Identification of linoleic acid,a main component of the n-hexane fraction from Dryopteris crassirhizoma,as an anti-Streptococcus mutans biofilm agent

Dryopteris crassirhizoma is a semi-evergreen plant. Previous studies have shown the potential of this plant as an agent for the control of cariogenic biofilms. In this study, the main antibacterial components of the plant were identified by correlating gas chromatography–mass spectrometry data with the antibacterial activity of chloroform and n-hexane fractions and then evaluating the activity of the most potent antibacterial component against Streptococcus mutans UA159 biofilms. The most potent antibacterial component was linoleic acid, a main component of the n-hexane fraction. Linoleic acid reduced viability in a dose dependent manner and reduced biofilm accumulation during initial and mature biofilm formation. Furthermore, when the biofilms were briefly treated with linoleic acid (10?min/treatment, a total of six times), the dry weight of the biofilms was significantly diminished. In addition, the anti-biofilm activity of the n-hexane fraction was similar to that of linoleic acid. These results suggest that the n-hexane fraction of D. crassirhizoma and linoleic acid may be useful for controlling cariogenic biofilms.     

Estimation of the Gelatinization Temperature of Noodles from Water Sorption Curves under Temperature-Programmed Heating Conditions

Both free and conjugated brassinosteroids (BRs) in the pollen and anthers of Erythronium japonicum Decne. were investigated. As a free form of BRs, typhasterol was identified by GC-MS. Polar conjugated BRs occurred only in the anther, and non-polar con-jugated BRs occurred both in the pollen and mainly in the anther.

BR parts of acid hydrolysis of the former were identified to be teasterone (major) and castasterone (minor). Those of alkaline hydrolysis of the latter were identified to be typhasterol (major) and teasterone (minor).     

Teasterone-3-O-β-D-glucopyranoside,A New Conjugated Brassinosteroid Metabolite from Lily Cell Suspension Cultures and Its Identification in Lily Anthers

The new brassinosteroid conjugate, teasterone-3-O-β-D-glucopyranoside, was found as a metabolite of teasterone in lily cell suspension cultures. Its structure was determined by means of FAB-MS and ¹H-NMR upon comparison with the authentic compound. Furthermore, its presence in lily anthers was confirmed by FAB-MS and LC-APCI-SIM data. This is the first natural brassinosteroid conjugate glucosylated at a hydroxyl group in ring A.     

Identification of Castasterone,Typhasterol and Teasterone from the Pollen of Zea mays

From the pollen of Zea mays, three brassinosteroids, castasterone, typhasterol and teasterone, were identified by GC/MS and/or ¹H NMR. Their concentrations in the pollen were shown by GC/SIM to be about 120 μg/kg fr.wt. (castasterone), 6.6 μg/kg fr.wt. (typhasterol) and 4.1 μg/kg fr.wt. (teasterone). It was also found that the anther contained a fairly large amount of brassinosteroids by a bioassay.     

Unusual wax esters and glycolipids: Biocatalytical formation and physico-chemical characterization

By aid of lipases, e.g. of Mucor michei, in n-hexane wax esters were produced from usual primary fatty alcohols and unusual hydroxy fatty acids (in part of microbial origin). Thus, (S)-17-hydroxyoctadecanoic acid dodecyl ester and (R)-3-hydroxy decanoic acid dodecyl ester were formed. In measurements of the film pressure using a LANGMUIR film balance the monolayers of both compounds indicated good stability compared to the non-hydroxy wax esters. Glycolipids de novo produced by microorganisms did not show suitable wetting properties, but they were able to lower ze surface tension of water to a higher extent than the unusual waxes.     

