Monomeric hexofuranoses having an acetylimino group in the hemiacetal ring

Acetolysis of methyl 4-acetamido-4-deoxyhexopyranosides of gluco and galacto configuration yields not only the corresponding 4-acetamido-4-deoxy-pyranoses, but also ring contraction products, i.e., 4-acetamido-1,2,3,5,6-penta-O-acetyl-4-deoxy-D-gluco- and -D-galactofuranose, respectively, with the ease of acetolysis and product distribution depending on the configuration at C-4. Mechanistic implications with respect to the formation of acylic and cyclic intermediates are discussed.     

The novel sorting nexin SNX33 interferes with cellular PrP formation by modulation of PrP shedding

The cellular prion protein (PrP^c) is a glycosyl-phosphatidylinositol (GPI)-anchored protein trafficking in the secretory and endocytic pathway and localized mainly at the plasma membrane. Conversion of PrP^c into its pathogenic isoform PrP^Sc is associated with pathogenesis and transmission of prion diseases. Intramolecular cleavage in the middle, the extreme C-terminal part or within the GPI anchor and shedding of PrP^c modulate this conversion process by reducing the substrate for prion formation. These phenomena provide similarities with the processing of amyloid precursor protein in Alzheimer's disease. Sorting nexins are a family of proteins with important functions in protein trafficking. In this study, we investigated the role of the newly described sorting nexin 33 (SNX33) in trafficking and processing of PrP^c. We found that overexpression of SNX33 in neuronal and non-neuronal cell lines resulted in increased shedding of full-length PrP^c from the plasma membrane and modulated the rate of PrP^c endocytosis. This was paralleled by reduction of PrP^Sc formation in persistently and newly infected cells. Using deletion mutants, we demonstrate that production of PrP fragment N1 is not influenced by SNX33. Our data provide new insights into the cellular mechanisms of PrP^c shedding and show how this can affect cellular PrP^Sc conversion.     

Properties and inhibition of the first two enzymes of the non-mevalonate pathway of isoprenoid biosynthesis

Enzymes of the 1-deoxy-D-xylulose 5-phosphate/2-C-methylerythritol 4-phosphate (DOXP/MEP) pathway are targets for new herbicides and antibacterial drugs. Until now, no inhibitors for the DOXP synthase have been known of. We show that one of the breakdown products of the herbicide clomazone affects the DOXP synthase. One inhibitor of the non-mevalonate pathway, fosmidomycin, blocks the DOXP reductoisomerase (DXR) of plants and bacteria. The I(50) values of plants are, however, higher than those found for the DXR of Escherichia coli. The DXR of plants, isolated from barley seedlings, shows a pH optimum of 8.1, which is typical for enzymes active in the chloroplast stroma.     

Dimerization of beta-site beta-amyloid precursor protein-cleaving enzyme

Cleavage of the beta-amyloid precursor protein (APP) by the aspartyl protease beta-site APP-cleaving enzyme (BACE) is the first step in the generation of the amyloid beta-peptide, which is deposited in the brain of Alzheimer's disease patients. Whereas the subsequent cleavage by gamma-secretase was shown to originate from the cooperation of a multicomponent complex, it is currently unknown whether in a cellular environment BACE is enzymatically active as a monomer or in concert with other proteins. Using blue native gel electrophoresis we found that endogenous and overexpressed BACE has a molecular mass of 140 kDa instead of the expected mass of 70 kDa under denaturing conditions. This suggests that under native conditions BACE exists as a homodimer. Homodimerization was confirmed by co-immunoprecipitation of full-length BACE carrying different epitope tags. In contrast, the soluble active BACE ectodomain was exclusively present as a monomer both under native and denaturing conditions. A domain analysis revealed that the BACE ectodomain dimerized as long as it was attached to the membrane, whereas the cytoplasmic domain and the transmembrane domain were dispensable for dimerization. By adding a KKXX-endoplasmic reticulum retention signal to BACE, we demonstrate that dimerization of BACE occurs already before full maturation and pro-peptide cleavage. Furthermore, kinetic analysis of the purified native BACE dimer revealed a higher affinity and turnover rate in comparison to the monomeric soluble BACE. Dimerization of BACE might, thus, facilitate binding and cleavage of physiological substrates.     

