Permeabilization of cultivated plant cells by electroporation for release of intracellularly stored secondary products

Summary Plant cell suspension cultures producing secondary metabolites have been permeabilized for product release by electroporation. The two cell cultures studied, i.e. Thalictrum rugosum and Chenopodium rubrum, require about 5 and 10 kV cm^–1, respectively, for complete permeabilization (release of all the intracellularly stored product). The number of electrical pulses and capacitance used had a relatively limited effect on product release while the viability of the cells was strongly influenced by the latter. Conditions for complete product release resulted in total loss of viability of the cells after treatment. The release of product from immobilized cells was also achieved by electroporation. Cells entrapped in alginate required less voltage for permeabilization than free or agarose entrapped cells.     

Permeabilization of plant cells for release of intracellularly stored products: viability studies

Summary The effects of various chemical substances on the permeability of plasma membranes and tonoplasts of three suspension cultures (Catharanthus roseus, Thalictrum rugosum and Chenopodium rubrum) have been studied. The permeability of the plasma membrane is monitored by measuring the activity of the cytosolic enzyme isocitrate dehydrogenase and the permeability of the tonoplast is measured by determining the release of substances stored in the vacuoles (inorganic phosphate, berberine and betanin for the three cell lines, respectively). The minimum concentration required for quantitative release of vacuolar products have been established for five different permeabilization agents. Cell viability is lost upon permeabilization except for treatment of Catharanthus roseus with DMSO and Triton X-100.Abbreviations DMSO dimethylsulfoxide - PEA phenethylalcohol - HDTMAB hexadecyltrimethylammonium bromide - ICDH isocitrate dehydrogenase     

Improvement in cell‐bound phytase activity of Pichia anomala by permeabilization and applicability of permeabilized cells in soymilk dephytinization

Aims: Whole cell permeabilization of Pichia anomala to ameliorate the cell‐bound phytase activity and usability of permeabilized cells in dephytinization of soymilk. Methods and Results: The cells of P. anomala were subjected to permeabilization using the surfactant Triton X‐100 to overcome the permeability barrier and prepare whole cell biocatalysts with high phytase activity. The statistical approach, response surface methodology (RSM) was used to optimize the operating conditions for permeabilization. The treatment of cells with 5% Triton X‐100 for 30 min resulted in c. 15% enhancement in cell‐bound phytase activity. The shrinkage of protoplast was observed, although cell viability and phytase stability were not significantly altered. The free as well as immobilized permeabilized cells hydrolysed soymilk phytate, and the latter could be reused over four consecutive cycles. Conclusions: Whole cell permeabilization of P. anomala using Triton X‐100 led to enhancement in cell‐bound phytase activity. The viability and integrity of yeast cells were not significantly affected because of permeabilization. The permeabilized P. anomala cells effectively dephytinized soymilk, and the permeabilized cells immobilized in alginate could be reused because of sustained phytase activity. Significance and Impact of the Study: This is the first report on the use of permeabilized yeast cells for mitigating phytate content of soymilk. Alginate entrapment of permeabilized P. anomala allows reuse of cells for soymilk dephytinization, thus suggesting a potential application in food industry.     

Structural requirements of anthocyanins in relation to inhibition of endothelial injury induced by oxidized low-density lipoprotein and correlation with radical scavenging activity

Anthocyanins may play an important role in atherosclerosis prevention. However, the structure-function relationships are not well understood. The objective of this study was to compare the inhibitory effect of 21 anthocyanins against oxidized low-density lipoprotein-induced endothelial injury to understand the relationship between anthocyanin chemical structure and the endothelial protective properties, measured as cell viability, MDA production and NO release. Additionally, the intracellular anti-radical activity of the selected anthocyanins was investigated to identify the correlation with endothelial protection. Our results provide evidence that the number of -OH in total or in B-ring, 3′,4′-ortho-dihydroxyl and 3-hydroxyl are the main structural requirements of anthocyanins in suppressing oxidative stress-induced endothelial injury and such inhibitory effect was significantly correlated with the intracellular radical scavenging activity.     