The extracellular pollen coat in members of the Brassicaceae: composition, biosynthesis, and functions in pollination

Summary. I have used cellular and molecular genetic and bioinformatic approaches to characterise the components of the pollen coat in plants of the family Brassicaceae, including Arabidopsis thaliana and several brassicas including Brassica napus, B. oleracea, and B. rapa. The pollen coat in these species is mostly made up of a unique mixture of lipids that is highly enriched in acylated compounds, such as sterol esters and phospholipids. These acyl lipids are characterised by an unusually high degree of saturation. The fatty acids typically contain 70–90% saturated acyl residues such as myristate, palmitate, and stearate. The major sterol components of the pollen coat are saturated fatty acyl esters of stigmasterol, campesterol, and campestdienol. In addition to lipids, the second major component of the pollen coat is a specific group of proteins that is dominated by a family of proteins that we term pollenins. Although pollenins are by far the major protein components of the pollen coat of members of the Brassicaceae, proteomic analysis reveals that there are several additional protein components, including lipases, protein kinases, a pectin esterase, and a caleosin. The biosynthesis of these lipids and proteins and their significance for overall pollen function are reviewed and discussed. Correspondence and reprints: Biotechnology Unit, School of Applied Sciences, University of Glamorgan, Pontypridd CF37 1DL, Wales, United Kingdom.     

Studies on the synthesis of short-chain geranyl esters catalysed by Fusarium oxysporum esterase in organic solvents

A novel esterase isolated from Fusarium oxysporum was investigated for the synthesis of short-chain esters of geraniol by alcoholysis and direct esterification reactions in organic solvents. The enzyme was used as a dried powder (i.e., not immobilized). The reaction parameters affecting the enzyme behavior such as the nature of organic solvent and acyl donor, the concentration of substrates and the water activity of the system were studied. High yields (80–90%) were obtained by both approaches (alcoholysis and direct esterification) at low values of water activity (a_w=0.11) in n-hexane. The enzyme retain its catalytic activity even after fifth reuse in n-hexane at a_w=0.11, demonstrating its stability and efficiency under the conditions of this study.     

Phytic Acid Metabolism in Lily (Lilium longiflorum Thunb.) Pollen

The accumulation of phytic acid during development of lily (Lilium longiflorum Thunb.) pollen and its degradation during germination have been studied. A substantial amount of phytic acid accumulates in lily pollen by 5 days before anthesis, and little change occurs during subsequent maturation. Mature lily pollen contains 7 to 8 micrograms phytic acid per milligram pollen. Considerable degradation of phytic acid occurs by 15 minutes of incubation in glucose culture medium, and very little is left by 3 hours. No partially phosphorylated myo-inositol accumulates during germination. The breakdown of phytic acid proceeds at a constant rate during this time period. The rate is calculated to be 0.037 microgram phytic acid/milligram pollen/minute. Two phytases are detected in germinated lily pollen extract using high performance liquid chromatography with an anion exchange column (diethylaminoethyl-5PW). The results suggest that one of the phytases is already present in mature ungerminated lily pollen and the other one is newly synthesized during germination from a long-lived, pre-existing mRNA.     

Molecular and physiological characterisation of a 14-3-3 protein from lily pollen grains regulating the activity of the plasma membrane H+ ATPase during pollen grain germination and tube growth

A 14-3-3 protein has been cloned and sequenced from a cDNA library constructed from mRNAs of mature pollen grains of Lilium longiflorum Thunb. Monoclonal antibodies (MUP 5 or MUP 15) highly specific against 14-3-3 proteins recognised a 30-kDa protein in the cytoplasmic fraction of many various lily tissues (leaves, bulbs, stems, anther filaments, pollen grains, stigmas) and in other plants (Arabidopsis seedlings, barley recombinant 14-3-3). In addition, 14-3-3 proteins were detected in a microsomal fraction isolated from pollen grains and tubes, and the amount of membrane-bound 14-3-3 proteins as well as the amount of the plasma membrane (PM) H⁺ ATPase increased during germination of pollen grains and tube growth. No change was observed in the cytoplasmic fraction. A further increase in the amount of 14-3-3 proteins in the microsomal fraction was observed when pollen grains were incubated in germination medium containing 1 μM fusicoccin (FC) whereas the number of 14-3-3s in the cytoplasmic fraction decreased. Fusicoccin also protected membrane-bound 14-3-3 proteins from dissociation after washing with the chaotropic salt KI. Furthermore, FC stimulated the PM H⁺ ATPase activity, the germination frequency and the growth rate of pollen tubes, thus indicating that a modulation of the PM H⁺ ATPase activity by interaction with 14-3-3 proteins may regulate germination and tube growth of lily pollen. Received: 20 June 2000 / Accepted: 2 October 2000     

