The reaction of acetylacetone with amino sugars: implications for the formation of glycosylpyrazole derivatives

Glycosylpyrazoles are efficiently formed by reaction of saccharide hydrazones with pentan-2,4-dione (acetylacetone), but in aqueous buffer, pyrazole derivatives of amino sugars couple with a further equivalent of acetylacetone affording high yields of ketoenamines. These ketoenamines were considerably more stable than the ketoenamines formed from 2-amino-2-deoxy aldoses that have been described as intermediates in the classical Elson-Morgan reaction. Moreover, high yields of perketoenamine derivatives were achieved with oligosaccharides derived from hydrolysis of chitosan. The removal of the ketoenamine moieties to regenerate the free amine was readily accomplished with aqueous hydrazine.     

Seasonal patterns of 13C partitioning between shoots and nodulated roots of N2- or nitrate-fed Pisum sativum L

The effect of nitrogen source (N(2) or nitrate) on carbon assimilation by photosynthesis and on carbon partitioning between shoots and roots was investigated in pea (Pisum sativum L. 'Baccara') plants at different growth stages using (13)C labelling. Plants were grown in the greenhouse on different occasions in 1999 and 2000. Atmospheric [CO(2)] and growth conditions were varied to alter the rate of photosynthesis. Carbon allocation to nodulated roots was unaffected by N source. At the beginning of the vegetative period, nodulated roots had priority for assimilates over shoots; this priority decreased during later stages and became identical to that of the shoot during seed filling. Carbon allocation to nodulated roots was always limited by competition with shoots, and could be predicted for each phenological stage: during vegetative and flowering stages a single, negative exponential relationship was established between sink intensity (percentage of C allocated to the nodulated root per unit biomass) and net photosynthesis. At seed filling, the amount of carbon allocated to the nodulated root was directly related to net photosynthesis. Respiration of nodulated roots accounted for more than 60 % of carbon allocated to them during growth. Only at flowering was respiration affected by N supply: it was significantly higher for strictly N(2)-fixing plants (83 %) than for plants fed with nitrate (71 %). At the vegetative stage, the increase in carbon in nodulated root biomass was probably limited by respiration losses.     

The lateral line of zebrafish: a model system for the analysis of morphogenesis and neural development in vertebrates

The lateral line of the zebrafish has many of the advantages that made the sensory organs of Drosophila a very productive model system: 1) it comprises a set of discrete sense organs (neuromasts) arranged in a defined, species-specific pattern, such that each organ can be individually recognized; 2) the neuromasts are superficial and easy to visualize, and the innervating neurons are easy to label; 3) the sensory projection is simple yet reproducibly organized. Here we describe some of the tools that can be used to investigate the development of this system, and we illustrate their usefulness with specific examples. We conclude that the lateral line is uniquely suited among vertebrate sensory systems for a molecular, cellular and genetic analysis of pattern formation and of neural development.     

Localisation of a carbohydrate epitope recognised by human IgE in pollen of Cupressaceae

The objectives of the present study were: (1) to localise, at the subcellular level, the allergens in pollen of Cupressaceae species, using a monoclonal antibody (mAb 5E6) that is specific for carbohydrate epitopes of allergenic components of Cupressus arizonica pollen extract; (2) to determine whether the glycidic epitope recognised by mAb 5E6 was present in pollen of allergenic species taxonomically unrelated to Cupressaceae; and (3) to determine whether human IgE purified from monosensitive patients recognises the same epitope as mAb 5E6 in Cupressaceae pollen. Immunogold labelling of mAb 5E6 showed a high density of gold particles on the orbicules, supporting the hypothesis that they are important vectors of allergens. A high density was also found on the exine and in the cytoplasm, with the latter finding confirming that fragments of pollen ruptured under humid conditions can represent a vector. The glycidic epitope recognised by mAb 5E6 was detected in all of the species taxonomically unrelated to Cupressaceae, although with varying density. Human IgE recognised the same epitope as mAb 5E6. These findings are consistent with observations of diffuse allergenic cross-reactivity among various allergens. The in situ localisation of a common epitope recognised by both a monoclonal antibody and human IgE could be of importance in immunotherapy.     