Chemical permeabilization and in situ removal of daidzein from biologically viable soybean (Glycine max) seeds

After 24 h of chemical permeabilization with 20% (v/v) methanol at 25 °C, the amount of daidzein released from soybean seeds is 15 to 20% of the amount (0.0423 ± 0.0045 mg/g seed dry wt) obtained by physical grinding. With this chemical permeabilization condition, 70% of the permeabilized seeds are still able to germinate. The release of daidzein is enhanced to 33% with the addition of XAD-4 to 20% (v/v) methanol without affecting seed viability. © Rapid Science Ltd. 1998     

Enhanced release of rosmarinic acid from Coleus blumei permeabilized by dimethyl sulfoxide (DMSO) while preserving cell viability and growth

Continuous permeabilization of preconditioned Coleus blumei cells with dimethyl sulfoxide (DMSO) is shown to be an effective strategy for the enhanced release of rosmarinic acid (RA) while preserving cell viability. When nonpreconditioned cells were permeabilized with DMSO, they lost their viability at DMSO concentrations higher than a critical value located between 0.1% and 0.5% DMSO. Product release was low [0.49 g RA/100 g dry cell weight (DCW)] at 0.1% DMSO. Preconditioning cells at 0.1% DMSO ensured high viability at DMSO concentrations of 0.5%, 1.0%, and 1.5%. Product release reached a maximum of 2.85 g RA/100 g DCW at 0.5% DMSO, which was 66.4% of the total rosmarinic acid produced. (c) 1992 John Wiley & Sons, Inc.     

Towards large scale methods for the selective release of periplasmic human cystatin C from E. coli

Summary Two promising methods, osmotic shock and guanidine treatment, for the selective release of human cystatin C from the periplasmic space of E. coli were developed at the laboratory scale. High release efficiencies were achievable by both approaches, however, the chemical membrane permeabilization proved to be much more selective. Both methods have excellent potential for scale up.     

Dependence of yeast permeabilization with Triton X-100 on the cell age

Summary Acid phosphatase activity and protein release were determined in cell suspensions ofYarrowia lipolytica andTorulospora delbrueckii at different stages of their growth by permeabilization with Triton X-100. The effect of the surfactant on the cell permeability did not depend on the cell age.     

Effects of industrial storage on the bioreduction capacity of brewer’s yeast

The effects of industrial storage on the changes of the cell viability and the activities of intracellular alcohol dehydrogenase (ADH) and glucose-6-phosphate dehydrogenase (G6PDH) in brewer’s yeast, and the corresponding capacity for the bioconversion of ethyl-3-oxobutanoate (EOB) to ethyl (S)-3-hydroxybutanoate ((S)-EHB), were investigated. The viability of fresh brewer’s yeast cells stored in industrial circulating cooling water at 1–2°C showed 4 and 15% drop after the storage of 7 and 15 days, respectively, after which cells died rapidly. The pretreatment of the stored brewer’s yeast cells by washing and screening significantly enhanced cell viability during industrial storage. The intracellular levels of ADH and G6PDH after permeabilization of these stored cells with cetyltrimetylammonium bromide (CTAB) were much higher, which showed only slight decrease within 2 weeks during the industrial storage. When the stored cells after the permeabilization treatment was used as the biocatalyst at 90–120 g/L, EOB was converted almost completely into enantiopure (S)-EHB with an enantiomeric excess (ee) more than 99% and a yield of over 96%, by fed-batch bioconversion of 560 mM EOB within 6 h. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Recovery of a foreign protein from the periplasm of Escherichia coli by chemical permeabilization.

