Antinuclear human autoantibodies as markers in Nicotiana tabacum pollen tubes

In the present paper we report on the use of antinuclear human autoantibodies as specific markers in <em>Nicotiana tabacum</em> pollen tubes. The antibodies have been tested by fluorescence techniques using a confocal laser scanning microscope. All the antibodies showed specifc labelling pattern and the results, although preliminary in nature, could open new perspectives of research.

Use of photoactivatable sphingolipid analogues to monitor lipid transport in mammalian cells

Photoactivatable derivatives of ceramide, glucosylceramide (GlcCer) and sphingomyelin {3-(p-azido-m-[125I]iodophenyl)propionylceramide, 3-(p-azido-m-[125I]iodophenyl)propionyl-GlcCer and 3-(p-azido-m-[125I]iodophenyl)propionylsphingomyelin} were synthesized in an attempt to identify compartment-specific proteins involved in sphingolipid sorting or metabolism. In HT29 and BHK cells the ceramide analogue entered the cell by monomeric diffusion, as evidenced by the probe's efficient internalization at low temperature (4 °C). In contrast, the photoactivatable GlcCer was internalized only at elevated temperatures (37 °C), presumably reflecting an endocytic mechanism of uptake. The photoactivatable ceramide was mainly metabolized to the corresponding sphingomyelin analogue, but small amounts of GlcCer and galactosylceramide were also synthesized. The newly synthesized photoreactive sphingomyelin was subsequently transported to the cell surface, a process that was effectively inhibited by the presence of brefeldin A. The incubation of cells with photoactivatable analogues at 4 °C, followed by illumination, led to the association of sphingolipid with a specific subset of proteins. The protein labelling pattern of ceramide differed from that of glucosylceramide. A further shift in labelling pattern was apparent when the cells were incubated with the lipid analogues at 37 °C. Moreover, most of the proteins labelled by photoreactive sphingomyelin seemed to be detergent-insoluble, which is indicative of a location in sphingolipid-rich microdomains at the plasma membrane. The potential of applying photoactivatable sphingolipids to further define and identify the role of distinct proteins in sphingolipid biosynthesis, transport and sorting, is discussed.

The biosynthesis of the new tremulane sesquiterpenes isolated from Phellinustremulae

The tremulane sesquiterpenes isolated from the wood-rotting fungus Phellinustremulae are irregular isoprenoids derived from the cyclization and rearrangement of farnesyl pyrophosphate (FPP). The most interesting feature in the formation of the tremulane skeleton seems to involve the migration of a methyl group from one of the isoprene units. To investigate the biosynthetic origin of this unique sesquiterpene skeleton, labelling experiments were carried out by adding 13C-labelled sodium acetate to liquid cultures of Phellinustremulae. The labelling pattern was determined by 13C NMR spectral analysis of labelled tremulenediol A, which was purified as its di-p-bromobenzoate. A possible route to the tremulane skeleton, which accounts for the observed labelling pattern, proceeds from FPP, via humulene. The stereochemical course of the tremulane biosynthesis is discussed. Keywords: fungal metabolites, aspen, perhydroazulene sesquiterpenes, labelling experiments.

Preparation of [14C]-Labelled 1,2,3,4,6-Penta-O-Galloyl-β-ᴅ-Glucose and Related Gallotannins

[U -14C]-Labelled 1,2,3,4,6-penta-O-galloyl-β-ᴅ-glucose was prepared by photoassimilation of 14CO2 with leaves from staghorn sumac (Rhus typhina) in the presence of the herbicide glyphosate. Extracts of the plant material were partitioned against ethyl acetate and chromatographed on Sephadex LH-20, yielding a series of crude tri-to decagalloylglucoses. The pentagalloylglucose fraction among these was further purified by HPLC to >99% purity and a specific radioactivity of 130 kBq (3.5 μCi) per μmol. The ratio of the radioactivities in the glucose and galloyl moieties, respectively, suggested a uniform labelling pattern of the product.

Accumulation and Biosynthesis of Solanapyrone Phytotoxins by Ascochyta rabiei

The biosynthesis of the phytotoxins solanapyrone A , B and C produced by the phytopathogenic fungus Ascochyta rabiei has been investigated. To optimize feeding conditons for the tracer experiments the growth of the fungus and the accumulation of the toxins in submers culture were determined. The accumulation kinetics revealed that formation of the toxins occurs in the stationary phase of the growth indicating that synthesis of solanapyrones follows a typical pattern of secondary metabolism. Incorporation experiments with sodium [1-14C]- and [2-13C]acetate were performed and the NMR-spectroscopically determined labelling pattern of the 13C-enriched solanapyrone A compound confirmed that the carbon skeleton of this compound is formed via the polyketide pathway. The biosynthetic route to solanapyrone B is discussed.

The biosynthesis of antibiotic F-244 in Fusarium sp. ATCC 20788: origin of the carbon, hydrogen, and oxygen atoms

Biosynthetic incorporation experiments were performed with carbon-13 labelled precursors including sodium [1-13C]-, [2-13C], and [1,2-13C2]-acetate as well as [methyl-13C]methionine into antibiotic F-244 (1) in growing cultures of Fusarium sp. ATCC 20788. After conversion to the methyl ester 2, analysis by NMR showed that the carbon skeleton of 1 derives from seven intact acetate units; the remaining four carbons are from methionine. Hence, the pathway is similar to that reported for 1 in Scopulariopsis. The biogenesis of the hydrogen atoms in 1 was also investigated. Incorporation of sodium [1-13C, 18O2]acetate gives 2, which exhibits 18O-induced isotope shifts at C-1 and C-3. The labelling pattern is consistent with formation of the β-lactone ring by nucleophilic attack of a C-3 hydroxyl group in the nascent polyketide precursor onto the C-1 carbonyl. Keywords: biosynthesis, polyketide, F-244, β-lactone, Fusarium.