Lipase-catalyzed synthesis of monoacylglycerol in a homogeneous system

The 1,3-regiospecifique lipase, Lipozyme IM, catalyzed the esterification of lauric acid and glycerol in a homogeneous system. To overcome the drawback of the insolubility of glycerol in hexane, which is extensively used in enzymatic synthesis, a mixture of n-hexane/tert-butanol (1:1, v/v) was used leading to a monophasic system. The conversion of lauric acid into monolaurin was 65% in 8 h, when a molar ratio of glycerol to fatty acid (5:1) was used with the fatty acid at 0.1 M, and the phenomenon of acyl migration was minimized.     

Hexane soluble non-volatiles in flowering to fruiting stages of Fatsia japonica

The n-hexane soluble non-volatile fraction of the acetone extracts from the flower buds, the flowers and the immature and the mature fruits of Fatsia japonica were all found to contain fatty acids, fatty acid methyl esters, squalene, β-amyrin and sterols. At all the stages between budding to the mature fruit, the major fatty acids were palmitic and linoleic acids and the major phytosterol was stigmasterol. In addition steryl and β-amyrenyl esters were found in the flowers and the immature and the mature fruits, but these esters were not present in the flower buds. Sitosteryl ester was the major constituent of the steryl ester fraction in the fruiting stages. Phytol was found in only the flowering stage and triglycerides in only the mature fruits. The variations in the lipid constituents is discussed in relation to the stages from budding to the mature fruit.     

Effects of Freeze-Drying on Permeability and Respiration of Germinating Lily Pollen

The germination of lily pollen (Lilium longiflorum cv. Ace) was impaired by freeze-drying. This loss of viability was associated with a modified pattern of respiration and an increased leakage of soluble carbohydrates, phosphate, and ninhydrin-positive material into the culture medium. 2,4-Dinitro-phenol, (DNP), an uncoupler of oxidative phosphorylation showed a decreased ability to stimulate O₂ uptake in freeze-dried pollen. The altered viability, respiration, and permeability resulted from drying under vacuum and not the initial freezing of the pollen. Mature lily pollen contained approximately 0.3 “Ai phosphate, of which 15 % was inorganic phosphate and about 50 %> was acid soluble organic phosphate of unknown identity.     

Identification of the endogenous apolar ecdysteroid conjugates present in newly-laid eggs of the house cricket (Acheta domesticus) as 22-long-chain fatty acyl esters of ecdysone

Newly-laid eggs of the house cricket Acheta domesticus contain significant amounts of apolar ecdysteroid conjugates, which can be hydrolysed by prolonged incubation with a mixture of Helix pomatia gut hydrolases. The ecdysteroid released on hydrolysis of the apolar conjugates has been purified and identified as ecdysone by co-chromatography on normal-phase and reversed-phase HPLC and by fast-atom bombardment mass spectrometry.Starting with only 22 g newly-laid eggs (containing 16 μg conjugated ecdysone), the ecdysone conjugates have been purified by open column chromatography and four successive HPLC purification steps to give essentially pure apolar conjugates with a yield of 57%. The conjugates are shown to be a mixture of ecdysone 22-fatty acyl esters by co-chromatography with authentic reference compounds and by fast-atom bombardment mass spectrometry. The fatty acyl composition of the conjugates is very similar to that produced by the ovaries of A. domesticus from [³H]ecdysone in vitro (Whiting and Dinan, Biochem. J.252, 95–103, 1988). The major fatty acyl esters are the 22-palmitate (C_16:0), 22-oleate (C_18:1) and 22-linoleate (C_18:2), with smaller amounts of the myristate (C_14:0), stearate (C_18:0) and arachidate (C_20:0) esters.This report constitutes the first identification from an insect source of endogenous ecdysteroid 22-fatty acyl esters, which have previously been identified in ticks and as metabolites of exogenous [³H]ecdysone in several arthropod species.     

Action of Poly-l-malic Acid on Various Proteolytic Enzymes

The steam volatile neutral fraction of tobacco smoke condensates was separated into n-hexane, nitromethane and 1:4 water-methanol soluble fractions by solvent partition.

