LaPSvS1, a (1-->3)-beta-galactan sulfate and its effect on angiogenesis in vivo and in vitro

LaPSvS1, a highly sulfated branched (1-->3)-beta-galactan was prepared from the arabino-galactan from Larix decidua Miller by partial hydrolysis and subsequent sulfation with SO(3)-pyridine in DMF. The molecular weight was analyzed by GPC and the sulfate content was determined by ion chromatography. LaPSvS1 exhibited good antiangiogenic and antiinflammatory effects in two different modifications of the known CAM-assay. In vitro results obtained in the FGF-2-trypsin-assay and in fluorospectrometric experiments revealed that LaPSvS1 interacts with the fibroblast growth factor 2 system. This interaction is correlated with the in vivo effect of LaPSvS1 on FGF-2 induced angiogenesis.     

Some new aspects of 17alpha-estradiol metabolism in man

17Alpha-estradiol (1,3,5(10)-estratriene-3,17alpha-diol) together with a tracer dose of the tritium-labeled compound was administered orally and sublingually to male volunteers. The serum concentrations of 17alpha-estradiol (free and liberated by enzymatic hydrolysis) were quantified by GC/MS, and the serum total radioactivity and urinary radioactivity excretion were determined. After oral administration, 17alpha-estradiol was rapidly and intensively conjugated; only tiny quantities of the free steroid (<1% of total) appeared in serum. Sublingual administration resulted in temporary (up to 3 h p.a.) higher serum levels of the free compound. The metabolite patterns obtained by TLC of extracts from serum and urine demonstrated that 17alpha-estradiol is the subject of a poor phase I metabolism in man. A great discrepancy was found in the serum concentrations of 17alpha-estradiol (free + conjugated) determined by GC/MS and the serum radioactivity expressed in 17alpha-estradiol equivalents. By TLC analysis of the steroid conjugates extracted from serum, various 17alpha-estradiol conjugate peaks were found. By enzymatic hydrolysis with beta-glucuronidase/aryl sulfatase from Helix pomatia they were only partially cleaved. Thus, the difference between the serum radioactivity and the 17alpha-estradiol levels determined by GC/MS had to be attributed to an incomplete conjugate hydrolysis. It has been shown with the synthesized 17alpha-estradiol sulfate conjugates that only the 3-sulfate is cleaved by enzymatic hydrolysis, whereas the 17-sulfate group resists enzymatic hydrolysis. The methanolysis procedure (acetyl chloride in MeOH) has proved to be an efficient method for cleaving both the 3-sulfate group and the 17-sulfate group. In contrast to the 17alpha-estradiol conjugates in serum, the urinary conjugates were intensively split by the enzyme preparation. From this, it has to be concluded that the serum conjugates were deconjugated and newly reconjugated before urinary excretion.     

Modification of tyrosine hydroxylase activity by chloral derived beta-carbolines in vitro

Beta-carbolines have been suggested to be involved in the pathogenesis of Parkinson's disease as a result of their structural similarity to the neurotoxin N -methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The chloral-derived beta-carboline derivative 1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo) causes cell loss in neuronal and glial cell cultures and induces a slowly developing neurodegenerative process in rats. In our experiments, effects of TaClo and its derivatives 2-methyl-TaClo (2-Me-TaClo), and 1-dichloromethylene-1,2,3,4-tetrahydro-beta-carboline (1-CCl(2) -THbetaC) on tyrosine hydroxylase (TH) activity were investigated in TH assays using homogenate preparations of the rat nucleus accumbens and recombinant human TH (hTH1). TH activity was determined in vitro by measuring l-DOPA production with HPLC-ECD. Using homogenate preparations, TaClo, 2-Me-TaClo, and 1-CCl(2) -THbetaC inhibited TH in concentrations of 0.1 mm, while 1-CCl(2) -THbetaC in low concentrations enhanced TH activity. When TH was activated by PACAP-27, TaClo, 2-Me-TaClo, or 1-CCl(2) -THbetaC also inhibited activated enzyme activity in high concentrations. However, in the case of 2-Me-TaClo and 1-CCl(2) -THbetaC a biphasic effect was observed with a marked increase of TH activity in the nanomolar range. In our experiments using recombinant hTH1, TaClo, 2-Me-TaClo, or 1-CCl(2) -THbetaC did not modify enzyme activity. After activation of hTH1 by PKA all the tetrahydro-beta-carbolines investigated in this study decreased l-DOPA formation. We suggest that these beta-carbolines modulate dopamine synthesis by interacting with a protein kinase TH-activating system.     

