Improved β-thujaplicin production in Cupressus lusitanica suspension cultures by fungal elicitor and methyl jasmonate

Production of a novel antimicrobial tropolone, beta-thujaplicin, in Cupressus lusitanica suspension cultures was studied by using a variety of chemicals and fungal elicitors. Sodium alginate, chitin, and methyl jasmonate resulted in 2-, 2.5-, and 3-fold higher beta-thujaplicin production, respectively, than in the control. Significantly improved beta-thujaplicin production (187 mg l(-1)) was obtained using a high cell density (180-200 g l(-1)) and fungal elicitor treatment [10 mg (g fresh cells)(-1)] in a production medium with a high ferrous ion concentration (0.3 mM). This improved volumetric productivity was 3- to 4-fold higher than obtained under standard conditions. A synergistic effect of fungal elicitor and ferrous ion on beta-thujaplicin production was also suggested by our study. Plant cell culture technology is a promising alternative for producing a large variety of secondary metabolites that are widely used as food additives, pharmaceuticals, and dairy products (Verpoorte et al. 1999). Thus, beta-thujaplicin production by plant cell cultures was developed with the goal of commercial application (Berlin and Witte 1988; Itose and Sakai 1997; Ono et al. 1998). However, the production of beta-thujaplicin by plant cell cultures is still not competitive for use in industrial applications. In this study, we assessed the effects of methyl jasmonate, alginate, chitin, and fungal elicitor on beta-thujaplicin production; we obtained a significantly elevated beta-thujaplicin production by using an improved culture strategy.     

O2− from elicitor-induced oxidative burst is necessary for triggering phenylalanine ammonia-lyase activation and catharanthine synthesis in Catharanthus roseus cell cultures

Elicitor, derived from the cell walls of Aspergillus niger, induced rapid generation of reactive oxygen intermediates (ROI), including superoxide anion (O₂⁻) and hydrogen peroxide (H₂O₂), sequentially followed by phenylalanine ammonia-lyase (PAL) activation and catharanthine biosynthesis in Catharanthus roseus suspension cells. The elicitor-induced PAL activation and catharanthine biosynthesis were blocked by NAD(P)H oxidase inhibitor, diphenylene iodonium (DPI). O₂⁻ generated by the reaction of xanthine/xanthine oxidase (X/XO) triggered PAL activation and catharanthine biosynthesis of C. roseus cells in the absence of elicitor and reversed the inhibitory effect of DPI on elicitor-induced PAL activation and catharanthine biosynthesis. External application of H₂O₂ and catalase had no effect on PAL activity and catharanthine contents of C. roseus cells. The results demonstrated a causal relationship between elicitor-induced oxidative burst and PAL activation in C. roseus suspension cells and suggested a sequence of signaling events from ROI production to PAL activation and catharanthine synthesis. Within this sequence, O₂⁻ rather than H₂O₂ appeared to trigger the subsequent reactions.     

Cytokinin-enhanced accumulation of indole alkaloids in Catharanthus roseus cell cultures — The factors affecting the cytokinin response

Summary Cytokinins were found to stimulate the alkaloid synthesis induced by removing auxin from the medium of a cell line of Catharanthus roseus. Diluting the mineral salts of the culture medium decreased the alkaloid production but increased the sensitivity of the cells. Addition of high levels of Ca²⁺, Mg²⁺ or Sr²⁺ to B5 media in which the mineral salts were diluted to 5–40%, increased the alkaloid production. The latter effect is related only partially to enhanced osmotic potential.Abbreviations BA 6-benzylaminopurine - CK cytokinin - 2,4D 2,4-dichlorophenoxyacetic acid - dw dry weight - Kin 6-furfurylaminopurine - TLC thin layer chromatography - SE standard error - Z trans-zeatin     

DIGE analysis of proteome changes accompanying large resveratrol production by grapevine (Vitis vinifera cv. Gamay) cell cultures in response to methyl-β-cyclodextrin and methyl jasmonate elicitors

