Vergleichende Untersuchungen zum Tryptophanstoffwechsel in Leberzellfraktionen bei Wirbeltieren

Zusammenfassung Der Tryptophanabbau in der Leber von Ratte (Mus rattus L. domest.), Kaninchen (Oryctolagus cuniculus L. domest.), Hahn (Gallus bankiva domest.), Taube (Columba livia L.), Frosch (Rana temporaria L.) und Fisch (Leuciscus rutilus L.) wurde in qualitativer und quantitativer Hinsicht untersucht. Es wurden die Aktivitäten des TryptophanPeroxydase-Systems, der Kynureninase und der Kynurenin-Transaminase bestimmt, sowie ihre Verteilung auf die einzelnen Zellfraktionen. Die Papierchromatographie wurde zur qualitativen Analyse der entstandenen Stoffwechselprodukte herangezogen.Außer beim Hahn wird Tryptophan praktisch ausschließlich über Kynurenin zu Kynurensäure und Anthranilsäure abgebaut. Die Verteilung der Enzyme auf die Zellfraktionen entspricht der vom Säugetier bekannten, die Aktivitäten liegen in der gleichen Größenordnung.Beim Hahn wird Kynurenin auch von Mitochondrien und Kernfraktion gebildet. Die Kynureninbildung im Cytoplasma wird durch Kombination von Cytoplasma + Mikrosomen oder Cytoplasma + Mitochondrien auf das Mehrfache erhöht. Auch scheint beim Hahn neben der Kynureninbildung noch ein zweiter Mechanismus des Tryptophanabbaus vorhanden zu sein. Durch Cyclophorasesystem wird aus Tryptophan eine gelbe Substanz gebildet, die aber nicht identifiziert werden konnte. Eine Kynurensäurebildung wird beim Hahn nicht gefunden, was im Einklang steht mit der Tatsache, daß er keine Kynurensäure ausscheidet.Fütterungsversuche an Ratten ergaben, daß bei tryptophanarmer Nahrung die Aktivität des Tryptophan-Peroxydase-Systems deutlich unter die Normalwerte absinkt, während Kynureninase und Kynurenintransaminase nicht beeinflußt werden.     

Size-fractionated biomass, photosynthesis and dark CO2 fixation in a tropical oceanic environment

This study examines the spatial distribution and size structureof phytoplankton biomass and productivity in relation to thevertical structrure of the Andaman Sea (northeastern IndianOcean). This region was characterized by low concentrationsof nutrients and high levels of insolation. Nitrogen availabilityappeared to control overall productivity with nitrate-based‘new’ production accounting for 8–24% of thetotal primary production. Euphotic column chlorophyll (chl a)averaged 52.5 mg m^–2 of which a major portion was locatedas a subsurface chl a maximum (SCM) at     

Studies on the catalytic mechanism of H2-forming methylenetetrahydromethanopterin dehydrogenase: para-ortho H2 conversion rates in H2O and D2O

H₂–forming N ⁵,N ¹⁰ –methylenetetrahydromethanopterin dehydrogenase is a novel type of hydrogenase that contains neither nickel nor iron-sulfur clusters. Evidence has been presented that the reaction mechanism catalyzed by the enzyme is very similar to that of the formation of carbocations and H₂ from alkanes under superacidic conditions. We present here further results in support of this mechanism. It was found that the purified enzyme per se did not catalyze the conversion of para H₂ to ortho H₂, a reaction catalyzed by all other hydrogenases known to date. However, it catalyzed the conversion in the presence of the substrate N ⁵,N ¹⁰ –methenyltetrahydromethanopterin (CH≡H₄MPT⁺), indicating that for heterolytic cleavage of H₂ the enzyme-CH≡H₄MPT⁺ complex is required. In D₂O, the formation of HD and D₂ from H₂ rather than a para–ortho H₂ conversion was observed, indicating that after heterolytic cleavage of H₂ the dissociation of the proton from the enzyme-substrate complex is fast relative to the re-formation of free H₂.     

Oligodeoxynucleotides enhance lipopolysaccharide-stimulated synthesis of tumor necrosis factor: dependence on phosphorothioate modification and reversal by heparin.

