Salvador Bergoñón

Effect of sucrose on growth and galanthamine production in shoot-clump cultures of Narcissus confusus in liquid-shake medium

In order to produce galanthamine, an alkaloid currently being tested in Alzheimers disease therapy, we have used in vitro organ cultures of Narcissus confusus (Amaryllidaceae) plants starting from two different explants: double scale segments with basal plate from bulbs (organogenic cultures), and mature seeds (callogenic-organogenic cultures). Shoot-clumps were induced from buds obtained from twin-scales and from organogenic calluses on a MS medium supplemented with 1 mg l−1 2,4-D and 5 mg l−1 BA. Shoot-clumps were then developed partially submerged in a liquid medium. After one month of precondition, the shoot-clumps were cultured in liquid media with different concentrations of sucrose, from 3% to 18% (w/v) for 14 days. The growth of the regenerated plants treated with 9% sucrose was significantly greater. Under a photoperiod 16 h light/8 h dark, the shoot-clump cultures subjected to the two highest sucrose concentrations gave rise to higher dry weight/fresh weight ratios. Different doses of sucrose affected not only the alkaloid profile in the shoot-clump tissues but also that excreted to the medium. In all cases, shoot cultures of N. confusus were capable of galanthamine biosynthesis, with the best results at 9% sucrose concentration.

Identification and characterization of a new human cDNA from chromosome 21q22.3 encoding a basic nuclear protein

Congenital heart disease (CHD) affects over 40% of Down syndrome (DS) patients. The region proposed to contain the gene(s) for DS CHD has been restricted to 21q22.2–22.3, from D21S55 to MX1. The identification and functional characterization of the genes mapping to this region is a necessary step to understand the pathogenesis of CHD in DS. In an effort to contribute to the construction of a transcriptional map of the DS CHD region we have performed direct cDNA selection using a YAC contig that maps between ETS2 and D21S15 and cDNAs synthetised from fetal heart structures. Here we describe the identification and characterization of a new gene, WRB, that maps to 21q22.3 between ACTL5 and HMG 14 and appears to be widely expressed in adult and fetal tissues. The new gene encodes a basic protein of unknown function containing a tryptophan-rich carboxyl-terminal region and a potential nuclear localization signal. Immunofluorescence analysis shows a predominant localization in the cell nucleus. The understanding of the biological function of the protein product should clarify the potential role of WRB in the pathogenesis of DS CHD.

Alkaloids from Narcissus muñozii-garmendiæ

Abstract Whole plants of Narcissus munozii-garmendiae were found to contain homolycorine, lycorenine and O -methyllycorenine. The structure of O -methyllycorenine has been completely assigned by means of 2D NMR studies. The isolation of these alkaloids is hitherto unreported from this plant source.

Alkaloids from Narcissus leonensis

Abstract The new alkaloids epinorgalanthamine epinorlycoramine have been isolated from whole plants of Narcissus leonensis . Their structures and stereochemistry were established by physical and spectroscopic methods.

Characterisation and expression analysis of the WDR9 gene, located in the Down critical region-2 of the human chromosome 21.

We report the isolation and characterisation of the gene WDR9 (WD Repeat 9), located in the Down Syndrome critical region-2 (DCR-2) from the human chromosome 21. This gene spans 125 kb of genomic sequence and is organised in 41 exons and 40 introns. The WDR9 cDNA has a size of 13 kb and encodes for a putative protein of 2269 amino acids with a potential location in the nucleus. Expression analysis in different human adult tissues and in cultured cell lines indicates that the gene has several tissue-specific transcripts. The more significant protein signatures in the WDR9 protein sequence are for WD repeats, bromodomain, beta-ketoacyl synthases, and ribonucleoprotein (RNP). The WDR9 protein has a high similarity with the Mus musculus neuronal differentiation protein (NDRP) and a region of similarity with the region of the Yotiao protein that has been proposed to bind the NR1 subunit of the NMDA receptor. The presence of protein-protein interaction domains as such the WD repeats, and the similarity of the WDR9 protein to regulatory proteins suggest a potential involvement in some of the clinical features associated to the DCR-2.

Galanthamine production in “shoot-clump” cultures of Narcissus confusus in liquid-shake medium

Galanthamine (GAL) is increasingly used in the treatment of Alzheimers disease. We have attempted to develop a method of producing this alkaloid using in vitro cultures of Narcissus confusus plants. The “shoot-clump” culture in liquid medium was shown to be an appropriate method for the micropropagation of this bulbous plant. The complete process included three steps:1.culture of “twin-scales” starting from the bulbs;2.culture of the newly formed shoots in a medium for bud proliferation (Murashige Skoog+1 mg l-1 of 2,4-dichlorophenoxyacetic acid+5 mg l-1 of benzyladenine), and3.culture of “shoot-clumps” in a liquid-shake medium. Here we describe the effect of the addition of trans-cinnamic acid, a precursor in the biosynthesis of the Amaryllidaceae alkaloids, on the production of galanthamine and related alkaloids, and also on the growth of the “shoot-clump” culture. The production of galanthamine was found to be inhibited by the addition of the precursor, which promoted the production of the other alkaloid in the same biosynthetic pathway, N-formyl-norgalanthamine. The total production of galanthamine in the control cultures in day-long photoperiod was 2.50 mg per culture, of which 1.97 mg per culture were released into the medium.