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Genetic variations in Lycoris radiata var. radiata in Japan.

The genetic variations of Lycoris radiata var. radiata, a completely sterile triploid from Japan, were examined by comparing the nucleotide sequences of genomic DNA regions in 11 triploid strains sampled from Japan and four triploid strains sampled from China, and in two diploid strains of Lycoris radiata var. pumila, which is endemic to China and fertile. For this purpose, two genes were analyzed, the lectin gene in the nuclear genome and the maturase gene in the chloroplast genome. A clear genetic constancy was observed in their DNA nucleotide sequences. For both genes, completely identical nucleotide sequences were detected in the 11 Japanese and four Chinese triploid strains and also between the two Chinese diploid strains. However, some genetic variations were observed between the Japanese and Chinese triploid strains, and between the triploid and diploid strains. These results are consistent with the findings obtained from previous chromosome karyotype analyses and allozyme analyses. In addition, in our preliminary FISH analysis of the physical mapping of the rRNA gene family, the 18S-5.8S-26S rRNA and 5S rRNA loci were localized on six and four chromosomes, respectively. Regarding the 18S-5.8S-26S rRNA loci, two were associated with two SAT chromosomes. The remaining four were distinguished by having no secondary constriction. Localization of 5S rRNA loci to chromosome spreads revealed three sites on the proximal part of the long arm of three acrocentric chromosomes and one site on the distal part of the long arm of the SAT chromosome; the latter site was juxtaposed to the 18S-5.8S-26S rRNA loci. These findings indicate that L. radiata var. radiata is not a typical autotriploid. The present paper discusses the possible origin of L. radiata var. radiata from a diploid variety of L. radiata var. pumila, based on the molecular cytogenetic analysis and DNA sequence analysis.

Base Sequence↗

[Determination of cadmium, chromium and lead in Lycoris radiata with microwave digestion technique by graphite furnace atomic absorption spectrometry].

A new method using microwave digestion technique was developed for the determination of cadmium, chromium and lead in Lycoris radiata by graphite furnace atomic absorption spectrometry (GFAAS). Digestion solvents were discussed for sample preparation by microwave digestion technique. The optimum condition of determination by GFAAS was studied in the presence of NH4H2PO4 matrix modifier. The detection limits of Cd, Cr and Pb are 0.066 7, 1.22 and 0.810 microg x L(-1), respectively. The method was applied to the determination of Cd, Cr and Pb in Lycoris radiata samples with satisfactory results. The RSD of determination were lower than 3.1%. The recoveries were from 83.8% to 118%.

Cadmium↗

Molecular cloning and characterization of a novel lectin gene from Lycoris radiata.

By the use of RACE-PCR with RNA extracted from Lycoris radiata young leaves, the full-length cDNA of L. radiata agglutinin (LRA) was cloned. The full-length cDNA of LRA was 669 bp and contained a 477 bp open reading frame encoding a 158 amino acid protein. Through comparative analysis of lra gene and its deduced amino acid sequence with those of other Amaryllidaceae species, it was found that lra encoded a precursor lectin with signal peptide. LRA was a mannose-binding lectin with three mannose-binding boxes like lectins from other Amaryllidaceae species. Southern blot analysis of genomic DNA revealed that lra belonged to a low copy gene family. Northern blot analysis demonstrated that lra constitutively expressed in various plant tissues including leaf, root, stem and inflorescence, and highly expressed in the inflorescence.

Amino Acid Sequence↗

Pharmacological studies of lycorenine, an alkaloid of Lycoris radiata Herb.: Vasodepressor mechanism in rats.