Epithelial cell proliferative activity and oral cancer progression

Accurate, predictive assessment of the behaviour and progression of oral cancers and precancers remains elusive in clinical practice. Archival tissue specimens from 10 previously treated patients with oral lesions of known clinical outcome (3 years post-treatment) were re-examined histopathologically, and proliferative cell labelling indices (LIs) determined for Ki67, cyclin A and histone mRNA cell cycle markers. While histone mRNA labelling ultimately proved unreliable, both Ki67 and cyclin A LIs demonstrated a clear trend for enhanced labelling to occur in increasingly dysplastic and neoplastic tissue, with particular emphasis on suprabasal labelling in abnormal tissue. Perhaps of greatest significance was the observation of increased LIs and suprabasal labelling in lesions with poor clinical outcome, such as patients developing recurrent disease or cervical lymph node metastasis. Measurement of cell proliferative activity in individual oral epithelial dysplastic lesions or invasive squamous cell carcinomas may thus provide unique, predictive information on clinical outcome.     

Localization of Fe-containing superoxide dismutase in cyanobacteria from the Baltic Sea: depth and light dependency

The abundance and cellular location of Fe-containing superoxide dismutase (Fe-SOD) in trichomes of Nodularia , Aphanizomenon and Anabaena collected from various depths in the Baltic Sea, and in trichomes of a cultured Nodularia strain, BC Nod-9427, isolated from the Baltic Sea, was examined by immunogold labelling. For trichomes collected from natural populations the areal concentration of Fe-SOD labelling decreased with depth: trichomes collected from surface accumulations had between 8 and 11 gold particles μm⁻² whereas trichomes collected from a depth of 18 m were unlabelled. When trichomes collected from a depth of 10 m (mean areal labelling density 0·5 gold particles μm⁻²) were exposed to the higher irradiances present at 1 m, the areal concentration of Fe-SOD increased to 3·5–4 gold particles μm⁻² within 4 h. When cultures of Nodularia strain BC Nod-9427, adapted to low light (10 μmol m⁻² s⁻¹), were transferred to an incident irradiance of 1350 μmol m⁻² s⁻¹, a doubling of the areal concentration of Fe-SOD gold label was observed within 1 h. Addition of 3-(3,4-dichlorophenyl)-1,1'-dimethylurea (DCMU) to cultures immediately before their transfer to increased illumination resulted in a decrease in areal Fe-SOD concentrations whereas addition of CdCl₂ caused an increase over and above that induced by the elevated irradiance. These results suggest that Baltic Sea cyanobacteria are able to modulate their Fe-SOD content but that this might be in response to oxidative stress rather than to light per se .     

A comparison of sequence resolution on plant chromosomes: PRINS versus FISH

 The resolution of the chromosomal positions of six high- and one low-copy sequences by oligonucleotide-primed in situ (PRINS) labelling was compared with corresponding data obtained after fluorescent in situ hybridization (FISH) on field-bean and barley chromosomes. While PRINS proved to be suitable for the rapid detection of high-copy tandem repeats at the same loci as those revealed by FISH, no clear PRINS signal was obtained for the low-copy family of vicilin genes at their locus on field-bean chromosome II. This indicates that localization of short target sequences by primer extension via Taq polymerase in situ does not yet provide a resolution equal, or superior, to FISH on plant chromosomes. Therefore, the use of a cocktail of chromosome-specific single-copy sequences as primers for PRINS is no alternative for the not as yet feasible chromosome painting in plants. Received: 21 April 1998 / Accepted: 12 May 1998     

Subunit-specific backbone NMR assignments of a 64 kDa trp repressor/DNA complex: A role for N-terminal residues in tandem binding

Deuterium decoupled, triple resonance NMR spectroscopy was used to analyze complexes of ²H,¹⁵N,¹³C labelled intact and (des2–7) trp repressor (2–7 trpR) from E. coli bound in tandem to an idealized 22 basepair trp operator DNA fragment and the corepressor 5-methyltryptophan. The DNA sequence used here binds two trpR dimers in tandem resulting in chemically nonequivalent environments for the two subunits of each dimer. Sequence- and subunit-specific NMR resonance assignments were made for backbone ¹HN, ¹⁵N, ¹³C positions in both forms of the protein and for¹³ C in the intact repressor. The differences in backbone chemical shifts between the two subunits within each dimer of 2–7 trpR reflect dimer-dimer contacts involving the helix-turn-helix domains and N-terminal residues consistent with a previously determined crystal structure [Lawson and Carey (1993) Nature, 366, 178–182]. Comparison of the backbone chemical shifts of DNA-bound 2–7 trpR with those of DNA-bound intact trpR reveals significant changes for those residues involved in N-terminal-mediated interactions observed in the crystal structure. In addition, our solution NMR data contain three sets of resonances for residues 2–12 in intact trpR suggesting that the N-terminus has multiple conformations in the tandem complex. Analysis of C chemical shifts using a chemical shift index (CSI) modified for deuterium isotope effects has allowed a comparison of the secondary structure of intact and 2–7 tprR. Overall these data demonstrate that NMR backbone chemical shift data can be readily used to study specific structural details of large protein complexes.     

