A three-step screening procedure to identify the mode of phloem loading in intact leaves

A three-step screening method was developed to identify the mode of phloem loading in intact leaves. Phloem loading of ¹⁴CO₂-derived photosynthate was challenged by p-chloromercuribenzenesulfonic acid (PCMBS) in leaves of dicotyledons with either a symplasmic (type 1, with intermediary cells as companion cells) or apoplasmic (type 2b, with transfer cells as companion cells) minor-vein configuration. Firstly, photosynthate export as the result of phloem loading was measured by collection of phloem exudate from the petiole. The PCMBS had virtually no effect on photosynthate export in representatives of type-1 families (Lamiaceae, Lythraceae, Onagraceae, Saxifragaceae). In contrast, photosynthate export was strongly reduced by PCMBS in representatives of type-2b families (Asteraceae, Balsaminaceae, Dipsacaceae, Linaceae, Tropaeolaceae, Valerianaceae) and type-2b members of polytypical families (Fabaceae, Scrophulariaceae). Secondly, densitometric measurements of leaf autoradiographs demonstrated that the contrast between the mesophyll and the lower-order veins was hardly affected by PCMBS treatment in type-1 species, whereas PCMBS strongly reduced the contrast in type-2b species. Thirdly, separate ¹⁴C-radioassays of vein and mesophyll tissues confirmed this observation. The three-step procedure thus revealed a strong and consistent reduction of phloem loading by PCMBS in type-2b species which was absent in type-1 species. In conclusion, phloem loading in type-2b species occurs via the apoplast and type-1 species execute an alternative — most likely symplasmic — mode of phloem loading.Abbreviations PCMBS p-chloromercuribenzenesulfonic acid - SE/CC-complex sieve element/companion cell complex We gratefully acknowledge the expert help of Dr. Maarten Terlou, Department of Image Processing and Design, University of Utrecht, in carrying out the densitometric measurements.     

In vitro binding of estrogens by dietary fiber and the in vivo apparent digestibility tested in pigs.

Within the framework of experiments related to the association between dietary fiber and breast cancer an in vitro test system was used to study the binding of estrogens to various fibers (e.g. cholestyramin, lignin and cellulose) and fiber sources (e.g. wheat bran, cereals, seeds and legumes). Furthermore, the in vivo apparent digestibility of the different fiber sources was tested using a mobile nylon bag technique in intestine-cannulated pigs. Estradiol-17 beta (E2) bound more strongly to the various fibers than did estrone (E1), estriol or estrone-3-glucuronide. At increasing pH (greater than 7) binding of both E1 and E2 to wheat bran decreased significantly. Cholestyramine and lignin bound almost all estrogens present in the medium. Linseed (91%), oats (83%), barley chaff (88%) and wheat bran (82%) are other excellent binders of E2. Corn, rye and white wheat flour showed lower binding capacity with a relatively low affinity. Cereals with the highest percentage of lignin in the fiber (greater than 3%) were also the fiber sources with the lowest apparent digestibility. Estrogens bound with the highest affinity (relative to bovine serum albumin) to these fiber sources. Together with wheat bran and lignin, oats, linseed and soybean seem to be products with good perspectives for in vivo evaluation of the lowering effect of dietary fiber on estrogen exposure of estrogen-sensitive tissues.     

Metabolic conversion of the biologically active phospholipid, lysophosphatidic acid, in fibroblasts.

Lysophosphatidic acid (3-sn-lysophosphatidic acid; LPA) can activate cells similar to hormones and growth factors. We have considered the question whether metabolic conversion of LPA taken up by the cell could be of any importance in this activation. Addition of [14C-glycerol]LPA to quiescent Rat-1 fibroblasts resulted in rapid formation of [14C]monoacylglycerol (MG), closely followed by accumulation of [14C]triacylglycerol. Only very little [14C]diacylglycerol and [14C]phosphatidic acid was formed (approx. 100-fold less than MG). MG, when added exogenously to cells, lacks detectable biological activity. The results suggest that LPA itself, rather than one of its metabolites is the biologically active principle.     