Vasodepressor mechanism of lycorenine (an alkaloid of Lycoris radiata Herb.) was investigated in anesthetized rats. Lycorenine (1--10 mg/kg i.v.) produced dose-related decreases in blood pressure and heart rate and tachyphylaxis developed with repeated injections. In the blood-perfused rat hindquarters, lycorenine (62.5--500 micrograms i.a.) produced dose-related decreases both in mean blood pressure and in perfusion pressure, and the lycorenine-induced decrease in perfusion pressure was abolished by phenoxybenzamine or hexamethonium. Lycorenine (more than 1 mg/kg i.v.) blocked the pressor response to sympathetic nerve stimulation, but failed to block the tachycardia induced by sympathetic nerve stimulation. Lycorenine (7.5 or 15 mg/kg i.v.) reduced the spontaneous splanchnic nerve activity. Lycorenine when given intracerebroventricularly produced decreases in blood pressure and heart rate only in large doses (over 500 micrograms). The maximal bradycardia induced by lycorenine was abolished by bilateral vagotomy. It is suggested that lycorenine may produce a decrease in blood pressure as the result of alpha-adrenergic blockade in conjunction with the reduction of the spontaneous sympathetic nerve activity, and produce bradycardia by modifying vagal activity.

Action Potentials↗

[Determination of galanthamine in bulb of Lycoris radiata by RP-HPLC].

OBJECTIVE: To establish a method for determination of galanthamine in Lycoris radiata. METHOD: HPLC separation was carried on a column of ODS (4.6 mm x 150 mm, 5 microm), with the mobile phase of phosphate buffer (pH = 3-4)-methanol (93:7) at the flow rate of 1.0 mL x min(-1), and the detection wavelength was set at 289 nm. RESULT: Galanthamine revealed linearity within the range of 3-30 microg x mL(-1) (r = 0.9997), the detection limit was 0.3 ng. The average recovery was 99.5% (RSD = 0.5%). CONCLUSION: The method is easy to operate and the results of the determination are accurate, it can be used to evaluate the quality of L. radiata.

Chromatography, High Pressure Liquid↗

A chromosome-level genome assembly of Lycoris radiata reveals the evolutionary origin of Amaryllidaceae alkaloids and elucidates the complete galanthamine biosynthetic pathway.

Amaryllidaceae alkaloids (AmAs) comprise a structurally diverse group of specialized metabolites produced almost exclusively by species of the Amaryllidoideae subfamily and are of substantial pharmacological importance. However, the limited availability of high-quality genomes from Amaryllidoideae plants has constrained systematic investigations of the genes and evolutionary processes underlying AmA biosynthesis. Here, we present a chromosome-level genome assembly of Lycoris radiata, which enabled the discovery of key downstream enzymes in the galanthamine biosynthetic pathway and uncovered reversible reactions between two critical metabolite pairs. These findings provide new mechanistic insight into pathway architecture and enable reconstruction of the galanthamine biosynthetic pathway in Yarrowia lipolytica. Comparative genomic analyses indicate that several core genes for AmA biosynthesis originated in ancestral angiosperms, whereas the complete pathway was likely assembled in the Amaryllidoideae subfamily through gene duplication and neofunctionalization. Furthermore, integrated metabolomic and transcriptomic analyses suggest that roots contribute actively to AmA metabolism in Lycoris. Together, these findings provide a genomic and biochemical framework for understanding the evolution and engineering of AmA biosynthesis.

Lycoris↗

Pharmacological studies of lycorenine, an alkaloid of Lycoris radiata Herb.: II. Effects of blood pressure in rats and dogs and the mechanism of tachyphylaxis to the vasodepressor action of lycorenine in rats.

Blood pressure responses to lycorenine were investigated in rats and dogs. Lycorenine (10--40 mg/kg p.o.) produced a decrease in blood pressure in conscious rats which was greater in hypertensive rats than in normotensive rats. In conscious dogs, lycorenine (5 or 10 mg/kg i.v.) produced rises in blood pressure and heart rate except for a few instances, while it produced a decrease in blood pressure in pentobarbital anesthetized dogs. Thus, there are species differences in cardiovascular effects of lycorenine between conscious rats and dogs. The development of tachyphylaxis to the vasodepressor actions of lycorenine and its related compounds was related to their alpha-adrenergic blocking activities at the time of the second injection in anesthetized rats. Desipramine which abolished or diminished the development of tachyphylaxis to the vasodepressor action of lycorenine (5 or 7.5 mg/kg i.v.), shortened the duration of its vasodepressor action and reduced its alpha-adrenergic blocking action. These results indicate that the development of tachyphylaxis to the vasodepressor action of lycorenine is ascribed to maintenance of its alpha-adrenergic blocking action.