We had previously shown that Vitis vinifera cv. Gamay cell suspension accumulates extracellularly large amounts of the phytoalexin trans-resveratrol (tR) in response to elicitation with methylated cyclodextrins (MBCD), which can be triplicated when the elicitor is combined with methyl jasmonate (MeJA). In parallel, new pathogenesis-related proteins accumulated in the apoplast-like extracellular space. The aim of this study was to investigate changes in the grapevine cell proteome potentially related to tR accumulation in response to the above elicitors. The DIGE technique was used to detect statistically significant changes in the cell's proteome. A total number of 1031 unique spots were detected, 67 of which were de-regulated upon elicitation. Sixty-four spots were successfully identified by nLC-MS/MS database search analysis. The tR biosynthetic pathway enzymes were up-regulated by MBCD alone or combined with MeJA, but not by treatment with MeJA alone, in agreement with tR accumulation pattern. Seven spots contained stilbene synthase encoded by four different isogenes. Likewise, four glutathione-S-transferases, potentially involved in tR trafficking within the cell and across membranes, were up-regulated in the same fashion as stilbene synthases. The relation of other de-regulated proteins with other effects caused by elicitors on grapevine cells, namely defense response and cell growth inhibition, is discussed.     

Effect of electrical stimulation on β-adrenergic receptor population and cyclic AMP production in chicken and rat skeletal muscle cell cultures

Summary Expression of the β-adrenergic receptor (βAR) and its coupling to cyclic AMP (cAMP) synthesis are important components of the signaling system that controls muscle atrophy and hypertrophy, and the goal of this study was to determine if electrical stimulation in a pattern simulating slow muscle contraction would alter the βAR response in primary cultures of avian and mammalian skeletal muscle cells. Specifically, chicken skeletal muscle cells and rat skeletal muscle cells that had been grown for 7d in culture were subjected to electrical stimulation for an additional 2 d at a pulse frequency of 0.5 pulses/sec and a pulse duration of 200 msec. In chicken skeletal muscle cells, the βAR population was not significantly affected by electrical stimulation; however, the ability of these cells to synthesize cyclic AMP was reduced by approximately one-half. In contrast, the βAR population in rat muscle cells was increased slightly but not significantly by electrical stimulation, and the ability of these cells to synthesize cyclic AMP was increased by almost twofold. The basal levels of intracellular cyclic AMP in neither rat muscle cells nor chicken muscle cells were affected by electrical stimulation.     

Methyl jasmonate increases silymarin production in Silybum marianum (L.) Gaernt cell cultures treated with β-cyclodextrins

Silymarin (Sm) from the fruit of Silybum marianum is an isomeric mixture of pharmacologically active flavonolignans which are formed by oxidative coupling of taxifolin (Tx) and coniferyl alcohol (CA). Suspension cultures of this plant constitutively secrete small amounts of Sm into the extracellular medium. Production can be increased by inclusion of cyclodextrins (CDs) in cultures. Both hydroxylated (RHCD) and dimethylated (RMCD) CDs strongly induced prompt accumulation of CA in the medium followed by a late production of flavonolignans. Simultaneous addition of methyl jasmonate (MJ) and RMCD to cells did not significantly modify CA release or flavonolignan accumulation. Delayed addition of MJ to cultures subcultivated in medium containing RMCD markedly influenced Sm production by promoting conversion of the previously formed CA precursor.     

Influence of production media on Cinchona cell cultures; spontaneous formation of β-carbolines from L-tryptophan

Evidence is presented that the β-carboline alkaloids norharman and harman are artifacts formed from L-tryptophan. Transfer of Cinchona ledgeriana suspension cultured cells to Zenk's alkaloid production medium (ZAP medium) resulted in cell death. No quinoline or terpenoid indole alkaloids were formed. However, the L-tryptophan present in the production medium was transformed to the β-carboline alkaloids, harman and norharman. It was demonstrated that norharman was also formed in ZAP medium without cells and in ZAP medium containing frost-killed cell material.     