Solid-phase synthesis of peptides with C-terminal asparagine or glutamine. An effective, mild procedure based on N alpha-fluorenylmethyloxycarbonyl (Fmoc) protection and side-chain anchoring to a tris(alkoxy)benzylamide (PAL) handle

Attempts to anchor Fmoc-asparagine or glutamine as p-alkoxybenzyl esters for solid-phase peptide synthesis are fraught with difficulties. A convenient and effective method to prepare peptides with C-terminal asparagine or glutamine involves quantitative attachment of N alpha-Fmoc-C alpha-tert.-butyl aspartate or glutamate via the free omega-carboxyl groups to a tris(alkoxy)benzylamino (PAL) support. Chain elongation proceeds normally by standard Fmoc chemistry, and treatment with acid, e.g., CF3COOH--CH2Cl2, 90 min at 25 degrees, releases the desired peptides in greater than 95% yields without side reactions at the C-terminus. Feasibility of the approach has been demonstrated by the syntheses of the C-terminal octapeptide from human proinsulin, H-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln-OH, and the serum thymic factor pGlu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn-OH.     

Characterization of the interaction of doxorubicin with (poly)phosphoinositides in model systems. Evidence for specific interaction with phosphatidylinositol-monophosphate and -diphosphate.

The anticancer drug doxorubicin penetrates into Langmuir monolayers containing phosphoinositides. Upon binding of doxorubicin to phosphoinositide-containing SUV, its fluorescence is self-quenched due to self-association. As compared to other anionic phospholipids, as much as 2- to 3-fold larger effects were obtained with PIP and PIP2, in mixtures of these lipids with DOPC. Doxorubicin competes efficiently with the non-penetrating antibiotic neomycin for binding to PIP2. According to its penetration, specific binding of doxorubicin was half-maximal at 5-15 microM. It is likely that also in biological membranes doxorubicin binds specifically to PIP and PIP2.     

Transferring sediment containing intact seed banks: a method for studying plant community ecology

The experimental study of the ecology of natural plant communities is necessary to demonstrate the effects of environmental factors on plant growth. It allows one to make predictions of the likely effects of environmental changes on plant communities. Manipulating an entire ecosystem is complicated, and is rarely attempted. An alternative method is applied here for the submerged plant communities of the seasonally-flooded oligohaline marshes of the Camargue. It consists of collecting undisturbed sediment samples with their contained intact seed bank, and submitting them to different salinities. The study of total biomass and of the biomass of the more frequent species was carried out using parametric tests. The precision of the results depended on the frequency of the species in the replicates. When the frequency was low, non-parametric statistics were necessary. The method seems to be particularly suitable for the study of communities of annual species in aquatic environments.

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Résumé L'étude expérimentale de l'écologie des communautés naturelles de plantes est nécessaire pour démontrer les relations de cause à effet entre les facteurs environnementaux et l'abondance des plantes. Elle l'est également pour faire des prédictions réalistes sur les effets de modifications envisagées de l'environnement sur des communautés de plantes. La manipulation de l'écosystème complet est complexe, ce qui rend son utilisation rare. Une méthode alternative a été utilisée pour l'étude de la composition spécifique des communautés de plantes submergées des marais temporaires de Camargue. Elle consiste à prélever des échantillons non perturbés de sédiment, avec leur stock de semences intact, et à les soumettre à des conditions de salinité. L'étude de la biomasse totale et de la biomasse des espèces les plus fréquentes est faite au moyen de test paramétriques. La précision attendue des résultats dépend de la fréquence de l'espèce dans les réplicats. Lorsque la fréquence n'est pas élevée l'emploi de statistiques non paramétriques est indispensable. La méthode se révéle particulièrement bien adaptée à l'étude des communautés d'annuelles en milieu aquatique.