2.methyl-4-hydroxy-2-hexenoic acid lactone, dihydroactinidiolide and phthalide were isolated from the 1:4 water-methanol soluble fraction, the highly polar portion of the steam volatile neutral fraction was designated as the M fraction.

By continuing analysis of the M fraction from a previous paper, benzyl alcohol, phenyl-ethyl alcohol, pyrrole-2-aldehyde, α-pyrrylmethylketone, α-pyrrylethylketone, α-carbomethoxypyrrole, pyrrole-2-carbonitrile, methyl-pyrrole-2-carbonitrile, 3-methyl-, 3-ethyl-, 3-n-propyl-, 2,3-dimethyl-, 2-ethyl-3-methyl-2-cyclopentene-1-one and norsolanadione were identified.

Identification of the compounds was based on the spectroscopic method (IR, MS, UV and GC-MS) and gas chromatographic analysis.     

21-kDa polypeptide,a low-molecular-weight cyclophilin,is released from pollen of higher plants into the extracellular medium in vitro

Calcium ions play a key role in the elongation and orientation of pollen tubes. We found that significant amounts of 21-kDa polypeptide were specifically released into the extracellular medium when pollen grains of lily, Lilium longiflorum Thunb., were incubated in the presence of EGTA or at low concentrations of Ca²⁺. This phenomenon was also dependent on pH and on the concentrations of MgCl₂ in the medium; the release of 21-kDa polypeptide from pollen was suppressed by increasing the MgCl₂ concentration and by lowering pH. Germination of pollen grains was inhibited in the medium into which the 21-kDa polypeptide had been released. This inhibition was irreversible; germination did not occur on transfer of the pollen grains into basal culture medium. Immuno-electron microscopy using an antibody against 21-kDa polypeptide showed that this polypeptide was present in the cytoplasm, vegetative nucleus and generative cell. When the pollen was treated with a medium containing EGTA, the density of 21-kDa polypeptide in the cytoplasm significantly decreased, but its density in vegetative nuclei and the generative cell did not, suggesting that only cytoplasmic 21-kDa polypeptide was released into the extracellular medium. The 21-kDa polypeptide was also present in the pollen of other higher-plant species, such as Tradescantia virginiana L., Nicotiana tabacum L. (angiosperms), and Cryptomeria japonica D. Don. (gymnosperm), and was also released into the medium in the presence of EGTA. In the case of C. japonica, however, it was released from pollen at alkaline pH above 8.5. The expression of 21-kDa polypeptide was not pollen-specific, because 21-kDa components immunoreactive with the anti-21-kDa polypeptide serum also existed in vegetative organs and cells of lily or tobacco. However, the 21-kDa polypeptide was not released into the extracellular medium from cultured tobacco BY-2 cells, even in the presence of EGTA. Amino acid sequences of two peptide fragments derived from 21-kDa polypeptide matched well those of low-molecular-weight cyclophilin (CyP). The antiserum against 21-kDa polypeptide recognized the CyP A from calf thymus and that in A431 carcinoma cells. The 21-kDa polypeptide fraction purified from lily pollen possessed peptidyl-prolyl cis-trans isomerase activity, which was suppressed by cyclosporin A (CsA), an inhibitor of enzyme activities of CyPs. From these results, we concluded that the 21-kDa polypeptide is a low-molecular-weight CyP. The present study showed that CyP in the pollen of higher plants is released into the extracellular matrix under unfavorable conditions.Abbreviations CaM Calmodulin - CBB Coomassie-brilliant-blue - CsA Cyclosporin A - CyP Cyclophilin     

Surfactant-modified yeast whole-cell biocatalyst displaying lipase on cell surface for enzymatic production of structured lipids in organic media

The cell surface engineering system, in which functional proteins are genetically displayed on microbial cell surfaces, has recently become a powerful tool for applied biotechnology. Here, we report on the surfactant modification of surface-displayed lipase to improve its performance for enzymatic synthesis reactions. The lipase activities of the surfactant-modified yeast displaying Rhizopus oryzae lipase (ROL) were evaluated in both aqueous and nonaqueous systems. Despite the similar lipase activities of control and surfactant-modified cells in aqueous media, the treatment with nonionic surfactants increased the specific lipase activity of the ROL-displaying yeast in n-hexane. In particular, the Tween 20-modified cells increased the cell surface hydrophobicity significantly among a series of Tween surfactants tested, resulting in 8–30 times higher specific activity in organic solvents with relatively high log P values. The developed cells were successfully used for the enzymatic synthesis of phospholipids and fatty acid methyl esters in n-hexane, whereas the nontreated cells produced a significantly low yield. Our results thus indicate that surfactant modification of the cell surface can enhance the potential of the surface-displayed lipase for bioconversion.     