Aided-efflux and high production of β-amyrin realized by β-cyclodextrin in situ synthesized on surface of Saccharomyces cerevisiae

As a plant-derived pentacyclic triterpenoid, β-amyrin has been heterogeneously synthesized in Saccharomyces cerevisiae. However, β-amyrin is intracellularly produced in a lower gram scale using recombinant S. cerevisiae, which limits the industrial applications. Although many strategies have been proven to be effective to improve the production of β-amyrin, the intracellularly accumulation is still a challenge in reaching higher titer and simplifying the extraction process. To solve this problem, the amphiphilic β-cyclodextrin (β-CD) has been previously employed to aid the efflux of β-amyrin out of the cells. Nevertheless, the supplemented β-CD in the medium is not consistent with β-amyrin synthesis and has the disadvantage of rather high cost. Therefore, an aided-efflux system based on in situ synthesis of β-CD was developed in this study to enhance the biosynthesis of β-amyrin and its efflux. The in situ synthesis of β-CD was started from starch by the surface displayed cyclodextrin glycosyltransferase (CGTase) on yeast cells. As a result, the synthesized β-CD could capture 16% of the intracellular β-amyrin and improve the total production by 77%. Furthermore, more strategies including inducing system remodeling, precursor supply enhancement, two-phase fermentation and lipid synthesis regulation were employed. Finally, the production of β-amyrin was increased to 73 mg/L in shake flask, 31 folds higher than the original strain, containing 31 mg/L of extracellular β-amyrin. Overall, this work provides novel strategies for the aided-efflux of natural products with high hydrophobicity in engineered S. cerevisiae.     

Increased podophyllotoxin production in Podophyllum hexandrum cell suspension cultures after feeding coniferyl alcohol as a β-cyclodextrin complex

Summary Cell suspension cultures, derived from roots of Podophyllum hexandrum Royle (Berberidaceae), accumulate podophyllotoxin. In this study the use of -cyclodextrin in feeding the poorly water-soluble precursor coniferyl alcohol to these cultures is described. By complexation with -cyclodextrin, a solution of 3 mM coniferyl alcohol could be fed, resulting in enhanced podophyllotoxin accumulation. The same concentration of non-complexed suspended coniferyl alcohol had only little effect on the podophyllotoxin accumulation. -Cyclodextrin itself was proven to be non-toxic for the cells. It did not influence the podophyllotoxin content and it was not metabolized or used as a carbon source by the cells. For comparison, coniferin, the water-soluble -D-glucoside of coniferyl alcohol, was also fed in the same concentration. The effect of coniferin on the podophyllotoxin accumulation was stronger than that of coniferyl alcohol complexed with -cyclodextrin, but coniferin is not commercially available.Abbreviations -CD -cyclodextrin - NAA naphthaleneacetic acid     

Effects of creatine and its analog, β-guanidinopropionic acid, on the differentiation of and nucleoli in myoblasts

The effects of supplementation with creatine (Cr) and its analog, β-guanidinopropionic acid (β-GPA), on the differentiation of myoblasts and the numbers of nucleoli were studied in C2C12 cells. The cells were cultured in differentiation medium for 4 d. Then Cr (1 mM) or β-GPA (1 mM) was added to the cells, and the mixture was cultured for an additional 2 d. Although the number of myotubes was not different among the groups, myotube diameters and nuclear numbers in myotubes were increased by Cr and β-GPA treatment respectively. The expression of differentiation marker proteins, myogenin, and the myosine heavy chain, was increased in the β-GPA group. Supplementation with β-GPA also increased the percentage of p21 (inhibitor for cell cycle progression)-positive myoblasts. Supplementation with Cr inhibited the decrease in nucleoli numbers, whereas β-GPA increased nucleolar sizes in the myotubes. These results suggest that β-GPA supplementation stimulated the differentiation of myoblasts into multi-nucleated myotubes through induction of p21 expression.     

Effects of the addition of laminaran on β-glucosidase production by Trichoderma viride

β-Glucosidase production by Trichoderma viride WU-36B was studied in media containing laminaran. By the addition of laminaran to the medium containing glycerol as a carbon source, extracellular activities of β-glucosidase and β-1,3-glucanase increased but CMCase activity was not detected during whole culture period. Extracellular activities of β-glucosidase, CMCase, and β-1,3-gluconase were higher in the medium containing both avicel and laminaran than in the media containing avicel or laminaran as a sole carbon source.     

