Hydroxycinnamoyltransferases Involved in the Accumulation of Caffeic Acid Esters in Gametophytes and Sporophytes of Equisetum arvense

Four hydroxycinnamoyltransferases from Equisetum arvense L. were studied that catalyze the formation of mono-O-caffeoyl-meso-tartrate, di-O-caffeoyl-meso-tartrate, 5-O-caffeoylshikimate (dactylifrate), and 5-O-caffeoylquinate (chlorogenate). The enzymes were classified as coenzyme A (CoA)-ester-dependent acyltransferases (EC 2.3.1), i.e. hydroxycinnamoyl-CoA:meso-tartrate hydroxycinnamoyltransferase (CTT), hydroxycinnamoyl-CoA:caf-feoyl-meso-tartrate hydroxycinnamoyltransferase (CCT), hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase (CST), and hydroxycinnamoyl-CoA:quinate hydroxycinnamoyltransferase. The CTT, CCT, and CST were partially purified and separated from E. arvense gametophytes by hydrophobic interaction chromatography on Fractogel TSK Butyl-650 followed by molecular exclusion on fast protein liquid chromatography-Superdex-75 with 87-, 62-, and 130- fold enrichments and 12, 8, and 11% yields, respectively. The enzyme activities obtained with caffeoyl-CoA were 95 (CTT), 74 (CCT), and 200 [mu]kat (CST) kg-1 protein. The apparent native relative molecular weight values were found to be approximately 45,000 (CTT), 52,000 (CCT), and 50,000 (CST). Each enzyme showed highest activities at pH 7.5, the CCT and CST in Tris-HCl (1.2 and 1.0 M) and the CTT in imidazole-HCl (1.25 M). Enzyme activities were stimulated more than 3-fold by 100 mM ascorbate. The apparent energies of activation (kilojoules mol-1) were calculated to be 56 (CTT), 69 (CST), and 76 (CCT). The enzymes accepted cinnamoyl-CoA and various hydroxycinnamoyl-CoAs. The time course of the transferase activities along with that of a fourth one, hydroxycinnamoyl-CoA:quinate hydroxycinnamoyltransferase, and the pattern of product accumulation were determined during a 1-year growth period of the E. arvense sporophytes.     

Inhibition of human in vitro osteoclastogenesis by Equisetum arvense

Objectives

Equisetum arvense has long been used in traditional medicines to treat different disorders, including bone pathologies. In this study a hydromethanolic extract of E. arvense was assessed for its effects on human osteoclastogenesis.

Materials and methods

Osteoclast precursors were maintained in non‐stimulated and stimulated (presence of M‐CSF and RANKL) conditions, or in co‐cultures with osteoblasts. Cell cultures were treated with 0.00016–0.5 mg/ml of a hydromethanolic E. arvense extract.

Results

The extract did not affect spontaneous osteoclastogenesis. In osteoclast precursors committed to osteoclastogenesis (stimulated or co‐cultured with osteoblasts), E. arvense caused dose‐dependent inhibitory effect that became statistically significant at concentrations ≥0.004 mg/ml. This was observed using different osteoclast differentiation and activation markers. Cell response was associated with changes in relative contribution of MEK and NFkB signalling pathways, as well as PGE2 production. As there were differences in the response of osteoclast precursors maintained in the presence of inductive factors, or co‐cultured with osteoblastic cells, it seems that E. arvense extract had the ability to modulate osteoclastogenesis, either by acting directly on osteoclast precursor cells, and/or via osteoblasts.

Conclusions

Equisetum appeared to have a negative effect on human osteoclastogenesis, which is in line with its putative beneficial role in pathophysiological conditions associated with increased osteoclastic activity, and might suggest potential utility for treatment with bone regeneration strategies.     

Enhancement of the Division of Equisetum arvense Protoplasts in Culture by Activated Charcoal and Their Further Development

Protoplasts were isolated from subcultured gametophytes of Equisetumarvense by treatment with Driselase and then cultured in vitro.Addition of activated charcoal (AC) to the culture medium enhancedthe rate of cell division, as well as the survival of both protoplastsand regenerated protoplasts. However, subsequent division ofcells was not observed after one or two cycles of replicationin cultures supplemented with AC. When regenerated protoplastswere transferred to fresh medium without AC 3 to 5 weeks afterthe first plating, the transferred cells formed rhizoids anddeveloped into small, young gametophytes without the prior formationof cell clusters or calluses. Furthermore, sprophytic shootsdifferentiated from the protoplast-derived gametophytes whenthey were cultured on medium supplemented with 6-benzylaminopurine(BA). (Received April 5, 1990; Accepted July 30, 1990)     

Sterol Composition of the Strobilus of Equisetum arvense L.

