Simple kinetic determination of trace amounts of ascorbic acid in plant extracts

Aa kinetic method of ascorbic acid assay based on the lag produced during the evolution of horse radish peroxidase-catalyzed oxidation of guaiacol or of homovanillic acid by hydrogen peroxide, is described and applied to ascorbic acid determination in Sedum album extracts.     

Antioxidative protection in the inducible CAM plant Sedum album L. following the imposition of severe water stress and recovery

The antioxidative protection during the C₃-CAM shift induced by water stress was investigated in the temperate succulent Sedum album L. The C₃-CAM shift was characterized in terms of CO₂ exchange, titratable acidity and phosphoenolpyruvate carboxylase activity. Well-watered plants displayed C₃-like patterns of gas exchange and exhibited a mild day-night acid fluctuation indicating that those plants were performing CAM-cycling metabolism. Imposed drought highly stimulated CAM cycling, decreasing the net CO₂ uptake during the day, eliminating net CO₂ efflux at night and stimulating tissue acid fluctuations. As water deficit developed, chlorophyll fluorescence measurements showed a decrease in the Fv/Fm ratio, indicating that photoinhibition could follow after severe drought. Protection might be performed by the increased activity of enzymes involved in the destruction of free radicals and oxidants, but their response depended on the water status of the plant. Ascorbate peroxidase and superoxide dismutase activities increased in plants subjected to mild stress but declined during severe water stress. Catalase activity, however, was quite stable under mild water stress and was clearly inhibited under severe water stress. At this stage, glutathione reductase and monodehydroascorbate reductase seemed to be very important in the protection against oxidants, both increasing considerably their activities under severe water stress. Even if recycling has been shown to alleviate photoinhibition, our results clearly demonstrate that antioxidative enzymes play an important role in the protection of plants from oxidants during the C₃-CAM shift induced by water stress.     

巨桉不同状态叶片浸提液对萝卜幼苗形态和抗性生理特性的影响

以珍珠岩作为基质,选择4年生巨桉(Eucalyptus grandis)嫩叶(T1)、老叶(T2)、表层凋落叶(T3)、腐解凋落叶(T4)4种状态的叶片,每种状态叶片设置3个浸提液浓度水平[分别称取风干叶片30g、15g和7.5g加入900mL蒸馏水进行浸提,以蒸馏水为对照(CK)],采用水培法研究了不同状态叶片浸提液对萝卜(Raphanus sativus)幼苗形态生长和抗性生理特性的影响。结果显示:(1)巨桉不同状态叶片浸提液显著抑制了萝卜幼苗的根长,其中嫩叶的抑制作用最强,腐解凋落叶抑制作用最弱。(2)各状态叶片浸提液处理后萝卜幼苗中过氧化氢酶(CAT)和过氧化物酶(POD)的活性均呈现升高趋势,嫩叶各浓度处理以及其他状态叶片的高浓度处理下超氧化物歧化酶(SOD)活性升高,而其余浓度处理的SOD活性降低。(3)各状态叶片浸提液处理萝卜幼苗的丙二醛(MDA)含量在低浓度处理时低于CK,其余处理下则高于CK。(4)嫩叶各浓度处理萝卜幼苗的可溶性糖(SS)含量显著高于CK,且随着老叶和表层凋落叶浸提液浓度的升高,幼苗SS含量先升后降,腐解凋落叶各浓度处理下则呈渐增的趋势;而可溶性蛋白(SP)含量则随浸提液浓度的增加而升高,且T2和T3两种状态叶片的各浓度处理与CK差异显著。研究表明,巨桉不同状态叶片浸提液对萝卜幼苗生长和抗性生理产生了强烈的抑制作用,其中以嫩叶最强,老叶和表层凋落叶次之,腐解凋落叶最弱。     

Wound healing and in vitro antiradical activity of five Sedum species grown within two sites of community importance in Emilia-Romagna (Italy)