Effects of curvature and cell–cell interaction on cell adhesion in microvessels

It has been found that both circulating blood cells and tumor cells are more easily adherent to curved microvessels than straight ones. This motivated us to investigate numerically the effect of the curvature of the curved vessel on cell adhesion. In this study, the fluid dynamics was carried out by the lattice Boltzmann method (LBM), and the cell dynamics was governed by the Newton’s law of translation and rotation. The adhesive dynamics model involved the effect of receptor-ligand bonds between circulating cells and endothelial cells (ECs). It is found that the curved vessel would increase the simultaneous bond number, and the probability of cell adhesion is increased consequently. The interaction between traveling cells would also affect the cell adhesion significantly. For two-cell case, the simultaneous bond number of the rear cell is increased significantly, and the curvature of microvessel further enhances the probability of cell adhesion.     

Effects of “skeleton” photoperiods and high frequency light-dark cycles on the rhythm of cell division in synchronized cultures of Euglena

Summary Two further lines of evidence support the contention (Edmunds, 1966) that the cell cycle in autotrophically grown Euglena can be coupled to an endogenous, circadian biological clock under certain conditions. So-called skeleton photoperiods (LD: 3,6,3:12 and LD: 4,4,4:12) following a complete photoperiod regime entrain the cell division rhythm in the population to a precise 24 hr period, although the step-sizes of the successive fission bursts are always less than 2.00, indicating that not all cells divide in any one 24 hr interval. These findings imply that the continuous action of light is not required for synchronization and suggest that the putative oscillation underlying the rhythm can be phased by discrete light (or dark) pulses or signals.The effects of high frequency LD cycles whose periods were integral submultiples of 24 hr were also investigated. In most regimes (LD:1/4,1/2; LD:1/2,1; LD: 1,2; LD: 1,3; LD: 2,4; LD: 2,6; LD: 4,4) synchronous cell division iccurred in the culture with an average period of 26–27 hr, although only a fraction of the cells divided during any one burst. Similar results were obtained if (i) a synchronized culture was exposed to certain high frequency cycles whose periods were not integral submultiples of 24 hr (e.g., LD: 5,5 or LD: 8,8); (ii) an asynchronous culture (grown in LL) was subsequently exposed to a high frequency cycle; or (iii) a synchronized culture was subjected to a random LD cycle. The synchrony does not break down as long as the given LD regime is imposed and shows some indications of persistence in certain ensuing conditions of continuous illumination.A general formula was derived which predicts the time of division, t _D , for an individual cell: t _D =k+n, where k is the initial phase delay, n is an integer, and is the free-running period of the rhythm observed in the population. These results are interpreted as indicating that the high frequency cycles employed were unable to entrain the circadian oscillation(s) hypothesized to underly and gate cell division, with the result that the rhythm reverted to its free-running period. Exposure to such cycles, however, apparently either initiates a rhythm or synchronizes the phases of the individual oscillations in the populations of cells. The possible direct interaction between energy supply and the observed somewhat variable period lengths is discussed; also, the relevance of stochastic models for the decay of division synchrony in the absence of a recurrent synchronizing procedure is considered.Some of these results were initially reported at the 5th International Congress on Photobiology, Hanover, N.H., U.S.A., August 26–31, 1968.This work was supported by NSF research grants #GB-4140 and #GB-6892 to L. Edmunds.     