“Process whey protein” was prepared by heating bovine milk whey protein isolate solution at neutral pH under salt-free conditions. The process whey protein solution, being clear, was heated at various pHs (2.0 to 11.0) and NaCl concentrations (0 to 200 mM), and the turbidity and gel properties of the products were then examined. For comparison, the properties of the whey protein isolate treated under the same conditions were measured. The whey protein isolate formed a transparent gel or sol below pH 3 and above pH 7 at low NaCl concentration after heating, but the process whey protein formed transparent gels and sols over a wider range of pH and NaCl concentrations than those of the whey protein isolate. More elastic, firmer, and denser gels were obtained from the process whey protein than from the whey protein isolate. The process whey protein provides a novel food material with useful properties.     

Anxiolytic effects of Equisetum arvense Linn. extracts in mice

The petroleum ether (PE), chloroform (CH), ethanol (ETH) and water extracts of E. arvense stems were evaluated for anti-anxiety activity in mice using elevated plus maze model. Ketamine induced hypnosis and actophotometer was used to evaluate sedative effect with various extracts in mice. The results were compared with standard drug diazepam. The ethanolic extract of E. arvense (50 and 100 mg/kg) significantly increased the time-spent and the percentage of the open arm entries in the elevated plus-maze model which was comparable to diazepam. Ethanolic extract (100 mg/kg) prolonged the ketamine-induced total sleeping time and decreased the locomotor activity in mice. The results suggest that the ethanolic extract of E. arvense seems to possess anxiolytic effect with lower sedative activity than that of diazepam. The results could be attributed to the flavonoid content of the ethanolic extract.     

问荆中三个新的酚甙化合物

从问荆（Equisetum arvense L.)营养茎的水溶性成分中分离到１２个化合物。经波谱解析和化学方法鉴定。其中３个新的酚甙化合物分别命名为equisetumosideA(3-methoxy-11,12-dihydroxy-phenylhexane-9-one-4-O-β－Ｄglucopyra-noside)、equisetumoside B(3-methoxy-4,11-dihdroxy-phenylhexane-9-one-12-O-β－Ｄ－glucopyranoside)和equisetumosideＣ（cis-ferulic acid potassium salt 4-O-β－Ｄ－glucopyranoside);其他成分鉴定为尿甙、次黄甙、２′－脱氧次黄甙、２′－脱氧胞甙、色氨酸、胸甙、５－羟基－２′－脱氧尿甙、松柏甙和莰非醇－３－O-β－Ｄ－槐糖基 －７－O-β－Ｄ－吡喃葡萄糖甙。这些化合物均为首次从该植物中分离得到。     

Roles of Inorganic Nitrogen in Gametophytic Growth and in Initiation and Development of Sporophytic Shoots of Equisetum arvense

The formation of sporophytic shoots, which is induced by treatmentwith benzylaminopurine of gametophyte tissue of Equisetum arvense,can be divided into initiation and developmental phases. Thenitrogen in MS medium was suitable for two phases as well as gametophytic growth, buta reduction in the concentration of available nitrogen was neededfor the development of shoots. ions alone were effective for gametophytic growth and the initiationof sporophytic shoots, but both and ions was required for the developmental phase. (Received February 18, 1992; Accepted April 14, 1992)     

Arrangement of microtubules during spore formation in Equisetum arvense (Equisetaceae)

The arrangement of cortical microtubules (MTs) during spore formation in Equisetum arvense was examined by immunofluorescence microscopy. The arrangement of MTs was observed to change during sporoderm formation. During exospore formation, the cortical MTs of the tapetum appeared along the tapetal plasma membrane that enclosed each developing spore cell. After exospore formation, the arrangement of the cortical MTs changed into one of separate bands of MTs arranged spirally (spiral bands of MTs). The spiral bands of MTs were superimposed on the developing elaters. This new pattern corresponded to the pattern of cellulose microfibrils deposited in the inner layer of the elater, suggesting that these spiral bands are involved in the deposition of the cellulose microfibrils in the elater. We conclude that the spiral bands of MTs are functionally equivalent to cortical MTs in secondary wall formation.     

