Cell proliferation and cell cycle alterations in oesophageal p53‐mutated cancer cells treated with cisplatin in combination with photodynamic therapy

Objectives: The major goal of anti‐cancer therapies is selective destruction of tumour cells with minimum side effects on normal cells. Towards this aim, combination of different therapeutic modalities has been evaluated for improving control of neoplastic diseases and quality of life for the patient. Photodynamic therapy (PDT) is a procedure for treatment of various types of cancer, but its combination with other established treatments has not been evaluated in detail. We have used KYSE‐510 cells from a human oesophageal carcinoma as an in vitro model to investigate whether cisplatin (CDDP) could be combined with PDT to increase cell death with respect to single treatments. Materials and methods: p53‐mutated KYSE‐510 cells were treated with CDDP alone or in combination with PDT. Analyses of cell viability, cell cycle progression and apoptosis induction were carried out at specific times after treatments. Results: Decrease in cell viability, cell cycle arrest at the G₂/M‐ and S‐phases boundary, and apoptosis induction were observed after single and combined treatments. Conclusions: Our results show that low CDDP doses (0.25–1 μm ) induce cell mortality and cell cycle perturbation, which were more evident when given in combination with PDT, but in contrast to work of other authors no synergistic activity was found. Apoptosis occurred via intrinsic pathways in treated cells, although it did not represent the predominant mode of cell death.     

Development of cell line preservation method for research and industry producing useful metabolites by plant cell culture

The cell culture ofAngelica gigas Nakai producing decursin derivatives and immunostimulating polysaccharides was preserved in liquid nitrogen after pre-freezing in a deep freezer at −70°C for 480 min. The effects of the cryoprotectant and pretreatment before cooling were investigated to obtain the optimal procedure for cyropreservation. When compared to mannitol, sorbitol, or NaCl with a similar osmotic pressure, 0.7M sucrose was found to be the best osmoticum for the cryopreservation ofA. gigias cells. In the pre-culture medium, the cells in the exponential growth phase showed the best post-freezing survival after cryopre-servation. A mixture of sucrose, glycerol, and DMSO was found to be an effective cryoprotectant and a higher concentration of the cryoprotectant provided better cell viability. When compared with the vitrification, the optimum cryopreservation method proposed in this study would seem to be more effective for the long-term storage of suspension cells. The highest relative cell viability established with the optimal procedure was 89%.     

MIR spectroscopic analysis on sugar metabolic and ethanol productive kinetics of suspension TBY-2 and rice cells pre-cultured in various media

The influence of sugars in pre-cultivation media suspended plant cells on the kinetics of the sugar uptake and the ethanol production was studied by mid-infrared spectroscopy using a Fourier transform infrared spectrometer (FT-IR) equipped with an attenuate total reflection accessory (ATR). We performed the plant cell cultivation with Nicotiana tabacum cv. Bright Yellow No.2 (TBY-2) cells and Oryza sativa L., Japonica, cv. Nipponbare (rice) cells, respectively, in pre-culture and culture media, which had various types of glucose, fructose, sucrose or glucose–fructose mixtures. The results confirmed the kinetic differences between the TBY-2 cells and rice cells. These results suggested that the TBY-2 cells consumed sugar before growth and the rice cells consumed sugar after growth, moreover, the ethanol content increased just after cell growth was activated based on the non-dimensional cultivation time for the cell growth behavior.     

The Growth of the Emt6 Tumour In the Lungs of Balb C Mice Following Intravenous Inoculation of Tumour Cells From Culture

