC-MS and by direct comparison with reference compounds obtained by partial synthesis. In total 28 different metabolites were identified, many of them reported here for the first time. All metabolites detected originated from phase I reactions (most probably catalysed by Cytochrom P-450-dependent monooxygenases) by hydroxylation, demethylation or reduction of the parent chromenes. The resulting metabolites can be regarded as detoxification products based on previous structure-activity studies. The increased polarity of the metabolites will furthermore facilitate their excretion by the larvae compared to the more apolar parent chromenes. The largest number of metabolites (eight for each compound) was detected following oral treatment with precocene II and encecalin respectively. 3-Monool as well as 3,4-trans-diol derivatives predominated in the frass of larvae treated with the latter compounds whereas the 6-hydroxyethyl derivatives were the major metabolites of the other chromenes investigated. The patterns of metabolites originating from precocene II or encecalin were the same following oral application or topical treatment.     

Proteomic Profiling of Mouse Helper T Cell Differentiation

Helper T cell differentiation is a key process in the regulation of adaptive immune responses. Here, mouse Th1 and Th2 cells are profiled using high‐throughput proteomics to increase the understanding of the molecular biology of Th differentiation to support the design of prophylactic and therapeutic intervention strategies for (infectious) diseases. Protein profiling of Th1/Th2 differentiated cells results in the quantification of almost 6000 proteins of which 41 are differentially expressed at FDR < 0.1, and 19 at the FDR < 0.05 level, respectively. Differential protein expression analysis identifies a number of the expected canonical Th differentiation markers, and gene set analysis using the REACTOME database and a hypergeometric test (FDR < 0.05) confirms that helper T cell pathways are the top sets that are differentially expressed. Additionally, by network analysis, many differentially expressed proteins are associated with the Th1 and Th2 pathways. Data are available via PRIDE database with identifier PXD004532.     

Three New Cutinases from the Yeast Arxula adeninivorans That Are Suitable for Biotechnological Applications

The genes ACUT1, ACUT2, and ACUT3, encoding cutinases, were selected from the genomic DNA of Arxula adeninivorans LS3. The alignment of the amino acid sequences of these cutinases with those of other cutinases or cutinase-like enzymes from different fungi showed that they all had a catalytic S-D-H triad with a conserved G-Y-S-Q-G domain. All three genes were overexpressed in A. adeninivorans using the strong constitutive TEF1 promoter. Recombinant 6× His (6h)-tagged cutinase 1 protein (p) from A. adeninivorans LS3 (Acut1-6hp), Acut2-6hp, and Acut3-6hp were produced and purified by immobilized-metal ion affinity chromatography and biochemically characterized using p-nitrophenyl butyrate as the substrate for standard activity tests. All three enzymes from A. adeninivorans were active from pH 4.5 to 6.5 and from 20 to 30°C. They were shown to be unstable under optimal reaction conditions but could be stabilized using organic solvents, such as polyethylene glycol 200 (PEG 200), isopropanol, ethanol, or acetone. PEG 200 (50%, vol/vol) was found to be the best stabilizing agent for all of the cutinases, and acetone greatly increased the half-life and enzyme activity (up to 300% for Acut3-6hp). The substrate spectra for Acut1-6hp, Acut2-6hp, and Acut3-6hp were quite similar, with the highest activity being for short-chain fatty acid esters of p-nitrophenol and glycerol. Additionally, they were found to have polycaprolactone degradation activity and cutinolytic activity against cutin from apple peel. The activity was compared with that of the 6× His-tagged cutinase from Fusarium solani f. sp. pisi (FsCut-6hp), also expressed in A. adeninivorans, as a positive control. A fed-batch cultivation of the best Acut2-6hp-producing strain, A. adeninivorans G1212/YRC102-ACUT2-6H, was performed and showed that very high activities of 1,064 U ml⁻¹ could be achieved even with a nonoptimized cultivation procedure.