BACKGROUND: Specific inhibition of target proteins by antisense oligodeoxynucleotides is an extensively studied experimental approach. This technique is currently being tested in clinical trials applying phosphorothioate-modified oligonucleotides as therapeutic agents. These polyanionic molecules, however, may also exert non-antisense-mediated effects. MATERIALS AND METHODS: We examined the influence of oligonucleotides on lipopolysaccharide (LPS)-stimulated tumor necrosis factor alpha (TNF alpha) synthesis in freshly isolated human peripheral blood mononuclear cells. Oligonucleotides (18 mer) with different degrees of phosphorothioate modification were studied. RESULTS: The addition of phosphorothioate oligonucleotides (5 microM) caused amplification of TNF synthesis of up to 410% compared with the control with LPS alone. Without LPS stimulation, phosphorothioate oligonucleotides did not induce TNF production. We demonstrate that the enhancement of LPS-stimulated TNF production by phosphorothioate oligonucleotides does not rely on the intracellular presence of oligonucleotides and is not mediated by LPS contamination. Partially phosphorothioate-modified oligonucleotides and unmodified oligonucleotides did not increase TNF synthesis. High concentrations of the polyanion heparin reversed the oligonucleotide-induced enhancement of TNF synthesis. CONCLUSIONS: The data suggest that amplification of TNF synthesis may be caused by binding of the polyanionic phosphorothioate oligonucleotide to cationic sites on the cell surface. Such binding sites have been proposed for polyanionic glycoaminoglycans of the extracellular matrix, which have also been described to augment LPS-stimulated TNF synthesis. The present results are relevant to all in vitro studies attempting to influence protein synthesis in monocytes by using phosphorothioate oligonucleotides. The significance of our findings for in vivo applications of phosphorothioates in situations where there is a stimulus for TNF synthesis, such as in sepsis, should be elucidated.     

Rp-phosphorothioate modifications in RNase P RNA that interfere with tRNA binding.

We have used Rp-phosphorothioate modifications and a binding interference assay to analyse the role of phosphate oxygens in tRNA recognition by Escherichia coli ribonuclease P (RNase P) RNA. Total (100%) Rp-phosphorothioate modification at A, C or G positions of RNase P RNA strongly impaired tRNA binding and pre-tRNA processing, while effects were less pronounced at U positions. Partially modified E. coli RNase P RNAs were separated into tRNA binding and non-binding fractions by gel retardation. Rp-phosphorothioate modifications that interfered with tRNA binding were found 5' of nucleotides A67, G68, U69, C70, C71, G72, A130, A132, A248, A249, G300, A317, A330, A352, C353 and C354. Manganese rescue at positions U69, C70, A130 and A132 identified, for the first time, sites of direct metal ion coordination in RNase P RNA. Most sites of interference are at strongly conserved nucleotides and nine reside within a long-range base-pairing interaction present in all known RNase P RNAs. In contrast to RNase P RNA, 100% Rp-phosphorothioate substitutions in tRNA showed only moderate effects on binding to RNase P RNAs from E. coli, Bacillus subtilis and Chromatium vinosum, suggesting that pro-Rp phosphate oxygens of mature tRNA contribute relatively little to the formation of the tRNA-RNase P RNA complex.     

Clinical ethics and nursing: "yes" to caring, but "no" to a female ethics of care

According to a contemporary school of thought there is a specific female approach to ethics which is based not on abstract "male" ethical principles or rules, but on "care". Nurses have taken a keen interest in these female approaches to ethics. Drawing on the views expounded by Carol Gilligan and Nel Noddings, nurses claim that a female "ethics of care" better captures their moral experiences than a traditional male "ethics of justice". This paper argues that "care" is best understood in a dispositional sense, that is, as sensitivity and responsiveness to the particularities of a situation and the needs of "concrete" others. While "care", in this sense, is necessary for ethics, it is not sufficient. Ethics needs "justice" as well as "care". If women and nurses excessively devalue principles and norms, they will be left without the theoretical tools to condemn some actions or practices, and to defend others. They will, like generations of nurses before them, be condemned to silence.     

Biochemical and immunocytochemical characterization of monoclonal antibody TOP 35 raised against purified tonoplast from cress root

The vacuolar membrane, the tonoplast, is a proteinrich membranehitherto only few proteins in it have been identified. As anapproach for the identification of tonoplast proteins by monoclonalantibodies (MABs), purified tonoplast from cress roots (Lepidiumsativum L.) were used for immunization and plasma membranesas a control membrane to test the absence of antigen. The MABTOP 35 identified a glycoprotein of about 35 kDa in purifiedtonoplast of cress roots. Triton X-114 phase separation showedthat it was a hydrophobic integral membrane protein. In immunocytochemistrythe MAB TOP 35 strongly labelled the vacuolar membrane. Theabsence of cell wall or plasma membrane labelling by TOP 35indicates a distinct biosynthetic pathway of this protein tothe vacuolar membrane in plants. Key words: Immnocytochemistry, Lepidium sativum, monoclonal antibody, secretion, vacuole