Tubers and rhizome fragments as propagules: competence for vegetative reproduction in Equisetum arvense

Rhizome fragments (referred to as “fragments”) and tubers of Equisetum arvense L. were cultured in order to investigate their competence with respect to vegetative reproduction. The starch concentration of the fragments was lower than that of the tubers, but the initial growth of new individuals from these fragments was superior to that from tubers obtained from the same dry mass. This superior growth was due to the large number of buds (grown from nodes) and aerial shoots on the fragments. The competence for vegetative reproduction depended on the relationship between the stored starch and the number of buds.     

Exploitation of the molecular potential of plants:Equisetum arvense (EQUISETACEAE)

After a search of the literature including computerized data bases on the economic potential of various plants, experiments designed to improve the extraction of silica from the horsetailEquisetum arvense L. were carried out. Improved yields of colloidal silica were obtained by sonication in a shorter time than by conventional extraction with boiling distilled water. However we conclude that commercial exploitation of silica (colloidal or solid) fromEquisetum arvense is not feasible at present because: growing conditions of this plant species are not yet well defined; the disappearance of the plant during the winter months limits its period of exploitation; the silica content of the plant can vary over a six-fold range depending on weather and soil conditions, and so its economic potential is unpredictable.     

A Suspension Culture Method for the Rapid Mass Culture of Cistella japonica Mycelium

Different methods were investigated for the rapid mass culture of Cistella japonica by using water extracts of some nutritional sources. In an agar culture test, there was little difference in mycelial growth in water extracts of wheat bran, rice bran and potato. In suspension culture with wheat bran extract, which is easily and cheaply available, the mycelium of C. japonica increased seven times more than that in agar culture after a month's incubation. C. japonica from suspension culture was pathogenic to Chamaecyparis obtusa. These results suggest that suspension culture in water extract of wheat bran can be adopted for the rapid mass culturing of C. japonica for use in inoculation tests.     

蕨类植物问荆DEAD-box RNA解旋酶基因的克隆与分析

DEAD-box RNA解旋酶参与RNA多方面的代谢,在植物生长发育和逆境反应中起重要作用。本研究从蕨类植物问荆（Equisetum arvense）中克隆到一条DEAD-box RNA解旋酶c DNA全长序列,命名为EaRH1,并在Gen Bank注册登记（KJ734026）。序列分析显示：该c DNA全长3 230 bp,包含一个从487 bp到2 799 bp编码770个氨基酸的开放读码框,其对应的蛋白序列包含9个保守模块结构。EaRH1与其它物种DEAD-box RNA解旋酶蛋白序列比对结果显示：模块Ⅰa、Ⅱ和Ⅲ序列几乎完全相同,模块Q、Ⅰ和Ⅳ序列存在一些差异。EaRH1与江南卷柏（Selaginella moellendorffii）基因组一条假定序列相似度高达69%,其中相似度最高的区域集中在包含9个保守模块的结构域。系统进化树分析显示：EaRH1与拟南芥（Arabidopsis thaliana）DEAD-box RNA解旋酶At3g22320在氨基酸序列上有相对较高的同源性。序列结构比较和进化分析可推测出EaRH1可能参与植物体生长发育、miRNA生物合成、与RNA结合蛋白的相互作用和非生物胁迫应答。本文的研究为探索问荆DEAD-box RNA解旋酶的进一步功能提供参考。     

A structural basis of Equisetum arvense ferredoxin isoform II producing an alternative electron transfer with ferredoxin-NADP+ reductase

We have determined the crystal structure, at 1.2-A resolution, of Equisetum arvense ferredoxin isoform II (FdII), which lacks residues equivalent to Arg(39) and Glu(28) highly conserved among other ferredoxins (Fds). In other Fds these residues form an intramolecular salt bridge crucial for stabilization of the [2Fe-2S] cluster, which is disrupted upon complex formation with Fd-NADP(+) oxidoreductase (FNR) to form two intermolecular salt bridges. The overall structure of FdII resembles the known backbone structures of E. arvense isoform I (FdI) and other plant-type Fds. Dramatically, in the FdII structure a unique, alternative salt bridge is formed between Arg(22) and Glu(58). This results in a different relative orientation of the alpha-helix formed by Leu(23)-Glu(29) and eliminates the possibility of forming three of the five intermolecular salt bridges identified on formation of a complex between maize FdI and maize FNR. Mutation of FdII, informed by structural differences with FdI, showed that the alternative salt bridge and the absence of an otherwise conserved Tyr residue are important for the alternative stabilization of the FdII [2Fe-2S] cluster. We also investigated FdI and FdII electron transfer to FNR on chloroplast thylakoid membranes. The K(m) and V(max) values of FdII are similar to those of FdI, contrary to previous measurements of the reverse reaction, from FNR to Fd. The affinity between reduced FdI and oxidized FNR is much greater than that between oxidized FdI and reduced FNR, whereas this is not the case with FdII. The pH dependence of electron transfer by FdI, FdII, and an FdII mutant with FdI features was measured and further indicated that the binding mode to FNR differs between FdI and FdII. Based on this evidence, we hypothesize that binding modes with other Fd-dependent reductases may also vary between FdI and FdII. The structural differences between FdI and FdII therefore result in functional differences that may influence partitioning of electrons into different redox metabolic pathways.     