Sedum genus includes more than 400 different species, many of which having ethnobotany interest. The skin healing is one of the most common therapeutic indication of Sedum spp. In this work, for the first time, we compared five different Sedum species grown in two sites of community importance in Emilia Romagna (Italy): Sedum acre L., Sedum album L., Sedum hispanicum L., Sedum rupestre L. and Sedum sexangulare L., analysing their total phenolic and flavonoid content, their antiradical capacity and the in vitro healing activity on human keratinocytes. Total phenolic content of the five species ranged from 35.41?±?1.18 to 90.22?±?1.03?µg gallic acid equivalent/mg of dry extract, being S. rupestre the richest one. Total flavonoid content ranged from 22.39?±?0.51 to 47.93?±?2.82?µg rutin equivalent/mg of extract and S. album resulted the species with the highest flavonoid content. Antiradical capacity was found to be related to the phenolic content of the extracts. All the extracts were active in wound healing assay and each one showed different kinetic of action and concentration-activity relationship. This study proposes few investigated Sedum species grown in Italy as promising agents for skin healing and suggests further phytochemical and biological investigations.     

Ascorbate-independent electron transfer between cytochromeb 561 and a 27 kDa ascorbate peroxidase of bean hypocotyls

Summary Cytochromeb ₅₆₁ (cytb ₅₆₁) is a trans-membrane cytochrome probably ubiquitous in plant cells. In vitro, it is readily reduced by ascorbate or by juglonol, which in plasma membrane (PM) preparations from plant tissues is efficiently produced by a PM-associated NAD(P)Hquinone reductase activity. In bean hypocotyl PM, juglonol-reduced cytb ₅₆₁ was not oxidized by hydrogen peroxide alone, but hydrogen peroxide led to complete oxidation of the cytochrome in the presence of a peroxidase found in apoplastic extracts of bean hypocotyls. This peroxidase active on cytb ₅₆₁ was purified from the apoplastic extract and identified as an ascorbate peroxidase of the cytosolic type. The identification was based on several grounds, including the ascorbate peroxidase activity (albeit labile), the apparent molecular mass of the subunit of 27 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the dimeric native structure, the typical spectral properties of a heme-containing peroxidase, and an N-terminal sequence strongly conserved with cytosolic ascorbate peroxidases of plants. Cytb ₅₆₁ used in the experiments was purified from bean hypocotyl PM and juglonol was enzymatically produced by recombinant NAD(P)H:quinone reductase. It is shown that NADPH, NAD(P)H:quinone reductase, juglone, cytb ₅₆₁, the peroxidase interacting with cytb ₅₆₁, and H₂O₂, in this order, constitute an artificial electron transfer chain in which cytb ₅₆₁ is indirectly reduced by NADPH and indirectly oxidized by H₂O₂.Abbreviations APX ascorbate peroxidase - b ₅₆₁PX cytochrome 6561 peroxidase - CPX coniferol peroxidase - cyt cytochrome - GPX guaia-col peroxidase - IWF intercellular washing fluid - MDHA monodehydroascorbate - PM plasma membrane     

Behavior of organelle nuclei (nucleoids) in generative and vegetative cells during maturation of pollen inLilium longiflorum andPelargonium zonale

Summary The behavior of organelle nuclei during maturation of the male gametes ofLilium longiflorum andPelargonium zonale was examined by fluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI) and Southern hybridization. The organelle nuclei in both generative and vegetative cells inL. longiflorum were preferentially degraded during the maturation of the male gametes. In the mature pollen grains ofL. longiflorum, there were absolutely no organelle nuclei visible in the cytoplasm of the generative cells. In the vegetative cells, almost all the organelle nuclei were degraded. However, in contrast to the situation in generative cells, the last vestiges of organelle nuclei in vegetative cells did not disappear completely. They remained in evidence in the vegetative cells during germination of the pollen tubes. InP. zonale, however, no evidence of degradation of organelle nuclei was ever observed. As a result, a very large number of organelle nuclei remained in the sperm cells during maturation of the pollen grains. When the total DNA isolated from the pollen or pollen tubes was analyzed by Southern hybridization with a probe that contained therbc L gene, for detection of the plastid DNA and a probe that contained thecox I gene, for detection of the mitochondrial DNA, the same results were obtained. Therefore, the maternal inheritance of the organelle genes inL. longiflorum is caused by the degradation of the organelle DNA in the generative cells while the biparental inheritance of the organelle genes inP. zonale is the result of the preservation of the organelle DNA in the generative and sperm cells. To characterize the degradation of the organelle nuclei, nucleolytic activities in mature pollen were analyzed by an in situ assay on an SDS-DNA-gel after electrophoresis. The results revealed that a 40kDa Ca²⁺-dependent nuclease and a 23 kDa Zn²⁺ -dependent nuclease were present specifically among the pollen proteins ofL. longiflorum. By contrast, no nucleolytic activity was detected in a similar analysis of pollen proteins ofP. zonale.     