The growth of the EMT6 tumour in the lungs of Balb C mice has been studied following intravenous inoculation of different numbers of tumour cells taken from culture. At various times after injection of cells into mice, cell suspensions have been prepared from pairs of lungs and the number of in vitro colony forming cells assayed by plating into petri dishes. Following intravenous injection of 10⁵ cells, the time required for doubling of the number of clonogenic tumour cells appearing in the cell suspension is around 17 hr until such time that the total tumour cell population per set of lungs reaches 10⁸ cells (at 10–12 days). This doubling time has to be corrected for changes in ability to extract cells from the lungs into the cell suspension at various times and also for possible changes in plating efficiency in vitro. When these correction factors are applied, the most likely value for the doubling time of clonogenic tumour cells in the lungs is in the range 20–24 hr. This is a similar figure to that previously deduced for the EMT6 flank tumour during its microscopic period of growth. After reaching a total size of 10⁸ tumour cells, the time for doubling of the number of clonogenic tumour cells in the lung increases. During the later stages of tumour growth a good correlation is seen between total lung tumour weight and the number of clonogenic cells present. For the final 3–4 days of the initial period of rapid tumour growth, it is possible to carry out a haemocytometer count of tumour cells in the lung suspension and hence surviving fraction experiments may be carried out after various forms of treatment. In this way the response to treatment of microscopic tumour foci may be determined.     

The influence of the developing bacterial spore on the mother cell

Mutants unable to develop a completely engulfed forespore do not lose their viability, i.e., their ability to resume cell division, for at least 10 hr after the end of exponential growth. In contrast, mutants, which are blocked at later stages in development and which are able to produce completely engulfed forespores, lose their ability to divide. The time course of this decrease in viability coincides with the time course for the appearance of completely enclosed forespores. Experiments with the sporulating standard strains of Bacillus subtilis and B. megaterium suggest that the mother cells also lose their viability at about the time of forespore enclosure. These results indicate that the forespore, as soon as it is completely engulfed and thus committed to continue differentiation, somehow prevents the mother cell (sporangium) from resumption of growth.     

Harvesting of Arthrospira platensis by flocculation with phytic acid from rice bran

ABSTRACT

The recovery of algal biomass is one of the critical steps involved in the commercial production of beneficial metabolites from Arthrospira platensis. Efficient and safe harvesting methods that do not sacrifice quality of final product are important for commercial application. Phytic acid (PA) is a natural non-toxic phytochemical widely distributed in plant tissues. Effect of PA from rice bran on the growth, trichome morphology such as spiral number and algal filament length, and harvesting efficiency of A. platensis were investigated. Cells aggregated into large cell flocs after the addition of PA in the medium, and algal spiral number and filament length increased. UV-vis spectra indicated the interactions between PA and algal cells. Adding PA at stationary growth phase is a good strategy for harvesting, since no adverse effect to biomass growth and harvesting efficiency. Harvesting efficiency of 95.69% at 0.5% (v/v) PA was superior to other conventional harvesting methodologies.     

Immobilization of Brevibacterium flavum cells on collagen for the production of glutamic acid in a recycle reactor

In this study, live cells of Brevibacterium flavum were immobilized for the production of glutamic acid. The reason for such a choice was that glutamic acid fermentation is an extensively studied fermentation and one which requires the viability of entire cellular faculties for the acid production. Brevibacterium flavum was chosen because it is an industrially used bacterium, and is very potent via a vis glutamic acid production. Studies were performed to find aeration and agitation conditions for optimal growth and glutamic acid productivity. Experiments were also done to find the optimum harvesting time. The cell activity peaks during the run of fermentation, and the time at which the peak occurs, was found. Conventional methods for immobilizing the cells on collagen were found to be lacking. The pH and drying were the two main reasons for loss of viability of the cells; the latter being more important. A modified immobilization procedure has been devised, which can immobilize live cells at any given pH and ionic strength, in contrast to the conventional method which requires the pH to be above 11 or below 3. This new method involves dialysis of collagen in suitable dialysis bags against water at pH7 (or buffer at any desired pH). The dialysed collagen blended at 20,000 rpm, resulted in a very smooth dispersion, unnoticeably different from collagen dispersion prepared at pH 11. The dispersed collagen was then cast and dried at an elevated temperature, and high air flow rate over the cast membrane, decreasing the time of drying from 6–8 hr ( in the conventional method) to 1.5–2 hr. The membrane has been tested for glutamic acid producing capabilities in a column reactor with the membrane spirally wound. The reactor has been operated under continuous conditions for 5–10 days with stable activities.     

