Purification and characterisation of a jacalin-related, coleoptile specific lectin from Hordeum vulgare.

A plant lectin was isolated from barley (Hordeum vulgare) coleoptiles using acidic extraction and different chromatographic methods. Sequencing of more than 50% of the protein sequence by Edman degradation confirmed a full-length cDNA clone. The subsequently identified open reading frame encodes for a 15 kDa protein which could be found in the soluble fraction of barley coleoptiles. This protein exhibited specificity towards mannose sugar and is therefore, accordingly named as Horcolin (Hordeum vulgare coleoptile lectin). Database searches performed with the Horcolin protein sequence revealed a sequence and structure homology to the lectin family of jacalin-related lectins. Together with its affinity towards mannose, Horcolin is now identified as a new member of the mannose specific subgroup of jacalin-related lectins in monocot species. Horcolin shares a high amino acid homology to the highly light-inducible protein HL#2 and, in addition to two methyl jasmonic acid-inducible proteins of 32.6 and 32.7 kDa where the jasmonic acid-inducible proteins are examples of bitopic chimerolectins containing a dirigent and jacalin-related domain. Immunoblot analysis with a cross-reactive anti-HL#2 antibody in combination with Northern blot analysis of the Horcolin cDNA revealed tissue specific expression of Horcolin in the coleoptiles. The function of Horcolin is discussed in the context of its particular expression in coleoptiles and is then compared to other lectins, which apparently share a related response to biotic or abiotic stress factors.

Human hydroxysteroid (17-beta) dehydrogenase 1 expression enhances estrogen sensitivity of MCF-7 breast cancer cell xenografts.

Hydroxysteroid (17-beta) dehydrogenase 1 (HSD17B1) catalyzes the conversion between estrone (E1) and estradiol (E2). The reaction is reversible in vitro, but the data in cultured cells suggest that E2 production is the predominant reaction in physiological conditions. However, the hypothesis has not been verified in vivo. In the present study, estrogen-dependent MCF-7 human breast cancer cells were stably transfected with an expression plasmid for human HSD17B1. The enzyme efficiently converted E1 to E2 and enhanced the estrogen-dependent growth of cultured MCF-7 cells in the presence of hormonally less active E1. The HSD17B1-expressing cells also formed estrogen-dependent tumors in immunodeficient nude mice. After treating the mice with an appropriate dose of the substrate (E1, 0.1 micromol/kg x d), a marked difference in tumor growth was observed between nontransfected and HSD17B1-transfected MCF-7 cells, mean tumor weights at the end of E1 treatment being 23.2 and 130.4 mg, respectively. Furthermore, estrogen-dependent growth of the HSD17B1-expressing xenografts in the presence of E1 was markedly inhibited by administering 5 micromol/kg x d of a specific HSD17B1 inhibitor. After a 4-wk treatment, the tumor size was reduced by 59.8% as compared with the nontreated tumors, whereas the uterine growth of the mice was not affected by the HSD17B1 inhibitor used. This was in line with the induction of apoptosis of the tumors. The results evidently show that estrogenic response for E1 is enhanced by the local action of HSD17B1 in vivo, and thus, the enzyme is a potential target for pharmacological inhibition of estrogen action.

Dydrogesterone (Duphaston) and its 20-dihydro-derivative as selective estrogen enzyme modulators in human breast cancer cell lines. Effect on sulfatase and on 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activity.

Estradiol (E2) is one of the main factors which control the growth and evolution of breast cancer. Consequently, to block the formation of E2 inside cancer cells has been an important target in recent years. Breast cancer cells possess all the enzymatic systems (e.g. sulfatase, aromatase, 17beta-hydroxysteroid dehydrogenase [17beta-HSD]) involved in the conversion of estrogen precursors into E2. Sulfotransferase, which converts estrogen to its sulfate, is also present in this tumoral tissue. Duphaston is a synthetic progestogen with properties similar to the natural progesterone. In the present study we examined the effect of Duphaston and its 20-dihydro-metabolite on the sulfatase and 17beta-HSD activities in MCF-7 and T-47D breast cancer cells. The cells were incubated with estrone sulfate (E1S) (5x10(-9)M) in the absence or presence of Duphaston or its 20-dihydro-metabolite (5x10(-5) to 5x10(-9)M) for 24h at 37 degrees C. In another series of experiments, estrone (E1) (5x10(-9)M) was incubated with T-47D cells in the absence or presence of the two progestogens (5x10(-5) to 5x10(-9)M) for 24h at 37 degrees C. E1S, E1 and E2 were characterized by thin layer chromatography and quantified using the corresponding standard. Duphaston and its 20-dihydro-metabolite, at concentrations of 5x10(-7) and 5x10(-5)M, inhibited the conversion of E1S to E2 by 14% and 63%, 65% and 74%, respectively, in MCF-7 cells; the values were 15% and 48% and 31% and 51%, respectively, in T-47D cells. In another series of experiments it was observed that, after 24-h incubation, E1 (5x10(-9)M) was converted in a great proportion to E2 in the T-47D cells and that this transformation was significantly inhibited by Duphaston and its 20-dihydro-metabolite. The IC50 value, corresponding to 50% of the inhibition in the conversion of 1 to E2, was 9x10(-6)M for 20-dihydro-metabolite in this cell line. It was concluded that the progestogen Duphaston and its 20-dihydro-metabolite are potent inhibitory agents on sulfatase and 17beta-HSD activities in breast cancer cells. Duphaston is a progestogen with properties similar to the endogenous progesterone. The data open interesting perspectives to study the biological responses of these progestogens in clinical trials of patients with breast cancer.

Phosphatidylcholine metabolism of rat trachea in relation to lung parenchyma and surfactant.

Pulmonary surfactant prevents alveolar collapse and contributes to airway patency by reducing surface tension. Although alveolar surfactant, consisting mainly of phospholipids (PL) together with neutral lipids and surfactant-specific proteins, originates from type II pneumocytes, the contribution of airway epithelia to the PL fraction of conductive airway surfactant is still debated. We, therefore, analyzed the composition, synthesis, and release of phosphatidylcholine (PC) molecular species as the main surfactant PL of the rat trachea compared with the lung. Analyses of individual PC molecular species with HPLC and electrospray ionization mass spectrometry revealed that the rat trachea contained and synthesized much more palmitoyloleoyl-PC, palmitoyllinoleoyl-PC, and palmitoylarachidonoyl-PC, together with increased amounts of alkylacyl-PC, and less surfactant-specific species such as dipalmitoyl-PC than the lung. Organ cultures with [methyl-3H]choline as precursor of PC revealed that, in the trachea, synthesized PC was retained in the tissue, rather than secreted. [Methyl-3H]choline-labeled dipalmitoyl-PC was a negligible component in the trachea, and, in contrast to the lungs, palmitoyloleoyl-PC was enriched in tracheal secretions. We conclude that the surfactant fraction in the airways does not originate from the airways but is produced in the alveolar space and transported upward.