Studies on carbon flow in Crassulacean acid metabolism during the initial light period

In the Crassulacean acid metabolism (CAM) plants Kalanchoë tubiflora and Sedum morganianum a shift in the pathways occurs by which external CO₂ enters the metabolism during the initial light period (phase II of the diurnal CAM cycle). At the beginning of phase II, CO₂ is fixed mainly by the C₄ pathway; during late phase II, however, it is fixed mainly via the C₃ pathway. The C₃ pathway contributes to the phosphoenolpyruvate-carboxylase-mediated CO₂ fixation by the provision of three-carbon skeletons. Since the shift in the carbon-flow pathway is delayed after a CO₂-free night when malic-acid accumulation in the vacuoles is prevented, it is very likely that the amount of malic acid in the vacuole is integrated in the mechanism which controls CAM during the initial light period. A light-on signal at the beginning of phase II is not required to bring about the shifts in the carbon-flow pathways, as is shown by the reaction of plants to a prolonged dark period. A model of carbon flow during phase II is proposed.Abbreviations CAM Crassulacean acid metabolism - PEP-Case phosphoenolpyruvate carboxylase     

Fluorescence microscopic study of the formation of giant mitochondrial nuclei in the young ovules ofPelargonium zonale

Summary The size of mitochondrial genomes in higher plants are known to range from 200 to 2400 kilobase pairs. However, we failed to identify cytochemically any mitochondria that contain an identifiable master mitochondrial genome. In the present experiments, we have found the giant mitochondrial nuclei which have the capacity for including the master mitochondrial genome in the young ovaries ofPelargonium zonale by use of a 4-6-diamidino-2-phenylindole (DAPI) epifluorescence microscopy, a Technovit embedding, and a video-intensified photon counting system.     

Cytoplasmic inheritance and ultrastructure of the male generative cell of higher plants

Summary The ultrastructure of the generative cell of the pollen grain has been studied in two different plants: Mirabilis jalapa L., where variegation is transmitted only by the egg cell, and Pelargonium zonale Ait., where variegation can be transmitted also by the pollen grain. It was found that only in Pelargonium zonale Ait. does the male generative cell possesses a great number of proplastids.     

Epidermal structures and composition of epicuticular waxes of Sedum album sensu lato (Crassulaceae) in Balkan Peninsula

Sedum album is a succulent plant, with phenotypic variability resulting in delimitation of a number of taxa with diverse taxonomic recognition. We analyzed the taxa from Sedum album in broad sense (S. album, S. micranthum, S. athoum and S. serpentini) from Balkan Peninsula, to check whether the variability of their epidermal structures and epicuticular wax composition corresponds to their taxonomic reliability. Epidermal structures and epicuticular waxes were analyzed using light and scanning electron microscopy, gas chromatography-mass spectrometry and gas chromatography-flame ionization detector chromatography and multivariate statistical techniques. Analyses of studied taxa have shown that significant systematic characters include adaxial epidermal and guard cell length, stomatal frequency and subsidiary cells area on abaxial epidermis. The content of n-alkanes C₂₉, C₃₀, C₂₇, C₃₂ and C₃₃ was shown to be valuable criterion for their phytochemical delimitation. The comparison of epicuticular wax microstructures has shown no significant differences. Results indicate presence of two (micromorphology) or three (phytochemistry) well-defined groups of populations, with S. serpentini manifesting a considerable level of separation toward the others. Sedum athoum have shown weak to moderate degree of differentiation, while S. micranthum has shown general resemblance to S. album.     

Re-examination of Mg-dechelation reaction in the degradation of chlorophylls using chlorophyllin a as a substrate