Bcl-2 overexpression in CHO cells improves polyethylenimine-mediated gene transfection

Transient gene expression (TGE) in Chinese hamster ovary (CHO) cells with polyethylenimine (PEI) as a transfection reagent has been considered as an attractive method to produce recombinant proteins rapidly for pre-clinical studies. A high level of transfection efficiency, which is required for high-level TGE in CHO cells, can be achieved by increasing the PEI concentration. However, PEI induces cytotoxicity in a dose-dependent manner. To overcome this problem, Bcl-2 protein, an anti-apoptotic protein, was overexpressed in CHO cells (DG44). At a ratio of PEI to DNA (an N/P ratio) of 10, there were no significant differences in transfection efficiency and cell viability between Bcl-2 overexpressing and non-overexpressing cells. The transfection efficiency and cell viability were 2–11% and 83–92%, respectively. However, there were significant differences (P < 0.05) in the transfection efficiency and cell viability between them at a higher N/P ratio. At an N/P ratio of 40, the transfection efficiency and cell viability of Bcl-2 non-overexpressing cells were 24–38% and 35–40%, respectively, while those of Bcl-2 overexpressing cells were 48–53% and 43–56%, respectively. Furthermore, compared with Bcl-2 non-overexpressing cells, more DNAs entered the Bcl-2 overexpressing cells, resulting in a higher rate of TGE per cell. PE-Annexin V apoptosis revealed that Bcl-2 overexpression suppressed PEI-induced apoptotic cell death at high N/P ratios. Taken together, Bcl-2 overexpression in CHO cells suppresses apoptotic cell death during PEI-mediated transient transfection, resulting in enhanced transfection efficiency and TGE.     

Real‐time cell analysis and heat shock protein gene expression in the TcA Tribolium castaneum cell line in response to environmental stress conditions

The rust red flour beetle, Tribolium castaneum (Herbst, 1797) (Coleoptera: Tenebrionidae), is a pest of stored grain and one of the most studied insect model species. Some of the previous studies involved heat response studies in terms of survival and heat shock protein expression, which are regulated to protect other proteins against environmental stress conditions. In the present study, we characterize the impedance profile with the xCELLigence Real‐Time Cell Analyzer and study the effect of increased temperature in cell growth and viability in the cell line BCIRL‐TcA‐CLG1 (TcA) of T. castaneum. This novel system measures cells behavior in real time and is applied for the first time to insect cells. Additionally, cells are exposed to heat shock, increased salinity, acidic pH and UV‐A light with the aim of measuring the expression levels of Hsp27, Hsp68a, and Hsp83 genes. Results show a high thermotolerance of TcA in terms of cell growth and viability. This result is likely related to gene expression results in which a significant up‐regulation of all studied Hsp genes is observed after 1 h of exposure to 40 °C and UV light. All 3 genes show similar expression patterns, but Hsp27 seems to be the most affected. The results of this study validate the RTCA method and reveal the utility of insect cell lines, real‐time analysis and gene expression studies to better understand the physiological response of insect cells, with potential applications in different fields of biology such as conservation biology and pest management.     

Silicon application promotes rice growth and negatively affects development of Spodoptera frugiperda (J. E. Smith)

Silicon (Si) has been reported to enhance plant resistance against biotic and abiotic stressors and also benefit plant growth. These effects are more pronounced in grass species, especially with soil‐applied Si. This study investigated the effects of Si application on rice resistance to Spodoptera frugiperda development and plant vegetative growth. Effects of Si on rice were assessed via soil and foliar applications and compared with untreated plants (control). Si was soil‐ and foliar‐applied as 1% silicic acid solution at a dosage equivalent to 1.4 t Si per ha. After application, leaf material was collected from Si‐treated and untreated plants and placed in Petri dishes with individual S. frugiperda neonate larvae, where development was followed to adult emergence and biological parameters recorded. Vegetative growth parameters recorded in rice plants were the height, chlorophyll content, fresh and dry weights of shoots, and shoot Si content. No effects of Si application were observed on the durations of larval and pupal stages, larval and pupal survival, and sex ratio of S. frugiperda. Insects fed leaves from Si‐treated plants exhibited lower leaf consumption, larval and pupal weights, longevity of males and females, number of eggs, and egg viability. The negative effects were correlated with higher rice Si content. Si application to rice increased plant height, chlorophyll content and dry weight. Our study demonstrates that foliar‐applied Si is as efficient as soil‐applied Si in negatively affecting S. frugiperda development and providing beneficial effects on rice plant growth.     