Effects of hydroxypropyl-β-cyclodextrin on the growth and morphology of Absidia coerulea

Cyclodextrin has been found to be an attractive novel solubilizer due to its unique material properties. Absidia coerulea is widely used in steroid bioconversion. The effects of hydroxypropyl-β-cyclodextrin (HP-β-CD) on the growth, morphology, and steroid-converting activity of A. coerulea CICC 40302 were systematically studied. HP-β-CD affected A. coerulea growth, resulting in changes in its spore morphology and mycelial morphology. It induced an increase in the spore germination rate and a decrease in cell biomass at the stationary phase. Optical microscopy revealed that HP-β-CD altered the mycelial morphology and reduced the pellet compactness of A. coerulea. A convenient and feasible computing method was used to measure pellet compactness, and it demonstrated that the compactness degree of the pellet decreased as HP-β-CD increased, which could be attributed to the modification of the physical properties of the fermentation medium. Moreover, the changing of mycelial morphology influenced steroid-converting activity. The results showed that HP-β-CD had multiple concentration-dependent effects on A. coerulea cells. HP-β-CD in the proper concentration range holds great potential as a biocompatible solubilizer.     

Effects of Progesterone, β-Estradiol,and Androsterone on the Changes of Inorganic Element Content in Barley Leaves

The present study was performed to determine the changes in inorganic element content in barley leaves of mammalian sex hormones (MSH). Barley leaves were sprayed with 10⁻⁴, 10⁻⁶, 10⁻⁹, 10⁻¹², 10⁻¹⁵ M concentrations of progesterone, β-estradiol, and androsterone at 7th day after sowing. The plants were harvested at the end of 18 days after treatment with MSH solutions. The inorganic element concentrations were determined using wavelength dispersive X-ray fluorescence spectroscopy technique. Although the all MSH concentrations significantly (p < 0.05) increased the concentrations of calcium, magnesium, phosphorus, sulfur, copper, manganese, aluminum, zinc, iron, potassium, and chlorine, it decreased those of sodium concentration in barley leaves. The maximum changes in the element concentrations were obtained at 10⁻⁹ M for plant leaves treated with progesterone, 10⁻⁶ M for plant leaves treated with β-estradiol and androsterone. The present study elucidated that MSH significantly (p < 0.05) affected the inorganic element concentrations in barley leaves.     

Effects of serum and serum-derived factors on the growth and production of α-fetoprotein and albumin by human hepatoma cell lines

A serum-free culture system of human hepatoma cell lines (HuH-6 and HuH-7) was used to investigate the activity of bovine serum (BS) and of serum-derived factors on the growth and production of -fetoprotein (AFP) and albumin. At higher concentrations, dialyzed BS was inhibitory to the growth of HuH-6 and caused reduction of the level of AFP production by the cells. AFP and albumin levels in HuH-6 and HuH-7 were reduced or unchanged by fetuin, bovine serum albumin (BSA) and transferin (TF), although no cytotoxicity was shown by any of them. Commercial preparations of platelet-derived growth factor exhibited cytotoxicity to HuH-6 and HuH-7 and induced a decrease of AFP and albumin levels in a dose-dependent manner. Transforming growth factor (TGF-) exhibited no cytotoxicity to HuH-6. AFP levels in HuH-6 were unchanged with 1000 pg/ml TGF-, but albumin levels were decreased. TGF-7 at a concentration of 1000 pg/ml was cytotoxic to HuH-7 and AFP levels were a little increased. Albumin levels, however, were unchanged. Following exposure to cycloheximide, AFP and albumin levels in HuH-6 were inhibited.Abbreviations AFP -fetoprotein - BS bovine serum - BSA bovine serum albumin - EDTA ethylenediaminetetraaceticacid - ELISA enzyme-linked immunosorbent assay - HBSS Hank's balanced salt solution - PBS phosphate buffered saline - PDGF platelet-derived growth factor - TF transferrin - TGF-\ transforming growth factor beta     

Inclusion complexation between baicalein and β-cyclodextrin and the influence of β-cyclodextrin on the binding of baicalein with DNA: a spectroscopic approach

This work deals with the commonly studied cyclic oligosaccharide and gains importance as it is entered on a drug delivering carbohydrate and provides insight into the oligosaccharide complex–biomolecular interaction. The binding of a flavone, baicalein, to β-cyclodextrin and calf thymus DNA is studied. The binding of baicalein to calf thymus DNA in the presence of β-cyclodextrin is analysed using the UV–vis absorption and fluorescence spectroscopy. The mode of binding and structure of the baicalein–β-cyclodextrin complex are reported. The role of the structure and the stoichiometry of the inclusion complex of baicalein–β-cyclodextrin in its influence on DNA binding are analysed.