We have applied the technique of protein release by chemical permeabilization to recover a foreign protein in active form from the periplasm of a recombinant strain of Escherichia coli. The two agents used in our chemical permeabilization scheme, guanidine hydrochloride and Triton X-100, have different modes of action, allowing selectivity in protein release based on intracellular location under different treatment conditions. Specifically, treatment of E. coli C600-1 cells by guanidine alone resulted in 40-fold purification of recombinant beta-lactamase, which is periplasmically expressed in this host. Achieving such high purification in the cell disruption stage could alleviate some of the problems associated with recovery of intracellular products, such as low expression or the need to solubilize cytoplasmic inclusion bodies. Recovery of periplasmic proteins by chemical permeabilization is simpler than by osmotic shock and is less expensive than using enzymatic digestion.     

Beta vulgaris L. suspension cultures permeabilized with triton X-100 retain cell viability and betacyanines production ability: a digital image analysis study

The influence of Triton X-100 on Beta vulgaris L. permeabilized cell culture viability, regrowth, and ability to produce betacyanines was evaluated in this study. A non-destructive method based on the analysis of images in the RGB (red, green, blue) system was developed to estimate betacyanines content. A treatment for 15 min with 0.7 mM Triton X-100 induced the release of 30% of betacyanines without loss of cell viability (>or=70%). After this permeabilization treatment, B. vulgaris cultures regrew normally, reaching a maximum biomass concentration of 48% higher than non-permeabilized cultures after 14 days of culture. Also, maximum betacyanines concentration was only 25% lower than that of non-permeabilized cultures.     

Permeabilization of metabolites from biologically viable soybeans (Glycine max)

Chemical permeabilization has been widely studied for the release useful metabolites from many types of plant cells and tissues. In this study, the effect of 0-30% (v/v) of aqueous methanol solutions were used to permeabilize soybeans for the release of two isoflavonoids: daidzein and genistein. The release of these metabolites increases with increasing methanol concentrations. The amounts of daidzein and genistein released can increase up to 40- and 86-fold, respectively, when incubated in a 30% (v/v) methanol solution for 24 h compared with those incubated with water only. The effect of methanol on the release rates is primarily due to an increase in solubility of the stored daidzein and genistein (14- to 18-fold) inside the seeds, thus maximizing the concentration gradients for metabolite release. However, the viability of the seeds dropped with increase in methanol concentrations and the incubation time. The viability of soybeans (indicated by their ability to germinate) after permeabilization treatment with 0-20% (v/v) methanol solutions was maintained above 80% throughout the 24 h, whereas no seeds were found to be viable when 30% (v/v) methanol solution was used. The permeability coefficients (P) of daidzein and genistein were found to increase as the methanol concentration used was increased. These P values were estimated to range from 1.1 x 10(-)(9) to 1.9 x 10(-)(8) m/s and 1.0 x 10(-)(9) to 1.7 x 10(-)(8) m/s, respectively. The increase in P can be attributed primarily to an increase in the partition coefficient of the metabolites in the soybean seedcoats. An empirical correlation is proposed in which the log P values are described as a function of the metabolite molecular weights and the partition coefficients of the metabolites between octanol and water, K(oct/water), which was modified to include the effect of methanol present. Knowledge obtained from this study will help provide useful selection criteria for chemical permeabilization of plant tissues, such as seeds, with minimal loss in their viability.     

Nanosecond electric pulses cause mitochondrial membrane permeabilization in Jurkat cells

Nanosecond, high‐voltage electric pulses (nsEP) induce permeabilization of the plasma membrane and the membranes of cell organelles, leading to various responses in cells including cytochrome c release from mitochondria and caspase activation associated with apoptosis. We report here evidence for nsEP‐induced permeabilization of mitochondrial membranes in living cells. Using three different methods with fluorescence indicators—rhodamine 123 (R123), tetramethyl rhodamine ethyl ester (TMRE), and cobalt‐quenched calcein—we have shown that multiple nsEP (five pulses or more, 4 ns duration, 10 MV/m, 1 kHz repetition rate) cause an increase of the inner mitochondrial membrane permeability and an associated loss of mitochondrial membrane potential. These effects could be a consequence of nsEP permeabilization of the inner mitochondrial membrane or the activation of mitochondrial membrane permeability transition pores. Plasma membrane permeabilization (YO‐PRO‐1 influx) was detected in addition to mitochondrial membrane permeabilization. Bioelectromagnetics 33:257–264, 2012. © 2011 Wiley Periodicals, Inc.     