The structure and diversity of retinal ganglion cells in the masked greenling Hexagrammos octogrammus Pallas, 1814 (Pisces: Scorpaeniformes: Hexagrammidae)

The authors studied the structure and diversity of retinal ganglion cells (GC) in the masked greenling Hexagrammos octogrammus. In vivo labelling with horseradish peroxidase revealed GCs of various structures in retinal wholemounts. A total of 154 cells were camera lucida drawn, and their digital models were generated. Each cell was characterized by 17 structural and topological parameters. Using nine clustering algorithms, a variety of clusterings were obtained. The optimum clustering was found using silhouette analysis. It was based on a set of three variables associated with dendritic field size and dendrite stratification depth in the retina. A total of nine cell types were discovered. A number of non-parametric tests showed significant pair-wise between-cluster differences in at least four parameters with medium and large effect sizes. Three large-field types differed mainly in dendritic field size, total dendrite length, level of dendrite stratification in the retina and position of somata. Six medium- to small-field types differed mainly in the structural complexity of dendritic arbors and level of dendrite arborization. Cells similar and obviously homologous to types 1–4 were identified in many fish species, including teleosts. Potential homologues of type 5 cells were identified in fewer teleost species. Cells similar to types 6–9 in relative dendritic field size and dendrite arborization pattern were also described in several teleostean species. Nonetheless, their homology is more questionable as their stratification patterns do not match so well as they do in large types. Potential functional matches of the GC types were identified in a number of teleostean species. Type 1 and 2 cells probably match spontaneously active units with the large receptive field centre, so-called dimming and lightening detectors; type 4 may be a counterpart of changing contrast detectors with medium receptive field centre size preferring fast-moving stimuli. Type 3 (biplexiform) cells have no obvious functional matches. Probable functional matches of types 6, 8 and 9 belong to ON-centre elements with small receptive fields such as ON-type direction-selective cells, ON-type spot detectors or ON-type spontaneously active units. Type 5 and 7 cells may match ON–OFF type units, in particular, changing contrast detectors or orientation-selective units. Potential functional matches of GC types presently described are involved in a wide spectrum of visual reactions related to adaptation to gradual change in illumination, predator escape, prey detection and capture, habitat selection and social behaviour.     

Specific proteins in the sieve-tube exudate of Ricinus commuais L. seedlings: separation,characterization and in-vivo labelling

Ricinus communis L. seedlings exuded pure phloem sap from the cut hypocotyl for several hours. Throughout the entire exudation period proteins were present in the phloem exudate at a constant concentration ranging from 0.11 to 0.41 mg·ml^–1 depending on the culture conditions and the age of the seedlings. Manipulation of the nutrient supply at the cotyledons after removal of the endosperm did not change the protein concentration in the exudate. Comparison of sieve-tube exudate proteins (STEPs) with soluble proteins extracted from the hypocotyl and the cotyledons showed a unique abundance of small proteins in the exudate, with molecular weights ranging from 10 to 25 kDa. Bands at 18, 19 and 20 kDa were especially dominant. The proteins found transiently in the xylem exudate, which might represent proteins secreted at the wound surface, were different in pattern. Two-dimensional separation of STEPs revealed that more than 100 distinct polypeptides occurred in the sieve-tube exudate, most of them slightly acidic with isoelectric points ranging from 4 to 6 and a few basic ones around 8. [³⁵S]Methionine fed to the cotyledons led to labelling of STEPs, demonstrating their rapid synthesis. It is concluded that there is a continuous synthesis and translocation of specific sieve-tube proteins, whose function is unknown.Abbreviations IEF isoelectric focussing - pI isoelectric point - STEP sieve-tube exudate protein - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid We wish to thank Pia Großmann and Libuse Badewitz for technical help.