Biosynthesis of isoprenoids in higher plant chloroplasts proceeds via a mevalonate-independent pathway

Isopentenyl diphosphate (IPP) is the biological C₅ precursor of isoprenoids. By labeling experiments using [1-¹³C]glucose, higher plants were shown to possess two distinct biosynthetic routes for IPP biosynthesis: while the cytoplasmic sterols were formed via the acetate/mevalonate pathway, the chloroplast-bound isoprenoids (β-carotene, lutein, prenyl chains of chlorophylls and plastoquinone-9) were synthesized via a novel IPP biosynthesis pathway (glyceraldehyde phosphate/pyruvate pathway) which was first found in eubacteria and a green alga. The dichotomy in isoprenoid biosynthesis in higher plants allows a reasonable interpretation of previous odd and inconclusive results concerning the biosynthesis of chloroplast isoprenoids, which so far had mainly been interpreted in the frame of models using compartmentation of the mevalonate pathway.     

Uptake of the Herbicide Diuron as Visualised by the Fluorescence Imaging Technique

A new fluorescence imaging system for monitoring the uptake of the PSII-herbicide diuron (OCMU) was tested in tobacco leaves. UV-laser-induced (Λ_exc = 355 nm) fluorescence images were collected for blue fluorescence F440 (Λ_em = 440 nm), green fluorescence F520 (Λ_em = 520 nm), red chlorophyll fluorescence F690 (Λ_em = 690 nm) and for far-red chlorophyll fluorescence F740 (Λ_em = 740 nm). Diuron-treated leaf parts exhibited a higher red and far-red chlorophyll fluorescence emission (F690 and F740) than untreated leaf halves, whereas the blue and green fluorescence, F440 and F520, remained unaffected. As a consequence, the fluorescence ratios blue/red (F440/F690) and blue/far-red (F440/F740) significantly decreased in diuron-treated leaf parts. The time course of diuron uptake into the leaf could be followed by fluorescence images taken 10 and 30 min after diuron application. The novel high resolution fluorescence imaging method supplies information on the herbicide uptake of each point of the leaf area. Its great advantage as compared to the point data fluorescence measurements applied so far is discussed.     

Studies on the Localization and Spectral Characteristics of the Fluorescence Emission of Differently Pigmented Wheat Leaves

Intensity, spectral characteristics and localization of the UV-laser (337 nm) induced blue-green and red fluorescence emission of green, etiolated and white primary leaves of wheat seedlings were studied in a combined fluorospectral and fluoromicroscopic investigation. The blue-green fluorescence of the green leaf was characterized by a maximum near 450 nm (blue region) and a shoulder near 530 nm (green region), whereas the red chlorophyll fluorescence exhibited maxima in the near-red (F690) and far-red (F735). The etiolated leaf with some carotenoids and traces of chlorophyll a, in turn, showed a higher intensity of the blue-green fluorescence with a shoulder in the green region and a strong red fluorescence peak near 684 to 690 nm, the far-red chlorophyll fluorescence maximum (F735) was, however, absent. The norfluorazone-treated white leaf, free of chlorophylls and carotenoids, only exhibited blue-green fluorescence of a very high intensity. In green and etiolated leaves the blue-green fluorescence primarily derived from the cell walls of the epidermis and the red fluorescence from the chlorophyll a of the mesophyll cells. In white leaves the blue-green fluorescence emanated from all cell walls of epidermis, mesophyll and leaf vein bundles. The shape and intensity of the blue-green and red fluorescence emission is determined by the reabsorption properties of chlorophylls and carotenoids in the mesophyll, thus giving rise to quite different values of the various fluorescence ratios F450/F690, F450/F530, F450/F735 and F690/F735 in green and etiolated leaves.     