The Mg-dechelating activity of extracts of Chenopodium album (goosefoot) was investigated using an artificial substrate, chlorophyllin a. The activity was measured spectrophotometrically by the formation of a reaction product, pheophorbin a (Mg-free chlorin), after release of the central Mg. The Mg-releasing protein was highly purified by successive DEAE, Butyl and HW-55 chromatographies. The molecular weight of the purified protein was 20 k by gel filtration. The protein showed a broad, but single, pH optimum at 7.5. The K _m value for chlorophyllin a was 95.1 nM at pH 7.5. The Mg-releasing protein was not active with chlorophyllide a, a native substrate, although it was active with Zn-chlorophyllin a. Similar results were obtained from horseradish peroxidase. Only a small molecular weight, metal-chelating substance (MCS) had Mg-dechelating activity for the native substrate. An inhibitor study showed involvement of radicals in the Mg-dechelation of the Mg-releasing protein. The purified Mg-releasing protein showed neither peroxidase activity nor absorption bands in the visible region, and this indicates that the Mg-releasing protein is clearly distinct from horseradish peroxidase, which is a heme-containing protein. A likely conclusion is that the Mg-releasing protein and horseradish peroxidase are not involved in the Mg-dechelation in the degradation pathway of chlorophylls. The relevance of the participation of MCS in Mg-dechelation in the breakdown of chlorophylls (Chls) is also discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Variations of chloroplast DNAs in the genus Pelargonium and their biparental inheritance

Summary The comparison of EcoRI patterns of chloroplast DNAs (ctDNAs) from five species of the genus Pelargonium and from 16 cultivars and varieties of Pelargonium zonale hort. demonstrates a remarkable inter- and intraspecific ctDNA (plastome) variation. The plastome of the P. zonale varieties could be differentiated into groups I, II and III. Reasons for this variation seem to be: occurrence of numerous spontaneous plastome mutations, intense hybridisation by gardeners and breeders, and biparental plastid inheritance.Crosses of P. zonale varieties with different ctDNA types lead to the direct evidence on the molecular level of biparental plastid inheritance and plastid sorting-out in F₁-hybrids.     

Escherichia coli expression and in vitro activation of a unique ligninolytic peroxidase that has a catalytic tyrosine residue

Heterologous expression of Trametes cervina lignin peroxidase (LiP), the only basidiomycete peroxidase that has a catalytic tyrosine, was investigated. The mature LiP cDNA was cloned into the pET vector and used to transform Escherichia coli. Recombinant LiP protein accumulated in inclusion bodies as an inactive form. Refolding conditions for its in vitro activation—including incorporation of heme and structural Ca²⁺ ions, and formation of disulfide bridges—were optimized taking as a starting point those reported for other plant and fungal peroxidases. The absorption spectrum of the refolded enzyme was identical to that of wild LiP from T. cervina suggesting that it was properly folded. The enzyme was able to oxidize 1,4-dimethoxybenzene and ferrocytochrome c confirming its high redox potential and ability to oxidize large substrates. However, during oxidation of veratryl alcohol (VA), the physiological LiP substrate, an unexpected initial lag period was observed. Possible modification of the enzyme was investigated by incubating it with H₂O₂ and VA (for 30 min before dialysis). The pretreated enzyme showed normal kinetics traces for VA oxidation, without the initial lag previously observed. Steady-state kinetics of the pretreated LiP were almost the same as the recombinant enzyme before the pretreatment. Moreover, the catalytic constant (k_cat) for VA oxidation was comparable to that of wild LiP from T. cervina, although the Michaelis–Menten constant (K_m) was 8-fold higher. The present heterologous expression system provides a valuable tool to investigate structure–function relationships, and autocatalytic activation of the unique T. cervina LiP.     

Activation of indole-3-acetic acid oxidase from horseradish and Prunus by phenols and H2O2

The enzyme-catalysed oxidation of indole-3-acetic acid (IAA) was sytematically investigated with respect to enzyme source and cofactor influence using differential spectrophotometry and oxygen uptake measurement. Commercially-available horseradish peroxidase (HRP) and a peroxidase preparation from Prunus phloem showed identical catalytic properties in degrading IAA. There was no lag phase of IAA oxidation with any of the reaction mixtures tested. Monophenols exhibited a much stronger stimulatory effect than inorganic cofactors, but during the incubation of IAA the phenols were also gradually oxidised. Hydrogen peroxide (H₂O₂) in combination with monophenols accelerated peroxidation of the monophenol and IAA oxidation simutaneously. Since photometric determination of IAA was affected by oxidation products of dichlorophenol or phenol contamination of the enzyme preparation used, the standard IAA absorption measurements appear to be susceptible to methodological errors. Under certain incubation conditions a catalase-like activity of HRP during the course of IAA oxidation was noted and substrate inhibition was observed above 1.5 × 10^\s-4 M IAA. Some concepts concerning the mode of activation of the enzyme-catalysed IAA oxidation are deduced from the experimental results.     