Der Bau der Canini bei den Paulchoffatiidae (Multituberculata; Ober-Jura)

Canines are preserved among Multituberculata only in the upper jaw of the Paulchoffatiidae, the Pinheirodontidae and the North American genusGliodon Engelmann &; Callison, 1999. They resemble the anterior premolars (p^1–3) in the morphology of their crown, but they differ from them by the presence of only one root. In the present paper, 126 isolated canines of the Paulchoffatiidae from the Guimarota coal-pit in Portugal are treated. They show a wide morphological variation, from bicuspid to pentacuspid expression, and they can be grouped into 18 morphological units (Tab. 1). The bicuspid canines (11 specimens) show one buccal and one lingual cusp, arranged side by side, the latter normally being larger than the former. In the tricuspid canines (32 specimens) the cusps (one buccal and two lingual) are arranged in a triangle, with a great variation in the position of the cusps to each other. Tetracuspid canines (69 specimens) are the dominating group. Two buccal and two lingual cusps are present, which differ markedly in their largeness and their position to each other. The teeth differ also much in the shape of their crown. The pentacuspid canines (14 specimens) show two buccal and three lingual cusps. The variation in the arrangement of the cusps and in the shape of the crown is similar as in the tetracuspid specimens. One or two small cuspules can be present at the anterior border of the crown additionally to the main cusps in the canines. Tricuspid and tetracuspid canines are present also in the skulls of the Paulchoffatiidae, whereas bicuspid and pentacuspid canines are known only as isolated specimens. In the Pinheirodontidae the canines are tricuspid or tetracuspid, inGlirodon they are unicuspid. The Paulchoffatiidae and Pinheirodontidae — Paulchoffatiid line sensuKielan-Jaworowska &; Hurum, 2001 — are characterized by increasing premolarization of the upper canines. With that they differ markedly from all other multituberculates where the canini become reduced.     

Nepenthes insignis uses a C2-portion of the carbon skeleton of L-alanine acquired via its carnivorous organs, to build up the allelochemical plumbagin

Tropical pitcher plants (Nepenthes) catch animals in their specialized cup-shaped leaves, digest the prey by secreting enzymes, and actively take up the resulting compounds. The benefit of this behaviour is the ability to grow and compete in nutrient-poor habitats. Our present in vitro study shows that not only the nitrogen of alanine fed to the carnivorous organs is used by the plant but that in addition intact C2-units derived from C-2 and C-3 of stable isotope labelled L-alanine serve as building blocks, here exemplarily for the synthesis of the secondary metabolite plumbagin, a potent allelochemical. This result adds a new facet to the benefit of carnivory for plants. The availability of plumbagin by a de novo synthesis probably enhances the plants' fitness in their defence against phytophagous and pathogenic organisms. A missing specific uptake or CoA activation mechanism might be the reason that acetate fed to the pitchers was not incorporated into the naphthoquinone plumbagin. The dihydronaphthoquinone glucosides rossoliside and plumbaside A, here isolated for the first time from Nepenthes, by contrast, showed no incorporation after feeding of any of the two precursors, suggesting these compounds to be storage forms with probably very low turnover rates.     