Highlights

? This paper deals with the binding of a flavone, baicalein to β-cyclodextrin and/or DNA.

? The inclusion complexation between baicalein and β-cyclodextrin is analysed.

? The stoichiometry and the binding strength of the inclusion complex is reported.

? The role of β-cyclodextrin in tuning the binding of baicalein to DNA is emphasized.

? Spectroscopic and docking analysis are used to articulate the results.     

Preparation of mixed alginate elicitors with high activity for the efficient production of 5′-phosphodiesterase by Catharanthus roseus cells

When various autoclaved microbial cells suspensions (exogenous elicitors) were added to Catharanthus roseus cell cultures, its growth was inhibited but 5′-phosphodiesterase (PDase) production was stimulated. The greatest effect was with autoclaved Alteromonas macleodii: the dry cell concentration decreased from 13 to 10.9 mg/ml while PDase production increased from 0.022 to 0.235 U/ml. A combination of A. macleodii (as exogenous elicitor) and 0.1%(w/v) alginate oligomers (AO: acting as both endogenous elicitor and scavenger of active oxygen species) minimized the cell growth inhibition but enhanced PDase production (0.474 U/ml) about 20 times higher than the control (no addition). The method for the preparation of mixed alginate elicitors with high activities containing exogenous elicitor (autoclaved A. macleodii), endogenous elicitor (AO), and trans-4,5-dihydroxy-2-cyclopenten-1-one was developed. The mixed alginate elicitors significantly promoted PDase production (2.67 U/ml) by C. roseus, and the productivity was increased 120-fold compared to the control without cell growth inhibition.     

DFT study on the effects of β-cyclodextrin in synthesis of 2-phenylbenzimidazole via benzaldehyde and o-phenylenediamine

The conversion of 2-phenylbenzimidazole using o-phenylenediamine and benzaldehyde can be improved significantly under β-cyclodextrin (β-CD). The density functional theory (DFT) method was applied to study the whole process. According to energy parameters (binding energy, deformation energy) and structural deformation, entry models and the reaction process can be pinpointed, with o-phenylenediamine embedding β-CD from a wide rim, and then benzaldehyde passing into the inclusion from the narrow rim. Subsequently, natural bonding orbital (NBO), Mulliken charge, frontier orbital, FuKui function and nuclear magnetic resonance (NMR) methods were employed to reveal the mechanism of electron transfer. The results illustrate that β-CD plays a catalytic role in synthesis reaction mechanism on the secondary side, improving the reactivity and selectivity of the process.

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Graphical Abstract Density functional theory study of the effects of β-cyclodextrin in synthesis of 2-phenylbenzimidazole via benzaldehyde and o-phenylenediamine

     

Methyl β-cyclodextrin reduces accumulation of reactive oxygen species and cell death in yeast

Stabilized F-actin structures have been shown to be detrimental to both mammalian and yeast cells. In yeast, stabilization of actin caused by addition of jasplakinolide, by point mutations in the act1 gene, or by deletion of certain genes that regulate F-actin leads to cell death with hallmarks of apoptosis. In particular, there is an elevation in the levels of reactive oxygen species, and we have shown the importance of the Ras/cAMP pathway for this effect. Here we show that in yeast cells deleted for end3, which functions to regulate actin organization during endocytosis, treatment of cells with methyl β-cyclodextrin reduces levels of reactive oxygen species and inhibits cell death progression. Methyl β-cyclodextrin is widely used to disrupt lipid rafts that contain cholesterol. The mechanism through which the rescue is achieved was investigated and we demonstrate that methyl β-cyclodextrin reduces accumulation of Ras2 at the plasma membrane in Δend3 cells. We use FRAP and live cell imaging to determine the possible mechanism through which methyl β-cyclodextrin functions to elicit this effect on Ras2 localization. Finally, we demonstrate that addition of methyl β-cyclodextrin to wild-type cells can act to protect cells from acute oxidative stress caused by addition of hydrogen peroxide.