Seed treatment with aqueous extract of Viscum album induces resistance to pearl millet downy mildew pathogen

Abstract

Downy mildew (Sclerospora graminicola [Sacc.] Schroet.) is a serious agricultural problem for pearl millet (Pennisetum glaucum [L.] R. Br.) grain production under field conditions. Six medicinally important plant species Azadirachta indica, Argemone mexicana, Commiphora caudata, Mentha piperita, Emblica officinalis and Viscum album were evaluated for their efficacy against pearl millet downy mildew. Seeds of pearl millet were treated with different concentrations of aqueous extract of the plants to examine their efficacy in controlling downy mildew. Among the plant extracts tested, V. album treatment was found to be more effective in enhancing seed quality parameters and also in inducing resistance against downy mildew disease. Germination and seedling vigor was improved in seeds treated with V. album extracts over control. Seeds treated with 10% concentration of V. album showed maximum protection against downy mildew disease under greenhouse and field conditions. The downy mildew disease protection varied from 44–70% with different concentrations. Leaf extract of V. album did not inhibit sporulation and zoospore release from sporangia of Sclerospora graminicola, indicating that the disease-controlling effect was attributed to induced resistance. Seed treatment with V. album extract increased pearl millet grain yield considerably. In V. album, treated pearl millet seedlings increased activities of peroxidase, and phenylalanine ammonia-lyase enzyme was detected. FTIR analysis of V. album extracts showed the presence of amides and other aromatic compounds which are antimicrobial compounds involved in plant defense.     

Water-soluble phenolic compounds in the coat control germination and peroxidase reactivation in <Emphasis Type="Italic">Triticum aestivum</Emphasis> seeds

In this work, we investigated the inhibitory effects of water-soluble phenolic compounds (WSPCs) in the coat of after-ripening wheat (Triticum aestivum L.) seeds on the processes of germination and peroxidase reactivation. Wheat bran has a WSPC content of 862.5 μg gallic acid equivalent g⁻¹ dry weight. When seeds were incubated in the water extract of bran, germination, peroxidase reactivation, and coleoptile and radicle growth were suppressed in a WSPC concentration-dependent manner. The inhibitory effects were significantly ameliorated by removing WSPCs from bran extract by treating with 1% insoluble polyvinylpolypyrrolidone. Pretreatment of seeds with 0.1% H₂O₂ reduced the WSPC content in the coat, which was confirmed using Fourier transform infrared microspectroscopy. With H₂O₂ pretreatment, seed germination, peroxidase reactivation, and post-germination seedling growth were significantly stimulated. Application of the known phenolics caffeic acid, feruic acid, or vanillin to the germination medium blocked seed germination and suppressed peroxidase reactivation. The results described here indicate that WSPCs act as endogenous inhibitors in the coat to control germination of Triticum aestivum seeds, and that inhibition of germination is at least partially caused by suppressing peroxidase reactivation.     

Modes of regeneration in <Emphasis Type="Italic">Pelargonium × hortorum (Geraniaceae)</Emphasis> and three closely related species

Summary Modes of regeneration from hypocotyl explants were studied in Pelargonium × hortorum ‘Scarlet Orbit,’ and three wild relatives, P. zonale, P. alchemilloides, and P. inquinans, on different cytokinin treatments [1 μM thidiazuron (TDZ), 4 μM TDZ, or 8 μM N⁶-benzylaminopurine (BA) and 1 μM indole-3-acetic acid (IAA)]. P. × hortorum ‘Scarlet Orbit’ and P. zonale showed similar high numbers of easily detached, embryo-like structures in response to 1 μM TDZ; P. alchemilloides and P. inquinans showed weak embryogenic responses to all treatments. To revisit whether P. × hortorum produces somatic embryos, and to examine modes of regeneration in the wild species, the histology of regenerating structures on hypocotyl explants in 1 μM TDZ was examined. Both P. × hortorum and P. zonale produced embryo-like structures from single cell derivatives of epidermal cells. Globular-shaped structures transitioned into heart-shaped structures that had loose attachments to explant surfaces and no vascular connection to the explant. Roots with direct vascular connections to the rest of the embryo-like structures were never observed; root organogenesis appeared to be secondary. We propose that P. × hortorum and P. zonale exhibit partial somatic embryogenesis, in which all of the criteria for somatic embryos are met except formation of a root pole. In both species, explants forming embryo-like structures could also undergo shoot organogenesis, where shoots exhibited a broad base of attachment to the explant and a vascular connection to vascular nodules within the explant. Epidermally derived embryo-like structures were not observed in P. alchemilloides or P. inquinans in response to 1 μM TDZ. Shoot organogenesis occurred in P. alchemilloides but not in P. inquinans.     