Light-enhanced caffeic acid derivatives biosynthesis in hairy root cultures of <Emphasis Type="Italic">Echinacea purpurea</Emphasis>

Light plays an important role in almost all plant developmental processes and provides the fundamental building blocks for growth, development, primary and secondary metabolism. The effects of light on growth rate and caffeic acid derivative (CADs) biosynthesis in hairy root cultures of Echinacea purpurea (Moench) were assessed. Light-grown hairy roots accumulated increased levels of anthocyanins, which became visible in outer cell layer of the cortex as a ring of purple color. The light-grown root cultures also had radially thickened morphology compared with the dark-grown controls. The growth rate and cell viability of the hairy root cultures in light did not show obvious difference in comparison with those in dark. However, biosynthesis of CADs including cichoric acid, caftaric acid, chlorogenic acid and caffeic acid was significantly increased in hairy root cultures grown in the light. The enhanced accumulation of CADs and anthocyanins in E. purpurea hairy root cultures was correlated to an observed light-stimulated activity of phenylalanine ammonium lyase (PAL).     

Protective effects of Anthocyanins against Amyloid beta-induced neurotoxicity in vivo and in vitro

Alzheimer's disease (AD) is one of the most common neurodegenerative disorders in recent world, characterized by increased production of amyloid beta in the nervous system with an ultimate effect of apoptotic neurodegeneration. This study was aimed to investigate the neuroprotective effect of black soybean anthocyanins in a neurodegenerative model of amyloid beta 1–42 (Aβ_1–42). Aβ_1–42 was treated to HT22 cell lines or adult male rats via intra-cerebro-ventricular injection to induce neurotoxicity in these experimental models. Anthocyanins were treated 0.2 mg/kg in case of cell lines or 4 mg/kg intragastrically to adult rats to protect against Aβ-induced neurodegeneration. Assay for cell viability, mitochondrial membrane potential (Ψm), intracellular free Ca²⁺ and apoptotic cells (fluoro-jade B and TUNEL) were performed in vitro while western blot analyses were performed to the hippocampal proteins of adult rats. Our results showed that Aβ_1–42 treatment reduced cell viability, disturbed the Ψm and Ca²⁺ homeostasis in and out of the cell, and increased neuronal apoptosis. Treatment with anthocyanins for 12 hr retained the cell viability, normalized Ψm and Ca²⁺ level, and decreased the neuronal cell death. In accordance, anthocyanins reversed Aβ-induced effect on protein expression of mitochondrial apoptotic pathway (Bax, cytochrome C, caspase-9 and caspase-3) and major Alzheimer's markers i.e. Aβ, APP, P-tau and BACE-1. Overall, our results showed that anthocyanins are potential candidates to treat neurodegenerative disorders like AD.     

Inactivation of Escherichia coli and Lactobacillus plantarum in relation to membrane permeabilization due to rapid chilling followed by cold storage

The relationship between membrane permeabilization and loss of viability by chilling depending on the chilling rate was investigated in two bacterial models: one Gram-positive bacterium, Lactobacillus plantarum, and one Gram-negative bacterium, Escherichia coli. Cells were cold shocked slowly (2°C/min) or rapidly (2,000°C/min) from physiological temperature to 0°C and maintained at this temperature for up to 1 week. Loss of membrane integrity was assessed by the uptake of the fluorescent dye propidium iodide (PI). Cell death was found to be strongly dependent on the rate of temperature downshift to 0°C. Prolonged incubation of cells after the chilling emphasized the effect of treatment on the cells, as the amount of cell death increased with the length of exposure to low temperature, particularly when cells were rapidly chilled. More than 5 and 3-log reductions in cell population were obtained with L. plantarum and E. coli after the rapid cold shock followed by 7-day storage, respectively. A correlation between cell inactivation and membrane permeabilization was demonstrated with both bacterial strains. Thus, loss of membrane integrity due to the chilling treatments was directly involved in the inactivation of vegetative bacterial cells.     