Intraperiplasmic growth of Bdellovibrio bacteriovorus on heat-treated Escherichia coli.

Heat treatment (55 degrees C for 40 min) of cell suspensions in buffer (ca. 3 x 10(9) cells per ml) of Escherichia coli ML35 caused a 4- to 4.5-log loss of cell viability. Similar results were found for several other E. coli strains that were examined. As a result of this heat treatment, 260-nm- and 280-nm-absorbing materials were released into the suspending buffer, along with about 10% of the total cellular radioactivity, when cells uniformly labeled with (14)C were used. In comparison with untreated cells, heat-treated E. coli ML35 cells showed (i) no significant changes in macromolecular composition other than ca. 22% less RNA content, (ii) an increased permeability to o-nitrophenyl-beta-d-galactopyranoside (a compound to which untreated cells are impermeable), (iii) almost complete loss of respiratory potential, and (iv) substantial losses of numerous glycolytic enzyme activities in cell extracts prepared from these cells. Intraperiplasmic development of Bdellovibrio bacteriovorus 109J with heat-treated E. coli ML35 as substrate cells appeared normal when observed microscopically, although bdellovibrio attachment and resultant bdelloplast formation were slightly retarded. No significant changes were observed in cell yields or in the ratios and contents of DNA, RNA, or protein between bdellovibrios harvested from untreated cells and those from heat-treated substrate cells after single-developmental-cycle growth on these cells. The average Y(ATP) values for intraperiplasmic growth on untreated and heat-treated substrate cells were 16.0 and 17.9, respectively. It is concluded that intraperiplasmic bdellovibrio growth on gently heat-treated E. coli substrate cells is very similar to growth on untreated substrate cells, even though the former substrate cells are nonviable and substantially impaired in many metabolic activities.     

Recovery of ionizing-radiation damage after high doses of gamma ray in the hyperthermophilic archaeon Thermococcus gammatolerans

The recently discovered hyperthermophilic and radioresistant archaeon Thermococcus gammatolerans is of great interest to compare and contrast the impact of its physiology on radioresistance and its ability to repair damaged chromosomes after exposure to gamma irradiation with radioresistant bacteria. We showed that, in contrast to other organisms, cell survival was not modified by the cellular growth phase under optimal growth conditions but nutrient-limited conditions did affect the T. gammatolerans radioresistance. We determined the first kinetics of damaged DNA recovery in an archaeon after exposure to massive doses of gamma irradiation and compared the efficiency of chromosomal DNA repair according to the cellular growth phase, nutrient availability and culture conditions. Chromosomal DNA repair kinetics showed that stationary phase cells reconstitute disrupted chromosomes more rapidly than exponential phase cells. Our data also revealed that this radioresistant archaeon was proficient to reconstitute shattered chromosomes either slowly or rapidly without any loss of viability. These results suggest that rapid DNA repair is not required for the extreme radioresistance of T. gammatolerans. Angels Tapias and Christophe Leplat contributed equally to this work.     

Effect of sodium butyrate on myoblast growth and differentiation.

Myoblasts from L₆ line, after a period of cell division, undergo differentiation into large multinucleated syncitia in 8–9 days. Butyrate was added for 24 hours at various times of culture. In all samples growth was strongly inhibited. After removal of butyrate, growth continued at the same rate for 2 days, afterwards the growth rate became the same as in control cells. Morphological and biochemical differentiations, estimated by creatine phosphokinase assay, occur with a 1–3 day delay according to the time of addition of butyrate, when compared to untreated cells. Only the M form of creatine phosphokinase was present in butyrate-treated cells as in untreated myoblasts.     