Extracts of the Brown Seaweed <Emphasis Type="Italic">Ascophyllum nodosum</Emphasis> Induce Gibberellic Acid (GA<Subscript>3</Subscript>)-independent Amylase Activity in Barley

Extracts of the brown seaweed Ascophyllum nodosum have been used as a biostimulant to promote growth and productivity in a number of agricultural production systems. Although the extracts have been shown to improve seedling emergence and vigor in a variety of plants, including barley, the mechanism(s) of this growth-promoting effect is(are) largely unknown. In our study, A. nodosum extract induced amylase activity in barley seed-halves; a significant difference in amylase activity was observed in seeds without an embryo. The addition of activated charcoal to the treatment media negated the bioactivity of the extracts suggesting the organic nature of bioactive compounds in A. nodosum extracts. The extracts induced amylase activity in a gibberellic acid (GA)-deficient barley mutant (grd2). LC-MS-MS analysis failed to detect the presence of GA₃ in the extracts. ABA supplementation of the medium caused a significant reduction of amylase activity in GA-treated seeds compared with those treated with the A. nodosum extract. Taken together, our results suggest that the organic components of A. nodosum extract induce amylase activity independent of GA₃ and might act in concert with GA-dependent amylase production leading to enhanced germination and seedling vigor in barley. Being derived from a renewable resource, the bioactive compounds from A. nodosum could be used to improve crop productivity in sustainable agricultural systems.     

Analysis of Early Embryogenesis in Rainbow and Banded Darters (Percidae: Etheostoma) Reveals Asymmetric Postmating Barrier

This study examines early embryogenesis in two species of darters, Etheostoma caeruleum and E. zonale (Teleostei: Percidae), and their hybrids. Results document the course of ontogeny from fertilization until the onset of pigmented eyes. Comparing fertilization and developmental success of conspecific versus heterospecific crosses revealed an asymmetric postmating reproductive barrier. E. caeruleum eggs treated with E. zonale sperm exhibited fertilization and developmental success similar to that of conspecific crosses. In contrast, E. zonale eggs treated with E. caeruleum sperm exhibited reduced fertilization relative to conspecific crosses and abnormal development. Development in this latter cross was compromised at all stages, but appeared to be concentrated around epiboly, or cell migration. As epiboly represents the stage of ontogeny when zygotic genes of both species jointly contribute to embryogenesis, results provide insight into the genetic mechanisms underlying postmating barriers in Etheostoma. Finally, the observed asymmetry in fertilization success is consistent with predictions based on the behavioral ecology of these species. Etheostoma zonale spawn in the open water column, whereas E.␣caeruleum bury their eggs under the substrate. The observed fertilization barrier may have therefore resulted from selection favoring increased fertilization specificity in E. zonale.     

Effects of extracts from sporoderm-broken spores of Ganoderma lucidum on HeLa cells

The effects of extracts from Ganoderma lucidum spores on the growth of human cervix uteri tumor HeLa cells as well as on the cell cycle and intracellular calcium level were investigated. Alcohol extracts were prepared from sporoderm-broken and sporoderm-nonbroken spores (termed extract I and extract II) of G. lucidum. Extract I was then subjected to silica gel chromatography to obtain extract III. Cytotoxicity was examined by means of trypan blue exclusion and MTT tests. It was found that extract I and extract III, but not extract II strongly inhibited the growth of HeLa cells, and that extract III was more effective than extract I. Moreover, extract III was shown to be capable of blocking the cell cycle at the transition from G₁ to S phase and inducing a marked decrease of intracellular calcium level, determined by flow cytometry and the specific fluorescent calcium probe Fura-2, respectively. These results imply that (1) the breaking of G. lucidum spores improves the release of cytotoxic activity and (2) the effective extract might influence the cell cycle and cellular signal transduction by altering the calcium transport system. This revised version was published online in July 2006 with corrections to the Cover Date.