Die Plastoglobuli von Spinat,ihre Grö\e,Isolierung und Lipochinonzusammensetzung

Zusammenfassung Die Grö\e und HÄufigkeit der osmiophilen Plastoglobuli nimmt mit steigendem Alter der Spinatchloroplasten zu. Die Plastoglobuli besitzen in ganzen oder aufgebrochenen Chloroplasten, in Sedimenten und in isolierter Form die gleiche Grö\enverteilung wiein situ. Es gibt keine Hinweise, da\ beim Ultraschallaufschlu\ ganzer Chloroplasten Plastoglobuli durch Entmischung der Thylakoide entstehen. Beim Zentrifugieren von broken chloroplasts wird nur ein kleiner Teil der Plastoglobuli freigesetzt. Der grö\ere Teil befindet sich mit den Chloroplastenbruchstücken im Sediment.Wie bei anderen Pflanzen enthalten auch die Plastoglobuli von Spinat vorwiegend die lipophilen Plastidenchinone vom Benzochinontyp (Plastochinon 45, Plastohydrochinon,-Tocopherol und-Tocochinon), geringe Mengen an Vitamin K₁ und Spuren an Carotinoiden, jedoch keine Chlorophylle. Zwischen dem Lipochinongehalt der Chloroplasten und der Grö\e und HÄufigkeit der osmiophilen Plastoglobuli besteht eine direkte Korrelation.

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The plastoglobuli of spinach: Their size, isolation, and lipoquinone composition

Summary The size and frequency of the osmiophilic plastoglobuli of spinach chloroplasts increase with increasing age of chloroplasts. The plastoglobuli of whole or broken chloroplasts, of sediments and in the isolated form show the same size distribution asin situ. There is no evidence that plastoglobuli may arise from thylakoid lipids by ultrasonic treatment of whole chloroplasts. During the centrifugation of broken chloroplasts only a small portion of the plastoglobuli is releazed, the greater portion remains in the sediment together with the chloroplast fragments.The plastoglobuli of spinach—like those of other plants—mainly contain the excess plastidquinones of the benzoquinone type (plastoquinone 45, plastoquinol,-tocopherol,-tocoquinone), little vitamin K₁ (a naphthoquinone), traces of carotenoids, but no chlorophylls. The lipoquinone content of spinach chloroplasts increases parallel to the augmentation of the size and frequency of the plastoglobuli.

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Der Deutschen Forschungsgemeinschaft danke ich für ihre Unterstützung und FrÄuleinMargarethe Wirtz für tüchtige technische Mitarbeit.     

The influence of continuous far-red and white light on prenyl chain synthesis in plastids of Raphanus seedlings

Summary The rate of prenyl chain accumulation (C₄₀ carotenoids; C₄₅ in plastoquinone-9; C₂₀ phytyl in chlorophylls, -tocopherol and vitamin K₁) in plastids of etiolated radish seedlings (Raphanus sativus L.) is determined in continuous darkness and after far-red and white light treatment. Continuous far-red light (active phytochrome P _fr ) stimulates the synthesis of all prenyl chains, but has no or only little effect on the dark pattern of the prenyl chain formation. White light enhances the accumulation of prenyl chains to a much higher degree than does far-red light. By a particularly strong promotion of the accumulation of phytyl chains, which are incorporated into chlorophyll, white light changes the percentage composition of prenyl chains to that of chloroplasts.     

Increase of the chlorophyll fluorescence ratio F690/F735 during the autumnal chlorophyll breakdown

Summary The chlorophyll content and the fluorescence induction kinetics at two wavelengths (690 nm and 735 nm) have been measured in leaves of nine common broadleaf tree species during the autumnal chlorophyll breakdown. The ratio of the chlorophyll fluorescence maxima F690/F735 was determined at fluorescence maximum (fm) and at steady-state conditions (fs) by the laser-induced fluorescence emission using the two-wavelength fluorometer. The ratio F690/F735 increases with the leaf discolouring during the autumnal chlorophyll breakdown. The relationship between the chlorophyll content and the ratio F690/F735 can be expressed by a power function (curvilinear relationship) which is valid for all the species examined. In most cases the ratio F690/F735 measured in the upper leaf side is lower than that in the lower leaf side, but the trend is the same along the decreasing chlorophyll content. The ratio F690/F735 is always higher at maximum fluorescence than at steady-state fluorescence in the upper as well as lower leaf side and these values are well fitted in a linear correlation. This study confirms the usefulness of the ratio F690/F735 as a suitable non-destructive indicator of the in-vivo chlorophyll content, especially at medium and low chlorophyll content.