Successful expression in pollen of various plant species of in vitro synthesized mRNA introduced by particle bombardment

Gold particles coated with -glucuronidase (GUS) mRNA with a 5 cap structure that had been synthesized in vitro were introduced, by use of a pneumatic particle gun, into pollen grains of lily (Lilium longiflorum), freesia (Freesia refracta) and tulip (Tulipa gesneriana). A fluorometric assay for the GUS activity indicated that in vitro synthesized GUS mRNA introduced into these pollen cells by particle bombardment was successfully expressed. GUS activity in extracts of the bombarded lily pollen became detectable fluorometrically within 30 min after bombardment, peaked at 6 h, then gradually decreased. This activity changed as a function of the developmental stage of the pollen cell of lily.     

Chemical profiling and in vitro biological effects of Cardiospermum halicacabum L. (Sapindaceae) aerial parts and seeds for applications in neurodegenerative disorders

Abstract

Cardiospermum halicacabum is widely used in traditional medicine. Previous studies have focused on the aerial parts, while the seeds have been poorly investigated. This work aimed to analyse the chemical composition of extracts from aerial parts and seeds obtained using Naviglio and Soxhlet (PN, PS, and SN, SS, respectively), the inhibitory properties against tyrosinase, acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) and the antioxidant effects. PN total extract showed significant anti-tyrosinase activity (IC₅₀ value of 10.8?µg/mL). After partitioning with n-hexane, an HPLC method for analysing chemical constituents was established. Apigenin, luteolin, and apigenin-7-O-glucoside are the predominant constituents. SN n-hexane fraction was the most active inhibitor of BChE (IC₅₀ of 57.9?µg/mL). Gas chromatography-mass spectrometry analysis revealed fatty acids, including eicosanoic acid, methyl 11-eicosenoate and oleic acid, as the major constituents. These findings suggest the potentiality of both seeds and aerial parts of C. halicacabum in the treatment of neurological disorders.     

Antimycotic activities of Cinnamon-derived compounds against <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> in vitro

In this study, the effects of medicinal plant extracts on the development of mycelium in the following phytopathogenic fungi were evaluated: Phytophthora capsici, Rhizoctonia solani, Fusarium solani, Colletotrichum gloeosprorioides, and Botrytis cinera. Of the 26 medicinal plants tested, six plant extracts showed antifungal activity against phytopathogenic fungi. The highest antifungal activity was exerted against R. solani by the n-hexane fraction of a Cinnamon (Cinnamomum cassia Blume) solvent extract. Therefore, the antifungal compound fractions I and II were purified from the n-hexane fraction by TLC on silica gel plates. When treated with solutions containing compound fractions I or II at a concentration of 2%, the mycelia growth rate of R. solani was reduced to 0.19 and 0.18, respectively. In addition, microscopic observation of the hyphal morphology of R. solani following treatment with compound fraction I revealed the presence of severely damaged hyphae. Specifically, the hyphal tips became swollen, collapsed or were completely destroyed in response to treatment with solution containing compound fraction I at concentration of 1%.     

Stability performance ofCynara cardunculus L. acid protease in aqueous-organic biphasic systems

Stability performance of the acid protease ofCynara cardunculus L. in biphasic systems containing ethyl acetate,n-hexane or isooctane was investigated and compared with that of pepsin. Activity retention was higher in the system containingn-hexane. In this system 100% retention was observed up to 144 hours. Pre-saturation of phases was found to increase enzyme stability in the cases ofn-hexane and isooctane and to be an absolute requirement in the case of ethyl acetate. The results obtained suggest also that, when dealing with pre-saturated phases, log P cannot be used straightforwardly to predict enzyme stability in biphasic systems.