Assessing the viability of a clumpy <Emphasis Type="Italic">mnn9</Emphasis> strain of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> used in the manufacture of recombinant pharmaceutical proteins

Demonstration of the viability of cryopreserved cell bank used to make a biopharmaceutical product is an important indicator of the ability to consistently manufacture over a long period of time, and is mandated in regulatory guidances. A mnn9 strain of Saccharomyces cerevisiae, chosen for its inability to hypermannosylate vaccine antigens, has a clumpy growth tendency due to the inactivation of the gene MNN9 (wild-type), complicating the interpretation of conventional viability measurements useful for single cells. Therefore, two growth-based measurements as well as staining by a membrane-impermeable dye were examined for their ability to reflect changes in viability of a clumpy mnn9 (defective) strain. The cell clumps proved to be stable to mixing, and variability of agar-plate-based viable counts (VC) of undisrupted suspensions of this clumpy mnn9 strain was consistent with variability observed for cell banks of a non-clumpy MNN9 strain. Both the VC and the growth times in an oxygen-sensing broth-based microplate assay corresponded well with shake-flask growth times for a set of stressed and unstressed samples, although the correlation was highest between the two broth-based systems. Counts of trypan-blue-stained cells within clumps also increased with time of stress, suggesting that this method could be adapted as a simple index of viability as well.     

Selective cytotoxicity and modulation of apoptotic signature of breast cancer cells by Pithecellobium dulce leaf extracts

We report the potent and selective cytotoxicity of the crude aqueous leaf extract from the medicinal plant, Pithecellobium dulce toward the human breast cancer cells (MCF‐7), but not the normal cells (MCF‐10A). The cytotoxicity was found to be dose and time dependent, as 300 µg/mL of the extract decreased the cell viability to 50% (IC₅₀) in 48 h. The induction of apoptosis in the breast cancer cells after treatment was confirmed by significant percentage (24.7%), of early apoptotic cells (AnnexinV ⁺Propidium Iodide^_) in treated cells as compared to control cells (3.5%). We observed a significant upregulation in the mRNA expression of various pro‐apoptotic gene such as Bax (21.1 folds), p21(14.4 folds), p53 (11.7 folds), TNF (10.2 folds) and fas (6.3 folds) after treatment as compared to untreated cells. On the other hand, the relative mRNA expression of anti‐apoptotic genes such as Bcl‐2, NF‐KB and Cdk was reduced. The selective upregulation of pro‐apoptotic gene and down regulation of specific anti‐apoptotic genes could be the inducing factor for apoptotic cell death in MCF‐7 cells after treatment with the herbal extract. We believe that our findings provide a foundation for further studies on this formulation as a potential therapeutic candidate for breast cancer. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:756–766, 2016     

Electro-enhancement of division of plant protoplast-derived cells

Summary Electric field pulses, ranging from 250 to 2000 V and of 10 to 50 sec duration, were assessed for their effect on the growth in culture of isolated protoplasts ofGlycine canescens, Prunus avium × pseudocerasus, Pyrus communis, Solanum dulcamara andSolanum viarum. Three successive voltage pulses between 250 and 1000 V caused a small decrease in protoplast viability, but promoted cell division and enhanced significantly the plating efficiency. A higher percentage of electro-pulsed protoplasts showed sustained growth in culture to the microcallus stage compared to untreated protoplasts. The rate of cell division was also stimulated in electro-treated protoplasts. These observations are discussed in relation to present knowledge of the effects of electrical treatments on plant and animal cells.     

Physiological manipulation and formulation of the biocontrol yeast Pichia anomala for control of Penicillium verrucosum and ochratoxin A contamination of moist grain