Adrenergic alpha-Antagonists↗

Genetic variations in the chloroplast genome and phylogenetic clustering of Lycoris species.

The genus Lycoris of Amaryllidaceae comprises approximately 20 species that are distributed only in the moist warm temperate woodlands of eastern Asia. The objectives of this study were: (1) to clarify the phylogeny of the Lycoris species by using the definitive DNA sequencing method and (2) to examine the possible maternal donor of the hybrid origin Lycoris species and the Japanese triploid strains of Lycoris radiata var. radiata. The nucleotide sequence of the maturase K (matK) gene and the noncoding intergenic spacer (IGS) between the atpB and rbcL genes in the chloroplast genome were determined in a total of 27 strains of 11 species of the genus Lycoris. Variation among taxa was mainly due to nucleotide substitution, although deletions and an insertion were found in the IGS. For two chloroplast regions, the phylogenetic trees showed essentially similar topology, indicating the existence of four clades, I, II, III, and IV. For all the species except L. radiata, intraspecific variation was smaller than interspecific variation. For L. radiata, triploid strains were divided into clades I and II, and diploid strains were divided into clades I and IV. This implies that the diploid species of L. radiata var. pumila is a probable ancestral species. The clustering indicated that the chloroplast genome has not evolved in parallel with the karyotype in genus Lycoris. Regarding the hybrid origin species, the maternal parents of L. squamigara, L. albiflora and L. rosea were revealed to be L. longituba, L. radiata and L. radiata var pumila, respectively. We also suggest that a diploid strain of L. radiata var. pumila in clade I might be a candidate of the maternal donor of the Japanese triploid strains. A possible model of the maternal donor of Lycoris species is proposed.

Base Sequence↗

Flow injection analysis of binding reaction between fluorescent lectin and cells.

A fluorometric binding assay for lectin and yeast cells using the avidin-biotin system was previously reported (Y. Oda, M. Kinoshita, and K. Kakehi, Anal. Biochem. 254, 41-48, 1997). However, the true amount of bound lectin could not be determined by this method due to difficulty in determination of the number of bound biotin molecules. In the present study, we have developed a method for assaying the binding reaction between fluorescent lectin and cells using a flow injection technique, which allows estimation of the amount of lectin bound to cells. An aliquot of the cell suspension was directly analyzed by injection into a flow injection system after the binding between the fluorescently labeled lectin and cells. The labeled lectins showed good linearity, at least over a range of 20-1000 ng as the injected amount. The intrinsic fluorescence of the labeled lectins did not change upon the binding. The binding reaction of the hydroxycoumarin-labeled lectins with yeast cells was rapid and reached an equilibrium state within 10 min. Scatchard analysis showed that Saccharomyces cerevisiae cells contained approximately 1. 3-1.6 x 10(8) binding sites per cell for Concanavalin A, Lycoris radiata agglutinin, and Tulipa gesneriana lectin with affinity constants of 3.2-4.7 x 10(6) M-1. The present method was applied to the study of binding between lectins and bacteria and mouse spleen cells. The assay method described here is highly sensitive and will be an alternative to assays using lectins labeled with radioisotopes. The procedure is quite simple and can be completed within 1 h.

Agglutination Tests↗

Characterisation of an isolate of Narcissus degeneration virus from Chinese narcissus (Narcissus tazetta var. chinensis).