Cross-talk in the A1-ATPase from Methanosarcina mazei Go1 due to nucleotide binding

Changes in the A(3)B(3)CDF-complex of the Methanosarcina mazei G?1 A(1)-ATPase in response to ligand binding have been studied by small-angle x-ray scattering, protease digestion, fluorescence spectroscopy, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and CuCl(2)-induced disulfide formation. The value of the radius of gyration, R(g), increases slightly when MgATP, MgADP, or MgADP + P(i) (but not MgAMP-PNP) is present. The nucleotide-binding subunits A and B were reacted with N-4[4-[7-(dimethylamino)-4-methyl]coumarin-3-yl]maleimide, and spectral shifts and changes in fluorescence intensity were detected upon addition of MgAMP-PNP, MgATP, MgADP + P(i), or MgADP. Trypsin treatment of A(1) resulted in cleavage of the stalk subunits C and F, which was rapid in the presence of MgAMP-PNP but slow when MgATP or MgADP were added to the enzyme. When A(1) was supplemented with CuCl(2) a clear nucleotide dependence of an A-A-D cross-linking product was generated in the presence of MgADP and MgATP but not when MgAMP-PNP or MgADP + P(i) was added. The site of cross-link formation was located in the region of the N and C termini of subunit D. The data suggest that the stalk subunits C, D, and F in A(1) undergo conformational changes during ATP hydrolysis.     

A novel biosynthetic pathway providing precursors for fatty acid biosynthesis and secondary metabolite formation in myxobacteria

Short chain carboxylic acids are well known as the precursors of fatty acid and polyketide biosynthesis. Iso-fatty acids, which are important for the control of membrane fluidity, are formed from branched chain starter units (isovaleryl-CoA and isobutyryl-CoA), which in turn are derived from the degradation of leucine and valine, respectively. Branched chain carboxylic acids are also employed as starter molecules for the biosynthesis of secondary metabolites, e.g. the therapeutically important anthelmintic agent avermectin or the electron transport inhibitor myxothiazol. During our studies on myxothiazol biosynthesis in the myxobacterium, Stigmatella aurantiaca, we detected a novel biosynthetic route to isovaleric acid. After cloning and inactivation of the branched chain keto acid dehydrogenase complex, which is responsible for the degradation of branched chain amino acids, the strain is still able to produce iso-fatty acids and myxothiazol. Incorporation studies employing deuterated leucine show that it can only serve as precursor in the wild type strain but not in the esg mutant. Feeding experiments using (13)C-labeled precursors show that isovalerate is efficiently made from acetate, giving rise to a labeling pattern in myxothiazol that provides evidence for a novel branch of the mevalonate pathway involving the intermediate 3,3-dimethylacryloyl-CoA. 3,3-Dimethylacrylic acid was synthesized in deuterated form and fed to the esg mutant, resulting in strong incorporation into myxothiazol and iso-fatty acids. Similar experiments employing Myxococcus xanthus revealed that the discovered biosynthetic route described is present in other myxobacteria as well.     

Rainforest air-conditioning: the moderating influence of epiphytes on the microclimate in tropical tree crowns

Epiphytes are often assumed to influence the microclimatic conditions of the tree crowns that they inhabit. In order to quantify this notion, we measured the parameters "temperature" (of the substrate surface and the boundary layer of air above it), "evaporative drying rate" and "evapotranspiration" at various locations within tree crowns with differing epiphyte assemblages. The host tree species was Annona glabra, which was either populated by one of three epiphyte species (Dimerandra emarginata, Tillandsia fasciculata, or Vriesea sanguinolenta) or was epiphyte-free. We found that during the hottest and driest time of day, microsites in the immediate proximity of epiphytes had significantly lower temperatures than epiphyte-bare locations within the same tree crown, even though the latter were also shaded by host tree foliage or branches. Moreover, water loss through evaporative drying at microsites adjacent to epiphytes was almost 20% lower than at exposed microsites. We also found that, over the course of several weeks, the evapotranspiration in tree crowns bearing epiphytes was significantly lower than in trees without epiphytes. Although the influence of epiphytes on temperature extremes and evaporation rates is relatively subtle, their mitigating effect could be of importance for small animals like arthropods inhabiting an environment as harsh and extreme as the tropical forest canopy.