The major hurdle in the production of commercial biocontrol agents (BCAs) has been the lack of production of appropriate formulations. Of particular importance is the conservation of viability and ecological competence after application. With this in mind studies were conducted to develop formulations of P. anomala which would have these attributes. Cells were grown in molasses-based medium modified with proline to different water availability levels (0.98 and 0.96) which significantly increased (up to 50%) the content of trehalose and arabitol in the yeast cells during liquid broth fermentation. The use of isotonic solutions for harvesting the yeast cells further increased the endogenous content of these compatible solutes as well as glycerol. Fluidised bed drying of cells at 30–80°C was carried out for 10 and 20 min and showed that viability was significantly decreased at 70–80°C. A temperature of 50°C for 20 min was found to be best for viability (70%) and moisture content of <10%. Several additives for conservation of viability showed that cotton seed flour+skimmed milk was the best treatment when dried at 50°C. The biocontrol efficacy of formulated P. anomala cells was tested in laboratory scale studies and this showed that they inhibited growth of Penicillium verrucosum and reduce ochratoxin A production in moist wheat grain under some combinations of water availability. Physiologically modified formulated yeast cells with increased levels of trehalose and arabitol gave similar efficacy as fresh cells. This suggests that ecophysiological manipulation of such BCAs can result in improved ecological competence of such formulations and effective biocontrol.     

Effect of partial defoliation during the vegetative phase on subsequent growth and grain yield of maize

Maize was grown in two densities, 2–47 or 4–94 plants m^-2, and the following treatments imposed: untreated, plants partly defoliated 51 days after sowing, and alternate plants in a row partly defoliated 44 days after sowing. Plants flowered about 82 days after sowing. Leaf area was decreased by 60–64% by defoliation on day 51. Defoliation resulted in decreases in grain yield and grain number of 6–17%, though when alternate plants were defoliated in the higher density there was a substantial decrease in yield and number of grains in defoliated plants, which was largely offset by an increase in adjacent intact plants. When plants were defoliated on day 51 subsequent growth in leaf area was similar to, and that in leaf weight nearly as large as that in untreated plants, while increase in stem weight was substantially less than in untreated plants. By the time of flowering untreated and defoliated plots differed by c. 30% in leaf area. Increments of dry matter after flowering differed by c. 15% between untreated and defoliated plots. The fraction of these increments which entered the grain was c. 90% in both untreated and defoliated plots. When alternate plants in the row were partly defoliated on day 44 their subsequent increase in leaf area was probably 5–16% less than that of the adjacent intact plants. Increments of dry matter after flowering of plots with alternate plants defoliated were 93–95 % of those of untreated plots; leaf efficiency after flowering was slightly greater than in untreated plots. The fraction of the dry matter increment after flowering which entered the grain was c. 88 % in both intact and defoliated plants of the small density, but was 94% in intact plants and 86% in defoliated plants of the large density.     

Improved enzymatic isolation of fibroblasts for the creation of autologous skin substitutes

Summary The number of medical applications using autologous fibroblasts is increasing rapidly. We investigated thoroughly the procedure to isolate cells from skin using the enzymatic tissue dissociation procedure. Tissue digestion efficiency, cell viability, and yield were investigated in relation to size of tissue fragments, digestion volume to tissue ratio, digestion time, and importance of other protease activities present in Clostridium histolyticum collagenase (CHC) (neutral protease, clostripain, and trypsin). The results showed that digestion was optimal with small tissue fragments (2–3 mm³) and with volumes tissue ratios ≥2 ml/g tissue. For incubations ≤10 h, the digestion efficiency and cell isolation yields were significantly improved by increasing the collagenase, neutral protease, or clostripain activity, whereas trypsin activity had no effects. However, a too high proteolytic activity of one of the proteases present in CHC digestion solution or long exposure times interfered with cell viability and cell culture yields. The optimal range of CHC proteases activities per milliliter digestion solutions was determined for digestions ≤10 h (collagenase 2700–3900 Mandl U/ml, neutral protease 5100–10,000 caseinase U/ml, and clostripain 35–48 BAEE U/ml) and for longer digestions (>14 h) (collagenase 1350–3000 U/ml, neutral protease 2550–7700 U/ml, and clostripain 18–36 U/ml). Using these conditions, a maximum fibroblast expansion was achieved when isolated cells were seeded at 1×10⁴ cells/cm². These results did not only allow selection of optimal CHC batches able to digest dermal tissue with an high cell viability but also significantly increased the fibroblast yields, enabling us to produce autologous dermal tissue in a clinically acceptable time frame of 3 wk.