A potyvirus from Chinese narcissus was transmitted mechanically to three species of Narcissus and to Lycoris radiata but not to 22 other test species. In western blot, the coat protein reacted strongly with Narcissus degeneration virus (UK isolate) antiserum. Antiserum raised to the Chinese virus did not react with eighteen other potyviruses. The complete nucleotide sequence (9816 nt) had the typical genome organisation for a member of the genus Potyvirus. Sequence comparisons and phylogenetic analysis showed that the Chinese virus was different from all previously sequenced potyviruses but distantly related to onion yellow dwarf and shallot yellow stripe viruses.

Amino Acid Sequence↗

Identification of natural compounds with antiviral activities against SARS-associated coronavirus.

More than 200 Chinese medicinal herb extracts were screened for antiviral activities against Severe Acute Respiratory Syndrome-associated coronavirus (SARS-CoV) using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay for virus-induced cytopathic effect (CPE). Four of these extracts showed moderate to potent antiviral activities against SARS-CoV with 50% effective concentration (EC50) ranging from 2.4 +/- 0.2 to 88.2 +/- 7.7 microg/ml. Out of the four, Lycoris radiata was most potent. To identify the active component, L. radiata extract was subjected to further fractionation, purification, and CPE/MTS assays. This process led to the identification of a single substance lycorine as an anti-SARS-CoV component with an EC50 value of 15.7 +/- 1.2 nM. This compound has a CC50 value of 14980.0 +/- 912.0 nM in cytotoxicity assay and a selective index (SI) greater than 900. The results suggested that four herbal extracts and the compound lycorine are candidates for the development of new anti-SARS-CoV drugs in the treatment of SARS.

Amaryllidaceae Alkaloids↗

Plants used in Chinese and Indian traditional medicine for improvement of memory and cognitive function.

In traditional practices of Ayurvedic and Chinese medicine, numerous plants have been used to treat cognitive disorders, including neurodegenerative diseases such as Alzheimer's disease (AD). An ethnopharmacological approach has provided leads to identifying potential new drugs from plant sources, including those for cognitive disorders. Many drugs currently available in Western medicine were originally isolated from plants, or are derived from templates of compounds isolated from plants. Some anticholinesterase (anti-ChE) alkaloids isolated from plants have been investigated for their potential in the treatment of AD, and are now in clinical use. Galantamine, isolated from several plants including Lycoris radiata Herb., which was used in traditional Chinese medicine (TCM), is licensed in the United Kingdom for the treatment of mild to moderate AD. Various other plant species have shown pharmacological activities relevant to the treatment of cognitive disorders, indicating potential for therapeutic use in disorders such as AD. This article reviews some of the plants and their active constituents that have been used in traditional Ayurvedic medicine and TCM for their reputed cognitive-enhancing or antiageing effects. Plants and their constituents with pharmacological activities that may be relevant for the treatment of cognitive disorders, including enhancement of cholinergic function in the central nervous system (CNS), anti-inflammatory and antioxidant activities, are discussed.

Animals↗

Evaluation of fluorescence polarization method for binding study in carbohydrate-lectin interaction.

The fluorescence polarization (FP) technique was evaluated to determine molecular interaction between plant lectins and polysaccharides, yeast cells and glycopeptide after labeling the lectins with fluorescein isothiocyanate. Use of Lycoris radiata agglutinin allowed determination of the molecular interactions with large biomolecules containing mannose oligomers and polymers. Another example using a fluorescein-labeled glycopeptide also indicated that use of the FP method would allow easy observation of the molecular interactions on the quantitative base. The present technique is highly sensitive and facile because it does not require any washing procedures before measurement.

Binding Sites↗

[Pharmacology of lycorine. 1) Effect on biliary secretion in the rat].

The Authors study pharmacological actions of lycorine, alkaloid of Lycoris radiata. On the basis that anti-inflammatory substances have a choleretic effect, the activity of lycorine on biliary secretion of rats has been investigated. Lycorine, at a dose of 1 mg/kg induces, in rats anaesthetized with urethane (1,2 g/kg i.m.), a marked choleretic effect. This experiment do not define the mechanism responsible for the observed choleresis.

Amaryllidaceae Alkaloids↗