蕨类植物问荆DEAD box RNA解旋酶基因的克隆与分析

DEAD box RNA解旋酶参与RNA多方面的代谢,在植物生长发育和逆境反应中起重要作用。本研究从蕨类植物问荆(Equisetum arvense)中克隆到一条DEAD box RNA解旋酶cDNA全长序列,命名为EaRH1,并在GenBank注册登记（KJ734026）。序列分析显示：该cDNA全长3230bp,包含一个从487bp到2799bp编码770个氨基酸的开放读码框,其对应的蛋白序列包含9个保守模块结构。EaRH1与其它物种DEAD box RNA 解旋酶蛋白序列比对结果显示：模块Ⅰa、Ⅱ和Ⅲ序列几乎完全相同,模块Q、Ⅰ和 Ⅳ序列存在一些差异。EaRH1与江南卷柏(Selaginella moellendorffii)基因组一条假定序列相似度高达69%,其中相似度最高的区域集中在包含9个保守模块的结构域。系统进化树分析显示：EaRH1与拟南芥(Arabidopsis thaliana)DEAD box RNA解旋酶At3g22320在氨基酸序列上有相对较高的同源性。序列结构比较和进化分析可推测出EaRH1可能参与植物体生长发育、miRNA生物合成、与RNA结合蛋白的相互作用和非生物胁迫应答。本文的研究为探索问荆DEAD box RNA解旋酶的进一步功能提供参考。     

The nucleotide sequence of the cytoplasmic 5S rRNA from the horsetail, Equisetum arvense

Using 3'- and 5'-end labelling sequencing techniques, the following sequence for the cytoplasmic 5S rRNA of the horsetail Equisetum arvense could be determined: (sequence in text). This sequence exhibits all features expected for higher plant cytoplasmic 5S rRNAs, and can be fitted to the secondary structure model for 5S rRNA proposed by De Wachter et al. (15).     

A Method for the Rapid Enumeration of Mycoplasma Species growing in Broth Culture

S ummary : A method is outlined whereby Mycoplasma species growing in broth culture can be rapidly counted, using the relationship between the number of organisms and the mean lactate dehydrogenase (LDH) content of each organism. The uses of this technique are discussed.     

Induction of Biplanar Growth in Fern Gametophytes

In constant white light, protonemata of different ages of Asplenium nidus produce a fixed number of cells before they become biplanar. Protonemata with a lower initial cell number require a longer time to become biplanar than those with a higher initial cell number. Increasing the photoperiod or the light intensity reduces the cell number in the filament at the time of initiation of biplanar morphology. It is suggested that inductive light acts directly on cell metabolism to induce biplanar morphology, rather than indirectly by inducing mitotic activity.     

Genetic diversity and population structure in the outcrossing populations of Equisetum arvense and E. hyemale (Equisetaceae)

An allozyme examination was conducted to study the mating systems and genetic differentiation of populations of Equisetum arvense and E. hyemale. The study revealed that the rate of intragametophytic selfing in these homosporous pteridophytes is very low, i.e., on average 0.020 and 0.019, respectively, despite the potential hermaproditism and selfing of the gametophytes. Most populations consisted of numerous genotypes, and the average heterozygosities of E. arvense and E. hyemale equalled 0.092 and 0.134, respectively. The commonly observed excess of the heterozygote genotypes indicates that there are interclonal differences in the frequency of vegetative reproduction. The level of genetic divergence among populations was considerable even within a limited geographic area. It is suggested that the life history of Equisetum, characterized by the inefficiency of spore germination and gametophyte reproduction in noncolonizing situations, limits the level of gene flow and leads to a great genetic divergence between populations.