Chitosan permeabilizes the plasma membrane and kills cells of Neurospora crassa in an energy dependent manner

Chitosan has been reported to inhibit spore germination and mycelial growth in plant pathogens, but its mode of antifungal action is poorly understood. Following chitosan treatment, we characterized plasma membrane permeabilization, and cell death and lysis in the experimental model, Neurospora crassa. Rhodamine-labeled chitosan was used to show that chitosan is internalized by fungal cells. Cell viability stains and the calcium reporter, aequorin, were used to monitor plasma membrane permeabilization and cell death. Chitosan permeabilization of the fungal plasma membrane and its uptake into fungal cells was found to be energy dependent but not to involve endocytosis. Different cell types (conidia, germ tubes and vegetative hyphae) exhibited differential sensitivity to chitosan with ungerminated conidia being the most sensitive.     

Electric Field Orientation for Gene Delivery Using High-Voltage and Low-Voltage Pulses

Electropermeabilization is a biological physical process in response to the presence of an applied electric field that is used for the transfer of hydrophilic molecules such as anticancer drugs or DNA across the plasma membranes of living cells. The molecular processes that support the transfer are poorly known. The aim of our study was to investigate the effect of high-voltage and low-voltage (HVLV) pulses in vitro with different orientations on cell permeabilization, viability and gene transfection. We monitored the permeabilization with unipolar and bipolar HVLV pulses with different train repetition pulses, showing that HVLV pulses increase cell permeabilization and cell viability. Gene transfer was also observed by measuring green fluorescent protein (GFP) expression. The expression was the same for HVLV pulses and electrogenotherapy pulses for in vitro experimentation. As the viability was better preserved for HVLV-pulsed cells, we managed to increase the number of GFP-expressing cells by up to 65?% under this condition. The use of bipolar HVLV train pulses increased gene expression to a higher extent, probably by affecting a larger part of the cell surface.     

Carbon monoxide prevents hepatic mitochondrial membrane permeabilization

Background  

Low concentrations of carbon monoxide (CO) protect hepatocytes against apoptosis and confers cytoprotection in several models of liver. Mitochondria are key organelles in cell death control via their membrane permeabilization and the release of pro-apoptotic factors.     

Catalases and thioredoxin peroxidase protect Saccharomyces cerevisiae against Ca(2+)-induced mitochondrial membrane permeabilization and cell death

The involvement of reactive oxygen species in Ca(2+)-induced mitochondrial membrane permeabilization and cell viability was studied using yeast cells in which the thioredoxin peroxidase (TPx) gene was disrupted and/or catalase was inhibited by 3-amino-1,2, 4-triazole (ATZ) treatment. Wild-type Saccharomyces cerevisiae cells were very resistant to Ca(2+) and inorganic phosphate or t-butyl hydroperoxide-induced mitochondrial membrane permeabilization, but suffered an immediate decrease in mitochondrial membrane potential when treated with Ca(2+) and the dithiol binding reagent phenylarsine oxide. In contrast, S. cerevisiae spheroblasts lacking the TPx gene and/or treated with ATZ suffered a decrease in mitochondrial membrane potential, generated higher amounts of hydrogen peroxide and had decreased viability under these conditions. In all cases, the decrease in mitochondrial membrane potential could be inhibited by ethylene glycol-bis(beta-aminoethyl ether) N,N, N',N'-tetraacetic acid, dithiothreitol or ADP, but not by cyclosporin A. We conclude that TPx and catalase act together, maintaining cell viability and protecting S. cerevisiae mitochondria against Ca(2+)-promoted membrane permeabilization, which presents similar characteristics to mammalian permeability transition.